Additional file 12: Table S9. File with FPKM values and DESeq2 statistics for all samples. The fi... more Additional file 12: Table S9. File with FPKM values and DESeq2 statistics for all samples. The file contains gene identifiers and various annotations along with the FPKM values for each sample and condition, the fold change and p adj values as calculated by DESeq2 for the comparison between the Glu 48Â h control and the other 26 conditions.
Additional file 8: Table S6. Analysis of genes encoding enzymes with auxiliary activities (AA) re... more Additional file 8: Table S6. Analysis of genes encoding enzymes with auxiliary activities (AA) relevant to lignin breakdown. Graphs of the total FPKM attributed to AA families relevant to lignin breakdown, number of induced AA members in each of the conditions compared to the Glu-48Â h control, Venn diagram comparing the induced genes on each of the time courses and the dataset of the AA FPKM values and Venn diagram membership of the induced AAs.
Additional file 11: Figure S3. Particle sizes of untreated and pretreated lignocellulosic substra... more Additional file 11: Figure S3. Particle sizes of untreated and pretreated lignocellulosic substrates. The figure contains images of the particle sizes of the untreated and pretreated straw substrates. The yellow scale bar in each image represents a length of 10Â mm.
Additional file 14: Table S11. CAZy, transcription factor and sugar transporter content of MFuzz ... more Additional file 14: Table S11. CAZy, transcription factor and sugar transporter content of MFuzz clusters from the HTS time-course. File with lists of genes from each cluster from the HTS time-course of all genes from the categories of CAZy, Pfam for sugar transporters and Pfam for transcription factors that met selection criteria. Only genes strongly associated with a clusterâ s predominant expression profile (membership value > 0.5 as well as an FPKM > 10 at the clusterâ s expression peak) were selected which led to the capture of between a half and a third of each category.
Additional file 8. Additional Methods. Determination of sugars, organic acids and monomeric aroma... more Additional file 8. Additional Methods. Determination of sugars, organic acids and monomeric aromatics in the hydrolysate.
Additional file 2. Isopentenol Production in C. glutamicum Strains Cultivated in Rich vs. Minimal... more Additional file 2. Isopentenol Production in C. glutamicum Strains Cultivated in Rich vs. Minimal Media. (A) Isopentenol production in minimal media: C. glutamicum was prepared for isopentenol production in two minimal media; M9 and CGXII supplemented with either 1% or 4% d-glucose as the carbon source in 5Â mL tubes. (B) Isopentenol production in rich media: C. glutamicum was prepared for isopentenol production in LB media supplemented with either 1% or 4% d-glucose as the carbon source in a 5Â mL culture tube. Data shown are production 48Â h after induction and is the average of biological triplicates; error bars represent standard error.
Additional file 1. Evaluation of Production Condition and C. glutamicum Properties as the Isopent... more Additional file 1. Evaluation of Production Condition and C. glutamicum Properties as the Isopentenol Production Chassis. (A-B) Growth assay of E. coli and C. glutamicum to ILs; 0–300 mM [C2C1im][OAc] and 0–40 mM [Ch][Lys] in LB in 96 well microtiter plates. (C) Analysis of isopentenol evaporation or consumption by C. glutamicum in a 5 mL cultivation format. (D) The same evaporation assay as in (C), but in a 24 well format.
Additional file 4: Figure S4. Correlation between EGFP and mRuby2 expression from promoter P9 in ... more Additional file 4: Figure S4. Correlation between EGFP and mRuby2 expression from promoter P9 in all 4 media and all 5 time points. Fluorescence expression values are on a Log2 scale. R2 = 0.5537 for a P value of 0.0002. Solid line is the linear regression and dashed lines are 95% confidence interval.
Additional file 3. XRD spectrum and crystallinity index (CrI) of agave bagasse under different co... more Additional file 3. XRD spectrum and crystallinity index (CrI) of agave bagasse under different conditions (untreated, Fresh IL pretreated and IL-recycled). XRD diffractograms of untreated and pretreated agave bagasse under different process conditions.
Additional file 2. Chemical changes tracked of fresh and recycled [C2C1Im][OAc] (up) and [C4C1Im]... more Additional file 2. Chemical changes tracked of fresh and recycled [C2C1Im][OAc] (up) and [C4C1Im][OAc] (down). FTIR spectra of recycled ionic liquids [C2C1Im][OAc] and [C4C1Im][OAc] from three different cycles.
