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TU Delft Library

Promethöuspiein 1

2628 ZC Delft

Development of a bioreactor with integrated on-line sensing

for batch and fed-batch cultivation on a 100 |jL-scale

Proefschrift

ter verl<rijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus, Prof. dr. ir. J.T. Fol<kema, voorzitter van het College voor Promoties,

in het openbaar te verdedigen op woensdag 11 juni 2008 om 10:00 uur

door

MIchiel VAN LEEUWEN

Ingenieur in de Bioprocestechnologie en

doctorandus in de Wijsbegeerte van een bepaald wetenschapsgebied geboren te Leidschendam

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Dit proefschrift is goedgekeurd door de promotor:

Prof. dr. ir. J. J. Heijnen

Samenstelling promotiecommissie:

Rector Magnificus Prof. dr. ir. J.J. Heijnen Prof. dr. S. de Vries Prof. dr. E. Heinzie Prof dr. ir. W. Soetaert Prof. dr. ir. J.C. Schouten Prof. dr. ir. J.G.E. Gardeniers Dr. H. Noorman

Prof. dr. J.T. Pronl<

Voorzitter

Technische Universiteit Delft, promotor Technische Universiteit Delft

Universitat des Saarlandes, Saarbrücl<en, Duitsland Universiteit Gent, België

Technische Universiteit Eindhoven Universiteit Twente

DSM Anti-lnfectives, Delft

Technische Universiteit Delft, reservelid

The studies performed in this thesis were performed at the section Bioprocess Technology, Department of Biotechnology, Delft University of Technology. The research was financially supported by the Dutch Science Foundation (NWO) through the ACTS program IBOS (Integration of Biosynthesis and Organic

Synthesis), with financial contributions from NWO, the Dutch Ministry of Economic Affairs, DSM Anti-lnfectives, Organon and Applikon.

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My [...] maxim was to be as firm and resolute in my actions as I was able, and not to adhere less steadfastly to the most doubtful opinions, when once adopted, than if they had been highly certain; imitating in this the example of travelers who, when they have lost their way in a forest, ought not to wander from side to side, far less remain in one place, but proceed constantly towards the same side in as straight a line as possible, without changing their direction for slight reasons, although perhaps it might be chance alone which at first determined the selection; for in this way, if they do not exactly reach the point they desire, they will come at least in the end to some place that will probably be preferable to the middle of a forest.

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Table of Contents

Chapter 1 Introduction

Chapter 2 A system for accurate on-line measurement of total gas consumption or production rate in microbioreactors Chapter 3 Development of a system for the on-line measurement

of carbon dioxide production in microbioreactors; application to aerobic batch cultivations of Candida

utilis

Chapter 4 The Hagen-Poiseuille pump for parallel fed-batch cultivations in microbioreactors

Chapter 5 Quantitative determination of glucose transfer between cocurrent, laminar flowing water streams in H-shaped microchannel

Chapter 6 Lab-scale fermentation tests of micro chip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration

Chapter 7 Aerobic batch cultivation in microbioreactor with integrated electrochemical sensors and off-gas measurement Chapter 8 Outlook Summary Samenvatting List of publications Curriculum vitae Dankwoord 33 45 61 83 101 123 145 153 159 165 169 171

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Chapter 1

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Chapter 1

1.1 Background

Micro-organisms (bacteria, yeast and fungi) and to a lesser extent animal and plant cells are widely exploited for the industrial production of both bulk and fine chemicals, such as ethanol, antibiotics, vitamins and therapeutic proteins, to name only a few. In such an industrial bioprocess, micro-organisms or cells are cultivated in bioreactors under well-controlled conditions with the aim to produce the desired product in a robust process in an efficient and cost effective manner. The cultivation is the cornerstone in any such bioprocess and the development and optimization of this process step is crucially important for the commercial success of the entire process. But even when the process is running, it generally requires continuous improvement to remain competitive. This development and improvement / optimization is by no means an easy task. Its cost accounts for a significant fraction of the cost to bring the product to the market.'^'

1.2 Bioprocess development and optimization 1.2.1 Screening and selection

The development of fermentation processes starts in most cases with the screening and selection of micro-organisms with a sufficiently high productivity and yield for the desired product. The micro-organisms that enter the screening phase may originate for example from nature, i.e. wild type strains, or from strain improvement programs. The current practice of screening for high producing strains has remained essentially unchanged for nearly 50 years'^' and typically starts with a fast and massive test for growth capacity on agar plates. From these agar plates the colonies are picked and the micro-organisms are further cultivated in submerged cultures to gain data on final product and biomass concentration. The best performers are transferred to the next step in the development phase. Traditionally shake flasks are used in the screening phase.''' However, there is a clear trend towards the use of deep well and microtiter plates. These plates are attractive for high-throughput cell cultivation because of their small working volumes, the high degree of parallelization and the already available robotics, shakers and liquid handling equipment. A completely automated screening setup can potentially handle more than 10.000 individual cultures at the same time.''"' It is expected that future advances in the automation of the screening and selection step will significant reduce costs and resources.''^'

Although these experiments yield valuable information at a high throughput, the process conditions under which the screening is generally performed, are very different from the conditions under which the micro-organisms ultimately should function i.e. in an industrial process. Because of the sheer amount of

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micro-Introduction

organisms that need to be screened, the fermentations in the screening and selection stage are typically performed under poor controlled batch conditions at low biomass and product concentration.

In many industrial-scale fermentations substrate limited fed-batch cultivation is the preferred mode of operation, yielding high biomass and product concentration. Furthermore, important environmental parameters like the pH, the dissolved oxygen and carbon dioxide concentration and temperature are all both measured and controlled tightly in an industrial process to ensure optimal performance. Because of the divergence in cultivation conditions between the screening and industrial-scale, potentially high producing candidates, which would perform well under industrial relevant conditions, are not identified in the screening. Furthermore a lot of false positives are selected in the screening phase.

