Dissertations / Theses on the topic 'Bioreactors design'

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2005
The white rot fungus, Phanerochaete chrysosporium, produces enzymes, which are capable of degrading chemical pollutants. It was detennined that this fungus has multiple growth phases. The study provided infonnation that can be used to classify growth kinetic parameters, substrate mass transfer and liquid medium momentum transfer effects in continuous secondary metabolite production studies. P. chrysosporium strain BKMF 1767 (ATCC 24725) was grown at 37 QC in single fibre capillary membrane bioreactors (SFCMBR) made of glass. The SFCMBR systems with working volumes of 20.4 ml and active membrane length of 160 mm were positioned vertically. Dry biofilm density was determined by using a helium pycnometer. Biofilm differentiation was detennined by taking samples for image analysis, using a Scanning Electron Microscope at various phases of the biofilm growth. Substrate consumption was detennined by using relevant test kits to quantify the amount, which was consumed at different times, using a varying amount of spore concentrations. Growth kinetic constants were detennined by using the substrate consumption and the dry biofilm density model. Oxygen mass transfer parameters were determined by using the Clark type oxygen microsensors. Pressure transducers were used to measure the pressure, which was needed to model the liquid medium momentum transfer in the lumen of the polysulphone membranes. An attempt was made to measure the glucose mass transfer across the biofilm, which was made by using a hydrogen peroxide microsensor, but without success.

Dissolved organics in wastewater samples were separated into three size fractions (0-1,000 amu, 1,000-10,000 amu, and 10,000 amu-0.22 m) using ultrafiltration (UF) membranes. The mass distribution within each fraction was adjusted by using a new permeation coefficient model to account for membrane rejection. Dissolved organic and soluble BOD (sBOD) removals in a trickling filter were studied for the different size fractions. The Logan trickling filter model was recalibrated and used to generate predicted removals by size fraction of sBOD, dissolved organic carbon (DOC), and biodegradable DOC (bDOC) for a given influent. Although there was moderate agreement between observed and predicted removals, more investigation is needed to explain shifts in material between different size fractions. Of the three parameters, bDOC may offer a better parameter for modelling trickling filter performance than sBOD.

L'ingegneria tissutale rappresenta un nuovo approccio che integra cellule e matrici ingegnerizzate per la formazione di nuovi tessuti. In questa strategia, tre componenti essenziali costituiscono la cosiddetta triade della Tissue Engineering: segnali regolatori, cellule e scaffold tridimensionali (3D) biodegradabili e porosi. Tali elementi sono combinati per sviluppare un tessuto funzionale organizzato e 3D che simula la matrice extracellulare (ECM) del tessuto da rigenerare. Le funzioni specifiche dei tessuti nativi sono correlate agli ambienti complessi che, all'esterno del corpo, possono essere imitati usando degli strumenti chiamati bioreattori. Questi sistemi forniscono un ambiente in cui i parametri specifici possono essere controllati per raggiungere le condizioni biologiche desiderate. In questa tesi, tutti questi componenti sono stati impiegati per lo sviluppo di modelli in vitro in diverse applicazioni dell'ingegneria tissutale. In particolare, sono stati analizzati e discussi i temi relativi a: scaffold a base di poli-(acido L-lattico) (PLLA), fabbricazione di scaffold tramite separazione di fase, colture cellulari statiche e colture cellulari dinamiche utilizzando bioreattori di perfusione. Due sezioni principali compongono questa tesi: diverse configurazioni sperimentali che utilizzano scaffold a base di PLLA per vari sistemi in vitro; e la progettazione e la modellazione di un bioreattore di perfusione utilizzando fluidodinamica computazionale (CFD) ed equazioni matematiche. In primis, un rigoroso quadro teorico è stato investigato per studiare le proprietà del biomateriale PLLA, l'uso di bioreattori a perfusione per la medicina rigenerativa e i modelli sviluppati per studiare la crescita delle cellule su matrici 3D coltivate all'interno di un sistema dinamico. Negli esperimenti, la morfologia di diversi scaffold in PLLA prodotti attraverso vari protocolli della tecnica di separazione di fase indotta termicamente (TIPS) è stata analizzata in base alle proprietà desiderate per scaffold adatti agli scopi dell’ingegneria tissutale, in termini di porosità, interconnessione dei pori e dimensione dei pori. Le colture cellulari sono state eseguite in questi costrutti per creare un ambiente 3D in modo che le cellule seminate potessero crescere sia in coltura statica 3D che nel bioreattore a perfusione. La proliferazione e l'adesione delle cellule sono state osservate fino a 7 giorni di coltura in vitro, dimostrando che la morfologia degli scaffold può indurre la crescita delle cellule sia in condizioni statiche che dinamiche. Per la seconda parte, si è seguito un approccio combinato di modellazione e sperimentazione. Il sistema di perfusione usato è un bioreattore airlift (precedentemente progettato dal mio gruppo di ricerca) che fornisce un ambiente a basso sforzo di taglio e una buona miscelazione, risolvendo i limiti del trasporto di massa e fornendo stimoli fisici Sommario v vantaggiosi per la proliferazione e la differenziazione delle cellule. L'idrodinamica (gas holdup, velocità superficiale del liquido e sforzo di taglio) e il trasferimento di massa (in termini di coefficiente di trasferimento di massa) sono stati modellati e determinati da analisi CFD per esaminare l'influenza di questi parametri sulla crescita delle cellule e dei tessuti. I risultati della simulazione hanno indicato che l'idrodinamica, i dati matematici e la validazione sperimentale erano in linea tra di loro. In seguito, cellule osteoblastiche sono state coltivate su scaffold posti su un supporto all’interno del bioreattore perfuso con terreno di coltura a 10ml/ min per un massimo di 6 giorni. Combinando i risultati della proliferazione e l'analisi statistica, è stata quantificata e analizzata la crescita cellulare in funzione dello spazio all'interno del sistema bioreattore. Data la natura gerarchica del sistema bioreattore-scaffold, tale sistema è stato considerato dalla scala della matrice extracellulare alla scala del bioreattore. Le proprietà dipendenti dal flusso di una matrice ingegnerizzata e coltivata all'interno di un bioreattore a perfusione sono state studiate teoricamente e valutate sperimentalmente, sottolineando l'influenza delle dipendenze inter-scala. I bioreattori a perfusione sono sistemi in vitro utili per testare famaci poiché imitano l'ambiente in vivo. A questo scopo, è stato modellato e validato sperimentalmente un sistema ottimizzato del bioreattore airlift in grado di indurre un doppio flusso su uno scaffold fabbricato con un canale al suo interno. In particolare, il sistema è stato testato per la diffusione di carriers e per simulare un sistema aria-liquido-interfaccia (ALI) tale da riprodurre l'ambiente della mucosa nasale. Il razionale di tale sistema è il potenziale legato alla combinazione di un flusso interno ed uno esterno di fluidi indipendenti al fine di diffondere i carriers in tutta la matrice ingegnerizzata per pre-screening di farmaci o reindirizzare il mezzo di coltura nel canale dello scaffold per alimentare le cellule seminate. In conclusione, questo progetto di tesi si è concentrato sui principali aspetti dell'ingegneria tissutale e della medicina rigenerativa, spaziando da test in vitro per la crescita delle cellule su scaffold, a modelli per studiare sia le caratteristiche multi-scala di un sistema atto a replicare un tessuto sia l'efficacia della fluidodinamica di un sistema nuovo destinato a validare test farmacologici o mimare al meglio la fisiologia di un tessuto.
Tissue engineering (TE) represents a novel approach that uses cells integrated with matrices to achieve the formation of new tissues. In this strategy, three essential components constitute the so-called triad of Tissue Engineering: regulatory signals, cells, and three-dimensional (3D) biodegradable porous scaffolds. They are combined to develop an organized 3D functional tissue that mimics the extracellular matrix (ECM) of tissue to be regenerated. The tissue-specific functions of native tissues are linked to complex environments that can be replicated outside the body by using special devices called bioreactors. These systems provide an environment where specific parameters can be controlled to match desired biological conditions. In this thesis, all these components are accounted for developing in vitro models for various applications in the field of Tissue Engineering. Specifically, poly-(L-lactic acid) (PLLA)-based scaffold, scaffold fabrication via phase separation, static cell cultures, and dynamic cell cultures using perfusion bioreactors are analyzed and discussed. Two main sections compose this thesis: several experimental setups using PLLA-based scaffolds for various in vitro systems; and the design and modeling of a custom perfusion bioreactor using computational fluid dynamics (CFD) and mathematical equations. A rigorous theoretical framework is developed to study the properties of PLLA biomaterial, the use of perfusion bioreactor for regenerative medicine, and models developed for investigating cells growth on 3D matrices cultured within a dynamic system. In the experiments, the morphology of different PLLA scaffolds produced through different protocols of the thermally induced phase separation technique (TIPS) is analyzed according to the targeted properties of TE scaffolds, i.e., porosity, pore interconnectivity, and pore size. Cell cultures are performed in these constructs to create a 3D environment so that seeded cells can grow both in static 3D culture and the perfusion bioreactor. Cell proliferation and adhesion are observed up to 7 days of in vitro culture, demonstrating that scaffold morphology can induce cell growth under both static and dynamic conditions. For the second part, a combined modeling and experimental approach is followed. The custom-made perfusion apparatus is an existing airlift bioreactor that provides a low-shear environment with good mixing, resolving mass transport limitations and providing physical stimuli beneficial for overall cells proliferation and differentiation. The hydrodynamics (gas holdup, superficial liquid velocity, and shear rate) and mass transfer (kLa and the volumetric mass transfer coefficient) are modeled and determined by CFD to examine the influence of Abstract iii these features on cell and tissue growth. The simulation results indicate that the hydrodynamics matched the mathematical data and experimental validation. Then, osteoblast cells are cultured on a support in the bioreactor perfused with culture medium at 10mL/min for up to 6 days. An evaluation combining proliferation results and statistical analysis allows the quantification of cell growth as a function of the space inside the system. Given the hierarchical nature of the bioreactor-scaffold system, its multi-scale nature will be considered, ranging from the extracellular matrix scale to the bioreactor scale. The flow-dependent properties of an engineered matrix cultured within a perfusion bioreactor are studied theoretically and evaluated experimentally, emphasizing the influence of inter-scale dependencies. Perfusion bioreactors are in vitro systems beneficial for drug screening because they mimic the in vivo environment. For this purpose, an optimized design of the airlift bioreactor that can induce a double-flow on a hollow scaffold is theoretically and experimentally validated. Specifically, the system is tested for carriers diffusion and air-liquid-interface (ALI) model to reproduce the nasal mucosa environment. The rationale is to combine an internal and an external flow of independent fluids for either diffusing the carriers throughout the engineered matrix for drug prescreening or redirecting the culture medium to feed the cells seeded into the channel of the hollow scaffold. In conclusion, this thesis project focuses on the major aspects of tissue engineering and regenerative medicine, varying from in vitro tests for growing cells on scaffolds toward models to study the multi-scale nature of a tissue-like system or recreate the physiology of a native tissue.

Bioengineering
M.S.
The rotating wall vessel (RWV) bioreactor is a well-established cell culture device for the simulation of microgravity for suspension cells and the generation of spheroids and organoids. The key to the success of these systems is the generation of a delicately maintained fluid dynamics system which induces a solid body rotation capable of suspending cells and other particles in a gentle, low-shear environment. Despite the unique capabilities of these systems, the inherently delicate nature of their fluid dynamics makes the RWV prone to multiple failure modes. One of the most frequently occurring, difficult to avoid, and deleterious modes of failure is the formation of bubbles. The appearance of even a small bubble in an RWV disrupts the crucial laminar flow shells present in the RWV, inducing a high-shear environment incapable of maintaining microgravity or producing true spheroids. The difficulty of eliminating bubbles from the RWV substantially increases the learning curve and subsequent barrier-to-entry for the use of this technology. The objective of this study is to create a novel RWV design capable of eliminating the bubble formation failure mode and to demonstrate the design’s efficacy. The tested hypothesis is: “The addition of a channel capable of segregating bubbles from the fluid body of the RWV will protect its crucial fluid dynamics system while enabling the growth of consistently sized and properly formed cell spheroids, improving ease of use of the RWV and decreasing experimental failure.”
Temple University--Theses

Bioreactors are used to carry out bioprocesses and are commonly used in e.g. biogas production and wastewater treatment. Two common hydraulic models of bioreactors are the continuous stirred tank reactor (CSTR) and the plug-flow reactor (PFR). In this paper, a differential equation system that describes the substrate, biomass and inert biomass in the bioreactors is presented. It is used in a steady-state analysis and design of CSTRs in series. Monod kinetics were used to describe the specific growth rate and the decay of biomass was included. Using the derived systems of differential equations, two optimization problems were formulated and solved for both CSTRs in series and for a CSTR+PFR. The first optimization problem was to minimize the effluent substrate level given a total volume, and the second was to minimize the total volume needed to obtain a certain substrate conversion. Results show that the system of differential equations presented can be used to find optimal volume distributions that solves the optimization problems. The optimal volume for N CSTRs in series decreases as N increases, converging towards a configuration of a CSTR followed by a PFR. Analyzing how the decay rate affects the results showed that when the total volume was kept constant, increasing the decay rate caused less difference between the configurations. When the total volume was minimized, increasing the decay rate caused the configurations to diverge from each other. The presented model can be used to optimally divide reactors into smaller zones and thereby increasing the substrate conversion, something that could be of interest in e.g. existing wastewater treatment plants with restricted space. A fairly accurate approximation to the optimal design of N CSTRs in series is to use the optimal volume for the CSTR in the configuration with a CSTR+PFR and equally distribute the remaining volumes.
Bioreaktorer används för att utföra olika biologiska processer och används vanligen inom biogasproduktion eller för rening av avloppsvatten. Två vanliga hydrauliska modeller som används vid modellering av bioreaktorer är helomblandad bioreaktor (på engelska continuous stirred tank reactor, CSTR) eller pluggflödesreaktor (på engelska plug-flow reactor, PFR). I den här rapporten presenteras ett system av differentialekvationer som används för att beskriva koncentrationerna av substrat, biomassa och inert biomassa i både CSTR och PFR. Ekvationssystemet används för analys och design av en serie CSTRs vid steady-state. Tillväxten av biomassa beskrivs av Monod-kinetik. Avdödning av biomassa är inkluderat i studien. Från ekvationssystemet formulerades två optimeringsproblem som löstes för N CSTRs i serie och för CSTR+PFR. Det första optimerinsproblemet var att minimera substrathalten i utflödet givet en total volym. I det andra minimerades den totala volymen som krävs för att nå en viss substrathalt i utflödet. Resultaten visade att ekvationssystemet kan användas för att hitta den optimala volymsfördelningen som löser optimeringsproblemen. Den optimala volymen för N CSTRs i serie minskade när antalet CSTRs ökade. När N ökade konvergerade resultaten mot de för en CSTR sammankopplad med en PFR. En analys av hur avdödningshastigheten påverkade resultaten visade att en ökad avdödningshastighet gav mindre skillnad mellan de två olika konfigurationerna när den totala volymen hölls konstant. När den totala volymen istället minimerades ledde en ökad avdödningshastighet till att de två konfigurationerna divergerade från varandra. Modellen som presenteras i studien kan användas för att fördela en total reaktorvolym i mindre zoner på ett optimalt sätt och på så vis öka substratomvandlingen, något som kan vara av intresse i exempelvis befintliga avloppsreningsverk där utrymmet är begränsat. En relativt bra approximation till den optimala designen av N CSTRs i serie är att optimera volymerna för en CSTR+PFR, använda volymen för CSTR som första volym i konfigurationen med N CSTR i serie, och sedan fördela den kvarvarande volymen lika mellan de övriga zonerna.

