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In July 2016, a severe coral reef invertebrate mortality event occurred approximately 200km southeast of Galveston, Texas at the East Flower Garden Bank wherein upwards of 80% of corals in a 0.06 km2 area died. Based on surveys of dead corals and other invertebrates shortly after this mortality event, responders hypothesized that localized hypoxia was the most likely direct cause. However, no dissolved oxygen data were available to test this hypothesis because oxygen is not continuously monitored within the Flower Garden Banks sanctuary. Here we quantify microbial plankton community diversity based on four cruises over two years at the Flower Garden Banks, including a cruise just 5-8 days after the mortality event was first observed. In contrast with observations collected during baseline conditions, microbial plankton communities in the thermocline were differentially enriched with taxa known to be active and abundant in oxygen minimum zones or that have known adaptations to oxygen limitation shortly after the mortality event (e.g. SAR324, Thioglobaceae, Nitrosopelagicus, and Thermoplasmata MGII). Unexpectedly, these enrichments were not localized to the East Bank, but were instead prevalent across the entire study area, suggesting there was a widespread depletion of dissolved oxygen concentrations in the thermocline around the time of the mortality event. Hydrographic analysis revealed the southern East Bank coral reef (where the localized mortality event occurred) was uniquely within the thermocline at this time. Our results demonstrate how temporal monitoring of microbial communities can be a useful tool to address questions related to past environmental events.IMPORTANCEIn the northwestern Gulf of Mexico in July 2016, upwards of 80% of corals in a small area of the East Flower Garden Bank coral reef suddenly died without warning. Oxygen depletion is believed to have been the cause. However, there was considerable uncertainty as no oxygen data is available from the time of the event. Microbes are sensitive to changes in oxygen and can be used as bioindicators of oxygen loss. In this study, we analyze microbial communities in water samples collected over several years at the Flower Garden Banks, including shortly after the mortality event. Our findings indicate that compared to normal conditions, oxygen depletion was widespread in the deep-water layer during the mortality event. Hydrographic analysis of water masses further revealed some of this low oxygen water likely upwelled onto the coral reef.

Following oil spills in aquatic environments, oil-associated flocculants observed within contaminated waters ultimately lead to the sedimentation of oil as marine oil snow (MOS). To better understand the role of aggregates in hydrocarbon degradation and transport, we experimentally produced a MOS sedimentation event using Gulf of Mexico coastal waters amended with oil or oil plus dispersant. In addition to the formation of MOS, smaller micrometer-scale (10- to 150-μm) microbial aggregates were observed. Visual inspection of these microaggregates revealed that they were most abundant in the oil-amended treatments and frequently associated with oil droplets, linking their formation to the presence of oil. The peak abundance of the microaggregates coincided with the maximum rates of biological hydrocarbon oxidation estimated by the mineralization of 14C-labeled hexadecane and naphthalene. To elucidate the potential of microaggregates to serve as hot spots for hydrocarbon degradation, we characterized the free-living and aggregate-associated microbial assemblages using 16S rRNA gene sequencing. The microaggregate population was found to be bacterially dominated and enriched with putative hydrocarbon-degrading taxa. Direct observation of some of these taxa using catalyzed reporter deposition fluorescence in situ hybridization confirmed their greater abundance within microaggregates relative to the surrounding seawater. Metagenomic sequencing of these bacteria-oil microaggregates (BOMAs) further supported their community’s capacity to utilize a wide variety of hydrocarbon compounds. Taken together, these data highlight that BOMAs are inherent features in the biological response to oil spills and likely important hot spots for hydrocarbon oxidation in the ocean. IMPORTANCE Vast quantities of oil-associated marine snow (MOS) formed in the water column as part of the natural biological response to the Deepwater Horizon drilling accident. Despite the scale of the event, uncertainty remains about the mechanisms controlling MOS formation and its impact on the environment. In addition to MOS, we observed micrometer-scale (10- to 150-μm) aggregates whose abundance coincided with maximum rates of hydrocarbon degradation and whose composition was dominated by hydrocarbon-degrading bacteria with the genetic potential to metabolize a range of these compounds. This targeted study examining the role of these bacteria-oil microaggregates in hydrocarbon degradation reveals details of this fundamental component of the biological response to oil spills, and with it, alterations to biogeochemical cycling in the ocean.

International Ocean Discovery Program Expedition 360 drilled Hole U1473A at Atlantis Bank, an oceanic core complex on the Southwest Indian Ridge, with the aim of recovering representative samples of the lower oceanic crust. Recovered cores were primarily gabbro and olivine gabbro. These mineralogies may host serpentinization reactions that have the potential to support microbial life within the recovered rocks or at greater depths beneath Atlantis Bank. We quantified prokaryotic cells and analyzed microbial community composition for rock samples obtained from Hole U1473A and conducted nutrient addition experiments to assess if nutrient supply influences the composition of microbial communities. Microbial abundance was low (≤10(4) cells cm(−3)) but positively correlated with the presence of veins in rocks within some depth ranges. Due to the heterogeneous nature of the rocks downhole (alternating stretches of relatively unaltered gabbros and more significantly altered and fractured rocks), the strength of the positive correlations between rock characteristics and microbial abundances was weaker when all depths were considered. Microbial community diversity varied at each depth analyzed. Surprisingly, addition of simple organic acids, ammonium, phosphate, or ammonium plus phosphate in nutrient addition experiments did not affect microbial diversity or methane production in nutrient addition incubation cultures over 60 weeks. The work presented here from Site U1473A, which is representative of basement rock samples at ultraslow spreading ridges and the usually inaccessible lower oceanic crust, increases our understanding of microbial life present in this rarely studied environment and provides an analog for basement below ocean world systems such as Enceladus. IMPORTANCE The lower oceanic crust below the seafloor is one of the most poorly explored habitats on Earth. The rocks from the Southwest Indian Ridge (SWIR) are similar to rock environments on other ocean-bearing planets and moons. Studying this environment helps us increase our understanding of life in other subsurface rocky environments in our solar system that we do not yet have the capability to access. During an expedition to the SWIR, we drilled 780 m into lower oceanic crust and collected over 50 rock samples to count the number of resident microbes and determine who they are. We also selected some of these rocks for an experiment where we provided them with different nutrients to explore energy and carbon sources preferred for growth. We found that the number of resident microbes and community structure varied with depth. Additionally, added nutrients did not shape the microbial diversity in a predictable manner.

