FEMS Microbiology Ecology, 94, 2018, fiy044

doi: 10.1093/femsec/fiy044
Advance Access Publication Date: 14 March 2018
Minireview

MINIREVIEW

Biofilm growth and control in cooling water industrial

systems

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F. Di Pippo1,2, *, L. Di Gregorio1,3 , R. Congestri3 , V. Tandoi1 and S. Rossetti1
1
CNR-IRSA, National Research Council, Water Research Institute, Via Salaria Km 29.300, Monterotondo 00015,
Rome, Italy, 2 CNR-IAMC, National Research Council, Institute for Coastal Marine Environment, Località Sa
Mardini, Torregrande, 09170 Oristano, Italy and 3 University of Rome Tor Vergata, Department of Biology, Via
Cracovia 1, 00133 Rome, Italy
*Corresponding author: Via Salaria Km 29.300, Monterotondo 00015, Rome, Italy. Tel: +39-0690672771; E-mail: dipippo@irsa.cnr.it
One sentence summary: This review critically examines the main factors affecting biofilm growth in cooling water systems together with the
monitoring and control strategies currently used or potentially useful in full scale plants.
Editor: Marcus Horn

ABSTRACT
Matrix-embedded, surface-attached microbial communities, known as biofilms, profusely colonise industrial cooling water
systems, where the availability of nutrients and organic matter favours rapid microbial proliferation and their adhesion to
surfaces in the evaporative fill material, heat exchangers, water reservoir and cooling water sections and pipelines. The
extensive growth of biofilms can promote micro-biofouling and microbially induced corrosion (MIC) as well as pose health
problems associated with the presence of pathogens like Legionella pneumophila. This review examines critically biofilm
occurrence in cooling water systems and the main factors potentially affecting biofilm growth, biodiversity and structure. A
broad evaluation of the most relevant biofilm monitoring and control strategies currently used or potentially useful in
cooling water systems is also provided.

Keywords: biofilm; cooling water system; monitoring and control measures; biodiversity

INTRODUCTION and the water source (make up water), which is mainly seawa-
ter, freshwater and groundwater. Here, the presence of nutri-
Cooling water systems are widely used to dissipate the heat
ents and organic matter, concentrated by evaporation, together
generated by process operations in many industries including
with anti-scalants and corrosion inhibitors, normally used in
refineries, steel mills, food, petrochemical, chemical and power
the treatment of cooling waters, favour rapid microbial prolifer-
plants (Venugopalan, Rajagopal and Jenner 2012). Efficient heat
ation. The availability of surfaces in the evaporative fill material,
removal requires the optimal circulation of cooling water, which
heat exchangers, water reservoir and cooling water pipelines
is often compromised by microbial overgrowth (Rao 2012; Venu-
contributes to the extensive growth of biofilms (Liu et al. 2011;
gopalau et al. 2012). These cooling systems provide an excellent
Rao 2012).
environment for planktonic and benthonic growth, supported
Biofilms are complex surface-attached microbial commu-
by temperatures ranging between 25◦ C and 35◦ C, a pH close
nities whose cells are embedded in a self-produced matrix
to neutrality, sunlight exposure and continuous aeration (Liu
of extracellular polymeric substances (EPS) responsible for the
et al. 2009). Microbes enter cooling systems from the atmosphere
integrity of their three-dimensional structure (Flemming and
Wingender 2010). The biofilm matrix is arranged in a gel-like

