Heterotrophic Culture of <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> in Various Nitrogen Sources for Lipid Production

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO₃ medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.     

Production of Astaxanthin from Corn Fiber as a Value-Added Co-product of Fuel Ethanol Fermentation

Five strains of the yeast Phaffia rhodozyma, NRRL Y-17268, NRRL Y-17270, ATCC 96594 (CBS 6938), ATCC 24202 (UCD 67-210), and ATCC 74219 (UBV-AX2) were tested for astaxanthin production using the major sugars derived from corn fiber. The sugars tested included glucose, xylose, and arabinose. All five strains were able to utilize the three sugars for astaxanthin production. Among them, ATCC 74219 was the best astaxanthin producer. Kinetics of sugar utilization of this strain was studied, both with the individual sugars and with their mixtures. Arabinose was found to give the highest astaxanthin yield. It also was observed that glucose at high concentrations suppressed utilization of the other two sugars. Corn fiber hydrolysate obtained by dilute sulfuric acid pretreatment and subsequent enzyme hydrolysis was tested for astaxanthin production by strain ATCC 74219. Dilution of the hydrolysate was necessary to allow growth and astaxanthin production. All the sugars in the hydrolysate diluted with two volumes of water were completely consumed. Astaxanthin yield of 0.82 mg/g total sugars consumed was observed.     

Cassava starch maltodextrinization/monomerization through thermopressurized aqueous phosphoric acid hydrolysis

Kinetic conditions were established for the depolymerization of cassava starch for the production of maltodextrins and glucose syrups. Thin-layer chromatography and high-performance liquid chromatography analyses corroborated that the proper H₃PO₄ strengthand thermopressurization range (e.g., 142–170°C; 2.8–6.8 atm) can be successfully explored for such hydrolytic purposes of native starch granules. Because phosphoric acid can be advantageously maintained in the hydrolysate and generates, after controlled neutralization with ammonia, the strategic nutrient triplet for industrial fermentations (C, P, N), this pretreatment strategy can be easily recognized as a recommended technology for hydrolysis and upgrading of starch and other plant polysaccharides. Compared to the classic catalysts, the mandatory desalting step (chloride removal by expensive anion-exchange resin or sulfate precipitation as the calcium-insoluble salt) can be avoided. Furthermore, properly diluted phosphoric acid is well known as an allowable additive in several popular soft drinks such as colas since its acidic feeling in the mouth is compatible and synergistic with both natural and artificial sweeteners. Glycosyrups from phosphorolyzed cassava starch have also been upgraded to high-value single-cell protein such as the pigmented yeast biomass of Xanthophyllomyces dendrorhous (Phaffia rhodozyma), whose astaxanthin (diketo-dihydroxy-β-carotene) content may reach 0.5–1.0 mg/g of dry yeast cell. This can be used as an ideal complement for animal feeding as well as a natural staining for both fish farming (meat) and poultry (eggs).

     

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.     

Kinetic study of growth,lipid and carotenoid formation in β-carotene producing <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The oleaginous and red yeast Rhodotorula glutinis CCY 20-2-26 is known for its high-storage lipids accumulation in cells as well as for β-carotene biosynthesis. The work is focused on the study of lipogenesis and carotenoid pigments formation by the yeast grown in media with four different C/N ratios (20:1, 50:1, 70:1 and 100:1). Total fatty acids accumulation in cells reached the maximal value of 48% and yielded up to 8.9 g fatty acid/L media under C/N 70:1. On contrary, while the highest pigment accumulation in the yeast (1268 μg/g of cells) was found at the C/N ratio of 20:1, the maximal carotenoid yield (mainly β-carotene) of 12.7 mg/L was obtained under 50:1 ratio. Calculation of kinetic parameters of metabolites production revealed the metabolic correlations among glucose consumption, lipogenesis and carotenoid biosynthesis. It was demonstrated that glucose exhaustion resulted in reduction of growth and lipid accumulation in cells. Then, storage fatty acids, especially palmitic and oleic acids, begun to be degraded by β-oxidations and formed acetyl-CoA which was especially used for biosynthesis of β-carotene. This is the first study providing the new hypothesis about the metabolic connection between fatty acids and β-carotene metabolism in the red yeasts.     

