Identification of 4-O-5-Units in Softwood Lignins via Definitive Lignin Models and NMR.

Lignins are complex and heterogeneous natural polymers in which the major units are characterized by certain prominent interunit linkages. Previous attempts to identify and quantify 4-O-5-linked units in softwood lignins by NMR were not successful. In this work, various lignin model compounds, including the tetramers formed by the 4-O-5-coupling of ß-O-4-, ß-ß-, and ß-5-model dimers, were synthesized. Such compounds are better able to model the corresponding structures in lignins than those used previously. 4-O-5-Linked structures could be clearly observed and identified in real softwood lignin samples by comparison of their 2D HSQC NMR spectra with those from the model compounds. When comparing NMR data of phenol-acetylated versus phenol-etherified model compounds with those of acetylated lignins, it was apparent that most of the 4-O-5-linked structures in softwood lignins are present as free-phenolic end units.

The effect of ethanol on hydroxyl and carbonyl groups in biopolyol produced by hydrothermal liquefaction of loblolly pine: (31)P-NMR and (19)F-NMR analysis.

The goal of this study was to investigate the role of ethanol and temperature on the hydroxyl and carbonyl groups in biopolyol produced from hydrothermal liquefaction of loblolly pine (Pinus spp.) carried out at 250, 300, 350 and 390°C for 30min. Water and water/ethanol mixture (1/1, wt/wt) were used as liquefying solvent in the HTL experiments. HTL in water and water/ethanol is donated as W-HTL and W/E-HTL, respectively. It was found that 300°C and water/ethanol solvent was the optimum liquefaction temperature and solvent, yielding up to 68.1wt.% bio-oil and 2.4wt.% solid residue. (31)P-NMR analysis showed that biopolyol produced by W-HTL was rich in phenolic OH while W/E-HTL produced more aliphatic OH rich biopolyols. Moreover, biopolyols with higher hydroxyl concentration were produced by W/E-HTL. Carbonyl groups were analyzed by (19)F-NMR, which showed that ethanol reduced the concentration of carbonyl groups.

Laboratory Evaluations of Durability of Southern Pine Pressure Treated With Extractives From Durable Wood Species.

Extracts from sawdust of four naturally durable wood species [Alaskan yellow cedar, AYC, Cupressus nootkanansis D. Don 1824; eastern red cedar, ERC, Juniperus virginiana L.; honey mesquite, HM, Prosopis glandulosa Torr.; and black locust, BL, Robinia pseudoacacia L.] were used to treat southern pine, Pt, Pinus taeda L. sapwood blocks. Extractive treated blocks were evaluated for decay resistance in standard soil bottle fungal assays challenged with brown and white rot decay fungi. Results showed that extractives did impart some improvement to decay resistance of Pt blocks. BL- and HM-treated Pt blocks were also used in choice and no-choice assays to determine feeding preference and damage by eastern subterranean termites (Reticulitermes flavipes) Kollar. Minimal feeding on treated blocks was seen in both choice and no-choice assays. In choice assays, there was similar mortality between HM and BL arenas; however, in no-choice assays, complete mortality was recorded for HM-treated Pt and high mortality was seen with BL-treated Pt. Subsequent dose mortality termite assays showed HM to be effective in killing R. flavipes at low concentrations. Both HM and BL show promise as deterrents or termiticidal protectants and will be further evaluated in field studies.

Microbial lipid production by oleaginous Rhodococci cultured in lignocellulosic autohydrolysates.

Metabolic synthesis of single cell oils (SCOs) for biodiesel application by heterotrophic oleaginous microorganisms is being hampered by the high cost of culture media. This study investigated the possibility of using loblolly pine and sweetgum autohydrolysates as economic feedstocks for microbial lipid production by oleaginous Rhodococcus opacus (R. opacus) PD630 and DSM 1069. Results revealed that when the substrates were detoxified by the removal of inhibitors (such as HMF-hydroxymethyl-furfural), the two strains exhibited viable growth patterns after a short adaptation/lag phase. R. opacus PD630 accumulated as much as 28.6 % of its cell dry weight (CDW) in lipids while growing on detoxified sweetgum autohydrolysate (DSAH) that translates to 0.25 g/l lipid yield. The accumulation of SCOs reached the level of oleagenicity in DSM 1069 cells (28.3 % of CDW) as well, while being cultured on detoxified pine autohydrolysate (DPAH), with the maximum lipid yield of 0.31 g/l. The composition of the obtained microbial oils varied depending on the substrates provided. These results indicate that lignocellulosic autohydrolysates can be used as low-cost fermentation substrates for microbial lipid production by wild-type R. opacus species. Consequently, the variety of applications for aqueous liquors from lignocellulosic pretreatment has been expanded, allowing for the further optimization of the integrated biorefinery.

