Robust paths to net greenhouse gas mitigation and negative emissions via advanced biofuels.

Biofuel and bioenergy systems are integral to most climate stabilization scenarios for displacement of transport sector fossil fuel use and for producing negative emissions via carbon capture and storage (CCS). However, the net greenhouse gas mitigation benefit of such pathways is controversial due to concerns around ecosystem carbon losses from land use change and foregone sequestration benefits from alternative land uses. Here, we couple bottom-up ecosystem simulation with models of cellulosic biofuel production and CCS in order to track ecosystem and supply chain carbon flows for current and future biofuel systems, with comparison to competing land-based biological mitigation schemes. Analyzing three contrasting US case study sites, we show that on land transitioning out of crops or pasture, switchgrass cultivation for cellulosic ethanol production has per-hectare mitigation potential comparable to reforestation and severalfold greater than grassland restoration. In contrast, harvesting and converting existing secondary forest at those sites incurs large initial carbon debt requiring long payback periods. We also highlight how plausible future improvements in energy crop yields and biorefining technology together with CCS would achieve mitigation potential 4 and 15 times greater than forest and grassland restoration, respectively. Finally, we show that recent estimates of induced land use change are small relative to the opportunities for improving system performance that we quantify here. While climate and other ecosystem service benefits cannot be taken for granted from cellulosic biofuel deployment, our scenarios illustrate how conventional and carbon-negative biofuel systems could make a near-term, robust, and distinctive contribution to the climate challenge.

Compaction effects on greenhouse gas and ammonia emissions from solid dairy manure.

Waste management practices of solid dairy manures were evaluated under controlled conditions to study gas transport and emission inside manure piles. Three applied stresses and three moisture contents were tested to represent manure conditions managed at various pile depths. A Fourier-transform infrared spectroscopy monitor measured concentrations of greenhouses gases [methane, carbon dioxide, and nitrous oxide] and ammonia as part of gas flux rate calculations. Results showed that carbon dioxide dominated the greenhouse gas emissions under all test conditions. Gas transfer, primarily diffusion, was facilitated by manure with high mechanical strength and high permeability. Gas emission rates reduced dramatically when moisture content increased in manure with high water holding capacity, while compaction treatments did not as strongly affect the gas emission rates. Results provide fundamental insights into management strategies for reducing gas emissions from solid dairy manure.

Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

Lignin is a complex polyphenolic heteropolymer that is abundant in the secondary cell walls of plants and functions in growth and defence. It is also a major barrier to the deconstruction of plant biomass for bioenergy production, but the spatiotemporal details of how lignin is deposited in actively lignifying tissues and the precise relationships between wall lignification in different cell types and developmental events, such as flowering, are incompletely understood. Here, the lignin-detecting fluorogenic dye, Basic Fuchsin, was adapted to enable comparative fluorescence-based imaging of lignin in the basal internodes of three Brachypodium distachyon ecotypes that display divergent flowering times. It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls. Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon. Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses. This study provides a new comparative tool for imaging lignin in plants and helps inform our views of how lignification proceeds in grasses.

Development of a clickable designer monolignol for interrogation of lignification in plant cell walls.

Lignin is an abundant and essential polymer in land plants. It is a prime factor in the recalcitrance of lignocellulosic biomass to agricultural and industrial end-uses such as forage, pulp and papermaking, and biofuels. To better understand lignification at the molecular level, we are developing a lignin spectroscopic and imaging toolbox on one "negligible" auxiliary. Toward that end, we describe the design, synthesis, and characterization of a new designer monolignol, 3-O-propargylcaffeyl alcohol, which contains a bioorthogonal alkynyl functional group at the 3-O-position. Importantly, our data indicate that this monolignol does not alter the fidelity of lignification. We demonstrate that the designer monolignol provides a platform for multiple spectroscopic and imaging approaches to reveal temporal and spatial details of lignification, the knowledge of which is critical to reap the potential of energy-rich renewable plant biomass for sustainable liquid fuels and other diverse economic applications.

