1 Introduction
Cave environments on Earth have long provided shelter to a variety of organisms, from microbes to humans. Though their scales of interest differ vastly, these two examples have sought the same comfort from caves: a stable and sheltered environment, protected from the woes of the surface world. Since the discovery of lava caves on Mars (see (Sauro et al., 2020) for a review), they have become of renewed interest as targets for human shelter in future missions, as well as areas of astrobiological interest, with the potential of harboring traces of extant or extinct extraterrestrial life.
Recent years have seen a huge advancement in the development and miniaturization of autonomous mobility systems and exploration technologies for robotic missions to planetary caves, including instrument suites for in situ astrobiological studies (summarized in (Blank et al., 2020)). All aspects of cave mission preparation, from robotics development to astronaut training and scientific advancement, are currently being tested in terrestrial analogue sites, namely lava tubes in the Azores, Hawai’i, Iceland, Lanzarote, the western continental United States, and other volcanic areas.
Astrobiological studies in terrestrial lava tubes focus on the characterization of the microbe-mineral continuum and the identification of biosignatures in the form of biologically mediated speleothems (secondary cave minerals) and other geochemical fingerprints that may remain preserved on geological timescales (Boston et al., 2001; Léveillé & Datta, 2010; Northup et al., 2011; Northup & Lavoie, 2001). A suite of analyses is required to distinguish a biologically mediated secondary mineral from one that is abiotic (Uckert et al., 2017), presenting a difficult challenge. Nonetheless, a variety of biologically mediated speleothems has been reported in the literature, including filamentous manganese ”snow”, ”crisco” moonmilk, lithified U-loops and living sulfuric acid ”snotties”, and pool fingers in limestone caves in New Mexico and Mexico (Boston et al., 2001).
Microbes in caves on Earth attach to minerals on cave walls, ceilings, or floors and initiate biomineralization reactions, creating biofilms. Biofilm formation is controlled by several processes, starting by initial microbe adhesion to the surface, governed by fluid flow and charging of the substratum. The initial colonizers excrete exopolysaccharides, which increase the surface irregularity and allow the biofilm to grow. Thus, the location of biofilm growth in caves depends not only on where particles and microbes can be transported to, but also be allowed to accumulate. A detailed review of the dynamics of biofilm formation on mineral surfaces and their spatial distribution is given by Little & Wagner (2018).
As the biofilm grows in layers away from the surface, it becomes a microbial mat of great complexity. Within its structure, chemical environments can exist that are radically different from that of the surrounding, allowing for the growth of minerals and microbes that would otherwise not be expected. Concentration of organic and inorganic particles can sustain a consortium of microorganisms of different nutritional modes. The putatively high microbial diversity within the biofilm may create local changes in pH or redox conditions, which can facilitate the precipitation of minerals that are unstable outside of the biofilm. Microbes can control the precipitation of these minerals either passively, where microbial cells act as nucleation sites, or actively, where bacterially produced enzymes control mineralization (Northup & Lavoie, 2001).
The majority of life in the Universe is thought to be unicellular (Schulze-Makuch & Irwin, 2018). Moreover, the tendency to form biofilms and mats may well be an adaptation to be expected on other planets, with other biologies, and perhaps other fundamental chemistries (Boston et al., 2001). Regardless of their specific chemistries, metabolisms relying on differences in redox potentials in elements present in the lava rock can be expected for any initial colonizers in Martian lava caves, due to the absence of light and the oligotrophic (low nutrient) quality of caves on Mars. The chemolithoautotrophic nature of this hypothetical life may eventuate the production of similar speleothems as found in terrestrial caves. Basaltic terrestrial lava tubes are most similar in mineralogy to those posited on Mars and may thus provide analogous potential for chemolithoautotrophy, resulting in similar, recognizable molecular markers and biologically mediated speleothems.
To prepare a multidisciplinary sampling protocol for future astrobiological missions to caves on Mars, we set up the Planetary Analogues and Exobiology Lava Tube Expedition (PELE). Since 2017, we have explored lava fields and lava tubes on Hawai’i, the Azores, and Iceland. In this paper we focus on data from three lava tubes in two distinct regions of Iceland during a PELE field expedition in the summer of 2018, during which we collected samples of microbial mats and their geological substrates. We obtained in situ elemental data with a portable X-ray fluorescence (XRF) spectrometer, analyzed biological samples with 16S rRNA gene sequencing methods, biogeochemical samples with Raman spectroscopy, and geological samples with scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). In synergizing these data sets, we attempted to describe the biogeochemical fingerprints of microbial life in lava tubes and define their validity as biosignatures. While the microbial mats collected in the caves we visited came in a variety of colors, the blue samples are used here as a case study to exemplify our protocols, describe our workflow, and show the data that can be gleaned from such a study.