Analysis of saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis
© Gomez et al; licensee BioMed Central Ltd. 2008
Received: 31 July 2008
Accepted: 22 October 2008
Published: 22 October 2008
Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials.
The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana. Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis. Monosaccharide and oligosaccharide analysis showed that the hemicellulose fraction is the main target of the enzymatic activity under the modest hydrolytic conditions used in our assays. Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments.
Results presented here indicate that under mild conditions cellulose and hemicellulose are hydrolysed to differing extents, with hemicellulose hydrolysis predominating. We anticipate that the sub-optimal conditions for hydrolysis identified here will provide a sensitive assay to detect variations in saccharification among Brachypodium plants, providing a useful analytical tool for identifying plants with alterations in this trait.
Although the contribution of agricultural waste to the generation of transportation fuels has been negligible, in recent years there has been an upsurge of interest in the use of grass straw from agricultural waste as well as dedicated energy crops for the production of biofuels . Since grasses of agricultural importance have complex genomes and growth requirements that make them difficult for use in research at the molecular level, there is a need for model grass species in basic research. This need for grass model species is particularly evident in the area of cell wall research, where dicots and grasses differ substantially in the composition and organisation of the component polymers [2, 3]. A growing range of genetic tools and the growth characteristics of Brachypodium distachyon make it potentially a good model for grass research [4, 5]. A 4× draft of the genome sequence has been released and an 8× assembly will be available http://www.brachypodium.org.
The high costs associated with the three biological steps involved in the conversion of lignocellulose to biofuels (enzyme production, biomass hydrolysis and fermentation of the released sugars) have driven numerous efforts to make the overall biochemical conversion more efficient . These efforts have been directed not only to reduce the costs of enzymes, but also to optimise the configuration of all the steps of the process . Any approach for improving the process of saccharification of lignocellulosic plant biomass requires, or at least will benefit from, a thorough understanding of the structure and biosynthesis of plant cell walls . Using Brachypodium as a model for understanding the characteristics of the grass cell wall involved in the process of saccharification will require a detailed characterisation of the saccharification in this material under different conditions .
The conversion of lignocellulose into sugars depends on variables present at different levels of organisation in the cell wall, from the diversity of the glycosidic bonds involved in the synthesis of the wall polymers, to the organisation and the relative abundance of each component . Indeed, alfalfa mutants with altered lignin levels present improved digestibility, and saccharification in maize and sorghum is affected by ferulic acid cross-links [11, 12].
Industrial procedures for the conversion of lignocellulose into sugars for fermentation use chopped biomass, in which the structure and consequently the architecture of lignocellulose represent another factor affecting conversion. The development of improved lignocellulose saccharification assays, relevant to industrial considerations, should include materials where tissue structure is intact in order to take in account features of the cell wall that will influence digestibility in 'real-life' conditions.
Here we present a characterisation of the saccharification of Brachypodium stem segments in order to understand the modifications occurring in lignocellulose under different pretreatment and hydrolysis conditions. We have carried out analyses of the composition of Brachypodium stems and the products released, as well as structural changes, occurring in the stem segments under mild conditions of hydrolysis.
Monosaccharide composition of Brachypodium stems
Brachypodium is closely related to agronomically important grasses, making it a potential model species from a phylogenetic point of view . To examine whether Brachypodium relatedness to important crop grasses is maintained at the level of cell wall composition, we compared the monosaccharide composition of stem cell wall from several grass species with that of Arabidopsis, a well-characterised dicot model.
Whilst the cell walls of grasses and dicot plants are both largely polysaccharide composites based around a framework of cellulose microfibrils, there are substantial differences between these groups of plants with regard to the composition of the matrix polysaccharides that associate with the cellulose. The primary cell walls of most dicots are relatively rich in pectins, and xyloglucans form the major hemicellulose coating the cellulose microfibrils. In contrast, the primary cell walls of grasses have much less pectin and xyloglucan, and glucuronoarabinoxylan (GAX) and mixed-linkage glucans form the major hemicelluloses . Dicots and grasses also show differences in the composition of secondary cell walls. The hemicellulose of dicot secondary cell walls is typically dominated by simple arabinoxylans with only occasional side chains . In contrast, the GAX of grass secondary cell walls has more complex and numerous side chains, and the arabinosyl side chains may serve as connections to lignin through feruloyl esters [1, 14].
