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Title: Title Page: The Aquatic Environment

Title Page The Aquatic Environment
The Aquatic Environment
  • Surface Fresh Water Systems Rivers, Lakes and
  • Surface Saline Water Systems Oceans, marshes,
  • Groundwater Systems (fresh)
  • Groundwater Systems (saline)
  • Ice, glaciers and compressed snowpack
  • Permafrost

  • Surface Fresh Water Systems Rivers, Lakes and

The main problem in studies of aquatic
microbiology is methodological.  It is
extremely difficult to obtain a "representative"
sample of water and it is difficult to extract
and examine the microorganisms. 
A suitable sampling regime in freshwater or
marine systems would be complex and
time-consuming this is because of the nature of
the environment. There are two types of
characteristics of the environment to be
considered in the design of any sampling
  • MORPHOMETRIC characters have to do with depth,
    dimension, geology of shores, sediment
    distribution, currents, inflow and outflow of
    water, etc.
  •  PHYSICOCHEMICAL characters are those such as
    temperature profiles, pH, inorganic ion content
    and distribution, pO2, oxygen profiles with
    depth, etc.

Lake Morphology 1
Sampling Lake Systems 1
Lake Morphology
Littoral Zone
Lake Morphology 2
Sampling Lake Systems 2
Lake Morphology
Light Compensation Point (about 1 of light
intensity of sunlight)
Lake Morphology 3
Sampling Lake Systems 3
Lake Morphology
Benthic Zone
Lake Morphology 4
Sampling Lake Systems 4
Lake Morphology Holomictic Lakes (turnover and
Lake Morphology 5
Sampling Lake Systems 5
Lake Morphology Meromictic Lakes ( no turnover
and mixing)
Nutrient Status
  • Another division of lakes into different "types"
    is based on nutritional status. 
  • OLIGOTROPHIC lakes have low nutrient status and
    relatively low primary productivity.
  • EUTROPHIC lakes have high nutrient status and
    high productivity.

All of these factors have to be taken into
account when designing a sampling scheme that
tries to provide a representative set of samples
for the entire lake. The sampling stations
should be spaced to include all of the various
zones.   This entails sampling around the
littoral region and taking depth profiles to
include the limnetic and profundal zones. 
Separate methods have to be used to sample the
sediment or benthic zone. 
Lake Sampling
Core Sampler taking sediment samples from the
Benthic Zone
Bottle Sampler taking water samples from
various depths
Sampling Cruise
Lake St. Clair St Clair River
Niagara River
Lake Erie Typical Sampling Cruise Scheme
Lake Erie Hot Spots
Lake St. Clair St Clair River
Niagara River
Lake Erie Typical Sampling Cruise Scheme and
Major Contaminated Regions
Toxics Distribution in Great Lakes
Toxics Distribution in Great Lakes 2
Phenols and Organics
PCBs, PAHs and other organics
Toxics Distribution in Great Lakes 3 Pesticides
Pesticides Mirex and DDT
Toxics Distribution in Great Lakes 4Summary
Summary Overview
Mercury. Metals, Asbestos
Organics, PAHs, pesticides, phenols
Photographs Lake Erie 1970s
Lake Erie in the 1970s and early 80s
Photographs Lake Erie 1990s
Lake Erie in the 1990s
Methodology Biomass Determinations
  • Attempts can be made to count the number of
    viable bacteria of various kinds in water (e.g.
    total coliforms, fecal coliforms, Pseudomonas
    species, etc.)  These tests are usually performed
    to count and identify pathogens or to indicate
    the possible presence of pathogens (e.g. coliform
    tests).  See Module 4
  • Similar, but less selective methods can also be
    used to count the viable bacteria, fungi,
    protozoa in water or sediment samples in efforts
    to understand the ecology and population dynamics
    of the microbial populations.
  • Other methods attempt to count the "total" (as
    opposed to "viable") numbers of bacteria and
    other microorganisms.  These methods (such as the
    Direct Method of fluorescence staining with
    acridine orange stain) do not distinguish between
    living and dead microorganisms in most cases.

Remote Sensing
  • Yet other methods use a "surrogate" technique to
    estimate biomass.  They would, for example, use
    the amount of chlorophyll as a measure of the
    presence of algal biomass in a water sample.

Using specific wavelengths, satellites can also
measure and locate chlorophyll concentrations
over large areas. The satellite image to the
right is the Eastern Coast of the U.S. showing
different chlorophyll concentrations (higher near
the coast line)
Link to Remote Sensing and Satellite Imagery
US geological Service Website for satellite
images from many parts of the world
Direct Counts
Direct methods Typical Procedure Membrane
filtration of standard volumes onto 8 µM, 2 µM,
0.45 µM and 0.2 µM Millipore or Nucleopore
filters followed by direct microscopic
observation with or without staining. 
Ultraviolet fluorescence of chlorophyll can be
used to detect and count algal cells
Fluorescence staining methods (e.g. acridine
orange, anilinonaphthalene sulfonic acid (ANS)
dyes, fluorescein isothiocyanate, etc.) can be
used for bacteria and fungi.  In these cases, a
black filter is required.  The black filters can
be produced from the normal cellulose filters by
dyeing the filter with a black dye such as
Irgalan black or Dylon 44. 
Bacteria in colony
Actinomycete mycelium
Fungal Mycelium
Filtered sample stained with ANS dyes
The microscope used for many of these direct
methods uses ultraviolet light directed onto the
surface of the materials (epi-illumination) being
examined rather than passing through it as in
normal microscopy
Biomass Methods
Other Methods for Measuring Biomass
  • Most Probable Number (MPN) technique
  • Viable counting procedures
  • Biochemical methods for estimating biomass such
  • Protein levels
  • ATP and adenylate charge
  • Lipopolysaccharide (LPS)
  • Muramic acid
  • Chlorophyll
  • Chlorophyll concentration is particularly
    important in the aquatic habitat because of the
    occurrence of photosynthetic algae and bacteria. 
  • The chlorophyll can be determined
    spectrophotometrically or fluorometrically. 
  • Chlorophyll a is usually measured but the
    chlorophylls b and c can be assayed selectively
    at different wavelengths with a

Activity Measurements 1
Activity Measurements
  • In many cases it is preferable to assay the
    microbial activity rather than the biomass. 
  • There are innumerable assays for microbial
    activity, but most rely on some measure of
    metabolic activity such as respiration,
    photosynthesis or biochemical pathway or product.
  • The main categories are
  • Rate of increase in the number of colonies on
  • Direct increase in numbers on membrane filters
  • Estimation of rates of multiplication in
    continuous culture systems
  • Measurement of metabolic activities such as
    photosynthesis, respiration, substrate
    utilization or product accumulation

Activity Measurements 2
  • Rate of increase in the number of colonies on
  • Sample at intervals, count viable cells,
    calculate growth rate and thereby the activity.
  • Very difficult to do accurately.
  • Very difficult to ensure most cells are counted.
  • Media is ALWAYS selective.
  • Many microorganisms in water grow very slowly and
    do not grow at all on high nutrient media.

