The pangenome of sulfate reducing bacteria represents a genetic reservoir that deciphers the intricate interplay of conserved and variable elements driving their ecological dominance, evolutionary adaptability, and industrial relevance. This study introduces the most comprehensive pangenome analysis of the genus Desulfovibrio till date, incorporating 63 complete and high-quality genomes using the Partitioned Pangenome Graph of Linked Neighbors (PPanGGOLiN) pipeline. The structure and dynamics of core gene families were investigated through gene ontology, KEGG pathway mapping, and gene network analyses, shedding light on the functional organization of the Desulfovibrio genomes. The analysis categorized 799, 4053, and 43,581 gene families into persistent, shell, and cloud groups, respectively. A core set of 326 gene families, conserved across Desulfovibrio genomes, highlights their essential role in community functionality. Genome plasticity analysis identified 4,576 regions of genome plasticity, with 1,322 hotspots enriched in horizontally acquired genes (89% in the cloud partition). Key gene families in these regions included glpE, fdhD, petC, and cooF, linked to sulfur metabolism. Out of 29 hypothetical genes, one was linked to actin nucleation, another contained a TRASH domain, while the other regulates filopodium assembly. Other predicted functions included lnrL, folE, RNA binding, and pyrG/pyrH involvement in CTP biosynthesis. Additionally, genomic islands revealed evolutionary events, such as cheY acquisition in Oleidesulfovibrio alaskensis G20. This study provides a genus-wide view of Desulfovibrio, emphasizing genome plasticity, hypothetical gene functions, and adaptation mechanisms.

Biofilms are common in water systems and can lead to mechanical failure or illness of water system users. Methods for evaluating anti-fouling coatings have largely been informed by the medical industry and have not been tailored to industrial or spacecraft water systems. The goal of the paper is to help guide researchers in designing experiments to evaluate coatings that accurately represent the system under investigation. This review identified eight experimental design considerations when evaluating coatings in water systems: biofilm reactor operation, microorganism selection, reinoculation, coating surface area, liquid medium, experiment duration, coating performance evaluation, and the use of microgravity. The impact of each decision made within each of these considerations is presented. Further, the methods featured in eight studies investigating coatings for Earth-based or spacecraft water systems are discussed. This review serves to guide researchers toward improved experimental design to enable successful technology transfer from the lab bench to Earth and beyond.

Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO 2 mitigation processes. Combinations of bicarbonate (HCO 3 − ) salt addition and application of CO 2 to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana , to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO 2 during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO 2 for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO 2 on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.

Selenium release to the environment as a result of industrial activities such as coal mining can impact receiving water. Saturated rock fill (SRF) technology is a robust and sustainable approach to reduce selenate to sorbed selenite and/or insoluble elemental selenium using native bacteria. Following SRF closure, the native microbial community and water chemistry is anticipated to return to conditions representing pre-SRF baseline chemistry in equilibrium with the backfilled rock, maintaining neutral pH, low oxygen, and reduced conditions that support stability of the accreted selenium. Mineralogical analyses of selenium in biomineralized samples recovered from the full-scale Elkview Operations SRF trial identified selenite strongly sorbed to ferrihydrite and reduced SenSn-8 minerals consistent with previous analyses of column residues. Batch tests showed sequestered selenium was not remobilized from active SRF treatment zone samples when nutrient amendment was stopped. Column tests run to accumulate selenium were subsequently shut down and allowed to remain static for 5 years with intermittent flow to mimic post-closure SRF conditions. Selenium was not remobilized from these columns under post-closure conditions. A series of comprehensive selenium adsorption-desorption experiments indicated selenite is strongly sorbed to ferrihydrite in coal mine rock with minimal desorption. Zero valent selenium remained stable when stored sub-aqueously and when air dried and exposed to atmospheric oxygen for over a year in the lab. These results suggest that selenium in SRF treatment reactors will likely be very stable and unlikely to negatively affect surface water or groundwater post closure.