Enhancing anaerobic digestion of lignocellulosic biomass by mechanical cotreatment.

BACKGROUND: The aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanical cotreatment: milling during fermentation, as an alternative to conventional pretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the 'status quo' control. RESULTS: The results indicate 0.5 - 10 min of cotreatment increased sugar solubilization by 5- 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production. CONCLUSIONS: Cotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.

Simultaneous cellulose degradation and electricity production by Enterobacter cloacae in a microbial fuel cell.

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047(T) based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 +/- 0.01 mW/m(2), compared to 5.4 +/- 0.3 mW/m(2) for strain ATCC 13047(T). These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.

Anaerobic bioprocessing of wastewater-derived duckweed: Maximizing product yields in a biorefinery value cascade.

This study integrated the sugar and carboxylate platforms to enhance duckweed processing in biorefineries. Two or three bioprocesses (ethanol fermentation, acidogenic digestion, and methanogenic digestion) were sequentially integrated to maximize the carbon-to-carbon conversion of wastewater-derived duckweed into bioproducts, through a series of laboratory-scale experiments. Reactors were fed either raw (dried), liquid-hot-water-pretreated, or enzymatically-saccharified duckweed. Subsequently, the target bioproduct was separated from the reactor liquor and the residues further processed. The total bioproduct carbon yield of 0.69 ±â€¯0.07 g per gram of duckweed-C was obtained by sequential acidogenic and methanogenic digestion. Three sequential bioprocesses revealed nearly as high yields (0.66 ±â€¯0.08 g of bioproduct-C per duckweed-C), but caused more gaseous carbon (dioxide) loss. For this three-stage value cascade, yields of each process in conventional units were: 0.186 ±â€¯0.001 g ethanol/g duckweed; 611 ±â€¯64 mg volatile fatty acids as acetic acid/g VS; and 434 ±â€¯0.2 ml methane/g VS.

Enzymatic hydrolysis of cellulose coupled with electricity generation in a microbial fuel cell.

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from a variety of biodegradable substrates, including cellulose. Particulate materials have not been extensively examined for power generation in MFCs, but in general power densities are lower than those produced with soluble substrates under similar conditions likely as a result of slow hydrolysis rates of the particles. Cellulases are used to achieve rapid conversion of cellulose to sugar for ethanol production, but these enzymes have not been previously tested for their effectiveness in MFCs. It was not known if cellulases would remain active in an MFC in the presence of exoelectrogenic bacteria or if enzymes might hinder power production by adversely affecting the bacteria. Electricity generation from cellulose was therefore examined in two-chamber MFCs in the presence and absence of cellulases. The maximum power density with enzymes and cellulose was 100 +/- 7 mW/m(2) (0.6 +/- 0.04 W/m(3)), compared to only 12 +/- 0.6 mW/m(2) (0.06 +/- 0.003 W/m(3)) in the absence of the enzymes. This power density was comparable to that achieved in the same system using glucose (102 +/- 7 mW/m(2), 0.56 +/- 0.038 W/m(3)) suggesting that the enzyme successfully hydrolyzed cellulose and did not otherwise inhibit electricity production by the bacteria. The addition of the enzyme doubled the Coulombic efficiency (CE) to CE = 51% and increased COD removal to 73%, likely as a result of rapid hydrolysis of cellulose in the reactor and biodegradation of the enzyme. These results demonstrate that cellulases do not adversely affect exoelectrogenic bacteria that produce power in an MFC, and that the use of these enzymes can increase power densities and reactor performance.

Engineering motility as a phenotypic response to LuxI/R-dependent quorum sensing in Escherichia coli.

The repertoire of functional outputs interfaced with the LuxI/LuxR quorum sensing system in engineered Escherichia coli has been expanded to include motility via inducible expression of motB. Appropriate choice of ribosome binding site controlling MotB translation was crucial to achieving control over motility.

Ensiled Wet Storage Accelerates Pretreatment for Bioconversion of Corn Stover.

