Biofilm basics: Section 1

What are biofilms?

A quick overview

Biofilm in pipe Biofilm in stream in Biofilm scraped from Dental plaque is

section Yellowstone National a reverse osmosis biofilm
N. Zelver Park membrane, Courtesy, ASM Image
D. Davies C. Wend Library
AN INTRODUCTION TO THE BIOFILM LIFE CYCLE:
1) Free-floating, or planktonic, bacteria encounter a submerged surface and
within minutes can become attached. They begin to produce slimy extracellular
polymeric substances (EPS) and to colonize the surface.
2) EPS production allows the emerging biofilm community to develop a complex,
three-dimensional structure that is influenced by a variety of environmental
factors. Biofilm communities can develop within hours.
3) Biofilms can propagate through detachment of small or large clumps of cells,
or by a type of "seeding dispersal" that releases individual cells. Either type of
detachment allows bacteria to attach to a surface or to a biofilm downstream of
the original community.

In aqueous systems, microbial cells

are found as both "planktonic"
(floating) cells and "sessile"
(attached) cells on surfaces. For
generations, microbiologists
studied microbial cells only in their
planktonic state or grown in
laboratories as single-species
colonies on nutrient media. Today's
antibiotics, for example, were
developed by testing their efficacy
on cells in suspension or grown on
agar. The research of recent years
has revealed, however, that
bacteria preferentially attach to a
variety of surfaces, and that
bacterial communities exhibit
properties, behaviors and survival
strategies that far exceed their
capabilities as individual bacteria.
For instance, microbial biofilms
are naturally tolerant of antibiotic
doses up to 1,000 times greater
than doses that kill planktonic
bacteria.

Aggregations of microbes were

noticed long before people had the
tools to study them in detail. In
1684 Anthony van Leewenhoek
remarked on the vast accumulation
of microorganisms in dental plaque
in a report to the Royal Society of
London: "The number of these
animicules in the scurf of a man's
teeth are so many that I believe
they exceed the number of men in
a kingdom."
In a 1940 issue of the Journal of Bacteriology, authors H. Heukelekian
and A. Heller wrote, Surfaces enable bacteria to develop in substrates
otherwise too dilute for growth. Development takes place either as
bacterial slime or colonial growth attached to surfaces. It was not
until the late decades of the 20th century, however, that scientists and
engineers possessed adequate technology to effectively study microbial
communities and began to understand the significant implications of
the biofilm mode of growth.

The study of biofilms has skyrocketed in recent years due to increased

awareness of the pervasiveness and impact of biofilms on natural and
industrial systems, as well as human health. Biofilms cost the U.S.
literally billions of dollars every year in energy losses, equipment
damage, product contamination and medical infections. But biofilms
also offer huge potential for bioremediating hazardous waste sites,
biofiltering municipal and industrial water and wastewater, and
forming biobarriers to protect soil and groundwater from
contamination. The complexity of biofilm activity and behavior
requires research contributions from many disciplines such as
biochemistry, engineering, mathematics and microbiology. New
insights into the mysteries of biofilm are being published daily in a
wide variety of science and engineering journals. We welcome you to
the exploration of this rapidly expanding area of study.
Where do biofilms grow?

Natural environments
Biofilms grow virtually everywhere, in almost
any environment where there is a
combination of moisture, nutrients, and a
surface. Biofilms grow in rain forests and in
deserts, as "desert varnish." They have been
found at the bottom of the ocean as early
colonizers of new deep-sea vents and living
on glaciers in the Antarctic. Bacteria that live
in these very hot or very cold environments
are called extremophiles. Yellowstone
National Park in the United States is home to
an amazing array of colorful communities of
extremophiles. In fact, the entire globeboth
above and below groundis "seeded" with
the bacteria that form biofilms, and bacterial
communities flourish, disperse or become
dormant depending on changing
environmental conditions.

Biofilms are an important life-link in many natural

communities. These organisms form the basis for
food webs that nourish larger organisms such as
insect larvae, which are consumed by fish, that are
in turn consumed by birds like eagles. Biofilms
attached to particles of contaminated soils and
aquatic sediments can help degrade soil-bound
contaminants that occur from accidental chemical
releases into the environment.

