Biofilm Formation in The Industry: A Review: Food Reviews International January 1992

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Biofilm formation in the industry: A

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Tiina Mattila‐Sandholm & Gun Wirtanen
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Food Reviews International, 8(4), 573-603 (1992)
BIOFILM FORMATION IN THE

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INDUSTRY: A REVIEW
TIINA MATTILA-SANDHOLM and GUN WIRTANEN
VTT Food Research Laboratory
PO Box 203
SF-02151 Espoo, Finland
ABSTRACT
Biofilm and biofouling refer to biological deposits on any surface.
Biofilms consist of both microbes and their extracellular products,
usually polysaccharides. The purpose of biofilm is to protect the
microbes from hostile environments or to act as a trap for nutrient
acquisition. Biofilm formation causes problems in many branches
of industry, such as in industrial water systems and the medical and
process industries. Besides causing problems in cleaning and hygiene,
biofilm may cause energy losses and blockages in condenser tubes,
cooling fill materials, water and wastewater circuits, and heat ex-
change tubes, and on ship hulls. Biofilm can also present micro-
bial risks due to the release of pathogens from cooling towers or
by reducing water quality in drinking water distribution systems. In
the medical industry biofilm is referred to as glycocalyx when dis-
eases of the lungs or the gastrointestinal or urinary tract are involved.
573
Copyright © 1992 by Marcel Dekker, Inc.

574 MATTILA-SANDHOLM AND WIRTANEN
INTRODUCTION
This review describes the diversity of biofilm as a phenomenon and assesses its
significance in the process industry. Only a brief section on methodology in bio-
film research is included; this area has been covered in several recent articles
(1-3). Furthermore, industrial water systems and the medical industry are only
briefly discussed because books and literature surveys have recently been pub-
lished in these areas (4, 5). The main effort has been focused on drawing to-
gether the considerable amount of industrial experience in the area of biofilms.
Many industries are in fact facing similar problems and most likely could profit
from a better knowledge of the results obtained in different fields.
BIOFILM FORMATION AND ITS CONSEQUENCES
Biofilms consist of both microbes and their extracellular products, usually poly-
saccharides (6-11). Microbes have a tendency to form these protective polysac-
charide matrices when they have adhered to surfaces. The formation of biofilm
complexes requires only some water and a surface material. In fact, a deficiency
of nutrients may increase the biofilm formation capacity of microbes (12,13). Bio-
film can generally be produced by almost all microbes under suitable conditions,
but some microbes have a higher tendency to produce biofilm than others. The
most common biofilm producers belong to the genera Pseudomonas, Entero-
bacter, Flavobacterium, Algaligenes, Staphylococcus, and Bacillus. There are
also anaerobes that can produce corrosive biofilms (14-18).
Biofilm serves to protect the producer microbes from hostile environments.
In practice this often means a combat between microbes and the preservatives,
disinfectants, antibiotics, or biocides used against them. Microbial resistance to
antibiotics depends on their metabolic activity. When microbes achieve the static
growth phase, they react totally differently than when they are in the active grow-
ing phase (19-21). The effect of many antibiotics is based on inhibition of active
growth. It has been pointed out that most of the bacteria in biofilms are no longer
growing actively and that their resistance is therefore high. It has also been stated
that the resistance of biofilm cannot be demonstrated without controlling the
growth rate of the microbes (22). In addition to the possible importance of growth
rate, other factors such as the permeability barrier also affect the practical effi-
ciency of antibiotics (23). Biofilm also acts as a trap for acquiring nutrients, which
may be important when bacteria grow on surfaces and their nutrient acquisition
is limited (6, 24-26).
In addition to causing problems in cleaning and hygiene, biofilm can cause
energy losses and blockages in condenser tubes, cooling fill materials, water and
BIOFILM FORMATION 575
Table 1 Effects and Relevance of Biofilms in Various Processes (27)

Effect in industrial fields Biofilm problems and applications
Heat Transfer Reduction
Power industry Biofilm formation in condenser tubes and on cooling tower fill
Chemical process industry material causes energy losses.
U.S. Navy
Solar energy systems
Increase in Fluid Frictional Resistance
Municipal utilities Biofilm formation in water and wastewater conduits as well as
Power industry condenser and heat exchange tubes causes increased power con-
Chemical process industry sumption in pumped systems, reduced capacity in gravity sys-
Solar energy systems tems, and causes energy losses.
U.S. Navy Biofilm formation on ship hulls results in increased fuel con-
Shipping industry sumption.
Mass Transfer and Chemical Transformations
Power industry Accelerated corrosion due to processes in the lower layers of
U.S. Navy the biofilm results in material deterioration in metal condenser
Municipal utilities tubes, sewage conduits, and cooling tower fill.
Chemical process industry
U.S. Navy Biofilm formation on remote sensors, submarine periscopes,
Water quality control sight glasses, etc., results in reduced effectiveness.
Public health Detachment of microorganisms from biofilms in cooling towers
releases pathogenic organisms (e.g., Legionella in aerosols).
Municipal utilities Biofilm formation and detachment in drinking water distribu-
Public health tion systems changes water quality in distribution systems.
Dental health Biofilm formation on teeth causes dental plaque and caries.
Human health Attachment of microbial cells to animal tissue causes diseases
of lungs, intestinal tract, and urinary tract.
Wastewater treatment Extraction and oxidation of organic and inorganic compounds
Water treatment from water and wastewater (e.g., rotating biological contact -
Stream analysis ers, biologically aided carbon adsorption and benthal stream
activity) required to reduce pollutant load.
Pulp and paper industry Biofilm formation in industrial production processes reduces
product quality.
Chemical process industry Immobilized organisms or communities of organisms for con-
ducting specific chemical transformations.
Desalination Fouling biofilm accumulation reduces effectiveness of ion ex-
Industrial water treatment change and membrane processes used for high-quality water
treatment.
wastewater circuits, and heat exchange tubes, and on ship hulls (27). Biofilm
can also cause corrosion or present microbial risks due to the release of pathogens
from cooling towers (Legionellá) or by reducing water quality in drinking water
distribution systems (28). In the medical industry, bio film (usually caused by
Pseudomonas species) is referred to as glycocalyx when diseases of the lungs or
the gastrointestinal or urinary tracts are involved (8, 9). Dental plaque is also a
typical form of biofilm (27). In addition, biofilm (usually of Staphylococcus
species) is one of the main contamination problems in foreign-body instruments
such as cardiac pacemakers, catheters, prothèses, artificial valves, etc. (6, 7,29-
35). Biofilm problems are also encountered in granular activated carbon columns,
reverse osmosis membranes, ion exchange systems, degasifiers, RO/DI water
storage tanks, and microporous membrane filters (36). Table 1 (27) lists the di-
versity of biofilm problem areas. It should be noted, however, that the biofilm
formation can also be utilized to advantage (37, 38).
The process industry is faced with biofilm problems at many stages. Biofilm
causes reduced effectiveness in ion exchange and membrane processes (39-41).
It accelerates corrosion and material deterioration, for example, in sensors and
detectors (4). The bacterial level of purified water used has been directly corre-
lated with the number of defects occurring in microelectronic devices (36). Con-
ductance, electromigration, and corrosion in the oxide layers have been noticed
in the manufactured devices (36). The paper industry has been fighting biofilm
(slime) for many years (42, 43). One of the most important factors in prevent-
ing biofilm in the process industry is the quality of surfaces and especially their
smoothness. Dead ends, corners, cracks, crevices, gaskets, and joints are the
most vulnerable points for biofilm accumulation. It is somewhat alarming that
pathogens such as Salmonella spp. and Yersinia spp. and also the famous Listeria
have been shown to produce biofilm complexes and thus create severe disinfec-
tion and cleaning problems on the surface materials used in the food industry
(44).
Microbe-induced corrosion is sometimes encountered in processing equip-
ment. This may appear in association with biofilm formation or without bio-
film. However, the corrosion often occurs with biofilm of microbial origin (4,
28), leading to enormous losses in different industrial areas, for example, piping
and cooling water systems. The corrosion is an electrochemical process brought
about by a potential difference between two different metals or two zones within
the metal. Consequently, an electrical circuit with cathodic and anodic areas is
formed (45). The microbes take part in the corrosion directly in the anodic-
cathodic reactions and indirectly in changing the surface properties through their
metabolic activities or through accumulation of electrochemical cells. The pres-
ence of microbial cells can also change the environment so that it corrodes the
metal surface (46, 47).
THE OCCURRENCE OF BIOFILM IN INDUSTRIAL SYSTEMS

