Isotopic tracking of condensation, infiltration and interstitial water to detect microbial activity in caves
Authors: Fernandez-Cortes Angel , Martin-Pozas Tamara , Cuezva Soledad , Gazquez Fernando , Benavente David , Cañaveras Juan Carlos , Saiz-Jimenez Cesareo , Sanchez-Moral Sergio 
Published: February 2023

Water plays a key role in the colonization and ecology of microorganisms in natural subterranean environments since it controls their metabolic activity and the microbe-mineral interactions. In turn, microbial activity leaves its signature in groundwater. For instance, the aerial hyphae of Actinobacteria colonizing cave-rock surfaces and sediments act as vapour condensation nuclei and facilitate the retention of interstitial water in the porous system of rocks, sediments and speleothems.Here, we present the results of exhaustive monitoring of cave waters from Pindal Cave (northern Spain), a shallow and well-ventilated cave on the coastline, with extensive microbial colonization on the sediments and walls surfaces. This study sheds light on the detection of biosignatures based on stables isotopes of cave waters and controlled by underground-dwelling microorganisms. The isotopic composition (d18O and dD) of seepage waters (fast drips, soda straws and gours) and condensation droplets were determined by cavity ring-down spectroscopy (CRDS). An analytical set-up based on a CRDS analyser coupled to an Induction Module enabled the step-heating of moonmilk deposits and other substrates with distinct degrees of microbial colonization, and the selective release of their interstitial water for isotope analyses.The &#948;18O and &#948;D values of the in-cave waters correlate linearly (&#948;D=7.71&#183;&#948;18O+12.28; R2=0.97), like the Local Meteoric Water Line (&#948;D=7.00&#183;&#948;18O+6.77; R2=0.92, based on the of isotopes composition of regional rain). The offset of d-excess between the cave waters and the LMWL would indicate a significant recharge by occult precipitation linked to local coastal fogs. The isotopic composition of dripping water,&#160; -5.7&#8240; [-6.7 to -4.8 &#8240;] for &#160;d18O and -31.3&#8240; [-39.0 to -22.7 &#8240;] for dD, agree with the water samples from the gours located far from the cave entrance, with some slight deviations indicating evaporation processes or sudden contributions of meteoric water due to flash-flood events.The &#948;18O and &#948;D values of condensation water collected from non-colonized surfaces are -5.4&#8240; [-5.9 to -4.5 &#8240;] and &#8211;28.4&#8240; [-32.9 to -21.3 &#8240;], respectively. This isotope composition is similar to the contemporary infiltration water, which suggests that condensation mostly comes from autochthonous vapour generated from in-cave waters. The Actinobacteria mats quickly absorb the condensation water and it is then retained during long periods on the rock surface. Thus, in the case of microbial induced deposits like moonmilk, condensate water becomes a reservoir of interstitial water exposed to isotopes fractionation linked to the microbial metabolism processes. Indeed, the &#948;18O and &#948;D values of this interstitial water correlate linearly with the rest of the in-cave waters but show a distinctive composition with higher &#948;18O and &#948;D values compared to the infiltration water and condensation droplets; -3.6&#8240; [-4.4 to -2.0 &#8240;] and &#8211;18.9&#8240; [-27.2 to -10.8 &#8240;], respectively. These findings lead to exploring the potential use of water isotopes as a tool for indirectly assessing microbial activity and its role in the water balances in underground ecosystems.Research funded by PID2019-110603RB-I00 &#8211; SUBSYST and PID2020-114978GB-I00 projects