Received: 30 November 2017; Accepted: 13 March 2018


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1
 2 FEMS Microbiology Ecology, 2018, Vol. 94, No. 5
structure (Mayer et al. 1999; Sutherland 2001), consisting of a net-
work of polymeric chains stabilised by intra- and intermolecu-
lar linkages, generating a high water retaining capability (Decho
2016). This polymeric material can have a heterogeneous com-
position and includes a wide range of biopolymers of differ-
ing chemical and physical properties (Flemming et al. 2016).
These are mainly polysaccharides, proteins, amyloids, extracel-
lular nucleic acids and amphiphilic compounds such as gly-
colipids and peptidolipids (Neu and Laurence 2016; Sutherland
2016). The polymeric matrix encloses and binds together the
microbes in the biofilm, thus providing considerable mechan-
ical stability (Mayer et al. 1999; Sutherland 2016), recognised
as one of the major advantages of the biofilm mode of life
(Flemming, Neu and Wozniak 2007; Flemming 2016). The sorp-
tion properties of the matrix facilitates the exchange of nutri-
ents, gases and other molecules with the environment (Flem-
ming and Wingender 2010). Biofilms are the prevailing mode
of microbial life in most natural and manmade environments
including industrial cooling water systems, where they can
promote micro-biofouling and thus interfere with operational
requirements (Fig. 1). Biofilms reduce conductive heat trans-
fer across surfaces and may clog hydraulic systems with con-
sequent energy losses and possible production cutbacks and
shutdowns (Rao et al. 2012; Venugopalau et al. 2012). Further-
more, microbes occupying niches in the deeper layers of biofilm
communities can promote microbially induced corrosion (MIC)
(Fig. 1) that increases the corrosion rate of the metal/alloy by
altering its surface electrochemical properties (Cloete and Flem-
ming 2012). Besides high economic loss, biofilm formation may
also pose serious health problems associated with the presence
there of pathogens including thermotolerant Legionella species
and free-living protozoa and may enable their widespread sur-
vival and propagation (Brouse, Brouse and Brouse 2017) over long
distances by the aerosol dispersal in air conditioner cooling sys-
tems.
Therefore, biofilm monitoring and control are essential to
ensure optimal cooling water system reliability and efficiency.
Practical experience has led to the development of strate-
gies principally based on the addition of chemical additives
to the water, including corrosion inhibitors, dispersants, scale
inhibitors and biocides (Cloete, Jacobs and Brozel 1998). How-
ever, antimicrobial compounds may not be fully effective in their
removal resulting from the protection provided by the biofilm
matrix (Stewart 2002; Hall-Stoodley, Costerton and Stoodley
2004; Fux et al. 2005). High concentrations of biocide, mainly
chlorine, are therefore used currently to control microbiofouling,
despite increased levels of environmental awareness and addi-
tional pressure from tighter legislation that suggest that alter-
native control strategies should be explored (Bognolo, John and
Evans 1992; Turnpenny et al. 2012).
Interest in this topic is high because of the serious prob-
lems that biofilms may cause in industrial settings worldwide.
Despite the considerable information now available on the diver-
sity, structure and ecology of biofilms in many natural habitats
(Stal 2010; Peter et al. 2011; Besemer et al. 2012; Battin et al. 2016;
Proia, Romanı́ and Sabater 2017) and mechanisms involved in
biofilm formation in medical fields (Donlan and Costerton 2002;
Costerton, Montanaro and Arciola 2005; Fux et al. 2005; Landini
et al. 2010; Archer et al. 2011), an integrated approach as to how
future investigations should be conducted in cooling systems
is still missing. This review aims to analyse critically the avail-
able literature on biofilm occurrence in such systems to pinpoint
crucial aspects that deserve further investigations, in the hope
that the biofouling process in industrial water systems will be
better understood. This review updates what is known already
about the factors affecting biofilm development and biodiversity,
the mechanisms involved in biofilm formation, highlighting the
complexity of genetic and environmental features involved.
The review also describes the relevant monitoring sys-
tems currently used in cooling water systems and provides an
overview of novel monitoring approaches with potential use in
industrial plants. Finally, the review discusses the most common
practices in use to control biofilm development and evaluates
several novel approaches now being applied.
OCCURRENCE OF BIOFILMS IN COOLING
WATER INDUSTRIAL SYSTEMS
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Biofilm diversity and structure in cooling water
industrial systems
Complex, multi-species biofilm communities colonise exten-
sively the available surfaces of most cooling water industrial sys-
tems. Although they may cause serious operational issues, little
information is available on their microbial community compo-
sition and structure (Hauer 2010; Balamurugan, Hiren Joshi and
Rao 2011; Wang et al. 2013; Hauer, Čapek and Böhmová 2016; Di
Gregorio et al. 2017; Pereira et al. 2017). Such scarcity of informa-
tion is attributable largely to the difficulties in collecting sam-
ples arising from the limited accessibility to the towers and to
authorisation constraints.
Analysis of the available literature reveals that Proteobac-
teria, particularly members of the Alpha and Beta subgroups,
Cyanobacteria and Acidobacteria dominate climax communities
of these biofilms (Wang et al. 2013; Di Gregorio et al. 2017)
(Table 1). Thus, populations, mainly affiliated to Rhodobacteri-
aceae and Sphingomonadaceae, dominated communities grown in
‘enclosed’ counter-flow cooling towers located in China (Wang
et al. 2013) and in Italy (Di Gregorio et al. 2017). On the other
hand, biofilms grown in two cross-flow cooling towers ‘open’
to the atmosphere and hence light, located in Eastern Europe,
were dominated by Cyanobacteria together with Alpha- and
Beta-Proteobacteria. The Cyanobacteria (mainly Calothrix, Leptolyn-
gbya, Microcoleus and Phormidium) coexisted with photosynthetic
eukaryotes as diatoms, principally Achanthes, Diadesmis and
Gomphonema species, and green algae, mainly Cladophora and
Stigeoclonium spp. (Di Gregorio et al. 2017). Similar communi-
ties were reported in 18 ‘open’ natural, draft cooling towers at
nine sites in different geographic areas of the Czech Republic
(Hauer 2010; Hauer, Čapek and Böhmová 2016), suggesting that
biofilm community composition was influenced markedly by
their exposure to light. Hauer, Capek and Bohmova (2016) also
showed that the phototrophic population compositions changed
following establishment of a light gradient from the periphery
to the centre of the studied ‘open’ cooling towers. Sulphate-
reducing bacteria, mainly Delta-Proteobacteria, were also detected
in biofilms colonising carbon steel pipelines of the water cool-
ing system of a nuclear test reactor (Balamurugan, Hiren Joshi
and Rao 2011), as well as in communities on galvanised steel
surfaces of an open, recirculating cooling tower of a hotel plant
in Istanbul, Turkey (Minnos et al. 2013). This information, albeit
scant, would seem to suggest that biofilm communities devel-
oped in the screened cooling systems are affected markedly by
existing environmental physical factors (i.e. irradiance), cooling
tower design and their construction material.
Several methodologies developed, or adapted for multi-
species biofilm studies, are now available (see the review
Azeredo et al. 2017), and these will provide data that contribute
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Figure 1. Examples of biofouling (A and B), https://www.drydenaqua.com/water-treatment; http://amsainc.com/cooling-tower-maintenance/) and MIC (C and D) in
cooling water industrial systems

Table 1. List of the main species found in biofilms occurring in different cooling water systems (Hauer 2010; Wang et al. 2013; Hauer, Čapek and
Böhmová 2016; Di Gregorio et al. 2017; Kadaifciler and Demirel 2017).

Bacteria Eukarya

Alphaproteobacteria Cyanobacteria Gammaproteobacteria Diatoms

Aliihoefleasp. Aphanothece spp. Acinetobactersp. Achnanhes sp.
Bosea sp. Brasilonema sp. Erwiniasp. Amphora sp.
Brevundimonassp. Calothrix sp. Pseudoxanthomonassp. Cymbella sp.
Devosiasp. Chroococcidiopsistermalis Rhizobactersp. Diadesmis sp.
Erythrobactersp. Chroococcus spp. Gomphonema sp.
Hyphomicrobiumsp. Cyanobium sp. Firmicutes Navicula sp.
Methylobacteriumsp. Cyanothece sp. Clostridium sp. Nitzschia sp.
Paracoccussp. Cyanosarcina sp. Exiguobacterium sp. Pinnularia sp.
Porphyrobactersp. Geitlerinema sp. Surirella sp.
Sphingomonassp. Gloeocapsa spp. Actinobacteria Green algae
Sphingopyxissp. Gloeocapsopsis sp. Microbacteriumsp. Cladophora sp.
Paracoccussp. Gloeothece palea Microcella sp. Chlorella sp.
Parvularculasp. Hassallia sp. Serinicoccussp. Cosmarium sp.
Rhodobactersp. Leptolyngbya spp. Gloeocystis sp.
Roseococcussp. Merismopedia sp. Planctomycetes Monodopsis sp.
Rubellimicrobiumsp. Microcoleus sp. Planctomycetes Pseudococcomyxa simplex
Nodularia sp. Scenedesmus sp.
Nostoccf.microscopicum Acidobacteria Stigeoclonium sp.
Betaproteobacteria Phormidium spp. Acidobacterium sp. Ulothrix variabilis
Cupriavidussp. Pleurocapsa sp. Vischeria sp.
Delftiasp. Pseudanabaenacf.minima Bacteroidetes Fungi
Hydrogenophagasp. Scytonema sp. Arenibactersp. Aspergillus versicolor
Massiliasp. Symploca muralis Flavobacteriumsp. Aspergillus niger
Rubrivivaxsp. Tolypothrix sp. Flexibactersp. Penicillium dipodomyicola.

