Dhrubojyoti D. Laskar

Phenylalanine Biosynthesis in Arabidopsis thaliana IDENTIFICATION AND CHARACTERIZATION OF AROGENATE DEHYDRATASES

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had kcat/Km values of 1050, 7650, and 1560 m-1 s-1 for arogenate versus 38, 240, and 16 m-1 s-1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had kcat/Km values of 1140, 490, and 620 m-1 s-1, with prephenate not serving as a substrate unless excess recombinant protein (>150 μg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1–ADT6.

Controlling Porosity in Lignin‐Derived Nanoporous Carbon for Supercapacitor Applications

Low-cost renewable lignin has been used as a precursor to produce porous carbons. However, to date, it has not been easy to obtain high surface area porous carbon without activation processes or templating agents. Here, we demonstrate that low molecular weight lignin yields highly porous carbon with more graphitization through direct carbonization without additional activation processes or templating agents. We found that molecular weight and oxygen consumption during carbonization are critical factors to obtain high surface area, graphitized porous carbons. This highly porous carbon from low-cost renewable lignin sources is a good candidate for supercapacitor electrode materials.

Noble-metal catalyzed hydrodeoxygenation of biomass-derived lignin to aromatic hydrocarbons

Conversion of biomass derived lignin to liquid fuels has the promising potential to significantly improve carbon utilization and economic competitiveness of biomass refineries. In this study, an aqueous phase catalytic process was developed to selectively depolymerize the lignin polymeric framework and remove oxygen via hydrodeoxygenation (HDO) reactions. Efficient methods (ethanol and dilute alkali extraction) for selectively producing reactive lignin oligomers with high yields from corn stover were established. Characteristic structural features of the technical lignins employed for hydrocarbon production were elucidated with the aid of advanced analytical techniques, such as 2D HSQC NMR spectroscopy and gel permeation chromatography (GPC). Combinations of noble metal catalysts in the presence of various solid acid zeolites were tested for HDO activity of the oligomeric technical lignins predominantly containing 8–O–4′ inter-unit linkages. Results showed 35%–60% conversion of lignin with 65%–70% product selectivity for aromatic hydrocarbons (e.g. toluene) under various HDO conditions in the presence of noble metals (Ru, Rh and Pt) over Al2O3 (or C) supports and solid acid zeolites (e.g., NH4+ Z-Y 57277-14-1) catalyst matrices.

Structural and Thermal Characterization of Wheat Straw Pretreated with Aqueous Ammonia Soaking

Production of renewable fuels and chemicals from lignocellulosic feedstocks requires an efficient pretreatment technology to allow ready access of polysaccharides for cellulolytic enzymes during saccharification. The effect of pretreatment on wheat straw through a low-temperature and low-pressure soaking aqueous ammonia (SAA) process was investigated in this study using Fourier transform infrared (FTIR), pyrolysis-gas chromatography/mass spectroscopy (Py-GC/MS), solid and liquid state nuclear magnetic resonance (NMR), and thermogravimetry/differential thermogravimetry (TG/DTG) to demonstrate the changes in lignin, hemicellulose, and cellulose structure. After treatment of 60 mesh wheat straw particles for 60 h with 28-30% ammonium hydroxide (1:10 solid/liquid) at 50 °C, sugar recovery increased from 14% (untreated) to 67% (SAA treated). The FTIR study revealed a substantial decrease in absorbance of lignin peaks. Solid and liquid state NMR showed minimal lignin structural changes with significant compositional changes. Activation energy of control and pretreated wheat straw was calculated according to the Friedman and ASTM methods and found to be decreased for SAA-treated wheat straw, from 259 to 223 kJ/mol. The SAA treatment was shown to remove significant amounts of lignin without strongly affecting lignin functional groups or structure.

Biodegradation of Hardwood Lignocellulosics by the Western Poplar Clearwing Borer, Paranthrene robiniae (Hy. Edwards)

Lignin in plant cell wall is a source of useful chemicals and also the major barrier for saccharification of lignocellulosic biomass for producing biofuel and bioproducts. Enzymatic lignin degradation/modification process could bypass the need for chemical pretreatment and thereby facilitate bioprocess consolidation. Herein, we reveal our new discovery in elucidating the process of hardwood lignin modification/degradation by clearwing borer, Paranthrene robiniae . The wood-boring clearwing borer, P. robiniae , effectively tunnels hardwood structures during the larval stage; its digestion products from wood components, however, has not yet been investigated. A series of analysis conducted in this study on tunnel walls and frass produced provided evidence of structural alterations and lignin degradation during such hardwood digestion process. The analysis included solid state (13)C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflectance Fourier transform infrared (ATR-FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermogravimetric (TG) analysis; the results strongly suggest that the structural alteration of lignin primarily involved a preferential degradation of syringyl units accompanied by oxidation on the side chains of lignin guaiacyl moieties. This study also further indicated that unlike the wood-feeding termite the clearwing borer does not target cellulose as an energy source, and thus its lignin degradation ability should provide potential information on how to disassemble and utilize hardwood lignin. Overall, this biological model with an efficient lignin disruption system will provide the new insight into novel enzyme system required for effective plant cell wall disintegration for enhanced cellulose accessibility by enzymes and production of value-added lignin derived products.

