Respiration

Background & Extensions

Why?

Photosynthesis transfers carbon dioxide from the atmosphere to plants, releasing oxygen in the process. Respiration runs this reaction backwards, releasing CO₂ while consuming oxygen.

C₆H₁₂O₆ + 6O₂ = 6CO₂ + 6H₂O

glucose + oxygen = carbon dioxide + water

Globally, photosynthesis removes 120 Pg (120 billion metric tons) of carbon every year from Earth’s atmosphere and turns it into the carbohydrates that make up living plants. Respiration and decay within ecosystems return that same amount of carbon to the atmosphere every year. We can quantitatively measure this flux in any outdoor environment, and compare it with anthropogenic carbon fluxes. Global climate change - especially rising temperature - can perturb the carbon cycle in both the short and long term.

What?

The movement of carbon dioxide from the atmosphere into biomass is part of the global carbon cycle (Figure 1).

• On very long timescales (thousands to millions of years) the geologic carbon cycle moves carbon from the Earth’s mantle and crustal rocks into the atmosphere through volcanic eruptions. Carbon is removed from the atmosphere by chemical reactions that weather and decompose rocks.
• On shorter timescales carbon moves between biomass to the atmosphere via photosynthesis and respiration.
• In the ocean carbon also cycles to the atmosphere and back through exchange with sea water.

Each of these three parts of the carbon cycle runs in a steady state, with all the inputs and outputs in balance. The fluxes can be very large, but as long as they are all balanced there is no overall accumulation in any one place. In contrast, the human contribution to the carbon cycle is one-way: humans burn fossil fuels and biomass releasing CO₂ to the atmosphere.  There is no equivalent return process, thus humans upset the natural balance.

C-Cycle Fluxes: Data Collection

The first step of this experiment is to make the one-liter CO₂ flux chambers.  This requires collecting empty plastic bottles ahead of time to have enough for your class.  There is a separate YouTube video that takes you and your students step-by-step through the process of cutting up the bottles to make the chambers.

Graphing & Analysis

Students should plot their measured concentrations as a time series, either by hand or using any graphing software. Based on the behavior of their particular experiment, students can then select a time segment to calculate the slope of a straight-line fit to their data. The slope of the line is the rate of change of CO₂ concentration due to soil respiration:

ΔCO₂ (ppmv) / ΔTime (min) = Flux (ppmv/min)

The data collected by the CO₂ probe is in units of concentration – parts per million.  It is often more useful to work in units of mass: grams, kilograms or metric tons of CO₂.  If students compare their own measurements to those published in the literature they will have to do some unit conversion.  For example, in Figure 1 the fluxes are given as gigatons of carbon per year.  In the Unit Conversion Chart below we will convert the measured concentration change into a mass flux of CO₂ – the mass of CO₂ per unit volume per unit time.  We will use metric (mks) units, so we should end up with kg/s (or kg/min). 

Flux is an extensive property; one that changes with the size of the system.  For example, the longer we observe, the more CO₂ moves through the flux chamber, or, the larger the chamber the more CO₂ will move through it.  Therefore, we need to normalize our measurement with respect to both time and the cross-sectional area of the flux chamber.  The rate calculation already accounts for the time of observation.  We still need to account for the basal area of the flux chamber.

Table 1: Unit Conversion from Concentration to Mass Flux of CO₂

CO₂ Experiments Indoors and Outdoors

The simplest version of this experiment—one flux chamber on a patch of bare soil—is the same in both indoor and outdoor environments. But each setting offers different opportunities to ask additional questions. For example, working indoors with small flower pots allows students to manipulate some of the environmental parameters that control respiration, like temperature and moisture. Working outdoors, students can investigate the balance between photosynthesis and respiration.