Biomass logistics analysis for large scale biofuel production: case study of loblolly pine and switchgrass.

The objective of this study was to assess the costs, energy consumption and greenhouse gas (GHG) emissions throughout the biomass supply chain for large scale biofuel production. Two types of energy crop were considered, switchgrass and loblolly pine, as representative of herbaceous and woody biomass. A biomass logistics model has been developed to estimate the feedstock supply system from biomass production through transportation. Biomass in the form of woodchip, bale and pellet was investigated with road, railway and waterway transportation options. Our analysis indicated that the farm or forest gate cost is lowest for loblolly pine whole tree woodchip at $39.7/dry tonne and highest for switchgrass round bale at $72.3/dry tonne. Switchgrass farm gate GHG emissions is approximately 146kgCO2e/dry tonne, about 4 times higher than loblolly pine. The optimum biomass transportation mode and delivered form are determined by the tradeoff between fixed and variable costs for feedstock shipment.

Discovering candidate genes that regulate resin canal number in Pinus taeda stems by integrating genetic analysis across environments, ages, and populations.

Genetically improving constitutive resin canal development in Pinus stems may enhance the capacity to synthesize terpenes for bark beetle resistance, chemical feedstocks, and biofuels. To discover genes that potentially regulate axial resin canal number (RCN), single nucleotide polymorphisms (SNPs) in 4027 genes were tested for association with RCN in two growth rings and three environments in a complex pedigree of 520 Pinus taeda individuals (CCLONES). The map locations of associated genes were compared with RCN quantitative trait loci (QTLs) in a (P. taeda × Pinus elliottii) × P. elliottii pseudo-backcross of 345 full-sibs (BC1). Resin canal number was heritable (h(2) ˜ 0.12-0.21) and positively genetically correlated with xylem growth (rg ˜ 0.32-0.72) and oleoresin flow (rg ˜ 0.15-0.51). Sixteen well-supported candidate regulators of RCN were discovered in CCLONES, including genes associated across sites and ages, unidirectionally associated with oleoresin flow and xylem growth, and mapped to RCN QTLs in BC1. Breeding is predicted to increase RCN 11% in one generation and could be accelerated with genomic selection at accuracies of 0.45-0.52 across environments. There is significant genetic variation for RCN in loblolly pine, which can be exploited in breeding for elevated terpene content.

An effective chemical pretreatment method for lignocellulosic biomass with substituted imidazoles.

Lignocellulosic biomass is the most abundant naturally renewable organic resource for biofuel production. Because of its recalcitrance to enzymatic degradation, pretreatment is a crucial step before hydrolysis of the feedstock. A variety of pretreatment methods have been developed and intensively studied to achieve optimal yield without imposing significant adverse impact on the environment. Herein, we present a novel chemical pretreatment method using substituted heterocycles with low temperature and short residence time requirements. 1-Methylimidazole (MI) is a precursor to some imidazolium-based ionic liquids. In this study, its potential utilization as a biomass pretreatment agent is being investigated for the first time. At mild conditions, such as 25°C for 5 min at ambient pressure, a substantial increase in the hydrolysis rate throughout the entire course of conversion for cellulose substrate was obtained. Furthermore, the pretreatment effectiveness of MI on both untreated and steam-exploded lignocellulosic biomass including loblolly pine, switchgrass, and sugarcane bagasse has been studied and MI was found to be an efficient delignifier. Remarkable rate enhancement was also observed for the non-woody lignocellulosic substrates after a short period of MI pretreatment at ambient conditions. The mechanism of MI pretreatment is explored through analysis of cellulose physical properties including crystallinity index, degree of polymerization, accessibility, and lignin dissolution quantification.

SO2 -catalyzed steam explosion: the effects of different severity on digestibility, accessibility, and crystallinity of lignocellulosic biomass.

