Using low-shear aerated and agitated bioreactor for producing two specific laccases by trametes versicolor cultures induced by 2,5-xylidine: Process development and economic analysis.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.

Xylan, Xylooligosaccharides, and Aromatic Structures With Antioxidant Activity Released by Xylanase Treatment of Alkaline-Sulfite-Pretreated Sugarcane Bagasse.

Xylanase enzymes are useful to fractionate plant biomass, producing xylan, xylooligosaccharides (XOS), and antioxidant-derived XOS. In a biorefinery, pretreated biomass can be digested with xylanase prior to cellulose saccharification, enhancing the product portfolio in the process. With this vision, this study highlighted a wide range of new products attainable from alkaline-sulfite-pretreated sugarcane bagasse by treatments with endo-xylanase under controlled conditions. The developed process provided a crude extract corresponding to 29.7% (w/w) of pretreated sugarcane bagasse. The crude extract included a relatively polymeric glucuronoarabinoxylan fraction, DP2-DP6 xylooligosaccharides, and aromatic compounds. The enzymatically produced extract was fractionated with increasing ethanol concentrations [up to 90% (v/v)], providing precipitation of varied polymeric xylan fractions (48% (w/w) of the crude extract) with average molar masses ranging from 28 kDa to 3.6 kDa. The fraction soluble in 90% ethanol was subjected to adsorption on 4% (w/v) activated charcoal and eluted with an ethanol gradient from 10% to 70% (v/v), thus providing xylooligosaccharides and aromatic fractions. Most of the xylooligosaccharides (74% of the eluted sugars) were washed out in 10%-30% ethanol. DP2 and DP3 structures predominated in the 10% ethanol fraction, while DP5 structures were significantly enriched in the 30% ethanol fraction. Higher ethanol concentrations desorbed xylooligosaccharides associated with higher amounts of aromatic compounds. Total aromatics, phenolic structures, and p-hydroxycinnamates predominated in the fractions desorbed with 60% and 70% ethanol. The antioxidant activity of produced fractions correlated with their phenolic contents. Compiled results indicate that a wide variety of products can be prepared from pretreated biomass using xylanase-aided extraction procedures. Recovered fractions presented different features and specific application prospects. Beyond polymeric xylan with low lignin contamination, xylooligosaccharides or even lignin-carbohydrate complexes with antioxidant activity can be included in the biorefinery portfolio based on the currently developed fractionation studies.

High-solid enzymatic hydrolysis of sugarcane bagasse and ethanol production in repeated batch process using column reactors.

Alkaline sulfite pretreated sugarcane bagasse was enzymatically hydrolyzed in a packed-bed column reactor and a bubble column reactor was evaluated to produce ethanol from the hydrolysate. Initial solid loadings of 9-16% were used in column reactor in the hydrolysis step, and the use of lower value (9%) resulted in 41 g L-1 of glucose in the hydrolysate, corresponding to 87% of cellulose hydrolysis yield. This yield was reduced to 65% for a solid loading of 16%, corresponding to a glucose concentration of 54 g L-1. Subsequently, Saccharomyces cerevisiae and Scheffersomyces stipitis were used for ethanol production in medium based on hydrolysate previously obtained, using different aeration flowrates (0.3, 0.5 and 0.7 vvm). In simple batch fermentation using S. cerevisiae, higher ethanol yield (0.40 g.g-1) and productivity (1.58 g.L-1.h-1) were achieved using 0.5 vvm. When S. stipitis was used in simple batch co-fermentations, the maximum ethanol productivities were obtained using 0.5 and 0.7 vvm (0.64 and 0.63 g.L-1.h-1, respectively). Successive repeated batches resulted in average ethanol concentration of 38 g.L-1 and fermentation efficiency of 82%, when using S. cerevisiae. For S. stipitis, those values were, respectively, 36 g.L-1 and 50%, with volumetric productivity increased along the cycles. Thus, the potential of the bioreactors as simple systems for use in the biological steps of biorefineries was demonstrated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02932-3.

The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials.

The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78-86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.

Co-production of xylo-oligosaccharides, xylose and cellulose nanofibrils from sugarcane bagasse.