Organic acids produced during ensiled wet storage are beneficial during the storage process, both for biomass preservation, and to aid in mild in-situ pretreatment. However, there is concern these acids could later have negative impacts on downstream processes, especially microbial fermentation. Organic acids can inhibit microbial metabolism or growth, which in turn could affect biofuel productivity or yield. This study investigated the interaction of organic acids produced during ensiled storage with subsequent pretreatment of the resulting corn stover silage, as well as the potential for interference with downstream ethanol fermentation. Interaction with pretreatment was observed by measuring xylan and glucan removal and the formation of inhibitors. The results indicated that organic acids generally do not impede downstream processes and in fact can be beneficial. The levels of organic acids produced during 220 days of storage jar tests at 23°C or 37°C, and their transformation during pretreatment, remained below inhibitory levels. Concentrations of individual acids did not exceed 6 g per liter of the pretreated volume, and < 5% on a dry matter basis. Whereas, unensiled corn stover required 15 min of 190°C pretreatment to optimize sugar release, ensiled corn stover could be treated equally effectively at a lower pretreatment duration of 10 min. Furthermore, the different organic acid profiles that accumulate at various storage moisture levels (35-65%) do not differ significantly in their impact on downstream ethanol fermentation. These results indicate biorefineries using ensiled corn stover feedstock at 35-65% moisture levels can expect as good or better biofuel yields as with unensiled stover, while reducing pretreatment costs.

Effect of pH and temperature on microbial community structure and carboxylic acid yield during the acidogenic digestion of duckweed.

BACKGROUND: Duckweeds (Lemnaceae) are efficient aquatic plants for wastewater treatment due to their high nutrient-uptake capabilities and resilience to severe environmental conditions. Combined with their rapid growth rates, high starch, and low lignin contents, duckweeds have also gained popularity as a biofuel feedstock for thermochemical conversion and alcohol fermentation. However, studies on the acidogenic anaerobic digestion of duckweed into carboxylic acids, another group of chemicals which are precursors of higher-value chemicals and biofuels, are lacking. In this study, a series of laboratory batch experiments were performed to determine the favorable operating conditions (i.e., temperature and pH) to maximize carboxylic acid production from wastewater-derived duckweed during acidogenic digestion. Batch reactors with 25 g/l solid loading were operated anaerobically for 21 days under mesophilic (35 °C) or thermophilic (55 °C) conditions at an acidic (5.3) or basic (9.2) pH. At the conclusion of the experiment, the dominant microbial communities under various operating conditions were assessed using high-throughput sequencing. RESULTS: The highest duckweed-carboxylic acid conversion of 388 ± 28 mg acetic acid equivalent per gram volatile solids was observed under mesophilic and basic conditions, with an average production rate of 0.59 g/l/day. This result is comparable to those reported for acidogenic digestion of other organics such as food waste. The superior performance observed under these conditions was attributed to both chemical treatment and microbial bioconversion. Hydrogen recovery was only observed under acidic thermophilic conditions, as 23.5 ± 0.5 ml/g of duckweed volatile solids added. More than temperature, pH controlled the overall structure of the microbial communities. For instance, differentially abundant enrichments of Veillonellaceae acidaminococcus were observed in acidic samples, whereas enrichments of Clostridiaceae alkaliphilus were found in the basic samples. Acidic mesophilic conditions were found to enrich acetoclastic methanogenic populations over processing times longer than 10 days. CONCLUSIONS: Operating conditions have a significant effect on the yield and composition of the end products resulting from acidogenic digestion of duckweed. Wastewater-derived duckweed is a technically feasible alternative feedstock for the production of advanced biofuel precursors; however, techno-economic analysis is needed to determine integrated full-scale system feasibility and economic viability.

The Maize Corngrass1 miRNA-Regulated Developmental Alterations Are Restored by a Bacterial ADP-Glucose Pyrophosphorylase in Transgenic Tobacco.

