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Biochemical Engineering Journal (v.67, #)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

Addition of protease during starch liquefaction affects free amino nitrogen, fusel alcohols and ethanol production of fermented maize and whole and decorticated sorghum mashes by Esther Perez-Carrillo; Sergio O. Serna-Saldivar; Cristina Chuck-Hernandez; M. Luisa Cortes-Callejas (pp. 1-9).

► Decortication of sorghum improves protein hydrolysis and FAN contents. ► Protease addition during liquefaction improves FAN contents in sorghum. ► The use of protease improved fusel alcohol concentrations after yeast fermentation. ► Sorghum decortication and protease addition had a synergistic effect on ethanol. ► Decorticated sorghum treated with protease yielded more ethanol compared to maize.The aim of this research was to study the effect of the dual treatment of sorghum decortication and protease addition during liquefaction with α-amylase, on the concentration of free amino nitrogen (FAN), fusel alcohols and ethanol during yeast fermentation. A bifactorial experiment was designed to revise the differences among grains (maize, whole and 9.7% decorticated sorghum) and the effectiveness of protease addition during liquefaction. The decorticated sorghum was more susceptible to protein hydrolysis compared to the whole kernel sorghum due to its lower fiber content. The protease improved the levels of FAN approximately to 60% and 30% in the maize and sorghum mashes, respectively. The maize mash contained the highest amount of FAN followed by the decorticated and whole sorghum mashes. The protease treatment improved the fusel alcohol concentration in both sorghum beers and did not affect ethanol concentration in the maize mash. Both sorghum decortication and protease addition during liquefaction are therefore recommended treatments to obtain mashes with a higher FAN level. The decorticated protease-treated sorghum mashes yielded higher amounts of ethanol compared to the maize treatments and produced the maximum ethanol after only 20h of fermentation. This research proved that a positive synergistic effect on FAN concentration, fusel alcohols and bioethanol yields can be achieved through the proposed proceeding of sorghum decortication and protease addition.

Keywords: Sorghum; Decortication; Protease; Free amino nitrogen; Fusel alcohols; Bioethanol

Stability and performance of two GSBR operated in alternating anoxic/aerobic or anaerobic/aerobic conditions for nutrient removal by Ahlem Filali; Angéla Mañas; Myriam Mercade; Yolaine Bessière; Béatrice Biscans; Mathieu Spérandio (pp. 10-19).

► Good physical properties for granules were obtained with anoxic/aerobic cycles. ► Pre anoxic phase diminishes the microbial competition for oxygen but limits the SND. ► Instability of GSBR with anaerobic/aerobic phases was due to the filamentous growth. ► Maximizing COD fraction stored anaerobically is a key parameter for process stability. ► Comparable P removal efficiency was observed in both systems. ► P removal is mainly due to precipitation in anoxic/oxic and EBPR in anaerobic/oxic process.Two granular sludge sequencing batch reactors (GSBR) with alternating anoxic/aerobic (R1) and anaerobic/aerobic (R2) conditions were operated with a 4-carbon-source synthetic influent. The physical properties of the granular sludge were very good (SVI≈20mLg⁻¹) and high solid concentrations (up to 35gL⁻¹) were obtained in the bioreactor operated with a pre-anoxic phase with additional nitrate (R1). In contrast, performance and granule settleability were lower in R2 due to the development of filamentous heterotrophic bacteria on the surface of granules. These disturbances were linked to the fact that a fraction of COD remained during the aerobic phase, which was not stored during the anaerobic period. To stabilize a GSBR with a mixture of organic carbon sources, it is thus necessary to maximize the amount of substrate used during the non-aerated, anaerobic or anoxic, phase. Comparable phosphate removal efficiency was observed in both systems; enhanced biological P removal being greater in anaerobic/aerobic conditions, while the contribution of precipitation (Ca–P) was more significant in anoxic/aerobic conditions.

Keywords: Aerobic granulation; Nitrification; Denitrification; Phosphorus; Filamentous; Precipitation

Enzymatic characterization of highly stable human alpha-galactosidase A displayed on magnetic particles by José L. Corchero; Rosa Mendoza; Neus Ferrer-Miralles; Anna Montràs; Lluís M. Martínez; Antonio Villaverde (pp. 20-27).

► The human α-galactosidase A enzyme has been coated onto magnetic microparticles. ► His-tag directed anchoring results in better performance than covalent conjugation. ► Immobilized enzyme shows better activity and stability than the soluble form. ► Coating onto magnetic carriers allows the recycling of the immobilized catalyst.The human α-galactosidase A (EC 3.2.1.22, GLA), a lysosomal enzyme with important biotechnological and biomedical applications, has been successfully immobilized for the first time onto different versions of micro-sized magnetic particles by means of alternative coupling chemistries (covalent and metal affinity adsorption). The immobilized enzyme shows higher specific activity than its soluble counterpart and its enhanced stability as well as the magnetic-controlled positioning and reusability provided by coupling make these new bioconjugates excellent platforms for the presentation of highly active and pure versions of human GLA for both in vitro catalysis and therapeutic applications.

Keywords: Immobilized enzymes; Human alpha-galactosidase A; Magnetic microparticles; Biocatalysis; Microcarriers; Enzyme activity; Operational stability

Immobilization of soybean peroxidase on aminopropyl glass beads: Structural and kinetic studies by Tatiana Marchis; Giuseppina Cerrato; Giuliana Magnacca; Valentina Crocellà; Enzo Laurenti (pp. 28-34).

.Display Omitted► We immobilized soybean peroxidase (SBP) on aminopropyl glass beads. ► The system was characterized by different spectroscopic techniques. ► The immobilization process slightly modify the Fe(III)-heme catalytic site of SBP. ► The system retain a good percentage of catalytic activity and resist to storage.The enzyme soybean peroxidase (SBP) is able to catalyze the oxidation of a large number of substrates and is characterized by high resistance to both chemical and thermal denaturation. In this contribution SBP was covalently immobilized on aminopropyl glass beads (APG) in order to obtain a solid biocatalyst, useful either in degradation of pollutants or in specific oxidative reactions. Several samples of immobilized SBP were first synthesized and then characterized by means of some experimental techniques (FT-IR, ESR, and UV–visible spectroscopies, gas-volumetric adsorption of nitrogen at 77K, SEM). Moreover, different kinetic measurements were carried out to determine activity and stability properties of these biocatalysts. Our data indicate that (i) the SBP catalytic site was partially modified during the immobilization process, but the enzyme retained about 35% of its specific activity after immobilization, and (ii) the biocatalyst exhibits a significant improvement of SBP stability over time, preserving up to 50% of its initial activity after 70 days of storage and 85% when used in ten consecutive reaction cycles.

Keywords: Immobilization; Immobilized enzymes; Heterogenous biocatalysis; Enzyme activity; Soybean; Peroxidase

Textural characteristics, physiochemical properties and adsorption efficiencies of Caribbean alga Turbinaria turbinata and its derived carbonaceous materials for water treatment application by S. Altenor; M.C. Ncibi; E. Emmanuel; S. Gaspard (pp. 35-44).

