A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin.

BACKGROUND: Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route. RESULTS: We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3-72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained ß-ether interunit linkages, providing an opportunity for conversion into saleable co-products. CONCLUSIONS: This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.

Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading.

In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2FPU/g of substrate and milling time of 120min gave a total monomeric sugar yield of 306g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood.

Nanoscale interactions of polyethylene glycol with thermo-mechanically pre-treated Pinus radiata biofuel substrate.

Non-productive adsorption of cellulose degrading enzymes on lignin is a likely reason for reduced rate and extent of enzymatic conversion of lignocellulosic substrate to sugars. Additives such as polyethyleneglycol (PEG) may act as blocking agents in this non-productive interaction. However, the exact molecular level interactions of PEG with lignin in pre-treated lignocellulosic substrates are not known. We have used confocal fluorescence microscopy combined with Förster resonance energy transfer (FRET) to reveal molecular level interactions between lignin present in thermo-mechanically pre-treated Pinus radiata substrate, and fluorescently labeled PEG. It is demonstrated that PEG interaction with lignin is mainly associated with particles derived from secondary walls, with little or no penetration into fragments derived from the middle lamella. This nanoscale information on the PEG-substrate interaction will assist in rationalizing pre-treatment methods to reduce the recalcitrance of softwood biofuel substrates.

Wide-angle x-ray scattering and solid-state nuclear magnetic resonance data combined to test models for cellulose microfibrils in mung bean cell walls.

A synchrotron wide-angle x-ray scattering study of mung bean (Vigna radiata) primary cell walls was combined with published solid-state nuclear magnetic resonance data to test models for packing of (1→4)-ß-glucan chains in cellulose microfibrils. Computer-simulated peak shapes, calculated for 36-chain microfibrils with perfect order or uncorrelated disorder, were sharper than those in the experimental diffractogram. Introducing correlated disorder into the models broaden the simulated peaks but only when the disorder was increased to unrealistic magnitudes. Computer-simulated diffractograms, calculated for 24- and 18-chain models, showed good fits to experimental data. Particularly good fits to both x-ray and nuclear magnetic resonance data were obtained for collections of 18-chain models with mixed cross-sectional shapes and occasional twinning. Synthesis of 18-chain microfibrils is consistent with a model for cellulose-synthesizing complexes in which three cellulose synthase polypeptides form a particle and six particles form a rosette.

A mathematical model for the inhibitory effects of lignin in enzymatic hydrolysis of lignocellulosics.

A new model for enzymatic hydrolysis of lignocellulosic biomass distinguishes causal influences from enzyme deactivation and restrictions on the accessibility of cellulose. It focuses on calculating the amount of unreacted cellulose at cessation of enzyme activity, unlike existing models that were constructed for calculating the time dependence of conversion. There are three adjustable parameters: (1) 'occluded cellulose' is defined as cellulose that cannot be hydrolysed regardless of enzyme loading or incubation time, (2) a 'characteristic enzyme loading' is sufficient to hydrolyse half of the non-occluded cellulose, (3) a 'mechanism index' measures deviations from first-order kinetics. This model was used to predict that the optimal incubation temperature is lower for lignocellulosics than for pure cellulose. For steam-exploded pine wood after 96h incubation, occluded cellulose was 24% and 26% at 30°C and 50°C, and the characteristic enzyme loadings were 10 and 18FPU/g substrate, respectively.

Optimizing the enzyme loading and incubation time in enzymatic hydrolysis of lignocellulosic substrates.

A mathematical model for costing enzymatic hydrolysis of lignocellulosics is presented. This model is based on three variable parameters describing substrate characteristics and three unit costs for substrate, enzymes and incubation. The model is used to minimize the cost of fermentable sugars, as intermediate products on the route to ethanol or other biorefinery products, by calculating optimized values of enzyme loading and incubation time. This approach allows comparisons between substrates, with processing conditions optimized independently for each substrate. Steam-exploded pine wood was hydrolyzed in order to test the theoretical relationship between sugar yield and processing conditions.

Effects of cooking on the cell walls (dietary fiber) of 'Scarlet Warren' winter squash ( Cucurbita maxima ) studied by polysaccharide linkage analysis and solid-state (13)C NMR.

