Search

The technical viability was investigated for the conversion of Pinus pinaster stumps to value-added products via a chain of pretreatments and simultaneous saccharification and fermentation (SSF) steps to obtain bioethanol. Sequential steam explosion (SE), organosolv (OS) pretreatment and soda-anthraquinone pulping (Na/AQ) were performed resulting in an unbleached pulp (UBP), which was successfully converted via SSF to bioethanol with concentrations up to 79 g l-1, which corresponds to a conversion yield of 97% and productivity of 1.09 g l-1 h-1 at 15% total solids. Accordingly, delignification steps by oxygen bleaching are not necessary for bioethanol production. Different industrial softwood (SW) pulps were also tested as reference materials. Total lignin contents up to 4.5% in SW-based pulps had no adverse effects on SSF efficiency.

Enzymatic hydrolysis of pre-treated wheat straw has been studied. Several additives have been checked as potential improvers of glucose yield in the enzymatic hydrolysis, trying to avoid the cellulases inhibition by the lignin as a cause of the decrease of the saccharification rate of lignocellulosic biomass. Non-ionic surfactants, such as Tween 80 (poly(oxyethylene)20-sorbitan-monooleate) and polyethylene glycol-6000, and proteins, as bovine serum albumin, casein and skimmed milk powder, have been used to improve the sugars yield in the enzymatic hydrolysis. Two pre-treated wheat straw materials, one with high delignification and another with low delignification, have been tested. Three concentrations of each additive have been considered. The results obtained point out that the lignin content of the pre-treated lignocellulosic biomass has a decisive influence in reaching a high yield in the enzymatic saccharification. The additives checked have a moderate effect on the cellulose hydrolysis rate. The more favorable option combines high delignification with the use of a cheap protein such as casein, reaching a glucose yield of 88.6% when the best conditions are employed.

The ability of the bacteria Gluconacetobacter sucrofermentans to produce high-quality cellulose is well known. This bacterial cellulose has unique properties, as the result of an ultrathin crosslinked structure. It is almost chemically pure (until 98% cellulose), has a high crystallinity, mechanical strength, elasticity and durability. It is compatible with plant cellulose and could be grown on a vegetal fibrous network what makes bacterial cellulose an excellent candidate to reinforce damaged papers. The objective of this work is to promote the production of bacterial cellulose on the surface of a variety of papers by the direct application of a culture of G. sucrofermentans and to test if this procedure could be an economic and feasible possibility in reinforcing papers or cellulosic surfaces. After 7 days of cultivation, G. sucrofermentans deposits a thin layer (ca. 10 µm) of bacterial cellulose on the papers surfaces. Microscopic observations have shown a heterogeneous covering of the surfaces, while the modified papers maintain their thickness and their basic weight. The treatment has not increased the papers mechanical properties, but maintains them after artificial aging. The changes in optical properties of the reinforced papers, such as opacity and colorimetric coordinates, are minor even after further aging. Air permeance is the paper characteristic that experiences more variations because the layers of bacterial cellulose inhibit the airflow through the papers. The changes in the static and dynamic contact angles suggest that the bacterial cellulose structure is more compact and make the surface more hydrophobic than the original paper surface. © 2017, Springer Science+Business Media New York.

One of the most common methods used to reinforce damaged paper is to apply a lining, using Japanese paper (JP). The reinforcing material must consolidate the paper without modifying its visual appearance. The unique properties of bacterial cellulose (BC) suggest that it could be efficiently used to reinforce degraded paper documents. The changes in the visual appearances of the printed commercial papers lined with BC and JP were examined in this study. Four commercial papers, coated and uncoated, were printed with cyan, magenta, yellow, and black offset inks. The printed samples were lined with BC and JP sheets. Print density, gloss, and CIELab coordinates were tested in the lined and unlined samples before and after aging. Lining with JP notably affected the print density and CIELab coordinates. The lining with BC resulted in lower decrements in color intensity. The gloss values of samples lined with BC differed widely amongst the papers, whereas in papers reinforced with JP these values never exceeded 6%. Subjecting the samples to an aging process did not markedly modify the results except for the BC-lined samples, in which color differences increased.

