Natural Microbial Assemblages Research Articles

Extracted Spectral Signatures from the Water Column as a Tool for the Prediction of the Structure of a Marine Microbial Community

In this communication, we present an innovative approach leveraging advanced Machine Learning (ML) and Artificial Intelligence (AI) techniques, specifically the Non-Negative Matrix Factorization (NMF) method, to analyze downward and upward light spectra collected by Hyperspectral Ocean Color Radiometer (HyperOCR, HOCR) sensors in the water column. Our work focuses on the development of a robust and efficient tool for unraveling the structure and activities of natural microbial assemblages in the ocean. By applying the NMF method to HyperOCR data, we successfully extracted five spectral signatures, representing unique patterns in the data. These signatures were instrumental in predicting the abundances of various microbial components, including bacteria, heterotrophic nanoflagellates, and picoeukaryotes, showcasing the potential of ML and AI in advancing oceanographic studies. To validate these methods, the study area included a shallow coastal area under the influence of freshwater inflow and an open offshore area with a depth of 100 m. The study sites in coastal and offshore waters (Kaštela Bay and Stončica Vis, respectively) had significantly different hydrographic and microbiological characteristics. Kaštela Bay had lower temperatures and salinity than the site on Vis. We have demonstrated prediction of the structure of the microbial community through application of different AI and ML methods with specific HOCR sensors.

Transitivity and intransitivity in soil bacterial networks.

Competition can lead to the exclusion of bacterial taxa when there is a transitive relationship among competitors with a hierarchy of competitive success. However, competition may not prevent bacterial coexistence if competitors form intransitive loops, in which none is able to outcompete all the rest. Both transitive and intransitive competition have been demonstrated in bacterial model systems. However, in natural soil microbial assemblages competition is typically understood as a dominance relationship leading to the exclusion of weak competitors. Here, we argue that transitive and intransitive interactions concurrently determine the structure of soil microbial communities. We explain why pairwise interactions cannot depict competition correctly in complex communities, and propose an alternative through the detection of strongly connected components (SCCs) in microbial networks. We finally analyse the existence of SCCs in soil bacterial communities in two Mediterranean ecosystems, for illustrative purposes only (rather than with the aim of providing a methodological tool) due to current limitations, and discuss future avenues to experimentally test the existence of SCCs in nature.

Direct Production of Bio-Recalcitrant Carboxyl-Rich Alicyclic Molecules Evidenced in a Bacterium-Induced Steroid Degradation Experiment.

Carboxyl-rich alicyclic molecules (CRAM) are highly unsaturated compounds extensively distributed throughout aquatic environments and sediments. This molecular group is widely referred to as a major proxy of recalcitrant organic materials, but its direct biosynthesis remains unclear. Steroids are a typical anthropogenic contaminant and have been previously suggested to be precursors of CRAM; however, experimental evidence to support this hypothesis is lacking. Here, a steroid-degrading bacterium, Comamonas testosteroni ATCC 11996, was incubated in a liquid medium supplemented with testosterone (a typical steroid) as the sole carbon source for 90 days. Testosterone-induced metabolites (TIM) were extracted for molecular characterization and to examine the bioavailability during an additional 90-day incubation after inoculation with a natural coastal microbial assemblage. The results showed that 1,775 molecular formulas (MFs) were assigned to TIM using ultrahigh-resolution mass spectrometry, with 66.99% categorized as CRAM-like constituents. A large fraction of TIM was respired or transformed during the additional 90-day seawater incubation; nevertheless, 638 MFs of the TIM persisted or increased during incubation. Among the 638 MFs, 394 were commonly assigned in natural deep seawater samples (depths of 500 to 2,000 m) from the South China Sea. Compared to the catabolites of the well-established testosterone degradation pathway, we compiled a list of bio-refractory MFs and potential chemical structures, some of which shared structural homology with CRAM. These results demonstrated direct microbial production of bio-refractory CRAM from steroid hormones and indicated that some of the biogenic CRAM resisted microbial decomposition, potentially contributing to the aquatic refractory dissolved organic matter (DOM) pool. IMPORTANCE CRAM are an operationally defined DOM group comprising a complex mixture of carboxylated and fused alicyclic structures. This DOM group is majorly characterized as refractory DOM in the marine environment. However, the origins of the complex CRAM remain unclear. In this study, we demonstrated that testosterone (a typical steroid) could be transformed into bio-refractory CRAM by a single bacterial strain and observed that some of the CRAM highly resisted microbial degradation. Through molecular comparison and screening, potential chemical structures of steroid-induced CRAM were suggested. This study established the biological connection between steroids and bio-refractory CRAM, and it provides a novel perspective explaining the fate of terrestrial contaminants in aquatic environments.

