The accuracy of the pepsin-cellulase technique for estimating the dry matter digestibility in vivo of grasses and legumes

A study was made of the accuracy of predicting dry matter digestibility (DMD) in vivo of 32 legume samples and 50 grass samples from the dry matter disappearance (DMS) of the samples in pepsin (48 h) followed by Onozuka cellulase (48 h). The effects of grinder screen size, incubation temperature and sample size on the accuracy of predicting dry matter digestibility were examined.The DMD in vivo of legumes and grasses was significantly correlated with DMS at 39° C (r = 0.91 and 0.94) and increasing the incubation temperature to 50° C had no effect on the correlations, although DMS was greater. There was no significant difference between the regression equations for grasses and legumes. The regression equation for the combined 82 samples had an RSD of ± 3.0 digestibility units. The corresponding RSD for regressions based on fermentation with rumen fluid in vitro was ± 2.6 digestibility units. Grinding the grass samples through a 0.4 mm screen prior to analysis resulted in regressions with a larger RSD (± 3.3) than when a 1.0 mm screen was used to prepare the samples (P < 0.001).It was concluded that the digestibility in vivo of both legumes and grasses could be predicted by the pepsin-cellulase method by using the same equation, but to eliminate biased results, samples of known digestibility in vivo similar to those being tested should be included as standards in each study. The analytical time could be reduced by 48 h if shorter incubation periods (24 h) were used, but only at the expense of slightly larger errors in predicting DMD in vivo (P < 0.01).     

Prediction of the day matter digestibility of tropical grasses using a pepsin-cellulase assay

A study was made of the accuracy of predicting dry matter digestibility in vivo from the dry matter disappearance of forage samples in pepsin (48 h) followed by Onozuka cellulase (48 h). Forty-five samples representing four tropical pasture species, Cenchrus ciliaris, Chloris gayana, Digitaria spp., Setaria spp. and one temperate grass, Lolium perenne were used.Dry matter digestibility in vivo was significantly correlated with the disappearance in pepsin-cellulase (r = 0.94) and the regression predicting dry matter digestibility in vivo had a residual standard deviation of ± 2.7 digestibility units. Separate regressions for the five grasses had RSD values ranging from ± 1.3 to ± 2.6.It was concluded that the digestibility in vivo of both tropical and temperate grasses in the range 40–76% digestibility could be accurately predicted by the pepsin-cellulase assay, provided samples of known digestibility in vivo similar to those being tested were included as standards in each run.     

The analytical and biological accuracy of estimating the dry matter digestibility of different legume species

A comparison was made of the accuracy of 7 laboratory techniques for estimating dry matter digestibility in vivo of 5 legume species represented by 32 samples of known digestibility in vivo. The legumes studied were Desmodium spp., Lablab purpureus, Macroptilium atropurpureum, Vigna sinensis and Medicago sativa. Regressions were calculated relating digestibility in vivo to the laboratory analysis, and the residual standard deviation (RSD) of these regressions was used to calculate the error in predicting digestibility in vivo.For regressions based on all 32 samples the RSD's were: modified acid detergent fibre ± 2.1, acid detergent fibre ± 2.2, dry matter digestibility in vitro ± 2.7, crude fibre ± 3.7, feed nitrogen ± 4.0, Christian's lignin ± 2.8, and Van Soest's Lignin ± 3.7 digestibility units. The cause of the high RSD of these regressions was discussed and it was concluded that it was mainly associated with differences between legume samples grown in different ways, analytical error being only ± 0.5 percentage units or less and the estimation of digestibility in vivo ± 0.7 percentage units.It was concluded that the error in predicting digestibility in vivo from a laboratory analysis is best expressed as the RSD of regressions relating digestibility in vivo to the laboratory analysis, and that this error will not be materially altered by increased replication.     

A note on Onozuka 3S cellulase as a replacement for Onozuka SS (P1500) cellulase when estimating forage digestibility in vitro

To test the suitability of Onozuka 3S cellulase as a replacement for the superceded Onozuka SS (P1500) for estimating forage digestibility, a comparison was made using 50 grass and 30 legume samples of known dry matter digestibility in vivo (means of 55.4% and 58.4%, respectively).Mean solubility of dry matter (DMS) following acid pepsin pretreatment and Onozuka 3S or SS (P1500) was 51.3 and 52.8%, respectively for grasses, and 60.2 and 60.7%, respectively, for legumes. The differences in DMS between the two cellulases for both grasses and legumes were not significant (P>0.05).Regressions were calculated relating DMD in vivo to the DMS in pepsin-cellulase with the two cellulase preparations. Errors for the Onozuka 3S and SS (P1500) were ± 2.6 and ± 2.7, respectively, for grasses, and ± 2.6 and ± 2.9, respectively, for legumes.It was concluded that 3S is a satisfactory replacement for SS (P1500) for estimating forage digestibility.     

