1. Bioprocess Technology-Immobilization of Cells

2. Process variables
Cell immobilisation

3. Introduction In Continuous Culture-
We know that higher dilution rates can lead to
- higher biomass productivity
But
- higher substrate concentrations in the
effluent and lower biomass concentrations in
the reactor
When the dilution rate exceeds the critical dilution rate then washout occurs.

4. Introduction These factors result in a number of problems.
E.g in continuous wastewater treatment processes:
Minimum reactor volume is set by the critical dilution rate.
High dilution rates will lead to an effluent containing high concentrations of "substrate" and the effluent will therefore contain not have been treated properly.
Low cell concentrations at high dilution rates will also make the reactor sensitive to inhibitors in the feed.
Inhibitors would cause the specific growth rate of the cells to drop and cause the cells to washout.
The lower the concentration of cells, then the faster the cells will washout.

5. Introduction
In chemostat processes similar consequences can occur. If the substrates are expensive, e.g animal cell culture, high dilution rates can dramatically affect process profitablility.
Immobilizing cells in the fermenter ensures that cells do not washout when the critical dilution rate is exceeded.
By immobilizing the cells in the fermenter, high cell numbers can be maintained at dilution rates which exceed µm.
Therefore in an immobilised continuous fermenter system high cell counts can be maintained leading to higher biomass productivity as compared to a normal chemostat.

6. Advantages over suspension cultures
(1). Immobilisation provides high cell concentration
(2). Immobilisation provides cell reuse and eliminates the costly processes of cell recovery and cell recycle
(3). Immobilisation eliminates cell washout problems at high dilution rates
(4). Combination of high cell concentrations and high flow rates allows high volumetric productivities
(5). Favourable microenvironmental conditions
(6). Improves genetic stability
(7). Protects against shear damage

7. Advantages of immobilised cell reactors
Being able to maintain high cell concentrations in the reactor at high dilution rates provides immobilised cell bioreactors with advantages over chemostats.
More biomass means that the fermenter contains more biocatalysts, thereby high bioconversion rates can be achieved.
Immobilised cell bioreactors are also more stable than chemostats.

8. A higher cell concentration in the immobilised bioreactor prevents the microbial population from completely washing out.
Inhibitor enters
inlet feed
Immobilised
bioreactor
Biomass
Chemostat
Time

9. Advantages of immobilised cell reactors
In a chemostat, a temporary (transient) increase in the dilution rate will cause a rapid drop in cell numbers.
The entry of a slug of toxic substances in the feed will have the same effect.
It will take time for the cells numbers to build up again
. Since the cells are not as easily washed out of an immobilized cell reactor, the recovery time will be more quicker and fall in biomass concentration will be smaller.

10. Advantages of immobilised cell reactors
If the toxic substance is a substrate (eg. in the waste treatment of toxic wastewaters), high cell concentrations will be able to more rapidly utilize any slug of toxins which may enter the reactor.
The resultant sag in biomass concentration will be smaller and the rise in concentration of the inhibitory substance will also be much smaller with immobilized cell reactors.

11. Advantages of immobilised cell reactors
The higher productivity and greater stability of immobilized fermenters leads to smaller fermenter requirements and considerable savings in capital and energy costs.

12. Limitations
(1). Often the product of interest has to be excreted from the cell
(2). Complications with diffusional limitations
(3). Control of microenvironment conditions is difficult due to heterogeneity in the system
(4). Growth and gas evolution can lead to mechanical disruption of the immobilised matrix

13. Types of Cell immobilisation
Active immobilisation
Passive immobilisation

14. Active immobilisation
Entrapment or binding of cells by physical or chemical forces
Physical entrapment within porous matrices is the most widely used method of cell immobilisation
Immobilised beads should be porous enough to allow transport of substrates and products in and out of the beads

15. Active immobilisation
Beads can be prepared by
1) Gelation of polymers
2) Precipitation of polymers
3) Ion exchange gelation
4) Polycondensation
5) Polymerisation
6) Encapsulation

