Matching Items (31)

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Over-expression of a putative multi-heme cytochrome c from Heliobacterium modesticaldum

Description

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can fix nitrogen (N2) and produce molecular hydrogen (H2). Recently, the Redding and Jones labs created a microbial photoelectrosynthesis cell that utilized these properties to produce molecular hydrogen using electrons provided

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can fix nitrogen (N2) and produce molecular hydrogen (H2). Recently, the Redding and Jones labs created a microbial photoelectrosynthesis cell that utilized these properties to produce molecular hydrogen using electrons provided by a cathode via a chemical mediator. Although this light-driven creation of fuel within a microbial electrochemical cell was the first of its kind, its production rate of hydrogen was low. It was hypothesized that the injection of electrons into H. modesticaldum was a rate-limiting step in H2 production. Within the H. modesticaldum genome, there is a gene (HM1_0653) that encodes a multi-heme cytochrome c that may be directly involved in this step. From past transcriptomic experiments, this gene is known to be very poorly expressed in H. modesticaldum. Our hypothesis was that increasing its expression with a strong promoter could result in faster electron transfer, and thus, increased H2 production in the photoelectrosynthesis cell. In order to test this hypothesis, different promoters that could lead to high expression in H. modesticaldum were included with a copy of HM1_0653 in various plasmid constructs that were first cloned into E. coli before being conjugated with H. modesticaldum. Cloning in E. coli was possible with the newly derived transformation system and by reducing the copy-number of the vector system. When overexpressed in E. coli, the protein appeared to be expressed, but its purification proved to be difficult. Moreover, conjugation with H. modesticaldum was not achieved. Our results are consistent with the idea that high level overexpression in H. modesticaldum was toxic. An inducible promoter may circumvent these issues and prove more successful in future experiments.

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2018-05

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Assessment of the Vertical Stratification of Microbial Community Structure in Permafrost Peatlands

Description

Peatlands are a type of wetlands where the rate of accumulation of organic matter exceed the rate of decomposition and have accumulated more than 30 cm of peat (Joosten and Clark, 2002). Peatlands store approximately 30% of all terrestrial carbon

Peatlands are a type of wetlands where the rate of accumulation of organic matter exceed the rate of decomposition and have accumulated more than 30 cm of peat (Joosten and Clark, 2002). Peatlands store approximately 30% of all terrestrial carbon as recalcitrant peat, partially decomposed plant and microbial biomass, while simultaneously producing almost 40% of the globally emitted methane (Schmidt et al., 2016), making peatlands an important component of the carbon budgets. Published research indicates that the efficiency of carbon usage among microbial communities can determine the soil-carbon response to rising temperatures (Allison et al. 2010). By determining carbon consumption in peatland soils, total community respiration response, and community structure change with additions, models of carbon use efficiency in permafrost peatlands will be well-informed and have a better understanding of how the peatlands will respond to, and utilize, increased availability of carbon compounds due to the melting permafrost. To do this, we will sequence Lutose deep core samples to observe baseline microbial community structure at different depths and different age-gradients, construct substrate incubations of glucose and propionate and observe community respiration response via a gas chromatography flame ionization detector, track the glucose and propionate additions with high-performance liquid chromatography (HPLC), and sequence the samples once more to determine if there was a deviation from the initial community structure obtained prior to the incubations. We found that our initial sequencing data was supported by previous work (Lin et al., 2014), however we were unable to sequence samples post-incubation due to time constraints. In this sequencing analysis we found that the strongest variable that made samples biologically similar was the age-gradient site in which they were extracted. We found that the group with glucose additions produced the most carbon dioxide compared with the other treatments, but was not the treatment that dominated the production of methane. Finally, in the HPLC samples that were analyzed, we found that glucose is likely forming the most by-product accumulation from mass balance calculations, while propionate is likely forming the least. Future experimentation should focus on the shortcomings of this experiment. Further analysis of 16S rRNA sequencing data from after the incubations should be analyzed to determine the change in microbial community structure throughout the experiment. Furthermore, HPLC analysis for the several samples need to be done and followed up with mass balance to determine where the added glucose and propionate are being allocated within the soil. Once these pieces of the puzzle are put into place, our original question of how the microbial community structure changes at different depths and age-gradients within permafrost peatlands will be conclusively answered.

