Reichel Samuel

Structural Characterization and Comparison of Switchgrass Ball-milled Lignin Before and After Dilute Acid Pretreatment

To reduce the recalcitrance and enhance enzymatic activity, dilute H2SO4 pretreatment was carried out on Alamo switchgrass (Panicum virgatum). Ball-milled lignin was isolated from switchgrass before and after pretreatment. Its structure was characterized by 13C, HSQC, and 31P NMR spectroscopy. It was confirmed that ball-milled switchgrass lignin is of HGS type with a considerable amount of p-coumarate and felurate esters of lignin. The major ball-milled lignin interunit was the β-O-4 linkage, and a minor amount of phenylcoumarin, resinol, and spirodienone units were also present. As a result of the acid pretreatment, there was 36% decrease of β-O-4 linkage observed. In addition to these changes, the S/G ratio decreases from 0.80 to 0.53.

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

BackgroundDown-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.ResultsGCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.ConclusionsDown-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Solid-state NMR characterization of switchgrass cellulose after dilute acid pretreatment

Background: Switchgrass (Panicum virgatum L.) is being considered as a potential feedstock for bioethanol production and extensive research is ongoing to establish the optimum pretreatment conditions for switchgrass. Cellulose comprises the most abundant biopolymer in the biosphere and its crystalline/ultrastructure is considered to be related to biomass recalcitrance. This study investigates the effects of dilute acid pretreatment on ultrastructural features of switchgrass cellulose. Results: In this study, switchgrass was pretreated in a pilot-scale reactor at 190°C (0.05 g sulfuric acid per gram of dry switchgrass) with 25% total solid loading and a reactor residence of 1 min. Cellulose was isolated from the pretreated and untreated switchgrass. The impact of pretreatment on the ultrastructure of cellulose was determined by solid-state cross polarization/magic angle spinning 13C NMR spectroscopy. Switchgrass demonstrated a preferable degradation of amorphous cellulose regions during dilute acid pretreatment. The pretreated switchgrass cellulose showed an 18% increase in crystallinity index when compared with the untreated switchgrass. Line-fitting analysis of the C-4 region of 13C NMR spectra revealed that the relative proportion of crystalline and paracrystalline celluloses in switchgrass was observed to increase after dilute acid pretreatment, accompanied with a concurrent decrease of the relative abundance of fibril surface cellulose. Conclusion: After dilute acid pretreatment, most of the hemicellulose in switchgrass was removed. The amorphous cellulose regions in switchgrass were degraded preferably during dilute acid pretreatment and the cellulose crystallinity index of pretreated switchgrass increased. Pretreated switchgrass had an increase in relative proportion of crystalline and paracrystalline cellulose in comparison to the starting material.

Investigation of the fate of poplar lignin during autohydrolysis pretreatment to understand the biomass recalcitrance

Lignocellulosic biomass is the most abundant renewable resource for the potential replacement of fossil fuels, though to fully realize this vision, an improved understanding of the chemical structures of its components within the biomass and after bioprocessing is critical. In this study, we investigated the fate of isolated poplar lignin during autohydrolysis pretreatment at different temperatures and subsequently analyzed the structural changes by gel permeation chromatography, 13C–1H HSQC and phosphorylation/31P NMR. Our results suggested that an increase in temperature and time of autohydrolysis of lignin resulted in an increase in phenolic hydroxyl groups coupled with a decrease in aliphatic hydroxyl groups. This may be attributed to the cleavage of β-O-4 linkages via acidolysis. Molecular weight determination revealed that lignin depolymerization predominates over condensation. Our results also highlight that the cleavage of lignin side-chain units is relatively fast in lignin autohydrolysis compared to the autohydrolysis of biomass. This study provides an enhanced understanding of the fundamental autohydrolysis pretreatment lignin chemistry and will facilitate improved methodology to reduce biomass recalcitrance.

