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WO2011072065A2 - Capteurs utilisant des microbes pour la surveillance de l'environnement - Google Patents

Capteurs utilisant des microbes pour la surveillance de l'environnement Download PDF

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Publication number
WO2011072065A2
WO2011072065A2 PCT/US2010/059554 US2010059554W WO2011072065A2 WO 2011072065 A2 WO2011072065 A2 WO 2011072065A2 US 2010059554 W US2010059554 W US 2010059554W WO 2011072065 A2 WO2011072065 A2 WO 2011072065A2
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Prior art keywords
nitrate
sensor
target compound
oxygen
bio
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WO2011072065A3 (fr
WO2011072065A8 (fr
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Justin Buck
Matthew Silver
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Intact Labs LLC
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Intact Labs LLC
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Priority claimed from PCT/US2010/025224 external-priority patent/WO2010147683A1/fr
Priority to CA2783256A priority Critical patent/CA2783256C/fr
Priority to US13/514,817 priority patent/US9046478B2/en
Priority to EP10836646.9A priority patent/EP2510345A4/fr
Priority to AU2010328173A priority patent/AU2010328173B9/en
Priority to JP2012543263A priority patent/JP5763092B2/ja
Application filed by Intact Labs LLC filed Critical Intact Labs LLC
Publication of WO2011072065A2 publication Critical patent/WO2011072065A2/fr
Publication of WO2011072065A3 publication Critical patent/WO2011072065A3/fr
Anticipated expiration legal-status Critical
Publication of WO2011072065A8 publication Critical patent/WO2011072065A8/fr
Priority to US14/694,082 priority patent/US9551685B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/4166—Systems measuring a particular property of an electrolyte
    • G01N27/4168—Oxidation-reduction potential, e.g. for chlorination of water
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/186—Water using one or more living organisms, e.g. a fish
    • G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/404—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/0037—NOx
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/0044—Sulphides, e.g. H2S
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/0054—Ammonia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/182—Specific anions in water
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present invention relates to the field of environmental monitoring, and in particular to the development bio-electrochemical sensors for monitoring chemical compound levels in surface water.
  • UV spectrophotometric devices
  • ISE ion-selective electrodes
  • UV sensors are the industry standard for continuous monitoring. They operate by scanning for dissolved nitrate molecules using a 21 Onm wavelength light and have a 95% confidence interval for a thirty second scan of 0.2uM (Johnson and Coletti, 2002). Thus, these systems are typically used for deployments which require high sensing precision with minimal maintenance schedules. They have been used for long-term monitoring in drinking water, freshwater, watershed, marine, and wastewater applications where they are deployed for their high sensitivity (Hach, 2009). There are a variety of UV sensor systems available, along with customized packages, such as sondes, in which they can be installed, but the common
  • ISE sensors cost less than the UV sensors; at less than $500 (NexSense WQSensors' Nitrate ISE sensor, Hach ISE sensor).
  • They have several drawbacks which make them less applicable in critical environments. Their main limitation is sensitivity to temperature and a severe interference by ionic compounds. This leads to one of the most limiting factors for widespread use: their poor performance in marine environments. Since ISEs need to be calibrated often and readings quickly become a function of other ionic compounds'
  • Electrode tip ($75-$ 150) needs to be replaced twice a year, quickly adding to over 30% of the initial capital used for upkeep within the first year.
  • the present invention provides novel, low-cost, real-time bio-electrochemical sensors for environmental monitoring, particularly surface water monitoring.
  • the sensors of the invention are based on the metabolism of one or more types of electricigenic/electrogenic microorganisms and provide an inexpensive, durable, and accurate tool useful to monitoring agencies, research institutions, watershed management organizations, and farmers concerned with monitoring and controlling nutrient run-off over large coverage areas.
  • the sensors of the invention utilize one or more electrogenic microbes which selectively interact with a working electrode in the sensor to produce an electrical current in the absence of the target compound to be detected.
  • electrogenic microbes which selectively interact with a working electrode in the sensor to produce an electrical current in the absence of the target compound to be detected.
