As the largest available freshwater resource, groundwater provides water for drinking and agricultural irrigation to billions of people worldwide. Subsurface environments are estimated to host over 30% of all microorganisms on Earth, with microbial communities having important roles in various groundwater processes. Discoveries of diverse and novel lineages of archaea, bacteria, microeukaryotes and viruses in groundwater have highlighted the key contributions of groundwater microbiomes to elemental cycling, contaminant degradation, pathogenicity and antimicrobial resistance. In this Review, we summarize the diversity and composition of prokaryotic, microeukaryotic and viral communities in groundwater, and describe the complex interactions between these different microbial groups. Groundwater microbiomes exhibit distinct biogeographic patterns, with key differences and similarities across groundwater types and other habitats. The community assembly of microorganisms across different groundwater environments is driven primarily by stochastic processes, whereas deterministic processes caused by environmental stress further modulate community structure and function. With regard to their function, groundwater microbiomes have crucial roles in shaping ecosystem functioning, including biogeochemical cycling, water quality and One Health. Finally, we highlight several future prospects for research to harness microbiomes as eco-sustainable solutions for groundwater restoration and protection in a changing world. Groundwater hosts a vast array of microbiomes that are essential to global water quality and ecosystem function. This Review discusses the diversity, biogeography and community assembly of the groundwater microbiome, and explores its role in biogeochemical cycling and sustainable groundwater management.

Abstract Replicating deep subsurface conditions remains a major challenge for advancing subterranean biotechnologies. Microbially enhanced coal bed methane production offers a promising approach to increase biogenic gas recovery from coal seams, yet experimental progress has been limited by the difficulty of simulating in situ conditions. A key question is whether nutrient amendments stimulate coal-dependent methanogenesis or simply convert injected nutrients to methane. Here, we report the use of a large-scale (325 L) reactor to investigate 13 C-labeled microalgae amendments on microbially-enhanced coal bed methane production at in situ pressure. Labeled methane was detected only during the initial stimulation phase, while most of the methane produced over five months originated from the coal. These field-relevant results demonstrate that microalgae can accelerate early methanogenesis and enhance coal-derived methane production over time. This study provides a critical proof-of-concept with broad implications for advancing subsurface biotechnologies toward field-scale deployment.

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.

We report 26 subsurface sediment and 9 groundwater metagenomes from the Oak Ridge Reservation at Oak Ridge, TN, USA. Samples were collected from various depths and phases (attached vs planktonic) to study subsurface microbial metabolism, the effect of contamination on microbial communities, and differences across groundwater and sediment microbial communities.

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.

Phycosomes, or algal microbiomes, can impact algal productivity, and the influence of phycosome succession and turnover may be magnified during stresses such as low temperature, low solar irradiance, and/or high pH. The combined effects of abiotic (e.g., temperature, irradiance) and ecological factors on biomass productivity in outdoor, open raceway cultivation were assessed on Chlorella SLA-04 in summer and fall conditions. Raceway ponds (200L) L ) were used to grow outdoor SLA-04 cultures under high pH conditions (i.e., pH 8 to pH 12) in five sequential runs. SLA-04 cultures had the highest biomass productivity in early runs (R1, R2, and R3) when maximum and minimum temperatures as well as radiative flux were higher while both total dry weight and lipid levels declined with declining temperatures and light (R4 and R5). Despite lower overall biomass, chlorophyll did not decline to the same extent, and nitrate was still depleted albeit at a slower rate. The overall diversity of microbial communities (both bulk phase and algal associated) increased during the successive runs, and predominant bacterial groups shifted as temperature and light declined (R3). During R1 and R2, specific amplicon sequencing variants (ASVs) predominated (related to Paracoccus, , Brevundimonas, , Rhodococcus, , and Rhizobium) ) and these ASVs declined in R3 as the light and temperature declined. These results are corroborated by decreased growth of a Paracoccus isolate at lower temperatures compared to SLA-04. As microbial growth slowed in R4 and R5, the following bacterial ASVs increased: Roseivivax, , Porphyrobacter, , unclassified GpV, Gemmata, , Rhizobacter, , and Luteolibacter. . Overall, the results demonstrated that SLA-04 could tolerate transitions to lower temperatures (and light) compared to the present bacterial populations during outdoor cultivations, but total biomass productivity (ash free dry weight) and lipids (Nile Red fluorescence) decreased significantly. Coinciding with these changes, Flavobacteria and Deinococci ASVs that were associated with higher algal biomass and higher temperatures declined and different ASVs ( Planctomycetes , Clostridia, , Gemmatimonadetes, , Chlamydia) ) were enriched during lower algal productivity and lower temperatures. Changes in the microbial community could be directly related to observed temperature shifts as well as indirectly related to SLA-04 growth dynamics during the temperature and/or light shifts. Weather (i.e., light and temperature) is predicted to have major impacts on large-scale algal production (Kleiman et al., 2021), and the presented results provide insight into the relationships between declining light/temperature, algal growth, and phycosome composition during successive, outdoor cultivations.