This thesis focuses on the design and characterisation of miniature bioreactors and evaluates their potential as a scale-down device for microbial cultivation processes. Miniature bioreactors, such as the one detailed in this work, have been developed by many research groups and companies, and seek to increase throughput at the early stages of bioprocess development. Power input was measured in two prototype stirred-tank miniature bioreactors (10 ml and 25 ml) as a function of impeller speed and the vessels were characterised alongside a 7 L bioreactor. The results show that both miniature bioreactors used in this study were able to be characterised using the same methods developed for larger vessels and that the key engineering parameters of volumetric oxygen transfer coefficient and mixing time compared favourably with those of a conventionally-sized bioreactor when expressed as a function of specific power input. An Escherichia coli plasmid DNA cultivation was successfully scaled down to the 10 ml miniature bioreactor from a 7 L bioreactor on the basis of equal specific power input, and demonstrated equivalent performance under oxygen-rich and oxygen- limited conditions. An intermittently-fed process to produce a Fab' antibody fragment using E. coli and a batch cultivation of the filamentous bacterium Saccharopolyspora erythraea producing erythromycin were also evaluated in the 25 ml miniature bioreactor and three other small scale cell cultivation devices (i.e. microtitre plate, miniature bubble column reactor and shake flasks). Their relative performances in terms of growth and product formation were related to that of the 7 L bioreactor. The results obtained demonstrated the ability of the 25 ml miniature stirred tank bioreactor to perform both of these technically-demanding, industrially-relevant bioprocesses to a comparable degree as the 7 L vessel that was not achievable using the other miniature devices tested. The results shown in this thesis highlight the potential of miniature bioreactors to be used to deliver a fully-integrated, high-throughput solution for cell cultivation process development.

Modern landfilling constitutes an unavoidable final step in solid waste management. It aims to close the “Material Cycle” bringing elements back to the non-mobile state they were in before their extraction. At the same time, the application of Sustainability Principle to landfills prescribes to guarantee environmental protection and health safety, ensuring that the disposed waste will be chemically and biochemically stable within a reasonable amount of time. A “Sustainable Landfill” must combine these two fundamental purposes, balancing the efforts to obtain a “sustainable closure of material loop”. The enhancement of biochemical processes in a landfill, with the purpose of reaching faster environmentally safe conditions and terminate the post closure care, is one of the main debated topics in waste management scientific literature. The general aim of the PhD project was giving a contribution to this debate through the lab-scale testing of systems able to simulate landfills behaviour and the analysis of the long-term expectable chemical status of waste undergone to sustainable landfilling. The first part of the work is an overview on the basic biochemical processes in landfills and on the laboratory-scale landfill simulation tests. The approach used by the PhD student is mainly experimental, starting from the design and the management of several laboratory-scale landfill simulation tests. The elaboration of the obtained data was useful for evaluating the performances of the tested bioreactor concepts as well as for comparing the results to other scientific data derived from a thorough bibliographic research. The original work produced by the student can be subdivided in three different arguments. The Semi-aerobic, Anaerobic, Aerated (S.An.A. ®) hybrid bioreactor is an innovative landfill concept, lab-scale run with promising results concerning the enhancement of methane production and the reduction of the long-term emissions. The effects of the recirculation of reverse osmosis leachate concentrate inside the landfill have been analysed to check if the potential accumulation of contaminants in waste body can turn this practice unsustainable. The Final Storage Quality (FSQ) procedure, for endorsing the landfill Post Closure Care termination, was tested on an over-stabilized waste of which total emissions and chemical speciation of main elements were calculated.
Il moderno sistema di deposito finale dei rifiuti in discarica costituisce un passaggio inevitabile nella gestione dei rifiuti solidi. Il suo scopo è chiudere il “ciclo della materia” riportando gli elementi allo stato di immobilità in cui erano prima di essere estratti. Contemporaneamente, l’applicazione del principio di sostenibilità alle discariche prescrive di garantire la salvaguardia ambientale e della salute, assicurando che il rifiuto smaltito diventi chimicamente e bio-chimicamente stabile entro un tempo “ragionevole”. Una “Discarica Sostenibile” deve combinare questi due principi, bilanciando i contributi per ottenere una “chiusura sostenibile del ciclo della materia”. Il potenziamento dei processi biochimici in discarica, con lo scopo di raggiungere più velocemente condizioni che garantiscano la salvaguardia ambientale e terminare la fase di post-chiusura, è uno degli argomenti più dibattuti nella letteratura scientifica inerente alla gestione dei rifiuti. Lo scopo generale del progetto di dottorato è stato contribuire a questo dibattito, mediante lo svolgimento di test in scala di laboratorio utili a simulare l’andamento dei processi in discarica e analizzando lo stato biochimico finale dei rifiuti trattati. La prima parte del lavoro consiste in una panoramica sui processi biochimici in discarica e sulla metodica dei test biochimici in scala di laboratorio. L’approccio usato dallo studente in questa tesi è principalmente sperimentale, basato sulla progettazione, l’esecuzione e la rielaborazione dei dati di svariate simulazioni di discarica in laboratorio. La discussione dei risultati ottenuti è stata propedeutica alla valutazione delle performance dei modelli concettuali testati così come al confronto con altri risultati ottenuti grazie a una approfondita ricerca bibliografica. Il lavoro originale svolto dallo studente può essere diviso in tre progetti principali. Il reattore ibrido Semi-aerobico, Anaerobico, Aerato (S.An.A ®) è una concetto innovativo testato in scala di laboratorio con promettenti risultati per quanto concerne la stimolazione della produzione di metano e la riduzione delle emissioni di lungo termine. Gli effetti del ricircolo del concentrato di percolato da osmosi inversa all’interno del corpo rifiuti di una discarica sono stati analizzati per verificare se possano esistere potenziali accumuli di contaminanti che rendano insostenibile tale pratica. La procedura di Final Storage Quality (FSQ) per determinare la chiusura della fase di aftercare di una discarica è stata testata su un rifiuto sovra-stabilizzato di sui sono state calcolate emissioni totali e la speciazione chimica degli elementi principali.

Cell cultivation is the process of cultivating cells in an artificial environment outside of their natural body. GE Healthcare develops products for cell cultivation, both the actual cell cultivation instrument and the software for controlling it. Presently GE's bioreactor system can only be controlled and monitored from a screen on amachine beside the cultivation. If the people working with cell cultivation could monitor the cultivations from a smartphone application, they would always know what is going on in the cultivation and could even change parameters if necessary, without having to go to the cultivation. Time is saved and the cultivation can be monitored more often, thus can problems in the cultivation be discovered earlier and the risk of the cultivation perishing is decreased. An iterative design process with focus on usability was used to explore what functions the cell cultivators need in a smartphone application to support their work, and different design prototypes were explored together with the cell cultivators and usability experts. The result showed that the necessary functions in the application mainly involves monitoring of the most important values, a list of elevated alarms and the history chart for the values, but controlling of the system such as turning functions on/off and changing setpoints is also useful in some situations. This report can be used as groundwork for future development of smartphone applications for cell cultivation.

The establishment of a high productivity microbial fermentation process requires the experimental investigation of many interacting variables. In order to speed up this procedure a novel miniature stirred bioreactor system is described which enables parallel operation of 4-16 independently controlled fermentations. Each miniature bioreactor is of standard geometry (100 mL maximum working volume) and is fitted with a magnetically driven six-blade miniature turbine impeller (dj = 20 mm, dj/dj = 1/3) operating in the range 100 - 2000 rpm. Aeration is achieved via a sintered sparger at flow rates in the range of 0 - 2 vvm. Continuous on-line monitoring of each bioreactor is possible using miniature pH, dissolved oxygen and temperature probes, while PC-based software enables independent bioreactor control and real-time visualisation of parameters monitored on-line. Initial characterisation of the bioreactor involved quantification of the volumetric oxygen mass transfer coefficient as a function of agitation and aeration rates. The maximum kLa value obtained was 0.11 s" The reproducibility of E. coli TOP10 pQR239 and B. subtilis ATCC6633 fermentations was shown in four parallel fermentations of each organism. For E. coli (1000 rpm, 1 vvm) the maximum specific growth rate, umax, was 0.68 0.01 h"1 and the final biomass concentration obtained, Xr,nai, was 3.8 0.05 g.L"1. Similarly for B. subtilis (1500 rpm, 1 vmm) umax was 0.45 0.01 h"1 and Xrinai was 9.0 0.06 g.L"1. Biomass growth kinetics increased with increases in agitation and aeration rates and the implementation of gas blending for control of DOT levels enabled umax and Xfmai values as high as 0.93 h"1 and 8.1 g.L"1 respectively to be achieved. The value of the miniature bioreactor design for high throughput experimentation was further demonstrated when Design of Experiments (DoE) techniques were employed to assess three variables temperature, pH and inducer concentration, for the optimisation of CHMO expression in E. coli TOP 10 pQR239. The optimised regression model derived from the results of 20 fermentations concluded that only temperature and inducer concentration had a significant influence, predicting a maximum specific CHMO activity of 105.9 U.g"1 at 37.1 C and 0.11 %w/v. This was in good agreement with the experimentally determined results at these conditions. In order to enable the predictive scale-up of miniature bioreactor results, the engineering characterisation of the miniature turbine impeller predicted a Power number of 3.5 based on experimental ungassed power consumption measurements. As a result of the numerous literature correlation relating kLa, gassed power per unit volume and superficial gas velocity being designed for much larger scale bioreactors, a similar correlation has been specifically derived for the miniature bioreactor scale. Constant ki,a has been shown to be the most reliable basis for predictive scale-up of fermentation results from the miniature bioreactor to conventional laboratory scale. This was confirmed over a range of kLa values (0.04-0.11 s"1), with good agreement between final biomass concentrations and maximum specific growth rates. In addition successful scale-up of the DoE results for optimum CHMO expression in E. coli at a constant kLa value of 0.04 s"1 yielded final biomass concentrations of 4.9 g.L"1 and 5.1 g.L"1 respectively in the miniature bioreactor and the 2 L vessel, and the CHMO activity obtained was 105.9 U.g"1 and 105.2 U.g"1 respectively. Finally, alternative on-line methods for monitoring cell growth within the miniature bioreactors without the need for repeated sampling have been described. The application of a novel optical density probe for monitoring biomass growth kinetics on-line has shown that comparable results for calculated maximum specific growth rates were obtained from off-line and on-line OD measurements 0.67 and 0.68 h"1 respectively. Thermal profiling techniques were also investigated as an alternative means for monitoring cell growth based on the natural heat generated by a microbial culture. Initial results showed that the heat generated during E. coli TOP 10 pQR239 fermentations followed the same pattern as the off-line growth curve. The maximum specific growth rates calculated from off-line and on-line thermal data were also in good agreement, 0.66 0 0.04 h"1 and 0.69 0 0.05 h"1 respectively. In summary the miniature bioreactor system designed and evaluated here provides a useful tool for the rapid optimisation and scale-up of microbial fermentation processes.