16. Passive immobilisation
Biological films
The multilayered growth of cells on solid support surfaces
The support material can be inert or biologically active
Biofilm formation is common in natural and industrial fermentation systems, i.e biological wastewater treatment and mold fermentations

17. Description of support material
The Hydrogels
Natural
Carrageenan
Alginate
Agar
Gelatin
Synthetic
Polyvinyl alcohol
Polyurethane
Polyethylene glycol

18. Carrageenan
Extracted from seaweed and a gel is derived by stabilisation with K+ ions or by thermogelation (reducing the temperature at low ion concentration)
Carrageenan consists of alternating structures of D-galactose 4-sulphate and 3,6-anhydro-D-galactose 2-sulphate
Carrageenan matrix becomes weak when disturbing ions are present

19. The seaweed Chondrus crispus. Image width ca 15 cm.

20. Alginate
Alginate is derived from algae and is stabilised by divalent cations
Most commonly used cation is Ca2+
It consists of 1-4 bonded D-mannuronic and L-guluronic acids groups
Gels are formed due to binding of divalent metal cations to the guluronic acids groups

21. Laminaria hyperborea forest. Image width ca 3 m.

22. General characteristics of natural hydrogels
Cells survive mild immobilisation methods
Cells grow easily in matrix
The diffusion coefficients of substrates are high
Relatively cheap
The matrices are soluble
Relatively weak
Biodegradable

23. Synthetic gels
Lately several gel-forming synthetic pre-polymers have been developed
Polymerisation or crosslinking is carried out in the presence of the microorganism
Rather hostile process leads to activity losses

24. General characteristics of synthetic gels
Low or no solubility
Low or no biodegradability
Strong
Diffusivity of substrates relatively low
Recovery of immobilised cells relatively low
Relatively expensive

25. Bioencapsulation or Gel Immobilised cells
Process intensification
results in high level of biomass which improves productivity
cells recovered easily
higher flow-through rates in continuous systems
Protection
cells protected from stress e.g. pH, temp etc. useful in inoculum delivery

26. Immobilised vs free cells
YEASTS - immobilised produce more ethanol
RECOMBINANT CULTURE - plasmid stability improved on immobilisation

27. Bead entrapment - gel matrix and products
Non-toxic
Agarose
Calcium alginate
Carrageenan
can be toxic
Polyacrylamide -
Polyvinyl alcohol
PRODUCTS
antibiotics
ethanol
citric acid
penicillin
phenol degradation

28. Types of immobilized cell reactors
There are many types of immobilized cell reactors either in use or under development.
In this section we will look at four major classes of immobilized cell reactors:
Cell recycle systems
Fixed bed reactors
Fluidized bed reactors
Flocculated cell systems

29. Cell recycle systems
In a fermenter with cell recycle the cells are separated from the effluent and then recycled back to the fermenter; thus minimising cell removal from the fermenter:

30. Cell recycle system Fresh feed Biomass Effluent separation system
Fermenter
Biomass recycle

31. Cell recycle systems Cell recycle is used in activated sludge systems.
A portion of the cells are separated in a settling tank and returned to the activated sludge fermenter.
Biomass recycling for product or biomass production is more difficult due to the need for maintaining sterility during cell separation.
Centrifugation which is a faster process than settling may be used to separate the cells.
Biomass recycle systems can be easily modeled.

32. Fixed bed reactors
In fixed bed fermenters, the cells are immobilized by absorption on or entrapment in solid, non-moving solid surfaces.

33. Fixed bed reactors
In one type of fixed bed fermenter, the cells are immobilized on the surfaces of immobile solid particles such as
plastic blocks
concrete blocks
wood shavings or
fibrous material such as plastic or glass wool.
The liquid feed is either pumped through or allowed to trickle over the surface of the solids where the immobilized cells convert the substrates into products.

