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Chemical and physical methods for characterisation of biofilms

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Microchim Acta 158, 1–27 (2007)
DOI 10.1007/s00604-006-0688-5
Printed in the Netherlands
Review
Chemical and physical methods for characterisation of biofilms
Evelin Denkhaus1 , Stefan Meisen1 , Ursula Telgheder1;
, and Jost Wingender2
1
Department of Analytical Chemistry, University of Duisburg – Essen, Lotharstr. 1, D-47057 Duisburg, Germany
2
Biofilm Centre, University of Duisburg – Essen, Geibelstr. 41, D-47057 Duisburg, Germany
Received April 10, 2006; accepted August 5, 2006; published online December 11, 2006
# Springer-Verlag 2006
Abstract. Research on biofilms has developed into
interdisciplinary work, in which scientists from different
fields are involved. This review summarizes the state-
of-the-art including models due to different aspects of
biofilms. Techniques for characterization of surfaces
and interfaces, e.g. microscopic, spectroscopic and
microsensoric methods will be presented. In addition,
procedures that involve sampling of biofilms and sub-
sequent separation of the components for their analy-
sis are a part of this article. Analytical methods are
summarized for the investigation of extracellular poly-
meric substances (EPS), mainly polysaccharides be-
side proteins in the microbial host. Finally, procedures
for the analysis of minerals as components of biofilms
are presented.
Key words: Biofilm; spectroscopy; surface characterising tech-
niques; separation-molecular-hyphenated-techniques; microsensors.
Biofilms are populations of micro-organisms that ac-
cumulate at interfaces and are typically surrounded by
a matrix of extracellular polymeric substances (EPS).
Attachment of micro-organisms to surfaces and the
development of microbial biofilms at phase bound-
aries are frequently encountered in natural, technical
and medical environments [1]. Biofilms exist for more
than 3.8 billion years and today are ubiquitous on
Earth. Biofilms are a common mode of growth for

Author for correspondence. E-mail: uschi@lims.uni-duisburg.de
microorganisms as multicellular communities which
colonize all types of surfaces, where growth is possi-
ble. Biofilms represent a protected mode of existence
which allows cells to survive over a wide range of
different and sometimes extreme environmental con-
ditions, e.g. at temperatures ranging from 5 to
120  C, at pH values from 0 to 13 and at pressures
as high as 100 MPa. In nature, biofilms can be found
in soil, groundwater and surface water environments.
In terrestrial and aquatic ecosystems, they are impor-
tant components of food chains, and are involved in
self-purification processes in soil, water and sediments
and the biodegradation of organic compounds includ-
ing environmental pollutants [2–4]. These properties
offer opportunities for useful industrial and environ-
mental applications such as technical waste water puri-
fication and drinking-water treatment by biofiltering,
bioremediation of hazardous waste sites, and forma-
tion of biobarriers for the protection of soil and ground-
water [5]. On the other hand, biofilms can be a threat
to human health, e.g. by the growth of biofilms on
medical implants such as prostheses and heart valves,
leading to infections [1, 6, 7], and biofilms as sources
of pathogens causing contaminations in food proces-
sing and drinking-water distribution systems [8, 9].
Moreover, biofilms show an increased resistance to anti-
microbial agents such as antibiotics and biocides [10].
Biofilms can also have detrimental effects in engineered
systems due to biofouling (undesired development of
2 E. Denkhaus et al.
biofilms on surfaces) [11, 12] or biocorrosion (destruc-
tion of materials) [13]. For example, biofilms can in-
crease the resistance to heat transfer in cooling-water
systems, increase the frictional resistance in technical
water systems and on ship hulls, and they can cause
disturbances in paper manufacturing [14], oil recovery
and mining [9]. Therefore, the industries have yearly
losses in billion extents. As a consequence, industry
and governments initiated biofilm-related projects,
which have exponentially increased during the last
decade [http:==www.erc.montana.deu=default.htm].
The research of biofilms developed into interdis-
ciplinary work, in which scientist from different fields
such as microbiology, chemistry, physics, engineering
and computer-based modelling are involved. Inves-
tigations of biofilms are generally focused on struc-
tural, functional as well as ecological aspects; these
include biofilm development from the very beginning
(primary adhesion of a single microbial organism to
a substratum) to growth into a mature biofilm and
detachment processes, the biochemical and microbial
composition, biofilm architecture, gene expression and
physiological activity of biofilms depending on the
surrounding conditions, the complex multicellular in-
teractions based on cell-to-cell signalling, the exchange
of genetic material between biofilm organisms, the
sorptive and protective properties of biofilms upon
exposure to inorganic and organic pollutants, chemi-
cal substances like biocides, detergents, synthetic sur-
factants and xenobiotics in combination with heavy
metals, and reactive oxygen species, and the function
of biofilms in biodegradation processes.
Fig. 1. Research fields of biofilms and
summary of analytical techniques pre-
sented. AAS atomic absorption spectro-
metry, AFM atomic force microscopy,
ATR attenuated total reflectance, CE
capillary electrophoresis, CLSM con-
focal laser scanning microscopy,
DGGE denaturing gradient gel electro-
phoresis, FFF field-flow fractionation,
FISH fluorescence in situ hybridiza-
tion, GC gas chromatography, GE gel
electrophoresis, GFP green fluorescent
protein, IR infrared, LC liquid chro-
matography, NMR nuclear magnetic
resonance, PCR polymerase chain re-
action, PFGE pulsed-field gel elec-
trophoresis, SEC size exclusion
chromatography, SEM scanning elec-
tron microscopy, STXM scanning
transmission X-ray microscopy
Chemical and physical methods for characterisation of biofilms
Approaches to biofilm analysis include microscop-
ical, microbiological, molecular biological, (bio)-
chemical and physical methods. In Fig. 1, methods
of biofilm analysis are presented with respect to their
application in the biofilm research fields of interest.
In the following we come to the conclusion that on
one side the importance and the attractiveness of bio-
film analysis is undisputed and the heterogeneity of
biofilms and their complex functioning offers research
perspectives in different fields, but on the other side the
analysis of a biofilm is a scientific challenge and re-
quires interdisciplinary cooperation. This article sum-
marizes the state-of-the-art of analytical research of
biofilms but does not claim completeness of literature
citation. Above all, this paper intends to reveal the fields
for analytical tasks in the frame of biofilm analytics.
Definition, architecture, function and models
of biofilms
A multitude of definitions of biofilm exists in the lit-
erature [15]. They all have in common that biofilms
are described as populations of micro-organisms of
different types (bacteria, algae, protozoa and fungi)
which accumulate at interfaces and excrete EPS that
form a highly hydrated slime in which the cells are
embedded [1, 15, 16]. In contrast to single-celled or-
ganisms which freely float or swim in liquid medium
(planktonic form), biofilms form predominantly in
aqueous systems on a solid surface (substratum), but
they can also be found at water–air and solid–air inter-
faces [17, 15]. In recent years, it has become obvious
that other differences between a biofilm and its plank-
tonic counterparts occur such as reduced growth rates
and changes in the expression of numerous genes, so
that the biofilm mode of growth results in the adoption
of a characteristic biofilm phenotype. Quorum sensing
(activation of a sensory system for bacterial com-
munication signals) is one of the triggers for the induc-
tion of biofilm-specific properties and phenotypic shifts
based on the the up- and down-regulation of specific
genes [18, 19].
Although the details of biofilm development pro-
cesses vary depending on the microbial species, gen-
eral distinct developmental steps have been recognized
in bacterial biofilm formation [1, 18, 20, 21]. The first
step is the adhesion of a single micro-organism to a
substratum through weak, reversible van der Waals
forces. Initially, association with a surface is transient,
followed by a firm and irreversible attachment. The
3
extent of microbial colonization depends on the rough-
ness and the hydrophobicity of the substratum and also
on the cell surface composition and hydrophobicity
(presence of cell adhesion proteins, pili, curli, flagella,
and (lipo)polysaccharides). In general, micro-organisms
attach more rapidly onto surfaces that are rougher,
more hydrophobic, and conditioned by the medium
(bulk fluid). Micro-organisms with cell surface poly-
mers with nonpolar sites prefer hydrophobic surfaces,
while EPS and lipopolysaccharides attach to hydro-
philic surfaces. The attachment is also influenced by
hydrodynamic forces (laminar and turbulent flows,
stream velocity etc.) as well as nutrient supply. The
next stage in biofilm development is growth of cells
and their aggregation into microcolonies, facilitated by
the adhesion sites of the first colonizers and the pro-
duction of EPS that hold the biofilm together. Further
increase in cell numbers by cell division and addi-
tional cell recruitment from the environment results
in mature biofilms. The maximal biomass yield as well
as biofilm composition and architecture depend large-
ly on the hydrodynamic conditions and nutritient avail-
ability. The last stage is characterised by detachment
and release of cells into the surrounding environment.
Biofilm cells can be actively dispersed by shedding of
divided and motile cells or passively detached by ero-
sion or shearing in form of clumps or small aggregates
or massive removal and abrasion (caused by particle
collision of the bulk fluid). The biofilm bacteria can
also move collectively by rippling or rolling across the
substratum.
Two grossly different conceptual models dealing
with the structure and function of biofilms can be found
in the literature [22]. The first one, the continuum
model, is a conventional diffusion-based concept with
division of the biofilm in specific compartments, the
substratum, the biofilm (subdivided in a surface and a
base film), the bulk liquid and a possible head space.
However, observations in natural aquatic environments
and technical water systems as well as laboratory studies
on submerged biofilms indicate that biofilms frequent-
ly display structural heterogeneities to varying degrees.
Thus, the microcluster model, a complex three-dimen-
sional model, was established, describing mushroom-
shaped biofilm clusters separated by channels and with
streamers on their ‘‘mounds’’. As a consequence, a
quantification of biofilm properties is unrealistic with-
out mathematical models due to the complexity of a
biofilm. Attempts at quantifying and modelling biofilm
structures are presented in the literature [23–25].
4 E. Denkhaus et al.
In contrast to planktonic microorganisms, biofilms
secrete multiple EPS, which can make up between
50–90% of the total organic matter of biofilms. Poly-
saccharides are characteristic components of the EPS
[26], but proteins, nucleic acids, (phospho)lipids and
humic substances have also been identified, some-
times in substantial amounts [27]. EPS determine the
structural and functional integrity of microbial bio-
films, and contribute significantly to the organization
of the biofilm community [16]. EPS are involved in
the formation and maintenance of a three-dimensional,
gel-like, highly hydrated and locally charged (often
anionic) biofilm matrix, in which the microorganisms
are more or less immobilized. The biofilm matrix may
trap inorganic particles (e.g. corrosion products, cal-
cium carbonate deposits) and biogenic material (detri-
tus), it may incorporate multivalent cations which can
be involved in the cross-linking of anionic EPS and
thus in polymer network formation, and it may contain
colloidal and dissolved compounds.