This thesis is based on diagnosing, assessing and optimising the design and operation of membrane bioreactors (MBR) used for treating municipal wastewater. Specifically, this thesis has been carried out within the framework of seven municipal MBR wastewater treatment plants (WWTP) located in Catalonia with the collaboration of the Catalan Water Agency. Firstly, the design and operational issues of MBRs have been diagnosed, together with determining the main operational problems related to this technology. Secondly, the optimisation strategies applied in each full-scale MBR and the resulting costs were assessed. Finally, two of the operational problems identified were exhaustively evaluated: The ragging phenomenon and the biological nitrogen removal and operational costs optimisations. The research carried out in this thesis has enabled the design and operation of the municipal MBRs to be assessed while, at the same time, presenting several optimisation strategies which will improve the operation and costs of this technology
Aquesta tesi es centra en la diagnosi, avaluació i optimització del disseny i l’operació dels bioreactors de membranes (BRM) pel tractament d’aigües residuals. Concretament, l’estudi s’ha realitzat dins del marc de treball de set estacions depuradores d’aigües residuals (EDARs) municipals amb tecnologia BRM presents a Catalunya en col·laboració amb l’Agència Catalana de l’Aigua. Primerament s’ha dut a terme la diagnosi de l’estat del disseny i operació dels BRMs i s’han determinat les problemàtiques associades a aquesta tecnologia. Així mateix, s’han avaluat les estratègies d’optimització realitzades i els corresponents costos d’operació. A partir de la diagnosis realitzada, dos dels principals problemes operacionals observats s’han caracteritzat i optimitzat: El ragging i l’optimització de l’eliminació biològica de nitrogen i dels costos d’operació. La recerca presentada en aquesta tesi ha permès diagnosticar l’estat dels BRMs, alhora que ha mostrat possibles vies d’optimització que permetran millorar l’operació i els costos associats a aquesta tecnologia

[EN] Anaerobic MBRs (AnMBRs) can provide the desired step towards sustainable wastewater treatment, broadening the range of application of anaerobic biotechnology to low-strength wastewaters (e.g. urban ones) or extreme environmental conditions (e.g. low operating temperatures). This alternative technology gathers the advantages of anaerobic treatment processes (e.g. low energy demand stemming from no aeration and energy recovery through methane production) jointly with the benefits of membrane technology (e.g. high quality effluent, and reduced space requirements). It is important to highlight that AnMBR may offer the possibility of operation in energy neutral or even being a net energy producer due to biogas generation. Other aspects that must be taken into account in AnMBR are the quality and nutrient recovery potential of the effluent and the low amount of sludge generated, which are of vital importance when assessing the environmental impact of a wastewater treatment plant (WWTP). The main aim of this Ph.D. thesis is to assess the economic and environmental sustainability of AnMBR technology for urban wastewater treatment at ambient temperature. Specifically, this thesis focusses on the following aspects: (1) development of a detailed and comprehensive plant-wide energy model for assessing the energy demand of different wastewater treatment systems at both steady- and unsteady-state conditions; (2) proposal of a design methodology for AnMBR technology and identification of optimal AnMBR-based configurations by applying an overall life cycle cost (LCC) analysis; (3) life cycle assessment (LCA) of AnMBR-based technology at different temperatures; and (4) evaluation of the overall sustainability (economic and environmental) of AnMBR for urban wastewater treatment. In this research work, a plant-wide energy model coupled to the extended version of the plant-wide mathematical model BNRM2 is proposed. The proposed energy model was used for assessing the energy performance of different wastewater treatment processes. In order to propose a guidelines for designing AnMBR at full-scale and to identify optimal AnMBR-based configurations, the proposed energy model and LCC were used. LCA was used to assess the environmental performance of AnMBR-based technology at different temperatures. An overall sustainability (economic and environmental) assessment was conducted for: (a) assessing the implications of design and operating decisions by including sensitivity and uncertainty analysis and navigating trade-offs across environmental and economic criteria.; and (b) comparing AnMBR to aerobic-based technologies for urban wastewater treatment. This Ph.D. thesis is enclosed in a national research project funded by the Spanish Ministry of Science and Innovation entitled "Using membrane technology for the energetic recovery of wastewater organic matter and the minimisation of the sludge produced" (MICINN project CTM2008-06809-C02-01/02). To obtain representative results that could be extrapolated to full-scale plants, this research work was carried out in an AnMBR system featuring industrial-scale hollow-fibre membrane units that was operated using effluent from the pre-treatment of the Carraixet WWTP (Valencia, Spain).
[ES] El reactor anaerobio de membranas sumergidas (AnMBR) puede proporcionar el paso deseado hacia un tratamiento sostenible del agua residual, ampliando la aplicabilidad de la biotecnología anaerobia al tratamiento de aguas residuales de baja carga (ej. agua residual urbana) o a condiciones medioambientales extremas (ej. bajas temperaturas de operación). Esta tecnología combina las ventajas de los procesos de tratamiento anaerobio (baja demanda energética gracias a la ausencia de aireación y a la recuperación energética a través de la producción de metano) con los beneficios de la tecnología de membranas (ej. efluente de alta calidad y reducidas necesidades de espacio). Cabe destacar que la tecnología AnMBR permite la posibilidad del autoabastecimiento energético del sistema debido a la generación de biogás. Otros aspectos que se deben considerar en el sistema AnMBR son el potencial de recuperación de nutrientes, la calidad del efluente generado y la baja cantidad de fangos producidos, siendo todos ellos de vital importancia cuando se evalúa el impacto medioambiental de una planta de tratamiento de aguas residuales urbanas. El objetivo principal de esta tesis doctoral es evaluar la sostenibilidad económica y medioambiental de la tecnología AnMBR para el tratamiento de aguas residuales urbanas a temperatura ambiente. Concretamente, esta tesis se centra en las siguientes tareas: (1) desarrollo de un modelo de energía detallado y completo que permita evaluar la demanda energética global de diferentes sistemas de tratamiento de aguas residuales tanto en régimen estacionario como en transitorio; (2) propuesta de una metodología de diseño e identificación de configuraciones óptimas para la implementación de la tecnología AnMBR, aplicando para ello un análisis del coste de ciclo de vida (CCV); (3) análisis del ciclo de vida (ACV) de la tecnología AnMBR a diferentes temperaturas; y (4) evaluación global de la sostenibilidad (económica y medioambiental) de la tecnología AnMBR para el tratamiento de aguas residuales urbanas. En este trabajo de investigación se propone un modelo de energía acoplado a la versión extendida del modelo matemático BNRM2. El modelo de energía propuesto se usó para evaluar la eficiencia energía de diferentes procesos de tratamiento de aguas residuales urbanas. Con el fin de proponer unas directrices para el diseño de AnMBR a escala industrial e identificar las configuraciones óptimas para la implementación de dicha tecnología, se aplicaron tanto el modelo de energía propuesto como un análisis CCV. El ACV se usó para evaluar la viabilidad medioambiental de la tecnología AnMBR a diferentes temperaturas. En este trabajo se llevó a cabo una evaluación global de la sostenibilidad (económica y medioambiental) de la tecnología AnMBR para: (a) evaluar las implicaciones que conllevan ciertas decisiones durante el diseño y operación de dicha tecnología mediante un análisis de sensibilidad e incertidumbre, y examinar las contrapartidas en función de criterios económicos y medioambientales; y (b) comparar la tecnología AnMBR con tecnologías basadas en procesos aerobios para el tratamiento de aguas residuales urbanas. Esta tesis doctoral está integrada en un proyecto nacional de investigación, subvencionado por el Ministerio de Ciencia e Innovación (MICINN), con título "Modelación de la aplicación de la tecnología de membranas para la valorización energética de la materia orgánica del agua residual y la minimización de los fangos producidos" (MICINN, proyecto CTM2008-06809-C02-01/02). Para obtener resultados representativos que puedan ser extrapolados a plantas reales, esta tesis doctoral se ha llevado a cabo utilizando un sistema AnMBR que incorpora módulos comerciales de membrana de fibra hueca. Además, esta planta es alimentada con el efluente del pre-tratamiento de la EDAR del Barranco del Carraixet (Valencia, España).
[CAT] El reactor anaerobi de membranes submergides (AnMBR) pot proporcionar el pas desitjat cap a un tractament d'aigües residuals sostenible, i suposa una extensió en l'aplicabilitat de la biotecnologia anaeròbia al tractament d'aigües residuals amb baixa càrrega (p.e. aigua residual urbana) o a condicions mediambientals extremes (p.e. baixes temperatures d'operació). Aquesta tecnologia alternativa reuneix els avantatges dels processos de tractament anaerobi (baixa demanda d'energia per l'estalvi de l'aireig i possibilitat de recuperació energètica per la producció de metà), conjuntament amb els beneficis de l'ús de de la tecnologia de membranes (p.e efluent d'alta qualitat, i reduïdes necessitats d'espai). Cal destacar que la tecnologia AnMBR permet la possibilitat de l'autoabastiment energètic del sistema degut a la generació de biogàs. Altres aspectes que s'han de considerar en el sistema AnMBR són el potencial de recuperació de nutrients, la qualitat de l'efluent i la baixa quantitat de fang generat, tots ells de vital importància quan s'avalua l'impacte mediambiental d'una planta de tractament d'aigües residuals urbanes. L'objectiu principal d'aquesta tesi doctoral és avaluar la sostenibilitat econòmica i mediambiental de la tecnologia AnMBR per al tractament d'aigües residuals urbanes a temperatura ambient. Concretament, aquesta tesi se centra en les tasques següents: (1) desenrotllament d'un detallat i complet model d'energia per al conjunt de la planta a fi d'avaluar la demanda d'energia de diferents sistemes de tractament d'aigües residuals tant en règim estacionari com en transitori; (2) proposta d'una metodologia de disseny i identificació de les configuracions òptimes de la tecnologia AnMBR mitjançant l'aplicació una anàlisi del cost de tot el cicle de vida (CCV) ; (3) anàlisi del cicle de vida (ACV) de la tecnologia AnMBR a diferents temperatures; i (4) avaluació global de la sostenibilitat (econòmica i mediambiental) de la tecnologia AnMBR per al tractament d'aigües residuals urbanes. En aquest treball d'investigació es proposa un model d'energia a nivell de tota la planta acoblat a la versió estesa del model matemàtic BNRM2. El model d'energia proposat s'ha utilitzat per a avaluar l'eficiència energètica de diferents processos de tractament d'aigües residuals urbanes. A fi de proposar unes directrius per al disseny d'AnMBR a escala industrial i identificar les configuracions òptimes de la tecnologia AnMBR, s'ha aplicat tant el model d'energia proposat, com el cost del cicle de vida (CCV). L'anàlisi del cicle de vida (ACV) s'ha utilitzat per a avaluar el rendiment mediambiental de la tecnologia AnMBR a diferents temperatures. En aquest treball s'ha dut a terme una avaluació global de la sostenibilitat (econòmica i mediambiental) de la tecnologia AnMBR per a: (a) avaluar les implicacions de les decisions de disseny i operació per mitjà d'una anàlisi de sensibilitat i incertesa i examinar les contrapartides en funció de criteris econòmics i mediambientals; i (b) comparar la tecnologia AnMBR amb tecnologies basades en processos aerobis per al tractament d'aigües residuals urbanes. Aquesta tesi doctoral està integrada en un projecte nacional d'investigació, subvencionat pel Ministerio de Ciencia e Innovación (MICINN), amb títol "Modelación de la aplicación de la tecnología de membranas para la valorización energética de la materia orgánica del agua residual y la minimización de los fangos producidos" (MICINN, projecte CTM2008-06809-C02-01/02). Per a obtenir resultats representatius que puguen ser extrapolats a plantes reals, aquesta tesi doctoral s'ha dut a terme utilitzant un sistema AnMBR que incorpora mòduls comercials de membrana de fibra buida. A més, aquesta planta és alimentada amb l'efluent del pretractament de l'EDAR del Barranc del Carraixet (València, Espanya).
Pretel Jolis, R. (2015). Environmental and economic sustainability of submerged anaerobic membrane bioreactors treating urban wastewater [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58864
TESIS
Premiado

Les bioréacteurs à biofilm représentent une technologie prometteuse pour la production continue de biosurfactants microbiens grâce à la robustesse naturelle des cellules immobilisées et à la conception possible de procédés évitant la formation de mousse. La souche bactérienne B. subtilis 168 a le potentiel de produire de la surfactine, un biosurfactant puissant qui possède des activités biologiques exceptionnelles ayant des applications industrielles diverses. Cependant, B. subtilis 168 ne présente que de faibles capacités de formation de biofilms et donc entraîne des capacités d'adhésion cellulaire limitées. Afin d'améliorer l'immobilisation cellulaire naturelle de B. subtilis 168 et pour mieux adapter cette souche à la culture de biofilms, des mutants filamenteux avec une production d'exopolysaccharides (EPS) restaurée ont été générés. Les impacts des modifications génétiques ont été évalués par des tests de colonisation et en mesurant la capacité de formation de biofilm sous faible contrainte de cisaillement dans un réacteur à écoulement goutte à goutte (DFR). Par la suite, les souches les plus performantes ont été sélectionnées et cultivées dans un bioréacteur à biofilm à film tombant continu contenant des éléments de garnissage métallique structurés pour la formation de biofilm. De plus, un modèle de croissance bactérienne a été développé pour décrire la dynamique de croissance des cellules planctoniques et du biofilm dans le système. Le développement des colonies a été fortement affecté par la croissance des cellules filamenteuses et la production d'EPS ce qui s’est manifesté par une capacité accrue d'étalement de surface et de colonisation. Dans le DFR et le bioréacteur à biofilm à film tombant, les mutants EPS+ ont montré des performances significativement augmentées concernant la formation de biofilm et les capacités de production de surfactine. La filamentation cellulaire a eu un impact mineur sur le procédé mais a contribué à une meilleure cohésion cellulaire dans le biofilm et a également conduit à un détachement cellulaire réduit pendant la culture. Ainsi, la production d'EPS et la croissance des cellules filamenteuses ont considérablement contribué à l'amélioration des performances du procédé dans le système. De plus, la culture en mode continu s'est révélée favorable à une production élevée en surfactine. Les données expérimentales du bioréacteur à biofilm à film tombant sont concordantes avec celles obtenues par des simulations avec le modèle de croissance développé. Par conséquent, le modèle de croissance a été validé avec succès et pourrait être utilisé pour une optimisation ultérieure de procédés à biofilm
Biofilm bioreactors show promise for continuous microbial biosurfactant production due to the natural robustness of self-immobilized cells and the possible design of processes avoiding foam formation. The widely used bacterial strain B. subtilis 168 has the potential to produce surfactin, a powerful biosurfactant with exceptional biological activities and various industrial applications. However, B. subtilis 168 exhibits only poor biofilm formation capacities and thus entails limited cell adhesion capacities. In order to improve the natural cell immobilization of B. subtilis 168 to adapt this strain better to biofilm cultivation, filamentous mutant strains with restored exopolysaccharide (EPS) production were generated. The impacts of the genetic modifications were evaluated through colonization assays and by measuring the biofilm formation capacity under low shear stress in a drip-flow reactor (DFR). Subsequently, the most performant strains were selected and cultivated in a newly designed continuous trickle-bed biofilm bioreactor containing highly structured metal packing elements for biofilm formation. Moreover, a bacterial growth model was built able to describe the growth dynamics of the planktonic cell and biofilm population in the system. The colony development was strongly affected by filamentous cell growth and EPS production which was manifested through an enhanced surface spreading and colonization capacity. In the DFR and trickle-bed biofilm bioreactor, the EPS+ mutants showed significantly increased performances regarding the biofilm formation and surfactin production capacities. Whereas cell filamentation had a minor impact on the processes, but contributed to a better cell cohesion in the biofilm and led to reduced cell detachment during the cultivation. Thus, EPS production and filamentous cell growth contributed considerably to an improved process performance in the system. In addition, continuous fermentation has shown to be favorable for a high surfactin productivity. The experimental data from the trickle-bed biofilm bioreactor were in good accordance with those obtained by simulations with the developed growth model. Hence, the growth model has been successfully validated and could be used for further process optimization