to a deeper understanding of biofilm structure and composi-
tion. For example, improved microscope imaging technology,
biochemical methods and biomolecular tools now offer the pos-
sibility of resolving at nano-scale level an overall view of the
three-dimensional biofilm structure (Neu and Lawrence, 2015,
2016) and provide a deeper understanding of the physiology of
biofilm cells and their genotypic and phenotypic variation (Raes
and Bork 2008).
Only a few of these techniques have been applied to study
biofilms in cooling towers. High-throughput next-generation
DNA sequencing protocols have exposed the full breadth and
complexity of their community biodiversities in these systems,
 4 FEMS Microbiology Ecology, 2018, Vol. 94, No. 5
revealing the presence of an elevated number of taxa, where a
few species dominate and rare taxa form a long tail (Di Gregorio
et al. 2017). In addition, the three-dimensional reconstruction of
biofilms from two ‘open’ cooling towers, obtained with Confo-
cal Laser Scanning Microscopy (CLSM) after Catalyzed Reporter
Deposition Fluorescence in situ Hybridisation (CARD-FISH) (Figs
2, 3 and 4) show them to be highly complex and multi-stratified
communities, where bacterial micro-colonies, closely associ-
ated with other prokaryotic and/or eukaryotic photosynthetic
microbes, have a patchy distribution and appear to be embed-
ded in matrix material mainly composed of proteins and α- and
β-glucans (Di Gregorio et al. 2017).
The few available studies have also suggested that the com-
munities of biofilms growing in cooling systems are substan-
tially different from the corresponding planktonic communities,
sharing only a few taxa (Wang et al. 2013; Di Gregorio et al. 2017).
Thus, it appears that only certain members of the suspended
communities can adhere to the surfaces available in these cool-
ing systems. It seems likely that existing operating factors like
constant high temperatures and pH, together with the presence
of biocides, would determine which populations in the source
water can participate in the initial colonisation of such habitats.
Of these, pioneer biofilm microbes (e.g. Sphingopyxis, see Di Gre-
gorio et al. 2017, Acidovorax, see Wang et al. 2013) are possible
candidates, able to attach to the appropriate substrata and initi-
ate biofilm formation, thus modifying the surfaces and allowing
further colonisation by other populations. Subsequent biofilm
development to a mature/climax community, characterised by
high species diversity composition, then occurs together with
the characteristic structural arrangement of biofilms.
Biofilm formation mechanisms and their molecular
basis
Although biofilm formation has been studied extensively with
cultured mono-specific communities, little is known about the
molecular mechanisms involved in the regulation of mixed-
species biofilm adhesion and development in either natural or
man-made environments, because of the complexities of such
dynamic communities.
It is well known that biofilm formation is a complex process
that includes several stages. These are (i) attachment of individ-
ual cells to surfaces, (ii) reversible, followed by irreversible, bind-
ing of these to the surface (iii) development of microcolonies
and (iv) maturation of biofilm architecture (Donlan 2001). A large
body of literature emphasises that biofilm formation and disper-
sal are highly controlled processes regulated at the genetic level,
and by a range of environmental cues and signals (Burmølle et al.
2014; Wolska et al. 2016). These include changes in nutrient con-
centrations, oxygen levels and temperature, as well as organic
carbon source and predatory stresses (Bassler et al. 1993; Matz
and Kjielleberg 2005; McDougald et al. 2011; Mitri, Xavier and Fos-
ter 2011).
Genetic and molecular approaches have identified genes
and regulatory circuits important for initial cell–surface inter-
actions and subsequent biofilm maturation, and also the return
of biofilm microbes to a planktonic mode of growth (Davey and
O’Toole 2000; Wolska et al. 2016). Current knowledge points to
bis-(3 -5 )- cyclic diguanosine monophosphate (c-di-GMP) and
quorum sensing (QS) as the main regulators of bacterial biofilms
(Boyd and O’Toole 2012; Fazli et al. 2014; Liang 2015).
The crucial phase in the formation of bacterial biofilms is the
transition from a motile to a sessile cell lifestyle (Jenal, Reinders
and Lori 2017), mediated by the intracellular secondary mes-
senger c-di-GMP, mainly in response to environmental stresses
(Jenal, Reinders and Lori 2017).
Generally, high c-di-GMP levels induce the biosynthesis of
adhesins and matrix polysaccharides, and inhibit cell motil-
ity therefore initiating biofilm formation. With low c-di-GMP
levels, the synthesis of adhesins and exopolysaccharide mate-
rial is downregulated, and bacterial motility enhanced, lead-
ing to biofilm dispersal. These processes have been discussed
in more detail by Hengge (2009), Liang (2015) and Jenal, Rein-
ders and Lori (2017). Two classes of enzymes control the level
of c-di-GMP in bacteria. Diguanylate cyclases, which contain
the typical domain, CGDEF, produce this nucleotide from two
molecules of GTP, whereas c-di-GMP is broken down into 5 -
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phosphoguanylyl-(3 -5 ) guanosine (pGpG) by specific photodi-
esterases (PDEs), whose activities are associated with EAL or HD-
GYP specific domains (Liang 2015).
Given the role played by c-di-GMP in integrating environ-
mental inputs and controlling the motile–sessile transition in
both directions, it seems highly likely that c-di-GMP signalling
is important in these mixed-species biofilms colonising cooling
water systems. Several methods have been developed to quan-
tify c-di-GMP levels (Schleheck et al. 2009; Rybtke et al. 2012), and
these can be used to follow changes in biofilm c-di-GMP levels.
Studies with a range of single-species biofilms have estab-
lished the importance of intra- and interspecies cell-to-cell com-
munication systems, QS, in controlling biofilm development
(see the reviews Papenfort and Bassler 2016; Wolska et al. 2016;
Whiteley, Diggle and Greenberg 2017). QS relies on the pro-
duction, secretion, accumulation and population-wide detec-
tion of signal molecules, called autoinducers. When a sufficient
number of bacteria is present and autoinducer concentrations
reach a threshold level, the bacteria start to sense their criti-
cal mass and respond by repressing or activating target genes
(Burmølle et al. 2014; Papenfort and Bassler 2016; Wolska et al.
2016). Although these systems seem to play no clear role in
cell attachment and initial biofilm developmental stages, they
are essential for further biofilm development and also regulate
biofilm cell dispersal (Wolska et al. 2016). In general, the N-acyl
homoserine lactone (AHL)-based systems impact biofilms com-
posed of Gram-negative species including the model P. aerugi-
nosa biofilm system (de Kievit 2009; Papenfort and Bassler 2016),
while peptide-based QS systems regulate biofilm formation
in Staphylococcus aureus and other Gram-positive populations
(Parsek and Greenberg 2005; Suntharalingam and Cvitkovitch
2005; Novick and Gelsinger 2008).
Quorum sensing research has led to important advances in
the understanding of the genetics, genomics, biochemistry and
signal diversity of QS (Whiteley, Diggle and Greenberg 2017), but
under laboratory cultures and with growth conditions very dif-
ferent to those found in the natural environment. Despite this,
in vitro systems have provided fundamental knowledge of the
role of QS molecules in modulating the composition and func-
tion of natural multispecies biofilms, where their levels play a
similar role in formation of microbial complex natural commu-
nities seen in stromatolites and microbial mats. Here, AHL levels
vary with the diurnal cycle driven by pH changes as a reflection
of community metabolism and photosynthesis activity (Decho
et al. 2009). The role of AHL-mediated QS in the formation and
assembly of microbial granules has been demonstrated in mem-
brane bioreactors (Yeon et al. 2009) and sequencing batch reac-
tors (Tan et al. 2014; Tan et al. 2015). Despite these findings,
it is still unclear how such interactions can be achieved and
coordinated among phylogenetically very different populations
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Figure 2. CLSM 3D reconstructions of winter biofilm samples from a cooling tower located in Eastern Europe. Structural complexity, biofilm thickness and diversity are
evident after CARD-FISH, DAPI staining and photosynthetic pigment excitation. Multichannel mode acquisition of a biofilm fragment signals. Biofilm bacteria targeted
by EUB338mix probe (green signal, (A) appear enmeshed with clusters of green algae cells (red signal of chlorophyll a autofluorescence, (B) and total biofilm members
are visible after DAPI staining in blue (C) while spatial relationships between all biofilm components are clearly evidenced in the merged image (D).

