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DOE OSTI2017

High Performance Polymers from Biomass

The objective of this study was to demonstrate feasibility and scale-up of cellulosic feedstocks for the production of a commercial C6 lactone. Visolis, Inc. has developed a fermentation platform for production of the lactone at high titers and yields. The lactone is used in high-end cosmetics industry and also serves as a valuable intermediate for production of biopolymers and biofuels. Strains employed by Visolis are capable of metabolizing both glucose and xylose, enabling full utilization of cellulosic hydrolysates as feedstock. Visolis and ABPDU conducted a 2L proof-of-concept fermentation campaign followed by a 50L scale-up campaign.

09 BIOMASS FUELS↗

DOE OSTI2015

The Catalysis of Uniform Metal Nanoparticles Deposited onto Oxide Supports: The Components of a Catalyst that Control Activity and Selectivity

Model materials consisting of metal nanoparticles loaded onto oxide supports were synthesized, characterized, and investigated in a number of catalytic chemical reactions. By varying the size, shape, and composition of nanoparticle, as well as the material used to support the nanoparticles, it was found that small changes to the catalyst can have enormous changes to the reaction activity and selectivity. Investigation of these carefully synthesized catalysts via in situ characterization, and reaction studies, leads to a deeper understanding of the molecular level parameters that govern catalysis. Through study of the properties of the nanoparticles it was discovered that nanoparticle size and shape have a dominant role in the chemoselective catalysis of furfural over platinum nanoparticles. When vapor phase furfural and hydrogen gas were passed over Pt nanoparticles ranging in size from 1.5 to 7.1 nm, the catalytic selectivity was found to be dominated by the size of the nanoparticle. Large nanoparticles promoted hydrogenation of furfural to furfuryl alcohol, while smaller nanoparticles favored decarbonylation to furan. The same size specific selectivity was found in the hydrogenative reforming (the transformation of hydrocarbons to branched isomers) of C6 hydrocarbons, in which Pt nanoparticle size controls isomerization selectivity. Methylcyclopentane was found to be extremely size dependent at lower temperatures (553 K). It was found that smaller sized nanoparticles favored isomer formation, while larger sizes catalyzed the aromatization reaction more efficiently. n-hexane was found to be much less dependent on particle size, but still showed an increase in isomerization with small particles over larger sized Pt nanoparticles. The composition of PtxRh1-x bimetallic nanoparticles was also studied. These catalysts were characterized under hexane reforming conditions with Ambient Pressure X-ray Photoelectron Spectroscopy (AP-XPS), in order to find the actual surface atomic composition under real catalytic working conditions. By using AP-XPS and catalytic data in tandem, it was found that an optimum Rh loading occurred when the surface ensemble statistically favored one Rh atom surrounded by Pt atoms. By utilizing different oxide materials for catalytic supports the flow of charge can play a role in the reaction at the surface or interface in a phenomenon known as the strong metal-support interaction (SMSI). When Pt nanoparticles were loaded onto mesoporous supports made of Co3O4, NiO, MnO2, Fe2O3, and CeO2 it was found that their activity for carbon monoxide oxidation was greatly enhanced relative to the support alone or Pt loaded onto inert mesoporous silica. This finding demonstrates that the interface of the metallic Pt nanoparticle and the oxide 2 support is able to produce turnovers that are orders of magnitude higher than the two materials separately. When the same type of experiments were investigated with n-hexane as the reactant and macroporous Al2O3, TiO2, Nb2O5, Ta2O5, and ZrO2 were utilized as supports, it was found that the reaction selectivity was greatly altered depending on the catalytic support material. TiO2, Nb2O5, and Ta2O5 (all of which are strong Lewis acids) were found to be much more selective for isomer production than the standard SiO2 mesoporous silica supported Pt nanoparticle catalyst. Finally, an acidified mesoporous silica material was utilized as the support. This material was synthesized by using AlCl3 to modify the surface of mesoporous silica. This support was found to have no activity for hexane isomerization alone. However, when Pt nanoparticles were supported on the material, the activity and isomer selectivity in hexane reforming was increased several orders of magnitude as compared to the same nanoparticles supported on unmodified mesoporous silica. This dissertation builds on the existing knowledge of known concepts in catalysis science such as structure sensitive reactions, the metal-support interaction, and acid-base chemistry. The results show how small changes in the active sites of a catalyst can create large changes in the catalytic chemistry. This research demonstrates how careful material control, characterization and reaction study can help to elucidate the molecular level components necessary to design efficient catalysts.

