1

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: cthuang@ntu.edu.tw
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Introduction
Environmental factors: pH, Temp, nutrients
Microorganisms in the soil, water and air.
Microbial activity at interfaces: between solid
and liquid phases.

Liquid Phase

_
O2 OH

P
Biofilm

Substratum S
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Microbial Attachment (Adsorption, Adhesion)

Definition: intimate interaction of a cell with the substratum

Sorption

Substratum Biofilm
Adsorption Attachment

Irreversible Reversible

Chemisorption Physical
(Adhesion) adsorption
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Relevance of Biofilms
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
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Biofilm Formation Process

4
2

3 7
1

6
5

Substratum

1. Transport 4. Lysis
2. Duplication 5. Attachment
3. Extracellular polymer 6. Duplication
production 7. Detachment
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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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Biofilm Formation Process

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
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Biofilm Formation Process

Pre-conditioned film
Organic molecules transported from the bulk fluid to
the substratum, where some of them adsorbed,
resulting in conditioned substratum.

Biofilms, Characklis and Marshall, 1990.

Cell transport
Planktonic cells transported to the conditioned
substratum.
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Biofilm Formation Process

Adsorption
Some 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

Biofilms, Characklis and Marshall, 1990.

movie
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Net Biofilm Formation

Biofilm net Deposition rate Growth Detachment
accumulation = from liquid + rate - rate
rate phase

dB/dt = katt X + μB - kdet B

4
2

3 7
1

6
5

Substratum
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Net Biofilm Formation

Deposition (Attachement)

Total adsorbed cells = reversibly adsorbed cells + irrevesibly

adsorbed cells
No. 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.
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Net Biofilm Formation

Growth
mm S
Growth rate (m) = B
KS + S

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 formation
Substratum, aquatic
environment, microbial cells,
nutrients, attachment 4
2
Biofilms can be found
everywhere in natural 3 7
1
environment
Mixed culture, various 6
5
(uncontrolled) condition,
Substratum
difficulty in sampling
Provide an appropriate
situation to enhance biofilm
formation
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Biofilm Culture Considerations

Source: www.erc.montana.edu
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Experimental Systems
The reactor
Provide substratum for biofilms to accumulate.
The reactor feed
Provide an aquatic environment containing all
cellular growth requirements (energy, carbon, trace
elements at appropriate concentration)
Environmental control system
Provide controlled flow velocity, temperature, pH
Specific treatment
Provide specific treatment such application of
antimicrobial agents or microelectrodes.
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System Consideration

Accumulate maximal biofilms at minimal

medium and time
Allow the least invasion
Provide the most samples
Reduce variables
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Experimental Variables
Chemical Physical
Substrate type Temperature
Substrate concentration Fluid shear stress
pH Heat flux
Inorganic composition Surface composition
Dissolved oxygen Surface texture (roughness)
Microbial inhibitors Hydraulic residence time

Biological
Organism type (mixed or pure cultures; aerobic or
anaerobic)
Organism concentration
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Reactor Design
Balance equation
Net rate of = Net rate of input + Net rate of production
Accumulation by transport 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)

dC
V dt = Vr

C: concentration (mass L-3)

r: conversion rate (mass L-3 time-1)
V
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Mass Balance
Continuously Stirred Tank Reactor (CSTR)
Chemostat
Well-mixed; no gradient; Qin = Qout
dC
Qin Cin Qout V dt = Q (Ci – C) + V  r

at Steady State
dC
=0
dt
C, V Q
(Ci – C) = - r
Q: flow rate (L3 time-1) V
C: concentration (mass L-3) D: dilution rate (time-1)
r: conversion rate (mass L-3 time-1) : 1/D, residence time
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dC
V dt = Q (Ci – C) + V  r r = m X growth rate
0 if sterile feeding
dC Q dX
= (Ci – C) + r = (m-D) X
dt V dt
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Mass Balance
Plug Flow Reactor (PFR)
Not well-mixed; the composition changes with distance
through the reactor

Qin Cin r C Qout

z

V = AL
Dz
C C
+V = r rate of conversion
t z
C C 2C
+V = D + r
t z z 2
rate of transport
by diffusion
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Biofilm Culture
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
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Schematic Diagram
Inoculation Without circulation

With circulation
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Biofilm Reactor
Roto Torque (Rotating Annular Reactor)

Source: BioSurface Technologies Corp.

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Biofilm Reactor
Parallel plate flow cell reactor

Flow cell reactor

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Source: www.erc.montana.edu

Source: BioSurface Technologies Corp.

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Biofilm Reactor
Tubular Reactor

Robbin’s
Devices
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Biofilm Reactor

Radial flow reactor Rotating disk reactor

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Source: erc.montana.edu
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Biofilm Reactor

Stirred disk reactor Drip-flow plate reactor

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Biofilm Reactor
Propeller reactor Fixed film reactor