in complex biofilm communities. Working in natural microbial
habitats is challenging and requires integration of a comprehen-
sive understanding of QS action derived from laboratory stud-
ies together with relevant ecological principles before the role
of intercellular communication in natural mixed multispecies
biofilms can be clarified.
Factors affecting the development of biofilm communities in
cooling water systems would not be dissimilar to those expe-
rienced by mixed species biofilms in other natural and biopro-
cess systems. It is therefore likely that QS signalling has an
equally important role in biofilm formation in cooling water
industrial systems, in mediating cooperative microbial commu-
nity behaviour.
In perspective, the analysis of QS molecules and c-di GMP
on biofilm communities grown on purpose artificial removable
substrata placed in cooling systems would enable their role to be
better understood and may allow the definition of appropriate
specific control actions.
Health related issues: occurrence of biofilm-associated
Legionella pneumophila in cooling water systems
Biofilm formation in cooling water systems poses a public health
risk associated with the presence there of pathogens. Biofilms
can favour the presence, survival and proliferation of thermo-
tolerant pathogenic bacteria, especially Legionella pneumophila,
held responsible for about 90% of worldwide cases of Legion-
naires’ disease (Wéry et al. 2008; Walser et al. 2014; Pereira et al.
2017). This is a severe pneumonia like illness with a case fatal-
ity rate of 10%–15% (Walser et al. 2014). As cooling towers are the
major sources (Walser et al. 2014; Pereira et al. 2017), surveillance
of Legionella in such systems is highly relevant to human health.
Public health authorities like the European Working Group for
Legionella Infections (EWGLI) and World Health Organization
(WHO) have published guidelines aimed at preventing and con-
trolling the risk of Legionella proliferation in cooling towers (Mou-
chotouri et al. 2010). However, the current approach to monitor
and control its presence remains haphazard, with inconsistent
approaches to disinfection and unsatisfactory endpoints to eval-
uate success of control countermeasures (Yu 2008). Monitoring
is usually based on colony plate counts of Legionella (Walser et al.
2014), and biocide dosages, mainly chlorine and/or glutaralde-
hyde, are applied subsequently to control it. The problems of
relying on plate counts to quantify this pathogen in cooling
towers has been discussed (Yu 2008). Consequently, alternative
approaches have been suggested (Collins et al. 2017; Gong et al.
2017). Quantitative real-time polymerase chain assays (qPCR) are
a widely accepted alternative to detect Legionella in environmen-
tal samples (Yang et al. 2010; Kao et al. 2013; Collins et al. 2017),
and its use in cooling water has been recommended as a valu-
able tool for timely facilitating control and public health protec-
tion (Collins et al. 2017). A combination of mip (Molecular Inver-
sion Probe), SBT (Sequence-Based Typing) and FAFLP (Fluores-
cence Amplified Fragment Length Polymorphism) methods have
also been proposed (Gong et al. 2017).
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Figure 3. 3D renderings of four different biofilm fragments: in (A) dominance of Bacteria over photosythetic microorganisms (chlorophyll a signal in red) is evidenced
here after hybridisation with EUB338mix probes and DAPI staining (green and blue signals, respectively); (B) large filamentous green algae in red (chlorophyll a autoflu-
orescence); (C) photosynthetic biofilm members are discriminated by excitation of chlorophyll a (grey signal) and phycobiliprotein (red) autofluorescence along with
total bacteria after hybridisation with EUB338mix probe. Diatoms, large desmids and filamentous green algae (grey) appeared scattered among cyanobacterial unicells
of different size (red) and bacteria (green signal). All community members were homogeneously distributed through biofilm thickness as shown in the profiles; in
(D) a wide and diffuse blue signal, after DAPI staining, is particularly evident indicating high amounts of compact, matrix material enmeshing bacteria (in red) and
phototrophs