Pseudomonas putida KT2440, a promising microbial platform for industrial biotechnology was toleri... more Pseudomonas putida KT2440, a promising microbial platform for industrial biotechnology was tolerized to low-cost biomass decomposing ionic liquids via the adaptive laboratory evolution.
eign oil, finite oil and gas resources, and the negative consequences associated with global warm... more eign oil, finite oil and gas resources, and the negative consequences associated with global warming due to continued greenhouse gas emissions have prompted significant interest in the development of low carbon and sustainable biofuels. Lignocellulosic biomass has the potential to be a low-cost feedstock for biofuel production, with costs estimated at US$50–70 per ton for certain types of biomass as being cost-competitive on a per gallon basis with current fossil fuels. The rapid creation and deployment of cost effective and scalable processes for converting this biomass into infrastructure-compatible, low carbon and sustainable transportation biofuels is critical to the economic vitality and energy security of the world. The majority of first-generation biofuels produced commercially to date are those derived from starches (e.g., corn) and vegetable oils (e.g., rapeseed oil). While these biofuels have reached relatively significant production levels over a comparatively short period of time, and have succeeded in establishing that biofuels in general are a feasible alternative to fossil fuels in terms of performance, there are several challenges to the current approach of biofuel production, distribution and infrastructure integration (e.g., the blend wall) that could be avoided with the appropriate selection and p roduction of the next wave of advanced biofuels. In the USA, there has been significant public and private investment into the development of these advanced biofuels; for example, the US Department of Energy has funded three Bioenergy Research Centers (total program funding of US$402 million over 5 years) to develop new approaches to advanced biofuel production: the BioEnergy Science Center (led by Oak Ridge National Laboratory, TN, USA) [101], the Great Lakes Bioenergy Research Center (led by the University of Wisconsin – Madison, WI, USA) [102], and the Joint BioEnergy Institute (led by Lawrence Berkeley National Laboratory, CA, USA) [103]. In the private sector, BP has invested US$500 million at the University of California – Berkeley (CA, USA) and has established the Energy Biosciences Institute [104]. ExxonMobil recently invested US$300 million in Synthetic Genomics Inc. for research and development into the engineering of algae designed to produce advanced biofuels. There are several start-up companies in the USA and the world actively engaged in the rapid commercialization and scale-up of advanced Opportunities and challenges in advanced biofuel production: the importance of synthetic biology and combustion science Editorial
ABSTRACTClathrate hydrates are crystalline inclusion compounds of water and a guest molecule (e.g... more ABSTRACTClathrate hydrates are crystalline inclusion compounds of water and a guest molecule (e.g., methane) that form at temperatures below ambient but above the freezing point of water. There are three known crystalline structures of hydrates (structure I, II, and H) in which cavities within the hydrogen bonded water molecule lattice trap the hydrate-forming species. The clathrate structure excludes dissolved solutes, such as sodium chloride, from the aqueous phase and thereby offers a possible means to produce potable water from seawater or brackish water. The concept of using clathrate hydrates for desalination is not new. However, before clathrate hydrate desalination becomes a viable technology, fundamental issues of controlled hydrate formation, hydrate size and morphology, agglomeration, amount of entrapped salt, and the efficient recovery of hydrates must be understood. This paper will report structural characterization of hydrates formed with several guest molecules over a...
Molecular dynamics simulations were performed on methane clathrate hydrates at ambient conditions... more Molecular dynamics simulations were performed on methane clathrate hydrates at ambient conditions. Thermal expansion results over the temperature range 60-300 K show that the unit cell volume increases with temperature in agreement with experiment. Power spectra were obtained at 273 K from velocity autocorrelation functions for selected atoms, and normal modes were assigned. The spectra were further classified according to individual atom types, allowing the assignment of contributions from methane molecules located in small and large cages within the structure I unit cell. The symmetric C-H stretch of methane in the small cages occurs at a higher frequency than for methane located in the large cages, with a peak separation of 14 cm-1. Additionally, we determined that the symmetric C-H stretch in methane gas occurs at the same frequency as methane in the large cages. Results of molecular dynamics simulations indicate the use of power spectra obtained from the velocity autocorrelation function is a reliable method to investigate the vibrational behavior of guest molecules in clathrate hydrates.
