Intro

This experiment was designed to test a specific point brought up in the final figure of the paper, where we propose a model for phenazine electron transfer. Specifically the question is: Do reduced PCA and PCN react more quickly with PYOox or oxygen?

If the inter phenazine reactions are faster, then most of the O2 reduction will be completed by PYOred, which actually has the fastest O2 reduction rate of all the phenazines. That seems inherently efficient and is satisfying. Furthermore, because PYOox seems to be retained, it may serve as a very effective barrier to quickly reoxidize PCA and PCN, promoting their back diffusion (to the interior) before they diffuse very far into the oxic region and beyond.

The design of these experiments was to see if reduced PCA/PCN was mixed with PYOox under oxic conditions if PYOred was generated in this reaction. If significant PYOred accumulates, it suggests that there is a separation of time scales where the interphenazine reaction is indeed fast compared to O2 reduction by the phenazines.

The approach was to monitor absorbance at 690nm at fast time intervals, since this wavelength is specific to PYO and varies in the oxidized and reduced states (Oxidized has higher 690 abs).

Methods

These reactions were performed in plastic cuvettes in the old aerobic spec that people use for od500 etc. This instrument was capable of recording one cuvette at 1.5sec intervals at 690nm. This setup was also convenient, because a baseline of the oxidized phenazine (or PBS) was acquired for 10 seconds, then the reduced phenazine (or PBS) was injected directly into the cuvette. This instrument simply covers the cuvette with a door that I could open and close rapidly (as opposed to the plate reader). This meant that I could record data before and after the reaction started, which is important because the O2 reaction with PYO does happen fast.

These experiments used the same reduced phenazines stocks made for the earlier inter phenazine experiments. Therefore these stocks were 100uM reduced (electrochemically) PYO, PCA or PCN (or PBS stored in the same way) and stored anoxically in sealed serum bottles. Oxidized PYO was freshly made 100uM stock. Everything was done in PBS pH 7.0, 50mM NaCl.

The following reactions were performed in triplicate with 90 second acquisitions:

1. PYO_ox + PBS
2. PBS + PYO_red
3. PYO_ox + PCA_red
4. PYO_ox + PCN_red

I also acquired PCA_red and PCN_red by themselves to confirm that they do not absorb at 690. That would be:

• PBS + PCA_red
• PBS + PCN_red

Whenever PBS was used in place of the oxidized phenazine an aerobic PBS solution was used and whenever PBS was used in place of the reduced phenazine an anoxic PBS solution from a serum bottle was used.

Note that the reduced phenazine stocks looked reasonably similar to the last time, but I did notice that there are particles in the PYOred stock and it is not perfectly clear. This matches with previous results that some of the PYO may have crashed out of solution and some of it may be oxidized. Regardless this stock contains significant reduced PYO that has worked for other experiments and I think it has been sufficient.

Reduced phenazines were drawn from the serum bottles using a 1mL syringe with a needle. This is not as accurate as pipetting, and occasionally there were bubbles that could not be removed, so I estimated 500uL as best I could.

Results

First let’s import the data.

source("text_file_import.R")

# divide swv into rep and subrep and then subtract 1 from rep
# to match with GC
paths <- dir(path = "data", pattern = ".+[csv]$", recursive = T, 
    full.names = T)

filenames <- basename(paths)

data_cols <- c("t", "blank", "abs690")

filename_cols = c("year", "month", "day", "PHZox", "ox", "PHZred", 
    "red", "rep")

skip_rows = 48  # or 48
n_max = 61


df <- import_to_df(filenames = filenames, file_paths = paths, 
    data_cols = data_cols, skip_rows = skip_rows, n_max = n_max, 
    filename_cols = filename_cols, rep = T, PHZadded = F) %>% 
    mutate(phz_redox = paste(PHZox, ox, " + ", PHZred, red, sep = ""))

df %>% kable() %>% kable_styling() %>% scroll_box(height = "300px")