Exopolysaccharides are high-molecular-weight poly-
mers with molecular masses varying between 500 and
2000 kDa [27]. They can associate together and can
interact with other components of the EPS matrix like
proteins, lipids, lectins, ions, and other macromolecules
of the bacterial cell surface, so that they can form a
polymer network, which determines the viscosity and
gelation of the biofilm.
EPS may consist of neutral molecules; more fre-
quently, EPS contain ionic groups which confer net
negative or positive charges on the polymers at near
neutral pH values. Extracellular polysaccharides owe
their negative charge either to carboxyl groups of uron-
ic acids or to non-carbohydrate substituents such as
phosphate, sulfate, glycerate, pyruvate or succinate [26].
Extracellular polysaccharides often contain variable
proportions of hexuronic acids such as glucuronic acid,
galacturonic acid or mannuronic acid. Exceptions are
polysaccharides, which completely consist of uronic
acid residues such as the well-studied extracellular
polysaccharide alginate of Pseudomonas and Azoto-
bacter species; alginates are composed of mannuronate
and guluronate residues, with mannuronate being par-
tially O-acetylated [28]. Sometimes, sulfate groups
have been described to occur in polysaccharide mole-
cules; cationic groups in polysaccharides may be due to
the presence of amino sugars.
Proteins can also contribute to the anionic proper-
ties of EPS. The negative charge of proteins is due to
the presence of diacid amino acids such as aspartic
and glutamic acid. Nucleic acids are polyanionic due
to the phosphate residues in the nucleotide moiety of
the poymer molecule. Uronic acids, acidic amino acids
and phosphate-containing nucleotides as negatively
charged components of EPS can be crucial structural
elements of flocs and biofilms, since they are expected
to be involved in electrostatic interactions with multi-
valent cations (e.g., Ca2þ , Mg2þ , Fe3þ ), thus mediat-
ing the formation and=or the stabilization of the
network of the EPS matrix by cross-linking polymer
strands. Additional studies of EPS expressed by other
bacteria, including teichoic acid or colanic acid (nega-
tively charged) besides cellulose and polyglucosamine
(positively charged) led to an alternative model – the
formation of polyelectrolyte complexes. They are
generated when bacteria synthesize positively and
negatively charged polymers which interact [29]. The
controlling of the matrix cohesiveness can be regu-
lated by the amount of polymers produced or by adjust-
ing the charge density on each polymer. The amount of
synthesis of exopolysaccharides also depends on the
bioavailability of carbon substrates and the balance be-
tween carbon substrates and limiting nutrients such as
nitrogen or phosphate.
EPS do not only play a role in keeping the biofilm
bacteria together and in mechanical stability of the
biofilm, but also can have other functions [27]. EPS
form a medium for communication processes and
genetic transfer. EPS are also involved in nutrition
(EPS as a nutrient reserve and accumulation of nutri-
ents by EPS), and interaction of micro-organisms with
their environment, acting as a protective barrier against
biocides, disinfectants and antibiotics as well as an
accumulator for xenobiotics, and toxic metal ions.
Additionally, exopolysaccharides interact with extra-
cellular enzymes, resulting in the accumulation, reten-
tion and stabilization of the secreted enzymes.
The whole activities, including the influx of substrates
as well as the efflux of waste products and the organisa-
tion of the microbial communities underlie regulation
processes. In the literature two hypotheses are pre-
sented. The first one is mechanistically based on biofilm
growth under the aspect of differences in local substrate
availability. The second one is morphologically based
on expression of genes that participate in cell-to-cell
communication and directly control the organisation
of the community [16–18]. All things considered, it
can be noted that the research of microbial metabolic
and regulatory activities has to be forced for a com-
plete understanding of structured bacterial populations.
Chemical and physical methods for characterisation of biofilms
Table 1. Overall composition of microbial biofilms
Extracellular polymeric substances (EPS)
– Cationic groups in amino sugars and proteins
(e.g. NH3 þ )
– Anionic groups in uronic acids, proteins, and nucleic acids
(e.g. COO ; HPO4  )
– Apolar groups from proteins (such as in aromatic amino acids),
(phospho) lipids, and humic substances
Microbial cell
Outer membrane: lipopolysaccharides of Gram-negative cells
– Cell wall consisting of N-acetylglucosamine and N-acetylmuramic
acid, offering cationic and anionic sites, and the lipoteichoic acids
in Gram-positive cells
Cytoplasmic membrane, offering a lipophilic region
– Cytoplasm, as a water phase separated from the surrounding water
Minerals
– Precipitates (sulfides, carbonates, phosphates, hydroxides)
– Free and bound metals (Ca2þ , Fe3þ , Mg2þ )
Biogenic particulate materials (degradation products)
Environmentally relevant substances
– Organic pollutants (e.g. biocides, detergents, xenobiotics)
– Inorganic pollutants (e.g. heavy metals)
Fundamental to characterisation of biofilms
As mentioned before analytical questions with regard
to biofilms can be divided into information about the
components, the architecture and the processes occur-
ring within biofilms. Apart from the high water con-
tent of up to 95%, the main constituents of biofilms
are microbial cells and EPS (Table 1). Depending on
the environment, biofilms can also contain variable
amounts of trapped particles and sorbed substances of
organic or inorganic nature. The spectrum of biofilm
systems to be investigated ranges from pure to mixed
cultures, from batch to continuous cultures and from
laboratory to environmental, technical and medical bio-
films. The characterisation of biofilms includes a wide
range of different analytical methods, which must be
adapted to the aim of the investigation (structural and=
or functional analysis).
Growth of biofilms in laboratory-based systems
The unique character of a natural biofilm makes inves-
tigations of different aspects of biofilms such as for-
mation, growth, metabolic activities, regulation, and
resistance difficult. For such investigations laboratory
devices are developed, which allow biofilm formation
under controlled conditions of physical (flow velocity,
shear stress, temperature, properties of the substratum
etc.), chemical (composition and amount of nutrients,
organic and inorganic particles, ions, etc.), and bio-
5
logical (composition of the microbial community –
single or mixed – type of micro-organism) parameters.
Principally, the laboratory devices can be divided in
flow-through-cells and batch systems. Different types
of bioreactors such as fluidised-bed reactors and ro-
tating annular reactors etc. are summarized in [30].
Submerged biofilms can also be formed on the surface
of microtiter dish walls under standing culture condi-
tions, as one example of a batch system [16]. The ad-
vantage of the microtiter dishes is the possibility of
high-throughput screens. A pellicle is a biofilm that
forms at the air–liquid interface of an agar-solidified
media, usually used for the cultivation of micro-organ-
isms in microbiology, with the disadvantage of biofilm
formation with more complex structure than that grow-
ing in continuous flow cell or on microtiter dish walls.
Surface and interface characterising techniques
The first papers published as far as the analysis of
biofilms concerned are more concentrated on micro-
biological parameters e.g. biofilm thickness, total dry
weight and total cell count [31–33]. A review about
biofilm characterisation and activity analysis in water
and wastewater treatment is written by Lazarova and
Manem [34]. These parameters are however, not suf-
ficient to describe biofilm activity in microscale. Soon
it was realised that improved analytical methods and
procedures are needed in order to understand the phys-
iological behaviour of the fixed biomass. Several opti-
cal methods that allow the study of undisturbed living
biofilms have been developed and are claimed to have
high potential in the analysis of biofilms. However,
natural biofilms can develop into thick packages of
cells that may limit light penetration into the biofilm
matrix hindering the use of optical methods.
Structure characterising methods for biofilm exam-
ination and monitoring are reviewed in [35]. Chen
et al. reviewed microscopic and spectroscopic tech-
niques separately and in combination [36]. They listed
methods and techniques applied to detection and mon-
itoring of microbiologically influenced corrosion.
Janknecht and Melo [37] presented a review of on-
line biofilm techniques focusing on methods based on
differential turbidimetry, light scattering, heat transfer,
pressure drop, real-time measurement of metabolic
products, image analysis, radiation signals, electric
and mechanical (vibration) signals. The detection of
radiation signals encloses bioluminescence fluorome-
try, FTIR, ATR, NMR and photoacoustic spectroscopy.
6 E. Denkhaus et al.
Lawrence and Neu reviewed aspects of biofilm culti-
vation, laser scanning microscopy, molecular probes and
digital image analysis [38]. Chen et al. gives a review
on developments for in situ observation of membrane
processes [39]. They summarized techniques including
direct observation through membrane and related tech-
niques, refractometric and interferometric techniques,
photo-interrupt sensors, video, fluorescence, and parti-
cle image velocimetry. Also techniques including im-
pedance spectroscopy, ultrasonic reflectometry, radio
isotope technique, NMR, computer aided tomography,
electrochemical methods and constant temperature an-
emometry are discussed. George et al. summarised var-
ious biofilm monitoring techniques that are currently
being used in water cooling systems [40].
The mainly used techniques and methods for char-
acterising the structure of biofilms have been sum-
marized in the following sections. It has been found
impossible to include each type of technique.
Confocal laser scanning microscopy (CLSM)
Microscopy plays a paramount role in the understand-
ing of any biological process, as it is the basic tool for
assessing microbial populations. It enables the detec-
tion of problems at an early stage, such as infections
and diseases, contamination in food production, mal-
functioning of biotechnological processes in general,
and deterioration of biological waste and wastewater
treatment performance. There has been an increasing
application of microscopic methods in general over
the past years due to the possibility of coupling them
with automated digital on-line image acquisition and
image analysis methods, and extending them to in situ
analysis [35].
Confocal laser scanning microscopy (CLSM) has
been preferred to other types of microscopy, because
it allows the study of live, fully hydrated biofilms
[38]. CLSM provides simultaneous information about
the 3-dimensional (3D) structure of the biofilms and
the identification of the different components, either
by autofluorescence (algae) or by using specific fluo-
rescent dyes for bacterial DNA or EPS glycoconjugates
[41, 42]. CLSM has been used for the assessment
of lectin-binding analysis for in situ detection of gly-
coconjugates in fully hydrated river biofilms [43].
