Session 2 biological research

Inedible plant material, generated in a Controlled Ecological Life Support System (CELSS), should be recycled preferably by bioregenerative methods that utilize enzymes or micro-organisms. This material consists of hemicellulose, cellulose, and lignin with the lignin fraction representing a recalcitrant component that is not readily treated by enzymatic methods. Consequently, the white-rot fungus,Pleurotus ostreatus, is attractive since it effectively degrades lignin and produces edible mushrooms. This work describes an unstructured model for the growth ofP. ostreatus in a solid-state fermentation system using lignocellulosic plant materials fromBrassica napus (rapeseed) as a substrate at three different particle sizes. A logistic function model based on area was found to fit the surface growth of the mycelium on the solid substrate with respect to time, whereas a model based on diameter, alone, did not fit the data as well. The difference between the two measures of growth was also evident for mycelial growth in a bioreactor designed to facilitate a slow flowrate of air through the 1.5 cm thick mat of lignocellulosic biomass particles. The result is consistent with the concept of competition of the mycelium for the substrate that surrounds it, rather than just substrate that is immediately available to single cells. This approach provides a quantitative measure ofP. ostreatus growth on lignocellulosic biomass in a solid-state fermentation system. The experimental data show that the best growth is obtained for the largest particles (1 cm) of the lignocellulosic substrate.     

Cellulolytic enzymes produced by the edible mushroom,Pleurotus sajor-caju

Pleurotus sajor-caju grows efficiently and degrades all the components present in lignocellulosic residues. Production of cellulase and xylanase enzymes in submerged culture and during solid state cultivation has been studied. An initial pH of 5.0 was found to be optimal for the production of cellulase in shake flasks; this was attained in about 6–8 d in a medium containing either cellulose or rice straw as the sole source of carbon. On the cellulose medium, the maximum filter paper activity attained was 0.15 IU/mL in 7 d whereas the endoglycanase activity of 1.0 IU/mL, xylanase activity of 1.55 IU/mL, and Β-glucosidase activity of 0.57 IU/mL were acheived after 9 d fermentation. The reducing sugars were absent in the culture medium. The cellulases (filter paper activity and endoglucanases) were most active at pH 5.0 and 45‡C. Xylanase had maximum activity at pH 4.8 and 45‡C, and Β-glucosidase at pH 5.5 and 40‡C. In shake cultures,P. sajor-caju produced dispersed suspension of short mycelial threads and various sizes of pellets. The profile and extent of enzyme biosynthesis during submerged cultivation on rice straw was found to be of the same nature as obtained on cellulose. During solid state cultivation ofP. sajor-caju on rice straw beds for 36 d, the elaboration of enzyme activities did not appear to follow any definite pattern. However, filter paper activity, which is representative of cellulase action in hydrolyzing cellulose, remained more or less constant during the period of about the first 20 d of cultivation after the appearance of fruit bodies on the surface of rice straw beds. All the activities attained their minimum values after 23 d of cultivation, during which approximately 1 kg of fresh fruit bodies had been harvested. The total fruit bodies harvested till 36th days were approx. 1.1 kg. ThroughT. sajor-caju elaborates cellulase and xylanse extracellularly, the activity values were not as high as those of other cellulase producers such asTrichoderma reesei.     

Induction and Purification by Three-Phase Partitioning of Aryl Alcohol Oxidase (AAO) from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289 U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67 kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40 °C, respectively. From the LB plot, K _m value of AAO for oxidizing veratryl alcohol was determined to be 0.6 mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.     

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.     

Mechanism and potential applications of bio-ligninolytic systems in a CELSS

A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms.Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.     

Solid-state fermentation of lignocellulosic plant residues from Brassica napus by Pleurotus ostreatus

Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratry alcohol (3,4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-flow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.     

