Encyclopedia of Environmental Microbiology

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Biofilms
Hans-Curt Flemming,
Biofilm Centre, University of Duisburg-Essen, Duisburg, Germany
Most microorganisms on earth live in aggregates such as films, flocs, mats, granules or
sludge –this form of life is referred to as ‘biofilms’, and it is involved in the
biogeochemical cycles of all major elements including metals. Encased in a highly
hydrophilic matrix of extracellular polymeric substances, biofilm organisms can develop
stable microconsortia and complex interactions, resulting in features of multicellular
organisms.
Introduction
It is only few decades since microorganisms, sitting at the
walls of microbiological liquid cultures, on rocks, sedim-
ents, in soil, on leaves, skin, teeth, implants or in wounds
turned from a nuisance which could not be investigated by
classical microbiological methods into a highly active field
of research in which biofilms were acknowledged as the
dominant form of life for microorganisms on earth. It be-
came obvious that microorganisms on earth generally do
not live as single cells and in pure cultures but do so in
aggregates of mixed species. Such aggregates can consist of
microcolonies as well as patchy or confluent films on sur-
faces, but also as thick mats, sludge or flocks in suspension.
By convention, all these phenomena are subsumed under
the (somehow vague) term ‘biofilm’ (Donlan, 2002). It was
just a shift of point of view which made it evident that this
form of life could be found everywhere. In fact, biofilms are
the first form of life recorded on earth, dating back 3.5
billion years (Schopf et al., 1983), and the most successful
one: biofilms are found even in extreme environments, such
as the walls of pores in glaciers, in hot vents, under pressure
of 1000 bar (100 MPa) at the bottom of the ocean, in ultra
pure water as well as highly salty solutions, and on elec-
trodes active through the entire range of thermodynamic
water stability. Biofilms occur as endolithic populations in
minerals, on the walls of disinfectant concentrate pipes or
even in highly radioactive environments such as nuclear
power plants. The surface of almost all living organisms is
colonized by biofilms which provide in many cases a pro-
tective and supportive flora, e.g. skin flora, whereas in other
cases they cause transient, acute, chronic and even fatal
diseases. Biofilms are substantially involved in the biogeo-
chemical cycles of carbon, oxygen, hydrogen, nitrogen,
sulphur, phosphorus and many metals. Enhancing mineral
weathering processes by microbial leaching, they mobilized
metal ions which were vital for further evolution. In bio-
films, photosynthetic organisms evolved from originally
anaerobic conditions on earth, providing oxygen as a
‘waste gas’ from photosynthesis to the atmosphere of this
planet and restricting the space for living of anaerobic or-
ganisms, which first dominated life on earth, to oxygen-
depleted areas. Predation among biofilm organisms is
thought to have led to endosymbionts and, eventually, to
the evolution of eukaryotic organisms. See also:Biogeo-
chemical Cycles
One of the reasons for the late acknowledgement of bio-
films is certainly the insufficient suitability of conventional
microbiological methods to investigate biofilms. The in-
troduction of fluorescence microscopy and confocal laser
scanning microscopy, microelectrodes, advanced chemical
analysis with particular respect to protein analysis, and,
most powerful, molecular biology, has allowed revealing
biofilms biology in much greater detail. As a consequence,
the literature in this field has virtually exploded with at least
a hundred thousand publications on biofilms currently.
The advance of knowledge is immense and fast, and this
brief chapter can only superficially cover it. From a life
science point of view, the most exciting aspect is that mi-
croorganisms today cannot be viewed as blind little indi-
viduals which compete as much as they can but as complex
communities with division of labour and many aspects of
multicellular life. This is certainly a new understanding of
microbiology with big consequences for biotechnology,
medicine and handling of microbial problems in technical
processes.
Development of Biofilms
The development of biofilms has been investigated for
many different organisms. The most used model is biofilm
formation by Pseudomonas aeruginosa, an organism which
colonizes the lungs of patients suffering from cystic fibrosis.
Although P. aeruginosa has evolved almost as a standard
biofilm organism, it must be kept in mind that it is not a
Advanced article
Article Contents
.
Introduction
.
Development of Biofilms
.
Extracellular Polymeric Substances
.
Intercellular Communication
.
Ecological Advantages of the Biofilm Mode of Life
.
Biofilms as Habitat for Pathogens
.
Biofouling
.
Biofilms in Biofiltration
.
