Preparation of levoglucosenone through sulfuric acid promoted pyrolysis of bagasse at low temperature

Fast pyrolysis of bagasse pretreated by sulfuric acid was conducted in a fixed bed reactor to prepare levoglucosenone (LGO), a very important anhydrosugar for organic synthesis. The liquid yield and LGO yield were studied at temperatures from 240 to 350 °C and sulfuric acid loadings from 0.92 to 7.10 wt.%. An optimal LGO yield of 7.58 wt.% was obtained at 270 °C with a sulfuric acid pretreatment concentration of 0.05 M (corresponding to 4.28 wt.% sulfuric acid loading). For comparison, microcrystalline cellulose pretreated by 0.05 M sulfuric acid solution was pyrolyzed at temperature from 270 °C to 320 °C, and bagasse loaded with 3-5 wt.% phosphoric acid was pyrolyzed at temperature from 270 °C to 350 °C. The highest yield of LGO from bagasse was 30% higher than that from microcrystalline cellulose, and treatment with sulfuric acid allowed a 21% higher yield than treatment with phosphoric acid.     

Combination of steam explosion and laccase-mediator treatments prior to Eucalyptus globulus kraft pulping

The effect of a pretreatment consisting of steam explosion (SE) followed by a laccase mediator system (LMS) stage on Eucalyptus globulus kraft pulping has been evaluated and compared with fungal pretreatments. Pretreatment with SE and LMS was more efficient than pretreatments using Pycnoporus sanguineus and Trametes sp. I-62. Steam explosion not only improved the enzyme penetration into the wood chips and shortened the pulping process by 60%, but also extracted around 50% of the hemicelluloses which could be converted into value-added products. The optimal conditions for the LMS treatment were 3 h, 3 UA/g and 40 °C. Compared to SE, the SE/LMS treatment yielded an increase in delignification of 13.9% without affecting pulp properties, provided a similar screened kraft yield, and reduced consumption of chemical reagents Na₂S and NaOH by 11.5% and 6.3%, respectively. Therefore, SE/LMS is a promising pretreatment for converting the pulp mill into a forest bio-refinery.     

Pyrolysis of grape bagasse: effect of pyrolysis conditions on the product yields and characterization of the liquid product

In this study, pyrolysis of grape bagasse was investigated with the aim to study the product distribution and their chemical compositions and to identify optimum process conditions for maximizing the bio-oil yield. Particular investigated process variables were temperature (350-600 °C), heating rate (10-50 °C/min) and nitrogen gas flow rate (50-200 cm³/min). The maximum oil yield of 27.60% was obtained at the final pyrolysis temperature of 550 °C, sweeping gas flow rate of 100 cm³/min and heating rate of 50 °C/min in a fixed-bed reactor. The elemental analysis and heating value of the bio-oils were determined, and then the chemical composition of the bio-oil was investigated using chromatographic and spectroscopic techniques such as column chromatography, ¹H NMR and FTIR. The fuel properties of the bio-oil such as flash point, viscosity and density were also determined. The bio-oils obtained from grape bagasse were presented as an environmentally friendly feedstock candidate for bio-fuels.     

Physicochemical properties of extrusion-modified konjac glucomannan

Konjac glucomannan was extruded and subsequently ground under four conditions denoted: KGM1 (33% solids, 90 °C), KGM2 (24% solids, 90 °C), KGM3 (24% solids, 90 °C, die restriction), and KGM4 (24% solids, 110 °C). SEM and particle size analysis showed that extruded KGM had slightly larger and rougher particles. The water absorption index was decreased to 47.92-128.80, as compared to 153.64 for the control (KGMC). The crystallinity index increased to 2.97 and 3.42 for KGM3 and KGM4 samples, as compared to 1.72 for the control. Zero-shear viscosity of 0.5% solutions decreased to 0.36-3.01 Pa s. All samples were shear-thinning and data were best fitted by the Cross model. Based on capillary viscometry, molecular weights were decreased to 2.7 × 10⁵-9.9 × 10⁵, as compared to 1.2 × 10⁶ for the control. In most cases, properties were most altered by higher temperature and shear, and to a lesser extent by lower solids in the feed.     

Identification of Paenibacillus sp. 2S-6 and application of its xylanase on biobleaching

A Paenibacillus sp. strain 2S-6 was isolated from the black liquor of the first brownstock washing stage of kraft pulping process and identified by its 16S rDNA sequence. This bacterial strain utilized a variety of saccharides and polysaccharides as carbon source, but neither lignin nor lipids. Crude xylanase from Paenibacillus sp. 2S-6 was produced in a 5 L laboratory fermenter at 37 °C, pH 7. After 24 h, up to 10.5 IU xylanase per mg of protein in the crude extract of fermentation broth was obtained. After two-stage ultrafiltration, the optimal activity of partially purified xylanase reached 60.51 IU/mg at 50 °C, pH 6. A major band indicating molecular weight of 33 kDa was shown on SDS-PAGE for the partially purified xylanase. After 4 h at 60 °C, 48.99% and 31.25% residual xylanase activities were demonstrated at pH 7 and 9, respectively. Efficacy of its xylanase on the bleaching agent saving was demonstrated by using 5 IU xylanase per gram oven-dried pulp prior to bleaching, referred as biobleaching. Identical levels of brightness and higher levels of viscosity were obtained for the xylanase pretreated eucalypt kraft pulps followed by a 20% reduction of the bleaching agent dosage in the first step of a commercial C₇₀/D₃₀-E_o-D bleaching sequence.     

