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Immobilized cells

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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.

IntroductionThese 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 incapital and energy costs.

Limitations(1). Often the product of interest has to be excreted from the cellComplications 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 immobilisationBeads can be prepared by1) 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 materialThe Hydrogels Natural Carrageenan Alginate Agar Gelatin

SyntheticPolyvinyl 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 cellsProcess 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 productsNon-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 methodOpposite 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 scaleDropping 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 concret

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