1.2.2 Strain improvement

Nature provides a wide variety of micro-organisms that can produce interesting compounds or that have other desirable features. Most often the productivity and yield of these so called wild-type strains is very low and improvement is necessary before they become commercially attractive to use. Strain improvement programs aim to enhance the productivity and yield for a certain desirable product by making changes in the genetic material of the cell.

The classical way of strain improvement is via mutagenesis techniques, e.g. by inducing random mutations by irradiation or chemical agents in a (production or wild type) strain and the subsequent selection of high producing variants. Because of the random nature of this technique large numbers of mutants are created that must be screened and selected for rapid growth and increased productivity or for other favorable features. Therefore screening, selection and strain improvement go hand in hand and form an iterative cycle.'^^"'

Modern genetic technologies that were developed in the past decades, nowadays allow precise, rather than random modifications in the DNA of a cell and can be (and are) used to enhance productivity or even let a cell produce a non-native compound. The use of recombinant DNA techniques, generally referred to as metabolic engineering, allows a more rational approach to strain improvement. Hereto gene targets should be identified whose modification would bring improvement, such as removing a bottleneck or inhibition in the metabolic network or removing or slowing down an undesired side route. However, finding these targets requires fundamental and reliable knowledge on the stoichiometric, kinetic and regulatory processes of all the reactions that occur in the cell. Although the knowledge in this field is increasing rapidly, it is still far from complete. For the

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Chapter 1

rational improvement of a strain the selection of gene targets is to a large extent an educated guess, to say the least.

To elucidate the properties of the metabolic network of the strain of interest, and thereby improving the selection of targets for strain improvement, high-throughput perturbation experiments are of great importance. In perturbation experiment either a gene (or set of genes) is changed, deleted or amplified or the environmental conditions under which the strain is grown are changed. Secondly

V Screening and selection

(

\ . Strain improvement

Figure 1.1: Schematic representation of the iterative cycles in the development of an industrial fermenation process.

the strain is characterized, preferably on multiple organizational levels of the cells, e.g. the transcriptome,'^^' the metabolome,™ fluxome'^'^^' and the proteome'^'' Thirdly the data is evaluated with the help of in silico modeling and simulations, new perturbations are proposed and targets for strain improvement are appointed. These steps result in an iterative framework for strain improvement and elucidating the complex landscape of cellular processes.'^''"^''

Whether the costly investment in analytical equipment and highly specialized personnel to improve industrial strains from a rational, but still iterative approach, can out-compete the classical approach for strain improvement, remains to be seen. What is safe to state is that both approaches greatly rely on high-throughput fermentation techniques. Clearly the ability for tight measurement and control of environmental conditions as well as the high-throughput is crucial for success.

1.2.3 Optimization of growth medium and setting of process

conditions

At present the divergence in cultivation conditions between the screening phase in microtiter and deep well plates and the actual production process reduces the selectivity of the entire selection procedure. Therefore potentially high producing candidates from the screening and strain improvement stage need to be tested under well controlled process conditions, in order to verify if they also perform well

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Introduction

under these conditions. Furthermore the process conditions are not set on forehand, but need to be optimised for the selected strains. Optimization is critical important because it can significantly affect the yield and the productivity of the strain and can also affect the ease and costs of downstream processing, the stage of product separation and recovery.

Well controlled bioreactor setups are required to carry out fermentations under industrial relevant conditions. Important process variables that need to be optimized are the temperature, the pH, the dissolved oxygen concentration, feeding strategy and harvest time. Robustness of the process is here probably as or even more important than optimal performance. Setting the process parameters is probably the simplest task in process development, because there are relatively few variables.'^*'

The growth medium should provide the micro-organism with all that is needed for optimal growth and/or production; this includes sources for energy, carbon, nitrogen and sulfur as well as supplements such as amino acids, vitamins, trace metals, minerals and sometimes hormones and specific precursors for product formation. The endless amount of combinations in medium composition makes the optimization a costly and laborious task. Many tools are available for this, each with its own advantages and disadvantages,'^*" but all include cultivation experiments preferably under well controlled conditions and at high-throughput.

1.2.4 The iterative cycle & integration of process development

The different stages in the development of an industrial-scale fermentation process, as were discussed above, are highly interconnected as is depicted in Figure 1.1. As was already discussed, strain improvement heavily relies on high-throughput screening facilities and the productivity of the best producers in the screening program could further be enhanced by improvement of the cultivation conditions. Furthermore medium design is also intrinsically linked to screening and selection: "[Y]ou can't choose the best strain until you have the best medium, and you can't design the best medium until you have the best strain".'^^' The same is true for tuning the process parameters: if the screening is performed under substrate excess batch conditions, it is very likely that interesting candidates that produce best under substrate limited fed-batch conditions are not identified. To cope with these interdependencies instead of a sequential development, i.e. screening, strain improvement, medium optimization, and tuning of the process conditions, the development is most often highly iterative.

These iterative cycles are unavoidable not only because of lack of fundamental knowledge and the shear amount of possibilities, but also, 1 would like to argue, because of equipment limitations. At the moment there is a strong inverse

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Chapter 1

relationship between the level of experimental throughput and the amount of measurement and control possibilities. Bench-scale bioreactors are equipped with sophisticated measurement and control equipment, with the use of which an industrial-scale bioprocess can be mimicked. Cultivations performed in

bench-control over cultivation conditions

Figure 1.2: Schematic representation o f t e relation between experimental throughput and the control over the cultivation conditions.

scale bioreactors yield reliable and information rich data, however they are laborious and time consuming to operate, which limits the amount of experiments that can be carried out. In the first stages of the process development the sheer number of experiments that is required enforces the use of high-throughput systems with less measurement and control features, like microtiter plates, at the expense of the quality of the data as well as the selectivity of the selection (see Figure 1.2). The possibilities and limitations of both systems are at least part of the causes for the iterative cycles in the development of a fermentation process.

