The search for life beyond the Earth is the overarching goal of the NASA Astrobiology Program, and it underpins the science of missions that explore the environments of Solar System planets and exoplanets. However, the detection of extraterrestrial life, in our Solar System and beyond, is sufficiently challenging that it is likely that multiple measurements and approaches, spanning disciplines and missions, will be needed to make a convincing claim. Life detection will therefore not be an instantaneous process, and it is unlikely to be unambiguous-yet it is a high-stakes scientific achievement that will garner an enormous amount of public interest. Current and upcoming research efforts and missions aimed at detecting past and extant life could be supported by a consensus framework to plan for, assess and discuss life detection claims (c.f. Green et al., 2021). Such a framework could help increase the robustness of biosignature detection and interpretation, and improve communication with the scientific community and the public. In response to this need, and the call to the community to develop a confidence scale for standards of evidence for biosignature detection (Green et al., 2021), a community-organized workshop was held on July 19-22, 2021. The meeting was designed in a fully virtual (flipped) format. Preparatory materials including readings, instructional videos and activities were made available prior to the workshop, allowing the workshop schedule to be fully dedicated to active community discussion and prompted writing sessions. To maximize global interaction, the discussion components of the workshop were held during business hours in three different time zones, Asia/Pacific, European and US, with daily information hand-off between group organizers.

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Differentiating biotic and abiotic processes in nature remains a persistent challenge, specifically in evaluating microbial contributions to geochemical processes through time. Building on previous work reporting that biologically-influenced systems exhibit stronger long-range correlation than abiotic systems, this study evaluated the relationship between long-range correlation of redox potential and oxidation rates of circumneutral microaerophilic bacterial Fe(II) oxidation using a series of batch microcosms with bacteriogenic iron oxides (BIOS). Initial detrended fluctuation analysis (DFA) scaling exponents of the abiotic microcosms were lower (ca. 1.20) than those of the biotic microcosms (ca. 1.80). As Fe(II) oxidation proceeded, correlation strength decayed as a logistic function of elapsed reaction time, exhibiting direct dependence on the free energy of reaction. Correlation strength for all microcosms decayed sharply from strong correlation to uncorrelated fluctuations. The decay rates are greater for abiotic microcosms than biotic microcosms. The Δ G m relaxation edges for biotic microcosms were lower, indicating less remaining free energy for Fe(II) oxidation than abiotic systems, with the implication that biologically-catalyzed reactions are likely more energetically efficient than abiotic reactions. These results strengthen the case for employing novel DFA techniques to distinguish in situ microbial metabolic activity from abiotic processes, as well as to potentially differentiate metabolisms among different chemoautotrophs.

Preprints — scholarly papers that precede publication in a peer-reviewed journal — speed the delivery and accessibility of academic work and lead to faster reuse by the scientific community, as evidenced by rapid citations and social media discussions. In the past year, EarthArXiv has emerged as a community-led initiative devoted to open scholarly communication. EarthArXiv became the first preprint server for the Earth Sciences on October 23rd, 2017, during the 10th International Open Access Week when it began accepting preprints and also postprints — the non-typeset version of a published article — from all subdomains of Earth science and related domains of planetary science. In our first year, EarthArXiv accepted ~500 submissions which had a total of ~60,000 downloads — we are taking these milestones as an opportunity to discuss the growth and next steps for EarthArXiv, as well as encourage others to join us in this movement toward openness.

Geophysical surveys add value to biogeochemical studies because of their ability to characterize systems remotely, and their precise time resolution. One limitation, however, is their lack of biogeochemical process specificity. Here, electrochemical time series from an oxic-anoxic cyclical bioreactor experiment were reanalyzed with detrended fluctuation analysis (DFA) to distinguish dominant biogeochemical processes. Measurements of EH, pH, dissolved oxygen (DO) were recorded every 20 minutes for 74 days. The time series were divided by geochemical environment (aerobic respiration, NO3- reduction, mixed Fe(III), Mn(IV), SO42- reduction, and anoxic-oxic transition), and analyzed for correlation strength using DFA. Correlation strength varied systematically by environment over five oxic-anoxic cycles. This repetition makes it clear that electrode fluctuations are not random, nor are they noise. In fact, electrode fluctuations are a system-specific measurement of dominant geochemical conditions. The results of this study in a well-constrained environment with a complex microbial community support the potential to use galvanic and electrochemical approaches to remediation as a viable, cost-effective, and simple long-term monitoring strategy. The information provided by time series analysis requires no special sensors or additional data collection, just a short computational analysis for new, valuable information about ongoing geochemical reactions. This approach could be valuable in any application where remote, long-term monitoring of ongoing biogeochemical processes is desirable, such as agriculture, bioreactors, or in long-term remediation and monitoring programs where inexpensive, consistent data sets could provide valuable insight into degradation and environmental stability.