AND RELATED PROBLEMS
Drinking Water Systems
Biofilm formation can occur in many different water ecosystems (48-51), and
in these environments biofilm is very evident. The number of microbial cells
adhering to surfaces may be 500 to 50,000 times the amounts of planktonic cells
in water (52).
The adherance of microbial cells to surfaces in piping and water distribution
systems is well known (14, 53-55). In drinking water systems sufficient nutrients
are present for planktonic cells to form biofilm, since the amounts needed are
not large. Microbes of the genera Flavobacterium, Moraxella, Acinetobacter,

Bacillus, Pseudomonas, Alcaligenes, and Achromobacter are common in drink-
ing water distribution systems. Coliforms indicate that the water is of poor hy-
gienic quality. The most common coliforms found in water ecosystems are Escheri-
chia coli, Klebsiella sp., Enterobacter cloacae, and Serrâtia marcescens (14, 15).
Biofilm formation appears in lavatories, sinks, taps, valves, and in different
joint areas throughout the piping systems. This effect should be taken into ac-
count when systems are designed and the water quality is to be ensured at a cer-
tain level (44). When the effects of biofilm are observed, mechanical rinsing and
shock treatment with chlorine or monochloramine should be performed.
The amount of free planktonic cells in the water does not necessarily correlate
with the amount of biofilm on the pipe surfaces. Free chlorine (1.0 mg/L) had
no effect on coliforms growing in the biofilm and even greater amounts (2.0
mg/L) did not kill Escherichia coli in the biofilm (55). Stressing factors can actually
induce biofilm buildup. Therefore, the possibility exists that chlorination may
induce formation of biofilm (56). It has in fact been reported that chlorination
induces certain bacteria such as Pseudomonas species to grow in biofilm (15,
57). Pseudomonas spp. are rather resistant to chlorine treatments and can even
multiply when chlorine has been used.
From a hygienic and health point of view, biofilm can cover organisms that
cause infections and diseases, and these organisms can also form biofilms. Le-
gionella pneumophila is a dangerous organism that is able to form biofilm. Le-
gionella is a major problem in houses with hot water systems (28, 58, 59). It has
been found that rubber is the best material for supporting growth and biofilm
formation by Legionella and that copper is the most resistant (60).
The material on which microbes form biofilm has a major influence on the
effects of the disinfectants used. The capsular Klebsiellapneumoniae was shown
to have a 150-fold greater resistance to chlorine when it grew on glass surfaces
rather than in suspension. When a low-nutrient liquid was used, this ratio was
further increased to about 600. On metal and carbon surfaces the correspond-
ing resistance ratios were about 2400 and 3000 times the value in suspension (56,
60, 61).
Biofilm has also been found in swimming pools and whirlpools. Microbes
such as Pseudomonas attach easily to surfaces of hydrophobic materials such
as polystyrene. Hoses, tubes, filters, etc., containing Polyvinylchloride increase
the risks of contamination (62). The pH value of water often increases when
contamination occurs. Pseudomonas aeruginosa has been isolated from water
even with a chlorine concentration of 3-5 ppm.
Cooling Water Systems

Cooling water systems may be either open or closed. In open systems the water
is taken directly from natural water sources such as seas and rivers, and the water
is returned after circulation in the cooling system. In closed systems there is a
circulation system with heat transfers between the process and the water source.
It is clear that closed systems are, for both functional and hygienic reasons, better
than open ones. The levels of microbes can be controlled by regular mechanical
and chemical cleaning and treatment with biocides. In contrast, the hygiene in
open systems is difficult to control and special filtration treatments must be used.
Treatment with biocides cannot be used since the water is returned to the water
source (44).
Biofilm formation in cooling systems has been investigated widely (4, 63-69).
The average temperature in these systems is 35°C, which is close to the optimum
temperature for most microbial growth. It has been found that temperatures
below 50°C promote biofilm formation (28, 69, 70). The primary colonization
of bacteria and molds is often followed by accumulation of algae and clams.
The microbes obtain nutrients from the algae mass, thus initiating a vicious circle.
The most common microbes in cooling waters are the slime-forming Pseudo-
monas and Gallionella, and the most common algae are Ulothrix, Spirogyra,
and Chloronella. Macroorganisms such as the Asiatic clam Corbicula have also
been found in cooling water systems (71).
The most evident problem caused by biofilm in cooling systems is that the heat
transfer may be decreased to only 10% of maximum efficiency. Other problems
include microbial corrosion, increased flow resistance, blockages and increased
accumulation of particles on surfaces, decreased efficiency of biocides and cor-
rosion inhibitors, and increased health risks (28, 72, 73).
Studies simulating real industrial situations have been performed using mi-
crobes isolated from water circulation systems. The formation of biofilm takes
at least days; and in low-nutrient water systems, even weeks. The flow rate of
water is a critical factor. If the flow rate of water is high, the biofilm formation
is rapid. When the rate exceeds 1 m/s, the nutrients are not a critical factor (26,
74). The biofilm formed in slow-flowing systems is structurally different from
that formed in systems with high flow rates. In low-nutrient systems a higher
flow rate is needed for the formation of deep films as compared to nutrient-
rich environments. A higher flow rate also removes the biofilm more efficiently
than slow rates (69, 74, 75). If the amount of oxygen is minimized in the water
system, the amount of biofilm formed can be decreased by more than 50% (69).
When the amounts of both oxygen and nutrients are minimized, biofilm forma-
tion may be decreased by more than 80%. However, anaerobic conditions favor
anaerobic organisms, the growth of which can cause increased corrosion (4, 5,
25, 69, 75, 76).
Process Industry
Problems with biofilms are very common in those branches of industry in which
organic materials are processed. However, since most of the biofilm consists of
water and cannot be readily seen on dry surfaces, biofilm problems have not
been widely recognized, attention being mainly focused on contamination prob-
lems. In contrast, biofilm is a well-recognized problem in the pulp and paper
industry, and in water circulating systems, where large amounts of slime occur.
The choice of surface materials is of great importance when designing and
building equipment and processing lines for industrial use (77). If the surface
materials are smooth and in good condition (i.e., no cracks and no dead cor-
ners), they are easier to clean. Bends, pockets, and extended joints form dead
zones, where microbial growth is favored. Problems such as accumulation of
particulates and cells will occur where the cleaning is for any reason inappro-
priate. If glass surfaces are used they should be even, transparent, and hygienic.
Unfortunately, glass is expensive, has a low pressure tolerance, and breaks easily.
The most useful material in processing equipment is steel, which can be treated,
for example, by mechanical grinding and electrolytic polishing. In electrolytic
polishing a preground surface is treated in an electrolytic bath to produce an
even surface (44). Many equipment faults can be avoided by using good design
practice guidelines. Table 2 lists the most important factors of good design prac-
tice (44).
Gaskets in equipment are favorable places for biofilm growth, because dirt
and different kinds of nutrients accumulate on the gasket material (78). Experi-
ments with Pseudomonas fluorescens, Listeria monocytogenes, and Yersinia
enterocolitica, known to cause contamination of gaskets in industrial use, have
been carried out under controlled conditions (79). Rubber- and teflon-based
materials often used in gaskets are easily contaminated. Some microbes are also
able to decompose rubber and use it as an energy source (78, 80).
Table 2 Good Design Practice (44)

