GENERAL LAB INTRODUCTION

By now, you know that primary producers, or autotrophs, are responsible for the vast majority of all energy inputs to an ecosystem.  Exceptions to this rule include ecosystems that are “powered” by organic matter flowing in from outside their boundaries as well as by carbon fixation by their own autotrophs.  An example of this is a lake ecosystem, where rivers draining into the lake provide allochthonous inputs of organic matter while phytoplankton and fringing wetland plants produce autochthonous inputs of organic matter.  Given the strong dependence of ecosystem energetics on primary production, it is instructive to investigate what controls rates of primary production in ecosystems.  Controls internal to the ecosystem are largely biotic processes, such as limitations on the growth rates of plants or competitive interactions among plants of different energetic efficiencies.  Exogenous, or external controls on ecosystem metabolism are often called forcing functions.  Two very important controls on autotrophic production are light availability and nutrient availability.  In today’s lab, we will use light-dark bottle experiments to study how both may control primary productivity, and thus whole-system metabolism, in aquatic ecosystems.

The control light has on primary productivity should be obvious.  Since photosynthesis is the biochemical process by which plants harness solar energy and us it to produce organic molecules from inorganic ones (namely CO₂ and nutrients), the amount of light available to a plant must obviously control the rate of production of organic matter.  In terrestrial ecosystems, light availability is controlled by shading, and plants compete for light with a number of strategies.  In aquatic ecosystems, shading is also a factor but the main control on light availability is the fact that water absorbs light energy.  In fact, there is an exponential decline in available light as you proceed deeper in a water column—light extinction--such that:

            I_Z = I₀e^(-kz)

Where I_z is the amount of light at some depth=z, I_o is the amount of light at the surface (often expressed simply as 100%), and k is the attenuation coefficient (in units of 1/z, or m^-1).  Clear water has a low attenuation coefficient, meaning that light penetrates relatively far.  Turbid or colored water bodies tend to have higher attenuation coefficients.  A quick and easy way to measure the attenuation coefficient of a body of water is with a Secchi Disk (you will learn how to do this in lab).  The Secchi Disk Depth (D_s) relates to k as:

            k = 1.7/D_s

In a very simple model system, you could assume that the rate of primary production is directly proportional to the amount of light at a given depth.  Thus, GPP should decline exponentially with depth in the same way that light availability declines:

            GPP_Z = GPP₀e^(-kz)

Notably, this extrapolation of the first equation assumes a direct relationship between GPP and I, and thus assumes that nothing else limits primary production and whole-system metabolism.

In fact, the availability of inorganic nutrients also very frequently control primary productivity in ecosystems as well.  Leibig’s Law of the Minimum says that the element or nutrient that is least available relative to its requirements by primary producers is the limiting nutrient.  The nutrients that most often limit ecosystem metabolism through their controls on primary productivity are nitrogen and phosphorus (these are known as macronutrients).  Inorganic nitrogen is a key component of amino acids—thus protiens—DNA, and RNA.  Inorganic phosphorus is a key component of ATP compounds, DNA, and bones.  The ratios at which C, N, and P are required by plants varies.  For unicellular algae, such as phytoplankton and the algae that make up periphyton, this ratio is very close to the Redfield Ratio, which is 106:16:1 for C:N:P (a molar ratio).  If the availability of N is 20 times greater than the availability of P, then P will limit carbon fixation by phytoplankton and periphyton.  If the concentration of N is 10 times that of P, then N availability will limit primary production.

The equation that relates how a limiting nutrient controls primary production is known as the Michaelis-Menten equation:

The key variables in this equation are the actual uptake rate of the limiting nutrient, U, at a given concentration of that nutrient, [S], given the known maximum uptake rate of that nutrient, U_max and the half-saturation constant, K_s.  The maximum rate of uptake is determined by the physiology of the plant in question.  The half-saturation constant is the concentration of limiting nutrient at which U = 0.5(U_max).  A high U_max suggests rapid production if this nutrient is not limiting and a low K_s suggests very efficient production at very low concentrations of the limiting nutrient.

