institute of chemical process fundamentals casold-biomikro.vscht.cz/vyuka/ifm/bioreactors.pdf ·...

BIOREACTORS

The State of the Art

Prof. Ing. František Kaštánek, DrSc. Ing. Petr Kaštánek, PhD.

Institute of Chemical

Process Fundamentals

CAS

Definitions

• Bioreactor is a vessel in which a chemical process is carried out which involves organisms (mainly microbes-viruses or bacteria, fungi and yeasts –traditionally designated as „Fermenters“) or biochemically active substances (enzymes, e.g.) derived from such organisms –in opposite to fermenters frequently considered as „true“ bioreactors. This process can either be aerobic or anaerobic.

• An apparatus, such as a large fermentation chamber, for growing

organisms such as bacteria or yeast that are used in the biotechnological production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste.

• A bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. Cell culture is the process by which cells are grown under cultivated conditions (animal cells, plant cells, algae).

Introduction

• The bioreactor´s environmental condition like gas (oxygen, nitrogen, carbon dioxide) and liquid flow rates, temperature, pH, concentration of substrate and products, cells number and their composition (proteins and nucleic acids), dissolved oxygen levels,, and agitation speed (or circulation rate) need to be closely and continuously monitored and controlled. In many cases, strictly aseptic conditions have to be maintained.

• In an aerobic process, optimal oxygen transfer is perhaps the most difficult task to accomplish.

• There are, limits to the speed of agitation, due both to high power consumption and to the damage to organisms caused by excessive tip speed.

Classification

• On the basis of mode of operation, a bioreactor may be classified as batch, fed batch or continuous.

• On the basis of mode of flow of fluids a bioreactor may be classified as CSTR bioreactor (continuous flow stirred reactor-the content of the bioreactor is ideally mixed), bioreactor with piston flow and bioreactors with non- ideal flow of fluids (cascade of ideal mixtures, dispersed flow of fluids). The quality of flow of fluids significantly influences the rate of grow of cells and the degree of conversion of substrate and consequently, the yield and selectivity of the products.

• On the basis of a number of phases treated into the bioreactor we can distinguished homogeneous bioreactors (e.g. one phase tubular bioreactor with enzyme diluted in the liquid substrate) and heterogeneous bioreactors (e.g. two phase solid-liquid bioreactor like the column type bioreactor with immobilized enzyme and liquid substrate and/or three phase bioreactors with submersed culture: gas (air bubbles)-liquid (substrate)-solids (cells).

Selected basic mathematical relations

• Enzymatic kinetics Non-inhibited kinetic in homogeneous (liquid) system: rp = dp/dt = ν a / (KM + a) = keoa/(KM + a) Reaction rate in immobilized enzymes, steady state conditions: Basic relations for prism-shape particles: DA (d

2a/dx2) = Ra = kétá´/ ( KM´+ a´),

x = 0, a´ = a* ´; x = δ, a´= a*´. If a´<< KM

´: Ra = 2 két(cosh Mδ -1) a*´/ (KM´ Mδ sinh Mδ). If a´>> KM

´: Ra = két

´.

Selected basic mathematical relations

• Microbial kinetics

Basic mass balances in batch aerobic fermenter dX/dt = μX , μ = μmax s /(Ks + s), at t = 0, X = Xo -ds/dt = ((1/Yx/s) μ + ms), at t = 0, s = so kLa([O*] - [O]) = (1/Yx/o) dX/dt + (1/Yp/o) dp/dt + moX + d[O]/dt at t = 0, O = O*, p = 0 Balances of the product: dp/dt = α dX/dt , or dp/dt (1/X) = αμ = q, or dp/dt = βX

Oxygen mass transfer Film theory of the mass transfer of oxygen to the cell is schematically depicted at Fig.1. In the case of aerobic fermentation, the main resistence to the transfer of oxygen is situated into the liquid film surrounding the bubble.

