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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

Influence of corn, switchgrass, and prairie cropping systems on soil microbial communities in the upper Midwest of the United States

Ederson da C. Jesus; Chao Liang; John F. Quensen; Endang Susilawati; Randall D. Jackson; Teresa C. Balser; James M. Tiedje

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2016

Because soil microbes drive many of the processes underpinning ecosystem services provided by soils, understanding how cropping systems affect soil microbial communities is important for productive and sustainable management. We characterized and compared soil microbial communities under restored prairie and three potential cellulosic biomass crops (corn, switchgrass, and mixed prairie grasses) in two spatial experimental designs - side by side plots where plant communities were in their second year since establishment (i.e., intensive sites) and regionally distributed fields where plant communities had been in place for at least 10 years (i.e., extensive sites). We assessed microbial community structure and composition using lipid analysis, pyrosequencing of rRNA genes (targeting fungi, bacteria, archaea and lower eukaryotes), and targeted-metagenomics of nifH genes. For the more recently established intensive sites, soil type was more important than plant community in determining microbial community structure, while plant community was the more important driver of soil microbial communities for the older extensive sites where microbial communities under corn were clearly differentiated from those under switchgrass and restored prairie. Bacterial and fungal biomasses, especially biomass of arbuscular mycorrhizal fungi, were higher under perennial grasses and restored prairie, suggesting a more active carbon pool and greater microbial processing potential, which should be beneficial for plant acquisition and ecosystem retention of carbon, water, and nutrients. This article is protected by copyright. All rights reserved.

Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

Rebecca G. Ong; Alan Higbee; Scott Bottoms; Quinn Dickinson; Dan Xie; Scott A. Smith; Jose Serate; Edward Pohlmann; Arthur D. Jones; Joshua J. Coon; Trey K. Sato; Gregg R. Sanford; Dustin Eilert; Lawrence G. Oates; Jeff S. Piotrowski; Donna M. Bates; David Cavalier; Yaoping Zhang

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2016

Integrating winter annual cereal rye or triticale into a corn forage biofuel production system

Pavani Tumbalam; Kaitlyn Hard; Kurt D. Thelen

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2016

Integrating cover crops into the corn (Zea mays L.) forage-production system could enhance growers? profitability and improve ecosystem services. Field plots were established at two locations in Michigan during 2012--2014 to evaluate ethanol production, estimate carbon and energy balance, and the economics of corn forage plus cover crop, cropping systems. In the 2012--2013 crop cycle, which was characterized by a summer drought, the cereal rye (Secale cereale L.), and triticale (Triticale hexaploide Lart.), winter annual cover crops increased cropping system biomass yield by 44%, and total ethanol yield by 28% relative to the no-cover control. During the 2012--2013 cycle, cover crops provided sufficient biomass (6.5 and 8.1 Mg ha?1, respectively, for cereal rye and triticale) to result in a profitable harvest as a biofuel feedstock. However, following a harsh winter, such as that experienced in the 2013--2014 cycle, cover crop yield (1.6 and 1.2 Mg ha?1, respectively, for cereal rye and triticale) was compromised to the point that harvest was not economically justified. Incorporating cover crops into a corn forage cropping system increased total biomass and potential biofuel yield and generated a very favorable net ecosystem carbon and energy balance.

Investment risks in bioenergy crops

Theodoros Skevas; Scott M. Swinton; Sophia Tanner; Gregg Sanford; Kurt D. Thelen

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2016

Isolation and characterization of new lignin streams derived from extractive-ammonia (EA) pretreatment

Leonardo da Costa Sousa; Marcus Foston; Vijay Bokade; Ali Azarpira; Fachuang Lu; Arthur J. Ragauskas; John Ralph; Bruce Dale; Venkatesh Balan

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2016

Lactobacillus casei as a biocatalyst for biofuel production

Elena Vinay-Lara; Song Wang; Lina Bai; Ekkarat Phrommao; Jeff R. Broadbent; James L. Steele

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2016

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.

