This document discusses microbial communities and biofilms. It begins by explaining that microbes thrive in diverse ecosystems under a range of conditions. Microbial communities are heterogeneous mixtures that interact. Biofilms provide advantages like nutrient sharing and protection. The document then discusses techniques to analyze microbial communities, including genetic methods. It covers positive and negative impacts of biofilms in areas like infections, food production, and wastewater treatment. Stress can impact microbial diversity by selecting certain organisms. Modern techniques allow direct analysis of constituent populations in communities.

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Nature of Microbial Communities
All natural ecosystems host a diverse array of microorganisms. Some of these
microbes tend to thrive under moderate living conditions (i.e. moderate temperature,
tolerable pH, ample nutrient supply, etc.) while there are others, known as
extremophiles, which “love” extreme conditions such as high acidity or alkalinity,
extremely high or low temperatures, high pressures, etc.
These organisms flourish under conditions that are normally fatal to most other
organisms. Hence it is without doubt that every inch of the planet Earth is inhabited by
microorganism communities, from the highest mountain summits to the abyssal depths
of the oceans, from the freezing cold of the Polar Regions to the boiling hot water
springs, from simple freshwater bodies to hypersaline environments.
But these microbial communities do not live apart from one another, as previously
thought, modern research indicates that these populations intermingle in the
environment to produce a heterogeneous mixture.
Spatial Organization
The distribution of microbes in the environment is mostly affected by certain
physiological gradients, i.e. temperature, pH, nutrient availability, chemicals released
by other microbes, etc. These gradients may be vertical or horizontal or both at once,
and hence affect the distribution likewise.
The result is a heterogeneous mixture of microbial species, with each constituent
population being present in the range most favorable for it. But they are in no way
isolated, since many microbes depend upon one another due to symbiotic
relationships, their populations tend to overlap.
In fact in small-sized "micro" environments, even microbes with conflicting interests
may be pinned close together. There is a very high degree of organization of microbial
communities, which appears to integrate the growth conditions and the species

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composition in such a way that resources available in the environment are used
effectively (at an optimum rate).
Biofilms
Biofilm is an aggregate of microorganisms in which cells that are frequently embedded
within a self-produced matrix of extracellular polymeric substances adhere to each
other and/or to a surface.
 Extracellular Polymeric Substances
The cells within the biofilm produce the extracellular polymeric substances (EPS)
components, which are typically a polymeric conglomeration of extracellular
polysaccharides, proteins, lipids, DNA, and mostly water. This matrix makes up around
50 – 95% of the dry weight of any biofilm.
Figure: Structure of a microbial biofilm.
 Advantages of Biofilms to Microbes
Spatial organization of microbes in biofilms and similar situations grants them many of
the advantages of multicellular life such as co-metabolism, cross-feeding, increased

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resilience to stress (i.e. electromagnetic radiations such as UV, heat shocks, pH
fluctuations, inhibitory substances, and most importantly dehydration – owing to the
large amount of water in the matrix). Biofilms are not just bacterial slime layers but
biological systems; the bacteria organize themselves into a coordinated functional
community. Biofilms can attach to a surface such as a tooth, rock, or surface, and may
include a single species or a diverse group of microorganisms. The biofilm bacteria
can share nutrients and are sheltered from harmful factors in the environment, such
as desiccation, antibiotics, and a host body's immune system.
Figure: A summary of how members of a microbial community in a biofilm (analogous
to a city) receive several advantages.
 Effect of environment on biofilms
Biofilms may form on living or non-living surfaces and can be prevalent in natural,
industrial, and hospital settings. The microbial cells growing in a biofilm are
physiologically distinct from planktonic cells of the same organism (planktonic cells are
single-cells that may float or swim in a liquid medium). The thickness of the biofilm
varies, depending upon the environmental conditions, i.e. biofilms in nutrient-deficient
environments tend to be only a few micrometers thick whereas soil crumbs, microbial
mats, sewage flocs, and biofilms in nutrient-rich environment are comparatively much

