CA2116706A1 - Gas analyzer - Google Patents
Gas analyzerInfo
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- CA2116706A1 CA2116706A1 CA002116706A CA2116706A CA2116706A1 CA 2116706 A1 CA2116706 A1 CA 2116706A1 CA 002116706 A CA002116706 A CA 002116706A CA 2116706 A CA2116706 A CA 2116706A CA 2116706 A1 CA2116706 A1 CA 2116706A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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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
- G01N27/403—Cells and electrode assemblies
- G01N27/406—Cells and probes with solid electrolytes
- G01N27/407—Cells and probes with solid electrolytes for investigating or analysing gases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N37/00—Details not covered by any other group of this subclass
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N7/00—Analysing materials by measuring the pressure or volume of a gas or vapour
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- G—PHYSICS
- G05—CONTROLLING; REGULATING
- G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
- G05D11/00—Control of flow ratio
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Abstract
An apparatus for measuring the concentration of minor gaseous components of a flowing gas stream is described. The apparatus, which is preferably computer controlled, includes gas mixing means and means to measure the concentration of at least two gases, including the minor component, in the gas stream. In order to calibrate the apparatus an auxiliary unit, which may include a gas generator, meters delivery of the minor component from a second source into the gas stream. The apparatus has many industrial and scientific applications and is particularly useful for measuring hydrogen concentrations from which a measure of nitrogenase activity in nitrogen fixing plants can be obtained.
Description
2~1670G
AN APPARATUS FOR MEASURING LOW CONCENTRATIONS
OF GASES IN A FLOWING GAS STREAM
Field of Invention The invention relates to an instrument for measuring the concentration of a gas present in low concentration in a flowing gas stream and more particularly to an instrument for measuring H2 concentration in a flowing gas stream and the use of this instrument to measure nitrogenase activity, N2 fixation rate, electron allocation coefficient of nitrogenase and degree of O -limitation of nitrogenase activity in N2-fixing org~ni~ms and N -fixing components thereo The instrument differs from previous apparatus developed for these purposes in that it is compact, easily calibrated, self-checking, relatively inexpensive and provides an immediate read-out and continuous data log of each physiological parameter it is designed to measure. The instrument therefore overcomes the difficulties and delays involved in the calibration of prior instruments designed for measuring nitrogenase activity, and also circumvents the need for manipulation of data derived from such prior instruments in order to obtain measurements of nitrogenase activity and related physiological parameters. Other applications of the invention include industrial gas detectors and alcohol detection and measuring instruments.
Background of Invention and Prior Art The yield of many crop species is limited by the amount of nitrogen available in the soil. To alleviate this limitation farmers supplement the soil with nitrogenous fertilizers which are expensive and hazardous to the environment. Some important crop 211670~
species such as the legumes soybean, pea, alfalfa, clover and bean, do not rely entirely on soil nitrogen but are able to meet their requirements by reducing atmospheric N2 to ammonia. This process is called N2 fixation and it is carried out by bacteria which form a symbiotic association with the roots of the host plant. The bacteria inhabit outgrowths of the root, termed nodules, which provide them with a suitable environment for N2 fixation. Biological N2 fixation is likely to become an essential component of sustainable agricultural systems, and a great deal of research is currently in progress to determine the factors which regulate this process in leguminous crops. Consequently, a simple, accurate method for measuring N2 fixation rate is required.
The reduction of atmospheric N2 to amrnonia is catalyzed by the enzyme nitrogenase and the activity of this enzyme therefore determines N2 fixation rate.
Several methods have been devised to measure N2 fixation rate and/or nitrogenase activity. These methods include:
(a) Measurement of whole plant nitrogen increment in which plants are harvested at different times during development and their nitrogen content is measured.
This method is destructive, labour intensive, requires complex and expensive equipment for the nitrogen assay and does not distinguish between nitrogen derived from the soil and that derived from N2 fixation.
(b) Measurement of the 15N and 14N content of plants tissues and comparison of this with that of the atmosphere and the soil (the 1sN natural abundance assay).
Alternatively, nodulated roots may be fed with an atmosphere enriched in 1sN2 and the rate of incorporation of this 15N can be determined (the 15N enrichment assay). Both of 2~16706 these assays are destructive, time-consuming and require the use of expensive and complex analytical instruments. Also, like the nitrogen-increment method, they provide only a time-integrated measurement of nitrogen incorporation and do not show how nitrogenase activity and N2 fixation rate may vary over the short term.
(c) An acetylene reduction assay in which the nodulated roots of the legume are supplied with a gas cont:~ining 10% acetylene and the reduction of this acetylene to ethylene is monitored with time. In the presence of 10% acetylene, all electron flow through nitrogenase is diverted to acetylene reduction and the rate of ethylene production therefore provides a measure of total nitrogenase activity. The assay can be performed by sealing the nodulated root in a closed cuvette containing 10% acetylene (the closed system assay) or by passing 10~o acetylene continuously through the cuvette cont~3ining the nodulated root (the open system assay). In the former case the assay provides only a time integrated measurement of nitrogenase activity, while in the latter case taking discrete samples of effluent gas from the cuvette allows a time-course of nitrogenase activity to be measured. However, both methods suffer from the fact that in vivo nitrogenase activity is inhibited by exposure of nodulated roots to acetylene, so the assays often greatly underestimate true activities. Also, the assays provide only a measure of total nitrogenase activity and cannot be used to measure N2 fixation rate.
(d) The H2 evolution assay which depends on the fact that during N2 fixation the nitrogenase enzyme also reduces protons to H2 gas which is evolved from the nodule. H2 evolution rate may be measured by sealing a nodulated root in a cuvette and measuring the ~ccllm~ tion of H2 in the cuvette with time (the closed system H2 assay), or by passing gas through the cuvette continuously and monitoring H2 concentration in the effluent gas stream (the open system H2 assay). H2 may be monitored in discrete sample of the effluent gas by gas chromatography, or H2 concentration may be monitored colltilluously using a semi-conductor H2 analyzer such as that described by Layzell et al.
(Plant Physiol 75: 582-585, 1984). The rate of H2 evolution in air provides a measurement of apparent nitrogenase activity (ANA) since only a proportion of the electron flow through nitrogenase is used for proton reduction. In order to measure total nitrogenase activity (TNA) it is necessary to expose nodulated roots to a gas ll~ixlure l~cking N2, such as Ar:02. In the absence of N2 all electron flow is diverted to proton reduction and the rate of H2 evolution from the nodule provides a measure of TNA (FIG.1). The difference between the rates of H2 evolution in N2:02 and in Ar:02 at the same P02 can be used to estimtate N2 fixation rate thus:
N2 Fixation Rate = (TNA - ANA) / 3 Equation 1 since 3 electron pairs are used in the reduction of N2 compared to 1 electron pair for the reduction of H+. A comparison of the rates of H2 evolution in N2:02 and in Ar:02 at the same P02 can also be used to estimate the electron allocation coefficient (EAC) of nitrogenase thus:
EAC = 1 - (ANA / TNA) Equation 2 This coefficient indicates the relative flow of electrons through nitrogenase which are used for reduction of N2 and protons. Since at least one H2 molecule is evolved for eveIy N2 fixed, EAC has a m~ximllm value of 0.75, though the value is often lower than this.
2~1670G
The measurement of H2 evolution using a H2 analyzer in the open system assay has several advantages over other methods for measuring nitrogenase activity and N2 fLxation rate. These include:
(1) The H2 analyzer is extremely sensitive and it is the only instrument which allows continuous real-time measurement of nitrogenase activity.
(2) The H2 analyzer is the only instrument which allows measurement of ANA, TNA, EAC and N2 fixation rate on the same plant material.
AN APPARATUS FOR MEASURING LOW CONCENTRATIONS
OF GASES IN A FLOWING GAS STREAM
Field of Invention The invention relates to an instrument for measuring the concentration of a gas present in low concentration in a flowing gas stream and more particularly to an instrument for measuring H2 concentration in a flowing gas stream and the use of this instrument to measure nitrogenase activity, N2 fixation rate, electron allocation coefficient of nitrogenase and degree of O -limitation of nitrogenase activity in N2-fixing org~ni~ms and N -fixing components thereo The instrument differs from previous apparatus developed for these purposes in that it is compact, easily calibrated, self-checking, relatively inexpensive and provides an immediate read-out and continuous data log of each physiological parameter it is designed to measure. The instrument therefore overcomes the difficulties and delays involved in the calibration of prior instruments designed for measuring nitrogenase activity, and also circumvents the need for manipulation of data derived from such prior instruments in order to obtain measurements of nitrogenase activity and related physiological parameters. Other applications of the invention include industrial gas detectors and alcohol detection and measuring instruments.