Area of concern Factors to consider
Process and material handling Process parameters
Installations
Maintenance of hygiene
Equipment Materials
Surface finishing
Accessories and joints
Checking and cleanability
Clearing and cleaning
Insultion
Pipelines Accessories (valves, etc.)
Materials
Surface finishing
Clearing and cleaning
Consoles
Insulation
Equipment layout Cleanability and cleaning of equipment
Servicing of equipment
Rationalization and systematization of operations
Systematic layout
Automation Control of production
Reliability of process
Production control Working plan
Follow-up of material flow
Disturbance reports
Electrification Shielding of equipment
Lighting
Buildings and structures Surface materials
Painting
Waste disposal
Tipping
Lead-throughs
Cleanability of places and surfaces
Layout of connections joining different production units
Air conditioning
Organization and personnel Functions, tasks
Responsibility
Training
Valves are also good sites for microbial growth. Due to their construction,
globe valves are a hygienic risk. Therefore membrane and conduit valves should
be used in processing lines because the risks are lower. Poorly designed sam-
pling valves can destroy the whole process or give incorrect information about
the process due to the effects of biofilm. Furthermore, any probes measuring
environments that include biofilm will not provide an accurate description of
the process. Measurement probes and the inner parts of tanks, etc., are usually
difficult to clean, and biofilm is easily formed on them. Thick layers of biofilm
and crust on the inner surfaces protect the microbes from disinfectants. The
crust on the surface is porous and together with air hampers the penetration of
steam and decreases the heat transfer, and therefore the effect of sterilization
(44).
In fermentation processes biofilm formation causes hygienic problems on sur-
faces, in membranes, and on probes and other devices. Fouling and accumula-
tion of proteins are common problems in filtration techniques, for example, in
ultrafiltration equipment and on membranes (81). Blockages in filtration equip-
ment are usually not caused by microbes but by biopolymers and sediments con-
taining proteins. The process may change the rheological properties of the sedi-
ments and lead to gel formation. These problems are solved by changing flow
rates and acidity (pH values), according to a program which is designed differently
for each process. Microbial accumulation is known to be a problem in reverse
osmosis membranes. The cleaning process associated with reverse osmosis is
more complex because the cellulose acetate membranes are destroyed when treated
with strong chemicals, acids, and enzymes, and when the temperature is increased
(39, 82).
Food Industry
The cleanliness of surfaces, instruments, and equipment in the food industry
affects the quality of the products processed. If the cleaning and sanitation pro-
cesses are inadequate, the products may be contaminated. When biofilm is formed
around microbes, they are protected against sanitizers and disinfectants. The
biofilm also affects heat transfer and thus the sterilization time is increased (83).
For example, slime production affected the heat resistance of a Bacillus strain
so that the time required for autoclave sterilization was increased to several hours.
Bio film can also alter the resistance to steam and formalin (44). Other common
contaminants are lactic acid bacteria, micrococci, streptococci, enterobacteria,
Pseudomonas fragi, and Listeria monocytogenes. Frank and Koffi found that
L. monocytogenes in biofilm was resistant to all the disinfectants tested and also
to heat treatment (83). Cationic and anionic surface active agents did not affect
the microbes growing at the surface. Listeria species adhere to equipment and
processing line surfaces over a wide temperature range (10-35°C) (83, 84).
On the basis of empirical experience, the biofilm phenomenon has a long his-
tory in the food industry. The occurrence of slime-forming microbes is in many
ways a problem in the sanitation and disinfection of process equipment. The
slime appearing on any surface is one type of biofilm, for example, slime on
sausage (85) and on meat surfaces (86-88). However, research into the bio film
phenomenon has begun only recently in the food industry. Pseudomonas fragi
(ATCC 4973), Pseudomonas fluorescens, and Acinetobacter spp. have been re-
ported to produce biofilm (89-100). Salmonella typhimurium and Yersinia enter-
ocolitica were reported as biofilm producers at the IAMFES conference (Inter-
national Association of Milk, Food, and Environmental Sanitarians) in 1990.
Pulp and Paper Industry

The pulp and paper process is an industrial area in which biofilm formation causes
major problems. Paper machines are connected to water circulation systems, in
which the environment favors rapid biofilm growth (101). Biofilm formation
and microbial corrosion on steel surfaces in paper machines have been studied
and anaerobic sulfate-reducing bacteria have been found in these systems. Cor-
rosion is caused by these bacteria and also by excessive concentrations of chlorine
(6, 43,102,103). When the acidity is increased, sedimentation and crust accumu-
lation favor the growth of microbes (104).
Practical experiments show that biofilm in the pulp and paper processing equip-
ment detaches easily from the surface. The problem in paper manufacturing is
that the loose slime causes holes in the paper. It has been reported that, even
when machines are all fabricated from the same steel, the problems that occur
may still be different (103). The most common group of bacteria found in paper
machines belongs to the Enterobactericae, but Pseudomonas sp., Clavibacter
sp., and Bacillus sp. have also been isolated from the slime. The slime may con-
tain traces of iron, chromium, copper, and chlorine (42, 103).
Chain Conveyor Lubricants
Lubricants used in conveyors can create problems, especially for the food in-
dustry, particularly dairies and breweries (105,106). The lubricants can be com-
pletely oil based but they may also contain water. If the lubricant contains water
it is very susceptible to microbial growth. Pseudomonas sp., Acinetobacter sp.,
Algaligenes sp., and sulfate-reducing bacteria have been isolated from lubri-
cants. The biofilm in the lubricant can also promote corrosion indirectly (17,
107).
Listeria monocytogenes is a risk organism which has been isolated from bio-
film in lubricants in dairies (105). In breweries, lubricants are very severe sources
of contamination and the biofilm formed increases the resistance of the microbes
to disinfectants (108). Nowadays synthetic lubricants containing biocides are

used and the problems are decreasing in severity (109). Problems with biofilm
are also encountered in engineering works, where lubricants based on vegetable
oils are used. Legionella has also been found in lubricants (28, 107).
Air Conditioning Systems
The microbial population in air channels depends on the environment, filtra-

tion membranes, and air openings. The cleaning of these channels is effected
most easily by good design practice. The structures and filters should be easily
cleaned. When the air conditioning system is cleaned and disinfected it is very
important that the disinfection medium penetrates the film on the surface and
does not simply flow through the system with the air stream (44).
The formation of biofilm in air conditioning systems does not occur without
a water reservoir of some kind or condensed water. Normally, water does not
exist in the air conditioning systems of houses, but it may get in because of tem-
perature differences and condensation (44). When the air conditioning is con-
nected to a cooling, heating, or humidifying system, there is usually an open
circulating water system. The membranes in the air conditioning system and the
walls in the air conditioning channels are places where biofilms start to grow
(110).
Biofilm can cause severe problems if the microbes in the biofilm are harmful
to health. The dangerous Legionella pneumophila has been isolated from water
systems connected to an air conditioner (28, 108). However, Legionella sp. can
also occur in air conditioning systems without biofilm. If the biofilm is formed,
it is very difficult to remove the microbe from the system (73).
Medical and Instrument Industry
Antibiotics used for disinfection in medicine usually affect only planktonic mi-
crobes and leave those embedded in the biofilm intact. The microbes are thus
able to retain their viability although the patient is receiving antibiotics. The mea-
surement of resistance to antibiotics is usually made in suspensions, and the true
effect on microbial growth may not be measured (44). Tobramycine is effective
against Pseudomonas aeruginosa in suspension at a concentration of only 50
¡ig/mL, but even if the concentration is increased to 1000 /¿g/mL it has no effect
on the microbes in biofilm. When the biofilm is broken, the effective concen-
tration needed is again only 50 ¿ig/mL (6, 35). In another experiment, a well-
established biofilm of P. aeruginosa withstood a treatment with 200 ¿ig/mL to-
bramycine whereas the corresponding concentration needed for a newly formed
biofilm was only 20 ¿tg/mL. In a suspension test the concentration needed to

