In this review, selected examples are presented to demonstrate how microfluidic approaches can be utilized for investigating microbial life from deep geological environments, both from practical and fundamental perspectives. Beginning with the definition of the deep underground biosphere and the conventional experimental techniques employed for these studies, the use of microfluidic systems for accessing critical parameters of deep life in geological environments at the microscale is subsequently addressed (high pressure, high temperature, low volume). Microfluidics can simulate a range of environmental conditions on a chip, enabling rapid and comprehensive studies of microbial behavior and interactions in subsurface ecosystems, such as simulations of porous systems, interactions among microbes/microbes/minerals, and gradient cultivation. Transparent microreactors allow real-time, noninvasive analysis of microbial activities (microscopy, Raman spectroscopy, FTIR microspectroscopy, etc.), providing detailed insights into biogeochemical processes and facilitating pore-scale analysis. Finally, the current challenges and opportunities to expand the use of microfluidic methodologies for studying and monitoring the deep biosphere in real time under deep underground conditions are discussed.

Diet profoundly influences the composition of an animal's microbiome, especially in holometabolous insects, offering a valuable model to explore the impact of diet on gut microbiome dynamics throughout metamorphosis. Here, we use monarch butterflies (Danaus plexippus), specialist herbivores that feed as larvae on many species of chemically well-defined milkweed plants (Asclepias sp.), to investigate the impacts of development and diet on the composition of the gut microbial community. While a few microbial taxa are conserved across life stages of monarchs, the microbiome appears to be highly dynamic throughout the life cycle. Microbial diversity gradually diminishes throughout the larval instars, ultimately reaching its lowest point during the pupal stage and then recovering again in the adult stage. The microbial composition then undergoes a substantial shift upon the transition from pupa to adult, with female adults having significantly different microbial communities than the eggs that they lay, indicating limited evidence for vertical transmission of gut microbiota. While diet did not significantly impact overall microbial composition, our results suggest that fourth instar larvae exhibit higher microbial diversity when consuming milkweed with high concentrations of toxic cardenolide phytochemicals. This study underscores how diet and developmental stage collectively shape the monarch's gut microbiota.

Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, the microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum, to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO2% present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport and catabolism based on COG categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to a NADH/NAD+ redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD+ redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally-relevant soil strains.

Thermal springs harbour microorganisms, often dominated by cyanobacteria, which form biofilms and microbial mats. These phototrophic organisms release organic exudates into their immediate surroundings, attracting heterotrophic bacteria that contribute to the diversity and functioning of these ecosystems. In this study, the microbial mats from a hydrothermal pool in the Ksar Ghilane oasis in the Grand Erg Oriental of the DesertTunisia, were collected to obtain cyanobacterial cultures formed by single cyanobacterial species. High throughput analysis showed that while the microbial mat hosted diverse cyanobacteria, laboratory cultures selectively enriched cyanobacteria from the Leptolyngbya, Nodosilinea and Arthronema. Per each of these genera, multiple non-axenic uni-cyanobacterial cultures were established, totalling 41 cultures. Cyanobacteria taxa mediated the assembly of distinct heterotrophic bacterial communities, with members of the Proteobacteria and Actinobacteria phyla dominating. The bacterial communities of uni-cyanobacterial cultures were densely interconnected, with heterotrophic bacteria preferentially co-occurring with each other. Our study highlighted the complex structures of non-axenic uni-cyanobacterial cultures, where taxonomically distinct cyanobacteria consistently associate with specific groups of heterotrophic bacteria. The observed associations were likely driven by common selection pressures in the laboratory, such as cultivation conditions and specific hosts, and may not necessarily reflect the microbial dynamic occurring in the spring microbial mats.