Everglades wetland and aquatic ecosystems are characterized by extreme nutrient limitation, and are thus known as oligotrophic systems.  Phosphorus is the limiting nutrient in Everglades ecosystems.  Although Hennington Pond is not exactly a pristine Everglades aquatic setting, we will assume that phosphorus limitation is controlling primary productivity and whole-system metabolism there as well.  In lab this week, you will use experimental additions of phosphorus and a light-dark bottle experiment to determine whether or not phosphorus availability is, in fact, controlling ecosystem metabolism.

PRE-FIELD LAB INSTRUCTIONS

1.  Generate several testable hypotheses as a class that you can test with today’s exercise.

2.  Set up field data sheets for today’s work, remembering the importance of noting exact times of readings, oxygen concentration, water temperature, bottle numbers, treatment type, and habitat location.

3.      Divide into groups and work as teams in the field.  Work should be divided up so that all team members get to experience each aspect of the exercise.  In other words, don’t make one person record data for the entire lab exercise!

4.      Be sure that you have all field sampling equipment that you will need.  Read below and make a list before you leave the lab.

5.      After sampling, return to the lab and your TA will pool data from all teams.

FIELD LAB INSTRUCTIONS

1.      The lab group before yours has already made the initial measurements in the bottles and set them in the appropriate places to incubate.  The light experiment has bottles incubated at the surface and at depth in the deep part of Hennington Pond.  The nutrient addition experiment was done in the wetland portion of the pond.   Retrieve the bottles, carefully remove the ground glass caps (you do not want to generate any bubbles in the bottles!), and insert the self-stirring oxygen probe.  Note the time, and measure the oxygen concentration and water temperature.  Your TA will give you the initial oxygen values for all bottles when you return to the lab.

2.      Now you have to set up an incubation for the next lab.  Empty all bottles, rinse them, refill them carefully, and organize them by treatment (water, water+periphyton, light, dark, surface vs. deep, nutrient vs. no nutrient, etc.).  Note which bottle numbers correspond to which treatment.  Carefully add a small piece of periphyton to the appropriate bottles.  Add the designated volume of NaHPO₄ solution to the appropriate bottles.  Take initial oxygen and temperature readings (and the exact time of each).   Carefully replace the bottles in the appropriate location in each habitat.

3.      Now make some measurements about the environment in which your bottles were incubating.  If possible, measure the depth of water over your bottles and total water depth.  Measure water clarity as Secchi Depth.  Make some generalities about the gross amount of solar energy during your incubation.  Note anything else that seems important to you.

Answer/address all of the following questions on your own paper.  In some cases, this will require computer printouts of spreadsheets, graphics, or statistical output.

1.       Present all of your system metabolism for both labs and all experiments in a spreadsheet.  Your spreadsheet should include calculations of GPP, NPP, and R (in mg O₂ hour^-1).

2.      From the first week’s lab, did you see a difference in either production or respiration rates with versus without periphyton?  Use a one-way ANOVA to test this “treatment” effect.  Do the statistics back up your conclusion?

3.      From the second week’s lab, did you see a difference in either production or respiration rates with phosphorus additions or with high vs. low light?  Use a one-way ANOVA to test both “treatment” effects.  Do the statistics back up your conclusions?

4.      Were you able to conclude that phosphorus is the nutrient limiting whole system metabolism in Hennington Pond?

5.      Take the Secchi Depth (D_s) that you measured for the deep water incubations you did in the second week’s lab.  What percent of surface light do these readings tell you was available at your incubation depth?  Now, using the attenuation coefficient (k) that you calculate from D_s and your known depth of incubation (z), calculate GPP at that depth (GPP_z) using your actual measured GPP at the surface (GPP₀).  How does it compare to your actual measured GPP at depth?  Explain any differences.

6.      Your measurements of whole system metabolism, using the light-dark bottle method, actually neglect several major components of the Hennington Pond ecosystem.  What are they?  How might you experimentally include these components in more inclusive measures of system metabolism?