2. Direct increase in numbers on membrane
  • Method
  • A water sample is taken, immediately filtered
    onto 2 sets of membrane filters. Half the filters
    are fixed to stop growth and half are incubated
    in the water sample itself.
  • This is intended to reproduce the conditions in
    the habitat so the temperature, pH, oxygen levels
    should remain stable throughout the incubation
  • After 4 hours (typically) the incubated filters
    are then fixed and stained.
  • The bacteria are counted on both sets of filters
    and the growth rates can be calculated from the
    differences seen.

Continuous Culture
3. Estimation of rates of multiplication in
continuous culture systems. The growth rates or
generation times of various microorganisms in
water systems can be estimated using continuous
culture systems.   If a chemostat is operated at
very slow growth rates, the growth rate of
bacteria from natural waters can be estimated
using those waters as the medium, even if the
dilution rate of the chemostat is higher than
their growth rates. This is done by measuring
the washout rate of the bacteria and calculating
the growth rate from the difference between this
(the washout rate) and the dilution rate of the
chemostat. Even if only very slow growth is
occurring, the bacteria will wash out more slowly
than the dilution rate
Constant volume pump
Collection Vessel
Constant Volume Culture Vessel withgrowing cells
Medium Reservoir Growth rate of cells in
culture vessel islimited by a limiting nutrient
in the medium
Carbon and Energy
Microorganisms classified by their Carbon and
Energy Requirements
Autotroph Organism which uses carbon dioxide as
the sole carbon source. Chemoautotroph Organism
that obtains energy from the oxidation of
chemical, generally inorganic, compounds and
carbon from carbon dioxide Phototroph Organism
that uses light as the energy source to drive the
electron flow from the electron donors, such as
water, hydrogen, or sulfide Heterotroph Organism
capable of deriving carbon and energy for growth
and cell synthesis from organic compounds
generally also obtain energy and reducing power
equivalents from organic compounds
Chemolithotroph Organism that obtains energy
from the oxidation of inorganic compounds and
uses inorganic compounds as electron donors
Metabolic Activities
  • 4. Measurement of metabolic activities such as
    photosynthesis, respiration, substrate
    utilization or product accumulation.
  • Any metabolic activity can be used as a measure
    of activity if it can be measured.
  • For example
  • Measurement of l4CO2 uptake into algal or
    bacterial tissues has long been used as a measure
    of photosynthetic activity and thus of primary
    production. The aquatic ecosystem is exposed to
    14Clabelled CO2 for given time periods in the
    dark and in the light.
  • A typical experimental outline would be
  • Take samples of the water, place in glass
    bottles. Place duplicate samples in dark glass
  • Add the radioactive isotope 14C in the form of
    NaHCO3 (approximately 50 100 microcuries in
    total, 2 5 microcuries per 125 mL of sample).
  • Suspend the bottles at the sampling depth to
    expose them to the same light and temperature
    conditions as the original sample. Leave for 20
    min to 6 hours (depending upon activity).
  • Remove bottles and filter through a 0.45 µM
    membrane filter.
  • Place filters in liquid scintillation fluid
    ("cocktail") and measure radioactivity
    incorporated into cells with a scintillation
    counter. The dark bottle gives an estimate of
    "dark fixation'' or heterotrophic CO2 fixation
    (see later). The light bottle gives an estimate
    of photosynthetic activity plus heterotrophic CO2
  • Note all photosynthetic organisms (plants and
    bacteria) will be incorporated into this assay
    for photosynthesis and primary productivity.
    However, all photosynthetic bacteria except for
    the Cyanobacteria ("bluegreen algae") are
    obligate anaerobes, so their photosynthetic
    activity will not occur in aerobically incubated

Heterotrophic Fixation
  • Heterotrophic CO2 fixation ("dark fixation")
  • The heterotrophic microbial assimilation of CO2
    in water bodies is the result of the activity of
    different groups of microorganisms.
  • Group 1. Heterotrophic bacteria which decompose
    proteins generally get 3 5 of their total
    carbon from external CO2 by heterotrophlc
    fixation. This group is the main contributor to
    the "dark fixation'' observed in the surface
    layers of water.
  • Group 2. Some bacteria have an intermediary
    metabolism between true heterotrophs and true
    chemoautotrophs and usually oxidize low molecular
    weight compounds such as methane, methanol, and
    formic acid produced during the anaerobic
    metabolism of organic matter. They can assimilate
    between 30 90 of their total carbon from
    external CO2
  • Group 3. The true chemoautotrophs are not
    involved in "dark fixation'' to the same extent
    as the above but, in certain environments, they
    can be of significance. They derive all of their
    cell material carbon from CO2 and utilize the
    energy gained from the oxidation of reduced
    compounds such as H2, H2S, NH4 and Fe and
    Fe .
  • The last two groups are most active in the
    boundary layer between the aerobic and anaerobic
    zones in the water column and in the interface
    between the sediment and the water.

