Lignocellulosic Plant Biomass Research Articles

Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion.

BackgroundPlant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX).ResultsThe transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants.ConclusionsThis study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0639-2) contains supplementary material, which is available to authorized users.

Purification and concentration of lignin from the spent liquor of the alkaline oxidation of woody biomass through membrane separation technology

Lignocellulosic plant biomass is a renewable and abundant source for the production of bio-based fuels, chemicals and chemical building blocks. Efficient fractionation and conversion of these feedstocks are an essential step in the valorization of the cellulose, hemicellulose and lignin fractions. The use of a new two-stage alkaline oxidation (AlkOx) process has been investigated for the pretreatment of softwood in presence of sodium carbonate.Within this study, the use of commercial polymeric and ceramic ultrafiltration membranes for the purification and concentration of lignin from the spent liquor of the AlkOx process has been evaluated enabling further valorization thereof. Higher permeation fluxes were observed, ranging from 30 to 139L/m2h, depending on the hydrophilicity, pore size and structure/chemistry of the membrane. High lignin retentions have been obtained for all membranes. Diafiltration of the spent liquor using the ESP04 membrane enables the purification of the lignin fraction with an efficient removal of the impurities originating from both the lignocellulosic material and the pretreatment.The integration of both processes allows the co-valorization of the lignin fraction, besides the primary C5/C6 sugar fraction produced in the two-stage alkaline oxidation of softwood. Also some preliminary techno-economic calculations have been realized on the membrane separation process to asses the economic potential of this technology.

ChemInform Abstract: Homogeneous Catalyzed Reactions of Levulinic Acid: To γ‐Valerolactone and Beyond

AbstractReview: 56 refs.

Application of Molecular Techniques to Better Understand the Roles of Rumen Microbiota in Cattle Feed Efficiency

Feed efficiency, simply expressed as less feed inputs versus animal production outputs, can be measured in several ways, such as feed conversion ratio (FCR) and residual feed intake (RFI). FCR is a common measurement in beef cattle operations, and is the ratio of feed intake to live-weight gain. RFI is defined as the difference between actual and predicted feed intake after taking into account variability in maintenance and growth requirements. Rumen microbiota, which inludes bacteria, archaea, protozoa, and fungi, play an essential role in the digestion of lignocellulosic plant biomass, and can provide more than 70% of the host ruminants energy requirements via the production of volatile fatty acids (VFAs). Methane, a potent greenhouse gas (GHG), is produced in large quantities by the rumen microbiota, and is a known contributor to the global increase in GHG emissions. Studies have shown a negative relationship between methane emission and feed efficiency. Therefore, there is a need to study the feed efficiency from a rumen microbiome perspective and explore the probability of improving feed efficiency and hence reduce methane production in cattle by manipulating the rumen microbiome. The development of high-throughput sequencing technologies incuding metagenomics and metatranscriptomic analyses in the past decade has led to a sharp increase in understanding the rumen microbiota and associated function. As such, this mini-review will focus on the new findings during the last decade in cattle feed efficiency and the rumen microbiome.

Homogeneous Catalyzed Reactions of Levulinic Acid: To γ-Valerolactone and Beyond.

Platform chemicals derived from lignocellulosic plant biomass are viewed as a sustainable replacement for crude oil-based feedstocks. Levulinic acid (LA) is one such biomass-derived chemical that has been widely studied for further catalytic transformation to γ-valerolactone (GVL), an important 'green' fuel additive, solvent, and fine chemical intermediate. Although the transformation of LA to GVL can be achieved using heterogeneous catalysis, homogeneous catalytic systems that operate under milder reactions, give higher selectivities and can be recycled continuously are attracting considerable attention. A range of new homogeneous catalysts have now been demonstrated to efficiently convert LA to GVL and to transform LA directly to other value-added chemicals such as 1,4-pentanediol (1,4-PDO) and 2-methyltetrahydrofuran (2-MTHF). This Minireview covers recent advances in the area of homogeneous catalysis for the conversion of levulinic acid and levulinic ester derivatives to GVL and chemicals beyond GVL.

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii These strains showed resistance to kanamycin at concentrations >50 μg · ml(-1), whereas wild-type C. bescii was sensitive to kanamycin at 10 μg · ml(-1) In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.

Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation.

Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses.

Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition.

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.

Microbial Production of Short Chain Fatty Acids from Lignocellulosic Biomass: Current Processes and Market.

Biological production of organic acids from conversion of biomass derivatives has received increased attention among scientists and engineers and in business because of the attractive properties such as renewability, sustainability, degradability, and versatility. The aim of the present review is to summarize recent research and development of short chain fatty acids production by anaerobic fermentation of nonfood biomass and to evaluate the status and outlook for a sustainable industrial production of such biochemicals. Volatile fatty acids (VFAs) such as acetic acid, propionic acid, and butyric acid have many industrial applications and are currently of global economic interest. The focus is mainly on the utilization of pretreated lignocellulosic plant biomass as substrate (the carbohydrate route) and development of the bacteria and processes that lead to a high and economically feasible production of VFA. The current and developing market for VFA is analyzed focusing on production, prices, and forecasts along with a presentation of the biotechnology companies operating in the market for sustainable biochemicals. Finally, perspectives on taking sustainable product of biochemicals from promise to market introduction are reviewed.

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria.

Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.

Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Lignocellulosic plant biomass is the target feedstock for production of second-generation biofuels. Ionic liquid (IL) pretreatment can enhance deconstruction of lignocellulosic biomass into sugars that can be fermented to ethanol. Although biomass is typically washed following IL pretreatment, small quantities of residual IL can inhibit fermentative microorganisms downstream, such as the widely used ethanologenic yeast, Saccharomyces cerevisiae. The aim of this study was to identify yeasts tolerant to the IL 1-ethyl-3-methylimidazolium acetate, one of the top performing ILs known for biomass pretreatment. One hundred and sixty eight strains spanning the Ascomycota and Basidiomycota phyla were selected for screening, with emphasis on yeasts within or closely related to the Saccharomyces genus and those tolerant to saline environments. Based on growth in media containing 1-ethyl-3-methylimidazolium acetate, tolerance to IL levels ranging 1-5% was observed for 80 strains. The effect of 1-ethyl-3-methylimidazolium acetate concentration on maximum cell density and growth rate was quantified to rank tolerance. The most tolerant yeasts included strains from the genera Clavispora, Debaryomyces, Galactomyces, Hyphopichia, Kazachstania, Meyerozyma, Naumovozyma, Wickerhamomyces, Yarrowia, and Zygoascus. These yeasts included species known to degrade plant cell wall polysaccharides and those capable of ethanol fermentation. These yeasts warrant further investigation for use in saccharification and fermentation of IL-pretreated lignocellulosic biomass to ethanol or other products.

Brachypodium Cell Wall Mutant with Enhanced Saccharification Potential Despite Increased Lignin Content

Plant lignocellulosic biomass, mostly composed of cell walls, is one of the largest, mostly untapped, reserves of renewable carbon feedstock on the planet. Energy-rich polysaccharide polymers of plant cell walls can be broken down to produce fermentable sugars used to produce bioethanol. However, the complex structure of plant cell walls, and in particular, the presence of lignin, makes them recalcitrant to enzymatic degradation. Reducing this recalcitrance represents a major technological challenge. Brachypodium distachyon is an excellent model to identify parameters underlying biomass quality of energy grasses. In this work, we identified a mutant line spa1 with a so far undescribed phenotype combining brittleness with increased elasticity of the internodes. Mutant cell walls contain less crystalline cellulose and changes in hemicellulose and lignin quality and quantity. Using a dedicated reactor to follow in real-time, the evolution of straw particle size and sugar release during enzymatic digestion, we show that, despite the increased lignin content, the spa1 mutant has a dramatic reduced recalcitrance to saccharification compared to the WT. These observations demonstrate that other parameters besides lignin content are relevant for the improvement of biomass recalcitrance in energy grasses.