Strathmann et al. have been used fluorescently labelled
lectins in combination with epifluorescence micros-
copy and CLSM to allow the visualisation and char-
acterisation of carbohydrate-containing EPS in biofilms
of Pseudomonas aeruginosa [44]. Barranguet et al.
published a paper where the light attenuation by the
biofilm is measured and compared with the signal
attenuation when using pulse amplitude modulation
(PAM) fluorimetry and CLSM [45]. In algal biofilms,
PAM fluorimetry allows the measurement of algal
photosynthetic activity, maximum quantum yield and
algal biomass in biofilm samples. The results of algal
biomass and EPS production determines by chemical
and optical methods on mixed autotrophic biofilms of
different ages are compared. The advantages and lim-
itations of both types of technologies are discussed
with the aim of clarifying when which method should
be preferred.
A microscopy-based method for visualizing the
structure and dynamics of microbial biofilms, indivi-
dual fluorescent microbial cells, and inorganic col-
loids within a model porous medium is described by
Leis et al. [46]. Biofilms growing in flow cells packed
with granules of an amorphous fluoropolymer could
be visulalised as a consequence of refractive index
matching between the solid fluoropolymer grains and
the aqueous immersion medium. In conjunction with
the capabilities of CLSM for optical sectioning, the
use of amorphous fluoropolymers as a solid matrix per-
mits observation of organisms and dynamic processes
to a depth of 2–3 mm, whereas sediment biofilms grow-
ing in sand-filled flow cells can only be visualised in
the region adjacent to the flow cell wall.
Scanning electron microscopy (SEM)
The combination of a conventional optical microscope
with a specially designed glass flow cell was used to
visualize ‘‘in situ’’ biofilms formed on opaque thin
biomaterials through a simple non-invasive way [47].
Comparisons of optical microscopy of thin biofilms
with SEM images were made. Also Stewart et al. de-
monstrated the structural heterogeneity of a microbial
biofilm by SEM, CLSM and cryoembedding followed
by sectioning and microscopic examination [32]. They
compared the three different microscopic methods
with respect to the information they provide concern-
ing biofilm structural heterogeneity.
Scanning transmission X-ray microscopy (STXM)
Scanning transmission X-ray microscopy, which uses
near-edge X-ray absorption spectroscopy (NEXAFS)
as its contrast mechanism, is a powerful new tool that
Chemical and physical methods for characterisation of biofilms
can be applied to fully hydrated biological materials.
This is possible due to the ability of soft X-rays to
penetrate water, the presence of suitable analytical
core edges in the soft X-ray region, and reduced radia-
tion damage (compared to that caused by electron beam
techniques). Lawrence et al. have been used STXM,
CLSM and Transmission Electron Microscopy (TEM)
to map the distribution of macromolecular subcom-
ponents (e.g. polysaccharides, proteins, lipids, and
nucleic acids) in a river biofilm [48]. They demon-
strated that this combination of multimicroscopy ana-
lysis can be used to create a detailed correlative map
of biofilm structure and composition. STXM also is
capable of mapping metals in biofilms. It can provide
quantitative maps of chemical species at environmen-
tally relevant concentrations (i.e. mg  kg1 ) with a
spatial resolution of better than 50 nm [49]. Dynes
et al. presented a method for a detailed quantitative
analysis of ferrous and ferric iron in a river biofilm in
concert with mapping Ni(II) and Mn(II) species in the
same region.
Atomic force microscopy (AFM)
AFM is one of the most important and most widely
used technique among the scanning probe microscopy
(SPM) techniques. AFM can be used to image features
with dimensions at the atomic scale up to approxi-
mately 100 mm and it reveals the surface topography
with direct depth information. Therfore it is suitable
for probing biopolymers.
Hansma et al. [50] presented a short review of
atomic force microscopy (AFM) of biopolymers and
specific examples of some of the biopolymers that
have been analysed by AFM. These specific examples
include extracellular polymeric substances (EPS) on
the surfaces of bacterial biofilms, condensed DNA,
DNA constructs, and DNA-protein interactions.
Infrared and raman spectroscopy
ATR-IR spectroscopy is a technique that can readily
be used to acquire in situ data from bacterial cells
attached to a substratum. Furthermore, it has the ad-
vantage that whole bacterial cells and biofilms can
be studied, thereby elimination the possibility of arte-
facts that can arise during the processes required to
isolate specific cellular components. Therefore this
technique is widely used in biofilm analysis [51–54].
Even Suci et al. combined reflected differential inter-
7
ference contrast microscopy and ATR-FT-IR spectro-
scopy to characterise the chemistry and architecture of
biofilms colonizing surfaces in vitro [52]. The depth
of penetration of the evanescent wave of IR radiation
restricts the region over which chemical information
is obtained to within approximately 1 mm of the prism.
With the described approach, it should be possible to
relate specific structural change reported for antibiotic
challenged biofilms to the concentrations of antibio-
tics within the biofilms. Also Chengdar et al. used the
combination of microscopy and FT-IR spectroscopy
in order to understand the mechanisms of microbially
influenced corrosion=corrosion inhibition. Mild steel
electrodes were incubated in phosphate-buffered basal
salt solution having two different aerobic bacteria, viz.
Pseudomonas alcaligenes and Pseudomonas cichoril.
The exposed surfaces were examined using scanning
electron micrographs (SEM), energy dispersive spec-
troscopy (EDS) and electron spectroscopy for chemi-
cal analysis (ESCA). The scraped surface film was
also examined using FT-IR spectroscopy [55].
Diffuse reflectance infrared Fourier transform
(DRIFT) spectrometry was applied to a set of sedi-
ment samples collected by sediment traps on a weekly
basis as part of a river sediment contamination mon-
itoring campaign lasting one and a half years [56].
The FT-IR-DRIFT mode offers some important ad-
vantages, such as the ability to assess both mineral
and organic structures in particles, good sensitivity
and high throughput. The main focus rested on the
ability of DRIFT to detect differences in sediment
composition reflecting frequent dynamic changes in
hydrological of life-cycle situations characteristic of
river systems. The impact of these changes on metal
binding by sediment particles was further investigated
and the consequences for metal bioavailability and
speciation modelling were discussed. Pink et al. pub-
lished an FT-IR study of Pseudomonas aeruginosa
PA01 biofilm development [57]. They gave an interpre-
tation of the ATR-FT-IR data in the 1500–1180 cm1
region. The experimental results provide the first
detection via a non-perturbative technique of an ex-
cess of protein in the neighbourhood of the surface.
The mathematical analysis applied to the data sup-
ports the conclusion that the observations cannot be
completely accounted for by the changing character-
istics of bacterial proteins but, rather shows that an
accumulation of excess protein near the attachment
surface is taking place. Kang et al. developed in situ
infrared spectroscopic methodologies to monitor chem-
8 E. Denkhaus et al.
ical processes in model bacterial biofilms and ap-
plied these methodologies to probe metal ion binding
by Pseudomonas aeruginosa [58]. Single layers of
Pseudomonas aeruginosa were prepared on ZnSe
ATR-IR prisma. The binding of Cr(III), Ni(II) and
Co(II) ions at pH 5.0 to such single-layer biofilms
of Pseudomonas aeruginosa has been investigated.
Photoacoustic spectroscopy (PAS)
PAS is based on the absorption of electromagnetic ra-
diation inside a sample. It combines features of optical
spectroscopy and ultrasonic tomography and allows – in
contrast to other spectroscopic techniques – a depth –
resolved analysis of both optically and acoustically
inhomogeneous media. Schmid et al. presented a photo-
acoustic sensor system for depth-resolved analysis of
biofilms [59, 60]. The research group has been used this
system for the on-line and in situ observation of the
sorption of suspended iron(III)oxide particles on the
outer and inner surfaces of the biofilm [61]. A PAS-
system containing three photoacoustic sensor heads
has been used for the investigation of the efficacy of
diverse biocides [62]. Monitoring of the photoacoustic
signal amplitude in the visible spectral range allows the
observation of biofilm detachment caused by biocides.
X-ray spectroscopy
X-ray spectroscopic and scattering techniques have
found widespread application in the study of envi-
ronmental and geochemical interfacial phenomena
[63]. The large penetration depth and inherent molec-
ular scale sensitivity of X-rays make them ideal
probes for investigating the structural details of en-
vironmental materials under in situ conditions. Trainor
et al. give a review of the application of grazing-angle
X-ray fluorescence techniques to investigate the dis-
tribution and speciation of heavy elements at envi-
ronmental interfaces including the presentation of a
model formalism that allows for quantitative analysis
of fluorescent yield profiles [63]. Al-Bataineh et al.
investigated the surface immobilisation of antibacter-
ial furanones using X-ray photoelectron spectroscopy
(XPS) and tapping mode AFM after each step of the
immobilisation sequence [64].
Nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopic techniques have provided de-
tailed metabolic information for live prokaryotic cell
suspensions, gel-immobilized cells and extracts as
wells as eukaryotic cell and tissue samples [65]. 1H
and 1H-detected 13C NMR, in particular, provide for
the direct, time-resolved and non-invasive monitoring
of metabolite concentrations, metabolic pathways and
flux rates for in situ studies of live cell suspensions
[66]. NMR techniques for the non-invasive study of
live biofilms have been restricted to the measurement
of water properties. These studies include e.g. the
imaging of biofilms in porous media [67], the mea-
surement of convective flow of media in and near in
situ biofilm surfaces [68, 69]. Further, liquid- and
solid-state NMR methods have been used to study
the chemical composition and molecular mobility of
biofilm extracellular polymeric substances and ex-
tracts [70–72].
Wind et al. described novel procedures and instru-
mentation for NMR spectroscopy and imaging stud-
ies of live, in situ microbial film [73]. Localised
NMR techniques were developed and used to non-
invasively monitor time-resolved metabolite con-
centrations and to image the biomass volume and
distribution.
Reflectance spectroscopy
Broschat et al. presented an optical reflectance assay
for measuring biofilm formation on both opaque and
nonopaque surfaces [74]. Time series reflectance mea-
surements are presented for two different bacteria on
two different substrata, glass and polystyrene. The
reflectance assay can be used for rapid quantitative
screening of mutant libraries, as well as rapid assess-
ment of phenotypic variation among isolates.
Combined surface characterising techniques
Several surface science techniques are often combined
to increase the level of knowledge of the external part
of the cell.
Beech et al. presented a review about the under-
standing of microbe=surface interactions from the
perspective of gaining insight into mechanisms of bio-
fouling and biocorrosion using atomic force spectro-
scopy (AFS) techniques in elucidating cell adhesion
to solid substrata and modern mass spectrometry tech-
niques in characterising biofilm populations and bio-
film matrix [75].