Leveraging genetic background effects in Saccharomyces cerevisiae to improve lignocellulosic hydrolysate tolerance

Maria Sardi; Nikolay Rovinskiy; Yaoping Zhang; Audrey P. Gasch

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2016

A major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variation in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene over-expression in different yeast strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six different Saccharomyces cerevisiae strains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to implicate common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene over-expression in three different strains, we revealed the breadth of background-specific effects of gene-fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms. IMPORTANCE Recent studies on natural variation within Saccharomyces cerevisiae have uncovered substantial phenotypic diversity. Here, we take advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated cells' physiological state when exposed to this stress. We also explored strain-specific effects of gene overexpression to further implicate strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

Leveraging genetic background effects in Saccharomyces cerevisiae to improve lignocellulosic hydrolysate tolerance

Maria Sardi; Nikolay Rovinskiy; Yaoping Zhang; Audrey P. Gasch

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2016

A major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variation in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene over-expression in different yeast strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six different Saccharomyces cerevisiae strains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to implicate common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene over-expression in three different strains, we revealed the breadth of background-specific effects of gene-fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms. IMPORTANCE Recent studies on natural variation within Saccharomyces cerevisiae have uncovered substantial phenotypic diversity. Here, we take advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated cells' physiological state when exposed to this stress. We also explored strain-specific effects of gene overexpression to further implicate strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

Long-term nitrous oxide fluxes in annual and perennial agricultural and unmanaged ecosystems in the upper Midwest USA

Ilya Gelfand; Iur Shcherbak; Neville Millar; Alexandra N. Kravchenko; Philip Robertson

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2016

Differences in soil nitrous oxide (N2O) fluxes among ecosystems are often difficult to evaluate and predict due to high spatial and temporal variabilities and few direct experimental comparisons. For 20 years, we measured N2O fluxes in 11 ecosystems in southwest Michigan USA: four annual grain crops (corn–soybean–wheat rotations) managed with conventional, no-till, reduced input, or biologically based/organic inputs; three perennial crops (alfalfa, poplar, and conifers); and four unmanaged ecosystems of different successional age including mature forest. Average N2O emissions were higher from annual grain and N-fixing cropping systems than from nonleguminous perennial cropping systems and were low across unmanaged ecosystems. Among annual cropping systems full-rotation fluxes were indistinguishable from one another but rotation phase mattered. For example, those systems with cover crops and reduced fertilizer N emitted more N2O during the corn and soybean phases, but during the wheat phase fluxes were ~40% lower. Likewise, no-till did not differ from conventional tillage over the entire rotation but reduced emissions ~20% in the wheat phase and increased emissions 30–80% in the corn and soybean phases. Greenhouse gas intensity for the annual crops (flux per unit yield) was lowest for soybeans produced under conventional management, while for the 11 other crop × management combinations intensities were similar to one another. Among the fertilized systems, emissions ranged from 0.30 to 1.33 kg N2O-N ha−1 yr−1 and were best predicted by IPCC Tier 1 and ΔEF emission factor approaches. Annual cumulative fluxes from perennial systems were best explained by soil NO3− pools (r2 = 0.72) but not so for annual crops, where management differences overrode simple correlations. Daily soil N2O emissions were poorly predicted by any measured variables. Overall, long-term measurements reveal lower fluxes in nonlegume perennial vegetation and, for conservatively fertilized annual crops, the overriding influence of rotation phase on annual fluxes.