Oxygen Uptake
Oxygen uptake measurements. In eutrophic waters
the overall respiration rate of the plankton and
bacteria can be measured simply by incubating the
samples in the dark (to preclude 02 evolution by
photosynthesis) and determining the 02 levels at
various times. The bacteria can be separated
partially from the plankton by differential
filtration through 8 or 5 µM Millipore filters. A
typical procedure would be 1. A sample of water
is split into two.   Half is filtered to remove
phyto and zooplankton. The samples are placed in
sterile dark glass oxygen bottles. 2. The
initial levels of 02 and initial numbers of
bacteria are measured (membrane filter method for
bacteria and Winkler titration for oxygen
3. The bottles are incubated in the dark (often
in situ) for 24 or 48 hours and the final
bacterial numbers and oxygen concentrations are
measured. If it is assumed that the bacteria grow
exponentially during the incubation period, it is
possible to calculate the oxygen uptake per
bacterial cell per hour during the incubation.
End of Section
Microorganisms in Aquatic Systems
Microorganisms in Aquatic Systems
Halophiles in salt collection ponds near San
Francisco Bay in California- Photo by NASA
  • Bacteria are involved in the geochemical cycling
    of nitrogen, sulphur, carbon, phosphorus and
    other elements in many different aquatic
  • Some aquatic environments have such extreme
    physical or chemical properties (such as low or
    high pH levels, high salinity, temperature or
    extremely low oxygen concentrations) that certain
    bacteria (called extremophiles) come to dominate
    those environments.
  • In more typical environments, the microorganisms
    coexist with many other groups of organisms.
  • We will restrict our treatment of microorganisms
    in aquatic environments to a few extreme
    environments and then to a freshwater lake system
    and the role of bacteria in the cycling of
  • This will serve as the example to show the
    typical functions of bacteria in nutrient cycling.

Extremophilic Microorganisms
Extremophilic Microorganisms
Organisms that thrive in what to humans are
extreme conditions are called extremophiles. The
organisms do not merely tolerate these
conditions they do best in the habitats and, in
many cases, require one or more extremes in order
to reproduce at all. Although some have been
identified for more than 40 years, the search for
them has intensified recently, as scientists have
recognized that places once assumed to be sterile
are not. Many industries have also found that the
properties of these organisms can potentially
serve in an number of pharmaceutical, industrial,
remediation and reclamation applications. For
more details see the article which provided most
of the basic information for this section on
extremophiles From Scientific American
Article Extremophiles April 1997
  • Thermophiles
  • Thermophiles reproduce, or grow, readily in
    temperatures greater than 45 degrees Celsius (113
    degrees Fahrenheit), and some of them, referred
    to as hyperthermophiles, favor temperatures above
    80 degrees C (176 degrees F). Some
    hyperthermophiles thrive in environments hotter
    than 100 degrees C (212 degrees F), the boiling
    point of water at sea level. In comparison, most
    bacteria grow fastest in temperatures between 25
    and 40 degrees C (77 and 104 degrees F).
  • No multicellular animals or plants have been
    found to tolerate temperatures above about 50
    degrees C (122 degrees F), and no microbial
    eukarya yet discovered can tolerate long-term
    exposure to temperatures higher than about 60
    degrees C (140 degrees F).
  • Thermophiles that are content at temperatures up
    to 60 degrees C have been known for a long time,
    but true extremophiles--those able to flourish in
    greater heat--were first discovered only about 30
    years ago in hot springs and other waters of
    Yellowstone National Park in Wyoming.

  • Thermophiles (contd)
  • In the late 1960s the first extremophile capable
    of growth at temperatures greater than 70 degrees
    C was found. It was a bacterium, now called
    Thermus aquaticus.
  • The first hyperthermophile in an extremely hot
    and acidic spring. This organism, the archaean
    Sulfolobus acidocaldarius, grows at temperatures
    as high as 85 degrees C. They also showed that
    microbes can be present in boiling water.
  • To date, more than 50 species of
    hyperthermophiles have been isolated

Smoker vents
Hydrothermal vents, the so-called smokers, are
essentially natural undersea rock chimneys
through which erupts superheated, mineral-rich
fluid as hot as 350 degrees C. See Module 2
Marine Microbiology
  • Thermophiles (contd)
  • The most heat-resistant of these microbes,
    Pyrolobus fumarii, grows in the walls of smokers.
    It reproduces best in an environment of about 105
    degrees C and can multiply in temperatures of up
    to 113 degrees C. It does not grow below 90
    degrees C (194 degrees F).
  • What is the maximum temperature limit? Can some
    extremophiles grow at 200 or 300 degrees C ?
    Evidence suggests the limit will be about 150
    degrees C. Above this temperature, probably no
    life-forms could prevent dissolution of the
    chemical bonds that maintain the integrity of DNA
    and other essential molecules.

Yellowstone Park
Yellowstone Park Hot Springs - Zonation
  • Psychrophiles
  • The oceans maintain an average temperature of
    one to three degrees C (34 to 38 degrees F)
  • Large land areas of the Arctic and Antarctic are
    permanently frozen or are unfrozen for only a few
    weeks in summer.
  • Many bacteria can only grow at temperatures below
    20C the psychrophiles. Others have an optimum
    below 20 but also grow above 20 - the
  • Microbial communities populate Antarctic sea
    ice--ocean water that remains frozen for much of
    the year. These communities include
    photosynthetic eukarya, notably algae and
    diatoms, as well as a variety of bacteria.
  • Polaromonas vacuolata, is a good example of a
    psychrophile its optimal temperature for growth
    is four degrees C, and it finds temperatures
    above 12 degrees C too warm for reproduction.

Acidophiles and alkaliphiles
  • Acidophiles and alkaliphiles
  • Most natural environments on the earth are
    essentially neutral, having pH values between
    five and nine. Acidophiles thrive in the rare
    habitats having a pH below five, and alkaliphiles
    favor habitats with a pH above nine.
  • Highly acidic environments can result naturally
    from geochemical activities (such as the
    production of sulfurous gases in hydrothermal
    vents and some hot springs) and from the
    metabolic activities of certain acidophiles
  • Acidophiles are also found in the debris left
    over from coal mining. Interestingly, acid-loving
    extremophiles cannot tolerate great acidity
    inside their cells, where it would destroy such
    important molecules as DNA. They survive by
    keeping the acid out. But the defensive molecules
    that provide this protection, as well as others
    that come into contact with the environment, must
    be able to operate in extreme acidity. Indeed,
    extremozymes that are able to work at a pH below
    one--more acidic than even vinegar or stomach
    fluids--have been isolated from the cell wall and
    underlying cell membrane of some acidophiles.
  • Alkaliphiles live in soils laden with carbonate
    and in so-called soda lakes, such as those found
    in Egypt, the Rift Valley of Africa and the
    western U.S. Above a pH of eight or so, certain
    molecules, notably those made of RNA, break down.
    Consequently, alkaliphiles, like acidophiles,
    maintain neutrality in their interior, and their
    extremozymes are located on or near the cell
    surface and in external secretions.

