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Magnetic materials can be easily separated from reaction media by application of an external magnetic field. On the other hand, nanomaterials are innovative platforms which present high surface-to-volume ratio allowing low mass transfer limitations. Magnetic nanoparticles (MNPs) can be considered as supports for catalysts immobilization since they greatly improve their reutilization avoiding the need of energy and time consuming centrifugation steps. Enzyme immobilization processes providing high biocatalysts stability are very desirable due to enzyme associated costs. Laccase (EC 1.10.3.2), an oxidative enzyme with numerous industrial applications, requires new technologies for its immobilization in order to improve its biocatalytic activity with reduced costs. In this study, the conditions of laccase immobilization on magnetic nanoparticles were optimised by box-Benhken experimental design. Laccase was successfully bound on functionalized MNPs according to FTIR spectroscopy. At the optimal conditions, the highest recovery activity of immobilized laccase reached 36.3 U/L. Compared to free laccase, thermal stability of immobilized laccase was improved. The immobilized laccase was able to retain above 75% of activity after 6 consecutive cycles of reaction. MNPs can be used for immobilization of important enzymes at industrial level, as these nanomaterials can improve both enzymatic application properties and easy and fast recovery for reutilization.

Developments in nanotechnology have led to the discovery of new materials, namely, magnetic nanoparticles (MNPs), that present easy surface functionalization and high surface-to-volume ratios. These properties allow a high mass transfer rate and easy removal from a reaction matrix. Simple separation under an external magnetic field makes them a promising immobilization support for enzymes. In this work, new MNPs were prepared by functionalization with EDTA-TMS and characterized by TEM, FTIR and BET analytical techniques, among others. These MNPs were applied as support for laccase immobilization to create a promising biocatalyst. Despite the known chelating nature of EDTA-TMS, its use for surface modification of MNPs for laccase immobilization is a rather unexplored strategy and is reported here for the first time. At pH 3.5, the immobilization process showed approximately 97% of enzymatic activity recovery. The Michaelis-Menten kinetic properties of immobilized laccase showed a lower V max and a similar K M compared to free laccase. Regarding operational stability, the immobilized enzyme presented approximately 73% of its initial activity after five sequential reactive cycles. The immobilized enzyme was successfully applied to the biocatalysis of Indigo Carmine dye degradation. These MNPs with immobilized laccase showed important advantages compared to other materials for application in industrial biochemical processes, biocatalysis and biosensors.

Biofuels are suitable alternatives to conventional and fossil fuels that pose serious environmental adverse effects to society. Bioethanol is the most promising biofuel and can be generated from lignocellulosic biomasses. Forest residues are outside the human food chain and inexpensive raw materials, but they need a previous pretreatment, in order to improve the cellulose accessibility for further bioconversion. The study of bioethanol production from rockrose pretreated by steam explosion (SE) was carried out employing separate enzymatic hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) approaches. Saccharification of untreated rockrose attained only 0.9% of sugars yield. However, the steam explosion pretreatment promoted the disruption of interfibrillar surfaces of fibers with partial degradation of lignin thus enhancing the accessibility of polysaccharides toward enzymatic hydrolysis. Alkaline extraction after steam explosion pretreatment of rockrose residue (R-SE-OH) led to the partial removal of lignin, hemicelluloses, and other degradation products from fibre surface allowing an increase of 75% in the glucose yield. Bioethanol production in SSF mode was faster and slightly more efficient process than SHE providing the best results: ethanol concentration of 16.1 g L-1, fermentation efficiency of 69.8% and a yield of 22.1 g ethanol/100 g R-SE-OH. (C) 2015 Elsevier B.V. All rights reserved.

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3(3) experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 L L-1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70 degrees C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid). Kinetic studies showed that the values of Michaelis-Menten constant and maximum reaction rate decreased upon immobilization (9.9- and 1.6-fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.

Hardwood spent sulphite liquors (HSSLs) are by-products from the pulping industry rich in pentoses, which are not yet fully exploited for bioprocessing, namely for the production of bioethanol. The sustainable fermentation of pentoses into bioethanol is a challenge to overcome. Besides sugars, HSSLs contains inhibitors that decrease the possibility of bioprocessing of these by-products. Nevertheless, recent studies have brought new insights in using HSSLs for bioethanol production. This paper reviews the results of relevant studies carried out with HSSLs towards bioprocessing to bioethanol. The composition of SSLs was compared and related with the wood origin stressing specificity of microbial inhibitors from HSSL and their anti-microbial effect. The different fermentative processes, the microorganisms used, and the strategies to improve yield and productivity used so far were also reviewed. This review allowed concluding that research is still needed in several areas, including optimization of detoxification processes, fermentation strategies and selection of suitable microbial strains in order to achieve the integration of the different steps needed for HSSLs bioconversion into ethanol thus contributing for sustainability of pulping mills within biorefinary concept. (C) 2012 Elsevier Ltd. All rights reserved.

