Scholarly Work - Center for Biofilm Engineering
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Item Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics(Springer Science and Business Media LLC, 2022-02) Schweitzer, Hannah D.; Smith, Heidi J.; Barnhart, Elliott P.; McKay, Luke J.; Gerlach, Robin; Cunningham, Alfred B.; Malmstrom, Rex R.; Goudeau, Danielle; Fields, Matthew W.Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.Item In Situ Enhancement and Isotopic Labeling of Biogenic Coalbed Methane(American Chemical Society, 2022-02) Barnhart, Elliott P.; Ruppert, Leslie; Hiebert, Randy; Smith, Heidi J.; Schweitzer, Hannah D.; Clark, Arthur C.; Weeks, Edwin P.; Orem, William H.; Varonka, Matthew S.; Platt, George; Shelton, Jenna L.; Davis, Katherine J.; Hyatt, Robert J.; McIntosh, Jennifer C.; Ashley, Kilian; Ono, Shuhei; Martini, Anna M.; Hackley, Keith C.; Gerlach, Robin; Spangler, Lee; Phillips, Adrienne J.; Barry, Mark; Cunningham, Alfred B.; Fields, Matthew W.Subsurface microbial (biogenic) methane production is an important part of the global carbon cycle that has resulted in natural gas accumulations in many coal beds worldwide. Laboratory studies suggest that complex carbon-containing nutrients (e.g., yeast or algae extract) can stimulate methane production, yet the effectiveness of these nutrients within coal beds is unknown. Here, we use downhole monitoring methods in combination with deuterated water (D2O) and a 200-liter injection of 0.1% yeast extract (YE) to stimulate and isotopically label newly generated methane. A total dissolved gas pressure sensor enabled real time gas measurements (641 days preinjection and for 478 days postinjection). Downhole samples, collected with subsurface environmental samplers, indicate that methane increased 132% above preinjection levels based on isotopic labeling from D2O, 108% based on pressure readings, and 183% based on methane measurements 266 days postinjection. Demonstrating that YE enhances biogenic coalbed methane production in situ using multiple novel measurement methods has immediate implications for other field-scale biogenic methane investigations, including in situ methods to detect and track microbial activities related to the methanogenic turnover of recalcitrant carbon in the subsurface.Item Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidenc(Frontiers Media SA, 2022-01) Pettygrove, Brian A.; Smith, Heidi J.; Pallister, Kyler B.; Voyich, Jovanka M.; Stewart, Philip S.; Parker, Albert E.The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.Item Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid—Novel Approaches to Fight Food-Borne Pathogens(MDPI, 2021) Chlumsky, Ondrej; Smith, Heidi J.; Parker, Albert E.; Brileya, Kristen; Wilking, James N.; Purkrtova, Sabina; Michova, Hana; Ulbrich, Pavel; Viktorova, Jitka; Demnerova, KaterinaIn the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-L-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.Item Janthinobacterium CG23_2: comparative genome analysis reveals enhanced environmental sensing and transcriptional regulation for adaptation to life in an Antarctic supraglacial stream(2019-10) Dieser, Markus; Smith, Heidi J.; Ramaraj, Thiruvarangan; Foreman, Christine M.As many bacteria detected in Antarctic environments are neither true psychrophiles nor endemic species, their proliferation in spite of environmental extremes gives rise to genome adaptations. Janthinobacterium sp. CG23_2 is a bacterial isolate from the Cotton Glacier stream, Antarctica. To understand how Janthinobacterium sp. CG23_2 has adapted to its environment, we investigated its genomic traits in comparison to genomes of 35 published Janthinobacterium species. While we hypothesized that genome shrinkage and specialization to narrow ecological niches would be energetically favorable for dwelling in an ephemeral Antarctic stream, the genome of Janthinobacterium sp. CG23_2 was on average 1.7 ± 0.6 Mb larger and predicted 1411 ± 499 more coding sequences compared to the other Janthinobacterium spp. Putatively identified horizontal gene transfer events contributed 0.92 Mb to the genome size expansion of Janthinobacterium sp. CG23_2. Genes with high copy numbers in the species-specific accessory genome of Janthinobacterium sp. CG23_2 were associated