Bias when predicting crude protein,dry matter digestibility and voluntary intake of tropical grasses by near-infrared reflectance

A study was made of the bias (non-random error) of regressions for predicting crude protein (CP) content, dry matter digestibility (DMD) in vivo and voluntary intake (VI) of forage from its near-infrared reflectance (NIRR) properties measured at 19 wavelengths. The regression was derived from 97 samples including six tropical grasses, leaf and stem fractions, pelleted and chopped forms and regrowths varying from 28 to 150 days.Bias associated with differences in feed type was proved to exist in regression equations for predicting nutritive value when it was shown that the residual errors of these regressions were significantly reduced by including the combined effects of grass species, fraction, form and age. Measures of the bias which might be expected were calculated for two contrasting situations. The internal measure of bias (IMB) was ‘estimated’ as a possible average bias of a feed type when predicting nutritive value for samples of similar types to those used when deriving the calibration equation, while the extrapolation measure of bias (EMB) was ‘estimated’ as that associated with prediction of the nutritive value for feed types not included in the calibration equation.Crude protein was estimated with a residual standard deviation (RSD) of ± 0.77% when regressed against the 19 reflectance values, but when information on grass species, parts and regrowth was also included in the regression the RSD was reduced to ± 0.46%. The maximum difference in the IMB between grass species was 0.80% crude protein, while for EMB it was 3.35% crude protein. When separated leaf and stem were considered the differences were larger.Dry matter digestibility was estimated with an RSD of ± 2.7% which decreased to ± 1.8% when grass species, age, form and study were taken into account. Grass species had only a small effect on IMB but the maximum difference for EMB between species reached 5.6%. The EMB indicated that the largest differences between deviations were associated with age of regrowth but this effect was absent with the IMB.Voluntary intake was estimated with an RSD of ± 7.2 g/kg W^0.75/day, which decreased to ± 2.5 g/kg W^0.75/day when grass species, form, part and age were taken into account. The largest bias was for pelleted and chopped forage with mean differences of 14.5 for IMB and 28.8 g/kg W^0.75/day for EMB, respectively.It was concluded that when nutritive value is predicted by NIRR the estimates may often be biased according to the grass species, plant part or physical form of the sample tested and that the pattern of bias may be complicated. In order to obtain accurate predictions from NIRR full account must be taken of this bias. This might be achieved by correcting the nutritive value estimates made from an NIRR regression, based on a large number of feed types, by addition of the bias associated with the feed type being estimated.     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Cellulase insoluble fibre as a measure of unavailable organic matter in cattle compounds containing alkalitreated straw

A laboratory method for determination of fibres insoluble by cellulase is suggested as a routine analysis for cattle compound feeds with varying proportion and quality of chemically treated straw and other fibrous components, in order to measure whether actual energy content corresponds to that declared.By a two-step analytical procedure, the total fibre is isolated by boiling with neutral detergent solution for 1 h, which removes the readily soluble carbohydrates, proteins and fat. By incubation of the isolated fibre in a solution of buffers and a commercial cellulase reagent for 48 h at 40°C the digestible fibre is removed. The organic matter in the residue, which is termed cellulase insoluble fibre (CIF), is a measure of unavailable organic matter for ruminants.The CIF content, total NDF and proximate constituents were determined in 47 compounds of extreme properties. Compared with digestibility in vitro (IVDOM), more organic matter was dissolved by the CIF-analyses in highly digestible feeds and less in feeds with low digestibility (IVDOM + fat = 87 ? 0.70 CIF).     