34. Fixed bed reactors
Once steady state has been reached there will be a continuous cell loss from the solid surfaces.
Fixed Bed types of fermenters are widely used in waste treatment
In other types of fixed-bed fermenters, the cells are immobilized in solidified gels such as agar or carrageenin

35. Fixed bed reactors
In these fermenters, the cells are physically trapped inside the pores of the gels and thus giving better cell retention and a large effective surface area for cell entrapment.
In order to increase the surface area for cell immobilization, some researchers have investigated the use of hollow fibres and pleated membranes as immobilization surfaces.
Industrial applications of fixed bed reactors include
waste water treatment
production of enzymes and amino acids
steroid transformations

36. Fixed bed reactors
One advantage of fixed bed reactors is that non-growing cells can be used.
In such systems, the cells enzymatically act on substrates in the feed.
The cells can be either inactivated or not fed nutrients required for growth.

37. Fluidised bed reactors
In fluidized-bed fermenters the cells are immobilized on or in small particles.
The use of small particles increases the surface area for cell immobilization and mass transfer.
Because the particles are small and light, they can be easily made to flow with the liquid (ie. fluidised).

38. Fluidised bed reactors
Small
moving
particles

39. Fluidized bed reactors
The fluidisation of the particles in the reactor leads to the surface of the particles being continuously turned over. This also increases the mass transfer rate.
Fluidised beds are typically categorized as either being a
2 phase system which are not aerated and
3 phase system which is aerated by sparging
Fluidized bed bioreactors are used widely in wastewater treatment.

40. Fluidized bed reactors
Fluidized bed bioreactors are also used for animal cell culture.
Animal cells are trapped in gels or on the surface of special particles known as "microcarriers".
Fluidized bed reactors are one example of perfusion culture technology used for animal cell culture.

41. Comparing fluidised bed and fixed reactors
Fluidised bed reactors are considerably more efficient than fixed bed reactors for the following reasons:
1) A high concentration of cells can be immobilized in the reactor due to the larger surface area for cell immobilization is available
2) Mass transfer rates are higher due to the larger surface area and the higher levels of mixing in the reactor.
3) Fluidised bed reactors do not clog as easily as fixed bed reactors.

42. Comparing fluidised bed and fixed reactors
Fluidised bed reactors are however more difficult to design than fixed bed reactors.
Design considerations include:
Setting the flow rate to achieve fluidisation
Ensuring that bubble size remains small during the fermentation.
Prevention of the cells from falling or "sloughing" off the particles.
Minimising particle damage.

43. Flocculated cell reactors
In flocculated cell reactors, the cells are trapped in the reactor due to an induced or natural flocculation process.
In flocculation cells tend to group together causing them to come out of solution and to sink towards the base of the reactor.
Flocculated cell reactors are used widely in anaerobic waste treatment processes.
In these reactors, the methanogenic and other bacteria form natural flocs.
The flocs move due to the release of methane and carbon dioxide by the cells.

44. Flocculated cell reactors
Large scale anaerobic flocculated cell systems, known as Upflow Anaerobic Sludge Blanket processes are widely used in Europe for the anaerobic digestion of high strength industrial wastewaters.
The reactors are typically egg-shaped.

45. Flocculated cell reactors
Cells form flocs which
gently fall and rise with
gases they produce

46. Some applications
ENCAPSEL - retained antibody plus mamalian cells in capsule during growth in bioreactor
Artificial seeds - polymer coating protects plant embryo

47. Artifical cells/organs
Leukocytes & antibodies cannot penetrate into membranes  immunological rejection avoided
Encapsulated hepatocytes placed into rat with defect in bilirubin uridine diphosphate glucuronyltransferase
Genetically engineered encapsulated E. coli into rats with renal failure (lowered plasma urea)

48. Biosorbents Remove heavy metals
AlgaSORB - immobilised algae cells in silica gel beads
S. cerevisiae and Zoogleoa ramigera immobilised in calcium alginate capsules used in removal of lead and cadmium

49. Summary Why use immobilisation; advantages over suspension cultures
Some limitations of ICT
Types of immobilisation
Immobilisation matrixes
Types of immobilised cell reactors
Applications of immobilised cells

50. Conclusions ICT ADVANTAGES
1. Increased reaction rates e.g. higher flow rates
2. Higher cell densities
3. Repeated use of biocatalyst
4. Minimal cost for cell separation