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O problema relacionado a deposicao segura dos rejeitos radioativos, sobretudo aqueles de alto nivel de radiacao, caso do combustivel nuclear usado dos reatores do Complexo Nuclear de Angra dos Reis, no estado do Rio de Janeiro, e ainda um gargalo no Brasil. No mundo inteiro, a principal solucao para manter em definitivo e em seguranca esses materiais perigosos e o repositorio geologico. Ambientes geologicos adequados, entretanto, relacionam-se a tipos de rochas especificas. Neste artigo, a importância dos depositos geologicos como depositos finais de rejeitos radioativos de alto nivel de radiacao e abordada, apontando as principais formacoes geologicas para o seu confinamento, bem como as diretrizes gerais relacionadas a esses depositos finais, incluindo os requisitos de seguranca e protecao radiologica aplicaveis ao projeto estrutural, a construcao, a operacao, ao encerramento, as salvaguardas e a garantia da qualidade. Sao, tambem, apresentados os principais aspectos a serem levados em consideracao para o projeto e construcao de um deposito geologico no Brasil.

The pulp and paper industry faces an emerging challenge for valorising wastes and side-streams generated according to the biorefinery concept. Eucalyptus globulus bark, an abundant industrial residue in the Portuguese pulp and paper sector, has a high potential to be converted into biobased products instead of being burned. This work aimed to evaluate the ethanol production from E. globulus bark previously submitted to kraft pulping through separate hydrolysis and fermentation (SHF) configuration. Fed-batch enzymatic hydrolysis provided a concentrated hydrolysate with 161.6 g L−1 of cellulosic sugars. S. cerevisiae and Ethanol Red® strains demonstrated a very good fermentation performance, despite a negligible xylose consumption. S. passalidarum, a yeast known for its capability to consume pentoses, was studied in a simultaneous co-culture with Ethanol Red®. However, bioethanol production was not improved. The best fermentation performance was achieved by Ethanol Red®, which provided a maximum ethanol concentration near 50 g L−1 and fermentation efficiency of 80%. Concluding, kraft pulp from E. globulus bark showed a high potential to be converted into cellulosic bioethanol, being susceptible to implementing an integrated biorefinery on the pulp and paper industrial plants.

In the context of the naturally occurring radioactive material associated with petrol exploitation, it is important to determine the concentrations of 226Ra and 228Ra in contaminated sludge originated from this activity and stored in packages. Although measurements based on chemical analyses of samples from package content can be done, difficulties associated with the collection and analyses of samples may be avoided by employing theoretical models based on the emitting radiation levels from the mentioned packages. This article presents a methodology to estimate 226Ra and 228Ra concentrations in sludge stored in cylindrical drums, employing a model developed in the Mathematica software. The model takes into consideration the radiation levels measured at one meter from the top surface of the waste package containing the contaminated sludge. It also takes into consideration packaging dimensions, waste density, time of measurement, among other factors, and allows the determination of all radionuclides concentrations in the sludge, helping to minimize the costs associated with sampling and analyses. Moreover, this proposed model is an alternative solution to pre-existing methods and can also assist operators, regulators, and decision-makers in the prediction of short- and long-term radiological impacts on human beings and on the environment, due to the disposal of waste containing naturally occurring radioactive materials.

Os rejeitos radioativos de alto nível e sua deposição final segura é um problema complexo e poucos países conseguiram dar uma solução adequada para os mesmos. Neste contexto, os repositórios geológicos apresentam-se como a única alternativa para a deposição definitiva desse tipo de passivo, cuja complexidade de seleção, construção, licenciamento ambiental, entre outros aspectos, são enormes. Os rejeitos de alto nível são gerados durante a fissão do urânio no combustível dos reatores provenientes das usinas nucleares. O combustível irradiado pode ainda ser reprocessado para a retirada de alguns produtos de fissão, como é o caso do césio-137, utilizado tanto na medicina quanto na indústria, quando então são chamados de rejeitos de alto nível. Atualmente, o Brasil não possui a parte do ciclo combustível que permitiria a retirada desses produtos de fissão e uma decisão precisa ser tomada, pois caso o combustível irradiado não seja reprocessado, o mesmo se torna um rejeito de alto nível. Os repositórios geológicos, segundo a International Atomic Energy Agency (IAEA), são considerados a principal e mais adequada solução para a deposição dos rejeitos radioativos de alto nível que permanecem perigosos por milhões de anos e precisam ser isolados completamente da biosfera. Portanto, este artigo, baseado em documentos da IAEA e da Comissão Nacional de Energia Nuclear (CNEN), aponta os principais aspectos que devem ser considerados para o licenciamento de depósitos geológicos, incluindo os aspectos de proteção radiológica mais importantes, os requisitos de avaliação de segurança necessários, além de outros aspectos que devem ser levados em consideração durante a seleção, planejamento, construção, operação e fechamento de um depósito geológico.