destroy the cells collected from the surface of the broken biofílm was only 5
(111-113).
Disinfectants
Disinfectants have been developed to destroy microbes. Despite this aim, con-
taminants are found in disinfectants. As early as 1967 Burdon and Whitby found
that chlorhexidine and savlon (a mixture consisting of chlorhexidine and cetri-
mide) were contaminated with microbes of Pseudomonas species. This problem
was temporarily overcome when 4% isopropylalcohol was added. Later, Pseudo-
monas species such as P. aeruginosa and P. pickettii were found both in savlon
and iodine solutions (33, 114-116). The amount of P. aeruginosa in an iodine-
based disinfectant used for disinfection of water pipes has been as high as 104
cfu/mL (114). According to these investigators, the Pseudomonas microbe was
still viable after a period of 96 days in a concentrated iodine solution.
Serratia marcescens was viable after a period of 27 months in a disinfectant
containing 2% chlorhexidine, although in suspension a concentration of 0.1% is
sufficient to kill the cells (33, 52). Microbial contamination has also been found
in solutions of aldehydes, quaternary compounds, and amfotensides (108). In
most investigations the biofilm has been detected using electron microscopy.
Other microbes that have been isolated from disinfectants include Alcaligenes
faecalis, Enterobacter cloacae, Escherichia coli, Flavobacterium meningosepti-
cum, and Enterobacter agglomérons. Gram-positive organisms have not been
found. It is alarming that even when efforts have been concentrated on hygiene,
the contamination rate has not decreased (117).
Instruments and Facilities

Many materials used in medicine consist of plastics, rubber, and metals. The
use of these materials has improved both surgical and medicine treatments enor-
mously. Unfortunately, foreign pieces in the body such as catheters, pacemakers,
medullar nails, and different kinds of prostheses make microbial colonization
possible, and this can be the origin of severe inflammations and sepsis. These
inflammations are often very resistant to antibiotics. Bacteria causing inflam-
mations often belong to the genera Staphylococcus or Pseudomonas (118-128).
The microbes are embedded under the layers of the biofilm and are therefore
difficult to isolate and eliminate. Biofilm has been found on many different sur-
faces such as:
1. Transparent dressings and surgical sutures (6, 7, 9).
2. Wound drainage tubes and air/water syringes (AWS) (6, 7, 9, 129).
3. Intra-arterial and intravenous catheters, silastic cardiac catheters, pulmonary

artery catheters, peritoneal catheters, urinary catheters (6, 7, 9, 29, 30, 35).
4. Hemodialysis buttons and tubes (6, 7, 9, 34).
5. Water reservoirs of unheated nebulizers (130).
6. Endocardial pacemakers (32).
7. Bioprosthetic and mechanical hearth valves (6, 7, 9, 34).
8. Prostheses and prosthetic hip joints (6, 7, 9, 34, 131).
9. Intrauterine contraceptive devices (31).
10. Contact lens storage cases and solutions (132).
METHODS OF STUDYING BIOFILM

Formation of biofilm in the laboratory environment is rather difficult mainly

because biofilm is not readily produced in laboratory media, which are rich in
nutrients. Biofilm is a protective matrix with the purpose of acquiring nutrients,
and when nutrients are present in excess, bacteria usually do not produce bio-
film and are unprotected (6, 111). Many reviews have been written on this topic
and methods are now available for producing biofilm in the laboratory (1-3).
Hitherto, circulating systems such as the Robbins device or Rototorque systems
have been the most popular (15, 52,133-135). This equipment and its modifica-
tions have been used both in laboratory and field assessments (52, 133-137). In
biofilm detection studies, it should be borne in mind that about 85-96% of bio-
film consists of water. If biofilms are detected on dry surfaces, the total volume
is only 2-5% of the initial volume (9, 70,138). The sessile mode of growth should
always be remembered when detecting biofilm, and the counts of planktonic
cells in the circulating fluids should be interpreted carefully because they do not
always represent the sessile organisms (63, 70, 106, 139).
Methods of studying biofilm formation include microbiological methods and
physical and chemical methods (19, 69, 138, 140-145). When organisms from
extreme environments are cultured, standard plate counts may not give accurate
estimates. The classical evaluation methods relying on microbiological plating
may therefore prove unreliable (36). However, microscopic techniques such as
fluorescence microscopy and especially electron microscopy are very informa-
tive (1, 134, 146-148). Interference reflection microscopy (IRM) has also been
used for investigation of biofilms (149). Investigating microbial growth with
lasers at frequencies between 190 and 260 nm causes shifts in wavelength of the
scattered photons and can thus be detected as a resonance Raman spectrum
(150). Bacterial biofilm formed on crystals of zinc selenide and germanium has
been detected by Fourier transformation infrared spectrometry (FT/IR) (151).
Recent trials with nuclear magnetic resonance (NMR) and scanning confocal
laser microscopy have also been carried out and appear promising (38).
ELIMINATION OF BIOFILM
The elimination of biofilm is a very difficult and demanding task. Mechanical
cleaning is the most efficient way of removing biofilm, but frequently the struc-
ture of the equipment makes this very difficult. The use of brush balls for cir-
culating systems also has many drawbacks because the circulating balls with
abrasive surfaces must have the same diameter as the tubes. Furthermore, high-
pressure washing systems have limitations because they cannot be used for closed
systems. Cleaning-in-place (CIP) systems have not been designed to eliminate
biofilms, although CIP can prevent biofilm formation if the equipment design
and materials are suitable (94-96, 99, 152).
The choice of materials and their surface treatments such as grinding and
polishing are the most important factors in inhibiting biofilm formation (24,
77). The material should be corrosion resistant. Treatment of surface materials

to reject biofilms can be divided into two different types: surfaces actively re-
moving the biofilm and surfaces passively rejecting the biofilm. The former cat-
egory may be based on electrochemical reactions such as the hydrolysis of poly-
saccharides or oxidation of biofilm layers. Surfaces rejecting biofilm can also
be divided according to the agents used in the surface finishing, such as copper
oxides, organotin alloys, and nonpoisonous compounds (153,154). The system
should be designed without dead ends and it should be cleaned frequently to
avoid the accumulation of biofilm. The cleanliness of surfaces, training of per-
sonnel, and good manufacturing and design practices are the most important
tools in combating biofilm formation.
Biocides have frequently been used in cooling systems and in the pulp and
paper industry (73). It should be borne in mind that oxidants and other chemi-
cals added for disinfection purposes may increase the amount of biodegradable
material in treated water. In consequence, biocides designed to prevent biofilm
formation may actually increase the amount of nutrients available for growth.
Coagulants, corrosion inhibitors, and ammonia may also introduce nutrients to
circulating systems (155). Generally the problem lies in increased amounts of
carbon, nitrogen, or phosphorous (155).
It is also important to remember the multicomplexity of biofilm when de-
signing methods for its elimination: biofilm consists of both the microbes and
their surrounding polysaccharides (6, 10). This means that satisfactory results
with biocides or disinfectant treatments alone cannot be achieved even if their
penetration capacity is very efficient. Breakage of the polysaccharide matrix is
essential, because the matrix protects the remaining microbes and can even act
as a nutrient source. Enzymatic approaches to the removal of biofilms have not
been widely utilized due to the complexity of the polysaccharide matrix (44).
Drinking Water Systems

