Fall Green Roof Research Intro

My focus of our project is on soil carbon content. I have two experimental questions. First: will added live versus sterile inoculum promote an increase in soil carbon? Second: do sedum species versus native prairie plants lead to higher soil carbon levels? To answer these questions I must be able to accurately measure the carbon in the soil. My goal this semester is just that: get reliable soil carbon data for our experimental trays by using the Flash CN Analyzer. 

Before I can run our real samples, I have to practice preparing samples and running standards and samples with the analyzer using excess soil. This coming week, Susanna and I will collect the end-of-season soil samples for our experiment. From there I will need to grind the soil, finalize the Flash analyzer protocol, and begin running samples.  The Flash Analyzer, which I described in detail in a previous post, is intimidating, but I am excited about the potential data sets we could get; that is if I can get good at using the machine.

This semester, I also want to work on scoring the MIP root slides for our mychorrizal data, but this is secondary because the slides are easily preserved where as I need the carbon data to continue my project. I am also looking forward to helping Susanna with her project which deals in part with soil water retention. 

Summer Green Roof Research explanation

Susanna and I assisted with Kelly Ksiazek’s experiment that had two main questions. The first: does adding live versus sterilized prairie inoculum increase mycorrhizal fungal relationships and promote plant growth. The second: do native prairie plants function better as green roof organisms than traditional sedum species in terms of growth and green roof services provided.

We set up a grid of forty trays filled with soil substrate on the Quinlan Life Science terrace. Life prairie inoculum was added twenty trays and sterilized prairie inoculum to the remaining twenty trays. The inoculum was collected from a local prairie, half of which was sterilized to kill all bacteria and fungi.

Of the forty trays, ten were planted with sedum species, ten with prairie species mix A, ten with prairie species mix B, and ten remained unplanted as the control group. The ten trays of sedum were ordered from the green roof distributer and re planted in the experimental trays. Prairie mixes A and B contain different species of native prairie plants, grown in a greenhouse before planted in the trays. The control trays contain live and sterile inoculum but no plants.

Over the course of the summer we took measurements of plant growth, substrate temperature, soil stability, and soil carbon content. For the plant growth data, we measured month to month the individual plant’s max height, tray coverage area, and flower and fruit number. To measure the tray substrate temperature we inserted a thermochron in the center of each tray. These thermochrons were calibrated to take a temperature reading every hour for an extended period of time. Soil stability was measured using the soil slake test procedure. Soil carbon content was measured by ashing the samples in a muffle furnace. Future soil samples will be measured using the Flash soil carbon and nitrogen analyzer.

Possessed beaker!

 

After staining the roots, I had to sterilize the tissue cassettes by boiling them in water for a while. I added a stirring rod to the mix so the cassettes would stir themselves. It took some careful adjusting, but i got that beaker spinning like nobody's business. Also, first gif ever!

Root Staining

Yesterday, we began staining our MIP roots we had collected and washed over the last few weeks. Luckily for us, the new arbuscular ink and vinegar procedure is really simple compared to old fungi staining techniques. At its core, the procedure is only two steps. The first being boiling the roots (in tissue cassettes) in 10% potassium hydroxide (KOH) for 3-5 minutes. This clears the root cells of their contents, leaving the cell walls and fungi unharmed.

Boiling the roots in the 10% KOH solution.

The second major step is boiling the roots in a vinegar and ink solution for 3 minutes. This stains the fungal tissue within the cells, making it possible to see the hyphae (regular fungal body tissue) and vesicles (reproductive capsules).

10% KOH on the left hotplate and vinegar/ink on the right.

Intermediate steps include removing the cassettes from the hot solutions and rinsing them with water several times. Washing after the first step rinses the remaining KOH from the roots. Washing after the second step initially rinses the excess ink which will leach from the roots.

Susanna removing the cassettes from the solution into water.

Because we are boiling the solutions, the beakers and hot plates obviously get really hot. We got the chance to open and use our brand new thermal safety gloves. They are thick, orange, and terry cloth. Embarrassingly, this is the first pair of elbow length gloves I've ever worn.