Lignocellulosic biomass is the most promising feedstock for biofuels production. To enhance the efficiency of enzymatic hydrolysis, lignocellulosics needs to be pretreated to lower their recalcitrance. SO(2) -catalyzed steam explosion is an efficient and relatively cost-efficient pretreatment method for softwood. This work investigates the effects of steam explosion severity on the digestibility, accessibility, and crystallinity of Loblolly pine. Higher severity was found to increase the accessibility of the feedstock while also promoting nonselective degradation of carbohydrates. The adsorption behavior of Celluclast® enzymes on steam-exploded Loblolly pine (SELP) can be described by a Langmuir isotherm. Cellulose crystallinity was found to first increase and then decrease with increasing pretreatment severity. A linear relationship between initial hydrolysis rates and crystallinity index (CrI) of pretreated Loblolly pine was found; moreover, a strong correlation between X-ray diffraction intensities and initial rates was confirmed. The findings demonstrate the significance of CrI in enzymatic hydrolysis of pretreated lignocellulosic biomass.

Distinct roles of residual xylan and lignin in limiting enzymatic hydrolysis of organosolv pretreated loblolly pine and sweetgum.

The interactions between xylan/lignin and cellulase enzymes play a key role in the effective hydrolysis of lignocellulosic biomass. Organosolv pretreated loblolly pine (OPLP) and sweetgum (OPSG) were used to quantitatively elucidate the distinct roles of residual xylan and lignin on enzymatic hydrolysis, based on the initial hydrolysis rates and the final hydrolysis yields. The initial hydrolysis rates of OPLP and OPSG were 1.45 (glucose) and 1.19 g/L/h (glucose), respectively, under the enzyme loading of 20 FPU/g glucan. The final glucan hydrolysis yields of OPLP and OPSG at 72 h were 76.4 and 98.9%, respectively. By correlating the amount of residual lignin and xylan to the initial hydrolysis rate and the final hydrolysis yield in OPLP and OPSG, a more accurate fundamental understanding of the roles of xylan and lignin in limiting the enzymatic hydrolysis has been developed. The higher amount of residual xylan (9.7%) in OPSG resulted in lower initial hydrolysis rate (1.19 g/L/h). The higher amount of residual lignin in OPLP (18.6%) resulted in lower final hydrolysis yield of glucan (76.4%). In addition, we observed in the simultaneous saccharification and fermentation (SSF) that ethyl xyloside was produced by the enzymatic catalysis of xylose/xylan and ethanol.

Effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on PCDD/F emissions from open burning of biomass.

To understand the effect of leaf-surface pesticides on emissions of PCDD/F during biomass burns, nine combustion experiments simulating the open burning of biomass were conducted. Needles and branches of Pinus taeda (Loblolly pine) were sprayed with the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) at 1 and 10 times the manufacturer's recommended application concentration. The biomass was then dried overnight, burned in an open burn test facility, and emission samples were collected, analyzed, and compared against emission samples from burning untreated biomass. Blank tests and analysis of PCDD/F in the raw biomass were also performed. Emission results from burning a water-sprayed control show a ~20-fold increase in PCDD/F levels above that of the raw biomass alone, implicating combustive formation versus simple volatilization. Results from burns of pine branches sprayed with pesticide showed a statistically significant increase in the PCDD/F TEQ emissions when burning biomass at ten times the recommended pesticide concentration (from 0.22 to 1.14 ng TEQ/kg carbon burned (C(b)), both ND = 0). Similarly, a 150-fold increase in the total PCDD/F congener mass (tetra- to octa-chlorinated D/F) above that of the control was observed (from 52 to 7800 ng/kg C(b)), confirming combustive formation of PCDD/F from 2,4-D. More replicate testing is needed to evaluate effects at lower pesticide concentrations.

Producing high sugar concentrations from loblolly pine using wet explosion pretreatment.