1.3 The promise of microbioreactor technology

In the emerging field of the development of miniature bioreactors the aim is to combine the small working volume and the high-throughput possibilities of microtiter plates with the monitoring and control features of lab-scale (fed-) batch / chemostat bioreactors.'^^°' The thus obtained arrays of small-scale bioreactors would be highly valuable to gain better quality information both faster and cheaper.'^^' The screening can benefit from microbioreactor development because it enables the screening under conditions more similar to the final industrial process, thus increasing the selectivity of the screening and selection phase. Process optimization, both for the medium optimization and the parameter tuning, could benefit from the increased throughput, while maintaining the essential

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Introduction

measurement and control features. A high-throughput platform with good measurement and control features could ultimately enable the integration of the development stages in strain development, for example by simultaneous screening of different micro-organisms in different media, or at different feed strategies. Furthermore microbioreactor development could be valuable for other fields in which cell cultivation are used, such as fundamental studies on the functioning of micro-organisms at nutrition, genetic and molecular level.'^^^

1.4 Generic and technical considerations for microbioreactor development

1.4.1 Industrial relevant conditions

In order to carry out micro-scale cultivations under similar conditions as of the industrial-scale process, important process parameters like the temperature, dissolved oxygen concentration (DO), dissolved carbon dioxide concentration (DCO2) and pH need to be tightly controlled in the microbioreactor. At least equally important but receiving less attention is the possibility of continuous feeding of substrate to the microbioreactors to achieve substrate limited conditions. As was already pointed out before, in the majority of the industrial fermentation processes fed-batch cultivation is the preferred mode of operation. With this technique high biomass and product concentrations can be achieved. The term 'fed-batch' refers to the technique where one or more substrates are supplied at a controlled, limiting rate to the bioreactor. Fed-batch cultivation is the preferred mode of operation if too high substrate concentrations affect the productivity and the yield of biomass and/or desired product in a negative way, or if the rate of product formation is maximal at a certain specific growth rate of the cells. The reason for this is that fed-batch operation makes it possible to control the growth rate and to maintain the substrate concentration in the bioreactor at a constant low level during cultivation.'^^"^''' Maintaining a low substrate concentration is important for the cultivation of industrial relevant micro-organisms like Escherichia coll and

Saccharomyces cerevisiae in order to reduce excess by-product formation, like

acetate and ethanol as is caused by overflow metabolism, also known as the

Crabtree or glucose effecP^^' and to avoid catabolic repression of the product

pathway.

1.4.2 Development of sensors for miniaturized bioreactors

Advances in the development of miniature sensors allows the measurement of important process parameters in very small bioreactors. Especially the development and commercialization^*''^'^'" of the optical sensors or optodes for QQ(39-45i g^^ p|^i43,45-47i really boostod the miniaturization of bioreactor systems.

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Chapter 1

However, optical sensors typically suffer from a small measurement range. The optodes for the measurement of pH are most sensitive around pH 7, whereas pH 5 or lower generally falls outside their measurement range. The optical sensors for oxygen are most sensitive at low oxygen concentrations.•'• An alternative to optical sensors are the electrochemical sensors. For the measurement of the pH the ISFET is an attractive alternative especially because it can cover a wide pH spectrum.'''*' Ultra micro electrode arrays (UMEA)'''^' are an electrochemical alternative for the measurement of the dissolved oxygen concentration in small volumes although they are not yet applied in microbioreactors.

1.4.3 Microtiuidics

The proper control of both the pH and the substrate concentration in a microbioreactor, using a feeding system, remains a challenge. Because of the small scale, the flow rates that needs to be accomplished should be extremely small, i.e. in the order of microliters per hour. Although syringe pumps suffice for a proof of principle, they are too cumbersome and costly for highly parallelized systems. The most obvious choice for controlling liquid flows in microbioreactor setups is the integration of microfluidic connections, microvalves and/or -pumps. Extensive research has been carried out on the development and improvement of individual components like micropumps'^°' and -valves,'^^' but the integration of these components into a functional system, has proven to be a complex tasks,'®^' especially because reliability and robustness are crucial.

7.4.4 Mixing and oxygen transfer

Insufficient oxygen supply is one of the most frequently occurring problems associated with the use of high-throughput cell cultivation systems like shake flasks, microtiter and deep well plates.''^' When oxygen is limiting the organisms may switch partially to anaerobic metabolism and will excrete by-products (acids, alcohols, etc.), and therefore oxygen limitation can decrease productivity.'^^''' An advantage of small-scale reactor systems is the increased area-to-volume ratio. Surface aeration could thereby potentially suffice the oxygen demand in small-scale bioreactors, simplifying their design. A great deal of attention has been devoted to increasing the mixing and 02-mass transfer capacity of shake flasks''^^*^"' and shaken microtiter plates."''™' It should be realized here that forces, like interfacial tension, that can normally be neglected when the mixing of a liquid is investigated, become important in miniaturized systems."*^'"' Especially in 96-well plates these forces need to be overcome for efficient mixing and increased mass transfer. High shaking frequencies (1000 rpm) ''^"'''' or increased shaker orbits (50 mm)'^^^^^^' where shown to be necessary to reach k|a values above 100 h~' in 96-(deep)well plates. These rather violent shaking conditions might become

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Introduction

problematic when optical, electrochemical or microfiuidic connections are introduced.

1.4.5 Monitoring ttie performance of the cells

Besides measuring environmental parameters, e.g. temperature and pH, also the performance of the cells in the reactor needs to be monitored. The biomass concentration is an important process indicator for the quantitative evaluation of the performance of the cultivation. Several techniques exist to measure the biomass concentration in small volumes, but all have limitations. In principle the on-line measurement of the biomass concentration in miniaturized systems is possible by both optical (i.e. turbidity measurement) and electrochemical means. However the turbidity measurement suffers from a decrease in sensitivity when miniaturized. An electrochemical alternative is impedance spectroscopy. The major advantage of this technique is that only viable cells, i.e. cells with an intact cell membrane, are detected.''^' Nevertheless impedance spectroscopy is sparsely applied in lab-scale bioreactors, let alone in miniaturized systems.