Biofilms are surface-attached microbial communities that play vital roles in natural ecosystems and contribute to persistent problems in medicine and industry. These communities exhibit heterogeneous chemical, physical, and physiological properties, which are governed by reciprocal structure-function relationships. Linking structure to function is crucial for understanding biofilm physiology but remains challenging due to the structural complexity of naturally formed biofilms. Bioprinting offers exquisite control over biofilm structure and holds potential for systematically exploring these relationships; however, the microscale colony distributions that emerge within hydrogel-based print resins remain unexplored. To address this, we use light-based bioprinting to create single-layer hydrogel films containing homogeneously dispersedPseudomonas fluorescensbacteria and characterize the spatiotemporal distribution of colonies that develop within these films. We systematically vary the concentration of bacteria over nearly three orders of magnitude, track colony growth using microscopy, and quantify structural features with image analysis. We observe empirical relationships between initial cell concentration and key structural features: colony size, colony volume, total biovolume, and characteristic gradient length scale. This knowledge can be used to print microbial communities with well-defined features, is readily applicable to more complex three-dimensional shapes, and provides a tool for advancing our understanding of microbial communities.

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in sub-optimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, nitrate or metal concentrations is underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimes of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse pH (3.5 to 5) and nitrate levels (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptation to survive and grow at low pH. Biofilms intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation warranting further investigation. Through RB-TnSeq, proteomics, use of specific mutants and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed absence of flagella and chemotaxis genes, and presence of putative Type VI secretion system in the high biofilm-forming strain FW021-MT20. This study identifies genetic determinants associated with biofilm growth in a predominant environmental genus, Rhodanobacter, under metal stress and identifies traits aiding survival and adaptation to contaminated subsurface environments.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Engineered living materials (ELMs) are an emerging class of materials with the potential for transformative impacts in sustainability across sectors (e.g., water, energy, health). Progress toward producing ELMs with tailorable and/or stimuli-responsive functionalities has occurred in recent years, along with advances in materials manufacturing with increased complexity and scale. While a few ELMs have been commercialized, important barriers must be surmounted before their broader integration into society. These social, ethical, legal, and regulatory barriers, as well as barriers to collaboration between stakeholders, were identified in a workshop combining academic, industry, and government agency participants that was convened as part of the annual Montana Biofilm Meeting (Bozeman, MT) in July 2023. The ELM research community finds itself at a defining moment. Urgent action is needed to realize the societal benefits of ELMs while decreasing the likelihood of negative perception, and actual consequences, of their commercialization.

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and ‘drift’ related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.

Algal-bacterial interactions are ubiquitous in both natural and industrial systems, and the characterization of these interactions has been reinvigorated by potential applications in biosystem productivity. Different growth conditions can be used for operational functions, such as the use of low-quality water or high pH/alkalinity, and the altered operating conditions likely constrain microbial community structure and function in unique ways. However, research is necessary to better understand whether consortia can be designed to improve the productivity, processing, and sustainability of industrial-scale cultivations through different controls that can constrain microbial interactions for maximal light-driven outputs. The review highlights current knowledge and gaps for relevant operating conditions, as well as suggestions for near-term and longer-term improvements for large-scale cultivation and polyculture engineering.