Figure 4. 3D reconstructions after excitation of photosynthetic pigments, clorophyll a in red and phycobiliproteins in green, showing the variability in taxonomy, size
and morphology of biofilm photototrophs.

Long-term studies conducted on the bulk water of cooling
towers (Wery et al. 2008; Pereira et al. 2017) have demonstrated
that the structure and species richness of microbial communi-
ties associated with Legionella species can be affected in these
systems by environmental and/or operational conditions (Wery
et al. 2008; Pereira et al. 2017). The distinct temporal changes
of the microbial communities and the substantial variation in
the abundance of L. pneumophila highlight the need for contin-
uous monitoring of the cooling towers to prevent outbreaks of
Legionellosis. However, because of its ecology, reducing the risk
related to Legionella remains a challenge. Its ability to exist in
biofilms in association with protozoa complicate its monitoring
and control. Although the literature, reviewed by Declerck (2010),
has focused on the survival and replication of L. pneumophila
in cultured biofilms, little is known so far about the mecha-
nisms and factors affecting its life cycle in multispecies biofilms
colonising anthropogenic systems like cooling towers. Many

questions remain to be answered, because of the lack of reli-
able data from studies using pilot-scale or on-site experiments
and naturally occurring biofilm communities. The main chal-
lenge should be to elucidate whether Legionella can survive and
replicate outside a protozoan host in environmental biofilms.
It is already known that Legionella spp. have evolved a capabil-
ity to replicate intracellularly in 14 species of biofilm-associated
free-living amoebae (Molmeret et al. 2005; Declarck 2010). On
the other hand, evidence exists of adhesion of L. pneumophila
in pre-established biofilms as a secondary coloniser (Declerck
2010). Data from some studies have demonstrated that Legionella
is able to replicate and thrive in the biofilm matrix (Vervaeren
et al. 2006; Dechlerck et al. 2009), because it has the capacity to
obtain nutrients either directly from other living microbes, like
algae and heterotrophic bacteria, and/or indirectly from decay-
ing organic matter (Declerck 2010). Under laboratory conditions,
L. pneumophila showed it could grow on extracellular exudates
from the cyanobacterium Fisherella sp. (Tison et al. 1980) and
the green algae Scenedesmus spp. and Chlorella spp. (Hume and
Hann 1984). Necrotrophic growth of L. pneumophila has also been
shown (Temmerman et al. 2006). It is clearly important to under-
stand the mechanisms regulating interactions between L. pneu-
mophila and populations in pre-established biofilms. The discov-
ery of intracellular signalling molecules Legionella autoinducer-
1 (LAI-1) (Spirig et al. 2008) and AHKs (Abdel-Nour et al. 2013)
suggests some involvement of QS mechanisms in interactions
between L. pneumophila and biofilm primary colonising popula-
tions.
BIOFILM MONITORING IN COOLING WATER
SYSTEMS
The operational and environmental issues related to biofilm
development in these systems tend to be addressed separately
though it is desirable to adopt more innovative and holistic
approaches, taking account of all fouling factors with the pri-
mary aim of preventing and/or reducing cell adhesion and
biofilm formation in addition to minimising their impact on the
recipient water bodies, where cooling water is discharged via
blow-down (Venugopalau et al. 2012).
Traditional methods of monitoring cooling water
systems
Microbial monitoring is an integral part of any cooling water
treatment program, and it is essential to determine the effec-
tiveness of any antifouling and/or anti-MIC treatments, and to
establish the most cost effective level of treatment in terms of
energy, water and chemical usage. Conventionally, biofouling
and MIC are monitored and diagnosed indirectly, by determin-
ing the number of free-living bacteria in bulk water samples
by plate-count methods (Flemming 2011). However, attached
bacterial numbers can exceed planktonic numbers by three to
four logarithm units in water systems (Cloete, Jacobs and Brozel
1998). Moreover, free-living cell number determinations do not
provide reliable or robust information about the biofilm site or
the extent and the composition of biofilms in cooling system.
Although biofilms and planktonic communities from dispersed
biofilms may share populations (Di Gregorio et al. 2017), such
samples taken from the same systems are notably different
(Wang et al. 2013; Di Gregorio et al. 2017). Thus, biofilm surface
sampling is mandatory for proper monitoring of biofouling in
such systems (Cloete, Jacobs and Brozel 1998; Schaule, Griebe
Di Pippo et al. 7
and Flemming 2000; Flemming 2002). Sampling approaches
utilised in cooling water systems, consist either of biofilm scrap-
ing from defined, representative surface areas or test substrata
known as ‘coupons’, located in situ. Coupons have been used
to monitor MIC values (Videla and Herrera 2005) by evaluating
the surface corrosion rate and by enumerating the sessile bac-
teria. Viable cell counts (‘colony-forming units,’ cfu) are used to
quantify microbes on the substrata, but with the drawback of
estimating only a fraction of the biofilm community, as the per-
centage of cultivable organisms there is usually less than 1%
of the total number (Rompre´ et al. 2002). A modified Robbins
device (Ruseska et al. 1982) can be used. It tests surfaces exposed
to the flowing bulk fluid, for given periods and data analysed
in the laboratory. Its main disadvantage is the lag between
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the time-consuming investigations and availability of the
results.
Online methods to detect biofilms in cooling water
systems
From the discussion above, it appears clear that, to obtain
more accurate data faster, non-destructive monitoring provid-
ing online and real time information are necessary for imple-
menting preventative measures against micro-biofouling and
MIC. Many online methods have been described, some of which
have been applied successfully, mainly for detecting the kinet-
ics of material deposition and changes of biofilm layer thick-
ness. These have been reviewed (Flemming 2011) and include
methods based on measurements of thermal or mechani-
cal variations induced by fouling (Fillaudeau 2003; Pavanello
et al. 2011), or with DTM (differential turbidity measurement)
devices (Klahre and Flemming 2000), rotoscope device, detect-
ing changes in light absorption in response to deposit forma-
tion (Cloete and Maluleke 2005) and systems measuring the fric-
tion resistance airing from biofilm roughness. However, none
can discriminate between biotic and abiotic fouling, and in
most cases, they cannot detect thin biofilm layers (Pavanello
et al. 2011). It would be better to utilise early warning systems,
which allow biofilm detection at its initial stage of development,
before it becomes a complex, multi-stratified and EPS-enclosed
structure, much more difficult to remove. Thus, electrochemi-
cal sensors, including the ALVIM device (Pavanello et al. 2011)
and BIoGEORGE sensors (Bruijs et al. 2001) have been devel-
oped, which exploit natural cathodic depolarisation induced by
biofilm growth on alloys exposed to natural, aerated waters.
These can provide information on biofilm development, mea-
suring cathodic currents of samples polarised at a fixed poten-
tial, generating a signal when the biofilm exceeds a predeter-
mined threshold in its surface area (Pavanello et al. 2011). In
full-scale cooling water systems, these devices have given rapid
and accurate information on formation and activity of biofilms,
even at early stages of their surface colonisation, allowing an
optimal, antimicrobial dosing regimen to be implemented that
keeps biofilm development under control.
