Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase Secreted from Phanerochaete sordida YK-624

In vitro bleaching of an unbleached hardwood kraft pulp was performed with manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624. When the kraft pulp was treated with partially purified MnP in the presence of MnSO₄, Tween 80, and sodium malonate with continuous addition of H₂O₂ at 37°C for 24 h, the pulp brightness increased by about 10 points and the kappa number decreased by about 6 points compared with untreated pulp. The pulp brightness was also increased by 43 points to 75.5% by multiple (six) treatments with MnP combined with alkaline extraction. Our results indicate that in vitro degradation of residual lignin in hardwood kraft pulp with MnP is possible.     

Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase from Phanerochaete sordida YK-624 without Addition of MnSO(inf4)

In vitro bleaching of an unbleached hardwood kraft pulp was performed with partially purified manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624 without the addition of MnSO(inf4) in the presence of oxalate, malonate, or gluconate as manganese chelator. When the pulp was treated without the addition of MnSO(inf4), the pulp brightness increased by about 10 points in the presence of 2 mM oxalate, but the brightness did not significantly increase in the presence of 50 mM malonate, a good manganese chelator. Residual MnP activity decreased faster during the bleaching with MnP without MnSO(inf4) in the presence of malonate than in the presence of oxalate. Oxalate reduced MnO(inf2) which already existed in the pulp or was produced from Mn(sup2+) by oxidation with MnP and thus supplied Mn(sup2+) to the MnP system. The presence of gluconate, produced by the H(inf2)O(inf2)-generating enzyme glucose oxidase, also improved the pulp brightness without the addition of MnSO(inf4), although treatment with gluconate was inferior to that with oxalate with regard to increase of brightness. It can be concluded that bleaching of hardwood kraft pulp with MnP, using manganese originally existing in the pulp, is possible in the presence of oxalate, a good manganese chelator and reducing reagent.     

Selective Delignification of Aspen Wood Blocks In Vitro by Three White Rot Basidiomycetes

Aspen wood blocks were selectively delignified in the laboratory by Ischnoderma resinosum, Poria medulla-panis, and Xylobolus frustulatus. After 8 weeks only the outer surfaces of wood blocks were selectively delignified. The percentages of weight loss obtained after 4, 8, and 12 weeks showed that decay occurred at a relatively constant rate. Selectively delignified wood could be identified by using scanning electron microscopy only when lignin had been extensively removed from cell walls. X. frustulatus was able to form pockets of delignified wood throughout blocks after 12 weeks.     

Decrease in Cryptosporidium parvum Oocyst Infectivity In Vitro by Using the Membrane Filter Dissolution Method for Recovering Oocysts from Water Samples

Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.     

Manganese Is Not Required for Biobleaching of Oxygen-Delignified Kraft Pulp by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 extensively delignified and bleached oxygen-delignified eucalyptus kraft pulp handsheets. Biologically mediated brightness gains of up to 14 ISO (International Standards Organization units) were obtained, providing high final brightness values of up to 80% ISO. In nitrogen-limited cultures (2.2 mM N), manganese (Mn) greatly improved manganese-dependent peroxidase (MnP) production. However, the biobleaching was not affected by the Mn nutrient regimen, ranging from 1,000 (mu)M added Mn to below the detection limit of 0.26 (mu)M Mn in EDTA-extracted pulp medium. The lowest Mn concentration tested was at least several orders of magnitude lower than the K(infm) known for MnP. Consequently, it was concluded that Mn is not required for biobleaching in Bjerkandera sp. strain BOS55. Nonetheless, fast protein liquid chromatography profiles indicated that MnP was the predominant oxidative enzyme produced even under culture conditions in the near absence of manganese. High nitrogen (22 mM N) and exogenous veratryl alcohol (2 mM) repressed biobleaching in Mn-deficient but not in Mn-sufficient culture medium. No correlation was observed between the titers of extracellular peroxidases and the biobleaching. However, the decolorization rate of the polyaromatic dye Poly R-478 was moderately correlated to the biobleaching under a wide range of Mn and N nutrient regimens.     

Changes in Molecular Size Distribution of Cellulose during Attack by White Rot and Brown Rot Fungi

The kinetics of cotton cellulose depolymerization by the brown rot fungus Postia placenta and the white rot fungus Phanerochaete chrysosporium were investigated with solid-state cultures. The degree of polymerization (DP; the average number of glucosyl residues per cellulose molecule) of cellulose removed from soil-block cultures during degradation by P. placenta was first determined viscosimetrically. Changes in molecular size distribution of cellulose attacked by either fungus were then determined by size exclusion chromatography as the tricarbanilate derivative. The first study with P. placenta revealed two phases of depolymerization: a rapid decrease to a DP of approximately 800 and then a slower decrease to a DP of approximately 250. Almost all depolymerization occurred before weight loss. Determination of the molecular size distribution of cellulose during attack by the brown rot fungus revealed single major peaks centered over progressively lower DPs. Cellulose attacked by P. chrysosporium was continuously consumed and showed a different pattern of change in molecular size distribution than cellulose attacked by P. placenta. At first, a broad peak which shifted at a slightly lower average DP appeared, but as attack progressed the peak narrowed and the average DP increased slightly. From these results, it is apparent that the mechanism of cellulose degradation differs fundamentally between brown and white rot fungi, as represented by the species studied here. We conclude that the brown rot fungus cleaved completely through the amorphous regions of the cellulose microfibrils, whereas the white rot fungus attacked the surfaces of the microfibrils, resulting in a progressive erosion.     