Without doubt the most important cellular performance indicator is the product concentration. In bench-scale bioreactors the product concentration is most often determined by taking samples of the broth and off-line analysis of these samples. For miniaturized bioreactor systems, the sample volume that is required for the analysis severely limits and generally excludes off-line analysis using conventional equipment. The integration and miniaturization of analytical techniques or sensors into a miniaturized bioreactor platform is a complex task, especially because the solution is product specific.

Because of the complexities that surrounds the direct, on-line measurement of the product and biomass concentration, indirect measurment techniques could also be explored. On-line measurement of O2 consumption and CO2 production has been state of the art in bench-scale bioreactors for many years. With the use of elemental balancing'"' these techniques provide essential, quantitative data on the performance of the cultivation including biomass growth and productivity.'"'

1.4.6 Confinement and sterility

The bioreactor should confine the cultivation from the outside world. The uncontrolled transport of substances, including micro-organisms into or out from the vessel should be prevented. In this respect water evaporation can, because of the large area-to-volume-ratio, cause problems in miniaturized bioreactors. A second important aspect in this respect is the biocompatibility of all the materials that are used in the design. Especially plastics are notohous for leaching toxic softeners into the culture. Poly(dimethylsiloxane) (PDMS) is widely applied in micro devices that are designed for biological experiments, mainly because its

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Table 1.1: Examples of miniaturized bioreactors that are Company Dasgip'^"' Applikon'"! BioProcessors'^^^' BioXplore>^2=> Fluorometrix'^^' Infors'^^^i PreSensP^i H+P Labortechnik'^'i AC Bioted^^i IVlodel Fed-batch pro 24l"3-1151 Simcell'^^'i Xplore Cellstation Sixfors Oxydish'^"'" Hydrodish Oxyplate'^°^i Hydroplate Sepromat®'^°> RAMOS COSBIOS Based upon SF ST MTP CC ST ST ST MTP MTP MTP MTP SF SF SF

SF=Shake Flask; ST= Stirred Tank; MTP= MicroTiter Oxygen Uptake Rate; CER= Carbon dioxide Reported; #=amount of cultivation vessels per

Evolution jnit

either commercially available or used in commercial services.

Volume (mL) 50-500 150-300 3-5 0.7 30 35 450/750 0.6-1.2 0.6-1.2 0.1-0.2 0.1-0.2 50 NR NR Mixing shal<ing stirring shaking rotation'"''" stirring stirring stirring shaking shaking shaking shaking stirring shaken shaken

Plate; CC= Credit Card; SA= Rate; T= Temperature; DO=

Gas transfer SA SP SP SA SP MA SP SA SA SA SA SA SA SA Sensing pH pH, DO pH, DO, T pH, DO, 0D'"=^'" T, pH, DO, DO T, pH, DO, OD T, pH, DO DO pH DO'""' pH OUR, DO OUR, CER, pH OUR, CER, pH

Surface Aeration; SP=Sparging; Dissolved Control pH,T pH, DO,T pH, DO, T p H 1"=^' T T, pH, DO T,pH MA= Membra Fluid addition Yes Yes No Yes'" 5^ NR Yes Yes No No No No Yes Yes Yes ne Aeration Oxygen concentration; 0D= Optical Density;

1 o|

# 16 16 24 1260 8 12 6 24 24 96 96 12 8 8 OUR= NR=Not O •o CD - 1

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Introduction

biocompatibility and the fact that micro- and nano-scaie structures can easily be made in this material with soft-lithographic techniques.'^^"'"' It must be noted however that PDMS is permeable to gases and components with a low molecular weight, which could be problematic when quantification of these substances is required. Finally, aseptic operation of the bioreactor should be guaranteed and cross-contamination from neighboring microbioreactors should be prevented. To ensure aseptic conditions the microcultivation should not only be carefully confined, but ail the parts, including pumps, valves and sensors, should also be able to withstand sterilization procedures like y-irradiation or autoclavation.

1.5 Miniaturized and parallelized bioreactors for high-throughput cell cultivation

Several research groups explored possibilities for miniaturized and parallelized bioreactor systems for the cultivation of micro-organisms with working volumes of several milliliters down to nanoliters. The published reviews'^ ^''''^''*'' on this topic give a good, but sometimes a somewhat biased, overview on the rapidly progressing field of miniaturized bioreactor development. Several interesting results that were reported in the scientific and patent literature as well as information on commercial products are summarized in Table 1.1 and 1.2 and are shortly discussed below to give a comprehensive view on the recent developments, the trends and the problem areas of this innovative research field.

1.5.1 Shake flasks

Traditionally shake flasks with typical working volumes of 25 mL to 6 L are used as a high-throughput cultivation platform; they are around for decades and are used extensively, mainly because of their simple and cheap design. Detailed description and assessment of shake flasks technology falls outside the scope of this introduction, but some of the modifications made to shake flasks in order to enable better measurement and control possibilities are worthwhile to mention here.

Shake flasks can nowadays be equipped with sensors for pH'^^' dissolved oxygen

^QQy56.59j9-8i] ^^^ dissolved COz.'"^' Also systems for the on-line measurement of

the O2 uptake rate (OUR)'"''''^'" and the CO2 evolution rate (CER)''"«'''''' were developed and are commercially applied.'^^^^' The OUR measurement especially when combined with the CER measurement and elemental balancing was shown to provide valuable data on the performance of the culture.'"'

