An alkalophilic laccase from Rheinheimera species isolate: Production and biobleaching of kraft pulp

Medium optimization was carried out to enhance laccase production from a novel Rheinheimera species, isolated from industrial effluent. Out of the 15 variables tested by Placket–Burman design (PBD)—yeast extract, soyabean meal, and peptone were the positively significant ones, enhancing laccase production. Both simple and complex sugars showed a negative effect on laccase production. Central composite design (CCD) of experiments, using the three positively significant variables in combinations, showed that laccase production was not affected by molar carbon, molar nitrogen levels or molar C/N ratio. Maximum laccase yield of 2.5 × 10⁵ nkat L^?1, 31 fold enhancement over the unoptimized medium, was achieved when soyabean meal (0.6%) was used alone as medium showing that laccase production was substrate dependent. Laccase was used, in the presence of 2 mM ABTS, for the biobleaching of eucalyptus kraft pulp resulting in kappa number reduction by 20% and brightness increase by 2.9%. Biobleaching improved further by sequential application of an alkalophilic xylanase (X) and laccase‐ABTS system (LAS) that decreased kappa number by 10, 15, and 35%, increased brightness by 2.7, 3.2, and 5.9% as compared to X treated, LAS treated and untreated control, respectively. XLAS treatment resulted in 15, 13, 10.9% increase in burst factor, tear factor, and viscosity with a 20% reduced consumption of elemental chlorine and hypochlorite. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Variability in the composition of tissue-specific galactan from flax fibers

Tissue-specific galactan of sclerenchyma fibers, with cell walls of the gelatinous type, was examined in flax plants (Linum usitatissimum L.) of 23 various genotypes. The content and average degree of polymerization of side chains of galactan were estimated before its deposition into the cell wall. The variability of the analyzed parameters of tissue-specific galactan from flax fibers was high; within the same genotype, the scope of paratypic variability between replicates and years of research was comparable to variability between different genotypes. The average length of side chains in the studied samples ranged from 5 to 41 galactose residues. The average degrees of polymerization of galactan side chains in flax fibers was found to be discrete, which could be explained by block assemblage of the polymer in the Golgi apparatus.     

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

Phenolic constituents in flax bast tissue and inhibition of cellulase and pectinase

Flax bast tissue was sequentially extracted using hexane, propanol, methanol and water as solvents and extracts were analyzed using reverse phase HPLC and ¹³C NMR. Results indicated a large variety of aromatic constituents including flavonoids and hydroxy-methoxy cinnamic acids linked to oligosaccharides and hydroxy acids through glycosidic linkages. The extracts inhibited cellulase and pectinase activities and can thus influence retting.     

Use of thermogravimetric analysis to monitor the effects of natural laccase mediators on flax pulp

The effects of 1-hydroxybenzotriazole (HBT) and the natural laccase mediators gallic acid, caffeic acid and p-hydroxybenzoic acid, on the enzymatic bleaching of flax pulp were compared. The treatment was performed under atmospheric air and oxygen pressure, and, for the first time, monitored by thermogravimetric analysis (TGA) for comparison with chemical analysis, FTIR and X-ray diffraction (XRD) spectroscopies. Thermogravimetric data were simulated by applying a nucleation kinetic equation to a combustion model based on four pseudo-components (hemicellulose, amorphous and crystalline cellulose, and lignin). The results thus obtained show that the effects of the natural mediators are similar to those of HBT and lead to increased order in cellulose microfibril surfaces. An increase in pulp crystallinity was additionally exposed by the XRD and FTIR techniques, and a reduction in microfibril equatorial size by the XRD patterns. Simulated DTG curves were used to determine the kinetic parameters for thermal degradation.     

Use of laccase in pulp and paper industry

Laccase, through its versatile mode of action, has the potential to revolutionize the pulping and paper making industry. It not only plays a role in the delignification and brightening of the pulp but has also been described for the removal of the lipophilic extractives responsible for pitch deposition from both wood and nonwood paper pulps. Laccases are capable of improving physical, chemical, as well as mechanical properties of pulp either by forming reactive radicals with lignin or by functionalizing lignocellulosic fibers. Laccases can also target the colored and toxic compounds released as effluents from various industries and render them nontoxic through its polymerization and depolymerization reactions. This article reviews the use of both fungal and bacterial laccases in improving pulp properties and bioremediation of pulp and paper mill effluents.     

Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

Climatic effects in Central Europe on the frequency of medical treatments of dairy cows

In the present study, the relationship between the temperature–humidity index (THI) and the incidence of medical treatments in lactating dairy cows in Lower Saxony, Germany, was investigated. Records of all veterinary-treated cases over 2 years (2003 and 2005) from eight Holstein–Friesian dairy herds raised in loose-housing systems (55 to 170 cows per herd) were evaluated. After exclusion of management-dependent and climate-independent cases, a total of 5547 treatments were analyzed. Treatments were clustered into the following groups: metabolism, fertility, udder and foot/leg. Meteorological data were compiled from the nearest weather station (average distance ± s.d. 39 ± 13 km). Hourly temperatures and relative humidity values were used to calculate the THI, which was divided into classes. Out of the total number of treatments, 37.4%, 32.9%, 21.6% and 8.1% belonged to metabolism, udder, fertility and foot/leg, respectively. Data were analyzed with a mixed model that included THI class, season and year as fixed effects and farm as random effect. In general, incidences were neither affected by the year (P > 0.05) and season (P > 0.05) nor by THI classes (P > 0.05). In tendency, incidences of metabolic treatments increased with increasing THI and incidences of udder treatments increased with decreasing THI. In conclusion, indications of moderate heat stress during summer months in Central Europe were found in the present study, although THI and season did not affect the different disease complexes significantly.     

漆酶固定化及其在小型酶反应器中的应用

以尼龙网为载体,戊二醛为交联剂,固定化真菌漆酶。用固定化酶在自行设计的小反应器中降解低浓度造纸废水,试验结果如下：当废水COD的浓度控制在3000 mg/L左右,降解时间为26h,废水的COD去除率达到35%。     

Indoleacetic acid oxidase activity in main stem homogenates of flax genotrophs and genotypes

The IAA-oxidase and peroxidase capabilities along the length of the main stem tissues of two flax genotrophs L and S and two flax genotypes R and M were examined in vitro. Stem gradients for peroxidase activity increased basipetally in all plant types, as did IAA-oxidase activity gradients at non-rate-limiting concentrations of Mn²⁺. Correlations between peroxidase activity and non-rate-limited IAA-oxidase activity supported the contention of dual activities on the same molecule. At rate-limiting concentrations of Mn²⁺, IAA-oxidase activity did not correlate with peroxidase activity. Plant type differences were detected in rate-limited IAA-oxidase activity. This activity was higher in the stem region immediately above the cotyledons (axillary buds) of the more branched types, L and R, than in the sparsely branched types, S and M.     

气候变化对宁夏固原地区胡麻发育进程和产量的影响

基于观测数据分析了宁夏固原地区平均温度和降水的年际变化,探讨了气候变化对当地胡麻发育进程和产量的影响状况.结果表明： 1957—2012年,固原地区年均气温呈上升趋势,年降水量呈下降趋势,气候倾向率分别为0.3 ℃·(10 a)^-1、-20 mm·(10 a)^-1;胡麻生长季平均温度的上升趋势更明显,降水的下降趋势则与年趋势类似.气温升高和降水减少加快了胡麻的发育速度,导致其生育期天数呈显著减少趋势.胡麻播种至出苗期温度每上升1 ℃,出苗期提前0.7 d;出苗至二对针叶期,温度每上升1 ℃,发育天数缩短0.8 d,降水量每减少1 mm,发育天数缩短0.1 d;工艺成熟至成熟期温度每上升1 ℃,成熟期提前1.8 d,降水量每减少1 mm,成熟期提前0.1 d.胡麻营养生长阶段平均温度升高、降水减少使发育加速是胡麻产量逐年降低的主要原因之一;生殖生长阶段温度升高会抑制花芽分化及正常授粉,对蒴果数和结实率产生影响而导致产量降低.调整胡麻品种种植布局、扩大中晚熟或晚熟品种比例是当地减少气候变化影响的重要措施.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.     

Studying the effects of laccase-catalysed grafting of ferulic acid on sisal pulp fibers

Functionalization of sisal specialty pulp fibers by laccase-catalysed grafting of ferulic acid (FRC) was investigated. To this end, the extent of phenol coupling to fibers under different reaction conditions (laccase and FRC rates, and time) was evaluated in terms of pulp properties including kappa number (expressed as the combined contributions of lignin and hexenuronic acids), brightness, Klason lignin and surface anionic charge after Soxhlet extraction of acetone-treated pulp. The specific treatment resulting in the highest degree of grafting was then used in a comparative study of the effects of applying the laccase-FRC system to refined and unrefined pulp with a view to confirming whether the increased surface area obtained by effect of fibrillation would lead to enhanced grafting. Based on the results, refining the pulp prior to the enzyme treatment resulted in increased grafting which in turn led to handsheets with improved strength-related properties (particularly wet tensile strength) relative to control samples.     