Outline of the reactors • Microbial Cell Aerobic Bioreactors

– Immobilized Cells – Suspended Cells, Mechanically Agitated – Suspended Cells, Pneumatically agitated

• Bioreactors for Animal and Plant Cells Growth („single use“ bioreactors) – Suspended Cells, Pneumatically Agitated – Suspended Cells, Bubble-free – Suspended Cells, Rocking Motion – Adherent Cells

• Microbial Cell Anaerobic Bioreactors • Bioreactors with Immobilized Enzymes and Cells

– Mechanically Agitated – Fluidized Bed – Package Bed – Membranes

• Photobioreactors – Suspended algae – Thin layer Flat Desks – Plastic Bags – Race-ways ponds

Microbial cell aerobic bioreactors

Reactors with biofilms • These types of bioreactors have been mainly uses for treatment of

water, contaminated with hazardous components. • The bacteria and protozoa consume biodegradable soluble organic

contaminants (as well as non-easily biodegradable –like chlorinated compounds, nitrates, pesticides, etc. under the addition of co-substrates).

• Although bacteria in nature are found suspended in water, an important part of them is attained to surfaces. These bacteria's often begin to form biofilm. Biofilms are compound of the bacteria itself and extracellular substances such as polysaccharides and proteines, which are produced by the bacteria. Bacteria immobilized in the biofilm contribute to the reaction with substrate together with the bacteria suspended in the water surrounding the biofilm.

Moving Bed Biofilm Reactors

• The moving bed biofilm reactor can operate as 2-anoxic or 3-aerobic phase system with buoyant-free moving plastic biofilm carriers. These systems can be used for municipal and industrial wastewater treatment, potable water denitrification, etc. Liquid solid separation can be achieved with a variety of processes. MBBR requires a smaller tank volume then activated sludge processes.


Trickling filters

• Trickling filters consist of a fixed bed of rocks, coke, ceramic, gravel, polyurethane foam, wood chips, plastic media, over which sewage or other wastewater and/or contaminated off gases flows and causes a layer of microbial biofilm to grow covering the bed of media. Aerobic conditions are maintained by forces air flowing through the bed (generally in counter-current way of flow) or natural convection of air. Modifications of these types of bioreactors are vessels with pararelly situated vertical desks form plastic polymers and/or expanded metals.


Trickling filters


Reactors with suspended cells (fermenters), mechanically agitated

• The main feature of aerobic fermentation is the provision for adequate aeration; in some cases, the amount of air needed per hour is about 60-times the medium volume.

• These bioreactors are of closed or batch type, but continuous flow reactors are also used. Such reactors provide a continuous source of cells and are also suitable for product generation when the product is released into the medium.

• The size of fermenters ranges from 1-21 laboratory fermenters to 500,000 1 or, occasionally, even more; fermenters of up to 1.2 million liters have been used. Generally, 20-25% of fermenter volume is left unfilled with medium as "head space" to allow for splashing, foaming and aeration. The fermenter design varies greatly depending on the type of fermentation for which it is used.

Scheme of the mixed tank bioreactor


• The most important parameter in the processes accomplished in the aerobic fermenters is the volumetric mass transfer coefficient kLa, see Eq. (13). Immense efforts to elucidate the mass transfer processes in the gas-liquid systems have been done in the past. It is possible to find the correlations of this parameter in the literature, however, for specific composition of the growth media it is indispensable to measure this parameter experimentally.

• Volumetric mass transfer coefficient for mechanically stirred fermenters (using Rushton turbine, water) the relation has been recently published:

kLa = 1.081. 10-3 (P/V)1.19w0.549, kLa volumetric mass transfer coefficient per unit volume of liquid, s-1

• P impeller power input, in W, V volume of liquid, m3, W aeration gas superficial velocity, m/s, n agitation speed, min-1, D diameter of impeller, m, ƍ density of liquid, kg/m3, Po power number = P / ƍ n3D5


• The principal functions of the impellers are a) homogenization of the gas-liquid dispersion, b) desperation and break-up of air bubbles with the aim to enhance the mass transfer area of bubbles and to increase a turbulence in the liquid.

• Both phenomena have the positive effect on the interfacial mass transfer. To achieve a perfect homogenization it has a vital impact on the growth and physiological stage of animal and plant cells. In opposite to oxygen demanding microorganisms, the demand on the supply of excess of oxygen is less essential in the case of cultivation of animal (mammalian) and plant cells.

• In the case of massive cultivation of bacterial cells (yeast, molds, etc.) which are big consumers of the oxygen and ask for huge continuous supply of oxygen, the massive transfer of oxygen across the interface gas-liquid area is absolutely essential for the growth of microorganisms and to achieve the high yield of pertinent metabolites.