Low temperature hydrogenation of pyrolytic lignin over Ru/TiO2: 2D HSQC and 13C NMR study of reactants and products

Wen Chen; Daniel J. McClelland; Ali Azarpira; John Ralph; Zhongyang Luo; George W. Huber

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2016

Pyrolytic lignin and hydrogenated pyrolytic lignin were characterized by 2D 1H–13C HSQC and quantitative 13C NMR techniques. The pyrolytic lignin was produced from a mixed maple wood feedstock and separ- ated from the bio-oil by water extraction. p-Hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatics were the basic units of pyrolytic lignin. The native lignin β-aryl ether, phenylcoumaran and resinol struc- tures were not present in the pyrolytic lignin. The hydrogenation was conducted with a Ru/TiO2 catalyst at temperatures ranging from 25–150 °C with higher temperatures exhibiting higher levels of hydrogenation. Solid coke formed on the catalyst surface (1% coke yield) even for hydrogenation at 25 °C. The carbon yield of pyrolytic lignin to coke increased from 1% to 5% as the hydrogenation temperature increased from 25 to 150 °C. A single-step hydrogenation at 150 °C resulted in a reduction from 65% to 39% aromatic carbons. A three-step hydrogenation scheme at this same temperature resulted in a reduction of aromatic carbons from 65% to 17%. The decrease in the aromatic carbon corresponded with an increase in the aliphatic carbon. Coke formation reduced from a 5% carbon yield of pyrolytic lignin in the first hydrogenation step to a 1% carbon yield in each of the second and third hydrogenation steps. The pyrolytic lignin could be sep- arated into a high and low molecular weight fraction. The coke yield from the high molecular weight frac- tion was twice as much as that from the low molecular weight fraction.

Low temperature hydrogenation of pyrolytic lignin over Ru/TiO2: 2D HSQC and 13C NMR study of reactants and products

Wen Chen; Daniel J. McClelland; Ali Azarpira; John Ralph; Zhongyang Luo; George W. Huber

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2016

Pyrolytic lignin and hydrogenated pyrolytic lignin were characterized by 2D 1H–13C HSQC and quantitative 13C NMR techniques. The pyrolytic lignin was produced from a mixed maple wood feedstock and separ- ated from the bio-oil by water extraction. p-Hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatics were the basic units of pyrolytic lignin. The native lignin β-aryl ether, phenylcoumaran and resinol struc- tures were not present in the pyrolytic lignin. The hydrogenation was conducted with a Ru/TiO2 catalyst at temperatures ranging from 25–150 °C with higher temperatures exhibiting higher levels of hydrogenation. Solid coke formed on the catalyst surface (1% coke yield) even for hydrogenation at 25 °C. The carbon yield of pyrolytic lignin to coke increased from 1% to 5% as the hydrogenation temperature increased from 25 to 150 °C. A single-step hydrogenation at 150 °C resulted in a reduction from 65% to 39% aromatic carbons. A three-step hydrogenation scheme at this same temperature resulted in a reduction of aromatic carbons from 65% to 17%. The decrease in the aromatic carbon corresponded with an increase in the aliphatic carbon. Coke formation reduced from a 5% carbon yield of pyrolytic lignin in the first hydrogenation step to a 1% carbon yield in each of the second and third hydrogenation steps. The pyrolytic lignin could be sep- arated into a high and low molecular weight fraction. The coke yield from the high molecular weight frac- tion was twice as much as that from the low molecular weight fraction.

Maize tricin-oligolignol metabolites and their implications for monocot lignification

Wu Lan; Kris Morreel; Fachuang Lu; Jorge Rencoret; Jose Carlos Del Rio; Wannes Voorend; Wilfred Vermerris; Wout Boerjan; John Ralph

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2016

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots.

