Microbial Community Dynamics Provide Evidence for Hypoxia during a Coral Reef Mortality Event.

In July 2016, a severe coral reef invertebrate mortality event occurred approximately 200 km southeast of Galveston, Texas, at the East Flower Garden Bank, wherein ∼82% 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 2 years at the Flower Garden Banks, including a cruise just 5 to 8 days after the mortality event was first observed. In contrast with observations collected during nonmortality 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. IMPORTANCE In the northwestern Gulf of Mexico in July 2016, ∼82% 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 were 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.

Mesopelagic microbial community dynamics in response to increasing oil and Corexit 9500 concentrations.

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.

Bacteria-Oil Microaggregates Are an Important Mechanism for Hydrocarbon Degradation in the Marine Water Column.

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.

Niche Partitioning between Coastal and Offshore Shelf Waters Results in Differential Expression of Alkane and Polycyclic Aromatic Hydrocarbon Catabolic Pathways.

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.

Exoenzymes as a Signature of Microbial Response to Marine Environmental Conditions.

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 ß-glucosidase, leucine aminopeptidase (LAP), and lipase were significantly correlated with dissolved nutrient concentrations. We measured correlations between carbon- and nitrogen-acquiring enzymes (ß-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 ß-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.

Biological response to dissolved versus dispersed oil.

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.

Role of Polysaccharides in Diatom Thalassiosira pseudonana and its Associated Bacteria in Hydrocarbon Presence.

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.

Metagenomic analysis of basal ice from an Alaskan glacier.

BACKGROUND: Glaciers cover ~ 10% of land but are among the least explored environments on Earth. The basal portion of glaciers often harbors unique aquatic microbial ecosystems in the absence of sunlight, and knowledge on the microbial community structures and their metabolic potential is very limited. Here, we provide insights into the microbial lifestyle present at the base of the Matanuska Glacier, Alaska. RESULTS: DNA and RNA were extracted from samples of the Matanuska Glacier basal ice. Using Illumina MiSeq and HiSeq sequencing, we investigated the microbial diversity with the metagenomic shotgun reads and 16S ribosomal RNA data. We further assembled 9 partial and draft bacterial genomes from the metagenomic assembly, and identified key metabolic pathways such as sulfur oxidation and nitrification. Collectively, our analyses suggest a prevalence of lithotrophic and heterotrophic metabolisms in the subglacial microbiome. CONCLUSION: Our results present the first metagenomic assembly and bacterial draft genomes for a subglacial environment. These results extend our understanding of the chemical and biological processes in subglacial environments critically influenced by global climate change.

Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill.

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 ß-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.

Rapid Formation of Microbe-Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit.

During the Deepwater Horizon (DWH) oil spill, massive quantities of oil were deposited on the seafloor via a large-scale marine oil-snow sedimentation and flocculent accumulation (MOSSFA) event. The role of chemical dispersants (e.g., Corexit) applied during the DWH oil spill clean-up in helping or hindering the formation of this MOSSFA event are not well-understood. Here, we present the first experiment related to the DWH oil spill to specifically investigate the relationship between microbial community structure, oil and Corexit®, and marine oil-snow in coastal surface waters. We observed the formation of micron-scale aggregates of microbial cells around droplets of oil and dispersant and found that their rate of formation was directly related to the concentration of oil within the water column. These micro-aggregates are potentially important precursors to the formation of larger marine oil-snow particles. Therefore, our observation that Corexit® significantly enhanced their formation suggests dispersant application may play a role in the development of MOSSFA events. We also observed that microbial communities in marine surface waters respond to oil and oil plus Corexit® differently and much more rapidly than previously measured, with major shifts in community composition occurring within only a few hours of experiment initiation. In the oil-amended treatments without Corexit®, this manifested as an increase in community diversity due to the outgrowth of several putative aliphatic- and aromatic-hydrocarbon degrading genera, including phytoplankton-associated taxa. In contrast, microbial community diversity was reduced in mesocosms containing chemically dispersed oil. Importantly, different consortia of hydrocarbon degrading bacteria responded to oil and chemically dispersed oil, indicating that functional redundancy in the pre-spill community likely results in hydrocarbon consumption in both undispersed and dispersed oils, but by different bacterial taxa. Taken together, these data improve our understanding of how dispersants influence the degradation and transport of oil in marine surface waters following an oil spill and provide valuable insight into the early response of complex microbial communities to oil exposure.