Protist communities in the southern Pacific Ocean make a major contribution to global biogeochemical cycling, but remain understudied due to their remote location. We therefore have limited understanding of how large-scale physical gradients (e.g. temperature) and mesoscale oceanographic features (e.g. fronts) influence microeukaryote diversity in this region. We performed a high-resolution examination of protist communities along a latitudinal transect (>3000 km) at 150°W in the central southern Pacific Ocean that encompassed major frontal regions, including the sub-tropical front (STF), the sub-Antarctic front (SAF), and the polar front (PF). We identified distinct microbial communities along the transect that were primarily delineated by the positions of the STF and PF. Some taxa were not constricted by these environmental boundaries and were able to span frontal regions, such as the colonial haptophyte Phaeocystis. Our findings also support the presence of a Latitudinal Diversity Gradient (LDG) of decreasing diversity of the protist community with increasing latitude, although some individual taxa, notably the diatoms, do not adhere to this rule. Our findings show that oceanographic features and large-scale physical gradients have important impacts on marine protist communities in the southern Pacific Ocean that are likely to strongly influence their response to future environmental change.

Despite covering less than 5% of Earth's terrestrial area, peatlands are crucial for global carbon storage and are hot spots of methane cycling. This study examined the dynamics of aerobic and anaerobic methane oxidation in two undisturbed peatlands: a fen and a spruce swamp forest. Using microcosm incubations, we investigated the effect of ammonium addition, at a level similar to current N pollution processes, on aerobic methane oxidation. Our findings revealed higher methane consumption rates in fen compared to swamp peat, but no effect of ammonium amendment on methane consumption was found. Members of Methylocystis and Methylocella were the predominant methanotrophs in both peatlands. Furthermore, we explored the role of ferric iron and sulfate as electron acceptors for the anaerobic oxidation of methane (AOM). AOM occurred without the addition of an external electron acceptor in the fen, but not in the swamp peat. AOM was stimulated by sulfate and ferric iron addition in the swamp peat and inhibited by ferric iron in the fen. Our findings suggest that aerobic methane oxidizers are not N-limited in these peatlands and that there is an intrinsic potential for AOM in these environments, partially facilitated by ferric iron and sulfate acting as electron acceptors.

Drylands' poly-extreme conditions limit edaphic microbial diversity and functionality. Furthermore, climate change exacerbates soil desiccation and salinity in most drylands. To better understand the potential effects of these changes on dryland microbial communities, we evaluated their taxonomic and functional diversities in two Southern African dryland soils with contrasting aridity and salinity. Fungal community structure was significantly influenced by aridity and salinity, while Bacteria and Archaea only by salinity. Deterministic homogeneous selection was significantly more important for bacterial and archaeal communities' assembly in hyperarid and saline soils when compared to those from arid soils. This suggests that niche partitioning drives bacterial and archaeal communities' assembly under the most extreme conditions. Conversely, stochastic dispersal limitations drove the assembly of fungal communities. Hyperarid and saline soil communities exhibited similar potential functional capacities, demonstrating a disconnect between microbial structure and function. Structure variations could be functionally compensated by different taxa with similar functions, as implied by the high levels of functional redundancy. Consequently, while environmental selective pressures shape the dryland microbial community assembly and structures, they do not influence their potential functionality. This suggest that they are functionally stable, and that they could be functional even under harsher conditions, such as those expected with climate change.

The Southern green shield bug, Nezara viridula, is an invasive piercing and sucking pest insect that feeds on crops and poses a threat to global food production. Insects live in close relationships with microorganisms providing their host with unique capabilities, such as resistance to toxic plant metabolites. In this study, we investigated the resistance to and detoxification of the plant metabolite 3-nitropropionic acid by core and transient members of the N. viridula microbial community. Microbial community members showed a different tolerance to the toxin and we determined that six out of eight strains detoxified 3-nitropropionic acid. Additionally, we determined that 3-nitropropionic acid might interfere with the biosynthesis and transport of L-leucine. Moreover, our study explored the influence of diet on the gut microbial composition of N. viridula, demonstrating that switching to a single-plant diet shifts the abundance of core microbes. In line with this, testing pairwise microbial interactions revealed that core microbiota members support each other and repress the growth of transient microorganisms. With this work, we provide novel insights into the factors shaping the insect gut microbial communities and demonstrate that N. viridula harbours many toxin-degrading bacteria that could support its resistance to plant defences.

