Scientific novelty beyond the experiment.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

Aqueous alteration processes in Jezero crater, Mars-implications for organic geochemistry.

The Perseverance rover landed in Jezero crater, Mars, in February 2021. We used the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument to perform deep-ultraviolet Raman and fluorescence spectroscopy of three rocks within the crater. We identify evidence for two distinct ancient aqueous environments at different times. Reactions with liquid water formed carbonates in an olivine-rich igneous rock. A sulfate-perchlorate mixture is present in the rocks, which probably formed by later modifications of the rocks by brine. Fluorescence signatures consistent with aromatic organic compounds occur throughout these rocks and are preserved in minerals related to both aqueous environments.

Water Activities of Acid Brine Lakes Approach the Limit for Life.

Water activity is an important characteristic for describing unusual waters and is a determinant of habitability for microorganisms. However, few empirical studies of water activity have been done for natural waters exhibiting an extreme chemistry. Here, we investigate water activity for acid brines from Western Australia and Chile with pH as low as 1.4, salinities as high as 32% total dissolved solids, and complex chemical compositions. These acid brines host diverse communities of extremophilic microorganisms, including archaea, bacteria, algae, and fungi, according to metagenomic analyses. For the most extreme brine, its water activity (0.714) was considerably lower than that of saturated (pure) NaCl brine. This study provides a thermodynamic insight into life within end-member natural waters that lie at, or possibly beyond, the very edge of habitable space on Earth.

Astrobiology of life on Earth.

Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub-fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low-temperature environments and chemically diverse saline- and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock-associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints.

Lipid Biomarkers in Ephemeral Acid Salt Lake Mudflat/Sandflat Sediments: Implications for Mars.

Sedimentary strata on Mars often contain a mix of sulfates, iron oxides, chlorides, and phyllosilicates, a mineral assemblage that is unique on Earth to acid brine environments. To help characterize the astrobiological potential of depositional environments with similar minerals present, samples from four naturally occurring acidic salt lakes and adjacent mudflats/sandflats in the vicinity of Norseman, Western Australia, were collected and analyzed. Lipid biomarkers were extracted and quantified, revealing biomarkers from vascular plants alongside trace microbial lipids. The resilience of lipids from dead organic material in these acid saline sediments through the pervasive stages of early diagenesis lends support to the idea that sulfates, in tandem with phyllosilicates and iron oxides, could be a viable target for biomarkers on Mars. To fully understand the astrobiological potential of these depositional environments, additional investigations of organic preservation in ancient acidic saline sedimentary environments are needed.

NaCl-saturated brines are thermodynamically moderate, rather than extreme, microbial habitats.

NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.

Microbial communities and their predicted metabolic functions in a desiccating acid salt lake.

The waters of Lake Magic in Western Australia are among the most geochemically extreme on Earth. This ephemeral saline lake is characterized by pH as low as 1.6 salinity as high as 32% total dissolved solids, and unusually complex geochemistry, including extremely high concentrations of aluminum, silica, and iron. We examined the microbial composition and putative function in this extreme acid brine environment by analyzing lake water, groundwater, and sediment samples collected during the austral summer near peak evapoconcentration. Our results reveal that the lake water metagenome, surprisingly, was comprised of mostly eukaryote sequences, particularly fungi and to a lesser extent, green algae. Groundwater and sediment samples were dominated by acidophilic Firmicutes, with eukaryotic community members only detected at low abundances. The lake water bacterial community was less diverse than that in groundwater and sediment, and was overwhelmingly represented by a single OTU affiliated with Salinisphaera. Pathways associated with halotolerance were found in the metagenomes, as were genes associated with biosynthesis of protective carotenoids. During periods of complete desiccation of the lake, we hypothesize that dormancy and entrapment in fluid inclusions in halite crystals may increase long-term survival, leading to the resilience of complex eukaryotes in this extreme environment.

Acidophilic halophilic microorganisms in fluid inclusions in halite from Lake Magic, Western Australia.

