Steam pretreatment of Salix with and without SO2 impregnation for production of bioethanol

In the wood-to-ethanol process, pretreatment of the material is necessary prior to enzymatic hydrolysis to obtain high overall yields of sugar and ethanol. Steam pretreatment of fast-growing Salix either with or without SO₂ impregnation has been investigated by varying different parameters. Overall glucose yields of above 90% and overall xylose yields higher than 80% were obtained both with and without impregnation. However, the most favorable pretreatment conditions for the separate yields of glucose and xylose differed to a lower degree using SO₂-impregnated wood chips, resulting in higher total sugar yield than that obtained with non-impregnated wood chips.     

Extrusion Pretreatment of Pine Wood Chips

Pretreatment is the first step to open up lignocellulose structure in the conversion of biomass to biofuels. Extrusion can be a viable pretreatment method due to its ability to simultaneously expose biomass to a range of disruptive conditions in a continuous flow process. Extruder screw speed, barrel temperature, and feedstock moisture content are important factors that can influence sugar recovery from biomass. Hence, the current study was undertaken to investigate the effects of these parameters on extrusion pretreatment of pine wood chips. Pine wood chip at 25, 35, and 45?% wb moisture content were pretreated at various barrel temperatures (100, 140, and 180?°C) and screw speeds (100, 150, and 200?rpm) using a screw with compression ratios of 3:1. The pretreated pine wood chips were subjected to standard enzymatic hydrolysis followed by sugar and byproducts quantification. Statistical analyses revealed the existence of significant differences in sugar recovery due to independent variables based on comparing the mean of main effects and interaction effects. Pine wood chips pretreated at a screw speed of 150?rpm and a barrel temperature of 180?°C with a moisture content of 25?% resulted in a maximum cellulose, hemicellulose, and total sugar recoveries of 65.8, 65.6, and 66.1?%, respectively, which was about 6.7, 7.9, and 6.8 fold higher than the control (unpretreated pine chips). Furthermore, potential fermentation inhibitors such as furfural, hydroxyl methyl furfural, and acetic acid were not found in any of the treatment combinations.     

Measuring mercury in wood: challenging but important

Mercury (Hg) in tree wood has been overlooked, in part because concentrations are so low as to be below detection limits of some analytical methods, but it is potentially important to forest ecosystem processes and budgets. We tested methods for the preparation and determination of Hg in tree wood by analysing samples of four tree species at the Hubbard Brook Experimental Forest, New Hampshire, USA, using thermal decomposition, catalytic conversion, amalgamation and atomic absorption spectrophotometry (USEPA Method 7473). Samples that were freeze-dried or oven-dried at 65°C were suitable for determination of Hg, whereas oven-drying at 103°C resulted in Hg losses, and air-drying resulted in Hg gains, presumably due to sorption from indoor air. Mean (±SE) concentrations of Hg tree bole wood were 1.75 ± 0.14 ng g^?1 for American beech, 1.48 ± 0.23 ng g^?1 for sugar maple, 3.96 ± 0.19 ng g^?1 for red spruce and 4.59 ± 0.06 ng g^?1 for balsam fir. Based on these concentrations and estimates of wood biomass by species based on stand inventory, we estimated the Hg content of wood in the reference watershed at Hubbard Brook to be 0.32 g ha^?1, twice the size of the foliar Hg pool (0.15 g ha^?1). Mercury in wood deserves more attention and is feasible to measure using appropriate techniques.     

Molecular and Computational Approaches to Characterize Thermostable Laccase Gene from Two Xerophytic Plant Species

Laccases are blue multicopper oxidases that carry out single electron transfers in the oxidation of phenols to quinones. In plants, they confer structural stability to the cell wall. Thermostable laccases were identified in xerophytes Cereus pterogonus and Opuntia vulgaris that could be used in biotechnology and industrial processes. Polyclonal anti-laccase antibodies were generated against purified laccase enzyme isoforms capable of 98–99 % inhibition of the catalytic activity. Antibodies raised against lower molecular weight isoforms inhibited 70 % of the catalytic activity of higher molecular forms. Only 20 % inhibition was noted when assayed in reverse. A partial gene sequence of thermostable xerophytic laccase comprising 712 and 880 bp was identified employing cDNA as template. The nucleotide sequence was submitted to GenBank. The gene sequence was in silico translated into protein sequence and a 3-D structure was predicted using I-Tasser and Genesilico online servers that justified the experimental observations. Anti-laccase antibodies and nucleotide gene sequence of this thermostable plant laccase can be utilized for predicting laccase antigenic sequences and for cloning and expression of the thermostable eukaryotic laccase.     

Thermokinetic Comparison of Trypan Blue Decolorization by Free Laccase and Fungal Biomass

Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k ₁′, value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ～22 kJ mol^?1, while energy for laccase inactivation was 18 kJ mol^?1. Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT?=?40 K) as opposed to fungal biomass (ΔT?=?15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.     

