Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments

Attempts were made to enhance cellulose saccharification by cellulase using cellulose dissolution as a pretreatment step. Four cellulose dissolution agents, NaOH/Urea solution, N-methylmorpholine-N-oxide (NMMO), ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and 85% phosphoric acid were employed to dissolve cotton cellulose. In comparison with conventional cellulose pretreatment processes, the dissolution pretreatments were operated under a milder condition with temperature <130 °C and ambient pressure. The dissolved cellulose was easily regenerated in water. The regenerated celluloses exhibited a significant improvement (about 2.7- to 4.6-fold enhancement) on saccharification rate during 1st h reaction. After 72 h, the saccharification yield ranged from 87% to 96% for the regenerated celluloses while only around 23% could be achieved for the untreated cellulose. Even with high crystallinity, cellulose regenerated from phosphoric acid dissolution achieved the highest saccharification rates and yield probably due to its highest specific surface area and lowest degree of polymerization (DP).     

Microwave pretreatment of lignocellulosic material in cholinium ionic liquid for efficient enzymatic saccharification

We demonstrated that the enzymatic hydrolysis of cellulose after microwave pretreatment of lignocellulosic material in ionic liquids (ILs) is drastically enhanced compared with that after conventional thermal pretreatment in ILs. Three types of cholinium ILs, choline formate (ChFor), choline acetate (ChOAc), and choline propionate (ChPro), were examined. The cellulose saccharification percentage was approximately 20% for kenaf powders pretreated in ChFor, ChOAc, and ChPro by conventional heating at 110 °C for 20 min. In contrast, approximately 60–90% of cellulose was hydrolyzed to glucose after microwave pretreatment in the same ILs at 110 °C for 20 min.     

Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf

Chitin in the α and the β forms has been extracted from different marine crustacean from the Arabian Gulf. The contents of the various exoskeletons have been analyzed and the percent of the inorganic salt (including the various elements present), protein and the chitin was determined. Deacetylation of the different chitin produced was conducted by the conventional thermal heating and by microwave heating methods. Microwave heating has reduced enormously the time of heating from 6–10 h to 10–15 min, to yield the same degree of deacetylation and higher molecular weight chitosan. This technique can save massive amount of energy when implemented on a semi-industrial or industrial scale. The chitin and the obtained chitosan were characterized by elemental analysis, XRD, NMR, FTIR and thermogravimetric measurements. XRD analysis showed that chitosan has lower crystallinity than its corresponding chitin; meanwhile its thermal stability is also lower than chitin.     

Organosolv fractionation process with various catalysts for improving bioconversion of triploid poplar

Triploid poplar (Populus tomentosa Carr.) was fractionated with mild organosolv process, and the effects of varying catalysts (formic acid, triethylamine and sodium hydroxide) and solvents (methanol, ethanol, n-propanol and n-butanol) on the substrate's physicochemical characteristics were examined. The data showed that neither the degree of polymerization (DP), nor the relative crystallinity was significantly influenced under the seven runs. In comparison, the addition of NaOH was more efficient on hemicelluloses dissolution, resulting in lower yields and higher glucose content of the cellulosic fractions than formic acid and triethylamine. Correspondingly, the final ethanol concentration was significantly increased from 1.83 g/L to 3.86–5.09 g/L. Besides, a certain amount of xylose (704.4–962.8 mg/L) was released in the hydrolyzates during the simultaneous saccharification and fermentation (SSF), which could be further isolated to improve the multiple utilization of raw material. This research provided some useful data for the application of mild organosolv fractionation on the utilization of whole biomass, especially for the recovery of hemicellulosic components.     

Temporal changes in wood crystalline cellulose during degradation by brown rot fungi

The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2θ = 22.2°. These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure.     

