Abstract:
A method for measuring an evolution rate of a gas from a sample includes equilibrating a sample with an alkaline solution and a pH indicator and permitting the alkaline solution to absorb formed carbon dioxide in an enclosed headspace. From the pH indicator at equilibrium is determined a time increment at which an increment of the alkaline solution is consumed by the CO 2 . Carbon dioxide evolution rate is calculated from the time increment, the volume increment, and the alkaline solution concentration. A device for performing this measurement includes a sample vial and a reaction chamber having an opening adapted for mating with a sample vial opening and an opening for receiving the solution. The reaction chamber is dimensioned for equilibrating the sample with the alkaline solution and for determining the time increment required for an increment of the alkaline solution to be consumed by CO 2 .

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application claims priority from commonly owned provisional application Ser. No. 60/249,771, filed Nov. 17, 2000, “Carbon Dioxide Microrespirometer.” 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates to a device and method for determining gas evolution rates in solids and liquids, and, more particularly, to such a device and method for determining carbon dioxide evolution rates in a sample.  
           [0004]    2. Description of Related Art  
           [0005]    Respiration is a common indicator of biological activity. Respirometry, the measurement of respiration rates, has been applied to a broad spectrum of applied and environmental microbiology, such as toxicity, with treatability, process control, and prediction of biological oxygen demand (BOD 5 ) in wastewater treatment, assessment of metal toxicity, living soil microbial biomass, and food quality.  
           [0006]    Respiration rates can be measured either by rates of oxygen consumption or CO 2  evolution. Rapid oxygen consumption rate can be measured by using an oxygen probe or a quantitative electrolytic cell. Most oxygen respirometers, however, are applicable only to liquid samples. Oxygen respirometers with an electrolytic cell can be used to determine respiration of solid or semisolid samples, but their sensitivity is compromised.  
           [0007]    Sensitive and rapid CO 2  respirometers based on infrared (ir) detectors have been developed in the past three decades and can handle solid samples with high speed and sensitivity. Instrumental respirometers are technically complicated and expensive if accuracy and sensitivity are needed. Noninstrumental CO 2  respirometers operated by an alkaline trap and acid-base titration have been in existence for many years. They are simple but relatively slow, with a measurement time in days, and less sensitive, with a detection limit in mL CO 2 /day. Sensitive and rapid determination of respiration rates is highly desirable in monitoring microbial activity in food and environmental samples. A desired sensitivity, for example, would comprise one in the microliter CO 2  per hour level, and a rapidity of determination within about an hour.  
         SUMMARY OF THE INVENTION  
         [0008]    It is therefore an object of the present invention to provide a device and method for determining gas evolution rates rapidly and sensitively.  
           [0009]    It is another object to provide such a device and method for determining CO 2  evolution rates directly.  
           [0010]    It is an additional object to provide such a device and method for use with solid or liquid samples.  
           [0011]    It is a further object to provide such a device and method having a modest cost.  
           [0012]    It is also an object to provide such a device and method operable under laboratory or remote site conditions.  
           [0013]    These and other objects are attained by the present invention, a first aspect of which is a method for measuring an evolution rate of a gas from a sample. The method comprises the steps of pre-equilibrating a sample with a solution comprising an alkaline solution and a pH indicator and permitting the alkaline solution to absorb formed carbon dioxide in an enclosed headspace. After the CO 2  absorption/evolution equilibrium is attained, from a change in the pH indicator is determined a time increment at which a small increment of the alkaline solution is substantially consumed by the CO 2  evolved. A calculation is made of a carbon dioxide evolution rate from the time increment, the small increment volume and concentration of the alkaline solution.  
           [0014]    Another aspect of the invention is a device for measuring an evolution rate of a gas from a sample. The device comprises a sample vial having an opening into an interior space for containing a sample therein. The device further comprises a reaction chamber having an opening adapted for mating with the sample vial opening and a solution-receiving opening for receiving a solution comprising an alkaline solution and a pH indicator. The reaction chamber is dimensioned for receiving a predetermined amount of the alkaline solution to absorb formed CO 2  from a sample within the headspace.  
