Atomic-Level Structure Characterization of Biomass Pre- and Post-Lignin Treatment by Dynamic Nuclear Polarization-Enhanced Solid-State NMR.

Lignocellulosic biomass is a promising sustainable feedstock for the production of biofuels, biomaterials, and biospecialty chemicals. However, efficient utilization of biomass has been limited by our poor understanding of its molecular structure. Here, we report a dynamic nuclear polarization (DNP)-enhanced solid-state (SS)NMR study of the molecular structure of biomass, both pre- and postcatalytic treatment. This technique enables the measurement of 2D homonuclear 13C-13C correlation SSNMR spectra under natural abundance, yielding, for the first time, an atomic-level picture of the structure of raw and catalytically treated biomass samples. We foresee that further such experiments could be used to determine structure-function relationships and facilitate the development of more efficient, and chemically targeted, biomass-conversion technologies.

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population.

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.

Reduction of volatile fatty acids and odor offensiveness by anaerobic digestion and solid separation of dairy manure during manure storage.

Volatile fatty acids (VFA) play an important role in the biodegradation of organic wastes and production of bioenergy under anaerobic digestion, and are related to malodors. However, little is known about the dynamics of VFA during dairy manure storage. This study evaluated the characteristics of VFA in dairy manure before and after anaerobic co-digestion in a laboratory experiment using eight lab-scale reactors. The reactors were loaded with four different types of dairy manure: (1) liquid dairy manure from a freestall barn, (2) mixture of dairy manure and co-digestion food processing wastes at the inlet of an anaerobic digester, (3) effluent from the digester outlet, and (4) the liquid fraction of effluent from a solid separator. Four VFA (acetic, propionic, butyric, and 2-methylbutyric acids) were identified and quantified in weekly manure samples from all reactors. Results showed that the dominant VFA was acetic acid in all four manure sources. The off-farm co-digestion wastes significantly increased the total VFA concentrations and the proportions of individual VFA in the influent. The dairy manure under storage demonstrated high temporal and spatial variations in pH and VFA concentrations. Anaerobic digestion reduced the total VFA by 86%-96%; but solid-liquid separation did not demonstrate a significant reduction in total VFA in this study. Using VFA as an indicator, anaerobic digestion exhibited an effective reduction of dairy manure odor offensiveness.

Severity factor coefficients for subcritical liquid hot water pretreatment of hardwood chips.

Single stage and multi-stage liquid hot water pretreatments of mixed hardwood pinchips were investigated at various severities (log R0 = 3.65-4.81) to assess the efficiencies of the pretreatments with respect to achieving high pentose sugar yields and improved enzymatic digestibility of pretreated cellulose. We investigate the effect of pretreatment parameters that is, temperature, and time, as expressed in the severity factor, on the recovery of sugars and hydrolyzability of pretreated cellulose. We find the severity factor, in its widely used form, is an incomplete measure for evaluating the pretreatment efficiencies and predicting overall sugar yields when pretreatment temperatures above 200°C are used. Corrections to the severity factor and its correlation to the measured pretreatment responses (% xylan solubilization, xylan recovery as fermentable sugars, cellulose enzymatic digestibility) indicate a greater influence of temperature on the pretreatment efficiencies than predicted by the commonly used severity factor. A low temperature, long residence time is preferred for hemicellulose dissolution during the pretreatment since the condition favors oligosaccharide and monomeric sugar formation over sugar degradation. On the contrary, high cellulose hydrolyzability is achieved with a high temperature (>200°C), high severity pretreatment when pretreatment is followed by enzyme hydrolysis. In multi-stage pretreatment, the first low-severity pretreatment is optimized for solubilizing fast-hydrolyzing hemicellulose while minimizing formation of furans. The subsequent pretreatment is carried out at over 200°C to recover the difficult-to-hydrolyze hemicellulose fraction as well as to increase susceptibility of pretreated cellulose to enzymes. High recovery (>92%) of hemicellulose-derived pentose sugars and enhanced enzymatic hydrolysis of pretreated cellulose (where >80% glucose yield results with 20 FPU = 32 mg protein/g glucan or 10-13 mg/g initial hardwood) are achieved by applying a multi-stage pretreatment. This work shows how the severity equation may be used to obtain a single characteristic curve that correlate xylan solubilization and enzymatic cellulose hydrolysis as a function of severity at pretreatment temperatures up to 230°C.

