Cellulase Enzyme Preparations Research Articles

Protein preparations from rapse processing waste: A review of the current status and development prospects of existing technologies

The demand for protein products is increasing due to the demographic growth of the world’s population. As an alternative to traditional sources of protein, waste from plant raw material processing is becoming increasingly popular. An important place in the global economy is occupied by oilseeds, in particular rapeseed, which production volumes are increasing in the Russian Federation every year. Rapeseed (Brassicaceae napus) is of great interest due to its high oil content (39.80–46.00%) and rich fatty acid composition, while cake and meal formed in the process of oil production are characterized by a significant content of crude protein (35.00–45.00%) and crude fiber (8.20–17.50%); however, they are used mainly as a feed additive. Recent studies on the processing of rapeseed waste indicate the value of this raw material as a source of dietary protein, which has a balanced amino acid profile and a high degree of digestibility (up to 85%). To obtain protein, rapeseed processing is envisaged: cleaning, grinding, cold pressing at a temperature of ≤ 40 °C, fat extraction with a solvent. At the next stages, the protein is extracted with 0.1–0.5 M NaCl at pH 5.3–12.0 and a temperature of 5–30 °C for 1 hour. The extracted protein is precipitated at the isoelectric point (pH 4.0) with HCl, separated from the mixture and neutralized. The result is a protein isolate with a protein content of 90.0–98.7%. It is possible to increase the quality and yield of the protein product due to the additional stage of processing the defatted cake with cellulolytic enzyme preparations. In this case, additional studies are required to determine the substrate specificity of commercial cellulase enzyme preparations and the optimal hydrolysis conditions. The parameters of extraction and precipitation of the protein in the case of using the stage of enzymatic lysis should also be specified.

Use of Endoglucanase and Accessory Enzymes to Facilitate Mechanical Pulp Nanofibrillation

Although selective enzyme treatments have been used to successfully fibrillate chemical pulps, high lignin-containing mechanical pulps have proven to be more recalcitrant. When a bleached chemi-thermomechanical pulp (BCTMP) was sulfonated prior to enzymatic treatment, relatively good fibrillation was achieved, although some pulp hydrolysis occurred after 6 h hydrolysis when using a commercial cellulase enzyme preparation (Cellic CTec 3). To try to minimize pulp losses, various enzyme cocktails, including endoglucanase (EG), xylanase, mannanase, and lytic polysaccharide monooxygenase (LPMO), were assessed for their ability to enhance fibrillation while minimizing cellulose hydrolysis. It was apparent that the yield as well as the zeta potential of the lignin-containing cellulose nanofibrils increased with enzyme treatment. This was likely due to an increase in surface charge and a decrease in particle size after LPMO and hemicellulase treatments, respectively. When carbohydrate-binding modules (CBMs) were used to quantify fiber changes, it was apparent that sulfonation had increased the accessibility of enzymes, while the combined action of the hemicellulases and LPMO increased EG accessibility to the less-ordered regions of the mechanical pulp, resulting in enhanced fibrillation. This work described, for the first time, the synergistic action of EG and various accessory enzymes enhancing mechanical pulp nanofibrillation.

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30

BackgroundMajor cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted.ResultsHydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus.ConclusionsThe results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.

Characterization and Deconstruction of Oligosaccharides in Black Liquor From Deacetylation Process of Corn Stover

The NREL developed, atmospheric pressure, low severity Deacetylation and Mechanical Refining (DMR) process under atmospheric pressure, generates highly fermentable, low toxicity sugar syrups and highly reactive lignin streams. The dilute alkali deacetylation step saponifies acetyl groups from the hemicellulosic fraction of biomass into a black liquor waste stream that also contains solubilized acetic, ferulic, p-coumaric acids and lignin-carbohydrate complexes (LCCs), oligosaccharides, and solubilized lignin. Valorization of the soluble components in the deacetylation black liquor waste stream requires characterization of the black liquor. WeOur analyses show that glucan, xylan and lignin were three main components in black liquor and the oligosaccharides in the black liquor were mainly xylan dimers and trimers with arabinofuranose branch groups mainly on the xylooligomers. GPC chromatograms showed that the black liquor also contained oligomeric lignin moieties with molecular weights ranging between 1800 to ~10,000 Da. Enzymatic saccharification of the black liquor was conducted based on the compositional and structural information. Incorporation of Novozymes Cellic® CTec2 and HTec2 commercial cellulase and hemicellulase enzyme preparations with various accessary enzymes showed up to 57% hydrolysis of the xylooligosaccharides in the black liquor to monomeric sugars, which could be used to improve biorefinery carbon utilization in biological fermentations to improve end product fuel or bioproduct volumes per metric tonne of biomass processed. This is so far the first study about characterizing DMR black liquor and exploring breaking down the oligosaccharides in black liquor for potential use.