FT-IR, XPS, and time-of-flight (TOF) secondary-
ion mass spectrometry (SIMS), three techniques hav-
Chemical and physical methods for characterisation of biofilms
ing different sensitivities and analysis depths, were
combined to characterise the chemical composition
of the topmost layers of the micro-organisms [76].
Separation, molecular and hyphenated
techniques
As mentioned before the main components of a bio-
film are water, the microbial cells the extracellular
polymeric substances, and organic particles. Corre-
spondingly, a subsequent separation of the biomass
(microbial cells) and the other extracellular compo-
nents are needed. Thus, it is a huge task to identify
and quantify each component because minimal cell
lysis, and no disruption or altering of the extracellular
biopolymers are basic requirements. Principally, the
separation method must be selected for each case,
considering the specific needs and constraints. The
aim of this part of the review is to give an overview
of analytical methods preferable according to the
investigation with the main focus on separation and
analysis of components of EPS. The investigation of
EPS is helpful for the implementation of analytical
methods which are suitable for biofilm analysis. EPS
can be handled without following legal regulations
according to laboratory equipment and safety ac-
tivities in comparison to the handling of pathogenic
microbes.
The choice of a suitable separation method corre-
sponds to the analytical and microbiological problem,
respectively. Principally, most of the regular separa-
tion techniques used in analytical separation, such
as extraction, derivatization, field flow fractionation,
chromatography, and electrophoresis, can be applied
for isolating components of the biofilm, like EPS,
polysaccarides, proteins, nucleic acids, phospholipids,
humic substances or individual organic or inorganic
compounds.
Extraction as a separation method
The extraction as a sample pre-treatment technique in
biofilm analysis is employed for the quantification of
the extracellular polymeric substances, mostly in form
of proteins, carbohydrates, nucleic acids, (phospho)
lipids, and humic acids or environmental relevant
organic and inorganic compounds or metal ions. Sev-
eral physical as well as chemical and combined meth-
ods have been proposed to extract EPS from biofilms
or activated sludge floes [78] such as the high-speed
9
centrifugation, ultrasonication, heating, and the use of
additives. An overview of the extraction methods are
presented in a review by Jahn and Nielsen [80]. They
have clearly represented multitude of diverse extrac-
tion methods and their application in biofilm analysis
and a useful extraction check list including sampling,
storage, washing, homogenisation, extraction itself,
and purification. The extraction efficiency given as
the amount of organic matter extracted depend on
the method used, the biological sample and the analyte.
Zhang et al. compare five different extraction methods
on basis of centrifugation, steaming, and chelatization
for the separation of EPS from defined biofilm sam-
ples evaluating the yields of carbohydrate and protein
concentrations [79]. They found that regular centrifu-
gation with formaldehyde is most suitable for carbo-
hydrate separation and steaming extraction for protein
separation [1a]. Li et al. [77] published the results of
experiments, where EPS using either EDTA or NaOH
was enhanced by alternating current especially within
the first 0.5 h of current application. All listed meth-
ods have the main disadvantage of high rate of cell
lysis. This can be circumvented applying a cation-
exchange resin (Dowex) for extraction of e.g. humic
substances and proteins in considerable amounts [80].
This method is based on the removal of calcium ions,
destabilizing the EPS structure, so a separation from
the microbial cells is possible. Wuertz et al. present a
suitable extraction method for the separation of EPS
for quantification of organic and inorganic contami-
nants, especially sorbed alkaline and alkaline earth
metals via a crown ether [81]. Additionally, they could
show better effectiveness for crown ether extrac-
tion than for separation using cation exchange resin.
Azeredo et al. circumvent the promotion of leakage of
intracellular material via pre-treatment of the biofilm
with glutaraldehyde, which shows protective proper-
ties against cell lysis [82]. Within the application of
solid phase extraction two publications of recent date
are quoted, the removal of a technical nonylphenol
ethoxylate surfactant and the formation of degrada-
tion products under oxic and anoxic conditions in a
miniaturised biofilm reactor were monitored after
solid phase extraction following reversed phase-high
performance liquid chromatography (RP-HPLC) by
fluorescence detection [83] and the quantification
of atrazine in a freshwater biofilm by chromatogra-
phy-mass spectrometric detection after immediate
extraction with acetonitrile following solid phase
extraction [84].
10
Concluding it can be ascertained that cation ex-
change resin can be applied in routine for the separa-
tion of EPS from the microbial cells.
Field-flow fractionation as a separation method
An interesting separation method with future perspec-
tives seems to be the field-flow fractionation in their
variants, up to now applied for the characterization of
natural colloids, particles and biotechnological tasks.
The biotechnological applications including separa-
tion of proteins, nucleic acids, and macromolecules
as well as sorting and fingerprinting of bacterial cells,
are reviewed in an article by Reschiglian et al. [85].
One variant of the field-flow fractionation (FFF) is the
sedimentation-FFF (Sd-FFF) well suitable for the de-
termination of particle size distribution or for the
investigation of parameters depending on the particle
size [86, 87]. Other technical constructions of FFF are
the flow field-flow fractionation (FIFFF) [88–90] with
the sub-technique of asymmetrical flow field-flow
fractionation (AF4) [91, 92] and the hollow-fiber flow
field-flow fractionation (HF FIFFF) [93]. All listed
FFF techniques have the main advantages of mini-
mized sample alteration using carrier with low ionic
strength, and of easy hyphenation to usual analyti-
cal detectors, e.g. element specific detectors such as
electrothermal atomic absorption spectrometer [87],
inductively coupled plasma-mass spectrometer [90,
91, 94], inductively coupled plasma atomic emis-
sion spectrometer [95], and X-ray spectrometer [94].
Additionally, the coupling of FIFFF to multi-angle
laser light scattering (MALLS), transmission electron
microscopy (TEM) [88, 89] and atomic force micro-
scopy (AFM) [96] is described. Recently, HF FIFFF is
interfaced with ESI=MS for protein separation and
simultaneous characterization [97]. This hyphenated
technique seems to be a powerful tool for the charac-
terization of biofilms, especially due to the composi-
tion of EPS, but isn’t described up to now.
Molecular techniques
Principally, a reconstruction of near complete gen-
omes or genome sequences of microorganisms is pos-
sible using genomic datasets in combination with
molecular techniques [98]. Further interesting reviews
deal with microbial genomics and metagenomics and
bioinformatics systems for data interpretation [99,
100]. Molecular techniques including nucleic acid
E. Denkhaus et al.
probes (oligonucleotide probe, DNA microarray) of
known nucleic acid molecules (specific bacterial DNA
sequences from target microorganisms), pulse field
gel electrophoresis (PFGE), denaturing-gradient gel
electrophoresis (DGGE), and polymerase chain reac-
tion (PCR) for amplification of DNA in combination
with different analytical methods, e.g. mass spectro-
metry and microscopic techniques are mainly used
in the identification of the different bacterial cells
forming a natural biofilm, organized in a microbial
ecosystem [101–104]. The application of ribosomal
RNA-targeted oligonucleotide probes (also known
as ribotyping or molecular fingerprinting: analysis of
DNA fragments generated from restriction enzyme
digestion of genes encoding their 16S rRNA) visu-
alized in different hybridisation formats, e.g. as dot
blot, fluorescence in situ hybridisation (FISH), and
green fluorescent protein (GFP), allows a quantifi-
cation of the composition of a complex microbial
community [103–105]. Combining FISH and micro-
autoradiography an in situ uptake and subsequent
incorporation of radioactively labelled substrate into
individual microbial cells is possible [106]. Addition-
ally, the results can be combined with results obtained
by microsensors [107]. An interesting approach to a
combination of electrochemical and molecular techni-
que is presented in a publication of Lassalle et al.
[108]. They construct an electrosensor based on an
electronically conductive polymer grafted with oli-
gonucleotides for real time detection of DNA hy-
bridisation using photocurrent spectroscopy and a
quartz crystal microbalance. New developments in
the rRNA-targeted probes are rRNA-based phyloge-
netic DNA microarrays consisting of several oligo-
nucleotide probes for microbial specification. The
application of such a DNA microarray offers the pos-
sibility to determine the up-and-down regulation of
genes that are involved in different steps of biofilm
formation or in pathogensis of microbial infections.
Boyce et al. present technologies of DNA microar-
rays that applied in the determination of response of
bacterial pathogens to different environments [109].
Sauer discusses in a minireview the limitation of
microarray studies during biofilm development [110].
A comprehensive review presents DNA microarray
analysis due to the gene expression profile of biofilms
[111]. The author comes to the conclusion that bio-
films may have a unique pattern of gene expression.
On the other side, the major disadvantage using oli-
gonucleotide probes is the requirement of specific DNA
Chemical and physical methods for characterisation of biofilms
fragments that recognize complementary specific bac-
terial DNA sequences from target microorganisms. In
praxis a multitude of oligonucleotide probes exists.
Therefore data bases are needed [105, 112].
Pulse field gel electrophoresis (PFGE) is similar to
ribotyping but instead of rRNA, the whole genome is
analysed [104]. In the first step the genome is cutted
by restriction enzymes into large genomic fragments.
In the second step the fragments are separated by
subjecting them to alternatively pulsed, perpendicu-
larly oriented electrical fields following electropho-
resis with different detection.
Based on the polymerase chain reaction (PCR) var-
ious analytical techniques can be used to identify bio-
film organisms in their natural habitat (cultivation is
not longer needed). Beginning with the extraction of
DNA from the microbial cells following cell lysis the
rDNA is amplified by DNA polymerase in a three
stage process. The amplification products are then
separated and analysed using DNA sequences capil-
lary electrophoresis, image processing and data base
with reference pattern [108]. One technique coupling
PCR amplification of rDNA genes and denaturing-
gradient gel electrophoresis (DGGE) is now in a devel-
opmental phase [104, 113–115]. The electrophoretic
separation is based on the melting properties of the
amplified DNA sequences. Each band observed on
the gel represents a specific sequence of a gene. The
PCR-amplified DNA can be directly determined by
matrix-assisted laser desorption-ionization time-of-
flight mass spectrometry (MALDI-TOF) [116]. This
combination has two main advantages: analysis can be
rapidly done and only small amounts of DNA are
needed.