Newly developed medium and strategy for bacterial cellulose production

Bacterial cellulose (BC) has unique properties, such as high crystallinity, a high degree of polymerisation, high tensile strength and high purity, compared with native cellulose. In this study, a previously determined BC production medium was improved in static culture, and the production cost was evaluated and compared with molasses and with other defined media, such as Hestrin–Schramm, Zhou, Yamanaka and Park, using Gluconacetobacter xylinus. In addition to this analysis, because the surface area/volume ratio is an important parameter in static culture, different surface area/volume ratios were analysed in the range of 0.2–1.46. The defined medium (M1A05P5) and culture type contained glucose (10 g/L), yeast extract (10 g/L), peptone (7 g/L), acetic acid (1.5 ml/L), and ethanol (5 ml/L), and the pH was adjusted to 5.0 in static culture. The highest productivity was observed in the M1A05P5 medium that was 5-fold higher than either molasses or Park's medium. Although the molasses medium was proposed as a cost-effective medium, the production price of BC was the lowest in the M1A05P5 medium. Therefore, the newly developed medium and strategy were highly promising candidates for the industrial-scale production of BC.     

Thermoimaging as a tool for studying light-induced heating of leaves: Correlation of heat dissipation with the efficiency of photosystem II photochemistry and non-photochemical quenching

Thermoimaging – a highly sensitive and non-invasive method of temperature measurement – was applied to explore the role of changing photosynthetic efficiency in light-induced heating of tobacco (Nicotiana tabacum cv. Samsun) leaves. In the absence of evaporative cooling through the stomata, which was achieved by covering leaves with Vaseline, illumination with 50–1400 μM photons m^?2 s^?1 intensity of photosynthetically active radiation resulted in ≈1–5 °C leaf temperature increase in about 2 min. The heating effect showed a non-linear correlation with the extent of non-photochemical quenching (NPQ) resulting in higher leaf temperatures at higher NPQ values. When leaves were adapted to excessive irradiance (1300 μM photons m^?2 s^?1 for 6 h), which resulted in reduction of photosynthetic efficiency and amplification of NPQ the light-induced heating effect was enhanced. The experimental results have been explained on the basis of a simple theoretical model characterizing the balance of energy fluxes in leaves in relation to the efficiency of photosystem II photochemistry and non-photochemical quenching. The role of alternative energy dissipation pathways outside of PSII in the phenomenon of light-induced leaf heating is also discussed.     

Study of microwave effects on the lipase-catalyzed hydrolysis

The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris–HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.     

Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study

A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101 °C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, ¹H NMR and ¹³C–¹H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30 min. When double time (60 min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with β-O-4′ ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL.     

Cellulose whiskers extracted from mulberry: A novel biomass production

A kind of cellulose whiskers were extracted from the branch-barks of mulberry (Morus alba L.) by an alkali treatment at 130 °C and subsequently a sulfuric acid hydrolysis. AFM image showed that the diameter of obtained whiskers was ranged from 20 to 40 nm. The chemical compositions analysis, FT-IR, XRD results indicated that the hemicellulose and lignin were removed extensively in the cellulose whiskers, with a crystallinity of 73.4%. The TGA curves implied a two-stage thermal decomposition behavior of cellulose whisker due to the introduction of sulfated groups into the crystals in the sulfuric acid hydrolysis process. The obtained whiskers may have the potential applications in the fields of composites as a reinforcing phase, as well as in pharmaceutical and optical industries as additives.     

Effect of microwave irradiation on the structure of glucoamylase

In the present study, we describe changes in the primary and secondary structural patterns of glucoamylase during starch hydrolysis under microwave irradiation using SDS-PAGE and circular dichroism (CD) spectroscopy. Our SDS-PAGE results show that the primary structure of glucoamylase did not change after microwave irradiation. According to the CD spectra, the positive peak height (λ = 193 nm) of the microwave-irradiated samples decreased by 36.4–68.2% compared to those without irradiation, whereas the double negative peak height (λ = 206 nm, λ = 220 nm) increased by 10.8–31.4%. In addition, the positive peak (λ = 193 nm) shifted by 0.2–3 nm. After treatment of glucoamylase with microwave irradiation, the α-helical content of glucoamylase decreased sharply, whereas the β-sheet, β-turn and random coil content increased gradually. The conformational changes of glucoamylase after microwave irradiation provide theoretical support for the mechanism whereby microwave irradiation accelerates starch hydrolysis catalyzed by glucoamylase.     