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2018-05

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Biotic vs. Abiotic Processes in Hyperarid Exoplanetary Atmospheres

Description

Exoplanetary research is a key component in the search for life outside of Earth and the Solar System. It provides people with a sense of wonder about their role in the evolution of the Universe and helps scientists understand life's

Exoplanetary research is a key component in the search for life outside of Earth and the Solar System. It provides people with a sense of wonder about their role in the evolution of the Universe and helps scientists understand life's potential throughout a seemingly infinite number of unique exoplanetary environments. The purpose of this research project is to identify the most plausible biosignature gases that would indicate life's existence in the context of hyperarid exoplanetary atmospheres. This analysis first defines hyperarid environments based on known analogues for Earth and Mars and discusses the methods that researchers use to determine whether or not an exoplanet is hyperarid. It then identifies the most relevant biosignatures to focus on based on the scientific literature on analogous hyperarid environments and ranks them in order from greatest to least biological plausibility within extreme hyperarid conditions. The research found that methane (CH4) and nitrous oxide (N2O) are the most helpful biosignature gases for these particular exoplanetary scenarios based on reviews of the literature. The research also found that oxygen (O2), hydrogen sulfide (H2S) and ammonia (NH3) are the biosignatures with the least likely biological origin and the highest likelihood of false positive detection. This analysis also found that carbon dioxide (CO2) is a useful companion biosignature within these environments when paired with either CH4 or the pairing of hydrogen (H2) and carbon monoxide (CO). This information will provide a useful road map for dealing with the detection of biosignatures within hyperarid exoplanetary atmospheres during future astrobiology research missions.

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2018-05

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Evaluating the Consumption Rates of Primary Versus Secondary Fermentation Substrates and Methane Production of Northern Peatlands

Description

Northern peatland carbon cycling is under close observation and is critical to include in models projecting the future effects of climate change as these ecosystems represent a significant source of atmospheric methane (CH4). Changes in the in situ conditions, brought

Northern peatland carbon cycling is under close observation and is critical to include in models projecting the future effects of climate change as these ecosystems represent a significant source of atmospheric methane (CH4). Changes in the in situ conditions, brought upon by the warming climate, could alter the rates of organic matter decomposition and accelerate the emissions of greenhouse, changing northern peatland’s status as a carbon sink. In order to develop a better understanding of the climate’s effect on the microbial community composition, carbon decomposition cascade, and flux of CH4 and CO2, anoxic soil microcosms were supplemented with either glucose or propionate to test the distinct intermediary metabolism of four northern peatland sites with statistically similar geochemistry that exist across a climate gradient. Lutose (LT) and Bog Lake (BL) consumed the supplemented glucose at the highest rates, 42.6 mg/L per day and 39.5 mg/L per day respectively. Chicago Bog (CB) and Daring Lake (DL) consumed the supplemented propionate at the highest rates, 5.26 mg/L per day and 4.34 mg/L per day respectively. BL microcosms showed low levels of methanogenesis as CH4 concentrations reached a maximum of 2.61 µmol/g dry soil in the treatments. In DL, the site with the highest production of CH4, the low abundance of hydrogenotrophic methanogens (Methanocellaceae and Methanoregulaceae) and relatively steady concentrations of acetate and formate could indicate that these are the more desired methanogenic substrates. These findings are indicative of the differences in metabolic potential found across these geochemically similar peatlands, lending to climate variables being a major driver in microbial community potential. To further characterize the intermediary metabolism and the effect of the climate gradient in these sites, future experimentations should incorporate 13C DNA-stable isotope probing data, establish a mass balance of the system, and incubate the microcosms at their respective in situ temperatures.

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2020-05

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Trait-Based Modeling of Peatland Methanogen Communities

Description

Methanogens are methane-producing archaea that play a major role in the global carbon cycle. However, despite their importance, the community dynamics of these organisms have not been thoroughly characterized or modeled. In the majority of methanogenesis models, the communities are

Methanogens are methane-producing archaea that play a major role in the global carbon cycle. However, despite their importance, the community dynamics of these organisms have not been thoroughly characterized or modeled. In the majority of methanogenesis models, the communities are approximated as a chemical reaction or divided into two populations based on the most common methanogenic pathways. These models provide reasonable estimate of methanogenesis rates but cannot predict community structure. In this work, a trait-based model for methanogenic communities in peatlands is developed. The model divides methanogens commonly found in wetlands into ten guilds, with divisions based on factors such as substrate affinity, pH tolerance, and phylogeny. The model uses steady-state, mixotrophic Monod kinetics to model growth and assumes peatlands operate as a semi-batch system. An extensive literature review was performed to parameterize the model. The acetoclastic module of the model was validated against experimental data. It was found that this portion of the model was able to reproduce the major result of an experiment that examined competition between Methanosaeta and Methanosarcina species under irregular feeding conditions. The model was analyzed as a whole using Monte Carlo simulation methods. It was found that equilibrium membership is negatively correlated with a guild's half-substrate constant, but independent of the guild's yield. These results match what is seen in simple pairwise competition models. In contrast, it was found that both the half-substrate constant and yield affected a guild's numerical dominance. Lower half-substrate constants and higher yields led to a guild accounting for a greater fraction of community biomass. This is not seen in simple pairwise competitions models where only yield affects final biomass. As a whole, the development of this model framework and the accompanying analyses have laid the groundwork for a new class of more detailed methanogen community models that go beyond the two compartment acetoclastic-hydrogenotrophic assumption. .

