Marcos S. Buckeridge

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

Mobilisation of storage cell wall polysaccharides in seeds.

Plants store carbohydrate polymers in a number of forms. Starch is the principal form, followed by fructans and cell wall storage polysaccharides (CWSP). The latter are present mainly in seeds and consist of magnifications of one of the polysaccharides present in one of the cell wall matrices. CWSPs are classified as mannans, xyloglucans and galactans, the first being subdivided into pure mannans, galactomannans and glucomannans. The present review updates the principal discoveries concerning occurrence, structure, metabolism and eco-physiological significance of the seed storage cell wall polysaccharides with emphasis on their importance for plant metabolism and adaptation to their respective environments during evolution. The properties of CWSPs as storage polysaccharides are compared with starch and fructans and the current knowledge on the catabolism (including control) of mannan/galactomannan, xyloglucan, and galactan is reviewed. On the basis of these data, the idea that the CWSPs are multifunctional molecules is proposed and this feature is used as evidence for the hypothesis that, during evolution, the CWSPs were involved in cycles of transference of functions which led them to become storage polysaccharides, yet preserving some of their previous primary cell wall functions.

Elevated CO2 increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (approximately 370 ppm) and elevated (approximately 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production.

The structure and fine structure of leaf and culm cell walls of sugarcane plants were analyzed using a combination of microscopic, chemical, biochemical, and immunological approaches. Fluorescence microscopy revealed that leaves and culm display autofluorescence and lignin distributed differently through different cell types, the former resulting from phenylpropanoids associated with vascular bundles and the latter distributed throughout all cell walls in the tissue sections. Polysaccharides in leaf and culm walls are quite similar, but differ in the proportions of xyloglucan and arabinoxylan in some fractions. In both cases, xyloglucan (XG) and arabinoxylan (AX) are closely associated with cellulose, whereas pectins, mixed-linkage-β-glucan (BG), and less branched xylans are strongly bound to cellulose. Accessibility to hydrolases of cell wall fraction increased after fractionation, suggesting that acetyl and phenolic linkages, as well as polysaccharide–polysaccharide interactions, prevented enzyme action when cell walls are assembled in its native architecture. Differently from other hemicelluloses, BG was shown to be readily accessible to lichenase when in intact walls. These results indicate that wall architecture has important implications for the development of more efficient industrial processes for second-generation bioethanol production. Considering that pretreatments such as steam explosion and alkali may lead to loss of more soluble fractions of the cell walls (BG and pectins), second-generation bioethanol, as currently proposed for sugarcane feedstock, might lead to loss of a substantial proportion of the cell wall polysaccharides, therefore decreasing the potential of sugarcane for bioethanol production in the future.

