Laboratories for Measuring Physiological
Processes in Plants and Animals

Nitrogen Fixation

Plants require a number of different mineral elements for healthy growth, elements which may already be available in the soil or may need to be added in the form of fertilizers. Some of these elements are needed in relatively large amounts (the macro-nutrients) and others in very small amounts (the micro-nutrients). One of the most important macro-nutrients, one that is frequently in short supply, is nitrogen. This may seem strange when the atmosphere contains 80% nitrogen. Unfortunately this nitrogen is in a gaseous form (N₂) that plants are unable to use. Plants must obtain nitrogen in the form of nitrate (NO₃^-) or ammonia (NH₃) and these forms are much less abundant.

There are organisms that can "fix" or convert gaseous nitrogen into ammonia and they are referred to as "nitrogen fixers". They are able to make large amounts of the nitrogen in the air available to plants through this activity. All of these nitrogen-fixing organisms are prokaryotes; some are free-living in soil or water and others live in a symbiotic relationship with higher organisms. The most well-studied symbiotic association exists between legumes (beans, peas) and the bacterium Rhizobium. The Rhizobium cells live in nodules on the roots of the plant. It is within the bacteria that live inside these nodules that the following reaction occurs:

N₂ + 8H⁺ + 8e^- + 16ATP   →  2NH₃ + H₂ + 16ADP + 16P_i

This reaction is catalyzed by the enzyme, nitrogenase. As you can see from this equation, the reduction of N₂ to ammonia is a very expensive process energetically. It requires a large number of electrons and ATP molecules to reduce a single molecule of N₂. The process is made more expensive by an unavoidable side reaction which simultaneously reduces protons (H⁺) to molecular H₂. While the latter reaction is a wasteful one for the plant, it does provide us with a way to indirectly measure the activity of the nitrogenase enzyme using a hydrogen sensor. By pumping air through the nodulated root system of a legume in a sealed pot, we can measure the amount of H₂ evolved by the activity of nitrogenase. This is not a direct measurement of the rate of N₂ fixation but it gives us the Apparent Nitrogenase Activity (ANA) expressed as µmol H₂/hr. This is because a minimum of 2 of the 8 electrons used by nitrogenase actually go to H₂ production; the remaining 6 electrons are used to reduce N₂ to NH₃. Thus by measuring H₂ production we can estimate the proportion of enzyme activity that is being used to reduce protons. While this proportion should represent at least 25% of the total nitrogenase activity (according to the equation above), the allocation of electrons between N₂ and H⁺ reduction can vary. To measure the Total Nitrogenase Activity (TNA), expressed as µmol H₂/hr, we must replace the N₂ in the air passing through our system with an inert gas such as argon (Ar). In the presence of Ar (and the absence of N₂) all of the electrons previously used to reduce N₂ will now be used to produce H₂ and the rate of H₂ production should increase. The maximum rate of H₂ production after the switch to argon will represent the TNA. The rate of nitrogen fixation that was occurring in air can be calculated as follows:

N₂ fixation rate = (TNA -- ANA)/3

The portion of electrons going to N₂ reduction (rather than H₂ production) is determined by subtracting the ANA from the TNA. This number is then divided by 3 because N₂ reduction requires 3 pairs of electrons compared to H⁺ reduction which requires only one pair of electrons. The N₂ fixation rate would be expressed in µmoles N₂ reduced per hour.

The other important parameter of nitrogen fixation is the Electron Allocation Coefficient (EAC) which describes the allocation of electrons between proton (H⁺) reduction and N₂ reduction.

EAC = 1 -- (ANA/TNA)

Remember that at least 25% of total electron flow through the nitrogenase enzyme is used to reduce H⁺ to H₂ but this value is not constant. Often more than 25% of the electrons are used to reduce H⁺, which is energetically very wasteful for the plant. Thus it is important to determine the EAC as an indicator of the efficiency of nitrogenase activity. Higher values of the EAC indicate that a higher proportion of nitrogenase activity is being used to provide ammonia to the plant.

The purpose of this experiment is to demonstrate that nitrogenase activity and the rate of N₂ fixation in legumes can be determined by measuring the rate of H₂ evolution from nodulated roots. It will also investigate how N₂-fixing nodules regulate their activity with changing physiological and environmental conditions. The nitrogenase enzyme is extremely O₂ labile and a very low O₂ concentration is maintained in the infected cells of the nodule to prevent nitrogenase inhibition. This experiment will show that the nodule is capable of regulating its internal O₂ concentration to maintain active nitrogenase.

