Non-cellulosic Polysaccharides Research Articles

A review on chimeric xylanases: methods and conditions.

Multi-functional enzymes are one of the nature's solutions to facilitate metabolic pathways, thus several reactions are regulated and performed simultaneously on one polypeptide chain. Inspired by nature, artificial chimeric proteins have been designed to reduce the production costs and improve the performance. One of the interesting applications of this method is in the plant-based industries such as feed additive, waste treatment, biofuel production, and pulp and paper bleaching. In fact, the heterogeneous texture of plants needs using a combination of different enzymes to achieve an optimal quality in the manufacturing process. Given that xylans are the most abundant non-cellulosic polysaccharides in nature, xylanases are widely utilized in the mentioned industries. In this regard, several studies have been conducted to develop the relevant chimeric enzymes. Despite the successes that have been attained in this field, misfolding, functional or structural interference, and linker breakage have been reported in some cases. The present paper reviews the research to introduce the prerequisites to design an appropriate chimeric xylanase.

Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover

BackgroundInefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans.ResultsIn this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues.ConclusionTo our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future.

Flax fibers: assessing the non-cellulosic polysaccharides and an approach to supramolecular design of the cell wall

Due to their particular tensile properties and composition, flax cellulosic fibres are widely used in various applications and are important in the stem architecture, as a source of mechanical support. Here, we have examined how various chemical extractions of cell-wall polysaccharides might improve our understanding of the structure and composition of these fibres. A set of chemical extractions, which had previously been independently studied on fibres of different origins and by different laboratories, was run on the same batch of technical fibres, variety Eden, and the released non-cellulosic polysaccharides were analyzed in terms of sugar composition and size exclusion chromatography. No single-step protocol was able to extract all the non-cellulosic polysaccharides estimated to be in the range of 130–140 mg per g of dry fibres. The major polymers consisted of rhamnogalacturonan and glucomannan, while the minor ones were xyloglucan and xylan. Several types of stepwise extractions helped to elucidate the subfractions of each polymer that are differently retained within cell wall. From our data, we have pointed out a new model which might help to approach the supramolecular design of the flax cellulosic fibres and, afterwards, the relationships between their chemical composition and their mechanical properties.

Insights into cell wall structure of Sida hermaphrodita and its influence on recalcitrance

The perennial plant Sida hermaphrodita (Sida) is attracting attention as potential energy crop. Here, the first detailed view on non-cellulosic Sida cell wall polysaccharide composition, structure and architecture is given. Cell walls were prepared from Sida stems and sequentially extracted with aqueous buffers and alkali. The structures of the quantitatively predominant polysaccharides present in each fraction were determined by biochemical characterization, glycome profiling and mass spectrometry. The amounts of glucose released by Accellerase-1500® treatment of the cell wall and the cell wall residue remaining after each extraction were used to assess the roles of pectin and hemicellulose in the recalcitrance of Sida biomass.4-O-Methyl glucuronoxylan with a low proportion of side substitutions was identified as the major non-cellulosic glycan component of Sida stem cell walls. Pectic polysaccharides and xylans were found to be associated with lignin, suggesting that these polysaccharides have roles in Sida cell wall recalcitrance to enzymatic hydrolysis.

Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR

Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

Differences in glycosyltransferase family 61 accompany variation in seed coat mucilage composition in Plantago spp.

Xylans are the most abundant non-cellulosic polysaccharide found in plant cell walls. A diverse range of xylan structures influence tissue function during growth and development. Despite the abundance of xylans in nature, details of the genes and biochemical pathways controlling their biosynthesis are lacking. In this study we have utilized natural variation within the Plantago genus to examine variation in heteroxylan composition and structure in seed coat mucilage. Compositional assays were combined with analysis of the glycosyltransferase family 61 (GT61) family during seed coat development, with the aim of identifying GT61 sequences participating in xylan backbone substitution. The results reveal natural variation in heteroxylan content and structure, particularly in P. ovata and P. cunninghamii, species which show a similar amount of heteroxylan but different backbone substitution profiles. Analysis of the GT61 family identified specific sequences co-expressed with IRREGULAR XYLEM 10 genes, which encode putative xylan synthases, revealing a close temporal association between xylan synthesis and substitution. Moreover, in P. ovata, several abundant GT61 sequences appear to lack orthologues in P. cunninghamii. Our results indicate that natural variation in Plantago species can be exploited to reveal novel details of seed coat development and polysaccharide biosynthetic pathways.

