Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.

Purification of complex biopharmaceuticals with new processes, advanced analytics and computer-aided process design tools

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Engineering design of an integrated sustainable process system for cassava biobased materials

Cassava contributes significantly to biobased material development. Conventional approaches for its bio-derivative-production and application cause significant wastes, tailored material development challenges, with negative environmental impact and application limitations. Transforming cassava into sustainable value-added resources requires redesigning new approaches. Harnessing unexplored material source, and downstream process innovations can mitigate challenges. The ultimate goal proposed an integrated sustainable process system for cassava biomaterial development and potential application. An improved simultaneous release recovery cyanogenesis (SRRC) methodology, incorporating intact bitter cassava, was developed and standardized. Films were formulated, characterised, their mass transport behaviour, simulating real-distribution-chain conditions quantified, and optimised for desirable properties. Integrated process design system, for sustainable waste-elimination and biomaterial development, was developed. Films and bioderivatives for desired MAP, fast-delivery nutraceutical excipients and antifungal active coating applications were demonstrated. SRRC-processed intact bitter cassava produced significantly higher yield safe bio-derivatives than peeled, guaranteeing 16% waste-elimination. Process standardization transformed entire root into higher yield and clarified colour bio-derivatives and efficient material balance at optimal global desirability. Solvent mass through temperature-humidity-stressed films induced structural changes, and influenced water vapour and oxygen permeability. Sevenunit integrated-process design led to cost-effectiveness, energy-efficient and green cassava processing and biomaterials with zero-environment footprints. Desirable optimised bio-derivatives and films demonstrated application in desirable in-package O2/CO2, mouldgrowth inhibition, faster tablet excipient nutraceutical dissolutions and releases, and thymolencapsulated smooth antifungal coatings. Novel material resources, non-root peeling, zero-waste-elimination, and desirable standardised methodology present promising process integration tools for sustainable cassava biobased system development. Emerging design outcomes have potential applications to mitigate cyanide challenges and provide bio-derivative development pathways. Process system leads to zero-waste, with potential to reshape current style one-way processes into circular designs modelled on nature's effective approaches. Indigenous cassava components as natural material reinforcements, and SRRC processing approach has initiated a process with potential wider deployment in broad product research development. This research contributes to scientific knowledge in material science and engineering process design.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Produção biotecnológica de poli-hidroxialcanoatos para a geração de polímeros biodegradáveis no Brasil

In recent years, several studies have been developed in Brazil to produce biodegradable materials. A particular family of bacterial polymers, the polyhydroxyalkanoates (PHA), has received special attention. PHAs are thermoplastic, biodegradable, biocompatible, are synthesised from renewable resources and can substitute petrochemical plastics in some applications. Different aspects have been focused to increase productivity and to reduce the cost of PHA production: bacterial improvement, use of industrial by-products as raw material, bioreactor design, process operation strategies, downstream process, mathematical modelling, polymer characterisation, application and biodegradability of blends. A production process was transferred to industry and studies to produce new PHA by controlling monomer composition are in progress. All these aspects are presented in this review.

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Purification of human albumin by the combination of the method of Cohn with liquid chromatography

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60ºC for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 ± 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were <=5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.

High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography

In order to obtain intravenous immunoglobulin G (iv IgG) of high quality from F-I+II+III or F-II+III pastes prepared by the Cohn method, we developed a chromatography process using ion exchange gels, Q-Sepharose FF and CM-Sepharose FF, and Sephacryl S-300 gel filtration. Viral inactivation was performed by incubating the preparation with pepsin at pH 4.0 at 35oC for 18 h. The characteristics of 28 batches produced by us were: yield 4.3 ± 0.2 g/l plasma, i.e., a recovery of 39.1 ± 1.8%; IgG subclasses distribution: IgG1 = 58.4%, IgG2 = 34.8%, IgG3 = 4.5% and IgG4 = 2.3%; IgG size distribution was 98.4% monomers, 1.2% dimers and 0.4% polymers and protein aggregates; anticomplement activity was less than 0.5 CH50/mg IgG, and prekallikrein activator activity (PKA) was less than 5 IU/ml. These characteristics satisfied the requirements of the European Pharmacopoea edition, and the regulations of the Brazilian Health Ministry (M.S. Portaria No. 2, 30/10/1998).

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

BIOPROCESS OPTIMIZATION AND CHARACTERIZATION OF PHYTASE FROM AN ENVIRONMENTAL ISOLATE BACILLUS MCCB 242

A study was undertaken to isolate phytase producers from environment and to segregate the most highly efficient phytase producer and to develop a bioprocess technology for commercial application. During this process, a potential phytase producer Bacillus MCCB 242 was isolated and characterized phenotypically and genotypically. Subsequently, phytase production was optimized, the enzyme purified and characterized and an appropriate downstream process also could be standardized.Precisely, through this work an environmental isolate Bacillus MCCB 242 could be brought out as phytase producer for commercial application. The enzyme production could be optimized and characterized, and an appropriate downstream process standardized. Cytotoxicity studies revealed the enzyme safe for feed application, especially in fish.