Marine microbial communities play an important role in biodegradation of subsurface plumes of oil that form after oil is accidentally released from a seafloor wellhead. The response of these mesopelagic microbial communities to the application of chemical dispersants following oil spills remains a debated topic. While there is evidence that contrasting results in some previous work may be due to differences in dosage between studies, the impacts of these differences on mesopelagic microbial community composition remains unconstrained. To answer this open question, we exposed a mesopelagic microbial community from the Gulf of Mexico to oil alone, three concentrations of oil dispersed with Corexit 9500, and three concentrations of Corexit 9500 alone over long periods of time. We analyzed changes in hydrocarbon chemistry, cell abundance, and microbial community composition at zero, three and six weeks. The lowest concentration of dispersed oil yielded hydrocarbon concentrations lower than oil alone and microbial community composition more similar to control seawater than any other treatments with oil or dispersant. Higher concentrations of dispersed oil resulted in higher concentrations of microbe-oil microaggregates and similar microbial composition to the oil alone treatment. The genus Colwellia was more abundant when exposed to multiple concentrations of dispersed oil, but not when exposed to dispersant alone. Conversely, the most abundant Marinobacter amplicon sequence variant (ASV) was not influenced by dispersant when oil was present and showed an inverse relationship to the summed abundance of Alcanivorax ASVs. As a whole, the data presented here show that the concentration of oil strongly impacts microbial community response, more so than the presence of dispersant, confirming the importance of the concentrations of both oil and dispersant in considering the design and interpretation of results for oil spill simulation experiments.

On July 25, 2016, turbid water and dead corals, sponges and other invertebrates were discovered at the East Bank (EB) of the Flower Garden Banks (FGB) National Marine Sanctuary. Mortality was spread over 0.06 km2, with up to 80% coral mortality reported in some areas. Within days, response efforts were underway to investigate the potential mechanisms leading to the mortality event. Hydrographic surveys, moored buoy data, and a regional hydrodynamic model were used to characterize water chemistry, hydrography, and microbial communities within the FGB. Low salinity (~ 31–33), total alkalinity (~ 2284–2330 µmol kg−1), and dissolved inorganic carbon (DIC, ~ 1968–2011 µmol kg−1) were detected in surface waters over the EB and eastern stations, revealing the presence of river-derived water. The Mississippi/Atchafalaya rivers were the primary sources of freshwater during the event, although Texas rivers, all of which had unusually high discharge during 2016, contributed approximately one-fifth to the total freshwater mass. At 75 m depth, high density, salinity, DIC, ammonium, and abundance of microbial taxa associated with deep waters were coincident with low temperature and aragonite saturation state at the northern and eastern stations, indicating a deeper source water at these stations. Cross-slope density gradients were also consistent with an upwelling circulation pattern. Using these observations and data, we hypothesize that the mortality event was most likely caused by the combination of two processes. The turbid freshwater layer inhibited photosynthesis, leading to net respiration of coral reef organic matter. Additionally, deep, dense waters upwelled onto the bank and formed a stratified bottom layer, which prevented re-oxygenation from the overlying water column and led to localized areas of hypoxia within pockets on the reef. Hypoxia likely formed rapidly, within two days. Moving forward, high-frequency temporal measurements of oxygen and carbonate chemistry are critical for monitoring risks (e.g., hypoxia and acidification) associated with freshwater discharge and upwelling, since these processes may adversely affect coral reef health.

Diatoms secrete a significant amount of polysaccharides, which can serve as a critical organic carbon source for bacteria. The 2010 Deepwater Horizon oil spill exposed the Gulf of Mexico to substantial amounts of oil that also impacted the phytoplankton community. Increased production of exopolymeric substances was observed after this oil spill. Polysaccharides make up a major fraction of exopolymeric substances; however, their physiological role during an oil spill remains poorly understood. Here, we analyzed the role of polysaccharides in the growth and physiology of the oil-sensitive diatom Thalassiosira pseudonana and how they shape the surrounding bacterial community and its activity in the presence of oil. We found that inhibition of chrysolaminarin synthesis had a negative effect on the growth of T. pseudonana and intracellular monosaccharide accumulation, which in turn suppressed photosynthesis by feedback inhibition. In addition, by acting as a carbon reserve, chrysolaminarin helped in the recovery of T. pseudonana in the presence of oil. Inhibition of chrysolaminarin synthesis also influenced the bacterial community in the free-living fraction but not in the phycosphere. Exposure to oil alone led to increased abundance of oil-degrading bacterial genera and the activity of exoenzyme lipase. Our data show that chrysolaminarin synthesis plays an important role in the growth and survival of T. pseudonana in the presence of oil, and its inhibition can influence the composition and activity of the surrounding bacterial community.