Background of Invention and Prior Art The yield of many crop species is limited by the amount of nitrogen available in the soil. To alleviate this limitation farmers supplement the soil with nitrogenous fertilizers which are expensive and hazardous to the environment. Some important crop 211670~
species such as the legumes soybean, pea, alfalfa, clover and bean, do not rely entirely on soil nitrogen but are able to meet their requirements by reducing atmospheric N2 to ammonia. This process is called N2 fixation and it is carried out by bacteria which form a symbiotic association with the roots of the host plant. The bacteria inhabit outgrowths of the root, termed nodules, which provide them with a suitable environment for N2 fixation. Biological N2 fixation is likely to become an essential component of sustainable agricultural systems, and a great deal of research is currently in progress to determine the factors which regulate this process in leguminous crops. Consequently, a simple, accurate method for measuring N2 fixation rate is required.
The reduction of atmospheric N2 to amrnonia is catalyzed by the enzyme nitrogenase and the activity of this enzyme therefore determines N2 fixation rate.
Several methods have been devised to measure N2 fixation rate and/or nitrogenase activity. These methods include:
(a) Measurement of whole plant nitrogen increment in which plants are harvested at different times during development and their nitrogen content is measured.
This method is destructive, labour intensive, requires complex and expensive equipment for the nitrogen assay and does not distinguish between nitrogen derived from the soil and that derived from N2 fixation.
(b) Measurement of the 15N and 14N content of plants tissues and comparison of this with that of the atmosphere and the soil (the 1sN natural abundance assay).
Alternatively, nodulated roots may be fed with an atmosphere enriched in 1sN2 and the rate of incorporation of this 15N can be determined (the 15N enrichment assay). Both of 2~16706 these assays are destructive, time-consuming and require the use of expensive and complex analytical instruments. Also, like the nitrogen-increment method, they provide only a time-integrated measurement of nitrogen incorporation and do not show how nitrogenase activity and N2 fixation rate may vary over the short term.
(c) An acetylene reduction assay in which the nodulated roots of the legume are supplied with a gas cont:~ining 10% acetylene and the reduction of this acetylene to ethylene is monitored with time. In the presence of 10% acetylene, all electron flow through nitrogenase is diverted to acetylene reduction and the rate of ethylene production therefore provides a measure of total nitrogenase activity. The assay can be performed by sealing the nodulated root in a closed cuvette containing 10% acetylene (the closed system assay) or by passing 10~o acetylene continuously through the cuvette cont~3ining the nodulated root (the open system assay). In the former case the assay provides only a time integrated measurement of nitrogenase activity, while in the latter case taking discrete samples of effluent gas from the cuvette allows a time-course of nitrogenase activity to be measured. However, both methods suffer from the fact that in vivo nitrogenase activity is inhibited by exposure of nodulated roots to acetylene, so the assays often greatly underestimate true activities. Also, the assays provide only a measure of total nitrogenase activity and cannot be used to measure N2 fixation rate.
(d) The H2 evolution assay which depends on the fact that during N2 fixation the nitrogenase enzyme also reduces protons to H2 gas which is evolved from the nodule. H2 evolution rate may be measured by sealing a nodulated root in a cuvette and measuring the ~ccllm~ tion of H2 in the cuvette with time (the closed system H2 assay), or by passing gas through the cuvette continuously and monitoring H2 concentration in the effluent gas stream (the open system H2 assay). H2 may be monitored in discrete sample of the effluent gas by gas chromatography, or H2 concentration may be monitored colltilluously using a semi-conductor H2 analyzer such as that described by Layzell et al.
(Plant Physiol 75: 582-585, 1984). The rate of H2 evolution in air provides a measurement of apparent nitrogenase activity (ANA) since only a proportion of the electron flow through nitrogenase is used for proton reduction. In order to measure total nitrogenase activity (TNA) it is necessary to expose nodulated roots to a gas ll~ixlure l~cking N2, such as Ar:02. In the absence of N2 all electron flow is diverted to proton reduction and the rate of H2 evolution from the nodule provides a measure of TNA (FIG.1). The difference between the rates of H2 evolution in N2:02 and in Ar:02 at the same P02 can be used to estimtate N2 fixation rate thus:
N2 Fixation Rate = (TNA - ANA) / 3 Equation 1 since 3 electron pairs are used in the reduction of N2 compared to 1 electron pair for the reduction of H+. A comparison of the rates of H2 evolution in N2:02 and in Ar:02 at the same P02 can also be used to estimate the electron allocation coefficient (EAC) of nitrogenase thus:
EAC = 1 - (ANA / TNA) Equation 2 This coefficient indicates the relative flow of electrons through nitrogenase which are used for reduction of N2 and protons. Since at least one H2 molecule is evolved for eveIy N2 fixed, EAC has a m~ximllm value of 0.75, though the value is often lower than this.
2~1670G
The measurement of H2 evolution using a H2 analyzer in the open system assay has several advantages over other methods for measuring nitrogenase activity and N2 fLxation rate. These include:
(1) The H2 analyzer is extremely sensitive and it is the only instrument which allows continuous real-time measurement of nitrogenase activity.
(2) The H2 analyzer is the only instrument which allows measurement of ANA, TNA, EAC and N2 fixation rate on the same plant material.
(3) Measurements of ANA and short-term measurements of TNA are not inhibitory to nitrogenase so that measurements can be performed on the same plant material either continuously or intermittently over virtually any experimental period.
(4) The method is not labour intensive and the H2 analyzer is much cheaper than the mass spectrophotomter required for 'sN measurements and the gas chromatograph required to measure ethylene production.
Despite these advantages, relatively few researchers use the H2 evolution assay to measure nitrogenase activity. This is because the method has some disadvantages.
These include:
(1) The assay can only be used on legume symbioses which lack the enzyme uptake hydrogenase (HUP) of which there are many which are agriculturally important. This enzyme recycles some or all of the H2 produced by nitrogenase. H2 analysis cannot be used to measure nitrogenase activity in HUP+ symbioses.
(2) Extended exposure of nodulated roots to Ar:02 causes inhibition of nitrogenase.
However, short-term exposures are not inhibitory and repeated assays of TNA can be 2' 16706 -made on the same plant material.
(3) The output of the H2 analyzer changes with PO2, with differences in water content of the gas stream and with the nature of the balance gas (N2 or Ar). The equipment is, therefore, difficult and time-consuming to calibrate.
Object of the Invention It is one object of the invention to provide an integrated gas analysis system for determining the concentration of minor gas constituents in a flowing gas stream.
It is another object of the present invention to provide an integrated, compact, easily calibrated and self-checking instrument for the measurement of nitrogenase activity in N2-fixing legumes, and N2-fixing parts thereof, which measures H2 evolution rate from the material under study and automatically calculates values of ANA, TNA, EAC, N2 fixation rate and 02 limitation coefficient of nitrogenase.
Brief Statement of Invention By one aspect of this invention there is provided an integrated gas analysis system for measuring low concentrations of a selected gas contained in a gas stream comprising a plurality of gases, comprising;
(a) gas flow path means having inlet and outlet means;
(b) means for mixing said plurality of gases in any selected ratio thereof for delivery to said inlet means;
(c) means to measure gas concentration of said selected gas and at least one 211670~
other gas in said gas stream;
(d) means to deliver said selected gas at a selected rate to said flow path whereby said means to measure the concentration of said selected gas may be calibrated;
(e) means for monitoring gas flow rate through said flow path; and (f) means to monitor and control said means for mixing gases, said means to measure gas concentration and said means for monitoring gas flow rate, so as to generate output signals from which values representative of at least one of: (i) concentrations of said selected gas and said at least one other gas and (ii) production rate of said selected gas passing through said flow path means, can be determined.
By a preferred aspect of this invention there is provided claim an integrated nitrogenase assay analysis system comprising:
sealable gas-tight container means having gas inlet and outlet means and adapted to receive a sample of a nitrogen fixing plant;
means for mixing selected gases in any selected ratio thereof and delivery to said gas inlet means;
means to measure hydrogen and oxygen concentration in a gas stream passing through said container means;
means to deliver hydrogen at a selected rate to said container means whereby said means to measure hydrogen may be calibrated:
means for monitoring gas flow rates through said container means; and means to monitor and control said means for mixing gases, said means to measure hydrogen and oxygen concentration and said means for monitoring gas flow rates, so as to generate output signals indicative of production rate of hydrogen from said hydrogen delivery means and said sample, from which a measure of nitrogenase activity in said sample can be derived.