Crop-based bioethanol has raised concerns about competition with food and feed supplies, and technologies for second- and third-generation biofuels are still under development. Alternative feedstocks could fill this gap if they can be converted to biofuels using current sugar- or starch-to-ethanol technologies. The aim of this study was to enhance carbohydrate accumulation in transgenic Nicotiana benthamiana by simultaneously expressing the maize Corngrass1 miRNA (Cg1) and E. coli ADP-glucose pyrophosphorylase (glgC), both of which have been reported to enhance carbohydrate accumulation in planta. Our findings revealed that expression of Cg1 alone increased shoot branching, delayed flowering, reduced flower organ size, and induced loss of fertility. These changes were fully restored by coexpressing Escherichia coli glgC. The transcript level of miRNA156 target SQUAMOSA promoter binding-like (SPL) transcription factors was suppressed severely in Cg1-expressing lines as compared to the wild type. Expression of glgC alone or in combination with Cg1 enhanced biomass yield and total sugar content per plant, suggesting the potential of these genes in improving economically important biofuel feedstocks. A possible mechanism of the Cg1 phenotype is discussed. However, a more detailed study including genome-wide transcriptome and metabolic analysis is needed to determine the underlying genetic elements and pathways regulating the observed developmental and metabolic changes.

Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times.

BACKGROUND: Anaerobic fermentation of lignocellulose occurs in both natural and managed environments, and is an essential part of the carbon cycle as well as a promising route to sustainable production of fuels and chemicals. Lignocellulose solubilization by mixed microbiomes is important in these contexts. RESULTS: Here, we report the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times and moderately high (55 °C) temperatures. Anaerobic microbiomes derived from a digester inoculum were incubated at 55 °C and fed semi-continuously with medium containing 30 g/L mid-season harvested switchgrass to achieve residence times (RT) of 20, 10, 5, and 3.3 days. Stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids were achieved for all RTs. Fractional carbohydrate solubilization was 0.711, 0.654, 0.581 and 0.538 at RT = 20, 10, 5 and 3.3 days, respectively, and glucan solubilization was proportional to xylan solubilization at all RTs. The rate of solubilization was described well by the equation r = k(C - C0fr), where C represents the concentration of unutilized carbohydrate, C0 is the concentration of carbohydrate (cellulose and hemicellulose) entering the bioreactor and fr is the extrapolated fraction of entering carbohydrate that is recalcitrant at infinite residence time. The 3.3 day RT is among the shortest RT reported for stable thermophilic, methanogenic digestion of a lignocellulosic feedstock. 16S rDNA phylotyping and metagenomic analyses were conducted to characterize the effect of RT on community dynamics and to infer functional roles in the switchgrass to biogas conversion to the various microbial taxa. Firmicutes were the dominant phylum, increasing in relative abundance from 54 to 96% as RT decreased. A Clostridium clariflavum strain with genetic markers for xylose metabolism was the most abundant lignocellulose-solubilizing bacterium. A Thermotogae (Defluviitoga tunisiensis) was the most abundant bacterium in switchgrass digesters at RT = 20 days but decreased in abundance at lower RTs as did multiple Chloroflexi. Synergistetes and Euryarchaeota were present at roughly constant levels over the range of RTs examined. CONCLUSIONS: A system was developed in which stable methanogenic steady-states were readily obtained with a particulate biomass feedstock, mid-season switchgrass, at laboratory (1 L) scale. Characterization of the extent and rate of carbohydrate solubilization in combination with 16S rDNA and metagenomic sequencing provides a multi-dimensional view of performance, species composition, glycoside hydrolases, and metabolic function with varying residence time. These results provide a point of reference and guidance for future studies and organism development efforts involving defined cultures.

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana).

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility.

Modeling the temperature kinetics of aerobic solid-state biodegradation.