Terrestrial runoff can negatively impact marine ecosystems through stressors including excess nutrients, freshwater, sediments, and contaminants. Severe storms, which are increasing with global climate change, generate massive inputs of runoff over short timescales (hours to days); such runoff impacted offshore reefs in the northwest Gulf of Mexico (NW GoM) following severe storms in 2016 and 2017. Several weeks after coastal flooding from these events, NW GoM reef corals, sponges, and other benthic invertebrates ∼185 km offshore experienced mortality (2016 only) and/or sub-lethal stress (both years). To assess the impact of storm-derived runoff on reef filter feeders, we characterized the bacterial communities of two sponges, Agelas clathrodes and Xestospongia muta, from offshore reefs during periods of sub-lethal stress and no stress over a three-year period (2016—2018). Sponge-associated and seawater-associated bacterial communities were altered during both flood years. Additionally, we found evidence of wastewater contamination (based on 16S rRNA gene libraries and quantitative PCR) in offshore sponge samples, but not in seawater samples, following these flood years. Signs of wastewater contamination were absent during the no-flood year. We show that flood events from severe storms have the capacity to reach offshore reef ecosystems and impact resident benthic organisms. Such impacts are most readily detected if baseline data on organismal physiology and associated microbiome composition are available. This highlights the need for molecular and microbial time series of benthic organisms in near- and offshore reef ecosystems, and the continued mitigation of stormwater runoff and climate change impacts.

Polymetallic sulfide (PMS) deposits produced at hydrothermal vents in the deep sea are of potential interest to miners. Hydrothermally active sulfide ecosystems are valued for the extraordinary chemosynthetic communities that they support. Many countries, including Canada, Portugal, and the United States, protect vent ecosystems in their Exclusive Economic Zones. When hydrothermal activity ceases temporarily (dormancy) or permanently (extinction), the habitat and associated ecosystem change dramatically. Until recently, so-called "inactive sulfide" habitats, either dormant or extinct, received little attention from biologists. However, the need for environmental management of deep-sea mining places new imperatives for building scientific understanding of the structure and function of inactive PMS deposits. This paper calls for actions of the scientific community and the emergent seabed mining industry to i) undertake fundamental ecological descriptions and study of ecosystem functions and services associated with hydrothermally inactive PMS deposits, ii) evaluate potential environmental risks to ecosystems of inactive PMS deposits through research, and iii) identify environmental management needs that may enable mining of inactive PMS deposits. Mining of some extinct PMS deposits may have reduced environmental risk compared to other seabed mining activities, but this must be validated through scientific research on a case-by-case basis. FCT: IF/00029/2014/CP1230/CT0002/ UID/05634/2020/ CEECIND005262017/ UID/MAR/00350/2019; Direcao-Geral de Politica do Mar (DGPM) Mining2/2017/005/ Mining2/2017/001 info:eu-repo/semantics/publishedVersion

In the wake of the Deepwater Horizon oil spill, the taxonomic response of marine microbial communities to oil and dispersants has been extensively studied. However, relatively few studies on the functional response of these microbial communities have been reported, especially in a longitudinal fashion. Moreover, despite the fact that marine oil spills typically impact thousands of square kilometers of both coastal and offshore marine environments, little information is available on how the microbial response to oil and dispersants might differ between these biomes. The results of this study help fill this critical knowledge gap and provide valuable insight into how oil spill response efforts, such as chemically dispersing oil, may have differing effects in neighboring coastal and offshore marine environments., Marine oil spills can impact both coastal and offshore marine environments, but little information is available on how the microbial response to oil and dispersants might differ between these biomes. Here, we describe the compositional and functional response of microbial communities to different concentrations of oil and chemically dispersed oil in coastal and offshore surface waters from the Texas-Louisiana continental shelf. Using a combination of analytical chemistry and 16S rRNA amplicon and metatranscriptomic sequencing, we provide a broad, comparative overview of the ecological response of hydrocarbon-degrading bacteria and their expression of hydrocarbon-degrading genes in marine surface waters over time between two oceanic biomes. We found evidence for the existence of different ecotypes of several commonly described hydrocarbon-degrading bacterial taxa which behaved differentially in coastal and offshore shelf waters despite being exposed to similar concentrations of oil, dispersants, and nutrients. This resulted in the differential expression of catabolic pathways for n-alkanes and polycyclic aromatic hydrocarbons (PAHs)—the two major categories of compounds found in crude oil—with preferential expression of n-alkane degradation genes in coastal waters while offshore microbial communities trended more toward the expression of PAH degradation genes. This was unexpected as it contrasts with the generally held view that n-alkanes, being more labile, are attacked before the more refractory PAHs. Collectively, our results provide new insights into the existence and potential consequences of niche partitioning of hydrocarbon-degrading taxa between neighboring marine environments. IMPORTANCE In the wake of the Deepwater Horizon oil spill, the taxonomic response of marine microbial communities to oil and dispersants has been extensively studied. However, relatively few studies on the functional response of these microbial communities have been reported, especially in a longitudinal fashion. Moreover, despite the fact that marine oil spills typically impact thousands of square kilometers of both coastal and offshore marine environments, little information is available on how the microbial response to oil and dispersants might differ between these biomes. The results of this study help fill this critical knowledge gap and provide valuable insight into how oil spill response efforts, such as chemically dispersing oil, may have differing effects in neighboring coastal and offshore marine environments.

The lithified oceanic crust, lower crust gabbros in particular, has remained largely unexplored by microbiologists. Recently, evidence for heterogeneously distributed viable and transcriptionally active autotrophic and heterotrophic microbial populations within low-biomass communities was found down to 750 m below the seafloor at the Atlantis Bank Gabbro Massif, Indian Ocean. Here, we report on the diversity, activity and adaptations of fungal communities in the deep oceanic crust from ~10 to 780 mbsf by combining metabarcoding analyses with mid/high-throughput culturing approaches. Metabarcoding along with culturing indicate a low diversity of viable fungi, mostly affiliated to ubiquitous (terrestrial and aquatic environments) taxa. Ecophysiological analyses coupled with metatranscriptomics point to viable and transcriptionally active fungal populations engaged in cell division, translation, protein modifications and other vital cellular processes. Transcript data suggest possible adaptations for surviving in the nutrient-poor, lithified deep biosphere that include the recycling of organic matter. These active communities appear strongly influenced by the presence of cracks and veins in the rocks where fluids and resulting rock alteration create micro-niches.

