Ricardo de Andrade Medronho

Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger

In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.

Cell retention devices for suspended-cell perfusion cultures.

Perfusion cultures of animal cells have several advantages over batch or fed-batch cultures. They give, for instance, higher productivities and a consistent product quality, and allow steady state operation and better cell physiology control. However, one of the main aspects limiting performance and scale-up of perfusion processes is the need for an adequate cell retention device. The devices currently in use for stirred perfusion bioreactors are continuous centrifuges, tangential flow membrane filters, dynamic filters, spin-filters, ultrasonic and dielectrophoretic separators, gravity settlers and, more recently, hydrocyclones. The advantages and disadvantages of each of these methods will be discussed.

Recovery of pectolytic enzymes produced by solid state culture of Aspergillus niger

Abstract Pectinases are enzymes with a wide range of applications in the food and drink industries. In the present work, the extraction of pectinases produced by Aspergillus niger in a solid state fermentation system was investigated. The purpose was to reduce enzyme losses in the fermented solids and at the same time obtain a crude extract as concentrated as possible. Initially the performances of stirred tank and fixed bed extractors were compared. Polygalacturonase activity and viscosity reducing capacity obtained in the stirred tank system were 105% and 15% superior, respectively. Repeated extractions and multiple stage countercurrent extraction were studied, employing stirred tanks. It was possible to observe that three stages were enough for total recovery of the enzymes contained in the solids. The final enzyme extract obtained by counter-current extraction with three stages showed a polygalacturonase activity 81% higher than the one obtained by one-stage extraction.

Biofilm development and ammonia removal in the nitrification of a saline wastewater

Abstract The present work investigated biofilm development and process parameters in the nitrification of a synthetic saline wastewater (50 g l−1 NaCl) in reactors known as ‘submerged aerated biological filters’. The support used for biofilm development was made of PVC wrinkled plates with large specific area (138 m2 m−3) and high porosity (> 95%). Biofilm development on the support was investigated by cultivating the mixed culture batchwise. In the first week of operation, a brown biofilm was macroscopically evident on the support surface. After 10 days the biofilm activity became stable, an ammonia removal rate of 2 g NH3 m−3 h−1 was observed and the attached biomass reached a maximum value of 172 μg cm−2. When the system was operated continuously, with a hydraulic retention time of 15 h, the removal efficiency observed was 94% in the absence of NaCl and 48% in the presence of 50 g l−1 NaCl.

The Use of Hydrocyclones for Mammalian Cell Retention in Perfusion Bioreactors

A new type of hydrocyclone was designed and used in continuous mammalian cell cultivation processes. The apparatus gave cell separation efficiencies as high as 99%. Due to the fact that a 5-L-bioreactor was used, a sub-optimal configuration of overflow and underflow diameters was used in order to obtain high cell viabilities combined with separation efficiencies suitable for cell retention. Viability was always above 90% during the 23 days run. The addition of 0.1% Pluronic F68 was found to be necessary for guaranteeing high growth rates and productivity.

CFD simulation of an internal spin-filter: evidence of lateral migration and exchange flow through the mesh

In the present work Computational Fluid Dynamics (CFD) was used to study the flow field and particle dynamics in an internal spin-filter (SF) bioreactor system. Evidence of a radial exchange flow through the filter mesh was detected, with a magnitude up to 130-fold higher than the perfusion flow, thus significantly contributing to radial drag. The exchange flow magnitude was significantly influenced by the filter rotation rate, but not by the perfusion flow, within the ranges evaluated. Previous reports had only given indirect evidences of this exchange flow phenomenon in spin-filters, but the current simulations were able to quantify and explain it. Flow pattern inside the spin-filter bioreactor resembled a typical Taylor-Couette flow, with vortices being formed in the annular gap and eventually penetrating the internal volume of the filter, thus being the probable reason for the significant exchange flow observed. The simulations also showed that cells become depleted in the vicinity of the mesh due to lateral particle migration. Cell concentration near the filter was approximately 50% of the bulk concentration, explaining why cell separation achieved in SFs is not solely due to size exclusion. The results presented indicate the power of CFD techniques to study and better understand spin-filter systems, aiming at the establishment of effective design, operation and scale-up criteria.

Particle image velocimetry (PIV) study of rotating cylindrical filters for animal cell perfusion processes

In the present work, the main fluid flow features inside a rotating cylindrical filtration (RCF) system used as external cell retention device for animal cell perfusion processes were investigated using particle image velocimetry (PIV). The motivation behind this work was to provide experimental fluid dynamic data for such turbulent flow using a high‐permeability filter, given the lack of information about this system in the literature. The results shown herein gave evidence that, at the boundary between the filter mesh and the fluid, a slip velocity condition in the tangential direction does exist, which had not been reported in the literature so far. In the RCF system tested, this accounted for a fluid velocity 10% lower than that of the filter tip, which could be important for the cake formation kinetics during filtration. Evidence confirming the existence of Taylor vortices under conditions of turbulent flow and high permeability, typical of animal cell perfusion RCF systems, was obtained. Second‐order turbulence statistics were successfully calculated. The radial behavior of the second‐order turbulent moments revealed that turbulence in this system is highly anisotropic, which is relevant for performing numerical simulations of this system.

Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments.

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10‐100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000‐L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large‐scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions).

Brazilian meningococcal C conjugate vaccine: scaling up studies

Several outbreaks caused by Neisseria meningitidis group C have been occurred in different regions of Brazil. A conjugate vaccine for Neisseria meningitidis was produced by chemical linkage between periodate-oxidized meningococcal C polysaccharide and hydrazide-activated monomeric tetanus toxoid via a modified reductive amination conjugation method. Vaccine safety and immunogenicity tested in Phase I and II trials showed satisfactory results. Before starting Phase III trials, vaccine production was scaled up to obtain industrial lots under Good Manufacture Practices (GMP). Comparative analysis between data obtained from industrial and pilot scales of the meningococcal C conjugate bulk showed similar execution times in the scaling up production process without significant losses or alterations in the quality attributes of purified compounds. In conclusion, scale up was considered satisfactory and the Brazilian meningococcal conjugate vaccine production aiming to perform Phase III trials is feasible.

ESTUDO DA DINÂMICA DE FLUIDIZAÇÃO DE UM SISTEMA COMPOSTO AREIA- GÁS-BIOMASSA

Leitos fluidizados podem ser utilizados em inumeros processos industriais, dentro dos quais, destacam-se a combustao e/ou gaseificacao de biomassa, estes equipamentos promovem um maior contato solido-gas. Objetivando entender a dinâmica das particulas durante a fluidizacao, foram realizadas simulacoes para dois sistemas contendo gas-biomassa-areia, onde variou-se o diâmetro das particulas da fase solida, verificando a influencia desta alteracao na fluidizacao das particulas. Para realizar as simulacoes foi utilizado o software ANSYS FLUENT 15.0, foi adotada a abordagem Euleriana, com a teoria cinetica granular. A velocidade de entrada do gas foi mantida a 0,38 ms. Entre os sistemas simulados, o sistema A apresentou fluidizacao dificil quando comparado com o sistema B, onde a fluidizacao ocorreu mais facilmente e os efeitos da segregacao foram atenuados. Compreender a hidrodinâmica nestes sistemas e importante para otimizar os processos.