ABSTRACT Sulfate-reducing bacteria (SRB) exhibit versatile metabolic adaptability with significant flexibility influenced by pH fluctuations, which play a critical role in biogeochemical cycles. In this study, we used a model SRB, Oleidesulfovibrio alaskensis G20, to determine the temporal effects of pH variations (pH 6, 7, and 8) on both growth dynamics and metabolic gene expressions. The specific growth rate at pH 6 (0.014 h −1 ) closely matched that at pH 7 (0.016 h −1 ), while pH 8 exhibited a lower growth rate (0.010 h −1 ). Lactate consumption peaked at pH 7 (0.35 mM lactate.h −1 ) and declined at pH 8 (0.09 mM lactate.h −1 ). Significant hydrogen production was evident under both acidic and alkaline conditions. Gene expression studies revealed that ATPases function as proton pumps, while hydrogenases mediate reversible proton-to-hydrogen conversion. Sulfate and energy metabolism act as electron acceptors and donors, while amino acid synthesis regulates basic and acidic amino acids to mitigate pH stress. Downregulation of FtsZ at pH 6 suggests impaired division, correlating with slightly longer lengths (~2 µm), while upregulation of divisome proteins at pH 8 suggests efficient division processes, aligning with shorter lengths (~1.8 µm). This study will facilitate the employment of O. alaskensis G20 in extreme pH environments, enhancing its effectiveness in optimizing bioremediation and anaerobic digestion processes. IMPORTANCE Sulfate-reducing bacteria (SRB) play essential roles in global sulfur and carbon cycling and are critical for bioremediation and anaerobic digestion processes. However, detailed studies on the genotypic and phenotypic responses of SRB under varying pH conditions are limited. This study addresses this gap by examining the pH-dependent genetic and metabolic adaptations of Oleidesulfovibrio alaskensis G20, revealing key mechanisms regulating hydrogenase and ATPase activities, cell division, and extracellular polymeric substance formation. These findings provide new insights into how SRB maintains pH homeostasis, showcasing their ability to survive and function in both acidic and alkaline environments. Furthermore, this study reveals critical genetic and phenotypic characteristics that will directly aid to engineer industrial effluent management systems, bioremediation, and dissolved heavy metal recovery. By elucidating the dynamic response of O. alaskensis G20 to varied pH environments, the research provides a foundation for enhancing the resilience and performance of SRB-based systems, paving the way for improved environmental and industrial applications.

Nitrogen and iron cycling are key drivers of biogeochemical processes, particularly in anoxic environments where they can influence contaminant mobility and bioavailability. Nitrate-dependent iron oxidation (NDFO) is a microbially-mediated process in which nitrate reduction is coupled to Fe(II) oxidation, forming solid Fe(III) minerals. This metabolism, detected in diverse environments such as sediments and aquifers, can proceed autotrophically, mixotrophically, or heterotrophically. NDFO represents a potential in situ bioremediation strategy due to the widespread environmental presence of capable microbial communities and the capacity of resulting iron oxides to adsorb and immobilize contaminants. These iron minerals can incorporate or reduce various metals, metalloids, and radionuclides, including arsenic, nickel, copper, and uranium, which may also accumulate in microbial biomass. NDFO-capable microorganisms may also transform some contaminants to less mobile oxidation states. Elevated nitrate and iron concentrations at contaminated sites, such as mines, offer conditions for NDFO induction. This review examines the microbial physiology and ecology underlying NDFO, the mineralogy and contaminant-binding properties of its iron oxide products, and current research into its application for environmental remediation. Key knowledge gaps and future research directions are highlighted to support further understanding of NDFO organisms, impacts on mineral phases, and development of NDFO-based strategies for contaminant management.

The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.