Brief Description of Drawings FIG.1 is a graph illustrating the typical voltage output of the H2 analyzer during measurements of ANA, TNA and PNA when the composition of the pN2, pAr and PO2 of the gas flowing past the N2-fixing plant material in the cuvette is altered.
FIG.2 is a schematic diagram of an open gas exchange system for measuring nitrogenase activity by monitoring H2 evolution from N2 fixing plant material maintained in a sealed cuvette according to the prior art.
FIG.3A and 3B are block diagrams illustrating the relationship between the voltage outputs of the H2 analyzer and the pH2 and PO2 Of the gas passing through the H2 analyzer when the values for voltage output, pH2 and PO2 are expressed as natural logs, and the balance of the gas is either N2 (Figure 3A) or A6 (Figure 3B).
FIG.4 is a schematic diagram of the embodiment of an apparatus according to the present invention.
Detailed Description of Preferred Embodiments Calibration problems deter most researchers from using the H2 evolution assay.
211 670~
In order to calibrate and use the H2 analyzer to its full potential an open flow gas çYch:~nge system is required such as that decribed by Hunt et al. (Plant Physiol. 91: 315-321 1989) and as illustrated in FIG 2. Electronic mass flow controllers 1 are used to miY gases from tanks 2 and produce any desired gas mixture for supply to a nodulated root system enclosed in a cuvette 3 and from there to a H2 analyzer 4 via an ice bath 7 and a m~gnesium perchlorate column 6 required for drying the gas. The flow rate of the gas from the mass flow controllers to the cuvette is controlled by needle valves (A,B,C) associated with a variable area flow meter S, and the flow rate to the H2 analyzer is controlled by a pump 6. Excess gas is vented and the PO2 Of the gas flushing through the system is monitored by an 02 electrode 9.
For calibration of the H2 analyzer 4 the cuvette 3 would not contain any plant material or would be detached from the system. Gas mixing is controlled by a computer D which both monitors and regulates the outputs from the mass flow controllers 1. The computer program has the ability to mix any combination of H2, Ar, 2 and and to alter the proportion of each gas in a mixture either immediately or gradually, and in a linear manner, with time. During calibration the operator would select a pH2 in :02 at a known PO2 and monitor the output of the H2 analyzer using either computer D or chart recorder means. When the output is stable the PO2 can be changed linearly with time between the desired limits and the H2 analyzer output monitored simultaneously. The procedure would then be repeated at a range of pH2 values in :02, and subsequently the entire procedure would be repeated with Ar, instead of, as the balance gas.
2l16706 Two sets of data would be collected during the calibration, one for 2 gas lures and one for Ar:02 gas mixtures. Each data set would consist of 3 columns of numbers relating pH2 to the voltage output of the H2 analyzer at a range Of PO2. Since the voltage output of the H2 analyzer is not linear with respect to pH2 or PO2, the relationship between voltage, pH2 and PO2 is linearized by converting the values to their natural logs. A multiple linear regression is then performed on each data set to calculate the equation of the plane which defines the relationship between ln voltage, ln PH2 and ln PO2 (see FIG.3). The equation of each plane has the form:
ln PH2 = (ln Volts 1~ a) + (ln PO2 * b) + c. Equation 3 where a and b are coefficients from the multiple linear regression and c is the constant.
To measure nitrogenase activity in a nodulated root system, or other -fixing system, the plant material is sealed in the cuvette and :02 at a known PO2 is flushed through the system at a known flow rate. H2 evolution from the plant material is monitored as the voltage output from the H2 analyzer until steady conditions are attained (FIG.1). The voltage output at this point is related to ANA. To measure TNA, the gas stream is switched from :02 to Ar:02 at the same PO2 and the voltage output from the H2 analyzer is monitored until a maximum value is attained (FIG.1). This maximum voltage value is related to TNA. Long term exposure to Ar:02 leads to nitrogenase inhibition so the gas stream must be switched back to 2 if this inhibition is to be avoided. Alternatively, after the maximum voltage output is attained in Ar:02 at the initial PO2, the computerized gas mixing system can be used to increase PO2 in Ar 21167û6 gradually at a defined rate and the voltage output from the H2 analyzer monitored continuously. Such an experiment may determine the m~ximllm potential nitrogenase activity (PNA) of the plant material since it has been shown that 2 concentration limits nitrogenase activity under normal conditions and severely limits this activity under many adverse conditions.
After an experiment of the type summ~rized in FIG.1, the user must calculate ANA, ~NA, EAC, fixation rate and PNA by converting the H2 analyzer output voltages measured at the appropriate points in the experiment to values of pH2 using the form of Equation 3 derived in 2 or Ar:02. This will require that the PO2 at each point in the experiment is also known. The pH2 calculated from this equation can be converted to a rate of H2 production from the plant material if the flow rate through the gas exchange system is known. Values of ANA and TNA can the be used to calculate fixation rate and EAC according to Equations 1 and 2 respectively. Also an index of the degree of 2 limitation of nitrogenase can be estimated by calculating the 2 limitation coefficient of nitrogenase (OLCN) thus:
OLCN = TNA / PNA Equation 4.
It will be appreciated that the calibration and use of the H2 analyzer for measuring nitrogenase activity and associated physiological parameters is a complex operation requiring the use of much ancillary, and expensive, equipment. The calculation of equations to describe the calibration planes of the H2 analyzer requires the use of spreadsheet programs such as Lotus 1,2,3 or QuattroPro, and other computer programs are necessary to control gas mixing and, ideally, to log voltage data from the 2~ 16706 H2 analyzer and 2 sensor. Even with such computational support, use of the H2 analyzer is difficult and time-consuming. Calibrations must be performed regularly since the voltage output at a given PO2 and pH2 drifts with time. Also, the data manipulation required to calculate ANA, TNA and PNA at the end of each experiment is complex and tedious. It is apparent, therefore, that there is a need for a H2 analyzer which measures nitrogenase activity and associated physiological parameters, but which is relatively compact, easy to calibrate, has integrated ability to control gas mixing, monitor pH2, PO2 and flow rate, provides immediate read out of H2 production by the plant material in the cuvette and automatically calculates ANA, TNA, fixation rate, EAC, PNA and OLCN.
In the open flow gas exchange system for determining nitrogenase activity illustrated in FIG.2 the electronic mass flow controllers 1 are functional only if a pressure drop of 15 psi, or greater, is maintained between their input and output ports.
They must therefore be supplied with pressurized gases from cylinders 2. The use of such mass flow controllers to supply a gas of predetermined composition to the cuvette 3 and/or H2 analyzer 4 limits the degree of compactness and adaptability of the gas exchange system. The method for regulating the flow of dry gas through the system using a needle valve, variable area flow meter 5, pump 6 ice bath 7 and magnesium perchlorate column 8 also adds to the physical size of the gas exchange apparatus.
Compactness is further limited by the computer D, which may be of the IBM PC or Macintosh type, that is used to regulate the mass flow controllers and to log the voltage outputs from the H2 analyzer and 02 sensor. The system is difficult and time-consuming to operate since the computer cannot be used to manipulate the voltage outputs from -the H2 analyzer and 2 sensor to provide measurements of nitrogenase activity while an experiment is in progress. Also, the system has no automated calibration procedure and no procedure for determining if recalibration is necessary. The user must therefore check calibration each time the instrument is used and repeat the calibration when significant drift from a previous calibration occurs.
To overcome these limitations of the prior art there is the need for a single integrated instrument which has the following features:
(1) A system for mixing user-determined combinations Of, 2 Ar and Air for supply to a cuvette cont~ining -fixing plant material and/or to a H2 sensor and 2 sensor.
(2) A system for generating a user-defined rate of H2 production, and thereby pH2 in the aforementioned gas mixture, in order to calibrate the H2 sensor.
(3) An electronic system for monitoring flow rates of gases within the instrument.
(4) A central on-board computer which:
(i) controls the gas mixing system.
(ii) monitors the voltage outputs of the H2, 2 and flow sensors .
(iii) presents the voltage outputs of the H2 and 2 sensors on a screen in graphical form in real time.
(iv) stores calibrations of the H2 sensor and uses these to convert the voltage outputs from the H2, 2 and flow sensors to calculate the production rate of H2 from either the H2 generating system or the plant material in the cuvette.
(v) stores the equations for the calculation of ANA, TNA, EAC, PNA, OLCN and fixation rate and provides the user with calculated values of these parameters.
21167q6 (vi) contains sub-routines for calibration of the H2 sensor, 2 sensor and flow meter within the instrument.