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Chapter 1

flasks with two compartments that were separated by a dialysis membrane. This enabled diffusive supply of a substrate from the feed- to the cultivation-compartment.®*''^^^''' Because diffusion kinetics govern the feed rate, a constant feed rate is only possible when the concentration difference over the membrane is maintained constant.'^°' This is hard to achieve in long-term experiments because of the depleting substrate in the feed-compartment. Furthermore many low molecular products are also transported over the membrane and removed from the cultivation-compartment.

The latter could be minimized by the slow release of a substrate from a solid carrier. Although this technique is not new,'^''^'' the recent commercialization of the Feedbead by AC Biotec'^^' addresses the increased interest in fed-batch cultivations in shake flasks'''^' and potentially in microtiter plates.'^"' Because the glucose supply by the Feedbead is based on diffusion, a tight control of the feed-rate, which is highly desirable, was shown to be problematic. It was reported that in shake flasks experiments with Feedbeads, the glucose supply initially exceeded the demand, resulting in batch growth; while after 6 - 1 2 hours growth became glucose limited and fed-batch growth set in with a very slow feed-rate.'^"'

A more conventional approach to achieve fed-batch conditions in shake flasks is the use of (syringe) pumps. Early attempts data back to the 1950s.'''^' More recently, a system has been developed for intermittent feeding of glucose and pH control to 16 shake flasks of 100 mL.'^°°' One syringe pump could feed the substrate to the 16 individual shake flaks via 2/2-way miniature valves, one for each flask. The substrate could be added to one flask at a time by opening the appropriate valve while closing all others. A second liquid handling assembly was used for controlling the pH. Compared to conventional shake flask cultivations, a higher biomass concentration was reached in these fed-batch and pH controlled shake flasks, the intermittent feeding regime however resulted in undesirable fast oscillations between glucose limited and glucose excess conditions every time the reactor was fed.

7.5.2 Microtiter and deep well plates

Microtiter plates were originally developed for analytical purposes, but are nowadays also frequently used for the cultivation of micro-organisms. Refraining from a detailed discussion on the recent advances made in microtiter plates cultivation platforms, it is, without question, a very powerful cultivation platform. The high degree of automation and standardization in this technology should not be underestimated. Therefore compatibility of any newly developed microbioreactor systems to this format is highly desirable. Many have recognized this advantage and integrated new features like optical sensors for pH and dissolved oxygen concentration'^"'"" in the wells. This trend increases the

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Introduction

versatility of this tool for physiological studies®^'®^^''°""°' as well as for a better understanding of the mass transfer in the wells."''®®"^"^'

Recently a batch bioreactor system based on the standard 24-well plate format became commercially available.'""^' In this system liquid mixing is accomplished by orbital shaking of the reactor plate. Each of the 24 cultivation wells contains optical sensors for the measurement of DO and pH, a thermistor for the measurement of temperature and a heater for individual temperature control. Gas mixtures (O2/CO2/NH3) can be fed at the bottom of each well, facilitating oxygen supply and pH control. The system was found to be a considerable promise especially for culture media and process optimization.'"""^'

At the University College London (UCL) several milliliter size bioreactor were fabricated and used to carry out batch cultivations. Several techniques for mixing and gas transfer, i.e. shaking,'"^' stirring'"^' and air sparging'"^"^' were investigated. Interestingly, it was demonstrated that the existing (empirical) models for estimating the mass transfer rate in large scale systems could also be applied with reasonable success for both miniaturized stirred reactor and bubble columns. The bubble column design (2 mL) that was developed at the UCL lacked sensors whereas the stirred reactor (6 mL) was equipped with optical sensors for pH, DO and optical density and in the shaken bioreactors (7 mL) only the pH was measured and controlled. All these miniaturized bioreactor types that are under development had a diameter equal to that of a single well of a standard 24-well plate. The compatibility of the bioreactor systems to microtiter plates gives these systems a major advantage over miniature bioreactor designs with a different format.

At the University of California the research group of professor Keasling developed an array of 8 microbioreactors with a working volume of 250 pL each.'^^°' Oxygen was generated electrochemically and permeated the microbioreactors from below via a PDMS seal. Batch growth was monitored via measurement of the optical density of the culture at varying oxygen generation rates. Although an ISFET was applied for the measurement of the culture pH, limited data was presented on the results of the pH measurement and nothing was said about compensation of the drift of the ISFET sensor, which is a well known problem of these sensors.'^^^^"'

1.5.3 Scale down of lab-scale bioreactors

Besides the implementation of more functionality in and compatibility to microtiter plate platforms, as was discussed above, an opposite trend is the down-scaling

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Chapter 1

Table 1.2: Overview of reported cultivations in miniaturized bioreactors

Developer of bioreactor and/or bioreactor name Volume (mL) Mode of operation (A CO

!^

0} (0 c E o Ü n o m

2

o

S

T3 0)

shake flak + sensing shake flask + feeding AC Biotec.Feedbead® dialysis flask RAMOS'^=«=-«^' -100-250 10-100 B pp(99,100] pg[88,95,961 Q[93.94] p[53,83-89] p p a [ 8 8 ] jy|^[132,133,161,162l 2 0 0 ' " ^ ' , 5 0 0 ' " ^ '

University College London'"'"'' 2 Universidade do Minho'^^^' 4.5 j y | ^ [ 1 3 6 - 1 3 9 . 1 6 4 1 5 . ^ 2 Space biology'^^^""' 3 TUM'^'^^' 200 University of Maryland "^•^^' 1 -2 Institute Pasteur'^'"'^"^' 70 University College London'"'' 6

g[133l p g I 1 3 2 l B B D[135,164] pD[135-137] C B B B B

a.

h-CD Ü C/5 Applikon, p-24''"^-"^' University College London'"'"

