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In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L-1.h-1. Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW-1) and volumetric productivity (0.32 g.L-1.h-1). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW-1) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L-1, in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.

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Polygalacturonases (EC 3.2.1.15) hydrolyze the α-1,4-glycosidic linkages in polygalacturonic acid chains. The interest on specific inhibitors of pectinase and the versatility of magnetic support for enzyme immobilization endorsed the preparation of an immobilized enzyme reactor (IMER). This work presents the synthesis of CoFe(2)O(4) amino-derivatives, which was employed as the support for the immobilization of pectinases from Leucoagaricus gongylophorus. Amino-functionalized CoFe(2)O(4) was obtained from glyceryl-derivatized CoFe(2)O(4) and was characterized by infrared spectroscopy and electronic microscopy. The immobilized enzyme maintained the same thermal, chemical and kinetic behaviour of the free enzyme (T(opt) 60 °C; pH(opt) 5.0; K(app)(M) = 0.5 mg min(-1); V(app)(M) ≈ 5.0 μmol min(-1) mL(-1)). The straightforward synthesis of CoFe(2)O(4) derivatives and the efficiency of immobilization offer wide perspectives for the use of the developed new IMER.

In this paper, we present the optimization of porous anodic alumina membranes for ultrafiltration prepared by anodically oxidized aluminum foils. The membranes were characterized by field-emission scanning electron microscopy to measure the pore diameter and the membrane thicknesses. The liquid fluxes were estimated through gas permeability measurements using Darcy's and Forchheimerś equations. A 2(3) factorial design we used to optimize the membrane properties: pore diameter, membrane thickness, and liquid flux using as control variables the applied current density, solution composition and concentration. It was observed that the most import variables to control the pore diameter were current density and electrolyte composition. After the anodization both, metallic aluminum substrate and the barrier layer of alumina were removed using adequate solutions to obtain the free standing membrane. Then, Escherichia coli a common bacterial contamination of drinking water was removed using these PAA membranes with 100% of efficiency to obtain bacteria-free water.

Bone regenerative medicine (BRM) aims to overcome the limitations of conventional treatments for critical bone defects by developing therapeutic strategies, based on temporary bioactive substitutes, capable of stimulating, sustaining, and guiding tissue regeneration. The aim of this study was to validate the “proof of concept” of a cellularized bioactive scaffold and establish its potential for use in BRM. For this purpose, three-dimensional scaffolds of poly-(lactic acid) (PLA), produced by the additive manufacturing technique, were incorporated into a human platelet-rich plasma (PRP-h) fibrin matrix containing human infrapatellar fat pad mesenchymal stem cells (hIFPMSC). The scaffolds (PLA/finbrin-bioactive) were kept under ideal culture conditions in a medium free from fetal bovine serum and analyzed at 5 and 10 days by Scanning Electron Microscopy (SEM), Fourrier Transform Infrared (FTIR), Circular Dichroism and fluorescence microscopy. The results demonstrated the feasibility of obtaining a rigid, cytocompatible, and cellularized three-dimensional structure. In addition, PRP platelets and leukocytes were able to provide a bioactive environment capable of maintaining the viability of hIFPMSC into scaffolds. The results validate the concept of a customizable, bioactive, cellularized, and non-immunogenic strategy for application in BRM.

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