Effect of pelleting on the enzymatic digestibility of corn stover.

Pelleting of lignocellulosic biomass to improve its transportation, storage and handling impacts subsequent processing and conversion. This work reports the role of high moisture pelleting in the enzymatic digestibility of corn stover prior to pretreatment, together with associated substrate characteristics. Pelleting increases the digestibility of unpretreated corn stover, from 8.2 to 15.5% glucan conversion, at 5% solid loading using 1 FPU Cellic® CTec2 per g solids. Compositional analysis indicates that loose and pelleted corn stover have similar non-dissolvable compositions, although their extractives are different. Enzymatic hydrolysis of corn stover after size reduction to normalize particle sizes and removal of extractives confirms that pelleting improves corn stover digestibility. Such differences may be explained by the decreased particle size, improved substrate accessibility, and hydrolysis of cross-linking structures induced by pelleting. These findings are useful for the development of processing schemes for sustainable and efficient use of lignocellulose.

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: enzymatic hydrolysis (part 1).

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue have a measurable effect on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The first part of our work combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. The extent of enzyme hydrolysis follows the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Physical fractionation of corn stalks or other C(4) grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses such as thermal processing, combustion, or recycle to the land from which the corn was harvested.

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2).

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.

Structure-property-degradability relationships of varisized lignocellulosic biomass induced by ball milling on enzymatic hydrolysis and alcoholysis.

BACKGROUND: Valorization of lignocellulosic biomass to obtain clean fuels and high-value chemicals is attractive and essential for sustainable energy and chemical production, but the complex structure of biomass is recalcitrant to catalytic processing. This recalcitrance can be overcome by pretreating biomass into deconstructable components, which involves altering the structural complexities and physicochemical properties. However, the impact of these alterations on biomass deconstruction varies considerably, depending on the pretreatment and subsequent conversion type. Here, we systematically describe the changes in structure and properties of corn stover after ball milling as well as their influence on the following enzymatic saccharification and acid-catalyzed alcoholysis, with the aim of elucidating the relationships between structures, properties and deconstructable potential of lignocellulosic biomass. RESULTS: Ball milling causes dramatic structural changes, since the resistant plant cell walls are destroyed with size reduction to a cellular scale, leading to the increase in surface area and reducing ends, and decrease in crystallinity and thermal stability. As a result, ball-milled corn stover is more susceptible to enzymatic saccharification to fermentable sugars and provides more industrially viable processing approaches, as it is effective at high solids loading and minor enzyme loading, without any other pretreatment. Acid-catalyzed alcoholysis of corn stover to biofuels, on the other hand, is also enhanced by ball milling, but additional processing parameters should be tailored to the needs of efficient conversion. Further, a detailed examination of process variables coupled with a kinetic study indicates that acid-catalyzed alcoholysis is limited by the process variables rather than by the substrate parameters, whereas ball milling facilitates this reaction to some extent, especially under mild conditions, by lowering the activation energy of corn stover decomposition. CONCLUSIONS: The efficient catalytic conversion of biomass is closely related to its structure and properties, an understanding of which offers prospects for the rational improvement of methods aimed at more economic commercial biorefineries.

Hormesis-Inducing Essential Oil Nanodelivery System Protects Plants against Broad Host-Range Necrotrophs.

Botrytis cinerea, a broad host-range necrotrophic (BHN) phytopathogen, establishes compatible interactions with hosts by deploying multigene infection strategies, rendering simply inherited resistance ineffective to fight off this pathogen. Since essential oils (EOs) serve as intermediators during phytobiome communication, we hypothesize that they have the potential to enhance the quantitative disease resistance against BHN by eliciting the adaptive stress response (hormesis) in plants. However, using EOs is challenging due to their poor solubility in water. Nanoemulsification of EOs enhances not only the solubility of EOs but also their potency and stability. Here, we demonstrate the potential use of essential oil nanoemulsions (EONEs) to control infections caused by BHN. Using basic engineering principles of nanocarrier design, we demonstrate the efficacy of a robust EONEs design for controlling B. cinerea infection in a model plant, Arabidopsis thaliana. Our nanoemulsion delivery system significantly enhanced the disease resistance of the host by reducing the necrotic area by up to 50% compared to untreated plants. RNA-seq analysis indicated that successful treatments upregulated autophagy, ROS scavenging, and activation of the jasmonic acid signaling pathway.