           [0015]    A further aspect of the invention is a system for measuring an evolution rate of a gas from a sample. The system comprises a respirometer device as described above and means for determining from a change in color in the pH indicator a time increment at which a small increment of the alkaline solution is substantially consumed by the CO 2  from the sample.  
           [0016]    The features that characterize the invention, both as to organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description used in conjunction with the accompanying drawing. It is to be expressly understood that the drawing is for the purpose of illustration and description and is not intended as a definition of the limits of the invention. These and other objects attained, and advantages offered, by the present invention will become more fully apparent as the description that now follows is read in conjunction with the accompanying drawing. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    [0017]FIG. 1 is a schematic illustration of a microrespirometer of the present invention.  
         [0018]    [0018]FIG. 2 is a graph of CO 2  absorption versus shaking rate of the microrespirometer.  
         [0019]    [0019]FIG. 3 is a graph of CO 2  absorption and concentration of an alkaline solution in the microrespirometer.  
         [0020]    [0020]FIG. 4 is a graph of CO 2  absorption rate versus CO 2  concentration in the headspace of the microrespirometer.  
         [0021]    [0021]FIG. 5 is a graph of the percent of equilibrated value versus pre-equilibration time in the microrespirometer for a range of evolution rates  
         [0022]    [0022]FIG. 6 is a graph of CO 2  evolution rate versus the rate determined by the microrespirometer. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0023]    A description of the preferred embodiments of the present invention will now be presented with reference to FIGS.  1 - 6 .  
         [0024]    The basis of the system  10  and method of the present invention is to establish a carbon dioxide absorption/evolution equilibrium between an alkaline solution and a sample. After the equilibrium is attained, an indicator comprising, for example, phenolphthalein, is used to indicate the end point of a small increment of the alkaline solution being consumed by the CO 2  evolved.  
         [0025]    The system  10  of the present invention comprises a microrespirometer device  11  (FIG. 1), which in turn comprises a substantially transparent reaction chamber  12  and sample vial  13 . The reaction chamber  12  comprises a small alkaline trap with a total headspace of 6-7 mL having a small septum hole  14 . The sample vial  13  size is variable, and exemplary sizes include 25, 30, 40, and 75 mL (e.g., Fisherbrand EPA bottles, Suwanee, Ga.). The reaction chamber  12  and sample vial  13  are coupled through a standard threaded screw  15  and septum liner  16  to form a closed headspace  17 .  
         [0026]    An alkaline solution  21  is injectable, such as using a syringe  18 , into the reaction chamber  12  via a solution-receiving opening  18 , and a sample  19  is placeable in the sample vial  13 . The alkaline solution absorbs the CO 2  in the headspace  17 . The indicator in the alkaline solution changes color when the alkaline solution is “consumed” by CO 2 . Preferably the microrespirometer  11  is shaken at a fixed rate (e.g., 240 rpm) on an orbital shaker  20  to enhance CO 2  absorption.  
         [0027]    The alkaline solution of the present invention comprises a solution of NaOH, BaCl 2 , and indicator, with an equal molar ratio of NaOH and BaCl 2  and 0.5 mL indicator solution, with 0.5% phenolphthalein in 50% ethanol solution, per 50 mL alkaline solution. BaCl 2  in the alkaline solution precipitates the absorbed CO 2 , which ensures the stoichiometry of 2 moles of alkaline spent per mole of CO 2  absorbed:  
         CO 2 +2NaOH+BaCl 2 =BaCO 3 ( s )+2NaCl   (1)  
         [0028]    BaCl 2  also sharpens the change of color at the end point when a very low level of respiration is being determined. The alkaline solution is stored in a septum-capped vial to prevent absorption of CO 2  from the air. The alkaline solution is transferred through, for example, a syringe  18 .  
         [0029]    Optimal operating conditions for the system  10  were determined with a series of experiments. The effect of shaking on the CO 2  absorption of the microrespirometer  11  was investigated by coupling microrespirometers  11  with empty 25-mL sample vials  13  in a glove box having a known CO 2  concentration, as determined with an ir CO 2  analyzer.  