Mixed linkage (1→3), (1→4)-β-D-glucans of grasses

Cereal Chem. 81(1):115–127 The mixed-linkage (1 3),(1 4)-D-glucans are unique to the Poales, the taxonomic order that includes the cereal grasses. (1 3), (1 4)-Glucans are the principal molecules associated with cellulose microfibrils during cell growth, and they are enzymatically hydrolyzed to a large extent once growth has ceased. They appear again during the developmental of the endosperm cell wall and maternal tissues surrounding them. The roles of (1 3),(1 4)-glucans in cell wall architecture and in cell growth are beginning to be understood. From biochemical experiments with active synthases in isolated Golgi membranes, the biochemical features and topology of synthesis are found to more closely parallel those of cellulose than those of all other noncellulosic -linked polysaccharides. The genes that encode part of the (1 3),(1 4)-glucan synthases are likely to be among those of the CESA/CSL gene superfamily, but a distinct glycosyl transferase also appears to be integral in the synthetic machinery. Several genes involved in the hydrolysis of (1 3),(1 4)-glucan have been cloned and sequenced, and the pattern of expression is starting to unveil their function in mobilization of -glucan reserve material and in cell growth. The starchy endosperm, a special trait of the Poales, is the fundamental reason that cereals are of such central importance in human nutrition (Langenheim and Thimann 1982). The world harvests over 1 billion tons of cereal grains annually. Rice and wheat alone provide at least half of the calories that humans ingest. The cell walls of the grasses also figure heavily in human and animal nutrition, from the mixed-linkage (1 3),(1 4)glucans that constitute a major portion of the endosperm walls to the vast amounts of xylanand cellulose-rich walls that are consumed by grazing animals. Scientific interest in (1 3),(1 4)glucans is associated partly with problems they cause in brewing and animal feed industries and partly from benefits they offer to human diets (as reviewed by Stone and Clarke 1992). The (1 3),(1 4)-glucans are the wall constituents responsible for the enhanced ability of barley and oat brans to reduce serum cholesterol in hypercholesterolemic individuals (for example, Braaten et al 1994) and to modulate the glycemic index in diabetics (Wood et al 1990). The (1 3),(1 4)-glucans and xylans of the endosperm cell walls in flours are contributing factors to bread quality (Girhammerar et al 1986). Incomplete hydrolysis of the viscous (1 3),(1 4)-glucans during the brewing process is a major production problem and contributes to hazing of beers upon storage (for review see Woodward and Fincher 1983). Because of these practical aspects of (1 3), (1 4)-glucans, they have been the focus of many studies describing their unique structure and their content and dynamics in cereal grains. These studies of (1 3),(1 4)-glucan structure and physiology and the special cell wall of grasses have been covered by several reviews and book chapters, and the readers are directed to these for more extensive coverage of the early literature (Wilkie 1979; Bacic et al 1988; Carpita and Gibeaut 1993; Carpita 1996). Grass species use (1 3),(1 4)-glucans as structural elements of the walls of growing cells and as an endosperm storage material that is hydrolyzed during germination to provide an extra source of carbon during early seedling establishment (Meier and Reid 1982). (1 3),(1 4)-Glucan synthase represents one of the few biosynthetic machines whose activity can be preserved in vitro, and the polymer synthesized is identical in size and structure to that produced by the plants. This feature gives us a unique opportunity to study all of the interacting proteins and enzymes that comprise this system. Although plant biologists have finally identified and characterized a few genes that encode the synthesis machinery, we still don’t understand the biochemical mechanisms of synthesis very well. Identification of a (1 3),(1 4)-glucan synthase genes and determination of the three-dimensional structures of their active sites will ultimately yield important clues to understanding the chemical mechanism of glycosyl transfer, not only of this specific synthase, but also for cellulose synthase and all other plant polysaccharide synthases. In this review, we summarize the features that make cell walls of cereals different from all other flowering plants and give a special focus to the special role that (1 3),(1 4)-glucans play in wall architecture and plant development. We will highlight recent advances in structurefunctional relationships, including the characterization of the enzymes that participate in the hydrolysis of (1 3),(1 4)glucan during germination, reserve mobilization, and cell growth, and the cloning of their genes. Our central effort will be to provide new insights and speculations into the mechanisms of synthesis at the Golgi apparatus and the progress toward identification of the genes that encode the synthetic machinery. Special Cell Wall of Grasses and Cereals All plant cell walls are composites of at least three independent but coextensive, interwoven networks of polymers. The cellulose and cross-linking glycan framework embedded in a matrix of pectic substances form two of them, and these networks are eventually cross-linked into a firm, inextensible structure by structural proteins or polyphenolic substances (McCann and Roberts 1991; Carpita and Gibeaut 1993). A vast majority of flowering plants possess a type I wall in which the principal cellulose cross-linking glycan is xyloglucan and as much as 35% of the wall mass is pectin (Carpita and Gibeaut 1993). The grasses and cereals possess a type II cell wall (Carpita and Gibeaut 1993; Carpita 1996), and at least two important evolutional changes resulted in their vastly different cell wall 1 Secao de Fisiologia e Bioquimica de Plantas, Instituto de Botânica CP 4005 CEP 01061-970, Sao Paulo, SP Brazil. 2 Department of Botany and Plant Pathology, Purdue University West Lafayette, IN 47907–1155. 3 Present address: UMR CNRS-UPS 5546, Pole de Biotechnologie Vegetale, BP 17, Auzeville, F-31326 Castanet Tolosan, France. 4 Present address: Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, NY 14853. 5 Corresponding author. Phone: +1-765-494-4653. Fax: +1-765-494-0393. Email: carpita@purdue.edu Publication no. C-2003-1216-01R.