Me, looking snazzy and being safe.

Our final product is a beaker full of stained roots, all ready to be mounted on slides. We are temporarily storing them in a beaker of water with a splash of vinegar in the fridge.

Purple haze, all in my roots.

Flash C&N Combustion Analyzer

Last week, we got a surprise present for the lab (surprise for me at least). A brand spanking new Flash Carbon and Nitrogen Combustion Analyzer!  The C&N combustion analyzer does just that: it analyzes the carbon and nitrogen in a sample by burning it at 980 degrees celsius and then 850 degrees celsius until the sample is completely vaporized. I know, pretty hot, huh?

The Flash C&N Combustion Analyzer.

Those two shiny cylinders are the furnaces. They get very hot, as hot as an iron at high temperature. I should know, I couldn't resist (everyone was doing it!). The white tube with red caps is the moisture trap, and the large box behind it is the detector.

Our auto-sampler with two carousels.

This is the auto-sampler. It holds the samples and drops them into the first furnace. Our auto-sampler can hold four carousels- 100 total samples! In the main part of the sampler, there is a piston that pushes the sample into the chrome tube underneath that leads to the furnace.

A carousel with 11 samples loaded.

The Flash analyzer can only process samples that are between about 5-100mg. That's very small. Unfortunately, to get acurate results, we have to buy a new balance for the lab that is accurate to the 0.01mg. Our current balance, that we just got at the beginning of the summer, is only accurate to the 1mg which is apparently not good enough for the Flash analyzer (well, la dee da). Once the sample is massed out in a small tin cartage, it must be balled up to fit into the machine.

Susanna, making a perfect sample ball.

In the first 15 seconds after the sample is dropped into the first furnace, an eerie red light flashes and glows from the eye of the auto-sampler. This is the reflection of the sample combusting at almost a thousand degrees celsius.

The burning eye of the auto-sampler is watching.

When the samples have finally dropped into the furnace, vaporized, and gone through the detector, the computer spits out this:

The not-so-impressive-looking C&N data.

It doesn't look like much, but the area under the peaks (the integral) tells us quantifiably how much nitrogen and carbon was in the sample. The first peak is the amount of nitrogen detected and the second peak is the amount of carbon detected. Once you understand the process and what the graph means, it actually is pretty awesome. You know, for nerds like us.

IButton fever

For the last few weeks we have been trying to understand how to use iButtons for our experiment. We got pretty frustrated because someone forgot to include a manual with the equipment (I'm looking at you, Maxum Integrated). IButtons remotely record the temperature, allowing us to gather multiple data points over a period of time. We were  eventually able to set them up to record the temperature once every hour, on the hour.

Susanna showing off her hard work.

We then planted the iButtons in a randomly selected trays of each experimental group. Each iButton was double bagged of course to prevent water damage.

Me, getting ready to plant an iButton.

Digging the hole.

A well planted iButton.

We also placed three iButtons on the bare roof to measure background roof temperature. Through these temperature measurements, we expect to see lower temperatures in the green roof soil compared to the bare roof temperature.

One of three hot roof iButtons.

After 2000 hours (that's all the storage the iButtons have) we will collect them and finally be able to see some data. After a few days, we downloaded the measurements from one of the bare roof iButtons to see if they were working. We were so excited to see beautiful oscillating data between night and day temperatures.

Corn harvest and root washing

Last week, we harvested the MIP corn. We had to do so carefully as to preserve the stalk and root tissues while also separating them for individual analysis.

Dr. Chaudhary demonstrating the harvest method.

We used clippers to cut the stalk at the very base and put it in a paper bag for later. We then put the conetainer, containing the roots, into plastic bag and into the freezer. We put them in the freezer to stop all biological activity, especially decomposition. 

We want to weigh and record the root tissue mass, but we first must wash all of the soil off of the roots (The roots are very dirty). We do this very carefully.

Gaze in wonder at my clean roots

We then collect some of the tissue to be made into slides and save the rest for weighing.