Screening Factors Influencing the Production of Astaxanthin from Freshwater and Marine Microalgae

Astaxanthin, a carotenoid pigment found in several aquatic organisms, is responsible for the red colour of salmon, trout and crustaceans. In this study, astaxanthin production from freshwater microalga Chlorella sorokiniana and marine microalga Tetraselmis sp. was investigated. Cell growth and astaxanthin production were determined spectrophotometrically at 620 and 480 nm, respectively. Astaxanthin was extracted using acetone and measured subsequent to biomass removal. Aerated conditions favoured astaxanthin production in C. sorokiniana, whereas Tetraselmis sp. was best cultured under unaerated conditions. C. sorokiniana produced more astaxanthin with the highest yield reached at 7.83 mg/l in 6.0 mM in nitrate containing medium compared to Tetraselmis sp. which recorded the highest yield of only 1.96 mg/l in 1.5 mM nitrate containing medium. Production in C. sorokiniana started at the early exponential phase, indicating that astaxanthin may be a growth-associated product in this microalga. Further optimization of astaxanthin production was performed using C. sorokiniana through a 2³ full factorial experimental design, and a yield of 8.39 mg/l was achieved. Overall, the study has shown that both microalgae are capable of producing astaxanthin. Additionally, this research has highlighted C. sorokiniana as a potential astaxanthin producer that could serve as a natural astaxanthin source in the current market.     

Cellulosic fuel ethanol

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The “Iogen Process” for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C₅ and C₆ sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C₆:C₅ = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.     

Lipid Production by Culturing Oleaginous Yeast and Algae with Food Waste and Municipal Wastewater in an Integrated Process

Food waste and municipal wastewater are promising feedstocks for microbial lipid biofuel production, and corresponding production process is to be developed. In this study, different oleaginous yeast strains were tested to grow in hydrolyzed food waste, and growths of Cryptococcus curvatus, Yarrowia lipolytica, and Rhodotorula glutinis in this condition were at same level as in glucose culture as control. These strains were further tested to grow in municipal primary wastewater. C. curvatus and R. glutinis had higher production than Y. lipolytica in media made from primary wastewater, both with and without glucose supplemented. Finally, a process was tested to grow C. curvatus and R. glutinis in media made from food waste and municipal wastewater, and the effluents from these processes were further treated with yeast culture and phototrophic algae culture; 1.1 g/L C. curvatus and 1.5 g/L R. glutinis biomass were further produced in second-step yeast cultures, as well as 1.53 and 0.58 g/L Chlorella sorokiniana biomass in phototrophic cultures. The residual nitrogen concentrations in final effluents were 33 mg/L and 34 mg/L, respectively, and the residual phosphorus concentrations were 1.5 and 0.6 mg/L, respectively. The lipid contents in the produced biomass were from 18.7% to 28.6%.     

Enzymatic Fractionation of SAA-Pretreated Barley Straw for Production of Fuel Ethanol and Astaxanthin as a Value-Added Co-Product

Barley straw was used to demonstrate an integrated process for production of fuel ethanol and astaxanthin as a value-added co-product. Barley straw was pretreated by soaking in aqueous ammonia using the previously determined optimum conditions, which included 77.6 °C treatment temperature, 12.1 h treatment time, 15 wt% ammonia concentration, and 1:8 solid-to-liquid ratio. In the newly developed process, the pretreated barley straw was first hydrolyzed with ACCELLERASE® XY (a commercial hemicellulase product) to generate a xylose-rich solution, which contained 3.8 g/l glucose, 22.9 g/l xylose, and 2.4 g/l arabinose, with 96 % of the original glucan being left intact. The xylose-rich solution was used for production of astaxanthin by the yeast Phaffia rhodozyma without further treatment. The resulting cellulose-enriched solid residue was used for ethanol production in a fed-batch simultaneous saccharification and fermentation using ACCELLERASE® 1500 (a commercial cellulase product) and the industrial yeast Saccharomyces cerevisiae. At the end of the fermentation, 70 g/l ethanol was obtained, which was equivalent to 63 % theoretical yield based on the glucan content of the solid substrate.     