Outlook and Perspectives
Online posting date: 30
th
April 2008
ELS subject area: Microbiology
How to cite:
Flemming, Hans-Curt (April 2008) Biofilms. In: Encyclopedia of Life
Sciences (ELS). John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0000342.pub2
ENCYCLOPEDIA OF LIFE SCIENCES &2008, John Wiley & Sons, Ltd. www.els.net
1

representative for all biofilm organisms. Different organ-
isms form different biofilms on different substrata under
different conditions, therefore results obtained with P. ae-
ruginosa represent examples of how biofilms evolve but
have taken with great care when used for understanding
biofilms. Heterogeneity in space and time is a characteristic
of biofilms, and one of the most important features is the
fact that biofilms outside of laboratories usually do not
occur as pure culture but in mixed populations, forming
synergistic microconsortia. Biofilm research is in the same
dilemma as entire biology, which is the dichotomy between
holistic and reductionist approaches in which P. aeruginosa
represents the reductionist part.
Considering all these caveats, it is possible to take the
results from P. aeruginosa research as examples. Sauer et al.
(2002) in an elegant and comprehensive flow-cell study have
10 µm
(a)
10 µm
(b)
10 µm
(c)
10 µm
(d)
10 µm
(e)
10 µm
(f)
Figure 1 Transitional episodes in biofilm development by Pseudomonas aeruginosa strain PAO1 examined by transmitted light microscopy. Each panel
represents a distinct episode in biofilm development. (a) Reversible attachment. Initial event in biofilm development, bacteria are attached to substratum at cells
pole (arrow). (b) Irreversible attachment. Cells were cemented to the substratum and formed nascent cell clusters (arrow) with all cells in contact with the
substratum. (c) Maturation-1. Cell clusters matured (arrow) and were several cells thick, embedded in the EPS matrix. (d) Maturation-2. Cell clusters reached
maximum thickness, approximately 100 mm. (e and f) Dispersion. Cells evacuated interiorportions of cell clusters (arrow), formingvoid spaces (figure supplied by
D. Davies, Binghamton University). Reproduced with permission from Sauer et al. (2002).
Biofilms
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shown that biofilm development can be divided into five
stages which coincide with changes in global gene expression
patterns. They followed biofilm development of P. aerugi-
nosa from primary adhesion to glass surfaces until eventual
partial dispersion processes which were observed after 9–12
days of culture. These stages are depicted in
Figure 1a
–
f
.
The first stage is defined as ‘initial attachment’, within
minutes of first contact to the substratum. Planktonic bac-
teria contact the glass surface, some via the cell pole, and
become transiently fixed. The initial attachment is revers-
ible, since some cells were observed to detach during this
development stage (
Figure 1a
).
The second stage, ‘irreversible attachment’, was
observed to occur when the remaining cells commenced
their development into clusters, as visualized by multiple
cells in contact to each other and the substratum (
Figure 1b
).
During the second stage, mobility ceased in attached cells.
The cell clusters which were formed during this stage re-
mained attached through to the last stage of biofilm devel-
opment (9–12 days of incubation).
The third stage of development, ‘maturation 1’, was
observed when cell clusters became progressively layered
(
Figure 1c
). This was defined as the point in time at which cell
clusters are thicker than 10 mm.
The penultimate stage is reached when cell clusters attain
their maximum average thickness at approximately 100 mm
and referred to as ‘maturation 2’ (
Figure 1d
). During mat-
uration 2, cells within clusters were observed to be non-
motile and the majority of cells are segregated within cell
clusters, and clusters are virtually growing away from the
glass surface.
After 9–12 days, cell clusters were observed to undergo
alterations in their structure due to the dispersion of bac-
teria from their interior portions (
Figure 1e
and
f
, ‘disper-
sion’). These bacteria were motile and were observed to
swim away from the inner portions of the cell cluster
through openings in the cluster and entering the bulk liq-
uid.
Figure 1e
is an image of a cell cluster taken from the side
(grown on the transverse wall of the flow cell), showing the
opening through which bacteria were observed to evacuate
the cluster centre. Bacteria remaining in the void space were
motile. The ability of bacteria to swim freely within the void
spaces as observed by microscopy indicated the absence of
dense polymer or other gel-like material in the void space.
Figure 1f
shows the wall of a cell cluster after a complete
dispersion event, where the remaining bacteria in the wall
of the cell clusters were nonmotile.
The development of life cycle is completed when dis-
persed biofilm cells revert to the planktonic mode of
growth.
Parallel to these phases, differences in the proteome were
observed which could be attributed to the individual
phases. Proteins showed differential regulation during the
course of biofilm development which could be categorized
into four general classes:
Class I included proteins that encode factors for metabo-
lic processes, such as amino acid metabolism, carbon
catabolism and cofactor biosynthesis. The majority of
these proteins were found to be upregulated following ad-
hesion. However, dihydrolipoamide dehydrogenase was
downregulated, suggesting some degree of differential ex-
pression of metabolic proteins following attachment.