(vii) contains sub-routines which check voltage outputs of the H2 and 2 sensors against standards and warns the user if recalibration of the sensors is necessary.
(viii) provides interactive screens which guide the user through each stage of the calibration and use of the instrument.
(ix) saves raw and/or calculated data from each experimental run and allows the user to export the data to a separate computer via an RS232 port.
A Nitrogenase Assay Analysis System A preferred embodiment of the integrated nitrogenase assay analysis system (NAAS) is shown in FIG.4. The instrument has 4 gas inlet ports 10 which are connected to external supplies of pure, 2 and Ar and to an air source. The gases can be supplied from tanks or from gas bags. 2~ Ar and air are connected within the NAAS to solenoid valves Soll, Sol2, Sol3 and Sol4, respectively, and the switching of each solenoid valve is controlled by a separate digital output (Dol, Do2, Do3 and Do4) from the computer.
The solenoid valves (Sol, So2 and So3) controlling the flow Of, 2 and Ar are normally closed and that controlling the flow of air is (So4) normally open. The rate at which the valves are opened and closed determines the composition of the mixed gas stream supplied to the NAAS. An electronic vacuum pressure guage 11 monitors the gas pressure within the NAAS at the point where the gases are combined. The analogue output of the vacuum pressure guage is monitored by the computer AI1. Downstream of the solenoid valves the gases are combined in a mixing vessel 12 of _ ml volume.
2~16706 After the mixing vessel the gas mixture is separated into an analysis stream which is drawn into the NAAS by pump 1 ( ) and a reference stream which enters an electronic 3-way valve 13 controlled by a digital output (DoS) from the computer. When the 3-way valve is in the on position, the reference stream is drawn into the analysis compartment of the NAAS by pump 14, otherwise all gas flow passes from the mixing vessel 12 into the NAAS via pump 15. The flow rates of the analysis and reference gas streams are controlled by analogue outputs from the computer (Aol, Do6) to pumps 15 and 14, respectively, and by needle valves (NV1 and NV2) downstream of each pump.
The flow rate of the analysis gas stream is measured by an electronic flow sensor 16, the analogue output A14 of which is monitored by the computer. Downstream of the needle valve NV2 and the flow sensor 16, the analysis gas is humidified by passage through a column 17 cont~ining a moist absorbent material such as glass wool. Humidification is required to retard drying of plant material as the analysis gas flushes through the plant cuvette 21. After humidification the analysis gas leaves the NAAS via a gas port 18.
For calibration purposes, and occasionally during experiments, the analysis gas outlet port 18 may be connected to an inlet port 34 of an electrolytic H2 generating system 19 which is described in detail in our copending patent application Serial No.
, filed concurrently herewith. The function of the H2 generating system 19 is to produce H2 at a rate controlled by the computer (Ao2) and to add a known amount of H2 to the analysis gas stream as it passes through the electrolysis unit at a flow rate measured by an electronic flow sensor 20 and monitored by the computer A16. After exiting from the H2 generating system 19 via outlet port 35 the analysis gas enters the plant cuvette 21 via 2~167~6 port 36 or, as is more usual during calibration, by-passes this cuvette and is dried by passage through an ice water bath external to the NAAS. If the H2 generating system is not used, the analysis gas from outlet port 18 is directed to inlet port 36 so that it enters the plant cuvette immediately after leaving the NAAS via the outlet gas port 18.
After flushing through the plant cuvette 21 the analysis gas stream is partially vented to atmosphere at 23, and the remainder is dried by passage through a sealed tube immersed in the ice water bath 22. Condensation of water from the analysis gas occurs within the tube, and condensation efficiency may be improved by immersing the tube in a liquid gas, such as liquid nitrogen, rather than in ice water. The dry gas issuing from the ice water bath re-enters the NAAS via a gas input port 24 at a rate determined by pump 14 and its associated needle valve NV1. Analysis gas only re-enters the NAAS if the 3-way valve 13 is in the off position, otherwise all the gas vents to atmosphere via the vent 23.
Either the analysis gas stream or the reference gas stream enters the analysis system of the NAAS dependent on whether the 3-way valve 13 is in the off or the on position, respectively. After passage through pump 14 and its associated needle valve NV1, the gas flows across the surface of an 2 sensor 25, the voltage output A12 of which is monitored by the computer. The gas then leaves the body of the NAAS by an outlet port 26 connected 27 directly to a column of magnesium perchlorate mounted on the side of the instrument. Flow of gas through this column removes any residual water vapor the gas may contain and the water content of the gas is detected by a humidity sensor 28 after it re-enters the body of the NAAS via a gas inlet port 29. The voltage output AI5 of the humidity sensor 28 is monitored by the computer. After movement of gas through the humidity sensor 28 it passes across the surface of a semi-conductor H2 sensor 30, the voltage output AI3 of which is monitored by the computer. The gas then vents to atmosphere via a gas outlet port 31.
All of the sensor outputs monitored by the computer are stored in a "current run"
file. During an experiment when N2-fixing plant material is enclosed within the plant cuvetter, voltage outputs from the H2 sensor are converted to rates of H2 production and may, but not essentially, be displayed graphically, in real-time, on a liquid crystal screen (not shown) on the front of the NAAS. A graphical display on the same screen can also show the composition of the analysis gas stream at all times during the experiment. The time elapsed during the experiment can also be shown in alphanumeric form, as can the flow rate of the gas through the plant cuvette 21, the PO2 f the analysis gas stream and the rate of H2 evolution. These, and other data collected and processed by the computer may be exported to a remote computer via an RS232 port 32 on the NAAS if desired.
The NAAS also has an analogue output port 33 through which sensor outputs may be monitored on conventional analogue recording devices (not shown).
A screen on the front of the NAAS allows the user to access all the calibration, experimental and data-handling functions of the instrument. In the preferred embodiment described in more detail hereinafter this is achieved by touching command buttons displayed on the screen. The act of touching the screen interrupts a specific point in a network of lights which divides the screen horizontally and vertically into a series of grids. The computer recognises the command associated with the interruption of light at a specific grid and activates the process selected by the user. The computer program also recognises when inappropriate command grids are selected and provides the user with error messages when this occurs. The user may also enter data into the computer in alph:~nllmeric form by ~ lling this data to the screen using a knob located on the front panel of the NAAS. The knob has different functions depending on the particular screen accessed by the user. Several of these screens have data input fields which can be filled by turning the knob to enter either a written or numerical input. The specific functions of the NAAS and the way in which they are accessed by the user are described below.
It will be appreciated from the above description of the NAAS that it possesses all the functions of the prior art gas exchange system illustrated if FIG.l, and incorporates several other features. It also comprises, in a single compact instrument, all the fuctional units required for open system measurement of nitrogenase activity.
However, the major advantage of the NAAS is its ease of use and calibration as will be described hereinafter.
Use of the Nitrogenase Assay Analysis System.
The invention has been described hereinabove with particular reference to a nitrogenase assay system, but it will be readily apparent to those skilled in the art that this invention is readily adaptable to the measurement of low concentrations of gas contained in a mixed gas stream in numerous different environments and situations. For example, the system could easily be adapted to determine alcohol concentration in exhaled air from a human subject and hence could be used as a breathalyzer by law enforcement agencies. Other breathalyzer applications include a hydrogen breath test for lactose intolerance in humans. The system may also be used in industrial or commercial applications, for example to detect and monitor hydrogen production in hydrogen/alllmimlm batteries.
The computer program for running the NAAS is written in and is downloaded to the instrument via the RS232 port 32. Modifications and up-grades of the program can be downloaded from a remote computer via the RS232 port 32.
Preferably, all operations of the NAAS are controlled by the on-board computer and accessed by a touch screen which has several different configurations dependent on the current operation selected. An on-screen "help" function describing how to use the NAAS in each of its modes may be provided for use at all times.
Calibration Before the NAAS can be used for the first time, all of its sensors must be calibrated. A description of the calibration methods is normally provided on the help screen, and the calibration procedure for each sensor is initiated via a main calibration menu. Calibration involves the following procedures:
(i) The H2 sensor is conditioned by exposure to a high partial pressure of H2. This is provided automatically by the electrolysis unit 19.
(ii) The time delay between selecting a change in gas composition and the response to ~116706 ~that change by the gas sensors is measured. The user supplies air and O2 (10) to the NAAS, and attaches the NAAS to the gas exchange system which will be used for experiments. The time delay associated with the particular experimental set-up is measured automatically by monitoring the time required for the O2 sensor to respond to a change in PO2 from air level to 100~o O2. This delay time must be recalibrated whenever the gas exchange system is reconfigured and/or the volume of the sample cuvette 21 is changed.