This study evaluated three models of microbial temperature kinetics using CO2 respiration data from aerobic solid-state biodegradation experiments. The models included those of Andrews and Kambhu/Haug, Ratkowsky et al., and the Cardinal Temperature Model with Inflection (CTMI) of Rosso et al. A parameter estimation routine implemented the Complex-Box search method for each model on 48 data sets collected during the composting of synthetic food waste or sewage-sludge (biosolids) mixed with maple wood chips at different oxygen concentrations and extents of decomposition. Each of the three nonlinear temperature kinetic functions proved capable of modeling a wide range of experimental data sets. However, the models differed widely in the consistency of their parameters. Parameters in the CTMI model were more stable over the course of the degradation process, and that variability which did arise was directly related to changes in the microbial process. Additional benefits of the CTMI model include the ease of parameter determinations, which can be approximated directly from laboratory experiments or full-scale system analysis, and the direct value of its parameters in engineering design and process control under a wide range of biodegradation conditions.

Effects of oxygen on aerobic solid-state biodegradation kinetics.

Oxygen is a critical control variable for composting and other solid-state biodegradation processes. In this study we examined the effect of varying oxygen concentrations (1%, 4%, and 21% O2 (v/v)) on biodegradation kinetics under different substrate (sewage sludge and synthetic food waste), temperature (35, 45, 55, and 65 degrees C), and moisture (36-60% H2O) conditions. Three forms of a saturation or Monod-type model and one form of an exponential model were evaluated against data from extensive experiments under carefully controlled environmental conditions. The exponential model performed well at temperatures from 35 to 55 degrees C but had problems at higher temperatures. The Monod-type models yielded the best fit based on R2 values. Multiple linear regression was used to express the oxygen half-saturation coefficient as a function of temperature and moisture. For a modified one-parameter saturation model the half-saturation coefficient varied from -0.67% to 1.74% v/v O2 under the range of conditions typical of composting systems. While the positive correlation of biodegradation rate with oxygen concentration reported by previous researchers held true for temperatures below 55 degrees C, an inverse relationship was found at 65 degrees C. Although this study did not directly examine anaerobic conditions, the results under microaerophilic conditions suggest oxygen may not offer kinetic advantages for extreme thermophilic biodegradation processes.

Ensiling corn stover: effect of feedstock preservation on particleboard performance.

Ensilage is a truncated solid-state fermentation in which anaerobically produced organic acids accumulate to reduce pH and limit microbial activity. Ensilage can be used to both preserve and pretreat biomass feedstock for further downstream conversion into chemicals, fuels, and/or fiber products. This study examined the ensilage of enzyme-treated corn stover as a feedstock for particleboard manufacturing. Corn stover at three different particle size ranges (<100, <10, and <5 mm) was ensiled with and without a commercial enzyme mixture having a cellulase:hemicellulase ratio of 2.54:1, applied at a hemicellulase rate of 1670 IU/kg dry mass. Triplicate 20 L mini-silos were destructively sampled and analyzed on days 0, 1, 7, 21, 63, and 189. Analysis included produced organic acids and water-soluble carbohydrates, fiber fractions, pH, and microorganisms, including Lactobacillus spp. and clostridia were monitored. On days 0, 21, and 189, the triplicate samples were mixed evenly and assembled into particleboard using 10% ISU 2 resin, a soy-based adhesive. Particleboard panels were subjected to industry standard tests for modulus of rupture (MOR), modulus of elasticity (MOE), internal bonding strength (IB), thickness swell (TS), and water absorption at 2 h boiling and 24 h soaking. Enzyme addition did improve the ensilage process, as indicated by sustained lower pH (P < 0.0001), higher water-soluble carbohydrates (P < 0.05), and increased lactic acid production (P < 0.0001). The middle particle size range (<10 mm) demonstrated the most promising results during the ensilage process. Compared with fresh stover, the ensilage process did increase IB of stover particleboard by 33% (P < 0.05) and decrease water adsorption at 2 h boiling and 24 h soaking significantly (P < 0.05). Particleboard panels produced from substrate ensiled with enzymes showed a significant reduction in water adsorption of 12% at 2 h boiling testing. On the basis of these results, ensilage can be used as a long-term feedstock preservation method for particleboard production from corn stover. Enzyme-amended ensilage not only improved stover preservation but also enhanced the properties of particleboard products.