How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion

The utilisation of lignocellulosic plant biomass as an abundant, renewable feedstock for green chemistries and biofuel production is inhibited by its recalcitrant nature. In the environment, lignocellulolytic fungi are naturally capable of breaking down plant biomass into utilisable saccharides. Nonetheless, within the industrial context, inefficiencies in the production of lignocellulolytic enzymes impede the implementation of green technologies. One of the primary causes of such inefficiencies is the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite repression. Fungi coordinate metabolism, protein biosynthesis and secretion with cellular energetic status through the detection of intra- and extra-cellular nutritional signals. An enhanced understanding of the signals and signalling pathways involved in regulating the transcription, translation and secretion of lignocellulolytic enzymes is therefore of great biotechnological interest. This comparative review describes how nutrient sensing pathways regulate carbon catabolite repression, metabolism and the utilisation of alternative carbon sources in Saccharomyces cerevisiae and ascomycete fungi.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii.

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.

The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan

Xylan, a major polysaccharide in plant lignocellulosic biomass, is acetylated at O-2 and/or O-3 and its acetylation impedes the use of biomass for biofuel production. Currently, it is not known what genes encode acetyltransferases that are responsible for xylan O-acetylation. In this report, we demonstrate an essential role for the Arabidopsis gene ESKIMO1 (ESK1) in xylan O-acetylation during secondary wall biosynthesis. ESK1 expression was found to be regulated by the secondary wall master regulator SND1 (secondary wall-associated NAC domain protein1) and specifically associated with secondary wall biosynthesis. Its encoded protein was localized in the Golgi, the site of xylan biosynthesis. The esk1 mutation caused reductions in secondary wall thickening and stem mechanical strength. Chemical analyses of cell walls revealed that although the esk1 mutation did not cause apparent alterations in the xylan chain length and the abundance of the reducing end sequence, it resulted in a significant reduction in the degree of xylan acetylation. The reduced acetylation of esk1 xylan rendered it more accessible and digestible by endoxylanase, leading to generation of shorter xylooligomers compared with the wild type. Further structural analysis of xylan showed that the esk1 mutation caused a specific reduction in 2-O- and 3-O-monoacetylation of xylosyl residues but not in 2,3-di-O-acetylation or 3-O-acetylation of xylosyl residues substituted at O-2 with glucuronic acid. Consistent with ESK1's involvement in xylan O-acetylation, an activity assay revealed that the esk1 mutation led to a significant decrease in xylan acetyltransferase activity. Together, these results demonstrate that ESK1 is a putative xylan acetyltransferase required for 2-O- and 3-O-monoacetylation of xylosyl residues and indicate the complexity of the biochemical mechanism underlying xylan O-acetylation.

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

BackgroundSecond-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance.ResultsAlthough lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield.ConclusionsAlthough the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell wall composition and its influence on saccharification yield, and provide new potential targets for genetic improvement.

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

Hyperthermophilic endoglucanase for in planta lignocellulose conversion

BackgroundThe enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass.ResultsHere we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions.ConclusionThe hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

Refinery of Biomass by Utilization of Specific Effects of Microwave Irradiation

The results of application of microwave (MW) irradiation to achieve refinery of various kinds of recalcitrant biomass were summarized with special emphasis on recent innovative utilization of sensitizers for MW irradiation. The saccharification rates of lignocellulosic plant biomass consisting of softwoods, hardwoods and monocotyledons attained by the simple MW irradiation treatment were in the orders of 35-65% (softwoods), 70-80% (hardwoods) and around 80% (monocotyledons), respectively. The corresponding values of cellulose were around 69-81% (230-240°C), while xylan was susceptible to the MW energy and easily decomposed into oligomers including xylose by heating at >150°C, optimally at 204°C, to give 99% solubility rate. However, the effects of the MW treatment on the agro-food byproducts varied highly depending upon their origins. These results indicate necessity of troublesome specification of the optimum condition for each case. For development of more versatile and comprehensive refinery method we tried to use sensitizers for MW irradiation.We found that hydrogen peroxide was remarkably effective for biomass refinery. Utilization of activated carbon as a sensitizer for MW irradiation was also effective for saccharification of starchy materials. Enhancement of the surface charge of the activated carbons by oxidation and preparation of microwave absorption solid acid catalysts were recommended to further enhance their effectiveness. The overall results showed the importance of four factors, less affinity toward oligosaccharides in relation with pore size, quantity of surface negative charges in addition to electro-conductivity, faster filtration ability and unknown hot spot productivity. Due to removal of the coloured materials by the activated carbons, clear saccharified solution produced by one pot conversion could be directly used for further fermentation to produce biofuels such as ethanol.