Inhibition of cellulases by phenols

Enzyme hydrolysis of pretreated cellulosic materials slows as the concentration of solid biomass material increases, even though the ratio of enzyme to cellulose is kept constant. This form of inhibition is distinct from substrate and product inhibition, and has been noted for lignocellulosic materials including wood, corn stover, switch grass, and corn wet cake at solids concentrations greater than 10 g/L. Identification of enzyme inhibitors and moderation of their effects is of considerable practical importance since favorable ethanol production economics require that at least 200 g/L of cellulosic substrates be used to enable monosaccharide concentrations of 100 g/L, which result in ethanol titers of 50 g/L. Below about 45 g/L ethanol, distillation becomes energy inefficient. This work confirms that the phenols: vanillin, syringaldehyde, trans-cinnamic acid, and hydroxybenzoic acid, inhibit cellulose hydrolysis in wet cake by endo- and exo-cellulases, and cellobiose hydrolysis by β-glucosidase. A ratio of 4 mg of vanillin to 1 mg protein (0.5 FPU) reduces the rate of cellulose hydrolysis by 50%. β-Glucosidases from Trichoderma reesei and Aspergillus niger are less susceptible to inhibition and require about 10× and 100× higher concentrations of phenols for the same levels of inhibition. Phenols introduced with pretreated cellulose must be removed to maximize enzyme activity.     

Microwave-assisted drying of paclitaxel for removal of residual solvents

In this study, the residual solvent in final purified paclitaxel was effectively removed using microwave-assisted drying. When the sample final purified by silica-HPLC was concentrated using a rotary evaporator, the residual methanol easily met the ICH-specified value (3000 ppm), but methylene chloride did not meet the ICH-specified value (600 ppm). Thus, the efficiency of microwave-assisted drying according to microwave power (100, 200, and 300 W) and drying time was investigated using the sample (methylene chloride conc.: 26,000 ppm, methanol conc.: 50 ppm) concentrated by rotary evaporation. A higher microwave power was effective in removing methylene chloride, and the ICH requirements were met by drying at 300 W for 21 h. In addition, when the sample concentrated by rotary evaporation was vacuum dried (35 °C, 24 h), the concentration of methylene chloride could be reduced to 8500 ppm. When the vacuum-dried sample was subjected to microwave-assisted drying, the ICH requirements could be met by drying for 10 h at 200 W and 8 h at 300 W. The lower the initial concentration of the solvent and the higher the microwave power, the greater the improvement in the efficiency of microwave-assisted drying.     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Effects of intermittent watering on water balance and feed intake in male Ethiopian Somali goats

The objective of this study was to investigate the regulation of water balance and dry matter (DM) intake of male Ethiopian Somali goats. Twenty-eight goats were grouped into four treatments; goats watered every day (W1), every 2nd day (W2), every 3rd day (W3) and every 4th day (W4) for a total period of 72 days. Plasma sodium and potassium were analyzed by flame photometry, plasma osmolality by freezing point depression, plasma protein concentration by TS refractometry, and the plasma vasopressin concentration by radioimmunoassay. Goats were fed grass hay ad libitum and were given 200 g of concentrates daily. The calculated daily water intake (ml/kg BW^0.75) of W1, W2, W3 and W4 was 128 ± 4, 86 ± 4, 88 ± 4 and 78 ± 4, respectively, (P < 0.001, W1 versus the other groups). Calculated total concentrates intake did not change, whereas Group W2 and W4 consumed less hay on days of water deprivation (W1 versus W2, P < 0.01; W1 versus W4, P < 0.001). Plasma sodium concentration of W1 was 143 ± 1 mmol/l. Intermittent watering increased plasma sodium concentration from 140 or 141 ± 1 mmol/l to 145 or 146 ± 1 mmol/l every 2nd day in group W2. In W3, it increased from 139 ± 1 to 146 ± 1 mmol/l and in W4 from 137 ± 1 to 149 ± 1 mmol/l (P < 0.001 for all) on days after watering compared to last day of water deprivation, respectively. Corresponding changes were observed in plasma osmolality. Total plasma protein concentration increased only in W3 from 65 ± 1 to 67 ± 1 mmol/l on day 3 (P < 0.01) and in W4 from 65 ± 1 to 67 and 68 ± 1 mmol/l on days 3 and 4, respectively (P < 0.05). The plasma vasopressin concentration of W1 was close to 0.80 ± 0.28 pmol/l on all days, but it increased on dehydration days in W2 (P < 0.05). In W3, the vasopressin concentration increased from 0.95 or 1.19 pmol/l the day after watering to 3.11 ± 0.28 pmol/l on day 3 (P < 0.01). In W4, it was 0.82 ± 0.31 pmol/l the day after watering and reached 6.06 ± 0.31 pmol/l on day 4 (P < 0.001). We conclude that male Ethiopian Somali goats can be watered once every 3rd days, and that longer interval is not recommended because it demands marked mobilization of water saving mechanisms and causes decreased DM intake.     