(iii) The flow sensor (FM1) in the analysis gas stream is calibrated in air and in Ar.
The user attaches a bubble flow meter (or similar, low resistance, flow meter) to the vent from the NAAS and the computer generates a range of flow rates while monitoring the voltage output of the flow sensor. Using the bubble flow meter, the user measures the flow rates generated by the computer and dials in these values to correspond with the voltage outputs from the flow sensor. The calibrations thus attained for air and Ar can be applied, with automated corrections, to any gas mixture used in the NAAS.
(iv) The 2 sensor is calibrated by exposure to pure N2, pure 2 and air in a sequence controlled automatically by the computer. The output of the 2 sensor is monitored in each gas and a linear relationship is calculated between voltage output and PO2.
(v) The H2 sensor is calibrated in both N2:O2 and Ar:O2 atomospheres. The user selects the range of pH2 and PO2 values over which the sensor is to be calibrated in each atmosphere. The electrolysis unit then generates these pH2 and the sensor is calibrated in N2:O2 and the Ar:O2 as PO2 is varied between the set limits. The computer records the voltage outputs of the sensor at each pH2 and PO2 in both N2:O2 and Ar:O2 and generates two look-up tables which relatethese outputs to the pH2 and PO2 in each atmosphere.
Once these initial calibrations have been completed the NAAS is ready for use.
Occasional recalibration of the H2 and 02 sensors may be necessary and the user can check this by an automatic calibration check procedure during which the computer generates a specific pH2 and PO2 and checks the voltage outputs of the H2 and 02 sensors against the expected outputs of the H2 and 02 sensors against the expected oulpu~s calculated from current calibrations. If the expected and measured outputs differ, the computer warns the user than recalibration may be necessary. The NAAS
also has an automatic system check function which monitors the gas pressure (vacuum gauge X) as each solenoid valve is opened sequentially, and warns the user if any of the four gases are not supplied to the system. A warning is also given if the output from the H2 sensor is significantly above zero when no H2 is supplied to the sensor from the electrolysis unit or from plant material in the cuvette. This alerts the user that H2, or another combustible gas, may be cont~min~ting the gas stream. The output from the humidity sensor is also checked automatically throughout the calibration and experimental procedures, and a warning is given if humidity rises beyond an acceptable threshold value.
Measurement of Nitrogenase Activity At the beginning of each experiment the user dials in the current time, date atmosphere pressure and temperature. Plant material is then sealed into the cuvette 21 ~116706 and the NAAS measures H2 evolution rate in either a m~nll~l, or one of several automatic, modes. In m~ml~l mode, the user selects the flow rate and gas composition supplied to the cuvette and the rate at which data collected by the computer is sampled to the current run file. The user may also specify time-dependent changes in the gas composition supplied to the cuvette. Throughout the run, H2 evolution rate, experimental time, measured flow rate and measured PO2 are displayed on the screen.
These data are stored in a current run file and may be downloaded to a remote computer via an RS232 port on the NAAS.
The NAAS has the ability to perform automatic measurements of total nitrogenase activity (TNA) and potential nitrogenase activity (PNA). Both of these involve changes in the gas stream supplied to the cuvette from one composed of N2:02 to one composed of Ar:02. TNA is measured as the peak rate of H2 evolution in Ar:02, whereas the measurement of PNA involves a gradual increase in PO2 during exposure of the plant material to Ar:02 and measurement of maximum H2 evolution rate at optimum PO2. The duration that the plant material is exposed to N2:02, Ar:02 and increasing PO2 in Ar:02 is selected by the user, but the computer recognises the H2 evolution rates corresponding to TNA and PNA. Values for apparent nitogenase acivity (ANA; H2 production rate in N2:02), TNA and PNA are calculated automatically after each automated run is complete. Values of N2 fixation rate, EAC and OLCN are automatically calculated from the ANA, TNA and PNA values. These calculated values may be saved in addition to, or instead of, the entire data set collected during the automated runs.
From the above description of the NAAS operations it will be appreciated that this instrument performs all the functions of the prior art gas exchange system (FIG.2), but overcomes the problems of calibration, expense and physical size associated with that, and similar, large-scale systems. Furthermore, the NAAS is self-checking to ensure that its components and calibrations are functional, and is able to measure and calculate ANA, TNA, PNA, OLCN, EAC and N2 fixation rate automatically. It is the first insllulllent with such capabilities. It should also be noted that the NAAS may also be used in any situation which requires the measurement of pH2 in a flowing gas stream. It could therefore function as, for example, a H2 breath analyzer to test for lactose intolerance in humans or be used in industrial applications to detect H2 gas production from non-living systems.
Despite these advantages, relatively few researchers use the H2 evolution assay to measure nitrogenase activity. This is because the method has some disadvantages.
These include:
(1) The assay can only be used on legume symbioses which lack the enzyme uptake hydrogenase (HUP) of which there are many which are agriculturally important. This enzyme recycles some or all of the H2 produced by nitrogenase. H2 analysis cannot be used to measure nitrogenase activity in HUP+ symbioses.
(2) Extended exposure of nodulated roots to Ar:02 causes inhibition of nitrogenase.
However, short-term exposures are not inhibitory and repeated assays of TNA can be 2' 16706 -made on the same plant material.
(3) The output of the H2 analyzer changes with PO2, with differences in water content of the gas stream and with the nature of the balance gas (N2 or Ar). The equipment is, therefore, difficult and time-consuming to calibrate.
Object of the Invention It is one object of the invention to provide an integrated gas analysis system for determining the concentration of minor gas constituents in a flowing gas stream.
It is another object of the present invention to provide an integrated, compact, easily calibrated and self-checking instrument for the measurement of nitrogenase activity in N2-fixing legumes, and N2-fixing parts thereof, which measures H2 evolution rate from the material under study and automatically calculates values of ANA, TNA, EAC, N2 fixation rate and 02 limitation coefficient of nitrogenase.
Brief Statement of Invention By one aspect of this invention there is provided an integrated gas analysis system for measuring low concentrations of a selected gas contained in a gas stream comprising a plurality of gases, comprising;
(a) gas flow path means having inlet and outlet means;
(b) means for mixing said plurality of gases in any selected ratio thereof for delivery to said inlet means;
(c) means to measure gas concentration of said selected gas and at least one 211670~
other gas in said gas stream;
(d) means to deliver said selected gas at a selected rate to said flow path whereby said means to measure the concentration of said selected gas may be calibrated;
(e) means for monitoring gas flow rate through said flow path; and (f) means to monitor and control said means for mixing gases, said means to measure gas concentration and said means for monitoring gas flow rate, so as to generate output signals from which values representative of at least one of: (i) concentrations of said selected gas and said at least one other gas and (ii) production rate of said selected gas passing through said flow path means, can be determined.
By a preferred aspect of this invention there is provided claim an integrated nitrogenase assay analysis system comprising:
sealable gas-tight container means having gas inlet and outlet means and adapted to receive a sample of a nitrogen fixing plant;
means for mixing selected gases in any selected ratio thereof and delivery to said gas inlet means;
means to measure hydrogen and oxygen concentration in a gas stream passing through said container means;
means to deliver hydrogen at a selected rate to said container means whereby said means to measure hydrogen may be calibrated:
means for monitoring gas flow rates through said container means; and means to monitor and control said means for mixing gases, said means to measure hydrogen and oxygen concentration and said means for monitoring gas flow rates, so as to generate output signals indicative of production rate of hydrogen from said hydrogen delivery means and said sample, from which a measure of nitrogenase activity in said sample can be derived.
Brief Description of Drawings FIG.1 is a graph illustrating the typical voltage output of the H2 analyzer during measurements of ANA, TNA and PNA when the composition of the pN2, pAr and PO2 of the gas flowing past the N2-fixing plant material in the cuvette is altered.
FIG.2 is a schematic diagram of an open gas exchange system for measuring nitrogenase activity by monitoring H2 evolution from N2 fixing plant material maintained in a sealed cuvette according to the prior art.
FIG.3A and 3B are block diagrams illustrating the relationship between the voltage outputs of the H2 analyzer and the pH2 and PO2 Of the gas passing through the H2 analyzer when the values for voltage output, pH2 and PO2 are expressed as natural logs, and the balance of the gas is either N2 (Figure 3A) or A6 (Figure 3B).
FIG.4 is a schematic diagram of the embodiment of an apparatus according to the present invention.
Detailed Description of Preferred Embodiments Calibration problems deter most researchers from using the H2 evolution assay.