[EN] As the fungal laccase production depends on some com-ponents which induce it, we have examined the indepen-dent effects of ethanol, wheat straw and copper sulphate and their combinations on the volumetric laccase activity of Pycnoporus sanguineus. Wheat straw and copper sulphate gave the best results showing a synergic effect after 13 days of incubation (lac-case activity was 3 and 4.5 times greater than the activ-ity generated when they were used alone). Ethanol on the contrary, was found to have a negative effect on laccase production.The laccase produced by the fungus Pycnoporus sanguin-eus maintains a good stability in a wide range of tempera-tures; a stability which also depends on the pH. By choos-ing the adequate pH, the level and stability of the laccase activity are satisfactory in a range of temperatures from 25 to 70ºC, which would allow the diversification of laccase use in industrial applications., [ES] Como la producción de lacasa fúngica depende de al-gunos componentes que la inducen, hemos estudiado el efecto del etanol, la paja de trigo y el sulfato de cobre de forma independiente y combinada sobre la actividad volu-métrica de la lacasa de Pycnoporus sanguineus. La paja de trigo y el sulfato de cobre han dado los mejores resultados, mostrando un efecto sinérgico en el décimo tercer día de incubación (la actividad lacasa fue 3 y 4,5 veces mayor que la actividad obtenida con cada uno de ellos por separado). Por el contrario, el etanol afectó nega-tivamente a la producción de lacasa. La lacasa producida por Pycnoporus sanguineus mantie-ne su estabilidad en un amplio intervalo de temperatura; estabilidad que depende también del pH. Eligiendo un pH adecuado, el nivel y la estabilidad de la actividad lacasa se mantienen satisfactoriamente a temperaturas entre 25 y 70ºC, lo que diversificaría el uso industrial de esta lacasa, [CAT] Donat que la producció de lacasa fúngica depèn d’alguns components que la indueixen, s’estudia l’efecte de l’eta-nol, la palla de blat i el sulfat de coure de forma indepen-dent i combinada sobre l’activitat volumètrica de la lacasa de Pycnoporus sanguineus. La palla de blat i el sulfat de coure donen els millors resul-tats, mostrant un efecte sinèrgic en el tretzè dia d’incuba-ció (l’activitat lacasa és 3 i 4,5 cops superior que l’activitat obtinguda amb cadascun d’ells per separat). Per contra, l’etanol afecta negativament a la producció de lacasa. La lacasa produïda per Pycnoporus sanguineus manté la seva estabilitat en un ampli interval de temperatura; esta-bilitat que depèn també del pH. Elegint un pH adequat, el nivell i l’estabilitat de l’activitat lacasa es mantenen satis-factòriament a temperatures entre 25 i 70ºC, el que diver-sificaria l’ús industrial d’aquesta lacasa.

Preservation of documentary heritage is one of the biggest challenges facing paper conservators today. The singular properties of bacterial cellulose (BC) lead us to propose to reinforce paper with BC sheets. In the first part of this study, the reinforcing capability of BC was tested on model papers of well-known fiber composition. The aim of the present study was to verify the suitability of rebuilding degraded old papers with BC. The degraded papers were characterized before and after the reinforcement. In addition, lined samples were characterized before and after an aging process in order to study the stability in time. The same methodology was used with Japanese paper (JP), a material commonly used by paper conservators, in order to compare both materials as reinforcement. Mechanical properties of paper lined with BC are as good as those obtained with JP. Papers lined with BC have more marked modifications on their optical properties than those restored with JP. Nevertheless, letters in books lined with BC are more legible. Moreover, only the papers restored with BC show high changes in porosity. The aging process leads to a slight decrement in burst index. Changes on tear index and optical properties with the aging process depend on the paper to be restored. This study suggests that BC improves deteriorated paper quality, without altering the information contained therein, and that this improvement is maintained over time. Hence, BC is a promising alternative material for the restoration of paper. © 2015, Springer Science+Business Media New York.