Oxidation of regenerated cellulose with NaClO2 catalyzed by TEMPO and NaClO under acid-neutral conditions

A new TEMPO-mediated oxidation with catalytic amounts of TEMPO and NaClO, and NaClO₂ as the primary oxidant under aqueous conditions at pH 3.5–6.8 was used to prepare water-soluble β-(1 → 4)-linked polyglucuronic acid Na salts (cellouronic acids, CUAs) with high molecular weight in good yield. When regenerated cellulose with original degree of polymerization (DP) of 680 was oxidized by the 4-acetamide-TEMPO/NaClO/NaClO₂ system at pH 5.8 and 40 °C for 3 days, CUA with weight average DP (DPw) of 490 was obtained quantitatively. No peaks other than six signals from β-(1 → 4)-linked anhydroglucuronic acid units of CUA were detected in the solution-state ¹³C NMR spectra of the oxidized products. Although the oxidized product prepared under the above conditions contained about 20% unoxidized cellulose particles, the non-CUA fraction was separable from CUAs by filtration or salt precipitation. The DPw values and yields of CUAs after the filtration or salt precipitation treatment were 250–380 and 45–71%, respectively.     

Warneckea parvifolia (Melastomataceae–Olisbeoideae), a new “sand-forest” endemic from northeastern KwaZulu-Natal (South Africa) and southernmost Mozambique,and a phylogenetic analysis of eastern and southern African representatives of W. section Warneckea

Warneckea populations from “sand-forest” or “sand-thicket” habitats in Tembe Elephant Park, South Africa, and Licuati Forest Reserve in adjacent southern Mozambique were previously thought to be a small-leaved form of W. sousae, which typically includes larger-leaved plants ranging from central Mozambique northward to Tanzania. We examine this hypothesis using molecular and morphological evidence. Maximum-likelihood phylogenetic analysis of combined nrDNA ETS and ITS sequence data failed to resolve W. sousae and the Maputaland populations as an exclusively monophyletic group. Instead, the Kenyan endemic W. mouririifolia was strongly supported as the sister species of W. sousae, and the Maputaland plants were resolved in a separate, strongly supported clade together with populations of an as-yet undetermined Warneckea species from northern Mozambique. A hypothesis of exclusive monophyly for the plants from Tembe and Licuati had moderate support in separate ETS and ITS1 analyses (bootstrap proportions of 88% and 81%, respectively). Statistically significant differences in leaf dimensions and internode length were found between the Maputaland plants and typical W. sousae. We conclude that the populations from Tembe and Licuati represent a distinct species, which we describe as W. parvifolia. The species differs from W. sousae in having shorter internodes (mostly 5–25 mm not 10–60 mm long), smaller leaves (mostly 14–32 × 8–19 mm not 40–76 × 22–52 mm), shorter petioles (mostly 1–1.5 mm not 1.5–6 mm long), smaller flowers (hypanthium 1 × 1.5–1.75 mm not 1.5–2 × 2 mm; calyx lobes 0.5 mm not 0.75 mm long; staminal filaments 3–4 mm not 5 mm long; style 4–5 mm not 9 mm long), and globose fruit (not obovoid). An IUCN conservation status of Endangered (EN) B1a, b(ii, iii) is indicated for W. parvifolia, due to its limited distribution and projected declines in its habitat quality and area of occupancy.     