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2017-05

Isolation of a Significant Fraction of Non-Phototroph Diversity From a Desert Biological Soil Crust

Description

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

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Date Created
2015-03-19

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Exometabolite Niche Partitioning Among Sympatric Soil Bacteria

Description

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13−26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. These results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.

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Date Created
2015-09-22

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Complete Genome Sequence of Methanosphaerula Palustris E1-9CT, a Hydrogenotrophic Methanogen Isolated From a Minerotrophic Fen Peatland

Description

Here, we report the complete genome sequence (2.92 Mb) of Methanosphaerula palustris E1-9CT, a methanogen isolated from a minerotrophic fen. This is the first genome report of the Methanosphaerula genus, within the Methanoregulaceae family, in the Methanomicrobiales order. E1-9CT relatives

Here, we report the complete genome sequence (2.92 Mb) of Methanosphaerula palustris E1-9CT, a methanogen isolated from a minerotrophic fen. This is the first genome report of the Methanosphaerula genus, within the Methanoregulaceae family, in the Methanomicrobiales order. E1-9CT relatives are found in a wide range of ecological and geographical settings.

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Date Created
2015-11-05

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Investigation of the effect of efflux pumps on the toxicity of phenol, 2-phenylethanol, and styrene to E. coli

Description

Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol,

Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol, and styrene was investigated, in particular the limitation in yield and accumulation that results from high product toxicity. This paper examines two methods of product toxicity mitigation: the use of efflux pumps and the separation of pathways which produce less toxic intermediates. A library of 43 efflux pumps from various organisms were screened for their potential to confer resistance to phenol, 2-phenylethanol, and styrene on an E. coli host. A pump sourced from P. putida was found to allow for increased host growth in the presence of styrene as compared to a cell with no efflux pump. The separation of styrene producing pathway was also investigated. Cells capable of performing the first and latter halves of the synthesis were allowed to grow separately and later combined in order to capitalize on the relatively lower toxicity of the intermediate, trans-cinnamate. The styrene production and yield from this separated set of cultures was compared to that resulting from the growth of cells containing the full set of styrene synthesis genes. Results from this experiment were inconclusive.

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Date Created
2015-05

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Early Assessment of Phage Communities in Amazon Peatland Soils

Description

Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published assessment of phage abundance on diversity in a key tropical

Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published assessment of phage abundance on diversity in a key tropical ecosystem such as Amazon peatlands. To better understand phage assemblages in terrestrial ecosystems and how bacteriophages influence organic C cycling to final products like CO2 and CH4, phage communities and phage-like particles were recovered, quantified, and viable phage particles were enriched from pore water from contrasting Amazon peatlands. Here we present the first results on assessing Amazon bacteriophages on native heterotrophic bacteria. Several steps to test for methodological suitability were taken. First, the efficiency of iron flocculation method was determined using fluorescent microscopy counts of phage TLS, a TolC-specific and LPS-specific bacteriophage, and Escherichia coli host pre- and post-extraction method. One-hundred percent efficiency and 0.15% infectivity was evidenced. Infectivity effects were determined by calculating plaque forming units pre and post extraction method. After testing these methods, fieldwork in the Amazon peatlands ensued, where phages were enriched from pore water samples. Phages were extracted and concentrated by in tandem filtering rounds to remove organic matter and bacteria, and then iron flocculation to bind the phages and allow for precipitation onto a filter. Phage concentrates were then used for overall counts, with fluorescent microscopy, as well as phage isolation attempts. Phage isolations were performed by first testing for lysis of host cells in liquid media using OD600 absorbance of cultures with and without phage concentrate as well as attempts with the cross-streaking methods. Forty-five heterotrophic bacterial isolates obtained from the same Amazon peatland were challenged with phage concentrates. Once a putative host was found, steps were taken to further propagate and isolate the phage. Several putative phages were enriched from Amazon peatland pore water and require further characterization. TEM imaging was taken of two phages isolated from two plaques. Genomes of selected phages will be sequenced for identification. These results provide the groundwork for further characterizing the role bacteriophage play in C cycling and greenhouse gas production from Amazon peatland soils.

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2016-05