Musselwhite, Nathan↗

DOE OSTI2018

Ediesel: Diesel Additive production from ethanol and bio-diesel coproducts

This project has focused on supporting and expanding the U.S. ethanol industry by developing new conversion routes and products from ethanol and the byproducts made in ethanol fermentation. The work is relevant to the DOE program goals of developing high-margin chemical products in the biorefinery to economically support large-scale biofuel production. The higher alcohols from condensation of ethanol, alkylated amine compounds made by electrocatalytic alkyation, and fusel alcohols produced by modified fermentation microorganisms are all existing or potentially large-scale commodity or specialty chemicals with well-defined uses in industry. The formation of higher alcohols from ethanol in the condensed phase developed several catalysts, first in stirred batch autoclave reactors and then in a continuous flow reactor system, to achieve significant yields of n-butanol and higher alcohols. The best selectivity to higher alcohols exceeded 80% of theoretical at ethanol conversions of 30-40%. From the laboratory experiments and analysis of the phase equilibria of the species involved, a process concept was generated and simulated in AspenPlus to determine the specifications of the various equipment involved in the process. Results show that for the conversion and selectivity described above, the required n-butanol selling price of ~$1.60/kg is in the range of current n-butanol selling price in the marketplace. Several additional permutations of the process, along with evaluation of higher conversion and selectivities to desired alcohol products, showed that the production of n-butanol from ethanol should be competitive in the marketplace as the science and engineering behind the process improves with continued development. A second aspect of the project focused on producing fusel alcohols in increased quantities during ethanol fermentation. Fusel alcohols, which are composed primarily of isoamyl alcohol and minor quantities of C3-C6 alcohols, have potential as higher value feed stocks for specialty chemical production. Several strains of yeast were evaluated for fusel alcohol production, and the most efficient process for separating and purifying fusel alcohols was developed from both experiments and simulation. Finally, model fusel alcohol mixtures were subjected to condensation reactions with ethanol to prepare higher alcohols that again have potential for specialty chemical or solvent applications. In this preliminary work, measurable quantities of several species were observed, including some species that were previously unknown or uncharacterized in the literature. The third aspect of the project involved the electrocatalytic alkylation of amines with alcohols to form alkyl amines. Ethanol has been demonstrated to serve as an effective ethyl group transfer agent to the nitrogen centers of amines, a reaction with only water as a byproduct. Mild reaction conditions (60 °C, aqueous solution) are enabled by the novel use of simple, undivided electrochemical cell reactors that include long-lived, low cost catalytic electrodes. Direct polyalkylation of ammonia and isolation of the resulting triethylamine product was readily achieved in high yield. Though classical high temperature and pressure industrial scale alkylations reactions will likely continue to be used for production of simple product such as triethylamine, this approach has opened the door to the low cost, low waste use of ethanol and related primary and secondary alcohols to alkylate more delicate amines that were heretofore difficult to synthesize.

02 PETROLEUM↗

DOE OSTI2021

A Novel Platform for Algal Biomass Production Using Cellulosic Mixotrophy (CeMix) (Final Technical Report)

