Microbial preparations have a long history of applications in broad and diverse areas of biotechnology, including medical and food biotechnologies; as active plant-growth-promoting components of biofertilisers in agrobiotechnology; in bioremediation (and phytoremediation) of contaminated soils and water, etc., many of which are currently in rapid development. A crucial issue for such preparations is their shelf life and long-term preservation (mainly in frozen or dry states). To control their safety and to optimise storage conditions, molecular-level analyses of the cell biomass and monitoring its composition in situ or in vivo is of primary importance. Over the last decade, along with microbiological, biochemical and molecular biological approaches, modern molecular spectroscopy techniques have been increasingly applied in this challenging area of bioanalysis.
In a series of our recent studies using in-situ transmission 57Fe Mössbauer spectroscopy (see, e.g. [1, 2] and references therein), it was noticed that, while assimilating iron(III) from the medium by its reduction to iron(II) with a notable FeII accumulation (which is typical of many microorganisms [2]), two Azospirillum species (widely studied phytostimulating rhizobacteria) showed a dramatic decrease in the relative content of iron(II) in dry biomass obtained by lyophilisation [1]. Note that this method (freeze-drying under vacuum) is widely used in biotechnology to obtain dry microbial preparations for long storage, in which the cells remain alive (in the dormant state, with very low metabolic rates). It is essential that active cells can metabolically control the formation of cell-damaging reactive oxygen species (ROS) as a result of Fenton-type reactions (FTRs) involving cellular FeII, whereas in dry (dormant) cells such a comprehensive metabolic control is impossible. Hence, it was suggested [1] that the possibility for a spontaneous oxidation of assimilated cellular FeII to FeIII upon lyophilisation could be a natural strategy to avoid cell damage owing to FTRs. In our further 57Fe Mössbauer spectroscopic studies ([3] and papers in preparation), this hypothesis has been confirmed on three other bacteria (another Azospirillum species, Enterobacter cloacae and Bacillus sp., which are all used in agriculture). Thus, all of them accumulated significant amounts of 57Fe(II) from the medium (over 20% of the total 57Fe cellular pool), while in their lyophilised samples, either no or only a minor part of 57FeII was detected. Low-temperature Mössbauer spectroscopic measurements (at T ≈ 5 K) allowed various states of cellular 57Fe(III) species to be distinguished.
In these experiments, it was of importance to control the state of cellular biomass in the fresh and lyophilised states. This was realised using FT-Raman spectroscopy (with the 1064-nm excitation wavelength to avoid fluorescent noise), which showed that, for each bacterium, all the main vibrational bands typical of cellular constituents were similar in both fresh (wet) biomass and lyophilised samples obtained thereof. FTIR spectroscopy, widely applied in microbiology [4], was also used to compare vibrational spectra of the lyophilised bacterial samples [5].
A combination of molecular spectroscopic techniques can thus provide a realistic picture of biotechnologically relevant processes occurring in live microbial cells upon drying. Note that desiccation is also one of natural states of soil microorganisms, in which they can survive. Transmission 57Fe Mössbauer spectroscopy is a unique probe for in-situ cellular iron speciation and redox transformations [1–3], and vibrational spectroscopic techniques are modern instrumental tools highly sensitive to the composition and fine structural features of macromolecular cellular constituents [4–6].
Funding. This work has been supported by the Russian Science Foundation (grant no. 24-26-00209).
References
[1] A.A. Kamnev, A.V. Tugarova, A.G. Shchelochkov, K. Kovács, E. Kuzmann. Spectrochim. Acta Part A, 229 (2020) 117970.
[2] A.A. Kamnev, A.V. Tugarova. Russ. Chem. Rev., 90 (2021) 1415–1453.
[3] A.A. Kamnev, K.V. Frolov, S.S. Starchikov, Yu.A. Dyatlova, S.A. Klimin, I.S. Lyubutin, A.V. Tugarova. In: A.A. Kamnev (Ed.), Plants and Microorganisms: Biotechnology of the Future. Cambridge Scholars Publ., Newcastle upon Tyne, U.K., 2025 (in press).
[4] A.A. Kamnev, A.V. Tugarova. J. Analyt. Chem., 78 (2023) 1320–1332.
[5] O.A. Kenzhegulov, Yu.A. Dyatlova, S.A. Klimin, A.V. Tugarova, A.A. Kamnev. Microbiology, 93 (2024) S153–S156.
[6] A.V. Tugarova, A.A. Vladimirova, Yu.A. Dyatlova, A.A. Kamnev. Spectrochim. Acta Part A, 329 (2025) 125463.
