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

2

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

3

Microbial Attachment (Adsorption, Adhesion)

Sorption

Adsorption Attachment

Chemisorption

(Adhesion)

Physical

adsorption

Biofilm

ReversibleIrreversible

Substratum

Definition: intimate interaction of a cell with the substratum

4

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

5

1. Transport2. Duplication3. Extracellular polymer

production

5. Attachment6. Duplication7. Detachment

4. Lysis

Biofilm Formation Process

Substratum

1

2

3

4

56

7

6

Bryers, J. D. Biotechnology and Bioengineering, Vol. 100, No. 1, May 1, 2008

Biofilm animation 1, 2

7

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

8

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.

9

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.

10

Biofilm Formation Process

Growth

Extracellular

polymer production Detachment

movie

Biofilms, Characklis and Marshall, 1990.

11

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

56

7

12

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)

13

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

14

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,

15

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

1

2

3

4

56

7

16

Biofilm Culture Considerations

Source: www.erc.montana.edu

17

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.

18

Accumulate maximal biofilms at minimal

medium and time

Allow the least invasion

Provide the most samples

Reduce variables

System Consideration

19

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

20

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)

21

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)

22

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

23

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

24

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

25

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

26

Schematic DiagramInoculation

With circulation

Without circulation

27

Roto Torque (Rotating Annular Reactor)

Biofilm Reactor

Source: BioSurface Technologies Corp.

28

Parallel plate flow cell reactor

Flow cell reactor

Biofilm Reactor

29

Source: www.erc.montana.edu

Source: BioSurface Technologies Corp.

30

Tubular Reactor

Robbin’s

Devices

Biofilm Reactor

31

Radial flow reactor Rotating disk reactor

Biofilm Reactor

32

Sourc

e: e

rc.m

onta

na.e

du

33

Biofilm Reactor

Drip-flow plate reactorStirred disk reactor

34

Biofilm Reactor

Fixed film reactorPropeller reactor