Eicosapentaenoic acid (EPA) Production byMortierella alpina ATCC 32222

Mortierella alpina ATCC 32222 grew well at 11 degrees C, as well as at 25 degrees C in a liquid medium containing glucose or linseed oil and yeast extract. High Eicosapentaenoic acid (EPA) yield was obtained at 11 degrees C. M. alpina cells did not produce EPA at 25 degrees C in the absence of linseed oil, whereas at 11 degrees C, EPA accumulation was noted in the absence of linseed oil. When grown at 11 degrees C for 10 d in a medium containing 2% linseed oil as carbon source, the mycelium yielded 435 mg/L EPA (20 mg EPA/g dry mycelia) with 5.1% in lipid fraction. By gradually increasing the concentration of linseed oil to 4%, yield of biomass and EPA were increased to 43 g/L and 596 mg/L, respectively.     

Effect of yeast extract on growth kinetics of Monascus purpureus

Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast extract, caused an increase in cell yield on glucose (Y_s/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

Kinetics of Lime Pretreatment of Sugarcane Bagasse to Enhance Enzymatic Hydrolysis

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2³ central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60–90°C). The delignification was highly dependent on temperature and duration of pretreatment.     

Oxidative lime pretreatment of high-lignin biomass

Lime (Ca[OH]₂) and oxygen (O₂) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150°C, 6 h, 0.1 g of Ca(OH)₂/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of −10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140°C, 3 h, 0.3 g of Ca(OH)₂/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO₂ carbonation.     

Effects of Plant Elicitors on Growth and Gypenosides Biosynthesis in Cell Culture of Giao co lam (Gynostemma pentaphyllum)

Giao co lam (Gynostemma pentaphyllum (Thunb.) Makino) is used in Northeast and Southeast Asia countries for the treatment of various diseases, including hepatitis, diabetes, and cardiovascular disease. G. pentaphyllum saponins (gypenosides) are the major components responsible for the pharmacological activities. In this study, different concentrations of abiotic (25–200 μM methyl jasmonate-MeJA and salicylic acid-SA) or biotic elicitors (1–5 g/L yeast extract-YE and Fusarium biomass) were used as plant elicitors, in order to investigate their influences on cell growth and gypenosides accumulation in G. pentaphyllum suspension cells. Suspension cells were grown on a MS medium containing 2.0 mg/L KIN and 0.5 mg/L IBA, with initial inoculum sizes of 3 g and shaking speeds of 120 rpm for 18 days. Gypenoside and Rb1 contents were measured by colorimetric and HPLC methods. Among three elicitors, SA was suitable for gypenosides accumulation in individual treatment. The cell biomass had the same values in elicitated and control suspension cells. Gypenosides content in cells treated with 100 μM salicylic acid after 6 days of culture reached a maximum value of 79.721 mg gypenoside/g dry biomass (including 0.093 mg ginsenoside Rb1/mg dry weight), which was 2.18-folds higher than that of the natural product. The elicitation promises an efficiency strategy for the production gypenosides in Gynostemma pentaphyllum suspension cells.     

Screening and Selection of Marine Isolate for l-Glutaminase Production and Media Optimization Using Response Surface Methodology

The current work details the screening of about 400 marine isolates from various marine niches, from which one isolate was finally selected based on the productivity of glutaminase (71.23 U/l). Further, biochemical identification tests and 16S rRNA sequencing identified this isolate to be Providencia sp. This isolate was taken up for further media optimization studies by using one-factor-at-a-time approach and subsequently by response surface methodology. A face centered central composite design was employed to investigate the interactive effects of four variables, viz., concentrations of glucose, methionine, urea, and succinic acid on glutaminase production. A significant influence of urea on glutaminase production was noted. Response surface methodology showed that a medium containing (g/l) glucose 10.0, urea 5.15, methionine 3.5, succinic acid 6.0, ammonium sulfate 2.5, and yeast extract 6.0 to be optimum for the production of glutaminase. The applied methodology was validated using this optimized media and enzyme activity 119 ± 0.12 U/l and specific activity of 0.63 U/mg protein after 28 h of incubation at 25 °C was obtained.     

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30?g?g^?1) and productivity (0.19?g?L^?1?h^?1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3?g?L^?1 resulted in high biomass production. The highest biomass concentration (21?g?L^?1), yield (0.45?g?g^?1) and productivity (0.31?g?L^?1?h^?1), as well as ribonucleotide production (13.13?mg?g^?1), were observed at 700?rpm and 0.5?vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.     