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Microbial transformation of distinct exogenous substrates into analogous composition of recalcitrant dissolved organic matter.

Oceanic dissolved organic matter (DOM) comprises a complex molecular mixture which is typically refractory and homogenous in the deep layers of the ocean. Though the refractory nature of deep-sea DOM is increasingly attributed to microbial metabolism, it remains unexplored whether ubiquitous microbial metabolism of distinct carbon substrates could lead to similar molecular composition of refractory DOM. Here, we conducted microbial incubation experiments using four typically bioavailable substrates (L-alanine, trehalose, sediment DOM extract, and diatom lysate) to investigate how exogenous substrates are transformed by a natural microbial assemblage. The results showed that although each-substrate-amendment induced different changes in the initial microbial assemblage and the amended substrates were almost depleted after 90 days of dark incubation, the bacterial community compositions became similar in all incubations on day 90. Correspondingly, revealed by ultra-high resolution mass spectrometry, molecular composition of DOM in all incubations became compositionally consistent with recalcitrant DOM and similar toward that of DOM from the deep-sea. These results indicate that while the composition of natural microbial communities can shift with substrate exposures, long-term microbial transformation of distinct substrates can ultimately lead to a similar refractory DOM composition. These findings provide an explanation for the homogeneous and refractory features of deep-sea DOM.

Ecosystem Capacity for Microbial Biodegradation of Munitions Compounds and Phenanthrene in Three Coastal Waterways in North Carolina, United States.

Munitions compounds (i.e., 2,4,6-trinitrotoluene (TNT), octahy-dro-1,3,5,7-tetranitro-1,3,5,7-tetrazocin (HMX), and hexadydro-1,3,5-trinitro-1,3,5-triazin (RDX), also called energetics) were originally believed to be recalcitrant to microbial biodegradation based on historical groundwater chemical attenuation data and laboratory culture work. More recently, it has been established that natural bacterial assemblages in coastal waters and sediment can rapidly metabolize these organic nitrogen sources and even incorporate their carbon and nitrogen into bacterial biomass. Here, we report on the capacity of natural microbial assemblages in three coastal North Carolina (United States) estuaries to metabolize energetics and phenanthrene (PHE), a proxy for terrestrial aromatic compounds. Microbial assemblages generally had the highest ecosystem capacity (mass of the compound mineralized per average estuarine residence time) for HMX (21–5463 kg) > RDX (1.4–5821 kg) ≫ PHE (0.29–660 kg) > TNT (0.25–451 kg). Increasing antecedent precipitation tended to decrease the ecosystem capacity to mineralize TNT in the Newport River Estuary, and PHE and TNT mineralization were often highest with increasing salinity. There was some evidence from the New River Estuary that increased N-demand (due to a phytoplankton bloom) is associated with increased energetic mineralization rates. Using this type of analysis to determine the ecosystem capacity to metabolize energetics can explain why these compounds are rarely detected in seawater and marine sediment, despite the known presence of unexploded ordnance or recent use in military training exercises. Overall, measuring the ecosystem capacity may help predict the effects of climate change (warming and altered precipitation patterns) and other perturbations on exotic compound fate and transport within ecosystems and provide critical information for managers and decision-makers to develop management strategies based on these changes.