Class II included b-hydroxydecanoyl-acyl carrier protein
(ACP) dehydrogenase, which is involved in various lipid
biosynthesis reactions, including that of lipopolysaccha-
rides and acyl-homoserine lactones (AHLs) biosynthesis.
Class III contained membrane proteins primarily in-
volved in molecular transport, such as the bacterial ex-
tracellular solute-binding proteins, and poring E1 which
forms a small channel in the outer membrane. The
membrane proteins were found to be upregulated fol-
lowing adhesion.
Class IV included proteins involved in adaptation and
protection such as alkyl hydroxyperoxide reductase
subunit C and superoxide dismutase.
Generally, the authors’ conclusion is justified that the
analysis of two-dimensional protein patterns reveals mul-
tiple stages in the physiology of biofilm bacteria exist over
time of biofilm development (Sauer et al., 2002). The idea
that biofilm formation is a process of microbial develop-
ment and is not unlike that observed in cell-cycle controlled
swarmer-to-stalk cell transition in Caulobacter crescentus,
sporulation in Bacillus subtilis and fruiting-body formation
by Myxococcus xanthus (O’Toole et al., 2000).
Extracellular Polymeric Substances
A characteristic feature of biofilm organisms is that they
are kept together and attached to surfaces by means of their
extracellular polymeric substances (EPS). An example is
shown in
Figure 2
, which is a scanning electron micrograph
of Pseudomonas putida on a mineral surface. The sheet-like
material that surrounds the cells is EPS, dehydrated by
sample preparation for scanning electron microscope
(SEM) observation.
The EPS determine the immediate conditions of life of
biofilm cells living in this microenvironment by affecting
porosity, density, water content, charge, sorption proper-
ties, hydrophobicityand mechanical stability – all belonging
to the parameters on which the conditions of life in a biofilm
depend (Branda et al., 2005). Thissection represents a recent
synopsis of the actual state of understanding of the EPS role
(Flemming et al., 2007).
EPS are biopolymers of microbial origin in which biofilm
microorganisms are embedded. In fact, the biopolymers are
produced by archaea, bacteria and eukaryotic microbes.
Contrary to common belief, they are certainly more than
only polysaccharides. Additionally, they comprise a wide
variety of proteins, glycoproteins, glycolipids and in some
cases surprising amounts of extracellular deoxyribonucleic
acid (e-DNA). In environmental biofilms, polysaccharides
are frequently only a minor component. All EPS biopoly-
mers are highly hydrated and form a matrix which keeps the
Biofilms
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biofilm cellstogether and retainswater. This matrix interacts
with the environment, e.g. by attaching biofilms to surfaces
and by its sorption properties, which allows for sequestering
dissolved and particulate substances from the environment
providing nutrients for biofilm organisms. The EPS influ-
ence predat or–prey int eractions as demonstrated in a system
of a predatory ciliate and yeast cells. Grazing led to an in-
crease in biofilm mass and viability with EPS as preferred
food source.
Some EPS components deserve particular attention. Al-
ginate is a polyanionic polysaccharide, which is the best-
investigated component of mucoid P. aeruginosa biofilms.
However, several recent reports have shown that other po-
lysaccharides contribute to biofilms formed by nonmucoid
P. aeruginosa strains, which are believed to be the first to
colonize cystic fibrosis patients. A recent example: The ex-
pression of the psl operonwhichwasfoundtoberequiredto
maintain the biofilm structure at steps postattachment.
Overproduction of the Psl polysaccharide led to enhanced
cell surface and intercellular adhesion of P. aeruginosa,which
translated into significant changes in the architecture of the
biofilm. Nevertheless, other polysaccharides produced by
Pseudomonas such as levan may have a role in biofilm for-
mation. Environmental biofilms contain surprisingly low
contents of alginate. Even charged polysaccharides seem to
be relatively rare in nature as determined by uronic acid
analysis. In environmental biofilms, it is extremely difficult to
isolate and characterize specific polysaccharides in detail,
which is the experience of many researchers. The production
of EPS in natural biofilms is dynamic and can follow cyclic
patterns as demonstrated in marine stromatolites. Currently,
the only in situ approach to EPS glycoconjugates is achieved
by means of fluorescently labelled lectins of which the
specifity has to be verified in every case. See also:
Polysaccharides: Bacterial and Fungal
Curli as proteinaceous fibrils have gained more interest
beyond infection as curli-like fibrils have been found to
play an important role also in natural biofilms produced by
a variety of different microorganisms. An abundance of
amyloid adhesions in natural biofilms has been found
which may contribute considerably to their mechanical
properties. Strengthening of biofilm structure is crucial for
the stability of the ‘house’ and the continuation of synergis-
tic interactions based on spatial proximity of various bio-
film organisms.