This work investigated the integration of a two-stage hydrothermal treatment for production of xylooligosaccharides (XOS), a high-value product, into the isolation process of cellulose nanofibrils (CNF) from sugarcane bagasse. Under optimized conditions, the first stage yielded a XOS-rich, high purity hydrolysate and in the second stage only a xylose-rich hydrolysate could be obtained at high purity. The resulting solid cellulosic fraction was delignified and bleached to obtain a cellulose-rich pulp, which was mechanically defibrillated by disc ultra-refining to CNF. Except for the viscosity, the sugarcane CNF showed properties (i.e., thermal stability, crystallinity and diameter size) comparable or superior to the CNF prepared from commercial bleached eucalyptus Kraft pulp. In conclusion, the integration of the two-stage hydrothermal treatment is an efficient and promising strategy to obtain hemicellulose-derived high-value co-products in the process of isolating CNF. In addition, lignin was also recovered as a co-product with yield comparable to other biomass fractionation approaches.

Enzyme-aided xylan extraction from alkaline-sulfite pretreated sugarcane bagasse and its incorporation onto eucalyptus kraft pulps.

Hemicellulose-rich substrates produced in the lignocellulose biorefinery context can yield macromolecular xylan structures with assorted application in the chemical industry. Xylan presents natural affinity to cellulose and its incorporation onto fibers increases the physical processability of pulp; however, current studies diverge on how molar mass affects xylan interaction with cellulose. In the current work, xylans with varied structural characteristics were prepared from alkaline-sulfite pretreated sugarcane bagasse with aid of an alkaline-active xylanase and selective precipitations using different ethanol concentrations. Prepared xylan fractions, containing low levels of lignin contamination (4-9%) and molar masses ranging from 2.3 kDa to 34 kDa, were incorporated onto eucalyptus pulp fibers up to 4.7 g xylan/100 g pulp. The efficiency of xylan incorporation onto cellulosic fibers was dependent on the xylan structures, where low molar mass and low substitution degree favored high incorporation levels.

Fate of p-hydroxycinnamates and structural characteristics of residual hemicelluloses and lignin during alkaline-sulfite chemithermomechanical pretreatment of sugarcane bagasse.

BACKGROUND: Preparing multiple products from lignocellulosic biomass feedstock enhances the profit and sustainability of future biorefineries. Grasses are suitable feedstocks for biorefineries as they permit a variety of possible by-products due to their particular chemical characteristics and morphology. Elucidating the fate of p-hydroxycinnamates (ferulates-FAs and p-coumarates-pCAs) and major structural components during bioprocessing helps to discriminate the sources of recalcitrance in grasses and paves the way for the recovery of p-hydroxycinnamates, which have multiple applications. To address these subjects, we assessed sugarcane bagasse biorefining under alkaline-sulfite chemithermomechanical (AS-CTM) pretreatment and enzymatic saccharification. RESULTS: The mass balances of the major bagasse components were combined with 2D-NMR structural evaluation of process solids to advance our understanding of sugarcane bagasse changes during biorefining. AS-CTM pretreatment provided a high yield and thoroughly digestible substrates. The pretreated material was depleted in acetyl groups, but retained 62 and 79% of the original lignin and xylan, respectively. Forty percent of the total FAs and pCAs were also retained in pretreated material. After pretreatment and enzymatic hydrolysis, the residual solids contained mostly lignin and ester-linked pCAs, with minor amounts of FAs and non-digested polysaccharides. Saponification of the residual solids, at a higher alkali load, cleaved all the ester linkages in the pCAs; nevertheless, a significant fraction of the pCAs remained attached to the saponified solids, probably to lignin, through 4-O ether-linkages. CONCLUSION: AS-CTM pretreatment provided soundly digestible substrates, which retain substantial amounts of xylans and lignin. Acetyl groups were depleted, but 40% of the total FAs and pCAs remained in pretreated material. Ester-linked pCAs detected in pretreated material also resisted to the enzymatic hydrolysis step. Only a more severe saponification reaction cleaved ester linkages of pCAs from residual solids; nevertheless, pCAs remained attached to the core lignin through 4-O ether-linkages, suggesting the occurrence of an alkali-stable fraction of pCAs in sugarcane bagasse.