Terrestrial runoff can negatively impact marine ecosystems through stressors including excess nutrients, freshwater, sediments, and contaminants. Severe storms, which are increasing with global climate change, generate massive inputs of runoff over short timescales (hours to days); such runoff impacted offshore reefs in the northwest Gulf of Mexico (NW GoM) following severe storms in 2016 and 2017. Several weeks after coastal flooding from these events, NW GoM reef corals, sponges, and other benthic invertebrates -185 km offshore experienced mortality (2016 only) and/or sub-lethal stress (both years). To assess the impact of storm-derived runoff on reef filter feeders, we characterized the bacterial communities of two sponges, Agelas clathrodes and Xestospongia muta, from offshore reefs during periods of sub-lethal stress and no stress over a three-year period (2016-2018). Sponge-associated bacterial communities were altered during both flood years. Additionally, we found evidence of wastewater contamination (based on 16S rRNA gene libraries and quantitative PCR) in offshore sponges following these flood years, but not during the no-flood year. We show that flood events from severe storms have the capacity to reach offshore reef ecosystems and impact resident benthic organisms. Such impacts are most readily detected if baseline data on organismal physiology and associated microbiome composition are available. This highlights the need for molecular and microbial time series of benthic organisms in near- and offshore reef ecosystems, and the continued mitigation of stormwater runoff and climate change impacts.

Microbes release exoenzymes into the environment to break down complex organic matter and nutrients into simpler forms that can be assimilated and utilized, thereby addressing their cellular carbon, nitrogen, and phosphorus requirements. Despite its importance, the factors associated with the synthesis of exoenzymes are not clearly defined, especially for the marine environment. Here, we found that exoenzymes associated with nitrogen and phosphorus acquisition were strongly correlated with inorganic nutrient levels, while those associated with carbon acquisition depended on the type of organic carbon available. We also show a linear relationship between carbon- and nitrogen-acquiring exoenzymes and a strong correlation between microbial biomass and exoenzymes, highlighting their significance to microbial productivity. Last, we show that changes in microbial community composition are not strongly associated with changes in exoenzyme activity profiles, a finding which reveals a redundancy of exoenzyme activity functions among microbial community. These findings advance our understanding of previously unknown factors associated with exoenzyme production in the marine environment., Microbial heterotopic metabolism in the ocean is fueled by a supply of essential nutrients acquired via exoenzymes catalyzing depolymerization of high-molecular-weight compounds. Although the rates of activity for a variety of exoenzymes across various marine environments are well established, the factors regulating the production of these exoenzymes, and to some extent their correlation with microbial community composition, are less known. This study focuses on addressing these challenges using a mesocosm experiment that compared a natural seawater microbial community (control) and exposed (to oil) treatment. Exoenzyme activities for β-glucosidase, leucine aminopeptidase (LAP), and lipase were significantly correlated with dissolved nutrient concentrations. We measured correlations between carbon- and nitrogen-acquiring enzymes (β-glucosidase/lipase versus LAP) and found that the correlation of carbon-acquiring enzymes varies with the chemical nature of the available primary carbon source. Notably, a strong correlation between particulate organic carbon and β-glucosidase activity demonstrates their polysaccharide depolymerization in providing the carbon for microbial growth. Last, we show that exoenzyme activity patterns are not necessarily correlated with prokaryotic community composition, suggesting a redundancy of exoenzyme functions among the marine microbial community and substrate availability. This study provides foundational work for linking exoenzyme function with dissolved organic substrate and downstream processes in marine systems. IMPORTANCE Microbes release exoenzymes into the environment to break down complex organic matter and nutrients into simpler forms that can be assimilated and utilized, thereby addressing their cellular carbon, nitrogen, and phosphorus requirements. Despite its importance, the factors associated with the synthesis of exoenzymes are not clearly defined, especially for the marine environment. Here, we found that exoenzymes associated with nitrogen and phosphorus acquisition were strongly correlated with inorganic nutrient levels, while those associated with carbon acquisition depended on the type of organic carbon available. We also show a linear relationship between carbon- and nitrogen-acquiring exoenzymes and a strong correlation between microbial biomass and exoenzymes, highlighting their significance to microbial productivity. Last, we show that changes in microbial community composition are not strongly associated with changes in exoenzyme activity profiles, a finding which reveals a redundancy of exoenzyme activity functions among microbial community. These findings advance our understanding of previously unknown factors associated with exoenzyme production in the marine environment.