Naegleria fowleri has been detected in drinking water distribution systems (DWDS) in Australia, Pakistan and the United States and is the causative agent of the highly fatal disease primary amoebic meningoencephalitis. Previous small scale field studies have shown that Meiothermus may be a potential biomarker for N. fowleri. However, correlations between predictive biomarkers in small sample sizes often breakdown when applied to larger more representative datasets. This study represents one of the largest and most rigorous temporal investigations of Naegleria fowleri colonisation in an operational DWDS in the world and measured the association of Meiothermus and N. fowleri over a significantly larger space and time in the DWDS. A total of 232 samples were collected from five sites over three-years (2016-2018), which contained 29 positive N. fowleri samples. Two specific operational taxonomic units assigned to M. chliarophilus and M. hypogaeus, were significantly associated with N. fowleri presence. Furthermore, inoculation experiments demonstrated that Meiothermus was required to support N. fowleri growth in field-collected biofilms. This validates Meiothermus as prospective biological tool to aid in the identification and surveillance of N. fowleri colonisable sites.

Microbial diversity estimation involves extracting nucleic acids from intricate sample matrices. Preparing nucleic acid samples is time-consuming, necessitating effective cell lysis and obtaining pure, inhibitor-free nucleic acid purifications before further use. An automated system offers advantages for field deployment due to its ease of use and quick autonomous results. This is especially beneficial for rapid measurement of in situ microbial diversity in remote areas. Our study aimed to assess microbial diversity of Yellowstone hot springs using a field-deployable lab in a resource-limited remote setting and demonstrate on-site nucleic acid sample processing and sequencing. We collected microbial mat and sediment samples from several Yellowstone National Park hot springs, focusing on the Five Sister Springs (FSS), spring LNN010, and Octopus Spring (OS). The samples were processed for DNA extraction on-site and further sequenced in the lab for microbial diversity. In addition, DNA extracted from one sample was sequenced and analyzed on-site as proof-of-concept. Using either Illumina or Oxford Nanopore Technology sequencing, we found similar microbial diversities. Bacteria (over 90%) were predominant at the FSS and OS sites, with archaea accounting for less than 10%. Metagenomic results were taxonomically categorized based on the closest known organism with a sequenced genome. The dominant archaeal community member was Candidatus Caldiarchaeum subterraneum, and among bacteria, Roseiflexus sp. RS-1 was abundant in mat samples. Interestingly, Bacterium HR17 was also frequently found, suggesting the need for more research on this newly recognized bacterial community member. The presence of Bacterium HR17 in these hot springs suggests its potential role in nitrogen cycling, informing both ecological understanding and industrial potential. This pioneering study assessed the microbiome of Yellowstone hot springs in about 8-9 hours using an automated system for nucleic acid extraction. By its deployment, the system’s value in elucidating the microbial diversity of extreme environments without the need to bring samples to the lab for processing had been highlighted. Sample processing and sequencing had been included in the benefits of the field-deployable lab, and the Nanopore platform had been utilized.

Removing selenium (Se) from mine effluent is a common challenge. A long-term, in situ experiment was conducted to bioremediate large volumes (up to 7500 m (c) d(-1)) of Se(VI)-contaminated water (mean 87 mu g L-1) by injecting the water into a saturated waste rock fill (SRF) at a coal mining operation in Elk Valley, British Columbia, Canada. To stimulate/maintain biofilm growth in the SRF, labile organic carbon (methanol) and nutrients were added to the water prior to its injection. A conservative tracer (Br-) was also added to track the migration of injected water across the SRF, identify wells with minimal dilution and used to quantify the extent of bioreduction. The evolution of the Se species through the SRF was monitored in time and space for 201 d. Selenium concentrations of <3.8 mu g L-1 were attained in monitoring wells located 38 m from the injection wells after 114 to 141 d of operation. Concentrations of Se species in water samples from complementary long-term (351-498 d) column experiments using influent Se(VI) concentrations of 1.0 mg L-1 were consistent with the results of the in situ experiment. Solid samples collected at the completion of the column experiments confirmed the presence of indigenous Se-reducing bacteria and that the sequestered Se was present as insoluble Se(0), likely in Se-S ring compounds. Based on the success of this ongoing bioremediation experiment, this technology is being applied at other mine sites.