The stability and activity of laccase from Trametes versicolor in two water-soluble ionic liquids (ILs), namely 1-butyl-3-methylimidazolium methyl sulfate, [bmim][MeSO4] and 1,3-dimethylimidazolium methyl sulfate, [mmim][MeSO4], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first-order kinetics was followed. Inactivation rate constant (k), half-life time (t1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.

Laccase production from Trametes versicolor was improved in the presence of the inducers ligninosulphonates, veratryl alcohol, and xylidine respectively two-, four-, and eightfold. The thermal inactivation of the produced laccase, after partial purification with ammonium sulfate was kinetically investigated at various temperatures (60-70 °C) and pH values (3.5, 4.5, and 5.5). The inactivation process followed first-order kinetics for all conditions tested, except for veratryl alcohol, for which a constant activity level was observed at the end of the inactivation, also after first-order decay. Enzyme thermostability was affected by the type of inducer used in the culture medium for the production of laccase and also by the pH of incubation mixture. Generally, laccase stability increased with pH increment, being more stable at pH 5.5, except with xylidine. At pHs 4.5 and 5.5, the three inducers significantly increased laccase thermal stability, with the higher effect being observed for pH 5.5 and ligninosulphonates, where increment of half-life times ranged from 3- to 20-fold, depending on the temperature.

Summary Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium-alginate double-layer beads (ProMalic®) was studied in Erlenmeyer flasks and in a total recycle fixed-bed reactor operating in batch mode. The reaction is pseudo-first order with respect to l-malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol−1) in comparison with the free cells (126 ± 19 kJ mol−1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale-up of a recycle reactor to process large volumes of grape must.

The spent liquor from acidic sulphite pulping of Eucalyptus globulus (HSSL) is the side product from sulphite pulp production and besides sulphonated lignin contains sugars from degraded hemicelluloses, mainly pentoses. Pichia stipitis fermentation of these sugars for bioethanol production was the primary goal of this work. The increasing of HSSL proportion in fermentation media affected negatively the ethanol yield. Thus with 20% of HSSL (v/v) attained maximum ethanol yield was 0.15 g of ethanol by g of sugar consumed (g(e) g(s)(-1)) and with 60% (v/v) only 0.08 g(e) g(s)(-1). Biological removal of acetic acid from HSSL improved fermentation though the complete removal of acetic acid and polyphenolics (including sulphonated species) by treatment with ion-exchange resins was required for highly successful bioethanol production. Accordingly, the fermentative metabolic pathway of P. stipitis has been promoted allowing fair ethanol productivity and yield (Yp/s = 0.49 g(e) g(s)(-1)) at relatively low maximum of cell growth rate (micro(max) = 0.21 h(-1)).

New polyoxometalate–laccase integrated system (PLIDS) employing polyoxometalate [SiW 11 V V O 40 ] 5− and laccase of Trametes versicolor for the continuous delignification of eucalypt kraft pulp has been developed. Pulp was delignified in a batch reactor containing catalytic amounts of [SiW 11 V V O 40 ] 5− at about 90 °C under atmospheric pressure. Re-oxidation of reduced polyoxometalate (POM) with laccase was carried out at 45 °C in a separate aerated bioreactor coupled with an ultrafiltration tubular ceramic membrane. This allowed the separation of laccase from re-oxidized POM, which was supplied in turn continuously to the delignification reactor. Proposed PLIDS allowed sustainable pulp delignification with minimal degradation of polysaccharides. The implementation of PLIDS, instead the fist chlorine dioxide stage (D) in conventional DEDED bleaching sequence, showed almost 60% of chlorine dioxide savings with strength properties of the bleached pulp (90% ISO) similar to those obtained after the conventional bleaching.

Coriolus versicolor is a medicinal fungus producing exopolysaccharides (EPS). Five well-defined culture media were studied to select the medium that maximizes production of EPS by C. versicolor. Biomass, reducing sugars and EPS concentrations along with the rheological behaviour of the broth were followed during fermentations lasting 9 days. The yeast malt extract medium (YM) was shown to yield the highest production of EPS. Fermentation conditions with YM medium were further investigated to optimize EPS production by C. versicolor. An experimental design to do this was adopted, in which the effects of pH and initial substrate concentration were considered. The effects of initial glucose concentration (5, 15 and 25 g l−1) and pH (4.0, 5.5 and 7.0) were evaluated. The initial glucose concentration was found to be the most important factor in EPS production and also cell growth.