PreSense, Oxydish'^^'^"'''"'' PreSense, Hydrodish"''^"^" PreSense, Oxyplate'^^^'^^'^"'"""^' PreSense, Hydroplate'^'^"''^''^' University of California'^^°' 3-5 4 0.6-1.2 0.6-1.2 0.1-0.2 0.1-0.2 0.25 B B B B B B B CD •o CD E E _o to O |^|J[19,142-150,152] California Institute of Technology'^=^^''°' O.OOS"""' B 0 05'^''2'""'''^''' B Q -|[152] Q -jc[19.146,147.149] Q1149] g[19,146,147,149l 16*10'* C

MIT=Massachusetts Institute of Technology TUM=Technische Universitat München, B=Batch cultivation, FB=Fed-batch cultivation, D= Dialysis, C=Continuous cultivation

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Introduction ! Gas transfer shaking shaking shaking shaking shaking bubbles bubbles osc. flow impellor stirrer impellor stirrer sparging impellor shaking shaking shaking shaking shaking shaking diffusion diffusion peristaltic mixer stirring fluid circulation Gas transfer SA SA SA SA SA bubbles bubbles bubbles SA MA S S S S SA SA SA SA SA SA MA MA MA MA N/R k,a (h-^) 57611621 220""^',324'"'' N/R 720I135.1371 N/R 1224 44.4'=',68-300" 750 68 & 128 32.6-51.6'""' N/R 602'^""' -360'^"' 20-75'^"^' N/R Sensing+ Control pH"^',DO'^'''=^''^-«^OD''^' pHiiM Q|jp^[53,83-891 Q|jp^[84,86.88] DO QH^mm OD pHUaa, DO, OD pH, T, redox DO pH,DO,OD pH, DO, OD pH, DO, OD eJH, DO pH DO pH DO pH pH, OD , pH, OD""''""'''"'^' OD # 1 211321J g[133l 25 48 1 1 [31 24121 8 1 24 6 24 24 96 96 8 1-8 6

SA=Surface Aeration, SP=Sparging, MA=Membrane Aeration, SA= Surface aeration, OUR=Oxygen uptake rate, CUR=Carbon dioxide uptate rate, DO=Dissolved oxygen concentration; NR=Not Reported, #=amount of cultivation vessels per unit

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Chapter 1

and parallelization of proven lab-scale bioreactors. Lab-scale bioreactors have typical working volumes ranging from a few hundred milliliters up to several liters and are in most cases fully equipped with sensors and fluid and gas-handling systems for the on-line measurement and tight control of important parameters like pH, dissolved oxygen concentration and temperature. Feed and effluent pumps make it possible to control the substrate supply.

Both the systems from Dasgip'^"' and Infers''^"' are basically systems of parallelly placed lab-scale bioreactors with a working volumes down to approximately 200 mL. A similar concept is offered by others, like BioXplore.'^^^' Most likely the robustness and reliability of these scaled down and parallelized system are important factors governing the choice for conventional sensors and equipment and proven bioreactor design, but the scaling to multiple small reactors has, apart from the saving of space, hardly any advantage for fabrication and operation costs.

One of the first successful attempts to fabricate a minibioreactor equipped with pumps, sensors, and controls in a highly integrated fashion came from space research. The need for miniaturization originated from lack of space on the space missions rather than need for parallelization or cost reduction. The minibioreactor system was very advanced, even for today's standards, on microfluidic components for handling the feed-supply. S. cerevisiae was cultivated in this bioreactor with a working volume of 2 mL under pH controlled, chemostat conditions during a space flight.'^^'^'"°'

At the University of Maryland, the group of professor Rao pioneered the field of microbioreactor development with a cuvette-based microbioreactor (2 mL working volume).'^' In a standard disposable cuvette E. coli was cultivated under batch conditions while monitoring the pH, the dissolved oxygen concentration and the optical density. Although the bioreactor was equipped with a small stirrer and sparger, the k.a in the microbioreactor was reported to be only 27.5 h'^ at a gas flow rate as high as 2 vvm. The microbioreactor was later parallelized to a platform of 24 microbioreactors, either in the format of a 24-well plate or as stand alone systems.'^•^^^^'' Professor Rao is co-founder of the company Fluorometrix which offers a cell cultivation platform of 12 stirred bioreactors of 35 mL, equipped with optical sensors.'^''

The group of professor Weuster-Botz (Technische Universitat MCinchen) is very active in the development of miniaturized bioreactor systems with an excellent

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Introduction

INTERLUDE: Scale, units and orders of magnitude ^^Ê

To miniaturize' is an often used verb in this thesis and simply means 'to make smaller than it normally is', while very little is implied on the actual dimensions of the miniature. Scalars like mill! (10"^), micro (*10"^) or nano (10") are generally used to indicate those dimensions, but most often they are used in a very loose and somewhat confusing sense. A microbioreactor generally means a bioreactor with a volume of microlitres (pL), implying a typical length-scale of millimetres (mm). However bioreactors with volumes over 1 millilitre (mL) are also sometimes called microbioreactors.'^"*'

Besides the loose usage of already hard to conceive scalars for indicating the dimensions of the microbioreactor equipment, the usage of non-metric units can, especially for those who do not regularly use these units, also hinder envisagement. Especially when dealing with tubes and capillaries, i.e. small tubes, the dimensions are generally stated in inches, where one inch (1") equals 25.4 millimetres (mm). Tubing made from stainless steel or polymers for microfluidic connections commonly have an outer diameter of 1/32" (0.79 mm) or 1/16" (1.57 mm), whereas the outer diameter of capillaries made out of fused silica is typically 360 micrometers (pm).