  • concentration of the target compound increases, the electrical current produced by the bio-electrochemical sensor decreases.
  • a biofilm comprising a plurality of electrogenic microbes is used in the sensor.
  • the working electrode in the sensors of the invention can be either an anode or a cathode. Where the working electrode is an anode, the sensors of the invention further comprise a controlled chemical cathode, and said target compound is selectively reduced in the sensor.
  • target compounds examples include electron acceptors such as oxygen, nitrite (N0 2 " ), nitrate (N0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ) (e.g., oxidized arsenic), uranium (VI), Molybdenum (VI), Vanadium (V or IV), Vanadate, selenate, selenite, Sulfate (S0 4 " ), elemental sulfur (S ), chromium (VI), phosphate, phosphite, and/or iron (III).
  • the target compound for detection by the sensors of the invention is nitrate.
  • the sensor further comprises a controlled chemical anode, and said target compound is selectively oxidized in the sensor.
  • target compounds that can be detected in such a configuration include electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide, iron (II), arsenite, elemental sulfur (S°), chromium (III), uranium (III), molybdenum (V), Vanadium (IV or III), vanadate, reduced phosphate a carbohydrate, and glucose.
  • electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide, iron (II), arsenite, elemental sulfur (S°), chromium (III), uranium (III), molybdenum (V), Vanadium (IV or III), vanadate, reduced phosphate a carbohydrate, and glucose.
  • the electrogenic microbe utilized in the sensors of the invention can vary, depending on the desired target compound(s) to be monitored.
  • known or genetically engineered electrogenic microbes incapable of using one or more of the following compounds can be used in the sensors of the invention: oxygen, nitrite (N0 2 " ), nitrate (N0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 , uranium (VI), Molybdenum (VI), Vanadium (V or IV), Vanadate, selenate, selenite, Sulfate (S0 4 " ), elemental sulfur (S°), chromium (VI), phosphate, phosphite, iron (III), hydrogen (H 2 ), methane (CH 4 ), ammonium ((NH 4 + ), ammonia (NH 3 ), sulfide, iron (II), arsenite, chromium (III), uranium (III), molyb
  • an electrogenic microbe having an inability to utilize nitrite (N0 2 " ) and/or nitrate (N0 3 " ) is used in a sensor according to the invention.
  • An example of an electrogenic microbe that is unable to utilize (N0 2 ⁇ ) and/or nitrate (N0 3 " ) includes, but is not limited to Shewanella oneidensis ⁇ ' .
  • At least two electrogenic microbes are utilized in a sensor according to the invention.
  • the two electrogenic microbes each have a different respiratory phenotype.
  • a mutant strain of an electrogenic microbe having an altered respiratory phenotype, and the corresponding wildtype strain of the electrogenic microbe can be used together in the sensor of the invention.
  • a non-limiting example of such mutant and wildtype strains includes, without limitation, wildtype Shewanella oneidensis and a mutant Shewanella oneidensis such as Shewanella oneidensis
  • the bio-electrochemical sensors of the invention may include a region for
  • sensing/detecting a first target compound a separate region for sensing/detecting a second target compound, a sample region, and/or a media re-circulation system.
  • the sample region and the first and second sensor regions are separated by a selectively permeable barrier, such as a filter or a porous membrane.
  • the first and/or second target compounds for detection are preferably in either a gaseous or an aqueous phase.
  • Each of the sensing regions include a unique anode chamber and a unique cathode chamber.
  • the bio-electrochemical sensor of the invention is designed for detecting/monitoring nitrate. In another particular embodiment, the bio-electrochemical sensor of the invention is designed for detecting/monitoring nitrate and oxygen.
  • Figure 1 is a schematic illustrating the design of a microbially-based sensor according to the invention.
  • Figure 2 is a schematic illustrating the sensor region of a microbially-based sensor according to the invention.
  • Figure 3 is a schematic illustrating the media recirculation system of a microbially-based sensor according to the invention.