Seed Cell Wall Storage Polysaccharides: Models to Understand Cell Wall Biosynthesis and Degradation

Cell wall storage polysaccharides (CWSPs) are found as the principal storage compounds in seeds of many taxonomically important groups of plants. These groups developed extremely efficient biochemical mechanisms to disassemble cell walls and use the products of hydrolysis for growth. To accumulate these storage polymers, developing seeds also contain relatively high activities of noncellulosic polysaccharide synthases and thus are interesting models to seek the discovery of genes and enzymes related to polysaccharide biosynthesis. CWSP systems offer opportunities to understand phenomena ranging from polysaccharide deposition during seed maturation to the control of source-sink relationship in developing seedlings. By studying polysaccharide biosynthesis and degradation and the consequences for cell and physiological behavior, we can use these models to develop future biotechnological applications.

Seed storage hemicelluloses as wet-end additives in papermaking☆

Xyloglucans and galactomannans are examples of hemicelluloses that can be accumulated in seeds of many plants, being extensively studied and used for industrial applications. Guar gum and starch are polysaccharides currently used as wet-end additives in papermaking, whereas xyloglucans have never been reported to improve paper quality. In this work we show that different types of xyloglucans improved the mechanical properties of paper sheets without affecting the optical ones. Addition of 1% (w/w) of hemicelluloses to cellulosic pulp was able to increase by about 30% the mechanical properties such as burst and tear indexes. Seeds of several species could be used as source for the production of wet-end additives, since the results did not vary with the source of polysaccharides. Even if the utilisation of these hemicelluloses will not cost less than starch or guar gum, it might represent an important strategy for sustainable use of rainforest species.

Novo método enzimático rápido e sensível de extração e dosagem de amido em materiais vegetais

A new rapid and sensitive enzymatic method for extraction and quantification of starch in plant material). In this work we compare methods normally used for starch determination in plant materials. The comparison between chemical (McCreadys method) and an enzymatic method proposed here showed that although McCreadys method is appropriate for most plant materials, in certain cases where cell wall polysaccharides (pectins and hemicelluloses) are present, the results may be significantly altered. However, using the enzymatic method described here afforded accurate estimation of starch content in such tissues. The enzymatic method proposed in this work is an affordable option for precise determination of starch contents in several plant tissues.

Mobilisation of the raffinose family oligosaccharides and galactomannan in germinating seeds of Sesbania marginata Benth. (Leguminosae-Faboideae)

Abstract Seeds of the tropical species Sesbania marginata present a living endosperm whose cells possess thickened walls which contain galactomannan. Oligosaccharides of the raffinose family and protein bodies are stored intracellularly. Between the seed coat and the endosperm an aleurone layer is present. The anatomy of the endosperm of Sesbania marginata is intermediate amongst other galactomannan-containing seeds that have been anatomically examined (Trigonella foenum-graecum and Ceratonia siliqua). Before radicle protrusion, oligosaccharides from the raffinose family are broken down concomitantly in the endosperm and embryo whereas galactomannan (apparently restricted to the endosperm) is degraded after germination. Three galactomannan hydrolases: α-galactosidase, e ndo-β-mannanase and β-mannosidase were detected in isolated endosperms and their activities peaked during the exponential phase of galactomannan degradation, suggesting their close correlation with the breakdown of this cell wall polysaccharide following germination. Our results indicate that unlike Trigonella foenum-graecum enzymes, the hydrolases of seeds of Sesbania marginata seem not to work in a concerted fashion, since the mannose: galactose ratio of the polysaccharide increased significantly at the end of the degradation process, suggesting that α-galactosidase attacks the polymer before the other hydrolases. The products of galactomannan degradation (galactose and mannose) do not accumulate either in the endosperm or in the embryo, where they are probably metabolised and used as a source of energy for the early growth of the plantlet.

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass.

Background Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the worlds second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant. Results Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source. Conclusion Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.