Biofouling in water systems - Cases, causes and countermeasures

Abstract

Biofouling is referred to as the unwanted deposition and growth of biofilms. This phenomenon can occur in an extremely wide range of situations, from the colonisation of medical devices to the production of ultra-pure, drinking and process water and the fouling of ship hulls, pipelines and reservoirs. Although biofouling occurs in such different areas, it has a common cause, which is the biofilm. Biofilms are the most successful form of life on Earth and tolerate high amounts of biocides. For a sustainable anti-fouling strategy, an integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.

Full text

Abstract Biofouling is referred to as the unwanted de-
position and growth of biofilms. This phenomenon can
occur in an extremely wide range of situations, from the
colonisation of medical devices to the production of ul-
tra-pure, drinking and process water and the fouling of
ship hulls, pipelines and reservoirs. Although biofouling
occurs in such different areas, it has a common cause,
which is the biofilm. Biofilms are the most successful
form of life on Earth and tolerate high amounts of bio-
cides. For a sustainable anti-fouling strategy, an integrat-
ed approach is suggested which includes the analysis of
the fouling situation, a selection of suitable components
from the anti-fouling menu and an effective and repre-
sentative monitoring of biofilm development.
Introduction
Biofilm is an expression which applies to microbial life
in aggregates. Biofilms can occur at solid–liquid, sol-
id–air and liquid–air interfaces. Most micro-organisms
can form biofilms and more than 99% of all micro-
organisms on Earth are living in such aggregates
(Costerton et al. 1987). A feature they all have in com-
mon is that the organisms are embedded in a matrix of
microbial origin, consisting of extracellular polymeric
substances (EPS). The EPS comprise mainly polysac-
charides and proteins, which form hydrogel matrices
(Wingender et al. 1999). Fossilised biofilms are the first
records of life on Earth, dating back 3.5 billion years
(Schopf et al. 1983) and represent by far the most suc-
cessful form of life, colonising soils, sediments, mineral
and plant surfaces in Nature, including extreme environ-
ments, such as pore systems in glaciers, hot vents, elec-
trodes and even highly irradiated areas of nuclear power
plants (Satpathy 1999). Biofilms are involved in the bio-
geochemical pathways of carbon, nitrogen, hydrogen,
sulphur, phosphorus and most metals. Self-purification
processes in Nature are performed by biofilm organisms;
and the biotechnology in drinking and wastewater treat-
ment is based on biofilms.
Life embedded in the EPS matrix offers important ad-
vantages for biofilm organisms. They can maintain sta-
ble arrangements of synergistic microconsortia of differ-
ent species and, thus, orchestrate the degradation of
complex substrates (Wimpenny 2000). The matrix can
sequester nutrients from the environment and is, thus,
part of a general microbial strategy for survival under
oligotrophic conditions (Decho 1990, 2000). Some of the
ecological advantages are listed in Table 1.
However, from a human point of view, biofilms can
occur in the wrong place and at the wrong times. Then,
they can cause biofouling. In such cases, two aspects of
biofilms are of particular importance: (1) the mechanical
stability of the EPS matrix, because this is what has to be
overcome in the cleaning process, and (2) the increased
tolerance of biofilm organisms to biocides, which can be
two or three orders of magnitude higher than that of sus-
pended cells.
Biofouling
Biofouling is referred to as the undesired development of
microbial layers on surfaces. The term has been adapted
from heat exchanger technology, where fouling is de-
fined generally as the undesired deposition of material
on surfaces (Epstein 1981), including:
1. Scaling, mineral fouling: deposition of inorganic ma-
terial precipitating on a surface
2. Organic fouling: deposition of organic substances
(e.g. oil, proteins, humic substances)
H.-C. Flemming (
✉
)
University of Duisburg, Institute for Interfacial Biotechnology,
Geibelstrasse 41, 47057 Duisburg, Germany
e-mail: HansCurtFlemming@compuserve.com
Tel.: +49-208-40303400, Fax: +49-208-4030384
H.-C. Flemming
IWW Centre for Water Research, Moritzstrasse 26,
45476 Mülheim, Germany
Appl Microbiol Biotechnol (2002) 59:629–640
DOI 10.1007/s00253-002-1066-9
MINI-REVIEW
H.-C. Flemming
Biofouling in water systems – cases, causes and countermeasures
Received: 18 December 2001 / Revised: 4 June 2002 / Accepted: 7 June 2002 / Published online: 26 July 2002
© Springer-Verlag 2002

3. Particle fouling: deposition of silica, clay, humic sub-
stances and other particles
4. Biofouling: adhesion of micro-organisms to surfaces
and biofilm development
In the first three kinds of fouling, the increase of a foul-
ing layer arises from the transport and abiotic accumula-
tion of material from the water phase on the surface.