Solid-State Fermentation of Rapeseed Meal with the White-Rot Fungi Trametes versicolor and Pleurotus ostreatus

Rapeseed meal is valuable high-protein forage, but its nutritional value is significantly reduced by the presence of a number of antinutrients, including phenolic compounds. Solid-state fermentation with white-rot fungi was used to decrease the sinapic acid concentration of rapeseed meal. After 7 days of growth of Trametes versicolor and Pleurotus ostreatus, the sinapic acid content of rapeseed meal was reduced by 59.9 and 74.5 %, respectively. At the end of the experiment, sinapic acid concentration of T. versicolor cultures decreased by 93 % of the initial value; in the case of cultures of P. ostreatus, 93.2 % reduction was observed. Moreover, cultivation of white-rot fungi on rapeseed meal resulted in the intensive production of extracellular laccase, particularly strong during the late phases of growth of T. versicolor. The obtained results confirm that both fungal species may effectively be used to decompose antinutritional phenolics of rapeseed meal. Rapeseed meal may also find use as an inexpensive and efficient substrate for a biotechnological production of laccase by white-rot fungi.     

Bioconversion of different agro-residues into edible protein byPleurotus sajor-caju

Pleurotus sajor-caju, an edible mushroom, is a source of protein-rich food.Pleurotus sajor-caju has broad temperature (15–25‡C) and pH (4-6) optima for its growth, thus making it suitable for village-level applications, where it can be cultivated in a generally artificially uncontrolled environment. In the present investigations, the following agro-residues have been used for cultivation of this mushroom: paddy straw, wheat straw, bagasse,Cyamposis tetragonoloba, Sorghum vulgare, Pennesitum typhoideum, mango leaves, banana leaves,Zea mays, etc. and some residues mixed with other materials such as cotton seeds, fiscus fruits,Azadirachta indica leaves, etc. All the substrates (400 g) were pasteurized at 60‡C, soaked in water, packed in polythene bags, and inoculated with 8% wheat spawn on a dry weight basis. The cultivation was carried out for 2 months, during which four crops were harvested. The temperature was 10–25‡C and relative humidity was maintained at 80–90%. The maximum yields in percent of fruit bodies obtained were as follows: banana leaves, 125 (91.8), paddy straw, 124.2 (90.6), wheat straw, 111.7 (90),Sorghum vulgare, 108.8 (92.9),Cyamposis tetragonoloba, 108.3 (91.5), followed by others. The maximum yields in percent of fruit bodies obtained in combinations of some substrates were as follows: paddy straw + cotton seeds, 148.3 (91.3),Cyamopsis tetragonoloba + Azadirachta indica leaves, 105.8 (91.5),Sorghum vulgare + Azadirachta indica leaves, 103.8 (91.8); the figures in parentheses represent the percent moisture content. The biochemical changes effected as a result of mushroom growth in terms of utilization of cellulose, hemicellulose, and lignin were presented.     

Production of 2,3- butanediol from pretreated corn cob byKlebsiella oxytoca in the presence of fungal cellulase

A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol byKlebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100°C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced byK. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.     

Radiation and NRSP effect on protein,filtration efficiency and crude fiber degradation after sugar cane bagasse fermentation by mushroom fungi

The upgrading of sugar cane bagasse with natural rubber waste by irradiation and three mushroom fungi was studied in order to produce economical animal feed and mushrooms. The protein concentrations changed a little at 0, 1% of NRSP but greatly increased at 5% NRSP concentration with irradiation and nonirradiation in liquid fermentation. The filtration rate decreased at 5% NRSP concentration after liquid fermentation but it increased by 2.7, 10.2, 11.1 times with irradiation for P. sajor-caju, C. phlytidosprorus and P. flavellatus. The amounts of crude fiber were decreased due to the irradiation and the different concentration of NRSP after 42 days of solid fermentation by P. sajor-caju.     

Fungal Pretreatment by Phanerochaete chrysosporium for Enhancement of Biogas Production from Corn Stover Silage

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.     