Tips & Tricks

Two important aspects of conducting respiration experiments:

• Student-grade CO₂ gas probes can be somewhat fussy instruments
• Soil respiration is heterogeneous, both in a natural outdoor environment and when using potting soil indoors

For both of these reasons, if students conduct paired experiments, it is important to hold constant as many variables as possible. Let the CO₂ probes sit still and equilibrate with the ambient environment for 5-10 minutes before beginning an experiment. Make measurements as closely-spaced in time as possible (e.g. don't conduct the measurements on two separate days). If using flowerpots, use the same soil sample and CO₂ probe for each of the heating and chilling experiments.

Don't let students breathe on the probes (until after they're done) and for the same reason they shouldn't hover over the apparatus while the probe is measuring CO₂ flux. When working outdoors, wind can significantly disturb the CO₂ concentration in the flux chamber. Use a sweater or jacket to shield the chambers on a windy day.

When you do the experiment the magnitude and rate of change will vary depending on your local conditions and whether you are running the experiment outdoors or indoors.  An amenable environment for respiration will produce better results, for example, the soil shouldn’t be too cold, too wet, or too dry.  If you want the students to graph the results by hand, make the sampling interval long enough that they are not overwhelmed with data, ex. at 15-second intervals for 3-4 minutes.  The rates in Figures 4 & 5 are typical.

When working with gases students need to understand the concept of the mole as a unit of molecular mass.  The units that a laboratory CO₂ gas probe measures – parts per million – are parts per million by volume (ppmv), rather than the parts per million by mass that is common in aquatic chemistry.  In order to convert measurements made in units of ppmv to mass units like grams of carbon, students will need to first convert ppmv to moles of gas, then moles to grams of gas. For middle school students, or others who have not yet encountered moles as units of molecular mass, the simple rate calculation can substitute for the mass flux in most situations, and can be used to compare the behavior of different systems.

Links

• Student Handout - Indoor Experiment
• Student Handout - Outdoor Experiment

Indoor Experiment

Prepare enough soil for your class. If you are using commercial potting soil you will need to inoculate it with soil fungi/microbes, as potting soil is sterilized. You can mix in some soil from outside, or add any kind of decomposing organic matter, or a fermented food like yogurt or kombucha. Keep the soil warm and moist to support its biological community ahead of your experiment. Mix your soil sample as well as you can. Soil is very heterogeneous, and your students will get more consistent results is the soil samples are as homogenous as possible. Set up and run the experiment following the instructions in the handout.

The relationship between temperature and respiration

You can run a simple experiment with a single chamber—or—you can investigate how temperature controls soil respiration rate. The data below are for two side-by-side pots of soil, one in an ice water bath, one in a hot water bath. Note that you will need a container without drainage holes if you use a water bath to control the temperature, as flooding the soil will also change the respiration rate.

A second way to examine the role of changing temperature on soil respiration rate is to use a single experimental set-up that begins chilled, and then warms up (Figure 7). If the soil samples can be stored in a fridge overnight, students can measure the initial respiration rate when they are removed from the fridge, then gradually warm the pots on the stove to monitor the changing respiration. This procedure has the advantage of working with a single system, instead of assuming that the two systems in Figure 6 would behave identically under identical conditions.

Outdoors: Conducting a Paired Experiment with opaque and clear flux chambers

An example of the outdoor experiment is shown in the video, "Respiration: Reuse, Recycle"

On a grassy outdoor site such as the one shown in the video both photosynthesis and respiration contribute to the CO₂ flux. For this reason, we run a paired experiment with an opaque chamber and a clear chamber. The darkness of the opaque chamber suppresses the photosynthesis of the grass, allowing students to measure the respiration flux. The magnitude of that difference can be seen by conducting the same experiment in the adjacent clear chamber (Figures 8 & 9).

Figure 8 above is a typical result for an outdoor measurement in a grassy field on a warm, sunny day. The different results in the clear chamber and the dark chamber each provide for interesting analysis and discussion.