Tracheid cell-wall structures and locations of (1 → 4)-β-d-galactans and (1 → 3)-β-d-glucans in compression woods of radiata pine (Pinus radiata D. Don)

BackgroundCompression wood (CW) forms on the underside of tilted stems of coniferous gymnosperms and opposite wood (OW) on the upperside. The tracheid walls of these wood types differ structurally and chemically. Although much is known about the most severe form of CW, severe CW (SCW), mild CWs (MCWs), also occur, but less is known about them. In this study, tracheid wall structures and compositions of two grades of MCWs (1 and 2) and SCW were investigated and compared with OW in slightly tilted radiata pine (Pinus radiata) stems.ResultsThe four wood types were identified by the distribution of lignin in their tracheid walls. Only the tracheid walls of OW and MCW1 had a S3 layer and this was thin in MCW1. The tracheid walls of only SCW had a S2 layer with helical cavities in the inner region (S2i). Using immunomicroscopy, (1 → 4)-β-D-galactans and (1 → 3)-β-D-glucans were detected in the tracheid walls of all CWs, but in only trace amounts in OW. The (1 → 4)-β-D-galactans were located in the outer region of the S2 layer, whereas the (1 → 3)-β-D-glucans were in the inner S2i region. The areas and intensities of labelling increased with CW severity. The antibody for (1 → 4)-β-D-galactans was also used to identify the locations and relative amounts of these galactans in whole stem cross sections based on the formation of an insoluble dye. Areas containing the four wood types were clearly differentiated depending on colour intensity. The neutral monosaccharide compositions of the non-cellulosic polysaccharides of these wood types were determined on small, well defined discs, and showed the proportion of galactose was higher for CWs and increased with severity.ConclusionThe presence of an S3 wall layer is a marker for very MCW and the presence of helical cavities in the S2 wall layer for SCW. The occurrence and proportions of (1 → 4)-β-D-galactans and (1 → 3)-β-D-glucans can be used as markers for CW and its severity. The proportions of galactose were consistent with the labelling results for (1 → 4)-β-D-galactans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0884-3) contains supplementary material, which is available to authorized users.

The inside and outside: topological issues in plant cell wall biosynthesis and the roles of nucleotide sugar transporters

The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered.

Activation of lignocellulosic biomass for higher sugar yields using aqueous ionic liquid at low severity process conditions.

BackgroundConcerns around greenhouse gas emissions necessitate the development of sustainable processes for the production of chemicals, materials, and fuels from alternative renewable sources. The lignocellulosic plant cell walls are one of the most abundant sources of carbon for renewable bioenergy production. Certain ionic liquids (ILs) are very effective at disrupting the plant cell walls of lignocellulose, and generate a substrate that is effectively hydrolyzed into fermentable sugars. Conventional ILs are relatively expensive in terms of purchase price, and the most effective imidazolium-based ILs also require energy intensive processing conditions (>140 °C, 3 h) to release >90 % fermentable sugar yields after saccharification.ResultsWe have developed a highly effective pretreatment technology utilizing the relatively inexpensive IL comprised tetrabutylammonium [TBA]+ and hydroxide [OH]− ions that generate high glucose yields (~95 %) after pretreatment at very mild processing conditions (50 °C). The efficiency of [TBA][OH] pretreatment of lignocellulose was further studied by analyzing chemical composition, powder X-ray diffraction for cellulose structure, NMR and SEC for lignin dissolution/depolymerization, and glycome profiling for cell wall modifications. Glycome profiling experiments and computational results indicate that removal of the noncellulosic polysaccharides occurs due to the ionic mobility of [TBA][OH] and is the key factor in determining pretreatment efficiency. Process modeling and energy demand analysis suggests that this [TBA][OH] pretreatment could potentially reduce the energy required in the pretreatment unit operation by more than 75 %.ConclusionsBy leveraging the benefits of ILs that are effective at very mild processing conditions, such as [TBA][OH], lignocellulosic biomass can be pretreated at similar efficiency as top performing conventional ILs, such as 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc], but at much lower temperatures, and with less than half the IL normally required to be effective. [TBA][OH] IL is more reactive in terms of ionic mobility which extends removal of lignin and noncellulosic components of biomass at the lower temperature pretreatment. This approach to biomass pretreatment at lower temperatures could be transformative in the affordability and energy efficiency of lignocellulosic biorefineries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0561-7) contains supplementary material, which is available to authorized users.

