The immobilization of whole cells can be defined as “the physical confinement or localization of intact cells to a certain region of space, without loss of desired biological activity.” In other words, cell immobilization means to freeze an entire cell in a state of suspended animation, such that its metabolism stops and hence does not die. Biological films are the multilayer growth of cells on solid support surfaces ; community of micro-organisms enclosed in a polymeric matrix and adhered on inert or living surface  These attached cells are embedded in a self-produced exopolysaccharide matrix, and exhibit different growth and bioactivity compared with suspended cells. Biofilm consists of three components: microorganism, extracellular polymeric substances (EPS), surface for attachment. The excreted polymeric substances hold the biofilm together and cement it to a surface. The thickness of a biofilm is an important factor affecting the performance of the biotic phase. Thin biofilms - low rates of conversion due to low biomass concentration. Thick biofilms - may experience diffusionally limited growth, which may or may not be beneficial depending on the cellular system and objectives

1. PASSIVE IMMOBILIZATION : BIOLOGICAL FILMS
PRESENTED BY:
ELIZABETH PHILIP

2. CELL
IMMOBILIZATION:
INTRODUCTION:
• The immobilization of whole cells can be defined as “the physical
confinement or localization of intact cells to a certain region of space,
without loss of desired biological activity.”
• In other words, cell immobilization means to freeze an entire cell in a
state of suspended animation, such that its metabolism stops and
hence does not die.
• It can be applied to basically all types of biocatalysts including
enzymes, cellular organelles, animal and plant cells.
• The cells are fixed in a suitable matrix to immobilize them and are
used in bioconversion, production of genetically engineered
proteins, vaccines, etc.
• By immobilizing the cells in the fermenter, high cell numbers can be
maintained.
• Immobilising cells in the fermenter ensures that cells do not wash out
when the critical dilution rate is exceeded.
• Therefore, in an immobilized continuous fermenter system high cell
counts can be maintained leading to higher biomass productivity.

3. 1. ACTIVE CELL IMMOBILISATION:
Entrapment or binding of cells by physical or
chemical forces
The two major methods of active immobilization
are entrapment and binding.
Physical entrapment within porous matrices is
the most widely used method of cell
immobilization
Various matrices can be used for the
immobilization of cells. Among these are porous
polymers (agar, alginate, k-carrageenan,
polyacrylamide, chitosan, gelatin,), polyurethane,
silica gel, polystyrene etc.
Cell entrapment methods include-
polymerization, encapsulation
Immobilization of cells on the surfaces of
support materials can be achieved by physical
adsorption or covalent binding.
TYPES OF CELL IMMOBILISATION:
ACTIVE AND PASSIVE
IMMOBILIZATION

4. PASSIVE CELL IMMOBILIZATION
BIOFILMS:
• Biological films are the multilayer growth of cells on
solid support surfaces ; community of micro-organisms
enclosed in a polymeric matrix and adhered on inert or
living surface
• These attached cells are embedded in a self-produced
exopolysaccharide matrix, and exhibit different growth
and bioactivity compared with suspended cells.
• Their use in bioreactors provides many advantages
including lesser tendencies to develop membrane
fouling and lower required capital costs, their higher
biomass density and operation stability, contribution to
resistance of microorganisms, etc.
• Biofilm formation occurs naturally by the attachment of
microbial cells to the support without use of any
chemicals agent in biofilm reactors.
Biofil
m

5. The support material can be inert or biologically active
Biofilm formation is common in natural and industrial fermentation systems, i.e
biological wastewater treatment, mould fermentation and also in ethanol,
butanol, lactic acid, fumaric acid, and succinic acid production.
Biofilm consists of three components:
o microorganism,
o extracellular polymeric substances (EPS)
o surface for attachment.
The excreted polymeric substances hold the biofilm together and cement it to a
surface.

6. BIOFILM FORMATION
VARIOUS TYPES OF BIOFILMS:
• In industrial applications, usually two types of biofilms are employed:
1. Biofilms that grow onto supports such as charcoal, resin, bonechar, concrete, clay brick, or sand
particles
 On the above supports, biomass grows all around the particles and the size of the biofilm particles
grows with time usually to several mm in diameter.
 The density of the support particles is usually higher than the fermentation broth and for this
reason bioparticles tend to remain in the lower section of the reactor.
2. Biofilms that are formed as a result of flocs and aggregate formation (form biomass granules )
 This type of biofilm is called granular biofilm and the reactor where this biofilm is used is called
granular biofilm reactor.
 The cells produce extracellular polymeric substances (EPS) binds the cells firmly in the form of
flocs and aggregates.
 The most commonly used bioreactors that fall in this category are upflow anaerobic sludge blanket
(UASB) reactors that are used to treat domestic and industrial wastewater anaerobically.