.Display Omitted► From low cost Caribbian alga, Turbinaria turbinata, several adsorbents were produced. ► The biomass and its thermo-chemically derived materials were characterized. ► Kinetics and isotherms assays were performed and modeled. ► Biomass modification enhanced significantly the sorption capacity. ► Production yield was estimated for a more reliable assessment of sorbent efficiency.The adsorptive removal capacities of highly available Turbinaria turbinata alga and its derived carbonaceous products (i.e. pyrolyzed, physically and chemically activated carbons) were investigated in this study. Several textural and chemical characterizations were performed on the alga and its activated carbons (ACs). Besides, kinetics and isotherms assays were performed and modeled in order to monitor the sorption capacities and dynamic behaviors.The main results showed that the raw Turbinaria biomass has a non porous structure. Then, after thermo-chemical treatments, a porous matrix starts to develop and the total pore volume drastically increased from 0.001cm³/g for the algal precursor (turb-raw) to 1.316cm³/g for its derived chemically AC (turb-P1). As well, the specific surface area improved from m²/g for (turb-raw) to 1307m²/g for (turb-P1). Consequently, the maximum sorption capacity went from 63mg/g for the algal biomass up to 411mg/g for the chemically ACs.Moreover, the removal rate was taken into consideration in order to set a more reliable and realistic approach to figure out the most efficient AC.Thus, based on those criteria, it was found that the chemically activated carbon “turb-P1” is the most efficient Turbinaria-derived sorbent to adsorb and remove methylene blue (MB) molecules from aqueous solutions with 169g of the dye using 1kg of raw alga (considering an AC production yield of 49%).

Keywords: Alga; Activated carbons; Characterization; Adsorption; Modeling; Waste-water treatment

Mechanistic study of on-site sludge reduction in a baffled bioreactor consisting of three series of alternating aerobic and anaerobic compartments by Feng Quan; Yu Anfeng; Chu Libing; Chen Hongzhang; Xin-Hui Xing (pp. 45-51).

► A bioreactor repeated coupling of aerobic and anaerobic (rCAA) was constructed. ► Sludge on-site minimization in the rCAA was realized in long time running process. ► Mechanism of sludge reduction was systematically elucidated by multi-approaches. ► Microbial community along the flow direction in rCAA was spatially diversified.A baffled reactor with repeated coupling of aerobic and anaerobic conditions (rCAA) was constructed and the mechanism of the on-site sludge reduction was analyzed systematically. During the long-term operation of 3 years, the chemical oxygen demand (COD) removal efficiency reached over 90% on average and the sludge yield in the rCAA system was 0.098kg suspended sludge (SS) kg⁻¹ COD, which was approximately 36.3% of that in a conventional activated sludge process at an influent COD of 700–800mgL⁻¹ and hydraulic retention time (HRT) of 15h. Analysis of soluble components, sludge characteristics, microorganism community and metabolic activities in the rCAA system showed that, when the sludge in the aerobic zone flowed into the anaerobic zone, sludge destruction and degradation occurred. Intracellular organic substances were then released and further hydrolyzed to small molecular compounds. These intermediate substances were degraded in subsequent aerobic compartments. In this way, cryptic growth of the sludge was realized that could contribute to sludge reduction. Microbial ecological analysis by denaturing gradient gel electrophoresis (DGGE) further showed that the rCAA process spatially domesticated the different microbial communities along the flow direction of the bioreactor, which enhanced excess sludge lysis and transformation, cryptic growth and bacterial predation.

Keywords: On-site sludge reduction; Baffled bioreactor; Aerobic processes; Anaerobic processes; Waste-water treatment; Environmental preservation

The effect of organic loading rates and nitrogenous compounds on the aerobic granules developed using low strength wastewater by Yet Nee Peyong; Yan Zhou; Ahmad Zuhairi Abdullah; Vel Vadivelu (pp. 52-59).

► Aerobic granules (AG) can be developed and maintained using low organic loading rate (OLR) of 0.6kg/m³ day. ► AG immediately responded to the increase in substrate after starvation for 2 months. ► The OLR and type of wastewater influence the morphology and structure of AG. ► AG was more sensitive to FNA, than FA.The changes in microbial morphologies and structures of aerobic granules under different organic loading rates (OLR) and their tolerance toward free ammonia (FA) and free nitrous acid (FNA) were studied in this research. The development of aerobic granules was carried out in 3 stages using sequencing batch reactor, (i) synthetic wastewater with OLR of 1.2kg/m³day, (ii) low strength real domestic wastewater with OLR of 0.13kg/m³day, and (iii) domestic wastewater with added external carbon source to give an OLR of 0.6kg/m³day. It was found that OLR as low as 0.6kg/m³day was able to develop and maintain the integrity of aerobic granules. After a starvation period, aerobic granules were capable to immediately responded to the increase in substrate. Microscopic images showed that different microbial morphologies and structures were found in aerobic granules at the three different stages. The inhibitory studies of FA and FNA on the activity of aerobic granules reveals that FA at a concentration up to 36.9mg NH₃-N/L did not inhibit the oxygen uptake rate (OUR) of aerobic granules. However, FNA started to inhibit the OUR at 0.015mg HNO₂-N/L and completely stopped the oxygen consumption at around 0.134mg/L HNO₂-N/L.

Keywords: Low strength wastewater; Aerobic granules; Free ammonia and free nitrous acid inhibition; Granular morphology

Digital image analysis and fractal-based kinetic modelling for fungal biomass determination in solid-state fermentation by Duan Yingyi; Wang Lan; Chen Hongzhang (pp. 60-67).

► Influence of mycelia growth on the morphological change of matrix was explained. ► Kinetics of the variation of fractal dimension and fungal biomass were modeled. ► Model parameters of fractal kinetics showed specificity for fungal growth. ► Combined dynamic imaging and kinetic models is feasible in determining biomass.This work deals with a non-destructive method involving image analysis and kinetic modelling to determine fungal biomass in solid-state fermentation (SSF). Fractal dimension, quantifying the morphological changes of mycelia-matrix from culture images, showed correlations with Penicillium decumbens biomass on lignocelluloses substrates. Kinetic models were constructed to describe the variation of fractal dimension of mycelia-matrix along with fungal growth. Fermentations on straw substrates with different particle lengths and moisture contents were carried out to validate the proposed models. Relative errors of the models were 0.541–5.221% for biomass and 0.454–3.885‰ for fractal dimension. Parameters δ and η in fractal kinetic models, which indicated the variation rates of fractal dimension, presented significant specificity for the specific growth rate of P. decumbens, thus can be used to predict fungal biomass in SSF. With advantages of low cost, reasonable accuracy and well adjustability, the coupling of dynamic imaging and computational modelling show potential in the on-line determination of fungal biomass in SSF.

Keywords: Image analysis; Fractal dimension; Modelling; Growth kinetics; Biomass; Solid-state fermentation

Characterization of aqueous biphasic systems composed of ionic liquids and a citrate-based biodegradable salt by Helena Passos; Ana R. Ferreira; Ana Filipa M. Cláudio; João A.P. Coutinho; Mara G. Freire (pp. 68-76).