Plants rely on a diverse rhizobiome to regulate nutrient acquisition and plant health. With increasing severity and frequency of droughts worldwide due to climate change, untangling the relationships between plants and their rhizobiomes is vital to maintaining agricultural productivity and protecting ecosystem diversity. While some plant physiological responses to drought are generally conserved, patterns of root exudation (release of small metabolites shown to influence microbes) and the consequential effects on the plant rhizobiome can differ widely across plant species under drought. To address this knowledge gap, we conducted a greenhouse study using blue grama (Bouteloua gracilis), a drought-tolerant C4 grass native to shortgrass prairie across North American plains, as a model organism to study the effect of increasing drought severity (ambient, mild drought, severe drought) on root exudation and the rhizobiome. Our previous results demonstrated physiological effects of increasing drought severity including an increase in belowground carbon allocation through root exudation and shifts in root exudate composition concurrent with the gradient of drought severity. This work is focused on the rhizobiome community structure using targeted sequencing and found that mild and severe drought resulted in unique shifts in the bacterial + archaeal and fungal communities relative to ambient, non-droughted controls. Specifically, using the change in relative abundance between ambient and drought conditions for each ZOTU as a surrogate for population-scale drought tolerance (e.g., as a response trait), we found that rhizobiome response to drought was non-randomly distributed across the phylogenies of both communities, suggesting that Planctomycetota, Thermoproteota (formerly Thaumarchaeota), and the Glomeromycota were the primary clades driving these changes. Correlation analyses indicated weak correlations between droughted community composition and a select few root exudate compounds previously implicated in plant drought responses including pyruvic acid, D-glucose, and myoinositol. This study demonstrates the variable impacts of drought severity on the composition of the blue grama rhizobiome and provides a platform for hypothesis generation for targeted functional studies of specific taxa involved in plant-microbe drought responses.

Copper is known to have toxic effects on bacterial growth. This study aimed to determine the influence of copper ions on Oleidesulfovibrio alaskensis G20 biofilm formation in a lactate-C medium supplemented with variable copper ion concentrations. OA G20, when grown in media supplemented with high copper ion concentrations of 5, 15, and 30 µM, exhibited inhibited growth in its planktonic state. Conversely, under similar copper concentrations, OA G20 demonstrated enhanced biofilm formation on glass coupons. Microscopic studies revealed that biofilms exposed to copper stress demonstrated a change in cellular morphology and more accumulation of carbohydrates and proteins than controls. Consistent with these findings, sulfur (dsrA, dsrB, sat, aprA) and electron transport (NiFeSe, NiFe, ldh, cyt3) genes, polysaccharide synthesis (poI), and genes involved in stress response (sodB) were significantly upregulated in copper-induced biofilms, while genes (ftsZ, ftsA, ftsQ) related to cellular division were negatively regulated compared to controls. These results indicate that the presence of copper ions triggers alterations in cellular morphology and gene expression levels in OA G20, impacting cell attachment and EPS production. This adaptation, characterized by increased biofilm formation, represents a crucial strategy employed by OA G20 to resist metal ion stress.