What is deposited on the surface originates quantitative-
ly from the water. In these cases, fouling can be con-
trolled by eliminating the foulants from the liquid phase.
However, the case of biofouling is different: micro-
organisms are pseudo-particles which can multiply.
Even if 99.9–99.99% of all bacteria are eliminated by
pretreatment, a few will enter the system to become pro-
tected, adhere to surfaces and multiply at the expense of
biodegradable substances. In most cases, biofouling is
caused by heterotrophic organisms; and thus, micro-
organisms convert dissolved organic material into bio-
mass locally. This is the same mechanism which sup-
ports biofilm technology – biofouling can be considered
as a biofilm reactor in the wrong place at the wrong
time. Substances suitable as nutrients, which would not
act as foulants per se, will support fouling indirectly. As
most anti-fouling measures target the micro-organisms,
the role of nutrients as a potential source of biomass is
overseen and biocides tend not to decrease the nutrient
level. In contrast, nutrients supplied by the oxidation of
recalcitrant organics can support rapid after-growth (Le-
Chevallier 1991). As it is virtually impossible to keep a
typical industrial system completely sterile, micro-
organisms on surfaces will always be present, waiting
for nutrients which, in the case of lithotrophic organ-
isms, can even be inorganic. Thus, all biotransformable
substances must be considered as potential biomass. It
should be emphasised that not only organic substances
can serve as nutrients. For example, lithotrophic and au-
totrophic organisms can use anaerobic Fe(II) oxidation
coupled to nitrate reduction, in which the organisms fix
carbon dioxide as a carbon source for biomass genera-
tion (Straub et al. 2001). Another example can be seen
in the hydrogen-based communities identified in the
deep subsurface (Chapelle et al. 2002; Stevens and
McKinley 1996). Although these examples do not origi-
nate from technical systems, similar processes may also
occur in such systems.
Usually, the different kinds of fouling occur together.
The extent of biofouling can be considerable. An exam-
ple is the development of dental plaque, i.e. mineral de-
positions on teeth which are favoured by biofilms. In al-
gal biofilms, precipitation of calcium carbonate is in-
creased, mainly due to the rise in pH resulting from pho-
tosynthesis (Callow et al. 1988). However, other mecha-
nisms may also play a role, such as the alteration of wa-
ter activity by EPS molecules. Generally, biofouling has
to be considered as a biofilm-based problem. In order to
understand the effects and dynamics of biofouling and to
design appropriate countermeasures, it is important to
understand the natural processes of biofilm formation
and development.
From a microbiological point of view, there is no typ-
ical fouling organism. If excessive biomass or a non-spe-
cific contamination of the water phase is the problem, it
will be the most abundant organism in a given site which
will be the main fouling organism. If metabolic products
such as low-chain fatty acids, hydrogen sulphide or inor-
ganic acids cause the problem, the organisms producing
these substances will cause the fouling. Again, fouling is
an operational expression which is defined by the de-
mands of a system.
Nearly all micro-organisms are capable of forming
biofilms, because this is a universal way of microbial
life. Practical observations revealed that particularly
slimy strains of environmental bacteria may prevail in
water system biofilms (Wingender and Flemming, per-
sonal observation). Usually, the composition of fouling
biofilms is dominated by the autochthonous flora, which
can differ profoundly with different fouling sites and
conditions, including responses to the effect of biocides.
Biofouling in the sense of the given definition can
occur in extremely diverse situations, ranging from
space stations (Koenig et al. 1997) to religious miracles
like that of Bolsena (Cullen et al. 1998), the latter being
attributed to the growth of Serratia marcescens on sac-
ramental bread and polenta. Communion cups have
been identified as potential infection risks, due to bio-
films on the chalices (Fiedler et al. 1998). Dental water-
630
Table 1 Ecological advantages of the biofilm mode of growth.