The cell walls of dicot species such as Arabidopsis are generally much richer in pectic components such as homogalacturonan and rhamnogalacturonan than are grasses, and this is clearly apparent in the higher levels of galacturonic acid released from Arabidopsis cell walls. The similarities in non-cellulosic monosaccharide composition between Brachypodium and commercially important grasses lend support to the use of Brachypodium as a model to explore the conversion of lignocellulose from grasses into ethanol.
Effect of H2SO4concentration and temperature during pretreatment on the hydrolysis of Brachypodium stem segments
Many studies have shown the need for pretreatment of the lignocellulosic material in order to produce an efficient hydrolysis . Here we studied different conditions of diluted acid pretreatment in the release of sugars from Brachypodium stem segments.
Analysis of sugars released during pretreatment at different temperatures
Standard conditions of hydrolysis and effect on Brachypodium stems
Time course of hydrolysis and products released during enzymatic digestion
In contrast, the Brachypodium stems have only been exposed to the gentle pretreatment conditions used in our assay and hence the more readily digestible hemicellulose components are releasing greater quantities of sugar than the more recalcitrant cellulose (Figure 9B). Saccharification analysis of dilute H2SO4-impregnated wheat straw produced large amounts of xylose after pretreatment in enzymatic hydrolysis . Since some of the main candidate energy crops are grasses, high levels of xylose are expected to be released for fermentation, underlying the need for pentose-fermenting organisms in the production of liquid biofuels .
Brachypodium is being considered as a suitable experimental model species to investigate various parameters related to the use of fast-growing biomass grasses in the context of biofuels and bioenergy [3, 6]. Work presented in this paper shows that the composition of non-cellulosic polysaccharides in Brachypodium appears broadly similar to that of biomass grasses and cereals, and that all of these differ to that seen in Arabidopsis, indicating that from a perspective of cell wall composition Brachypodium appears to be a suitable model. In order to underpin the development of high-throughput saccharification screens that can be used to identify Brachypodium mutants with altered digestibility characteristics, we have undertaken an examination into the effects of a range of pretreatment and enzymatic hydrolysis conditions. Dilute acid pretreatment has been shown to be an efficient method of increasing efficiency in the digestion of lignocellulosic materials in general. Here we show the effect of particular combinations of pretreatment and enzymatic hydrolysis in the saccharification of Brachypodium stems.
We found that both pretreatment temperature and acid concentration had effects on subsequent enzymatic saccharification. Temperatures above 110°C combined with acid in the pretreatment led to hydrolysis of cell wall polysaccharides directly, decreasing the amount of sugars released by subsequent enzymatic hydrolysis. The monosaccharide composition of the sugars released during pretreatment corresponds to hemicellulosic materials. SEM images of the segments after pretreatment and enzymatic attack revealed changes in stem structure associated with changes in sugar release. Our data support the use of Brachypodium as a model species for studying lignocellulose from cereals and biomass grasses, and suggest a combination of modest temperature and dilute acid pretreatment may form the basis of a sensitive screening method to identify genes affecting grass biomass saccharification potential.
Plant materials and growth conditions
Seed of Brachypodium distachyon Bd21 were sown in 4-inch pots, watered, and kept at 4°C for two weeks prior to growth. Plants were grown in a glasshouse under a 16-hours light regime, at 22°C. After setting seed, plants were left to dry in the pots. For studying saccharification, stem segments of 4 mm length were taken from the middle section of internodes 5, 6, and 7. Preliminary studies showed that there were no differences in saccharification using material taken from these internodes. In all the saccharification assays 4 mg samples were used and assays were carried out in quadruplicates. In the case of paper used for comparative purposes with Brachypodium stems, samples were 4 mg of 5 mm discs of 80 g/m2 recycled paper (M-Real, Amsterdam, The Netherlands).