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.

Second-generation bioethanol production’s main bottleneck is the need for a costly and technically difficult pretreatment due to the recalcitrance of lignocellulosic biomass (LCB). Chemical pulping can be considered as a LCB pretreatment since it removes lignin and targets hemicelluloses to some extent. Chemical pulps could be used to produce ethanol. The present study aimed to investigate the batch ethanol production from unbleached Kraft pulp of Eucalyptus globulus by separate hydrolysis and fermentation (SHF). Enzymatic hydrolysis of the pulp resulted in a glucose yield of 96.1 ± 3.6% and a xylose yield of 94.0 ± 7.1%. In an Erlenmeyer flask, fermentation of the hydrolysate using Saccharomyces cerevisiae showed better results than Scheffersomyces stipitis. At both the Erlenmeyer flask and bioreactor scale, co-cultures of S. cerevisiae and S. stipitis did not show significant improvements in the fermentation performance. The best result was provided by S. cerevisiae alone in a bioreactor, which fermented the Kraft pulp hydrolysate with an ethanol yield of 0.433 g·g−1 and a volumetric ethanol productivity of 0.733 g·L−1·h−1, and a maximum ethanol concentration of 19.24 g·L−1 was attained. Bioethanol production using the SHF of unbleached Kraft pulp of E. globulus provides a high yield and productivity.

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.

Hardwood spent sulfite liquor (HSSL) is a by-product from pulp industry with a high concentration of pentose sugars, besides some hexoses suitable for bioethanol production by Scheffersomyces stipitis. The establishment of optimal aeration process conditions that results in specific microaerophilic conditions required by S. stipitis is the main challenge for ethanol production. The present study aimed to improve the ethanol production from HSSL by S. stipitis through a two-stage aeration fermentation. Experiments with controlled dissolved oxygen tension (DOT) in the first stage and oxygen restriction in the second stage were carried out. The best results were obtained with DOT control at 50% in the first stage, where the increase of oxygen availability provided faster growth and higher biomass yield, and no oxygen supply with an agitation rate of 250 rpm, in the second stage allowed a successful induction of ethanol production. Fermentation using 60% of HSSL (v/v) as substrate for S. stipitis provided a maximum specific growth rate of 0.07 h−1, an ethanol productivity of 0.04 g L h−1 and an ethanol yield of 0.39 g g−1, respectively. This work showed a successful two-stage aeration strategy as a promising aeration alternative for bioethanol production from HSSL by S. stipitis.

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.

Bioethanol production potential from cardoon ( Cynara cardunculus ) and rockrose ( Cistus ladanifer ), pretreated by diluted acid hydrolysis (DAH), was studied and compared by both, separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. DAH pretreatment allowed an increase of 5 fold to cardoon and 16 fold to rockrose in cellulose accessibility towards enzymatic hydrolysis. Both plants showed better overall fermentation efficiency and ethanol yield in SSF than in SHF also with shorter processing periods, 24 h against 72 + 24 h, respectively. Results obtained for rockrose are about half the concentration and volumetric productivity of ethanol and 60% of the overall fermentation efficiency of those obtained for cardoon. The recalcitrance of rockrose to enzymatic hydrolysis was explained by higher abundance of lignin and extractives than in cardoon. Comparing the steam explosion (SE) and DAH pretreatments, the former was more advantageous for the recalcitrant rockrose in terms of enzymatic saccharification yield., This research was funded by INALENTEJO 2007.2013/QREN (BioEcos Project – Valorização Integrada de Biomassa (ALENT-09-0140-FEDER-000705 (QREN)). The author’s thanks, FCT (Fundação para a Ciência e Tecnologia) under projects CICECO-Aveiro Institute of Materials (Ref.FCTUID/CTM/50011/2013) and ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas (Ref.FCTUID/AGR/00115/2013). Agrarian School from Instituto Politécnico de Beja and Associação de Defesa do Património de Mértola, are greatly acknowledged for kindly supplying cardoon and rockrose, respectively.

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)).

BACKGROUND: A sequential batch reactor (SBR) was used for eucalypt kraft pulp effluent treatment with Trametes versicolor. A 23 full factorial design and response surface methodology were applied to optimise the batch fermentation conditions. Effluent concentration, culture medium and inoculum age were the factors selected for this study in order to optimise the reduction of chemical oxygen demand (COD). RESULTS: The presence of Trametes Defined Medium (TDM) in the fermentation was required to obtain a significant COD reduction. Experiments in the batch reactor confirmed, in general, the predicted results of optimisation developed from Erlenmeyer batch assays. The T. versicolor culture remained active during 42 days of study in the SBR, providing approximately 80% of COD reduction. CONCLUSION:Trametes versicolor may be considered as very promising for the biological treatment of effluents from kraft pulp mills in an SBR system instead of the activated sludge mixed cultures traditionally used. Copyright © 2008 Society of Chemical Industry

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.