EPS Extracellular polymeric substances
Function Relevance
Adhesion to surfaces Primary biofilms and microcolonies
Prerequisite for further biofilm
development
Aggregation of cells, Immobilisation of cells
formation of flocs High cell density possible
and biofilms
EPS as a structural Mechanical stability
element of biofilms Development of microconsortia
Concentration gradients
Retention of extracellular enzymes
Polysaccharide–exoenzyme interactions
Prevention of loss of lysed cell
components
Convective mass transport through
channels
Pool of genes, easy horizontal gene
transfer
Matrix for exchange of signalling
molecules
Light transmission into biofilm depth
Protective barrier Tolerance against biocides, metals, toxins
Protection against phagocytosis
Protection of exoenzymes by
complexation
Protection against some predator species
Sorption properties Accumulation of nutrients
Water retention, protection against
desiccation
Accumulation of pollutants in sludge

631
lines can be seriously contaminated by pathogens (Bar-
beau et al. 1998) and also by microbial metabolites,
such as low-chain fatty acids or hydrogen sulphide in
the case of the anaerobic conditions which can occur in
such environments. In general, it is acknowledged now
that biofilms are a common cause of infections (Coster-
ton et al. 1999).
Biofilms growing on building walls can significantly
influence the heat uptake of buildings, leading to an in-
creased energy demand for air conditioning equipment in
hot countries (Garty 1990). In water systems, in general
(Melo and Bott 1997) and during the filtration of seawa-
ter, biofouling is frequently observed. It also represents a
serious problem in fish farms where the cage netting
fouls rapidly (Hodson et al. 2000). Little (1988) has giv-
Fig. 1a–c Biofouled reverse osmosis membrane. a Scanning elec-
tron micrograph of an irreversibly biofouled reverse osmosis
membrane. The biofilm has completely covered the spacer.
b Magnification of the same view. Bacteria are visible under a
blanket of extracellular polymeric substances (EPS). c Magnifica-
tion of the same view, looking inside the bacterial layer. The fibril-
lar structures represent artefacts formed by dehydrated EPS in
which the cells are embedded and protected against biocides ap-
plied to sanitise the system
en an excellent overview on marine biofouling. Micro-
organisms can contribute to calcareous deposits, adding
scaling to biofouling (Dexter and Lin 1991). Massive de-
position of manganese and iron minerals is frequently
due to microbial activity (Tuhela et al. 1997). Water
treatment plants may include granular filters with a large
surface area, which are predestined for biofilm growth.
Biofilms can grow on piping material, insulation, fit-
tings, elastic filling materials, etc. and develop signifi-
cant tolerance against disinfectants. A change in nutrient
concentration, shear forces, temperature or other factors
can cause either biomass production or sloughing of bio-
films, which leads to increasing contamination of the
water. In ion exchangers, biofilms clog the material and
lead to an increasing pressure drop and in membrane
processes, such as reverse osmosis, ultrafiltration and
others, biofouling represents a serious problem (Ridgway
and Flemming 1996; Vrouwenfelder et al. 1998). Fig-
ure 1 shows a scanning electron micrograph of the sur-
face of an irreversibly biofouled reverse osmosis mem-
brane, with a biofilm which has survived many cleaning
cycles.
Investigations into the beginning of the fouling pro-
cess revealed that the micro-organisms settle on the
membrane during the first hours of operation (Flemming
1994). This means that every operating plant possesses a
biofilm from its beginning. Only the extent of biofilm
growth decides whether biofouling occurs or not: only
when a certain threshold of interference is exceeded does
biofouling take place; and thus, it is an operationally de-
fined expression.
Heat exchanger systems
The efficiency of a heat exchanger can be seriously de-
creased by biofilms (Characklis 1990). Forming a gel
layer, they insulate the heat exchanger surface from the
liquid phase. Although the heat transfer resistance of
biofilms is similar to that of water, the gel allows only

632
diffusive heat transport. Thus, heat transfer by convec-
tive transport is inhibited. In addition, friction resistance
is increased, which increases energy consumption. This
aspect is considered to be an even more costly problem
in heat exchange (Melo and Bott 1997). Similar to
membrane systems, heat exchangers in aqueous systems
operating in the temperature range 5–60 °C always carry
biofilms which remain unnoticed, because their effects
stay below the threshold level of interference. Biofoul-
ing problems do not arise from micro-organisms which
have suddenly invaded the system, but are much more
likely to be caused by an increase in nutrient concentra-
tion or an absence of inhibiting factors. The additives
can represent nutrients. As an example, chromate has
been replaced by other corrosion inhibitors because of
its environmentally undesirable properties. A new or-
ganic inhibitor was introduced which had excellent
properties, including full biological degradability. The
system protected by this substance failed after 3 months.
