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Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.

The pulp and paper industry is a major source of lignocellulose-containing streams. The components of lignocellulose material are lignin, hemicellulose, and cellulose that may be hydrolyzed into their smaller components and used as feedstocks for valorization efforts. Much of this material is contained in underutilized streams and waste products, such as black liquor, pulp and paper sludge, and wastewater. Bacterial fermentation strategies have suitable potential to upgrade lignocellulosic biomass contained in these streams to value-added chemicals. Bacterial conversion allows for a sustainable and economically feasible approach to valorizing these streams, which can bolster and expand applications of the pulp and paper industry. This review discusses the composition of pulp and paper streams, bacterial isolates from process streams that can be used for lignocellulose biotransformations, and technological approaches for improving valorization efforts. KEY POINTS: • Reviews the conversion of pulp and paper industry waste by bacterial isolates. • Metabolic pathways for the breakdown of lignocellulose components. • Methods for isolating bacteria, determining value-added products, and increasing product yields.

Laccases are multicopper enzymes present in plants, fungi, bacteria, and insects, which catalyze oxidation reactions together with four electron reduction of oxygen to water. Plant, bacterial, and insect laccases have a polymerizing role in nature, implicated in biosynthesis of lignin, melanin formation, and cuticle hardening, respectively. On the other hand, fungal laccases carry out both polymerizing (melanin synthesis and fruit body formation) as well as depolymerizing roles (lignin degradation). This bifunctionality of fungal laccases can be attributed to the presence of multiple isoforms within the same as well as different genus and species. Interestingly, by manipulating culture conditions, these isoforms with their different induction patterns and unique biochemical characteristics can be expressed or over-expressed for a targeted biotechnological application. Consequently, laccases can be considered as one of the most important biocatalyst which can be exploited for divergent industrial applications viz. paper pulp bleaching, fiber modification, dye decolorization, bioremediation as well as organic synthesis. The present review spotlights the role of fungal laccases in various antagonistic applications, i.e., polymerizing and depolymerizing, and co-relating this dual role with potential industrial significance.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etcaEuro broken vertical bar) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

Switching between metabolically active and dormant states provides bacteria with protection from environmental stresses and allows rapid growth under favorable conditions. This rapid growth can be detrimental to the environment, e.g., pathogens in recreational lakes, or to industrial processes, e.g., fermentation, making it useful to quickly determine when the ratio of dormant to metabolically active bacteria changes. While a rapid increase in metabolically active bacteria can cause complications, a high number of dormant bacteria can also be problematic, since they can be more virulent and antibiotic-resistant. To determine the metabolic state of Escherichia coli and Salmonella Typhimurium, we developed two paper-based colorimetric assays. The color changes were based on oxidoreductases reducing tetrazolium salts to formazans, and alkaline phosphatases cleaving phosphates from nitrophenyl phosphate salt. Specifically, we added iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate to metabolically active bacteria on paper and INT and para-nitrophenyl phosphate salt to dormant bacteria on paper. The color changed in less than 60 min and was generally visible at 103 CFU and quantifiable at 106 CFU. The color changes occurred in both bacteria, since oxidoreductases and alkaline phosphatases are common bacterial enzymes. On one hand, this feature makes the assays suitable to a wide range of applications, on the other, it requires specific capture, if only one type of bacterium is of interest. We captured Salmonella or E. coli with immobilized P22 or T4 bacteriophages on the paper, before detecting them at levels of 102 or 104 CFU, respectively. Determining the ratio of the metabolic state of bacteria or a specific bacterium at low cost and in a short time, makes this methodology useful in environmental, industrial and health care settings.

Colorimetric, fluorescence, and paper-based method were developed to measure the Hg

Industrially produced bleached recycled pulp (R) comprising essentially hardwood fibres was subjected to enzymatic treatment with endo-xylanase from Thermomyces lanuginosus with or without ultra-high hydrostatic pressure (UHP) pre-treatment at 300–600 MPa for 10 min. The kinetics and the extent of enzymatic hydrolysis after UHP pre-treatment under different conditions have been evaluated by released reducing sugars and the analysis of neutral sugars in pulps, respectively. The changes in surface chemical composition of pulps were assessed by UV-vis diffuse reflectance spectroscopy. UHP-pre-treated R under optimal conditions (400 MPa), with or without posterior enzymatic treatment, was used for the production of handsheets and evaluation of its mechanical properties. It was suggested that enzymatic modification improves significantly the papermaking properties of recycled pulp. These improvements were related with selective removal of xylan bound to impurities and to aggregated cellulose fibrils on the fibre surface, thus favouring the ensuing swelling and inter-fibre bonding in paper. UHP pre-treatment and posterior enzymatic treatment revealed a synergetic effect on the mechanical properties of recycled pulp. This fact was assigned to enhanced accessibility of fibres towards xylanase and by forced hydration and favourable rearrangement of cellulosic fibrils in fibres after UHP pre-treatment. The increase of basic strength properties after UHP-promoted xylanase treatment was up to 30 % being the most pronounced for the tensile strength and the burst resistance.