Investigating Biochemical and Developmental Dependencies of Lignification with a Click-Compatible Monolignol Analog in Arabidopsis thaliana Stems.

Lignin is a key structural component of plant cell walls that provides rigidity, strength, and resistance against microbial attacks. This hydrophobic polymer also serves a crucial role in water transport. Despite its abundance and essential functions, several aspects of lignin biosynthesis and deposition remain cryptic. Lignin precursors are known to be synthesized in the cytoplasm by complex biosynthetic pathways, after which they are transported to the apoplastic space, where they are polymerized via free radical coupling reactions into polymeric lignin. However, the lignin deposition process and the factors controlling it are unclear. In this study, the biochemical and developmental dependencies of lignification were investigated using a click-compatible monolignol analog, 3-O-propargylcaffeyl alcohol (3-OPC), which can incorporate into both in vitro polymerized lignin and Arabidopsis thaliana tissues. Fluorescence labeling of 3-OPC using click chemistry followed by confocal fluorescence microscopy enabled the detection and imaging of 3-OPC incorporation patterns. These patterns were consistent with endogenous lignification observed in different developmental stages of Arabidopsis stems. However, the concentration of supplied monolignols influenced where lignification occurred at the subcellular level, with low concentrations being deposited in cell corners and middle lamellae and high concentrations also being deposited in secondary walls. Experimental inhibition of multiple lignification factors confirmed that 3-OPC incorporation proceeds via a free radical coupling mechanism involving peroxidases/laccases and reactive oxygen species (ROS). Finally, the presence of peroxide-producing enzymes determined which cell walls lignified: adding exogenous peroxide and peroxidase caused cells that do not naturally lignify in Arabidopsis stems to lignify. In summary, 3-OPC accurately mimics natural lignification patterns in different developmental stages of Arabidopsis stems and allows for the dissection of key biochemical and enzymatic factors controlling lignification.

Winter rye as a bioenergy feedstock: impact of crop maturity on composition, biological solubilization and potential revenue.

BACKGROUND: Winter annual crops such as winter rye (Secale cereale L) can produce biomass feedstock on seasonally fallow land that continues to provide high-value food and feed from summer annuals such as corn and soybeans. As energy double crops, winter grasses are likely to be harvested while still immature and thus structurally different from the fully senesced plant material typically used for biofuels. This study investigates the dynamic trends in biomass yield, composition, and biological solubilization over the course of a spring harvest season. RESULTS: The water soluble fraction decreased with increasing maturity while total carbohydrate content stayed roughly constant at about 65%. The protein mass fraction decreased with increasing maturity, but was counterbalanced by increasing harvest yield resulting in similar total protein across harvest dates. Winter rye was ground and autoclaved then fermented at 15 g/L total solids by either (1) Clostridium thermocellum or (2) simultaneous saccharification and cofermentation (SSCF) using commercial cellulases (CTec2 and HTec2) and a xylose-fermenting Saccharomyces cerevisiae strain. Solubilization of total carbohydrate dropped significantly as winter rye matured for both C. thermocellum (from approximately 80% to approximately 50%) and SSCF (from approximately 60% to approximately 30%). C. thermocellum achieved total solubilization 33% higher than that of SSCF for the earliest harvest date and 50% higher for the latest harvest date. Potential revenue from protein and bioethanol was stable over a range of different harvest dates, with most of the revenue due to ethanol. In a crop rotation with soybean, recovery of the soluble protein from winter rye could increase per hectare protein production by 20 to 35%. CONCLUSIONS: Double-cropping winter rye can produce significant biomass for biofuel production and feed protein as coproduct without competing with the main summer crop. During a 24-day harvest window, the total carbohydrate content remained relatively constant while the early-harvest yielded much higher carbohydrate solubilization for both C. thermocellum fermentation and SSCF. C. thermocellum fermentation achieved higher carbohydrate solubilization than SSCF across all growth stages tested. Although winter rye's yield, composition, and biological reactivity change rapidly in the spring, it offers a substantial and stable income across the harvest season and thus flexibility for the farmer.