A study was conducted to determine in situ rates of Fe(II) oxidation and Fe(III) precipitation along a 5.0m reach of a ferruginous groundwater discharge zone under two distinct conditions; (i) the natural state featuring abundant flocculent mats of bacteriogenic iron oxides (BIOS) produced by Fe(II)-oxidizing bacteria, and (ii) after a manual washout of the streambed to remove the microbial mat. Examination of mat samples by differential interference contrast light microscopy revealed tangled meshworks of filamentous Leptothrix sheaths and helical Gallionella stalks intermixed with fine-grained hydrous ferric oxide (HFO) precipitates. The greatest accumulation of BIOS mat was 1.0m downstream of the groundwater spring. Redox potential (Eh) increased sharply from 200mV to over 300mV over the last 2.0m of the reach. Similarly, dissolved oxygen increased from < 10% saturation to almost 100% saturation over the last 2.0m of the reach, whereas pH increased from 6.4 to 7.3. Pseudo-first-order rate constants determined on the basis of analytical solutions to sequential partial differential advection-dispersion-reaction equations for the linear Fe(II)Fe(III)HFO reaction network yielded in situ Fe(II) oxidation rate constants (k(ox)) of 1.70 +/- 0.20min(-1) in natural conditions and 0.48 +/- 0.14min(-1) after washout. Corresponding Fe(III)-precipitation rates (k(p)) before and after washout were 3.45 +/- 0.10min(-1) and 0.90 +/- 0.01min(-1), respectively. These values for k(ox) and k(p) are higher than estimates obtained from closed batch microcosm and laboratory experiments, underscoring the crucial dependence of in situ Fe(II) oxidation and Fe(III) precipitation rates on advective and dispersive mass transport. The results also highlight the influence that BIOS microbial mats exert on the reaction kinetics of the multiple heterogeneous reactions contributing not only to Fe(II)/Fe(III) redox transformations in groundwater discharge zones, but also the precipitation of HFO.

This investigation evaluates spatial relationships between summer (July) groundwater temperatures and Fe(II)/Fe(III) biogeochemical cycling over a five year period in a shallow pristine sand aquifer at Meilleurs Bay near Deep River, Ontario, Canada. A warm subsurface thermal island of 12.5-16.1 °C, compared to background conditions of 10-11 °C, was manifest in contour maps of average groundwater temperature over the study period. The warm zone coincided with an area of convergent groundwater flow, implicating horizontal heat transfer by advective convection as the reason for elevated temperatures. Additionally, high concentrations of dissolved Fe(II) and Fe(III) overlapped the warm thermal island, indicative of increased rates of bacterial Fe(II)-oxidation and Fe(III)-reduction. A depletion in the modal abundance of Fe(II)-bearing minerals, notably amphibole and biotite, inside the area of the warm thermal island was also observed, suggesting enhanced mineral dissolution owing to chemoautotrophic Fe(II)-oxidation coupled to the reduction and fixation of dissolved inorganic carbon as biomass. Throughout the aquifer, redox conditions were poised in terms of Eh and pH close to equilibrium with respect to the Fe(II)/Fe(OH)3 couple, feasibly enabling simultaneous bacterial Fe(II)-oxidation and Fe(III)-reduction with an adequate supply of electron acceptors and donors, respectively. The significance of higher groundwater temperature as a determinant of elevated dissolved Fe(II) and Fe(III) concentrations induced by thermal intensification of microbial biogeochemical activities yielded Pearson product-moment correlations in which temperature alone, as a single independent variable, explains almost 30 to nearly 60 percent of the variation in the measured dissolved Fe(II) and Fe(III) concentrations in the groundwater. These results emphasize the important influence of thermal conditions on biogeochemical processes in aquifers coupled to the development of steep gradients in groundwater quality over short distances in shallow unconfined groundwater systems.

We developed a novel method for distinguishing abiotic and biological iron oxidation in liquid media using oxidation-reduction (redox) potential time series data. The instrument and processing algorithm were tested by immersing the tip of a Pt electrode with an Ag-AgCl reference electrode into an active iron-oxidizing biofilm in a groundwater discharge zone, as well as in two abiotic systems: a killed sample and a chemical control from the same site. We used detrended fluctuation analysis to characterize average root mean square fluctuation behavior, which was distinct in the live system. The calculated α value scaling exponents determined by detrended fluctuation analysis were significantly different at p < 0.001. This indicates that time series of electrode response data may be used to distinguish live and abiotic chemical reaction pathways. Due to the simplicity, portability, and small size, it may be suitable for characterization of extraterrestrial environments where water has been observed, such as Mars and Europa. Key Words: Oxidation-reduction potential-Detrended fluctuation analysis-Iron-oxidizing bacteria. Astrobiology 16, 846-852.