► Novel ABS composed of ionic liquids and potassium citrate are presented. ► 15 ionic liquids were evaluated toward their ability to form ABS. ► The effect of the structural features of the ionic liquid is discussed. ► The ionic liquids aptitude to form ABS is controlled by their hydrophobicity. ► The partitioning ofl-tryptophan among the two aqueous phases was addressed. ► Single-step extraction efficiencies range between 72% and 99%.Albeit ionic-liquid-based aqueous biphasic systems (ABS) have been largely explored as liquid–liquid extractive approaches for a large array of (bio)molecules, the application of biodegradable and nontoxic salts as phase constituents of these systems has been seldom investigated. In this work 15 ionic liquids were evaluated toward their ability to form ABS in the presence of a common biodegradable organic salt: potassium citrate. The ternary phase diagrams, tie-lines, and respective tie-line lengths, were determined at 25°C. The gathered data allowed the evaluation of the effects of the ionic liquid cation core, of the cation side alkyl chain length, and of the anion nature, to form two-phase systems. It is shown that the ionic liquids aptitude to undergo liquid–liquid demixing is mainly controlled by their hydrophobicity. The large differences observed between the phase diagrams behavior suggest the possibility of tailoring the aqueous phases’ polarities for a specific extraction. Therefore, the partitioning of a hydrophobic amino acid produced by bacteria fermentation,l-tryptophan, was also addressed aiming at exploring the applicability of the proposed systems in the biotechnology field. Single-step extraction efficiencies ofl-tryptophan for the ionic-liquid-rich phase range between 72% and 99%.

Keywords: Aqueous biphasic system; Aqueous two-phase system; Ionic liquid; Potassium citrate; Phase diagram; l; -Tryptophan; Partition coefficient; Extraction efficiency

Production of single-chain Fv–Fc fusion protein in stably transformed insect cells by Hiroyuki Sonoda; Yoichi Kumada; Tomohisa Katsuda; Hideki Yamaji (pp. 77-82).

► Stably transformed lepidopteran insect cells secreting scFv–Fc fusion protein. ► Use of a Drosophila Bip secretion signal resulted in a high production level. ► Yield of 60mg/l scFv–Fc was achieved in a shake-flask culture. ► Supplementing the medium with sericin improved culture viability and product yield.We describe the secretory production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in stably transformed lepidopteran insect cells. Use of the insect-derived Bip and melittin signal peptides resulted in higher yields of the secreted scFv–Fc fusion protein than use of the baculovirus gp64 signal peptide. After cotransfection with an expression vector that contains the Bip signal sequence upstream of the DNA encoding the scFv–Fc and a selection vector that carries a neomycin resistance gene, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418. Colonies of resistant cells were obtained around 2 weeks after adding G418, and clonal cells were screened by enzyme-linked immunosorbent assay (ELISA) of the culture supernatant. The yield of the scFv–Fc protein secreted from the most productive clone was around 60mg/l in a shake-flask culture. To improve the productivity, we investigated the effect of medium supplements of sodium butyrate (NaBu), dimethyl sulfoxide (DMSO), and sericin. Supplementing culture medium with sericin increased the scFv–Fc protein yield to 82mg/l, but productivity was not increased by either NaBu or DMSO. These results indicate that the stably transformed insect cells may allow for the efficient production of scFv–Fc and other Fc fusion proteins.

Keywords: Insect cell culture; Recombinant protein production; Stable expression; Single-chain variable fragment; Fc fusion protein; Medium supplement

Increase in lipids production by Pichia angusta DL-1 utilizing the chemostat under double limitation of heterologous nutrients by Romina Álvarez; Fernando Acevedo (pp. 83-87).

► The double limitation of heterologous nutrients can increase the lipid content of the yeast Pichia angusta DL-1. ► The largest accumulation of lipids occurred under nitrogen/phosphorus limitation and carbon/magnesium limitation. ► The content of lipids was twice the one obtained in batch culture and 15% larger than under simple limitation. ► Carbon and magnesium double limitation as the most adequate condition to obtain the highest lipid and cell volumetric productivities. ► The double limited chemostat can be used as an important tool for significantly improve the industrial production of SCO.For many years there has been industrial interest to produce microbial oils through oleaginous microorganisms. Its fatty acids composition is similar to vegetable oils, so that the use of microbial polyunsaturated fatty acids has been very important in animal nutrition and is currently considered the potential feedstock for biodiesel, therefore any significant improvement in the process of lipid synthesis will reduce costs of production at industrial level. We investigated the use chemostat under different heterologous double nutrient limitations, that is, nutrients that meet different physiological requirements, in order to achieve a higher production lipid accumulation in oleaginous yeast. We used Pichia angusta DL-1 grown in a chemostat under carbon/phosphorus, carbon/magnesium, nitrogen/phosphorus and nitrogen/magnesium double limitations at five different dilution rates. The largest accumulation of lipids occurred under nitrogen/phosphorus limitation at a dilution rate of 0.03h⁻¹ and carbon/magnesium limitation at a dilution rate of 0.07h⁻¹. At that condition lipid content was twice the one obtained in batch culture and 15% larger than under simple limitation. Finally, under carbon/magnesium limitation the highest lipid productivity (0.011g/Lh) was obtained with a cell productivity of 0.033g/Lh.

Keywords: Pichia angusta; Chemostat; Lipid accumulation; Double nutrient limitations; Fatty acids; Biodiesel

High-yield and scalable cell-free assembly of virus-like particles by dilution by Mervyn W.O. Liew; Yap P. Chuan; Anton P.J. Middelberg (pp. 88-96).

► VLP yield and quality by dialysis and dilution assembly compared quantitatively. ► Process intensification leading to unit operation elimination. ► New process reduces buffer use 9× and increases product concentration 5×. ► VLP yield improved to 54%, 22 percentage points more than conventional methods. ► High-quality VLPs assembled using this scalable, high-yield cell-free method.Virus-like particles (VLPs) have been developed as safe and efficacious vaccines, and are increasingly used as carriers for foreign peptide epitopes in modular architectures. An emerging technology for low-cost and rapid-response VLP vaccine manufacture is based on controlled cell-free assembly of capsomeres, which are expressed in Escherichia coli, into VLPs presenting pharmaceutically relevant antigenic modules. A key bioprocessing challenge in this technology is VLP self-assembly, which has until now been studied using qualitative laboratory methods and without sufficient quantitation. In this work, the yield and size distribution of VLPs assembled by dialysis or by ten-fold dilution were compared using quantitative metrics. Membrane-based steps used for dialysis and particle concentration were identified as the key inefficiencies in each method, resulting in 13–18% protein loss. Key inefficiencies were circumvented through process intensification that led to development of a two-fold dilution assembly method. The new process eliminated a unit operation, improved the final concentration of assembly products by a factor of five and reduced buffer consumption nine-fold. Using this process, modular capsomeres presenting a group A Streptococcus (GAS) antigenic module were assembled into high-quality VLPs with a final yield of 54%, which was 18–22 percentage points higher than obtained using conventional methods described in the literature. This study demonstrates the feasibility of manufacturing VLP vaccines in cell-free reactors at high yield, with high structural integrity, using a scalable and simple process. Ongoing development based on these results will be conducive to process-intensified VLP bioprocessing for low-cost vaccine delivery at global scale.