Halophiles and Barophiles
  • Halophiles
  • Tolerate highly saline conditions. Because water
    tends to flow from areas of high solute
    concentration to areas of lower concentration, a
    cell suspended in a very salty solution will lose
    water and become dehydrated unless its cytoplasm
    contains a higher concentration of salt (or some
    other solute) than its environment.
  • Halophiles contend with this problem by producing
    large amounts of an internal solute or by
    retaining a solute extracted from outside.
  • For instance, Halobacterium salinarum
    concentrates potassium chloride in its interior.
    The enzymes in its cytoplasm will function only
    if a high concentration of potassium chloride is
  • As you would expect, any proteins in H. salinarum
    cell structures that are in contact with the
    environment require a high concentration of
    sodium chloride
  • Barophilic Microorganisms
  • Barophiles tolerate or require high barometric
    pressures to survive and grow. They are found at
    great depth in marine environments and can grow
    under thousands of atmospheres of pressure.
  • See Module 2 on Marine Microbiology.

Normal Variation in Environmental Conditions
An example of the variation in normal
environmental conditions (ie not those that
support extremophilic microorganisms) is that
seen in an estuarine environment where rivers
enter the ocean. If we examine the variation in
oxygen levels and pH levels in an estuary, it is
clear that there is a large (and constantly
varying) set of conditions. Oxygen levels are
indicated as Eh (in millivolts) plotted on the Y
axis against the pH on the X axis. Bacteria and
other organisms can tolerate different ranges of
Eh and pH The typical bacteria in estuarine
conditions are indicated
Can we make some generalizations about aquatic
  • Freshwater systems
  • The pH, Eh, temperature and oxygen concentrations
    of freshwater systems tends to change over fairly
    short time periods.
  • Many of the bacteria are the same as terrestrial
    (soil) microorganisms
  • Many are also adapted to life in aquatic low
    nutrient conditions (oligotrophs)
  • Most microorganisms in lakes and rivers grow
    slowly (because of low nutrient levels)
  • Many have temperature optima of 15 to 25C or
    growth ranges from 0 to 25C
  • Biofilms of microorganisms are common and an
    important biomass in aquatic systems
  • Many aquatic microorganisms grow and survive in
    mixed communities with other microorganisms,
    algae and higher plants
  • The number of microorganisms mL 1 (bacteria,
    especially) in a lake profile is highest near the
    surface and decreases with depth. At the
    sediment-water interface the numbers increase
  • Anaerobic conditions are relatively common where
    oxygen has been utilized (e.g. in the sediment of
    eutrophic lakes). Anaerobes and facultative
    anaerobes are found in those environments (see
    Module 7 Groundwater Microbiology for more
    details of such a system)

Marine Systems
  • Marine systems (points of difference)
  • The pH, Eh, and temperature of marine systems
    tend to be stable
  • Many of the bacteria are similar to freshwater or
    terrestrial species but can tolerate salinity
  • Many are also adapted to life in aquatic low
    nutrient conditions (oligotrophs)
  • Most microorganisms in marine systems grow slowly
    (because of low nutrient levels)
  • Many have temperature optima of 0 to 20C
  • Biofilms of microorganisms are less common than
    in freshwater (volume effects) but can still play
    an important
  • Many marine aquatic microorganisms grow and
    survive in mixed communities with other
    microorganisms, algae and higher plants
  • The number of microorganisms mL 1 (bacteria,
    especially) in a ocean profile is highest near
    the surface and decreases with depth. At the
    sediment-water interface the numbers are still
    low if the environment is cold and there is no
    light penetration
  • Anaerobic conditions are relatively common where
    oxygen has been utilized (e.g. in the sediment).
    Anaerobes and facultative anaerobes are found in
    those environments
  • The deep sea black-smoker volcanic vents are a
    unique habitat in marine systems

End of Section
This biofilm formed from mixed culture of
Pseudomonas aeruginosa, P. fluorescens and
Klebsiella pneumoniae. The image was taken with
a confocal laser microscope and was generated as
27 overlaid optical sections of 6 micrometer
  • Background and Definition
  • Biofilms are composed of populations or
    communities of microorganisms adhering to
    environmental surfaces.
  • They are usually encased in an extracellular
    polysaccharide that they themselves synthesize.
  • Biofilms may be found on essentially any
    environmental surface where there is sufficient
  • Their development is most rapid in flowing
    systems where adequate nutrients are available

Microbial Mats
Microbial Mats, on the other hand, are
specialized microbial communities composed mainly
of photosynthetic procaryotes. The principle
distinction between microbial mats and other
biofilms is their dependence on photosynthetic
primary productivity as their source of energy.
Microbial Mat
Biofilms may form
  • on solid substrates in contact with moisture
  • on soft tissue surfaces in living organisms
  • at liquid air interfaces..
  • Typical locations for biofilm production include
    rock and other substrate surfaces in marine or
    freshwater environments.