• The efficiency of homogenization is characterized by the time of mixing, t m. For individual types of impellers (which are characterized by a Power number Po) is expressed as a function of Reynolds number of mixing Re, Re = n d2/,in then form of function

n tm = f (Re, Po), or, n tm = (Po), for the case when Re 103

• To create the great interface area it is necessary to guarantee sufficient shear-stresses in the batch of the gas-liquid dispersion.

• The shear-stress greatly contributes to the break-up of the bubbles but, simultaneously, could be mortal for some fibrous microorganisms and specially for very stress-sensible animal (hybridomal) and plant cells and tissues.

= , where means the viscosity of the liquid in the suspension and represents the rate of deformation (or shear velocity).

• In the cae of the rotatory impellers, the shear-stress is proportional to the peripheral velocity of the impeller :

= f (), = nd


• Scale- up procedure a) if we want to maintain an approximately equal value of

maximum deformation rates , then we opt for nd = const.

b) If it seems decisive to maintain in both model and scaled fermenters equal conditions for dispergation, we opt for P/V const., which influence on the value of kLa.

c) If it will be decisive to maintain the same degree of homogenization we opt for n const.

d) Basic dimension of the fermenters: H/D = 1 or more, d/D = 0.25-0.35, the last value is valid for filamentous (non-newtonian liquids) microorganisms.


Example: • Production of L-carnitine (see A.P. Mayer and Karen T.Robins. in Montashefte fur Chemie 136, 1269-1277 (2005). Only physiological L-carnitine should be used by food manufacturers. In Lonza (Swiss) came to the conclusion that the most attractive process for the production of L-carnitine is a biotransformation. The first task was to find bacteria that transferred an achiral precursor 4-butyrobetaine into chiral (optically active) L-carnitine.

Production of L-carnitine

The top left picture

and the top right

picture show the

interior of a 15 m3

and 75 m3 fermenter

with 500 kW motor

for the stirrer.

Pneumatically mixed reactors (air-lift reactor)

• Air-lift bioreactors are similar to bubble column reactors, but differ by the fact that they contain a draft tube.

• The draft tube is always an inner tube (this kind of air-lift bioreactor is called "air-lift bioreactor with an internal loop) or an external tube (this kind of air-lift bioreactor is called "air-lift bioreactor with an external loop) which improves circulation and oxygen transfer and equalizes shear forces in the reactor.

Pneumatically mixed reactors

• Features – Pneumatic agitation of broth by

stream of air – Ideal for shear sensitive

Mammalian & Plant Cell cultures. – External Tempering Jacket. – Efficient suspension & circulation

of carrier bound immobilized cells.

– High OTR (oxygen transfer rates). – No mechanical agitation, thus low

power consumption & good aseptic environment

– In-situ Sterilizable upto Temperature 130°C

Pneumatically mixed reactors

Laboratory –scale air-lift reactor (with external loop) for mammalian cells cultivation

Air-lift fermenter

Bioreactors for Animal and Plant Cells Growth („single use“ bioreactors)

• In bioreactors where the goal is to grow cells or tissues for therapeutic purposes, the design is significantly different from industrial bioreactors.

• Cells are grown either in suspension or in adherent cultures.

• Many cells and tissues, especially mammalian ones, must have a structural support in order to grow and agitated environments are often destructive to these cell types and tissues.Higher organisms also need more complex growth media. (More in: E.L. Decker, R. Reski (2008).Bioprocees and Biosystems Engineering 31, 3-9. Individual cells exert individual conditions of a treatment.)

Bioreactors for Animal and Plant Cells

• Growth of animal cells in a suspension – The CELLine bioreactor is a disposable, two-compartment

cultivation device suitable for many cell culture applications, e.g. the production of monoclonal antibodies on a laboratory scale. Efficient cell cultivation is dependent on an optimal supply of oxygen and nutrients.

– The two-compartment bioreactor is designed by dividing the bioreactor into a medium compartment and a cell compartment. A semi-permeable membrane between the compartments allows small molecules to diffuse from one compartment to the other. Higher molecular weight molecules secreted by the proliferating cells are retained within the cell compartment.