  • Microbial fuel cells take advantage of bacteria that normally use insoluble metal deposits as electron sinks during anaerobic consumption of reduced substrates. By substituting an electrode for the metal deposits, current can be collected, induced to flow through external circuitry and do electrical work before ultimately arriving in the cathode chamber where electrons reduce a terminal electron acceptor, often oxygen. MFCs generally fall under the category of proton exchange membrane fuel cells (PEMFCs) because a membrane divides the anode and cathode half reactions and allows only selective passage of cations to balance the charge carried by electrons arriving at the cathode through the circuitry.
  • PEMFCs proton exchange membrane fuel cells
  • the driving force for electron flow or current is derived from the electrochemical redox difference between the oxidation reaction in the anodic half-cell and the reduction reaction of the terminal electron acceptor at the cathode.
  • Electrogenic microbes seem to be ubiquitous in nature, as sediments in many locations provide the microbes to generate electrical currents (Holmes et al, 2004).
  • Microbes utilize several mechanisms to release electrons to the anode: (i) through dissolved exogenous mediator or electron shuttle compounds; (ii) by interspecies electron transfer involving membrane
  • Microbial fuel cell performance varies according to a number of physical and chemical parameters of the device, including substrate concentration, temperature and redox state. While perturbations to voltage or amperage output may be deleterious in some applications, this quality also allows MFCs to function as biological sensors for parameters that affect their performance. Anode or cathode substrate concentration is probably the most convenient sensor input.
  • MFCs have been utilized in a number of studies to report on concentration of biological oxygen demand (BOD) or lactate.
  • BOD biological oxygen demand
  • a two-chamber MFC containing a potassium ferrocyanide electron acceptor in the cathode was dosed with lactate in the anode containing Shewanella sp. bacteria; the rate of increase of current production was correlated to lactate dosage (Kim et al., 1999).
  • a more generalized sensor utilized an air- cathode MFC colonized by Shewanella sp. bacteria was used as a sensor for BOD concentration in starch processing plant wastewater by correlated total Coulombs generated in batch mode performance to the starting BOD of the wastewater (Kim et al., 2003).
  • this sensor used an MFC device that had been established for over 4 years, and showed reproducibility of +/- 13% in sensing BOD concentrations over the course of almost 1 year, demonstrating that MFC- based sensors have a long usable lifetime (Kim et al., 2003).
  • the senor of the present invention is capable of targeting the availability of electron acceptors in a liquid or gaseous solution.
  • the basic concept behind the sensor is the preferential and hierarchical use of terminal electron acceptors or electron donors in solution (aqueous or gaseous) by microorganisms at either the anode or cathode region, depending on the configuration of the sensor.
  • the sensors of the invention further comprise a controlled chemical cathode, and said target compound is selectively reduced in the sensor.
  • target compounds that can be detected in such a configuration include electron acceptors such as oxygen, nitrite (N0 2 " ), nitrate (N0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), uranium (VI), Molybdenum (VI), Vanadium (V or IV), Vanadate, selenate, selenite, Sulfate (S0 4 " ), elemental sulfur (S°), chromium (VI), phosphate, phosphite, and/or iron (III).
  • electron acceptors such as oxygen, nitrite (N0 2 " ), nitrate (N0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), uranium (VI), Molybdenum (VI), Vanadium (V
  • the sensor further comprises a controlled chemical anode, and said target compound is selectively oxidized in the sensor.
  • target compounds include electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide (e.g., H 2 S, HS. and S " ), iron (II), arsenite, elemental sulfur (S ), chromium (III), uranium (III), molybdenum (V), Vanadium (IV or III), vanadate, reduced phosphate a carbohydrate, and glucose.
  • electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide (e.g., H 2 S, HS. and S " ), iron (II), arsenite, elemental sulfur (S ), chromium (III), uranium (III), molybdenum (V), Vana
  • microbes have the ability to respire with numerous electron acceptors, including but not limited to, oxygen, nitrite (N0 2 " ), nitrate ( ⁇ 0 3 ' ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), and uranium.