Simultaneous conversion of all cell wall components by an oleaginous fungus without chemi-physical pretreatment

Lignin utilization during biomass conversion has been a major challenge for lignocellulosic biofuel. In particular, the conversion of lignin along with carbohydrate for fungible fuels and chemicals will both improve the overall carbon efficiency and reduce the need for chemical pretreatments. However, few biomass-converting microorganisms have the capacity to degrade all cell wall components including lignin, cellulose, and hemicellulose. We hereby evaluated a unique oleaginous fungus strain, Cunninghamella echinulata FR3, for its capacity to degrade lignin during biomass conversion to lipid, and the potential to carry out consolidated fermentation without chemical pretreatment, especially when combined with sorghum (Sorghum bicolor) bmr mutants with reduced lignin content. The study clearly showed that lignin was consumed together with carbohydrate during biomass conversion for all sorghum samples, which indicates that this organism has the potential for biomass conversion without chemical pretreatment. Even though dilute acid pretreatment of biomass resulted in more weight loss during fungal fermentation than untreated biomass, the lipid yields were comparable for untreated bmr6/bmr12 double mutant and dilute acid-pretreated wild-type biomass samples. The mechanisms for lignin degradation in oleaginous fungi were further elucidated through transcriptomics and chemical analysis. The studies showed that in C. echinulata FR3, the Fenton reaction may play an important role in lignin degradation. This discovery is among the first to show that a mechanism for lignin degradation similar to those found in white and brown rot basidiomycetous fungi exists in an oleaginous fungus. This study suggests that oleaginous fungi such as C. echinulata FR3 can be employed for complete biomass utilization in a consolidated platform without chemical pretreatment or can be used to convert lignin waste into lipids.

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

BackgroundCurrently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation.ResultsWe discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate.ConclusionCollectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion.

DEGRADATION OF NATIVE WHEAT STRAW LIGNIN BY STREPTOMYCES VIRIDOSPORUS T7A

Lignin is one of the major contributing factors toward the recalcitrance of lignocellulosic biomass. Understanding the process of lignin degradation in natural biological processes will provide useful information to develop novel biomass conversion technologies. Functional group changes in the lignin entities during the process may contribute to the cellulose degradation (utilization) by the microorganisms. In this study, compositional and advanced Fourier transform infrared, pyrolysis gas chromatography/mass spectrometry and 13C cross polarization/magic angle spinning nuclear magnetic resonance analysis were performed to explore the mechanism of biodegradation of wheat straw by Streptomyces viridosporus T7A. The results indicated that S. viridosporus T7A removed lignin and hemicelluloses as indicated by the increased carbohydrate/lignin ratio. Significant modification of carbonyl and methoxyl groups in the complex lignin structure was also evident. Most importantly, the quantitative results showed that lignin degradation was featured by deduction of guaiacyl unit. The results provide new insight for understanding lignin degradation by bacteria.

Aqueous phase catalytic conversion of agarose to 5-hydroxymethylfurfural by metal chlorides

The production of 5-hydroxymethylfurfural (5-HMF) from agarose catalyzed by metal chlorides was studied in aqueous phase. A series of metal chlorides, including NaCl, CaCl2, MgCl2, ZnCl2, CrCl3, CuCl2 and FeCl3, were comparatively investigated to catalyze agarose degradation for the production of 5-HMF at temperatures of 180 °C, 200 °C and 220 °C, catalyst concentration of 0.5% (w/w), 1% (w/w) and 5% (w/w), time of 0–50 min, and substrate concentration of 2% (w/w). Results revealed that alkali and alkaline earth metal chlorides, including NaCl, CaCl2 and MgCl2, resulted in higher 5-HMF yields from agarose with negligible amount of byproducts, such as levulinic acid and lactic acid, derived from further degradation reactions. 1% (w/w) MgCl2 was the most efficient catalyst among the tested metal chlorides for 5-HMF production from agarose and resulted in both the highest yield of 40.7% and highest selectivity of 49.1% at 200 °C for 35 min. The cleavage of C–O–C bond in agarose with subsequent isomerization of galactose to its ketose was considered as a possible mechanism for formation of 5-HMF under MgCl2 catalyzed conditions.

System integration for producing microalgae as biofuel feedstock.

Although a promising technology, using microalgae as feedstock for biofuel production faces a broad range of grand challenges to become technologically and economically viable. Growing algae for fuel production involves altering the culture conditions and processes toward maximum accumulation of biomass, especially lipid. Commercial success of such targeted use requires optimal combination of processes and culture environments so that maximum value from algae biomass will be achieved with high productivity, minimal inputs, sustainable resources and lowest possible costs. A systematic approach and process integration are critical factors in a successful future for algal biorefineries. This article presents opinions and supporting literature on: employing physiological characteristics of algae for increasing biomass productivity and lipid accumulation, opportunities for producing co-products, water resource conservation, nutrient recycling, CO2 capture and delivery and process integration with downstream processing. While various information gaps still need to be filled, the available knowledge base clearly demonstrates the need for system integration.