1. The dark chamber data are the result of active soil respiration. Students can either use the software that supports the CO₂ probe to find a linear best fit to the data, or can draw a straight line by hand through the data points.  The slope of the line gives the rate of CO₂ flux in units of ppm/minute (or ppm/sec if the experiment was set up with x-axis units of seconds).
2. The clear chamber is even more interesting. Everything about the two experiments (clear and opaque) is the same —except—that the clear chamber transmits sunlight.  Here the grass is photosynthesizing, thus consuming the CO₂ that the soil respires.  The sum of these two processes is no significant change in the CO₂ in the chamber.

The advantage of conducting this experiment outdoors is that students are measuring an actual environmental flux. When designing this experiment we collaborated with colleagues at Cornell University, and calibrated the measurements made with our Vernier Go-Direct CO₂ probe and plastic bottle flux chamber to data collected with a research-grade LiCOR 6400 CO₂ flux system.  The student results were identical to the research-grade data.  This result gives us confidence that interesting and powerful observations can be made with very simple tools.

When students quantify their observations they are then able to compare the data that they collect themselves with other local, regional, and global data sets.  For this experiment we designed the gas flux chamber to have a volume of one liter.  Additionally, we measured the cross-sectional area of the chamber so that we know the area of the soil that we are sampling.  These two parameters allow us to convert the units of the student-collected data and compare them with published data for soil CO₂ flux.

Take it Beyond the Classroom

Figure 11: Global mean annual soil carbon flux (MgC/km2/yr), Raich et al., 2003. Figure from Carbon Dioxide Information Analysis Center., U.S. Department of Energy Lawrence Berkeley National Laboratory.

The map above (Figure 11) shows the global variation in annual soil CO₂ flux in units of megagrams (10⁶ grams = 1000 kg = 1 metric ton ) of carbon per square kilometer.  In order for students to compare their own data to the global map here they will need to do a multi-step unit conversion.  The table below (Table 2) walks students through this process step-by-step.  Alternately, you can download an Excel spreadsheet that automates the unit conversion process.

Download Excel unit conversion spreadsheet (780 kB)

Table 2: Additional Unit Conversion

If students can conduct the indoor temperature-control experiments in addition to the outdoor experiment then they will have experience both with the effects of photosynthesis as well as temperature. Even without doing the direct temperature experiments the influence of temperature on respiration rate can be inferred from the global flux map. On the handout we suggest asking questions such as -

1. How does your result compare? Is it the same/higher/lower?
2. Since the map shows annual values, how might your result be different if you could make measurements all year long?
3. Examine the map. Where are C fluxes high (generally)? Where are they low?
4. What conclusions can you draw about how environmental conditions control the rates of soil respiration and decomposition?
5. If climate is warming globally, how might that change these results several decades into the future?
6. Look again at the global carbon cycle in Figure 1. Consider the three terrestrial reservoirs for carbon:
   1. Atmosphere 800 Gt
   2. Biosphere 550 Gt
   3. Soil 2300 Gt

Beyond that, these experiments have implications for the global carbon cycle, and particularly for our climate as the Earth warms. The terrestrial soil carbon reservoir is much larger that the carbon reservoir of terrestrial biomass and that atmospheric carbon reservoir. Increased global temperatures lead to increased soil respiration rates that will transfer carbon from soil to the atmosphere. This is an example of a "positive" or reinforcing feedback. Warmer temperatures lead to faster transfer of CO₂ from soil to the atmosphere, where it causes increased warming, which reinforces the faster respiration, etc. The fact that the soil reservoir is almost three times as large as the atmosphere means that the effect of this feedback is potentially very large.

If this conclusion is a source of anxiety for students, encourage them to think about photosynthesis (especially if they have done the paired outdoor experiment) and how simple actions like planting trees actually can have a real impact on climate.

Note about soil respiration map: as of March 2023, the website that hosts these data has been moved here – https://data.ess-dive.lbl.gov/view/doi:10.3334/CDIAC/LUE.NDP081 .Unfortunately, this is a much less user-friendly site than the original – https://cdiac.ess-dive.lbl.gov/epubs/ndp/ndp081/map_yrmn.html. As long as the original site remains on-line, it’s a better resource.