  • electron acceptors including but not limited to, oxygen, nitrite (N0 2 " ), nitrate ( ⁇ 0 3 ' ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), and uranium.
  • microbes In the presence of multiple electron acceptors, microbes typically regulate respiratory pathways to utilize the most energetically favorable electron acceptors first. This hierarchical use of electron acceptors often has several levels. For example, E. coli, a well- studied model organism, respires to oxygen even in the presence of all other suitable electron acceptors. If oxygen is depleted and nitrate is present, nitrate will be used before other electron
  • E. coli is not an optimal organism for use in a bio-electrochemical cell-based sensor due to a limited ability to utilize an electrode for respiration and inclination for fermentation.
  • Shewanella oneidensis (S. oneidensis), on the other hand, has been shown to be effective for conductive anodic biofilm formation (Biffinger et al, 2007) and extracellular transfer at 500 W/m 3 (Ringseisen et al 2006).
  • High power density is an important feature as sensing performance will depend on attenuation of the electrical output of the cell.
  • S. oneidensis is capable of utilizing a wide range of terminal electron acceptors (Bretschger et al, 2007; Kim et al, 1999; DiChristina 1992; Arnold, et al 1990) and incapable of fermentation (Kostka and
  • S. oneidensis has also been shown to produce a differential response in electron acceptor utilization (Belleav et al, 2005). These factors make S. oneidensis a model organism for bioelectrochemical cell-based sensing of electron acceptors.
  • the sensors of the invention utilize one or more electrogenic microbes having an altered respiratory phenotype.
  • the altered phenotype can be an altered electron acceptor utilization capacity, or an altered electron donor utilization capacity, depending on the configuration of the sensor, as previously described.
  • the electrogenic microbe can be a known wildtype or mutant microbe, or a genetically engineered microbe that is incapable utilizing electron acceptors such as oxygen, nitrite (N0 2 " ), nitrate ( 0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), uranium (VI), Molybdenum (VI), Vanadium (V or IV), Vanadate, selenate, selenite, Sulfate (S0 4 " ), elemental sulfur (S°), chromium (VI), phosphate, phosphite, and/or iron (III).
  • electron acceptors such as oxygen, nitrite (N0 2 " ), nitrate ( 0 3 " ), fumarate, dimethyl sulfoxide, Arsenate (As0 4 "3 ), uranium (VI), Molybdenum (VI), Vanadium (V or IV), Vanadate, selenate
  • the electrogenic microbe can be a known wildtype or mutant microbe, or a genetically engineered microbe that is incapable of utilizing electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide (e.g., H 2 S, HS. and S"), iron (II), arsenite, elemental sulfur (S ), chiomium (III), uranium (III), molybdenum (V), Vanadium (IV or III), vanadate, reduced phosphate a carbohydrate, and/or glucose.
  • electron donors such as hydrogen (H 2 ), methane (CH 4 ), ammonium (NH 4 + ), ammonia (NH 3 ), sulfide (e.g., H 2 S, HS. and S"), iron (II), arsenite, elemental sulfur (S ), chiomium (III), uranium (III), molybdenum (V
  • Suitable electrogenic microbes that can be used in the sensors of the invention include strains of Shewanella, Psuedemonas, Geobacter, and Cyanobacteria, including known or genetically engineered mutants thereof.
  • FIG. 1 One embodiment of a microbially-based sensor of the invention is depicted in Figure 1.
  • the sensor shown in Figure 1 is designed to detect the amount of oxygen and nitrate present in a sample by determining the distance over which they are capable of diffusing in a controlled chamber before being consumed in respiration. It should be noted that the sensors of the invention are not limited to detecting nitrate and/or oxygen.
  • the sensor depicted in Figure 1 and described in Example 1 below is merely one example of a sensor designed in accordance with the invention.
  • the sensor is divided into two distinct regions on the interior; the first is the oxygen sensor, and the second is the nitrate sensor. Each region shares a common diffusion chamber, but contains unique anode and cathode chambers. Diffusion of oxygen and nitrate through the sensor is shown from left to right.