We present quantitative analysis of pretreatment for obtaining high conversion and release of sugars from loblolly pine. We use wet explosion (WEx): wet oxidation followed by steam explosion and enzymatic hydrolysis (EH) at high dry matter to solubilize sugars. WEx was conducted at 25% (w/w) solids in presence of oxygen at pressures 6.5-7.2 bar, temperatures 170-175°C and residence time from 20 to 22.5 min. EH of pretreated samples was performed by Cellic® Ctec2 (60 mg protein/g cellulose) and Cellic® Htec2 enzymes (10% of Ctec2) at 50°C for 72 h. At the optimal WEx condition 96% cellulose and nearly 100% hemicellulose yield were obtained. The final concentrations of monomeric sugars were 152 g/L of glucose, 67 g/L of xylose, and 67 g/L of minor sugars (galactose, arabinose and mannose). Compared to previous work WEx seems to be superior for releasing high concentrations of monomeric sugars.

Semiochemical emission from individual galleries of the southern pine beetle, (Coleoptera: Curculionidae: Scolytinae), attacking standing trees.

We collected, identified, and quantified volatiles arising from individual gallery entrances of the monogamous bark beetle Dendroctonus frontalis Zimmermann. Samples were collected while the insects were mass attacking mature loblolly pines (Pinus taeda L.) in an established infestation in western Mississippi, 1 August through 3 October 2005. Following volatile sample collection, the entrances were dissected and categorized according to those that 1) contained a solitary female (the gallery initiating sex), 2) contained a pair that had not yet produced an egg gallery, 3) led to an egg gallery with niches and/or eggs, or 4) represented failed attacks (either abandoned or containing dead beetles). The greatest mean release rate of the female-produced aggregation pheromone components frontalin (74 ng/h) and trans-verbenol (0.35 microg/h) was detected from entrances of solitary females, whereas the highest mean quantities of the male-produced multifunctional pheromone components endo-brevicomin (18 ng/h) and verbenone (0.15 microg/h) were detected from entrances of preoviposition beetle pairs. Alpha-pinene, a host-produced monoterpene that functions as a synergist for the aggregation attractant for D. frontalis, was detected from entrances of solitary females and preoviposition pairs at a rate of 0.6 mg/h, or 3-4 orders of magnitude greater than the insect-produced components of the attractant. Our results indicate that the release rates of pheromone components used in published field studies of the chemical ecology of D. frontalis (generally > 0.1 mg/h) represent thousands of 'attack equivalents' or production rates on the scale of a beetle mass attack on a single host. Additionally, our data suggest that the loss in attractiveness of host tissue fully colonized by D. frontalis is because of the disappearance of attractants rather than an increase in inhibitors.

Characterization of milled wood lignin (MWL) in Loblolly pine stem wood, residue, and bark.

Milled wood lignin samples from Loblolly pine stem wood, forest residue, and bark were isolated and characterized by quantitative (13)C and (31)P nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC) for molecular weight determination. Results from (13)C NMR show the stem wood and forest residue samples have similar functional group contents. However, the bark has fewer methoxyl groups, ß-O-4 structures, dibenzodioxocin, and side chains than the other two lignins. The bark lignin has the highest amounts of p-hydroxyphenyl (h) and C-5 condensed lignin, stem wood has the lowest, and the residue lies between. (31)P NMR analysis indicates that bark lignin contains more C-5 substituted phenolics and fewer aliphatic hydroxyl groups than the lignin isolated from stem wood or residue. The molecular weight distribution analysis indicates the bark lignin has higher weight-average molecular weight (M(w)) and polydispersity index than the lignin recovered from stem wood or residue.

Selective detection of crystalline cellulose in plant cell walls with sum-frequency-generation (SFG) vibration spectroscopy.

The selective detection of crystalline cellulose in biomass was demonstrated with sum-frequency-generation (SFG) vibration spectroscopy. SFG is a second-order nonlinear optical response from a system where the optical centrosymmetry is broken. In secondary plant cell walls that contain mostly cellulose, hemicellulose, and lignin with varying concentrations, only certain vibration modes in the crystalline cellulose structure can meet the noninversion symmetry requirements. Thus, SFG can be used to detect and analyze crystalline cellulose selectively in lignocellulosic biomass without extraction of noncellulosic species from biomass or deconvolution of amorphous spectra. The selective detection of crystalline cellulose in lignocellulosic biomass is not readily achievable with other techniques such as XRD, solid-state NMR, IR, and Raman analyses. Therefore, the SFG analysis presents a unique opportunity to reveal the cellulose crystalline structure in lignocellulosic biomass.

Effect of moisture, charge size, and chlorine concentration on PCDD/F emissions from simulated open burning of forest biomass.