The bioavailability of riverine dissolved organic matter in coastal marine waters of southern Texas

To examine the bioavailability of dissolved organic carbon (DOC) and nitrogen (DON) in riverine dissolved organic matter (DOM) discharged to the coastal ocean, we conducted a series of month-long (24 days) incubation experiments with filtered samples collected from five southern Texas rivers (Lavaca, San Antonio, Mission, Aransas, and Nueces) inoculated using the same natural coastal microbial assemblages during summer (June) and winter (January) in 2016. The bioavailable fractions of DOC and DON (BDOC% and BDON%) varied substantially in different rivers and seasons, ranging respectively from 6–11% and 15–38% during winter, and 0–6% and 9–15% during summer. Relatively higher BDOC% and BDON% occurred in the San Antonio and Aransas Rivers, which are impacted more by human activities through discharge from wastewater treatment plants. Seasonally, the riverine DOM was more bioavailable in winter than in summer when DOM may have been extensively degraded in situ due to the low base flow (or long residence time) and the elevated temperature in river water in summer. The principal component analysis on amino acid composition further confirmed that DOM was less degraded in winter than in summer. Functional gene abundance data revealed that winter riverine DOM was relatively labile as evidenced by an increase in N-metabolism pathways and functional genes during the winter incubation, whereas the opposite pattern was observed in summer. The findings of the varying bioavailability of DOM among rivers and seasons have important implications about the fate of riverine DOM and their potential contributions to nutrient supplies as southern Texas bays and estuaries are often nitrogen limited.

AFISsys - An autonomous instrument for the preservation of brackish water samples for microbial metatranscriptome analysis

Microbial communities are the main drivers of biogeochemical cycling of multiple elements sustaining life in the ocean. The rapidity of their response to stressors and abrupt environmental changes implies that even fast and infrequent events can affect local transformations of organic matter and nutrients. Modern molecular techniques now allow for monitoring of microbial activities and functions in the environment through the analysis of genes and expressed genes contained in natural microbial assemblages. However, messenger RNA turnover in cells can be as short as 30 seconds and stability varies greatly between transcripts. Sampling of in situ communities involves an inevitable delay between the collection of seawater and the extraction of its RNA, leaving the bacterial communities plenty of time to alter their gene expression. The characteristics of microbial RNA turnover make time-series very difficult because samples need to be processed immediately to limit alterations to the metatranscriptomes.To address these challenges we designed an autonomous in situ fixation multi-sampler (AFISsys) for the reliable sampling of microbial metatranscriptomes at frequent intervals, for refined temporal resolution. To advance the development of this instrument, we examined the minimal seawater volume necessary for adequate coverage of community gene expression, and the suitability of phenol/ethanol fixation for immediate and long-term preservation of transcripts from a microbial community. We then evaluated the field eligibility of the instrument itself, with two case studies in a brackish system. AFISsys is able to collect, fix, and store water samples independently at a predefined temporal resolution. Phenol/ethanol fixation can conserve metatranscriptomes directly in the environment for up to a week, for later analysis in the laboratory. Thus, the AFISsys constitutes an invaluable tool for the integration of molecular functional analyses in environmental monitoring in brackish waters and in aquatic environments in general.

Specificity Re-evaluation of Oligonucleotide Probes for the Detection of Marine Picoplankton by Tyramide Signal Amplification-Fluorescent In Situ Hybridization.

Oligonucleotide probes are increasingly being used to characterize natural microbial assemblages by Tyramide Signal Amplification-Fluorescent in situ Hybridization (TSA-FISH, or CAtalysed Reporter Deposition CARD-FISH). In view of the fast-growing rRNA databases, we re-evaluated the in silico specificity of eleven bacterial and eukaryotic probes and competitor frequently used for the quantification of marine picoplankton. We performed tests on cell cultures to decrease the risk for non-specific hybridization, before they are used on environmental samples. The probes were confronted to recent databases and hybridization conditions were tested against target strains matching perfectly with the probes, and against the closest non-target strains presenting one to four mismatches. We increased the hybridization stringency from 55 to 65% formamide for the Eub338+EubII+EubIII probe mix to be specific to the Eubacteria domain. In addition, we found that recent changes in the Gammaproteobacteria classification decreased the specificity of Gam42a probe, and that the Roseo536R and Ros537 probes were not specific to, and missed part of the Roseobacter clade. Changes in stringency conditions were important for bacterial probes; these induced, respectively, a significant increase, in Eubacteria and Roseobacter and no significant changes in Gammaproteobacteria concentrations from the investigated natural environment. We confirmed the eukaryotic probes original conditions, and propose the Euk1209+NChlo01+Chlo02 probe mix to target the largest picoeukaryotic diversity. Experiences acquired through these investigations leads us to propose the use of seven steps protocol for complete FISH probe specificity check-up to improve data quality in environmental studies.