Plants commonly have microbial populations
associated with their external tissues. One
beneficial type of plant-microbe relationship
occurs in the rhizosphere between the plant
roots, root hairs and a complex microbial
community. Plant roots secrete significant
amounts of sugars, amino acids, vitamins and
plant hormones that serve as nutrients for
microbes to grow on root hairs. This microbial
growth may facilitate the plant's ability to
absorb nutrients from the soil.
This streambed in Yellowstone National Park is
coated with biofilm that is several inches thick in
places. The warm, nutrient-rich water provides an
ideal home for this biofilm, which is heavily
populated by green algae. The microbes colonizing
thermal pools and springs in the Park give them
their distinctive and unusual colors. More
examples of these extremophilic bacterial
communities can be viewed in the slide show
below. Photo, above, courtesy of D. Davies.
Manufactured materials and systems
Microbes like to grow on surfaces, whether natural or manmade. When it comes to industrial
surfaces, bacteria are just doing "what comes naturally" by attaching to the surfaces with which
they come into contact. However, their propensity for attachment causes many problems for a
wide range of industries. Biofilm contamination and fouling occur in nearly every industrial water-
based process, including water treatment and distribution, pulp and paper manufacturing and the
operation of cooling towers. Biofilms are responsible for billions of dollars in lost industrial
productivity, as well as product and capital equipment damage each year. Biofilms are notorious
for causing pipe plugging, corrosion and water contamination.

Click to see Table 1-1, a summary of manufactured surfaces and systems affected by biofilm formation,
associated impacts, and journal references.

Health and the human body

This may come as something of a shock to you, but about 90 percent of the cells in a human body
are not human! In fact, the human body is heavily colonized by microbes that have found it a great
place to live. We have communities of microbes living on all mucous surfaces and in our digestive
tract, as well as on and in layers of our skin. For the most part we all get along; in fact, we depend
on some of our gut microbes to help with digestion. Sometimes, however, the microbial load
causes problems of infection. Dentists now understand, for instance, that dental caries (cavities)
are the result of bacterial infection (and biofilms!). When the normal balance of microbial
populations is upset or when our immune system is overwhelmed, we can have a real battle with
microbial opportunists.

In the 1990s, as the biofilm concept was being introduced to the medical community, doctors began to make
the connection between chronic, low-grade infections and the biofilm mode of growth. Dental professionals
made the connection easily, as teeth could readily be scraped for microscopic examination. Internal cases of
chronic infection have taken longer to prove, but testing has shown that many troublesome diseases have
entrenched microbial populations at their core. Peptic ulcers, once thought to be caused by stress, have been
proved to be caused by bacterial communities of Heliobacter pylori. The cyclical flare-up and subsidence of
otitis mediathe most common cause of children's recurrent earachesis an example of a typical biofilm-
based infection. Click to see Table 1-2, a summary of biofilms in medical contexts, the associated impacts,
and journal references.

How do biofilms impact our world?

Biofilms profoundly affect human health and industrial productivity. Their pervasive effects on
human health, water quality, corrosion, and power generation efficiency cost the U.S. billions of
dollars annually. Other related issues include deterioration of dental surfaces, contamination of
surfaces in the food processing industry, and the deterioration of air quality in ventilation and
air handling systems.

However, it is very important to point out that biofilms are an integral part of the natural
environment and can also serve very beneficial purposes, such as in the treatment of drinking
water, wastewater and detoxification of hazardous waste.

Both the beneficial and detrimental aspects of biofilms are summarized below.
In the natural environment
In bodies of water both large and small, on land, underground, as well as on and within higher
organisms, biofilms are an integral component of the natural environment. The report, "Global
Environmental Change: Microbial Contributions, Microbial Solutions," points out: ". . .the basic
chemistry of Earth's surface is determined by biological activity, especially that of the many
trillions of microbes in soil and water. Microbes make up the majority of the living biomass on
Earth and, as such, have major roles in the recycling of elements vital to life." Bacteria and
other microorganisms have also been associated with the formation of many types of
sedimentary rocks and minerals.

Bacteria are early colonizers of clean surfaces submerged in water. Scientists have been able to document
the predictable sequential colonization of surfaces in ocean waters by series of organisms, beginning with
microbial biofilms. Whether the surface is a boat hull at the surface or a new deep sea vent at the bottom
of the ocean, microbes are available and capable of rapid community development on surfaces. Microbes
have evolved along with other organisms, including human beings. While some bacteria produce effects
that are detrimental to surrounding organisms or hosts, most bacteria are harmless or even beneficial.
When it comes to bacteria, higher organisms are just another environment to colonize.

Water and wastewater treatment

Engineers have taken advantage of
natural biofilm environmental activity in
developing water-cleaning systems.
Biofilms have been used successfully in
water and wastewater treatment for
over a century. English engineers
developed the first sand filter treatment
methods for both water and wastewater
treatment in the 1860s. In these filtration
systems the surfaces of the filter media
act as a support for microbial attachment
and growth, resulting in a biofilm
adapted to using the organic matter
found in that particular water. The end
result of biological filtration is a
conversion of organic carbon in the
water into bacterial biomass. Ideally, this
biomass is immobilized on the filter
media and removed during the backwash
cycle.

