P1252122141OYSwg

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Industrial Microbiology INDM 4005 Lecture
15 24/03/04
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Process variables

• Cell immobilisation

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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

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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.

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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.

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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.

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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

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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.

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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
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• 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.

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• 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.

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

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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

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Types of immobilisation

• Active immobilisation
• Passive immobilisation

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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

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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

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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

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Description of support material

• The Hydrogels
• Natural
• Carrageenan
• Alginate
• Agar
• Gelatin
• Synthetic
• Polyvinyl alcohol
• Polyurethane
• Polyethylene glycol

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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

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

• Alginate is derived from algae and is stabilised
  by divalent cations
• 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
• Most commonly used cation is Ca2

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Laminaria hyperborea forest. Image width ca 3 m.
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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

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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

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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

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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

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Immobilised vs free cells

• YEASTS - immobilised produce more ethanol
• RECOMBINANT CULTURE - plasmid stability improved
  on immobilisation

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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

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Entrapment (beads) vs encapsulation (capsules)

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

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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

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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

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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

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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

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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

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

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Fixed bed reactors

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

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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.

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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

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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

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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.

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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).

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Fluidised bed reactors
Small moving particles
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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.

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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.

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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.

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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.

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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.

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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.

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Flocculated cell reactors
Cells form flocs which gently fall and rise with
gases they produce
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Some applications

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

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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)

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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

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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

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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

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CONVENTIONAL STRATEGY FOR STABILIZATION OF
BIOTECHNOLOGICAL PROCESSES (e.g. STR)

• Eliminate contamination / competition
• Regulate process environment

HOMOGENEITY PARADIGM OFTEN DOMINATES MICROBIOLOGY
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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
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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

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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
  

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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

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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
• Cell release in defined / controlled patterns

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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

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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

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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.

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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)

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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

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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