Feed intake and milk production in dairy cows fed different grass and legume species: a meta-analysis

The aim of this meta-analysis was to compare feed intake, milk production, milk composition and organic matter (OM) digestibility in dairy cows fed different grass and legume species. Data from the literature was collected and different data sets were made to compare families (grasses v. legumes, Data set 1), different legume species and grass family (Data set 2), and different grass and legume species (Data set 3+4). The first three data sets included diets where single species or family were fed as the sole forage, whereas the approach in the last data set differed by taking the proportion of single species in the forage part into account allowing diets consisting of both grasses and legumes to be included. The grass species included were perennial ryegrass, annual ryegrass, orchardgrass, timothy, meadow fescue, tall fescue and festulolium, and the legume species included were white clover, red clover, lucerne and birdsfoot trefoil. Overall, dry matter intake (DMI) and milk production were 1.3 and 1.6 kg/day higher, respectively, whereas milk protein and milk fat concentration were 0.5 and 1.4 g/kg lower, respectively, for legume-based diets compared with grass-based diets. When comparing individual legume species with grasses, only red clover resulted in a lower milk protein concentration than grasses. Cows fed white clover and birdsfoot trefoil yielded more milk than cows fed red clover and lucerne, probably caused by a higher OM digestibility of white clover and activity of condensed tannins in birdsfoot trefoil. None of the included grass species differed in DMI, milk production, milk composition or OM digestibility, indicating that different grass species have the same value for milk production, if OM digestibility is comparable. However, the comparison of different grass species relied on few observations, indicating that knowledge regarding feed intake and milk production potential of different grass species is scarce in the literature. In conclusion, different species within family similar in OM digestibility resulted in comparable DMI and milk production, but legumes increased both DMI and milk yield compared with grasses.     

A method using solubility to predict dry matter digestibility of cellulosic materials.

A rapid chemical procedure based on the solubility of a holocellulose sample in a system of dimethyl sulfoxide with paraformaldehyde has been developed to provide a laboratory method for predicting dry matter digestibility of cellulose containing samples. The amount of dry matter solubilized by the chemical procedure was closely correlated with anaerobic, in vitro, rumen fluid digestion and with digestibility as measured by aerobic Cellulomonas, sp. bacteria. The quantity of solvent and dissolving time had little effect on solubility over a wide range. The method is rapid, well suited for various cellulosic materials, and may be carried out with simple equipment and facilities.     

Increasing cellulose accessibility is more important than removing lignin: A comparison of cellulose solvent‐based lignocellulose fractionation and soaking in aqueous ammonia

While many pretreatments attempt to improve the enzymatic digestibility of biomass by removing lignin, this study shows that improving the surface area accessible to cellulase is a more important factor for achieving a high sugar yield. Here we compared the pretreatment of switchgrass by two methods, cellulose solvent‐ and organic solvent‐based lignocellulose fractionation (COSLIF) and soaking in aqueous ammonia (SAA). Following pretreatment, enzymatic hydrolysis was conducted at two cellulase loadings, 15 filter paper units (FPU)/g glucan and 3 FPU/g glucan, with and without BSA blocking of lignin absorption sites. The hydrolysis results showed that the lignin remaining after SAA had a significant negative effect on cellulase performance, despite the high level of delignification achieved with this pretreatment. No negative effect due to lignin was detected for COSLIF‐treated substrate. SEM micrographs, XRD crystallinity measurements, and cellulose accessibility to cellulase (CAC) determinations confirmed that COSLIF fully disrupted the cell wall structure, resulting in a 16‐fold increase in CAC, while SAA caused a 1.4‐fold CAC increase. A surface plot relating the lignin removal, CAC, and digestibility of numerous samples (both pure cellulosic substrates and lignocellulosic materials pretreated by several methods) was also developed to better understand the relative impacts of delignification and CAC on glucan digestibility. Biotechnol. Bioeng. 2011; 108:22–30. © 2010 Wiley Periodicals, Inc.     

Prediction of the digestibility of ruminant feeds from their solubility in enzyme solutions

The solubility of 24 diets containing grain and 23 forages was measured in solutions of either cellulase, pepsin or amylase. Sequential incubations in pepsin and cellulase (from Aspergillus niger) yielded solubility values which were significantly correlated with the dry matter digestibility in vivo of the diets containing grain (r = 0.85) and the forages (r = 0.82). When cellulase from Trichoderma viride was used instead of the cellulase from Aspergillus niger, the correlation was lower for the feeds containing grain (r = 0.60) but not for the forages (r = 0.84). The former method was comparable in accuracy to the two-stage technique for measuring digestibility in vitro with rumen fluid and pepsin, for which correlation coefficients of 0.89 and 0.83 were observed for the diets containing grain and the forages, respectively.     

