Comparative in-situ Mössbauer and vibrational spectroscopic monitoring of changes in cells of the rhizobacterium Azospirillum lipoferum Sp59b upon lyophilisation.

The redox states of the cellular Fe in the agriculturally important rhizobacterium Azospirillum lipoferum Sp59b (grown with 57FeIII complex with nitrilotriacetate as an iron source) were compared in fresh wet biomass (rapidly frozen) and in lyophilised samples using transmission 57Fe Mössbauer spectroscopy (measurements were performed at T = 90 K and 4.7 K). The overall biomacromolecular composition of the samples was monitored using in-situ Raman and FTIR spectroscopic techniques. The two 57FeII components (21% of the total cellular Fe pool), detected in the fresh biomass as a result of reductive iron assimilation, were found to be completely re-oxidised to 57FeIII upon lyophilisation. Thus, the lyophilised biomass at T = 90 K contained two 57FeIII forms only (with Mössbauer parameters different from those observed in the fresh biomass). At T = 4.7 K, a significant part of the iron(III) showed magnetic splitting (sextet) characteristic of bacterial ferritin-like FeIII-containing species. Raman spectra of fresh and lyophilised biomass and FTIR spectrum of the latter showed similar typical bands of main cellular bio(macro)molecular constituents. This is in line with the known fact that bacteria in lyophilised biomass remain viable (in the metabolically inactive state), which is of importance for long-term storage of dry microbial preparations, e.g., used in biofertilisers. Re-oxidation of cellular iron(II) to iron(III) upon lyophilisation, observed for A. lipoferum (similar to the result reported earlier for two other Azospirillum species), confirms the hypothesis that this process is a common natural strategy to avoid cell damage due to Fenton-type reactions involving iron(II) in dormant dry cells.