Biodegradation of the inhibitor had started during oper-
ation and coated the exchanger surfaces with a thick mi-
crobial layer (Fig. 2). Thus, an environmentally reason-
able step has to be harmonised with all other effects in
the system.
If light has access, substantial algal growth can occur.
This is frequently the case in cooling towers. The instal-
lations can be overgrown so heavily that they physically
break down (Characklis 1990). In ocean thermal energy
conversion, biofouling is addressed as the most difficult
problem, seriously limiting the use of an otherwise
promising technology (Darby 1984).
Ship hulls
A classic case of surfaces prone to biofouling are ship
hulls. The speed of ships can be significantly reduced
even by thin biofilms. Prior to colonisation by higher or-
ganisms, such as mussels, a microbial biofilm develops.
D.C. White (personal communication) has assessed that
the United States Navy has to spend more than U.S.
$ 500 million for additional fuel, which is required to
overcome the additional friction resistance caused by
biofilms. A biofilm of only 100 µm thickness leads to a
5–15% increase in friction resistance (Characklis 1990).
The attempts at effective anti-fouling coatings go back to
the mediaeval ages when copper plating was introduced,
without long term effect. After a certain time, some or-
ganisms can tolerate the toxicity of copper, colonise it,
complex the ions and shield other organisms to which
copper would be poisonous. Successively colonising
species do not encounter the copper surface but the pri-
mary biofilm. The problem has been transiently solved
by modern anti-fouling paints containing tributyl tin
compounds (Fig. 3). Unfortunately, they are so poison-
ous and recalcitrant to biological degradation (Cooney
and Wuertz 1989) that many countries have resorted to a
partial ban. Thus, the problem remains unresolved.
Drinking-water reservoirs and distribution systems
The maintenance of the hygienic and aesthetic quality of
water during transport in the distribution systems still
challenges drinking-water technology (Percival et al.
Fig. 2 Heat exchanger of a pump, protected by a biodegradable
corrosion inhibitor, after 6 months of operation. The corrosion in-
hibitor is completely degraded and causes massive biofouling
Fig. 3 Structure of tributyl tin compounds which are used for anti-
fouling. Red Sn, green (X) may be F, Cl, Br, O-(
n
Bu)
3
Sn, or others

633
1998). The bottom of drinking-water reservoirs is coloni-
sed by biofilms (Uhlmann et al. 1998). Herb et al. (1995)
investigated cases where brown and grey spots unexpect-
edly developed on mineral coatings in drinking-water
reservoirs (Fig. 4a). Intact concrete coatings display a
highly smooth surface texture, which is provided by mi-
croscopically small calcium carbonate crystals (Fig. 4b).
Microbiological investigation of those spots, in which
the mechanical integrity of the concrete was strongly de-
creased, revealed an extensive microbial colonisation
(Fig. 4c) by pigmented organisms. The damage was
caused by a two-step process: the first step could be ex-
plained by abiotic hydrolysis processes, which led to lo-
cal pits and a decrease in pH values. In fresh concrete,
these range between 12 and 13 – conditions under which
no microbial growth was observed. Next, organic addi-
tives (ca. 0.5% w/w) were released from the matrix.
These provided nutrients for an autochthonous flora
which was subsequently dominated by pigmented bacte-
ria. Only the abundant development of those bacteria
made the spots visible, representing aesthetic damage
which would not have occurred if the biofilm had re-
mained colourless.