7. MECHANISM OF BIOFILM
FORMATION
I. ATTACHMENT:
The attachment step could be further categorized as a two-
stage process: initial reversible attachment and irreversible
attachment
a) Initial reversible attachment : In order for a free floating cell
to attach to a surface, it must first interact with the surface.
• Surfaces immersed - acquire a surface charge which attracts
and concentrates inorganic solutes, and charged or highly
polar organic molecules - provide a relatively nutritious zone
for bacteria.
• When at the interface, the cell will form a temporary
association with the surface or microbes already present on
the surface
b) Irreversible attachment: After initial association with the
surface, the cell can dissociate from the surface or become
irreversibly attached to the surface.
• Irreversible attachment involves the production of
extracellular polymeric substances (EPS)- can be composed
of polysaccharides, proteins, nucleic acids, or phospholipids.
• EPS - bind the cell to the surface and protect it from the
surrounding environment.

8. II MATURATION:
 Over time, the biofilm thickens and matures together with the reproduction of
the microbes and secretion of additional polymers.
 Once the first layer of the biofilm is established, cells of the same species or
other species are recruited to the biofilm from the bulk fluid.
 Water and nutrient diffusion into the interior of a biofilm is highly limited.
 As biofilms mature, water channels can develop that allow water and nutrient
access deeper into the biofilm.
 These channels partially relieve the diffusion limitation within the biofilm. The
architecture of the biofilm develops in response to shear forces.
 In low shear environments, biofilms can form as thick mushroom-like masses.
 In high shear environments, biofilms may be flatter or form long strands
III DETACHMENT:
 Final stage in the life of a biofilm is reversion of part of the cells to the
planktonic state.
 When cells living in biofilm take up nutrients, they channel much of that
energy towards production of EPS rather than to cell growth and division.
 When nutrients become scarce, cells must escape the EPS matrix or be
trapped in an unfavorable environment.

9. FACTORS ENHANCING BIOFILM
FORMATION
• Several parameters affect how quickly biofilms form and mature,
including surface, cellular, and environmental factors.
Surface: rough surface and porous material work well for biofilm
formation. Shear forces are lower near a rough surface and inside
pores
 Amount of nutrients: Biofilms tend to form more readily in the
presence of ample nutrients. In a stagnant biological film,
nutrients diffuse into the biofilm and products diffuse out into
liquid nutrient medium.
Temperature: Depending upon the species involved, high
temperature increases the rate of cell growth, EPS production, and
surface adhesion, all of which enhance biofilm formation
Cellular factors: A hydrophobic cell will be more able to overcome
the initial electrostatic repulsion with the solid surface and adhere
more readily. Flagellated cells show increased ability to attach to
surfaces. Schematic representation of a
biofilm.

10. • The thickness of a biofilm is an important factor
affecting the performance of the biotic phase.
Thin biofilms - low rates of conversion due to low
biomass concentration.
Thick biofilms - may experience diffusionally
limited growth, which may or may not be beneficial
depending on the cellular system and objectives.
• In many cases, an optimal biofilm thickness resulting in the maximum rate of bioconversion exists
and can be determined.
• In some cases, growth under diffusion limitations may result in higher yields of products as a result
of changes in cell physiology and cell–cell interactions
• In this case,improvement in reaction stoichiometry (e.g., high yield) may overcome the reduction in
reaction rate, and it may be more beneficial to operate the system under diffusion limitations
• Usually, the most sparingly soluble nutrient, such as dissolved oxygen, is the rate-limiting nutrient
within the biofilm

11. TYPES OF BIOFILM
REACTORS
• Biofilm reactors can be assembled in a number of
configurations including :
 Continuous stirred tank reactor(CSTR): used for the
production of butanol and lactic acid
 Packed bed (PBR): usually fed at the bottom, getting product
at the top of the reactor ; prone to blockade due to excessive
cell growth.
 Trickling bed (TBR): fed at the top of the reactor thus
obtaining product at the bottom; in anaerobic waste water
treatment and acetic acid production, these reactors have
been used at large scale successfully.
 Fluidized bed (FBR): degradation of toxic phenolic chemicals
and butanol production
 Upflow anaerobic sludge blanket (UASB): used for anaerobic
treatment of wastewater/industrial effluents.
 Airlift reactors (ALR)
 Expanded bed reactors. Schematic diagram of biofilm particles.

12. THANK
YOU
REFERENCES :
1. Bioprocess Engineering-Basic Concepts
by Shuler and Kargi
http://137.184.111.112/bioprocess_eng
ineering_basic_concepts_pdf.pdf
2. https://microbialcellfactories.biomedce
ntral.com/articles/10.1186/1475-2859-
4-24#Fig1
3. https://www.researchgate.net/publicati
on/273452184_Characterisitcs_of_biofil
ms_in_bioreactors-A_review
4. https://slideplayer.com/slide/10631381
/