Fundamentals and Applications of Biofilms Bacterial...
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Fundamentals and
Applications of Biofilms
Bacterial Biofilm
Formation and Culture
Ching-Tsan Huang (黃慶璨)
Office: Agronomy Building, Room 111
Tel: (02) 33664454
E-mail: [email protected]
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IntroductionEnvironmental factors: pH, Temp, nutrients
Microorganisms in the soil, water and air.
Microbial activity at interfaces: between solid
and liquid phases.
O OH2
_
P
S
Liquid Phase
Substratum
Biofilm
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Microbial Attachment (Adsorption, Adhesion)
Sorption
Adsorption Attachment
Chemisorption
(Adhesion)
Physical
adsorption
Biofilm
ReversibleIrreversible
Substratum
Definition: intimate interaction of a cell with the substratum
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Detrimental effects
Increase the transfer resistance of mass, heat and
momentum
Deteriorate attached materials due to biological and
chemical reactions
Health risks
Beneficial effects
Increase biomass concentration
Facilitate biomass-liquid separation
Improve overall productivity
Relevance of Biofilms
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1. Transport2. Duplication3. Extracellular polymer
production
5. Attachment6. Duplication7. Detachment
4. Lysis
Biofilm Formation Process
Substratum
1
2
3
4
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Bryers, J. D. Biotechnology and Bioengineering, Vol. 100, No. 1, May 1, 2008
Biofilm animation 1, 2
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In liquid phase
Dissolution of organic macromolecules
Pre-conditioning adhesion
Cell transport
Replication
Production of extracellular polymers
Death
Attachment to the substratum
Within biofilms
Replication
Production of extracellular polymers;
Detachment
Biofilm Formation Process
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Pre-conditioned filmOrganic molecules transported from the bulk fluid to
the substratum, where some of them adsorbed,
resulting in conditioned substratum.
Biofilm Formation Process
Cell transportPlanktonic cells transported to the conditioned
substratum.
Biofilms, Characklis and Marshall, 1990.
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Biofilm Formation Process
AdsorptionSome cells adsorbed to the substratum for a while
and then desorb due to biological, chemical and
physical factors (reversible adsorption); some
remained immobilized (irreversible adsorption).
Biofilms, Characklis and Marshall, 1990.
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Biofilm Formation Process
Growth
Extracellular
polymer production Detachment
movie
Biofilms, Characklis and Marshall, 1990.
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Biofilm net Deposition rate Growth Detachment
accumulation= from liquid + rate - rate
rate phase
dB/dt = katt X + μ B - kdet B
Net Biofilm Formation
Substratum
1
2
3
4
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Total adsorbed cells = reversibly adsorbed cells + irrevesibly
adsorbed cellsNo. of cells adsorbed to the substratum
Sticking efficiency = No. of cells transported to the substratum
Transport velocity, concentration
Adsoprtion velocity, concentration, physical, chemical and
biological properties.
Net Biofilm Formation
Deposition (Attachement)
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mm S
Growth rate (m) = B
KS + S
Net Biofilm Formation
Growth
m specific growth rate
mm maximum specific growth rate
S substrate concentration
KS saturation constant
B biofilm density
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Net Biofilm Formation
Detachment
Erosion a continuous loss of small portion of
biofilms.
Sloughing a rapid, massive loss of biofilms.
Abrasion due to repeated collisions between
substratum and particles.
Detachment - kdet B
kdet Shear force, roughness,
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Culture
Essentials of biofilm formationSubstratum, aquatic
environment, microbial cells,
nutrients, attachment
Biofilms can be found everywhere in natural environmentMixed culture, various
(uncontrolled) condition,
difficulty in sampling
Provide an appropriate
situation to enhance biofilm
formation
Substratum
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2
3
4
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Biofilm Culture Considerations
Source: www.erc.montana.edu
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Experimental Systems
The reactorProvide substratum for biofilms to accumulate.
The reactor feedProvide an aquatic environment containing all
cellular growth requirements (energy, carbon, trace
elements at appropriate concentration)
Environmental control systemProvide controlled flow velocity, temperature, pH
Specific treatmentProvide specific treatment such application of
antimicrobial agents or microelectrodes.
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Accumulate maximal biofilms at minimal
medium and time
Allow the least invasion
Provide the most samples
Reduce variables
System Consideration
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Experimental Variables
ChemicalSubstrate type
Substrate concentration
pH
Inorganic composition
Dissolved oxygen
Microbial inhibitors
PhysicalTemperature
Fluid shear stress
Heat flux
Surface composition
Surface texture (roughness)
Hydraulic residence time
BiologicalOrganism type (mixed or pure cultures; aerobic or
anaerobic)
Organism concentration
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Reactor Design
Balance equation
Net rate of
Accumulation =
Net rate of input
by transport+
Net rate of production
by transformation
Mass balance
Batch culture
Continuous Stirred Tank Reactor (CSTR); Chemostat
Plug Flow Reactor (PFR)
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Mass Balance
Batch culture
Well-mixed; No input or out put (Q = 0)
V
V = V rdC
dt
C: concentration (mass L-3)
r: conversion rate (mass L-3 time-1)
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Mass BalanceContinuously Stirred Tank Reactor (CSTR)
ChemostatWell-mixed; no gradient; Qin = Qout
V = Q (Ci – C) + V rdC
dt
Q: flow rate (L3 time-1)
C: concentration (mass L-3)
r: conversion rate (mass L-3 time-1)
C, V
Qin CinQout
at Steady State
dC
dt= 0
Q
V(Ci – C) = - r
D: dilution rate (time-1)
: 1/D, residence time
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V = Q (Ci – C) + V rdC
dt
dX
dt= (m-D) X= (Ci – C) + r
dC
dt
Q
V
0 if sterile feeding
r = m X growth rate
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Mass Balance
Plug Flow Reactor (PFR)
Not well-mixed; the composition changes with distance
through the reactor
Qin Cin
zr QoutC
V = AL
C
t+ V
C
z= r
C
t+ V
C
z= D + r
2C
z2
rate of transport
by diffusion
rate of conversion
Dz
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CSTR
Provides advantages for observing, separating
and evaluating the kinetics and stoichiometry
of each biofilm process.
The bulk liquid phase is uniform.
The steady state condition is convenient and
reproducible
Constant shear stress
Biofilm Culture
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Schematic DiagramInoculation
With circulation
Without circulation
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Roto Torque (Rotating Annular Reactor)
Biofilm Reactor
Source: BioSurface Technologies Corp.
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Parallel plate flow cell reactor
Flow cell reactor
Biofilm Reactor
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Source: www.erc.montana.edu
Source: BioSurface Technologies Corp.
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Tubular Reactor
Robbin’s
Devices
Biofilm Reactor
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Radial flow reactor Rotating disk reactor
Biofilm Reactor
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Sourc
e: e
rc.m
onta
na.e
du
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Biofilm Reactor
Drip-flow plate reactorStirred disk reactor
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Biofilm Reactor
Fixed film reactorPropeller reactor