Enzymatic treatment of deinking sludge – effect on fibre and drainage properties

ABSTRACTThis work focused on the enzymatic treatment of deinking sludge (DS), a waste stream arising from German deinked pulp mills. Three industrial DS samples were characterised with respect to chemical composition and fibre morphology. In this study, four cellulase enzyme preparations were tested under neutral pH conditions and one cellulase was identified as suitable for DS conditioning with no need of pH adjustment. Additionally, our approach shows that inorganic particles contained in the DS samples do not negatively affect cellulase activity, as illustrated by respectable hydrolysis yields of up to 61%. Enzymatic treatment of DS led to fibre shortening and fibre fines generation with increasing enzyme dose and incubation time. The dewaterability of enzyme-treated DS samples was tested and the results showed that high amounts of fibre fines enhanced filter cake consolidation during dewatering, resulting in higher cake solids. A 10–14 percentage point increase in cake solids was obtained depending upon the enzyme dose applied, the origin of the DS sample, and the exposure period. Through the enzymatic treatment the annually generated amount of DS in Germany could be reduced by up to 20%, which would mean considerable disposal cost savings.

Simultaneous Saccharification and Fermentation of Sugar Beet Pulp for Efficient Bioethanol Production.

Sugar beet pulp, a byproduct of sugar beet processing, can be used as a feedstock in second-generation ethanol production. The objective of this study was to investigate the effects of pretreatment, of the dosage of cellulase and hemicellulase enzyme preparations used, and of aeration on the release of fermentable sugars and ethanol yield during simultaneous saccharification and fermentation (SSF) of sugar beet pulp-based worts. Pressure-thermal pretreatment was applied to sugar beet pulp suspended in 2% w/w sulphuric acid solution at a ratio providing 12% dry matter. Enzymatic hydrolysis was conducted using Viscozyme and Ultraflo Max (Novozymes) enzyme preparations (0.015–0.02 mL/g dry matter). Two yeast strains were used for fermentation: Ethanol Red (S. cerevisiae) (1 g/L) and Pichia stipitis (0.5 g/L), applied sequentially. The results show that efficient simultaneous saccharification and fermentation of sugar beet pulp was achieved. A 6 h interval for enzymatic activation between the application of enzyme preparations and inoculation with Ethanol Red further improved the fermentation performance, with the highest ethanol concentration reaching 26.9 ± 1.2 g/L and 86.5 ± 2.1% fermentation efficiency relative to the theoretical yield.

Enzymatic hydrolysis of steam-pretreated sweet sorghum bagasse by combinations of cellulase and endo-xylanase

Abstract Sweet sorghum bagasse (SSB) is considered a potential lignocellulosic raw material for the production of fuel ethanol by means of pretreatment, enzymatic hydrolysis and fermentation. Enzyme cocktail composition is critical to achieve maximum sugar yield per gram of enzyme used. Given that the enzyme composition relies on feedstock properties and pretreatment applied, the main objective of this study was to develop an optimised combination of cellulase (Cellic® CTec2) and endo-xylanase (Cellic® HTec2) enzyme preparations (cocktail) that is adapted to steam treated sorghum bagasse. This was done by using a central composite design by which combined effects of Cellic® CTec2 and Cellic® HTec2 were investigated further using response surface methodology (RSM). The optimum was defined as the minimum total enzyme dosage that is needed for a target glucan conversion of at least 80% for an incubation period of 72 h. The addition of Cellic® HTec2 proved to boost the glucan conversion to glucose by Cellic® CTec2, but synergistic interaction was only observed at early stages of the enzymatic hydrolysis and mainly for those combinations with lower cellulase content. The pretreatment and enzymatic hydrolysis with 0.15 mL/g WIS (water insoluble solids) of Cellic® CTec2 (24.9 mg protein/g WIS) and 0.32 mL/g WIS of Cellic® HTec2 (54 mg protein/g WIS), respectively, resulted in 364.8 kg sugar/ton of bagasse, 20% more than with a conventional enzyme combination.

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.

Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation

Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin. When grown on malt extract or minimal medium supplemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited high laccase activity, ∼3U/mL toward syringaldazine. This activity was 13–17 fold higher than the corresponding activities of the crude protein extracts of P. brumalis, P. ciliatus and T. versicolor. Native PAGE electrophoresis of the crude G. lucidum extract confirmed the presence of an active laccase. The G. lucidum laccase had a molecular weight of ∼62.5kDa, and a Km value of 0.107mM (determined on ABTS). A partial amino acid sequence analysis of four short de novo sequenced peptides, defined after trypsin digest analysis using MALDI-TOF MS/MS analysis, revealed 64–100% homology to sequences in related laccases in the UniProt database, but also indicated that certain sequence stretches had low homology. Addition of the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) together with a state-of-the-art cellulase enzyme preparation (Cellic™CTec1) produced significantly increased cellulolytic yields, which were also better than those obtained with a T. versicolor laccase addition, indicating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosic biomass conversion.

Product inhibition of enzymatic hydrolysis of cellulose: are we running the reactions all wrong?

Sweet sorghum bagasse (SSB) is considered a potential lignocellulosic raw material for the production of fuel ethanol by means of pretreatment, enzymatic hydrolysis and fermentation. Enzyme cocktail composition is critical to achieve maximum sugar yield per gram of enzyme used. Given that the enzyme composition relies on feedstock properties and pretreatment applied, the main objective of this study was to develop an optimised combination of cellulase (Cellic® CTec2) and endo-xylanase (Cellic® HTec2) enzyme preparations (cocktail) that is adapted to steam treated sorghum bagasse. This was done by using a central composite design by which combined effects of Cellic® CTec2 and Cellic® HTec2 were investigated further using response surface methodology (RSM). The optimum was defined as the minimum total enzyme dosage that is needed for a target glucan conversion of at least 80% for an incubation period of 72 h. The addition of Cellic® HTec2 proved to boost the glucan conversion to glucose by Cellic® CTec2, but synergistic interaction was only observed at early stages of the enzymatic hydrolysis and mainly for those combinations with lower cellulase content. The pretreatment and enzymatic hydrolysis with 0.15 mL/g WIS (water insoluble solids) of Cellic® CTec2 (24.9 mg protein/g WIS) and 0.32 mL/g WIS of Cellic® HTec2 (54 mg protein/g WIS), respectively, resulted in 364.8 kg sugar/ton of bagasse, 20% more than with a conventional enzyme combination.

Production of fermentable sugars from oil palm empty fruit bunch using crude cellulase cocktails with Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for bioethanol production

Utilization of oil palm empty fruit bunch (OPEFB) for bioethanol production with crude cellulase cocktails from locally isolated fungi was studied. Enzymatic saccharification of alkaline pretreated OPEFB was done using different cellulase enzyme preparations. Crude cellulase cocktails from Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 produced 8.37 g/L reducing sugars with 0.17 g/g yield. Production of bioethanol from OPEFB hydrolysate using Baker’s yeast produced approximately 0.59 g/L ethanol, corresponding to 13.8% of the theoretical yield. High reducing sugars concentration in the final fermentation samples resulted from accumulation of non-fermentable sugars such as xylose and cellobiose that were not consumed by the yeast. The results obtained support the possible utilization of OPEFB biomass for bioethanol production in the future.

D-(−)-Lactic acid production from cellobiose and cellulose by Lactobacillus lactis mutant RM2-24

Lactobacillus lactis mutant RM2-24 utilizes cellobiose efficiently, converting it into D-(−)-lactic acid. Cellobiose-degrading enzyme activities were determined for whole cells, cell extracts and disrupted cells. Aryl-β-glucosidase activity was detected in whole cells and disrupted cells, suggesting that these activities are confined to the cells. The mutant produced 80 g l−1 of lactic acid from 100 g l−1 of cellobiose with 1.66 g l−1 h−1 productivity. Production of D-lactic acid from different cellulose samples was also studied. The cellulose samples at high concentration (10%) were hydrolyzed by cellulase enzyme preparation (10 FPU g−1 cellulose) derived from Penicillium janthinellum mutant EU1 generated in our own laboratory. We obtained a maximum 72% hydrolysis, yielding glucose and cellobiose as the main end products. Lactic acid was produced from these cellulose samples by simultaneous saccharification and fermentation (SSF) in a media containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and cellobiose utilizing Lactobacillus lactis mutant RM2-24. A maximum lactic acid concentration of 73 g l−1 was produced from a concentration of 100 g l−1 of bagasse-derived cellulose, the highest productivity and yield being 1.52 g l−1 h−1 and 0.73 g g−1, respectively. Considering that bagasse is a waste material available in abundance, we propose to use this biomass to produce cellulose and then sugars, which can be fermented to valuable products such as ethanol and lactic acid.