211 670~
In order to calibrate and use the H2 analyzer to its full potential an open flow gas çYch:~nge system is required such as that decribed by Hunt et al. (Plant Physiol. 91: 315-321 1989) and as illustrated in FIG 2. Electronic mass flow controllers 1 are used to miY gases from tanks 2 and produce any desired gas mixture for supply to a nodulated root system enclosed in a cuvette 3 and from there to a H2 analyzer 4 via an ice bath 7 and a m~gnesium perchlorate column 6 required for drying the gas. The flow rate of the gas from the mass flow controllers to the cuvette is controlled by needle valves (A,B,C) associated with a variable area flow meter S, and the flow rate to the H2 analyzer is controlled by a pump 6. Excess gas is vented and the PO2 Of the gas flushing through the system is monitored by an 02 electrode 9.
For calibration of the H2 analyzer 4 the cuvette 3 would not contain any plant material or would be detached from the system. Gas mixing is controlled by a computer D which both monitors and regulates the outputs from the mass flow controllers 1. The computer program has the ability to mix any combination of H2, Ar, 2 and and to alter the proportion of each gas in a mixture either immediately or gradually, and in a linear manner, with time. During calibration the operator would select a pH2 in :02 at a known PO2 and monitor the output of the H2 analyzer using either computer D or chart recorder means. When the output is stable the PO2 can be changed linearly with time between the desired limits and the H2 analyzer output monitored simultaneously. The procedure would then be repeated at a range of pH2 values in :02, and subsequently the entire procedure would be repeated with Ar, instead of, as the balance gas.
2l16706 Two sets of data would be collected during the calibration, one for 2 gas lures and one for Ar:02 gas mixtures. Each data set would consist of 3 columns of numbers relating pH2 to the voltage output of the H2 analyzer at a range Of PO2. Since the voltage output of the H2 analyzer is not linear with respect to pH2 or PO2, the relationship between voltage, pH2 and PO2 is linearized by converting the values to their natural logs. A multiple linear regression is then performed on each data set to calculate the equation of the plane which defines the relationship between ln voltage, ln PH2 and ln PO2 (see FIG.3). The equation of each plane has the form:
ln PH2 = (ln Volts 1~ a) + (ln PO2 * b) + c. Equation 3 where a and b are coefficients from the multiple linear regression and c is the constant.
To measure nitrogenase activity in a nodulated root system, or other -fixing system, the plant material is sealed in the cuvette and :02 at a known PO2 is flushed through the system at a known flow rate. H2 evolution from the plant material is monitored as the voltage output from the H2 analyzer until steady conditions are attained (FIG.1). The voltage output at this point is related to ANA. To measure TNA, the gas stream is switched from :02 to Ar:02 at the same PO2 and the voltage output from the H2 analyzer is monitored until a maximum value is attained (FIG.1). This maximum voltage value is related to TNA. Long term exposure to Ar:02 leads to nitrogenase inhibition so the gas stream must be switched back to 2 if this inhibition is to be avoided. Alternatively, after the maximum voltage output is attained in Ar:02 at the initial PO2, the computerized gas mixing system can be used to increase PO2 in Ar 21167û6 gradually at a defined rate and the voltage output from the H2 analyzer monitored continuously. Such an experiment may determine the m~ximllm potential nitrogenase activity (PNA) of the plant material since it has been shown that 2 concentration limits nitrogenase activity under normal conditions and severely limits this activity under many adverse conditions.
After an experiment of the type summ~rized in FIG.1, the user must calculate ANA, ~NA, EAC, fixation rate and PNA by converting the H2 analyzer output voltages measured at the appropriate points in the experiment to values of pH2 using the form of Equation 3 derived in 2 or Ar:02. This will require that the PO2 at each point in the experiment is also known. The pH2 calculated from this equation can be converted to a rate of H2 production from the plant material if the flow rate through the gas exchange system is known. Values of ANA and TNA can the be used to calculate fixation rate and EAC according to Equations 1 and 2 respectively. Also an index of the degree of 2 limitation of nitrogenase can be estimated by calculating the 2 limitation coefficient of nitrogenase (OLCN) thus:
OLCN = TNA / PNA Equation 4.
It will be appreciated that the calibration and use of the H2 analyzer for measuring nitrogenase activity and associated physiological parameters is a complex operation requiring the use of much ancillary, and expensive, equipment. The calculation of equations to describe the calibration planes of the H2 analyzer requires the use of spreadsheet programs such as Lotus 1,2,3 or QuattroPro, and other computer programs are necessary to control gas mixing and, ideally, to log voltage data from the 2~ 16706 H2 analyzer and 2 sensor. Even with such computational support, use of the H2 analyzer is difficult and time-consuming. Calibrations must be performed regularly since the voltage output at a given PO2 and pH2 drifts with time. Also, the data manipulation required to calculate ANA, TNA and PNA at the end of each experiment is complex and tedious. It is apparent, therefore, that there is a need for a H2 analyzer which measures nitrogenase activity and associated physiological parameters, but which is relatively compact, easy to calibrate, has integrated ability to control gas mixing, monitor pH2, PO2 and flow rate, provides immediate read out of H2 production by the plant material in the cuvette and automatically calculates ANA, TNA, fixation rate, EAC, PNA and OLCN.
In the open flow gas exchange system for determining nitrogenase activity illustrated in FIG.2 the electronic mass flow controllers 1 are functional only if a pressure drop of 15 psi, or greater, is maintained between their input and output ports.
They must therefore be supplied with pressurized gases from cylinders 2. The use of such mass flow controllers to supply a gas of predetermined composition to the cuvette 3 and/or H2 analyzer 4 limits the degree of compactness and adaptability of the gas exchange system. The method for regulating the flow of dry gas through the system using a needle valve, variable area flow meter 5, pump 6 ice bath 7 and magnesium perchlorate column 8 also adds to the physical size of the gas exchange apparatus.
Compactness is further limited by the computer D, which may be of the IBM PC or Macintosh type, that is used to regulate the mass flow controllers and to log the voltage outputs from the H2 analyzer and 02 sensor. The system is difficult and time-consuming to operate since the computer cannot be used to manipulate the voltage outputs from -the H2 analyzer and 2 sensor to provide measurements of nitrogenase activity while an experiment is in progress. Also, the system has no automated calibration procedure and no procedure for determining if recalibration is necessary. The user must therefore check calibration each time the instrument is used and repeat the calibration when significant drift from a previous calibration occurs.
To overcome these limitations of the prior art there is the need for a single integrated instrument which has the following features:
(1) A system for mixing user-determined combinations Of, 2 Ar and Air for supply to a cuvette cont~ining -fixing plant material and/or to a H2 sensor and 2 sensor.
(2) A system for generating a user-defined rate of H2 production, and thereby pH2 in the aforementioned gas mixture, in order to calibrate the H2 sensor.
(3) An electronic system for monitoring flow rates of gases within the instrument.
(4) A central on-board computer which:
(i) controls the gas mixing system.
(ii) monitors the voltage outputs of the H2, 2 and flow sensors .
(iii) presents the voltage outputs of the H2 and 2 sensors on a screen in graphical form in real time.
(iv) stores calibrations of the H2 sensor and uses these to convert the voltage outputs from the H2, 2 and flow sensors to calculate the production rate of H2 from either the H2 generating system or the plant material in the cuvette.
(v) stores the equations for the calculation of ANA, TNA, EAC, PNA, OLCN and fixation rate and provides the user with calculated values of these parameters.
21167q6 (vi) contains sub-routines for calibration of the H2 sensor, 2 sensor and flow meter within the instrument.
(vii) contains sub-routines which check voltage outputs of the H2 and 2 sensors against standards and warns the user if recalibration of the sensors is necessary.
(viii) provides interactive screens which guide the user through each stage of the calibration and use of the instrument.
(ix) saves raw and/or calculated data from each experimental run and allows the user to export the data to a separate computer via an RS232 port.
A Nitrogenase Assay Analysis System A preferred embodiment of the integrated nitrogenase assay analysis system (NAAS) is shown in FIG.4. The instrument has 4 gas inlet ports 10 which are connected to external supplies of pure, 2 and Ar and to an air source. The gases can be supplied from tanks or from gas bags. 2~ Ar and air are connected within the NAAS to solenoid valves Soll, Sol2, Sol3 and Sol4, respectively, and the switching of each solenoid valve is controlled by a separate digital output (Dol, Do2, Do3 and Do4) from the computer.