Drinking water and treated wastewater that have been subjected to microbial activity in a
controlled manner in a treatment plant are more "biologically stable" and therefore less likely
to contribute to microbial proliferation downstream in distribution system or receiving water.
Biologically treated water typically has lower disinfectant demand and disinfection by-product
formation potential than conventionally treated water if the source water is high in organic
carbon. As drinking water utilities move to using ozone as a primary disinfectant and for
taste/odor/color control, biological filters may be necessary to reduce the concentrations of
biodegradable organic carbon entering the distribution system.
Remediation of contaminated soil and groundwater
In soil, biofilm morphology can be highly
variable, ranging from patchy
discontinuous colonies to thick
continuous films, depending on
environmental conditions. When toxic
organic contaminants (i.e. gasoline, fuel
oil, chlorinated solvents) are accidentally
released underground, the native soil
bacterial population will, to the degree
possible, adjust their ecological
composition in order to use the organic
contaminants as a food source. This
process is commonly referred to as
bioremediation and if successful,
potentially has the ability to render
initially toxic organic material into
harmless by-products. Typical biofilm cell
densities found in the vicinity of
contaminated ground water sites vary
from around 105 to 108 cells per gram of
soil.

Bioremediation has emerged as a technology of choice for remediating groundwater and soil at
many sites contaminated with hazardous wastes. Bioremediation results in 1) the reduction of
both contaminant concentration and mass for many subsurface contaminants (e.g., petroleum
hydrocarbons, chlorinated organics and nitroaromatics) and/or 2) a beneficial phase transfer or
speciation change (e.g., for heavy metals and radionuclides). Subsurface bioremediation is
controlled by abiotic geochemical and transport phenomena, including multiphase flow,
convective mass transport, adsorption/desorption, and phase partitioning, as well as biotic
processes, such as microbial biomass growth and contaminant metabolism.

Microbial leaching
Extraction of copper form ore deposits using acid solutions has been practiced for centuries, but
the role of bacteria in metal dissolution was not verified until the 1940s. Today approximately
1020% of copper mined in the U.S. is extracted by microbially assisted processing of low grade
ores. There is also a considerable investment in extending microbial leaching to the recovery of
other metals such as uranium, silver, gold cobalt, and molybdenum. Most microbial leaching
depends upon microbial oxidation of metal sulfides. Aqueous environments in association with
spent mineral produce very harsh conditions of low pH, high metal concentrations and high
temperatures, which select for a microbial flora with very specialized nutritional requirements.

Heap leaching is the most common process used to microbially extract copper and other minerals from
spent ore. The process consists of organizing the spent ore fragments into a packed bed configuration
which allows water to be trickled through. To initiate the process, acidified water (pH = 1.5 to 3.0) is
sprayed over the porous ore bed. Acidophilic bacteria, such as Thiobacillus ferrooxidans, actively oxidize
the soluble ferrous iron and attack the sulfide minerals, releasing the soluble cupric ion that can then be
recovered from aqueous solution. This oxidation process is similar in concept to corrosion of metal
surfaces.

Biological reaction and mass transfer rates presently limit the commercial application of microbial
leaching, however substantial improvements in process design have been made in recent years and the
method is viewed with promise by the mining industry.

Other biofilm technologies with promise

Microbial fuel cells

Biofilm "traps"

Microbial "canaries"

DETRIMENTAL BIOFILM IMPACTS

In natural environments
"Microbes can negatively impact environments on a global level including producing and
consuming atmospheric gases that affect climate; mobilizing toxic elements such as mercury,
arsenic and selenium; and producing toxic algal blooms and creating oxygen depletion zones in
lakes, rivers and coastal environments (eutrophication). Furthermore, the incidence of microbial
diseases such as plague, cholera, Lyme disease, and West Nile Virus are linked to global
change."

Industrial environments
Biofouling, biocorrosion, equipment damage and product contamination are constant and
expensive problems in industry.

Public health
Between 1980 and 1992, infectious disease deaths increased by 58% (39% after age
adjustment); the major contributors were HIV infection and AIDS, respiratory disease (primarily
pneumonia), and bloodstream infection. Infectious diseases are still broadly endemic and
maintain a large reservoir of agents that have the potential for rapid and widespread
dissemination. Infectious diseases remain the leading cause of death worldwide and the third
leading cause of death in the United States. In the United States, each year, approximately 25%
of physician visits are attributable to infectious diseases, with direct and indirect costs
estimated at more than $120 billion. Because recent research implicates biofilms as reservoirs
for pathogenic organisms and sources of disease outbreaks, biotechnology measures are being
created to control biofilms and/or sever the routes by which pathogenic organisms are
transmitted from biofilms to susceptible people.

