Confining the window-for-life: recovery and analysis of bio-signatures in CaCl2-saturated Don Juan Pond. Title (IT) Recupero ed analisi di tracce di vita nel lago Don Juan Pond, saturo di CaCl2.

As we mentioned above, since its discovery (Meyer et al., 1962), DJP has attracted extensive research as the one of the most extreme environment
on our planet (e.g. Dickson et al., 2013; Samarkin et al., 2010; Siegel et al., 1979; Webster, 1994). Despite initial biological investigations found
evidence for alive microbial communities in DJP (Meyer et al., 1962; Siegel et al., 1979), later studies questioned whether microbes actively grow
in the hypersaline core of the pond (Oren, 2013) and to date there is no conclusive evidence of microbes able to grow in CaCl2-dominated DJP
bine (Bell, 2012). From our previous experience in studying the MgCl2-saturated Mediterranean deep-sea brine lakes Discovery and Kryos, we
can be confident that the water activity of the DJP bine (aw=0.411) is far below the recognized minimum value (aw=0.585), where life is known to
cease. So, the core of the DJP pond is evidently dead, but could contain the signature(s) of precedent life, i.e. microbial assemblages, grown at
waterline during occasionally occurred high groundwater flows. As we demonstrated for the MgCl2-saturated Mediterranean deep-sea brine
lakes, the different types of nucleic acids differently behave in presence of huge amount of bivalent cations. DNA and, to some extent, ribosomal
RNA (rRNA) are stable in such aggressive hydrochemical settings, which is not the case for labile and easily degradable messenger RNA (mRNA)
(Hallsworth et al., 2007; Yakimov et al., 2015).
Recently the adoption of new and powerful metagenomic analysis and the development of long reads sequencing methodologies, such as PacBio,
Nanopore and MINION have contributed to enormously increase our knowledge on the metabolic potential of microbes thriving in hypersaline
environments including Great Salt Lake, the Dead Sea , saltern evaporation and crystallizer ponds and deep-sea brine pools . All the information
obtained by these new methodologies lead to rewrite a “new version of tree of life”, describing new lineages of bacteria and archaea with no
representatives currently isolated (Hug et al., 2016). These studies clearly show that many types of halophilic prokaryotes are awaiting isolation
and characterization.
The whole research project is subdivided in three phases = work packages according to the specific activities and expertise of the operating units
of the DON JUAN project:
Work Package 1 (0-12 months). Sampling activities:
In DON JUAN proposal we will sample all DJP sections (inner zone, waterline, sediments); extract any traces of bio-signatures (DNA, rRNA and
mRNA) and scrupulously analyse these material using sophisticated bioinformatics platform. Simultaneously, a comprehensive geochemical
analysis of DJP brine and sediments, including elemental analysis, stable isotopes (2H, 13C, 18O and 15N) profiling and characterization of
dissolved and particulate organic matter will be performed. The sampling activity is foreseen be done during two short-time (3-4 days)
expeditions, performed in the first and second year of project running. Optionally, if first sampling will be scheduled for beginning of December,
the second one will be performed one month later. In this case, the material collected during first sampling will give us first glimpse on eventual
troubles and difficulties, faced by many scientific teams before (Samarkin et al., 2010). During this initial phase of our permanence at MZS
(previewed to be 45 days-long), we will assess the quantity and quality of extracted nucleic acid and of some components of dissolved organic
matter (DOM). This will allow us to improve our sampling strategies and sample treatment protocols, in order to better react the objectives of
DON JUAN project during second sampling.
Work Package 2 (7-20 month). Sample analysis: Molecular biology, bioinformatics, hydro- and geochemistry and biophysics
In DON JUAN project we are proposing to perform for the first time a massive molecular survey (see Methodologies) on brine and sediments
samples collected from DJP environment and from nearby sites (for comparative reasons). High throughput New Generation Sequencing (NGS)
platform will be employed to obtain the first inside into diversity of nucleic acids (envDNA) in DJP habitats. Contemporaneous analysis of 16S
rRNA and mRNA will be applied to clarify which microbial fraction (if any) is metabolically active in the areas of the lake with aw values,
permissible for life. These niches will be detected by measuring water activity directly in situ using our previous experience in studying the
MgCl2-saturated Mediterranean deep-sea brine lakes (Hallsworth et al., 2007; Yakimov et al., 2015). Detection of metabolic activity is the key
issue for confirming the fact that microbes are thriving in some parts of DJP (where aw values allow) and do not present there in dormant or,
keeping in mind that high concentrations of divalent cations can play a relevant role in preservation of macromolecules, in mummified state.
To connect obtained biological data with environmental settings, all sampled sites will be comprehensively characterized using various
geochemical and biophysical approaches (see Methodologies). Briefly, basic parameters (temperature, pH, redox potential, dissolved oxygen,
conductivity/salinity and water activity) will be measured in situ by portable sensors and refractometer. Other analyses of organic/inorganic
matter and nutrients will be performed in inland laboratories upon samples will be received Italy. The stable isotope ratios 15N/14N and 13C/12C
in particulate organic matter will be used to better understand the origin of particulate matter also by comparing it with that found in the nearby
to DJP area. Organic matter in the lake sediments will be characterized to quantify the C:N:P in the sedimentary organic matter, to determine its
15N/14N and 13C/12C signatures. Total lipids, proteins and carbohydrates will be quantified in order to estimate the biopolymeric fraction
present in the sedimentary organic matter. Quantification of water activity and chaotropicity, as well as ultra-high resolution mass spectrometry of
dissolved organic matter will be performed as described elsewhere (Yakimov et al., 2015).
Work Package 3 (12-24 month). Data acquisition, systematization and processing.
To reveal metabolic activities that may be occurring in DJP hypersaline habitats with aw values > 0.585, we will perform metatranscriptomic
analyses in parallel with metagenomics. Transcripts (mRNA) will be extracted from all cells captured and preserved. Soluble, i.e. extracellular
fraction of envRNA (both rRNA and mRNA) will be also analyzed. All libraries will be submitted for sequencing. As sequencing technologies are
advancing rapidly, we will adapt methods to use the best platforms available at the time. It will be the same situation for bioinformatics
approaches for (meta)genome and (meta)transcriptome data analysis. They are improving rapidly, and we will use the best practices available at
the time of our analyses. Results, data, and collections will be made available to qualified researchers upon request. Required reports will
summarize all data. Environmental, DNA and RNA sequence data will be uploaded to IMG/M, MG-RAST, and at NCBI within 2 years of
acquisition. We anticipate improved community resources will be available during the term of DON JUAN project to maximize accessibility and
utility to the greater scientific community for all data.
It is crucial to underline that the proposed approach to study limits of life in hydrological formations, saturated with bivalent cations, has been
already successful in the past and new MgCl2–filled deep-sea lake Kryos has been found by our group (Yakimov et al., 2015).