In-depth comparative genomic analysis was conducted to predict carbon, nitrogen, and phosphate assimilation pathways in the halotolerant, acidophilic genus Acidihalobacter. The study primarily aimed to understand how the metabolic capabilities of each species can determine their roles and effects on the microbial ecology of their unique saline and acidic environments, as well as in their potential application to saline water bioleaching systems. All four genomes encoded the genes for the complete tricarboxylic acid cycle, including 2-oxoglutarate dehydrogenase, a key enzyme absent in obligate chemolithotrophic acidophiles. Genes for a unique carboxysome shell protein, csoS1D, typically found in halotolerant bacteria but not in acidophiles, were identified. All genomes contained lactate and malate utilization genes, but only Ac. ferrooxydans DSM 14175T contained genes for the metabolism of propionate. Genes for phosphate assimilation were present, though organized differently across species. Only Ac. prosperus DSM 5130T and Ac. aeolianus DSM 14174T genomes contained nitrogen fixation genes, while Ac. ferrooxydans DSM 14175T and Ac. yilgarnensis DSM 105917T possessed genes for urease transporters and respiratory nitrate reductases, respectively. The findings suggest that all species can fix carbon dioxide but can also potentially utilize exogenous carbon sources and that the non-nitrogen-fixing species rely on alternative nitrogen assimilation mechanisms.

Particle-attached bacterial (PAB) communities play pivotal roles in water organic matter decomposition, nutrient cycling, and the natural self-purification processes. However, we know little about their responses to seasonal environmental fluctuations, under eutrophication in reservoir ecosystems. In this study, we studied the shifts of PAB communities to seasonal environmental fluctuations in tropical China. Trophic state index (TSI) indicated that the studied reservoirs ranged from mesotrophic to eutrophic state with a gradual increase in TSI from 31 to 58. In eutrophic reservoirs, Cyanobacteria, especially Raphidiopsis raciborskii, significantly increased in its relative abundance from wet to dry season, but Synechococcales and Microcystaceae decreased. In contrast, the relative abundance of Clostridia, Bacilli, Coriobacteriia, Enterobacteriales, and Vibrionales were more susceptible to seasonal environmental fluctuations in mesotrophic than eutrophic reservoirs. PAB co-occurrence relationships in mesotrophic reservoirs varied more greatly in response to seasonal environmental fluctuations, compared with eutrophic reservoirs, in terms of topological properties of connectedness, average degree, robustness and vulnerability. Our results further demonstrated that the seasonal stability of PAB co-occurrence relationships was strongly correlative with TSI through mediating key bacterial taxa and community biodiversity. We proposed that eutrophication dramatically reduced the seasonal variation of PAB community compositions and co-occurring relationships in reservoir ecosystems.

Microbial dysbiosis is hypothesized to cause larval mass mortalities in New-Caledonian shrimp hatcheries. In order to confirm this hypothesis and allow further microbial comparisons, we studied the active prokaryotic communities of healthy Penaeus stylirostris larvae and their surrounding environment during the first 10 days of larval rearing. Using daily nutrient concentration quantitative analyses and spectrophotometric organic matter analyses, we highlighted a global eutrophication of the rearing environment. We also evidenced drastic bacterial community modifications in the water and the larvae samples using Illumina HiSeq sequencing of the V4 region of the 16S rRNA gene. We confirmed that Alteromonadales, Rhodobacterales, Flavobacteriales, Oceanospirillales and Vibrionales members formed the core bacteriota of shrimp larvae. We also identified, in the water and the larvae samples, several potential probiotic bacterial strains which could lead to rethink probiotic use in aquaculture (AEGEAN 169 marine group, OM27 clade, Ruegeria, Leisingera, Pseudoalteromonas and Roseobacter). Finally, investigating the existing correlations between the environmental factors and the major bacterial taxa of the water and the larvae samples, we suggested that deterministic and stochastic processes were involved in the assembly of prokaryotic communities during the larval rearing of P. stylirostris. Overall, our results showed that drastic changes mostly occurred during the zoea stages suggesting that this larval phase is crucial during shrimp larval development.