Aqueous Ammonia Soaking of Switchgrass Followed by Simultaneous Saccharification and Fermentation

Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment. Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid–solid ratios (5 and 10 ml/g) for either 5 or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40–50% delignification (Klason lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately 50%. The Sacccharomyces cerevisiae (D₅A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings (26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72. Liquid–solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method of pretreatment for lignocellulosic feedstocks.     

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high‐performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty‐seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii.     

Comparison of volatile compound production in fruit body and in mycelium of Pleurotus ostreatus identified by submerged and solid-state cultures

Comparative analyses of the production of volatile compounds by Pleurotus ostreatus JMO.95 fruit body and its corresponding mycelium grown in liquid, on agar surface, and on solid support cultures were carried out by dynamic headspace concentration using gas chromatography/mass spectrometry and gas chromatography sniffing. The aroma produced by fruit body was owing essentially to the presence of octan-3-one and, to a lesser extent, to octan-3-ol. Other compounds, such as oct-1-en-3-ol, oct-1-en, 2-methylbutanol, and α-pinene were also present in low concentrations. Comparison of aromatic spectra of the fruit body with that of mycelia obtained under different culture conditions indicated that the main aromatic compounds present in the P. ostreatus fruit body and mycelium were produced in the same proportions on agar surface and on solid support culture, but not under submerged conditions.     

Rapid Measurement of Cellulose,Hemicellulose, and Lignin Content in Sargassum horneri by Near-Infrared Spectroscopy and Characteristic Variables Selection Methods

Near-infrared (NIR) spectroscopy and characteristic variables selection methods were used to develop a quick method for the determination of cellulose, hemicellulose, and lignin contents in Sargassum horneri. Calibration models for cellulose, hemicellulose, and lignin in Sargassum horneri were established using partial least square regression methods with full variables (full-PLSR). The PLSR calibration models were established by four characteristic variables selection methods, including interval partial least square (iPLS), competitive adaptive reweighted sampling (CARS), correlation coefficient (CC), and genetic algorithm (GA). The results showed that the performance of the four calibration models, namely iPLS-PLSR, CARS-PLSR, CC-PLSR, and GA-PLSR, was better than the full-PLSR calibration model. The iPLS method was best in the performance of the models. For iPLS-PLSR, the determination coefficient (R²), root mean square error (RMSE), and residual predictive deviation (RPD) of the prediction set were as follows: 0.8955, 0.8232%, and 3.0934 for cellulose, 0.8669, 0.4697%, and 2.7406 for hemicellulose, and 0.7307, 0.7533%, and 1.9272 for lignin, respectively. These findings indicate that the NIR calibration models can be used to predict cellulose, hemicellulose, and lignin contents in Sargassum horneri quickly and accurately.     

Development and Characterization of an Environmentally Friendly Process Sequence (Autohydrolysis and Organosolv) for Wheat Straw Delignification

The present work describes the delignification of wheat straw through an environmentally friendly process resulting from sequential application of autohydrolysis and organosolv processes. Wheat straw autohydrolysis was performed at 180°C during 30 min with a liquid–solid ratio of 10 (v/w); under these conditions, a solubilization of 44% of the original xylan, with 78% of sugars as xylooligosaccharides of the sum of sugars solubilized in the autohydrolysis liquors generated by the hemicellulose fraction hydrolysis. The corresponding solid fraction enrichment with 63.7% of glucan and 7.55% of residual xylan was treated with a 40% ethanol and 0.1% NaOH aqueous solution at a liquid–solid ratio of 10 (v/w), with the best results obtained at 180°C during 20 min. The highest lignin recovery, measured by acid precipitation of the extracted lignin, was 3.25 g/100 ml. The lignin obtained by precipitation was characterized by FTIR, and the crystallinity indexes from the native cellulose, the cellulose recovered after autohydrolysis, and the cellulose obtained after applying the organosolv process were obtained by X-ray diffraction, returning values of 21.32%, 55.17%, and 53.59%, respectively. Visualization of the fibers was done for all the processing steps using scanning electron microscopy.     