Keywords: Virus-like particles; Self-assembly; Bioprocess design; Downstream processing; Process integration; Scale-up

Biodeinking of flexographic inks by fungal laccases using synthetic and natural mediators by Ursula Fillat; Alicia Prieto; Susana Camarero; Ángel T. Martínez; María Jesús Martínez (pp. 97-103).

► Removal of flexographic inks is a problem for the recycled paper industry. ► First report on decolorization of these inks by laccase–mediator systems. ► Synthetic mediators (HBT, ABTS) gave the best results with basidiomycete laccases. ► Natural mediators allowed ink decolorization by M. thermophila laccase. ► Biodeinking could be an alternative process in paper recycling.The use of new printing technologies based on flexographic inks hampers ink elimination during paper recycling, making necessary the development of alternative methods. Decolorization of four flexographic inks has been evaluated by using fungal laccases, three of them from basidiomycetes ( Trametes villosa, Coriolopsis rigida, and Pycnoporus coccineus), and one from the ascomycete Myceliophthora thermophila in the presence of synthetic and natural mediators.The results obtained showed a higher capacity of the three basidiomycete laccases to decolorize flexographic inks as compared with M. thermophila laccase, a low redox potential laccase. Basidiomycete laccases decolorized inks without mediators at long reaction times, although the presence of natural or synthetic mediators (above all HBT) accelerated the process. On the other hand, M. thermophila laccase was unable to decolorize the inks assayed. The addition of syringyl-type mediators led to medium levels of decolorization except for R48 ink, which was almost completely decolorized.Most decolorization was obtained during the first hours of treatment, when all the basidiomycete laccases were fully active in the presence of mediators. As opposed to other basidiomycete laccases, which become inactive in the presence of HBT after 24h, the enzyme of P. coccineus was not deactivated by this mediator even after 48h. A complete loss of M. thermophila laccase activity was observed at short times with acetosyringone and methyl syringate, the only two mediators able to promote ink decolorization with this enzyme.

Keywords: Flexographic ink; Biodeinking; Enzyme activity; Biodegradation; Filamentous fungi; Waste treatment

Immobilized heterologous Rhizopus oryzae lipase: A feasible biocatalyst for the production of human milk fat substitutes by Carla Tecelão; Marina Guillén; Francisco Valero; Suzana Ferreira-Dias (pp. 104-110).

► Rhizopus oryzae heterologous lipase (r-ROL) was immobilized in different supports. ► Immobilized r-ROL was able to catalyze the production of human milk fat substitutes. ► Results were comparable to those obtained with commercial immobilized lipases. ► Operational stability of r-ROL in Lewatit VPOC 1600 increased with rehydration.This work aims at evaluating the potentialities of a heterologous Rhizopus oryzae lipase (rROL) as a feasible biocatalyst, to be used as an alternative to commercial lipases, for the synthesis of Human Milk Fat Substitutes (HMFS). This enzyme was immobilized in three different supports (Accurel^® MP 1000, Eupergit^® C and Lewatit^® VP OC 1600) and tested as catalysts for the acidolysis between tripalmitin and oleic acid, batchwise, at 60°C, in solvent-free medium.The highest molar incorporations of oleic acid in the triacylglycerols were achieved with rROL in Lewatit^® VP OC 1600 and Accurel^® MP 1000, although the highest immobilization yields were observed with Eupergit^® C.Quasi-equilibrium was attained after 6h or 12h reaction, with about 30mol.% and 22mol.% of oleic acid incorporation, when rROL immobilized in Lewatit and Accurel were used, respectively.Also, 23h batch operational stability tests were performed: half-life times ( t_1/2) of 34.5h and 64.0h were achieved for rROL immobilized in Accurel^® MP 1000 and in Lewatit^® VP OC 1600, respectively. An important increase in the operational stability was observed for rROL immobilized in Lewatit after rehydration at the end of each batch ( t_1/2=202h). Thus, the loss of activity may be explained by a progressive dehydration occurring along the reaction.

Keywords: Acidolysis; Human milk fat substitutes; Immobilization; Recombinant lipase; Rhizopus oryzae; Operational stability

Biofunctionalization and self-interaction chromatography in PDMS microchannels by Kedar S. Deshpande; Shreyas Kuddannaya; Judith Staginus; Peter C. Thüne; Louis C.P.M. de Smet; Joop H. ter Horst; Luuk A.M. van der Wielen; Marcel Ottens (pp. 111-119).

► We demonstrate a miniaturized flexible version of self-interaction chromatography by immobilizing proteins on polydimethylsiloxane (PDMS) microchannels. ► Fabrication process for PDMS-based microstructures is relatively cheap, quick and requires limited lab expertise. ► The resulting microchip is successfully applied and the protein interaction results align well with existing literature data. ► This approach will help to bring high throughput cheap microchip protein interaction screening within practical reach.In this paper we present an experimental protocol for protein immobilization on polydimethylsiloxane (PDMS) polymer surfaces and the subsequent application of a chromatographic PDMS microfluidic chip to measure protein–protein interactions. The PDMS surface modification steps are quantitatively and qualitatively experimentally analyzed using an array of techniques (water contact angle measurement, fluorescence spectroscopy and X-ray photoelectron spectroscopy). The protocol involves PDMS acidic surface activation using a potassium disulfite/potassium peroxidisulfate/acrylic acid mixture, followed by amination with 3-aminopropyl diethoxymethylsilane, followed by glutaraldehyde grafting and subsequent covalent protein binding. The applicability of such a miniaturized PDMS-based microfluidic system has been exemplified by measuring protein–protein interactions in a fast and accurate fashion for three model proteins, namely: hen egg white lysozyme, bovine ribonuclease-A and α-chymotrypsinogen. The protein interaction results align well with existing literature data using different materials and techniques. As the fabrication process for PDMS-based microstructures is relatively cheap, quick and requires limited lab expertise/access to specialized equipments, we consider that the implementation of such a flexible, easy to fabricate, PDMS-based microfluidic system for estimating protein interactions an important step toward quickly mapping protein phase behavior and measuring protein (self/cross) interactions in complex biological systems.

Keywords: Protein immobilization; Self-interaction chromatography; PDMS; Osmotic second virial coefficient; Microfluidic chip

Direct binding glucoamylase onto carboxyl-functioned magnetic nanoparticles by Chen Guo; Ma Yunhui; Su Pengfei; Fang Baishan (pp. 120-125).