Hydrogen Limitation and Syntrophic Growth among Natural Assemblages of Thermophilic Methanogens at Deep-sea Hydrothermal Vents.

Thermophilic methanogens are common autotrophs at hydrothermal vents, but their growth constraints and dependence on H2 syntrophy in situ are poorly understood. Between 2012 and 2015, methanogens and H2-producing heterotrophs were detected by growth at 80°C and 55°C at most diffuse (7–40°C) hydrothermal vent sites at Axial Seamount. Microcosm incubations of diffuse hydrothermal fluids at 80°C and 55°C demonstrated that growth of thermophilic and hyperthermophilic methanogens is primarily limited by H2 availability. Amendment of microcosms with NH4+ generally had no effect on CH4 production. However, annual variations in abundance and CH4 production were observed in relation to the eruption cycle of the seamount. Microcosm incubations of hydrothermal fluids at 80°C and 55°C supplemented with tryptone and no added H2 showed CH4 production indicating the capacity in situ for methanogenic H2 syntrophy. 16S rRNA genes were found in 80°C microcosms from H2-producing archaea and H2-consuming methanogens, but not for any bacteria. In 55°C microcosms, sequences were found from H2-producing bacteria and H2-consuming methanogens and sulfate-reducing bacteria. A co-culture of representative organisms showed that Thermococcus paralvinellae supported the syntrophic growth of Methanocaldococcus bathoardescens at 82°C and Methanothermococcus sp. strain BW11 at 60°C. The results demonstrate that modeling of subseafloor methanogenesis should focus primarily on H2 availability and temperature, and that thermophilic H2 syntrophy can support methanogenesis within natural microbial assemblages and may be an important energy source for thermophilic autotrophs in marine geothermal environments.

Editorial: Anthropogenic Impacts on the Microbial Ecology and Function of Aquatic Environments.

Editorial: Anthropogenic Impacts on the Microbial Ecology and Function of Aquatic Environments.

Eutrophication and acidification: Do they induce changes in the dissolved organic matter dynamics in the coastal Mediterranean Sea?

Two mesocosms experiments were conducted in winter 2010 and summer 2011 to examine how increased pCO2 and/or nutrient concentrations potentially perturbate dissolved organic matter dynamics in natural microbial assemblages. The fluorescence signals of protein- and humic-like compounds were used as a proxy for labile and non-labile material, respectively, while the evolution of bacterial populations, chlorophyll a (Chl a) and dissolved organic carbon (DOC) concentrations were used as a proxy for biological activity. For both seasons, the presence of elevated pCO2 did not cause any significant change in the DOC dynamics (p-value<0.05). The conditions that showed the greatest changes in prokaryote abundances and Chl a content were those amended with nutrients, regardless of the change in pH. The temporal evolution of fluorophores and optical indices revealed that the degree of humification of the organic molecules and their molecular weight changed significantly in the nutrient-amended treatment. The generation of protein-like compounds was paired to increases in the prokaryote abundance, being higher in the nutrient-amended tanks than in the control. Different patterns in the magnitude and direction of the generation of humic-like molecules suggested that these changes depended on initial microbial populations and the availability of extra nutrient inputs. Based on our results, it is expected that in the future projected coastal scenarios the eutrophication processes will favor the transformations of labile and recalcitrant carbon regardless of changes in pCO2.

Predicting taxonomic and functional structure of microbial communities in acid mine drainage.