The structures formed in a four-component water-in-oil microemulsion consisting of deuterated wat... more The structures formed in a four-component water-in-oil microemulsion consisting of deuterated water added to sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and phosphatidycholine (lecithin) in 2,2,4trimethylpentane (isooctane) have been probed by small-angle neutron scattering (SANS). The total surfactant concentration in each of the five samples studied is fixed at 0.01 M in isooctane. The water content is fixed at Wt) 20 (Wt) moles water/moles total surfactant). The molar ratios of lecithin to AOT are 0, 1:7, 1:3, 3:5, and 1:1 in the five samples, respectively. The experimental SANS data from all five samples are fit well by a model containing polydispersed spheres. The mean size of the spheres increases, whereas the polydispersity decreases as the lecithin content is increased. The different packing parameters for AOT and lecithin drive the observed changes in droplet size, whereas the reduction in polydispersity is a consequence of the increased rigidity of the interface as lecithin is added. An alternate morphology, consisting of prolate ellipsoids, also produces good fits to the SANS data. For this model, the ellipsoid volume increases, whereas the ratio of the major to minor axis goes toward unity as the lecithin content is increased. Radii of gyration obtained from fits in the Guinier/Zimm region match calculations from the droplet dimensions produced by fitting the complete scattering profiles. For lecithin/AOT molar ratios of 3/5 and 1, SANS measurements reveal that an attractive interaction develops between these droplets. The interdroplet interaction is described using a square well potential model. Fits to the SANS data give well depths of 0.022 and 0.126 kT, whereas the widths are 538 and 450 Å, respectively. The weak interaction over such long length scales is consistent with the formation of loosely bound clusters of microemulsion droplets at these lecithin concentrations.
The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low su... more The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low sulfonate (LS)--before and after treatment with 1-ethyl-3-methylimidazolium acetate were studied using small-angle neutron scattering (SANS) and dynamic light scattering (DLS) over a concentration range of 0.3-2.4 wt %. The results indicate that each of these lignins is comprised of aggregates of well-defined basal subunits, the shapes and sizes of which, in D(2)O and DMSO-d(6), are revealed using these techniques. LS lignin contains a substantial amount of nanometer-scale individual subunits. In aqueous solution these subunits have a well-defined elongated shape described well by ellipsoidal and cylindrical models. At low concentration the subunits are highly expanded in alkaline solution, and the effect is screened with increasing concentration. OS lignin dissolved in DMSO was found to consist of a narrow distribution of aggregates with average radius 200 ± 30 nm. K lignin in DMSO consists of aggregates with a very broad size distribution. After ionic liquid (IL) treatment, LS lignin subunits in alkaline solution maintained the elongated shape but were reduced in size. IL treatment of OS and K lignins led to the release of nanometer-scale subunits with well-defined size and shape.
Additional file 12: Table S9. File with FPKM values and DESeq2 statistics for all samples. The fi... more Additional file 12: Table S9. File with FPKM values and DESeq2 statistics for all samples. The file contains gene identifiers and various annotations along with the FPKM values for each sample and condition, the fold change and p adj values as calculated by DESeq2 for the comparison between the Glu 48Â h control and the other 26 conditions.
Additional file 8: Table S6. Analysis of genes encoding enzymes with auxiliary activities (AA) re... more Additional file 8: Table S6. Analysis of genes encoding enzymes with auxiliary activities (AA) relevant to lignin breakdown. Graphs of the total FPKM attributed to AA families relevant to lignin breakdown, number of induced AA members in each of the conditions compared to the Glu-48Â h control, Venn diagram comparing the induced genes on each of the time courses and the dataset of the AA FPKM values and Venn diagram membership of the induced AAs.
Additional file 11: Figure S3. Particle sizes of untreated and pretreated lignocellulosic substra... more Additional file 11: Figure S3. Particle sizes of untreated and pretreated lignocellulosic substrates. The figure contains images of the particle sizes of the untreated and pretreated straw substrates. The yellow scale bar in each image represents a length of 10Â mm.
Additional file 14: Table S11. CAZy, transcription factor and sugar transporter content of MFuzz ... more Additional file 14: Table S11. CAZy, transcription factor and sugar transporter content of MFuzz clusters from the HTS time-course. File with lists of genes from each cluster from the HTS time-course of all genes from the categories of CAZy, Pfam for sugar transporters and Pfam for transcription factors that met selection criteria. Only genes strongly associated with a clusterâ s predominant expression profile (membership value > 0.5 as well as an FPKM > 10 at the clusterâ s expression peak) were selected which led to the capture of between a half and a third of each category.
Additional file 8. Additional Methods. Determination of sugars, organic acids and monomeric aroma... more Additional file 8. Additional Methods. Determination of sugars, organic acids and monomeric aromatics in the hydrolysate.
Additional file 2. Isopentenol Production in C. glutamicum Strains Cultivated in Rich vs. Minimal... more Additional file 2. Isopentenol Production in C. glutamicum Strains Cultivated in Rich vs. Minimal Media. (A) Isopentenol production in minimal media: C. glutamicum was prepared for isopentenol production in two minimal media; M9 and CGXII supplemented with either 1% or 4% d-glucose as the carbon source in 5Â mL tubes. (B) Isopentenol production in rich media: C. glutamicum was prepared for isopentenol production in LB media supplemented with either 1% or 4% d-glucose as the carbon source in a 5Â mL culture tube. Data shown are production 48Â h after induction and is the average of biological triplicates; error bars represent standard error.