Another possibility for the determination of target
microorganisms in a microbial community is the use
of immunological assays based on antigen-antibody
reactions. The visualisation can be performed by
direct or indirect immunofluorescent microscopy as-
says, flow cytometry, enzyme-linked immunosorbent
assay (ELISA), membrane assay and latex agglutina-
tion [117]. The main problem using immunological
assays is that they can only be applied for thin bio-
films because of the large molecule of an antibody. In
a multilayered biofilm the EPS acts as natural barrier
for large molecules. A further disadvantage is the lim-
ited number of antibodies tested.
Recent developments are integrated genetic anal-
ysis microsystems on basis of microscale PCR com-
bined with microchannel capillary electrophoresis
11
having the advantage of high analysis speed (20–
70 min instead of 5–12 h), low analysis cost and port-
ability [118]. Integrated optics, e.g. CCD camera,
photodiodes etc., allow a complete online analysis of
microbial composition in a biofilm in form of a ‘‘lab
on chip’’. The authors impressively reviewed the ad-
vances of technologies for micro- and nanoscale anal-
ysis of genetic material including design, fabrication,
and testing of the integrated microsystems. Addition-
ally they give a lot of useful references and show the
future perspectives in this field.
Chromatographic, electrophoretic,
and hyphenated techniques
In the following the sample pre-treatment, the sep-
aration, here chromatographic and electrophoretic sep-
aration, and characterization of polysaccharides (1)
and proteins (2) are mainly presented because of their
functional importance in biofilm formation, growth,
metabolic and regulatory activities. At the present
time, no single method exists that could be recom-
mended for the chromatographic and electrophoretic
separation and purification of polysaccharides and
proteins and the determination of their analytical char-
acteristics, generally. Dependent on the matrix, the
polysaccharides and the proteins have to be isolated
from the sample and dissolved before a characterisa-
tion can be done.
Polysaccharides
To prevent a degradation of the polysaccharides the
experimental conditions during the homogenisation of
the biological sample and the following isolation
of the polysaccharides have to be chosen as follows
[119]: (a) aqueous or buffer solutions (pH 5–7.5), (b)
low temperatures (0–4  C), (c) short extraction times,
and (d) addition of reduction reagents (e.g. Dithio-
threitol). Exopolysaccharides can be achieved by di-
verse extraction methods, described in the chapter
extraction as a separation method. The proteins ex-
tracted can be removed by thermal denaturation, pre-
cipitation with trichloroacetic acid (10–20%) [120],
with ammonium-sulphate, extraction with phenol=
acetic acid=water (2:2:1) or protease treatment [119].
For the extraction of polysaccharides from the
treated sample material extraction reagents, like (a)
water or buffer solutions for water soluble polysac-
charides, (b) dimethylsulfoxide for starch, (c) pectins
12
with Ca2þ bounding chelats e.g. EDTA, (d) hemicel-
lulose by NaOH or KOH, (e) methylmorpholino-N-
oxide for neutral polysaccharides like hemicellulose
or cellulose, and (f) cadoxen (1,2-diaminoethan=water=
cadmium oxide) for cellulose, can be used. After
the extraction, salts and other lower molecular com-
pounds have to be removed. Methods e.g. dialysis,
dialfiltration, tangential flow filtration, precipitation
with ethanol and GPC have been applied for this pur-
pose [119]. Mixtures of the isolated polysaccharides
can be separated by methods which permit a separa-
tion caused by charge and=or molecular size. The
most applied techniques are fractional precipitation
[119], preparative GPC, gel filtration [121] and pre-
parative ion exchange chromatography [122].
Using chromatographic methods for the char-
acterisation of polysaccharides mostly a reduction to
mono- or oligosaccharides is necessary, an exception
is the size-exclusion chromatography for the determi-
nation of the molecular weight. The quantitative and
qualitative composition of polysaccharides will be
carried out by acid catalyzed total hydrolysis of the
polysaccharide as a rule. Besides of further solvolysis
methods e.g. methanolysis, acetolysis, and formolysis
[119] alternative methods e.g. the enzymatic hydroly-
sis [123] have been described for the degradation of
polysaccharides.
Hydrolysis belongs to the most widely used meth-
ods, which are used for the degradation of polysac-
charides to mono- or oligosaccharides. In this case an
O-glycosidic bounding will be splitted by addition of
one molecule H2O. The reaction is carried out in aque-
ous solutions by catalysis with acids and high tem-
peratures. Commonly used acids are hydrochloric acid,
sulphuric acid and trifluoroacetic acid. The mechan-
ism of this reaction is reported in [124]. Some work-
ing off steps can be necessary after the hydrolysis
reaction depending on the chromatographic method.
Salts or acids can disturb the chromatographic separa-
tion and must be eliminated e.g. by participation of
the disturbing compound or neutralisation with a cor-
responding reagent [125].
Chromatographic methods have long been recog-
nized as a vital part of carbohydrate analysis, generally.
Two approaches exist to the use of chromatography
in investigation of polysaccharides. One approach is
the chromatographic separation of polysaccharides ac-
cording to their molecular masses. Traditionally, the
determination of molecular mass distribution of the
polysaccharides has been accomplished using size
E. Denkhaus et al.
exclusion chromatography (SEC), in which analytes
are separated based on their molecular size in solu-
tion, with refractive index or light scattering detection
[126]. Here, Churms gives an overview about carbo-
hydrate separation by HPLC based on size exclusion
[127]. This review surveys changes in SEC systems
and their application to the separation and molecular-
weight (MW) distribution analysis of carbohydrates
till 1996. Mistry et al. published an interesting method
based on size-exclusion capillary electrochromato-
graphic separation of polysaccharides using polymeric
stationary phases in 2003 [126]. They have demon-
strated the separation of polysaccharides with higher
molecular mass, up to 112 000 g  mol1 routinely,
although with limited detection sensitivity by two ap-
proaches with and without derivatization. The calibra-
tion has been carried out by using pullulan standards.
They did not observe aggregation, which could falsely
lead to high MW determinations. As far as the calibra-
tion problems concerned the use of a multiangle light
scattering detector is a promising opportunity to avoid
these problems. Light scattering is one of the few
absolute methods available for the determination of
molecular mass and structure and certainly is appli-
cable over the broadest range of molecular weights of
any method [128]. Especially for the investigation of
branched polysaccharides SEC coupled with a multi-
angle light scattering detector is recognized as one of
the most powerful techniques [129].
The second approach is the employment of chro-
matography for analysing polysaccharide degradation
products. But most monosaccharides exhibit only
slight differences in charge, hydrophobicity, and mole-
cular mass, and as with monosaccharides, the separ-
ation of oligosaccharides is a complex task because
they often comprise a mixture of similar chemical
structures and have only minor chemical differences
in features such as their anomeric configuration, link-
age points, and ring structure.
The first reviews summarized the main develop-
ments in the thin-layer chromatography (TLC), GLC
and HPLC techniques, owing to the application of
novel methods of derivatization. This includes also
the use of techniques, such as supercritical fluid chro-
matography and ion chromatography [130, 131]. In
the second half of the 1990s more and more liquid
chromatography methods have been established and
summarized by Zalyalieva et al. [132]. The possibility
of the separation of underivatized sugars and, there-
fore less sample preparation is one reason for this
Chemical and physical methods for characterisation of biofilms
development. In investigations of polysaccharides, the
development of silica gel sorbents with grafted-on non-
polar and polar phases has led to the appearance and
use of methods of high-performance liquid chromato-
graphy (HPLC): reversed-phase and ligand-exchange
variants. Classical methods of chromatography, based
on the use of soft gels and low pressures are gradually
being displaced by HPLC methods.
The importance of capillary electrophoresis as an
additional analytical tool has been illustrated by two
successive reviews from El Rassi in the 1990s [133,
134]. Davies and Hounsell have been concentrated
on the detection methods used in HPLC and CE for
carbohydrate chromatography in the first half decade
of the 1990s [135]. They summarized the important
developments of methods used UV absorbance, re-
fractive index, laser light scattering, electrochemical
detection, radioactivity and fluorescence as the detec-
tion method. Paulsen et al. published a review about
the chromatography and electrophoresis in separation
and characterization of polysaccharides from lichens
in 2002 [136]. Generally, the techniques of capillary
electrophoresis are promising for the characterisation
of natural polysaccharides [137].
High-performance anion-exchange chromatogra-
phy connected to pulsed amperometric detection
(HPAEC-PAD) seems to be another powerful tool
for the analysis of saccharides according to the sep-
aration efficiency and detector sensitivity [138–142].
Carbohydrates are weak acids that are ionized at high
pH (pKa values 12–14 [142]) and are therefore amen-
able to separation by anion exchange chromatography
with a high pH eluent. The carrier material of these
pellicular anion exchange resins form monodisperse,
not porous spherical particle of 10 mm which exist of
polystyrene=divinylbenzene-copolymers with extre-
mely high across interlinking.
The high across interlinking of the material leads to a
bigger solvent compatibility and a stability in the pH
area from 0 to 14. The carrier particles are covered
with a thin latex layer from micro pearls of functional
quaternary amino groups and form a surfaces-porous
structure. The functional latex layer is either electro-
static or covalent bounded [142]. For the pulsed am-
perometric detection mostly gold working electrodes
vs. Ag=AgCl electrodes have been used [143]. The
use of gold working electrodes is the best choice for
the detection of monosaccharides because their sur-
faces are able to catalyse the electrochemical oxida-
tion of polar aliphatic compounds in alkaline media.
13
A review about the separation and detection of
amino acid-carbohydrate mixtures by HPAEC-inte-
grated pulsed amperometric detection is presented
by Jandik et al. in 2004 [144]. They show the possi-
bilities and advantages of this technique in compari-
son to traditional methods.
A promising method is reported by Stroop et al.
using a combination of HPAEC with combined elec-
trochemical pulsed amperometric detection and in-
line detection of optical rotation with an in-line laser
polarimeter for analysis of a number of mono- and
oligosaccharides found in complex polysaccharides
[139]. They showed how information can be easily
obtained about both the monosaccharide composition
and the absolute configuration of the residues in one
analysis without chemical derivatization.
Of course, the coupling of HPLC to electrospray
ionization (ESI) mass spectrometry (MS) is suitably
very well for the separation and characterization of
saccharides. Liu et al. presented a method based on
this technique using a native cyclodextrin-based col-
umn for the separation and characterization of under-
ivatized oligosaccharide mixtures [145].
But also GC-MS methods are well established and
have been used routinely since the early 1980s. GC-
MSMS methods have been readily adapted from exist-
ing GC-MS technology [146]. A review according to
the carbohydrate profiling of bacteria by GC-MS and
their trace detection in complex matrices by GC-MS
MS is given by Fox [146].