A series of manganese substituted polyoxotungstates, [SiW11MnIII(H2O)O39]5− and [PW11MnIII(H2O)O39]4−, and [SiW11VVO40]5− were applied as catalysts for the oxygen delignification of unbleached eucalypt kraft pulp with laccase of Trametes versicolor. Unlike to modest results obtained in the Laccase-Mediator System (LMS) at 45–60 °C (lignin oxidation and catalyst re-oxidation occurred at the same stage), a sustainable delignification with removal of about 50% of residual lignin was achieved with [SiW11MnIII(H2O)O39]5− and [SiW11VVO40]5− when the kraft pulp treatment was carried out with polyoxometalate at 110 °C (lignin oxidation stage) and with laccase at 45 °C (catalyst re-oxidation stage) in separate stages. The use of [PW11MnIII(H2O)O39]4− in this multi-stage process was limited by the low re-oxidation rate with laccase. The best selectivity on the pulp delignification was found with polyoxoanion [SiW11MnIII(H2O)O39]5−, whereas [SiW11VVO40]5− was the most effective in the oxidative delignification. The influence of process factors on the POMs re-oxidation, such as the amount of laccase, oxygen pressure and temperature is discussed. UV–vis and 51V NMR studies indicated that POMs maintained stable after redox turnovers during the pulp delignification.

A novel approach for the catalytic oxygen bleaching of kraft pulp using the heteropolyanion [SiW 11 Mn III (H 2 O)O 39 ] 5− (SiW 11 Mn III ) and laccase of Trametes versicolor is proposed. The oxidation of the residual lignin in pulp with the heteropolyanion was followed by the catalyst re-oxidation by laccase in a separate stage. The alterative treatment in a multi-stage process with SiW 11 Mn III at 110 °C and with laccase at 45 °C allowed a sustainable eucalypt kraft pulp delignification with removal of about 50% of the residual lignin with minimal polysaccharides damage. Heteropolyanion maintained stable after redox turnovers during the treatments.

A series of phenolic/non-phenolic biphenyl model compounds mimicking 5-5′ type “condensed” lignin substructures were subjected to the oxidation with laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole (HBT) or [SiW 11 VO 40 ] 5− (SiW 11 V) as mediators. Phenolic models suffered a significant degradation in both the laccase-mediator systems (LMS), which was more pronounced, however, in the case of SiW 11 V-mediated oxidation. This result was explained, at least partially, by HBT decomposition and by the increased extent of competing radical coupling reactions of phenolic models in the HBT–laccase reaction system. The non-phenolic biphenyl model was non-reactive in the presence of SiW 11 V and degraded substantially in the presence of HBT. The main degradation pathways of lignin model compounds were deduced based on the analysis of the detected oxidation products.

Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH > 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied.

Hardwood spent sulfite liquor (HSSL) is a by-product from pulping industry with huge potential for second generation bioethanol production, owing to its high sugars content, namely xylose and glucose. Scheffersomyces stipitis can use this substrate since it is able to ferment either hexoses or pentoses. Nevertheless, this yeast requires microaerophilic conditions for ethanol production, so the establishment of optimal aeration conditions is the main challenge for a successful ethanol production. The objective of this work was the establishment of an aeration strategy that allowed an efficient bioethanol production from HSSL by S. stipitis C4, an adapted strain to this substrate. Fermentations were carried out in a bioreactor using two aeration stages, with dissolved oxygen tension (DOT, %) control in the first aeration stage and with oxygen limitation in the second aeration stage. With this strategy, DOT control at high values in the first aeration stage allowed to obtain high specific growth rates and biomass yields. In addition, the DOT control at high values in the first stage also contributed to increase the ethanol productivity during the second aeration stage. In turn, the oxygen limitation in the second aeration stage stimulated the ethanol production and suppressed the growth of S. stipitis C4 in this second stage. This approach of two aeration stages resulted in a better control between the biomass and ethanol production. Using 60% HSSL, a conversion to ethanol of 74.4% was reached. The two stages aeration approach is a promising strategy to obtain second generation bioethanol from HSSL.

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.

A simple method of assessing the viability of a lactic starter culture is presented. The method is based on the kinetics of growth of a culture during the lag and early exponential phases. The microorganisms used throughout this work were Lactobacillus spp. The method was tested with inocula samples of different ages and with samples taken during chemostat runs, at different dilution rates. The results obtained are similar to ones describes in the literature for similar situations. The method is very easy to operate once the intrinsic parameters have been established (χmax and μmax). It needs only standard laboratory equipment and is very economical.