This novel project, branded CeMIX for cellulosic mixotrophy, targets solutions for several technological barriers limiting the deployment of algae cultivation in sunny, arid regions (specifically the southwest United States). Opening up the region for algae cultivation could increase DOE’s resource assessments and biofuel potential for the U.S. Mixotrophic metabolism in support of liquid fuel production is completely dependent on the availability of waste organic carbon in order to avoid diversion of food resources for fuel production. The use of cellulosic sugar hydrolysate (CSH) produced via the NREL process provided a standardized carbon source for study of the underlying biochemistry and metabolic adjustments to mixotrophy. Mixotrophic metabolism uses both photosynthetic CO 2 fixation and sugar oxidation to improve biomass productivity and harvest yield. The project was high-risk, high-reward in the sense that adding organic carbon to algal cultivation is an invitation for rapid-growth, heterotrophic contaminants to overtake the culture and consume mineral nutrients needed for algal growth. To help evade this outcome the project focused on acidophilic red algae that require low pH conditions with 40-48°C optimal temperature profiles that define them as low-range thermophiles. This is the only project in the BETO portfolio to utilize extreme conditions of low pH and high operating temperatures to control pathogens and competitors in mass culture. The project yielded five major outcomes. 1) Harvest densities of 5-10 g ash-free dry weight are easily achieved, providing a 10-fold reduction in water required and dewatering demand. 2) Catabolic repression of photosynthesis in G. sulphuraria is conditionally repressed by low oxygen. 3) Mixotrophic growth on cellulosic hydrolysate consumes all C6 and C5 sugars concomitant with increases in floridean starch with little change in lipid content. Protein content can be manipulated between 35 and 54 wt% by varying the C:N molar ratio from 10:1 to 20:1, respectively. 4) Mixotrophic cultures with up to 50 mM total sugar are remarkably stable at pH 2.5 and daily average reactor temperatures of 40°C. 5) Capital costs for glass tubular photobioreactors are prohibitive for liquid fuel production using a hydrothermal liquefaction processing pathway. The economic outlook for covered raceway ponds is better with a project minimum fuel selling price at $3.32 dm 3 GE -1 .

09 BIOMASS FUELS↗

DOE OSTI2023

Carbon, Nitrogen, and Sulfur Analysis of the Cetama Viognier Standard Reference Material

The Viognier sample was analyzed for C, N, and S concentrations and stable isotope compositions using an Elementar Vario Isotope Cube Elemental Analyzer (EA) that is coupled to an IsoPrime PrecisION IRMS. Powder samples were placed in tin capsules and then loaded onto a rotary autosampler. The autosampler dropped samples into the EA, and samples were combusted at 1175 °C over tungsten oxide in a continuous stream of helium carrier gas. A pulse of oxygen is added to the gas stream resulting in flash combustion of the tin capsule containing the sample, which raises the reaction temperature to approximately 1800 °C for a few seconds. The combustion reaction produces SO 2 , N 2 and CO 2 from any sulfur, nitrogen and carbon present in the sample. The resulting gases were then passed through a reduced copper reactor that was heated to 850 °C, to reduce NO x to N 2 , reduce SO 3 to SO 2 , and trap any volatile halogen compounds on silver wool. Following water removal using an adsorption tube, the N 2 , CO 2 , and SO 2 analyte gases were separated and purified using purge-trap columns. The purified gases were then carried through a thermal conductivity detector. The detector signal was passed to software that calculated elemental abundances based on integrated peak areas. The sample gases were then passed to the IRMS and stable isotope ratios were measured. Raw sample peak areas were corrected by subtracting the average peak area from blanks consisting of empty tin capsules run using the same EA analysis method. Blanks were also run following each replicate to verify that all the material was combusted. The Viognier sample was analyzed in triplicate on two days (target sample masses: 30, 60 and 90 mg). The following standards were analyzed to calibrate EA-IRMS measurements: IAEA-C6, USGS-40, USGS-41, IAEA-S1, and IAEA-S2.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

DOE OSTI2025

Understanding and Harnessing the Robustness of Undomesticated Yarrowia lipolytica Strains for Biosynthesis of Designer Bioesters (Final Report)