Improvement of laccase production by Trametes versicolor was made by employing different operational strategies. In the cell growth medium, various glucose concentrations were compared for improving laccase production. A clear and significant stimulation of enzyme production under carbon limitation was obtained. Copper, 2,5-xylidine, and a phenolic mixture were also used as laccase inducers. A cooperative effect between the inducers on laccase production was identified. Mixtures of inducers produced higher laccase activities, reaching values of 5500Udm −3 . Further productivity enhancement can be obtained using the inducers along with the carbon limitation strategy. It is shown that low laccase concentrations are obtained by a primary metabolism of Tv ersicolor, and that phenolic compounds and carbon limitation induce a secondary metabolism, providing higher laccase concentrations. A mathematical model for laccase production based on a direct experimental measure of biomass, along with substrate consumption and enzymatic activity over time is proposed for non-homogeneous fermentations of Tv ersicolor.  2005 Society of Chemical Industry

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.

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L-1, fermentation efficiency of 66.6% and a yield of 26.6 g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon. (C) 2015 Elsevier Ltd. All rights reserved.

Background: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. Results: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C-4) was isolated and characterized in 60% undetoxified HSSL. C-4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C-4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C-4. Conclusion: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C-4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C-4 may represent a major advantage for the production of bioethanol using HSSL as substrate.

Mechanical stress due to micro/ultrafiltration has been suggested by a number of authors as one of the sources for cell deactivation in continuous cell-recycle reactors. This work introduces cell deactivation in the modelling of cell-recycle fermenters. A two-population based model is used. It is shown that the kinetic parameters obtained from chemostat fermentations describe accurately the experimental results and the apparent deviations to the Luedeking-Piret relationship at low growth rates are explained. The model is applied to the data of two authors with dilution rates (D) ranging 0.2 and 1.05h ˇ1 and using either Lactobacillus rhamnosus or Lactococcus cremoris cells. Although only data for lactic fermentation were used, the proposed model is of general applicability and not restricted to a type of fermentation, a given apparatus configuration or Newtonian fluids. The development of improved procedures for biological reactor modelling, such as presented in this work, wall allow more adequate and efficient design and operation of these reactors. # 2000 Society of Chemical Industry

Image analysis technique was applied to identify morphological changes of pellets from white-rot fungus Trametes versicolor on agitated submerged cultures during the production of exopolysaccharide (EPS) or ligninolytic enzymes. Batch tests with four different experimental conditions were carried out. Two different culture media were used, namely yeast medium or Trametes defined medium and the addition of lignolytic inducers as xylidine or pulp and paper industrial effluent were evaluated. Laccase activity, EPS production, and final biomass contents were determined for batch assays and the pellets morphology was assessed by image analysis techniques. The obtained data allowed establishing the choice of the metabolic pathways according to the experimental conditions, either for laccase enzymatic production in the Trametes defined medium, or for EPS production in the rich Yeast Medium experiments. Furthermore, the image processing and analysis methodology allowed for a better comprehension of the physiological phenomena with respect to the corresponding pellets morphological stages., The authors acknowledge Portucel-Empresa de Celulose e Papel, Cacia, Portugal, SA for the pulp and paper Kraft effluent used in this work. This work was funded by FEDER Funds through the Programa Operacional Factores de Competitividade-COMPETE, and national funds through FCT-Fundacao para a Ciencia e a Tecnologia under the projects PEst-C/CTM/LA/0011/2013 and PEst-C/EQB/LA0020/2013. A. P. M. Tavares acknowledge the financial support of (Programme Ciencia 2008) FCT, Portugal.

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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.

Fermentation with complete cell recycle on microfiltration tubular membranes was studied and its effect upon broth rheology evaluated. The increase in cell concentration was found to be responsible for a continuous rise in viscosity leading to changes of broth rheology and consequent operating problems. A strategy to control the broth rheological behaviour with the aim of achieving long-term stable fermentations is proposed. This strategy involves the use of a cell purge in order to maintain cell concentration at a manageable level. The results obtained show that the total cell concentration in the fermenter can be stabilised if the cell bleed rate is calculated to remove exactly the amount of cells produced. To obtain long-term stable fermentations, the time to initiate the bleeding procedure and the cell bleed rate need to be determined and optimised. © 1997 SCI.

An approach for the integrated utilization of grape skins from white grape pomaces has been proposed. This consists of consecutive or simultaneous extraction of grape skins with neutral organic solvent and water under reflux. Organic extract is a valuable raw material for the isolation of oleanolic acid. The aqueous extract (ca 50%, w/w) is composed of essentially hexoses and suitable for the high yield (till 51%) bioethanol production at a maximum specific cell growth rate (mu(max)) of 0.29 h(-1). The remained extracted grape skins are the complex of structural polysaccharides embedded into cutinous matrix. Extracted grape skins were shown to be a prospective raw material for the production of low-density boards (d

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.