Chemical composition and characterization of cellulose for Agave as a fast-growing, drought-tolerant biofuels feedstock

A major issue raised about development of cellulosic biomass derived fuels technologies is the concern about possible competition for land with agricultural crops and impacts on food and feed supply. However, because agave offers high productivity with low water and nutrient demands, it can thrive on semiarid lands not suitable for conventional agriculture, making it a promising lignocellulosic feedstock for biofuels production. Because agave composition will establish the maximum potential fuel yield that is vital to low cost conversion, detailed chemical composition data and cellulose characteristics were measured by standard biomass analysis procedures and solid-state NMR methods, respectively, for four agave samples: A. americana leaves, A. salmiana leaves, A. tequilana leaves, and A. americana heart. For the first time, we report substrate characteristics relevant to biochemical conversion for the tested agave species, specifically cell wall compositional data along with the relative proportions of cellulose ultra-structural components. The experimental results also provide an important baseline for further characterization and conversion of different agave species as biofuels feedstocks for semi-arid lands.

Structural Characterization of Lignin in Wild-Type versus COMT Down-Regulated Switchgrass

This study examined the chemical structural characteristics of cellulolytic enzyme lignin isolated from switchgrass focusing on comparisons between wild-type control and caffeic acid 3-O-methyltransferase (COMT) down-regulated transgenic line. Nuclear magnetic resonance (NMR) techniques including 13C, 31P, and two-dimensional 13C-1H heteronuclear single quantum coherence (HSQC) as well as gel permeation chromatography (GPC) were employed. Compared to the wild-type, the COMT down-regulated transgenic switchgrass lignin demonstrated a decrease in syringyl (S): guaiacyl (G) ratio and p-coumarate:ferulate ratio, an increase in relative abundance of phenylcoumaran unit, and a comparable content of total free phenolic OH groups along with formation of benzodioxane unit. In addition, COMT down-regulation had no significant effects on the lignin molecular weights during its biosynthesis process.

A review of whole cell wall NMR by the direct-dissolution of biomass

To fully realize the potential of lignocellulosic biomass as a renewable resource for the production of fuels, chemicals, and materials, an improved understanding of the chemical and molecular structures within biomass and how those structures are formed during biosynthesis and transformed during (thermochemical and biological) conversion must be developed. This effort will require analytical techniques which are not only in-depth, rapid, and cost-effective, but also leave native cell wall features intact. Whole plant cell wall nuclear magnetic resonance (NMR) analysis facilitates unparalleled structural characterization of lignocellulosic biomass without causing (or with minimal) structural modification. The objective of this review is to summarize research pertaining to solution- or gel-state whole plant cell wall NMR analysis of biomass, demonstrating the capability of NMR to delineate the structural features and transformations of biomass. In particular, this review will focus on the application of a two-dimensional solution-state NMR technique and perdeuterated ionic liquid based organic electrolyte solvents for the direct dissolution and analysis of biomass. We believe this type of analysis will be critical to advancing biofuel research, improving bioprocessing methodology, and enhancing plant bioengineering efforts.

Recalcitrance and structural analysis by water-only flowthrough pretreatment of (13)C enriched corn stover stem.

This study presents high temperature water-only continuous flowthrough pretreatment coupled with nuclear magnetic resonance (NMR) as a promising analytical tool to examine the plant cell wall, to understand its recalcitrance (i.e., cell wall resistance to deconstruction), and to probe the chemistry occurring during batch pretreatment of biomass. (13)C-enriched corn stover stems were pretreated at 170°C for 60min with a hot-water flow rate of 20mL/min to control fractionation of the cell wall. This approach helped elucidate the nature of plant cell wall chemical recalcitrance and biomass pretreatment chemistry by tracking cell wall fragmentation as a function of time. Fractions of the reactor effluent were collected in a time-resolved fashion and characterized by various NMR techniques to determine the degree and sequence of fragments released, as well as, the chemical composition, molecular structure, and relative molecular weight of those released fragments.