This project seeks to elucidate and harness the exceptional robustness of novel and undomesticated Y. lipolytica isolates, which were identified from a genetic diversity screening for compatibility with bioenergy development. Bioenergy-relevant isolates were further developed as microbial platforms for efficient conversion of undetoxified biomass hydrolysates into designer bio-esters continuously recovered by solvent extraction. The project has three major goals. Goal 1. Elucidate and enhance the endogenous metabolism of Y. lipolytica for superior growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates under hypoxic conditions. Goal 2. Understand and enhance the underlying mechanism of exceptional tolerance of Y. lipolytica to organic solvents. Goal 3. Elucidate and rewire endogenous metabolism of the most robust Y. lipolytica strains for effective conversion of accumulating lipids to designer bio-esters. Significant progress has been made toward completing all research goals. We elucidated and optimized the robustness of Y. lipolytica by utilizing mixed C5 and C6 sugars in switchgrass hydrolysates (SGH) for lipid production (Aim 1). We conducted extensive omics analysis to investigate how genetic diversity among Yarrowia strains, derived from natural isolates or developed through adaptive laboratory evolution, influences lipid production when utilizing SGH. In Aim 2, novel mechanisms and underlying genetics were discovered that enabled Yarrowia strains to thrive in cultures containing high ionic liquid (IL) concentrations. Amongst other novel findings, it was found that sterols strengthened cell membranes to confer IL toxicity resistance, specifically via increased ergosterol content upon exposure to IL. In Aim 3, mechanistic studies elucidated how Y. lipolytica utilized intracellular lipids and alkanes/alkenes, leading to our discovery of novel enzymes and pathways for making short-chain esters. Most notably, thermostable chloramphenicol transferases were repurposed to function as alcohol acetyltransferases in Y. lipolytica, as well as the Gram-negative and Gram-positive bacteria Escherichia coli and thermophile Clostridium thermocellum, respectively.

09 BIOMASS FUELS↗

DOE OSTI2025

Catalytic Activation of Pyran Ethers in Self-Solvating Saccharides with Alkaline Earth Metals (Final Report)

Catalytic activation of cellulose ethers occurs via hydroxyl-stabilized cleavage of inter-monomer glycosidic bonds. Cooperativity between two hydroxyl groups lowers the ether cleavage transition state necessary to break apart long carbohydrate chains to initiate small molecule formation as fuel precursors. Metals existing with lignocellulosic materials including alkaline earth metals (Ca 2+ , Mg 2+ ) or alkali (Na + , K + ) also can catalyze ether scission cooperatively. By bonding with carbohydrate hydroxyl groups, a metal cation disrupt the hydrogen bonding network and free carbohydrate functional groups to react; a second metal cation stabilizes the carbohydrate ether transition state and enhances the rate of polymer scission. In this work, we expanded our initial understanding of metal-catalyzed glycoside ether scission. Within polysaccharides such as cellulose, two ether groups exist: one between monomers (glycosidic linkages) and one within a pyran ring. The hydroxyl-group hydrogen bonding network and bound metal ions form low energy binding states that interact with both ether oxygens simultaneously. Based on this interaction, our primary hypothesis was that the competition between stabilizing the two ether scission transition states determines the extent of the major pathways. Metals that disrupt the hydrogen bonding network of the C6 of sugars and stabilize the glycosidic ether promote transglycosylation to levoglucosan; in contrast, metals that stabilize the pyran ether oxygen promote sugar ring fragmentation to furans such as furfural. Varying characteristics of metal cation catalysts and configurations of bound polysaccharide chains dictate the relative rates of ether scission.

09 BIOMASS FUELS↗

DOE OSTI2025

Development of emerging model microorganisms: Megasphaera elsdenii for biomass and organic acid upgrading to fuels and chemicals

The metabolic diversity of microorganisms in nature represents a largely untapped source of valuable compounds that are difficult or impossible to produce in the limited number of available model systems. Efforts to produce longer-chain alcohols, such as hexanol, in organisms like Escherichia coli have met with limited success; production of C6 and larger products remains low, highlighting the challenges of extending chain elongation pathways beyond a single cycle. Megasphaera elsdenii naturally condenses acetyl-CoA to efficiently generate C4–C8 organic acids, making it a promising candidate for producing fuels and chemicals from lactate and plant-derived carbohydrates. This high native flux through the chain elongation pathway offers the potential for higher yields and titers of medium-chain products, such as hexanol, compared to conventional hosts. Recent advances—most notably the development of a transformation method for M. elsdenii—have further opened the organism to detailed physiological studies and bioengineering. While full development of M. elsdenii as a hexanol-producing platform was not achieved, significant progress was made in understanding its metabolism and building foundational genetic tools for future engineering.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid Cytochrome c Assembly in the Green Alga Chlamydomonas reinhardtii

