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CO2 mineralization write up

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					        Protocol for obtaining values for soil CO2 mineralization over time

Data that must be collected in addition to the CO2 concentrations
    •   Volume of each jar
    •   Bulk density of soil (use with volume of jar and weight of soil to determine moles of air
        in jar when soil is in there, too)
    •   Air temperature at beginning and ending of each incubation time period
    •   With t=0 being the first incubation and the beginning of the experiment, the incubation
        time for each phase, as well as the time at which each incubation pahse begins (in the
        overall experiment scheme)
    •   Soil moisture content (g H2O/ g dry weight soil) (because soil is added to jars when wet,
        but dries out)
    •   Atmospheric pressure at beginning and end of each incubation period (if available;
        especially important if the weather is variable over the experiment time frame)


Data Collection
    1. Acid wash as many pint-size mason jars as you need. Double rinse the jars, and let them
       dry completely before using them. You need as many jars as you have samples, plus
       extras to insert between every ten jars (blanks for calibrating the analytical equipment).
    2. Collect your other supplies:
            a. 20ml syringes with stopcocks (as many as you have jars)
            b. needles for sampling (at least 2 or 3)
            c. lids for each of the jars, both the screw on cap and the replaceable lid. Each lid
               should have a rubber stopper (septum) in the lid.
            d. thermometer
            e. balance
            f. barometer
            g. paper bags or drying tins
            h. Sharpie marker for labeling (you don’t have to have tape – you can write on the
               glass jars
            i. lots of plastic wrap
    3. Go to your field site and collect soils. Be sure to collect enough to do all the tests you
       plan, as well as some to archive if you have freezer space!
    4. When you collect your soils, be sure to keep them cold – just above freezing temperatures
       – so you don’t experience any microbial activity before you begin the experiment.


CO2 mineralization protocol
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    5. Go to the lab.
        (If you can’t start right away, you can store your soils in the refrigerator for no more than
        24 hours after you collected them.)
        **Keep soils cold even when you remove them from the refrigerator – work with a cooler
        close by if necessary**
    6. Sieve the soil sample to remove any particles > 2mm.
    7. To obtain soil moisture content: add 10 g of soil to a drying tin or more to a paper bag,
       label, and place the sample in a drying oven for at least 48 hours at 60ºC. (After it is dry
       you can save the sample for C:N analysis.)
    8. Prepare each mineralization test jar this way:
            a. Label the jar for the soil sample. (write with the Sharpie on the glass)
            b. Add 20 g of soil to the jar.
            c. Cover the jar with plastic wrap, and use the screw-on lid to keep the plastic in
               place.
            d. Keep the jar cold until all the jars are prepared and you can begin the experiment!
    9. When all the jars are ready, and you have the appropriate number of control jars (which
       don’t get anything in them) you can begin the experiment!
    10. Set all the jars out on a counter, where they will stay during the incubation period.
    11. Ensure homogenized air over all the jars by fanning a large lunch tray or something
        similar about 1 foot above the jars.
    12. Then, being sure not to breathe at all (yes, hold your breath) so you don’t add CO2 to any
        jars, being capping the jars and screwing the lids on tight. When you have to exhale and
        inhale again, go somewhere else, like outside the room. Continue until all the jars have
        been capped.
    13. Make a note of the air temperature, the atmospheric pressure, and the time, which you
        should also denote as time = 0.
    14. 12 hours later (note the time exactly), you should extract air from each jar in the
        following manner. Be sure each of your syringes is labelled. Keep them in order!
            a. Open the stopcock on the syringe.
            b. Pump air in and out of the syringe a few times to clean it out.
            c. Add a needle to the end of the syringe.
            d. Pump air in and out of the needle at least twice to flush out the needle.
            e. Insert needle through the septum.
            f. Mix the air in the jar by pumping air in and out of the syringe while the needle is
               in the jar.
            g. Draw up 20 ml of air into the syringe, and turn the stopcock so air cannot be
               exchanged once the needle is removed from the jar.