It is now acknowledged that biofilms can provide a
habitat for pathogenic micro-organisms. Klebsiellae,
Mycobacteria, Legionellae, Escherichia coli and coli-
form organisms have been found in biofilms (LeChevallier
1990). In the presence of corrosion products, the organ-
isms seem to be particularly protected. This is the con-
clusion of the study by Emde et al. (1992), which found
a much higher variety of species in corrosion-product de-
posits, called tubercles, compared with the free-water
phase, even after extended periods of chlorination. The
fate of viruses in biofilms is still in question. Reasoner
(1988) reports very occasional incidents of pathogens in
drinking-water biofilms. This is confirmed by a large
study on drinking-water distribution system biofilms be-
ing carried out currently in Germany. First results indi-
cate that some pathogens even seem to be eliminated by
the autochthonous biofilms (Flemming et al., in prepara-
tion). In distribution systems, due to the surface to vol-
ume ratio, more than 95% of the entire biomass is locat-
Fig. 4a–c Mineral coating of a
drinking-water reservoir.
a Dark spots on a white
mineral coating of a drinking-
water reservoir. b Intact surface
of the mineral coating, covered
by a tight layer of calcium
carbonate crystals. c Scanning
electron micrograph of a
damaged site: calcium carbon-
ate crystals are dissolved and
bacteria are enmeshed in fibrils
of dried EPS (Herb et al. 1995)

634
ed at the walls, with less than 5% in the water phase
(Flemming 1998). These biofilms contribute consider-
ably to the overall purification process, because they de-
grade diluted organic matter. There is no correlation be-
tween the cell concentration in the water and in the bio-
film, although most of the cells found in the water phase
originate from biofilms. However, ongoing large-scale
field research reveals that biofilms developing on certi-
fied materials seem not to represent a threat to drinking
water and in general do not harbour potentially patho-
genic organisms (Flemming et al., in preparation). The
situation is different when materials are involved which
support microbial growth. An example is shown in
Fig. 5a: massive biofilm formation on synthetic elasto-
mers in drinking-water pipelines. Figure 5b shows a
scanning electron micrograph of the same biofilm. The
size of the cells indicates very good growth conditions,
in contrast to the starving microcolonies which are usu-
ally found on materials which do not leach nutrients.
This biofilm harboured coliform bacteria which were de-
tected in downstream drinking-water samples.
In medicine, mycobacteria have long been known as
trumpet bacteria, occurring in the mouthpieces of musi-
cal instruments of tuberculosic performers and, less ar-
tistically, in telephone handles (Müller 1946).
Countermeasures
In biofouling cases, it is reasonable to follow a three step
protocol: (1) identification of the cause and localisation of
the problem, (2) sanitation (cleaning is even more impor-
tant than killing the micro-organisms) and (3) prevention.
This has been described in detail earlier (Flemming
and Schaule 1996). Usually, when a problem arises in a
process, the diagnosis of biofouling will be made when
other causes do not explain the phenomenon. In order to
design the most effective countermeasures, it is impor-
Fig. 5a, b Biofilm develop-
ment. a Massive biofilm
development on an elastomer
coating of a valve in a drink-
ing-water system. b Scanning
electron micrograph of a
section of the biofilm
(Lange et al. 2002)

635
tant to verify this diagnosis. This has to be performed by
sampling the surfaces, which requires a set of techniques
(Schaule et al. 2000) more sophisticated than those for
sampling the water phase, although the latter is also car-
ried out in most cases. The most common countermea-
sure against unwanted microbial growth is the use of
biocides (Flemming and Schaule 1996). This line of
thinking expands a medical paradigm to technical sys-
tems: colonisation by bacteria is considered as a kind of
disease which has to be cured by some means of disin-
fectant, antibiotic or other biocide. However, it is well
known that biofilm organisms display a much higher tol-
erance to biocidal agents than their freely suspended
counterparts (LeChevallier 1991). Various mechanisms
which may protect biofilm organisms are discussed by
McBain and Gilbert (2001). The most plausible explana-
tion is based on a diffusion limitation of the biocide by
the EPS matrix. However, recent measurements have re-
vealed that this cannot be the case. Small molecules ex-
perience practically no diffusion limitation in a biofilm
matrix. Only when they react with EPS components, as
is the case with oxidising biocides such as chlorine or
ozone, is consumption of the biocide and, thus, a con-
centration gradient observed. Tolerance against hydrogen
peroxide is frequently accompanied by an enhanced cat-
alase activity. In general, enhanced biocide tolerance
must be taken into account in anti-fouling applications
(Morton et al. 1998).
An integrated anti-fouling strategy
In this review, a more complex approach is developed,
highlighted against the background of an increasingly re-
strictive legislation towards biocides, although the relevant
literature cannot be reviewed here exhaustively. It is im-
portant not to rely only on one “wonder weapon”, but to
analyse all fouling factors and to develop an integrated ap-
proach, based on detailed knowledge of biofilm develop-
ment. The basic idea is to live with biofilms, an approach
which may well inspire creativity in new directions.