In bacteria and energy generating organelles, c -type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III). System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants (cytochrome c synthesis), which display a block in the apo- to holo- form conversion of cytochrome f and c6, the thylakoid lumen resident c-type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the octatricopeptide-repeat motif (OPR) recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana . Here, we discuss the possible functions of CCS2 in the heme attachment reaction.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1978

Abiotic synthesis of fatty acids

The formation of fatty acids by Fischer-Tropsch-type synthesis was investigated with ferric oxide, ammonium carbonate, potassium carbonate, powdered Pueblito de Allende carbonaceous chondrite, and filings from the Canyon Diablo meteorite used as catalysts. Products were separated and identified by gas chromatography and mass spectrometry. Iron oxide, Pueblito de Allende chondrite, and Canyon Diablo filings in an oxidized catalyst form yielded no fatty acids. Canyon Diablo filings heated overnight at 500 C while undergoing slow purging by deuterium produced fatty acids only when potassium carbonate was admixed; potassium carbonate alone also produced these compounds. The active catalytic combinations gave relatively high yields of aliphatic and aromatic hydrocarbons; substantial amounts of n-alkenes were almost invariably observed when fatty acids were produced; the latter were in the range C6 to C18, with maximum yield in C9 or 10.

Leach, W. W.↗

NASA NTRS1981

The evolution of blue-greens and the origins of chloroplasts

All of the available molecular data support the theory that the chloroplasts of eukaryote cells were originally free-living blue-greens. Of great interest is what the relationships are between contemporary types of blue-greens and eukaryote chloroplasts and whether the chloroplasts of the various eukaryotes are the result of one or more than one symbiosis. By combining information from phylogenetic trees based on cytochrome c6 and 2Fe-2S ferredoxin sequences, it is shown that the chloroplasts of a number of eukaryote algae as well as the protist Euglena are polyphyletic; the chloroplasts of green algae and the higher plants may be the result of a single symbiosis.

Schwartz, R. M.↗

NASA NTRS1983

Evaluation of high-contact-ratio spur gears with profile modification

Scoring tests, surface fatigue tests, and single-tooth bending fatigue tests were conducted with four sets of spur gears of standard design and three sets of spur gears of the new tooth form (NTF) design. Scoring tests were conducted in a gear test rig at a speed of 10,000 rpm using a synthetic polyester C5, C6, C7 substituted pentaerythritol oil. Surface fatigue tests were conducted in the same rig at a speed of 10,000 rpm and Hertz stresses of 173 x 10 to the 7th power and 143 x 10 to the 7th power Pa (250,000 and 214,000 psi). Single tooth bending fatigue tests were conducted on both the standard and NTF gears at an initial load that produced a 10.35 x 10 to the eight power Pa (150,000-psi) bending stress. The gears were load cycled to failure or for 3 x 10 to the 6th power cycles, whichever occurred first. The load was increased after test until failure occurred at 3 x 10 to the 6th power cycles or less. Both the standard and NTF gears scored at a gear bulk temperature of approximately 409 K (277 F). At this temperature the load on the NTF gears was 22 percent less than the load on the standard gears. The scoring failure was a function of gear bulk temperature, where for a given lubricant the temperature is a function of gear design, operating load, and speed.

Townsend, D. P.↗

NASA NTRS1984

The alpha-form of the hydroxides of bivalent metals

X-ray analyses were made of the hydroxides of the bivalent metals. The freshly pptd. hydroxide is usually in the alpha-form, which on standing is converted to another form or other forms. The alpha and c grating dimensions of the alpha-form and the C6-type of Co, Zn, C, Co-Zn and Ni-Zn hydroxides are tabulated. Ni hydroxide does not exhibit an alpha-form. The alpha-Co(OH)2, the blue form, is stabilized by sugar or by the higher alcohols: these compounds do not stabilize alpha-Zn(OH)2.