Chemical derivatization methods allow the conver-
sion of saccharides into derivatives that can be detected
with higher sensitivity. Also chemical derivatization
often enhances selectivity. The derivatization of the
compounds can be carried out before injecting (pre-
column) or after the separation (post-column) of the
sample compounds on the chromatographic column.
Pre-column derivatization causes modification, to a
greater or lesser degree, of the structure of a sugar resi-
due. Two reactions are employed for the pre-chromato-
graphic derivatization of saccharides: carbonyl groups
are coupled with amines, giving Schiff bases, or
amines are formed by reductive amination [147, 148].
The advantage of carbonyl derivatization is that it is
specific to reducing sugars and is done only at one
position in the molecule, allowing sugar molecules
to be quantified stoichiometrically – a feature that is
very important for oligosaccharide structure analysis.
Post-column derivatization, however, has the ad-
vantage that sugars are directly injected into the
14 E. Denkhaus et al.

chromatograph. Artifact formation of the derivatiza- of colorimetric assays have been used to quantify
tion reaction plays a minor role; in contrast to pre- carbohydrates [149]. An overview is given in [150].
column derivatization, the derivatization reactions do The use of fluorescence compounds as derivatiza-
not need to be completed or fully defined. A number tion reagents is a good opportunity. The derivatives

Table 2. Analytical methods for the determination of polysaccharides

Sample Sample preparation Method of determination Remarks Reference
Biofilm Extraction by EDTA= Phenol-sulfuric acid [120]
dialyse; precipitation method
of proteins by pH
adjustment and TCA
Biofilm Extraction by EDTA Phenol-sulfuric acid method [53]
or NaOH and
alternating current
Corn syrup Hydrolysis on a cation HPAEC-PAD [140]
exchange resin Column: CarboPac PA1
(Ca-form) Eluent: NaOH=NaOCH3 gradient
Different food Enzyme digestion HPAEC Recoveries in a fortified [138]
samples Two Columns: CarboPac PA1 drink matrix: 94.9–105%
Eluent: NaOH=water gradient
Capsular Acid hydrolysis HPAEC-OR-PAD Simultaneous use of [139]
polysaccharides of with TFA Column: CarboPac PA1 electrochemical and optical
gram-positive and Eluent: NaOH isocratically rotation detectors permits
gram-negative Optical rotation detector quantitative evaluation
pathogenic bacteria Advanced Laser Polarimeter of the OR signal allowing
detection mixtures of
enantiomeric sugars
Standard mixture Autoclave HPAEC-colorimetric detection Detection limit 0.1 mg [149]
of galacturonic hydrolysis=HCl Column: CarboPac PA1 Method responds to the
acid Post-column derivatization mass of sugar rather than
Oligomers with KMnO4=sulphuric acid the molarity of sugar
Escherichia coli Acid treatment and SAX-HPLC-Fluorometric detection Detection limit: 20 ng [151]
K4 bacterium enzymatic digestion Column: Spherisorb 5-SAX
Eluent: NaCl
Post-column derivatization
with 2-cyanoacetamide
Standards of Dissolution in water HPLC-ESI-MS Detection limit: 50 pg [145]
underivatized Column: Cyclobond I 2000
oligosaccharides Eluent: Acetonitrile=formic acid=water
Natural Hydrolysis with TFA CE-UV-VIS [140]
polysaccharides and lyophilisation Column: uncoated fused-silica-
(plant gums) capillaries
Standards of Enzymatic digestion HPLC-UV Detection limit: 50 pmol; [148]
oligosaccharides Pre-column dervatization Column: LiChrosorb Si 60 Up to 11 sugar residues
with p-aminobenzoic Eluent: Acetonitrile-water-DAB can be separated
ethyl ester TLC; GC; GC-MS, FAB-MS
Glycoprotein Acid hydrolysis with RP-HPLC-UV Detection limit: 50 pmol; [147]
TFA. Pre-column Column: Pico-Tag The procedure can be used
derivatization with Eluent: Sodium acetate to analyze neutral and
p-aminobenzoic buffer-acetonitrile-methanol amino sugars in a single
ethyl ester chromatographic step
Pullulan and Derivatization with SE-capillary electro chromatography Aggregation of the PIC [126]
amylase Phenylisocyanat (CEC) tagged polymer did
Column: home made combination of not play a role in the
high capacity ion-exchanger and a analysis at the
neutral polystyrene-divinylbenzene, concentrations used
Indirect detection by adding an
absorbing mobile phase
Chemical and physical methods for characterisation of biofilms
achieved by reaction of monosaccharides e.g. with 2-
cyanoacetamide can be detected by UV or=and fluo-
rescence [151].
The most articles as far as polysaccharide analysis
concerned mentioned in this paper do not deal with
the analysis of polysaccharides from biofilms. Those
publications are missing up to now. But the described
methods can be transferred to this field and therefore
selected methods are listed in Table 2.
Proteins
Beside information from genomic and metagenomic
studies of bacterial communities the interest of scien-
tists working in the field of biofilm research is also
focused on proteomic and metaproteomic analysis,
respectively. Normally, the biomass of the biofilms
for proteome analysis is restricted. This restriction
can causes an enrichment process of the biomass or
biofilm generation. For this purpose alternative labora-
tory-assisted biofilm systems were developed for an
increase of biofilm mass. Best results could be ob-
served using glass wool as solid surface for biofilm
growth because of large surface-to-volume-ratio [152].
An additional advantage applying glass wool is the
possibility of simple separation of the biofilm mass
from the surrounding planktonic cells. On the other
side newly published biofilm investigations deal with
proteomic analysis of natural biofilms. Kan et al. pre-
sents the first metaproteomic study on natural aquatic
biofilms including description of the analytical meth-
ods used [153]. An additional interesting aspect of
proteins in biofilm research is the fact, that proteins
are also involved in cell-to-cell communication. The
attendance of specific proteins on signalling path-
ways are reviewed in a publication of Visick and
Fuqua [154].
The investigation of micriobial metabolic activity
due to proteins can be divided into two parts, intra-
cellular and extracellular protein expression. For the
comparison of protein expression in biofilms and their
planktonic counterparts and physiological changes
during biofilm formation the detection of the largest
possible number of proteins is desirable. Principally,
complete protein analysis (proteomics) is separated
into three areas: functional, structural and expression
proteomics. All of the protein analysis areas need
their special matching sample preparation procedure.
Classical protein sample preparation includes desalt-
ing, concentration, centrifugation, dialysis, filtration
15
and ultrafiltration, precipitation and lyophilization.
The protein separation of a biofilm initially begins
with cell lysis after homogenisation, excepting the
investigation of extracellular protein expression and
the investigation of proteins in EPS (see chapter
extraction as a separation method). The cell lysis can
be performed in a buffer solution (e.g. Tris–HCl at
approx. pH 6.5–8.0) by sonification, enzymatic, and
mechanical treatment, respectively. To prevent decom-
position of the proteins, RNA, and DNA during storage
a Dnase, Rnase and proteinase inhibitor cocktail can
be added. Pellets containing the insoluble fractions
can be separated by centrifugation. Nucleic acid re-
moval can be achieved by precipitation with e.g. tri-
chloroacetic acid and lipid removal with excess
detergent. Soluble fractions are sequentially denatured
with urea or thiourea or both, reduced (e.g. by tris(2-
carboxyethyl)-phosphine), methylated (e.g. by iodo-
acetamide), and digested with endoproteinase [155].
Using 2-dimensional gel electrophoresis (2-DGE) the
proteins can be enriched on basis of hydrophobic in-
teraction, heparin and hydroxyapatite chromatography
[156, 157]. In the case of phosphopeptides the enrich-
ment can be obtained by immobilized metal affinity
chromatography [158]. Histidine rich proteins can
also be pre-concentrated and purified by immobilized
nickel affinity chromatography [159]. For the purifica-
tion and further concentration of the proteins capillary
reversed phase chromatography can be applied [160].
A suitable development in sample preparation used in
proteomics is the introduction of in-gel digestion kits.
A summary of the sample preparation methods for
proteomics is compressed presented in a paper by
Wells and Weil as a perspective study including a
clearly arranged diagram of the protein separation pro-
cedures with focus on mass spectrometric detection
[161]. Additionally, they have listed preparation and
automation products of divers companies. For a first
quantification of the whole proteins the determination
of the total amount of proteins e.g. in cytosols of
microbial cells or EPS is helpful and can be carried
out by Lowry (alkaline copper), Bradford (Coomassie
stain) and Smith (bicinchoninic acid) protein assays
with spectrophotometric detection [162].
Traditionally, proteins are separated by 1-dimen-
sional polyacrylamide gel electrophoresis (1DGE),
visualized as spots coloured by dyes and labelled with
markers by immunoblotting and by chemical meth-
ods. In this way, the quantification of the proteins is
time-consuming and only a few proteins on each gel
16
can be analysed because of the lack of standards. A
new generation of gel electrophoretic methods have
been developed in the last two decades. The two-
dimensional gel electrophoresis (2DGE) is the elec-
trophoretic method of choice. The application in the
separation of hundred to thousand proteins is pre-
sented in [163]. Similar electrophoretic techniques
such as two dimensional fluorescence difference gel
electrophoresis (DIGE) can be applied for protein anal-
ysis according to their isoelectric point and molecular
mass with subsequent tryptogenic digestion of the
proteins of interest [164].
Mass spectrometric methods in combination with
gel electrophoresis make a complete protein analy-
sis possible. The mass spectrometric analysis can be
carried out by matrix-assisted laser desorption ioni-
sation or electrospray ionisation mass spectrometry
(MALDI-MS and ESI-MS). Lahm and Langen present
in their publication the application of mass spec-
trometry as a tool for the identification of proteins
separated by gels in general [165]. The analysis of
gel-separated proteins begins with the generation of
peptide fragments by electroelution and digestion in
solution, electrotransfer on a membrane and cleavage
on the membrane, direct digestion in the gel matrix,
and the digestion during blotting processes, respec-
tively. The identification of the peptides generated
can be achieved by peptide mass fingerprinting or
peptide sequence information. For detailed structural
information the data obtained by MALDI-MS analysis
in combination with data from MS=MS experiments
seems to be a potential possibility analysing the pep-
tides in the lower sub-pmol level.
Despite of automation in 2-DGE-MS the conver-
sion of individual proteins from their entrapped form
in the gel is time-consuming and does not meet the
demand of high-throughput in contrast to liquid form.