Priority question exercises are increasingly used to frame and set future research, innovation and development agendas. They can provide an important bridge between the discoveries, data and outputs generated by researchers, and the information required by policy makers and funders. Microbial biofilms present huge scientific, societal and economic opportunities and challenges. In order to identify key priorities that will help to advance the field, here we review questions from a pool submitted by the international biofilm research community and from practitioners working across industry, the environment and medicine. To avoid bias we used computational approaches to group questions and manage a voting and selection process. The outcome of the exercise is a set of 78 unique questions, categorized in six themes: (i) Biofilm control, disruption, prevention, management, treatment (13 questions); (ii) Resistance, persistence, tolerance, role of aggregation, immune interaction, relevance to infection (10 questions); (iii) Model systems, standards, regulatory, policy education, interdisciplinary approaches (15 questions); (iv) Polymicrobial, interactions, ecology, microbiome, phage (13 questions); (v) Clinical focus, chronic infection, detection, diagnostics (13 questions); and (vi) Matrix, lipids, capsule, metabolism, development, physiology, ecology, evolution environment, microbiome, community engineering (14 questions). The questions presented are intended to highlight opportunities, stimulate discussion and provide focus for researchers, funders and policy makers, informing future research, innovation and development strategy for biofilms and microbial communities.

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.

Microbial generation of coal bed methane (CBM) represents a significant source of natural gas on Earth. While biostimulation has been demonstrated in batch cultures, environmental parameters such as overburden pressure and formation water flow need to be tested at the laboratory scale to understand in situ potential. We designed and constructed a high-pressure (HP) flow-through reactor system that simulates in situ conditions of underground coal seams. Two stainless-steel columns contained coal from the Powder River Basin (PRB), USA, or a coal/sand mixture to represent the interface of coal seams with sandstone layers, which are hypothesized to exhibit higher methanogenesis rates in situ. The system was filled with CBM formation water, inoculated with a methanogenic enrichment from PRB coal beds, and stimulated with algal biomass as a nutrient. The reactors were incubated under pressure (5.4 atm) and flow of CBM water (0.01 mL/min), and control batch cultures were incubated at ambient pressure and without flow (± amendment). Dissolved and headspace methane concentrations were analyzed over time by gas chromatography for 75 days. The pressurized reactors exhibited longer latency periods than ambient pressure controls, but methane production did not reach a plateau phase, which might reflect the impact of scale on the inoculum. The coal/sand reactor exhibited higher methane production than the coal-only reactor, a pattern also observed in the corresponding controls, suggesting an interface effect on methanogenesis. This study indicates that the HP flow test system we designed is well suited for the study of methanogenesis and provides a successful demonstration of CBM generation from the PRB in field-relevant laboratory conditions as a precursor to meso‑scale demonstrations.

The addition of small amounts of algal biomass to stimulate methane production in coal seams is a promising low carbon renewable coalbed methane enhancement technique. However, little is known about how the addition of algal biomass amendment affects methane production from coals of different thermal maturity. Here, we show that biogenic methane can be produced from five coals ranging in rank from lignite to low-volatile bituminous using a coal-derived microbial consortium in batch microcosms with and without algal amendment. The addition of 0.1 g/l algal biomass resulted in maximum methane production rates up to 37 days earlier and decreased the time required to reach maximum methane production by 17–19 days when compared to unamended, analogous microcosms. Cumulative methane production and methane production rate were generally highest in low rank, subbituminous coals, but no clear association between increasing vitrinite reflectance and decreasing methane production could be determined. Microbial community analysis revealed that archaeal populations were correlated with methane production rate (p = 0.01), vitrinite reflectance (p = 0.03), percent volatile matter (p = 0.03), and fixed carbon (p = 0.02), all of which are related to coal rank and composition. Sequences indicative of the acetoclastic methanogenic genus Methanosaeta dominated low rank coal microcosms. Amended treatments that had increased methane production relative to unamended analogs had high relative abundances of the hydrogenotrophic methanogenic genus Methanobacterium and the bacterial family Pseudomonadaceae. These results suggest that algal amendment may shift coal-derived microbial communities towards coal-degrading bacteria and CO2-reducing methanogens. These results have broad implications for understanding subsurface carbon cycling in coal beds and the adoption of low carbon renewable microbially enhanced coalbed methane techniques across a diverse range of coal geology.