  • oxygen is consumed until completion resulting in different levels of current produced on the oxygen bioelectrodes.
  • the nitrate is unaltered in the first chamber because the mutant strain is incapable of using it as a terminal electron acceptor.
  • the second chamber it is consumed until completion resulting in different levels of current produced on the nitrate bioelectrodes.
  • the diffusion chamber is maintained sterile by excluding microbes from the external sample and bioanode chambers using a barrier with pore size of less than 0.2 microns, which simultaneously prevents cross-contamination of the sensor and sample.
  • this sensor design is an instantiation of a platform design that can be extended to additional electron acceptors. Through the incorporation of additional mutant strains, species, and chambers, a wide range of electron acceptors can be measured. While the first targets are oxygen and nitrate, in the future the sensor could be modified to address water contaminant such as arsenic and uranium for different applications. Genetically engineered microbes having altered respiratory phenotypes have been described. For example, S.
  • oneidensis carrying the mutation napA has been shown to be incapable of respiring or assimilating nitrate and/or nitrite (Cruz-Garcia 2007).
  • Other microbes can be genetically engineered to carry mutations which render them incapable of utilizing oxygen, arsenic, chromium, sulfate, fumarate, phosphate, and/or iron, and can be used in the sensors of the invention.
  • microbes having altered electron donor utilization capacity can be genetically engineered for use in the sensors of the invention.
  • One or more electricigenic/electrogenic microbes can be used in sensors of the invention.
  • the microbes utilized in the sensors of the invention can vary, depending on the desired target chemical/compound to be monitored. In a preferred embodiment, at least two microbes having different respiratory phenotypes are used in sensors of the invention.
  • the target chemical desired to be monitored is nitrate
  • S. oneidensis carrying the mutation napA ' can be used in the oxygen anode, which renders it incapable of respiring or assimilating nitrate and/or nitrite (Cruz-Garcia 2007), and a wild type strain of S.
  • oneidensis can be used in the nitrate anode, which renders it capable of using both oxygen and nitrate and/or nitrite.
  • oxygen and nitrate diffuse into the sensor.
  • the microbes at the beginning of the oxygen anode experience concentrations of oxygen and nitrate near levels outside the sensor. These microbes will readily make use of the oxygen as a preferred electron acceptor.
  • S. oneidensis decreases the amount of electron transfer to an external electrode, producing a measurable signal of oxygen presence.
  • the oxygen As the oxygen is consumed by microbes at the beginning of the oxygen anode, the amount of oxygen diffusing down the chamber decreases and subsequent microbes experience a reduced oxygen concentration. Progressing further into the sensor, oxygen becomes depleted and the microbes are dependent on respiration to the electrode to maintain metabolic activity. The nitrate level is unaltered by the oxygen anode because the napA strain is incapable of using nitrate and/or nitrite.
  • the concentration of electron acceptors within the diffusion chamber can be modeled using principles of transport phenomenon and reaction kinetics.
  • the generalized form of the mass transfer e uation is:
  • C is the concentration of chemical species (oxygen or nitrate)
  • D is the diffusivity of the chemical species in water
  • R is the rate of reaction (consumption) of the chemical species
  • t is time
  • x, y, and z are the Cartesian dimensions.
  • This equation is a second order linear homogeneous differential equation with a general solution of the form:
  • the length over which the electron acceptor is depleted can be fine tuned by altering several of the geometric parameters of the sensor, the most prominent being the height of the diffusion chamber. As the height of the diffusion chamber increases, the volume of liquid (and thus the total amount of electron acceptor) increases while the surface area for the consumption reaction (and thus the rate of reaction) remains constant. Alternatively, by altering the anode chamber volume and the surface area of the electrodes it is possible to modify the electron acceptor consumption rate that will affect the length over which the diffusion gradient is established. For simplicity, these factors have not been analyzed here, but will be addressed by future modeling efforts.
  • the present invention provides microbial fuel cell-based environmental sensors that also fulfills the power requirements needed to remotely transmit gathered data.