Predicting the dynamics of community composition and functional attributes responding to environmental changes is an essential goal in community ecology but remains a major challenge, particularly in microbial ecology. Here, by targeting a model system with low species richness, we explore the spatial distribution of taxonomic and functional structure of 40 acid mine drainage (AMD) microbial communities across Southeast China profiled by 16S ribosomal RNA pyrosequencing and a comprehensive microarray (GeoChip). Similar environmentally dependent patterns of dominant microbial lineages and key functional genes were observed regardless of the large-scale geographical isolation. Functional and phylogenetic β-diversities were significantly correlated, whereas functional metabolic potentials were strongly influenced by environmental conditions and community taxonomic structure. Using advanced modeling approaches based on artificial neural networks, we successfully predicted the taxonomic and functional dynamics with significantly higher prediction accuracies of metabolic potentials (average Bray–Curtis similarity 87.8) as compared with relative microbial abundances (similarity 66.8), implying that natural AMD microbial assemblages may be better predicted at the functional genes level rather than at taxonomic level. Furthermore, relative metabolic potentials of genes involved in many key ecological functions (for example, nitrogen and phosphate utilization, metals resistance and stress response) were extrapolated to increase under more acidic and metal-rich conditions, indicating a critical strategy of stress adaptation in these extraordinary communities. Collectively, our findings indicate that natural selection rather than geographic distance has a more crucial role in shaping the taxonomic and functional patterns of AMD microbial community that readily predicted by modeling methods and suggest that the model-based approach is essential to better understand natural acidophilic microbial communities.

Protistan diversity and activity inferred from RNA and DNA at a coastal ocean site in the eastern North Pacific.

Microbial eukaryotes fulfill key ecological positions in marine food webs. Molecular approaches that connect protistan diversity and biogeography to their diverse metabolisms will greatly improve our understanding of marine ecosystem function. The majority of molecular-based studies to date use 18S rRNA gene sequencing to characterize natural microbial assemblages, but this approach does not necessarily discriminate between active and non-active cells. We incorporated RNA sequencing into standard 18S rRNA gene sequence surveys with the purpose of assessing those members of the protistan community contributing to biogeochemical cycling (active organisms), using the ratio of cDNA (reverse transcribed from total RNA) to 18S rRNA gene sequences within major protistan taxonomic groups. Trophically important phytoplankton, such as diatoms and chlorophytes exhibited seasonal trends in relative activity. Additionally, both radiolaria and ciliates displayed previously unreported high relative activities below the euphotic zone. This study sheds new light on the relative metabolic activity of specific protistan groups and how microbial communities respond to changing environmental conditions.

Bacterial use of choline to tolerate salinity shifts in sea-ice brines

Abstract Bacteria within the brine network of sea ice experience temperature-driven fluctuations in salinity on both short and long temporal scales, yet their means of osmoprotection against such fluctuations is poorly understood. One mechanism used to withstand the ion fluxes caused by salinity shifts, well-known in mesophilic bacteria, is the import and export of low molecular weight organic solutes that are compatible with intracellular functions. Working with the marine psychrophilic gammaproteobacterium, Colwellia psychrerythraea 34H, and with natural microbial assemblages present in sackhole brines drained from sea ice in Kanajorsuit Bay (2013) and Kobbefjord (2014), Greenland, we measured the utilization of 14C-choline (precursor to glycine betaine, a common compatible solute) at −1°C upon salinity shifts to double and to half the starting salinity. In all cases and across a range of starting salinities, when salinity was increased, 14C-solute (choline or derivatives) was preferentially retained as an intracellular osmolyte; when salinity was decreased, 14C-choline was preferentially respired to 14CO2. Additional experiments with cold-adapted bacteria in culture indicated that an abrupt downshift in salinity prompted rapid (subsecond) expulsion of retained 14C-solute, but that uptake of 14C-choline and solute retention resumed when salinity was returned to starting value. Overall, the results indicate that bacteria in sea-ice brines use compatible solutes for osmoprotection, transporting, storing and cycling these molecules as needed to withstand naturally occurring salinity shifts and persist through the seasons. Because choline and many commonly used compatible solutes contain nitrogen, we suggest that when brines freshen and bacteria respire such compatible solutes, the corresponding regeneration of ammonium may enhance specific biogeochemical processes in the ice, possibly algal productivity but particularly nitrification. Measurements of potential nitrification rates in parallel sea-ice samples are consistent with a link between use of the compatible solute strategy and nitrification.