A conventional 2D (two-dimensional) culture, in T-flasks or multi-well plates, is commonly perfo med for the stem cell development; however, it is time and labour consuming process. Moreover, it is impractical to scale-up to high cell number production. Growing stem cells inside bioreactor might be a solution. 3D bioreactor is not only a solution for scalable production but also a mimic environment for in vivo system. Herein, sparged-type bioreactors (e.g. airlift bioreactor) were chosen as bioreactors to differentiate murine embryonic stem cells (mESCs) into type II pneumocytes in the lung. There are two main sections in this thesis: the design of airlift bioreactor using computational fluid dynamics (CFD) and the differentiation of mESCs into the alveolar progenitor cells in a sparged bioreactor. The airlift bioreactors provide a better environment, which theoretically has been known to simulate the gas-exchange interface encountered in the lung alveoli. They require a low power input and provide a low shear environment with good mixing. The hydrodynamics (gas holdup, superficial liquid velocity, and shear rate) and mass transfer (kLa, the volumetric mass transfer coefficient) features of different airlift designs were determined by CFD. The simulations were based on a 3D transient model, Eulerian-Eulerian approach, and two-phase liquid/gas model with all phases being treated as laminar flow. The superficial gas velocity was varied from 0.001 m/s to 0.02 m/s. The simulation results indicated that the hydrodynamics were corresponded to the data found in literatures and the gas holdup were agreed with an experiment validation. The CFD results also suggested that in which range of superficial gas velocity (ug) that the system can be operated without any fluctuation in terms of the hydrodynamics. In addition, the airlift bioreactor is suitable for shear sensitive cells with high mass transfer rate, e.g. kLa, = 180 hr-1 at ug= 0.01 m/s and normoxia (20% O2) condition. Hence, the results from these simulations have been initially utilised as a promising hypothesis to design an airlift bioreactor for the scalable and automatable culture in multiphase bioreactors. For the second part, mESCs were encapsulated in a calcium-alginate hydrogel to create a 3D environment then the encapsulated cells weregrown in both 3D static culture, in a T-flasks, and the sparged bioreactor. The gas, 5% CO2 and 20% O2, was directly sparged into the bioreactor. The A549 conditioned medium was used to induced the mESCs to the endodermal lineages, targeting for the alveolar type II cells, type II pneumocytes. The differentiated cells expressed lung cell markers: SPC (pneumocyte type II), and FoxA2 (endoderm marker). In experiments, the relative expression of SPC markers reached the maximum level, 10-fold increase, at day 14 and day 20 for 3D static culture and the sparged bioreactor, respectively. After day 20 of the differentiation process, the pneumocyte-like cells in static culture trend to lose their SPC expression whereas the cells in sparged bioreactor maintain relatively high SPC markers. At the end of a differentiation protocol, day 30, it was observed that both systems highly expressed the endodermal makers, FoxA2, i.e. approximately 2000-fold increase for static culture and 5000-fold increase for the sparged bioreactor. In conclusion, the direct gassing in the sparged bioreactor not only enhanced the differentiation of embryonic stem cells into type II pneumocytes but also mimicked the in vivo environment in the lung therefore the differentiated cells can maintain the lung phenotype for a long term culture, up to 5 weeks in vitro culture. This in vitro system would be beneficial for drug screening and regenerative medicine applications.

La production de protéines recombinantes, d’anticorps, de vaccins, … est de plus en plus réalisée par culture de cellules animales. Le bioréacteur classiquement utilisé en industrie pour réaliser ces cultures est le bioréacteur à cuve agitée. Ce bioréacteur présente un volume important ce qui rend difficile le développement d’un bioréacteur à usage unique dans l’optique de réduire les risques de contaminations entre deux cultures consécutives. L’intensification du procédé et le développement de bioréacteur à usage unique sont donc deux défis intéressants dans la réalisation de cultures cellulaires à l’échelle industrielle. Un bioréacteur particulièrement prometteur pour l’intensification de culture cellulaire est le bioréacteur lit-fixe. Dans cette thèse, nous étudions le bioréacteur lit-fixe à usage unique iCELLis développé par Artelis S.A.

Le lit-fixe du bioréacteur iCELLis est composé d’un empilement de porteurs maintenu entre deux grilles perforées. Ces porteurs sont utilisés comme support par les cellules au cours de la culture. Le bioréacteur est équipé d’un système de transfert gaz-liquide par film tombant afin d’oxygéner le milieu de culture en continu. Une pompe centrifuge plongée dans un bac d’immersion assure la circulation du milieu de culture à travers l’ensemble du bioréacteur. Les cultures se déroulent en trois phases :une phase d’adhérence des cellules aux porteurs du lit-fixe, une phase de croissance cellulaire et une phase de production.

Les avantages d’un bioréacteur lit-fixe sont nombreux :une concentration cellulaire élevée impliquant une productivité élevée, un petit volume de bioréacteur, une faible exposition des cellules aux contraintes de cisaillement, Les bioréacteurs lits-fixes présentent cependant certains inconvénients qui freinent leur développement à l’échelle industrielle. Le lit-fixe se présente comme un réacteur piston ce qui implique l’apparition de gradients de concentrations de cellule et d’espèces extracellulaires (nutriments et produits) le long du lit-fixe. L’intérieur du lit-fixe est également difficilement accessible au cours de la culture. Le suivi des concentrations de cellules et d’espèces dans cette zone est donc problématique.

Une modélisation globale du bioréacteur lit-fixe nous permet de mieux comprendre les différents phénomènes qui prennent place dans le bioréacteur. Grâce à cette modélisation, nous sommes donc capables d’identifier les phénomènes clés contrôlant le procédé et ainsi fournir des pistes de travail pour l’optimisation et la montée en échelle du bioréacteur, ceci sur base de critères rationnels.

Nous choisissons de développer un modèle à compartiments du bioréacteur lit-fixe. Dans ce type de modèle, le bioréacteur est représenté par un réseau de compartiments interconnectés. Nous définissons trois compartiments :un premier pour la pompe, un deuxième pour les cellules et le lit-fixe, et un troisième pour le système de transfert gaz-liquide.

Pour le premier compartiment, nous souhaitons caractériser divers paramètres identifiés comme pertinents pour une sélection adéquate de la pompe. Dans cette thèse, nous présentons une méthode pour caractériser ces paramètres pour une pompe de référence (celle du bioréacteur iCELLis) et pour une pompe en similitude géométrique à la pompe de référence (dans le but d’étudier la montée en échelle).

La pompe de référence est étudiée numériquement (grâce aux logiciels Gambit 2.4 et Fluent 6.3) et expérimentalement. Nous mettons en évidence les liens entre les paramètres de la pompe déterminés numériquement et ceux déterminés expérimentalement. Ces liens définissent notre modèle. En intégrant au modèle les résultats de la simulation numérique de l’écoulement du milieu de culture dans le bac d’immersion contenant la pompe en similitude géométrique à la pompe de référence, nous déterminons entièrement les paramètres recherchés de la seconde pompe sans avoir recours à un prototype. Ceci permet donc de tester différentes échelles avant de choisir la version finale de la seconde pompe.

Le deuxième compartiment du modèle caractérise les cellules et le lit-fixe. La sélection de certains paramètres opératoires dépend du métabolisme cellulaire. Nous souhaitons développer un outil de surveillance en ligne de l’évolution des concentrations de certaines espèces extracellulaires sur base de la connaissance de la concentration cellulaire dans le bioréacteur. Cet outil est développé sur base d’un modèle structuré du métabolisme des cellules animales. Dans un tel modèle, nous établissons des bilans de matière sur les espèces extra- et intracellulaires en considérant les voies métaboliques intracellulaires. Un paramètre requis pour l’emploi de cet outil est la connaissance de la concentration cellulaire au cours de la culture. Or, la surveillance de cette concentration est l’un d’un problème évoqué dans les bioréacteurs lits-fixes. Nous développons donc un modèle ségrégé de culture cellulaire en bioréacteur lit-fixe. Dans ce modèle, nous considérons l’entièreté du lit-fixe. Le modèle comprend différentes populations de cellules :les cellules en suspension dans le milieu au début de la culture et les cellules adhérentes au lit-fixe. Le modèle inclut une distribution spatiale de la concentration d’espèces extracellulaires dans le lit-fixe. Par conséquent, le modèle rapporte les gradients potentiels de concentration de cellules et d’espèces extracellulaires dans le lit-fixe.

Le troisième compartiment du modèle du bioréacteur caractérise le système de transfert gaz-liquide. L’oxygénation est très souvent un paramètre clé dans la conception d’un bioréacteur. Dans le bioréacteur iCELLis, le système de transfert gaz-liquide est un film liquide tombant turbulent. Dans cette thèse, nous proposons une méthode pour caractériser le transfert d’oxygène à travers ce type de film tombant. Notre méthode, basée sur une approche numérique (grâce à Gambit 2.4 et Fluent 6.3), est scindée en deux parties. Premièrement, nous calculons la forme de l’interface gaz-liquide. Une simulation de l'écoulement est réalisée avec le modèle Volume of Fluid (VOF). A partir de cette simulation, la forme de l'interface est traquée. Deuxièmement, la forme de l'interface est générée dans un nouveau domaine de calcul afin de simuler le transfert d’oxygène. Grâce à cette seconde simulation, le coefficient de transfert d’oxygène de la phase gazeuse vers le milieu de culture est déterminé. Grâce à notre méthode, nous caractérisons ce coefficient pour différentes conditions opératoires. Nous étudions notamment l’influence du débit et de la température du milieu de culture sur le coefficient de transfert d’oxygène.
Doctorat en Sciences de l'ingénieur
info:eu-repo/semantics/nonPublished

Many control systems of practical importance are multivariable. In such systems, each manipulated variable (input signal) may affect several controlled variables (output signals) causing interaction between the input/output loops. For this reason, control of multivariable systems is typically much more difficult compared to the single-input single-output case. It is therefore of great importance to quantify the degree of interaction so that proper input/output pairings that minimize the impact of the interaction can be formed. For this, dedicated interaction measures can be used. The first part of this thesis treats interaction measures. The commonly used Relative Gain Array (RGA) is compared with the Gramian-based interaction measures the Hankel Interaction Index Array (HIIA) and the Participation Matrix (PM) which consider controllability and observability to quantify the impact each input signal has on each output signal. A similar measure based on the norm is also investigated. Further, bounds on the uncertainty of the HIIA and the PM in case of uncertain models are derived. It is also shown how the link between the PM and the Nyquist diagram can be utilized to numerically calculate such bounds. Input/output pairing strategies based on linear quadratic Gaussian (LQG) control are also suggested. The key idea is to design single-input single-output LQG controllers for each input/output pair and thereafter form closed-loop multivariable systems for each control configuration of interest. The performances of these are compared in terms of output variance. In the second part of the thesis, the activated sludge process, commonly found in the biological wastewater treatment step for nitrogen removal, is considered. Multivariable interactions present in this type of bioreactor are analysed with the tools discussed in the first part of the thesis. Furthermore, cost-efficient operation of the activated sludge process is investigated.