Molecular Cloning,Characterization, and Dye-Decolorizing Ability of a Temperature- and pH-Stable Laccase from Bacillus subtilis X1

Laccases from fungal origin are typically unstable at high temperatures and alkaline conditions. This characteristic limits their practical applications. In this study, a new bacterial strain exhibiting laccase activity was isolated from raw fennel honey samples and identified as Bacillus subtilis X1. The CotA-laccase gene was cloned from strain X1 and efficiently expressed in Escherichia coli in a biologically active form. The purified recombinant laccase demonstrated an extensive pH range for catalyzing substrates and high stability toward alkaline pH and high temperatures. No loss of laccase activity was observed at pH 9.0 after 10 days of incubation, and approximately 21 % of the initial activity was detected after 10 h at 80 °C. Two anthraquinonic dyes (reactive blue 4 and reactive yellow brown) and two azo dyes (reactive red 11 and reactive brilliant orange) could be partially decolorized by purified laccase in the absence of a mediator. The decolorization process was efficiently promoted when methylsyringate was present, with more than 90 % of color removal occurring in 3 h at pH 7.0 or 9.0. These unusual properties indicated a high potential of the novel CotA-laccase for industrial applications.     

Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability

POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t_1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.     

Dilute acid hydrolysis of softwoods

Whole tree chips obtained from softwood forest thinnings were converted to ethanol via a two-stage dilute acid hydrolysis followed by yeast fermentation. The chips were first impregnated with dilute sulfuric acid, then pretreated in a steam explosion reactor to hydrolyze, more than 90% of the hemicellulose and approx 10% of the cellulose. The hydrolysate was filtered and washed with water to recover the sugars. The washed fibers were then subjected to a second acid im pregnation and hydrolysis to hydrolyze as much as 45% of the reamining cellulose. The liquors from both hydrolysates were combined and fermented to ethanol by a Saccharomyces cerevisiae yeast that had been adapted to the inhibitors. Based on available hexose sugars, ethanol yields varied from 74 to 89% of theoretical. Oligosaccharide contents higher than about 10% of the total available sugar appear to have a negative impact on ethanol yield.     

Detoxification of Organosolv-Pretreated Pine Prehydrolysates with Anion Resin and Cysteine for Butanol Fermentation

Bioconversion of lignocellulose to biofuels suffers from the degradation compounds formed during pretreatment and acid hydrolysis. In order to achieve an efficient biomass to biofuel conversion, detoxification is often required before enzymatic hydrolysis and microbial fermentation. Prehydrolysates from ethanol organosolv-pretreated pine wood were used as substrates in butanol fermentation in this study. Six detoxification approaches were studied and compared, including overliming, anion exchange resin, nonionic resin, laccase, activated carbon, and cysteine. It was observed that detoxification by anion exchange resin was the most effective method. The final butanol yield after anion exchange resin treatment was comparable to the control group, but the fermentation was delayed for 72 h. The addition of Ca(OH)₂ was found to alleviate this delay and improve the fermentation efficiency. The combination of Ca(OH)₂ and anion exchange resin resulted in completion of fermentation within 72 h and acetone–butanol–ethanol (ABE) production of 11.11 g/L, corresponding to a yield of 0.21 g/g sugar. The cysteine detoxification also resulted in good detoxification performance, but promoted fermentation towards acid production (8.90 g/L). The effect of salt on ABE fermentation was assessed and the possible role of Ca(OH)₂ was to remove the salts in the prehydrolysates by precipitation.     

Decolorization of Remazol Brilliant Blue R by a Purified Laccase of <Emphasis Type="Italic">Polyporus brumalis</Emphasis>

A white rot basidiomycete Polyporus brumalis has been reported to induce two laccase genes under degradation conditions of dibutylphthalate. When this fungus was grown in a minimal medium, one laccase enzyme was detected by the native polyacrylamide gel electrophoresis. A laccase was purified through ammonium sulfate precipitation and ion exchange chromatography, and the estimated molecular weight was 70 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The K _m value of the enzyme was 685.0 μM, and the V _max was 0.147 ODmin⁻¹ unit⁻¹ for o-tolidine. Purified laccase showed effective decolorization of a dye, Remazol Brilliant Blue R (RBBR), without any laccase mediator. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was directly involved in the decolorization of RBBR.     

Cotton Stalk Pretreatment Using <Emphasis Type="Italic">Daedalea flavida</Emphasis>, <Emphasis Type="Italic">Phlebia radiata</Emphasis>, and <Emphasis Type="Italic">Flavodon flavus</Emphasis>: Lignin Degradation,Cellulose Recovery,and Enzymatic Saccharification

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.     