Additional file 1. Evaluation of Production Condition and C. glutamicum Properties as the Isopent... more Additional file 1. Evaluation of Production Condition and C. glutamicum Properties as the Isopentenol Production Chassis. (A-B) Growth assay of E. coli and C. glutamicum to ILs; 0–300 mM [C2C1im][OAc] and 0–40 mM [Ch][Lys] in LB in 96 well microtiter plates. (C) Analysis of isopentenol evaporation or consumption by C. glutamicum in a 5 mL cultivation format. (D) The same evaporation assay as in (C), but in a 24 well format.
Additional file 4: Figure S4. Correlation between EGFP and mRuby2 expression from promoter P9 in ... more Additional file 4: Figure S4. Correlation between EGFP and mRuby2 expression from promoter P9 in all 4 media and all 5 time points. Fluorescence expression values are on a Log2 scale. R2 = 0.5537 for a P value of 0.0002. Solid line is the linear regression and dashed lines are 95% confidence interval.
Additional file 3. XRD spectrum and crystallinity index (CrI) of agave bagasse under different co... more Additional file 3. XRD spectrum and crystallinity index (CrI) of agave bagasse under different conditions (untreated, Fresh IL pretreated and IL-recycled). XRD diffractograms of untreated and pretreated agave bagasse under different process conditions.
Additional file 2. Chemical changes tracked of fresh and recycled [C2C1Im][OAc] (up) and [C4C1Im]... more Additional file 2. Chemical changes tracked of fresh and recycled [C2C1Im][OAc] (up) and [C4C1Im][OAc] (down). FTIR spectra of recycled ionic liquids [C2C1Im][OAc] and [C4C1Im][OAc] from three different cycles.
Pseudomonas putida KT2440, a promising microbial platform for industrial biotechnology was toleri... more Pseudomonas putida KT2440, a promising microbial platform for industrial biotechnology was tolerized to low-cost biomass decomposing ionic liquids via the adaptive laboratory evolution.
eign oil, finite oil and gas resources, and the negative consequences associated with global warm... more eign oil, finite oil and gas resources, and the negative consequences associated with global warming due to continued greenhouse gas emissions have prompted significant interest in the development of low carbon and sustainable biofuels. Lignocellulosic biomass has the potential to be a low-cost feedstock for biofuel production, with costs estimated at US$50–70 per ton for certain types of biomass as being cost-competitive on a per gallon basis with current fossil fuels. The rapid creation and deployment of cost effective and scalable processes for converting this biomass into infrastructure-compatible, low carbon and sustainable transportation biofuels is critical to the economic vitality and energy security of the world. The majority of first-generation biofuels produced commercially to date are those derived from starches (e.g., corn) and vegetable oils (e.g., rapeseed oil). While these biofuels have reached relatively significant production levels over a comparatively short period of time, and have succeeded in establishing that biofuels in general are a feasible alternative to fossil fuels in terms of performance, there are several challenges to the current approach of biofuel production, distribution and infrastructure integration (e.g., the blend wall) that could be avoided with the appropriate selection and p roduction of the next wave of advanced biofuels. In the USA, there has been significant public and private investment into the development of these advanced biofuels; for example, the US Department of Energy has funded three Bioenergy Research Centers (total program funding of US$402 million over 5 years) to develop new approaches to advanced biofuel production: the BioEnergy Science Center (led by Oak Ridge National Laboratory, TN, USA) [101], the Great Lakes Bioenergy Research Center (led by the University of Wisconsin – Madison, WI, USA) [102], and the Joint BioEnergy Institute (led by Lawrence Berkeley National Laboratory, CA, USA) [103]. In the private sector, BP has invested US$500 million at the University of California – Berkeley (CA, USA) and has established the Energy Biosciences Institute [104]. ExxonMobil recently invested US$300 million in Synthetic Genomics Inc. for research and development into the engineering of algae designed to produce advanced biofuels. There are several start-up companies in the USA and the world actively engaged in the rapid commercialization and scale-up of advanced Opportunities and challenges in advanced biofuel production: the importance of synthetic biology and combustion science Editorial
ABSTRACTClathrate hydrates are crystalline inclusion compounds of water and a guest molecule (e.g... more ABSTRACTClathrate hydrates are crystalline inclusion compounds of water and a guest molecule (e.g., methane) that form at temperatures below ambient but above the freezing point of water. There are three known crystalline structures of hydrates (structure I, II, and H) in which cavities within the hydrogen bonded water molecule lattice trap the hydrate-forming species. The clathrate structure excludes dissolved solutes, such as sodium chloride, from the aqueous phase and thereby offers a possible means to produce potable water from seawater or brackish water. The concept of using clathrate hydrates for desalination is not new. However, before clathrate hydrate desalination becomes a viable technology, fundamental issues of controlled hydrate formation, hydrate size and morphology, agglomeration, amount of entrapped salt, and the efficient recovery of hydrates must be understood. This paper will report structural characterization of hydrates formed with several guest molecules over a...