Hydrolysis of materials
Enzymatic hydrolysis was carried out using Celluclast (cellulase from Trichoderma reesei) and Novozyme 188 (cellobiase from Aspergillus niger) in an enzyme cocktail containing a 4:1 proportion of Celluclast: Novozyme 188. The enzymes were filtered using a Hi-Trap desalting column (GE Healthcare, Little Chalfont, UK) before use and the hydrolysis was carried out during the indicated times, at 30°C in buffer Na Acetate 25 mM at pH 4.5.
Total sugar content
The determination of total sugar content in untreated, pretreated, and no pretreatment stems was carried out following the protocol described by Fry . 4 mg of stem fragments were dissolved in 72% H2SO4 at 30°C and then hydrolysed in 4% H2SO4 at 120°C for 1 h. The resulting solution was neutralised with Ba(OH)2 and the sugars were measured as described below in sugar determination.
Scanning electro microscopy
Stems were incubated in buffer Na Acetate 25 mM pH 4.5 for 20 min at room temperature (untreated and no pretreatment), or incubated in 1% H2SO4 for 20 min at 90°C (pretreated). The segments were rinsed several times with buffer and then incubated with 0 or 0.5 mg of enzyme cocktail for 1 h. Segments were placed in 100% ethanol and then air-dried. For SEM, stems were mounted in SEM stubs and coated with gold/palladium. The mounted specimens were observed with a JEOL JSM 6490LV (Jeol Ltd, Tokyo, Japan) at an accelerating voltage of 5kV.
The determination of sugars released after hydrolysis was done using a modification of the method by Anton and Barrett  using 3-methyl-2-benzothiazolinone hydrozone (MTBH). Determination was carried out in a 1 ml final volume using 300 μl of sample. The detection reaction contained 0.25 N NaOH, 0.06% MTBH, 0.02% DTT, and colour was developed by adding 0.2% FeNH4(SO4)2, 0.2% Sulfamic acid and 0.1% HCl. The absorbance of the reaction was followed at 620 nm. This method was tested for the detection of a range of sugars that are released from the cell wall, showing no significant differences between reducing sugars.
Oligosaccharide analyses of the sugars released from stem segments after hydrolysis was carried out by taking 0.2 ml samples from digestions after 0.5, 1, 3, 4, 5, 12, and 24 h of enzymatic digestion. Samples were filtered using AG50W × 12 resin (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) and subsequently dried and re-suspended in 100 μl of deionised water. The samples were filtered using a Millex LH 0.45 μm pored filter (Millipore, Billerica, USA) and then separated by high-performance anion-exchange chromatography (HPAEC) on a Dionex Carbopac PA-100 column (Dionex Corporation, Sunnyvale, USA) with integrated amperometry detection. The separated oligosaccharides were quantified by using external calibration with an equimolar mixture of eleven monosaccharide and oligosaccharide standards (glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, xylose, xylobiose, xylotriose, xylotetraose, and xylopentaose).
Cell walls for non-cellulosic sugar composition from grasses and Arabidopsis were extracted from mature stems. Tissue was subsequently extracted with phenol, chlorophorm:methanol 4:1, 90% DMSO and 100% ethanol. 4 mg of dried material was digested with 2 M trifluoroacetic acid at 100°C for 4 h. After the acid was evaporated, samples were rinsed with isopropanol and resuspended in 100 μl of deionised water. Samples were filtered and analysed as described below.
For the determination of sugars during pretreatment, samples were taken after 20 min of pretreatment in the presence of 0 and 1% H2SO4at 4, 30, 60 and 90°C. Monosaccharide analyses were performed by HPAEC using a Dionex Carbopac PA-10 as described in . Samples (1.4 ml) were collected immediately after pretreatment and filtered using a column of Dowex 1 × 2 to eliminate the H2SO4 from the samples. The monosaccharide standards used for quantification were arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose.
M Bevan is acknowledged for the supply of Brachypodium seeds; A Viksø-Nielsen for the generous gift of cellulolytic enzymes. We thank D Ashford for support with glycan analysis.
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