Cell wall polysaccharides of 'Scarlet Warren' winter squash ( Cucurbita maxima ) were investigated before and after thermal processing. Linkage analysis of polysaccharides was done by gas chromatography coupled to mass spectrometry (GC-MS). The linkage analysis showed the cell wall polysaccharide compositions of raw and cooked squash were similar. The total pectic polysaccharides (galacturonan, rhamnogalacturonan, arabinan, and arabinogalactan) contents of the cell walls of both raw and cooked squash were 39 mol %. The amounts of pectic polysaccharides and xyloglucan in the cell walls of squash showed little alteration on heating. The cellulose content of the raw and cooked cell walls was relatively high at 47 mol %, whereas the xyloglucan content was low at 4 mol %. Solid-state (13)C nuclear magnetic resonance (NMR) spectroscopy techniques were used to examine the molecular motion of the polysaccharides in the cell walls. The mobility of highly flexible galactan depends on the water content of the sample, but no difference was seen between raw and cooked samples. Likewise, the mobility of semimobile pectic polysaccharides was apparently unaltered by cooking. No change was detected in the rigid cellulose microfibrils on cooking.

Using solid-state ¹³C NMR spectroscopy to study the molecular organisation of primary plant cell walls.

Studies of the mobilities of polysaccharides or parts of polysaccharides in a cell-wall preparation may give clues about the molecular interactions among the polysaccharides in the cell wall and the relative locations of polysaccharides within the cell wall. A number of solid-state (13)C NMR techniques have been developed that can be used to investigate different types of polysaccharide mobilities: rigid, semi-rigid, mobile, and highly mobile. In this chapter, techniques are described for obtaining spectra from primary cell-wall preparations using CP/MAS, proton-rotating frame, proton spin-spin, spin-echo relaxation spectra, and single-pulse excitation. We also describe how proton spin relaxation editing can be used to obtain subspectra for cell-wall polysaccharides of different mobilities.

Solid-state 13C NMR study of a composite of tobacco xyloglucan and Gluconacetobacter xylinus cellulose: molecular interactions between the component polysaccharides.

To investigate possible molecular interactions between xyloglucans (XGs) and cellulose in plant cell walls, a model composite was produced using cellulose from the bacterium Gluconacetobacter xylinus and XG from the walls of a tobacco cell-suspension culture that had been incubated with (13)C-labeled glucose. Solid-state (13)C NMR with cross-polarization (CP) and magic-angle spinning (MAS) was used in combination with proton spin-relaxation editing to separate signals from crystalline (rigid) and less rigid domains of the composite. Signals from XG were confined to subspectra of less rigid domains, with no detectable signals from XG attached to surfaces of cellulose crystallites. Signal displacements indicated XGs were more rigid than the mobile coil (twisted backbone) conformation expected for unattached XGs. Similar (13)C chemical shifts were observed in a single-pulse excitation experiment. The results were not compatible with extensive hydrogen bonding between XG and cellulose, but were consistent with a composite structure in which cellulose crystallites were embedded in a matrix of XG with a semirigid (straightened backbone) conformation, that is, a matrix that is partly ordered rather than amorphous.

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria.

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M (n)) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 x 10(6) and 2.5 x 10(6) g mol(-1), respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.

WAXS and 13C NMR study of Gluconoacetobacter xylinus cellulose in composites with tamarind xyloglucan.

- Model composites, produced using cellulose from stationary cultures of the bacterium Gluconoacetobacter xylinus and tamarind xyloglucan, were examined by wide-angle X-ray scattering (WAXS) and CP/MAS solid-state (13)C NMR spectroscopy. The dominant crystallite allomorph of cellulose produced in culture media with or without xyloglucan was cellulose I(alpha) (triclinic). The presence of xyloglucan in the culture medium reduced the cross-section dimensions of the cellulose crystallites, but did not affect the crystallite allomorph. However, when the composites were refluxed in buffer, the proportion of cellulose I(beta) allomorph increased relative to that of cellulose I(alpha). In contrast, cellulose I(alpha) remained the dominant form when cellulose, produced in the absence of xyloglucan, was then heated in the buffer. Hence the presence of xyloglucan has a profound effect on the formation of the cellulose crystallites by G. xylinus.

Variations in the fibre repeat between samples of cellulose I from different sources.

A powder X-ray diffractometer was used to measure the fibre repeat in cellulose I with sufficient precision to detect variations between samples from different sources. The variations were correlated with the lateral dimensions of the crystallites and were attributed to different minimum-energy fibre repeats for chains in the interiors and on the surfaces of crystallites. Results were interpreted in terms of a model for internal mechanical stress in which the interior chains were under compression and the surface chains under tension to ensure identical fibre repeats for all chains. The model was used to extrapolate the fibre repeat to a value of 1.043 nm for a hypothetical, infinitely large crystal, and to 1.029 nm for a crystallite so narrow that all chains were exposed on surfaces.