Marine oil spills can impact both coastal and offshore marine environments, but little information is available on how the microbial response to oil and dispersants might differ between these biomes. Here we describe the compositional and functional response of microbial communities to different concentrations of oil and chemically dispersed oil in coastal and offshore surface waters from the Texas-Louisiana continental shelf. Using a combination of analytical chemistry, 16S rRNA amplicon, and metatranscriptomic sequencing, we provide a broad, comparative overview of the ecological response of hydrocarbon degrading bacteria and their expression of hydrocarbon degrading genes in marine surface waters over time between two oceanic biomes. We found evidence for the existence of different ecotypes of several commonly described hydrocarbon degrading bacterial taxa which behaved differentially in coastal and offshore shelf waters despite being exposed to similar concentrations of oil, dispersants, and nutrients. This resulted in the differential expression of catabolic pathways for n-alkanes and polycyclic aromatic hydrocarbons (PAH)—the two major categories of compounds found in crude oil—with preferential expression of n-alkane degradation genes in coastal waters while offshore microbial communities trended more towards the expression of PAH degradation genes. This was unexpected as it contrasts with the generally held view that n-alkanes, being more labile, are attacked before the more refractory PAHs. Collectively, our results provide new insights into the existence and potential consequences of niche partitioning of hydrocarbon degrading taxa between neighboring marine environments.IMPORTANCEIn the wake of the Deepwater Horizon oil spill, the taxonomic response of marine microbial communities to oil and dispersants has been extensively studied. However, relatively few studies on the functional response of these microbial communities have been reported, especially in a longitudinal fashion. Moreover, despite the fact that marine oil spills typically impact thousands of square kilometers of both coastal and offshore marine environments, little information is available on how the microbial response to oil and dispersants might differ between these biomes. The results of this study help fill this critical knowledge gap and provide valuable insight into how oil spill response efforts, such as chemically dispersing oil, may have differing effects in neighboring coastal and offshore marine environments.

Oceanic crust comprises a vast but virtually unexplored habitat for life on Earth, characterized by massive global flows of water, heat, and dissolved chemicals. Uncovering where and how life exists in oceanic crust is important because chemical reactions occurring in this environment impact broader ocean systems and also because it is an earth analog for considering the possibility of extraterrestrial life on other ocean worlds. Over the past decade, several major oceanographic expeditions focused on characterizing the ocean crust microbiome, enabled by advances in seafloor drilling and observatory technologies. Here we review what is known about the crustal ocean microbial biosphere, focusing on a new synthesis of recent studies on the diversity of microbial life in oceanic crust to reveal common and unique taxa in this environment.

Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, cyc2, in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments., Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans. However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ, often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments. IMPORTANCE Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, cyc2, in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments.

Large-scale exopolymeric substances (EPS) formation was observed in contaminated surface waters of the Gulf of Mexico , after the Deepwater Horizon oil spill. The conditions under which EPS are produced can cause variations in their physico-chemical properties , affecting the fate of the oil through either emulsification or coagulation with particles, followed by aggregation and marine oil snow (MOS) sedimentation . Here, we report results from a four-day mesocosm experiment that examined EPS production, the change in the bulk chemical composition (mainly polysaccharides and proteins), as well as the partitioning into different size fractions (suspended particulate matter (SPM), colloidal fraction, and sinking MOS). The treatments included 1) natural coastal seawater (Control), 2) a water accommodated fraction of oil (WAF), 3) a chemically-enhanced WAF with addition of oil dispersant Corexit (CEWAF), and 4) a diluted CEWAF (DCEWAF). Generally, the presence of WAF stimulated more extracellular polysaccharide production, whereas the addition of Corexit promoted more protein production. Moreover, marine aggregates changed from a terrestrial signature (higher aromaticity, lower EPS content and more uniform monosaccharide distribution) in the early stages of the experiment, to a fresher autochthonous signature (less aromatic, higher EPS content and glucose-dominated monosaccharide distribution) in the later stage of the mesocosm experiment. Corexit promoted the association between oil and proteins, assisting to emulsify the oil in colloids and SPM and delay the settling of EPS and oil (MOS) for the duration of the mesocosm. Furthermore, when oil, especially Corexit-dispersed oil, was present, extracellular polysaccharides were preferentially incorporated into sinking MOS relative to proteins, as evidenced from a lower protein-C/carbohydrate-C ratio of the sinking MOS than those of SPM and colloidal fraction in WAF, CEWAF and DCEWAF, respectively. This is in contrast to a higher protein-C/carbohydrate-C ratio in the sinking marine snow than those of SPM and colloidal fraction in the control. Likely, microbially-mediated extracellular polysaccharides are the key component that anchors the mineral ballast until the aggregates become dense enough and overcome the buoyancy added to the aggregates as a result of their association with oil/Corexit. We conclude that interactions between Corexit and EPS components regulate petroleum hydrocarbon distribution between the water column and sinking MOS.

Sulfide mineral precipitation occurs at mid-ocean ridge (MOR) spreading centers, both in the form of plume particles and seafloor massive sulfide structures. A common constituent of MOR is the iron-bearing sulfide mineral pyrrhotite, which was chosen as a substrate for in-situ incubation studies in shallow waters of Catalina Island, CA to investigate the colonization of iron-oxidizing bacteria. Microbial community datasets were obtained from in-situ incubated pyrrhotite, allowing for direct comparison to microbial communities of iron-sulfides from active and inactive chimneys in deep-sea environments. Unclassified Gammaproteobacteria and Alphaproteobacteria (Magnetovibrio) largely dominated the bacterial community on pyrrhotite samples incubated in the water column while samples incubated at the surface sediment showed more even dominance by Deltaproteobacteria (Desulfobulbus), Gammaproteobacteria (Piscirickettsiaceae), Alphaproteobacteria (Rhodobacteraceae), and Bacteroidetes (Flavobacteriia). Cultivations that originated from pyrrhotite samples resulted in the enrichment of both, sheath-forming and stalk-forming Zetaproteobacteria. Additionally, a putative novel species of Thiomicrospira was isolated and shown to grow autotrophically with iron, indicating a new biogeochemical role for this ubiquitous microorganism.