Display Omitted► Carboxyl functioned magnetic Fe₃O₄ nanoparticles (CMNPs) as support. ► The effects of immobilization of condition on the activity of immobilized enzyme were investigated. ► Catalysis characteristics of immobilized glucoamylase were compared with free enzyme.A novel and efficient immobilization of glucoamylase from Aspergillus niger has been developed by using carboxyl functioned magnetic Fe₃O₄ nanoparticles (CMNPs) as support. The CMNPs were prepared by co-precipitation of Fe²⁺/Fe³⁺ with oleic acid as surfactant and consequent oxidation of CC intoCOOH by KMnO₄ solution in situ. And then glucoamylase was directly bonded onto the magnetic nanoparticles. The bonding was verified by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis. The immobilization process was investigated by examining immobilization time, enzyme concentration and pH. As a result, it could gain an enzyme loading with 120mg glucoamylase/g magnetic nanoparticles and specific activity of 90U/mg protein while 1mg/mL glucoamylase and 10mg/mL ferrofluid were mixed at pH 6.0 and 25°C for 16h. The immobilized enzyme presented a same or better storage, pH and thermal stability than the free enzyme and could be reused. And the residual activity was about 40% after 8 cycles. The Michaelis constant ( K_m) showed that affinity of immobilized enzyme for binding substrate was lower than free enzyme.

Keywords: Enzyme biocatalysis; Immobilized enzymes; Kinetic parameters; Magnetic nanoparticles; Glucoamylase

Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering by Zhikun Wang; Cuijuan Gao; Qian Wang; Quanfeng Liang; Qingsheng Qi (pp. 126-131).

► Combining adaptive evolution and cofactor engineering obtained engineered yeasts. ► G2U1-A₀ was able to produce 75.1gl⁻¹ pyruvate. ► Differential expression of noxE and udhA was investigated. ► Fine regulation of NADH/NAD⁺ ratio is critical to pyruvate production. ► A new strategy for improvement of the Pdc⁻ strain engineering platform is provided.Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been proven as a suitable metabolic engineering platform to produce organic acids. S. cerevisiae BY5419 Pdc⁻ strain cannot grow in batch cultures on synthetic medium with glucose as the sole carbon source, yet grows well on synthetic medium with ethanol or acetate. In this study, by combining adaptive evolution and cofactor engineering, we obtained a series of engineered yeasts that can produce pyruvate using glucose as sole carbon source. Differential expression of noxE, encoding a water-forming NADH oxidase from Lactococcus lactis, and udhA, encoding a soluble pyridine nucleotide transhydrogenase from Escherichia coli, was investigated. Of all the constructed recombinant strains, G2U1-A₀ was able to produce 75.1gl⁻¹ pyruvate, increased 21% compared with the original strain A₀. The production yield of this strain reached 0.63gofpyruvategofglucose⁻¹. This study demonstrates that the fine regulation of intracellular NADH/NAD⁺ ratio is critical for cell metabolism and pyruvate production. Combining the adaptive evolution and fine regulation of intracellular NADH/NAD⁺ ratio provides a new strategy for improving the Pdc⁻ strain engineering platform.

Keywords: NADH/NAD; ⁺; Pyruvate; Saccharomyces cerevisiae; Metabolic engineering; Adaptive evolution; Cofactor metabolic engineering

Effects of ratio of manganese peroxidase to lignin peroxidase on transfer of ligninolytic enzymes in different composting substrates by Meihua Zhao; Zhuotong Zeng; Guangming Zeng; Danlian Huang; Chongling Feng; Cui Lai; Chao Huang; Zhen Wei; Ningjie Li; Piao Xu; Chen Zhang; Zhifeng Liu; Gengxin Xie (pp. 132-139).

► Laboratory simulation tests of the transfer of ligninolytic enzymes in composting substrates (soil, vegetable leaf, rice straw and chaff) were studied. ► The physico-chemical properties of composting substrates had effect on the transfer of ligninolytic enzymes in composting substrates. ► The results of transfer experiment were consistent with the FTIR spectrometry. ► The transfer of ligninolytic enzymes in different substrates changed as the MnP/LiP ratio changed.The transfer of ligninolytic enzymes in composting substrates (soil, vegetable leaf, rice straw and chaff) was investigated using a series of column elution experiments. Environmental scanning electron microscope (ESEM) and Fourier transform infra-red spectroscopy (FTIR) were used to analyze the mechanism of ligninolytic enzymes adsorption onto those substrates. The significant differences were found between substrates with adsorption of enzymes and those without enzymes by FTIR. Hydrophobic groups (methylene and alkane groups) and active groups (CH₂CO orCH₂NH) contents decreased in the substrates with adsorption of enzymes. The transfer abilities of total protein in the four composting substrates (soil, vegetable leaf, rice straw and chaff) were weakened as the ratio of manganese peroxidase (MnP) to lignin peroxidase (LiP) increased from 4 to 6, and were enhanced when the MnP/LiP ratio increased to 8. The transfer abilities of LiP in the four composting substrates were enhanced continually with the increasing MnP/LiP ratio. The transfer of MnP was similar to that of total protein with the change of MnP/LiP ratio. These results indicated that the change of MnP/LiP ratio could affect the transfer of ligninolytic enzymes in different composting substrates.

Keywords: Ligninolytic enzymes; Protein; Composting substrate; Transfer

Enhancement of Dichomitus squalens tolerance to copper and copper-associated laccase activity by carbon and nitrogen sources by Ranjani Kannaiyan; Nader Mahinpey; Thilakavathi Mani; Robert J. Martinuzzi; Victoria Kostenko (pp. 140-147).

► Copper has a dual effect on laccase activity, inducing and suppressing. ► Copper for maximum laccase activity depends on carbon/nitrogen sources and their ratios. ► Laccase suppression and eradication due to copper depends on nutrients. ► There are two types of copper toxicity: growth-related and laccase-related. ► Casein reduces growth related copper toxicity and enables high laccase activity.This study revealed a dual effect of copper on D. squalens laccase activity, inducing and suppressing. The addition of copper sulfate led to enhancement of the enzyme activity until it reached a maximum value at peak copper sulfate concentration (PC) followed by suppression and even complete termination of the enzyme activity. Maximum laccase yield is a function of fungi tolerance and media composition. Nutrients had a significant impact on copper-independent and copper-induced laccase activity. Some nutrients also improved tolerance of cells and laccase-producing system to copper load. The effect of specific nutrient depended on the ratio of carbon and nitrogen sources. This research can be used to predict the effective copper concentration and nutrient composition to stimulate the laccase production without compromising the fungi growth. The methylated substrates (3- O-methylglucose, methylcellulose) in combination with casein and copper demonstrated high potential to support fungi tolerance and enhanced laccase activity.

Keywords: Copper; Tolerance; Laccase activity; Carbon/nitrogen ratio

Biosynthesis and characterization of violacein, deoxyviolacein and oxyviolacein in heterologous host, and their antimicrobial activities by Haisheng Wang; Fengzhong Wang; Xiaofei Zhu; Yanchun Yan; Xianghong Yu; Peixia Jiang; Xin-Hui Xing (pp. 148-155).