Growth ofschizosaccharomyces pombe on glucose-malte mixtures in continuous cell-recycle cultures kinetics of substrate utilization

The aerobic growth ofSchizosaccharomyces pombe on mixtures of glucose and malate was investigated during continuous high cell density cultures with partial cell-recycle using a membrane bioreactor. Determination of the specific metabolic rates relative to substrates and products allowed the capacity of the yeast to metabolize malic acid under both oxidative metabolism (carbon limited cultures) and oxidofermentative metabolism (carbon sufficient cultures) situations to be characterized. Under carbon limiting conditions, the specific rate of malate utilization was dependent on the residual concentration and a limit for a purely oxidative breakdown without ethanol formation was observed for a characteristic ratio between the rates of substrate consumption qm/qg of 1.63 g.g^-1. In addition, the mass balance analysis revealed the incorporation of malic acid into biomass. In carbon excess environments, the specific rate of malate utilization was dependent on both the residual malate and the specific rate of glucose consumption indicating that in addition to its conversion into ethanol malate can be respiratively metabolized for qm/qg ratios higher than 0.4 g.g^-1.     

Ammonia Fiber Expansion (AFEX) Pretreatment,Enzymatic Hydrolysis,and Fermentation on Empty Palm Fruit Bunch Fiber (EPFBF) for Cellulosic Ethanol Production

Empty palm fruit bunch fiber (EPFBF), a readily available cellulosic biomass from palm processing facilities, is investigated as a potential carbohydrate source for cellulosic ethanol production. This feedstock was pretreated using ammonia fiber expansion (AFEX) and enzymatically hydrolyzed. The best tested AFEX conditions were at 135 °C, 45 min retention time, water to dry biomass loading of 1:1 (weight ratio), and ammonia to dry biomass loading of 1:1 (weight ratio). The particle size of the pretreated biomass was reduced post-AFEX. The optimized enzyme formulation consists of Accellerase (84 μL/g biomass), Multifect Xylanase (31 μL/g biomass), and Multifect Pectinase (24 μL/g biomass). This mixture achieved close to 90% of the total maximum yield within 72 h of enzymatic hydrolysis. Fermentation on the water extract of this biomass affirms that nutrients solely from the pretreated EPFBF can support yeast growth for complete glucose fermentation. These results suggest that AFEX-treated EPFBF can be used for cellulosic biofuels production because biomass recalcitrance has been overcome without reducing the fermentability of the pretreated materials.     

Comparison of changes in cellulose ultrastructure during different pretreatments of poplar

One commonly cited factor that contributes to the recalcitrance of biomass is cellulose crystallinity. The present study aims to establish the effect of several pretreatment technologies on cellulose crystallinity, crystalline allomorph distribution, and cellulose ultrastructure. The observed changes in the cellulose ultrastructure of poplar were also related to changes in enzymatic hydrolysis, a measure of biomass recalcitrance. Hot-water, organo-solv, lime, lime-oxidant, dilute acid, and dilute acid-oxidant pretreatments were compared in terms of changes in enzymatic sugar release and then changes in cellulose ultrastructure measured by ¹³C cross polarization magic angle spinning nuclear magnetic resonance and wide-angle X-ray diffraction. Pretreatment severity and relative chemical depolymerization/degradation were assessed through compositional analysis and high-performance anion-exchange chromatography with pulsed amperometric detection. Results showed minimal cellulose ultrastructural changes occurred due to lime and lime-oxidant pretreatments, which at short residence time displayed relatively high enzymatic glucose yield. Hot water pretreatment moderately changed cellulose crystallinity and crystalline allomorph distribution, yet produced the lowest enzymatic glucose yield. Dilute acid and dilute acid-oxidant pretreatments resulted in the largest increase in cellulose crystallinity, para-crystalline, and cellulose-I_β allomorph content as well as the largest increase in cellulose microfibril or crystallite size. Perhaps related, compositional analysis and Klason lignin contents for samples that underwent dilute acid and dilute acid-oxidant pretreatments indicated the most significant polysaccharide depolymerization/degradation also ensued. Organo-solv pretreatment generated the highest glucose yield, which was accompanied by the most significant increase in cellulose microfibril or crystallite size and decrease in relatively lignin contents. Hot-water, dilute acid, dilute acid-oxidant, and organo-solv pretreatments all showed evidence of cellulose microfibril coalescence.