  • Several aspects of the sensor design make it amenable to such a system.
  • the MFC will function and produce power over a period of time likely measured in months or years.
  • a substrate-enhanced MFC was successfully operated for a period of 10 days with no loss in power output, and longevity could be improved at least linearly with the amount of chitin utilized (Rezaei et al., 2007).
  • the sensor of the invention operates so that relatively constant power is produced in the absence of an increased oxygen or nitrate stimulus, and is designed so that concentrations of these interfering chemicals will produce only an incomplete reduction in power output that is still sufficient to generate a transfer of information remotely from the sensor. In the event of an extreme and sustained oxygen or nitrate plume sufficient to completely ablate power production, such a situation would very likely warrant operator intervention to remediate the offending pollutants.
  • capacitors must be used to store the steady trickle of charge for a burst of signaling activity, but this technology is well developed and practical considerations have been previously addressed (Shantaram et al., 2005).
  • the magnitude of the sensor response will be correlated to increased current magnitude in one or several of the anode electrode circuits.
  • There are several potential methods available to sense current variations within a wire including Hall effect sensors that produce a charge when current is passed through a permanent magnet, or using a simple operational amplifier-based circuit, currents from 0-200 nA can be reliably sensed by a device costing under $10 to build (Linear Technology, 2010).
  • the configuration of electrical components and current meters may vary, depending on cost, performance and robustness of the components.
  • Circuit design for signal analysis - signal derived from the individual anode wires for each sensor are accumulated and processed to determine the concentration of oxygen and nitrate.
  • Microbe genetic drift - being essential to sensor performance, stability of the microbial community is preserved by preventing cross-contamination using impermeable barriers and seeding the anode with a genetically stable strain.
  • Biomass accumulation in the sensor - cellular debris may accumulate in the sensor and biofilms may form on barriers between chambers.
  • Microbial fuel cells can sustain long-term stable performance (Kim et al., 2003), but careful design is needed to prevent fouling.
  • the microbially-based sensors of the present invention are particularly useful for monitoring agricultural run-off, with an emphasis on regions with high usage subsurface tile drainage systems. Given the low cost, durability and sensitivity of the sensor design, the sensors of the invention are useful tools for government agencies, including regulators such as the EPA and the U.S. Department of Agriculture (USDA), agricultural consultants, and individual agricultural holders alike.
  • regulators such as the EPA and the U.S. Department of Agriculture (USDA), agricultural consultants, and individual agricultural holders alike.
  • the microbially-based sensors of the present invention are particularly useful for watershed monitoring and management.
  • Watershed monitoring and data collection appears to be growing as part of the core mission of watershed management associations across the USA.
  • District, city and/or state sponsored watershed management associations are now conducting monitoring activities of local streams, rivers, ponds, reservoirs, and lakes.
  • Such associations typically monitor monthly and rely on volunteers and external lab analysis.
  • the low cost, durability and sensitivity of the sensor design make the sensors of the present invention an ideal tool for use by such watershed management associations.
  • the microbially-based sensors of the invention can also be applied in the field of wastewater treatment.
  • Example 1 Development of a microbially-based nitrate sensor
  • the present example demonstrates the feasibility of using the preferential electron acceptor utilization of Shewanella oneidensis as the basis for a microbial sensor. Specifically, the present example aims to:
  • the sensor is developed in four parallel steps according to the method outlined below.
  • Step 1 Construct the test cells
  • test cells for Phase I is composed of two components, the sensor region and the media recirculation system ( Figure 2).
  • the general design of these components is the same for testing the oxygen and nitrate sensors.
  • Three test cells are fabricated: the oxygen sensor test cell, the nitrate sensor test cell, and the no-acceptor control cell.
  • the sensor region ( Figure 2) is composed of a sample chamber, a diffusion chamber, an anode chamber, and a cathode chamber separated by ion exchange membranes or sterile filters.
  • the structure for each of these chambers is made from a biocompatible polymer material with limited oxygen diffusivity.