Rheology of enzyme liquefied corn stover slurries: The effect of solids concentration on yielding and flow behavior.

The measurement of yield stress and shear thinning flow behavior of slurries formed from unpretreated corn stover at solids loadings of 100-300 g/L provides a key metric for the ability to move, pump, and mix this lignocellulosic slurry, particularly since corn stover slurries represent a major potential feedstock for biorefineries. This study compared static yield stress values and flow hysteresis of corn stover slurries of 100, 150, 200, 250, and 300 g/L, after these slurries were formed by adding pellets to a cellulase enzyme solution (Celluclast 1.5 L) in a fed-batch manner. A rotational rheometer was used to quantitate relative yield stress and its dependence on processing history at insoluble solids concentrations of 4%-21% (wt/vol). Key findings confirmed previous observations that yield stress increases with solids loadings and reaches ~3000 Pa at 25% (wt/vol) solids concentration compared to ~200 Pa after enzyme liquefaction. While optimization of slurry forming (i.e., liquefaction) conditions remains to be done, metrics for quantifying liquefaction extent are needed. The method for obtaining comparative metrics is demonstrated here and shows that the yield stress, shear thinning and shear thickening flow behaviors of enzyme liquefied corn stover slurries can be analyzed using a wide-gap rheometry setup with relative measuring geometries to mimic the conditions that may exist in a mixing vessel of a bioreactor while applying controlled and precise levels of strain.

New strategy for liquefying corn stover pellets.

The movement of solid material into and between unit operations within a biorefinery is a bottleneck in reaching design capacity, with formation of biomass slurries needed to introduce feedstock. Corn stover slurries have been achieved from dilute acid, pretreated materials resulting in slurry concentrations of up to about 150 g/L, above which flowability is compromised. We report a new strategy to liquefy corn stover at higher solids concentration (300 g/L) by initially cooking it with the enzyme mimetic maleic acid at 40 mM and 150 °C. This is followed by 6 h of enzymatic modification at 1 FPU (2.2 mg protein)/g solids, resulting in a yield stress of 171 Pa after 6 h and 58 Pa in 48 h compared to 6806 Pa for untreated stover. Mimetic treatment of corn stover pellets minimizes the inhibitory effect of xylo-oligomers on hydrolytic enzymes. This strategy allows for the delivery of solid lignocellulosic slurry into a pretreatment reactor by pumping, improving operability of a biorefinery.

Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae.

A current challenge of the cellulosic ethanol industry is the effect of inhibitors present in biomass hydrolysates. Acetic acid is an example of one such inhibitor that is released during the pretreatment of hemicellulose. This study examined the effect of acetic acid on the cofermentation of glucose and xylose under controlled pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engineered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 g L(-1), representing the range of concentrations expected in actual biomass hydrolysates, were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic acid in the fermentation media led to a significant decrease in the observed maximum cell biomass concentration. Glucose- and xylose-specific consumption rates decreased as the acetic acid concentration increased, with the inhibitory effect being more severe for xylose consumption. The ethanol production rates also decreased when acetic acid was present, but ethanol metabolic yields increased under the same conditions. The results also revealed that the inhibitory effect of acetic acid could be reduced by increasing media pH, thus confirming that the undissociated form of acetic acid is the inhibitory form of the molecule.

Nanoscale Drug Delivery Systems: From Medicine to Agriculture.

The main challenges in drug delivery systems are to protect, transport and release biologically active compounds at the right time in a safe and reproducible manner, usually at a specific target site. In the past, drug nano-carriers have contributed to the development of precision medicine and to a lesser extent have focused on its inroads in agriculture. The concept of engineered nano-carriers may be a promising route to address confounding challenges in agriculture that could perhaps lead to an increase in crop production while reducing the environmental impact associated with crop protection and food production. The main objective of this review is to contrast the advantages and disadvantages of different types of nanoparticles and nano-carriers currently used in the biomedical field along with their fabrication methods to discuss the potential use of these technologies at a larger scale in agriculture. Here we explain what is the problem that nano-delivery systems intent to solve as a technological platform and describe the benefits this technology has brought to medicine. Also here we highlight the potential drawbacks that this technology may face during its translation to agricultural applications, based on the lessons learned so far from its use for biomedical purposes. We discuss not only the characteristics of an ideal nano-delivery system, but also the potential constraints regarding the fabrication including technical, environmental, and legal aspects. A key motivation is to evaluate the potential use of these systems in agriculture, especially in the area of plant breeding, growth promotion, disease control, and post-harvest quality control. Further, we highlight the importance of a rational design of nano-carriers and identify current research gaps to enable scale-up relevant to applications in the treatment of plant diseases, controlled release of fertilizers, and plant breeding.

Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.

BACKGROUND: Low-temperature swelling of cotton linter cellulose and subsequent gelatinization in trifluoroacetic acid (TFA) greatly enhance rates of enzymatic digestion or maleic acid-AlCl3 catalyzed conversion to hydroxymethylfurfural (HMF) and levulinic acid (LA). However, lignin inhibits low-temperature swelling of TFA-treated intact wood particles from hybrid poplar (Populus tremula × P. alba) and results in greatly reduced yields of glucose or catalytic conversion compared to lignin-free cellulose. Previous studies have established that wood particles from transgenic lines of hybrid poplar with high syringyl (S) lignin content give greater glucose yields following enzymatic digestion. RESULTS: Low-temperature (- 20 °C) treatment of S-lignin-rich poplar wood particles in TFA slightly increased yields of glucose from enzymatic digestions and HMF and LA from maleic acid-AlCl3 catalysis. Subsequent gelatinization at 55 °C resulted in over 80% digestion of cellulose in only 3 to 6 h with high-S-lignin wood, compared to 20-60% digestion in the wild-type poplar hybrid and transgenic lines high in guaiacyl lignin or 5-hydroxy-G lignin. Disassembly of lignin in woody particles by Ni/C catalytic systems improved yields of glucose by enzymatic digestion or catalytic conversion to HMF and LA. Although lignin was completely removed by Ni/C-catalyzed delignification (CDL) treatment, recalcitrance to enzymatic digestion of cellulose from the high-S lines was reduced compared to other lignin variants. However, cellulose still exhibited considerable recalcitrance to complete enzymatic digestion or catalytic conversion after complete delignification. Low-temperature swelling of the CDL-treated wood particles in TFA resulted in nearly complete enzymatic hydrolysis, regardless of original lignin composition. CONCLUSIONS: Genetic modification of lignin composition can enhance the portfolio of aromatic products obtained from lignocellulosic biomass while promoting disassembly into biofuel and bioproduct substrates. CDL enhances rates of enzymatic digestion and chemical conversion, but cellulose remains intrinsically recalcitrant. Cold TFA is sufficient to overcome this recalcitrance after CDL treatment. Our results inform a 'no carbon left behind' strategy to convert total woody biomass into lignin, cellulose, and hemicellulose value streams for the future biorefinery.

Kinetic modeling analysis of maleic acid-catalyzed hemicellulose hydrolysis in corn stover.

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings.

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.

Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage.

DDGS and wet distillers' grains are the major co-products of the dry grind ethanol facilities. As they are mainly used as animal feed, a typical compositional analysis of the DDGS and wet distillers' grains mainly focuses on defining the feedstock's nutritional characteristics. With an increasing demand for fuel ethanol, the DDGS and wet distillers' grains are viewed as a potential bridge feedstock for ethanol production from other cellulosic biomass. The introduction of DDGS or wet distillers' grains as an additional feed to the existing dry grind plants for increased ethanol yield requires a different approach to the compositional analysis of the material. Rather than focusing on its nutritional value, this new approach aims at determining more detailed chemical composition, especially on polymeric sugars such as cellulose, starch and xylan, which release fermentable sugars upon enzymatic hydrolysis. In this paper we present a detailed and complete compositional analysis procedure suggested for DDGS and wet distillers' grains, as well as the resulting compositions completed by three different research groups. Polymeric sugars, crude protein, crude oil and ash contents of DDGS and wet distillers' grains were accurately and reproducibly determined by the compositional analysis procedure described in this paper.

Cellulose modification by recyclable swelling solvents.