Lactic acid production from waste sugarcane bagasse derived cellulose

Production of L(+)lactic acid from sugarcane bagasse cellulose, one of the abundant biomass materials available in India, was studied. The bagasse was chemically treated to obtain a purified bagasse cellulose sample, which is much more amenable to cellulase enzyme attack than bagasse itself. This sample, at high concentration (10%), was hydrolyzed by cellulase enzyme preparations (10 FPU g–1 cellulose) derived from mutants generated in our own laboratory. We obtained maximum hydrolysis (72%), yielding glucose and cellobiose as the main end products. Lactic acid was produced from this bagasse cellulose sample by simultaneous saccharification and fermentation (SSF) in a media containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and cellobiose utilizing Lactobacillus delbrueckii mutant Uc-3. A maximum lactic acid concentration of 67 g l–1 was produced from a concentration of 80 g l–1 of bagasse cellulose, the highest productivity and yield being 0.93 g l–1 h–1 and 0.83 g g–1, respectively. The mutant Uc-3 was found to utilize high concentrations of cellobiose (50 g l–1) and convert it into lactic acid in a homo-fermentative way. Considering that bagasse is a waste material available in abundance, we propose to valorize this biomass to produce cellulose and then sugars, which can be fermented to products such as ethanol and lactic acid.

Coupled acid and enzyme mediated production of microcrystalline cellulose from corn cob and cotton gin waste

Microcrystalline cellulose has applications in food, pharmaceuticals, and other industries. Most microcrystalline cellulose (MCC) is produced from dissolving pulp using concentrated acids. We investigated steam explosion treatment of corn cobs and cotton gin waste for the production of microcrystalline cellulose. The corn cob was converted into a coarse brown powder after steam explosion and the lignin and residual hemicellulose fractions were extracted respectively with sodium hydroxide solution and water. The residual cellulose was readily bleached with hydrogen peroxide and converted to microcrystalline cellulose using hydrochloric acid, sulfuric acid and cellulase enzyme preparation. The resulting microcrystalline cellulose samples had properties that were similar to commercial microcrystalline cellulose. Similarly, cotton gin waste was steam exploded and converted into microcrystalline cellulose, but this material was more difficult to bleach using hydrogen peroxide. The degree of polymerization for the MCC samples ranged from 188.6 to 549.8 compared to 427.4 for Avicel PH101 MCC.

Enzymatic Saccharification and Fermentation of Xylose-Optimized Dilute Acid–Treated Lignocellulosics

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180 degrees C, and 0.5 min for switchgrass and 1% acid, 180 degrees C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.

Treatment of recycled fiber with Trichoderma cellulases

Recycled fibers have become increasingly important as raw material in the pulp and paper industry. Although utilization of secondary fibers has environmental and economical advantages, unfortunately the deteriorated drainability of secondary fibers causes decreased productivity. In this work, two commercial Trichoderma cellulase enzyme preparations were used to improve pulp properties of recycled paper. A blend of various cellulase components (Pergalase A40) was compared with endoglucanase III (IndiAge Super L) based on their effect on drainage, water retention value, fiber length distribution and paper properties (air permeability, tensile index, burst index, tear index). Enzyme characteristics were investigated in order to establish the industrial applicability. IndiAge Super L containing only EG III proved to be more stable than Pergalase A40 against fluctuation of temperature and pH. Experimental design (cross-classification) was used to examine the influence of enzyme dosage and treatment time on drainage. The effects of enzymatic treatments on pulp and paper properties showed that the type of enzyme component is probably very important. Moreover, existence or absence of cellulose-binding domain might play a determinative role.

Cellulase fermentation on a novel substrate (waste cardboard) and subsequent utilization of home-produced cellulase and commercial amylase in a rabbit feeding trial

Cellulase and amylase enzymes are increasingly being used as feed additives in animal nutrition. Those are mainly applied in order to improve the nutritional value of feed by partially degrading its fiber content, which has major importance during the early post-weaning period of young animals possessing inadequate digestive enzyme activities. Due to their commercial availability at low cost, the utilization of amylases as animal feed additive can be highly recommended, while the high price of cellulases actually makes their use as a feed additive unreasonable. A promising way to gain cheap cellulases is to conduct the enzyme fermentation process on novel substrates, which are readily available at no cost. In this study, the utilization of old corrugated cardboard (OCC) as a novel fermentation substrate has been investigated for cellulase enzyme fermentation by Trichoderma reesei Rut C-30. Batch fermentation experiments were carried out in shake-flasks and in a 30 l laboratory fermenter. The OCC feedstock proved to be a suitable fermentation substrate, a filter paper activity (FPA) of as high as 2.27 FPU ml −1 was achieved in the fermenter, which corresponds to a yield of 227 FPU g −1 cellulose. The maximum productivity achieved was 35.7 FPU l −1 h −1. The subsequent utilization of this cellulase enzyme preparation was investigated as additive of commercial rabbit compound feed in order to improve its digestibility during the early post-weaning period. In parallel, commercial amylase preparations were also tested for the same purposes. The results of animal feeding experiments showed, that enzyme supplementation of the complete feed improved production traits, as it increased the digestibility of nutrients and thus reduced the mortality caused by digestive disorders.