Decolorization of Some Reactive Textile Dyes by White Rot Fungi Isolated in Pakistan

Summary Four white-rot fungi isolated in Pakistan were used for decolorization of widely used reactive textile dyestuffs. Phanerochaete chrysosporium, Coriolus versicolor, Ganoderma lucidum and Pleurotus ostreatus were grown in defined nutrient media for decolorization of Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR for 10 days in shake flasks. Samples were removed every day, centrifuged and the absorbances of the supernatants were read to determine percentage decolorization. It was observed that P. chrysosporium and C. versicolor could effectively decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR. Drimarene Orange K-GL was completely decolorized (0.2 g/l after 8 days) only by P.chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also showed good decolorization efficiencies (0.19–0.2 g/l) on all dyes except Remazol Brilliant Yellow (0.07 g/l after 10 days). G. lucidum did not decolorize any of the dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 8 days).     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

白腐菌对培养环境pH的调节及其产漆酶的相关性

研究了不同种属白腐菌多孔菌属C1(Polyporussp.)、侧耳属B2(Pleurotussp.)、香菇属A3(Lentinusedodes)对培养环境pH的调节,及其与菌株产漆酶状况的相关性。结果表明:3株白腐菌均可调节培养液酸化,培养环境的pH可分别由初始的4.5~5.0持续下降至培养结束时3.0~3.5水平,相应C1、B2、A3三者的漆酶合成与分泌在pH值较高(pH 4.5以上)时均表现较弱(酶活分别处于约1 500、200、50 U/mL以下的水平),在pH 3.2~4.5范围内的不同偏酸性条件下则得到较好表达(最高酶活分别达5 000、340、144 U/mL)。统计学分析指出,白腐菌调节环境pH状况与其产漆酶状况之间存在显著或极显著相关性,但不同白腐菌调节培养环境pH的能力之间并不具有显著性差异。     

In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology

In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model’s predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.     

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G₁ phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.     

糖脂类生物表面活性剂的特性及其对白腐真菌降解蒽的强化作用

【目的】对铜绿假单胞菌(Pseudomonas aeruginosa)所产生物表面活性剂的稳定性进行分析,考察该生物表面活性剂对乳白耙齿菌F17(Irpex lacteus F17)降解蒽的强化作用。【方法】采用三氯甲烷萃取的方法从铜绿假单胞菌的发酵液中提取生物表面活性剂,采用表/界面张力仪测定该生物表面活性剂在不同条件下的表面张力值,对其进行稳定性研究。在乳白耙齿菌F17降解蒽的过程中加入适量的生物表面活性剂,测定蒽的降解率,探讨其对蒽生物降解的强化作用。【结果】铜绿假单胞菌所产生物表面活性剂的临界胶束浓度为40 mg/L,在15-150°C及pH 6.0-13.0范围内表现出优良的稳定性,对盐浓度的耐受性也很高。在蒽的生物降解过程中,生物表面活性剂能极大地促进蒽的降解,在生物表面活性剂浓度为50 mg/L时,第15天蒽的降解率达到了82.9%。生物表面活性剂在接种乳白耙齿菌F17前1天加入培养基中,能更好地促进蒽的降解。与化学表面活性剂相比,生物表面活性剂对蒽降解的强化作用更显著。【结论】该生物表面活性剂性能优良、稳定性好,能够显著强化乳白耙齿菌F17对蒽的降解,具有良好的应用前景。     

Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively. The activity was strongly enhanced in the presence of Cu²⁺, Mn²⁺, and Mg²⁺ and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.     

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow^1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

In Vitro Cultivation and Antibiotic Susceptibility of a Cytophaga-Like Intracellular Symbiote Isolated from the Tick Ixodes scapularis

A Cytophaga-like organism (CLO), isolated from the tick Ixodes scapularis (IsCLO), was adapted to growth in insect cell lines and its antibiotic sensitivity was tested. IsCLO were introduced to four insect cell lines, and their growth was measured by quantitative polymerase chain reaction. IsCLO propagated well in a mosquito cell line, AeAl-2, and caused cytopathic effects in host cells. A lepidopteran cell line, HZ-AM1, was also suitable for propagation of IsCLO and kept a steady state with bacterial growth. Using IsCLO-infected AeAl-2, antibiotics effective against the bacteria included ampicillin, chloramphenicol, penicillin-G, rifampicin, and tetracycline. These antibiotics will be useful for eliminating CLO from host arthropods, which is necessary for in vivo studies of the intracellular facultative symbiotes.     

Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles

Background

Growing cells in simulated weightlessness condition might be a highly promising new technique to maintain or generate tissue constructs in a scaffold-free manner. There is limited evidence that microgravity condition may affect development of ovarian follicles. The objective of the present study was to investigate the effects of simulated microgravity on the in vitro development of mouse preantral follicles.

Methods and Results

Ovarian tissue from 14-day-old mice, or preantral follicles mechanically isolated from 14-day-old mouse ovaries were cultured at a simulated microgravity condition generated using a rotating wall vessel apparatus. Follicle survival was assessed quantitatively using H&E staining. Follicle diameter and oocyte diameter were measured under an inverted microscope. Ultrastructure of oocytes was evaluated using transmission electron microscopy. We observed that simulated microgravity compromised follicle survival in vitro, downregulated PCNA and GDF-9 expressions, and caused ultrastructural abnormalities in oocytes.

Conclusion

This study showed for the first time that three-dimensional culture condition generated by simulated microgravity is detrimental to the initial stage development of mouse preantral follicles in vitro. The experimental setup provides a model to further investigate the mechanisms involved in the in vitro developmental processes of oocytes/granulosa cells under the microgravity condition.     

Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.