BACKGROUND: The invention of efficient systems for lignocellulose conversion is essential for economically feasible production of bio-based chemicals and biofuels. One limiting step is highly selective processes to quickly decrystallize the compact cellulose structure for efficient hydrolysis. We evaluated the impact of trifluoroacetic acid (TFA) and phosphorous acid (PA)-induced swelling of crystalline cellulose on enhancement of enzymatic digestion. RESULTS: In this study, two swelling agents, TFA and PA, are compared and found to be highly efficient for cellulose decrystallization at low temperatures within 1 h. After treatment, the microfibril structure of swollen celluloses was observed to develop distinct microscopic morphology and subsequent enzymatic hydrolysis resulted over 90% cellulose conversion within 24 h. The crystalline cellulose change was determined by reduction of loss of X-ray diffractability, and loss of resistance to enzymatic hydrolysis. NMR results suggest that both TFA and PA efficiently converted most of the crystalline cellulose regions to amorphous regions through cellulose chain relocation that inhibits recrystallization. It was found that the swelling mechanism is different between TFA and PA. To the best of our knowledge, it is the first time to compare and quantify the cellulose regions transformation by swelling agents. CONCLUSION: This study shows the low-temperature swelling of different celluloses in TFA and PA reduces recalcitrance of crystalline cellulose to enzymatic hydrolysis. TFA and PA are both ideal candidate swelling agents for a closed system for ease of solvent recovery by either simple distillation or filtration. This study provides potentially useful agents in large-scale deconstruction of biomass.

Hydrolysis of untreated lignocellulosic feedstock is independent of S-lignin composition in newly classified anaerobic fungal isolate, Piromyces sp. UH3-1.

BACKGROUND: Plant biomass is an abundant but underused feedstock for bioenergy production due to its complex and variable composition, which resists breakdown into fermentable sugars. These feedstocks, however, are routinely degraded by many uncommercialized microbes such as anaerobic gut fungi. These gut fungi express a broad range of carbohydrate active enzymes and are native to the digestive tracts of ruminants and hindgut fermenters. In this study, we examine gut fungal performance on these substrates as a function of composition, and the ability of this isolate to degrade inhibitory high syringyl lignin-containing forestry residues. RESULTS: We isolated a novel fungal specimen from a donkey in Independence, Indiana, United States. Phylogenetic analysis of the Internal Transcribed Spacer 1 sequence classified the isolate as a member of the genus Piromyces within the phylum Neocallimastigomycota (Piromyces sp. UH3-1, strain UH3-1). The isolate penetrates the substrate with an extensive rhizomycelial network and secretes many cellulose-binding enzymes, which are active on various components of lignocellulose. These activities enable the fungus to hydrolyze at least 58% of the glucan and 28% of the available xylan in untreated corn stover within 168 h and support growth on crude agricultural residues, food waste, and energy crops. Importantly, UH3-1 hydrolyzes high syringyl lignin-containing poplar that is inhibitory to many fungi with efficiencies equal to that of low syringyl lignin-containing poplar with no reduction in fungal growth. This behavior is correlated with slight remodeling of the fungal secretome whose composition adapts with substrate to express an enzyme cocktail optimized to degrade the available biomass. CONCLUSIONS: Piromyces sp. UH3-1, a newly isolated anaerobic gut fungus, grows on diverse untreated substrates through production of a broad range of carbohydrate active enzymes that are robust to variations in substrate composition. Additionally, UH3-1 and potentially other anaerobic fungi are resistant to inhibitory lignin composition possibly due to changes in enzyme secretion with substrate. Thus, anaerobic fungi are an attractive platform for the production of enzymes that efficiently use mixed feedstocks of variable composition for second generation biofuels. More importantly, our work suggests that the study of anaerobic fungi may reveal naturally evolved strategies to circumvent common hydrolytic inhibitors that hinder biomass usage.

Production of cellulose nanofibers using phenolic enhanced surface oxidation.

In this study we demonstrate that lignin monomers formed as byproducts of pulping or bioprocessing of lignocellulosic biomass is an effective enhancer to oxidize cellulose surfaces with ozone for the production of cellulose nanofibers (CNF). Never dried softwood pulp with minimum mercerization was enzymatically treated leading to a homogeneous pulp slurry with a higher reactivity. The slurry was oxidized by ozone gas in the presence of syringic acid, a lignin degradation model compound, as an oxidation enhancer at room temperature and pH 11. Transmission electron microscopy (TEM) observations showed that stable CNF bundles with 3-10nm widths and lengths >100nm were obtained after ultrasonication of the oxidized product in water. Extensive characterization of the new CNF films revealed the nanofibers had carboxylate content similar to conventional carboxylated cellulose prepared by TEMPO-mediated oxidation. Based on NMR spectra, chemical conversion of the syringic acid during oxidation is proposed.