Every day objects can help to envision the scale and size of these miniatures and can help to comprehend the complexity of the aims as are stated in this thesis. The microbioreactor that is developed in the work and described in the following chapters has a volume of 100 pL. A typical rain droplet is roughly 5 pL,'^' whereas a droplet from a dripping tap can easily be 20 - 50 pL.'^' The steel capillaries that served as microfluidic connections in the first bioreactor prototype as is described in chapter 2 had an internal diameter of 150 pm. This is around 1.5-3 times the size of a black human hair, which is typically 50 - 100 pm thick.'"' The Hagen-Poiseuille micropump that is described in chapter 4, consist of a capillary with an internal diameter similar to the thickness of a wool fibre (20 pm)'"' and comparable to a silk fibre (15 pm).'"' This micropump was used for continuous feeding the bioreactor with a glucose solution to perform fed-batch experiments. The flow rate of the pump was maximally 150 nL/min; this is equivalent to approximately two rain droplets per hour that were pumped into the microbioreactor when the micropump was operated at full capacity. A garden snail could easily keep up

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Chapter 1

with the linear velocity of the liquid in the pump which is 8 mm/s whereas a speed of 13 mm/s was reported for the garden snail.'^^' The electrochemical sensor chip developed,'^"' tested (chapter 6) and integrated in the bioreactor (chapter 7), contained among others, a sensor for the measurement of the dissolved oxygen concentration (DO). The individual electrodes on the DO sensor had a diameter of 4 pm, which is smaller than the diameter of yeast cell (5-10 |jm).'^^' Although the microbioreactor that was developed in this project had a working volume of only 100 pL, still the number of yeast cells in this volume at a modest 7 g/L is equivalent to'^"' more then 4.5 times the Dutch population.'^^'

detail to the technical/engineering aspects of the system. Up to 16 parallel batch and fed-batch cultivations were reported in pH controlled bubble columns with a working volume of 200 mL.'^'^""'*' Further development and miniaturization let to a liquid handling robot capable of intermittently feeding substrate to custom made, miniature, parallel operated, stirred bioreactors of 5 - 10 mL.'^^^"'^^' Samples were taken from the bioreactors of which the pH and biomass concentration were measured by optical means. These pH measurements were used for the controlled addition of base to the bioreactors by the robot. The dissolved oxygen concentration was measured by an optical DO sensor that was mounted in the bioreactors. To optimize the oxygen transfer rate these small bioreactors were equipped with sophisticated impellers. Impressive oxygen transfer coefficients of 720 - 1440 h"^ were reported at stirring speeds over 2000 rpm.'^^^' While significant improvements in biomass and product concentrations were obtained in their system, this approach requires dedicated hardware and instrumentation.

The same is true for the system that was developed at the Institute Pasteur. A system with 8 tube-shaped-bioreactors, with a working volume of 70 mL each, placed in parallel was constructed. The temperature, dissolved oxygen concentration, biomass concentration (turbidity) and pH were monitored with conventional electrochemical, but miniaturized sensors. The reported system was completely automated and automatic fluid handling might be added in the future. An impressive k.a of 750 h^ was reported for this system at a gas flow rate of 1vvm. In batch cultivations a biomass concentration up to 1 0 - 2 0 g/L of E. coli was reported, producing significant amounts of recombinant protein.'^''°^''^' Unfortunately nothing was said about the prevention of foaming in this high-biomass, high-protein, intensely sparged environment.

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Introduction

The research groups of professor Sinskey and professor Jensen from the Massachusetts Institute of Technology, pioneered the downscaling of bioreactors and reported prototypes with working volumes of 5, 50, 100 and 150 pL. All their prototypes were equipped with optical sensors for the measurement of pH, dissolved oxygen and biomass concentration. Furthermore sensors for the measurement of bioluminescence and fluorescence were successfully implemented. These last sensors allow experimentation with genetically modified strains in which the green fluorescent protein (GFP) or lux operon was used as reporter for for example specific environmental conditions or gene expression.•^''^' In the smallest microbioreactors, with working volumes of 5 and 50 pL, oxygen supply was accomplished by diffusion through a PDMS membrane. Because the height of the bioreactor was only 300 pm, the measured k|a value was still reasonable, -60 h"\ Diffusion was the sole mechanism for mixing in the microbioreactors. Batch cultivations of E. coli were performed in these microbioreactors and compared to bench-scale bioreactors. In these experiments the on-line data for pH, DO and biomass concentration as well as off-line analysis of the glucose, acetate, formate and lactate profiles was shown to be very reproducible. When comparing the data that were obtained in the different cultivation volumes, no scaling effects were observed.'^''^"^''°'

The volume of their second prototype was increased to 150 pL for accommodating better sampling possibilities and a stirrer bar was introduced for better fluid mixing. Stirring at 800 rpm yielded a disappointing k.a of 75 hV'^"^' Results from both batch i^^su?] ^^^ chemostat'^"®^''^' cultivations were presented. The global gene expression analysis that were performed in both the 150 pL stirred'^^' and 50 pL nonstirred'^^"' microbioreactors showed the potential of microbioreactor technology.

Furthermore they showed successful control of the pH and the DO in a bioreactor with a working volume of 100 pL.''^^^^^' Reservoirs for base and acid were integrated together with pneumatically actuated pumps and valves. With what was called a "peristaltic oxygenating mixer", a k.a of -360 h"' was achieved in the microbioreactor. They performed pH controlled batch cultivations of E. coli up to a biomass dry weight concentration of 13 g/L. This strategy of pneumatically actuated valves and pumps enabled a high level of integration on the chip but at the expense of bulky equipment off-chip for controlling the pressures in the numerous air lines, that were needed for actuating the valves and pumps on the chip.

The company Bioprocessors'^^^' seems to provide services for screening, medium optimization and setting of process parameters with a tailored system called the

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Chapter 1

2Q SimCell™. Information on the system is sparse but it seems to be a powerful tool. — A credit card like module accommodates 6 cultivation chambers of approximately 700 pL medium'^^"' with optional measurement of pH and DO by optical means.'^^^' Liquids for fed-batch cultivation and pH control could be introduced by injecting them in the culture via a septum.'^^^•^"' IVIixing of the fluid was achieved by the displacement of an air bubble when moving the module.'^^*'

An impressive piece of work on the miniaturization of the bioreactors was achieved in the group of professor Quake at the California Institute of Technology.'^^^^®"' They successfully fabricated a bioreactor of 16 nL comprising of a circular tube with integrated peristaltic pumps and a series of micromechanical valves to add medium, remove waste and recover the cells. They were able to run chemostat cultivations at dilution rates varying from 0.072 to 0.37 h'^ in this bioreactor. Although this is a great achievement, the volume of the bioreactor is most likely too small for current analytical techniques, thus limiting its feasibility.