The kinetics of bacterial Fe(II) oxidation was investigated 297m underground at the Äspö Hard Rock Laboratory (near Oskarshamn, Sweden) under steady state groundwater flow conditions in a flow-through cell containing well-developed flocculent mats of bacteriogenic iron oxides (BIOS). Pseudo first-order rate constants of 0.004 min^-1 and 0.009 min^-1 were obtained for chemical and bacterial Fe(II) oxidation, respectively, based on the 104 min retention time of groundwater in the flow cell, inlet Fe(II) concentration of 21.0± 0.5μm, outlet Fe(II) concentration of 8.5 ± 0.7 μm, as well as constant pH = 7.42 ± 0.01, dissolved O2 concentration of 0.11 ± 0.01mg/L, and groundwater temperature of 12.4± 0.1°C. Redox potential was lower at the BIOS-free inlet (-135.4± 1.16mV) compared to inside BIOS within the flow cell (-112.6± 1.91mV), consistent with the Nernst relationship and oxidation of Fe(II) to Fe(III). Further evaluation of the redox potential time series data using detrended fluctuation analysis (DFA) revealed power law scaling in the amplitude of fluctuations over increasing intervals of time with significantly different (p<0.01) DFA α scaling exponents of 1.89 ± 0.03 for BIOS and 1.67 ± 0.06 at the inlet. These α values not only signal the presence of long-range correlation in the redox potential time series measurements but also distinguish between the slower rate of chemical Fe(II) oxidation at the inlet and faster rate accelerated by FeOB in BIOS.

The oxidation of Fe(II) at circumneutral pH by natural bacterial consortia capable of oxidizing Fe(II) in flocculent mats of bacteriogenic iron oxides (BIOS) was investigated in a series of microcosm experiments under constant pH and pO(2) conditions. The oxidation of Fe(II) was faster in the presence BIOS compared to chemical oxidation in solution with pseudo-first-order rate constants ranging from 0.036 to 0.248 min(-1) for BIOS compared to 0.004 min(-1) for the chemical control. The Fe(II)-oxidation rates exhibited a kinetic dependency on BIOS concentrations, measured in terms of a pseudo-second-order rate constant at 0.066 Lg(-1)min(-1). From the asymptotic decline in Fe(total) in the microcosms, apparent rate constants for HFO dissolution (zero-order values 0.001 to 0.046 mgL(-1)min(-1)) and precipitation (second-order values 0.007 to .297 min(-1)) were obtained from the BIOS and control microcosms, which yield an apparent equilibrium BIOS solubility of 0.157 mgL(-1) (i.e., 2.8 M). These results reflect the complex interplay of Fe(II) oxidation reactions associated with BIOS including homogenous chemical oxidation in solution, chemical oxidation on HFO surfaces, and bacterially mediated oxidation. Furthermore, BIOS precipitated in response to Fe(II) oxidation exhibit a greater apparent solubility than more stable varieties of HFO. The implication is that the competitive advantage of Fe(II)-oxidizing bacteria over autocatalytic surface chemical Fe(II) oxidation on HFO might be enhanced by a reduction in the total amount of BIOS that is precipitated.

The goal of this thesis was to assess the utility of using small-scale fluctuations in oxidationreduction (redox) potential to distinguish microbial from chemical iron oxidation. Fluctuations in potential arise from the motion of particles in a fluid; measuring fluctuations is therefore a systemscale observable property of micro-scale chemical behaviour, as such particle motion constitutes diffusion. Fluctuations are described by the strength of their correlation, as measured by scaling exponents. A method for the calculation of scaling-exponents of long-range correlation in redox potential measurements was developed, including new instrumentation and the modification of an existing physiological processing algorithm for use with environmental microbiological data sets. Steady-state biological and chemical systems were compared, and scaling exponents calculated from each system were found to differ significantly. In a final study, a series of microcosms were used to determine the relationship between scaling exponent, measuring correlation strength, and oxidation rate. The biological systems are governed by the rate of reaction, while the chemical systems appear to be diffusion-controlled. Because in these systems, Fe(II) is a metabolite, redox potential can then be interpreted as a physically-constrained proxy for metabolic activity. This allows the characterization of biological activity in situ.