Microalgae culture is currently receiving considerable attention for its potential in wastewater treatment and production of algal biomass from which high-value bioproducts and bioenergy can be obtained, as well as the consequent carbon dioxide sequestration via photosynthesis process. However, biomass harvesting is one of the bottlenecks in microalgae culture and microalgae-based wastewater treatment systems. Energy intensive technologies are required to separate the solid-liquid phase due the low density of microalgae. Low-cost processes, such as sedimentation, are not efficient enough due to the low settling velocities of the microalgae. Sedimentation coupled to coagulation and flocculation has been widely studied on lab-scale in order to increase the microalgae settling velocity. However, few studies have addressed the scaling up of these experimental results in order to industrializing the process. The thesis has been divided into two main parts. A first study addressed the physical and theoretical principles of sedimentation used for the operation and optimization of biomass harvesting from a microalgae culture for wastewater treatment at demonstration scale in the framework of the INCOVER research project "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), which aimed to validate innovative technologies at demonstration scale to convert wastewater into an alternative energy source and value-added products. A second study focused on the operation and optimization of the downstream thickening process of biomasspreviously harvested in the same facilities. Finally, the second part consist of a study and optimization of the behavior of mixed liquor in its transit through a high rate algae pond for wastewater treatment using Computational Fluid Dynamics modeling for its implementation in the city of Aligarh. This study has been carried out under the H2020 PAVITR project (http://www.pavitr.net; GA 821410), which aims at validation of sustainable natural and advanced technologies for water and wastewater treatment, monitoring and safe reuse of water in India. In the first study of the first part, the physical and theoretical principles of sedimentation were addressed to be used for the operation and optimization of harvesting biomass in lamella settler (700 L) from a microalgae culture for wastewater treatment on a three semi-closed tubular photobioreactor (11.7 m3 each) at demonstrative scale. During 6 months the inflow (6900 m3·day-1), coagulant dosage (1-12 mg·L-1) and purges of the biomass (60-240 L·day-1) were adjusted in order to achieve a proper separation of the solid-liquid phase. Results in this section evidenced the efficiency of the Lamella in the solid-liquid separation task obtaining an outlet turbidity below of 5 NTU after the optimization period. In the second part, two thickeners were operated and optimized in order to achieve a proper concentration (20 g·L-1) of previous harvested biomass for subsequent anaerobic digestion process at the same installations. The scrapers and purges were optimized in four periods during two months. Results showed an eventually concentrations of 26.5 g·L-1 in last period due a minimized use of scrapers in order to avoid the particles resuspension allowing a proper compression settling. In the second part, demonstrative-scale HRAP system was designed to be implemented in Aligarh (India) with a treatment capacity of 50 m3·day-1. The objective of the study was to assist, verify and optimize the conventional dimensioning of the High Rate Algae Ponds (HRAP) by means of biokinetic modelling and hydrodynamic analysis using Computational Fluid Dynamics (CFD). According to the biokinetic model simulations, 4 days was the optimal hydraulic retention time to enhance nutrient removal. A 3D model of the HRAP was built to analyze the hydrodynamic behavior of 36 different carousel designs. The different combinations of baffle numbers on the reversals, center wall widths and tear-shape sizes were simulated. The presence of low velocity zones as well as the useful area vs. the total occupied area were quantify. Two baffles and tear-shapes with a diameter equal to ¼ of the channel width was the most efficient configuration. In addition, a techno-economic assessment of the system determined an investment cost of € 483 per population equivalent (PE) and an operational cost of € 0.19 per m3 of treated wastewater.
El cultiu de microalgues està rebent actualment una atenció considerable pel seu potencial en el tractament d'aigües residuals i la producció de biomassa d'algues de la qual es poden obtenir bioproductes d'alt valor i bioenergia, així com el segrest consegüent de diòxid de carboni mitjançant el procés de fotosíntesi. Tot i això, la recol·lecció de biomassa és un dels colls d'ampolla del cultiu de microalgues i dels sistemes de tractament d'aigües residuals basats en elles. La separació de la fase sòlida-líquida requereix tecnologies d'alt consum energètic a causa de la baixa densitat de les microalgues. Els processos de baix cost, com la sedimentació, no són prou eficaços a causa de la baixa velocitat de sedimentació de les microalgues. La sedimentació combinada amb la coagulació i la floculació s'ha estudiat àmpliament a escala de laboratori per augmentar la velocitat de sedimentació de la biomassa algal. Tot i això, pocs estudis han abordat l'augment d'escala d'aquests resultats experimentals per industrialitzar el procés. La tesi ha estat dividida en dues parts. La primera està formada per dos estudis i és el tema principal d'aquesta tesi. Un primer estudi va abordar els principis físics i teòrics de la sedimentació que s'utilitzen pel funcionament i optimització de la recol·lecció de biomassa d'un cultiu de microalgues pel tractament d'aigües residuals a escala demostrativa en el marc del projecte de recerca INCOVER "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), l'objectiu del qual era validar tecnologies innovadores a escala demostrativa per convertir les aigües residuals en una font d’energia alternativa i en productes de valor afegit. Un segon estudi es va centrar en el funcionament i optimització del procés d'espessiment posterior de la biomassa prèviament collida a les mateixes instal·lacions mitjançant dos espessidors treballant en línia. Finalment, la segona part va consistir en l'estudi i l'optimització del comportament del licor barrejat en el trànsit per un estany d'algues d'alta taxa pel tractament d'aigües residuals mitjançant la modelització de la Dinàmica de Fluids Computacional per a la seva implantació a la ciutat d’Aligarh. Aquest estudi s'ha realitzat en el marc del projecte H2020 PAVITR (http://www.pavitr.net; GA 821410), l'objectiu del qual és la validació de tecnologies naturals i avançades sostenibles pel tractament de l'aigua i de les aigües residuals, control i la reutilització segura de l’aigua a l’Índia. Al primer estudi de la primera part, es van abordar els principis físics i teòrics de la sedimentació per utilitzar-los en el funcionament i l'optimització de la collita de biomassa en sedimentador de làmines (700 L) d'un cultiu de microalgues pel tractament d'aigües residuals en un fotobioreactor tubular semitancat de tres (11,7 m3 cadascun) a escala demostrativa. Durant 6 mesos es va ajustar el flux d'entrada (6900 m3-dia-1), la dosi de coagulant (1-12 mg·L-1) i les purgues de la biomassa (60-240 L·dia-1) per aconseguir una adequada separació de la fase sòlid-líquida. Els resultats d'aquest apartat van evidenciar l'eficàcia de les lamel·les en la tasca de separació sòlid-líquid obtenint una terbolesa de sortida inferior a 5 NTU després del període d'optimització. En el segon estudi, es van operar i optimitzar dos espessidors per aconseguir una concentració adequada (20 g·L-1) de la biomassa recol·lectada prèviament pel posterior procés de digestió anaeròbia a les mateixes instal·lacions. Els espessidors i les purgues es van optimitzar en quatre períodes durant dos mesos. Els resultats van mostrar una concentració final de 26,5 g·L-1 a l'últim període a causa d'un ús minimitzat dels rascadors per evitar la resuspensió de les partícules permetent una adequada sedimentació per compressió. A la segona part, es va dissenyar una Llacuna d'Alta Càrrega a escala demostrativa per ser implementada a Aligarh (Índia) amb una capacitat de tractament de 50 m3・dia-1. L'objectiu de l'estudi era assistir, verificar i optimitzar el dimensionament convencional de les llacunes d'alta càrrega mitjançant la modelització biocinètica i l'anàlisi hidrodinàmica mitjançant dinàmica de fluids computacional (CFD). Segons les simulacions del model biocinètic, el temps de retenció hidràulica òptim per millorar l'eliminació de nutrients va ser de 4 dies. Es va construir un model 3D de la llacuna per analitzar el comportament hidrodinàmic de 36 dissenys en forma de carrusel amb diferents configuracions. Es van simular les diferents combinacions de nombres de deflectors en les inversions, amples de paret central i mides de forma de llàgrima als extrems del mur central. Es va quantificar la presència de zones de baixa velocitat, així com l'àrea útil davant de l'àrea total ocupada. La configuració més eficient va ser la composta per dos deflectors i formes de llàgrima amb un diàmetre igual a . de l'amplada del canal. A més, una avaluació tecno-econòmica del sistema va determinar un cost d'inversió de 732 euros per població equivalent (PE) i un cost operatiu de 0,19 euros per m3 d'aigua residual tractada.
El cultivo de microalgas está recibiendo actualmente una atención considerable por su potencial en el tratamiento de aguas residuales y la producción de biomasa de algas de la que se pueden obtener bioproductos de alto valor y bioenergía, así como el consiguiente secuestro de dióxido de carbono mediante el proceso de fotosíntesis. Sin embargo, la recolección de biomasa es uno de los cuellos de botella del cultivo de microalgas y de los sistemas de tratamiento de aguas residuales basados en ellas. La separación de la fase sólida-líquida requiere tecnologías de alto consumo energético debido a la baja densidad de las microalgas. Los procesos de bajo coste, como la sedimentación, no son lo suficientemente eficaces debido a la baja velocidad de sedimentación de las microalgas. La sedimentación combinada con la coagulación y la floculación se ha estudiado ampliamente a escala de laboratorio para aumentar la velocidad de sedimentación de la biomasa algal. Sin embargo, pocos estudios han abordado el aumento de escala de estos resultados experimentales con el fin de industrializar el proceso. La tesis se ha dividido en dos partes principales. La primera está conforma por dos estudios y es el tema principal de esta tesis. Un primer estudio abordó los principios físicos y teóricos de la sedimentación que se utilizan para el funcionamiento y la optimización de la recolección de biomasa de un cultivo de microalgas para el tratamiento de aguas residuales a escala demostrativa en el marco del proyecto de investigación INCOVER "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), cuyo objetivo era validar tecnologías innovadoras a escala demostrativa para convertir las aguas residuales en una fuente de energía alternativa y en productos de valor añadido. Un segundo estudio se centró en el funcionamiento y optimización del proceso de espesamiento posterior de la biomasa previamente cosechada en las mismas instalaciones mediante dos espesadores trabajando en línea. Por último, la segunda parte consistió en el estudio y optimización del comportamiento del licor mezclado en su tránsito por un estanque de algas de alta tasa para el tratamiento de aguas residuales mediante la modelización de la Dinámica de Fluidos Computacional para su implantación en la ciudad de Aligarh. Este estudio se ha realizado en el marco del proyecto H2020 PAVITR (http://www.pavitr.net; GA 821410), cuyo objetivo es la validación de tecnologías naturales y avanzadas sostenibles para el tratamiento del agua y de las aguas residuales, el control y la reutilización segura del agua en la India. En el primer estudio de la primera parte, se abordaron los principios físicos y teóricos de la sedimentación para utilizarlos en el funcionamiento y la optimización de la cosecha de biomasa en sedimentador de láminas (700 L) de un cultivo de microalgas para el tratamiento de aguas residuales en un fotobiorreactor tubular semicerrado de tres (11,7 m3 cada uno) a escala demostrativa. Durante 6 meses se ajustó el flujo de entrada (6900 m3·día-1), la dosis de coagulante (1-12 mg·L-1) y las purgas de la biomasa (60-240 L·día-1) para conseguir una adecuada separación de la fase sólido-líquida. Los resultados de este apartado evidenciaron la eficacia de las lamelas en la tarea de separación sólidolíquido obteniendo una turbidez de salida inferior a 5 NTU tras el periodo de optimización. En el segundo estudio, se operaron y optimizaron dos espesadores para conseguir una concentración adecuada (20 g·L-1) de la biomasa recolectada previamente para el posterior proceso de digestión anaerobia en las mismas instalaciones. Los espesadores y las purgas se optimizaron en cuatro periodos durante dos meses. Los resultados mostraron una concentración final de 26,5 g·L-1 en el último periodo debido a un uso minimizado de los rascadores para evitar la resuspensión de las partículas permitiendo una adecuada sedimentación por compresión. En la segunda parte, se diseñó una Laguna de Alta Carga a escala demostrativa para ser implementada en Aligarh (India) con una capacidad de tratamiento de 50 m3·día-1. El objetivo del estudio era asistir, verificar y optimizar el dimensionamiento convencional de las Lagunas de Alta Carga mediante la modelización biocinética y el análisis hidrodinámico mediante Dinámica de Fluidos Computacional (CFD). Según las simulaciones del modelo biocinético, el tiempo de retención hidráulica óptimo para mejorar la eliminación de nutrientes fue de 4 días. Se construyó un modelo 3D de la laguna para analizar el comportamiento hidrodinámico de 36 diseños en forma de carrusel con diferentes configuraciones. Se simularon las diferentes combinaciones de números de deflectores en las inversiones, anchos de pared central y tamaños de forma de lágrima en os extremos del muro central. Se cuantificó la presencia de zonas de baja velocidad, así como el área útil frente al área total ocupada. La configuración más eficiente resultó ser la compuesta por dos deflectores y formas de lágrima con un diámetro igual a ¼ de la anchura del canal. Además, una evaluación técnico-económica del sistema determinó un coste de inversión de 732 euros por población equivalente (PE) y un coste operativo de 0,19 euros por m3 de agua residual tratada.
Enginyeria ambiental

Thesis (MTech (Chemical Engineering))--Peninsula Technikon, 2001
Certain fungi have been shown to excrete extracellular enzymes, including peroxidases, laccases, etc. These enzymes are useful for bioremediation of aromatic pollutants present in industrial effluents (Leukes, 1999; Navotny et aI, 1999). Leukes (1999) made recent significant development in the form of a capillary membrane gradostat (fungal) bioreactor that offers optimal conditions for the production of these enzymes in high concentrations. This system also offers the possibility for the polluted effluent to be treated directly in the bioreactor. Some operating problems relating to continuous production of the enzymes and scale-up of the capillary modules, were, however, indentified. In an attempt to solve the above-mentioned identified problems the research group at Peninsula Technikon considered a number of alternative bioreactor configurations. A pulsed packed bed bioreactor concept suggested by Moreira et at. (1997) was selected for further study. Their reactor used polyurethane pellets as the support medium for the fungal biofilm and relied upon pulsing of the oxygen supply and recycle of nutrient solution in order to control biomass accumulation. These authors reported accumulation due to the recycle of proteases that were believed to destroy the desired ligninases. We experimented with a similar concept without recycle to avoid backrnixing and thereby overcome protease accumulation. In our work, a maximum enzyme productivity of 456 Units.L1day·1 was attained. Since this was significantly greater than the maximum reported by Moreira et aI, 1997 (202 Units.L-1day-I) it appeared that the elimination of recycle had significant benefits. In addition to eliminating recycle we also used a length / diameter (L / D) ratio of 14: 1 (compared with 2.5: 1 used by Moreira et aI, 1997) in order to further reduce backrnixing. Residence time distributions were investigated to gain insight into mechanisms of dispersion in the reactor. It was found that the pulsed packed bed concept presented problems with regard to blockage by excess biomass. This led us to consider the advantages of a fluidized bed using resin beads. Accordingly, growth of fungi on resin beads in shake flasks was investigated with favorable results. An experimental program is proposed to further investigate the fluidized bed concept with a view to extending the operation time of the bioreactor. From our literature survey to date, packed bed fungal bioreactors are still the best reactor configuration for continuous production ofligninolytic enzymes. An interesting study of the application of laccases to the degradation of naphthalene and MTBE is described in an addendum to this thesis.