Current development has focused on the use of liquid-
phase separation techniques in combination with MS.
The most suitable LC-MS technique for proteome
analysis is the so called multidimensional protein
identification technology (MUDPIT) based of on the
separation of proteolytic peptides by LC and their
identification by directly coupled electrospray ioniza-
tion-tandem mass spectrometery (ESI-MS=MS) [109].
Applied mass spectrometers are time of flight (TOF),
quadrupole and ion trap MS [166]. The combination
of a quadrupole and a time of flight mass spectro-
meter is known as QTOF. The most sensitive MS tech-
nique is the Fourier-transform cyclotron resonance
E. Denkhaus et al.
mass spectrometry (FTICR-MS) showing the best
resolution and mass accuracy in comparison to other
mass spectrometer [166, 167]. Methodologies of mass
spectrometry and other analytical technologies are
reviewed in several papers [166–168]. Promising ap-
proaches are the liquid-junction interfacing of the sep-
aration column outlet and the inlet of the capillary
sprayer using nano-ESI, and the miniaturized ESI
techniques. Banfield et al. have detected more than
two thousand proteins in a natural acid mine drainage
microbial biofilm applying LC-MS=MS [169]. They
found proteins involved in protein refolding and re-
sponse to oxidative stress to be highly expressed.
Beside protein analysis the LC-MS=MS can also be
used for the characterisation of ubiquinones neutral
lipid extract of microbial cells and biofilm commu-
nities [170]. The separation of the microbial ubiqui-
nones was achieved using a C18 phase and a mobile
phase consisting of methanol-isopropyl alcohol con-
taining ammonium acetate. The advantage of the
method described is that no derivatisation is required
in comparison to the analysis by GC-MS [171].
Finally, it has to be noticed that 2-DGE and MUD-
PIT are complementary technologies, and to achieve
optimal coverage, both should be used in proteomics.
Microsensors in biofilm analysis
Microsensors suitable in biofilm analysis are minia-
turised electrochemical and fiber-optic sensors with
tip diameter smaller than 20 mm. The small tip size
provides a high spatial resolution and is a condition
precedent to in situ probing of laboratory assisted and
natural biofilms. Due to their small dimensions and
analyte consumption they are applied in the research
of formation of a biofilm, influx and efflux of sub-
stances and nutrients (e.g. rates of nutrient consump-
tion, depth of nutrient penetration, mass-transport rates,
diffusivity), microenvironmental conditions including
concentration profiles of dissolved substances and
biofouling processes [172, 173]. Furthermore micro-
sensors are suitable studying microbiologically in-
fluenced corrosion (MIC) of water transporting pipes
or cooling systems [174–178] and biocide testing on
biofilms [179].
The electrochemical microsensors can be divided
into three groups, the first one are sensors on basis
of potentiometric microelectrodes, the second one is
based on amperometric microelectrodes including a
biological reaction in the sensor tip, and the third
Chemical and physical methods for characterisation of biofilms
one are summarized under the commonly used term
micro-biosensors. Detailed reviews are presented by
[172, 173]. The potentiometric microsensors are based
on charge separation of ions across a membrane and
can be subdivided into sulphide microsensor working
as a silver half cell, ion-selective microsensors in form
of pH glass electrodes [172, 173], liquid ion-exchange
(LIX) microelectrode [172, 173, 180], redox micro-
electrodes (normally a platinum wire in a glass capil-
lary combined with a suitable reference electrode)
[172, 173, 181], and carbon dioxide microsensors with
(sodium) bicarbonate as internal electrolyte that equi-
librates with carbonic acid [172, 173]. As to new
developments further miniaturization of the potentio-
metric microsensors are described, e.g. a new carbon
dioxide microsensor with a tip diameter of less than
20 nm [180] and a redox microelectrode array with
four-probe glass electrodes each with a tip diameter
of 200 nm [181].
Amperometric microsensors measuring the cur-
rent, caused by electrochemical reactions of the ana-
lyte on the surface of the electrode, can be applied for
measurements of oxygen, sulphide, hydrogen, chlor-
ine, hydrogen peroxide, and mass-transport dynamics
[172, 173, 182]. Amperiometric microelectrodes used
are working without membranes and in different
experimental arrangement due to the biological pro-
blem: measurements of the local mass-transport coef-
ficient, the local effective diffusivity or the local flow
velocity.
The third group of electrochemical microsensors,
the micro-biosensors, are based on reaction of organic
molecules with the biological material immobilized
on the electrode tip transducing the chemical signal.
Miniaturised biosensors for the quantification of glu-
cose, methane, nitrate and nitrous oxide are described
in the literature [172, 173]. On the other side the min-
iaturization causes the problem of decreased sensitiv-
ity because the space for immobilizing the biological
materials is decreased in comparison to macroscale
biosensors. Furthermore the lifetime of the micro-bio-
sensors are very short (a few days to maximal several
month in the case of methane biosensor).
Another possibility to report biofilm growth on-
line and in real time is the use of optical micro-
sensors. One example of an optical microsensor is
an integrated illuminated fiber based optical sensor
(micro-optode), measuring the light scattered by the
material deposited on the tip [176]. The optical sys-
tem can contain light emitting diodes (LED) for fluor-
17
escence excitation, excitation filters, emission filters,
and a beam splitter. A tapered fiber-optic microsen-
sor with a tip diameter of 10 nm to measure profiles
of backscattered light in biofilms is presented by
Beyenal et al. [183]. Recently, a special fiber based
sensor, a photonic-crystal fiber (PCF) Bragg grating-
based biosensor is characterised for the detection of
selected single-stranded DNA molecules, hybridised
to a biofilm [184]. Keirsse et al. have published an
optical sensor operating in the mid-infrared (MIR)
spectral domain, using a chalcogenide glass fiber
[185]. A further example is the use of a laser trian-
gulation sensor for the measurement of the surface
roughness and thickness of a biofilm [186]. The main
advantages of optical over electrochemical sensors
are chemical inertness, internal reference, and geo-
metric versatility. The disadvantage is the restricted
tip diameter, usually 20–30 mm, because of coupling
with the optical device.
In conclusion, the application of microsensors in
biofilm characterization are restricted by their limited
commercial availability and their isolated information
caused by the heterogeneity on the microscale and the
lack of suitable mathematical models of biofilm accu-
mulation and activity. The isolated information can be
circumvented adding information provided by mole-
cular techniques.
Atomic spectrometry
Only a few papers have been published on the analysis
of free and bound metals in biofilms although the
elemental analysis in aquatic systems is a very impor-
tant task to understand uptake and accumulation pro-
cesses of trace elements into the trophic chain [187].
Van Hullebusch et al. presented a review about the
mechanisms and analytical tools according to the
metal immobilisation by biofilms [188]. This review
shows the advantage of using a combination of differ-
ent techniques to evaluate the fate of metals within
microbial biofilms. It has been demonstrated that an
interdisciplinary approach is required to study metal
fate within the biofilm matrix. The review gives an
overview about the different mechanisms involved in
the immobilisation of metals within biofilms e.g. bio-
sorption, sulphide precipitation, phosphate precip-
itation etc. Furthermore, extraction techniques and
methods for localisation and identification of metals
in the biofilm have been presented, focused on X-ray
absorption spectroscopy techniques.
18 E. Denkhaus et al.

X-ray spectrometry [196]. Also Ivena et al. has been investigated the sin-
gle and joint effects of increased phosphate and metal
To study the metal accumulation of biofilms, total-
(Cd, Zn) concentrations on benthic diatom com-
reflection X-ray fluorescence spectrometry (TXRF)
munities in microalgal biofilms by graphite furnace
[189–194] has been used following different oxidative
atomic absorption spectrometry (GFAAS) [197].
digestion procedures. TXRF is a highly sensitive
Dong et al. applied the classical Langmuir and
method for determining trace elements in the low
Freundlich adsorption isotherms to estimate equili-
absolute mg range and is therefore applicable to small
brium coefficients of Pb and Cd adsorption to biofilms
sample sizes. Friese et al. used a TXRF method to
and associated minerals as surface coatings [198].
analyse heavy metal contents of aquatic biofilms iso-
Correlation analysis between the maximum adsorp-
lated from stones collected from, and ceramic plates
tion of Pb and Cd and components in the surface
exposed in of the River Elbe [189]. Kr€opfl et al. devel-
coatings indicate that the adsorption of Pb to Mn oxi-
oped a TXRF method for elemental analysis of lead
des and the adsorption of Cd to Fe oxides are sta-
and nickel contaminated natural biofilms grown on
tistically higher than the adsorption of Pb and Cd to
polycarbonate substrates [191]. For elemental analysis
other components.
one part of the sample was freeze-dried and for tax-
onomical investigations the second part was stored in
Lugol-solution. A part of the freeze-dried biofilm Photospectrometric methods
sample was digested in concentrated nitric acid apply-
Only a few publications are available according to the
ing a microwave assisted digestion system. As internal
quantitative determination of polysaccharides in bio-
standard, gallium was added to each solution. It was
films [77, 120, 199]. They all used the phenol-sulfuric
established that the accumulation of the bivalent
acid method according to Dubois et al. [199] although
cations Pb and Ni were practically the same, and in
the development of the chemistry and biochemistry of
their presence the accumulation of zinc decreased by
polysaccharides is now connected mainly with the use
approximately 20% while the other elements (Ca, K,
of chromatographic methods.
Fe, Mn, Sr, Ti, Rb) showed smaller changes within the
Another application of photometric and fluorimet-
statistical interval. Mages et al. analysed natural fresh-
ric methods is the determination of enzyme activities
water and biofilm samples collected at different sites
in biofilms [200]. The analysis of enzyme activities
at the upper catchment area of the Tisza river system
in biofilms is mostly restricted to representative
in Hungary by TXRF [187, 193]. The biofilms were
enzymes involved in the degradation of a particular
taken from natural supports or from artificial supports.
biopolymer.
They used a portable TXRF spectrometer for in-field
These enzyme activities are considered as indica-
water analysis to get a rapid screening of trace ele-
tors of the microbial potential of polymer degradation
ments just before the confluence of the rivers [194].
and metabolism in their respective environments.
