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Carbon Dioxide (CO₂) Evolution From Sediments

Question of Interest:

What effects do different carbon sources and other amendments have on the rate of carbon dioxide evolution from microbial respiration?

Hypothesis:

If simple sugars are metabolized by microorganisms  more readily than more complex sugars, then the rate of carbon dioxide evolution should be increased more by the addition of simple sugars than by complex polysaccharides. Other amendments might also increase or decrease CO₂ evolution.

Materials:

Sediment samples
Pint size wide-mouth mason-type jars with lids, ~ 500 ml
50 ml beakers (one for each jar)
Amendments:  NH₄NO₃, glucose, sucrose, fructose, other sugars, be creative
NH₄Cl
NaOH (1.0N)
HCL (1.0N)
BaCl₂, 50% solution
Phenolphthalein
Pipettes, 1 ml
Burettes
Analytical balance
 

Procedure:

1. Weigh four 150 g samples of the sediment to be tested.  Place on sheets of paper and set aside until ready for use.
2. To each of the jars, add enough water to bring the soil sample to approximately 60% of field capacity (`15-20 ml for 150g sample).
3. Amend all but one of the samples with the additions of your choice.  Be sure to leave one sample unamended.  Mix the samples thoroughly and add to the separate jars containing the water.  The samples will moisten from the bottom by capillary action.
4. Add 15 ml or 1.0N sodium hydroxide (NaOH) to each of the 50 ml beakers.  Gently rest the beakers containing the solution on the surface of the soil in the jars and also place a solution containing beaker into an empty jar.  Seal the jars tightly, making sure to wipe all soil from the rim of the jar and slightly moisten the rubber seal in the lid prior to screwing it down.

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5. Incubate the jars at room temperature (25 C) or at the temperature of your choosing.  After 24 hours to 14 days (depending on your amendments), determine the amount of CO₂ evolved by the sediment sample.

Determining the amount of CO2 produced:

1. Open one jar at a time and carefully lift out the beaker containing the base solution.

2. Add 2-3 drops of phenolphthalein (watch for a color change as demonstrated in the figure below) and 1.0 ml of 50% BaCl₂ to the beaker to precipitate the carbonate as insoluble barium carbonate.

3.  Titrate the un-neutralized base with 1.0N HCl. Titrate slowly and stir gently with a glass rod until the pink coloring just disappears. Approach the endpoint slowly.

4. Record the exact volume of acid required.

5. Calculate the amount of CO₂ evolved using the following formula:

Milligrams (mg) C or CO₂ = (B-V)NE
Where:
V = Volume (ml) of acid to titrate the base in the CO₂ collectors from the samples
B = Volume (ml) of acid to titrate the base in the CO₂ collectors from the control.
N = the normality of the Acid
E = equivalent weight.  If results are expressed in terms of carbon, E = 6; if expressed as CO₂, E = 22.
* It is easiest to express the results as mg of CO₂ produced per 100 g of soil.

Results:
 

Conclusions:

Treatment 2, the sucrose amendment, showed a significantly increased amount of CO₂ evolution by the bacteria. This is surprising given that sucrose is a disaccharide. We had hypothesized that a monosaccharide would be more readily metabolized by the bacteria than a disaccharide.

Analysis Questions:

1. How much carbon dioxide is evolved from the control jar per gram of sediment?
2. Glucose is what percentage by weight carbon?
3. If you used another carbon amendment, what percentage of carbon by weight would it contain?
4. Calculate the percentage of added carbon that you recovered as carbon dioxide.
5. Assuming there are a billion cells/gram of sediment, how much carbon dioxide was respired by each bacterium? Why is this a gross oversimplification of what is actually occurring?
 

Modifications: for K-8 modifications
 

Extensions:

1. To measure carbon dioxide production as a function of time, one could make multiple samples using the same soil recipe throughout. These samples would be analyzed at different times to plot the changes in carbon dioxide production.

The shape of the graph produced from # 1, reveals details of bacterial growth (i.e. lag phase, exponential growth, etc.).

2. A "true baseline" experiment can be produced by creating, in addition to the control as described above, a control sample in which the environment is sterilized (microwaving or autoclaving the sediment, for example). Any CO₂ produced in this control is a result of "chemical turnover" rather than microbial metabolism.

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