Biofilms in drinking water systems have mainly been eliminated by using com-
pounds containing chlorine or hypochlorite. The action of chlorine-based biocides
can be divided into three phases: loosening of the biofilm from the surface, break-
ing of the biofilm, and disinfection by active chlorine (156). The effect of the
biocide and the penetration ability of chlorine-based biocides are both mainly
based on the oxidative properties of chlorine (7). However, Exner et al. (34) re-
ported that even a very high concentration of chlorine (10 mg/L) was not sufficient
to kill bacteria growing in the biofilm. In experiments carried out by Seidler et
al. (157), Klebsiella pneumoniae was found in water tanks after cleaning and
sanitation with water containing 200 mg chlorine per liter (14, 15, 34, 56).
Normally a rather low concentration of 3-5 mg/L active chlorine is sufficient
for biofilm elimination (158). The disinfection effect of chlorine is affected by
the age of the biofilm, the surface material, the encapsulation of microbes, and
nutritive factors (56). Oxidative biocides such as hypochlorite are more efficient
when a higher concentration is used for a short time. The amount of free chlorine
is also important for the breakdown of biofilm formations. A high flow rate
and rinsing improves the effect. An increased temperature is also favorable, but
the upper limit for heating is about 50-60°C. Most outbreaks of Legionella have
occurred when the temperature has been too low (28, 69, 159).
Practical experiments with Legionella growing in biofilms have been carried
out. Disinfection was carried out with 10 mg hypochlorite per liter for 240 min,
2% aldehyde for 80 min, 2% peroxide for 60 min, and 3% peracetic acid for 5
min. None of these treatments eliminated the test bacterium completely. Legion-
ella was normally found again in the water pipelines within 3 days of the treat-
ment. When peracetic acid was used, microbes were detected after only 56 h;
and the times for aldehyde, peroxide, mechanical, and hot water treatment were
46 h, 16 h, 16 h, and 2 h, respectively (160).
In drinking water systems, chloramine-based compounds (monochloramine)
have become poular due to their penetration capacity (54, 56). Monochloramine
is good because it penetrates the polysaccharides very well and the effect de-
pends only on the surface material, not on the age of the biofilm or on the char-
acteristics of the microbes (155). In trials with monochloramine, the amount of
coliforms was reduced from 56.1% to 18.2% (54, 56, 161). Although the effect
of hypochlorite on planktonic microbes is greater than the effect of monochlor-
amine, the monochloramine is better in practical use because of its penetration
qualities. As was stressed earlier, the effect on planktonic cells has nothing or
very little to do with the microbes in biofilms. Comparison of equal activities
(concentration and time) of hypochlorous acid, chlorine dioxide, and mono-
chloramine on the same bacteria grown on metal strips showed that monochlor-
amine was most effective for the inactivation of biofilm bacteria (155). On the
basis of concentration and time data of unattached cells, it would be predicted
that monochloramine would be 1200-fold less effective than hypochlorous acid.
However, experiments with biofilm bacteria revealed a totally different picture.
Mechanical cleaning is a decisive factor in combination with biocides in the
elimination of biofilms from water pipelines and circulating systems. The forces
achieved by high-pressure water rinsing are an alternative to mechanical clean-

ing. However, induced breakage of biofilms is essential for the effective use of
biocides. An extreme biofilm problem cannot be overcome using only shock
treatment with biocides. The effect is temporary without a combined treatment
using both biocides and mechanical cleaning. Otherwise, the microbes are back
on the surfaces within a week after the treatment (14). In daily life it is impos-
sible to clean pipelines completely by mechanical treatment. There will always
be bends, corners, pockets, cracks, etc., where biofilm will remain; and from these
places microbes are distributed to the whole system with planktonic cells in the
water flow (14). In all effective systems, both types of treatment are essential.
Cooling Water Systems
Different kinds of biocides have been tested in cooling water systems. The bio-
cides usually used in closed circulating systems are listed in Table 3 (73). The
effect of oxidizing biocides is usually good. The effect of l-bromo-3-chloro-
5,5-dimethylhydantoin was reported to be 10- to 20-fold that of chlorine com-
pounds. When treatment with this biocide was combined with zinc molybdate
compounds, the corrosive effect was lowered (67). A specific biocide containing
metronidazole and biodispersant for treatment of obligate anaerobes causing
corrosion has also been developed (162).
Table 3. Typical Chemicals Utilized in Cooling Water Systems (73)

Corrosion inhibitors Scale inhibitors Biocides
Ferrous metals Acrylates Oxidizing biocides
Anodic Lignosulfonates Bromine
Chromate Methacrylates Chlorinated isocyanurates
Molybdate Phosphonates Chlorine
Nitrite Polycarboxylic acid Chlorine dioxide
Orthophosphate Polyphosphates Halogenated hydantoins
Cathodic Sodium hypobromite
Phosphonates Dispersants Sodium hypochlorite
Polyphosphates Acrylates
Zinc Lignosulfonates Nonoxidizing biocides
Methacrylates Aldehydes
Copper and its alloys Polycarboxylic acid Halogenated bisphenols
Benzotriazole Isothiazalones
Mercaptobenzothiazole Biodispersants Organobromines
Tolytriazole Ethylene oxide copolymers Organosulfurs
Quaternary ammonium
Quaternary phosphonium
Different types of programs are available for the control and inhibition of
biofilm formation in cooling water systems. Especially in these systems, the costs
of maintenance and chemicals such as biocides must be considered. Mainten-
ance includes removal of oxygen from water, control of dirt accumulation, cir-
culation of chlorine, finishing of the surface material of the pipelines, ozone
treatment, use of ultrasonication, and UV light. The use of place-adjusted chlorine
treatment has been applied successfully in power plants (163). The maintenance
programs guarantee that excess biofilm accumulation does not occur. Elimina-
tion is successfully accomplished by using both mechanical cleaning (e.g., brush
balls) and biocides (76). The elimination of biofilm can also be performed by
flushing. Pressure washers are efficient in pipelines in which the pipes are straight.
In systems including bends, corners, etc., brush balls are more efficient.
In systems using seawater mechanical cleaning has often been carried out with
Tapproge rubber balls. The circulation of these rough balls in the water flow
cleans the inner surfaces of the pipelines. Successful sanitation requires treat-
ment with some kind of chlorine-based agent (44). Different kinds of combined
cleaning and sanitation systems for heat exchangers using Tapproge balls and
chlorine treatment have been investigated. The effectiveness of these treatments
was determined by measuring changes in heat exchange and in the rate of corro-
sion. The surface material is very important when cleaning programs are de-
signed. Aluminum surfaces need mechanical cleaning, whereas for titanium sur-
faces chlorine rinsing is usually sufficient. The cleaning of surfaces containing
both copper and nickel was performed with a combination of mechanical and
chemical treatments. The mechanical cleaning was too harsh, breaking the pro-
tective film on the surface and thus leading to corrosion (68). In the United States,
systems like Amertap with rough rubber balls and MAN flow-driven brushes
have been used (76).
In experiments carried out in cooling towers, the results obtained with methyl-
chlorine/methylisotiazolone in the elimination of biofilms formed by algae, fungi,
and bacteria have been promising. The results achieved using glutaraldehyde,
quaternary ammonium salts, tributyltinoxides, and méthylène bisthiocyanates
were not good. Glutaraldehyde was only effective against algae. Tributyltin-
oxides and quaternary ammonium salts were effective against algae and fungi
but not against bacteria. The méthylène bisthiocyanate used had no effect on
the biofilm formers (164). In power plant water systems, good results have been
obtained using hydrogen peroxide and iron sulfates (165). Biocides for the elim-
ination of microbes causing corrosion are being developed continuously (64).
The effects of biocides on microbes, both aerobic and anaerobic iron sulfate-
reducing, found in the oil processing industry have been investigated both in
the Robbins device and in suspension tests. The microbes were allowed to grow
in a flowing system with natural nutrients (137). The microbes on the surfaces
were 20-50 times more resistant to the biocide treatment than the cells in the
suspensions. The treatment using isothiazolone was the most effective against
microbes in biofilms, the aerobic microbes being killed in 6 h using a concentra-