Dynamics of an experimental microbial invasion

The ecological dynamics underlying species invasions have been a major focus of research in macroorganisms for the last five decades. However, we still know little about the processes behind invasion by unicellular organisms. To expand our knowledge of microbial invasions, we studied the roles of propagule pressure, nutrient supply, and biotic resistance in the invasion success of a freshwater invasive alga, Prymnesium parvum, using microcosms containing natural freshwater microbial assemblages. Microcosms were subjected to a factorial design with two levels of nutrient-induced diversity and three levels of propagule pressure, and incubated for 7 d, during which P. parvum densities and microbial community composition were tracked. Successful invasion occurred in microcosms receiving high propagule pressure whereas nutrients or community diversity played no role in invasion success. Invaded communities experienced distinctive changes in composition compared with communities where the invasion was unsuccessful. Successfully invaded microbial communities had an increased abundance of fungi and ciliates, and decreased abundances of diatoms and cercozoans. Many of these changes mirrored the microbial community changes detected during a natural P. parvum bloom in the source system. This role of propagule pressure is particularly relevant for P. parvum in the reservoir-dominated southern United States because this species can form large, sustained blooms that can generate intense propagule pressures for downstream sites. Human impact and global climate change are currently causing widespread environmental changes in most southern US freshwater systems that may facilitate P. parvum establishment and, when coupled with strong propagule pressure, could put many more systems at risk for invasion.

Substrate selection for heterotrophic bacterial growth in the sea

Growth of heterotrophic microbes requires the extraction of energy, electrons, carbon, and nutrients from a complex and dynamic reservoir of potential substrates. We employed a matrix of selected organic substrates with varying characteristics, and experimentally followed the kinetics of assimilation and respiration to explore the basic principles that govern selection and preferential use based on carbon, nitrogen, and energy content. We further competed these substrates in a combinatorial fashion to evaluate preferential substrate utilization in natural microbial assemblages. Several substrates displayed biphasic kinetic responses and variable respiration: assimilation ratios. Amino acids had the shortest turnover times and were taken up preferentially at ambient concentrations. We also identified a linear relationship between substrate uptake rates and affinity, suggesting that the microbial community optimizes the relative abundances of membrane transporters according to substrate demand. When competed against one another at saturating concentrations, substrate assimilation and respiration rates were enhanced or inhibited by up to two orders of magnitude, compared to competitor-free controls. Further, we describe an unexpected trend between the substrate energy density and turnover times, with more energetic, reduced carbon substrates turning over more slowly than more oxidized substrates.

Dose-dependent regulation of microbial activity on sinking particles by polyunsaturated aldehydes: Implications for the carbon cycle

Diatoms and other phytoplankton play a crucial role in the global carbon cycle, fixing CO2 into organic carbon, which may then be exported to depth via sinking particles. The molecular diversity of this organic carbon is vast and many highly bioactive molecules have been identified. Polyunsaturated aldehydes (PUAs) are bioactive on various levels of the marine food web, and yet the potential for these molecules to affect the fate of organic carbon produced by diatoms remains an open question. In this study, the effects of PUAs on the natural microbial assemblages associated with sinking particles were investigated. Sinking particles were collected from 150 m in the water column and exposed to varying concentrations of PUAs in dark incubations over 24 h. PUA doses ranging from 1 to 10 µM stimulated respiration, organic matter hydrolysis, and cell growth by bacteria associated with sinking particles. PUA dosages near 100 µM appeared to be toxic, resulting in decreased bacterial cell abundance and metabolism, as well as pronounced shifts in bacterial community composition. Sinking particles were hot spots for PUA production that contained concentrations within the stimulatory micromolar range in contrast to previously reported picomolar concentrations of these compounds in bulk seawater. This suggests PUAs produced in situ stimulate the remineralization of phytoplankton-derived sinking organic matter, decreasing carbon export efficiency, and shoaling the average depths of nutrient regeneration. Our results are consistent with a "bioactivity hypothesis" for explaining variations in carbon export efficiency in the oceans.

Wiretapping into microbial interactions by single cell genomics.

Wiretapping into microbial interactions by single cell genomics.

Re-defining microbial diversity from its single-celled building blocks.