Cellulose has been found to be a constituent EPS com-
ponent in amoebae, algae and bacteria. In agrobacteria,
cellulose is involved in attachment and it seems as if cel-
lulose plays an underestimated role in environmental EPS.
It is formed by a variety of organisms and influences biofilm
structure. Cellulose is important also in infectious proc-
esses when co-expressed with curli fimbriae in Escherichia
coli (Wang et al., 2007).
Biofilms are also an ideal place for exchanging genetic
material and maintaining a large and well accessible gene
pool. Horizontal gene transfer is facilitated as the cells
are maintained in proximity to each other, not fully im-
mobilized and can exchange genetic information. Signifi-
cantly higher rates of conjugation in bacterial biofilms
compared to planktonic populations have been reported.
However, recently nucleic acids have attracted more
attention. Although e-DNA has been reported as a com-
ponent of biofilms for quite some time, it is commonly
considered a remnant of lysed cells. However, e-DNA
occurs in sufficiently high enough quantities to raise some
doubt. In fact, the accumulation of DNA in the EPS
matrix of activated sludge and pure cultures of P. putida
was found. In P. aeruginosa biofilms, the e-DNA is likely
derived from whole genomic DNA. Surprisingly, e-DNA
was organized in distinct patterns in biofilms of this or-
ganism, forming grid-like structures and suggests a struc-
tural role for e-DNA. Further observations strongly
support and differentiate such considerations reporting
the formation of e-DNA as a spatial structure forming a
filamentous network in biofilms of an aquatic bacterium.
The e-DNA had similarities but also distinct differences
to genomic DNA. It seemed as if the cells could move
along these filaments using them as nanowires. e-DNA
was demonstrated as one of the major matrix compo-
nents in P. aeruginosa biofilms, functioning as an inter-
cellular connector and they supported the concept of the
stabilizing role of e-DNA for the biofilm matrix. In
P. aeruginosa, release of e-DNA is under the control of
quorum-sensing systems as well as iron regulation. In
Staphylococcus aureus biofilms, cidA-controlled cell lysis
plays a significant role during biofilm development and
releases genomic DNA, which served as an important
structural component for S. aureus biofilms. After all,
from an energetic point of view, DNA is an expensive
molecule and the question remains still to be resolved as
to what advantage makes the cells afford such an effort.
1 µm
Figure 2 Scanning electron micrograph of a biofilm of Pseudomonas putida
on a mineral surface. EPS (dehydrated for SEM sample preparation) are
surrounding the cells, keeping them together and on the surface.
Biofilms
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The EPS matrix is not only composed of a variety of
components but in addition, these are able to interact. One
example is the retention of extracellular proteins such as
lipase by alginate. Such mechanisms are crucial for pre-
venting the wash-out of enzymes, keeping them close to the
cells which produced them and allowing for effective deg-
radation of polymeric and particulate material. This leads
to the concept of an ‘activated matrix’. Activation is made
even more dynamic and versatile by the excretion of mem-
brane vesicles (MVs). These highly ordered nanostructures
act as ‘parcels’ containing enzymes and nucleic acids, sent
into the depth of the EPS matrix. Such vesicles, along with
phages and viruses, which are of similar size, can serve as
carriers for genetic material thereby enhancing gene ex-
change. Through their chemistry, the MVs may bind ex-
traneous components; their enzymes may help degrade
polymers, providing nutrients or inimical agents thereby
inactivating them. Furthermore, they seem to be part of
‘biological warfare’ within biofilms, occurring as predatory
vesicles, containing lytic enzymes. This biological warfare
is also long range as, in common with other matrix mate-
rial, they are shed from the biofilm and, in this respect,
vesicles are ‘missiles’ delivering, among others, virulence
factors and cell-to-cell signals (Schooling and Beveridge,
2006).
Composition, architecture and function of the EPS ma-
trix reveal a very complex, dynamic and biologically excit-
ing view. First of all, the matrix is a network providing
sufficient mechanical stability to maintain spatial arrange-
ment for microconsortia over a longer period of time. This
stability is provided by hydrophobic interactions, cross-
linking by multivalent cations and entanglements of the
biopolymers with e-DNA as a newly appreciated structural
component. A systematic approach to organize the infor-
mation on EPS components and functions is given in
Table 1
.