Types of Biofilms
Some other typical biofilm locations and types
From Am. Soc. Micro
Types of Biofilms
Biofilms are also commonly associated with living
organisms, both plant and animal. Tissue surfaces
such as teeth and intestinal mucosa which are
constantly bathed in a rich aqueous medium
rapidly develop a complex aggregation of
microorganisms enveloped in an extracellular
polysaccharide they themselves produce
Here, human dental plaque has been exposed to 5
sucrose for 5 minutes, after which Gram's iodine
(0.33 Iodine in 0.66 KI) was applied. The
sucrose solution was applied to the left central
incisor (which appears on the right) while the
right central incisor served as a control.
Iodine selectively binds to alpha-1,4 glucans
(iodophilic polysaccharide, i.e. glycogen or
amylose) which results in brown to purple
staining. The ability of oral bacteria to store
iodophilic polysaccharides or glycogen-like
molecules inside their cells is associated with
dental caries since these storage compounds may
extend the time during which lactic acid
formation may occur. It is this prolonged
exposure to lactic acid which results in
decalcification of tooth enamel.
From D. RubySchool of DentistryUniversity of
Alabama - BirminghamBirmingham, Alabama
35294john_ruby_at_cs1.dental.uab.eduVincent F.
GerencserHealth Sciences Center of West Virginia
UniversityMorgantown, West Virginia 26506
Dental Biofilm
A scanning electron micrograph of co-adhering
oral microorganisms in dental plaque, showing
so-called corncob structures. Bacteria in the
photograph have a typical corncob structure. Each
kernel is a bacterium, and what one sees is an
aggregate of organisms stacked on top of each
other. Scale bar 10 µm. Rolf Bos, H. J.
Busscher, W. L. Jongebloed, and H. C. van der
Mei, Laboratory for Materia Technica, University
of Groningen, Groningen, The Netherlands
From Am. Soc. Micro
Biofilms in a water pipe 1
Biofilm Development in a Water Supply Pipe (1)
These images show the colonization of opaque
surfaces in a water supply by a heterogeneous
microbial population in a nutrient limiting
environment using scanning electron
microscopy. Figure 1 shows the surface
topography of the biofilm present on a galvanized
iron surface (water pipe line material) at a
magnification of 543X. Figure 2 shows microbial
cells, exopolysachharide material and water
channels present in the boxed area from Figure 1
at 2459X magnification.
Biofilms in a water pipe 2
Biofilm Development in a Water Supply Pipe (2)
Figure 3 shows the presence of water channels
and rod shaped bacteria at a magnification of
2338X. Figure 4 shows a large and uneven clump
of microbial cells at 1302X with a central water
channel (arrow) going into clump of cells.
Sand Grain Biofilm
This image is a scanning electron micrograph of
the naturally occurring biofilm on sand grains in
the clog mat of a septic system infiltration
mound. Scale Bar 150 micrometers
Water treatment plants, waste water treatment
plants and septic systems associated with private
homes remove pathogens and reduce the amount of
organic matter in the water or waste water
through interaction with biofilms.
From Am. Soc. Micro
How do Biofilms Form ?
How do biofilms form?
  • Typically, within minutes, an organic monolayer
    adsorbs to the surface of the slide substrate.
    This changes the chemical and physical properties
    of the glass slide or other substrate.
  • These organic compounds are found to be
    polysaccharides or glycoproteins. These adsorbed
    materials condition the surface of the slide and
    appear to increase the probability of the
    attachment of planktonic bacteria

From Am. Soc. Micro Mark Wiencek, Rohm Haas
Company, Springhouse, Pennsylvania 19477
Formation 2
  • Free floating or planktonic bacteria encounter
    the conditioned surface and form a reversible,
    sometimes transient attachment often within
  • This attachment called adsorption is influenced
    by electrical charges carried on the bacteria, by
    Van der Waals forces and by electrostatic
    attraction although the precise nature of the
    interaction is still a matter of intense debate.
    In some instances, as for example, in the
    association between a pathogen and the receptor
    sites of cells of its host there may be a
    stereospecificity which though still reversible
    is stronger than that achieved strictly by ionic
    or electrostatic forces.
  • If the association between the bacterium and its
    substrate persists long enough, other types of
    chemical and physical structures may form which
    transform the reversible adsorption to a
    permanent and essentially irreversible

Formation 3
  • The final stage in the irreversible adhesion of a
    cell to an environmental surface is associated
    with the production of extracellular polymer
    substances or EPS. Most of the EPS of biofilms
    are polymers containing sugars such as glucose,
    galactose, mannose, fructose, rhamnose,
    N-acetylglucosamine and others.
  • This layer of EPS and bacteria can now entrap
    particulate materials such as clay, organic
    materials, dead cells and precipitated minerals
    adding to the bulk and diversity of the biofilm
    habitat. This growing biofilm can now serve as
    the focus for the attachment and growth of other
    organisms increasing the biological diversity of
    the community.