It would be an astonishing experience for an environmental microbiologist of 1985 to be teleported straight into 2015! Few things have remained the same in this dynamic research field, and our understanding of the abundance, diversity and activities of microorganisms on the planet Earth has been thoroughly revamped. Research technologies that were unimaginable in 1985 are producing data of unprecedented quantity and quality. But there may be a distressing moment as well: realizing that microbial community composition data collected with so much effort in 1985 is rarely looked at in 2015, even when trying to understand decadal and longer-term environmental changes. The reasons are deeper than science fads and difficulties accessing non-digitized records: most analyses of the composition of natural microbial assemblages of 1985 are considered incorrect and incompatible with the science of 2015. Now let us imagine that we can be teleported into 2045. Most likely we would see technological and conceptual advances exceeding those that took place between 1985 and 2015. Unfortunately, we would likely see Earth's environment continuing to undergo significant change, largely as a result of anthropogenic perturbations. Hopefully, we would also see human societies that are increasingly cognizant of their interdependence with the rest of Earth's biota, in particular with the predominant, microbial component of it. By 2045, microbiology may become one of the key areas of expertise required to understand, predict and mitigate global environmental changes. Historical records will be of key importance. Will suitable data exist? Will scientists of 2045 look back at the results of microbial community composition in oceans, soils and other environments collected in 2015 to draw meaningful conclusions about ongoing alterations? Chances are high that the answer to the question above will be ‘no’. Although today's studies of microbial diversity are no longer prone to cultivation limitations of 1985, they are still predominantly framed around operational, technique-based definitions rather than a deep understanding of the underlying biology. As research techniques change, operational definitions change, too. Therefore, many microbial species, genera and even broader taxonomic units of 1985 have no relevance today (Garrity and Lyons, 2003), while many of today's operational taxonomic units and associated data may lose relevance in a few years or decades from now despite the enormous (at least by 2015 standards) datasets produced around them. We can say that this is part of science progress. However, I cannot stop thinking that there must be a smarter way to go about it. How come we confidently recognize many animals and plants described in millennia-old texts using long-gone languages, while some microbial taxonomic units turn unintelligible in a matter of decades? There are good reasons to believe that the upcoming decade will place microbial diversity research on more solid footing. The recent emergence of single-cell genomics (SCG) will likely play a critical role (Lasken, 2012; Stepanauskas, 2012). Already, SCG offers routine recovery of genomic blueprints of microbial groups that used to be inaccessible because of the absence of their pure cultures (Marcy et al., 2007; Rinke et al., 2013; Swan et al., 2013). This may lead to a long-overdue reconciliation of the robust biological nomenclature of cultivated taxa (Garrity and Lyons, 2003) with the chaotic world of naming conventions for the remaining 99+% of microorganisms. However, the potential of SCG goes beyond descriptive analyses of uncultivated lineages. By focusing on the most fundamental units of biological organization – individual cells – SCG breaks free from the necessity to bin microbial community molecular data into arbitrary taxonomic units. Furthermore, unlike other cultivation-independent methods, SCG is well-suited to analyze genomic rearrangements, horizontal gene transfer and the organization of hereditary information in multiple DNA molecules in a cell, all of which likely play key roles in the non-Darwinian evolution of microorganisms and their rapid adaptation to a changing environment (Ochman et al., 2000; Shapiro, 2010). This is starting to offer a fresh, assumptions-free view of the microbial genomic diversity and underlying ecological and evolutionary processes at unprecedented detail (Kashtan et al., 2014; Engel et al., 2014). As SCG and complementary techniques grow in scale, biological patterns may emerge to inspire natural rather than operational definitions of microbial diversity. These definitions may differ substantially from the current biological species concept, which was specifically designed for multicellular eukaryotes that constitute only a few, relatively young branches of the tree of life. We may find out that the evolutionarily much older and more diverse world of unicellular Bacteria, Archaea and Eukarya is organized and evolves in ways that defy our current, anthropocentric conventions. Such prospects justifiably make environmental microbiology a very dynamic and futuristic field that leads the way for improved understanding and stewardship of Earth's predominant, unicellular biota.