Many aspects of EPS remain to be addressed. An exam-
ple is their function in biocide resistance. Also, the man-
ifold ways in which the biofilm cells can modify their
matrix, which include production of various EPS by var-
ious organisms, the extracellular enzymatic EPS turnover
and modification and the resulting spatial and temporal
heterogeneity, have not been addressed. In this context,
species dynamics is crucial for understanding because
different species produce different EPS.
In conclusion, it seems as if ‘slime’ has been very much
underestimated and it may turn out that the EPS matrix is
considerably more than simply the glue for biofilms.
Rather, it is a highly sophisticated system, which gives
the biofilm mode of life particular and successful features.
Intercellular Communication
Individual bacteria can alter their behaviour through
chemical interactions between organisms in microbial
communities. This is generally called ‘quorum sensing’,
excellently reviewed by Keller and Surette (2006).
Frequently, these interactions are interpreted in terms of
communication to mediate coordinated, multicellular be-
haviour. The signalling molecules, also referred to as au-
toinducers, bind to receptors on, or in, the bacterial cell,
which leads to changes in gene expression at some thresh-
old concentration. Quorum-sensing acts are generally
thought to act as a mechanism for coordinated regulation
of behaviour at the level of populations of cells. Currently,
there are three well-defined classes of molecules that serve
as the paradigms for chemical signalling in bacteria: oligo-
peptides, AHLs and the LuxS/autoinducer-2 class.
Oligopeptide signalling is the predominant mechanism
used by Gram-positive bacteria. Typically, a pre-protein is
generated, processed into the active signalling peptide and
exported from the cell. The chemical structure of the signal
is precisely defined by the sequence of the amino acids,
which might be further modified, such as the formation of a
thiolactone ring in the S. aureus strains, which are classified
according to their oligopeptide signals.
AHLs are often involved in a cell population density-
dependent regulatory system of Gram-negative bacteria
and represent the second system. AHLs are produced by
the protein LuxI and sensed by the protein LuxR. The
specificity of this system is only moderate, in that a typical
LuxI protein will make one predominant AHL, or more
minor AHLs. The receivers also show some relaxed
specificity, as different LuxR proteins differ in the AHL
molecules they recognize, as well as in the number of
Table 1 Systematic approach to the role of EPS components
in biofilms
Effect and nature of EPS
component Role in biofilm
Constructive
Neutral polysaccharides Structural component
Amyloids
Sorptive
Charged or hydrophobic
polysaccharides
Ion exchange, sorption
Active
Extracellular enzymes Polymer degradation
Surface-active
Amphiphilic Interface interactions
Membrane vesicles Export from cell, sorption
Informative
Lectins Specificity, recognition
Nucleic acids Genetic information,
structure
Redox-active
Bacterial refractory
polymers
Electron donor or acceptor
Nutritive
Various polymers Source of C, N, P
Source: From Flemming et al. (2007). Reproduced by permission
of ASM.
Biofilms
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variants that they can detect. In some cases, the LuxR
protein will recognize a wider range of AHLs, such as the
Agrobacterium tumefaciens and Chromobacterium viola-
ceum proteins, which have been exploited to generate gen-
eral sensors for a wide range of AHL molecules.
The third cell–cell signalling system in bacteria is gen-
erally referred to as the LuxS/AI-2 pathway. This system is
found in Gram-negative and Gram-positive bacteria. The
signal that is produced by all strains is thought to be an
identical product (4,5-dihydroxy-2,3-pentanedione) that is
in chemical equilibrium with several furanones. It seems
that this pathway is not specific at all and so cannot convey
precise information.
In the simplest case of bacterial colonization, microcol-
onies are probably established from single cells, as in in-
fections in which bacteria establish on mucosal surfaces
(some pathogens). In such cases, the expression of extra-
cellular enzymes and/or virulence factors can be controlled
by the signalling molecules. A more complex example is the
interaction within groups of bacteria, such as M. xanthus.
In these organisms, individual cells aggregate under star-
vation conditions and form fruiting bodies. Within these
fruiting bodies, some cells develop into spores, others die.
In M. xanthus, this process is mediated by two signalling
pathways: the first (A-signalling) leads to the aggregation
of cells and the second (C-signalling) involves the forma-
tion of a mound and, ultimately, fruiting-body formation.
The C-signalling requires cell– cell contact, as the signalling
molecule is on the surface of the cell.
An overview on some structures of quorum-sensing sig-
nals and their derivatives is given in
Figure 3
.