In this experiment you will also investigate the paradox faced by nodules with respect to O₂. Oxygen is required for aerobic respiration in support of nitrogenase activity, but it is also a potent inhibitor of the nitrogenase enzyme. Therefore, there must be stringent control of the O₂ status of the infected cells so that a sufficient flux of O₂ is maintained to support nitrogenase-linked respiration, while a low O₂ concentration is maintained to avoid nitrogenase inhibition. This control of O₂ concentration is achieved, in part, by the presence of a "diffusion barrier" in nodules that is able to vary its resistance to O₂ entry depending on changes in both O₂ supply and demand. The barrier restricts entry of O₂ into the nodule, and the high rate of infected cell respiration (O₂ consumption), helps to maintain a low O₂ concentration in the nodule central zone. In this experiment, you will observe how nodules respond to changes in external O₂ concentration.

You must read the file "Measurement of Nitrogen Fixation" in the N₂Fix Pack folder or on the web before proceeding with the experiment.

Materials

A nodulated soybean in a growth pot cuvette

Qubit Systems Nitrogen Fixation Package

Procedure - Part 1 Electron allocation efficiency

1. The computer screen will show two graphs, the upper graph displaying corrected H₂ in volts plotted against time, and the lower graph showing %O ₂ plotted against time. Adjust the x axis on both graphs to show a maximum time of 45 minutes by first clicking anywhere within the graph to activate it, then highlighting the maximum value showing and typing in "45". Then hit return.
2. The first step in determining rates of nitrogen fixation is to establish base lines of corrected H₂ values in air and in an argon:oxygen (Ar:O₂) mixture, without a plant attached to the system.
3. Attach a gas bag containing Ar:O₂ (80:20) to the inlet of the air pump and the purple flow restrictor (100 ml/min) to the outlet and flush this gas through the system. Click on the Start button to begin data collection.
4. A trace will appear on the graph that represents the ZERO value for your experiment in Ar:O₂. Note that this value is not 0.0, but a positive value that must be subtracted from your measured data during data analysis. Allow the zero reading to proceed for approximately 5 minutes, or until the trace is stable. Record this value in the Results and Discussion section.
5. Detach the bag of Ar:O₂ from the pump and flush room air through the system. Allow the H₂ trace to reach a new steady state value. This is your ZERO value for measurements made in air, and it may be different from that for measurements made in Ar:O₂. Record this value in the Results and Discussion section.
6. Seal the plant in its growth pot and attach the outlet of the pump, with the black flow restrictor (500 ml/min) attached, to the gas inlet port at the base of the pot. Attach the tubing from the gas outlet port on the lid of the pot to the inlet of the desiccator column.
7. When the pot is attached to the system, observe the increase in the voltage output of the H₂ sensor. This is shown numerically at the far right of the bottom of the screen and is labeled "Cor. H₂", for corrected hydrogen. When this reaches a steady state, record this value in the column labeled "Cor. H₂ in Air".
8. Reduce the flow rate through the system by attaching the red flow restrictor (200 ml/min) to the pump. Observe the change in output of the H₂ sensor. Does it increase or decrease?
9. Increase the flow rate through the system again by replacing the red flow restrictor with the black one and wait for the Cor. H₂ to reach a steady state.
10. Attach the bag of Ar:O₂ to the inlet of the pump and observe the increase in the Cor. H₂ value. When this reaches a steady-state, record the value in the column labeled "Cor. H₂ Ar:O₂". After you have completed this measurement, flush the plant with room air.
11. After measurement of H₂ evolution in Ar:O₂, stop data collection and save your data by selecting "Save as . ." from the File menu. Save your data in your designated folder. Now you may then clear your graph by selecting "Delete Run, Latest" from the Data menu.

Procedure - Part 2 Effects of oxygen

1. Remove the plant from the system and attach the purple flow restrictor to the pump. Attach a gas bag containing N₂:O₂ (70:30 or 30% O₂) to the inlet of the pump and measure zero. Wait until this reading is stable and record it. The trace on the graph represents the ZERO value for your experiment in 30% O₂. Note that this value is not 0.0, but a positive value that must be subtracted from your measured data during data analysis. Allow the zero reading to proceed for approximately 5 minutes or until the trace is stable. Record this value in Part 2 of the Results and Discussion section.
2. Detach the 30% O₂ bag from the pump and allow room air to flush through the system again. Allow the zero reading to proceed for approximately 5 minutes or until the trace is stable. This represents the ZERO value for your experiment in room air. Record this value in the Results and Discussion section.
3. Attach the plant in its sealed growth pot to the system with the black flow restrictor to observe the change in the Cor. H₂ value until a steady state is reached. Record this value in the column labeled "Stable at 20% O₂.
4. Attach the gas bag containing 30% O₂ to the inlet of the pump and note the time. You should see the Cor. H₂ value decline swiftly, and then recover slowly over the following 30 min. Record the minimum value attained, and the steady state value attained while the root system is exposed to 30% O₂.
5. When the output from the H₂ sensor has reached a steady value at 30% O₂and you have recorded it, detach the gas bag from the system and flush with room air. Stop data collection.

