Clase Textural

The toxicity of disulfiram to the chosen strains of bacteria was evaluated by comparing the optical density at 600 nm (OD600) over a range of time points and

disulfiram concentrations. As disulfiram is practically insoluble in water the drug was first dissolved in DMSO before adding to M9 minimal media and 0.1% glucose (w/v). Concentrations above 150 µM instantly precipitated when introduced to the medium and therefore experimental conditions were limited to concentrations below this amount. Investigations were then undertaken to see which concentration of disulfiram was tolerable by the strains chosen and their ability to grow in its presence.

Before testing a chemical for its growth-inhibiting abilities it was important to understand if there are any chemical changes to the structure caused by reactions with media constituents. With this in mind, all in vivo chemical experiments performed during this project included controls where an identical medium was made up and inoculated after 2 days incubation.

The exposure of the bacterial strains to disulfiram had markedly varied effects on growth across the different phyla. The Sphingobacterium strain showed complete growth inhibition after 96 h in the presence of disulfiram even at the lowest concentration of 5 µM. B. subtilis,R. opacus and R. jostii RHA1 displayed complete growth inhibition at concentrations of 50 µM and over. P. fluorescens and P. putida

and M. phyllosphaerae displayed almost normal growth patterns at all concentrations of disulfiram, compared with the experiments without any inhibitor (Figure 66).

Figure 66: Growth of selected bacteria observed at OD600 in incubations with M9 minimal media, 0.1% glucose (w/v) and 100 µM disulfiram

Samples taken from incubations in which growth was severely retarded were streaked on LB agar plates and failed to show signs of growth, this indicated disulfiram was acutely toxic to the primary metabolism of those species at these concentrations. As the purpose of these experiments was not to fatally harm the bacteria, it was decided that only the strains M. phyllosphaerae, P. fluorescens and P. putida would be selected for vdh inhibition experiments.

Incubations with M. phyllosphaerae, P. fluorescens and P. putida exhibited initial strong growth when incubated in growth media P1 supplemented with 0.1% (w/v) glucose and either 2.5% wheat straw lignocellulose or 0.5% industrial Kraft lignin in the presence of disulfiram at a final concentration of 100 µM. A drop in growth was observed after 24 h in the incubations with P. fluorescens and the wheat straw lignocellulose (in contrast to controls with just growth media P1 and disulfiram and the experiments with M. phyllosphaerae and P. putida). This was a promising observation as Narbad et al had shown in previous work that P. fluorescens growth could not be supported at concentrations of 10 mM and higher.[180]

The extracellular fractions after centrifugation were analysed by LC-MS and GC-MS. EICs for m/z 153.8 did not indicate any trace amounts of vanillin, in fact no detectable difference was seen between any of the incubations after close analysis of the UV spectra and base peak chromatograms.

Intracellular metabolites were then extracted from cell pellets using the boiling ethanol method (Section 5.2.3.2) and analysed by LC-MS. This time a distinctive broad peak in the base peak chromatogram (BPC) at 2.6 minutes was seen in the sample taken at 24 h from the P. fluorescens incubation. The mass spectrum showed dominant ions at m/z 120.0 (Figure 67).

Figure 67: The base peak chromatogram shows a peak with a retention time of 2.6 min obtained the sample taken at 24 h from the P. fluorescens incubation (a) in comparison with that of a control incubation without disulfiram (b) for the same time period. The dominant ions at m/z 120.0 and 134.0 are displayed in the spectrum (c). Incubations were performed in M9 minimal media, 2.5% wheat straw lignocellulose (w/v), 0.1% glucose (w/v) and with or without 100 µM disulfiram.

The peak was isolated by collecting the fraction at the retention time and HR-ESI-MS analysis by the University of Warwick Mass Spectrometry Service indicated the

presence of a compound with the molecular formula C5H12NO (calculated for

[C5H12NO]+: 102.0913 found 102.0912) (Figure 68).

Figure 68: HR-MS analysis identified a molecular formula of C5H12NO

A database search returned the possibility of glycine betaine aldehyde (GBA) (N,N,N-trimethyl-2-oxoethanaminium) for the C5H12NO and the hydrated form of

glycine betaine aldehyde, known as betaine aldehyde hydrate (BAH) (2,2-Dihydroxy-N,N,N-trimethylethanaminium) could account for the ions at m/z 120 for C5H12NO2 (Figure 69).

Figure 69: Glycine betaine aldehyde (N,N,N-trimethyl-2-oxoethanaminium) and betaine aldehyde hydrate (2,2 Dihydroxy-N,N,N-trimethylethanaminium) were identified as potential compounds isolated from the intracellular metabolite extraction. of P. fluorescens from a 24 hour incubation with 2.5% wheat straw lignocellulose (w/v) and 100 µM disulfiram in P1 growth media.

The use of a reference standard and spiking experiments to compare retention times confirmed the analyte to be GBA for m/z 102.1 with a retention time of 2.6 minutes (Figure 70).

Figure 70: An EIC chromatogram for m/z 102.1 (m+H)+ shows a peak with a retention time of 2.6 min obtained from the sample taken at 24 h from the P. fluorescens incubation (a) in comparison with that of an analytical standard (b) confirming the compound to be glycine betaine aldehyde.