1.6 Conclusions

As was outlined above, the main idea behind the development of miniaturized bioreactors is to combine the small working volumes and the high degree of parallelization of the current high-throughput cultivation systems with the sophisticated sensing and control features from the current lab-scale bioreactors, in one system. There is an increasing and widespread interest in these bioreactors and several areas in biotechnology could potentially benefit greatly from such a system. These areas include industrial bioprocess development as well as fundamental research on the functioning of micro-organisms on a nutrition, genetic and molecular level.

The scientific and patent literature, as was reviewed in the previous section, showed rapid progress in development and commercialization of these miniaturized bioreactors. However the literature also indicated difficulties with mixing and mass transfer in small scale systems that are hard to resolve as well as restrictions that are caused by supporting technologies. In particular the optical pH sensors. These sensors are very useful for the cultivation of for example animal cell and E. coli. However industrially relevant micro-organisms like yeast species, are generally cultivated at a pH around 5, which falls outside the working range of the optodes. Furthermore much work is still to be done on the integration of microfluidic components such as valves and pumps for continuous convective feeding and pH control in miniaturized systems. As was already pointed out before, in the majority of the industrial fermentation processes pH-controlled, fed-batch cultivation is the preferred mode of operation. High-throughput cultivation of

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Introduction

micro-organisms under fed-batch conditions could significantly increase the selectivity of the current selection and screening programs. Also the opportunities for the development of tools designed for quantitatively monitor the performance of the organisms, e.g. the specific growth (jj), O2 consumption {qo2), CO2 production (^002) arid product formation rates (qp), are largely unexplored.

1.7 A i m and outline of the thesis

A technological platform that allows for high-throughput cultivation of micro-organisms under controlled nutrient limiting and well controlled environmental conditions (T, pH, DO, etc.) can strongly improve the effectiveness of screening and selection for industrially relevant micro-organisms as well as speed up experimentation in fundamental research on the functioning of micro-organisms at a nutrient, molecular and genetic level. Sensing and control of important environmental conditions like the temperature, dissolved oxygen concentration and pH during the cultivation is of crucial importance, as is the ability to perform the cultivations under substrate limited, fed-batch / chemostat conditions. Although the development of miniaturized components, like vessels, sensors, pumps and -valves, progresses rapidly, an integrated platform with full measurement and control features is not available.

The research described in this thesis aims at the development of a system consisting of parallel bioreactors of 100 microliters working volume for the cultivation of micro-organisms under industrially relevant, fed-batch conditions. The idea is to equip each of the microbioreactors with sensors and measurment techniques for on-line measurement of important environmental parameters as wells as cellular performance (/j, qo2, ^002) indicators. Furthermore microfluidic components are important to facilitate substrate feeding as well as pH control. An important criterion in the design of the tools is the ability for integration and parallelization in the microbioreactor system.

We first developed an on-line technique for the quantification of the oxygen consumption rate and coupled it to a custom made microbioreactor of 100 pL. This technique enabled us to experimentally determine the k|a of our microbioreactor. The data is presented in chapter 2.

The on-line measurement of the oxygen consumption rate or carbon dioxide production rate are important cellular performance indicators and together with the use of elemental balancing these could be used for the quantification of the biomass growth and/or product formation even at high biomass concentrations. In

chapter 3 we tested and applied a novel conductometric method for the

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Chapter 1

cultivation of Candida utilis on a 100 microliter-scale. This quantitative approach immediately allowed better understanding on the negative effect of mechanical shear in stirred microbioreactors on the growth rate.

In chapter 4 we focussed on anaerobic fed-batch cultivations of Saccharomyces

cerevisiae in microtiter plates with the use of a new pulse-free micropump that

was developed for this purpose. The operating principle of this pump is based on the Hagen-Poiseuille law, thus giving it its name.

The characteristics of microfluidic channels for mass transfer were explored in

chapter 5. When two miscible streams come together in a microchannel, they

flow in parallel to each other forming a stable liquid-liquid interface over which components can be transferred. Because of the lack of turbulence in micrometer-scale channels diffusion is the predominant transfer mechanism in these channels. In this chapter the diffusive transfer of glucose between two cocurrent, laminar flowing water streams was estimated by CFD calculations and experimentally verified.

Small and reliable sensors are a first prerequisite for the measurement and control of important environmental parameters. Industrially relevant yeast species are generally cultivated at around pH 5, which is outside the working range of the commercially available optical sensor. Furthermore the turbidity measurement of determining the biomass concentration in miniaturized bioreactors fails at high biomass concentrations. Therefore a new sensor chip was developed that integrated electrochemical sensors for the measurement of the pH, the dissolved oxygen concentration, the temperature and the biomass concentration'^'" This sensor chip was tested in a bench-scale bioreactor under dynamic batch conditions as well as under prolonged continuous cultivation conditions of

Candida utilis. These test results are presented and discussed in chapter 6. Chapter 7 focuses on the integration of the different tools that were developed.

The sensor chip was integrated in the stirred microbioreactor and the device for the quantification of the carbon dioxide production rate was attached to its off-gas system. Successful aerobic batch cultivations of Candida utilis were performed, while monitoring the pH, dissolved oxygen concentration, the temperature, biomass concentration and the carbon dioxide production rate

Finally, in chapter 8 the possible perspectives are discussed for further development and future usage.

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Introduction

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