Measurement of intrinsic catalytic activity of Pt monometallic and Pt-MoOx interfacial sites over visible light enhanced PtMoOx/SiO2 catalyst in reverse water gas shift reaction

Insoo Ro; Canan Sener; Thomas M. Stadelman; Madelyn R. Ball; Juan M. Venegas; Samuel P. Burt; I. Hermans; James A. Dumesic; George W. Huber

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2016

Supported Pt-Mo catalysts were prepared with different Mo contents by a controlled surface reaction (CSR) method and studied for the reverse water gas shift (RWGS) reaction under dark and visible light irradiation conditions. Characterization results from Raman spectroscopy, scanning transmission electron microscopy (STEM), CO chemisorption, and inductively coupled plasma-absorption emission spectroscopy (ICP-AES) indicate that selective Mo deposition onto Pt was achieved at low Mo loading (Mo/Pt ratio

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

Quinn Dickinson; Scott Bottoms; Li Hinchman; Sean McIlwain; Sheena Li; Chad L. Myers; Charles Boone; Joshua J. Coon; Alexander Hebert; Trey K. Sato; Robert Landick; Jeff S. Piotrowski

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2016

Background
Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae.

Results
We found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effects of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2∆ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance.

Conclusions
This work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.

Methodological guidelines for accurate detection of viruses in wild plant species

Christelle Lacroix; Kurra Renner; Ellen Cole; Eric W. Seabloom; Elizabeth T. Borer; Carolyn M. Malmstrom

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2016

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

Methodologies for probing the metatranscriptome of grassland soil

Aaron Garoutte; Erick Cardenas; James Tiedje; Adina Howe

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2016

Metatranscriptomics provides an opportunity to identify active microbes and expressed genes in complex soil communities in response to particular conditions. Currently, there are a limited number of soil metatranscriptome studies to provide guidance for using this approach in this challenging matrix. Hence, we evaluated the technical challenges of applying soil metatranscriptomics to a highly diverse, low activity natural system. We used a non-targeted rRNA removal approach, duplex nuclease specific (DSN) normalization, to generate a metatranscriptomic library from field collected soil supporting a perennial grass, Miscanthus x giganteus (a biofuel crop), and evaluated its ability to provide insight into its active community members and their expressed protein-coding genes. We also evaluated various bioinformatics approaches for analyzing our soil metatranscriptome, including annotation of unassembled transcripts, de novo assembly, and aligning reads to known genomes. Further, we evaluated various databases for their ability to provide annotations for our metatranscriptome. Overall, our results emphasize that low activity, highly genetically diverse and relatively stable microbiomes, like soil, requires very deep sequencing to sample the transcriptome beyond the common core functions. We identified several key areas that metatranscriptomic analyses will benefit from including increased rRNA removal, assembly of short read transcripts, and more relevant reference bases while providing a priority set of expressed genes for functional assessment.

Methodology for the experimental measurement of vapor-liquid equilibrium distillation curves using a modified ASTM D86 setup

Alison M. Ferris; David A. Rothamer

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2016

A method has been developed to determine experimental equilibrium distillation curves using a modified ASTM D86 distillation apparatus.The method determines accurate equilibrium initial boiling points and accounts for the dynamic holdup inherent in distillation curves measured in accordance with the ASTM D86 standard.In this work, the ASTM D86 distillation setup has been modified to simultaneously measure liquid and vapor temperature using two resistance temperature detectors (RTDs) and a data acquisition system has been employed to record temperature data at one-second time intervals for the duration of each distillation.Additionally, the time for each volume recovery point is recorded.The method presented here uses the time-resolved liquid temperature data to identify the true initial boiling point (IBP) of four fuel mixtures of known composition; the IBPs are within 2 °C of the calculated equilibrium values.The time-resolved volume recovery information and the identified initial boiling point time are used to construct a volume evaporated versus time curve.The measured temperatures determined at the corresponding volume evaporated increments provide an experimental equilibrium distillation curve (EEDC). The EEDCs for the four fuel mixtures of known composition match the calculated equilibrium curves within a few degrees Celsius; a maximum mean absolute error of 2.2 ± 1.4 °C was observed.The dynamic holdup (volume difference between volume evaporated and volume recovered) associated with a distillation is found to correlate with the initial boiling point of the fuel being distilled and the temperature of the condenser bath used in the experiment.The method was also applied to measure EEDCs for a gasoline fuel and a diesel fuel, where the compositions were unknown, to investigate the differences between the EEDCs and the ASTM D86 distillation curves.The results highlight the large errors incurred when using ASTM D86 results to approximate equilibrium distillation curves.