► Violacein derivatives were synthesized by recombinant strains with 5-HTP as precursor. ► Violacein derivative was identified as oxyviolacein based on HPLC–MS and NMR analysis. ► Oxyviolacein and deoxyviolacein exhibited strong activities against G⁺ bacteria. ► Oxyviolacein displayed strong activity against Phytophthora capsici compared to violacein.Violacein is a natural bisindole pigment produced by bacteria, which has attracted much attention recently for its antibacterial, antiviral, antitumor, and antioxidant activities. To better understand the bioactive properties of violacein derivatives, deoxyviolacein and oxyviolacein were biosynthesized by genetically engineered strains of Citrobacter freundii(pComvio) and C. freundii(pComvioΔd), respectively, usingl-tryptophan or 5-OH-l-tryptophan as the precursor, respectively. The structure of oxyviolacein was confirmed based on HPLC–MS and NMR analysis. Deoxyviolacein and oxyviolacein were evaluated for their effectiveness against bacteria and phytopathogenic fungi. The two pigments exhibited strong activities against all Gram-positive bacteria tested except Streptococcus sp. Oxyviolacein showed a surprisingly high activity against Phytophthora capsici, a devastating pathogen of vegetable crops, and also showed strong antifungal activity against Fusarium oxysporum, Botrytis cinerea, and Verticillium dahliae at 0.5mgmL⁻¹. Deoxyviolacein showed strong antifungal activity against Rhizoctonia solani at concentrations exceeding 2mgmL⁻¹. The presence of a hydroxyl group appeared to be essential for their bioactivities. These findings provide important information for the further exploitation of violacein and its derivatives as fungicides.

Keywords: Violacein derivatives; Biosynthesis; Antimicrobial activities; Pathogenic fungi; Biological control

Substrate interactions and kinetics study of phenolic compounds biodegradation by Pseudomonas sp. cbp1-3 by Jiao Liu; Xiaoqiang Jia; Jianping Wen; Zhengxi Zhou (pp. 156-166).

► Isolation and characterization of Pseudomonas sp. cbp1-3 were performed. ► The strain degraded phenol, m-cresol and 4-chlorophenol as sore carbon source. ► Interactions of substrates in dual- and ternary-substrate systems were investigated. ► Kinetic models for ternary-substrate systems were proposed and evaluated.A new strain cbp1-3 was isolated from activated sludge of a coking plant in Tianjin and identified as Pseudomonas sp. based on physiological and 16S rRNA gene sequence analysis, which could completely degrade 1400mg/L phenol, 800mg/L m-cresol and 150mg/L 4-chlorophenol (4-CP) as sole carbon and energy source within 60h, 40h and 60h, respectively. Investigation on substrate interactions in the biodegradation of mixed phenols indicated that phenol and m-cresol exhibited a mutual inhibition to each other, and they (only below 300mg/L) both promoted the 4-CP biodegradation. However, 4-CP strongly inhibited the biodegradation of other phenols in their mixtures despite of the low concentration. Further analysis showed that the newly proposed kinetic model for cell growth on ternary substrates fitted the experimental data very well. And sensitivity analysis suggested that substrate interaction coefficientsfi ( i=12, 13 and 23) of the models were the most sensitive parameters. Finally, yield coefficient, calculated for all conditions, taken as a whole, followed an allometric decline pattern with the increase of substrate concentration.

Keywords: Biodegradation; Growth kinetics; Modeling; Substrate inhibition; Pseudomonas; sp.; Phenolic compounds

Desorption of butanol from zeolite material by A. Oudshoorn; L.A.M. van der Wielen; A.J.J. Straathof (pp. 167-172).

► Carbon dioxide can be used as displacement agent for 1-butanol recovery from zeolite. ► Heat effect of butanol desorption is slightly above its evaporation enthalpy. ► Desorption of butanol from CBV28014 shows a chemical reaction at 200°C. ► Desorption rate of butanol from CBV28014 is significantly slower than from CBV901.Sorption-based recovery of 1-butanol from aqueous solution has been investigated focusing on the recovery of butanol by desorption from the sorbate. Sorption isotherm, thermogravimetric adsorption and differential scanning calorimetry experiments have been used to determine the desorption behavior of butanol and water for two high-silica zeolite adsorbents, faujasite (CBV901) and ZSM-5 (CBV28014).Carbon dioxide can be used as displacement agent for butanol recovery, with the butanol–carbon dioxide equilibria determining the carbon dioxide mass requirement for such a process.For CBV901 desorption requires 2440J/g of water and 1080J/g of butanol. The heat effects for CBV28014 are 2730J/g (water) and 1160J/g (butanol). A significant difference in water content can be seen between both zeolite materials, with CBV28014 showing the least amount of water adsorption. The desorption rate of butanol from CBV28014 is significantly slower than from CBV901.A catalytic reaction, most probably dehydration, occurs around 200°C during temperature programmed desorption of butanol from CBV28014.

Keywords: Bioseparations; Desorption; Biofuels; Butanol; Zeolite

Lignin as natural radical scavenger. Effect of the obtaining and purification processes on the antioxidant behaviour of lignin by Araceli García; María González Alriols; Giorgia Spigno; Jalel Labidi (pp. 173-185).

► The effect of lignin isolation on its antioxidant capacity was studied. ► Several solvents and operation conditions were used for lignin obtaining. ► The purity of lignin had high influence on its antiradical behaviour. ► Ultrafiltration and differential precipitation improved lignin antioxidant power.In the present work, the antioxidant activity of 14 lignin samples obtained from apple tree pruning was studied. Different solvents (water, soda–water 7.5% (w/w), ethanol–water 60:40 (v/v) and acetic acid–water 60:40 (v/v)) under different operation conditions were used in order to extract the lignin and different techniques (ultrafiltration, differential precipitation and lignin purification) were applied with the aim of improving the lignin properties. The obtained lignin samples were characterized by different analytical techniques evaluating their chemical structure (ATR-IR), thermal behaviour (TGA and DSC) and molecular weight (GPC). Further analyses (¹³C NMR and Folin–Ciocalteu method) were carried out in order to determine the purity and the total phenolics content present in the lignin samples. The antioxidant activity of the analyzed lignins was evaluated by the radical ABTS assay. The results revealed the high influence that the lignin obtaining process had on the lignin properties, determining its possible commercial application. Results also indicated that autohydrolysis, organosolv and some ultrafiltrated soda lignins showed antioxidant efficiency comparable with a powerful natural antioxidant, as catechin, and a commercial one, Trolox^®.

Keywords: Lignin; Biorefinery; Antioxidant capacity; Agricultural wastes; Ultrafiltration; Purification

A novel efficient optimisation system for purification process synthesis by Eleftheria M. Polykarpou; Paul A. Dalby; Lazaros G. Papageorgiou (pp. 186-193).

► This paper presents an MILP modelling framework for the optimal purification process synthesis. ► The proposed models have been applied to three different example protein mixtures containing up to 13 contaminants and selecting from a set of up to 21 candidates. ► The results of the examples highlight the increase in computational efficiency.Protein purification through chromatographic processes has been broadly used in the biopharmaceutical industry over the last decades, but still remains a major bottleneck. In this work, we address the challenge of selecting appropriate chromatographic steps, along with product collecting timeline for separating the target protein from the contaminants in a multicomponent mixture. A novel mixed integer linear programming (MILP) model for purification process synthesis is proposed. The model allows product losses and is tested on three example protein mixtures, containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The results are compared with previous literature models attempting to solve the same problem and show that the proposed approach offers significant gains in computational efficiency without compromising the quality of the solution.