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            h. Remove needle from the jar and take needle off syringe.
    15. After collecting air from all of your jars, including the controls, collect information on the
        air temperature and air pressure.
    16. Take your syringes to the analysis equipment that you will use to measure CO2
        concentration. Be sure to insert a syringe from a blank jar every ten jars or so.
        ** At UAF, use the Forest Soils lab’s backpack Licor with the attached injection module.
        Call to find out if it is available. **
    17. At the analysis instrument, follow the instructions for using it very carefully! Be sure to
        set up the necessary standards. If a computer program is available, take a disk to save
        your data.
        ** At UAF, you will find instructions in the notebook to the right of the computer, but
        PLEASE have someone familiar with the module walk you through the process a couple
        of times. When you have collected your data, it will need to be transformed to a
        workable format using a separate program. Ask for help. **
    18. After collecting your data for the incubation period, clean up your jars. Remove the lids
        from each jar and cover them with plastic wrap, using the screw top lids to keep it place.
    19. You should begin incubations approximately
        every 24 hour period until you notice a steep                    4000
                                                            co2/gdw/hr



        decline in the CO2 concentration produced in                     3500
        each jar. The figure will look like this:
                                                                         3000                      Linbor over tim e

    20. Once the decline has flattened out incubations                              H
                                                                         2500
        can be approximately 3 days apart.
                                                                         2000
    21. When you are done, the soil can be used for                                 1 H

        NH4+ and NO3- extractions in KCl or K2SO4                        1500
                                                                                    3 1 H             H
                                                                                                      1
        (particularly informative if you also used 20 g                  1000
                                                                                    F 3
                                                                                        1
                                                                                                                        H
                                                                                                                                         H
                                                                                                                                         1
        of soil at t=0 to get the amounts of NH4+ and
                                                                                        3             3                 1
                                                                          500         F                                 3                3
                                                                                        F
        NO3-)
                                                                                                      F
                                                                                                                        F                F
                                                                            0
    22. To clean up, wash out the jars to remove all                            0       50   100    150   200    250   300   350   400   450
                                                                                                              tim e
        soil, and then wash in acid bath, rinse, and dry.
        Put away all materials used!
    23. Once you have collected data, it should be reworked and analyzed! Read on…


    Data reformatting and analysis

    24. Figure out the grams of dry weight for each sample:
        gdw = total wet weight of soil/(1+ gH2O/gdw)
    25. Use gdw along with soil bulk density to determine the volume of the jar occupied by soil,
        which will also reveal the volume of the jar occupied by air.
                 ml of soil in jar = gdw/(bulk density) = soil volume (cm3)

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    26. PV=nRT: use this to determine the number of moles of air in each jar at the start of the
        incubation and the end of the incubation.
        n=PV/RT, where P = air pressure (mm Hg), V = volume of jar (L), and T = air
        temperature (K); R = 8.3143
    27. calculate the number of moles (n) in the syringe when the sample was removed at the end
        of the incubation:
                 n in 10 ml syringe= (101*.010)/(8.3143*T at end)
    28. determine micromoles of CO2 present in the jar initially
        µmolCO2 initial = (n in syringe*initial[CO2]/100)*10000000
                       (unless you take a reading at t=0 when the incubation begins, initial CO2
        = average initial [CO2] of all jars)
    29. determine micromoles of CO2 present in the jar at the end of the incubation
        µmol CO2 final = (n in syringe*final[CO2]/100)*10000000
    30. calculate difference in CO2 concentrations (µmol CO2 final - µmol CO2 initial)
    31. µmol CO2/gdw/hr =
              (µmol CO2 final - µmol CO2 initial)/gdw soil in jar/incubation interval (hours)
    32. When you graph the µmol CO2/gdw/hr over time, you can get a decay curve, (as shown
        in the figure at #19). DeltaGraph has a curve fitting feature you can use with this, so for
        each sample you can establish its decay curve over the length of the experiment.
        With the decay curve, you can get decay constants (k). Also, you can integrate the
        equation to find the area under the curve (the total CO2 produced over the length of the
        minerlaization experiment).
        The decay curve has the equation y = m + y0e-kt,
        so the integral of the equation is ∫(m + y0e-kt) dx.
        Therefore the area under the curve can be calculated as [mx + (y0/-k)e-kt]0final t

    Other analysis tips:
       - Subtract the average of the control specimens from the values obtained for each
           sample to standardize based on the control.
       - Use ANOVA to determine the significance of differences between the k or the CO2
           production of samples




CO2 mineralization protocol
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