  • the sterile filters separating the diffusion chamber from the sample chamber and the anode chamber are made out of a filter of pore size 0.2 urn (Pall Supor filter) to ensure no microbial contamination from the anode chamber into the diffusion chamber, sample chamber, and media reservoir. All membranes and plastic pieces are sealed in the sensor assembly using rubber gaskets to prevent leakage and air introduction. All parts for the sensor region are sterilized prior to each experiment.
  • the sample chamber contains inlet and outlet ports to allow for the constant exchange of fresh media, replenishing both the nutrient supply and the level of the electron acceptor being studied (oxygen or nitrate). Flow through the sample chamber is tangential to the filter, which minimizes any convective flow in the anode chamber while maintaining constant sample concentrations for the establishment of the diffusion gradient.
  • the anode chamber design has a large length to height aspect ratio to ensure depletion of the electron acceptor and to increase the sensor sensitivity.
  • Anode wires are aligned parallel to the width dimension and extend across the entire chamber to ensure a unique but uniform environment at each wire. The sensitivity and statistical certainty and accuracy directly correlates to the number of wires but increases sensor complexity in cost; this issue is addressed during construction.
  • the anode wires are adjacent to the filter separating the diffusion chamber from the anode chamber. This allows the microbes colonizing the wires to experience the oxygen concentration directly across the filter in the diffusion chamber without consumption and interference from microbes living in the bulk.
  • the cathode directly opposes the anode and is equally separated from each anode wire.
  • Each anode wire corresponds to a directly opposing cathode lead. This uniform geometry, along with excess cathode capacity, ensures that only the composition of the media will cause variance in anode wire performance.
  • the ion exchange membrane separating the anode chamber from the cathode is made of National to mimmize oxygen permeation into the anode chamber and minimize MFC internal resistance.
  • the membrane is incorporated directly with the cathode in a membrane electrode assembly composed of a platinum catalyst.
  • the cathode is operated as a standard air cathode. If oxygen leakage into sensor is suspected as a problem, the platinum catalyzed air cathode is replaced with an anaerobic cathode half-cell.
  • Maintaining a constant concentration of electron acceptor and nutrients is essential to testing the sensor design. This is achieved using a large reservoir of media and re-circulating the media through the sample chamber at a high rate.
  • the size of the reservoir is sufficient to maintain a nearly uniform composition over the duration of the experiment, which can be confirmed by testing for the presence of electron acceptors and nutrients. If necessary, the media in the reservoir can be changed without drastically affecting the sensor component.
  • the reservoir itself may be a large media bottle with a cap modified to contain tubing fittings.
  • the reservoir is connected to the sensor with flexible tubing ( Figure 3).
  • a peristaltic pump drives flow from the reservoir into the sample chamber.
  • a separate tube returns the liquid from the sample chamber to the reservoir.
  • the reservoir is well mixed to avoid hydraulic short circuiting and maintain a uniform composition.
  • Step 2 Verify oxygen sensor principle
  • Step 2 is divided into three subtasks.
  • the first subtask is to confirm the differential response of the culture in electrode utilization with and without oxygen. Once this is confirmed, the second subtask is to utilize the culture in the sensor test cell to check for the establishment of an oxygen gradient and differential response down that gradient. Finally, varying levels of dissolved oxygen in the media are tested in the third subtask to verify the sensor operation.
  • a culture of Shewanella oneidensis MR-1 is inoculated in a conventional MFC.
  • This MFC is conditioned to establish an active anaerobic microbial community engaging in extracellular electron transport. Fresh nutrients are added (anaerobically) in batches to the reactor to maintain constant activity. Once a steady performance is reached, the media in the cell is continuously sparged with air to saturate with oxygen. The presence of excess oxygen should drastically reduce the respiration to the electrode as reported previously (Arnold 1990). As in the anaerobic case, fresh nutrients are added to the media to ensure constant activity.
  • Shewanella oneidensis MR-1 is used to verify the oxygen sensor principle.