FT-IR and FT-Raman characterization of non-cellulosic polysaccharides fractions isolated from plant cell wall

The purpose of this work was to reveal the structural changes of cell wall polysaccharides’ fractions during tomato fruit development by analysis of spectral data. Mature green and red ripe tomato fruit were taken into consideration. The FT-IR spectra of water soluble pectin (WSP), imidazole soluble pectin (ISP) and diluted alkali soluble pectin (DASP) contained bands typical for pectins. Whereas for KOH fraction spectra bands typical for hemicelluloses were present. The FT-IR spectra showed the drop down of esterification degree of WSP and ISP polysaccharides during maturation. The changes in polysaccharides structure revealed by spectra were the most visible in the case of pectic polysaccharides. The WSP and DASP fraction pectins molecules length were shortened during tomato maturation and ripening. Whereas the ISP fraction spectra analysis showed that this fraction contained rhamnogalacturonan I, but also for red ripe was rich in pectic galactan comparing with ISP fraction from mature green.

Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa.

The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.

Production of new cellulose nanomaterial from red algae marine biomass Gelidium elegans

Nanocellulose was successfully isolated from Gelidium elegans red algae marine biomass. The red algae fiber was treated in three stages namely alkalization, bleaching treatment and acid hydrolysis treatment. Morphological analysis was performed by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). TEM results revealed that the isolated nanocellulose had the average diameter and length of 21.8±11.1nm and of 547.3±23.7nm, respectively. Fourier transform infrared (FTIR) spectroscopy proved that the non-cellulosic polysaccharides components were progressively removed during the chemically treatment, and the final derived materials composed of cellulose parent molecular structure. X-ray diffraction (XRD) study showed that the crystallinity of yielded product had been improved after each successive treatments subjected to the treated fiber. The prepared nano-dimensional cellulose demonstrated a network-like structure with higher crystallinity (73%) than that of untreated fiber (33%), and possessed of good thermal stability which is suitable for nanocomposite material.

Role of UDP-Glucuronic Acid Decarboxylase in Xylan Biosynthesis in Arabidopsis

UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell-wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDP-glucuronic acid decarboxylase (UXS). Until now, little has been known about the physiological roles of UXS in plants. Here, we report that AtUXS1, AtUXS2, and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5, and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1 usx2 uxs4 triple mutant show no obvious phenotype, the uxs3 uxs5 uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in uxs3 uxs5 uxs6 and linkage analysis confirmed that the xylan content in uxs3 xus5 uxs6 declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel-permeation chromatography showed that the molecular weight of non-cellulosic polysaccharides in the triple mutants, mainly composed of xylans, is lower than that in the wild type, suggesting an effect on the elongation of the xylan backbone. Upon saccharification treatment stems of the uxs3uxs5uxs6 triple mutants released monosaccharides with a higher efficiency than those of the wild type. Taken together, our results indicate that the cytosol UXS plays a more important role than the Golgi-localized UXS in xylan biosynthesis.

Effect of Nixtamalized Maize with Lime Water (Nejayote) on Rheological and Microbiological Properties of Masa

Nixtamalization process generates a large amount of waste called nejayote. The purpose of this research was to evaluate the microbiological and rheological properties of masa obtained from nixtamalized maize with nejayote at processing and 4 days after processing. Rheological parameters (elastic and viscous modulus) showed no significant difference between days 1 and day 4 for masa with 2:1 diluted nejayote. Microbiological analyses showed an increase in Fusarium verticillioides in masa with diluted nejayote 1:1 (water/full-strength nejayote) at day 4. Penicillium aethiopicum showed no growth in masa with diluted nejayote 2:1 (water:full-strength nejayote). Rhodotorula and bacteria showed higher growth in control masa for day 1 and 4, respectively. Masa with 2:1 diluted nejayote showed no bacterial growth at day 1, and the day 4 bacterial growth was lower than control masa. Hence, the microbiological quality of masa nejayote 2:1 was better than control masa, which could increase shelf-life. Practical Applications A sub-product of nixtamalization is nejayote, a highly polluting waste. During nixtamalization, a large amount of nejayote is produced; therefore, in Mexico it is important to propose reuse alternatives. Nejayote contains 2 g/100 g of dry solids, including pericarp, cellulose, hemicellulose, germ, proteins, calcium, endosperm and noncellulosic polysaccharides.

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components.

UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.