The role of nitrogen cycling in submarine hydrothermal systems is far less studied than that of other biologically reactive elements such as sulfur and iron. In order to address this knowledge gap, we investigated nitrogen redox processes at Loihi Seamount, Hawaii, using a combination of biogeochemical and isotopic measurements, bioenergetic calculations and analysis of the prokaryotic community composition in venting fluids sampled during four cruises in 2006, 2008, 2009 and 2013. Concentrations of NH4+ were positively correlated to dissolved Si and negatively correlated to NO3− + NO2−, while NO2− was not correlated to NO3− + NO2−, dissolved Si or NH4+. This is indicative of hydrothermal input of NH4+ and biological mediation influencing NO2− concentrations. The stable isotope ratios of NO3− (δ15N and δ18O) was elevated with respect to background seawater, with δ18O values exhibiting larger changes than corresponding δ15N values, reflecting the occurrence of both production and reduction of NO3− by an active microbial community. δ15N-NH4+ values ranged from 0‰ to +16.7‰, suggesting fractionation during consumption and potentially N-fixation as well. Bioenergetic calculations reveal that several catabolic strategies involving the reduction of NO3− and NO2− coupled to sulfide and iron oxidation could provide energy to microbes in Loihi fluids, while 16S rRNA gene sequencing of Archaea and Bacteria in the fluids reveals groups known to participate in denitrification and N-fixation. Taken together, our data support the hypothesis that microbes are mediating N-based redox processes in venting hydrothermal fluids at Loihi Seamount.

Processes active within buoyant hydrothermal plumes are expected to modulate the flux of elements, such as Fe, to the deep ocean; however, they are yet to be described in a comprehensive manner through observations or models. In this study, we compare observed particulate Fe (pFe) speciation with thermodynamic (equilibrium) reaction path modeling for three vent fields in the Eastern Lau Spreading Center (ELSC). At each site, particles were collected from the buoyant rising portion of hydrothermal plumes using in situ filtration with a Remotely Operated Vehicle. Filter bound particles were analyzed by synchrotron micro-probe X-ray fluorescence mapping (XRF), X-ray diffraction (XRD), XRF spectroscopy, and X-ray absorption near edge structure (XANES) spectroscopy at the Fe 1 s edge, as well as XRF-based chemical speciation mapping for Fe. For buoyant plumes of the ELSC, diversity in solid-state chemistry was high, and poorly crystalline, meta-stable phases were common. We demonstrate that to fully describe the crystalline-to-noncrystalline character of plume pFe, a multi-modal XRD-XANES analytical approach is needed. We found that an equilibrium modeling approach worked well for pyrite but performed poorly for important families of meta-stable pFe, namely Fe (oxyhydr)oxides and monosulfides. Based on our findings, we recommend future field expeditions strategically explore sites representing a diversity of site-specific conditions to better capture the full range of processes active in plumes. We also recommend development of kinetic models, as well as expansion of thermodynamic databases to better reflect the solid-state composition of plumes. These steps should allow oceanographers to understand the processes controlling Fe speciation in plumes well enough to create realistic models of hydrothermal fluxes to the ocean.

Extracellular polymeric substances (EPS) released from marine microbial cells is a major response to stress. Most previous studies focus on the quantity of EPS released from microbes; however, the critical characteristics of EPS composition receive much less attention. Considering the complex nature and diversity of microbes in the marine system also impart distinct behaviors and reactions when exposed to stressful conditions, we cultured four phytoplankton and five bacterial species, to investigate the changes of the chemical composition of EPS under stress from a water-accommodated fraction (WAF) of oil or a chemically-enhanced WAF (CEWAF). Remaining viable cells (DNA amount) of nine marine microbes showed a higher linear negative correlation with increasing protein to carbohydrate (P/C) ratio under CEWAF than WAF treatments. The data also show that EPS with higher protein ratios was released under higher cellular stress levels induced by CEWAF, suggesting that higher protein ratio EPS, which is more hydrophobic, is secreted to physically or chemically ameliorate the hazardous agents. Our findings provide evidence that microbes can actively modify their EPS release and composition in responses to various adverse stress. Collectively, the results support the hypothesis that higher stress not only can trigger more EPS release, but also induce changes in its composition (P/C), thereby affecting environmental processes such as marine oil snow formation and the characteristic of marine organic matter.

The flavins (including flavin mononucleotide (FMN) and riboflavin (RF)) are a class of organic compounds synthesized by organisms to assist in critical redox reactions. While known to be secreted extracellularly by some species in laboratory-based cultures, flavin concentrations are largely unreported in the natural environment. Here, we present pore water and water column profiles of extracellular flavins (FMN and RF) and two degradation products (lumiflavin and lumichrome) from a coastal marine basin in the Southern California Bight alongside ancillary geochemical and 16S rRNA microbial community data. Flavins were detectable at picomolar concentrations in the water column (93-300 pM FMN, 14-40 pM RF) and low nanomolar concentrations in pore waters (250-2070 pM FMN, 11-210 pM RF). Elevated pore water flavin concentrations displayed an increasing trend with sediment depth and were significantly correlated with the total dissolved Fe (negative) and Mn (positive) concentrations. Network analysis revealed a positive relationship between flavins and the relative abundance of Dehalococcoidia and the MSBL9 clade of Planctomycetes, indicating possible secretion by members of these lineages. These results suggest that flavins are a common component of the so-called shared extracellular metabolite pool, especially in anoxic marine sediments where they exist at physiologically relevant concentrations for metal oxide reduction.

Earth’s subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were Propionibacterium, Aquabacterium, Ralstonia, and Acinetobacter. While the top five most frequently observed genera were Pseudomonas, Propionibacterium, Acinetobacter, Ralstonia, and Sphingomonas. The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA extraction and DNA cleanup methods. Thus, controls must be taken at every step of the collection and processing procedure when working with low biomass environments such as, but not limited to, portions of Earth’s deep subsurface. Taken together, we stress that the CoDL dataset is an incredible resource for the broader research community interested in subsurface life, and steps to remove contamination derived sequences must be taken prior to using this dataset.