Besides electrochemical sensors, optical techniques show
promise for monitoring online biofilm development (Fischer,
Wahl and Friedrichs 2012; Fischer, Triggs and Krauss 2016).
The current state of monitoring techniques based on opti-
cal sensors has been reviewed (Fischer, Triggs and Krauss
2016), and the increasing number of biofilm monitoring sys-
tems based on the detection of changes in light properties
discussed (Fischer, Triggs and Krauss 2016). These exploit
interactions between light irradiation and biofilms including
 8 FEMS Microbiology Ecology, 2018, Vol. 94, No. 5
absorption/transmission, reflection, fluorescence and biolumi-
nescence. In particular, methods based on fluorescence are
especially because of their high sensitivity and rapid response
time (Fischer, Wahl and Friedrichs 2012).
Biofilm monitoring systems based on detection of fluoresc-
ing molecules have also been detailed. These include amino and
nucleic acids, NAD(P)H, ATP and pyridoxine (Wolf, Crespo and
Reis 2002; Fischer, Wahl and Friedrichs 2012). Online systems
able to discriminate in situ biological deposits from chemical
fouling by measuring simultaneously fluorescence, light refrac-
tion, transmission and scattering, in real time, have also been
described (Strathmann et al. 2013). Several challenges still need
to be overcome to improve such monitoring systems, for exam-
ple in miniaturising the sensors using microfabrication technol-
ogy or functionality of the sensor surfaces (Fischer, Triggs and
Krauss 2016).
Monitoring systems able to detect the compounds responsi-
ble for biofilm adhesion and development including AHLs and c-
di-GMP and/or the EPS biopolymers or cell appendages, respon-
sible for the initial contact with the colonisable surface would
be particularly attractive.
BIOFOULING CONTROL STRATEGIES
Current practice to limit biofilm growth in cooling
water systems
The most common countermeasure adopted to control biofilm
development in cooling water systems is to use oxidis-
ing (mainly chlorine, ozone, hydrogen peroxide) and/or non-
oxidising (principally amines, aldehydes, isothiazolone) bio-
cides (see Table 2) often combined with synthetic dispersants
or enzymes for enhancing biocide activity (Cloete, Jacobs and
Brozel 1998; Bott 2009). Their mode of action depends on which
biocide is used (Russell 2003; Bridier et al. 2011), but their tar-
get sites are essentially cell wall components, cytoplasmic mem-
branes, functional and structural proteins, RNA, DNA and other
cytoplasmic components (Cloete, Jacobs and Brozel 1998). Their
use is predicated on the view that disinfection, by killing micro-
bial cells, will solve the biofouling problem (Flemming 2011).
However, biofilm cells display a higher tolerance to disinfec-
tants than their planktonic counterparts. Their increased resis-
tance is multi-factorial (Heinzel 1998; Bridier et al. 2011; Pal et al.
2015) resulting from phenotypic adaptation, gene transfers and
mutations (Kelly, Vespermann and Bolton 2009; Hannan et al.
2010). Increased resistance is also intimately related to their
tree-dimensional structures (Bridier et al. 2011), with the key role
most likely played by the EPS matrix. This compact structure
may hamper biocide efficacy by limiting their penetration to the
extent where microbial cells are not exposed to them (Cloete,
Jacobs and Brozel 1998; Flemming and Ridgway 2009; Bridier et al.
2011; Kundukad et al. 2016). Optimising the breakdown of this
matrix is therefore essential to improve the disinfection process,
by disrupting the EPS matrix and exposing microbial cells to the
biocide (Cloete, Jacobs and Brozel 1998). Dispersants and emul-
sifiers are also commonly used in combination with biocides
in cooling water systems. Their possession of both hydrophilic
and hydrophobic functional groups enable them to penetrate
the complex the EPS matrix and to pre-condition the biofilm sur-
face (Cloete, Jacobs and Brozel 1998). Sulphonates, sulphates and
quaternary ammonium compounds represent the most com-
monly used surfactants in industrial systems (Cloete, Jacobs and
Brozel 1998).
‘Green’ bio-dispersants, mainly polyglucosides, charac-
terised by their high biodegradability, lack of potential bioaccu-
mulation, non-toxicity and non-carcinogenicity have also been
proposed for cooling water systems (Di Pippo et al. 2017). Sim-
ilarly, the use of enzyme-based detergents, mainly proteases
and polysaccharide hydrolysing enzymes (Meyer 2003), can also
enhance the cleaning process (Bridier et al. 2011), exploiting
their activities against key EPS matrix components. Commer-
cial enzyme formulations containing mixtures of enzymes
with different substrate specificities are available, although
it is important to elucidate the biofilm matrix composition
first so that the appropriate enzymes, proteases, cellulases,
polysaccharide depolymerases, DNAse, can be applied (Bridier
et al. 2011). Indeed, the biofilm matrix composition will vary
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depending on different abiotic conditions (i.e. pH, temperature,
pressure and shear forces) and on biofilm community diversity
(Neu 1994; Di Pippo et al. 2009). Because biofilms in cooling water
systems are complex microbial communities (Wang et al. 2013;
Di Gregorio et al. 2017), their EPS matrix composition will reflect
the populations present there (Flemming, Neu and Wozniak
2007), and this may change in response to environmental
factors, such as irradiance in phototrophic biofilms, where
EPS composition is closely related to photosynthesis diurnal
fluctuations (Staats et al. 2000; Otero &Vincenzini 2003; Stal and
Defarge 2005).
For many years, chlorine was the biocide used in almost all
industries, being relatively cheap, largely available and easy to
apply. However, its detrimental effect on the quality of the water
discharged into the recipient environment severely restricts its
use. Furthermore, chlorine can form carcinogenic compounds
like chloromethanes in contact with organic material in nat-
ural environments (Bott 2009). Ozone has also been used to
sterilise cooling water. However, it too has a number of limi-
tations. Ozone decomposes rapidly in water, its half-life time
in pure water is a few hours but in waters used for cooling
purpose, the half-life is reduced to minutes by its oxidation
of any organic matter present. Its rate of decomposition also
depends on the water pH, being higher at increased pH values.
Moreover, ozone reacts with natural organic substances to pro-
duce low molecular weight oxygenated byproducts that gener-
ally are more biodegradable than their precursors. These pro-
mote biological growth and further limit the disinfection efficacy
of ozone (Cloete, Jacobs and Brozel 1998).
Dechlorination before disposal can be an alternative to the
discharge of chlorinated water. Since techniques for dechlori-
nation constitute additional operating costs, efforts should be
directed to finding more efficient, economic and environmen-
tally friendly biocides (Bott 2009).
Future perspectives for biofilm control
The need to manage biofouling in industrial systems beyond
the application of one-shot solutions in order to prevent and/or
limit biofilm growth has been already emphasised (Flemming
and Ridgway 2009; Flemming 2011). One of the most effective
and convenient methods to minimise biofilm development is to
focus on the initial developmental step of cell adhesion by using
low-fouling surfaces, not readily prone to microbial colonisa-
tion. It is possible now to treat surfaces of cooling water sys-
tems with paints or multi-layer coatings at different key loca-
tions, e.g. intake conduits, pipes or condenser waterboxes. Many
innovative and developing technologies come from the marine
fields, where biofouling on ship hulls is a serious problem and
is usually controlled by hull coatings releasing at controlled
 Di Pippo et al. 9