– The CELLine is perfectly suited for a wide range of applications involving suspension cell culture, like monoclonal antibody production or long-term continuous culture maintenance.


• Bioreactors with rocking motion – Disposable (plastic) are suitable for microbial and

mammalian cells (in suspension or adherent): Cell culture medium and cells contact only a pre-sterile, disposable chamber, the cell bag, that is placed on a special rocking platform. The rocking motion of this platform induces waves in the cell culture fluid. These waves provide mixing and oxygen transport, resulting in a perfect environment for cell growth. These cell culture devices are suitable for application in animal, virus, insect and plant cell cultures in suspension or on micro carriers, disposable cell culture systems for 0.1 –500 liters of the volumes.

Bioreactors with rocking motion


• Single use bubble-less bioreactors

• Its main feature is a membrane aeration stirrer which enables controlled and gentle mixing, bubble free aeration while avoiding foam generation. The gassing membrane ensures higher oxygen transfer and thus optimal growth conditions and higher cell densities compared to standard spinner flasks.

Industrial-size single use bioreactors for animal cell cultivation

PadReactor system provides enhanced gas exchange, due to its moving sparger. It also achieves low shear mixing due to the vertices of the square tank, which act as natural baffles. These advantages, plus the system’s scalability, make it ideal for the production of vaccines, monoclonal antibodies and other secreted proteins


• Single use membrane bioreactors – Growth of a hybridoma culture, along with production

of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules.

– Hollow fibers are tubular membranes with pore sizes ranging from 10 kD to 0.3µm. Cells grow on and around the large surface area provided by the network of hollow fibers. When perfused with culture media, the hollow fibers allow oxygen and nutrients to be supplied to the cells while metabolic waste products are eliminated.

Single use membrane bioreactors

Hollow fiber module and scheme of the live- mammalian cells cultivation

Bioreactors with immobilized enzymes and cells

• Immobilization means associating the biocatalysts with an insoluble matrix, so so that it can be retained for it economic reuse under stabilized conditions. Immobilization helps in the development of continuous processes.

• Some examples of the use of enzymes: – Removal urea from a wastewater stream – Bioreactor for blood detoxification (deheparization) by

heparinase immobilized to agarose particles – The most important application of immobilized enzymes in

industry is for the conversion of glucose syrups (from starch) to high fructose syrup by the enzyme glucose isomerase.

– One of a major application of immobilized enzyme in pharmaceutical industry is the production of 6-aminopenicillanic acid by deacylation of the side chain either penicillin G or V, using penicillin acylase .


• Immobilization of enzymes is useful if:

– enzymes of interest are intracellular, – extracted enzymes are unstable (are easily inactivated), – the product compounds are released into the medium – high operational stability is needed, – lower costs condition have to be maintained – application in multistep enzyme reaction is needed.

• Both immobilized enzymes and immobilized cells are acceptable as the biocatalysts essential for the attainment of rapid rate of bioconversion.


Scheme of the horizontal and vertical bioreactor with immobilized enzymes

Bioreactors with immobilized cells

• Cell immobilization may be achieved in one of the following ways: cells may be bound to water insoluble carriers e.g. cellulose, ion exchange resins, porous glasses, etc., by adsorption or covalent binding; they can be cross-linked e.g. with glutaraldehyde; polymere matrices may be used to entrapping cells (e.g. calcium alginate).

• The cells can be immobilized either in a viable or non-viable form. • The use of permeabilized cells (non-viable or non growing cells) as a

source of enzymes can be exploited in an immobilization form. The decision to immobilize cells in a viable or nonviable form is very important and depends on their application (e.g., nonviable convert lactose to glucose and galactose, whereas the viable cell convert the lactose to ethane.

• Synthesis of L-aspartic acid by the use the enzyme aspartase which catalyze a one-step addition of ammonia to the double bond of fumaric acid.The enzyme has been immobilized using the whole cells of E.coli.

Bioreactors with immobilized cells

Fluidized–bed bioreactor with immobilized cells

Membrane bioreactors

• It is supposed that the enzymes are freely entrapped in the cavities of the support part of asymetric mebranes. The substrate in the aqueous phase is forced to flow across the membrane an react with enzyme and consequently flow to the inner part of the hollow fiber and out of the membrane.