The effects of fungi pre-treatment of poplar chips on the kraft fiber properties

Poplar chips were pre-treated by Trametes versicolor for 1, 2 and 3 weeks. Pre-treated chips, after washing, have been air dried for kraft pulping to achieve pulp kappa number of about 20. Pulp samples have been analyzed by Bauer Mc Nett, Kajaani analyzer and SEM. The results indicated that fungi pre-treatment of chips can degrade lignin and carbohydrates and affect kraft pulping and fiber characteristics. Higher chemical charge in pulping, lower fine and higher long fiber fraction were observed in pre-treated pulp samples in comparison with others. Fiber length, cross sectional area, width, cell wall thickness and volume index were increased by increasing pre-treatment time while fine length, fiber coarseness and curl have been reduced. Based on the study findings, with respect to higher fiber length, lower fine, and lower fiber curl and coarseness, 2-weeks pre-treatment of chips was recommended to produce acceptable overall fiber properties in kraft pulping.     

Extraction and chemical characterization of starch from S. lycocarpum fruits

In this study the pulp from Solanum lycocarpum fruits was used as raw material for extraction of starch, resulting in a yield of 51%. The starch granules were heterogeneous in size, presenting a conical appearance, very similar to a high-amylose cassava starch. The elemental analysis (CHNS) revealed 64.33% carbon, 7.16% hydrogen and 0.80% nitrogen. FT-IR spectroscopy showed characteristic peaks of polysaccharides and NMR analysis confirmed the presence of the α-anomer of d-glucose. The S. lycocarpum starch was characterized by high value of intrinsic viscosity (3515 mPa s) and estimated molecular weight around 645.69 kDa. Furthermore, this starch was classified as a B-type and high amylose content starch, presenting 34.66% of amylose and 38% crystallinity. Endothermic transition temperatures (T_o = 61.25 °C, T_p = 64.5 °C, T_c = 67.5 °C), gelatinization temperature (ΔT = 6.3 °C) ranges and enthalpy changes (ΔH = 13.21 J g⁻¹) were accessed by DCS analysis. These results make the S. lycocarpum fruit a very promising source of starch for biotechnological applications.     

Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170

Microaeration (injecting air into the headspace) improved the fermentation of hemicellulose hydrolysates obtained from the phosphoric acid pretreatment of sugarcane bagasse at 170 °C for 10 min. In addition, with 10% slurries of phosphoric acid pretreated bagasse (180 °C, 10 min), air injection into the headspace promoted xylose utilization and increased ethanol yields from 0.16 to 0.20 g ethanol/g bagasse dry weight using a liquefaction plus simultaneous saccharification and co-fermentation process (L+SScF). This process was scaled up to 80 L using slurries of acid pretreated bagasse (96 h incubation; 0.6 L of air/min into the headspace) with ethanol yields of 312-347 L (82-92 gal) per tonne (dry matter), corresponding to 0.25 and 0.27 g/g bagasse (dry weight). Injection of small amounts of air into the headspace may provide a convenient alternative to subsurface sparging that avoids problems of foaming, sparger hygiene, flotation of particulates, and phase separation.     

Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L + SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190 °C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180 °C).     

Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant ��-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10 min at 90 °C and pH 8. The enzyme was stable over a broad range of pH (pH 6-10) and showed good thermal stability when incubated at 70 °C. Chemicals like EDTA, Hg²⁺, Cu²⁺ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K_M and V_max values of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (>70 mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.     

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.     

Biokraft pulping of European black pine with Ceriporiopsis subvermispora

European black pine (Pinus nigra Arn.) chips were treated with the white-rot fungus Ceriporiopsis subvermispora for periods ranging from 20 to 100 days. The effects of pretreatment on the chemical composition of wood and kraft pulping were investigated. The results showed that fungal pretreatment reduced the lignin and extractive content of wood chips. Also, weight losses occurred. Kappa number, viscosity, and reject ratio of biokraft pulps decreased. Biokraft pulps gave better response to beating, which led to significant energy saving during refining. The tear index, burst index, and tensile index of biokraft pulps were found to be lower than those of kraft pulps. However, the tensile index and burst index of 20-day biotreated and unbeaten pulp was higher than those of kraft pulp. Also, the tear index of 20-day biotreated and beaten pulp was higher than that of kraft pulp. The brightness of biokraft pulps decreased irregularly with increasing incubation time.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

Optimization of pulping conditions of abaca. An alternative raw material for producing cellulose pulp

