Engineering of an environmental isolate of bacillus megaterium for biochemical production under supercritical CO2

Continuous processing is a mainstay for chemical production but is far less common for biochemical processes. The increase in productivity and corresponding decrease in costs make continuous processing an intriguing option for bulk chemicals where price is a major consideration. Among the various challenges of continuous bioprocessing are the risks of contamination and the toxicity of the target products. Supercritical carbon dioxide (scCO2) may provide a means to address both of these issues. scCO2 is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones and aldehydes. scCO2 is also known for its broad microbial lethality. The isolation and engineering of a microbe that is capable of growth and production in the presence of scCO2 thus represents an opportunity to create a production environment that is both resist to contamination and capable of sequestering toxic products through phase separation. Using a targeted bioprospecting approach by sampling fluid from a natural, deep-subsurface scCO2 well, a strain of Bacillus megaterium was isolated that is able to germinate and grow in the presence of scCO2. Transformation is possible using a protoplast-based method, which permitted the identification of promoters capable of inducible heterologous protein expression in both aerobic and anaerobic conditions. A xylose-inducible promoter was evaluated under scCO2 and found to have similar expression under both conditions. We engineered the B. megaterium strain to produce isobutanol from 2-ketoisovalerate by introducing a two-enzyme pathway (2- ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)). Due to the strong partition of the aldehyde to the scCO2 phase, we tested five homologous Adh enzymes and found that YqhD from E. coli resulted in greater than 85% conversion when grown aerobically. Isobutanol production was also observed when our recombinant strain was cultured under scCO2. Finally, we have developed a process model for an integrated bioprocess and have found conditions that are comparable if not better than existing in situ extraction techniques such as gas stripping. Boock, J.T., A.J.E. Freedman, G.A. Tompsett, S.K. Muse, A.J. Allen, L.A. Jackson, B. Castro-Dominguez, M.T. Timko, K.L.J. Prather (co-corresponding author), J.R. Thompson. 2019. “Engineered microbial biofuel production and recovery under supercritical carbon dioxide.” Nat. Commun. 10:587. DOI: 10.1038/s41467- 019-08486-6

Characterization and reactivity of soot from fast pyrolysis of lignocellulosic compounds and monolignols

peer-reviewedThis study presents the effect of lignocellulosic compounds and monolignols on the yield, nanostructure and reactivity of soot generated at 1250  ° C in a drop tube furnace. The structure of soot was characterized by electron microscopy techniques, Raman spectroscopy and electron spin resonance spectroscopy. The CO2 reactivity of soot was investigated by thermogravimetric analysis. Soot from cellulose was more reactive than soot produced from extractives, lignin and monolignols. Soot reactivity was correlated with the separation distances between adjacent graphene layers, as measured using transmission electron microscopy. Particle size, free radical concentration, differences in a degree of curvature and multi-core structures influenced the soot reactivity less than the interlayer separation distances. Soot yield was correlated with the lignin content of the feedstock. The selection of the extraction solvent had a strong influence on the soot reactivity. The Soxhlet extraction of softwood and wheat straw lignin soot using methanol decreased the soot reactivity, whereas acetone extraction had only a modest effect

Optimizing the Shape Selectivity of Zeolite Catalysts for Biomass Conversion: The Kinetic Diameter

We have studied the influence of catalyst pore size and morphology on the conversion of glucose to aromatics by catalytic fast pyrolysis using over 15 different zeolite catalysts having a variety of shapes and pore sizes. The estimated kinetic diameter for the catalytic pyrolysis products and reactants was used to determine the optimal pore size for zeolite catalysts for catalytic fast pyrolysis. Smaller oxygenate pyrolysis products including furans, hydroxyaldehydes, and organic acids are sufficiently small in diameter to diffuse easily into ZSM-5 (6.3 Å). Of the aromatic products only benzene, toluene, indane, indene, naphthalene, ethylbenzene and xylenes are of a sufficiently small size compared to the ZSM-5 pore. Zeolites type catalysts with a range of pore size 3.9-7.4Å were used for catalytic testing. From these an optimum pore size range of 5.7-6.6Å is identified to maximize aromatic yield. In addition to pore window size, zeolite pore structure and intersections are critical for the reaction mechanism. It is likely that this small pore size also limits the formation of larger aromatics including coke in the pores. Key words: Zeolite, Catalytic Fast Pyrolysis, Kinetic Diameter, Aromatics

Spectroscopic Signatures of Nitrogen-Substituted Zeolites

Zeolites are important heterogeneous acid catalysts with pores the diameter of small molecules. Turning zeolites into bases would open up these unique materials to alkaline-catalyzed reactions, many of which are important in the synthesis of liquid fuels from biomass. One method of preparing alkaline zeolites is to replace some framework oxygen with nitrogen, producing an amine. Unfortunately, such replacements require a post-synthetic treatment, so the question of whether the resulting materials are intact is still open. It is, in general, difficult to characterize these new materials experimentally (since the actual structures can only be guessed), so we employ a mixture of experiment and theory to develop characterization methods suitable for nitrogen-substituted zeolites. We confirm the observation that high-temperature treatment produces new peaks in the silicon NMR spectrum, and present calculations suggesting that these new peaks correspond to framework substitutions. This suggests the possibility of using a zeolitic catalyst in base-catalyzed reactions