Lake Magic is one of the most extreme of hundreds of ephemeral acid-saline lakes in southern Western Australia. It has pH as low as 1.7, salinity as high as 32% total dissolved solids, temperatures ranging from 0°C to 50°C, and an unusually complex aqueous composition. Optical petrography, UV-vis petrography, and laser Raman spectrometry were used to detect microorganisms and organic compounds within primary fluid inclusions in modern bedded halite from Lake Magic. Rare prokaryotes appear as 1-3 µm, bright cocci that fluoresce green with UV-vis illumination. Dimpled, 5-7 µm yellow spherules that fluoresce blue with UV-vis illumination are interpreted as Dunaliella algae. Yellow-orange beta-carotene crystals, globules, and coatings are characterized by orange-red fluorescence and three distinct Raman peaks. Because acid saline lakes are good Mars analogues, the documentation of prokaryotes, eukaryotes, and organic compounds preserved in the halite here has implications for the search for life on Mars. Missions to Mars should incorporate such in situ optical and chemical examination of martian evaporites for possible microorganisms and/or organic compounds in fluid inclusions.

Molecular analysis of the microbial communities of Mars analog lakes in Western Australia.

Unique, shallow interdune lakes and groundwaters with extremely low pH and high salinity exist in Australia, along with nearby lakes that possess higher pH values. These acidic hypersaline environments are possibly the best modern terrestrial analogues for past martian environments. However, no previous microbiological analyses of these lakes have been conducted. During the Australian winter of 2005, water samples were taken from several hypersaline lakes located in southern Western Australia that possessed acidic to slightly alkaline pH. These samples were subjected to molecular analysis to identify bacterial communities. DNA extraction and polymerase chain reaction (PCR) amplification of the 16S rRNA gene sequences, by using universal bacterial primers, were also performed on the samples. Extracted DNA was amplified with 1070 forward and 1392 GC-clamped reverse primers and analyzed by using denaturant gradient gel electrophoresis (DGGE). In addition, libraries were developed from DNA retrieved from four lakes, including a marginal marine neutral lake, an inland neutral lake, and two inland acid lakes, and selected clones with distinct operational taxonomic units were sequenced. The DGGE profiles and clone sequence data indicate that there are distinct, abundant, and diverse microbial populations in these Australian hypersaline environments, especially the acidic ones. These results are significant for two reasons: (1) they provide the first microbiological survey of natural acid saline lakes and (2) they hint at the possibility that there could have been a diverse microbial population in acidic hypersaline environments on Mars.

"Hairy blobs:" microbial suspects preserved in modern and ancient extremely acid lake evaporites.

"Hairy blobs" are unusual clumps of organic bodies and sulfate crystals that have been found in evaporite minerals grown in acid saline lakes. Here, we document modern hairy blobs in halite and gypsum from 5 modern acid saline lakes in southern Western Australia, and Permian hairy blobs trapped in halite from the mid-Permian Opeche Shale in the subsurface of North Dakota. These are among the first microbial remains described from acid saline lake environments. They give clues about the role of microorganisms in the acidity, geochemistry, and mineralogy of these extreme environments. This study also may add to the inventory of life in extreme environments and help predict possible martian life-forms and the method of preservation.

Modern and ancient extremely acid saline deposits: terrestrial analogs for martian environments?

Extremely acid (pH <1) saline lakes and groundwaters existed in the mid-Permian of the mid-continent of North America. Modern counterparts have been found in acid saline lake systems throughout southern Australia. We compare and contrast the Permian Opeche Shale of North Dakota and Nippewalla Group of Kansas to modern Australian salt lakes in southern Western Australia and in northwest Victoria. With the exception of some minor variations in pH, evaporite mineralogy, and water geochemistry, the Permian and modern systems are similar and characterized by: (1) ephemeral saline continental playas hosted by red siliciclastic sediments, (2) evaporite minerals, including abundant sulfates, (3) Al-Fe-Si-rich waters with low pH values, (4) acidophilic microbes, and (5) paucity of carbonates. The composition of these terrestrial systems is strikingly similar to compositional data returned from the martian surface. Specifically, both Earth and martian systems have high amounts of iron oxides and sulfates, and little, if any, carbonates. We propose that the modern and ancient terrestrial acid saline environments may be good analogs for possible environments on Mars.