Chapter 12:Applications of Fourier-Transform Infrared Spectroscopy in Geomicrobiology

Chapter 12: Supplementary material

Infrared spectroscopy is a technique that has been around commercially since the 1940’s and was thought to be coming to an end of its usefulness in the early 1970’s. Infrared spectroscopy was even compared to Shakespeare’s play “As You Like It”, with the editor of Analytical Chemistry, Herbert Laitinen, likening the technique to the seventh (and final) age of man (read: old age) where infrared spectroscopy was entering a stage where it was becoming less useful and other techniques were rising up which were faster, more convenient, and more sensitive (Laitinen, 1973). What Laitinen didn’t know at the time was that soon the Fourier transform algorithm would be used to decompose difficult to interpret raw infrared data into more easily interpreted frequencies and advances in detector technologies would significantly improve the measurement times and sensitivities. In this textbook we discussed how we can use FTIR spectroscopy to analyse samples in geomicrobiolpgy. The following text explains how the bacterial cells (Pseudomonas putida) in Figure 12.1 of the text were grown and measured using an ATR-FTIR spectrometer, as a function of pH of the bacterial suspension.

Bacterial Growth

Bacterial cells were grown and washed according to Kenney and Fein (2011). Briefly, the P. putida cells were cultured aerobically at 32 °C in Luria–Bertani (LB) medium until they reached stationary growth phase (24 h), after which P. putida cells were harvested by centrifugation and washed using a 0.1 M NaCl electrolyte solution. After washing the bacteria were centrifuged (4200 x g) twice for 30 minutes to remove as much of the solution as possible and the resulting bacterial pellet was weighed. Enough bacterial biomass was then suspended into a 0.1 M NaCl electrolyte solution (to buffer the ionic strength) so that the biomass concentration was 30 g wet mass of cells/L. The biomass suspension was divided into 17 aliquots and the pH was adjusted with 0.1 M HCl and NaOH to range between pH 2.7 and 10.7. This mass of P. putida cells was chosen so that there was enough biomass in suspension to cover the ATR crystal in the analysis. Cells were allowed to equilibrate for 30 minutes and the final pH after equilibration is recorded in Figure 12.2. After equilibration, cells were centrifuged (4200 x g) for 5 minutes and then the supernatant was collected and reserved for analysis. The cells were then centrifuged (4200 x g) twice for 30 minutes to remove as much of the supernatant as possible and the resulting bacterial paste analysed using ATR-FTIR spectroscopy (section 12.4.2.3).

Recording the Spectra and Subtracting Water Growth

In order to analyse the wet bacterial paste we first took a background spectra of the empty ATR crystal from the average of 128 scans in the range of 400-4000 cm^-1. The worse your signal, the more scans you should run, as this should help to get rid of some possible noise from the spectrometer; studies in geomicrobiology have used anywhere between 30 to 5000 scans, so it really depends on what quality spectra are produced by the FTIR spectrometer and what kind of sensitivity is needed for your study. An example of a weak signal could be in the case of large water peaks, where the O-H vibration overwhelms most of the signal. If you are doing a subtraction of the water from your sample, hoping to analyse the resulting subtraction spectra, you should increase the number of scans run in order to increase the likelihood that the resultant subtraction spectra is data and not noise. Following the background spectra collection, the reserved supernatant was pipetted onto the diamond ATR crystal and we took a sample spectra (with the same parameters of the background spectra). Following this spectra collection, the crystal was wiped clean with wet cotton batting and bacterial paste of one of the sample (corresponding to the supernatant) was pressed against the diamond ATR crystal (Figure 12.6). Following this measurement the crystal was washed with water and then ethanol and the ethanol was allowed to evaporate completely before the next spectra recorded.

References

1. Laitinen, H.A., (1973), Editorial. The Seven Ages of an Analytical Method, Analytical Chemistry, v. 45, pp 2305-2305
2. Kenney, J. P. L. and Fein, J. B. (2011) Importance of extracellular polysaccharides in proton and Cd binding to bacteria: a comparative study, Chemical Geology. v 286, Issues 3-4, pp 109-117