Bioelectrochemical reactors (BERs) with a cathodic working potential of -0.6 or -0.8 V more efficiently degraded cellulosic material, i.e., filter paper (57.4-74.1% in 3 days and 95.9-96.3% in 7 days) than did control reactors without giving exogenous potential (15.4% in 3 days and 64.2% in 7 days). At the same time, resultant conversions to methane and carbon dioxide in cathodic working chamber of BERs by application of electrochemical reduction in 3 days of operation were larger than control reactors. However, cumulative methane production in cathodic BERs was similar to those in control reactors after 7 days of operation. Microscopic observation and 16S rRNA gene analysis showed that microbial growth in the entire consortium was higher after 2 days of operation of cathodic BERs as compared with the control reactors. In addition, the number of methanogenic 16S rRNA gene copies in cathodic BERs was higher than in control reactors. Moreover, archaeal community structures constructed in cathodic BERs consisted of hydrogenotrophic methanogen-related organisms and differed from those in control reactors after 2 days of operation. Specifically, the amount of Methanothermobacter species in cathodic BERs was higher within archaeal communities than in those control reactors after 2 days of operation. Electrochemical reduction may be effective for accelerating microbial growth in the start-up period and thereby increasing microbial treatment of cellulosic waste and methane production.

14 páginas, 7 figuras, Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching., This work was supported by the Spanish projects AGL2008-00709, BIO2005-03569 and BIO2008-01533, and the BIORENEW project of the European Union (contract NMP2-CT-2006-026456).

Increased interest in sustainable agriculture and bio-based industries requires that we find more energy-efficient methods for treating cellulose-containing wastewaters. We examined the effectiveness of simultaneous electricity production and treatment of a paper recycling plant wastewater using microbial fuel cells. Treatment efficiency was limited by wastewater conductivity. When a 50 mM phosphate buffer solution (PBS, 5.9 mS/cm) was added to the wastewater, power densities reached 501+/-20 mW/m2, with a coulombic efficiency of 16+/-2%. There was efficient removal of soluble organic matter, with 73+/-1% removed based on soluble chemical oxygen demand (SCOD) and only slightly greater total removal (76+/-4%) based on total COD (TCOD) over a 500-h batch cycle. Cellulose was nearly completely removed (96+/-1%) during treatment. Further increasing the conductivity (100 mM PBS) increased power to 672+/-27 mW/m2. In contrast, only 144+/-7 mW/m2 was produced using an unamended wastewater (0.8 mS/cm) with TCOD, SCOD, and cellulose removals of 29+/-1%, 51+/-2%, and 16+/-1% (350-h batch cycle). These results demonstrate limitations to treatment efficiencies with actual wastewaters caused by solution conductivity compared to laboratory experiments under more optimal conditions.

1Instituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080, Seville, Spain; and 2Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase-mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes - eucalypt kraft pulping, spruce thermomechanical pulping and flax soda-anthraquinone pulping - were treated with a high-redox potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically-treated pulps revealed that the laccase-mediator treatment completely (or greatly) removed most of the pitch-causing lipophilic compounds present in the different pulps including: i) free and conjugated sitosterol in eucalypt paper pulp; ii) resin acids, sterol esters and triglycerides in spruce pulp; and iii) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase-mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood and nonwoody plants., This study has been funded by the Spanish projects AGL2002-393 and BIO2002-1166, the EU projects QLK5-99-1357 and QLK3-99-590, and by ENCE-CSIC contracts.

Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K(m) of the enzyme changed from 5.9 mg ml(-1) to 9.1 mg ml(-1) upon immobilization. The V(max) of the immobilized enzyme showed an increase from 90.9 micro mol ml(-1) min(-1) (for the free enzyme) to 111.1 micro mol ml(-1) min(-1). The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.

An endo-beta-mannanase (EC 3.2.1.78) from Streptomyces ipomoea CECT 3341 was purified and applied to the biobleaching of pine kraft pulps. The maximum level of endo-beta-mannanase activity (0.6 units ml(-1)) was achieved after 4 days of growth in a medium containing locust bean gum and yeast extract. Zymograms revealed mannanase bands (Man) with high and low electrophoretic mobility on the second and seventh days of incubation (Man1, Man3) and three bands of high, medium and low mobility from the third to sixth days of growth (Man1, Man2, Man3). Although these exhibited different molecular masses, their amino-terminal sequences were identical. The action of proteases detected in the culture supernatant could be responsible for such events, suggesting that only one endo-beta-mannanase is produced by S. ipomoea. The purified Man3 exhibited a molecular mass of 40 kDa, an isoelectric point of 4.0 and an optimal temperature and pH reaction of 55 degrees C and 7.5, respectively. It was strongly inhibited by Ag+, Hg2+, Al3+ and Fe3+, and was strongly activated by Mn2+. The ability of the purified endo-beta-mannanase to improve the bleachability of pine kraft pulp, when applied with alkaline extraction, was demonstrated by an increase in the pulp brightness (1.7%, using the International Standards Organisation's test) and an absence of variations in the viscosity values. A relationship between the increase in pulp brightness and the presence of manganese in the pulps could be established.

Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on laboratory observations only. There are several critical challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)—synthetic or natural, pH, temperature, stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive production at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.

Biofilm-degrading enzymes are potential agents for slime control in paper machines. In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes. Polysaccharide yields of 1.5–3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium. Two K. pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid. Fructose-containing polysaccharides were the main products of B. licheniformis and P. fluorescens. Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening. None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides.

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known beta-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 degrees C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect.

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp.

The relative importance of each of three dechlorinating species to overall organochlorine removal from bleached kraft-mill effluents (BKME) was assessed. Ancylobacter aquaticus A7, Pseudomonas P1, and Methylobacterium CP13, strains indigenous to a BKME treatment system, were tested for growth on chlorinated acetic acids and alcohols, and for adsorbable organic halogen (AOX) reduction in batch cultures of sterile BKME from three sources. A. aquaticus A7 exhibited the broadest substrate range, but could only affect significant AOX reduction in softwood effluents. Methylobacterium CP13 exhibited a limited substrate range, but was capable of removing significant amounts of AOX from both hardwood and softwood effluents. By contrast, Pseudomonas sp. P1 exhibited a limited substrate range and poor to negligible reductions in AOX levels from both effluent types. Mixed inocula of all three species combined and inocula of sludge from mill treatment systems removed as much AOX from softwood effluents as did pure populations of Methylobacterium CP13. When BKME was hydrolysed prior to AOX analysis, the subsequent estimates of recalcitrant, or non-hydrolysable, AOX levels were far less variable than their counterpart total AOX measures. It is suggested that this is a relevant and useful measure of AOX for biodegradation studies.

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure L(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g(-1). By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l(-1) showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l(-1)) and glucose (19.2 g l(-1)) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l(-1) h(-1) and 0.5 g xylose l(-1) h(-1). This resulted mainly into the product lactic acid (6.8 g l(-1)) and ethanol (5.7 g l(-1)).

Cellulose (in the form of printed paper) has always been the prime medium for displaying information in our society and is far better than the various existing display technologies. This is because of its high reflectivity, contrast, low cost and flexibility. There is a major initiative to push for a dynamic display technology that emulates paper (popularly known as "electronic paper"). We have successfully demonstrated the proof of the concept of developing a dynamic display on cellulose. To the best of our knowledge, this is the first significant effort to achieve an electronic display using bacterial cellulose. First, bacterial cellulose is synthesized in a culture of Acetobacter xylinum in standard glucose-rich medium. The bacterial cellulose membrane thus formed (not pulp) is dimensionally stable, has a paper-like appearance and has a unique microfibrillar nanostructure. The technique then involves first making the cellulose an electrically conducting (or semi-conducting) sheet by depositing ions around the microfibrils to provide conducting pathways and then immobilizing electrochromic dyes within the microstructure. The whole system is then cased between transparent electrodes, and upon application of switching potentials (2-5 V) a reversible color change can be demonstrated down to a standard pixel-sized area (ca. 100 microm2). Using a standard back-plane or in-plane drive circuit, a high-resolution dynamic display device using cellulose as substrate can be constructed. The major advantages of such a device are its high paper-like reflectivity, flexibility, contrast and biodegradability. The device has the potential to be extended to various applications, such as e-book tablets, e-newspapers, dynamic wall papers, rewritable maps and learning tools.

International audience; Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l–1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied.

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l-1), improved FAEA activity 24.5-fold and a yield of 1 g l-1 of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification - close to 75% - and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector. Chemicals/CAS: hemicellulose, 63100-39-0, 63100-40-3, 9034-32-6; laccase, 80498-15-3; xylan endo 1,3 beta xylosidase, 37278-89-0, 9025-55-2; Carboxylic Ester Hydrolases, EC 3.1.1.-; Endo-1,4-beta Xylanases, EC 3.2.1.8; feruloyl esterase, EC 3.1.1.73; Laccase, EC 1.10.3.2; Recombinant Proteins

Suspended solids in the nutrient medium for Acetobacter xylinium in a rotating disk bioreactor become incorporated into the gelatinous mat of bacterial cellulose as it forms. Embedding fibers of ordinary cellulose creates composites with enhanced strength and the toughness of bacterial cellulose. Purified cellulose and elongated fibers from paper are incorporated differently than are spherical particles such as silica gel. About 90% of the final cellulose can come from scrap paper, and dried composite sheets were much stronger than plain bacterial cellulose per unit area.

Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 microg of biofilm sugar cm(-2) in 5 days in rich medium and 10 microg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 microg cm(-2)) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA. gave the most effective kappa number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

The gene ce1A, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The ce1A gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0. Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.