Pretty Good Philosophical Instruments

coming soon: an inexpensive kit to measure biological nitrogen fixation… and more!

The core function of the Pretty-Good Philosophical Instrument board (“PI” for short) is to estimate the rate of nitrogen fixation by legume root nodules. A user-supplied host computer (Raspberry Pi 4 or 5) is needed to process user commands, display graphs, and save data, using downloaded open-source Python code. The on-board Raspberry Pico (also running Python) controls pumps and reads sensors. The PI board can support additional measurements, most requiring added components. These include:

• optical density of bacterial cultures
• photosynthesis
• respiration cost of N fixation (Oono et al., 2020)
• nodule-interior oxygen relations (Denison & Layzell, 1991)

“Philosophical Instrument” was an old name for things like microscopes and barometers, when science was called “Natural Philosophy.”

Is “Pretty Good” Good Enough?

The PI’s software-centered approach reduces costs and expands potential uses, but this flexibility comes with tradeoffs. The PI may be less accurate or less portable than more-expensive devices optimized for some subset of the measurements the PI can make. For example, adding a $59 CO2 sensor would let you measure photosynthesis or respiration, but its accuracy of +/- 30 ppm makes it most suitable for relative measurements. The PI should not be used for medical diagnosis or other applications where limited accuracy could lead to health risks or economic loss.

Some specific limitations are addressed below. If you don’t understand these limitations (or are too busy to read on), the PI is not for you. Soldering is only required if you want to add something to the prototype area. But if downloading software, using a screwdriver, learning a little Python, or maybe rearranging the flexible-tubing connections on the back of the PI (to increase throughput or add gas mixing) sound like a hassles rather than opportunities, please don’t order a PI.

For example, the optical-fiber LED and photodetector on the PI make it fairly easy to add the capability to measure optical density of bacterial cultures. This only requires downloading a file, sending it to a 3D printer, and adding two prisms to direct light through the culture in a user-supplied cuvette. But using the same fiber-optics to study nodule-interior oxygen relations (nodule O2 permeability and nodule-interior respiration: Denison and Layzell, 1991) requires compressed gases, 3D printing a nodule-oximetry probe, and adding about $120 of components to the PI.

The PI depends on a user-supplied host computer, assumed to be a Raspberry Pi 4 or 5. It also needs a 12-volt power supply, which users must supply, based on the electrical standards in their country. Users must also supply a rack to support at least two 50-mL tubes: the hydrogen generator and a water trap, which can also be used as a cuvette for detached nodules or roots. Assays using intact plants are recommended, but require some way to contain the roots and connect them to the PI.

A PI measures production of hydrogen gas, a byproduct of nitrogen fixation (Layzell et al., 1984). This is faster, easier, and less dangerous than the old actetylene-reduction method. However, some root-nodule bacteria (rhizobia) and most cyanobacteria (including nitrogen-fixing symbionts in some lichens) recycle hydrogen, making hydrogen release (if any) an unreliable measure of nitrogen fixation. Even when nodules release hydrogen, uptake of hydrogen by soil bacteria could be a problem.

Host-imposed “sanctions” constrain both the hydrogen and acetylene methods. In saturating (10%) acetylene, 100% of nitrogenase activity goes to reduction of acetylene to ethylene, which can be measured by gas chromatography (about 5 minutes per gas sample). Similarly, in a nitrogen-free atmosphere (Ar:O2), 100% of nitrogenase activity goes to hydrogen production. In each case, measured nitrogenase activity should match nitrogen fixation in air, but only until the lack of N fixation triggers host “sanctions” (Kiers et al., 2003), which reduce nitrogenase activity (Minchin et al., 1983). Nitrogenase activity can be monitored continuously as hydrogen production in air or using subsaturating acetylene (Denison et al., 1983), but converting those to nitrogen fixation in air requires an assumed or measured Electron Allocation Coefficient (Maloney et al., 1994).

Furthermore, sensitivity of the PI’s sensor to hydrogen depends on the background gas (e.g., humidity and percent oxygen). Therefore, the PI supports electrolytic generation of hydrogen to calibrate the sensor (for each gas mix, if you add gas-mixing capability to measure N-fixation efficiency). The sensor also responds to methane, ethanol, or carbon monoxide, so it will only be useful when the gas mixture is dominated by only one of these gases. On the other hand, it might be possible to (for example) estimate methane production from soils where hydrogen production is negligible. Or, maybe you could use sensor response to a known hydrogen-production rate to measure oxygen concentration.

The PI board is designed to support additional functions, including ones designed and implemented by users who are competent to assess any risks of modifications to core functions. See the blog for examples.