Explosive pretreatment of lignocellulosic residues with high-pressure carbon dioxide for the production of fermentation substrates

A new pretreatment technique has been developed in which the lignocellulosic material is subjected to the action of steam and high-pressure carbon dioxide before being explosively discharged through a defibrating nozzle of novel design. Operating at 200 degrees C and gas pressures in the range of 3.45-13.8 MPa, exploded products with maximum in vitro cellulase digestibilities of 81, 78, and 75% were obtained from wheat straw, bagasse, and Eucalyptus regnans woodchips, respectively. The treatment times required to obtain substrates of maximum digestibility were 5 min for wheat straw and bagasse and 15 min for E. regnans. Analysis of the exploded products indicated that the pretreatment had substantially removed and solubilized the hemicellulose fraction of the feed materials, giving an autohydrolysis liquor rich in xylose and a fibrous residue primarily composed of alpha-cellulose and lignin. The fibrous residue was readily amenable to cellulase hydrolysis, with saccharification being completed within 48 h. The theoretical energy demand for gas compression in the pretreatment process has been calculated at 11 k Wh/ton raw material when the digester is operated at 3.45 MPa and a packing density of 250 kg raw material/m(3) digester volume.     

Application of cellulase and hemicellulase to pure xylan, pure cellulose, and switchgrass solids from leading pretreatments

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.     

Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover

Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.     

Rapeseed-straw enzymatic digestibility enhancement by sodium hydroxide treatment under ultrasound irradiation

In this study, we carried out sodium hydroxide and sonication pretreatments of rapeseed straw (Brassica napus) to obtain monosugar suitable for production of biofuels. To optimize the pretreatment conditions, we applied a statistical response-surface methodology. The optimal pretreatment conditions using sodium hydroxide under sonication irradiation were determined to be 75.0 °C, 7.0 % sodium hydroxide, and 6.8 h. For these conditions, we predicted 97.3 % enzymatic digestibility. In repeated experiments to validate the predicted value, 98.9 ± 0.3 % enzymatic digestibility was obtained, which was well within the range of the predicted model. Moreover, sonication irradiation was found to have a good effect on pretreatment in the lower temperature range and at all concentrations of sodium hydroxide. According to scanning electron microscopy images, the surface area and pore size of the pretreated rapeseed straw were modified by the sodium hydroxide pretreatment under sonication irradiation.     

Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO₂) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with β‐glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8–116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and β‐glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase–xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies

In order to investigate changes in substrate chemical and physical features after pretreatment, several characterizations were performed on untreated (UT) corn stover and poplar and their solids resulting pretreatments by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough, lime, and SO₂ technologies. In addition to measuring the chemical compositions including acetyl content, physical attributes determined were biomass crystallinity, cellulose degree of polymerization, cellulase adsorption capacity of pretreated solids and enzymatically extracted lignin, copper number, FT-IR responses, scanning electron microscopy (SEM) visualizations, and surface atomic composition by electron spectroscopy of chemical analysis (ESCA). Lime pretreatment removed the most acetyl groups from both corn stover and poplar, while AFEX removed the least. Low pH pretreatments depolymerized cellulose and enhanced biomass crystallinity much more than higher pH approaches. Lime pretreated corn stover solids and flowthrough pretreated poplar solids had the highest cellulase adsorption capacity, while dilute acid pretreated corn stover solids and controlled pH pretreated poplar solids had the least. Furthermore, enzymatically extracted AFEX lignin preparations for both corn stover and poplar had the lowest cellulase adsorption capacity. ESCA results showed that SO₂ pretreated solids had the highest surface O/C ratio for poplar, but for corn stover, the highest value was observed for dilute acid pretreatment with a Parr reactor. Although dependent on pretreatment and substrate, FT-IR data showed that along with changes in cross linking and chemical changes, pretreatments may also decrystallize cellulose and change the ratio of crystalline cellulose polymorphs (Iα/Iβ).     

A comparison of three fibre methods for predicting the metabolizable energy content of sorghum grain for poultry

An apparatus designed to simplify analytical procedures for determining fibre in food was used to measure fibre of similar composition to crude fibre, acid-detergent fibre and neutral-detergent fibre in 27 samples of sorghum grain. The metabolizable energy content of these grains for poultry was predicted from the three fibre methods with precision, respectively, of ± 0.49, ± 0.62 and ± 0.53 MJ/kg dry matter. These values corresponded to coefficients of variation of ± 3.0, ± 3.8 and ± 3.3%.The three fibre fractions were highly correlated with each other, and so a simple method that can measure a part of the total fibre is suitable for predicting the energy value of sorghum grain for poultry.     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.