Hydrothermal saccharification of cotton cellulose in dilute aqueous formic acid solution

Cotton cellulose subjected to a dilute aqueous formic acid solution, at acid concentrations up to 1% (w/w), under hydrothermal conditions in a semi-batch reactor was converted into glucose and oligomers with lower degrees of polymerizations (DP). After heating at 250 °C for 60 min in 0.1% (w/w) aqueous formic acid solution, yields of glucose and total sugar with DP = 1 to 9 were 36.6 and 83.8% (100 × gC/gC of initial cotton sample), respectively, and 5-hydroxymethylfurfural was almost as low as 1%. The yields of glucose and oligomers were significantly improved by adding the acid. The reaction was represented by first-order reaction kinetics with regard to (1 ?C x) where x is the conversion based on the total sugar or glucose yield. At 250 °C, the differences in the rate constants (k ? k _water) were proportional to the square root of formic acid concentration.     

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.     

Flavonoid-Rich Plants Used as Sole Substrate to Induce the Solid-State Fermentation of Laccase

High cost becomes the major obstacle for the industrial application of laccase. Many approaches have been applied to enhance the yield and decrease the cost of laccase. Since flavonoids are the natural inducers for laccase production, in this article, flavonoid-rich plants were taken as the sole substrate for the solid-state fermentation of Funalia trogii (Cui 3676). It indicated that flavonoid-rich plants can effectively promote the production of F. trogii laccase without the addition of inducers. The laccase activity was 42.5 IU g^?1 substrate when kudzu vine root was used as the substrate, which was enhanced by 4.46 times than that when bran was used as the substrate. Meanwhile, the solid-state fermentation of laccase could enrich flavonoids, benefiting their extraction. The content of flavonoids extracted from fermented kudzu vine root and Ginkgo biloba leaves was enhanced by 56.41 and 24.11 %, respectively, compared to the unfermented substrate, and the relative reductive ability and scavenging ability of hydroxyl radicals of flavonoids in the fermented residues were essentially unchanged. Thus, flavonoid-rich plants will become a kind of potential substrate for laccase fermentation which is beneficial in enhancing the yield and reducing the cost of laccase.     

Biochemical and Kinetic Study of Laccase from Ganoderma cupreum AG-1 in Hydrogels

In the present study, three different types of hydrogels i.e., (poly (?acrylamide)/alginate (P (AAm)/Alg), poly (acrylamide-N-isopropylacrylamide) (P (AAm-NIPA)), and poly (acrylamide-N-isopropylacrylamide)/alginate (P (AAm-NIPA)/Alg)) were synthesized by acrylamide, alginate, and N-isopropylacrylamide for the entrapment of laccase. The hydrogel-entrapped and free laccase showed optimum temperature of 50 °C for the oxidation of ABTS, but the entrapped laccase showed high temperature, pH, and storage stability as compared to the free enzyme. The K _m values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 0.13, 0.28, 0.33, and 0.50 mM, respectively. The V _max values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 22.22?×?10², 5.55?×?10², 5.0?×?10², and 4.54?×?10² mM/min, respectively. The entrapped laccase hydrogels were used for the decolorization of Reactive Violet 1 dye, with 39 to 45 % decolorization efficiency till the 10th cycle.     

Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

Enhanced Production of Xylitol from Poplar Wood Hydrolysates Through a Sustainable Process Using Immobilized New Strain <Emphasis Type="Italic">Candida tropicalis</Emphasis> UFMG BX 12-a

A new strain, Candida tropicalis UFMG BX 12-a, was found to produce higher yields of xylitol on poplar wood hemicellulose hydrolysate. The hemicellulose hydrolysate liquor was detoxified using a novel method we developed, involving vacuum evaporation and solvent separation of inhibitors which made the hydrolysate free of toxins while retaining high concentrations of fermentable sugars. The effect of the detoxification method on the fermentation was also reported and compared to well-known methods reported in literature. In this study, the new strain C. tropicalis UFMG BX 12-a was used on the detoxified hydrolysate to produce xylitol. It was also compared to Candida guilliermondii FTI 20037, which has been reported to be one of the best strains for fermentative production of xylitol. To further improve the efficiency of the fermentation process, these strains were immobilized in calcium alginate beads. The yield (0.92 g g^?1) and productivity (0.88 g L^?1 h^?1) obtained by fermenting the wood hydrolysate detoxified by our new detoxification technique using an immobilized new Candida strain were found to be higher than the values reported in literature.     

Defibration mechanisms of autohydrolyzed Eucalyptus wood chips

The objective of this study was to evaluate the influence of autohydrolysis on mechanical defibration of Eucalyptus wood chips. The autohydrolysis process changed notably the mechanical properties of Eucalyptus chips. The removal of mainly hemicelluloses undoubtedly decreased the overall pulp yield. Hemicellulose losses cannot be solely accounted for the changes in the wood and pulp properties, because the autohydrolysis also caused changes in lignin. When comparing the mechanical pulp fibers of the original wood chips with the fibers resulting from the autohydrolyzed wood material, it was clear that the rupture point shifted from the secondary wall to the middle lamella, confirmed by X-ray photoelectron spectroscopy measurements. This study revealed the mechanical behavior of autohydrolyzed wood chips and can provide useful information for integration of mechanical pulp mills into the biorefinery concept in the future.     

Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.