Table 2. Mainly chemical antifouling treatment methods commonly used in cooling systems (JEA 2005; Ludensky 2005; Rajagopal et al. 2012).

Recommended
maximum
Biocide type Form concentration Advantages Disadvantages

Oxidising biocides
Cl2 Gas 0.5 mg/L Economical, effectiveness Toxic gas Safety concerns Costly
equipment
NaOCl Liquid N/A Economical, effectiveness Low activity, limited stability
Calcium hypochlorite Solid N/A Economical, effectiveness Adds calcium to the system
NaOCl + NaBr Liquid N/A Effective, easily retrofitted to Requires feeding two chemicals
most chlorination systems
Bromine Chloride Liquid N/A Most economical form of Feeder cost, safety and handling

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bromine concern
Stabilised bromine 0.2 mg/L Good stability, less toxic Higher cost, less effective on
algae and at high organic load
Ozone Gas N/A Effective against spore-formers Very reactive and volatile,
and fungi corrosive, rapidly lost, low
solubility, safety concerns
Hydrogen peroxide Liquid N/A Degrades to water and oxygen Slower acting, needs higher
residual
Non-oxidising biocides
Target
Glutaraldehyde Bacteria Algae Fungi N/A High efficacy in various Inhalation toxicity
applications
Thiocyanates Bacteria N/A Low cost Low solubility
Isothiazolone Bacteria Algae Fungi N/A Persistent broad spectrum Irritation, Skin sensitisation
Quaternary ammonium Bacteria Algae Mollusks N/A Broad spectrum, Fast acting Not effective vs. fungi, Foam
compounds possible