         [0030]    A 0.2-mL portion of 0.002 M alkaline solution was injected into each reaction chamber  12 . The microrespirometers  11  were shaken at fixed rates of 100, 150, 200, 250, and 300 rpm. The time required to consume the alkaline solution in each microrespirometer  11 , as indicated by the indicator color change, was recorded. Each test was repeated in triplicate, and the results are plotted in FIG. 2. The CO 2  absorption is shown to increase as the shaking rate is increased from 100 to 250 rpm. The increase in CO 2  absorption levels off when the shaking rate exceeded 250 rpm. Shaking at 200 rpm or higher improves reproducibility of CO 2  absorption. A fixed shaking rate between 200 and 250 rpm is recommended for the microrespirometer  11  because the benefit of shaking is achieved while the difficulty of operation at higher rates is avoided.  
         [0031]    The effect of alkaline concentration on the absorption of CO 2  in a closed headspace  17  was investigated at 25° C. A 25-mL sample vial was connected to an ir analyzer so that the vial  13  and the ir detector formed a closed headspace  17  in which air circulated continuously. The 25-mL vial  13  was shaken at 240 rmp on an orbital shaker  20 . 1-mL portions of 0.2, 0.1, 0.01, and 0.0011 M were injected into the vial  13  through the solution-receiving opening  18  at the beginning of the experiment, and the concentration of CO 2  in the vial  13  was recorded periodically.  
         [0032]    The experiment was repeated twice, and the results are plotted in FIG. 3, where each dot represents a single measurement. It can be seen that as the concentration of alkaline solution decreases from 0.2 to 0.01 M, the CO 2  absorption rate decreases as well. The CO 2  absorption rate does not decrease further as the alkaline concentration is reduced from 0.01 to 0.001 M. It is not believed possible to have complete absorption of CO2 in the headspace  17  of the microrespirometer  11  in a matter of hours when the concentration of the alkaline solution is less than 0.01 M. The concentration of the alkaline solution has to be much less than 0.01 M in order to determine CO 2  evolution rate at a microliter per hour level. The microrespirometer  11  therefore does not work on the principle of complete CO 2  absorption, but on an absorption/evolution equilibrium principle that will be discussed in the following.  
         [0033]    An alkaline solution of less than 0.0005 M is not sufficiently stable to be used in the microrespirometer  11  because the possibility of contamination from ambient CO 2  is too large for such low alkalinity. Phenolphthalein is not stable in alkaline concentrations exceeding 0.01 M; the deep pink color fades away by itself within 1 h. Therefore, a preferred alkaline concentration range suitable for the microrespirometer  11  is between 0.01 and 0.001 M.  
         [0034]    The relationship between CO 2  absorption rate and the CO 2  concentration in the headspace  17  of the microrespirometer  11  was also investigated. Microrespirometers  11  with a 75-mL sample vial  13  were coupled in a glove box of known CO 2  concentration. Increments of 0.1 mL 0.002 M alkaline solution were injected into the reaction chamber  12 . The microrespirometers  11  were shaken at 240 rpm, and the time required to consume each increment of the alkaline solution was recorded. The consumption of each increment of the alkaline solution, for example, 0.2 μmol alkaline, or 0.1 μmol CO 2 , represents a 29.7-ppm (v/v) reduction of CO 2  concentration in the 82-mL microrespirometer  11  at 25° C. Each treatment was performed in triplicate, and the results are plotted in FIG. 4, with each dot representing a single measurement.  
         [0035]    In using the microrespirometer  11  of the present invention, a portion of solid or liquid sample  19  is placed in the sample vial  13 , and the vial  13  is coupled to the reaction chamber  12 . 0.8 mL alkaline solution of a desired concentration is injected into the reaction chamber  12  using a syringe  18 . The respirometer  11  is shaken at a fixed rate, for example, 240 rpm, for 30 min, which comprises the pre-equilibration period, ensuring that the alkaline solution is not completely consumed during this time. If the alkaline solution is about to be consumed, more alkaline solution is injected into the reaction chamber  12 . After the 30-min pre-equilibration the shaker  20  is stopped, and the alkaline solution in the chamber  12  is withdrawn to leave 0.1-0.2 mL. The respirometer  11  is continued to be shaken until the alkaline solution changes to a faint pink color. The shaker  20  is stopped immediately, and 0.1 mL alkaline solution is injected, shaking is resumed, and the time required to consume the alkalinity is recorded.  