tion of 15 mg/L. In order to inactivate the anaerobic microbes, a treatment with
30 mg/L was needed. In a flowing system a continuous concentration of 7.5
mg/L isothiazolone was sufficient to control the biofilm formation. The effects
obtained using 100 mg/L glutaraldehyde were only limited. Agents such as di-
bromonitrilo-proprionamide (DBNPA) and alcyldimethylbenzyl-ammonium-
chloride (ADBAC) were less effective than isothiazolone and glutaraldehyde
(52, 166). These results demonstrate that the microbes growing both on the sur-
face and in the biofilm must be considered in biocide research (167).
An alternative method for biofilm control is the use of ozone in cooling water
systems. PVC pipes coated with titanium, aluminum, and epoxy were used in
the trials. Ozone treatment with 5 mg/L for 5 min/day was sufficient for elim-
ination of biofilm. The results were not equally promising for all the microbes
tested and the ozone did not affect the algae at all (168). Disadvantages of ozone
treatment are high costs and the corrosion effect (76).
Other Circulation Systems
Hydrogen peroxide was very effective in removing biofilms from equipment

used in hospitals. It possessed biocide properties and was able to penetrate the
biofilms (34). The aldehydes did not break the biofilm but rather seemed to im-
prove its stability. Peracetic acid proved to be microbicidal but its effect on mi-
crobes in biofilms was limited (34). If mechanical treatment of biofilms cannot
be used in the water circulation systems used in hospital equipment, the pipe-
lines should be cleaned and sanitized with hydrogen peroxide. When the biofilm
is broken in some other way, chemical agents such as peracetic acid and alde-
hydes can be used (34). The effect of hydrogen peroxide is based on the produc-
tion of free radicals, which degrades the polysaccharides. Concentrations of
1-10 mM hydrogen peroxide are recommended, although these concentrations
are too low to break down cells. Negative results have also been obtained with
this compound (10, 44).
Process Industry
The elimination of biofilm in processing industry should be based on systematic

planning using suitable materials (169). Biofilm is a symptom of disturbances in
the process, and its elimination may remove the symptoms but not the under-
lying faults. The biofilm is a "disease" in the equipment and like all other dis-
eases, an ounce of prevention is worth a pound of cure (170). It is easier to de-
sign the process well rather than to repair it continuously during operation. The
approach to preventing the "disease" should be based on the use of the right
materials and structures. The design and layout of the process must also be con-
sidered.
Microbiological problems in the processing industry usually originate from
incorrect decisions in the design of equipment and plants. To prevent problems
in the industrial plant the design should follow commonly used technical pro-
cedures which have been shown to be effective. Quality assurance and control
are as important during planning as in plant operation. Most planning agencies
apply a quality system based on the ISO 9000 standards. A good-quality sys-
tem requires educated and skilled planning personnel, control of and guidance
in planning, application of project instructions, follow-up of quality directives,
use of norms and well-designed models, attention to government regulations,

etc. All this together is called good design practice (Table 2; Ref. 44).
The basis of hygienic maintenance is created when the process is planned. The
methods for cleaning, cleaning programs, cleaning and disinfection agents and
the use of biocides, cleaning equipment and accessories, and irradiation should
all be chosen before production begins. Attention should also be paid to the
quality of processing water, steam, and additives. The process may easily be
spoiled by using additives of poor quality. The growth of microbes can also be
inhibited by choosing processing conditions which differ from the optimum for
the growth of the organisms (44). It is noteworthy that the list of factors affect-
ing biofilm potential in industrial water systems, introduced by Flemming, could
be applied to the process industry rather easily (Table 4; Ref. 53).
Second only to the planning of the process and the design of the plant is the
program "introduction" and the education of the personnel. A basic factor in
the education should be the processing hygiene. The quality of products in a
factory where processing hygiene is poor cannot be good. The problem is to some
extent one of attitudes. Production in the pharmaceutical industry is based on
good manufacturing practice norms (GMP norms). The GMP norms provide
common rules that should be followed (44).
Food Industry
The elimination of biofilm from food processing equipment has not been widely
reported. Holah et al. utilized biofilms in their studies on cleaning and disinfec-
tion for the food industry (146, 147). Generally speaking CIP treatment is as-
sumed to protect the process line from biofilm formation. The surfaces and dead
zones in the line are, unfortunately, places where microbes may accumulate and
cause contamination of the process. When the biofilm has been left to grow it is
very hard to remove. The CIP systems used nowadays are based on a combina-
tion of acid-alkaline or alkaline-acid treatment and time-temperature treatment.
Table 4. Biofouling Potential in Industrial Water Systems (53)

High potential Low potential
Plant Design
Extended piping Short piping
Dead legs, fissures No dead legs or fissures
Armatures with dead zones Specially designed armatures
Access of light Exclusion of light
Materials leaching nutrients Inert materials
Rough surfaces Smooth surfaces
Nondisinfected holding tanks Disinfectable holding tanks
Feedwater Characteristics
Temperatures > 25 CC Low temperatures
High concentrations of: Low concentrations of:

Organic nutrients Organic nutrients
Inorganic nutrients Inorganic nutrients
Cells Cells
Particles Particles
Operational Parameters
Intermittent operation Continuous operation
Rare monitoring of biofouling Good monitoring of biofouling
Unattended ion exchanger and activated Limited and monitored filter systems
carbon filters
Changing raw water composition Constant and controlled raw water com-
position
Contaminated chemicals Clean chemicals
Cleaning only after failures Early and preventive cleaning
Poor access to surfaces Good access to surfaces
Insufficient efficiency of cleaning measures Good efficiency of cleaning measures
The CIP treatment can be improved by mechanical cleaning with brush and cloth.
Of course, CIP does not provide 100% certainty that the process is clean. Prob-
lems caused by equipment construction, valves, etc., and surface materials can-
not be eliminated with CIP (44).
Trials with small-scale circulation systems to which CIP has been connected
have been carried out by the dairy industry. In these investigations the amount
of living bacteria in the circulation water and of polysaccharides accumulated
on surfaces have been measured (94). The results showed that the amount of
living bacteria did not correlate with the accumulation of biofilm (94). It was
also apparent that the time between CIP treatments should be less than 8 h in
order to prevent biofilm accumulation. Furthermore, living cells at +4°C ap-
peared to be unable to form fibers of polysaccharides or biofilm. It is clear that
the frequency of cleaning is important because it is usually easier to remove young-
er biofilms than older ones.
Recent studies by Wirtanen and Mattila-Sandholm (171) showed that food