• Exact solution of the reaction rates in a hollow fiber membrane bioreactoers see in: Prenosil J.E., Hedinger T : Biotechnol. Bioeng. 31 (1988), 911.

• The term membrane bioreactor (MBR) frequently defines a combination of an activated sludge process and membrane separation. Due to recent technical innovations and significant cost reductions the applicability for the MBR technology in municipal wastewater treatment has sharply increased.

MBR Membrane bioreactors

Assymetric hollow-fibre membrane – perpendicular cut

Small – scale bioreactors

• Stem cells have great potential for use as a regenerative therapy for degenerative diseases, such as diabetes or Parkinson’s.

• Suspension bioreactors offer major advantages over traditional, static culture methods, including the ability to monitor and control important bioprocess parameters such as dissolved oxygen, pH, and temperature.

• The use of small-scale bioreactors potentially permits high-throughput experimentation to test operating and growth conditions (media components, agitation rate, cell density) and the resulting interactions

Bioreactor Working Volume

• Nanobioreactor < 1 μL • Microbioreactor 1 ≤ v < 1000 μL • Minibioreactor 1 ≤ v < 100 mL • Standard bioreactor 100 ≤ v < 500 mL • Large bioreactor ≥ 500 mL • Industrial sized bioreactors can reach sizes much greater than 1L,

but currently, large bioreactors of this size and beyond have not been needed for embryonic stem cell culture.

Cuvette-based microbioreactor.

• At the left cuvette wall, blue and UV LED together with 530 nm photodetector are used to measure pH; at the right cuvette wall, blue LED, oxygen sensing patch, and 590 nm photodetector are used to measure dissolved oxygen; red LED and 600 nm photodetector are used to measure optical density through the front and back wall. The air supply inlet and outlet are positioned at the corners of the cuvette. LEDs are fired in succession to prevent crosstalk (for clarity, figure not to scale).

Anaerobic bioreactors

• The longest established anaerobic treatment processes include:

– anaerobic suspended growth, – upflow and down-flow anaerobic attached growth, – fluidized-bed attached growth, – upflow anaerobic sludge blanket (UASB), – covered anaerobic lagoons, and – membrane separation anaerobic processes – dry process anaerobic digestion of Municipal Solid Waste (MSW).

• Tasks of bioreactors

– efficiently remove material (mostly organic) from liquid streams – to provide treatment of an MSW to make it suitable for diversion away

from landfill, with biogas generation

Anaerobic bioreactors

UASB anerobic digester – the most popular modification of the biogas producing reactor

Biogas station Renergie s.r.o.

Photobioreactors

• Micro-algae are source of unique metabolites that can be used to produce novel high-added value bioactive compounds with industrial potential in medical technologies or as food, feed or cosmetic ingredients or as potential source of biofuels.

• Among these substances play important role Poly-Unsaturated Fatty Acids (PUFAs). Production of novel PUFAs by micro algae is highly challenging as current production processes from fish oil threatens natural marine organism’s populations.

• Algae can accumulate large amounts of polysaccharides, lipids and proteins with potential as nutrients/energy or biofuel source

Photobioreactors - experimental

Algae does not need expensive reactors to be cultivated…

mfischer @t stanford.edu

Photobioreactors – plastic bags


Research photobioreactors,

Photon Systems Instruments

..but deserves them.


Research photobioreactors,

EcoFuel Labs.


Torus shape photobioreactor,

University of Nantes

Advantages

Controlled, optimized conditions

Contamination can be minimized

High rates of production

Disadvantages

Expensive

Photobioreactors

Flat panel photobioreactors

Tubular photobioreactors

Open ponds

Advantages

Economical

Relatively simple

High rates of production possible, but with raceway ponds usually

low harvesting concentrations = high downstream processing costs

Disadvantages

Potential for contamination (competitors, invaders)

Less control of conditions (e.g., pH, Temp)

Open ponds

Seambiotic, Israel, Tel Aviv

Open inclined Thin Layer Flat Plate Photobioreactor with high rates

of algae suspension on the top of the plates.

Harvest concentration 10-30 g/l.

„Czech“ way

„Czech“ way, 280 m2

And future…?

Future design to fit the environment…

institute of chemical process fundamentals casold-biomikro.vscht.cz/vyuka/ifm/bioreactors.pdf ·...

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