We set up series of stable isotope tracer assays (13C-glucose, 13C-acetate and 13CO2) in microcosms incubated at subzero temperatures (from -5C to -15C) and anoxic conditions (step no. 1, see Fig. 1) and focused on investigating three main aspects: biodiversity, biological activity and functionality.
The monitoring of the biological activity all through the duration of the experimental work (up to 1 ½ year) via measurement of methane production indicated very low levels of activity at the bulk population, thus suggesting that, under such extreme conditions, the number of active cells was little and their metabolic activity was reduced. Based on these first results, we preferred to implement an analytical approach strongly based on single cells, aiming to capture active microorganisms and better understand their physiological state.To maximise the number of target cells, we extracted microorganisms from the permafrost matrix and separated them on a Nycodenz gradient (step no. 2), then sorted and counted them by Fluorescence Automated Cell Sorting (FACS) using probes both generic (SYBR Green I) and specific to discriminate between live and dead cells (SYTO9/Propidium Iodide) (step no. 3). In this way, we could enumerate the biomass of this deep core of permafrost as approximately in the order of 106 cells per gram and also recover high numbers of microbial cells, which were deposited onto a gold-platinum filter ready for downstream analyses. The implementation of this procedure prior to the analytical work had very important advantages, enabling us to prepare samples with microbial cells in high numbers, clean from organic and inorganic contaminants typically abundant in sediments and suitable to be analysed with a variety of techniques.
We attained a first preliminary taxonomic identification using Halogen In Situ Hybridisation (HISH) with probes for both bacteria and archaea (step no. 4), and found a generally higher abundance of bacterial over archaeal organisms. Microscopic observation with a Scanning Electron Microscope (SEM) combined with Energy Dispersive X-ray Spectroscopy (EDX) to image at high resolution and map the elemental composition of target cells, revealed a relatively high proportion of very small microorganisms in the range of 200-400 nm in size, so called “nanobacteria”. These nanobacteria were found mostly occurring in aggregates, and our findings are well in line with similar observations made in other studies on permafrost microbiota.
This was followed by the analysis of the metabolic activity done with Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) (step no. 5). Here, we quantified the assimilation rates of the 13C-labelled substrates based on the 13C-isotopic enrichment of the biomass of individual cells. As anticipated by previous experiments, we detected a low number of metabolically active cells and most of them had reduced metabolic rates corresponding to a maintenance physiological state rather than active growth. To gain better insights on what metabolic functions are maintained under such reduced metabolic expenditures, we attempted to identify key metabolic fingerprinting via infrared spectroscopy (step. no. 6). The gradual decrease of the overall spectroscopic signature at increasingly lower incubation temperatures provided an additional line of evidence of reduced metabolic activity. Furthermore, the increase of spectroscopic features corresponding to proteins (amide I and amide II bands) indicated that translational activity, i.e. synthesis of proteins, is particularly important during the adaptation to subzero temperatures. However, this was a pioneering work, which will be further explored and refined in future research.
Last, we addressed the question of the eco-physiological role of bioactive metabolites such as extracellular enzymes (e.g. proteases and lipases) and biosurfactants in enhancing the habitability of cold environments. A large fraction of the microbial community that we studied was capable to produce lipolytic enzymes (85%) involved in the degradation and cycling of carbon substrates, while proteolytic enzymes for the nitrogen cycling were synthesised by only 33%. Using as model organism a strain of Psychrobacter, a typical inhabitant of permafrost and cold habitats, we also studied via cloning and heterologous expression the structural and physicochemical properties of a target cold-active protease. Beside the relevance in the ecosystem functioning, these cold-active enzymes and biosurfactants can have a marked applicative potential and our results could open up to further research in the field of microbial biotechnology.