microbes such as Listeria monocytogenes, Pseudomonas fragi, Enterococcus
hirae, and Bacillus subtilis had increased resistance toward disinfectants such
as sodium hydroxide-hydrochloric acid, hypochlorite, iodophores in phosphoric
acid, quaternary ammonium compounds, and anionic tenside containing peroxi-
sulfate when grown on surfaces rather than in suspensions. It was noteworthy
that all the disinfectants tested left the formed glycocalyx intact. The residual
glycocalyx could have a role in further biofilm buildup and could provide good
protection for cells remaining alive under the film (171, 172).
In the prevention of biofilm, some possibilities that can be used in practice
include cleaning with detergents such as alkaline chlorine compounds, alkaline
compounds, and acidic compounds. The effect of the detergent used is affected
by the ready-to-use concentration, the active agents in the compound, the type
and material of the surface, the frequency of treatments, temperature, wash-
ing time, flow rate, water quality, and the type and age of the biofilm treated
(173, 174). Better results in cleaning of food processing equipment and process-
ing lines have been achieved using alkaline chlorine compounds (0.4% v/v) than
with acidic treatments of corresponding concentration. Alkaline treatment with-
out chlorine has also been effective. Acidic treatment has proved to be ineffec-
tive, the effect being almost the same as with water (173). Detergents are more
effective in warm (>52CC) than in lukewarm (<41°C) water. When the washing
time was reduced from 15 min to 5 min and the flow rate was simultaneously
reduced from 2.1 m/s to 0.3 m/s, the effectiveness of treatments decreased radi-
cally (173).
Cleaning and sanitation procedures have also been performed with iodophores
and quaternary ammonium compounds (83,175). The research results have shown
that microbes in biofilm withstand the treatment better than microbes in suspen-
sions. The measurement of biofilm formation is very important when experiments
are performed. Have the cells only been allowed to adhere to the surface or has
real biofilm been formed? How long has the biofilm been allowed to deveop?
Older biofilms are usually more resistant to disinfectants than younger ones
(6, 10, 27).
Pulp and Paper Industry

Agents used in the pulp and paper industry for elimination of biofilm are divided
into three groups: oxidizing agents (e.g., chlorine, chlorine dioxide, hydrogen
peroxide, ozone), nonoxidizing agents (e.g., méthylène bisthiocyanate), and
enzymes. Oxidative and nonoxidative biocides have been used for a long time,
whereas the use of enzymes is currently on a trial basis. Enzymes such as glucose
oxidase, which form hydrogen peroxide, and levan hydrolase with the capacity
to degrade slime, have been tested (44, 71, 101, 176). It is very important that
the effects of biocides used are based on penetration of the biofilm. When biocides
are developed for the pulp and paper industry, the following aspects should be
remembered: many biocides are absorbed in the pulp and thus inactivated; addi-
tives used in paper production can neutralize the effects of biocides; the con-
tact time must be evaluated; trials should be performed as closely as possible to
practical conditions (substrates, fluids, mixing, etc.); and the biocide should be
effective on both aerobic and anaerobic microbes (101). The microbial quality
of the water circulating in the system should be checked and regulated, and suit-
able biocides should be used (44).
ALTERNATIVE METHODS AND CONCLUSIONS

Many combinations of different methods for eliminating biofilm are available.

Traditionally, detergents, biocides, and mechanical cleaning have been used in
the elimination of biofilm. However, it is important to remember the multi-
complexity of biofilm when designing methods for its elimination: biofilm con-
sists of both the microbes and their surrounding polysaccharides (6, 10). This
means that satisfactory results with biocides or disinfectant treatments alone
cannot be achieved even if their penetration capacity is very efficient. Break-
down of the polysaccharide matrix is essential, for example, by enzymes, be-
cause the matrix protects the remaining microbes and, furthermore, can act as
a nutrient source. However, applications of enzymes have been unsuccessful
because of the wide variety of polysaccharides formed and the specificities of
the enzymes used. In most cases the enzymatic treatment should be combined
with a microbicidal agent (44). Dali et al. (177) achieved good results using dex-
tranases for breaking biofilm formed on artificial heart valves. The biofilm orig-
inated from Streptococcus viridans, on which the pure antibiotic treatment had
no effect. Very good results were obtained with combined antibiotic and enzyme
treatment.
Metal chelaters (EDTA and EGTA) have been used for the breakdown of bio-
film layers in cutting oils and reactors (92, 108, 178-181). Chelaters have also
been used in the inhibition of biofouling-induced corrosion in processing of oils
(182). The chelaters bind ions of magnesium and calcium (Mg2+ and Ca2+), thus
destabilizing the outer membranes of the microbial cells. They are not them-
selves biocides and they should be combined with antibacterial compounds. Che-
laters in combination with quaternary compounds have shown a synergistic effect
(108). The effect of EDTA-chelaters on elimination of Pseudomonas fragi biofilm
was limited, but when EGTA was used, the effect was good. EGTA has also been
effective in eliminating biofilm in waters from waste treatment. The biofilm layers
were loosened within 5 min (92, 183).
A recent book edited by Flemming and Geesey considers many aspects of bio-
film elimination in various industrial water systems (4). The volume presents
several very valuable tables in which the elimination methods of biofilm have
been collected into a compact form (Tables 5 and 6). It can be seen from these
tables that an extensive battle against biofilm has been carried out without fully
controlling the system. However, it should be remembered that in the process
industry, equipment design plays the most important role in combatting biofilm.
A huge advantage can be achieved by careful selection and layout of equipment
based on a good understanding of biofilm formation sites, and by following the
good design practice rules. In the process industry empirical knowledge has been
gained over time in methods of prevention of fouling (152). It should be borne
in mind that microbes are present everywhere and that fouling is certainly a pre-
requisite for microbial attachment and growth, and biofilm formation.
The understanding that microbes grow differently on surfaces than in suspen-
sions is the first step in the development of advanced process hygiene regimens.
Table 5. Some Physical Methods of Cleaning Biofouled Surfaces (53)

Method Comments
Flushing Simple method; limited efficiency. Biofilms thinner than the viscous
sublayer are not sheared (184). Special case: flushing supported by
cleaners and/or after application of chemical agents which destabilize
the biofilm matrix and adhesion to the support.
Backwashing Effective for loosely adhering films in tubes, on filters, and to a cer-
tain extent in ion exchangers, but not below a certain level.
Air bumping Very limited efficiency.
Abrasive sponge Demonstrated efficiency, but possible problems because of abrasion
of protective oxide films.
Nonabrasive Extensively used in industry; problems with thick biofilms and with
smearing of organics by the sponge balls.
Sand scouring Difficult to control abrasive effects (185).
Brushing Very effective, but limited applicability and expensive; can lead to
the selection of firmly adhering species (186).
Hot water, steam Used in high-purity water systems in Japan with good results (187);
saves the use of expensive and possibly harmful and toxic chemicals.
Hot water systems may select for thermophiles and are reported to
carry biofilms including mycobacteria (188).
Ice nucleating Application of ethylene glycol at —12° destabilized the biofilm ma-
trix and detached it from the support (189); no practical experiences
reported.
Irradiation Very low efficiency against biofilms; entrapped particles and opaque
biofilms may shield bacteria; biofilms have even been reported on
protection mantles of UV irradiators (190).
Ultrasonic energy Promising method for soft biofilms; application limited to nonsensi-
tive material; some biofilms are extremely stable (191).
Table 6. Some Bioeides in Technical Use in Industrial Water Systems (53)