with environmental sensing, locomotion, response and transcriptional regulation, stress response, and mobile elements—functional categories which also showed molecular adaptation to cold. Our data suggest that genome plasticity and the abundant complementary genes for sensing and responding to the extracellular environment supported the adaptation of Janthinobacterium sp. CG23_2 to this extreme environment.Item DOM composition alters ecosystem function during microbial processing of isolated sources(2019-01) D'Andrilli, Juliana; Junker, James R.; Smith, Heidi J.; Scholl, Eric A.; Foreman, Christine M.Dynamics of dissolved organic matter (DOM) in ecosystems are controlled by a suite of interacting physical, chemical, and biological factors. Growing recognition of the associations between microbial communities and metabolism and intrinsic DOM characteristics, highlight the potential importance of microbe-DOM relationships to modulate the role and fate of DOM, yet these relationships are difficult to isolate because they often operate across confounding environmental gradients. In a controlled laboratory incubation (44 days), we integrated DOM bulk and molecular characterization, bacterial abundances, microbial assemblage composition, nutrient concentrations, and cellular respiration to discern the structural dynamics of biological processing among DOM sources from different allochthonous litters (grass, deciduous leaves, and evergreen needles). We identified two periods, consistent among DOM sources, where processing dynamics differed. Further, bulk fluorescent analyses showed shifts from low to high excitation and emission wavelengths, indicating the biological production of more complex/degraded materials over time. Molecular level analyses revealed similar temporal patterns among DOM sources in the production and consumption of individual chemical components varying in reactivity and heteroatomic content. Despite these similarities, total carbon (C) removed and carbon dioxide (CO2) accumulation differed by ~ 20% and 25% among DOM sources. This range in C processing was apparently tied to key chemical properties of the DOM (e.g., initial DOM composition, N content, and labile nature) as well as differential reorganization of the microbial populations that decomposed the DOM. We conclude that the production, transformation, and consumption of C in aquatic ecosystems is strongly dependent on the source and character of DOM as well as the structure of the microbial communities present, both of which change as DOM is processed over time. It is crucial that stream C processing models represent this complexity accurately.Item Ultrafast Excited-State Deactivation of the Bacterial Pigment Violacein(2017-08) Beckstead, Ashley A.; Zhang, Yuyan; Hilmer, Jonathan K.; Smith, Heidi J.; Bermel, Emily; Foreman, Christine M.; Kohler, BernThe photophysical properties of the natural pigment violacein extracted from an Antarctic organism adapted to high exposure levels of UV radiation were measured in a combined steady-state and time-resolved spectroscopic study for the first time. In the low-viscosity solvents methanol and acetone, violacein exhibits low fluorescence quantum yields on the order of 1 x 10(-4), and femtosecond transient absorption measurements reveal excited-state lifetimes of 3.2 +/- 0.2 and 4.6 +/- 0.2 ps in methanol and acetone, respectively. As solvent viscosity is increased, both the fluorescence quantum yield and excited-state lifetime of this intensely colored pigment increase dramatically, and stimulated emission decays 30-fold more slowly in glycerol than in methanol at room temperature. Excited-state deactivation is suggested to occur via a molecular-rotor mechanism in which torsion interring bond leads to a conical intersection with the ground state.Item Biogeophysical properties of an expansive Antarctic supraglacial stream(2017) SanClements, M. D.; Michael, D.; Smith, Heidi J.; Foreman, Christine M.; Tedesco, Marco; Chin, Yu-PingSupraglacial streams are important hydrologic features in glaciated environments as they are conduits for the transport of aeolian debris, meltwater, solutes and microbial communities. We characterized the basic geomorphology, hydrology and biogeochemistry of the Cotton Glacier supraglacial stream located