rates antimicrobial compounds that inhibit the growth of ben-
thic microbes (Ma et al. 2017). Organotin- and metal-based coat-
ings have been extremely successful in biofouling prevention
and are widely used in the antifouling paints of ships (How-
ell and Behrends 2010; Ten Haller-Tjabbes and Walmsley 2010).
However, their banning, because of their toxic effects on the
marine ecosystem, and the potential development of antibiotic
resistance among strains of the fouling bacteria, have necessi-
tated searches for alternative, sustainable and environmentally
safe antifouling approaches. Advances in nanotechnology and
polymer science meet the need for using ‘green’ technologies
to develop non-biocidal, non-fouling coatings for the maritime
industry. An example is the fabrication of amphiphilic surfaces
displaying both hydrophobic and hydrophilic domains to avoid
microbial attachment, foul release (FR) systems, consisting of
advanced composite silicon polymers and hydrogel nanolayers
that weaken foulant interfacial bond. These FR properties have
been combined with microencapsulated hydrolytic enzymes
(e.g. serin proteases) capable of preventing bioadhesion to effec-
tively replace traditional biocidal paints (Callow and Callow
2011; Ciriminna, Bright and Pagliaro 2015). In this context, Ma
et al. (2017) have synthesised novel coatings by incorporating
butenolide derived from the marine bacteria Streptomyces spp.
into biodegradable poly(ε-caprolactone)-based polyurethane.
Apart from seeking ‘green’ fouling resistant coatings, other
bio-inspired antifouling, long lasting solutions have sought to
mimic the surface topographies of certain marine fauna (shark,
pilot whale) and aquatic plants (lotus leaf) (Nir and Reches 2016).
Their unique surface roughness and wettability protect these
marine organisms from bio-adhesion, and these properties have
been reproduced using soft litography on polydimethylsiloxane,
and by polyelectrolyte layer-by-layer spray coating. The lotus
leaf superhydrophobic and self-cleaning properties have been
reproduced by thermal deposition of paraffin and fluorinated
waxes on a range of surfaces (e.g. copper, glass, and silicon)
that then exhibited durable resistance to biofilm formation and
development. Furthermore, Kreder et al. (2016) have designed
a new naturally derived antifouling solution, the ‘slippery liq-
uid infused porous surface’ (SLIPS), derived from Nepenthes car-
nivorous plant. It resists wetting by oil, water and even blood.
Moreover, SLIPS has proved effective in preventing the growth of
Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.
SLIPS also exhibits great durability under extreme environmen-
tal conditions including high temperatures, extreme pH values
and UV radiation.
The interest in mimicking some of the naturally evolved
antifouling strategies exhibited by aquatic organisms has also
led Sathe et al. (2017) to exploit reactive oxygen species, ROS,
used in some algae against biofouling. ROS production has been
achieved with fabrication of a ZnO photocatalytic nanocoating
on fishing nets. The authors compared biofilm communities
grown in mesocosms upon light activation, on ZnO nanorods
and antifouling (AF, copper) coated nets and control uncoated
nets, with high throughput DNA sequencing. Improved per-
formance was achieved with the nanostructured (ZnO) nets
over the AF coated net, with microbial abundances significantly
lower with the former. Furthermore, there was a preferential
removal of the pathogenic biofilm members with the AF coated
nets. The authors concluded that sunlight-responsive antifoul-
ing ZnO nanorods coating is more efficient at mitigating biofoul-
ing than commercial biocidal paints, and they emphasise the
scalability and low-cost potential of their approach as an exam-
ple of solar-assisted, environmentally friendly antifouling tech-
nologies (Sathe et al. 2017).
 10 FEMS Microbiology Ecology, 2018, Vol. 94, No. 5
Other innovative approaches have been developed, based
on fundamental understanding of the foulant adhesion strate-
gies leading to manufacture of less specific antifouling coat-
ings. Libraries of peptide anchors, including 3,4-dihydoxy-L-
phenyalanine (DOPA), a common constituent of adhesive pro-
teins in marine organisms, were designed and conjugated to a
range of polymers, especially saccharides, showing the capabil-
ity of adhering to different surfaces and thus conferring resis-
tance to non-specific interaction (Nir and Reches 2016). Further
development of this bio-molecular mimetic approach led to the
polymerisation of DOPA into sticky coatings to then be cast
as adhesive-activated layers on silicon surfaces. DOPA-based
tripeptides have also been developed that exhibit self-assembly
properties into antifouling films, thin transparent coatings of
3 nm in thickness, on range of surfaces, preventing protein
adsorption and thus biofilm formation (Nir and Reches 2016).
The understanding of the in vivo mechanisms involved in
biofilm initial adhesion and development led to the search for QS
inhibitors or quenchers as natural antifouling compounds (Jha
et al. 2013; Nir and Reches 2016). One approach was to design
structural analogues of the AHL QS autoinducers, on the basis
that these would exert the same biological function, thus abol-
ishing cell-to-cell communication. Indeed, a range of lactone
derivatives and furanones have now been explored as quorum
quenchers with some success, indicating it might be possible
with them to alter microbial biofilm morphology and function-
ing (Wu et al. 2004). Jha et al. (2013) have described a QS inhibitor
2-dodecanoyloxyethanesulfonate, a compound extracted from
the red alga Asparagopsis taxiformis.
Exploitation of natural substances isolated from a broad
range of biological sources, including terrestrial plants, algae,
corals and microbes shows promise in biofouling prevention and
mitigation, as these compounds exhibit a compatibility with bio-
logical systems and a higher specificity than heavy metals (Chen
and Qian 2017; Le Norcy et al. 2017; Ma et al. 2017; Dahms and
Dobretsov 2017; Wang et al. 2017). A wealth of reports exists
on their biological and antimicrobial activities in vitro. However,
progress in applying them as effective antifouling agents has yet
to be pursued, especially in terms of their cost effective mass
production, biosafety and antifouling action. Problems still to
be solved satisfactorily are the incorporation of these antifoul-
ing compounds into suitable polymers and their compatibility
and controlled release from new generation coatings (Ma et al.
2017). Furthermore, the potential use of natural product-based
antifouling coatings in industrial cooling water systems must be
tested on-site, or, at least, on multispecies biofilms grown in sys-
tems that mimic the real conditions, to verify their applicability
on treated surfaces, their antifouling effects and lifetime.
CONCLUSIONS
A thorough insight in the microbial community organisational
structure, with a major attention to microbial key players, is
necessary to encourage the development and the application
of sensitive and effective biofouling control measures. Unfor-
tunately, even though many aspects of the ecology of biofilms
in natural habitats and their formation in medical fields have
been disclosed, only scant information is available on those in
cooling water systems. A critical evaluation of the available lit-
erature here highlights the research questions urgently need-
ing answers and deserving further investigation. The scarce
evidences from long-term studies performed on multi-species
biofilms growing in cooling industrial systems and the lack of
data on biofilm formation processes in such systems have pre-
vented the elucidation of the key molecular and ecophysiolog-
ical details of their initiation and development. This situation
has clearly limited the development of reliable and robust early-
warning monitoring approaches. The need for better exploita-
tion of novel, sustainable and environmentally safe antifouling
control strategies as alternatives to toxic biocides, e.g. chlorine,
commonly used in cooling water systems has also been dis-
cussed.
ACKNOWLEDGEMENTS
The Authors wish to thank Prof. Robert Seviour for the criti-
cal review of the English version and Dr Elena Romano from
Downloaded from https://academic.oup.com/femsec/article/94/5/fiy044/4935158 by guest on 25 August 2022
the Centre of Advanced Microscopy ‘P. Albertano’ (CAM), Depart-
ment of Biology, University of Rome ‘Tor Vergata’, for her skillful
assistance in the use of the facility.
Conflict of interest. None declared.
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