Nematodes are common in most terrestrial environments, where populations are often known to undergo cycles of boom and bust. Useful in such scenarios, nematodes present developmental programs of diapause, giving rise to stress-resistant larvae and enabling dispersal in search of new resources. Best studied in Caenorhabditis elegans, stress resistant dauer larvae emerge under adverse conditions, primarily starvation, and migrate to new niches where they can resume development and reproduce. C. elegans is a bacterivore but has been shown to harbor a persistent and characteristic gut microbiome. While much is known about the gut microbiome of reproducing C. elegans, what dauers harbor is yet unknown. This is of interest, as dauers are those that would enable transmission of microbes between nematode generations and geographical sites, maintaining continuity of host-microbe interactions. Using culture-dependent as well as sequencing-based approaches we examined the gut microbiomes of dauers emerging following population growth on ten different natural-like microbially diverse environments as well as on two defined communities of known gut commensals and found that dauers were largely devoid of gut bacteria. These results suggest that host gut-microbiome interactions in C. elegans are not continuous across successive generations and may reduce the likelihood of long-term worm-microbe coevolution.

While numerous environmental factors contribute to the spread of antibiotic resistance genes (ARGs), quantifying their relative contributions remains a fundamental challenge. Similarly, it is important to differentiate acute human health risks from environmental exposure, versus broader ecological risk of ARG evolution and spread across microbial taxa. Recent studies have proposed various methods for achieving such aims. Here, we introduce MetaCompare 2.0, which improves upon original MetaCompare pipeline by differentiating indicators of human health resistome risk (HHRR, potential for human pathogens of acute resistance concern to acquire ARGs) from ecological resistome risk (ERR, overall mobility of ARGs and potential for pathogen acquisition). The updated pipeline's sensitivity was demonstrated by analyzing diverse publicly-available metagenomes from wastewater, surface water, soil, sediment, human gut, and synthetic microbial communities. MetaCompare 2.0 provided distinct rankings of the metagenomes according to both HHRR and ERR, with both scores trending higher when influenced by anthropogenic impact or other stress. We evaluated the robustness of the pipeline to sequence assembly methods, sequencing depth, contig count, and metagenomic library coverage bias. The risk scores were remarkably consistent despite variations in these technological aspects. We packaged the improved pipeline into a publicly-available web service (http://metacompare.cs.vt.edu/) that provides an easy-to-use interface for computing resistome risk scores and visualizing results.

Biodiversity, the source of origin, and ecological roles of fungi in groundwater are to this day a largely neglected field in fungal and freshwater ecology. We used DNA-based Illumina high-throughput sequence analysis of both fungal gene markers 5.8S and internal transcribed spacers region 2 (ITS2), improving taxonomic classification. This study focused on the groundwater and river mycobiome along an altitudinal and longitudinal transect of a pre-alpine valley in Austria in two seasons. Using Bayesian network modeling approaches, we identified patterns in fungal community assemblages that were mostly shaped by differences in landscape (climatic, topological, and geological) and environmental conditions. While river fungi were comparatively more diverse, unique fungal assemblages could be recovered from groundwater, including typical aquatic lineages such as Rozellomycota and Olpidiomycota. The most specious assemblages in groundwater were not linked to the input of organic material from the surface, and as such, seem to be sustained by characteristic groundwater conditions. Based on what is known from closely related fungi, our results suggest that the present fungal communities potentially contribute to mineral weathering, carbon cycling, and denitrification in groundwater. Furthermore, we were able to observe the effects of varying land cover due to agricultural practices on fungal biodiversity in groundwater ecosystems. This study contributes to improving our understanding of fungi in the subsurface aquatic biogeosphere.

The bacteria of a host's digestive tract play crucial roles in digestion and pathogen resistance. Hosts living in captivity often have more human interaction and antibiotic use, in addition to differences in diet and environment, compared to their wild counterparts. Consequently, wild and captive animals frequently harbour different bacterial communities. We tested whether diversity of diet provided in captivity shifts the gut bacteria of tuatara, an endemic New Zealand reptile, at three captive sites, and examined how the gut community of these tuatara compares to those in the wild. Dietary manipulation did not cause a strong overall shift in tuatara gut bacteria, but individual tuatara did experience bacterial shifts during manipulation, which subsequently reverted after manipulation. We found that Bacteroides, a genus common in most vertebrate guts but rare in tuatara, increased significantly in the gut during manipulation, then decreased post-manipulation. Finally, the gut bacteria of captive tuatara significantly differed from those of wild tuatara, though most of the dominant bacterial genera found in wild tuatara persisted in captive tuatara. This work represents a first investigation of the captive tuatara bacterial community and establishes the sensitivity of the gut community to dietary manipulation and captivity for this relict reptile.