Biofilms Image
From Costerton Stewart, Scientific American,
July 2001
Biofilm - mature
From Costerton Stewart, Scientific American,
July 2001
Scanning electron micrograph (SEM) of a
Pseudomonas aeruginosa PANO67 biofilm
Enlarged view of A showing details of
From Am. Soc. Micro
These images on the next slide are micrographs
of biofilm cross-sections composed of two
bacterial species (Klebsiella pneumoniae and
Pseudomonas aeruginosa) with progressive exposure
to disinfectant. Untreated biofilm samples
(control) and those following exposure to a low
level (4 mg/L) of chloramine were stained with
two fluorogenic compounds, frozen and cut into
thin (5 µm) sections that were observed by
fluorescence microscopy and photographed. The
base of the biofilm that rests on the substratum
is at the bottom of each image the biofilm
surface that is exposed to the overlying bulk
fluid is the upper aspect of each picture. A
combination of 4'6-diamido-2-phenylindole (DAPI)
and 5-cyano-2,3-ditolyl tetrazolium chloride
(CTC) was used to stain the bacterial cells.
This combination of stains distinguishes
individual cells with active respiration
(red-gold) from those that are nonrespiring
Biofilms may also play a role in the
biodegradation of resistant chemicals since they
can consist of stable aggregations of many
different organisms. An example might be that
of PCB degradation (below) carried out by a
consortium of different microorganisms
Perchloroethylene (PCE), used as a dry-cleaning
agent throughout the world, is one of the most
commonly encountered groundwater contaminants in
the United States. PCE is a priority pollutant
regulated by the Environmental Protection Agency.
Scientists in the laboratory have discovered that
certain bacteria can use PCE for food in the
absence of oxygen.
From Am. Soc. Micro
Biofilms are present in groundwater and may play
a role in microbial activities there.
William Ghiorse, Section of Microbiology,
Cornell University, Ithaca, N.Y., USA
Contact lens case contamination
From ASM - Louise McLaughlin-BorlaceDepartment
of PathologyInstitute of OphthalmologyLondon,
England EC1V 9EL, United Kingdomlmclaugh_at_hgmp.mrc
The scanning electron micrograph shows the inside
of a lens case, collected from a patient with
Acanthamoeba keratitis. Rod-shaped bacteria and
Acanthamoeba cysts are visible in dehydrated
form, a result of the electron microscopy
processing. Amoebal cysts and trophozoites were
cultured from the lens case. Scale bar1
micrometer. Studies have shown that contamination
of lens cases by bacteria, fungi, and amoebae is
common with 20 to 80 of lens wearers having a
contaminated lens case.
Dental equipment 1
A. These specimens comprise sections of tubing
from 30-year-old dental equipment. The tubing is
heavily colonized by bacteria which contaminate
the water used to perform intraoral procedures.
The bacteria, isolated from the water of dental
units, belong to the common water bacteria
community, which include the potential
opportunistic pathogenic microorganisms
Pseudomonas aeruginosa and Legionella
pneumophila. When we look at the structure of
these biofims, bacteria are found to be either
isolated or organized in microcolonies (arrow) of
tightly spaced bacteria embedded in the
glycocalyx B. A closer look at one
From ASM Jean BarbeauFaculty of
DentistryUniversity of MontrealMontreal, Quebec
H3C 3J7, Canadabarbeauj_at_medent.umontreal.ca
Dental Suction line
C. The specimen represents a section of the high
volume suction line from a dental unit. The
deposits on the inner wall () of the evacuation
line are mainly composed of heterogeneous
microcolonies (arrows) composed principally of
bacteria and extensive polysaccharide material
and tissue debris. The majority of recovered
bacteria are Pseudomonas species and
staphylococci. The presence of recognizable cells
and tissue elements in these biofilms shows that
human material can persist in these systems for
an extended period of time and can even be
trapped within biofilms. Since some blood-borne
pathogens can survive for a long time in the
environment, these systems must be cleaned and
disinfected after each patient.
C. Section of suction line from dental unit
Biofilms 3-dimensional view Accumulation
Detachment Simulation and Modelling
BACSIM Simulation Model
Biofilm Simulation Model BACSIM weblink
The model shows the development of a biofilm
consisting of nitrifying bacteria on a bioreactor
vessel. Nitrifying bacteria are an important
part of the nitrogen removal process from
wastewater, and an example of a simple two
species food-chain. The first species (red
circles) oxidises ammonia to nitrite (NO2).
NH4 NO2 Nitrosomonas The second
species (black circles) oxidises nitrite to
nitrate (NO3). NO2 NO3
Nitrobacter Oxygen is required for this process
and growth is usually limited by the oxygen
concentration in the microenvironment, as you can
see in this simulation. Oxygen is displayed as
blue color. Regions of oxygen depletion are
evident as lighter shades of blue. Oxygen
diffuses into the biofilm from the bulk liquid
(dark blue patch), separated from the wall
surface to the right of the hydrodynamic boundary
  • Why are biofilms important ?
  • Some examples
  • Protection of E. coli or pathogens from
    chlorination or chloramination in water
    distribution systems.
  • Protection of microorganisms against predation,
    parasitism or other adverse conditions
  • Maintenance of consortia of different species of
    microorganisms that allow sequential biochemical
  • Surface concentration effects (nutrients and
    toxic compounds)
  • Physical conditions may be different (pH, oxygen
    levels, etc.)
  • Protection against flow (maintain position in
    flowing systems)
  • Medical prevent antibiotic penetration or
    activity, contamination of catheters and drips in
    hospitals, contamination of contact lenses,
    peridontal disease, and possible involvement in
    Legionnaires disease, tuberculosis, kidney
    stones, and others.
  • Engineering biofilms in corrosion of pipes,
    fouling of bio-treatment equipment or transfer
    pipelines, cooling systems, etc.

Reference Sources
  • Reference Sources
  • Center for Biofilm Engineering, Montana State
  • http//www.erc.montana.edu
  • Community Structure and Cooperation in Biofilms.
  • Ed. By Alison, D.G, Gilbert, P, Lappin-Scott
    and Wilson, M.
  • Cambridge University Press 2001

End of Biofilms Section
Title Module 1C
  • Water Pollution and Microorganisms
  • The Phosphorus Cycle
  • The Great Lakes and Lake Simcoe
  • Water Pollution and Microorganisms
  • P cycle as example
  • reat Lakes
  • Control of Eutrophication
  • Lake Victoria, East Africa
  • Key Concepts (Summary)

The Phosphorus Cycle
  • The phosphorus cycle
  • Phosphorus is essential for biological activity
    because of its role in energy transfer (ATP, ADP,
    AMP, etc.) and as phosphodiester bonds in nucleic
  • The cycling of phosphorus does not involve a
    change in valence state (except in the rare case
    of phosphine (PH3) production).
  • The usual transformations are between inorganic
    and organic forms of phosphorous, and between
    insoluble and immobilized forms and soluble and
    mobile forms.
  • The main transformations in the phosphorus cycle
    are between
  • insoluble organic phosphate
  • soluble organic phosphate
  • insoluble inorganic phosphate
  • soluble inorganic phosphate

Some scientists declared Lake Erie dead in the
late 1950s
Phosphorus Cycle 2
  • Most other element cycling (for example - N, S
    and C) involves valence state changes
  • Phosphorus limitation often determines activity
    in aquatic systems.
  • It is the limiting nutrient for algal growth and
    proliferation in many lakes and has been
    recognized as the key element in eutrophication.
  • It is not a common element in the biosphere
    since it is easily precipitated from solution
    with calcium, magnesium and ferric ions.
  • A large quantity is sequestered in sediments and
    minerals and is unavailable to organisms. For
    instance, apatite minerals (typically
    3Ca3PO42.CaFeCl2) are ubiquitous in igneous
    rocks and are also found in metamorphic and
    sedimentary rocks. These rocks contain extremely
    large quantities of phosphorus.