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids.

Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and ß-glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70-100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944-951, 2018.

An ascomycota coculture in batch bioreactor is better than polycultures for cellulase production.

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, ß-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, ß-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and ß-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

BACKGROUND: New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. RESULTS: Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. CONCLUSIONS: Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin.

BACKGROUND: The effectiveness of the enzymatic hydrolysis of cellulose in plant cell wall is strongly influenced by the access of enzymes to cellulose, which is at least in part limited by the presence of lignin. Although physicochemical treatments preceding the enzymatic catalysis significantly overcome this recalcitrance, the residual lignin can still play a role in the process. Lignin is suggested to act as a barrier, hindering cellulose and limiting the access of the enzymes. It can also unspecifically bind cellulases, reducing the amount of enzymes available to act on cellulose. However, the limiting role of the lignin present in pretreated sugarcane bagasses has not been fully understood yet. RESULTS: A set of sugarcane bagasses pretreated by five leading pretreatment technologies was created and used to assess their accessibility and the unproductive binding capacity of the resulting lignins. Steam explosion and alkaline sulfite pretreatments resulted in more accessible substrates, with approximately 90% of the cellulose hydrolyzed using high enzyme loadings. Enzymatic hydrolysis of alkaline-treated (NaOH) and steam-exploded sugarcane bagasses were strongly affected by unproductive binding at the lowest enzyme loading tested. Analysis of the extracted lignins confirmed the superior binding capacity of these lignins. Sulfite-based pretreatments (alkaline sulfite and acid sulfite) resulted in lignins with lower binding capacities compared to the analogue pretreatments without sulfite (alkaline and acidic). Strong acid groups present in sulfite-based pretreated substrates, attributed to sulfonated lignins, corroborated the lower binding capacities of the lignin present in these substrates. A more advanced enzyme preparation (Cellic CTec3) was shown to be less affected by unproductive binding at low enzyme loading. CONCLUSIONS: Pretreatments that increase the accessibility and modify the lignin are necessary in order to decrease the protein binding capacity. The search for the called weak lignin-binding enzymes is of major importance if hydrolysis with low enzyme loadings is the goal for economically viable processes.

Tissue-specific distribution of hemicelluloses in six different sugarcane hybrids as related to cell wall recalcitrance.

BACKGROUND: Grasses are lignocellulosic materials useful to supply the billion-tons annual requirement for renewable resources that aim to produce transportation fuels and a variety of chemicals. However, the polysaccharides contained in grass cell walls are built in a recalcitrant composite. Deconstruction of these cell walls is still a challenge for the energy-efficient and economically viable transformation of lignocellulosic materials. The varied tissue-specific distribution of cell wall components adds complexity to the origins of cell wall recalcitrance in grasses. This complexity usually led to empirically developed pretreatment processes to overcome recalcitrance. A further complication is that efficient pretreatment procedures generally treat the less recalcitrant tissues more than necessary, which results in the generation of undesirable biomass degradation products. RESULTS: Six different sugarcane hybrids were used as model grasses to evaluate the tissue-specific distribution of hemicelluloses and the role of these components in cell wall recalcitrance. Acetylated glucuronoarabinoxylan (GAX) occurs in all tissues. Mixed-linkage glucan (MLG) was relevant in the innermost regions of the sugarcane internodes (up to 15.4 % w/w), especially in the low-lignin content hybrids. Immunofluorescence microscopy showed that xylans predominated in vascular bundles, whereas MLG occurred mostly in the parenchyma cell walls from the pith region of the hybrids with low-lignin content. Evaluation of the digestibility of sugarcane polysaccharides by commercial enzymes indicated that the cell wall recalcitrance varied considerably along the internode regions and in the sugarcane hybrids. Pith regions of the hybrids with high MLG and low-lignin contents reached up to 85 % cellulose conversion after 72 h of hydrolysis, without any pretreatment. CONCLUSIONS: The collective characteristics of the internode regions were related to the varied recalcitrance found in the samples. Components such as lignin and GAX were critical for the increased recalcitrance, but low cellulose crystallinity index, high MLG contents, and highly substituted GAX contributed to the generation of a less recalcitrant material.

Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification.

BACKGROUND: Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion. RESULTS: GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and ß-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-ß-glucosidases, and GH10-xylanases. CONCLUSIONS: The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-ß-glucosidases, and GH10-xylanases.

Recovery of Peniophora cinerea laccase using aqueous two-phase systems composed by ethylene oxide/propylene oxide copolymer and potassium phosphate salts.

Aqueous two-phase systems (ATPSs) composed by UCON (ethylene oxide/propylene oxide copolymer) and potassium phosphate salts were for the first time evaluated in the recovery of Peniophora cinerea laccase from complex fermented medium. The ATPSs were obtained by combining the random copolymer UCON with KH2PO4, potassium phosphate buffer pH 7 or K2HPO4. According to the results, protein partition occurred predominantly toward the saline phase (bottom phase) of the ATPSs, while some contaminants such as pigments partitioned mainly to the top phase. In preliminary tests, it was found that the salt with the lowest pH value (KH2PO4, pH 4.6) stimulated the enzyme activity, while the other salts (pH between 7.0 and 9.5) caused a strong inhibition. However, the salt inhibition was not observed in the equilibrium phases of the UCON-Potassium phosphate ATPSs. The laccase recovery was high for all the biphasic systems, but the highest value (134%) was obtained when using UCON combined with KH2PO4. When compared to conventional concentration and purification methods (lyophilization, ammonium sulfate precipitation, ultrafiltration, and ion exchange chromatography), ATPS was demonstrated to be an efficient alternative for P. cinerea laccase recovery from fermented medium.

Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae.

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.

Enzymatic digestion of alkaline-sulfite pretreated sugar cane bagasse and its correlation with the chemical and structural changes occurring during the pretreatment step.

Sugar cane bagasse is recalcitrant to enzymatic digestion, which hinders the efficient conversion of its polysaccharides into fermentable sugars. Alkaline-sulfite pretreatment was used to overcome the sugar cane bagasse recalcitrance. Chemical and structural changes that occurred during the pretreatment were correlated with the efficiency of the enzymatic digestion of the polysaccharides. The first 30 min of pretreatment, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly, and hemicellulose removal was not enhanced; however, acid groups continued to be introduced into the residual lignin. Water retention values were 145% to the untreated bagasse and 210% to the bagasse pretreated for 120 min and fiber widths increased from 10.4 to 30 µm, respectively. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92% with the 120 min pretreated sample.

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study.

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method.

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)2SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6H5O7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.

Enzymatic hydrolysis of chemithermomechanically pretreated sugarcane bagasse and samples with reduced initial lignin content.

Chemithermomechanical (CTM) processing was used to pretreat sugarcane bagasse with the aim of increasing cell wall accessibility to hydrolytic enzymes. Yields of the pretreated samples were in the range of 75-94%. Disk refining and alkaline-CTM and alkaline/sulfite-CTM pretreatments yielded pretreated materials with 21.7, 17.8, and 15.3% of lignin, respectively. Hemicellulose content was also decreased to some extent. Fibers of the pretreated materials presented some external fibrillation, fiber curling, increased swelling, and high water retention capacity. Cellulose conversion of the alkaline-CTM- and alkaline/sulfite-CTM-pretreated samples reached 50 and 85%, respectively, after 96 h of enzymatic hydrolysis. Two samples with low initial lignin content were also evaluated after the mildest alkaline-CTM pretreatment. One sample was a partially delignified mill-processed bagasse. The other was a sugarcane hybrid selected in a breeding program. Samples with lower initial lignin content were hydrolyzed considerably faster in the first 24 h of enzymatic digestion. For example, enzymatic hydrolysis of the sample with the lowest initial lignin content (14.2%) reached 64% cellulose conversion after only 24 h of hydrolysis when compared with the 30% observed for the mill-processed bagasse containing an initial lignin content of 24.4%.

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid.

Experiments based on a 2(3) central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar-alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.