亚麻籽粒中主要脂肪酸和蛋白质积累过程研究

以亚麻品种82(50)为材料,研究了亚麻籽粒中主要脂肪酸、蛋白质和糖积累的规律,结果表明:在亚麻籽粒发育成熟的过程中,棕榈酸、亚油酸含量持续下降,当籽粒完全成熟时降至最低;油酸和亚麻酸含量持续增加,当籽粒完全成熟时含量最高;硬脂酸在籽粒发育10～25d,其含量逐渐提高,以后逐渐下降。蛋白质随着籽粒发育成熟其含量持续提高,且蛋白质含量的提高主要发生在籽粒发育成熟的30d以前。总糖随籽粒发育成熟含量迅速下降,当种子完全成熟时降至最低。     

Effects of a pulp and paper mill on the ecology of the La Trobe River,Victoria, Australia

Four riverine animal communities were measured to assess the impact of extensively treated wastewater from a pulp and paper mill on the lower La Trobe River in Victoria, Australia. Benthic macroinvertebrates in channel and bankside habitats were sampled using a new air-lift corer. Population density was expressed in relation to substrate volume. Other communities measured at five sites upstream and downstream of the mill's wastewater outfall were the zooplankton, and the animals associated with submerged littoral vegetation. Ten environmental variables were also measured during the two sampling periods.A total of 50 benthic macro-invertebrate taxa were dominated by Oligochaeta, Chironomidae and Bivalvia. Benthic communities upstream and downstream of the outfall were very similar. Benthic samples showed large unexplained variation between stations and seasons, despite the similarity of stations and the stratified sampling design, but within-sample variation was small. There was some evidence that benthic faunal patchiness was associated with patterns of stream-bed scouring and deposition in periods of high flow. Littoral samples collected 28 taxa, dominated by Decapoda and Hemiptera. The benthic and littoral communities were quite distinct, with only three species common to both.Only two of the biological and environmental variables responded to wastewater from the mill: total dissolved solids rose by 20–25% over upstream levels; and zooplankton density increased by 2–3 orders of magnitude. It was concluded that wastewater treatment had successfully avoided the major environmental problems often associated with pulp and paper mills.     

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5–1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase. (Mol Cell Biochem 174: 87–90, 1997)     

Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure and arrangement of mitochondria in the red, white and intermediate striated muscle fibers of the rat were examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by means of the Osmium-DMSO-Osmium procedure.Beneath the sarcolemma, spherical or ovoid subsarcolemmal mitochondria show accumulations. The mitochondria are numerous and large in size in the red fibers, intermediate in the intermediate fibers, and few and small in the white fibers. Paired, slender I-band-limited mitochondria were located on both sides of the Z-line and partly embraced the myofibrils at the I-band level; they occurred in all three types of fibers. In the intermyofibrillar spaces, numerous mitochondria formed mitochondrial columns. These columns were classified into two types: 1) thick mitochondrial columns, formed by multiple mitochondria each with an intermyofibrillar space corresponding to one sarcomere in length, and 2) thin mitochondrial columns, established by single mitochondria corresponding to one sarcomere in length. In the red fibers mitochondrial columns were abundant and the ratio of the thick and thin columns was almost the same, while in the intermediate fibers most of the columns belonged to the thin type. The white fibers displayed rare, very thin columns.     

棉麻纤维作物雄性不育研究进展及展望

棉麻是我国重要天然纤维作物。本文对我国棉麻作物雄性不育的类型、雄性不育的选育及雄性不育的机理等研究进展进行了综述,并讨论了棉麻作物雄性不育研究发展方向与杂种优势利用有关问题,同时提出了雄性不育系选育的思路。     

Interactive effects of nutrients and contaminants from pulp mill effluents on riverine benthos

Bleached kraft mill effluents (BKME) contain both nutrients(nitrogen and phosphorus) and contaminants that can have a stimulatoryor inhibitory effect on riverine food webs. For the Northern RiverBasins Study (NRBS), separation of the effects of nutrients andcontaminants in BKME was an important issue because field biomonitoringwas unable to isolate these impacts. We addressed this problem by usingriverside mesocosm experiments, along with field observations, todetermine the effects of BKME on benthic food webs of the upperAthabasca River near Hinton, Alberta, Canada. Response variablesincluded periphyton biomass, benthic insect density and composition, andthe biomass of abundant invertebrates (i.e., caddisfly, mayfly andstonefly taxa). Experiments were conducted during autumn when nutrientenhancement was hypothesized to increase autochthonous primaryproduction as a result of low river discharge and high water clarity.Treatments included reference river water, 1% BKMEconcentrations, and nutrients (nitrogen plus phosphorus) equivalent tothe 1% BKME treatment. Although pulp mill effluents can inducesublethal toxicity in benthic biota, BKME concentrations equivalent tolevels in the Athabasca River did not cause measurable toxicity.Relative to reference water treatments, BKME-associated increases inavailable phosphorus produced enrichment effects including increases inperiphyton and insect biomass, and invertebrate abundance. Insect familyrichness was not affected by the treatments. The development andapplication of a field-based artificial stream system for the NRBSimproved our mechanistic understanding of the effects of BKME on benthicbiota of nutrient and contaminant stressors. By combining artificialstream results with field observations, we were also able to link thismechanistic understanding of stressor effects directly to impacts in theriver. We conclude that the response to BKME in the Peace-AthabascaBasin is largely one of nutrient enrichment rather than that oftoxicity.