Biofilms are implicated in otitis media, the most common acute ear infection in children in the U.S. Other
diseases in which biofilms play a role include bacterial endocarditis (infection of the inner surface of the
heart and its valves), cystic fibrosis (a chronic disorder resulting in increased susceptibility to serious lung
infection), and Legionnaire's disease (an acute respiratory infection resulting from the aspiration of
clumps of Legionnella biofilms detached from air and water heating/cooling and distribution systems).
Biofilms may also be responsible for a wide variety of nosocomial (hospital-acquired) infections. Sources
of biofilm-related infections can include the surfaces of catheters, medical implants, wound dressings, or
other types of medical devices.

Biofilms avidly colonize many household surfaces, including toilets, sinks, countertops, and cutting boards
in the kitchen and bath. Poor disinfection practices and ineffective cleaning products may increase the
incidence of illnesses associated with pathogenic organisms in the household environment.

What are key characteristics of biofilms?

1. Biofilms are complex, dynamic community structures
Bacterial biofilms are remarkably heterogeneous in virtually all parameters that can be measured
accurately and reproducibly. These heterogeneities: structural, physiological, chemical, ecological,
electrical, etc., have been implicated as the cause of many phenomena characteristic of the
attachedas opposed to the planktonicmode of growth. Pure cultures are virtually non-existent in
the world outside the laboratory. Microorganisms, like other organisms, exist in communities where
a variety of interactions exist.

One example of the effects of

microbial colonization on metal
surfaces is Microbially Influenced
Corrosion (MIC). The presence of
microorganisms modifies deposition
and dissolution rates of minerals, and
by this mechanism, influences the
electrochemical properties of the
metals or alloys. Pitting corrosion, as
seen at right in this example from a
stainless steel surface, results from
this activity.

Biofilms are also dynamic and

Pitting corrosion on 316S stainless steel, an example of
responsive to their environment.
microbially influenced corrosion. Image, courtesy of Z.
Bacterial cells may detach from
Lewandowski and W. Dickinson, MSU-CBE
biofilms individually or in clumps.
When they detach in clumps, they
retain the reduced susceptibility to
antimicrobials characteristic of
biofilms. In the right conditions,
biofilms can also migrate across
surfaces over a period of time in a
variety of ways, as illustrated below.
All materials have certain properties of elastic solids and viscous fluids. Biofilms appear to show
aspects of both solids and liquidsmuch like slug slimeand fall into a category called "viscoelastic."
However, as biofilms collect sediment, or become scaled with rust or calcium deposits, they become
less fluid and more like a brittle solid.

2. Genetic expression is different in biofilm bacteria when compared to

planktonic bacteria

The double-stranded helix structure

of molecular DNA (deoxyribonucleic
acid), discovered in 1953, has by now
become a familiar image. DNA
molecules, composed of units called
genes, carry the "instructions" that
determine characteristics of living
organisms and comprise the genetic
material passed along to offspring
through reproduction.

The genes that form DNA molecules also

play a crucial role in cellular activities.
Simple cells like bacteria control their
internal functions using various parts of
their genetic code to initiate chemical
activities. So, for instance, consuming
nutrients and getting rid of waste
products are processes that are carried
out under the influence of genetic
instructions. When genes are activated to SDS PAGE preparation of the outer membrane proteins
make chemical products (amino acids and (OMPs) of Pseudomonas aeruginosa cells in planktonic and
proteins), they are said to be biofilm states. Courtesy, Hongwei Yu
upregulated; when the genes are de-
activated, they are downregulated. The
proteins made by activated genes
constitute about half of the material
inside a cell, and are responsible for
numerous activities that keep a cell
viable.
Since not all of the genes in a cell are activated to make proteins all of the time, we can get a picture
of cellular activity by examining the proteins produced by cells at a particular time. One way to get
this kind of protein "snapshot" is by a technique called SDS-PAGE (for "Sodium Dodecyl Sulfate" and
"PolyAcrylamide Gel Electrophoresis"). This technique allows scientists to see large (nearer the top)
and small (nearer the bottom) cellular proteins as dark bands in an array of columns. In the SDS PAGE
gel above, we see proteins from the outer membranes of planktonic (outlined in blue, Lanes 1-4 and
6) and biofilm (outlined in red, Lane 5) bacteria, of a single strain of Pseudomonas aeruginosa. The
bands of proteins are strikingly different, telling us that the planktonic and biofilm forms of a single
species are expressing different genes, and therefore carrying out different activities.