         [0036]    In an alternate embodiment, all the alkaline solution in the chamber  12  is withdrawn, and a new 0.1 mL portion of alkaline solution is injected prior to resuming the shaking.  
         [0037]    In either case, once the first indicator change has been recorded, increments of 0.1 mL alkaline solution are injected a predetermined number of further times, for example, twice more, and the time required to consume each increment is recorded.  
         [0038]    The average of the times required to consume each 0.1-mL increment is used to calculate CO 2  evolution rate using the following formula:  
         carbon dioxide evolution rate (μ mol/h )=(0.1×10 3   ×M/ 2)/60 t   (2)  
         [0039]    where M is the concentration of the alkaline solution in mol/L and t is the time required to consume the 0.1-mL increment in min. The CO 2  evolution rate can be expressed in microliters per hour by multiplying the molar volume of CO 2  at a specific temperature.  
         [0040]    The relationships between the CO 2  absorption rate of a 0.002 Malkaline solution and the concentration of the CO 2  in the headspace  17  is shown in FIG. 4. In general, the CO 2  absorption rate has a positive curve-linear relationship with the concentration of CO 2 . The absorption rate of the respirometer  11  at a given temperature and shaking rate reflects the CO 2  concentration in the headspace  17 , which may not be the CO 2  evolution rate of the sample. However, if a sample is equilibrated with the alkaline solution in the respirometer at a given temperature and shaking rate, the concentration of CO 2  in the respirometer would eventually reach a constant value when the CO 2  absorption rate equals the CO 2  evolution rate. For example, if the starting CO 2  evolution rate of the sample  19  is 100 μL/h, the CO 2  concentration of the respirometer  11  is increased to about 660 ppm and remain there because an equilibrium of CO 2  absorption and evolution is established. If the CO 2  evolution rate of the sample is 20 μL/h, the CO 2  concentration of the respirometer  11  is decreased to about 150 ppm, where an absorption/evolution equilibrium is established. The CO 2  evolution rate of a sample  19 , therefore, can be determined by the CO 2  absorption rate of the microrespirometer  11  when an equilibrium or steady state is established. That is, after a sample is equilibrated with an alkaline solution in a microrespirometer  11  of the present invention, the CO 2  evolution rate can be determined by the time required to consume a small increment of the alkaline solution, as shown in Eq. (2).  
         [0041]    The minimum time required for a sample  19  in the respirometer  11  to reach an equilibrium is deduced from a computer simulation based on a relationship between the CO 2  absorption rate and the CO 2  concentration of the respirometer  11  and the CO 2  evolution rate of the sample  19 . That is, the concentration of CO 2  in the headspace  17  after being shaken for a small increment of time Δt is  
           C   i+Δt   =C   i +( E−A   Ci )Δ t/V   headspace    (3)  
         [0042]    where C i  and C i+t  are the CO 2  concentrations of the respirometer at time i and time i+Δt, respectively. A Ci  is the CO 2  absorption rate of the respirometer at time i and is a function of the CO 2  concentration C i . E is the CO 2  evolution rate of the sample  19 , and V headspace  is the volume of the headspace  17 .  