Kinetic studies on clavulanic acid degradation

Clavulanic acid (CA), a potent beta-lactamase inhibitor, is very sensitive to pH and temperature. It is produced by Streptomyces clavuligerus and to optimize both the fermentation step and the downstream process, the expression of the hydrolysis kinetics has to be determined. In the present work the CA degradation rate from various sources was investigated at temperatures of 10, 20, 25, 30 and 40degreesC and PH values of 6.2 and 7.0. The results showed that first-order kinetics explained very well the hydrolysis kinetics and the Arrhenius equation could be applied to establish a relationship between the degradation rate constant and temperature, at both pHs. It has been observed that CA from fermentation medium was much more unstable than that from standard solution and from a commercially available medicine. Also, it was observed that CA was more stable at PH 6.2 than at pH 7.0, irrespective of the CA source. (C) 2004 Elsevier B.V. All rights reserved.

A novel hybrid thermochemical-biological refinery integrated with power-to-X approach for obtaining biopolymers

In this study, a novel hybrid thermochemical-biological refinery integrated with power-to-x approach was developed for obtaining biopolymers (namely polyhydroxyalkanoates, PHA). Within this concept, a trilogy process schema comprising of, (i) thermochemical conversion via integrated pyrolysis-gasification technologies, (ii) anaerobic fermentation of the bioavailable products obtained through either thermochemistry or water-electrolysis for volatile fatty acids (VFA) production, (iii) and VFA-to-PHA bioconversion via an original microaerophilic-aerobic process was developed. During the first stage of proposed biorefinery where lignocellulosic (wooden) biomass was converted into, theoretically fermentable products (i.e. bioavailables) which were defined as syngas and water-soluble fraction of pyrolytic liquid (WS); biochar as a biocatalyst material; and a dense-oil as a liquid fuel. Within integrated pyrolysis - gasification process, biomass was efficiently converted into fermentable intermediates representing up to 66% of biomass chemical energy content in chemical oxygen demand (COD) basis. In the secondary stage, namely anaerobic fermentation for obtaining VFA rich streams, three different downstream process were investigated. First fermentation test was acidogenic bioconversion of WS materials obtained through pyrolysis of biomass within an original biochar-packed bioreactor, it was sustained up to 0.6 gCOD/L-day volumetric productivity (VP). Second, C1 rich syngas materials as the gaseous fraction of pyrolysis-gasification stage, was fermented within a novel char-based biofilm sparger reactor (CBSR), where up to 9.8 gCOD/L-day VP was detected. Third was homoacetogenic bioconversion within the innovative power-to-x pathway for obtaining commodities via renewable energy sources. More specifically, water-electrolysis derived H2 and CO2 as a primary greenhouse gas was successfully bio-utilized by anaerobic mixed cultures into VFA within CBSR system (VP: 18.2 gCOD/L-day). In the last stage of the developed biorefinery schema, VFA is converted into biopolymers within a new continuous microaerophilic-aerobic microplant, where up to 60% of PHA containing sludges was obtained.

Downstream processing development of enveloped viruses for clinical applications: innovative tools for rational process optimization

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Biotechnology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Optical study of a light diaphragm rupture process in an expansion tube

Rupture of a light cellophane diaphragm in an expansion tube has been studied by an optical method. The influence of the light diaphragm on test flow generation has long been recognised, however the diaphragm rupture mechanism is less well known. It has been previously postulated that the diaphragm ruptures around its periphery due to the dynamic pressure loading of the shock wave, with the diaphragm material at some stage being removed from the flow to allow the shock to accelerate to the measured speeds downstream. The images obtained in this series of experiments are the first to show the mechanism of diaphragm rupture and mass removal in an expansion tube. A light diaphragm was impulsively loaded via a shock wave and a series of images was recorded holographically throughout the rupture process, showing gradual destruction of the diaphragm. Features such as the diaphragm material, the interface between gases, and a reflected shock were clearly visualised. Both qualitative and quantitative aspects of the rupture dynamics were derived from the images and compared with existing one-dimensional theory.

Improving downstream processing for viral vectors and viral vaccines

Viral vectors are playing an increasingly important role in the vaccine and gene therapy elds. The broad spectrum of potential applications, together with expanding medical markets, drives the e orts to improve the production processes for viral vaccines and viral vectors. Developing countries, in particular, are becoming the main vaccine market. It is thus critical to decrease the cost per dose, which is only achievable by improving the production process. In particular advances in the upstream processing have substantially increased bioreactor yields, shifting the bioprocess bottlenecks towards the downstream processing. The work presented in this thesis aimed to develop new processes for adenoviruses puri cation. The use of state-of-the-art technology combined with innovative continuous processes contributed to build robust and cost-e ective strategies for puri cation of complex biopharmaceuticals.(...)