Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release.

BACKGROUND: Lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance. RESULTS: Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content. The control samples had an average of 29.6 %, the C3'H reduced lines had an average of 21.7 %, and the C4H reduced lines had an average of 18.9 % lignin from wet chemical analysis. The C3'H and C4H down-regulated lines had different lignin compositions with average S/G/H ratios of 48.5/33.2/18.3 for the C3'H reduced lines and 59.0/39.8/1.2 for the C4H reduced lines, compared to the control with 65.9/33.2/1.0. Both the C4H and C3'H down-regulated lines had reduced recalcitrance as indicated by increased sugar release as determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment. CONCLUSIONS: Lowering lignin content rather than altering sinapyl alcohol/coniferyl alcohol/4-coumaryl alcohol ratios was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.

Unraveling the 13C NMR chemical shifts in single-walled carbon nanotubes: dependence on diameter and electronic structure.

The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT (13)C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of (13)C labeling and density gradient ultracentrifugation (DGU) to produce an array of (13)C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic (13)C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the (13)C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.

Thermal degradation in a trimodal poly(dimethylsiloxane) network studied by (1)H multiple quantum NMR.

Thermal degradation of a filled, cross-linked siloxane material synthesized from poly(dimethylsiloxane) chains of three different average molecular weights and with two different cross-linking species has been studied by (1)H multiple quantum (MQ) NMR methods. Multiple domains of polymer chains were detected by MQ NMR exhibiting residual dipolar coupling () values of 200 and 600 Hz, corresponding to chains with high average molecular weight between cross-links and chains with low average molecular weight between cross-links or near the multifunctional cross-linking sites. Characterization of the values and changes in distributions present in the material were studied as a function of time at 250 degrees C and indicate significant time-dependent degradation. For the domains with low , a broadening in the distribution was observed with aging time. For the domain with high , increases in both the mean and the width in were observed with increasing aging time. Isothermal thermal gravimetric analysis reveals a 3% decrease in weight over 20 h of aging at 250 degrees C. Degraded samples also were analyzed by traditional solid-state (1)H NMR techniques, and off-gassing products were identified by solid-phase microextraction followed by gas chromatography-mass spectrometry. The results, which will be discussed here, suggest that thermal degradation proceeds by complex competition between oxidative chain scissioning and postcuring cross-linking that both contribute to embrittlement.

NMR metabolomics of planktonic and biofilm modes of growth in Pseudomonas aeruginosa.

Bacteria often reside in communities where the cells have secreted sticky, polymeric compounds that allow them to attach to surfaces. This sessile lifestyle, referred to as a biofilm, affords the cells within these communities a tolerance of antibiotics and antimicrobial treatments. Biofilms of the bacterium Pseudomonas aeruginosa have been implicated in cystic fibrosis and are capable of colonizing medical implant devices, such as heart valves and catheters, where treatment of the infection often requires the removal of the infected device. This mode of growth is in stark contrast to planktonic, free floating cells, which are more easily eradicated with antibiotics. The mechanisms contributing to a biofilm's tenacity and a planktonic cell's susceptibility are just beginning to be explored. In this study, we have used a metabolomic approach employing nuclear magnetic resonance (NMR) techniques to study the metabolic distinctions between these two modes of growth in P. aeruginosa. One-dimensional 1H NMR spectra of fresh growth medium were compared with spent medium supernatants from batch and chemostat planktonic and biofilms generated in continual flow system culture. In addition, 1H high-resolution magic angle spinning NMR techniques were employed to collect 1H NMR spectra of the corresponding cells. Principal component analysis and spectral comparisons revealed that the overall metabolism of planktonic and biofilm modes of growth appeared similar for the spent media, while the planktonic and biofilm cells displayed marked differences. To determine the robustness of this technique, we prepared cell samples under slightly different preparation methods. Both techniques showed similar results. These feasibility studies show that there exist chemical differences between planktonic and biofilm cells; however, in order to identify these metabolomic differences, more extensive studies would have to be performed, including 1H-1H total correlated spectroscopy.

Magnetic resonance microscopy analysis of advective transport in a biofilm reactor.

In this article we present magnetic resonance microscopy (MRM) characterization of the advective transport in a biofilm capillary reactor. The biofilm generates non-axial flows that are up to 20% of the maximum axial velocity. The presence of secondary velocities of this magnitude alters the mass transport in the bioreactor relative to non-biofilm fouled reactors and questions the applicability of empirical mass transfer coefficient approaches. The data are discussed in the context of simulations and models of biofilm transport and conceptual aspects of transport modeling in complex flows are also discussed. The variation in the residence time distribution due to biofilm growth is calculated from the measured propagator of the motion. Dynamical systems methods applied to model fluid mixing in complex flows are indicated as a template for extending mass transport theory to quantitatively incorporate microscale data on the advection field into macroscale mass transfer models.

Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor.

Microorganisms that colonize surfaces, biofilms, are of significant importance due to their role in medical infections, subsurface contaminant remediation, and industrial processing. Spatially resolved data on the distribution of biomass within a capillary bioreactor, the heterogeneity of the biofilm itself and the impact on transport dynamics for a Staphylococcus epidermidis biofilm in the natural growth state are presented. The data demonstrate the ability of magnetic resonance microscopy to study spatially resolved processes in bacterial biofilms, thus providing a basis for future studies of spatially resolved metabolism and in vivo clinical detection.