Bioreactors are devices used for the growth of tissues in a laboratory environment. They exist in many different forms, each designed to enable the production of high-quality tissues. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. Chondrocytes, the building blocks of articular cartilage, for example, are stimulated by mechanical stresses such as shear, as compared with those in tissues grown under static incubation conditions. On the other hand, high shear can damage cells. Consequently the shear-stress level has to be controlled in order to optimize the design and the operating conditions of bioreactors. Spinner flasks have been used for the production of articular cartilage in vitro. Assuming the existence of a relation between the cellular glycosaminoglycan (GAG) synthesis and the local shear stresses on the construct surfaces, this research focuses on the development of a model for cartilage growth in such devices. The flow produced in a model spinner flask is characterized experimentally using particle-image velocimetry (PIV). A computational fluid dynamic (CFD) model validated with respect to the laboratory measurements is constructed in order to predict the local shear stresses on the construct surfaces. Tissue growth experiments conducted in the prototype bioreactor permit construct histologies and GAG contents to be analyzed and then correlated with the shear-stress predictions. The integration of this relation into the CFD model enables the prediction of GAG synthesis through convective effects. Coupling this convective model to an existing diffusive model produces a complete cartilage-growth model for use in aiding the optimization of existing bioreactors, and in the design of new ones.

In questa tesi viene presentato un bioreattore in grado di mantenere nel tempo condizioni biologiche tali che consentano di massimizzare i cicli di evoluzione molecolare di vettori di clonazione fagici: litico (T7) o lisogeno (M13). Verranno quindi introdtti concetti legati alla Teoria della Quasispecie e alla relazione tra errori di autoreplicazione e pressioni selettive naturali o artificiali su popolazioni di virus: il modello naturale del sistema evolutivo. Tuttavia, mantenere delle popolazioni di virus significa formire loro un substrato dove replicare. Per fare ciò, altri gruppi di ricerca hanno giá sviluppato complessi e costosi prototipi di macchinari per la crescita continua di popolazioni batteriche: i compartimenti dei sistemi evolutivi. Il bioreattore, oggetto di questo lavoro, fa parte del progetto europeo Evoprog: general purpose programmable machine evolution on a chip (Jaramillo’s Lab, University of Warwick) che, utilizzando tecnologie fagiche e regolazioni sintetiche esistenti, sará in grado di produrre funzionalità biocomputazionali di due ordini di grandezza più veloci rispetto alle tecniche convenzionali, riducendo allo stesso tempo i costi complessivi. Il primo prototipo consiste in uno o piú fermentatori, dove viene fatta crescere la cultura batterica in condizioni ottimizzate di coltivazione continua, e in un cellstat, un volume separato, dove avviene solo la replicazione dei virus. Entrambi i volumi sono di pochi millilitri e appropriatamente interconnessi per consentire una sorta di screening continuo delle biomolecole prodotte all’uscita. Nella parte finale verranno presentati i risultati degli esperimenti preliminari, a dimostrazione dell’affidabilità del prototipo costruito e dei protocolli seguiti per la sterilizzazione e l’assemblaggio del bioreattore. Gli esperimenti effettuati dimostrano il successo di due coltivazioni virali continue e una ricombinazione in vivo di batteriofagi litici o lisogeni ingegnerizzati. La tesi si conclude valutando i futuri sviluppi e i limiti del sistema, tenendo in considerazione, in particolare, alcune applicazioni rivolte agli studi di una terapia batteriofagica.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2013.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 195-203).
The research objective is to design a micro-bioreactor for the culture of Chinese Hamster Ovary (CHO) cells. There is an increasing demand for upstream development in high-throughput micro-bioreactors specifically for the recombinant CHO cell line, an important cell line for producing recombinant protein therapeutics. In order to translate a micro-bioreactor originally designed by our group for bacteria to CHO cells, there would need to be significant modifications in the design of the micro-bioreactor due to the extreme sensitivity of CHO cells to physical and chemical stresses. Shear stresses inside the growth chamber will have to be reduced by three orders of magnitude. Moreover, the long doubling time of CHO cells requires a 2 weeks long culture. In a high surface to volume ratio micro-bioreactor, evaporation becomes a major problem. Contamination control is also vital for CHO cultures. In addition, the offline sampling volume required for validation necessitates a doubling of the working volume to 2mL. The newly designed Resistive Evaporation Compensated Actuated (RECA) micro-bioreactor is fully characterized in this thesis to ensure that the design meets the physical specifications of the required CHO cell culture conditions. The RECA micro-bioreactor will be tested with industrial recombinant CHO cell lines. This work is done in collaboration with Genzyme, USA and Sanofi-Aventis, Frankfurt. In this thesis, we also propose the use of dielectric spectroscopy electrodes for online cell viability sensing of CHO cells in micro-bioreactors. The electrodes are fabricated on polycarbonate, a biocompatible and optically clear thermoplastic that will be one of the future base material for microfluidic devices which can be rapidly prototyped. To demonstrate the viability of dielectric spectroscopy as an online viability sensor for CHO cells in a micro-bioreactor, the electrodes are used to characterize samples taken daily from a CHO shake flask batch culture without any sample modifications. Two different electrode geometries and correction methods will be compared to find the optimal system for viability measurements in a micro-bioreactor.
by Shireen Goh.
Ph.D.

The following thesis presents a computational framework that can capture inherently non-linear and emergent biocomplex phenomena. The main motivation behind the investigations undertaken was the absence of a suitable platform that can simulate, both the continuous features as well as the discrete, interaction-based dynamics of a given biological system, or in short, dynamic reciprocity. In order to determine the most powerful approach to achieve this, the efficacy of two modelling paradigms, transport phenomena as well as agent-based, was evaluated and eventually combined. Computational Fluid Dynamics (CFD) was utilised to investigate optimal boundary conditions, in terms of meeting cellular glucose consumption requirements and exposure to physiologically relevant shear fields, that would support mesenchymal stem cell growth in a 3-dimensional culture maintained in a commercially available bioreactor. In addition to validating the default bioreactor configuration and operational parameter ranges as suitable towards sustaining stem cell growth, the investigation underscored the effectiveness of CFD as a design tool. However, due to the homogeneity assumption, an untenable assumption for most biological systems, CFD often encounters difficulties in simulating the interaction-reliant evolution of cellular systems. Therefore, the efficacy of the agent-based approach was evaluated by simulating a morphogenetic event: development of in vitro osteogenic nodule. The novel model replicated most aspects observed in vitro, which included: spatial arrangement of relevant players inside the nodule, interaction-based development of the osteogenic nodules, and the dependence of nodule growth on its size. The model was subsequently applied to interrogate the various competing hypotheses on this process and identify the one that best captures transformation of osteoblasts into osteocytes, a subject of great conjecture. The results from this investigation annulled one of the competing hypotheses, which purported the slow-down in the rate of matrix deposition by certain osteoblasts, and also suggested the acquisition of polarity to be a non-random event. The agent-based model, however, due to being inherently computationally expensive, cannot be recommended to model bulk phenomena. Therefore, the two approaches were integrated to create a modelling platform that was utilised to capture dynamic reciprocity in a bioreactor. As a part of this investigation, an amended definition of dynamic reciprocity and its computational analogue, dynamic assimilation, were proposed. The multi-paradigm platform was validated by conducting melanoma chemotaxis under foetal bovine serum gradient. Due to its CFD and agent-based modalities, the platform can be employed as both a design optimisation as well as hypothesis testing tool.

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2017.
Page 206 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 196-205).
Biologic drug manufacturing is traditionally conducted in large-scale, industrial bioreactors. The emergence of interest in disposable, bench-top bioreactors as a viable alternative is due to potential advantages such as lower contamination risk, time and cost savings, and ease of handling. The challenges associated with disposable, bench-top bioreactors include poor mixing, limited oxygen transfer, and a scarcity of non-invasive sensors for process control. This thesis investigates multiple aspects of a disposable, perfusion-capable bioreactor, in order to facilitate an optimal design. In order to determine an impeller configuration that improves the mixing and mass transfer characteristics of a i-liter bioreactor, Computational Fluid Dynamics (CFD) was used. The potential benefits of switching to a dual-Marine impeller system was revealed, which was then validated during fermentation experiments. Further predictions of a merging flow pattern in the i-liter vessel was consistent with the literature based on the impeller spacing. A scaled-up 5-liter vessel was designed with Rushton impellers spaced so as to create a parallel flow pattern, which was later successfully predicted in the CFD simulations. Flow patterns were analyzed at various locations in both vessels to aid future design iterations. Monitoring of process parameters, including liquid level, is important for automated control in bioreactors. Three novel, non-invasive, optical liquid level sensing methods were conceptualized, prototyped, and successfully tested. These solutions relied on self-developed image processing algorithms. Additionally, a magnetic liquid level sensor was also developed and tested that relied on a magnetic float and a series of reed switches. In order to increase the perfusion membrane surface area and reduce complexity, the switch to a hollow-fiber harvest probe was examined. CFD studies guided design iterations by modeling the flow around the probe, giving insight into the stagnation properties and shear forces acting on the fibers. Additionally, experimental testing of the new harvest probe revealed its successful functionality and viability in the bioreactor.
by Craig Anthony Mascarenhas.
S.M.

The availability of large numbers of engineered organs would offer significant benefits to the clinical management of surgery. Tissue engineering offers the potential of providing tissues that can mimic the morphology, function and physiologic environment of native ones. Cells could grow in vitro within a biodegradable polymer to construct tissue for implantation. However no generic bioreactor design currently exists. There is now a need to establish a robust process for the production of engineered tissues using autologous cells. A key challenge will be the prediction of the supply of nutrients and removal of metabolites. Models of transport phenomena were developed in order to predict the fluid flow and mass transfer requirements of a prototype bioreactor for the formation of engineered tissues. These models were solved to generate windows of operation which relate key operating parameters with the feasibility of tissue preparation. Examples highlight how the windows of operation can be used to visualize rapidly the region of operating conditions that satisfy the design constraints. The impact of the cell concentration, tube geometry, alginate diffusivity, substrate and metabolite concentration levels, feed and recycle rate on the design of the bioreactor is illustrated. The result of this analysis determines the best configuration of the bioreactor which can meet the cellular transport requirements as well as being reliable in performance whist seeking to reduce the amount of valuable nutrients to be used. Micro scale experiments were designed in order to evaluate from measurements, effective diffusivities of substrates and metabolites in alginate matrices as well as substrate consumption and metabolite production rates in matrices with immobilized growing cells. The oxygen diffusivity and oxygen uptake rate of alginate immobilized neonatal fibroblasts were evaluated using integrated oxygen sensor spots. Additionally, alginate cylindrical constructs with immobilized neonatal fibroblasts were prepared in transwells in order to evaluate the effective diffusivities of glucose and lactate as well as the glucose consumption and lactate production rate. The advantage of such micro scale experiments was that greater data sets could be generated with the small number of cells available but in a way which predicts the larger scale. The database which was created was used to construct the windows of operation to give quantitative solutions of how engineered tissues may be prepared and to visualize process operability in a more explicit way.

Tissue engineering is an interdisciplinary field in which cell biology, biomaterials science, and surgery are combined and its main goal is to repair, replace and reproduce tissues and organs. Following this procedure, cells are seeded on proper scaffolds and induced in sequence to adhere, eventually differentiate, proliferate and finally to produce the wanted extracellular matrix (ECM). During cell culture, the usefulness of applying proper physiological-like stimuli, i.e., biochemical but also mechanical signals to drive and accelerate both cell differentiation and ECM production has been demonstrated. Tissue regeneration can be either conducted entirely in vivo or assisted by a previous in vitro phase. Considering the latter situation, a bioreactor can be defined as any apparatus that attempts to mimic physiological conditions in order to maintain and encourage tissue regeneration in dynamic conditions. Dynamic cell cultures using bioreactors can be considered a good intermediate step between the conventional in vitro static approach and in vivo studies. Therefore it is possible to promote the formation of the specific tissue by simulating physiological conditions via the application of specific mechanical and biochemical stimuli. The proposed work is focused on the design and development of bioreactors for bone and cartilage regeneration, in which optimal cell culture conditions are controlled (temperature, nutrients, carbon dioxide and oxygen levels), and mechanical stimuli are applied on the cell constructs. This study presents a wide investigation concerning these mechanical stimulations in order to understand the best cell culture parameters for the activations of cells, naturally accustomed to similar stresses inside the joint. In particular, direct compression, change in hydrodynamic pressure and perfusion modes are compared and analyzed.