The Chemical and Enzymatic Hydrolysis of Archaeological Wood Cellulose and Monosaccharide Purification by High Ph Anion Exchange Chromatography for Compound-Specific Radiocarbon Dating

Preliminary experiments were carried out on archaeological wood to investigate methods of cellulose hydrolysis and carbohydrate monomer purification for the purpose of compound-specific radiocarbon dating. The Chelford log, a known 14C dead source of wood cellulose, was selected for study in order to investigate the levels of contamination introduced during sample purification. Two methods of hydrolysis were examined, mineral acid hydrolysis and enzyme hydrolysis using cellulase from Penicillium funiculosum. Under the conditions described, enzymolysis was far superior to acid hydrolysis in terms of the glucose monomer yield. Glucose monomer purification was accomplished using high pH anion exchange chromatography with pulsed amperometric detection. This high performance liquid chromatography (HPLC) method does not require sample derivatization and the chromatography products can be collected in water. These characteristics make it potentially well suited to carbon dating applications. 14C dating of chromatographically purified glucose fractions revealed significant levels of contamination had accumulated during both protocols. Glucose contamination from the cellulase enzyme preparation was a major source of contamination within the enzymatically hydrolyzed samples. Ultrafiltration of the enzyme removed some but not all of this contamination. The contamination must be reduced 10-fold before the methodology could be viable for dating. This hydrolysis/HPLC method is also being investigated for 14C dating of other carbohydrate polymers such as chitin.

Simultaneous saccharification and cofermentation of dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fermenting Zymomonas mobilis.

Simultaneous saccharification and cofermentation (SSCF) was carried out at approximately 15% total solids using conditioned dilute-acid pretreated yellow poplar feedstock, an adapted variant of National Renewable Energy Laboratory (NREL) xylose-fermenting Zymomonas mobilis and either commercial or NREL-produced cellulase enzyme preparations. In 7 d, at a cellulase loading of 12 filter paper units per gram cellulose (FPU/g), the integrated system produced more than 3% w/v ethanol and achieved 54% conversion of all potentially available biomass sugars (total sugars) entering SSCF. A control SSCF employing Sigmacell cellulose and a commercial cellulase at an enzyme loading of 14 FPU/g achieved 65% conversion of total sugars to ethanol.

Effect of degradation on the porosity and surface area of forage cell walls of differing lignin content

Cell walls, prepared from the stems of wheat, maize, lucerne and rape and from timothy grass, were degraded using a commercial cellulase enzyme preparation. Timothy and lucerne were extensively degraded (60–70% loss of dry matter) while dry matter losses from the more lignified maize, rape and wheat samples were substantially less (30–40%). Residues obtained after 6 and 72 h degradation and original preparations were examined for pore structure and surface area by nitrogen adsorption. The pore regime of all of the samples fell within the range 0.5–5 nm radius with pores of 1–2 nm radius predominating. Degradation had little impact on pore regimes or mean pore radius which were essentially the same as the starting material. The exception was timothy where the mean pore radius fell from 2.80 to 2.36 nm (P<0.001) accompanied by a fall in available surface area (2.49 to 1.39 m2 g −1 P<0.001). Wheat showed a similar fall in available surface area after 72 h degradation (3.29 to 2.49 m2 g−1 P<0.05) but no significant differences were seen in the remaining samples. In a separate experiment, effect of lignification on pore structure was examined using sequential sections taken from a newly extended maize internode. The phenolic content differed two-fold between the youngest and oldest sections and there was an accompanying steady decrease in electronegativity which weakly correlated with the phenolic content (r2=0.504). No significant differences were seen in pore volume, mean pore radius or pore regimes along the internode, but the available surface area increased significantly with maturity (P<0.01) and was strongly correlated with the phenolic content (r 2=0.896). Since porosity is defined by the spacing between polymers of the wall, a considerable commonality of three-dimensional structure evidently existed amongst the cell walls of the plants examined which was little affected by lignification or degradation. This is consistent with exclusion of enzymes by the wall which ensures that bacterial attack in the rumen and elsewhere proceeds by a process of surface erosion. © 1999 Society of Chemical Industry