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thicker. Microbes form a biofilm in response to various factors, which may include
cellular recognition of specific or non-specific attachment sites on a surface, nutritional
cues, or in some cases, by exposure of planktonic cells to sub-inhibitory
concentrations of antibiotics.
Formation of a Biofilm
Biofilms are the product of a microbial developmental process. The process of biofilm
formation consists of the following stages:
1. Initial Attachment
The formation of a biofilm begins with the attachment of free-floating microorganisms
to a surface. The first colonist bacteria of a biofilm may simply adhere to the surface
due to physical factors such as van der Waal’s forces, hydrophobic effects, etc.
2. Irreversible Attachment
If the colonists are not immediately separated from the surface, they can anchor
themselves more permanently using cell adhesion structures such as pili. Some
bacteria species are not able to attach to a surface on their success due to their limited
motility but are instead able to anchor themselves to the matrix or directly to other,
earlier bacterial colonists. Non-motile bacteria cannot recognize surfaces or aggregate
together as easily as motile bacteria.
3. Maturation
Once the colonization has begun, the biofilm grows by a combination of cell division
and recruitment. Polysaccharide matrices typically enclose bacterial biofilms. In
addition to the polysaccharides, these matrices may also contain material from the
surrounding environment, including but not limited to minerals, soil particles, and blood
components, such as erythrocytes and fibrin.
4. Dispersion
The final stage of biofilm formation is known as dispersion and is the stage in which
the biofilm is established and may only change in shape and size. Dispersal enables
biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm

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extracellular matrix may contribute to biofilm dispersal, they may also be useful as
anti-biofilm agents. Cells dispersed from biofilms immediately go into the planktonic
growth phase. Hence, the dispersal process is a unique stage during the transition
from biofilm to planktonic lifestyle in bacteria.
Figure: Steps involved in biofilm formation.
Negative Impacts of Biofilms
Microbial biofilms have several negative impacts on the environment and directly on
humans as well, some of them are:
1. Infections
Biofilms are involved in a wide variety of microbial infections in the body, by one
estimate 80% of all infections. Infectious processes in which biofilms have been
implicated include common problems such as bacterial vaginosis, urinary tract
infections, catheter infections, middle-ear infections, formation of dental plaque,
gingivitis, coating contact lenses, and less common but more lethal processes such
as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling
devices such as joint prostheses, heart valves, and intervertebral disc.

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Infections associated with the biofilm growth usually are challenging to eradicate. This
is mostly because mature biofilms display tolerance towards antibiotics and the
immune response. Microbial infections can develop on all medical devices and tissue
engineering constructs. 60 to 70% of nosocomial or hospital-acquired infections are
associated with the implantation of a biomedical device.
2. Food Industry
Biofilms have become problematic in several food industries due to the ability to form
on plants and during industrial processes. Along with economic problems, biofilm
formation on food poses a health risk to consumers due to the ability to make the food
more resistant to disinfectants. During the washing process, biofilms resist sanitization
and allow bacteria to spread across the product. This problem is also found in ready-
to-eat foods because the foods go through limited cleaning procedures before
consumption.
 Dairy Products: Due to the perishability of dairy products and limitations in
cleaning procedures, resulting in the buildup of bacteria, dairy is susceptible to
biofilm formation and contamination. The bacteria can spoil the products more
readily and contaminated products pose a health risk to consumers.
 Poultry (Salmonella Infestation): One species of bacteria that can be found in
various industries and is a major cause of foodborne disease is Salmonella spp.
Large amounts of Salmonella contamination can be found in the poultry
processing industry as about 50% of Salmonella strains can produce biofilms
on poultry farms. Salmonella increases the risk of foodborne illnesses when the
poultry products are not cleaned and cooked correctly.
 Seafood (Salmonella Infestation): Salmonella is also found in the seafood industry
where biofilms form from seafood borne pathogens on the seafood itself as well
as in water. Shrimp products are commonly affected by Salmonella because of
unhygienic processing and handling techniques. The preparation practices of

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shrimp and other seafood products can allow for bacteria buildup on the
products.
3. Aquaculture
In shellfish and algae farms, bio-fouling microbial species tend to block nets and cages
and ultimately outcompete the farmed species for space and food. Bacterial biofilms
start the colonization process by creating microenvironments that are more favorable
for bio-fouling species. In the marine environment, biofilms could reduce the
hydrodynamic efficiency of ships and propellers, lead to pipeline blockage and sensor
malfunction, and increase the weight of appliances deployed in seawater. Biofilms can
be a reservoir for potentially pathogenic bacteria in freshwater aquaculture. As
mentioned previously, biofilms can be difficult to eliminate even when antibiotics or
chemicals are used in high doses.
Positive Roles of Biofilms
Microbial biofilms have also been employed in many positive scenarios, such as:
1. Sewage Treatment
Many sewage treatment plants include a secondary treatment stage in which
wastewater passes over biofilms grown on filters, which extract and digest organic
compounds.
Figure: Biological treatment of wastewater (microbes are involved in steps 2 & 3).