Solid-state 13C-NMR spectroscopy shows that the xyloglucans in the primary cell walls of mung bean (Vigna radiata L.) occur in different domains: a new model for xyloglucan-cellulose interactions in the cell wall.

Xyloglucans (XG) with different mobilities were identified in the primary cell walls of mung beans (Vigna radiata L.) by solid-state 13C-NMR spectroscopy. To improve the signal:noise ratios compared with unlabelled controls, Glc labelled at either C-1 or C-4 with 13C-isotope was incorporated into the cell-wall polysaccharides of mung bean hypocotyls. Using cell walls from seedlings labelled with d-[1-13C]glucose and, by exploiting the differences in rotating-frame and spin-spin proton relaxation, a small signal was detected which was assigned to Xyl of XGs with rigid glucan backbones. After labelling seedlings with d-[4-13C]glucose and using a novel combination of spin-echo spectroscopy with proton spin relaxation-editing, signals were detected that had 13C-spin relaxations and chemical shifts which were assigned to partly-rigid XGs surrounded by mobile non-cellulosic polysaccharides. Although quantification of these two mobility types of XG was difficult, the results indicated that the partly-rigid XGs were predominant in the cell walls. The results lend support to the postulated new cell-wall models in which only a small proportion of the total surface area of the cellulose microfibrils has XG adsorbed on to it. In these new models, the partly-rigid XGs form cross-links between adjacent cellulose microfibrils and/or between cellulose microfibrils and other non-cellulosic polysaccharides, such as pectic polysaccharides.

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan.

The primary walls of celery (Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state 13C nuclear magnetic resonance (CP/MAS 13C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state 13C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS 13C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS 13C NMR. From solid-state 13C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.

Conformational features of crystal-surface cellulose from higher plants.

Native cellulose in higher plants forms crystalline fibrils a few nm across, with a substantial fraction of their glucan chains at the surface. The accepted crystal structures feature a flat-ribbon 21 helical chain conformation with every glucose residue locked to the next by hydrogen bonds from O-3' to O-5 and from O-2 to O-6'. Using solid-state NMR spectroscopy we show that the surface chains have a different C-6 conformation so that O-6 is not in the correct position for the hydrogen bond from O-2. We also present evidence consistent with a model in which alternate glucosyl residues are transiently or permanently twisted away from the flat-ribbon conformation of the chain, weakening the O-3' - 0-5 hydrogen bond. Previous molecular modelling and the modelling studies reported here indicate that this 'translational' chain conformation is energetically feasible and does not preclude binding of the surface chains to the interior chains, because the surface chains share the axial repeat distance of the 21 helix. Reduced intramolecular hydrogen bonding allows the surface chains to form more hydrogen bonds to external molecules in textiles, wood, paper and the living plant.

Molecular ordering of cellulose after extraction of polysaccharides from primary cell walls of Arabidopsis thaliana: a solid-state CP/MAS (13)C NMR study.

Solid-state CP/MAS 13C NMR spectroscopy was used to determine the effects of three different sequential extraction procedures, used to remove non-cellulosic polysaccharides, on the molecular ordering of cellulose in a cell-wall preparation containing mostly primary cell walls obtained from the leaves of the model dicotyledon, Arabidopsis thaliana. The extractions were 50 mM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid (CDTA) and 50 mM sodium carbonate (giving Residue 1); 50 mM CDTA, 50 mM sodium carbonate and 1 M KOH (giving Residue 2); and 50 mM CDTA, 50 mM sodium carbonate and 4 M KOH (giving Residue 3). The molecular ordering of cellulose in Residue 1 was similar to that in unextracted walls: the cellulose was almost all crystalline, with 43% of molecules contained in crystallite interiors and similar proportions of the triclinic (I(alpha)) and monoclinic (I(beta)) crystal forms. Residue 2 was partly decrystallized and the remaining crystallites were mostly in the I(beta) form. Residue 3 was a mixture of cellulose II, cellulose I and amorphous cellulose. The presence of signals at 100.0 and 102.3 ppm in the spectra of Residues 1 and 2, but not of unextracted cell walls, suggested that the extractions giving these residues caused some of the non-cellulosic polysaccharides, possibly xyloglucans and galactoglucomannans, to become relatively well ordered, for example through interactions with cellulose crystallite surfaces.