  • the strain is cultured aerobically to a fixed density on a nitrate-free formulation of medi a described previously (Kim 1999). This culture is used to inoculate the anode chamber.
  • the same sterile media is saturated with oxygen and added to the media reservoir and circulated through the sample chamber.
  • the media is formulated to contain an excess of nutrients so as not to limit metabolic activity.
  • the media in the reservoir is sparged with sterile air to maintain oxygen saturation.
  • the media reservoir and the cell are maintained at a constant temperature of 30° C for this experiment.
  • the microbes in the anode chamber are allowed to reach a steady state after being introduced in the anode chamber.
  • the current of each individual wire is determined by measuring the voltage across drop across a resistor. Vary sample oxygen concentration
  • the amount of oxygen in the media is varied to test the response of the sensor. For these tests, the media is sampled and the amount of oxygen is measured using an oxygen electrode.
  • Step 3 Verify nitrate sensor principle (anaerobically)
  • Step 3 is divided into three subtasks to confirm 1) differential MFC output with and without nitrate as an electron acceptor, 2) nitrate gradient formation and detection within the sensor, and 3) response of the sensor dependent on sample nitrate concentration.
  • a culture of Shewanella oneidensis MR-1 is grown in media as previously described (Kim 1999), with a nitrate concentration of 50 mM, to exponential phase and inoculated into the anode chamber of the nitrate sensor.
  • Substantial variation in MFC performance has been noted at anodic nitrate concentrations below 8 mM, as such, a gradient response should be clearly visible (Sukkasem et al, 2008).
  • Anaerobic media with the same composition is added to the media reservoir and circulated through the sample chamber. As with the oxygen sensor test cell, excess nutrients are supplied in the media and a constant temperature is maintained to ensure uniform results. Performance is judged based on current produced by the electrodes.
  • the nitrate sensor cell is tested with media containing a wide range of nitrate concentration.
  • This study allows one to determine the detection threshold nitrate value using the microbial fuel cell as described herein, as well as the linearity of nitrate-response interaction. Characterization methods are the same as in baseline determination phase of the experiment, and sample nitrate concentration is confirmed using standard spectrophotometric methods (Clesceri, 1999).
  • Step 4 Integration of sensor design and Identification of design parameters and challenges
  • the final step in the initial development of a Shewanella oneidensis based sensor is focused on a detailed design based on the full integration of each of the components described above into a first generation sensor, and addresses additional design challenges that are not addressed in Steps 1-4. These additional issues include: oxygen diffusivity, nitrate diffusivity, metabolic and respiratory rates, dissolved oxygen rage, nitrate concentration range, and anode power density. In addition to accommodating the basic design parameters, the final design will also include features to address challenges with operational reliability and product lifespan.

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Abstract

La présente intervention concerne le domaine de la surveillance de l'eau de surface. Plus spécifiquement, la présente invention concerne des biocapteurs électrochimiques en temps réel, de faible coût, destinés à surveiller l'eau de surface en fonction du métabolisme d'un ou plusieurs microbes électrogènes.
PCT/US2010/059554 2009-12-08 2010-12-08 Capteurs utilisant des microbes pour la surveillance de l'environnement Ceased WO2011072065A2 (fr)

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CA2783256A CA2783256C (fr) 2009-12-08 2010-12-08 Capteurs utilisant des microbes pour la surveillance de l'environnement
US13/514,817 US9046478B2 (en) 2009-12-08 2010-12-08 Microbially-based sensors for environmental monitoring
EP10836646.9A EP2510345A4 (fr) 2009-12-08 2010-12-08 Capteurs utilisant des microbes pour la surveillance de l'environnement
AU2010328173A AU2010328173B9 (en) 2009-12-08 2010-12-08 Microbially-based sensors for environmental monitoring
JP2012543263A JP5763092B2 (ja) 2009-12-08 2010-12-08 環境モニタリングのための、微生物をベースとするセンサー
US14/694,082 US9551685B2 (en) 2009-12-08 2015-04-23 Microbially-based sensors for environmental monitoring

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