A 5 litre laboratory-scale upflow anaerobic sludge blanket (UASB) bioreactor was constructed and operated for approximately one year to reduce sulphate in water using an agricultural byproduct, silage, as carbon source. The purpose of this water treatment system was to test the suitability of the UASB design to treat simulated ground water with high sulphate concentrations destined to be used as cattle drinking water. The UASB reactor design was selected after performing an extensive literature review of all available sulphate-reduction processes. A previous MASc project (Amber Brown, 2007) demonstrated the suitability of silage as a carbon source for sulphate reducing bacteria and, furthermore, in this thesis, fate of the organic compounds in the silage leachate during sulphate-reduction was determined. Six particular tests were performed in order to quantify the type of organics in the feed and effluent: chemical oxygen demand (COD), total organic carbon (TOC), total carbohydrates, total alcohols, total phenols, and selected organic and volatile fatty acids (VFA). The reactor ran continuously for approximately one year with a constant silage leachate feed COD concentration of 10,000 mg L₋−¹, and sulphate feed concentrations varying from 2,000 to 3,200 mg L−¹. The flow rates for each feed stream were maintained at ~0.5 mL min−¹ for silage leachate and ~1 mL min−¹ for sulphate feed for most of the experiment. The sulphate reduction rates (SRR) ranged from 368 to 845 mg L−¹ d−¹ and the amount of organics consumed was between 80-90%. Sulphide levels in the UASB bioreactor were consistently high for most of the experiment, ranging from 600-800 mg L−¹. When the sulphate feed concentration was increased to a maximum of 3,282 (± 27.22) mg L−¹, the sulphide concentration within the bioreactor reached a maximum of 1,273 (± 473.5) mg L−¹. A sulphide stripping column was introduced midway through the experiment in an attempt to reduce the sulphide concentration in the system. Short-term results were promising, however, prolonged sulphide removal in the system could not be maintained due to operational problems. Interestingly, during the last month of operation, despite the high sulphide levels, the SRR was at its highest with an upward trend.

Intervertebral disc (IVD) degeneration is a prevalent health problem that is highly linked to back pain. To understand the disease and tissue response to therapies, ex-vivo whole IVD organ culture systems have recently been introduced. The goal of this study was to develop and validate a whole spinal segment culturing system that loads the disc in complex loading similar to the in-vivo condition, while preserving the adjacent endplates and vertebral bodies. The complex loading applied to the spinal segment was achieved with three pneumatic cylinders. The pneumatic cylinders were rigidly attached to two triangular alumni plates at each corner, comprising the loading mechanism. By extending or compressing the pneumatic cylinders, three modes of loading were achieved: flexion-extension, bi-lateral bending, and cyclic compression. The cylinders were controlled via microcontroller, and the entire system was fully automated. The culture container, which housed the spinal segment during culturing, was a flexible silicone container with an aluminum base and lid. The culture container attached to the loading mechanism allows for loading of the spinal segment. It had a vent attached to the aluminum lid that allowed for gas exchange in the system. The dynamic bioreactor was able to achieve physiologic loading conditions with 100 N of applied compression and approximately 2-4 N-m of applied torque. The function of the bioreactor was validated through testing of bovine caudal IVDs with intact endplates and vertebral bodies that were isolated within 2 hours of death and cultured for 14 days under a diurnal cycle. The resulting IVD cell viability following 14 days of loading was approximately 43% and 20% for the nucleus pulposus and annulus fibrosus respectively, which was significantly higher than the unloaded controls. The loading system accurately mimicked flexion-extension, bi-lateral bending, and compression motions seen during daily activities. Results indicate that this complex dynamic bioreactor may be appropriate for extended pre-clinical testing of vertebral mounted spinal devices and therapies.

ABSTRACT Implementation of Physiologic Pressure Conditions in a Blood Vessel Mimic Bioreactor System Kevin Mark Okarski Tissue engineering has traditionally been pursued as a therapeutic science intended for restoring or replacing diseased or damaged biologic tissues or organs. Cal Poly’s Blood Vessel Mimic Laboratory is developing a novel application of tissue engineering as a tool for the preclinical evaluation of intravascular devices. The blood vessel mimic (BVM) system has been previously used to assess the tissue response to deployed stents, but under non-physiologic conditions. Since then, efforts have been made to improve the vessel and bioreactor’s ability to emulate in vivo conditions. The ability to tissue engineer constructs similar to their native tissue counterparts is heavily reliant upon controlling the environment and mechanical stimuli the construct is exposed to. Mimicking physiologic conditions influences cellular growth, proliferation, and differentiation. Two important mechanical stimuli are cyclic strain and wall shear stress. Previous work sought to improve these factors within the BVM bioreactor and resulted in the implementation of pulsatile perfusion and increased fluid viscosity. These previous bioreactor design modifications generated pulsatile pressures of approximately 80 mmHg and a wall shear stress of 6.4 dynes/cm2. However, physiologic pressure waveforms were not achieved. Studies in this thesis were carried out to implement an effective means of establishing a more physiologic pressure wave within the bioreactor that is accurate, consistent, and easily adjustable. As a result of conducting the present studies, modifications to the bioreactor system were made that uphold the overall goals of efficacy and efficiency. The desired pressure wave was created by setting the degree of pump tubing occlusion on the 3-roller peristaltic pump head and using a water column to backpressure the bioreactor chamber. Maintaining a desired backpressure within the system necessitated the development of a new bioreactor chamber with increased extraluminal leak pressure resistance. The opportunity was also used to further improve upon the bioreactor chamber design to allow for 360° rotation to reduce cell sedimentation. Modifications to the bioreactor system required quantitative evaluation to assess their impact upon local flow dynamics to the tissue construct. A system model was created and evaluated using computational modeling. Through the work performed in this thesis, pulsatile pressure waves of approximately 120/80 mmHg were successfully established within the bioreactor. The ability to accurately model physiologic pressures will ultimately help yield tissue constructs more similar to native tissues – both healthy and pathological. The newly designed bioreactor chamber and computational model for the system will be helpful tools for implementing or evaluating future bioreactor developments or improvements. While the main objective of the thesis has been completed by creating a system capable of emulating physiologic pressure fluctuations, there still remains room for further improvements in back-pressuring and scaling the system, refining the rotational bioreactor chamber design, and building upon the complexity and accuracy of the computational model.

Bioreactors capable of subjecting cells and tissues to time-varying mechanical strain are one aspect of simulating in vivo conditions. A bioreactor to impart arbitrary strain waveforms on cells or tissue scaffolds for loading conditions found in the airway was designed and developed and, in the process, it was determined that there are sources of experimental error which could invalidate bioreactor experiments if not properly mitigated. Without effective design and validation, bioreactors can impart significantly different stimuli than the assumed experimental conditions. Cyclic strain is thought to play a role in airway remodeling by mediating cytoskeletal contraction of the airway smooth muscle. In vitro experiments have demonstrated varying changes to the cytoskeleton depending on experimental conditions. Based on literature review, the strain waveform, magnitude, mechanical properties of the substrate, and anisotropy of the strain stimulus may all affect airway smooth muscle (ASM) differentiation. A bioreactor capable of imparting a broad range of strain stimulus was developed using stepper motors as actuators to allow open-loop control. Any changes in the cells subjected to cyclic strain in these bioreactors would be assumed to correlate with cyclic strain, but a poorly designed bioreactor could introduce confounding experimental stimuli which could easily invalidate the experiment. Heat generated by the actuators can overheat the cell cultures. Vibration might alter the cytoskeletal response. Strain response across the substrate can drastically vary from modeling predictions depending on the loading conditions and how the substrate has been constrained. Methods of mitigating heat generation and transfer were developed. The vibrations emitted by the two stepper motor options were evaluated. A method of mapping the substrate was developed such that nonplanar strains across the substrate surface could be characterized to validate the experimental conditions prior to testing. Finally, ASM cells were subjected to cyclic and static strain on PDMS substrates and cell realignment evaluated. Cells were noted to realign in the cyclic strain tests, as has been reported in several earlier publications, but also realigned under static strain conditions. The bioreactor design objectives were met.
Applied Science, Faculty of
Graduate

Optimisation of a mammalian cell culture process requires the testing of many process parameters. High yielding processes can result in reduced batches, hence bringing the product to market quicker and increasing manufacturing capacity. To reduce the cost and duration of process optimisation a novel miniaturised stirred bioreactor system (MBR), the BioXplorer™, a prototype of a commercial MBR system initially developed for microbial fermentations is described here. The system enables the operation of 4-16, 500 mL, independently controlled bioreactors in parallel. Each bioreactor is a scale down model of a lab-scale stirred tank bioreactor (STR) and constructed from the same materials. Agitation of the bioreactor can be via a magnetically driven 4 blade marine impeller or a directly driven 3 blade marine impeller. Aeration can be achieved through a variety of sparger designs directly into the culture or via the headspace at a maximum flow rate of 200 mL/min. A detailed characterisation of the key engineering parameters has been conducted focusing on power input and the power to volume ratio (P/V), mixing time and the overall volumetric mass transfer coefficient (kLa). Successful scale comparison studies were conducted to 5L scale using constant P/V and mixing time, employing an industrially relevant GS-CHO cell line producing an IgG antibody. The growth kinetics and product titres compared favourably in both systems when conducting fed-batch operations. μ-max in the MBR was 0.024 h-1 and the maximum viable cell concentration was 10.4 x 106 cells/mL while in the 5L STR μ¬max was 0.029 h-1 and the maximum viable cell concentration was 9.8 x106 viable cells/mL. The product titres were also very similar in both the MBR (1.07 g/L) and the 5L STR (1.05 g/L). It has also been shown that the MBR can conduct continuous feeding using built-in peristaltic pumps, maintaining the glucose concentration in the culture at approximately 2.0 g/L after initiation of feeding. The MBR described here potentially provides a valuable and effective tool for process optimisation and is capable of performing complex feeding strategies.

Solid state fermentation (SSF) refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment to which the selected microorganisms, especially fungi, are naturally adapted. The current status of SSF research globally was discussed in terms of articles publication. This was followed by discussion of the advantages of SSF and the reason for interest in SSF as a notable bioprocessing technology to be investigated and compared to submerged fermentation (SmF) for the production of various added-value products. SSF also proved to be a potential technology to treat solid waste produced from food and agricultural industry and to provide environmental benefits with solid waste treatment. A summary was made of the attempts at using modern SSF technology for future biorefineries for the production of chemicals. Many works were carried out in the Satake Centre for Grain Process Engineering (SCGPE), University of Manchester, to prove the strategy of using SSF for the production of hydrolysate rich in nutrients for sequel microbial fermentation with or without adding any commercial nutrients. The research findings presented in this thesis are based on a series of SSF experiments carried out on systems varying in complexity from simple petri dishes to our own design of bioreactor systems. They were conducted to assess a solution for biomass estimation, enzymes production, and successful mass and heat transfer. A proper technique for inoculum transfer prior to the start of the fermentation process was developed. In SSF, estimation of biomass presents difficulties as generally the fungal mycelium penetrates deep and remains attached with the solid substrate particles. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become laborious, impractical and inaccurate. Essentially, this remains another critical issue for monitoring growth. In these studies, measurement of colour changes during SSF are presented as one of the potential techniques that can be used to describe growth, complementary to monitoring metabolic activity measurement, such as CER, OUR and heat evolution, which is directly related to growth. For the growth of Aspergillus awamori and Aspergillus oryzae on wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. This colourimetric technique was also proved to be a feasible approach for fungal biomass estimation in SmF. This new approach is an important complementation to the existing techniques especially for basic studies. The key finding is that the colourimetric technique demonstrated and provided information of higher quality than that obtained by visual observation or spores counting. The effect of aeration arrangements on moisture content, oxygen (O2), mass and heat transfer during SSF was investigated. A. awamori and A. oryzae were cultivated on wheat bran in newly designed four tray solid state bioreactor (SSB) systems. The new tray SSB systems were: (1) single circular tray SSB, (2) multi-stacked circular tray SSB, (3) Single rectangular tray SSB and (4) multi-square tray SSB. The purpose was to study the effect, on heat and water transfer, of operating variables, fermentation on the perforated base tray and internal moist air circulation under natural and forced aeration. Temperature, O2 and carbon dioxide were measured continuously on-line. Enzyme activity, moisture content and biomass were also measured. The results suggest that the air arrangements examined have a remarkable effect on the quantity of biomass produced using measurement of spores and enzymes production. The strategy presented in these studies allowed quantitative evaluation of the effect of forced internal moist air circulation on the removal of metabolic heat. With the proposed strategy, it was possible to maintain the bed temperatures at the optimum level for growth. However, the effect on moisture content was very different for the two fungi. It was found that the ability of A. oryzae to retain moisture was much higher than that of A. awamori. This is possibly due to the higher levels of chitin in A. oryzae. Greater spores and enzymes (glucoamylase, xylanase and cellulase) production was observed for A. awamori in multi-stacked circular tray and multi-square tray SSB systems compared to the conventional petri dishes and the other two systems. A. oryzae was excellent in producing protease in the same bioreactors. A direct technique of establishing a correlation between fungal growth and CER, OUR, heat evolved was proven successful in this work. The information obtained from CER and OUR led to the estimation of respiratory quotient (RQ). RQ describes the state of the fungal population in the tray SSB and gives an indication of fungal metabolic behaviour. RQ values < 1 were obtained from 38 experiments using four tray SSB systems for the two fungi. A kinetic model based on CO2 evolution instead of biomass concentration was examined in order to simplify the required experiments for kinetic model development. A Gompertz model was used to fit the integrated CO2 data and predict the quantity of CO2 evolution in all experiments. A correlation was found between the heat evolution and CER. The performances of tray SSB systems can be improved by constructing them as multi-trays. The multi-tray systems improved the mass transfer considerably compared with single tray systems. In addition, the multi-tray systems allowed precise measurement of the gradients of CO2, enzymes, spores and fungal biomass. In addition, the air arrangements using moistened air were successful in maintaining moisture content, adequate O2 supply and control of temperature, and hence, increased the productivity of both fungi. Overall A. awamori and A. oryzae have their own ability and performance to degrade and utilise the complex compositions contained in the solid substrate and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray and multi-square tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large scale operation, though considerable optimisation work remains to be done, for example the height/diameter ratio and total number of trays should be optimised.