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In such biofilms, bacteria are mainly responsible for the removal of organic matter,
while protozoa and rotifers are mainly responsible for the removal of suspended solids,
including pathogens and other microorganisms. Slow sand filters rely on biofilm
development in the same way to filter surface water from lake, spring, or river sources
for drinking purposes.
2. Removal of Petroleum from Marine Waters
Biofilms can help eliminate petroleum oil from contaminated oceans or marine
systems. The oil is eliminated by the hydrocarbon-degrading activities of microbial
communities.
3. Microbial Fuel Cells
Biofilms are used in microbial fuel cells to generate electricity from a variety of starting
materials, including complex organic waste and renewable biomass.
4. Bioleaching
Biofilms are also relevant for the improvement of metal dissolution in the bioleaching
industry. Bioleaching is the extraction of metals from their ores through the use of living
organisms. This is much cleaner than the traditional heap leaching using cyanide.
Bioleaching is one of several applications within biohydrometallurgy and several
methods are used to recover copper, zinc, lead, arsenic, antimony, nickel,
molybdenum, gold, silver, and cobalt.
Effect of Stress on Microbial Communities
Stress is an abiotic factor or a set of factors that limit the production of biomass. Stress
factors such as temperature variations, pH fluctuations, changes in nutrient availability
and water supply have a direct effect on the growth of microbes and the diversity of
microbial communities on a whole (as many species might be wiped out altogether
from an area because of their inability to resist such changes).
The degree of effectiveness of any stress factor is determined by three parameters:
(1) nature of the factor, (2) extent of damage done by the factor, and (3) rapidity of
application.

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If the stress factor is applied slowly, the microbes gain an opportunity to adapt (within
certain limits) to that factor, this may lead to the formation of a much more resilient
population than before. Stress may also provide a selective advantage to some
microbes, at the expense of others, for instance, extremophiles such as thermophiles
will be able to resist heat shocks, while all of their competitors (mesophiles) will be
wiped out. Alternatively, endospore-forming bacteria will be able to repopulate an
environment, after it has been cleared of all viable cells, owing to the germination of
the endospores. In many natural environments, cyanobacteria are among the first
organisms to recolonize barren areas, hence making them pioneers in ecological
succession.
Determination of the Structure & Diversity of Constituent Populations
The constituent populations of a microbial community are hard to determine via
conventional methods such as culturing them on nutrient media. These media fail to
emulate the conditions of the natural environment for the microbes, for instance, they
are mostly rich in nutrients, and hence only the copiotrophs from the sample will grow
properly whereas the oligotrophs will be inhibited simply by the high concentration of
nutrients. Similarly, no single medium is suitable for growing all microbial species.
1. Modern Techniques
Modern techniques have allowed to take a direct peek into the microbial communities
and hence determine the constituent populations, these techniques include:
Microscopy: Direct microscopic analysis, owing to the modern advances in
microscopes and microscopic techniques have allowed the diagnosis of many
microbial species based on their morphology (distinct features, such as trichomes and
akinetes of cyanobacteria).
Analysis of Cellular Constituents: Various cellular constituents such as types of fatty acids
are distinct for each species and if they are detected in a culture then the presence of
that particular species (or at least that genus) can be confirmed.

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Genetic Techniques: Analysis of nucleic acid sequences is perhaps the most reliable
and most modern of all techniques for determining the species of a microbe. Some of
these techniques include:
(1) Determination of GC Content
The GC content of the DNA of any organism is species-specific and hence a valuable
parameter for the determination of species, but this method is outdated.
(2) DNA Hybridization
In this technique, the DNA of one organism is labeled, then mixed with the unlabeled
DNA to be compared against. The mixture is incubated to allow DNA strands to
dissociate and then cooled to form renewed hybrid double-stranded DNA. Hybridized
sequences with a high degree of similarity will bind more firmly, and require more
energy to separate them: i.e. they separate when heated at a higher temperature than
dissimilar sequences, a process known as "DNA melting". The temperatures at which
labeled DNA comes off the sample DNA reflects the amount of similarity between
sequences (and the self-hybridization sample serves as a control). These results are
used to determine the degree of genetic similarity between organisms.
Figure: Procedure of DNA homology experiments.

