high-throughput-screening-recalcitrance-variations-lignocellulosic

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

National Renewable Energy Laboratory

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.

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

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 analysis of advective transport in a biofilm reactor.

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.

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

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 (<Omega(d)>) 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 <Omega(d)> values and changes in <Omega(d)> 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 <Omega(d)>, a broadening in the distribution was observed with aging time. For the domain with high <Omega(d)>, increases in both the mean <Omega(d)> and the width in <Omega(d)> 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.

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

Interventional medical devices based on thermally responsive shape memory polymer (SMP) are under development to treat stroke victims. The goals of these catheter-delivered devices include re-establishing blood flow in occluded arteries and preventing aneurysm rupture. Because these devices alter the hemodynamics and dissipate thermal energy during the therapeutic procedure, a first step in the device development process is to investigate fluid velocity and temperature changes following device deployment.

Magnetic resonance flow velocity and temperature mapping of a shape memory polymer foam device.

The average spatial dimensions between major biopolymers within the plant cell wall can be resolved using a solid-state NMR technique referred to as a (13)C cross-polarization (CP) SELDOM (selectively by destruction of magnetization) with a mixing time delay for spin diffusion. Selective excitation of specific aromatic lignin carbons indicates that lignin is in close proximity to hemicellulose followed by amorphous and finally crystalline cellulose. (13)C spin diffusion time constants (T(SD)) were extracted using a two-site spin diffusion theory developed for (13)C nuclei under magic angle spinning (MAS) conditions. These time constants were then used to calculate an average lower-limit spin diffusion length between chemical groups within the plant cell wall. The results on untreated (13)C enriched corn stover stem reveal that the lignin carbons are, on average, located at distances ∼0.7-2.0 nm from the carbons in hemicellulose and cellulose, whereas the pretreated material had larger separations.

Solid-state selective (13)C excitation and spin diffusion NMR to resolve spatial dimensions in plant cell walls.

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.

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

Large populations of potential cellulosic biomass feedstocks are currently being screened for fuel and chemical applications. The monomeric sugar content, released through hydrolysis, is of particular importance and is currently measured with time-consuming HPLC methods. A method for sugar detection is presented here that employs (1)H NMR spectra regressed against primary HPLC sugar concentration data to build partial least squares (PLS) models. The PLS2 model is able to predict concentrations of both major sugar components, like glucose and xylose, and minor sugars, such as arabinose and mannose, in biomass hydrolysates. The model was built with 65 samples from a variety of different biomass species and covers a wide range of sugar concentrations. Model predictions were validated with a set of 15 samples which were all within error of both HPLC and NMR integration measurements. The data collection time for these NMR measurements is less than 20 min, offering a significant improvement to the 1 h acquisition time that is required for HPLC.

Rapid determination of sugar content in biomass hydrolysates using nuclear magnetic resonance spectroscopy.

Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, "absent technological breakthroughs", it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold.

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production.

The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner.

Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock.

The creation of fuels, chemicals, and materials from plants can aid in replacing products fabricated from non-renewable energy sources. Before using biomass in downstream applications, it must be characterized to assess chemical traits, such as cellulose, lignin, or lignin monomer content, or the sugars released following an acid or enzymatic hydrolysis. The measurement of these traits allows researchers to gage the recalcitrance of the plants and develop efficient deconstruction strategies to maximize yields. Standard methods for assessing biomass phenotypes often have experimental protocols that limit their use for screening sizeable numbers of plant species. Raman spectroscopy, a non-destructive, non-invasive vibrational spectroscopy technique, is capable of providing qualitative, structural information and quantitative measurements. Applications of Raman spectroscopy have aided in alleviating the constraints of standard methods by coupling spectral data with multivariate analysis to construct models capable of predicting analytes. Hydrolysis and fermentation products, such as glucose and ethanol, can be quantified off-, at-, or on-line. Raman imaging has enabled researchers to develop a visual understanding of reactions, such as different pretreatment strategies, in real-time, while also providing integral chemical information. This review provides an overview of what Raman spectroscopy is, and how it has been applied to the analysis of whole lignocellulosic biomass, its derivatives, and downstream process monitoring.

Evaluating lignocellulosic biomass, its derivatives, and downstream products with Raman spectroscopy.

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.

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.

Erica Gjersing

BioEnergy Science Center

National Renewable Energy Laboratory

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

Erica Gjersing

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National Renewable Energy Laboratory

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

BioEnergy Science Center