Chlorine Advantages: broad spectrum of activity, residual effect, advanced
technology available, can be generated on site, active in low
concentrations, destroys biofilm matrix and supports detach-
ment.
Disadvantages: toxic by-products (192, 193), degradation of re-
calcitrant compounds to biodegradable products, development
of resistance, corrosiveness, reacts with extracellular polymer
substances (EPS) in biofilms, low penetration characteristics
in biofilms, oxidizes S2~ to elemental sulfur [extremely difficult
to remove from surfaces (194)].
Hypochlorite Advantages: cheap, effective, destabilizes and detaches the bio-
film matrix (6), easy to handle; useful for biofilm thickness con-
trol (195).
Disadvantages: poor stability, oxidizing, rapid aftergrowth ob-

served (69), toxic by-products, corrosive; does not control ini-
tial adhesion (195).
CIO2 Advantages: can be generated on site, activity less pH dependent,
less sensitivity against hydrocarbons, effective in low concen-
trations.
Disadvantages: explosive gas, safety problems, toxic by-products.
Chloramine Advantages: good penetration of biofilms (54,56,196), reacts spe-
cifically with microorganisms; less toxic by-products, higher resi-
dual effect because of lower reactivity with water ingredients (57).
Disadvantages: less effective than chlorine against suspended bac-
teria (57), resistance observed.
Bromine Advantages: very effective against a broad microbial spectrum.
Disadvantages: toxic by-products, development of resistance.
Ozone Advantages: efficiency similar to that of chlorine, decomposes
to oxygen, no residues, weakens biofilm matrix.
Disadvantages: oxidizes bromide in seawater, reacts with organics
and can form epoxides (197), degrades humic acids and makes
them bioavailable (198-200), corrosive, short half-life, sensitive
to water ingredients.
H2O2 Advantages: decomposes to water and oxygen, relatively non-
toxic, can easily be generated in situ; weakens biofilm matrix
and supports detachment and removal (201).
Disadvantages: high concentrations ( > 3 % ) necessary, resistance
commonly observed, corrosive (202).
Peracetic acid Advantages: very effective in low concentrations, broad spec-
trum, kills spores, decomposes to acetic acid and water (203),
no toxic by-products known, penetrates biofilms.
Disadvantages: corrosive, rather unstable, increases DOC.
Formaldehyde Advantages: low costs, broad antimicrobial spectrum, stability,
easy application.
Disadvantages: resistance in some organisms (188), toxicity, sus-
pected of promoting cancer, reacts with protein-fixing biofilms
on surfaces (34), legal restrictions.
Table 6. Continued
Glutaraldehyde Advantages: effective in low concentrations, cheap, nonoxidiz-
ing, noncorrosive.
Disadvantages: does not penetrate biofilms well (197), degrades
to formic acid, increases DOC.
Isothiazolones Advantages: effective at low concentrations, broad antibiotic
spectrum (204).
Disadvantages: problems of compability with other water ingre-
dients, inactivation by primary amines.
Quaternary ammonia Advantages: effective in low concentrations, surface activity sup-
compounds (QUACs) ports biofilm detachment, relatively nontoxic, adsorb to sur-
faces and prevent biofilm growth (205).
Disadvantages: inactivation by low pH, Ca 2+ , Mg2+ (204), de-
velopment of resistance (206).
Thereafter it is necessary to appreciate that biofilm prevention is as important

as its elimination. Preventive operations against biofilm in the process industry
depend on the choice of surface materials and their finishing, good design, con-
trol of the process, and training of the staff. In practice, training is very impor-
tant because it affects the whole process, although this might not always be easy
to demonstrate. Surface materials, their finishing, and design are also factors
that may be very expensive if not planned for in advance. Last but not least,
basic research into factors promoting biofilm accumulation is still urgently needed
in order to provide the basic knowledge needed for process development.
ACKNOWLEDGMENT
This work was supported by the Technology Development Center (TEKES).

The valuable comments from representatives of the steering group of the project
and from experts in the processing industry are gratefully acknowledged: Paula
Nybergh, TEKES; Esa Aittomäki, Rintekno Oy; Tarja Kärkkäinen, Finnish
Meat Research Center; Juhani Nordlund, Valió; Pertti Walliander, Cultor Oy;
Kyósti Orre, Oy Alko Ab; Julia Mentu, Enso-Gutzeit Oy; Auli Haikara, VTT;
Liisa Viikari, VTT; and Outi Hyökyvirta-Varjonen, VTT.
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Bioﬁlm formation in the industry: A Review

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Biofilm formation in the industry: A

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BIOFILM FORMATION IN THE

Copyright © 1992 by Marcel Dekker, Inc.

BIOFILM FORMATION AND ITS CONSEQUENCES

Table 1 Effects and Relevance of Biofilms in Various Processes (27)

THE OCCURRENCE OF BIOFILM IN INDUSTRIAL SYSTEMS

Drinking Water Systems

not large. Microbes of the genera Flavobacterium, Moraxella, Acinetobacter,

Cooling Water Systems

Table 2 Good Design Practice (44)

Pulp and Paper Industry

Chain Conveyor Lubricants

to disinfectants (108). Nowadays synthetic lubricants containing biocides are

Air Conditioning Systems

The microbial population in air channels depends on the environment, filtra-

Medical and Instrument Industry

biofilm was only 20 ¿tg/mL. In a suspension test the concentration needed to

Instruments and Facilities

3. Intra-arterial and intravenous catheters, silastic cardiac catheters, pulmonary

METHODS OF STUDYING BIOFILM

Formation of biofilm in the laboratory environment is rather difficult mainly

77). The material should be corrosion resistant. Treatment of surface materials

Drinking Water Systems

achieved by high-pressure water rinsing are an alternative to mechanical clean-

Cooling Water Systems

Table 3. Typical Chemicals Utilized in Cooling Water Systems (73)

microbes in biofilms, the aerobic microbes being killed in 6 h using a concentra-

Other Circulation Systems

Hydrogen peroxide was very effective in removing biofilms from equipment

The elimination of biofilm in processing industry should be based on systematic

use of norms and well-designed models, attention to government regulations,

Table 4. Biofouling Potential in Industrial Water Systems (53)

High concentrations of: Low concentrations of:

Recent studies by Wirtanen and Mattila-Sandholm (171) showed that food

Pulp and Paper Industry

ALTERNATIVE METHODS AND CONCLUSIONS

Many combinations of different methods for eliminating biofilm are available.

Table 5. Some Physical Methods of Cleaning Biofouled Surfaces (53)

Table 6. Some Bioeides in Technical Use in Industrial Water Systems (53)

Disadvantages: poor stability, oxidizing, rapid aftergrowth ob-

velopment of resistance (206).

Thereafter it is necessary to appreciate that biofilm prevention is as important

This work was supported by the Technology Development Center (TEKES).

13. G. A. McFeters, M. J. Bazin, J. D. Bryers, D. E. Caldwell, W. G. Characklis, D. B. Lund,

36. M. W. Mittelmann, in "Biofouling and Biocorrosion in Industrial Water Systems" (H.-C.

ilmiöt teollisuudessa" [Biofilm formation in the industry], TEKES, Helsinki, 1991.

68. R. O. Lewis, Mater. Perform., 21(9), 31 (1982).

Seattle, 1978, p. 147.

103. S. A. Marmo, E.-L. Nurmiaho-Lassila, O. Varjonen, and M. S. Salkinoja-Salonen, in

113. H. Anwar, M. Dasgupta, K. Lam, and W. Costerton, J. Antimicrob. Chemother., 34,

137. P. F. Sanders, Int. Biodeterior., 24, 239 (1988).

170. C. C. Gaylarde, Int. Biodeterior., 26, 11 (1990).

183. M. H. Turakhia, K. E. Cooksey, and W. G. Characklis, Appl. Environ. Microbiol., 46,

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