Biofouling is an operational definition, referring to
that amount of biofilm development which interferes
with technical or economic requirements. Research on
reverse membrane biofouling revealed that biofilms de-
velop within the first hours of operation, contributing un-
knowingly to the separation process (Flemming et al.
1998). Only after exceeding a certain loss of permeabili-
ty the level of interference is passed and biofouling initi-
ated. This can be transferred to other water systems –
they practically all carry biofilms, but not all suffer from
biofouling. Figure 6 shows schematically the develop-
ment of biofilms in a system. The threshold of interfer-
ence may be expressed as a critical hydraulic resistance
(as is the case in membrane systems), as a critical heat
transfer resistance (as in heat exchanger systems), or in
terms of other parameters. They all have in common that
they are operationally defined and apply specifically to a
given system.
What are the options for keeping biofilm development
in a system below the individual level of interference?
Basically, the extent of biofilm growth is ruled grossly
by the availability of nutrients and the shear forces.
Thus, nutrients must be considered as potential biomass.
This is an important issue, as biocidal approaches usual-
ly do not take this aspect into account and do not limit
nutrients. Indeed, some biocides increase the nutrient
content by oxidising recalcitrant organics, making them
more bioavailable (LeChevallier 1991). An overview of
the situation and possible modules available for the de-
sign of specific and effective countermeasures is given in
Fig. 7.
Surface design and primary adhesion
Primary adhesion of the micro-organisms is the first step
to biofilm formation. This has been extensively investi-
gated (see Fletcher 1996), indicating that no system could
be established to predict the results of the complex inter-
actions between surfaces, micro-organisms and the sur-
rounding medium. Marshall and Blainey (1991) already
considered the primary adhesion of micro-organisms as
being primarily driven by physico-chemical processes.
This was confirmed by Flemming (1994) who demon-
strated similar adhesion rates for dead and living cells of
Pseudomonas diminuta onto polysulfone surfaces.
Clearly, rough surfaces are more prone to microbial
colonisation than smooth surfaces. This has been demon-
strated in the case of stainless steel surfaces (Faille et al.
2000). However, even on the smoothest surface, bacteria
can attach. This has been observed in unsuccessful ap-
proaches to prevent biofouling in heat exchangers by
electropolishing. In order to understand what happens
when a bacterial cell comes into contact with a surface, it
is helpful to take the entire situation into account. As
shown in Fig. 8 with the example of a Gram-negative or-
ganism, cells are surrounded by EPS (Wingender et al.
1999), which may include lipopolysaccharides (LPS), al-
Fig. 6 Schematic depiction of biofilm development. The dotted
line indicates an arbitrary threshold of interference

636
though the LPS are anchored in the outer membrane. The
primary contact between a solid surface and a micro-
organism is mediated by the EPS, with their particular
properties. The cell wall is involved in the adhesion pro-
cess only in the later stages, if at all. Microbial adhesion
can be considered mainly as an abiotic process (Marshall
and Blainey 1991), which can also occur when the cells
are dead (Flemming 1994).
Also, within seconds, surfaces immersed in water be-
come covered with a so-called conditioning film consist-
ing of macromolecules, such as humic substances, poly-
saccharides and proteins, which are present in trace
amounts in water, as was recognised many years ago
(Loeb and Neihof 1975) but not taken into account. The
cells do not need to be viable for adhesion – the EPS al-
ready present are sufficient for adhesion (Flemming and
Schaule 1988)
Many approaches have been pursued in order to pre-
vent microbial adhesion. Until now, only three of them
have been successful:
1. Tributyl tin anti-fouling compounds (see Fig. 3).
However, these are so toxic to marine organisms that
they have been widely banned from use.
Fig. 7 Elements of an
integrated anti-fouling strategy
Fig. 8 Situation of a
Gram-negative bacterium
encountering a non-biological
surface in water

cessive biofilm development, although it does not pre-
vent it. Under high shear stress, there is a selection for
organisms which produce mechanically stable biofilms.
Limiting the access of micro-organisms will also be
helpful. However, it must be taken into account that cells
are particles which can multiply. The most important
fouling factors are nutrients.