Metatranscriptomic discovery of plant biomass-degrading capacity from grass carp intestinal microbiomes.

Despite the economic importance of fish, the ecology and metabolic capacity of fish microbiomes are largely unknown. Here, we sequenced the metatranscriptome of the intestinal microbiota of grass carp, Ctenopharyngodon idellus, a freshwater herbivorous fish species. Our results confirmed previous work describing the bacterial composition of the microbiota at the phylum level as being dominated by Firmicutes, Fusobacteria, Proteobacteria and Bacteriodetes. Comparative transcriptomes of the microbiomes of fish fed with different experimental diets indicated that the bacterial transcriptomes are influenced by host diet. Although hydrolases and cellulosome-based systems predicted to be involved in degradation of the main chain of cellulose, xylan, mannan and pectin were identified, transcripts with glycoside hydrolase modules targeting the side chains of noncellulosic polysaccharides were more abundant. Predominant 'COG' (Clusters of Orthologous Group) categories in the intestinal microbiome included those for energy production and conversion, as well as carbohydrate and amino acid transport and metabolism. These results suggest that the grass carp intestinal microbiome functions in carbohydrate turnover and fermentation, which likely provides energy for both host and microbiota. Grass carp intestinal microbiome thus reflects its evolutionary adaption for harvesting nutrients for an herbivore with a high-throughput nutritional strategy that is not dominated by cellulose digestion but rather the degradation of intracellular polysaccharides.

Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus II. Degradation of lignin and non-cellulosic polysaccharides in European ash wood

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) by the white rot fungus Pycnoporus sanguineus were investigated in European ash wood (hardwood) using transmission electron microscopy coupled with immunocytochemistry. Lignin was preferentially removed in secondary cell walls of ash xylem cells including fibers, vessel and (ray/axial) parenchyma cells prior to degradation of hemicelluloses (i.e. selective decay). Concomitantly, degradation of cell walls occurred by progressive thinning from the lumen surface outwards similar to simultaneous decay. Preferential degradation of middle lamellae before complete degradation of secondary cell walls (i.e. a typical feature of selective decay) was only observed in bi-/triseriate rays by initial attack from pit regions. Vessel and parenchyma cells showed greater resistance to decay than fibers with narrower decay zone produced. A comparison of decay between ash and spruce wood (Part I, softwood) demonstrated that P. sanguineus produces selective decay in both hard- and softwoods but more typical micromorphological features of selective decay are produced in spruce tracheids than ash fibers. This study highlights that degradation of lignin proceeds hemicelluloses in wood cell walls attacked by the selective white rot fungus P. sanguineus.

Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus I. preferential lignin degradation prior to hemicelluloses in Norway spruce wood

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) in Norway spruce wood (softwood) by the white rot fungus Pycnoporus sanguineus was investigated using transmission electron microscopy coupled with immunocytochemistry. P. sanguineus produced selective decay in xylem cells including tracheids, ray tracheids and ray parenchyma cells. Lignin was preferentially removed first from cell walls with hemicelluloses (xylan and mannan) and cellulose remaining. Lignin in compound middle lamella (CML) regions of tracheids was also preferentially degraded prior to degradation of xyloglucan (hemicellulose). This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. In contrast, no clear preferential degradation of lignin prior to pectins and hemicelluloses was observed in middle lamella cell corner (MLcc) regions of tracheids which were the last regions to be attacked by the fungus. Micromorphologically, prominent cell separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls was observed as evidence of selective decay. Variations in resistance to decay by the fungus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types were also observed.

Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study.

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like—rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47–50% w/w) and non-cellulosic polysaccharides (16–22% w/w), and whole leaves were low in lignin (9–13% w/w). Of the dry mass of whole Agave leaves, 85–95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41–48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.

Chemical characterization of hydrothermally pretreated and enzyme-digested wheat straw: An evaluation of recalcitrance

There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure. Compositional analysis portrayed a considerable loss of cross-linking di-ferulic acid phenolics, hemicellulosic and cellulosic sugars. The remaining cellulosic and non-cellulosic polysaccharides were much more readily extractable in alkali and molecular profiling revealed the presence of low Mw oligomers in the fractions suggesting the partial enzyme hydrolysis of hemicelluloses and cellulose. Simultaneous saccharification and fermentation of the pretreated and enzyme-digested residues surprisingly resulted in ethanol yields of up to 99% of the theoretical. This is discussed in relation to the “recalcitrant” nature of the original pretreated and enzyme digested biomass.