The influence of temperature (150-170 degrees C), pulping time (15-45 min) and soda concentration (5-10%) in the pulping of abaca on the yield, kappa, viscosity, breaking length, stretch and tear index of pulp and paper sheets, was studied. Using a factorial design to identify the optimum operating conditions, equations relating the dependent variables to the operational variables of the pulping process were derived that reproduced the former with errors lower than 25%. Using a high temperature, and a medium time and soda concentration, led to pulp that was difficult to bleach (kappa 28.34) but provided acceptable strength-related properties (breaking length 4728 m; stretch 4.76%; tear index 18.25 mN m2/g), with good yield (77.33%) and potential savings on capital equipment costs. Obtaining pulp amenable to bleaching would entail using more drastic conditions than those employed in this work.     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

Bio-refinery system of DME or CH4 production from black liquor gasification in pulp mills

There is great interest in developing black liquor gasification technology over recent years for efficient recovery of bio-based residues in chemical pulp mills. Two potential technologies of producing dimethyl ether (DME) and methane (CH₄) as alternative fuels from black liquor gasification integrated with the pulp mill have been studied and compared in this paper. System performance is evaluated based on: (i) comparison with the reference pulp mill, (ii) fuel to product efficiency (FTPE) and (iii) biofuel production potential (BPP). The comparison with the reference mill shows that black liquor to biofuel route will add a highly significant new revenue stream to the pulp industry. The results indicate a large potential of DME and CH₄ production globally in terms of black liquor availability. BPP and FTPE of CH₄ production is higher than DME due to more optimized integration with the pulping process and elimination of evaporation unit in the pulp mill.     

A natural shear-thickening water-soluble polymer from the fronds of the black tree fern, Cyathea medullaris: Influence of salt, pH and temperature

A water-soluble polymer was extracted from the fronds of the black tree fern (Cyathea medullaris or “Mamaku” in Māori) and characterized under various physico-chemical conditions. The rheological properties (in the semi-dilute region) and particle size and charge (in the dilute region) were investigated under various salt (0-1 M NaCl), pH (1-12) and temperature conditions (5-80 °C), using rheometry and dynamic light scattering techniques. A 7% (w/w) Mamaku crude extract showed strong shear-thickening properties at high salinity levels (1 M NaCl) and over the whole range of pH (1-12). However, the thickening properties disappeared above 50 °C. Apparent viscosity remained constant over the pH range 3-9, although the particle size systematically decreased with increasing pH. Overall, Mamaku solutions showed very good salt and pH resistance and exhibited strong temperature dependency. Hydrophobic and electrostatic interactions are not likely to be the cause of the shear-thickening phenomenon observed. Other forces such as hydrogen bonding may play a dominant role on the formation of shear-induced associations.     

Effect of water temperature on dietary protein requirement,growth and body composition of Asian catfish, Clarias batrachus fry

This study aimed to evaluate the protein requirement of Clarias batrachus fry, were estimated at two different water temperatures, 28 and 32 °C. The influence of dietary protein level and water temperature on body composition, weight gain, food and nutrient utilization were estimated. The Asian catfish, C. batrachus fry were fed four diets containing 28% (diet 1), 32% (diet 2), 36% (diet 3) and 40% (diet 4) protein levels and reared at two water temperatures 28 and 32 °C for 60 days. Fry fed with diet 3 containing 36% protein showed the highest mean final body weight at 32 °C. Final body weight was significantly (P<0.05) affected by dietary treatments and temperatures. Clarias batrachus fry raised at 28 °C had higher feed efficiency (93.20%) than the fry reared at 32 °C (87.58%) with 28% dietary protein level. Further, feed efficiency decreased with increase in dietary protein level. Higher daily protein retention (0.089%) observed at lower (0.0217 g) daily protein intake at 28 °C than 0.0283 g at 32 °C. While, optimal (0.0282 g) daily protein intake showed higher daily weight gain at 32 °C. Productive protein value (% PPV) was maximum (1.76%) at 32 °C than at 28 °C (0.76%). Final body lipid recorded higher value than initial body lipid at both the temperatures. Hepatosomatic index (HSI) observed to have been influenced (P<0.05) by diets and temperatures, while viscerosomatic index (VSI) affected (P<0.05) by only diets and not (P>0.05) by temperatures. The study concluded that the diet 3 containing 36% protein was optimal for growth of C. batrachus fry at both the temperatures.     

A new pulping process for wheat straw to reduce problems with the discharge of black liquor

Aqueous ammonia mixed with caustic potash as wheat straw pulping liquor was investigated. The caustic potash did not only reduce the NH3 usage and cooking time, but also provided a potassium source as a fertilizer in the black liquor. Excess NH3 in the black liquor was recovered and reused by batch distillation with a 98% recovery rate of free NH3. The black liquor was further treated for reuse by coagulation under alkaline conditions. The effects of different flocculation conditions, such as the dosage of 10% aluminium polychloride, the dosage of 0.1% polyacrylamide, the reaction temperature and the pH of the black liquor on the flocculating process were studied. The supernatant was recycled as cooking liquor by adding extra NH4OH and KOH. The amount of delignification and the pulp yield for the process remained steady at 82-85% and 48-50%, respectively, when reusing the supernatant four times. The coagulated residues could be further processed as solid fertilizers. This study provided a new pulping process for wheat straw to reduce problems of discharge black liquor.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.