Data Analysis

If you have not previously recorded them, determine the appropriate values from your experiment and record them in the Results and Discussion section by following the procedure described below:

1. Open your experimental file. A command box will appear asking you whether or not you wish to load the calibration stored with your data file. Answer "Yes". Your data will appear on the screen exactly as it appeared when you saved it at the end of the experiment.
2. Select "Examine" from the Analyze menu. A vertical line will appear on your graph which can be moved along the data by moving the mouse. Note that as you move the vertical line, the digital display on the bottom of the screen will change to show you the Cor. H₂ value from the H₂ sensor and the time value at the point on the graph where the line is situated.
3. The Cor. H₂ measurements are shown in volts and should be converted to rates of H₂ production using the following equation:

   H₂ production (µmol/hr) = voltage x K x flow rate

   K is a factor for converting voltage readings to measurements of H₂ concentration in units of µmol/liter. Flow rate has units of liters per hour. Since the H₂ sensor has a different response in N₂ and Ar, the value of K differs in each gas.

K = 0.75 µmol/liter per volt in air K = 1.0 µmol/liter per volt in Ar: O2 K = 0.95 µmol/liter per volt in N2:O2 (30% O2)

You must use the correct value of K in your calculations in air, Ar:O₂ and 30% O₂. Also, remember that you must subtract the appropriate zero reading from your H₂ voltage measurements before using the equation above.

Results and Discussion

Part 1

	Black Flow Restrictor Flow Rate	=	________ L/hour
	Cor. H2 Zero Value in Ar:O2	=	________ Volts
	Cor. H2 Zero Value in Air	=	________ Volts
	Cor. H2 Value in Air	=	________ Volts
	Cor. H2 Value in Ar:O2	=	________ Volts

1. Calculate values of ANA and TNA, in µmol H₂/hr, from your experiment using the formula and K values given on the preceding page. Use these to calculate the EAC of your plant. Show your calculations. Why does EAC have a maximum value of 0.75?

 ANA=

 TNA=

 EAC=

2. What happened to the H₂ content of the gas flowing from the pot when flow rate was reduced (red flow restrictor used)? Can you suggest a reason for this response? How did this affect ANA?
3. Given that 2 electrons are required to reduce 2 protons to H₂, and 6 electrons are required to reduce one N₂ molecule to 2 NH₃, use your data to calculate the rate of N₂ fixation in your plant in terms of µmol N₂ fixed/hr. Show your calculations.

   µmol N₂ fixed/hr =

4. Legumes have a maximum EAC value of 0.75. What would be the effect on plant yield if this value declined to a very low level? Explain.

Part 2

	Black Flow Restrictor Flow Rate	=	________ L/hour
	Cor. H2 Zero Value in Air	=	________ Volts
	Cor. H2 Zero Value in 30% O2	=	________ Volts

	Values of Cor. H2
	Stable at 20% O2	Minimum at 30%O2	Maximum at 30% O2
Plant # 1
Plant # 2

Using these values of Cor. H₂, calculate rates of H₂ production and record these in the table below. Make sure to subtract appropriate zeros from the Cor. H₂ values, to use the appropriate value of K, and to use the correct flow rate for each of your calculations.

	H2 Production Rate (µmoles H2/hour)
	Stable at 20% O2	Minimum at 30%O2	Maximum at 30% O2
Plant # 1
Plant # 2

1. When O₂ concentration is raised from 20% to 30%, the increased gradient of O₂ from the rhizosphere to the nodule interior causes an increase in internal O₂ concentration which, if maintained for a long period, would cause irreversible inhibition of nitrogenase activity. What changes in nitrogenase activity did you observe when O₂ concentration was raised in the roots of your plant? Explain your results.
2. Examine the nodules that are attached to the root system of your plant. Remove one of them and slice it in half to observe its structure. Note that its interior is a bright red color due to the presence of the blood-like protein leghemoglobin. What is a possible role of this protein in the nodule?