Antifungal potentiality of mycogenic silver nanoparticles capped with chitosan produced by endophytic Amesia atrobrunnea

This research reports the fabrication of silver nanoparticles (AgNPs) from endophytic fungus, Amesia atrobrunnea isolated from Ziziphus spina-christi (L.). Influencing factors for instance, thermal degree of incubation, media, pH, and silver nitrate (AgNO₃) molarity were optimized. Then, the AgNPs were encapsulated with chitosan (Ch-AgNPs) under microwave heating at 650 W for 90 s. Characterization of nanoparticles was performed via UV–visible (UV–vis) spectrophotometer, Fourier-transform infrared spectrophotometer (FTIR), zeta potential using dynamic-light scattering (DLS), and field-emission-scanning electron microscope (FE-SEM). Anti-fungal activity of Ch-AgNPs at (50, 25, 12.5, 6.25 mg/L) was tested against Fusarium oxysporum, Curvularia lunata, and Aspergillus niger using the mycelial growth inhibition method (MGI). Results indicated that Czapek-dox broth (CDB) with 1 mM AgNO₃, an acidic pH, and a temperature of 25–30 °C were the optimum for AgNPs synthesis. (UV–vis) showed the highest peak at 435 nm, whereas Ch-AgNPs showed one peak for AgNPs at 405 nm and another peak for chitosan at 230 nm. FTIR analysis confirmed that the capping agent chitosan was successfully incorporated and interacted with the AgNPs through amide functionalities. Z-potential was −19.7 mV for AgNPs and 38.9 mV for Ch-AgNPs, which confirmed the significant stability enhancement after capping. FES-SEM showed spherical AgNPs and a reduction in the nanoparticle size to 44.65 nm after capping with chitosan. The highest mycelial growth reduction using fabricated Ch-AgNPs was 93% for C. lunata followed by 77% for A. niger and 66% F. oxysporum at (50 mg/L). Biosynthesis of AgNPs using A. atrobrunnea cell-free extract was successful. Capping with chitosan exhibited antifungal activity against fungal pathogens.     

Timing of muscle activation of the lower limbs can be modulated to maintain a constant pedaling cadence

This study investigated changes in muscle activity when subjects are asked to maintain a constant cadence during an unloaded condition. Eleven subjects pedaled for five loaded conditions (220 W, 190 W, 160 W, 130 W, 100 W) and one unloaded condition at 80 rpm. Electromyographic (EMG) activity of six lower limb muscles, pedal forces and oxygen consumption were calculated for every condition. Muscle activity was defined by timing (EMG onset and offset) and level (integrated values of EMGrms calculated between EMG onset and EMG offset) of activation, while horizontal and vertical impulses were computed to characterize pedal forces. Muscle activity, pedal forces and oxygen consumption variables measured during the unloaded condition were compared with those extrapolated to 0 W from the loaded conditions, assuming a linear relationship. The muscle activity was changed during unloaded condition: EMG onset and/or offset of rectus femoris, biceps femoris, vastus medialis, and gluteus maximus muscles were delayed (p < 0.05); iEMGrms values of rectus femoris, biceps femoris, gastrocnemius medialis and tibialis anterior muscles were higher than those extrapolated to 0 W (p < 0.05). Vertical impulse over the extension phase was lower (p < 0.05) while backward horizontal impulse was higher (p < 0.05) during unloaded condition than those extrapolated to 0 W. Oxygen consumptions were higher during unloaded condition than extrapolated to 0W (750 ± 147 vs. 529 ± 297 mLO₂.min^?1; p < 0.05). Timing of activation of rectus femoris and biceps femoris was dramatically modified to optimize pedal forces and maintain a constant cadence, while systematic changes in the activation level of the bi-articular muscles induced a relative increase in metabolic expenditure when pedaling during an unloaded condition.     

Green synthesis of a novel biodegradable copolymer base on cellulose and poly(p-dioxanone) in ionic liquid

A new cellulose graft copolymer was synthesized in 1-N-butyl-3-methylimidazolium chloride ([Bmim]Cl) by the ring opening graft polymerization (ROGP) of p-dioxanone (PDO) onto cellulose. The structure of the copolymer was characterized by ¹³C and ¹H NMR, WAXD, DSC as well as SEM. Cellulose graft copolymers with a molar substitution (MS) in the range of 2.08–4.60 were obtained with 24 h at 80 °C in a completely homogeneous procedure. The obtained copolymers exhibited the clear glass transition temperatures (T_g) indicating the inter-molecular and intra-molecular hydrogen bonds in cellulose molecules had been destroyed. The reaction media applied can be easily recycled and reused.