Feitknecht, W.↗

NASA NTRS1986

The high-temperature oxidation of aromatic hydrocarbons

Chemical mechanisms of the atmospheric pressure, high-temperature (875-1500 K) gas-phase oxidation of benzene, toluene, ethylbenzene, and propylbenzene are described and discussed. Oxidation trends evident from turbulent flow reactor experiments serve as the basis for the mechanisms of the oxidation of benzene and alkylated aromatics. The potential effects of very high temperatures and pressures on the chemistry of oxidation of aromatics are described. The oxidation of benzene and phenyl radical has been found to proceed in a stepwise C6-C5-C4 sequence. Species profiles obtained from flow-reactor experiments suggest that the oxidation of benzene and phenyl radical follows the generalized route via phenoxy, cyclopentadienyl and butadienyl radical. The oxidation of the C4 species branches into multiple pathways that yield copious amounts of ethylene and acetylene. Certain major trends are evident: the alkylated aromatics on initial attack either form styrene, benzyl radical or benzene. The styrene reacts further to produce a benzyl radical or benzene. The oxidation of an alkylated aromatic hydrocarbon appears eventually to reduce to the oxidation of either phenyl radical or benzene.

Brezinsky, K.↗

NASA NTRS1986

XSST/TRC rocket observations of July 13, 1982 flare

The present analysis of UV filtergrams of the July 13, 1982 solar flare obtained by the XSST/TRC rocket experiments has used calibrated intensities of the flare components to directly estimate the Lyman-alpha line flux, C IV line flux, and excess 160-nm continuum temperature brighness over the underlying plage. The values obtained are small by comparison with other observed or calculated equivalent quantities from the Machado (1980) model of flare F1. The corresponding power required to heat up to the temperature minimum over the 1200 sq Mm area is found to be 3.6 x 10 to the 25th erg/sec for this small X-ray C6 flare, 7 min after the ground-based observed flare maximum.

Foing, Bernard H.↗

NASA NTRS1986

Variability of ultraviolet emission in the carbon star TX Piscium

Multiple low-resolution IUE observations of the cool carbon star TX Psc (N0; C6, 2) permit an analysis of the variations in strength of the strongest emission lines - the Mg II line at 2800 A, the C II line at 2330 A, and certain Fe II lines. The integrated flux of the Mg II line varied by at least a factor of eight, while that of the C II line varies by at least a factor of five. The variations in Fe II may be considerably larger. The lines appear to vary together. The continuous flux in the best observed range from 2800 to 3200 A does not vary noticeably.

Johnson, Hollis R.↗

NASA NTRS1988

Antarctic Meteorite Newsletter, Volume 11, Number 2, August 1988

Presented are classifications and descriptions of a large number of meteorites which include the last samples from the 1984 collection and the first samples from the 1987 collection. There is a particularly good selection of meteorites of special petrologic type in the 1987 collection. The achondrites include aubrites, ureilites, howardites, eucrites, and a diogenite. The howardites are particularly notable because of their size and previous scarcity in the Antarctic collection. Noteworthy among the 7 irons and 3 mesosiderities are 2 anamolous irons and 2 large mesosiderites. The carbonaceous chondrites include good suites of C2 and C4 meteorites, and 2 highly equilibrated carbonaceous chondrites tentatively identified as C5 and C6 meteorites. Also included are surveys of numerous meteorites for Al-26 and thermoluminescence. These studies provide information on the thermal and radiation histories of the meteorites and can be used as measures of their terrestrial ages.

Source record↗

NASA NTRS1986

Innervation of periesophageal region of cat's diaphragm - Implication for studies of control of vomiting

The extent of the region of the diaphragm around the esophagus that displays greatly reduced activity during the expulsive phase of vomiting was determined from electromyographic studies in cats to be about 0.75-1.0 cm from the esophagus. Horseradish peroxidase injected into this region retrogradely labeled motoneurons throughout most of the rostral-caudal extent of the phrenic nucleus, with the exception of caudal C6 and rostral C7. This widespread intermingling of motoneurons that innervate the region of reduced activity with other phrenic motoneurons creates a difficulty for needed follow-up studies of diaphragmatic control during vomiting.

Tan, L. K.↗