Characterizing microbial diversity and the potential for metabolic function at -15 °c in the Basal ice of taylor glacier, antarctica.

Measurement of gases entrapped in clean ice from basal portions of the Taylor Glacier, Antarctica, revealed that CO2 ranged from 229 to 328 ppmv and O2 was near 20% of the gas volume. In contrast, vertically adjacent sections of the sediment laden basal ice contained much higher concentrations of CO2 (60,000 to 325,000 ppmv), whereas O2 represented 4 to 18% of the total gas volume. The deviation in gas composition from atmospheric values occurred concurrently with increased microbial cell concentrations in the basal ice profile, suggesting that in situ microbial processes (i.e., aerobic respiration) may have altered the entrapped gas composition. Molecular characterization of 16S rRNA genes amplified from samples of the basal ice indicated a low diversity of bacteria, and most of the sequences characterized (87%) were affiliated with the phylum, Firmicutes. The most abundant phylotypes in libraries from ice horizons with elevated CO2 and depleted O2 concentrations were related to the genus Paenisporosarcina, and 28 isolates from this genus were obtained by enrichment culturing. Metabolic experiments with Paenisporosarcina sp. TG14 revealed its capacity to conduct macromolecular synthesis when frozen in water derived from melted basal ice samples and incubated at -15 °C. The results support the hypothesis that the basal ice of glaciers and ice sheets are cryospheric habitats harboring bacteria with the physiological capacity to remain metabolically active and biogeochemically cycle elements within the subglacial environment.

Draft genome sequence of Paenisporosarcina sp. strain TG-20, a psychrophilic bacterium isolated from the basal ice of Taylor Glacier.

We report the draft genome sequence of Paenisporosarcina sp. strain TG-20, which is 4.12 Mb in size and consists of 4,071 protein-coding genes and 76 RNA genes. The genome sequence of Paenisporosarcina sp. TG-20 may provide useful information about molecular adaptations that enhance survival in icy subsurface environments.

Draft genome sequence of Paenisporosarcina sp. strain TG-14, a psychrophilic bacterium isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica.

The psychrophilic bacterium Paenisporosarcina sp. TG-14 was isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica. Here we report the draft genome sequence of this strain, which may provide useful information on the cold adaptation mechanism in extremely variable environments.

Implications of subzero metabolic activity on long-term microbial survival in terrestrial and extraterrestrial permafrost.

The survival of microorganisms over extended time frames in frozen subsurface environments may be limited by chemical (i.e., via hydrolysis and oxidation) and ionizing radiation-induced damage to chromosomal DNA. In an effort to improve estimates for the survival of bacteria in icy terrestrial and extraterrestrial environments, we determined rates of macromolecular synthesis at temperatures down to -15°C in bacteria isolated from Siberian permafrost (Psychrobacter cryohalolentis K5 and P. arcticus 273-4) and the sensitivity of P. cryohalolentis to ionizing radiation. Based on experiments conducted over ≈400 days at -15°C, the rates of protein and DNA synthesis in P. cryohalolentis were <1 to 16 proteins cell(-1) d(-1) and 83 to 150 base pairs (bp) cell(-1) d(-1), respectively; P. arcticus synthesized DNA at rates of 20 to 1625 bp cell(-1) d(-1) at -15°C under the conditions tested. The dose of ionizing radiation at which 37% of the cells survive (D(37)) of frozen suspensions of P. cryohalolentis was 136 Gy, which was ∼2-fold higher (71 Gy) than identical samples exposed as liquid suspensions. Laboratory measurements of [(3)H]thymidine incorporation demonstrate the physiological potential for DNA metabolism at -15°C and suggest a sufficient activity is possible to offset chromosomal damage incurred in near-subsurface terrestrial and martian permafrost. Thus, our data imply that the longevity of microorganisms actively metabolizing within permafrost environments is not constrained by chromosomal DNA damage resulting from ionizing radiation or entropic degradation over geological time.

Macromolecular synthesis by yeasts under frozen conditions.

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8 degrees C min(-1)), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at -5 degrees C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [(3)H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15 degrees C and -15 degrees C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at -5 degrees C under frozen and liquid conditions revealed 2-3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of approximately 1.4 mol l(-1) of NaCl at -5 degrees C). The mesophile Saccharomyces cerevisae also incorporated [(3)H]leucine at -5 degrees C and -15 degrees C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.