Záray et al. studied freshwater biofilms grown on
Enzymes usually act on more than one substrate. This
polycarbonate substrata by applying TXRF and taxo-
allows for the use of synthetic substrate analogs in-
nomical techniques [192].
stead of natural substrates for detection of enzyme
Toner et al. have been examined the molecular spe-
activities. Hydrolytic activities in biofilms were mostly
ciation of Zn within the Pseudomonas putida biofilm
determined using model substrates consisting of a
with Zn K-edge extendes X-ray absorption fine struc-
chromogenic or fluorescent molecule bound to a sub-
ture (EXAFS) spectroscopy [195].
strate monomer via ester, glycoside, or peptide (amide)
bonds. Upon hydrolysis of the colourless or nonfluo-
rescent model substrate, the chromogenic of fluoro-
Atomic absorption spectrometry
genic groups are released, this can be monitored by
Especially in the beginning of biofilm analysis the an increase in absorbance or fluorescence.
atomic absorption spectrometry (AAS) has been used A summary of applications is given in [200].
for the determination of selected heavy metals (Cu, Table 3 summarizes selected methods for biofilm
Zn, Pb) [3]. Even Admiraal et al. discussed the gra- monitoring and examinations and gives informa-
dual changes in micro- and macro benthos under high tion about the performances and limitations of each
metal loads determining Zn, Cd, and Fe by AAS method.
Chemical and physical methods for characterisation of biofilms 19

Table 3. Comparison of methods for biofilm monitoring and examination

Method Type of information Performance criteria Limitations References
extracted
Surface and interface characterising techniques
Light microscopy Microbial morphology; – Simple – Depth examination is [33, 35, 37]
thickness and density – Rapid not possible
– No complex sample – Low resolution
pre-treatment – Often requires biofilm
removal (destructive)
Scanning electron Biofilm structure – High resolution – Preparatory techniques are [35–37]
microscopy (SEM) prone to artefact; interior
structure of the biofilm is
not accessible
Combination of Microstructural – Useful to compare images – Only thin biofilms can [47]
SEM and optical characteristics obtained in situ, without be investigated
microscopy of thin biofilms invasive pretreatments but at
low magnification with those
obtained ex situ, with pretreatments
and at higher magnification
Confocal laser – Quantification of – Simple and accurate, able to – Expense of the equipment [32, 35, 36,
scanning EPS by measuring distinguish cells from inorganic – Not applicable to thick 38, 41–46]
microscopy the total cell volume particulates and opaque biofilms
– In situ monitors – Enables the internal structure – Low resolution
biofilm structure of the biofilm to be visualized
Scanning – Mapping of the – Speciation and spatial distribution – low penetration depth [48, 49]
transmission distribution of of metals – only 2D-imaging
X-ray microscopy macromolecules in – can be applied to fully hydrated
(STXM) microbial biofilms biological materials
– Quantitative analysis
of ferrous and ferric
iron; Ni(II); Mn(II)
Atomic force – Topographical – High resolution, ability to capture – Some dehydration of sample [35, 36, 50]
microscopy information biomolecular behaviours during examination; no
in real time compositional information
Cryoembedding Internal structure; – Simple and applicable to biofilms – Sample pre-treatment [31, 32]
and sectioning thickness from a wide variety of systems, is necessary
methods variability even for opaque biofilms, any
fixation or dehydration
Infrared – Chemical – Non-invasive – Interference from water [35, 36,
spectroscopy information – in situ in IR absorption 51–53, 55–58]
Distinction between biotic – FT-IR-ATR: penetration
and abiotic deposit depth limited to thin
biofilms
ATR-leaky mode – Monitoring of – In situ – Low local resolution [201]
spectroscopy biofilm growth
Photoacoustic – Depth-resolved On-line – High technical effort [37, 59–62]
spectroscopy analysis of optically – In situ
(PAS) and acoustically – Distinguishing between different
inhomogeneous media, biofilm components is possible
detect growth or
detachment over time
X-ray – Structural information – Large penetration – High technical effort [63, 64]
spectroscopy – Speciation depth
– Inherent molecular
scale sensitivity
– In situ
Reflectance – Measurement of – Rapid screening – Semiquantitative [74]
spectroscopy biofilm formation – Investigation of opaque and
non opaque surfaces

(continued)
20 E. Denkhaus et al.

Table 3 (continued)
Method Type of information
extracted
M€ossbauer – Investigation of microbial-
spectroscopy mineral interactions
Nuclear magnetic – Metabolic pathways
resonance – Hydrodynamic and mass
transport phenomena
Separation techniques
Extraction – Separation of EPS from
microbial cells
Field-flow – Characterisation of
fractionation natural colloids, particles
– Determination of particle
size distribution
Molecular – Identification of different
techniques bacterial cells forming
a natural biofilm
– Determination of up-and-
down regulation of genes
PCR techniques- Determination of genetic
MALDI-TOF and phylogenetic properties
on subgenomic scale
Chromatographic and electrophoretic methods
HPLC techniques EPS; mono-, oligo- and
e.g. HPAEC-PAD, polysaccharides; proteins
HPLC-ESI-MS
LC-FTICR-MS Proteome analysis
CE Carbohydrates
2D gel Separation of proteins
electrophoresis-
MALDI-TOF
GC-MS; Carbohydrate profiling
GC-MS-MS of bacteria, fatty acids
GC-MS Identification and
quantification of atrazine
and acetochlor
GC-Flame Separation and quantification
photometric of butyltin compounds
detector (FPD)
Performance criteria Limitations References
– Differentiation between – High technical effort [203]
Fe2þ and Fe3þ species
– Compounds in small amounts
– Non-invasive – Low sensitivity [35, 68–73]
– Time consuming
– Diverse extraction methods – Low throughput [77, 79–84]
exist achieving high amounts – All lysis dependent on
of carbohydrate, proteins, the extraction method
humic substances, respectively
– Minimised sample alteration – Not applied on biofilms [86–91,
– Easy hyphenation 94–97]
– Miniaturisation is possible – Requirement of specific [99–102,
(PCR-CE) DNA fragments 104–115]
– Sensitive – Data bases are needed
– Ability to directly measure – Transient snapshoot
gene expression of gene expression
– In situ – Lack of standards [116]
– Rapidly – Limited sequence
– Small amount of DNA is needed information available
in databases
– High operating costs
Qualitative and quantitative – Sample preparation See Table 2
determination of the compounds is necessary [169]
– Low detection limits – Decomposition of
– Miniaturisation is possible the biofilm
– High sensitivity – Need of combinatorial [166, 167]
– High mass accuracy and resolution libraries
– Combination with MALDI – High technical effort
and ESI technique
Qualitative and quantitative – Sample preparation is See
determination of the compounds necessary Table 2
– Low detection limits – Decomposition of the
biofilm
– Huge number of proteins – Lack of standards [163, 165]
can be separated – Limited sequence
– High selectivity information available
in databases
– High operating costs
– Low throughput
Qualitative and quantitative – Sample preparation and [146]
determination of the compounds derivatization to volatile
– Low detection limits compounds are necessary
– Decomposition of the
biofilm
– High selectivity and sensitivity – Time-consuming [204]
procedure because of the
sample preparation
– High selectivity – Time-consuming [202]
procedure

(continued)
Chemical and physical methods for characterisation of biofilms 21

Table 3 (continued)
Method Type of information
extracted
Microsensors – Formation of a biofilm,
influx and efflux
– hygienic aspects
– Study of microbiologically
influence of corrosion
(MIC)
Spectrometric methods
Fluorescence – Biomass quantification
– Microbial activity
Atomic absorption – Determination of heavy
spectrometry metals and nutrients
Total-reflection X-ray – Determination of heavy
fluorescence metals and nutrients
spectrometry (TXRF)
Photospectrometric – Determination of the total
methods amount of polysaccharides
and proteins
– Determination of enzyme
activities
Electrochemical method
Amperometry Determination of phosphate
Performance criteria
– Miniaturised system
– Chemical inertness,
internal reference,
geometric versatility
of optical microsensors
– Non-invasive
– In situ
– Low detection limits
– Highly sensitive method;
applicable to small
sample sizes
– Simple
Cost effective, rapid in situ
measurements, easy
to operate good selectivity,
low detection limit
Limitations References
– Decreased sensitivity [172–186]
caused by miniaturising
– Short life-time of biosensors
– Restricted tip diameter
– Limited commercial
availability
– Isolated information caused
by the heterogeneity
on the microscale
– Fluorescence quenching [35]
– Cascade and inner filter effects
– Time consuming because of [4, 196–198]
single element analysis
– Decomposition of the biofilm
Digestion procedures are [187, 189–191,
necessary, direct analysis 193, 195]
of biofilms suffer from
limitations according to
the calibration, high
background intensity
– Overestimation [77, 120,
199, 200]
– Possible interference [205]
of silicate
– Batch injection analysis
suffers from non-stability
of the signal during repeated
measurements

Conclusion microscopic area in future, also under the point of

view of suitable reference materials covered to the
Research on biofilms offers many new activity fields
heterogeneity of a biofilm. For the quantification of
in analytical chemistry. The enterprise of projects,
a biofilm in the macroscopic area possibilities on the
realization and the evaluation of analytical results
base of mathematical and statistical models are
require interdisciplinary collaboration. In the liter-
already available, for the microscopic area, however,
ature different model attempts are discussed with
not. Additionally, a better transparency and coopera-
regard to architecture, activities and their accompany-
tion of diverse disciplines including databases with
ing functions of the biofilms and different experi-
unique formats are necessary for a complete under-
mental approaches. Investigations of the phenotypic
standing of biofilm formation and activities. As far as
differences between biofilms and their planktonic
the qualitative and quantitative analysis of carbohy-
counterparts are interesting according to medical
drates, proteins, (phosphor) lipids, humic substances
questions but less as far as questions with environ-
(see Table 1) concerned analytical methods with higher
ment relevance to biodegradation, biofouling and hy-
throughput should be developed. This requires inte-
gienic relevance concerned. Innovative experimental
grated microsystems of sample preparation and separa-
attempts are asked to examine biofilms in their natu-
tion units for nanoscale analysis and new hyphenated
ral habitat. The biggest challenge will be the quanti-
techniques of multidimensional separation systems
fication of the whole compounds of a biofilm in the
22
with specific detectors. First attempts have been made
with the coupling of LC-nano ESI-MS=MS and PCR-
CE (lab on a chip). Ideally, biofilm analysers would be
reagentless, field portable, reusable, of low cost and of
easy software for data acquisition and interpretation.
In summary, it could be shown that analytical ques-
tions have been extremely changed in biofilm research
by interdisciplinary during the last years. In conse-
quence, the time is coming for a new definition and
position of analytical chemistry. Analytical chemistry
should not be only a tool but more a scientific part in
this field. Hopefully, this article creates a platform for
discussion due to this aspect.
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