Molecular dynamics simulations were performed on methane clathrate hydrates at ambient conditions... more Molecular dynamics simulations were performed on methane clathrate hydrates at ambient conditions. Thermal expansion results over the temperature range 60-300 K show that the unit cell volume increases with temperature in agreement with experiment. Power spectra were obtained at 273 K from velocity autocorrelation functions for selected atoms, and normal modes were assigned. The spectra were further classified according to individual atom types, allowing the assignment of contributions from methane molecules located in small and large cages within the structure I unit cell. The symmetric C-H stretch of methane in the small cages occurs at a higher frequency than for methane located in the large cages, with a peak separation of 14 cm-1. Additionally, we determined that the symmetric C-H stretch in methane gas occurs at the same frequency as methane in the large cages. Results of molecular dynamics simulations indicate the use of power spectra obtained from the velocity autocorrelation function is a reliable method to investigate the vibrational behavior of guest molecules in clathrate hydrates.
The structures formed in a four-component water-in-oil microemulsion consisting of deuterated wat... more The structures formed in a four-component water-in-oil microemulsion consisting of deuterated water added to sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and phosphatidycholine (lecithin) in 2,2,4trimethylpentane (isooctane) have been probed by small-angle neutron scattering (SANS). The total surfactant concentration in each of the five samples studied is fixed at 0.01 M in isooctane. The water content is fixed at Wt) 20 (Wt) moles water/moles total surfactant). The molar ratios of lecithin to AOT are 0, 1:7, 1:3, 3:5, and 1:1 in the five samples, respectively. The experimental SANS data from all five samples are fit well by a model containing polydispersed spheres. The mean size of the spheres increases, whereas the polydispersity decreases as the lecithin content is increased. The different packing parameters for AOT and lecithin drive the observed changes in droplet size, whereas the reduction in polydispersity is a consequence of the increased rigidity of the interface as lecithin is added. An alternate morphology, consisting of prolate ellipsoids, also produces good fits to the SANS data. For this model, the ellipsoid volume increases, whereas the ratio of the major to minor axis goes toward unity as the lecithin content is increased. Radii of gyration obtained from fits in the Guinier/Zimm region match calculations from the droplet dimensions produced by fitting the complete scattering profiles. For lecithin/AOT molar ratios of 3/5 and 1, SANS measurements reveal that an attractive interaction develops between these droplets. The interdroplet interaction is described using a square well potential model. Fits to the SANS data give well depths of 0.022 and 0.126 kT, whereas the widths are 538 and 450 Å, respectively. The weak interaction over such long length scales is consistent with the formation of loosely bound clusters of microemulsion droplets at these lecithin concentrations.
The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low su... more The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low sulfonate (LS)--before and after treatment with 1-ethyl-3-methylimidazolium acetate were studied using small-angle neutron scattering (SANS) and dynamic light scattering (DLS) over a concentration range of 0.3-2.4 wt %. The results indicate that each of these lignins is comprised of aggregates of well-defined basal subunits, the shapes and sizes of which, in D(2)O and DMSO-d(6), are revealed using these techniques. LS lignin contains a substantial amount of nanometer-scale individual subunits. In aqueous solution these subunits have a well-defined elongated shape described well by ellipsoidal and cylindrical models. At low concentration the subunits are highly expanded in alkaline solution, and the effect is screened with increasing concentration. OS lignin dissolved in DMSO was found to consist of a narrow distribution of aggregates with average radius 200 ± 30 nm. K lignin in DMSO consists of aggregates with a very broad size distribution. After ionic liquid (IL) treatment, LS lignin subunits in alkaline solution maintained the elongated shape but were reduced in size. IL treatment of OS and K lignins led to the release of nanometer-scale subunits with well-defined size and shape.
Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bond... more Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzy-matic breakdown of aromatic oligomers linked via-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site ser-ine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because-aryl ether bonds account for 50 –70% of all interunit linkages in lignin, understanding the mechanism of enzymatic-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
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Papers by Blake Simmons