Keywords: Downstream processing; Mixed integer programming; Optimisation

Enzyme hydrolysis of soybean oil in a slug flow microsystem by Jiří Čech; Walter Schrott; Zdeněk Slouka; Michal Přibyl; Michal Brož; Gabriela Kuncová; Dalimil Šnita (pp. 194-202).

Display Omitted► A slug flow microfluidic system is used for enzyme hydrolysis of soybean oil. ► The system consists of a generator, reaction capillary, and two separators. ► A primary separator enables continuous separation of the oil product. ► 30% conversion of triglycerides was obtained within 10min. ► The system is characterized by low pressure drop and large interfacial area.In this paper, we report on the development of a continuous microfluidic reaction system for hydrolysis of soybean oil catalyzed with Thermomyces lanuginosus lipase (Lipolase 100L). The microfluidic reaction system generates water–oil emulsions in the form of a hydrodynamically well controlled slug flow and automatically separates the oil phase after the hydrolysis by employing two microfluidic separators. All elements of this reaction system were tested at different hydrodynamic conditions and showed the ability to operate in a wide range of reactant flow rates. 25–30% conversion of triglyceride was reached in setting the residence time of the emulsion mixture to 10min. This conversion increased to almost 50% for the residence time of 1h. These results are comparable with those published for the same enzyme reaction system. This feature along with the benefits stemming from the use of microfluidics make our developed system a useful, easy to control and easy to scale-up technology for fast production of fine chemicals. Moreover, our calculations indicate that our slug flow system allows for significant savings of the mechanical energy.

Keywords: Lipase; Microemulsion; Micromixing; Enzyme bioreactor; Microreactor; Soybean oil hydrolysis

Improved production of carotenes from synthetic medium by Blakeslea trispora in a bubble column reactor by Konstantina Nanou; Triantafyllos Roukas; Emmanuel Papadakis (pp. 203-207).

► The fungus B. trispora grown in a bubble column reactor formed pellets. ► This resulted in a mild oxidative stress in B. trispora. ► The oxidative stress increased significantly the carotene production. ► Bubble column reactor was a useful fermentation system for carotene production.The carotene production from a synthetic medium sterilized at initial pH 11.0 by Blakeslea trispora in a bubble column reactor was investigated. Under the above conditions the fungus formed compact aggregates (pellets). As a consequence, a mild oxidative stress in B. trispora was occurred indicated by the specific activities of superoxide dismutase (SOD) and catalase (CAT). The oxidative stress resulted in a significant increase in carotene production and a change in the biosynthesis of carotenes. The highest concentration of carotenes (85.0mg/g dry biomass) was obtained after 8 days of fermentation. In this case, the proportion of β-carotene, γ-carotene, and lycopene was 52.42%, 45.43%, and 2.15%, respectively. Bubble column reactor has a potential for carotene production from synthetic medium sterilized at initial pH 11.0.

Keywords: Carotenes; Blakeslea trispora; Synthetic medium; Bubble column reactor; Culture morphology; Oxidative stress

Bioleaching kinetics of a spent refinery catalyst using Aspergillus niger at optimal conditions by F. Amiri; S.M. Mousavi; S. Yaghmaei; M. Barati (pp. 208-217).

► The kinetics of bioleaching of metals from a spent catalyst, using Aspergillus niger was studied. ► Most effective bioleaching variables were selected in accordance with the Plackett–Burman design. ► Determinations of organic acid concentration showed noticeable variation during bioleaching. ► Predictions by the model showed that diffusion through the solid matrix is the rate-controlling step.The kinetics of bioleaching of Mo, Ni, and Al from spent hydrocracking catalyst, using Aspergillus niger was studied. The four most effective bioleaching variables were selected in accordance with the Plackett–Burman design and were further optimized via central composite design (CCD). The optimal values of the variables for maximum multi-metal bioleaching were as follows: particle size 150–212μm, sucrose 93.8g/L, pulp density 3%w/v, and pH 7. The maximum metal recoveries corresponding to these conditions were 99.5±0.4% Mo, 45.8±1.2% Ni, and 13.9±0.1% Al. The relatively low Ni extraction was attributed to the precipitation of Ni in the presence of oxalic acid. Under the optimal conditions, the fungus growth was found to be higher in the presence of spent catalyst than that in the catalyst-free medium. Determinations of the organic acid concentration showed noticeable variation during bioleaching, particularly for gluconic acid. Accordingly, a modified form of shrinking core model was used to take these variations into account. The predictions by the model showed good consistency with the experimental results, suggesting that diffusion of bioleaching agent through the solid matrix was the rate-controlling step.

Keywords: Bioleaching; Waste treatment; Extraction kinetics; Spent catalyst; Optimization; Filamentous fungi

Chloroperoxidase catalyzed oxidation of Cbz-ethanolamine to Cbz-glycinal by Milja Pešić; Carmen López; Gregorio Álvaro (pp. 218-224).

► An amino aldehyde was synthesized using chloroperoxidase from Caldariomyces fumago. ► Pulses of tert-butyl hydroperoxide yielded high conversion preserving CPO activity. ► Biphasic and cosolvent systems allowed an increase of amino aldehyde productivity. ► Use of 5% (v/v) dioxane permitted 6-fold higher production of Cbz-glycinal.Chloroperoxidase catalyzed oxidation of Cbz-ethanolamine to Cbz-glycinal was performed using hydrogen peroxide and tert-butyl hydroperoxide as oxidants. Frequency of the addition of the oxidants in aqueous medium was optimized. Tert-butyl hydroperoxide resulted as more appropriate oxidant when added at the rate of 3mM/h, while the high catalase activity of CPO towards hydrogen peroxide limited the application of this peroxide as oxidant. Looking for benefits in terms of reaction productivity, reaction medium engineering was further studied. Oxidation of Cbz-ethanolamine was investigated in the presence of miscible (acetone, acetonitrile, dioxane) and immiscible (ethyl acetate) organic solvents. Dioxane at 5% (v/v) was the most appropriate medium as a concentration of 47.6mM of Cbz-glycinal was produced, which was almost 6-fold higher than the value obtained in aqueous reaction (7.8mM).

Keywords: Chloroperoxidase from; Caldariomyces fumago; Cbz-ethanolamine oxidation; Cbz-glycinal synthesis; Reaction medium; Peroxide

A label-free immunosensor based on recordable compact disk chip for early diagnostic of the dengue virus infection by Igor T. Cavalcanti; Maria I.F. Guedes; Maria D.P.T. Sotomayor; Hideko Yamanaka; Rosa F. Dutra (pp. 225-230).

