Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

A new approach to heart valve tissue engineering: mimicking the heart ventricle with a ventricular assist device in a novel bioreactor

The `biomimetic` approach to tissue engineering usually involves the use of a bioreactor mimicking physiological parameters whilst supplying nutrients to the developing tissue. Here we present a new heart valve bioreactor, having as its centrepiece a ventricular assist device (VAD), which exposes the cell-scaffold constructs to a wider array of mechanical forces. The pump of the VAD has two chambers: a blood and a pneumatic chamber, separated by an elastic membrane. Pulsatile air-pressure is generated by a piston-type actuator and delivered to the pneumatic chamber, ejecting the fluid in the blood chamber. Subsequently, applied vacuum to the pneumatic chamber causes the blood chamber to fill. A mechanical heart valve was placed in the VAD`s inflow position. The tissue engineered (TE) valve was placed in the outflow position. The VAD was coupled in series with a Windkessel compliance chamber, variable throttle and reservoir, connected by silicone tubings. The reservoir sat on an elevated platform, allowing adjustment of ventricular preload between 0 and 11 mmHg. To allow for sterile gaseous exchange between the circuit interior and exterior, a 0.2 mu m filter was placed at the reservoir. Pressure and flow were registered downstream of the TE valve. The circuit was filled with culture medium and fitted in a standard 5% CO(2) incubator set at 37 degrees C. Pressure and flow waveforms were similar to those obtained under physiological conditions for the pulmonary circulation. The `cardiomimetic` approach presented here represents a new perspective to conventional biomimetic approaches in TE, with potential advantages. Copyright (C) 2010 John Wiley & Sons, Ltd.

O-2 uptake during solid-state fermentation in a rotating drum bioreactor

The maximum O-2 uptake by Rhizopus oligosporus grown in a 200 litre rotating drum bioreactor at 0.5 rpm ranged from 6.7 to 7.6 mmol per min per kg initial dry substrate (IDS), for runs done with 4 baffles each 17 cm wide, and 12 baffles each 5 cm wide. Without baffles, the maximum O-2 uptake rate at 5 rpm was 6.9 mmol/(min.kg IDS), compared to 5.1 mmol/(min.kg IDS) obtained at 0.5 rpm. Therefore O-2 supply is adequate in rotating drum bioreactors as long as slumping flow regimes of the substrate bed are avoided.

Exceptionally high-rate nitrification in sequencing batch reactors treating high ammonia landfill leachate

The nitrogen removal capacity of a suspended culture system treating mature landfill leachate was investigated. Leachate containing high ammonium levels of 300-900 mg N/L was nitrified in a bench scale sequencing batch reactor. Leachate from four different landfills was treated over a two year period for the removal of nitrogen. In this time, a highly specific nitrifying culture was attained that delivered exceptionally high rates of ammonia removal. No sludge was wasted from the system to increase the throughput and up to 13 g/L of MLSS was obtained. Settleability of the purely nitrifying biomass was excellent with SVI less than 40 mL/g, even at the high sludge concentrations. Nitrification rates up to 246 mg NI(L h) (5.91 g N/(L d)) and specific nitrification rates of 36 mg N/(gVSS h) (880 mg N/(gVSS d)) were obtained. The loading to the system at this time allowed complete nitrification of the leachate with a hydraulic retention time of only 5 hours. Following these successful treatability studies, a full-scale plant was designed and built at one of the landfills investigated.

Modelling and optimization of the ion exchange membrane bioreactor for removal of anionic pollutants from drinking water streams

Dissertação para obtenção do Grau de Doutor em Engenharia Química, especialidade de Engenharia Bioquímica

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Immature mosquitoes (Diptera: Culicidae) in a eutrophic landfill tank from State of Rio de Janeiro, Brazil

Introduction To determine the faunal composition of immature culicids inhabiting a percolation tank in the landfill of Sapucaia, State of Rio de Janeiro, Brazil, immature mosquitoes were collected over a two-day period during the third weeks of April, August and October 2011. Results The species found were Culex usquatus, Lutzia bigoti, Anopheles argyritarsis and Limatus durhamii. This study is the first to report the colonization of eutrophic breeding sites by these species. Conclusions The oviposition behavior observed in this study suggests a secondary adaptation or change in habit to select eutrophic environments during the developmental stages of the observed species.

Design and validation of a biomechanical bioreactor for cartilage tissue culture

Specific tissues, such as cartilage undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation - amplitude and frequency - to three dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

Page County Landfill Association, June 30, 2001 & June 30, 2002

Other Audit Reports

Fremont County Landfill Commission, June 30, 2002

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Adair County Sanitary Landfill Commission, June 30, 2002

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Page County Landfill Association, June 30, 2003

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Fremont County Landfill Commission, June 30, 2003

Other Audit Reports - 28E Organization