Note There are many other kinds of apatite
minerals but all have the general formula
(M)10(XO4)6(Z)2 where M is Ca, Na, K, or Mg(X)
is P, As, Si, S, Cr, or Ge and (Z) is F, OH, Cl
or Br. The calcium phosphates are the most
important form of apatites, but the roles of
apatite (predominantly hydroxyapatite
Ca10(PO4)6(OH)2 and fluorapatite
Ca10(PO4)6F2) in aquatic systems are not well
understood. It has been often stated that apatite
have no role in the phosphorus cycle of lakes
because of their low solubilitv. Many bacteria
and algae can liberate P from apatite when
growing in close proximity or on the surface of
apatite minerals.
The Scarborough Bluffs on Lake Ontario contain
large quantities of apatite minerals
Phosphorus Cycle 3
  • In the diagrams to follow, note
  • The very low exchange rates in most cases
  • The vastly different "sinks" of P in different
    levels of lakes
  • The very large amount of P available in the
    sediment even if the input is removed
  • Most of the cycling is in the water column
  • The "residence time" or "turnover time" can be
    very low one would expect very low
    orthophosphate levels in the water column if
    bacteria and phytoplankton are present
  • Inputs of orthophosphate (from sewage plants,
    detergents, etc.) are likely to have an immediate
    effect on the levels of phytoplankton in the lake
  • The importance of detritus in the system
    detritus is dead organic material, very often
    partially decomposed.
  • The low rates of sedimentwater exchange
  • The very high sediment levels of P

Phosphorus Cycle in Lake Erie 1
Rate of Phosphorus Cycle in Lake Erie 2
Cycling Rates of Phosphorus
Lake Simcoe 1
  • Another example Lake Simcoe
  • Lake Simcoe is rapidly becoming more eutrophic
  • The Lake Simcoe Conservation Authority is
    attempting to slow down the rate of phosphorus
    input using a Management Plan to persuade
    landowners, members of the public and municipal
    councils to reduce the phosphorus loading.
  • An average of 100 metric tonnes, or the
    equivalent of ten dump truck loads, of phosphorus
    enter Lake Simcoe every year.
  • Average Annual Phosphorus Loading into Lake
  • 6 Sewage Treatment Plants (STPs)
  • 25 urban stormwater runoff
  • 24 soil erosion from crops, streambanks and
    shorelines, and drainage from the Holland Marsh
  • 15 runoff from manure piles / livestock access
    to streams
  • 2 faulty septic systems
  • 28 natural inputs
  • More recently it has been shown that a large
    percentage of P input is via atmospheric
  • Lake Simcoe
    Conservation Authority Web Site

Lake Simcoe 2
  • The Conservation Authority is proposing a series
    of Remedial Projects to reduce P input
  • Remedial Projects
  • Phosphorus pollution is also being reduced with
    the help of landowners and a cooperative
    assistance program offered by the Lake Simcoe
    Region Conservation Authority.
  • The Landowner Environmental Assistance Program
    (LEAP) promotes conservation practices which
    reduce pollution, improve the environment,
    conserve our natural resources and generally
    improve the quality of living for everyone within
    the watershed.
  • Projects are targeted to address both urban and
    rural sources of phosphorus pollution.
  • Eligible projects include
  • constructing manure storage and runoff
    containment systems for feedlots installing
    milkhouse waste treatment systems fencing
    livestock access to a watercourse installing
    erosion control structures to reduce erosion on
    agricultural lands fixing streambank
    shoreline erosion problems tree planting in
    high priority areas controlling urban runoff.

Water Pollution and Microorganisms
Water Pollution and Microorganisms
  • Effects of pollution on microbiological processes
    in water.
  • This concerns the environmental toxicology of
    toxic compounds and elements, and the effects of
    nutrients on microorganisms and their activities
    in water systems.
  • 2. Pollution of water supplies by microorganisms
  • This is concerned with the role that pathogenic
    microorganisms play in making water unfit for
    consumption (human or animal) or making it unfit
    for crop irrigation purposes.

Effects of pollutants
1. Effects of pollutants on microbiological
processes in water
  • Organic compounds are degradable compounds that
    cause Biological Oxygen Demand (BOD) problems
    through high oxygen consumption as they are
    degraded. This causes oxygen deficits and
    consequent modification of other microbial
    processes and also  fish kills.
  • Inorganic compounds result from mineralization
    processes of organic compounds -   rock leaching,
    run off, fertilizers, etc. Most common are
    phosphate, nitrate sulfate, ammonium ions and
    carbon dioxide.
  • Recalcitrant or xenobiotic compounds such
    materials as herbicides, insecticides,
    larvicides, nematocides, molluscicides,
    fungicides, etc., can leach into or be directly
    added to aquatic systems. Other recalcitrant
    organics such as ABS (alkyl benzene sulfonate)
    detergents, fluorinated hydrocarbons,
    polyethylene, poly vinyl chloride (PVC),
    industrial solvents such as toluene, benzene,
    xylene, and trichloroethylene, polynuclear
    aromatic hydrocarbons such as naphthalene,
    phenanthrene, etc., may also enter aquatic
    systems from various point sources or from the
  • Heat input is not often regarded as pollution,
    but can have significant effects near large scale
    heat outputs such as those from hydro generating