Cleaning is an important issue in biofilm manage-
ment. For cleaning, two factors which describe the me-
chanical stability of biofilms have to be overcome: cohe-
sion of the biofilm and adhesion to surfaces. Koerstgens
et al. (2001) developed a film rheometer which allowed
for the quantification of biofilm stability with the appar-
ent elasticity module, ε, as a relevant parameter. This re-
search revealed that the EPS matrix is kept together by
weak physico-chemical interactions, which result in a
fluctuating network of adhesion points. In compression
experiments, it was shown that, until at yield point (σ),
biofilms behave as gels with constant partner groups re-
sponsible for the adhesion. After exceeding σ, the gel
breaks down, the partners of the adhesion points change
and the biofilm behaves as a highly viscous fluid. This is
why biofilms are slippery. In a model system with P. ae-
ruginosa, it was shown that Ca
2+
ions increase the stabil-
ity of the network by bridging alginate molecules, which
are the main component of P. aeruginosa EPS. Mg
2+
did
not show such an effect, but Fe
2+
, Fe
3+
and Cu
2+
did.
Most commercial cleaners and biodispersants, however,
proved ineffective in this testing system. An effective
weakening of the EPS matrix can be achieved by en-
zymes (Johansen et al. 1997). However, this is not a rap-
id effect and, in practice, it has proven ineffective in
many cases (Klahre et al. 1998). This is not surprising,
as EPS, like other structural biopolymers, are not readily
biodegradable. Also, the continuous use of enzymes will
select for organisms producing EPS which are not sus-
ceptible to these enzymes.
An important aspect in cleaning is the use of surfaces
to which biofilms do not attach strongly. Such materials
have been developed and tested for anti-fouling on ship
hulls and fishing nets, with silicones as a promising class
of compound (Estarlich et al. 2000; Hodson et al. 2000;
Holm et al. 2000; Terlezzi et al. 2000).
Electric fields have been used both to prevent micro-
bial adhesion and to inhibit biofilm growth (Matsunaga
et al. 1998; Kerr et al. 1999). Practical observation, how-
ever, has shown that all kinds of electrodes immersed in
water can be colonised and fouled by biofilms.
A common measure which can be considered as bio-
film management is an increase in temperature. This is,
of course, not possible in all cases, but it is successfully
applied in hot water (70–80 °C) circuits in ultra-pure wa-
ter systems. Another parameter which can be shifted in
order to control microbial growth is the pH value. How-
ever, this may lead to other problems. An example is the
paper industry, which has abandoned production at low
pH, due to technical and environmental reasons. Neutral
conditions have considerably contributed to increasing
biofouling (Klahre et al. 1996).
637
2. Natural anti-fouling compounds. Such compounds have
been isolated mainly from marine plants which are not
colonised by bacteria (Terlezzi 2000). Steinberg et al.
(1997) isolated signalling molecules from an Australian
seaweed which displays anti-colonisation activity
(Fig. 9). These molecules have been identified to repel
bacteria from the surface of the plant and are patented
for use as anti-foulants. More marine anti-fouling prod-
ucts have been investigated by Armstrong et al. (2000).
The problem with these compounds is that most of
them are scarcely available, they are difficult to apply to
a surface on a consistent basis and they select for organ-
isms which can overcome the effect. Apart from that,
they will have to undergo evaluation under the Europe-
an Union’s biocide guideline procedure, which has an
assessment cost of about 5 million Euro per substance.
3. Surfaces with lotus effect (Neinhuis and Barthlott
1997). This effect relates to the “purity of the sacred lo-
tus” (which is also shared by cabbage leaves), based on
the particular structure of the wax layers on the leaf
surface. A highly hydrophobic pattern of needles pre-
vents water from moistening the surface, due to the
physicochemical interactions of three phases: solid, liq-
uid and gaseous. In nature, this effect is not achieved
with immersed surfaces. Also, as soon as surface-ac-
tive substances cover the hydrophobic pattern, surface
tension decreases and water is no longer repelled.
Thus, the lotus effect only offers an advantage on sol-
id–air interfaces and only when no surfactants are used.
As a consequence, we will have to live with the fact that
most surfaces can and will be colonised by micro-organ-
isms which will cause fouling if given the right condi-
tions.