Microbial community analysis with ribosomal gene fragments from shotgun metagenomes

Jiarong Guo; James R. Cole; Qingpeng Zhang; Titus Brown; James M. Tiedje

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2016

Microorganisms and their residues under restored perennial grassland communities of varying diversity

Chao Liang; Jenny Kao-Kniffin; Gregg R. Sanford; Kyle Wickings; Teri C. Balser; Randall D. Jackson

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2016

Rising atmospheric CO2 concentration and global mean temperatures have stimulated interest in managing terrestrial systems to sequester more carbon and mitigate climate change. In a restored prairie experiment, we compared high diversity (HD, 25 species) with low diversity (LD, 6 species) prairies to investigate the effect of plant diversity on soil microbial communities and their residues with soil depth. We assayed lipid and amino sugar biomarkers for soil samples, taken after 9 years following the establishment of the prairie treatment, at 5 depth increment layers: 0–2 cm, 12–15 cm, 25–27 cm, 50–52 cm, and 98–100 cm. We found that the microbial biomass and residues decreased considerably with depth in both diversity treatments. Ordination analysis of lipid profiles indicated soil microbial communities were consistently distinct between the deeper and the upper layers, regardless of treatment, and also differed between the LD and HD treatments. Plant diversity effects on soil microbial communities strongly correlated with arbuscular mycorrhizal fungi (AMF), as indicated by the lipid marker 16:1ω5c. Soil microbial residues in deeper horizons were relatively more enriched in HD than LD treatments, suggesting that greater plant diversity might sustain higher soil carbon storage through relatively recalcitrant necromass inputs in the long term. Decreasing glucosamine/muramic acid (GluN/MurA) ratio in LD and increasing in HD with depth suggested that the new microbially-accumulated carbon was positively contributed by fungal-derived residues. Our results indicate that plant diversity drives soil microbial carbon sequestration through changes in AMF abundance in restored native tallgrass ecosystems. These findings have implications for understanding how the management of plant diversity can improve soil quality and sustainability in grasslands, and how efforts to conserve and restore diverse grasslands could mitigate greenhouse gas emissions.

Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling

Jonathan A. Stefely; Nicolas W. Kwiecien; Elyse C. Freiberger; Alicia L. Richards; Adam Jochem; Matthew J.P. Rush; Arne Ulbrich; Kyle P. Robinson; Paul D. Hutchins; Michael T. Veling; Xiao Guo; Zachary A. Kemmerer; Kyle J. Connors; Edna A. Trujillo; Jacob Sokol; Harald Marx; Michael S. Westphall; Alexander S. Hebert; David J. Pagliarini;

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2016

Mitochondria are complex organelles linked to diverse human diseases, often through incompletely characterized proteins and pathways. Here, toward systematically defining functions for poorly characterized mitochondrial proteins, we used mass spectrometry to map proteome, lipidome, and metabolome alterations across 174 single gene deletion yeast strains; 144 of these genes have human homologs, and 60 are associated with disease. We generated a dataset with over 3.5 million biomolecule measurements and developed a data analysis and visualization approach to enable biological hypothesis generation. Our multi-omic analysis reveals functionally-predictive molecule covariance networks, correlations between related genes, gene-specific perturbations, and a universal respiration deficiency response—each of which provides a foundation for numerous biological investigations. Here, we leveraged a subset of our data to elucidate uncharacterized features of mitochondrial coenzyme Q (CoQ) biosynthesis—an essential pathway disrupted in many human diseases. Our analyses link seven new proteins to this pathway, including Hfd1p and its human homolog ALDH3A1. Collectively, our results provide molecular insight into mitochondrial biology and establish a widely applicable approach for multi-omic analysis of diagnostic phenotypes and protein functions.

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