Douglas G. Hayes

Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom.

BackgroundAs a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.ResultsWe analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.ConclusionThe research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

Compatible Ionic liquid‐cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048.

Efficient Reduction of Chitosan Molecular Weight by High-Intensity Ultrasound : Underlying Mechanism and Effect of Process Parameters

The degradation of chitosan by high-intensity ultrasound (HIU) as affected by ultrasound parameters and solution properties was investigated by gel permeation chromatography coupled with static light scattering. The molecular weight, radius of gyration, and polydispersity of chitosan were reduced by ultrasound treatment, whereas chitosan remained in the same random coil conformation and the degree of acetylation did not change after sonication. The results demonstrate that (1) the degradation of chitosan by ultrasound is primarily driven by mechanical forces and the degradation mechanism can be described by a random scission model; (2) the degradation rate is proportional to M w (3); and (3) the degradation rate coefficient is affected by ultrasound intensity, solution temperature, polymer concentration, and ionic strength, whereas acid concentration has little effect. Additionally, the data indicate that the degradation rate coefficient is affected by the degree of acetylation of chitosan and independent of the initial molecular weight.

Fast classification and compositional analysis of cornstover fractions using Fourier transform near-infrared techniques.

The objectives of this research were to determine the variation of chemical composition across botanical fractions of cornstover, and to probe the potential of Fourier transform near-infrared (FT-NIR) techniques in qualitatively classifying separated cornstover fractions and in quantitatively analyzing chemical compositions of cornstover by developing calibration models to predict chemical compositions of cornstover based on FT-NIR spectra. Large variations of cornstover chemical composition for wide calibration ranges, which is required by a reliable calibration model, were achieved by manually separating the cornstover samples into six botanical fractions, and their chemical compositions were determined by conventional wet chemical analyses, which proved that chemical composition varies significantly among different botanical fractions of cornstover. Different botanic fractions, having total saccharide content in descending order, are husk, sheath, pith, rind, leaf, and node. Based on FT-NIR spectra acquired on the biomass, classification by Soft Independent Modeling of Class Analogy (SIMCA) was employed to conduct qualitative classification of cornstover fractions, and partial least square (PLS) regression was used for quantitative chemical composition analysis. SIMCA was successfully demonstrated in classifying botanical fractions of cornstover. The developed PLS model yielded root mean square error of prediction (RMSEP %w/w) of 0.92, 1.03, 0.17, 0.27, 0.21, 1.12, and 0.57 for glucan, xylan, galactan, arabinan, mannan, lignin, and ash, respectively. The results showed the potential of FT-NIR techniques in combination with multivariate analysis to be utilized by biomass feedstock suppliers, bioethanol manufacturers, and bio-power producers in order to better manage bioenergy feedstocks and enhance bioconversion.

Activation of lignocellulosic biomass by ionic liquid for biorefinery fractionation.

Fractionation of lignocellulosic biomass is an attractive solution to develop an economically viable biorefinery by providing a saccharide fraction to produce fuels and a lignin stream that can be converted into high value products such as carbon fibers. In this study, the analysis of ionic liquid-activated biomass demonstrates that in addition of decreasing crystallinity, the selected ILs (1-butyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate) deacetylate Yellow poplar under mild conditions (dissolution at 60-80 °C), and lower the degradation temperature of each biomass polymeric component, thereby reducing the recalcitrance of biomass. Among the three tested ILs, 1-ethyl-3-methylimidazolium acetate performed the best, providing a strong linear relationship between the level of deacetylation and the rate of enzymatic saccharification for Yellow poplar.

Transparent dispersions of milk-fat-based nanostructured lipid carriers for delivery of β-carotene.

Nanostructured lipid carriers (NLCs) are possible vehicles to incorporate lipophilic bioactive compounds in transparent functional beverages. In this work, anhydrous milk fat (AMF) and Tween 80 were used to prepare NLCs using a phase-inversion temperature method, and β-carotene was used as a model lipophilic bioactive compound. The phase-inversion temperature decreased from >95 to 73 °C, when NaCl increased from 0 to 1.0 M in the aqueous phase. At 0.8 M NaCl and phase inversion by heating at 90 °C for 30 min, transparent NLC dispersions were observed at AMF levels higher than 10% (w/w), corresponding to particles smaller than ~25 nm. The NLC dispersions were dilution- and dialysis-stable and maintained turbidity and particle size during 90 days of storage at room temperature. The degradation of β-carotene encapsulated in NLCs was much reduced when compared to its encapsulation in the soybean-oil-based nanoemulsion.