The disappearance of bibliographic heritage is one of the biggest problems facing libraries. One of the most common methods used to restore paper, lining, is to apply a reinforcing layer to the document. This study focuses on lining papers with bacterial cellulose (BC) sheets from Gluconacetobacter sucrofermentans. For this purpose, several model papers have been selected. They have been characterized before and after the lining with this BC and a specific Japanese paper (JP) to compare both materials. Taking into account the differences between bacterial and vegetal cellulose is expected that the results may be similar to other BC and JP. The samples have been characterized before and after an aging process. There are no significant differences in some of the characteristics studied. Nevertheless, BC-lined papers present higher gloss values and b* coordinate. The wettability decreases with both BC and JP. However, in papers lined with BC, the wettability decreases more markedly and independently of the model paper used. This is related to the sealing of the surface structure by BC, which also leads to a reduction of air permeability. When the lined papers go through an aging process, there are no significant changes in any characteristic, except in b* and L* color coordinates. Additionally, the wettability rate decreases in all cases. This study indicates that papers lined with BC are stable over time. Finally, the use of BC as reinforcing material may offer advantages for specific conservation treatments, being more suitable for certain types of paper than JP. © 2015, Springer Science+Business Media New York.

Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules.

A TCF bleaching sequence consisting of a urea pretreatment stage (U), laccase-mediator system stage (L), alkaline extraction stage (E), and hydrogen peroxide bleaching stage (P) was used to study the effect of five independent variables on the dependent variables pulp properties, hydrogen peroxide consumption, and residual enzyme activity. Results showed that the most influential variable was L stage pulp consistency, followed by mediator and laccase dosages. On the other hand, oxygen pressure did not have a significant effect. The optimal UL partial sequence significantly enhanced the EP bleaching sequence 49.8% vs. 33.4% delignification, up to 65.6% ISO vs. 56.3% ISO bleached brightness, and 50.3% vs. 89.9% peroxide consumption in the P stage. The ULE partial sequence also improved an ECF bleaching sequence (ULED0E1D1) 0.6 vs. 1.0 final kappa number; 82.7% ISO vs. 76.0% ISO brightness; and 54.1 N·m·g-1 vs. 51.9 N·m·g-1 and 3.3 KPa·m2·g-1 vs. 2.7 KPa·m2·g-1, tensile and burst indexes, respectively, when compared to the control D0E1D1 sequence.

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans CECT 7291 seems to be a good option for the restoration of degraded paper. In this work BC layers are cultivated and purified by two different methods an alkaline treatment when the culture media contains ethanol and a thermal treatment if the media is free from ethanol. The main goal of these tests was the characterization of BC layers measured in terms of tear and burst indexes, optical properties, SEM, X-ray diffraction, FTIR, degree of polymerization, static and dynamic contact angles, and mercury intrusion porosimetry. The BC layers were also evaluated in the same terms after an aging treatment. Results showed that BC has got high crystallinity index, low internal porosity, good mechanical properties and high stability over time, especially when purified by the alkaline treatment. These features make BC an adequate candidate for degraded paper reinforcement. © 2014 Elsevier Ltd.

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans has a high degree of crystallinity, durability, great resistance, and biocompatibility. This study evaluates the effect that carbon and nitrogen sources, present in HS (Hestrin-Schramm) culture medium, have on BC from G. sucrofermentans CECT 7291 used to restore damaged documents. The initial pH of the medium and the temperature were set for all assays, which were conducted in static conditions. The cellulose layers were obtained at four time points. The growth media were characterized at each time point (pH and carbon source consumption). Cellulose layers were washed, pressed, dried, and characterized by determining their pH, dry weight, and optical and mechanical properties. The best combination of carbon and nitrogen sources proved to be fructose plus yeast extract-corn steep liquor, with or without ethanol, which provided a good balance between BC production and carbon source consumption, and generated a resistant and homogeneous cellulose layer.

The effect of a pretreatment consisting of steam explosion (SE) followed by a laccase mediator system (LMS) stage on Eucalyptus globulus kraft pulping has been evaluated and compared with fungal pretreatments. Pretreatment with SE and LMS was more efficient than pretreatments using Pycnoporus sanguineus and Trametes sp. I-62. Steam explosion not only improved the enzyme penetration into the wood chips and shortened the pulping process by 60%, but also extracted around 50% of the hemicelluloses which could be converted into value-added products. The optimal conditions for the LMS treatment were 3h, 3UA/g and 40°C. Compared to SE, the SE/LMS treatment yielded an increase in delignification of 13.9% without affecting pulp properties, provided a similar screened kraft yield, and reduced consumption of chemical reagents Na2S and NaOH by 11.5% and 6.3%, respectively. Therefore, SE/LMS is a promising pretreatment for converting the pulp mill into a forest bio-refinery. © 2011 Elsevier Ltd.