Loblolly pine (Pinus taeda) was combusted at different charge sizes, fuel moisture, and chlorine content to determine the effect on emissions of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/Fs) as well as copollutants CO, PM, and total hydrocarbons. The experiments were performed in an enclosed chamber under conditions simulating open, prescribed burns of forest biomass. Burn progress was monitored through on line measurement of combustion gases and temperature while PCDD/F concentrations were determined by ambient sampling methods. PCDD/F toxic equivalency (TEQ) and total (tetra- to octa-CDD/F) emission factors were independent of charge size (1-10 kg) and moisture content (7-50%). However, the lower chlorinated, mono- to tri-CDD/F compounds were increased by poor combustion conditions: combustion efficiency lower than 0.919 was generally found when the moisture content was higher than 30%. The increase of fuel matrix chlorine from 0.04% to 0.8% using a brine bath resulted in about a 100-fold increase of PCDD/F to about 90 ng TEQ/kg of carbon burned, C(b). These emission factors were linearly dependent on Cl concentration in the biomass. PCDD 2,3,7,8-Cl-substituted congeners and homologue patterns were also influenced by the addition of chlorine resulting in emissions with a higher abundance of the most toxic congeners (TeCDD and PeCDD). When both chlorine and moisture content were increased in the fuel, a simultaneous effect of the two parameters was observed. The increased TEQ values expected from higher Cl concentrations were mitigated by the presence of water, giving MCE = 0.868, promoting formation of mono- to tri-PCDD/F, and lowering the TEQ value. Open burn simulations were used to study PCDD/F formation in different combustion conditions providing a mathematical correlation between PCDD/F emissions and chlorine and moisture content in the fuel.

Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy.

A quantitative approach to characterize lignin-carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

Galactoglucomannan Oligosaccharides (GGMO) from a molasses byproduct of pine ( Pinus taeda ) fiberboard production.

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a ß-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.

Chemical structure and heterogeneity differences of two lignins from loblolly pine as investigated by advanced solid-state NMR spectroscopy.

Advanced solid-state NMR was employed to investigate differences in chemical structure and heterogeneity between milled wood lignin (MWL) and residual enzyme lignin (REL). Wiley and conventional milled woods were also studied. The advanced NMR techniques included 13C quantitative direct polarization, various spectral-editing techniques, and two-dimensional 1H-13C heteronuclear correlation NMR with 1H spin diffusion. The 13C chemical shift regions between 110 and 160 ppm of two lignins were quite similar to those of two milled woods. REL contained much more residual carbohydrates than MWL, showing that MWL extraction more successfully separated lignin from cellulose and hemicelluloses than REL extraction; REL was also of higher COO, aromatic C-C, and condensed aromatics but of lower aromatic C-H. At a spin diffusion time of 0.55 ms, the magnetization was equilibrated through the whole structure of MWL lignin, but not through that of REL, indicating that REL is more heterogeneous than MWL.

An improved method of RNA isolation from loblolly pine (P. taeda L.) and other conifer species.

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

Polymerase chain reaction preparation of template for massively parallel pyrosequencing.

Massively parallel pyrosequencing of DNA fragments immobilized on beads has been applied to genome survey sequencing and transcriptome analysis of a variety of eukaryotic organisms, including laboratory model species, agricultural crops and livestock, and species of interest to population biologists and ecologists. Preparation of sufficient high-quality template for sequencing has been an obstacle to sequence analysis of nucleic acids from tissues or cell types available in limited quantities. We report that the use of a biotinylated primer for polymerase chain reaction amplification allows removal of excess primer and poly(A) tract fragments from the sequencing templates, providing much higher yields of useful sequence information from pyrosequencing of amplified templates. This advance allows deep sequencing analysis of nucleic acids isolated from very small tissue samples. Massively parallel pyrosequencing is particularly useful for preliminary investigations of species that have not yet been the subject of significant genomic research, as genomic survey sequences and catalogs of expressed genes provide a means of linking the biology of less intensively studied species to that of more intensively studied model organisms. We obtained over 220 Mb of transcript DNA sequences from Abies fraseri (Pursh) Poir., a conifer species native to the southern Appalachian Mountains of eastern North America. Comparison of the resulting assembled putative transcripts with similar data obtained by other sequencing methods from other conifers demonstrates the utility of the improved sequencing template preparation.