         [0043]    The mathematical relationship of A Ci  and C i  was generated by a nonlinear regression curve fitting program (Table Curve, Jandel Scientific, San Rafael, Calif.) using the data of FIG. 4. The regression enabled the calculation of A Ci  based on C i . The values of A Ci , C i , and C i+Δt , for each small time increment (0.5 min) of Δt were calculated and tabulated using a spreadsheet software (Excel, Microsoft, Redmond, Wash.) based on Eq. (3). An equilibrium is attained in the simulation when the CO 2  concentration in the respirometer approaches a constant, i.e., (E−A Ci ) approaches 0 and C i+t  approaches C i . The minimum time required to attain an equilibrium is the sum of all small time increments, Δt, during which CO 2  concentration approaches a constant. The ratio of the CO 2  absorption rate to evolution rate (i.e., A Ci /E) expressed as a percentage of the CO 2  evolution rate during the time source of reaching an equilibrium is presented in FIG. 5. Two headspace volumes of the respirometer, i.e., 12 mL (5 mL remaining headspace in the sample vial plus 7 mL in the reaction chamber) and 27 mL (20 mL remaining headspace in the sample vial plus 7 mL in the reaction chamber) were simulated in FIG. 5.  
         [0044]    The results indicate that the smaller the headspace  17 , the quicker an equilibrium is reached, and that the greater the CO 2  evolution rates, the quicker an equilibrium is reached. For example, in the 12-mL headspace case, 30 min pre-equilibration is sufficient for the measurement of all CO 2  evolution rates ≳1 μL/h. In the 27 mL headspace case, 100-107% of equilibrated value can be attained within 45 min for all CO 2  evolution rates, except the 1 μL/h case. The working range of the respirometer is designed to be 1-300 μL/h, which requires 30-45 min of pre-equilibration time, according to the condition of this study, to measure accurately the CO 2  evolution rate. If the CO 2  evolution rate is very low (≳5 μL/h), the headspace  17  of the respirometer  11  should be kept minimal to hasten the equilibration. The respirometer  11  was designed so that the size of the reaction chamber  12  stays the same while the size of the sample vial  13  may vary according to the need of samples and the requirement of a minimal headspace  17 .  
         [0045]    A validation experiment was performed by comparing results using the microrespirometer  11  with a method using an ir analyzer such as known in the art. Portions of soil samples of relatively low CO 2  evolution rates (2-5 μL/h/g), unfrozen processed meat samples of medium CO 2  evolution rates (10-100 μL/h/5 g), and room-temperature milk samples of high CO 2  evolution rates (80-280 μL/h/20 mL) were placed in 25-mL sample vials  13 . The CO 2  evolved by microorganisms associated with each sample was determined by the microrespirometer  11  method of the present invention. A duplicate sample in another 25-mL sample vial  12  was also placed in a 250-mL flask, and the CO 2  evolution rate was determined by the ir analyzer method known in the art. The sample vials  12  in the microrespirometers  11  and those in the 250-mL flasks of the ir analysis method were exchanged, and the CO 2  evolution rates determined again with the alternate methods.  
         [0046]    One of the advantages of the microrespirometer  11  is its ability to determine the CO 2  evolution rate accurately at the μL/h level in a short time. Determination of the CO 2  evolution rates at a μL/h level is quite a challenge even for a sophisticated ir method. The IR analyzer must be able to detect less than 10 ppm (v/v) changes of CO 2  concentration with certainty during a period of hours. The accuracy of an IR analyzer method is further limited by the uncertainty of the volume occupied by a solid sample, and, therefore, that of the headspace, in most cases. Variation of headspace humidity, pressure, and temperature all affect the accuracy and precision of an ir respirometer. Because the microrespirometer method is based on the principle of CO 2  absorption-evolution equilibrium, its accuracy is not affected by headspace volume, humidity, pressure, or initial CO 2  concentration. The simplicity, noninstrumental nature, and very modest costs of the microrespirometer  11  make it available to many laboratory and field applications where accurate and rapid determination of respiration rate is desired.  
         [0047]    In the foregoing description, certain terms have been used for brevity, clarity, and understanding, but no unnecessary limitations are to be implied therefrom beyond the requirements of the prior art, because such words are used for description purposes herein and are intended to be broadly construed. Moreover, the embodiments of the apparatus illustrated and described herein are by way of example, and the scope of the invention is not limited to the exact details of construction.  
         [0048]    Having now described the invention, the construction, the operation and use of preferred embodiment thereof, and the advantageous new and useful results obtained thereby, the new and useful constructions, and reasonable mechanical equivalents thereof obvious to those skilled in the art, are set forth in the appended claims.