As the fungal lacease production depends on some components which induce it, we have examined the independent effects of ethanol, wheat straw and copper sulphate and their combinations on the volumetric lacease activity of Pycnoporus sanguineus. Wheat straw and copper sulphate gave the best results showing a synergic effect after 13 days of incubation (laccase activity was 3 and 4.5 times greater than the activity generated when they were used alone). Ethanol on the contrary, was found to have a negative effect on lacease production. The lacease produced by the fungus Pycnoporus sanguineus maintains a good stability in a wide range of temperatures; a stability which also depends on the pH. By choosing the adequate pH, the level and stability of the lacease activity are satisfactory in a range of temperatures from 25 to 70°C, which would allow the diversification of lacease use in industrial applications.

The aim of this work was to obtain a LMS pre-treatment applicable to industrial TCF bleaching. Kraft pulp from Eucalyptus globulus was treated at 40 °C/pH 3 and 60 °C/pH 5 for 1 h using an extracellular fluid enriched in laccase produced by Pycnoporus sanguineus and acetosyringone as mediator (HBT was used as a control mediator) (L). Alkaline extraction (E) and hydrogen peroxide (P) stages were then assayed. The LEP alternative was an efficient sequence to bleach kraft pulp since the enzymatic pre-treatment boosted the subsequent chemical bleaching. The best L pre-treatment was obtained with laccase-acetosyringone at 40 °C/pH 3. It reduces kappa number and hexenuronic acids, increases pulp viscosity, lowers hydrogen peroxide consumption down to an 87.4% (94.0% without L) and enhances brightness up to a 59% ISO (51% ISO without L). © 2009 Elsevier Ltd.

Cellulose pulp bleaching is one of the main biotechnological applications of fungal laccases due to their capacity to degrade lignin from unbleached pulp. This application requires low cost enzyme production and higher enzyme concentrations than those obtained from the natural fungal producers. Heterologous expression of laccase in yeasts is an option for producing these enzymes on an industrial scale. In this work, we have demostrated the heterologous expression of the cglcc1 gene, responsible for laccase production in the basidiomicetous fungus Coriolopsis gallica, in the yeast Kluyveromyces lactis. In order to know if the transformed yeast has delignificant capability, a Pinus radiata kraft pulp has been incubated with it. After the treatment, a significant decrease in kappa number (13%) and in lignin content (22%) was observed. These results showed the delignificant capability of this transformed yeast. It can be concluded that the use of genetically modified microorganisms that do not demonstrate cellulolitic activity can produce high laccase levels and delignify cellulose pulps with a potential applications in cellulose pulp bleaching.

Pycnoporus sanguineus is a white-rot fungus that produces ligninolytic enzymes such as laccases. These enzymes can endure temperatures as high as 60 °C and are useful for pulp bleaching, dye decolorization and phenolic degradation. Laccase production by fungi depends not only on the carbon and nitrogen sources but also on the nitrogen concentration of the culture medium. In this work, we examined the effect of four carbon sources (maltose, glucose, fructose and sucrose) and four nitrogen sources (ammonium tartrate, sodium nitrate, asparagine and yeast extract) on the activity of laccase from Pycnoporus sanguineus. All carbon and nitrogen sources exhibited a strong influence on laccase activity, a sucrose-asparagine medium providing the best results (320 mU/ml). Moreover, using an asparagine concentration 5 times higher than the reference level increased laccase activity to 820 mU/ml. Higher asparagine concentrations, however, resulted in no further increase in activity. Consistent with previous results, the carbon and nitrogen sources, and the nitrogen concentration, had a strong impact on laccase activity, the optimum conditions depending on the particular fungus. The conditions of the culture medium had a marked effect on laccase activity, which increased up to 820 mU/ml. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kenaf (Hibiscus cannabinus L.) is a herbaceous annual plant amenable to use as a papermaking raw material. Kraft and soda pulping of kenaf have so far been done exclusively on the bark fraction (about 34-38% of the stem) or whole stem of the plant. Using kenaf bark exploits the higher quality of its bast fibers but reduces the typically high crop yields of this plant. In any case, core kraft pulp has acceptable properties some of which (e.g. tensile index, burst index) can even surpass those of bark pulp. Pulp made from both fractions has been found to exhibit better bonding properties than bark pulp. However, too high a proportion of core fibers can result in difficult drainage, a low tear strength or poor air permeability. These problems restrict the proportion of core that can be mixed with bast fibers, hinders separation of the two fractions and raises operational costs. The primary purpose of this study was to examine the influence of the core-bark ratio on the properties of mixed kenaf pulp. We used unrefined core pulp and refined bark pulp. Based on the results for kraft sacks, obtaining kenaf paper from both fractions has some advantages. Because Gurley air porosity changed dramatically with the proportion of core pulp used, it was used to determine the maximum amount of core fibers to be added to bast fibers. A proportion of up to 34% was found to have no adverse effect on air permeability. Such a proportion allowed paper strength to be preserved with an acceptable tear index (19.8 mN m2/g) and excellent tensile index (72 N m/g). Also, energy consumption was reduced if only the bark fraction was refined. The proposed strategy thus provides increased fiber yields of kenaf per hectare per year and valorizes the core fraction. © 2008 Elsevier B.V. All rights reserved.

Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins. © 2007 Elsevier Masson SAS. All rights reserved.

The capability of some ligninolytic fungi to degrade lignosulphonates has been studied. Three lignosulphonates concentrations, three culture media and seven different basidiomycetes in solid-cultures have been assayed to select the conditions for further experiments on submerged cultures. The best results of growth and lignosulphonate decolourization in solid-cultures were obtained with Pycnoporus sanguineus, Coriolus pubescens and Trametes sp. I-62 on Kirk's medium and 1% and 2% of lignosulphonate concentrations. In submerged cultures the lignosulphonate decolourization rate was generally higher when it was added on the 6th day, rather than when it was added from the beginning of the incubation and C. pubescens and P. sanguineus showed again the optimum results of decolourization. Extracellular laccase activity increased with lignosulphonate concentration in all assayed fungi, suggesting that lignosulphonate act as inductors of laccase activity. © 2007 Elsevier Ltd. All rights reserved.

Nine phenolic compounds (p-coumaric acid, ferulic acid, guaiacol, syringol, p-methoxyphenol, pyrocatechol, phloroglucinol, 3,5-dihydroxybenzoic acid, and syringaldazine) were tested for their ability to increase laccase production in the ligninolytic basidiomycete Trametes sp. I-62. All these compounds resulted in increases in laccase activity, with the highest levels being detected in the presence of p-coumaric acid (273-fold) and guaiacol (73-fold). The three laccase isozyme genes in this fungus lcc1, lcc2, and lcc3 are differentially expressed in the presence of some of these aromatics with total lcc transcript levels differing markedly depending on the aromatic compound tested. Guaiacol (the best inducer of lcc gene transcription) and p-coumaric acid selectively induced expression of lcc1 and lcc2; ferulic acid induced lcc3 expression, while 3,5-dihydroxybenzoic acid had no marked effect on laccase gene transcription. The results demonstrate that close-related aromatic compounds appear to have different effects on both laccase activity levels and lcc gene expression in this basidiomycete. © 2004 Elsevier Inc. All rights reserved.

The study of isozymes has to date been successfully applied in the identification of a number of different microorganisms, particularly fungi. Trametes sp. I-62 (CECT20197) is a white-rot fungus with potential biotechnological applications. It has been identified as Trametes on the basis of the mycelium morphology, but the features observed are not sufficient for its classification at the species level. Given that this strain produces a very characteristic laccase isozyme pattern, in the present work we compared its isozyme profile with the profiles produced by ten other very closely phylogenetically related fungi from the Polyporaceae family. The data indicates that Trametes sp. I-62 could not be included in any of the taxonomically related species which were analysed in the present work. Extracellular laccases were detected in nine of the fungi studied and in four of them namely Trametes subectypus, Trametes pavonia, Trametes ochracea and Trametes gallica, this is the first report of such activity. Given that phylogenetically related fungi growing in the same culture conditions exhibit different laccase patterns, these isozymes could be used as an additional criterion for fungal identification.

Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain 1-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain 1-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.