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium under solid-state fermentation

Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28±1 °C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.     

Bleach Enhancement of Mixed Wood Pulp by Xylanase–Laccase Concoction Derived Through Co-culture Strategy

Mixed enzyme preparation having both xylanase and laccase activity was evaluated for its bleach enhancing ability of mixed wood pulp. The enzyme was produced through co-cultivation of mutant Penicillium oxalicum SAU_E-3.510 and Pleurotus ostreatus MTCC 1804 under solid-state fermentation. Bleaching of pulp with mixed enzyme had resulted into a notable decrease in kappa number and increased brightness as compared to xylanase alone. Analysis of bleaching conditions had denoted that 8 IU g⁻¹ of mixed enzyme preparation (xylanase/laccase, 22:1) had led into maximal removal of lignin from pulp when bleaching was performed at 10% pulp consistency (55 °C, pH 9.0) for 3 h. An overall improvement of 21%, 8%, 3%, and 5% respectively in kappa number, brightness, yellowness, and viscosity of pulp was achieved under derived bleaching conditions. Process of enzymatic bleaching was further ascertained by analyzing the changes occurring in polysaccharide and lignin by HPLC and FTIR. The UV absorption spectrum of the compounds released during enzymatic treatment had denoted a characteristic peak at 280 nm, indicating the presence of lignin in released coloring matter. The changes in fiber morphology following enzymatic delignification were studied by scanning electron microscopy.     

Enzymes involved in cellulose and lignin degradation

A detailed presentation was given of the discovered and studied enzymes involved in degradation of cellulose and lignin by the white-rot fungus,Sporotrichum pulverulentum (Phanerochaete chrysosporium). The fungus utilizes, for the degradation of cellulose: (a) Five different endo-1,4-Β-glucanases (b) One exo-1,4-Β-glucanase (acting synergistically with the endoglucanases) (c) Two 1,4-Β-glucosidases The regulation, induction, and catabolite repression of the endoglucanases have been studied in depth and the results of these studies were also presented. In addition to the hydrolytic enzymes,S. pulverulentum also produces the oxidative enzyme cellobiose oxidase that is of importance for cellulose degradation. Another unconventional enzyme is cellobiose: quinone oxidoreductase, which is of importance for both cellulose and lignin degradation. It reduces quinones from the lignin under oxidation of cellobiose from the cellulose. It has recently been discovered thatS. pulverulentum produces two acidic proteases of importance for cellulose degradation since they enhance the endoglucanase activity, particularly in young cultures of the fungus grown on cellulose. The enzymes involved in lignin degradation are not known nearly as well as these involved in cellulose degradation. However, extracellular phenol oxidases, laccase, and peroxidase have been shown to be involved in and necessary for lignin degradation to take place. A phenol oxidase-less mutant ofS. pulverulentum cannot degrade lignin unless a phenol oxidase is added to the medium. Recently, an enzyme splitting the α—Β bond in the propane side chain has been discovered by Kirk and coworkers. Several enzymes involved in the metabolism of vanillic acid, always a metabolite in lignin degradation, have been discovered and studied in our laboratory. Presentations of the enzymes for decarboxylation, demethoxylation, methanol oxidation, ring cleavage, and intracellular quinone reduction by NAD(P)H: quinone oxidoreductase were given. A discussion of possibilities for a specific enzymic primary attack on the native lignin, as well as of the likeliness for an unspecific radical nature of this attack, was also given.     

Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood

The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58–64% cellulose, 2–16% hemicellulose, and 24–30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature of the pretreatment, the residual hemicellulose content of the substrate, and the type of the commercial cellulase preparation used were the most important factors affecting the enzymatic hydrolysis. The highest sugar yield in a 72-h hydrolysis, 79% of theoretical, was obtained using a pretreatment of 200°C for 10 min at neutral pH.