The world is facing a reduction of global fossil fuels resources, like petroleum, natural gas, or charcoal, while energy requirements are progressively growing up. Fossil fuels should be replaced, at least partially, by biofuels once the current fuel supply is suspected to be unsustainable in the foreseen future. In fact, the search for sustainable alternatives to produce fuel and chemicals from non-fossil feedstocks has attracted considerable interest around the world, to face the needs of energy supply and to response to climate change issues. Alternative resources of energy are being explored in order to reduce oil dependence and increase energy production by exploring of solar, wind, hydraulic and other natural phenomena. Besides these sources of energy, also biomass possesses a potential target for fuel and power production as well as for chemicals or materials feedstocks. Thus biomass can efficiently replace petroleum-based fuels for a long term. (Sanchez et al. 2008; AlvaradoMorales et al. 2009; Brehmer et al. 2009; Gonzalez-Garcia et al. 2009; Singhania et al. 2009; Mussatto et al. 2010; Sannigrahi et al. 2010). Many countries in Europe, North and South America and Asia are replacing fossil fuels by biomass-based fuels according to international regulations. One of the directives of European Union (2009/28/CE) imposes a quota of 10% for biofuels on all traffic fuel until 2020 (Rutz et al. 2008; Xavier et al. 2010). Also economic incentives for research on biofuels are being implemented all over the world. Bioethanol can be produced from different raw materials containing simple sugars, starch or more complex substrates as lignocellulosics. New methodologies for biofuels (e.g. ethanol and biodiesel) production have been developed in the last years, to achieve new and non cost-intensive technologies for bioconversion of lignocellulosic renewable resources. The most common renewable fuel is ethanol, which is produced from direct fermentation of sugars (e.g. from sucrose of sugarcane or sugar beet) or polysaccharides (e.g. starch from corn and wheat grains) (Gonzalez-Garcia et al. 2009; Mussatto et al. 2010). The selection of the best raw material is strongly dependent on the local conditions where feedstock is obtained. Evidently, ethanol in Brazil is produced from sugarcane, whereas, in North America or Europe the ethanol industry is based on starchy materials. Besides, energy considerations should be attained: not only the energy input required for ethanol production and the content in fermentable

Sulphite spent liquor (SSL) is a side product from acidic sulphite pulping of wood, which organic counterpart is composed mainly by lignosulphonates (LS) and sugars. The last are a prominent substrate for the bioprocessing although a previous purification step is necessary to eliminate microbial inhibitors. In this study a fractionation of hardwood SSL (HSSL) has been accomplished employing ion exchange resins in order to separate sugars fraction from concomitant inhibitors: LS, acetic acid, furan derivatives, phenolics, acetic acid and excess of inorganic salts. The fractionation of HSSL has been carried out using two fixed-bed ion exchangers in series (cationic+anionic). The first cation exchange column packed with Dowex 50WX2 resin was able to eliminate free cations and partially separate sugars from high molecular weight LS and furan derivatives. The second anion exchange column packed with Amberlite IRA-96 sorbed remaining LS, phenolics and acetic acid. Overall, the series arrangement under investigation has removed 99.99% of Mg(2+), 99.0% of Ca(2+), 99.6% of LS, and 100% of acetic acid, whereas the yield of recovered sugars was at least 72% of their total amount in HSSL.

The fermentation of reducing sugars from hardwood (eucalypt) spent sulphite liquor (HSSL) into ethanol by Pichia ( Scheffersomyces ) stipitis is hindered by concomitant inhibitors of microbial metabolism. The conditions for the HSSL biological treatment step by Paecilomyces variotii were evaluated and optimised. Two different strategies of reactor operation were compared using single batch (B) and sequential batch reactor (SBR). Biological treatment of HSSL in the SBR revealed the best results with respect to the removal of microbial inhibitors. Also, most of inhibitory compounds, acetic acid, gallic acid, pyrogallol, amongst others, were removed from HSSL by P. variotii before the ethanol fermentation. The bio-detoxified HSSL was subjected to a successful fermentation by P. stipitis , attaining a maximum ethanol concentration of 2.4 g L −1 with a yield of 0.24 g ethanol g sugars −1 .

The operation of cell recycle systems by membrane filtration is discussed, stressing the importance of the mutual interaction between the biological and the physical characteristics of the fermentation process. Broth circulation flow rate, dilution rate, and cell bleed rate are identified as being the most important process variables. Their effects on process performance and criteria for its adequate selection are analysed. Fermentation results for different acidogenic microorganisms are presented and the kinetic modelling of high cell density recycling systems is discussed.