in the McMurdo Dry Valleys of Antarctica. The distinctive geomorphology of the stream is driven by accumulated aeolian sediment from the Transantarctic Mountains, while solar radiation and summer temperatures govern melt in the system. The hydrologic functioning of the Cotton Glacier stream is largely controlled by the formation of ice dams that lead to vastly different annual flow regimes and extreme flushing events. Stream water is chemically dilute and lacks a detectable humic signature. However, the fluorescent signature of dissolved organic matter (DOM) in the stream does demonstrate an extremely transitory red-shifted signal found only in near-stream sediment leachates and during the initial flushing of the system at the onset of flow. This suggests that episodic physical flushing drives pulses of DOM with variable quality in this stream. This is the first description of a large Antarctic supraglacial stream and our results provide evidence that the hydrology and geomorphology of supraglacial streams drive resident microbial community composition and biogeochemical cyclingItem Microbial formation of labile organic carbon in Antarctic glacial environments(2017-04) Smith, Heidi J.; Foster, Rachel A.; McKnight, Diane M.; Lisle, John T.; Littmann, Sten; Kuypers, Marcel M. M.; Foreman, Christine M.Roughly six petagrams of organic carbon are stored within ice worldwide. This organic carbon is thought to be of old age and highly bioavailable. Along with storage of ancient and new atmospherically deposited organic carbon, microorganisms may contribute substantially to the glacial organic carbon pool. Models of glacial microbial carbon cycling vary from net respiration to net carbon fixation. Supraglacial streams have not been considered in models although they are amongst the largest ecosystems on most glaciers and are inhabited by diverse microbial communities. Here we investigate the biogeochemical sequence of organic carbon production and uptake in an Antarctic supraglacial stream in the McMurdo Dry Valleys using nanometre-scale secondary ion mass spectrometry, fluorescence spectroscopy, stable isotope analysis and incubation experiments. We find that heterotrophic production relies on highly labile organic carbon freshly derived from photosynthetic bacteria rather than legacy organic carbon. Exudates from primary production were utilized by heterotrophs within 24 h, and supported bacterial growth demands. The tight coupling of microbially released organic carbon and rapid uptake by heterotrophs suggests a dynamic local carbon cycle. Moreover, as temperatures increase there is the potential for positive feedback between glacial melt and microbial transformations of organic carbon.Item Biofilms on glacial surfaces: hotspots for biological activity(2016-06) Smith, Heidi J.; Schmit, Amber; Foster, Rachel A.; Littmann, Sten; Kuypers, Marcel M. M.; Foreman, Christine M.Glaciers are important constituents in the Earth’s hydrological and carbon cycles, with predicted warming leading to increases in glacial melt and the transport of nutrients to adjacent and downstream aquatic ecosystems. Microbial activity on glacial surfaces has been linked to the biological darkening of cryoconite particles, affecting albedo and increased melt. This phenomenon, however, has only been demonstrated for alpine glaciers and the Greenland Ice Sheet, excluding Antarctica. In this study, we show via confocal laser scanning microscopy that microbial communities on glacial surfaces in Antarctica persist in biofilms. Overall, ~35% of the cryoconite sediment surfaces were covered by biofilm. Nanoscale scale secondary ion mass spectrometry measured significant enrichment of 13C and 15N above background in both Bacteroidetes and filamentous cyanobacteria (i.e., Oscillatoria) when incubated in the presence of 13C–NaHCO3 and 15NH4. This transfer of newly synthesised organic compounds was dependent on the distance of heterotrophic Bacteroidetes from filamentous Oscillatoria. We conclude that the spatial organisation within these biofilms promotes efficient transfer and cycling of nutrients. Further, these results support the hypothesis that biofilm formation leads to the accumulation of organic matter on cryoconite minerals, which could influence the surface albedo of glaciers.