The role of the skin microbiome in resistance and susceptibility of wildlife to fungal pathogens has been examined from a taxonomic perspective but skin microbial function, in the context of fungal infection, has yet to be studied. Our objective was to understand effects of a bat fungal pathogen site infection status and course of invasion on skin microbial function. We sampled seven hibernating colonies of Myotis lucifugus covering three-time points over the course of Pseudogymnoascus destructans (Pd) invasion and white nose syndrome (pre-invasion, epidemic, and established). Our results support three new hypotheses about Pd and skin functional microbiome: (1) there is an important effect of Pd invasion stage, especially at the epidemic stage; (2) disruption by the fungus at the epidemic stage could decrease anti-fungal functions with potential negative effects on the microbiome and bat health; (3) the collection site might have a larger influence on microbiomes at the pre-invasion stage rather than at epidemic and established stages. Future studies with larger sample sizes and using meta-omics approaches will help confirm these hypotheses, and determine the influence of the microbiome on wildlife survival to fungal disease.

Over the next few years, it is planned to convert all or part of the underground gas storage (UGS) facilities used for natural gas (salt caverns, depleted hydrocarbon reservoirs, and deep aquifers) into underground dihydrogen (H2) storage reservoirs. These deep environments host microbial communities, some of which are hydrogenotrophic (sulfate reducers, acetogens, and methanogens). The current state of microbiological knowledge is thus presented for the three types of UGS facilities. In the mid-1990s, the concept of anaerobic subsurface lithoautotrophic microbial ecosystems, or SLiMEs, emerged. It is expected that the large-scale injection of H2 into subsurface environments will generate new microbial ecosystems called artificial SLiMEs, which could persist over time. These artificial SLiMEs could lead to H2 loss, an intense methanogenic activity, a degradation of gas quality and a risk to installations through sulfide production. However, recent studies on salt caverns and deep aquifers suggest that hydrogenotrophic microbial activity also leads to alkalinization (up to pH 10), which can constrain hydrogenotrophy. Therefore, studying and understanding these artificial SLiMEs is both a necessity for the development of the H2 industry and presents an opportunity for ecologists to monitor the evolution of deep environments in real time.

The strength of the microbial biogeographic patterns decreased along the increasing gradient of habitat specificity (from sediment to gut tissue) provided by a benthic sea urchin in the Southern Ocean.

Along the river-sea continuum, microorganisms are directionally dispersed by water flow while being exposed to strong environmental gradients. To compare the two assembly mechanisms that may strongly and differently influence metacommunity dynamics, namely homogenizing dispersal and heterogeneous selection, we characterized the total (16S rRNA gene) and putatively active (16S rRNA transcript) bacterial communities in the Pearl River-South China Sea Continuum, during the wet (summer) and dry (winter) seasons using high-throughput sequencing. Moreover, well-defined sampling was conducted by including freshwater, oligohaline, mesohaline, polyhaline, and marine habitats. We found that heterogeneous selection exceeded homogenizing dispersal in both the total and active fractions of bacterial communities in two seasons. However, homogeneous selection was prevalent (the dominant except in active bacterial communities during summer), which was primarily due to the bacterial communities' tremendous diversity (associated with high rarity) and our specific sampling design. In either summer or winter seasons, homogeneous and heterogeneous selection showed higher relative importance in total and active communities, respectively, implying that the active bacteria were more responsive to environmental gradients than were the total bacteria. In summary, our findings provide insight into the assembly of bacterial communities in natural ecosystems with high spatial connectivity and environmental heterogeneity.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup-  Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1h and group 3b are not.

Injecting H in deep underground to store this energy carrier will produce artificial subsurface lithoautotrophic microbial ecosystems that modify the taxonomic diversity of indigenous microbial communities and their metabolic activities.