Biofilm management
In such a situation, it is reasonable to assess the possibil-
ities to keep biofilm development under control. This
can be described as biofilm management; and it focuses
on the limitation of factors which support biofilm growth
above the level of interference. A thorough fouling-fac-
tor analysis is necessary. Initially, this has to include an
assessment of the nutrient situation. It has been ex-
plained earlier in this review that nutrients must be con-
sidered as potential biomass. High shear forces limit ex-
Fig. 9 Structure of furanones from the red alga Delisea pulchra
and the bacterial signal molecule N-butanouyl-
L-homoserine lac-
tone

Biofilm engineering
In cases where biofilm development cannot be sufficient-
ly prevented, other means of living with biofilms have to
be developed. This approach can be described as biofilm
engineering, which accepts a given biofilm for some rea-
son and is characterised by efforts to mitigate its unwant-
ed effects. For example, in reverse osmosis membrane
biofouling, it was shown that some cleaning formulations
did not remove the fouling layer, but increased perme-
ability ten-fold, allowing for a much thicker biofilm to be
tolerated than before treatment (McDonogh et al. 1994).
Creative approaches can also be applied in other
fields, e.g. if one succeeds in minimising the frictional
resistance of biofilms in pipelines. Sar and Rosenberg
(1987) report EPS from bacteria on fish skin which act
as drag reducers. Such polymers may be used as addi-
tives in closed systems when cleaning does not suffi-
ciently reduce the drag-increasing effects of biofilms.
Biofilm monitoring
It is of utmost importance to monitor biofilm develop-
ment, in order to optimise the time (frequency, duration)
and extent of countermeasures. This is not possible by
sampling the water phase. Such samples give no infor-
mation about the site, the extent and the composition of a
biofilm. Although biofilms contaminate the water phase,
they do so not on a constant basis but only very irregu-
larly. Biofilm cells may erode, but sloughing events may
also happen, leading to intermittently high cell numbers
in water. Thus, biofilm monitoring has to be performed
on representative surfaces.
Conventional methods rely on sampling defined sur-
face areas or on exposure of test surfaces (coupons) with
subsequent analysis in the laboratory. A classic example
is the so-called “Robbins device” (Ruseska et al. 1982),
which consists of plugs inserted flush with pipe walls,
thereby experiencing the same shear stress as the wall it-
self. After given periods of time, they are removed and
analysed in the laboratory for all biofilm-relevant param-
eters. The disadvantage of such systems is the time-lag
between analysis and result. Jacobs et al. (1996) de-
scribed a simple spectrophotometric monitoring method,
using a nucleotide-specific fluorescent stain (4′,6-diami-
dino-2-phenylindole) and automated measurement.
Other methods which report biofilm growth on-line,
non-destructively and in real time have been invented.
They are all based on physical methods. One example is
a fibre optic device, which has an illuminated fibre inte-
grated in the test surface and measures the scattering of
light by material deposited on the tip (Flemming et al.
1998). The principle of the sensor is schematically de-
picted in Fig. 10a; and a typical response graph is shown
in Fig. 10b (Tamachkiarow and Flemming 2002). The
detection of autofluorescence by biomolecules (using
spectroscopy) may allow differentiation between biolog-
ical material and abiotic material in the deposit.
638
Another method uses two turbidity-measuring devic-
es, one of which is constantly cleaned. The difference
between the signals is proportional to the biomass devel-
oping on the non-cleaned window (Klahre et al. 1998).
Nivens et al. (1995) gave an excellent overview of con-
tinuous, non-destructive biofilm-monitoring techniques,
including Fourier transform infra-red spectroscopy, mi-
croscopic, electrochemical and piezoelectric techniques.
Conclusions
An integrated anti-fouling strategy will not aim to kill all
organisms in a system but to keep them below a thresh-
old of interference. The strategy has to be based on:
1. Analysis of the fouling situation
2. Selection of suitable components from the anti-foul-
ing menu
3. Effective and representative monitoring of biofilm de-
velopment
Any step towards a better understanding of biofilm
growth and properties will add to the menu and expand
the possibilities for a flexible, effective and environmen-
tally suitable response to biofouling.
Fig. 10a, b Fibre optic device (FOS). a Schematic depiction of a
FOS. The tip of the fibre is integrated into the water-contacting
surface and light is conducted by the sending fibre. Material de-
posited on the tip scatters the light, which is collected by the read-
ing fibre. b Typical graph of intensity of back-scattered light as
provided by the FOS (Tamachkiarow and Flemming 2002)

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