Microorganisms make up most of the earth’s biomass, and most microbes exist in the form of biofilms, complex communities of microorganisms growing attached to surfaces. Biofilms are directly relevant to a large number of scientific disciplines, and are the subjects of growing multidisciplinary research. As such, there is a pressing requirement for information systems that specialize in biofilm knowledge. Realization of such systems will require a coherent approach to understanding and curating the language used to study biofilms; an ontology of biofilms-related terms offers a foundation for such systems. Here we present an ontology for the study of biofilms (BIFO), a tool that will provide precisely defined terms describing all aspects involved in the biofilms domain. We describe semi-automated methods for the identification of relevant terms from a body of literature, the selection of a set of important terms by domain experts, and the construction of the ontology. A generic approach for BIFO is presented, in which foundational biofilm-related entities and relationships are represented. This ontology reuses terms from other ontologies that provide biofilm knowledge from the Open Biological and Biomedical Ontologies (OBO) foundry.

Subsurface environments represent diverse microbial communities responsible for mediating biogeochemical cycles linked to the turnover of organic and inorganic carbon important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the different sediment environments, microorganisms typically reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the respective contributions of groundwater (planktonic) and sediment-associated (biofilm) cells to subsurface processes is largely unresolved. In order to directly compare the distribution of microbial biomass and activity in a shallow, subsurface environment, total cell numbers, translationally-active cell numbers (Bioorthogonal non-canonical amino acid tagging- BONCAT), and microbial activity ( 3 H-Leucine incorporation) were investigated for a low biomass pristine and contaminated groundwater and corresponding soil cores. The results demonstrated that cell numbers for the 0.2 um fraction were approximately an order of magnitude higher for the pristine groundwater compared to the contaminated groundwater (10 6 v. 10 5 ). When contaminated groundwater was compared to the pristine, there was a drastic reduction in the BONCAT activity and the contaminated groundwater was between 100-700-fold less. Additionally, the rate of leucine incorporation ( 3 H-leucine) on a per cell basis in pristine groundwater was up to 1,000 times greater than the contaminated groundwater, respectively. Overall, like total cell numbers, activity was lower (both per volume and per cell) in contaminated groundwater compared to pristine groundwater. In pristine soil, activity ( 3 H-leucine) displayed steep gradients of microbial activity in association with transition zones of water table height ( i.e ., vadose, capillary fringe, saturated). A similar trend was also observed for the contaminated soil; however, the contaminated soil displayed an overall gradient of decreasing activity with depth. The highest activity for pristine soil was 9,253 ng C/g/d located in the transition depth between the capillary fringe and the saturated zone. Conversely, the highest activity for the contaminated soil was 9,175 ng C/g/d located in the vadose zone, perhaps the zone that is least impacted by contaminant flux. The pristine groundwater had higher activity rates than pristine sediment (per cell), but the contaminated groundwater had slower activity rates than the contaminated sediment (per cell). However, for both pristine and contaminated samples on a per volume basis, sediments had the vast majority of microbial activity compared to groundwater (80-95%). In the absence of strong selection forces compared to the contaminated well, the uncontaminated samples demonstrated more phylogenetic differences between the viable and translationally active populations that could be attributed to growth rate differences. The contaminated groundwater sample was predominated by a single, persistent Rhodanobacter strain in the viable fraction, while Rhodococcus , Brevundimonas , and Pseudomonas species dominated the translationally active fraction. Overall, the top active ASVs were prevalent and persistent across the estimated landscape. This is the first quantitative comparison between corresponding groundwater and subsurface sediments as well as predictions of viable and active ASVs ( e.g ., stable analog probing- SAP) within commonly used sequencing methods. The results suggest that field sampling schemes should consist of both viability and activity-based assessments that can help delineate key microbial populations within diverse microbial communities across and within subsurface systems.