Sucrose monolaurate improves the efficacy of sodium hypochlorite against Escherichia coli O157:H7 on spinach.

It is well-recognized that chlorine has limited efficacy when applied to inactivate pathogens on fresh produce. One of the many factors limiting efficacy is the high interfacial tension of chlorine-based sanitizers that limits the access of chlorine to the microorganisms. In this work, we investigated the efficacy of sodium hypochlorite (200 ppm, pH 6.0) at 4 and 20 °C against Escherichia coli O157:H7 inoculated on baby spinach leaves as affected by the surfactant sucrose monolaurate (SML) at below (100 ppm), above (250 ppm), and well above (10,000 ppm) the critical micelle concentration (CMC) of ~200 ppm at 20 °C. The surfactant-containing chlorine treatments were compared to those with buffer only, surfactant only, and chlorine only. Significantly improved inactivation, as evidenced by survival of E. coli O157:H7 was achieved when 250 or 10,000 ppm SML was added with chlorine. This is attributed to the reduction of interfacial tension between the sanitizing solutions and spinach surface. Treatments at 20 °C resulted in greater mean inactivation than those at 4 °C but the difference was not significant. Comparisons of SML concentrations in treatment solutions before and after sanitization showed that SML decreased more at a lower temperature and when chlorine was present, resulting from adsorption of SML onto spinach matrix. Our work illustrates the importance of using surfactants at concentrations above the CMC to enhance the efficacy of chlorine sanitization.

Shearing Characteristics of Biomass for Size Reduction

Warner-Bratzler shearing device in a universal test machine evaluated the cutting response characteristics of single stems of hickory, switchgrass, and corn stover. Different knife bevel angles (30 ° and 45 °) at a fixed cutting speed of 254 mm/min were evaluated. Biomass cutting energy was determined on a stem cross-sectional area basis (specific cutting energy, kN/m). Biomass shear strength was based on peak load and actual cross-sectional area. Biomass variety significantly (p < 0.001) affected cutting energy for both knife bevel angles. Mean specific cutting energies for hickory, switchgrass, and corn stover were 41.1, 6.3, 53.1 kN/m, and 75.4, 10.1, 27.7 kN/m for 30 ° and 45 ° knife bevel angles, respectively. Thus, the shallower 30 ° bevel angle required less cutting energy when cutting hickory and switchgrass. Corn stover unexpectedly required less cutting energy for the increased bevel angle of 45 °, and may reflect different composition. Statistical differences between mean specific cutting energies for 30 ° and 45 ° knife bevel angles varied with biomass. Shear strengths were statistically different (p < 0.001) for biomass varieties at a given knife bevel angles. Mean shear strength due to cutting of hickory, switchgrass, and corn stover by 30 ° and 45 ° knife bevel angles were 10.5, 12.2, 3.0 MPa, and 14.9, 15.3, 2.6 MPa, respectively. In conclusion, magnitude of specific cutting energy and shear strength varied with biomass selection and sometimes varied with knife bevel angle. Optimum biomass cutting and equipment design may need to be tailored to the specific biomass application and conditions.

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregates increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the proteins affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process.

Expulsion of proteins from water-in-oil microemulsions by treatment with cosurfactant

A quick and simple method has been developed for the recovery of proteins from water-in-oil microemulsions (w/o-MEs), which is needed to further the use of liquid-liquid extraction in bioseparations. By adding a small portion (0.1 v/v or less) of cosurfactant (e.g., 1-alkanol) to w/o-ME solution, proteins were readily expelled, sometimes as solids, while most or all of the surfactant (Aerosol OT) remained in solution. The release of proteins increased with the further addition of cosurfactant and was greater when the molar ratio of protein to w/o-ME or fractional occupancy (f) was high. However, protein expulsion was also significant when f was small. The addition of cosurfactant released ribonuclease, lysozyme, alpha-chymotrypsin, pepsin, bovine serum albumin (BSA), and catalase from w/o-ME solution, but the expulsion was greater for BSA relative to chymotrypsin and lysozyme. Protein expulsion also increased with cosurfactant chain length for the homologous series of 1-alkanols starting at 1-butanol; however, water was also coexpelled in significant amounts. An exception to the latter rule was 1-butanol, which readily promoted the release of protein, but not encapsulated water. The addition of 1-butanol to a w/o-ME solution containing alpha-chymotrypsin and BSA selectively released the former protein, with chymotryptic activity occurring in the recovered protein. Possible mechanisms for the cosurfactant-mediated release of protein are discussed. Copyright 1998 John Wiley & Sons, Inc.