This thesis describes research carried out by the author between 1988 and 1991 at the Department of Chemical Engineering, University of Edinburgh, under the supervision of Dr Donald Glass and Dr Bruce Ward. The aim of the research was to design, build, operate and control a dual-hollow fibre bioreactor. The principle behind the design is that of the blood supply system in animals. The nutrients are supplied in one set of fibres to the growth region, similar to the arteries in the blood system, and another separate set of fibres takes waste products away from the growth region, in a manner analogous to the venous system. The design, construction and operation of the bioreactor is described. The development of novel building techniques are explained, covering new ground in fibre bioreactor construction. The monitoring equipment required is described with a number of successful experimental runs demonstrating the data collection capabilities of the apparatus. During the research, areas of work not initially envisaged were explored, with the aim to provide a basis for future control strategies. This included the development of a fibre testing rig, so that different fibres and various medium preparations could be tested outside a reactor system. This was done due to the lack of basic information available on fibre performance. This leads into work on the modelling the bioreactor by means of a numerical solution run on a computer. The model provides new areas of simulation, the fouling of fibres and the changing nutrient concentrations supplied to the bioreactor. The work is now at a stage where experimental work and modelling work should be brought closer together to help understand problems experienced in both areas.

An engineered bioreactor system was designed and constructed to bioremediate approximately 1,800 m$\sp3$ of petroleum-contaminated soil at CFB Petawawa, Petawawa, Ontario. The bioremediation facility operated between May-November 1993. The facility consists of four above ground bioreactors each incorporating aeration piping and a water/nutrient delivery system. The aeration piping is connected to a central vacuum pump which draws air through the bioreactor leachate collection system enables the leachate to be amended. The bioreactors are covered with an opaque vapour barrier. Monitoring involved the collection of soil, water and air samples on a weekly and bi-weekly basis and various field measurements. A detailed microbial monitoring program was also implemented. Total petroleum hydrocarbon concentrations in the bioreactor soils were found to have been reduced by 97%. Temperature had an effect on the rate of petroleum biodegradation. Little or no evidence suggested that the continuous addition of nutrients to the soil had a significant effect on the rate of biodegradation. The estimated treatment cost for this project was 70-$90 per tonne. This facility however, is reusable and hence the potential exists to lower the net treatment cost to 20--\$40 per tonne. This project has shown that diesel contaminated soil can be efficiently and effectively treated to meet the most stringent federal and/or provincial criteria in a cost effective manner over a typical Canadian summer. (Abstract shortened by UMI.)

Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.

Destructive respiratory diseases are predicted to become the 3rd global cause of morbidity and mortality within the next few decades. Despite their high prevalence, there are no treatments due to the limited regenerative capacity of lung tissues. End-stage sufferers of severe respiratory disease ultimately require tissue or whole organ transplantation, which is associated with low success rate and severely offset by donation shortages. The ever-growing field of tissue engineering offers the potential to not only regenerate human tissue equivalents of respiratory epithelia for surgical implantation, but also to provide an ethical research platform for respiratory pathology research, OH&S toxicology studies and more efficient pharmaceutical screening. However, progress has been hindered by difficulties in maintaining lung-specific cell phenotypes in vitro due to poorly understood or neglected cellular requirements. There is opportunity for a biomimetically-inspired bioreactor approach to provide an optimised culture environment for respiratory tissue engineering. This body of work details the development and validation of such an integrated bioreactor system that captures key in vivo conditions experienced by respiratory tissues during breathing. The system incorporates a unique magnetically driven linear actuator, a porous 3-D tissue scaffold and a scaffold straining mechanism that synergistically exposes cultured cells to air and culture medium at physiological strain rates. A modular compatible perfusion unit was designed and developed, and potential control of the immediate gas environment of a cell culture was conceptually developed. Biological studies with a human lung carcinoma cell line cultured on the integrated system successfully demonstrated that the scaffold-straining unit sustained cellular establishment, growth and proliferation in vitro under dynamic culture conditions. Furthermore, actuation at an air-liquid interface was shown to confer superior proliferation, scaffold infiltration and distribution compared to a static submerged control, thus meeting identified design and functional requirements and validating the underlying biomimetic design philosophy. This system overcomes major limitations of current lung tissue models by producing an organotypic, dynamic, air-liquid interfacing environment. In addition, this system is compatible with standard cell culture techniques, enabling potential large-scale use in research. Overall, the system presented shows great potential for use in regeneration of airway tissues in vitro.

This research project investigated a bioreactor system capable of high density cell growth intended for use in regenerative medicine and protein production. The bioreactor was based on a drip-perfusion concept and constructed with minimal costs, readily available components, and straightforward processes for usage. This study involved the design, construction, and testing of the bioreactor where the results showed promising three dimensional cell growth within a polymer structure. The accessibility of this equipment and the capability of high density, three dimensional cell growth would be suitable for future research in pharmaceutical drug manufacturing, and human organ and tissue regeneration.

Silage bunker runoff is a form of agricultural pollution that contributes to aquatic ecosystem degradation. Current handling and treatment methods for this process wastewater are often ineffective or expensive. A woodchip bioreactor is an emerging treatment technology designed to facilitate denitrification through the provision of an anaerobic, carbon rich environment. A wood chip bioreactor treatment system, consisting of three pre-treatment tanks, two wood chip bioreactors, and one infiltration basin, was constructed at the Miller Research Complex in South Burlington, Vermont in 2016. Runoff and leachate from an adjacent silage storage bunker is directed into the system. The pre-treatment tanks include two settling tanks and one aeration tank. The former allows for sedimentation of organic matter, while the latter is designed to allow for nitrogen transformations that will help maximize nitrogen removal in the bioreactors. During the summer and fall of 2017, sampling occurred at four points within the system in order to determine the efficacy of various treatment steps. Samples were analyzed for nitrate (NOx—N), ammonium (NH4+-N), total nitrogen (TN), soluble reactive phosphorus (SRP), and total phosphorus (TP) in order to compare inflow and outflow pollutant concentrations and loads. Results indicate that this treatment system significantly reduced nutrient loads in the runoff. Over the entirety of the sampling period, the influent TN and TP mass load were both reduced by approximately 44%.

A Tissue Engineering (TE) approach to heart valve replacement has the aim of producing an implant that is identical to healthy tissue in morphology, function and immune recognition. The aim is to harvest tissue from a patient, establish cells in culture from this tissue and then use these cells to grow a new tissue in a desired shape for the implant. The scaffold material that supports the growth of cells into a desired shape may be composed of a biodegradable polymer that degrades over time, so that the final engineered implant is composed entirely of living tissue. The approach used at Swinburne University was to induce the desired mechanical and functional properties of tissue and is to be developed in an environment subjected to flow stresses that mimicked the haemodynamic forces that natural tissue experiences. The full attainment of natural biomechanical and morphological properties of a TE structure has not as yet been demonstrated. In this thesis a review of Tissue Engineering of Heart Valves (TEHVs) is presented followed by an assessment of biocompatible materials currently used for TEHVs. The thrust of the work was the design and development of a Bioreactor (BR) system, capable of simulating the corresponding haemodynamic forces in vitro so that long-term cultivation of TEHVs and/or other structures can be mimicked. A full description of the developed BR and the verification of its functionality under various physiological conditions using Laser Doppler Anemometry (LDA) are given. An analysis of the fluid flow and shear stress forces in and around a heart valve scaffold is also provided. Finally, preliminary results related to a fabricated aortic TEHV-scaffold and the developed cell culture systems are presented and discussed. Attempts to establish viable cell lines from ovine cardiac tissue are also reported.

The differentiation of myosin into the respective heavy chain isoforms has shown a correlation with high mechanical stress. Aortic valve myosin expression has been reported; however, the characterization of the pressure response has yet to be fully developed. Thus, a cyclic pressure bioreactor was developed to elucidate the á/â-myosin heavy chain (MHC) expression in aortic valve leaflets subject to physiological and pathological transvalvular pressure loads. The pressure bioreactor achieved the desired pressure modulation via LabVIEW controlled solenoid valves. Results showed á/â-MHC expression on the fibrosal endothelium and minimal dispersal in the subendothelium, indicating the presence of smooth muscle cells. Endothelial layer denudation was evident with time progression while protein expression was limited to sites of excision or injury, indicating a causal relationship with high shear stress. In conclusion, á/â-MHC expression is limited by endothelium detachment and lack of smooth muscle cells, possibly on account of insufficient mechanical stimuli.

Development and Manufacture of a miniatured flow through-put (multiple) bioreactor system PhD-Thesis, University of Rome – Tor Vergata, 2015, 163/174 Pages, 73 Figures, 14 Tables, 123 References Bioreactor systems for cultivating cells in Life Sciences have been widely used for decades. Recently, there is a trend towards miniaturized and even microsized systems, fulfilling increasing demands strongly aiming for production and testing of novel pharmaceutical products. The aim of this PhD thesis is to develop and to manufacture such a disposable miniaturized multiple bioreactor system aiming for low cost mass production of such devices implementing artificial lymph nodes. A recursive strategy is necessary for optimizing the design and the manufacture of such artificial lymph nodes. The designs of the components and the final reactor system additionally have to be compatible to low costs manufacturing via injection moulding. Furthermore, the applied polymeric material has to be biocompatible as well as resistant to sterilisation by means of hard gamma-ray radiation. The final task includes the joining techniques in order to ensure a fluidic sealing of each single reactors as well as the whole reactor system.

One of the World Health Organization’s biggest concerns is meeting blood demand while ensuring safety and despite the efforts of world blood banks, the gap between supply through donations and demand continues to widen (Nolan, 2017). Hence, a practical and cost-effective alternative to conventional blood donation is essential to meet the demand and reduce patient risks. Previous attempts to recruit blood stem cells to produce blood cells in platforms have achieved limited success specifically in areas of cell-platform interaction, perfusion, and cell harvest. The PhD thesis presented here is aimed at bringing the Bone Marrow (BM) mimicry Bioreactor (BR) developed and patented by BioBlood project closer to physiological representation of the natural human BM niche which hosts blood cells production. This is achieved by focusing on modification and optimization of the synthetic materials used in the bioreactor. Firstly, the polyurethane (PU) scaffold which mimics the BM microenvironment and modulates cell expansion and fate. Secondly, the alumina hollow fibres (HF) representing the vascular system of BM which regulate nutrients and cellular constituents while harvesting mature blood cells. To augment PU bio-functionality and optimize signalling/interaction between cells and scaffold, a novel protocol of RGD surface modification of PU was developed targeting enhancing: cell adhesion, cellular infiltration, and differentiation into blood cell lineages. Adhesion of human umbilical stem cells (hUSC) was improved by more than 85% in RGD-modified PU scaffolds, whereas cell penetration was increased by 4-folds. Alumina hollow fibres’ (HF) structural and filtration characteristics, on the other hand, were improved to support a higher yield and purity of harvested RBC through manipulation and optimization of fabrication parameters. HF improved purity of harvested RBC from 30% to 80% and supported a 1.6 fold increase in cellular density when incorporated in a PU-bioreactor. Combining the two optimized materials in the 3D bioreactor (BR) set-up envisioned to support increased production and selective harvesting of clinically relevant quantities of red blood cells.

Doctor of Philosophy
Department of Grain Science and Industry
Praveen V. Vadlani
The purpose of this study was to investigate micro- and macro-scale aspects of solid state fermentation (SSF) for production of cellulolytic enzymes using fungal cultures. Included in the objectives were investigation of effect of physicochemical characteristics of substrate on enzymes production at micro-scale, and design, fabrication and analysis of solid-state bioreactor at macro-scale. In the initial studies response surface optimization of SSF of soybeans hulls using mixed culture of Trichoderma reesei and Aspergillus oryzae was carried out to standardize the process. Optimum temperature, moisture and pH of 30ºC, 70% and 5 were determined following optimization. Using optimized parameters laboratory scale-up in static tray fermenter was performed that resulted in production of complete and balanced cellulolytic enzyme system. The balanced enzyme system had required 1:1 ratio of filter paper and beta-glucosidase units. This complete and balanced enzyme system was shown to be effective in the hydrolysis of wheat straw to sugars. Mild pretreatments– steam, acid and alkali were performed to vary physicochemical characteristics of soybean hulls – bed porosity, crystallinity and volumetric specific surface. Mild nature of pretreatments minimized the compositional changes of substrate. It was explicitly shown that more porous and crystalline steam pretreated soybean hulls significantly improved cellulolytic enzymes production in T. reesei culture, with no effect on xylanase. In A. oryzae and mixed culture this improvement, though, was not seen. Further studies using standard crystalline substrates and substrates with varying bed porosity confirmed that effect of physicochemical characteristics was selective with respect to fungal species and cellulolytic activity. A novel deep bed bioreactor was designed and fabricated to address scale-up issues. Bioreactor’s unique design of outer wire mesh frame with internal air distribution and a near saturation environment within cabinet resulted in enhanced heat transfer with minimum moisture loss. Enzyme production was faster and leveled within 48 h of operation compared to 96 h required in static tray. A two phase heat and mass transfer model was written that accurately predicted the experimental temperature profile. Simulations also showed that bioreactor operation was more sensitive to changes in cabinet temperature and mass flow rate of distributor air than air temperature.

In several cases, current therapies available to treat a large number of musculoskeletal system diseases are unsatisfactory as they provide only temporary or partial restoration of the damaged or degenerated site. In an attempt to maintain a high standard of life quality and minimise the economic losses due to the treatments of these frequently occurring ailments and subsequent lost working days, alternative therapies are being explored. Contrary to the current treatments, tissue engineering aims to regenerate the impaired tissue rather than repair and alleviate the symptoms; thus offering a definitive solution. The annulus fibrosus (AF) of the intervertebral disc (IVD) is a musculoskeletal system component frequently subjected to degeneration and rupture, characterised by predominance of anisotropically arranged collagen fibres. In the present thesis, electrospinning technology is used to fabricate polycaprolactone (PCL) scaffolds intended to replicate the anisotropic structure of the AF.