The solenoid valves (Sol, So2 and So3) controlling the flow Of, 2 and Ar are normally closed and that controlling the flow of air is (So4) normally open. The rate at which the valves are opened and closed determines the composition of the mixed gas stream supplied to the NAAS. An electronic vacuum pressure guage 11 monitors the gas pressure within the NAAS at the point where the gases are combined. The analogue output of the vacuum pressure guage is monitored by the computer AI1. Downstream of the solenoid valves the gases are combined in a mixing vessel 12 of _ ml volume.
2~16706 After the mixing vessel the gas mixture is separated into an analysis stream which is drawn into the NAAS by pump 1 ( ) and a reference stream which enters an electronic 3-way valve 13 controlled by a digital output (DoS) from the computer. When the 3-way valve is in the on position, the reference stream is drawn into the analysis compartment of the NAAS by pump 14, otherwise all gas flow passes from the mixing vessel 12 into the NAAS via pump 15. The flow rates of the analysis and reference gas streams are controlled by analogue outputs from the computer (Aol, Do6) to pumps 15 and 14, respectively, and by needle valves (NV1 and NV2) downstream of each pump.
The flow rate of the analysis gas stream is measured by an electronic flow sensor 16, the analogue output A14 of which is monitored by the computer. Downstream of the needle valve NV2 and the flow sensor 16, the analysis gas is humidified by passage through a column 17 cont~ining a moist absorbent material such as glass wool. Humidification is required to retard drying of plant material as the analysis gas flushes through the plant cuvette 21. After humidification the analysis gas leaves the NAAS via a gas port 18.
For calibration purposes, and occasionally during experiments, the analysis gas outlet port 18 may be connected to an inlet port 34 of an electrolytic H2 generating system 19 which is described in detail in our copending patent application Serial No.
, filed concurrently herewith. The function of the H2 generating system 19 is to produce H2 at a rate controlled by the computer (Ao2) and to add a known amount of H2 to the analysis gas stream as it passes through the electrolysis unit at a flow rate measured by an electronic flow sensor 20 and monitored by the computer A16. After exiting from the H2 generating system 19 via outlet port 35 the analysis gas enters the plant cuvette 21 via 2~167~6 port 36 or, as is more usual during calibration, by-passes this cuvette and is dried by passage through an ice water bath external to the NAAS. If the H2 generating system is not used, the analysis gas from outlet port 18 is directed to inlet port 36 so that it enters the plant cuvette immediately after leaving the NAAS via the outlet gas port 18.
After flushing through the plant cuvette 21 the analysis gas stream is partially vented to atmosphere at 23, and the remainder is dried by passage through a sealed tube immersed in the ice water bath 22. Condensation of water from the analysis gas occurs within the tube, and condensation efficiency may be improved by immersing the tube in a liquid gas, such as liquid nitrogen, rather than in ice water. The dry gas issuing from the ice water bath re-enters the NAAS via a gas input port 24 at a rate determined by pump 14 and its associated needle valve NV1. Analysis gas only re-enters the NAAS if the 3-way valve 13 is in the off position, otherwise all the gas vents to atmosphere via the vent 23.
Either the analysis gas stream or the reference gas stream enters the analysis system of the NAAS dependent on whether the 3-way valve 13 is in the off or the on position, respectively. After passage through pump 14 and its associated needle valve NV1, the gas flows across the surface of an 2 sensor 25, the voltage output A12 of which is monitored by the computer. The gas then leaves the body of the NAAS by an outlet port 26 connected 27 directly to a column of magnesium perchlorate mounted on the side of the instrument. Flow of gas through this column removes any residual water vapor the gas may contain and the water content of the gas is detected by a humidity sensor 28 after it re-enters the body of the NAAS via a gas inlet port 29. The voltage output AI5 of the humidity sensor 28 is monitored by the computer. After movement of gas through the humidity sensor 28 it passes across the surface of a semi-conductor H2 sensor 30, the voltage output AI3 of which is monitored by the computer. The gas then vents to atmosphere via a gas outlet port 31.
All of the sensor outputs monitored by the computer are stored in a "current run"
file. During an experiment when N2-fixing plant material is enclosed within the plant cuvetter, voltage outputs from the H2 sensor are converted to rates of H2 production and may, but not essentially, be displayed graphically, in real-time, on a liquid crystal screen (not shown) on the front of the NAAS. A graphical display on the same screen can also show the composition of the analysis gas stream at all times during the experiment. The time elapsed during the experiment can also be shown in alphanumeric form, as can the flow rate of the gas through the plant cuvette 21, the PO2 f the analysis gas stream and the rate of H2 evolution. These, and other data collected and processed by the computer may be exported to a remote computer via an RS232 port 32 on the NAAS if desired.
The NAAS also has an analogue output port 33 through which sensor outputs may be monitored on conventional analogue recording devices (not shown).
A screen on the front of the NAAS allows the user to access all the calibration, experimental and data-handling functions of the instrument. In the preferred embodiment described in more detail hereinafter this is achieved by touching command buttons displayed on the screen. The act of touching the screen interrupts a specific point in a network of lights which divides the screen horizontally and vertically into a series of grids. The computer recognises the command associated with the interruption of light at a specific grid and activates the process selected by the user. The computer program also recognises when inappropriate command grids are selected and provides the user with error messages when this occurs. The user may also enter data into the computer in alph:~nllmeric form by ~ lling this data to the screen using a knob located on the front panel of the NAAS. The knob has different functions depending on the particular screen accessed by the user. Several of these screens have data input fields which can be filled by turning the knob to enter either a written or numerical input. The specific functions of the NAAS and the way in which they are accessed by the user are described below.
It will be appreciated from the above description of the NAAS that it possesses all the functions of the prior art gas exchange system illustrated if FIG.l, and incorporates several other features. It also comprises, in a single compact instrument, all the fuctional units required for open system measurement of nitrogenase activity.
However, the major advantage of the NAAS is its ease of use and calibration as will be described hereinafter.
Use of the Nitrogenase Assay Analysis System.
The invention has been described hereinabove with particular reference to a nitrogenase assay system, but it will be readily apparent to those skilled in the art that this invention is readily adaptable to the measurement of low concentrations of gas contained in a mixed gas stream in numerous different environments and situations. For example, the system could easily be adapted to determine alcohol concentration in exhaled air from a human subject and hence could be used as a breathalyzer by law enforcement agencies. Other breathalyzer applications include a hydrogen breath test for lactose intolerance in humans. The system may also be used in industrial or commercial applications, for example to detect and monitor hydrogen production in hydrogen/alllmimlm batteries.
The computer program for running the NAAS is written in and is downloaded to the instrument via the RS232 port 32. Modifications and up-grades of the program can be downloaded from a remote computer via the RS232 port 32.
Preferably, all operations of the NAAS are controlled by the on-board computer and accessed by a touch screen which has several different configurations dependent on the current operation selected. An on-screen "help" function describing how to use the NAAS in each of its modes may be provided for use at all times.
Calibration Before the NAAS can be used for the first time, all of its sensors must be calibrated. A description of the calibration methods is normally provided on the help screen, and the calibration procedure for each sensor is initiated via a main calibration menu. Calibration involves the following procedures:
(i) The H2 sensor is conditioned by exposure to a high partial pressure of H2. This is provided automatically by the electrolysis unit 19.
(ii) The time delay between selecting a change in gas composition and the response to ~116706 ~that change by the gas sensors is measured. The user supplies air and O2 (10) to the NAAS, and attaches the NAAS to the gas exchange system which will be used for experiments. The time delay associated with the particular experimental set-up is measured automatically by monitoring the time required for the O2 sensor to respond to a change in PO2 from air level to 100~o O2. This delay time must be recalibrated whenever the gas exchange system is reconfigured and/or the volume of the sample cuvette 21 is changed.
(iii) The flow sensor (FM1) in the analysis gas stream is calibrated in air and in Ar.
The user attaches a bubble flow meter (or similar, low resistance, flow meter) to the vent from the NAAS and the computer generates a range of flow rates while monitoring the voltage output of the flow sensor. Using the bubble flow meter, the user measures the flow rates generated by the computer and dials in these values to correspond with the voltage outputs from the flow sensor. The calibrations thus attained for air and Ar can be applied, with automated corrections, to any gas mixture used in the NAAS.
(iv) The 2 sensor is calibrated by exposure to pure N2, pure 2 and air in a sequence controlled automatically by the computer. The output of the 2 sensor is monitored in each gas and a linear relationship is calculated between voltage output and PO2.
(v) The H2 sensor is calibrated in both N2:O2 and Ar:O2 atomospheres. The user selects the range of pH2 and PO2 values over which the sensor is to be calibrated in each atmosphere. The electrolysis unit then generates these pH2 and the sensor is calibrated in N2:O2 and the Ar:O2 as PO2 is varied between the set limits. The computer records the voltage outputs of the sensor at each pH2 and PO2 in both N2:O2 and Ar:O2 and generates two look-up tables which relatethese outputs to the pH2 and PO2 in each atmosphere.