Although there are many examples showing that bacte-
ria respond to chemical substances that are produced by
other organisms, there are no conclusive examples of com-
munication systems that have specifically evolved for in-
terspecies communication. Interaction between bacteria
OO
ON
OO
O
ON
OOH
HO
N
O
HO
HO
OO
N
OH
O
O
COH3C
H3C
CH3
OO
−
O
O
O
OH
HO
HO
HO HO
HO
OH
B
AHLs and derivatives
Butyryl-homoserine lactone (C4 AHL)
3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12 AHL)
3-oxo-dodecanoyl homoserine
3-(1-hydroxydecylidene)-5-
(2-hydroxyethyl)pyrrolidine-2,4-dione
3-OH-palmitic acid methylester
AI-2 structures for S.typhimurium (left)
and V.harveyi (ri
g
ht)
H2NI
I
Hb
Ha
S
AABu
PG
AK
G
SS
S
S
G
ANMK
Bu
Bu
A
AH
H
Hn
KCOOH
V
I
A
M
L
A
Bu
S
L
Lactococcus lactis
Bacillus subtilis CSF
Bacillus subtilis ComX ADDPITRQWGD
ERGMT
Staphylococcus aureus AIP
ISP
D
H2N
O
O
O
TSY
OO
S
O
O
O
O
N
HN
H
N
H
HN
HO F
I
M
NH
NH
S
H
HO
OH
OH
N
Gram-positive peptide signals
Figure 3 Structures of quorum-sensing signals and their derivatives. Letter designations for the Gram-positive peptide signals indicate amino acids. For the
Lactobacillus lactis signal nisin, the structural abbreviations were Bu, dehydroxybutyrine with a lanthionine bridge; Ha, dehydroalanine; Hb, dehydrobutirine
(from Horswill et al., 2007, with kind permission of Springer Science and Business Media; figure supplied by Ale xander R. Horswill, University of Iowa and Matthew
R Parsek, University of Washington).
Biofilms
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and their host, coordinated by signalling, has been dem-
onstrated with Vibrio fischeri and its eukaryotic host, the
bobtail squid (Stevens and Greenberg, 1997). Bacterial
chemotaxis towards compounds found in the squid’s light
organ mucus and colonization of the light organ require
motility. Quorum-sensing pathways control genes that are
involved in motility, biofilm formation and colonization, in
addition to the genes for light production. Two other quo-
rum-sensing systems, AinS and LuxS, in addition to the
canonical LuxI/LuxR system, contribute to light produc-
tion and colonization. On the host side, it is known that
colonization by the bacterium is necessary for normal de-
velopment of the light organ.
It seems that the nature of interactions through quorum-
sensing chemicals is not simply cooperative communica-
tion, but involves other interactions such as cues (molecules
or acts which alter behaviour and gene expression of other
organisms but was not evolved for that purpose) and
chemical manipulations. Furthermore, these chemical sig-
nals might also have a role in conflicts, both within and
between species. Some bacteria possess dual flagella sys-
tems, such as Vibrio parahaemolyticus,Rhodospirillum
centenum or Azospirillum brasilense. These bacteria are
able to express both a constitutive polar flagellum required
for swimming motility and a separate lateral flagella system
that is induced in viscous media or on surfaces and is es-
sential for swarming motility. The latter system is, conse-
quently, induced when the organisms form biofilms or
integrate themselves into existing biofilms. See also:
Quorum Sensing
Ecological Advantages of the Biofilm
Mode of Life
The biofilm mode of life provides a range of advantages to
the single-cell planktonic mode of life. One of the biggest
advantages is the fact that the cells can develop stable
interactions, resulting in synergistic microconsortia. An
example is the close association of ammonia- and nitrite-
oxidizing bacteria. The ammonia oxidizers produce nitrite,
an inhibitory end product which is comfortably used as
substrate by the nitrite oxidizers. This process occurs in the
environment and has been employed in nitrification steps in
wastewater treatment since long and with great success.
There are many other examples for orchestrated degrada-
tion of substrates by cascades of organisms. The EPS ma-
trix provides an extra advantage by retaining extracellular
enzymes. Furthermore, it acts as a sorbent for dissolved
and particulate substances and retains water (Flemming
and Leis, 2002). See also:Bacterial Ecology; Stream and
River Community Structure
Another great advantage of spatial proximity is the
facilitated gene exchange which has been long observed
(Hausner and Wuertz, 1999). This offers access to a large
gene pool and results in high biodiversity, providing genes
for degradation, resistance and other useful abilities. The
biofilm matrix is highly hydrated and very heterogeneous.
Figure 4
shows an artists view of various aspects of evolving
and mature biofilms as compiled from many recent findings
in biofilm research.