What are the implications of this difference in genetic expression? One example is in the development of
antibiotics. These drugs have traditionally been developed to kill planktonic bacteria. We now know, however,
that planktonic bacteria are more susceptible to antimicrobials than biofilm bacteriaand also that many of
the infections plaguing humans are actually caused by bacteria in the biofilm mode of growth. So traditional
antibiotics have been targeting bacterial cells in their relatively unprotected state. We will need to develop new
classes of antibiotics that target bacteria without in the biofilm state. Understanding the genetic activity of
biofilm bacteria will help us to find new ways to target these cells and disrupt their functions.

3. Biofilm cells can coordinate behavior via intercellular "communication" using

biochemical signaling molecules
One of the fascinating aspects of bacterial community living is that it provides a setting for bacteria to
communicate using chemical signals. There is evidence that some of these chemical signals, produced
by cells and passed through their outer membranes, may be interpreted not just by members of the
same species, but by other microbial speciesand perhaps even by more complex organisms in some
cases.
In the cartoon above, various species of bacteria are represented by different colors. Bacteria can
produce chemical signals ("talk") and other bacteria can respond to them ("listen") in a process
commonly known as cell-cell communication or cell-cell signaling. This communication can result in
coordinated behavior of microbial populations. Courtesy, MSU-CBE.

In planktonic populations, chemical signals produced by cells are not concentrated enough to cause
changes in genetic expression. However, in biofilms, the matrix material (EPS) that holds cells in close
proximity allows concentrations of signal molecules to build up in sufficient quantity to effect
changes in cellular behavior. Bacterial populations will activate some genes only when they are able
to sense, via cell signaling, that their population is numerous enough to make it advantageous and/or
"safe" to initiate that genetic activity. For example, some bacterial pathogens will not produce toxins
until they sense that an adequate population has been established to survive host defenses. This
system of population recognition has been termed "quorum sensing." It was first observed in the
marine bacterium Vibrio fischeri, which can produce light after a sufficient population has developed.
Though planktonic cells secrete chemical signals (HSLs, for homoserine lactones), the low
concentration of signal molecules does not change genetic expression. Biofilm cells are held
together in dense populations, so the secreted HSLs attain higher concentrations. HSL molecules
then re-cross the cell membranes and trigger changes in genetic activity. Courtesy, MSU-CBE.

The discovery that simple cells are capable of coordinated behavior has given us a new appreciation of their
survival strategies. There is also good evidence that cell signaling can regulate the differentiation of cells into
sub-populations that carry out different activities within a microbial community of a single species. In the late
1990s an investigation of the marine bacterium Pseudoalteromonas revealed two physiologically distinct
subpopulations. In effect there was a cellular division of labor: one group stayed attached to the surface and
made nutrient available to the the second group, which reproduced and released daughter cells to the
surrounding water.

4. Biofilms make bacteria less susceptible to antimicrobial agents

Many studies have shown that the multicellular construction of biofilms affords protection for cells.
This protection is the result of intrinsic shifts in genetic expression when floating bacterial cells attach
to surfaces and begin to form biofilms. Some of the hypothesized mechanisms of protection from
antimicrobial agents are pictured in the diagram below.
A.
Free-floating cells utilize nutrients, but
do not have sufficient metabolic activity
to deplete substrates from the
neighborhood of the cells.
B.
Free-floating cells carry the genetic code for numerous protective
stress responses. Planktonic cells, however, are readily
overwhelmed by a strong antimicrobial challenge. These cells die
before stress responses can be activated.

In contrast, the collective metabolic In contrast, stress responses are effectively implemented in some
activity of groups of cells in the biofilm of the cells in a biofilm at the expense of other cells which are
leads to substrate concentration sacrificed.
gradients and localized chemical
microenvironments. Reduced metabolic
activity may result in less susceptibility
to antimicrobials.

C. D.
Free-floating cells neutralize the Free-floating cells spawn protected persister cells. But under
antimicrobial agent. The capacity of a permissive growth conditions in a planktonic culture,
lone cell, however, is insufficient to persisters rapidly revert to a susceptible state.
draw down the antimicrobial
concentration in the neighborhood In contrast, persister cells accumulate in biofilms because
of the cell. they revert less readily and are physically retained by the
biofilm matrix.
In contrast, the collective
neutralizing power of groups of cells
leads to slow or incomplete
penetration of the antimicrobial in
the biofilm.