Once these initial calibrations have been completed the NAAS is ready for use.
Occasional recalibration of the H2 and 02 sensors may be necessary and the user can check this by an automatic calibration check procedure during which the computer generates a specific pH2 and PO2 and checks the voltage outputs of the H2 and 02 sensors against the expected outputs of the H2 and 02 sensors against the expected oulpu~s calculated from current calibrations. If the expected and measured outputs differ, the computer warns the user than recalibration may be necessary. The NAAS
also has an automatic system check function which monitors the gas pressure (vacuum gauge X) as each solenoid valve is opened sequentially, and warns the user if any of the four gases are not supplied to the system. A warning is also given if the output from the H2 sensor is significantly above zero when no H2 is supplied to the sensor from the electrolysis unit or from plant material in the cuvette. This alerts the user that H2, or another combustible gas, may be cont~min~ting the gas stream. The output from the humidity sensor is also checked automatically throughout the calibration and experimental procedures, and a warning is given if humidity rises beyond an acceptable threshold value.
Measurement of Nitrogenase Activity At the beginning of each experiment the user dials in the current time, date atmosphere pressure and temperature. Plant material is then sealed into the cuvette 21 ~116706 and the NAAS measures H2 evolution rate in either a m~nll~l, or one of several automatic, modes. In m~ml~l mode, the user selects the flow rate and gas composition supplied to the cuvette and the rate at which data collected by the computer is sampled to the current run file. The user may also specify time-dependent changes in the gas composition supplied to the cuvette. Throughout the run, H2 evolution rate, experimental time, measured flow rate and measured PO2 are displayed on the screen.
These data are stored in a current run file and may be downloaded to a remote computer via an RS232 port on the NAAS.
The NAAS has the ability to perform automatic measurements of total nitrogenase activity (TNA) and potential nitrogenase activity (PNA). Both of these involve changes in the gas stream supplied to the cuvette from one composed of N2:02 to one composed of Ar:02. TNA is measured as the peak rate of H2 evolution in Ar:02, whereas the measurement of PNA involves a gradual increase in PO2 during exposure of the plant material to Ar:02 and measurement of maximum H2 evolution rate at optimum PO2. The duration that the plant material is exposed to N2:02, Ar:02 and increasing PO2 in Ar:02 is selected by the user, but the computer recognises the H2 evolution rates corresponding to TNA and PNA. Values for apparent nitogenase acivity (ANA; H2 production rate in N2:02), TNA and PNA are calculated automatically after each automated run is complete. Values of N2 fixation rate, EAC and OLCN are automatically calculated from the ANA, TNA and PNA values. These calculated values may be saved in addition to, or instead of, the entire data set collected during the automated runs.
From the above description of the NAAS operations it will be appreciated that this instrument performs all the functions of the prior art gas exchange system (FIG.2), but overcomes the problems of calibration, expense and physical size associated with that, and similar, large-scale systems. Furthermore, the NAAS is self-checking to ensure that its components and calibrations are functional, and is able to measure and calculate ANA, TNA, PNA, OLCN, EAC and N2 fixation rate automatically. It is the first insllulllent with such capabilities. It should also be noted that the NAAS may also be used in any situation which requires the measurement of pH2 in a flowing gas stream. It could therefore function as, for example, a H2 breath analyzer to test for lactose intolerance in humans or be used in industrial applications to detect H2 gas production from non-living systems.
Claims (12)
1. An integrated gas analysis system for measuring low concentrations of a selected gas contained in a gas stream comprising a plurality of gases, comprising;
(a) gas flow path means having inlet and outlet means;
(b) means for mixing said plurality of gases in any selected ratio thereof for delivery to said inlet means;
(c) means to measure gas concentration of said selected gas and at least one other gas in said gas stream;
(d) means to deliver said selected gas at a selected rate to said flow path whereby said means to measure the concentration of said selected gas may be calibrated;
(e) means for monitoring gas flow rate through said flow path; and (f) means to monitor and control said means for mixing gases, said means to measure gas concentration and said means for monitoring gas flow rate, so as to generate output signals from which values representative of at least one of: (i) concentrations of said selected gas and said at least one other gas and (ii) production rate of said selected gas passing through said flow path means, can be determined.
(a) gas flow path means having inlet and outlet means;
(b) means for mixing said plurality of gases in any selected ratio thereof for delivery to said inlet means;
(c) means to measure gas concentration of said selected gas and at least one other gas in said gas stream;
(d) means to deliver said selected gas at a selected rate to said flow path whereby said means to measure the concentration of said selected gas may be calibrated;
(e) means for monitoring gas flow rate through said flow path; and (f) means to monitor and control said means for mixing gases, said means to measure gas concentration and said means for monitoring gas flow rate, so as to generate output signals from which values representative of at least one of: (i) concentrations of said selected gas and said at least one other gas and (ii) production rate of said selected gas passing through said flow path means, can be determined.
2. A gas analysis system as claimed in claim 1, wherein said selected gas is hydrogen and said other gas is oxygen.
3. A gas analysis system as claimed in claim 2, wherein said gas flow path means is adapted to receive plant tissues.
4. A system as claimed in claim 3 wherein said plant tissues comprises at least part of a nitrogen-fixing plant.
5. A system as claimed in claim 1 wherein said means to monitor and control comprises computer means.
6. A system as claimed in claim 5 wherein said means to deliver said selected gas includes means to generate said selected gas.
7. A system as claimed in claim 6 wherein said means to generate said selected gas comprises electrolytic means.
8. A system as claimed in claim 7 wherein said means to monitor and control operates in real time.
9. An integrated nitrogenase assay analysis system comprising:
sealable gas-tight container means having gas inlet and outlet means and adaptedto receive a sample of a nitrogen fixing plant;
means for mixing selected gases in any selected ratio thereof and delivery to said gas inlet means;
means to measure hydrogen and oxygen concentration in a gas stream passing through said container means;
means to deliver hydrogen at a selected rate to said container means whereby said means to measure hydrogen may be calibrated:
means for monitoring gas flow rates through said container means; and means to monitor and control said means for mixing gases, said means to measure hydrogen and oxygen concentration and said means for monitoring gas flow rates, so as to generate output signals indicative of production rate of hydrogen from said hydrogen delivery means and said sample, from which a measure of nitrogenase activity in said sample can be derived.
sealable gas-tight container means having gas inlet and outlet means and adaptedto receive a sample of a nitrogen fixing plant;
means for mixing selected gases in any selected ratio thereof and delivery to said gas inlet means;
means to measure hydrogen and oxygen concentration in a gas stream passing through said container means;
means to deliver hydrogen at a selected rate to said container means whereby said means to measure hydrogen may be calibrated:
means for monitoring gas flow rates through said container means; and means to monitor and control said means for mixing gases, said means to measure hydrogen and oxygen concentration and said means for monitoring gas flow rates, so as to generate output signals indicative of production rate of hydrogen from said hydrogen delivery means and said sample, from which a measure of nitrogenase activity in said sample can be derived.
10. An integrated system as claimed in claim 9 wherein said hydrogen delivery means includes hydrogen generation means.
11. An integrated system as claimed in claim 10 wherein said hydrogen generation means comprises electrolytic means.
12. An integrated system as claimed in claim 9 wherein said means to monitor and control comprises computer means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002116706A CA2116706A1 (en) | 1993-03-04 | 1994-03-01 | Gas analyzer |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/027,513 | 1993-03-04 | ||
| CA002116706A CA2116706A1 (en) | 1993-03-04 | 1994-03-01 | Gas analyzer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2116706A1 true CA2116706A1 (en) | 1995-09-02 |
Family
ID=4152995
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002116706A Abandoned CA2116706A1 (en) | 1993-03-04 | 1994-03-01 | Gas analyzer |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2116706A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252421A (en) * | 2021-06-17 | 2021-08-13 | 西南石油大学 | Device and method for measuring trace carbon isotopes and heavy components in natural gas |
-
1994
- 1994-03-01 CA CA002116706A patent/CA2116706A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252421A (en) * | 2021-06-17 | 2021-08-13 | 西南石油大学 | Device and method for measuring trace carbon isotopes and heavy components in natural gas |
| CN113252421B (en) * | 2021-06-17 | 2021-09-21 | 西南石油大学 | Device and method for measuring trace carbon isotopes and heavy components in natural gas |
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| FZDE | Dead |