The picture reveals structural aspects which make life
in biofilms even more attractive. The porous architecture
allows for convectional flow through the depth of the bio-
film, while within the EPS matrix only diffusional transport
is possible. Organisms at the bottom of the biofilm, thus,
can get access to nutrients without competing with those at
the interface to the bulk water phase. Strong gradients can
occur in biofilms, e.g. by actively respiring aerobic het-
erotrophic organisms which consume oxygen faster than it
can diffuse through the matrix. This generates anaerobic
habitats just below highly active aerobic colonies in dis-
tances of less than 50 mm. Other gradients, such as pH
value, redox potential and ionic strength are known within
biofilms. The result is complex interactions and a func-
tionally structured system. The ecological relevance of this
heterogeneity has inspired Watnick and Kolter (2000) to
describe the biofilm as a ‘City of Microbes’.
Another feature of biofilm cells is the increased tolerance
to biocides, compared to planktonic cells. It must be taken
into consideration that biofilms exist since billions of years
and have survived all kinds of adverse conditions. There-
fore, many different mechanisms evolved for resistance,
and they are far from being fully understood (Lewis, 2001).
The fact is that resistance genes can be exchanged and that
biofilms have been observed even in disinfectant-concen-
trate pipes. The resistance of biofilms is particularly prob-
lematic in medicine where contaminations of implants,
catheters or bones result in long-term infections which in
many cases can only be overcome by radical measures such
as exchange of implants and removal of bone parts. In
drinking water systems, biofilms can harbour hygienically
relevant organisms which may even proliferate if nutrients
are provided. Even enhanced application of disinfectants
such as chlorine will not eradicate such biofilms.
The ecological advantages of the biofilm mode of life are
quite a few and can be summarized as follows:
.Formation of stable microconsortia
.Biodiversity: Gradients create different habitats
.Gene pool and facilitated genetic exchange
.Retention of extracellular enzymes in matrix
.Access to particulate biodegradable matter by
colonization
.Recycling of nutrients because lysed cells are retained in
the biofilm
.Protection against biocides and other stress
.High population density: Threshold concentration of
signalling molecules easily reached, facilitated intercel-
lular communication.
From an ecological point of view, it is interesting to con-
sider that the number of prokaryotic cells on earth is esti-
mated to be 4–6 10
30
cells, most of which (90–95%) occur
in sediments, and thus, live in biofilms. They are involvedin
the mobilization of minerals on a geological scale and pro-
vide metal ions for the biosphere. Mineral deposition is also
Biofilms
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performed by biofilms; especially impressive are the forma-
tions of calcium carbonate which are attributed mainly to
microbial activity. Biofilms not only occur on solid surfaces
but also at the interface between water and the atmosphere.
The uppermost microbial layer of surface waters is known as
the neuston. Here, hydrophobic substances accumulate and
provide nutrients for hydrophobic organisms, growing in
the neuston layer and forming biofilms. Owing to the pres-
ence of microbial products such as surfactants, they influ-
ence the surface tension and thus, the physico-chemical
conditions of the mass transfer of gases between atmosphere
and water. See also:Biocomplexity
Biofilms as Habitat for Pathogens
In a medical context, it is long known that biofilms play an
important role particularly in infections (Parsek and Singh,
2003). Such biofilms are very difficult to treat and frequently
lead to chronic infections which are fatal in many cases.
However, biofilms in the environment or in technical system
can harbour hygienically relevant organisms or pathogens.
Keevil (2002) gives a good overview on the role of Pseudo-
monas spp., Legionella spp., Klebsiella,Campylobacter,
Helicobacter and faecal indicator bacteria such as E. coli
and coliforms in drinking water systems. There, some of these
organisms can immigrate, persist and even proliferate, con-
taminating the water phase. Protozoa seem to be not only
carriers for Legionella spp. but for other organisms too, and
the survival of intraprotozoal organisms in biofilms is
extraordinary.
Biofouling
‘Biofouling’ is referred to as the unwanted deposition and
growth of biofilms. This phenomenon can occur in an ex-
tremely wide range of opportunities ranging from coloni-
zation of medical devices, production of ultrapure,
drinking and process water, fouling of ship hulls, pipelines
and reservoirs (Flemming, 2002). Although biofouling oc-
curs in such different areas, it has a common cause which is
the biofilm. Biofouling in the sense of the given definition
can occur in extremely diverse situations ranging from
space stations, to profane explanations for religious mir-
acles like that of Bolsena which is attributed to the growth
of red pigmented Serratia marcescens on sacramental
bread and polenta. In water systems in general and during
the filtration of seawater, biofouling is commonly ob-
served. It also represents a serious problem in fish farms
where the cage netting fouls rapidly. The submerged struc-
tural surfaces of offshore oil and gas production platforms
are covered by biofilms. Microorganisms can contribute to
calcareous deposits, adding scaling to biofouling. Massive
deposition of manganese and iron minerals is frequently
due to microbial activity. Boreholes and aquifers can be
clogged by excessive biofilm growth, leading to consider-
able technical problems. In some instances, unexpected
biofouling has led to problems such as the deterioration of
pH electrode response due to biofilm formation on the glass
membrane. Biofilms growing on the walls of houses can
influence the surface temperature of the building walls and,
thus, increase mechanical weathering process due to differ-
ential thermal expansion; in addition, such biofilms can
Figure 4 Artist’s view of biofilm architecture and processes. Reproduced by permission of P Dirckx, Center for Biofilm Engineering, MSU, Bozeman.