Along with the MgCl2-saturated Mediterranean deep-sea brine lakes, the Don Juan Pond habitat presents a legitimate earthy analog to putative
martian ecosystems, likely originated from dissolution of hydrated magnesium- and calcium-rich minerals. It is very likely, that similarly to earthy
analogs, hydrological formation on Mars possess extremely low aw values (approx. 0.400), i.e. far below the point where life is known to cease
(aw=0.585).
1. Thus, the first objective of DON JUAN proposal is to measure, monitor and map the aw value across the whole DJP area in order to localize the
eventual (micro)niches with higher aw values, allowing the life. We expect that this objective will provide a constraining “boundary conditions”
which are likely to support or have supported life.
2. Particularly for biological analyses, careful consideration should be made of the most prudent and unambiguous measurements to detect life or
its signature(s). As far as high amounts of bivalent cations in water solutions have strong capability of preserving many types of biomolecules
(lipids, proteins and DNA), the second objective of DON JUAN proposal is thoughtful examination of most efficient, sensitive and reliable
protocols to extract all types of nucleic acids.
3. Further biomolecular treatment and processing of extracted envDNA and envRNA molecules is the third objective of DON JUAN proposal.
Subsequent bioinformatics analysis will define the phylogenetic belonging of recovered bio-signatures. These findings could be helpful for future
exobiology investigations.
4. As far as the interpretation of biological data is inconceivable without hydrochemical characterization of studied habitats, the comprehensive
analysis of DJP environmental settings (see Methodologies) is the forth objective of DON JUAN proposal.

In totale sono state svolte 5 spedizioni verso il lago Don Juan Pond.

Durante la quarta e quinta spedizione inoltre grazie alla collaborazione con il personale ENEA UTA, (Riccardo Scipinotti, Riccardo Maso e Samuele Pierattini), è stato possibile effettuare due sorvoli dell’area interessata con drone dotato di camera RGB e termica, acquisendo importanti dati per la caratterizzazione chimico fisica del lago e del bacino idrografico circostante.

Sono stati prelevati campioni di brine, sedimenti superficiali sottostanti le brine e da siti limitrofi al corpo principale del lago.

E’ stato possibile anche prelevare campioni di ghiaccio (probabilmente generato da fenomeni di condensazione o anch’esso trasportato dal vento) nelle vicinanze del lago oltre a identificare e campionare piccole porzioni di microbial mat, formato da microalghe e batteri riscontrate nelle sorgenti effimere sopra descritte (Figura 2).

Questi campioni sono stati utilizzati per allestire degli arricchimenti a diverse salinità, al fine di cercare di identificare le condizioni chimico fisiche più estreme a cui la vita riesce ancora ad adattarsi e prosperare in presenza di questo particolare tipo di sale, denominato antarticite (CaCl2 x 6H2O).

Questi esperimenti sono di fondamentale importanza al fine di cercare di mantenere in vita ed eventualmente isolare e caratterizzare microorganismi poli estremofili adattati a vivere in queste proibitive condizioni.

Sono state effettuate operazioni di pretrattamento dei campioni per predisporre ed organizzare la loro spedizione in Italia e procedere successivamente con analisi chimico fisiche e molecolari.

In accordo agli obiettivi del progetto, sono stati prelevati e trattati campioni per effettuare analisi chimiche dei principali parametri quali: carbonio inorganico disciolto; misure di isotopo 13C del carbonio inorganico; carbonio organico disciolto; alcalinità totale; nutrienti; azoto e fosforo totale disciolto; metalli e salinità.