Inorganic Compounds
Inorganic compounds - details
Acid rain
Effects of Acid rain on Fresh water Systems -
Acid rain is actually an input of inorganic ions
such as sulfate and nitrate. The total
atmospheric input into an aquatic system such as
the Great Lakes can be very substantial a major
city may contribute 300,000 lbs/sq. mile of dust
per year.
Acid mine drainage
Acid mine drainage can contribute ferric and
ferrous iron and sulfate plus some trace
minerals (uranium, chromium, etc.).
Toxic industrial wastes
Acid Mine Drainage entering Creeks in
Toxic industrial wastes include cyanide,
chromium, arsenic, cadmium, lead and mercury.
Radioactive materials
Radioactive materials constitute a special case
and can include radioactive carbon, phosphorus,
radium, uranium, daughter fission products such
as strontium.
It is impossible to examine all of these
potential aquatic pollutants. Some of them will
be dealt with in more detail in different
sections of the course. Instead of examining a
wide range of pollutants, we will examine the
case where a nutrient becomes a pollutant. This
example is that of phosphorus in lake systems
leading to algal blooms, fish kills, "green"
lakes, etc. - the process of eutrophication. We
will also use this situation as a model for a
broader model of what pollution really is - a
measurable deleterious effect on an ecosystem.
Phosphorus as a pollutant in lake systems
Phosphorus as a pollutant in lake systems
In an ideal ecosystem, a balance between
photosynthesis and respiration is maintained
(i.e. between the production and destruction of
organic matter and therefore between the
production and consumption of oxygen and carbon
dioxide). This can be expressed as a
stoichiometric equation (equation 1)  
Phosphorus and P/R
In an ideal ecosystem, a balance between
photosynthesis and respiration is maintained
(i.e. between the production and destruction of
organic matter and therefore between the
production and consumption of oxygen and carbon
dioxide). This can be expressed as a
stoichiometric equation (equation 1)  
A. is the ionic composition of seawater B. is
the empirical formula for algal cells
For sea water, where the equation usually
describes the balance between P and R very well,
it is possible to examine the stoichiometric
relationships between the various elements in the
equation. The mole ratios between the elements
are N P C 16 1 106 O2 N 9
O2 P 138
N-P levels
In the system described above (in equation 1),
the elements are used in these ratios for
photosynthesis in the photic zone.
N-P levels
In the system described above (in equation 1),
the elements are used in these ratios for
photosynthesis in the photic zone.
Ratios CHONP
106263110161 The dead algae settle out and
are mineralized (respiration) and release the
same ratio of nutrients back into the water.
Oxygen is evolved during photosynthesis and
consumed during respiration in the ratios given.
The equation is a very simple one, but seems to
be generally true for marine ecosystems. The
result is that, as a result of photosynthesis,
both N and P become exhausted simultaneously.
Perhaps at some point P was limiting, but by N2
fixation and denitrification processes the N
level was adjusted to the present levels.
(Alternatively, the presently observed ratios of
elements in marine algae adjusted to the
preexisting levels of nutrients?).
Ocean NP ratios
We can also express these molar ratios in
graphical form by plotting correlations between
concentrations of N, P, O, etc. The figure below
shows the result of this correlation for oceanic
Water samples from many ocean sampling sites
Note that the line passes through the origin,
indicating that the stoichiometric relationship
holds and that neither N nor P is limiting growth
in the ocean they are exhausted simultaneously!
Lake NP ratios
This figure gives the same plot for a typical
Note that the line does NOT pass through the
origin indicating that there is an excess of N
in the water, therefore phosphorus content limits
growth in this lake. This is also indicated by
the fact that the NP ratio is higher 90 versus
16 for the oceanic waters.
NP ratios
Plotting these ratios from different parts of the
ocean and lakes at various times of the year
provides a very good measure of the nutritional
status of the lake and the nutrient actually
limiting growth can be determined. Of course
local variations occur,  but in general terms the
oceans do not exhibit P limitations while fresh
water lakes often do. Note This entire
argument is based on Equation 1. This equation
assumes that all nutrients are recycled. The
equation does not hold where the mixing of the
water is rapid compared with the settling time of
the dead cells. Spatial separation of the
photosynthesis and respiration zones (as in deep
lakes) also changes the observed stoichiometry.
Due to the flow rate of the water, rivers and
estuaries separate the two processes in time .
A consequence of these concepts is that it is
possible and often valuable to regard any water
pollution as simply a mechanism that causes an
imbalance between P (photosynthesis) and R
(respiration). This is an oversimplification
but can lead to some instructive insights into
the ecology of water pollution Phosphorusproduce
d eutrophication of lake systems is one example
of this. Adding phosphorus to the lake allows
additional algal biomass to be produced by
photosynthesis and so causes algal blooms and
oxygen deficits as the biomass decays. In a
very deep lake, the settling of the algal biomass
produces the hypolimnion a layer with low oxygen
Estuary 1
Another example is the estuary of a river This
is a natural concentration mechanism for
nutrients coming down the river system. It is
one of the most productive ecosystems known
because of this concentration effect and the by
the fact that the flow of the water separates the
P and R zones in the river estuary.
Estuary 2
Another example is the estuary of a river This
is a natural concentration mechanism for
nutrients coming down the river system. It is
one of the most productive ecosystems known
because of this concentration effect and the by
the fact that the flow of the water separates the
P and R zones in the river estuary.
Estuary 3
Another example is the estuary of a river This
is a natural concentration mechanism for
nutrients coming down the river system. It is
one of the most productive ecosystems known
because of this concentration effect and the by
the fact that the flow of the water separates the
P and R zones in the river estuary.
Nutrients Recycled and reused
Meditteranean Sea
The reverse situation (where there is a net loss
of nutrients due to current flows) can also
occur. The best example is in the Mediterranean
Sea. The pumping action removes nutrients from
the lower levels this effectively separates P
and R zones and leads to nutrientpoor water.  
Title Page Module 1D
  • Water Pollution and Microorganisms
  • The Phosphorus Cycle in Module 1C
  • The Great Lakes and Lake Simcoe in Module 1C
  • Water Pollution and Microorganisms in Module
  • P cycle as example in Module 1C
  • The Great Lakes
  • Control of Eutrophication
  • Lake Victoria, East Africa
  • Key Concepts (Summary)

Great Lakes
The Great Lakes
Phosphorus and its effects have been studied
mainly in freshwater lakes such as the Great
Lakes and many European lake systems. It has
become apparent that the lake cannot be
considered in isolation the surrounding drainage
basis must also be considered. When this is done,
it is possible to calculate the input of
phosphorus into a lake system and to try to
isolate the effects of humanity's activities in
generating extra phosphorus input into the lake.
Great Lakes 2
The Great Lakes
Phosphorus and its effects have been studied
mainly in freshwater lakes such as the Great
Lakes and many European lake systems. It has
become apparent that the lake cannot be
considered in isolation the surrounding drainage
basis must also be considered. When this is done,
it is possible to calculate the input of
phosphorus into a lake system and to try to
isolate the effects of humanity's activities in
generating extra phosphorus input into the lake.
Previous Diagram of Hydrologic Cycle in the Great
Lakes Drainage Basin
P in Great Lakes
It is then possible to model the results of
limiting these inputs and finding the most
efficient way to slow down or prevent the
eutrophication process. Typical sources of
phosphorus input into the lake system were
Lake Area effects
This table is based on a population density of
150,000/km2 with a total excretion each of 3
g/day (representing a major city). To obtain
some idea of the effect on a lake in the drainage
area, the input in g/m2/year is multiplied by
the factor drainage arealake surface
area This gives the total phosphorus loading
for the lake in g/m2year (as shown in the table).

Control of Eutrophication
  • Control of phosphorus eutrophication
  • Legislated restriction of phosphorus input by,
    for example, improving sewage treatment
    facilities or removing phosphorus detergents from
    the market. Improvement of farming practices so
    that excess fertilizer use is limited.
  • Ecosystem control this is a combination of
    methods which can apply to any "pollution
  • There are some very basic and simple ecological
    principles involved in ecosystem control
  • The background to the process is to consider the
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