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(3) PCR
Polymerase chain reaction has allowed us to amplify (not merely replicate, amplify to
millions of copies) a DNA sample so that it can be analyzed via gel electrophoresis
later on and be compared with the known sequence of a known species to judge the
degree of similarity.
(4) Hybridization Probes
Within the field of microbial ecology, oligonucleotide probes are used to determine the
presence of microbial species, genera, or microorganisms classified on a more broad
level, such as bacteria, archaea, and eukaryotes via fluorescence in situ hybridization
(FISH) – Fluorescence in situ hybridization (FISH) is a molecular cytogenetic
technique that uses fluorescent probes that bind to only those parts of a nucleic acid
sequence with a high degree of sequence complementarity.
(5) 16S & 23S rRNA sequencing
The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between
different species of bacteria and archaea. It is suggested that the 16S rRNA gene can
be used as a reliable molecular clock because 16S rRNA sequences from distantly
related bacterial lineages are shown to have similar functionalities. In some instances,
differentiation between species may be problematic when using 16S rRNA sequences
due to similarity. In such instances, 23S rRNA may be a better alternative. The global
standard library of rRNA sequences is constantly becoming larger and continuously
being updated.
Some Constituent Populations of Biofilms
Biofilms are formed by both prokaryotes and eukaryotes, modern techniques have
allowed us to identify many of these constituent populations:
1. Prokaryotic Biofilms
Many different bacteria form biofilms, including gram-positive (e.g. Bacillus spp.,
Listeria monocytogenes, Staphylococcus spp., and lactic acid bacteria, including

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Lactobacillus plantarum and Lactococcus lactis) and gram-negative species (e.g.
Escherichia coli, or Pseudomonas aeruginosa). Cyanobacteria also form biofilms in
aquatic environments.
Biofilms are also formed by bacteria that colonize plants, i.e. Pseudomonas putida,
Pseudomonas fluorescens, and related pseudomonads which are common plant-
associated bacteria found on leaves, roots, and in the soil, and the majority of their
natural isolates form biofilms. Several nitrogen-fixing symbionts of legumes such as
Rhizobium leguminosarum and Sinorhizobium meliloti form biofilms on legume roots
and other inert surfaces. Many bacterial species which cause diseases in animals such
as Streptococcus pneumoniae (causes community-acquired pneumonia and
meningitis in children and the elderly, and sepsis in HIV-infected persons),
Pseudomonas aeroginosa (causes chronic wounds, chronic otitis media, chronic
prostatitis, and chronic lung infections in cystic fibrosis patients), etc. also form
biofilms.
2. Eukaryotic Biofilms
Along with bacteria, biofilms are often initiated and produced by eukaryotic microbes.
The biofilms produced by eukaryotes are usually occupied by bacteria and other
eukaryotes alike, however the surface is cultivated and EPS is secreted initially by
eukaryotes. Both fungi and microalgae are known to form biofilms in such a way.
Biofilms of fungal origin are important aspects of human infection and fungal
pathogenicity, as the fungal infection is more resistant to antifungals.
In the environment, fungal biofilms are an area of ongoing research. One key area of
research is fungal biofilms on plants. For example, in the soil, plant-associated fungi
including mycorrhiza have been shown to decompose organic matter and protect
plants from bacterial pathogens.
Biofilms in aquatic environments are often founded by diatoms. The exact purpose of
these biofilms is unknown, however, there is evidence that the EPS produced by
diatoms helps them resist both cold and salinity stresses. These eukaryotes interact

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with a diverse range of other organisms within a region known as the phycosphere
(phycosphere is a microscale mucus region that is rich in organic matter and surrounds
a phytoplankton cell), but importantly are the bacteria associated with diatoms, as it
has been shown that although diatoms excrete EPS, they only do so when interacting
with certain bacterial species.