Master regulators of wood formation in Eucalyptus

With the current global focus on bioenergy, forest plantations are increasingly becoming important sources for second generation biofuel, where the whole plant lignocellulosic biomass is to be mobilized. The lignocellulosic biomass is mainly composed of secondary walls (SW) possessing unique characteristics (biochemical composition and tridimensional association of polymers), which govern the intrinsic properties of wood. They especially contain high amounts of lignins, hydrophobic phenolic polymers which constitute an obstacle to the optimal utilization of plant species in paper industry and for saccharification prior to bioethanol production. Among perennial species, Eucalyptus species grow very fast and produce high yields of lignocellulosic biomass. They represent the main industrial plantations in the worldand one of the most appealing lignocellulosic feedstock for bioenergy production. Dissection of the molecular switches controlling the coordinated lignin biosynthetic genes is therefore of utmost importance to understand the molecular mechanisms underlying tissue specific deposition of lignin and be able to improve secondary cell wall properties. With the objective of improving Eucalyptus wood quality to better-fit industrial applications, we are focusing our efforts towards the identification and functional characterisation of regulatory genes controlling the biosynthesis of the cell wall polymers (mainly lignins). We performed a precise mapping and functional characterization of the cis-regulatory elements contained in the promoters of two genes encoding key and consecutive steps of the lignin biosynthetic pathway i.e. Cinnamoyl CoA reductase (CCR) and Cinnamyl Alcohol dehydrogenase (CAD) (Rahantamala et al, 2010). Our results supported a major role for the MYB transcription factors (TF) consensus sites in the control of the coordinated expression of these two genes. The functional analysis of two MYB factors (EgMYB1 and EgMYB2) preferentially expressed in Eucalyptus xylem revealed that they are able to bind specifically to these promoters and regulate transcriptionin vivo. EgMYB1 behaves as a repressor whereas EgMYB2 is an activator (Goicoechea et al, 2005, Legay et al, 2010) of the lignin biosynthetic genes but also of the secondary wall biosynthesis. Indeed, both MYBs were shown recently to be master genes regulating the entire secondary wall biosynthetic program including cellulose, xylan and lignin genes (Zhong et al, 2010; Legay et al 2010). The presence of both positive and negative regulators in Eucalyptus xylem offers the possibility of a combinatorial control of gene expression that could provide the necessary flexibility to ensure tight temporal and spatial regulation of lignin biosynthesis or secondary cell wall. To address this question and get a deeper insight the complex regulation of the SW formation in Eucalyptus, we are now studying the regulation of these two MYBs including fine spatial and temporal expression, identification of their direct targets genes and of their protein partners. We have constructed a yeast-two-hybrid library from Eucalyptus xylem that will also be instrumental for deciphering the interactants of landmark genes for the International Eucalyptus community. Thanks to the recent release of the E. grandis genome (Eucalyptus grandis Genome Project 2010, http://www.phytozome.net/eucalyptus), we have performed a genome-wide survey of the large R2R3-MYB superfamily. The phylogenetic comparison of this family with Arabidopsis, rice, poplar and grapevine showed a marked expansion of some clusters putatively involved in wood-related processes. Some R2R3 MYB genes seem to be specific of woody plants. The spatiotemporal expression patterns of members of such clusters are currently being studied. Although Auxin is known as a key regulator of cambium activity and wood formation, the Auxin response mediators [Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors] extensively characterized in model plants, are still largely uncharacterized in tree species. We have identified 23 Aux/IAA and 17 ARF in the E. grandis genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted amongst the three dicotyledonous plants studied (Arabidopsis, Populus and Eucalyptus). Expression analysis and EST database surveys are currently underway to explore the transcript levels of each member in the different organs and tissues of Eucalyptus at key developmental stages as well as in response to hormonal treatments and to environmental stresses. Further functional genomics studies conducted on new candidate transcription factors, their regulation under the developmental or environmental stimuli will help identifying major factors underpinning the physicochemical properties of cell walls, the recalcitrance of which remains a key scientific challenge for establishing highly efficient, sustainably produced, second-generation biofuels.