Extracellular enzymes and extracellular polymeric substances (EPS) play a key role in overall microbial activity, growth and survival in the ocean. EPS, being amphiphilic in nature, can act as biological surfactant in an oil spill situation. Extracellular enzymes help microbes to digest and utilize fractions of organic matter, including EPS, which can stimulate growth and enhance microbial activity. These natural processes might have been altered during the 2010 Deepwater Horizon oil spill due to the presence of hydrocarbon and dispersant. This study aims to investigate the role of bacterial extracellular enzymes during exposure to hydrocarbons and dispersant. Mesocosm studies were conducted using a water accommodated fraction of oil mixed with the chemical dispersant, Corexit (CEWAF) in seawater collected from two different locations in the Gulf of Mexico and corresponding controls (no additions). Activities of five extracellular enzymes typically found in the EPS secreted by the microbial community - α- and β-glucosidase, lipase, alkaline phosphatase, leucine amino-peptidase - were measured using fluorogenic substrates in three different layers of the mesocosm tanks (surface, water column and bottom). Enhanced EPS production and extracellular enzyme activities were observed in the CEWAF treatment compared to the Control. Higher bacterial and micro-aggregate counts were also observed in the CEWAF treatment compared to Controls. Bacterial genera in the order Alteromonadaceae were the most abundant bacterial 16S rRNA amplicons recovered. Genomes of Alteromonadaceae commonly have alkaline phosphatase and leucine aminopeptidase, therefore they may contribute significantly to the measured enzyme activities. Only Alteromonadaceae and Pseudomonadaceae among bacteria detected here have higher percentage of genes for lipase. Piscirickettsiaceae was abundant; genomes from this order commonly have genes for leucine aminopeptidase. Overall, this study provides insights into the alteration to the microbial processes such as EPS and extracellular enzyme production, and to the microbial community, when exposed to the mixture of oil and dispersant.

A facultatively anaerobic bacterium, designated strain 1MBB1T, was isolated from basaltic breccia collected from 341 m below the seafloor by seafloor drilling of Rigil Guyot during Integrated Ocean Drilling Program Expedition 330. The cells were straight rods, 0.5 μm wide and 1–3 μm long, that occurred singly and in chains. Strain 1MBB1T stained Gram-positive. Catalase and oxidase were produced. The isolate grew optimally at 30 °C and pH 7.5, and could grow with up to 12 % (w/v) NaCl. The DNA G+C content was 40.5 mol%. The major cellular fatty acids were C16: 1ω11c (26.5 %), anteiso-C15: 0 (19.5 %), C16: 0 (18.7 %) and iso-C15: 0 (10.4 %), and the cell-wall diamino acid was meso-diaminopimelic acid. Endospores of strain 1MBB1T oxidized Mn(II) to Mn(IV), and siderophore production by vegetative cells was positive. Phylogenetic analysis of the 16S rRNA gene indicated that strain 1MBB1T was a member of the family Bacillaceae, with Bacillus foraminis CV53T and Bacillus novalis LMG 21837T being the closest phylogenetic neighbours (96.5 and 96.2 % similarity, respectively). This is the first novel species described from deep subseafloor basaltic crust. On the basis of our polyphasic analysis, we conclude that strain 1MBB1T represents a novel species of the genus Bacillus, for which we propose the name Bacillus rigiliprofundi sp. nov. The type strain is 1MBB1T ( = NCMA B78T = LMG 28275T).

The water-soluble compounds of oil (e.g. low molecular weight PAHs) dissolve as a function of their physicochemical properties and environmental conditions, while the non-soluble compounds exist as dispersed droplets. Both the chemical and physical form of oil will affect the biological response. We present data from a mesocosm study comparing the microbial response to the water-soluble fraction (WSF), versus a water-accommodated fraction of oil (WAF), which contains both dispersed and dissolved oil components. WAF and WSF contained similar concentrations of low molecular weight PAHs, but concentrations of 4- and 5-ring PAHs were higher in WAF compared to WSF. Microbial communities were significantly different between WSF and WAF treatments, primary productivity was reduced more in WSF than in WAF, and concentrations of transparent exopolymeric particles were highest in WSF and lowest in the controls. These differences highlight the importance of dosing strategy for mesocosm and toxicity tests.

Within hydrothermal plumes, chemosynthetic processes and microbe–mineral interactions drive primary productivity in deep-ocean food webs and may influence transport of elements such as iron. However, the source of microorganisms in plumes and the factors governing how these communities assemble are poorly understood, in part due to lack of data from early stages of plume formation. In this study, we examined microbial community composition of rising hydrothermal plumes from five vent fields along the Eastern Lau Spreading Center. Seafloor and plume microbial communities were significantly dissimilar and shared few phylotypes. Plume communities were highly similar to each other with significant differences in community membership only between Kilo Moana and Mariner, two vents that are separated by extremes in depth, latitude and geochemistry. Systematic sampling of waters surrounding the vents revealed that species richness and phylogenetic diversity was typically highest near the vent orifice, implying mixing of microbial communities from the surrounding habitats. Above-plume background communities were primarily dominated by SAR11, SAR324 and MG-I Archaea, while SUP05, Sulfurovum, Sulfurimonas, SAR324 and Alteromonas were abundant in plume and near-bottom background communities. These results show that the ubiquitous water-column microorganisms populate plume communities, and that the composition of background seawater exerts primary influence on plume community composition, with secondary influence from geochemical and/or physical properties of vents. Many of these pervasive deep-ocean organisms are capable of lithotrophy, suggesting that they are poised to use inorganic electron donors encountered in hydrothermal plumes.