Biofilms
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increase the heat uptake, leading to an increase of energy
demand for air conditioning. Biofilms can grow on piping
material, insulations, fittings, elastic filling materials, etc.,
and develop significant tolerance against disinfectants.
A change in nutrient concentration, shear forces, temper-
ature or other factors can cause either mass production
or sloughing of biofilms which leads to an increasing
contamination of the water. In ion exchangers, biofilms
clog the material and lead to an increased pressure drop,
and in membrane processes such as reverse osmosis, ultra-
filtration and others, biofouling represents a serious
problem.
Current countermeasures are not effective in many cases.
One of the reasons is that detection is usually performed by
analysis of water samples although these do not reveal site
or extent of fouling biofilms. Instead, surface sampling
is required. The common countermeasure to biofouling is
based on a quasi-medical paradigm in which the solution is
seen in ‘disinfection’. Numerous biocides are applied, usu-
ally with only transient success but considerable environ-
mental burden. However, even if it is possible to kill all
organisms in a technical system, the dead biomass will re-
main and still cause problems, as killing is not equivalent to
cleaning. Instead, biocides represent a potential threat to
system components, e.g. in terms of accelerated corrosion.
If biofouling is acknowledged to be a biofilm problem, it
makes sense to consider biofilm processes. Biofilms grow at
the expense of nutrients, therefore, nutrients in a system
have to be considered as contributing to potential biomass.
Nutrient limitation is part of advanced antifouling strat-
egies, and the same is true for early warning systems as they
allow for timely countermeasures. An integrated antifoul-
ing strategy includes nutrient limitation, cleaning-friendly
design, low-adhesion surface materials and monitoring
systems with early warning capacity (Flemming, 2002).
Biofilms in Biofiltration
Biofiltration is a technology which is successfully employed
for drinking water purification (Gimbel et al., 2006) and
wastewater treatment (Wuertz et al., 2003). Biological
wastewater treatment is a simulation of the self-purifica-
tion processes occurring in rivers by biodegradation of or-
ganic carbon performed by microorganisms in sediments
and flocs. The floc model is adopted in activated sludge
plants where biomass is suspended but the organisms are
still organized in these flocks, displaying a defined structure
which allows the development of different zones in the
matrix. Although aerobic processes occur in the border
zones of the floc, anaerobic processes can happen in the
central zone where oxygen is depleted due to diffusion lim-
itation. The film model is adopted in biofilm reactors.
Many technological solutions have been employed to sup-
port sufficient biomass with trickling filters as one of the
oldest and most successful technologies. This is a textbook
material which is excellently presented by Bryers (2000).
Outlook and Perspectives
Biofilm research has provided many surprises for micro-
biologists. One of the most interesting aspects is the diver-
sity of a genetically homogenous population. This leads to
a variety of phenotypes within the biofilm which was un-
expected. As biofilms have now more widely been accepted
as the dominant form of microbial life, the mechanisms of
their formation and their functions will increasingly be in-
vestigated. The concept of biofilm research is quite exactly
opposed to the conventional microbiological approach in-
vestigating pure cultures under laboratory conditions. In
biofilms, microorganisms show different behaviour from
their isolated and suspended state. Thus, biofilm research
can be considered as a very important field of environ-
mental microbiology, revealing the actual properties, in-
teractions and activity of microorganisms in their natural
environment. Many methods from other scientific fields
have been creatively applied to biofilm research, leading to
a much better understanding of biofilm processes. How-
ever, biofilms are hard to investigate as this form of life is
characterized by huge variations in space and time and an
almost incontrollable set of variables which influence bio-
film development and properties. As indicated before,
many efforts to provide standard biofilms have been made,
but they always could cover only limited sets of conditions.
Therefore, biofilms still represent a major challenge for re-
search. As this challenge has been acknowledged now, we
will witness major changes in our understanding of the mi-
crobial world in the coming years.
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