► Dengue (DENV) is a major public health concern globally that can lead to hemorrhagic fever or shock. ► An inexpensive immunoelectrode built from recordable compact disk chip was developed for NS1 based on the self-assembled protein A. ► It was achieved a real early diagnostic of the dengue virus by serum samples.The non-structural protein 1 (NS1) of dengue virus is abundantly circulating in the blood during the acute phase of the dengue infection being correlated with viremia levels and hence can be used to early diagnostic of the dengue hemorrhagic fever. An electrochemical immunosensor based on gold film electrode obtained from a recordable compact disk (CD-trode) was developed for NS1 protein. Anti-NS1 monoclonal antibodies were immobilized on the CD-trode via protein A. The stepwise immobilization of the anti-NS1 was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analytical response to the NS1 interaction with anti-NS1 immobilized on CD-trode was detected by applying the differential pulse voltammetry technique. The immunosensor showed a linear response from 1 to 100ng/mL of NS1 and a detection limit of 0.33ng/mL. This label-free immunosensor exhibited a good reliability for NS1 detection in serum samples presuming an early diagnostic of the dengue virus.

Keywords: Electrochemical immunosensor; NS1; Dengue; CD-trode

Separation of phenylalanine and tyrosine by ion-exchange using a strong-base anionic resin. I. Breakthrough curves analysis by Maria João A. Moreira; Licínio M. Gando-Ferreira (pp. 231-240).

► A strong anionic resin was used to selectively separate phenylalanine and tyrosine. ► The resin exhibited a better selectivity for tyrosine over phenylanine. ► Breakthrough curves were well described by a model with non-constant selectivity data. ► Saturation experiments were significantly affected by the operating conditions.Dynamics of separations of the amino acids phenylalanine and tyrosine in fixed-bed ion-exchange column using the strongly basic resin PA 316 was investigated for different operating conditions. The flow rate, feed amino acid concentration and pH solution affect significantly the breakthrough time of the saturations curves. A mathematical model which takes into account axial dispersed plug flow, external and intraparticle mass transfer resistances, as well as non-constant selectivity coefficient data, enabled successfully to simulate the experimental breakthrough curves. The model was satisfactorily extended to prediction of the experimental dynamic behaviour of phenylalanine (Phe) and tyrosine (Tyr) mixtures where the ratio of the feed concentrations of both amino acids was changed. A most effective separation of the amino acids was achieved when the concentration of Phe in the feed solution is higher than that of Tyr.

Keywords: Amino acids; Separation; Ion-exchange resin; Adsorption; Breakthrough curves; Modelling

Separation of phenylalanine and tyrosine by ion-exchange using a strong-base anionic resin. II. Cyclic adsorption/desorption studies by Maria João A. Moreira; Licínio M. Gando-Ferreira (pp. 241-250).

► Sequential bed saturations of Phe and Tyr were analysed under different pH values. ► Regeneration of resin loaded with amino acid at high pH is effective with 0.14M NaOH. ► A top product enriched in amino acid was obtained via pH parametric pumping runs. ► Experimental data were suitably simulated with a model with linear driving force.Cyclic adsorption and desorption dynamics of phenylalanine (Phe) and tyrosine (Tyr) was investigated in a column packed with a strong anionic resin, Diaion PA316, under different pH conditions. Experiments were performed in order to study the effect of the pH in two sequential bed saturations keeping constant the feed concentration. A adsorption step at isoelectric pH (≈6) followed by a desorption step at high pH (12–13) causes an overshoot in the breakthrough curves due to lower affinity resin by amino acid under alkaline conditions. Contrarily to this behaviour, the solution that leaves the column reaches a zero concentration of amino acid during a certain time in two consecutive saturations at high pH and isoelectric pH. The regeneration bed with 0.14M NaOH is almost effective for conditions less favourable of sorption of amino acid by the resin. Satisfactory agreement was achieved between experimental and simulated results using a mathematical model that considers axial dispersed plug flow and intraparticle mass transfer described by the linear driving force model. Preliminary pH parametric pumping experiments, in semi-continuous mode, enabled to achieve a concentration factor of the top product of ≈2.3 and 2.0 for Phe and Tyr during 10 cycles of operation.

Keywords: Cyclic adsorption/desorption; Ion-exchange resin; Amino acids; Phenylanine and tyrosine separation; Modeling; pH parametric pumping

Development of nitrate elimination by autohydrogenotrophic bacteria in bio-electrochemical reactors – A review by Seyyedalireza Mousavi; Shaliza Ibrahim; Mohamed Kheireddine Aroua; Shahin Ghafari (pp. 251-264).

► We focus in this review on new innovative designs of BERs to eliminate of nitrate. ► The review explains the effects of operational and environmental factors on BERs. ► Selecting a suitable structure and configuration increases the efficiency of BERs. ► The environmental parameters play key role in efficiency of BERS.In recent years, the removal of nitrate as a global soil and water contaminant has been increasingly considered. Various methods have been applied to remove this inorganic pollutant from water and wastewater. Among them is an integrated “bio-electrochemical reactor” system, which is a novel method for water and wastewater denitrification. The novelty of this system is improved biological denitrification by immobilizing autohydrogenotrophic bacteria directly on the surface of a cathode, providing easy access to NO₃⁻ and H₂ as the electron acceptor and electron donor, respectively. The system effectiveness depends on the configuration of reactor, and the operational and environmental parameters. Identification and optimization of these parameters are important to enhance the efficiency of the system. In this review following the introduction and explanation of the process microbiology, the bio-electrochemical process is described focusing on new configurations, and the impact of important variables, namely, hydraulic retention time, electric current, pH, and carbon source.

Keywords: Nitrate; Denitrification; Autohydrogenotrophic bacteria; Bio-electrochemical; Electrolysis; Reactor configuration

Batch operational stability of immobilized heterologous Rhizopus oryzae lipase during acidolysis of virgin olive oil with medium-chain fatty acids by P.A. Nunes; P. Pires-Cabral; M. Guillén; F. Valero; S. Ferreira-Dias (pp. 265-268).

► Rhizopus oryzae heterologous lipase (r-ROL) was immobilized in different supports. ► r-ROL was able to catalyze acidolysis of olive oil with caprylic or capric acid. ► r-ROL was reused, either without or with biocatalyst rehydration between batches. ► Operational stability of r-ROL in Lewatit VPOC 1600 increased with rehydration.Structured triacylglycerols containing medium-chain fatty acids (M) at positions sn-1,3 and long-chain fatty acids (L) at the sn-2 position (MLM type), were obtained by acidolysis of virgin olive oil with caprylic or capric acid, in solvent-free media, at 40°C, catalyzed by a heterologous Rhizopus oryzae lipase (r-ROL) immobilized in Eupergit^® C or in Lewatit VP OC 1600. The biocatalyst immobilized in Eupergit^® was reused in consecutive 23-h batches with rehydration of the biocatalyst between batches. A first-order deactivation was observed (half-life time, t_1/2=39.0h for caprylic; t_1/2=54.3h for capric acid). During acidolysis with capric acid catalyzed by r-ROL immobilized in Lewatit VP OC 1600, without rehydration, a first-order deactivation was observed ( t_1/2=49.1h); with rehydration, a considerable increase in stability was observed ( t_1/2=234h; Sadana's series-type inactivation kinetics model).

Keywords: Key words; Acidolysis; Biocatalysis; Enzyme deactivation; Heterologous; Rhizopus oryzae; lipase; Immobilization; Structured lipids

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Biochemical Engineering Journal (v.67, #)