Nutrients delivered by the Mississippi and Atchafalaya Rivers to the Louisiana continental shelf cause local eutrophication and feed phytoplankton growth, resulting in a massive region of bottom water hypoxia each summer. Much of what is known about the drivers of this hypoxia is based on extensive datasets of inorganic nutrients. We measured dissolved organic and particulate organic carbon, nitrogen and phosphorus in surface water along the Louisiana continental shelf between the Mississippi and Atchafalaya Rivers during three cruises in March, May and July 2004 to develop a more comprehensive understanding of interactions between nutrients and eutrophication. We specifically sought to determine if sufficient organic P is present to relieve the inorganic P limitation that occurs during the spring and early summer. DOP is added to shelf waters in situ, but drawdown of DOP concentrations below conservative mixing lines indicate DOP is also being used as a source of P for growth. Calculations of the amount of DOP potentially utilized towards primary production reveal there is not enough to relieve P-limitation. Total nitrogen to total phosphorus ratios were high, averaging 86, 72 and 51 in March, May and July, respectively. Additionally, correlations between DIN and total nitrogen (TN) were identical to those between DIP and total phosphorus (TP), indicating that the use of either TN:TP or DIN:DIP ratios is equally relevant in assessing nutrient limitation in this region, but DIN:TP ratios are clearly underestimates. Organic matter on the shelf is best correlated to TN and TP loads measured upstream in the Mississippi River one month prior. The combination of low organic P concentrations and high total N:P ratios indicated P-limitation of phytoplankton biomass on the Louisiana shelf during the spring and early summer of 2004. This P-limitation occurs during the period of hypoxia formation as a result of excess N loads delivered by the rivers, supporting dual controls of both N and P to reduce the hypoxia.

Sulfide mineral precipitation occurs at mid-ocean ridge (MOR) spreading centers, both in the form of plume particles and seafloor massive sulfide structures. A common constituent of MOR is the iron-bearing sulfide mineral pyrrhotite, which was chosen as a substrate for in-situ incubation studies in shallow waters of Catalina Island, CA to investigate the colonization of iron-oxidizing bacteria. Microbial community datasets were obtained from in-situ incubated pyrrhotite, allowing for direct comparison to microbial communities of iron-sulfides from active and inactive chimneys in deep-sea environments. Unclassified Gammaproteobacteria and Alphaproteobacteria (Magnetovibrio) largely dominated the bacterial community on pyrrhotite samples incubated in the water column while samples incubated at the surface sediment showed more even dominance by Deltaproteobacteria (Desulfobulbus), Gammaproteobacteria (Piscirickettsiaceae), Alphaproteobacteria (Rhodobacteraceae), and Bacteroidetes (Flavobacteriia). Cultivations that originated from pyrrhotite samples resulted in the enrichment of both, sheath-forming and stalk-forming Zetaproteobacteria. Additionally, a putative novel species of Thiomicrospira was isolated and shown to grow autotrophically with iron, indicating a new biogeochemical role for this ubiquitous microorganism.

International Ocean Discovery Program (IODP) Expedition 360 was the first leg of Phase I of the SloMo (shorthand for "The nature of the lower crust and Moho at slower spreading ridges") Project, a multiphase drilling program that proposes to drill through the outermost of the global seismic velocity discontinuities, the Mohorovicic seismic discontinuity (Moho). The Moho corresponds to a compressional wave velocity increase, typically at ∼7 km beneath the oceans, and has generally been regarded as the boundary between crust and mantle. An alternative model, that the Moho is a hydration front in the mantle, has recently gained credence upon the discovery of abundant partially serpentinized peridotite on the seafloor and on the walls of fracture zones, such as at Atlantis Bank, an 11-13 My old elevated oceanic core complex massif adjacent to the Atlantis II Transform on the Southwest Indian Ridge. Hole U1473A was drilled on the summit of Atlantis Bank during IODP Expedition 360, 1-2 km away from two previous Ocean Drilling Program (ODP) holes: Hole 735B (drilled during ODP Leg 118 in 1987 and ODP Leg 176 in 1997) and Hole 1105A (drilled during ODP Leg 179 in 1998). A mantle peridotite/gabbro contact has been traced by dredging and diving along the transform wall for 40 km. The contact is located at ∼4200 m depth at the drill sites but shoals considerably 20 km to the south, where it was observed in outcrop at 2563 m depth. Moho reflections have, however, been found at ∼5-6 km beneath Atlantis Bank and

We deployed sediment traps adjacent to two active hydrothermal vents at 9°50'N on the East Pacific Rise (EPR) to assess the variability in bacterial community structure associated with plume particles on the timescale of weeks to months, to determine whether an endemic population of plume microbes exists, and to establish ecological relationships between bacterial populations and vent chemistry. Automated rRNA intergenic spacer analysis (ARISA) indicated that there are separate communities at the two different vents and temporal community variations between each vent. Correlation analysis between chemistry and microbiology indicated that shifts in the coarse particulate (>1 mm) Fe/(Fe+Mn+Al), Cu, V, Ca, Al, (232) Th, and Ti as well as fine-grained particulate (

Exploration of the microbiology in igneous, 'hard rock' oceanic crust represents a major scientific frontier. The igneous crust harbours the largest aquifer system on Earth, most of which is hydrologically active, resulting in a substantial exchange of fluids, chemicals and microorganisms between oceanic basins and crustal reservoirs. Study of the deep-subsurface biosphere in the igneous crust is technically challenging. However, technologies have improved over the past decade, providing exciting new opportunities for the study of deep-seated marine life, including in situ and cross-disciplinary experimentation in microbiology, geochemistry and hydrogeology. In this Progress article, we describe the recent advances, available technology and remaining challenges in the study of the marine intraterrestrial microbial life that is harboured in igneous oceanic crust.