Industrial Microbiology INDM 4005 Lecture 15 24/03/04

Process variables
Cell immobilisation

Introduction
In 1995 the symposium, Immobilised Cells: Basics and Applications was organised under the auspices of the working party of applied catalysis of the European Federation of Biotechnology
Symposium covered the path from basic physiological research to bioprocess applications Immobilised cells, Springer lab manual Wijffels, R.H

Introduction
In a previous lecture we learnt 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.

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.

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 m. Therefore in an immobilised continuous fermenter system high cell counts can be maintained leading to higher biomass productivity as compared to a normal chemostat.

Advantages over suspension cultures

(1). (2).
(3). (4).

(5). (6). (7).

Immobilisation provides high cell concentration Immobilisation provides cell reuse and eliminates the costly processes of cell recovery and cell recycle Immobilisation eliminates cell washout problems at high dilution rates Combination of high cell concentrations and high flow rates allows high volumetric productivities Favourable microenvironmental conditions Improves genetic stability Protects against shear damage

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.

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

Biomass

Immobilised bioreactor

Chemostat

Time

 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.

 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.

 The higher productivity and greater stability of immobilized fermenters thus leads to smaller fermenter requirements and considerable savings in
capital and energy costs.

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

(2). (3).

(4).

Types of immobilisation
Active immobilisation Passive immobilisation

Active immobilisation
Is 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

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

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

Description of support material

The Hydrogels Natural Carrageenan Alginate Agar Gelatin

Synthetic
Polyvinyl alcohol Polyurethane Polyethylene glycol

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 Dgalactose 4-sulphate and 3,6-anhydro-D-galactose 2sulphate
Carrageenan matrix becomes weak when disturbing ions are present

The seaweed Chondrus crispus. Image width ca 15 cm.

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

Laminaria hyperborea forest. Image width ca 3 m.

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 matrixes are soluble Relatively weak Biodegradable

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

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

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

Immobilised vs free cells

YEASTS - immobilised produce more ethanol

RECOMBINANT CULTURE - plasmid stability improved on immobilisation

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

Entrapment (beads) vs encapsulation (capsules)

Entrapment
cells leak
large beads, surface layer of growth biomass disrupts matrix (limits to 25% by volume)

Pregel dissolving 2 step method

calcium alginate bead containing cells formed first then coated in poly-L-lysine crosslinked with sodium alginate finally calcium alginate core dissolved using sodium citrate method

Liquid-droplet one step method

Opposite of conventional bead formation
cells + curing solution (calcium chloride) dropped into sodium alginate solution results in a gel skin formed on surface of the drop with cells contained in liquid centre cells are allowed to grow to increase level of biomass encapsulated

Mass production - industrial scale

Dropping methods have limitation - can be improved by increasing number of needles liquid jet-based method - form drops by vibration cut with wires Centrifugal force vs gravity concentric - cells, polymer and air extruded separately

 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

Types of immobilized cell reactors

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:

Fresh feed

Cell recycle system

Biomass separation system Fermenter

Effluent

Biomass recycle

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 would be used to separate the cells. Biomass recycle systems can be easily modelled.

Fixed bed reactors

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

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.

Fixed bed reactors

Once steady state has been reached there will be a continuous cell loss from the solid surfaces. These 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

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

Fixed bed reactors

One advantage 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.

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).

Fluidised bed reactors

Small moving particles

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.

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.

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.

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.

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.

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.

Flocculated cell reactors

Cells form flocs which gently fall and rise with gases they produce

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

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)

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

RECOMBINANT DNA TECHNOLOGY and PROCESS INSTABILITY

Smallest unit of reaction is gene / plasmid
Specialist cultures have been developed Non-robust nature e.g. plasmid instability

Generalist cultures represent the competing contamination

BIOLOGICAL PROCESSES AND DYNAMIC ENVIRONMENTS (changing environmental conditions)

Waste-treatment, Bioremediation Biological control Oil-breakdown

Agricultural e.g. rhizobium, mycorrhiza, silage

Food e.g. meat fermentation, yogurt

CONVENTIONAL STRATEGY FOR STABILIZATION OF BIOTECHNOLOGICAL PROCESSES (e.g. STR) Eliminate contamination / competition

Regulate process environment

HOMOGENEITY PARADIGM OFTEN DOMINATES MICROBIOLOGY

STRATEGIES TO OVERCOME PERTURBATIONS

1. Modification of cell physiology And biochemistry to produce a Supercompetitor
= Genetic 2. Create microenvironments to help the inocula = Microbial ecology

ECOLOGICAL COMPETENCE OF ANY INOCULA is influenced by

INTRACELLULAR PERTURBATION Modification of replicon Modification of extrinsic factors e.g nutrient limitation, selective agents etc.
EXTRACELLULAR PERTURBATIONS Modification of process factors Growth in bioreactor Harvesting, storage and transport conditions Delivery to ultimate site of action

COMBINED STRATEGIES TO OVERCOME PERTURBATIONS

Modification of cell physiology Create microenvironments - to optimize activity of desired culture = Protect - to optimize adaptation and release to new environment = Controlled / sustained delivery

CONSIDER SPATIALLY ORGANIZED MICROBIAL POPULATIONS and STABILITY (in nature)

DIMENSIONS MAY BE; Vertical peat, soil, water etc.

Horizontal colony formation Radial activated sludge flocs, yeast flocs

STABILISATION FROM ICT

Protective effect from encapsulation within a matrix Manipulation of diffusion rates
Co-immobilization of different phases, food sources, selective chemicals and/or protectants

GEL-IMMOBILIZED CELLS AND ARTIFICIAL MICROCOSMS / MICROENVIRONMENTS

Combine ecological mechanisms based on; enhanced stress resistance juxtapositioning of cells protection afforded within aggregates/flocs Are based on space and/or time dimensions targeted delivery controlled release

MICRONICHES existing in ALGINATE GELS

PROFILE INFLUENCED BY; Removal rate and diffusivity of molecules Bead characteristics; Matrix properties Homogeneity of matrix Diameter Microbial characteristics Morphology Biomass density Biomass activity

CHARACTERISTICS OF GEL
PROPERTIES OF BOTH LIQUID AND SOLID Shape retention, Resistance to mechanical stress.

PHYSICALLY IMMOBILIZED WATER Similar to semi-permeable membrane, Water soluble molecules can diffuse, Water moves in / out (dry) depending on external environment.

SUSTAINED / CONTROLLED DELIVERY

DEGREE OF PROTECTION Profiles of substrate, end-products, metabolites Co-immobilisation of beneficial cultures, complex nutrients, protectants, selective chemicals and pH or osmotic regulators. DEGREE OF CELL RELEASE Rate of outgrowth, polymer characteristics, gelation process, particle characteristics, biomass activity, macroenvironment. (McLoughlin, A.J., Adv. Biochem. Eng. / Biotechnol., 1994)

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

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