Nitrate-dependent iron oxidation (NDFO) is a novel mechanism for microbial bioremediation of metal and metalloid contaminants. During NDFO, microbes catalyze a redox reaction wherein nitrate is reduced to nitrite and nitrogen gas while Fe(II) is oxidized to solid Fe(III) hydroxide minerals. Metalloid contaminants such as selenium and arsenic have a propensity for adsorption to iron minerals produced during NDFO; some contaminants may also be concurrently bioreduced. A number of bacterial isolates have been shown to be capable of NDFO (e.g., Kappler et al. (2005), Kiskira et al. (2017)), but little work has been done to date characterizing mixed microbial communities performing NDFO. Some autotrophic communities have been characterized, with high relative abundances for strains of Gallionellaceae in both a freshwater sediment enrichment culture and an activated sludge culture (Blöthe and Roden 2009, Tian et al. 2020). In mixotrophic activated sludge cultures, the concentration of Fe(II) amendment was found to significantly impact microbial community composition; these cultures were fed with methanol in addition to Fe(II), and the dominant community members were Methyloversatilis and other methylotrophic strains (Liu et al. 2018). The work presented here examines microbial communities performing NDFO in the context of remediation, and in particular how differences between NDFO and heterotrophic communities may influence remediation effectiveness. This research characterizes and compares microbial communities performing NDFO versus heterotrophic denitrification during removal of selenium and nickel from mining wastewater. Sediment and influent water from a subsurface bioreactor treating mining wastewater were used to construct batch bioreactors, which were amended with selenium and nickel as well as either Fe(II) or methanol to investigate contaminant removal and microbial community composition in NDFO versus heterotrophic microbial communities. Both Fe(II) and methanol reactors removed total aqueous selenium to below the quantification limit, but Fe(II) reactors removed it more rapidly, likely due to adsorption of selenite. For nickel, removal to below the detection limit was achieved with methanol amendment, while Fe(II) amendment resulted in 42-95% removal. This was likely due to precipitation of nickel sulfide during sulfate reduction in methanol-amended reactors. DNA from the batch bioreactors will be sequenced and the results analyzed for differences among communities. Permutational multivariate analysis of variance and non-metric multidimensional scaling will be used to determine significant correlations of community composition with experimental variables, selenium and nickel removal, and NDFO (Roberts 2023, Kruskal 1964). Indicator species analyses (De Cáceres et al. 2010) will be applied to identify taxa found significantly more often (i.e., at a higher relative abundance) in one group of microbial communities than in any other group. The indicator species analysis may reveal whether there are groups of denitrifiers that predominate in NDFO conditions vs. groups that predominate during heterotrophic denitrification. The results of these microbial community analyses, in combination with the geochemical analyses, will improve our understanding of microbial communities performing NDFO in remediation environments.

Water is not only essential to sustain life on Earth, but also is a crucial resource for long-duration deep space exploration and habitation. Current systems in space rely on the resupply of water from Earth, however, as missions get longer and move farther away from Earth, resupply will no longer be a sustainable option. Thus, the development of regenerative reclamation water systems through which useable water can be recovered from “waste streams” (i.e., used waters) is sorely needed to further close the loop in space life support systems. This review presents the origin and characteristics of different used waters generated in space and discusses the intrinsic challenges of developing suitable technologies to treat such streams given the unique constrains of space exploration and habitation (e.g., different gravity conditions, size and weight limitations, compatibility with other systems, etc.). In this review, we discuss the potential use of biological systems, particularly biofilms, as possible alternatives or additions to current technologies for water reclamation and waste treatment in space. The fundamentals of biofilm reactors, their advantages and disadvantages, as well as different reactor configurations and their potential for use and challenges to be incorporated in self-sustaining and regenerative life support systems in long-duration space missions are also discussed. Furthermore, we discuss the possibility to recover value-added products (e.g., biomass, nutrients, water) from used waters and the opportunity to recycle and reuse such products as resources in other life support subsystems (e.g., habitation, waste, air, etc.).

While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly clean-rooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.

Plant survival during environmental stress greatly affects ecosystem carbon (C) cycling, and plant-microbe interactions are central to plant stress survival. The release of C-rich root exudates is a key mechanism plants use to manage their microbiome, attracting beneficial microbes and/or suppressing harmful microbes to help plants withstand environmental stress. However, a critical knowledge gap is how plants alter root exudate concentration and composition under varying stress levels. In a greenhouse study, we imposed three drought treatments (control, mild, severe) on blue grama (Bouteloua gracilis Kunth Lag. Ex Griffiths), and measured plant physiology and root exudate concentration and composition using GC-MS, NMR, and FTICR. With increasing drought severity, root exudate total C and organic C increased concurrently with declining predawn leaf water potential and photosynthesis. Root exudate composition mirrored the physiological gradient of drought severity treatments. Specific compounds that are known to alter plant drought responses and the rhizosphere microbiome mirrored the drought severity-induced root exudate compositional gradient. Despite reducing C uptake, these plants actively invested C to root exudates with increasing drought severity. Patterns of plant physiology and root exudate concentration and composition co-varied along a gradient of drought severity.