Title Page: The Aquatic Environment

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Title Page The Aquatic Environment
The Aquatic Environment

• Surface Fresh Water Systems Rivers, Lakes and
  Wetlands
• Surface Saline Water Systems Oceans, marshes,
  estuaries
• Groundwater Systems (fresh)
• Groundwater Systems (saline)
• Ice, glaciers and compressed snowpack
• Permafrost

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Sampling

• Surface Fresh Water Systems Rivers, Lakes and
  Wetlands

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
procedure

• 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.

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Lake Morphology 1
Sampling Lake Systems 1
Lake Morphology
Littoral Zone
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Lake Morphology 2
Sampling Lake Systems 2
Lake Morphology
Light Compensation Point (about 1 of light
intensity of sunlight)
PROFUNDAL ZONE
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Lake Morphology 3
Sampling Lake Systems 3
Lake Morphology
Benthic Zone
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Lake Morphology 4
Sampling Lake Systems 4
Lake Morphology Holomictic Lakes (turnover and
mixing)
EPILIMNION
Thermocline
HYPOLIMNION
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Lake Morphology 5
Sampling Lake Systems 5
Lake Morphology Meromictic Lakes ( no turnover
and mixing)
MYXOLIMNION
Chemocline
MONOLIMNION
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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. 
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Lake Sampling
Core Sampler taking sediment samples from the
Benthic Zone
Bottle Sampler taking water samples from
various depths
EPILIMNION
Thermocline
HYPOLIMNION
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Sampling Cruise
Lake St. Clair St Clair River
Niagara River
Detroit
Buffalo
Toledo
Cleveland
Lake Erie Typical Sampling Cruise Scheme
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Lake Erie Hot Spots
Lake St. Clair St Clair River
Niagara River
Detroit
Buffalo
Toledo
Cleveland
Lake Erie Typical Sampling Cruise Scheme and
Major Contaminated Regions
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Toxics Distribution in Great Lakes
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Toxics Distribution in Great Lakes 2
Phenols/Organics
Phenols and Organics
PCBs, PAHs and other organics
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Toxics Distribution in Great Lakes 3 Pesticides
Pesticides Mirex and DDT
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Toxics Distribution in Great Lakes 4Summary
Summary Overview
Mercury. Metals, Asbestos
Organics, PAHs, pesticides, phenols
Mixtures
Mixtures
Mixtures
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Photographs Lake Erie 1970s
Lake Erie in the 1970s and early 80s
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Photographs Lake Erie 1990s
Lake Erie in the 1990s
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Biomass
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.

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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
WebLink
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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
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Microscope
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
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Biomass Methods
Other Methods for Measuring Biomass

• Most Probable Number (MPN) technique
• Viable counting procedures
• Biochemical methods for estimating biomass such
  as
• 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
  spectrophotometer.  

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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
  media
• 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

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Activity Measurements 2

• Rate of increase in the number of colonies on
  media
• 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
filters

• 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
  period.
• 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.

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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
Chemostat
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
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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
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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
  bottles.
• 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
  fixation.
• 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
  samples.

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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.

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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
levels).
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
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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
  environments.
• 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
  phosphorus.
• This will serve as the example to show the
  typical functions of bacteria in nutrient cycling.

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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
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Thermophiles

• 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.
•  

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Thermophiles

• 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

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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
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Thermophiles

• 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.

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Yellowstone Park
Yellowstone Park Hot Springs - Zonation
37
Psychrophiles

• 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
  psychrotrophs.
• 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.

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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
  themselves.
• 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.

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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
  present
• 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.

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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
41
Can we make some generalizations about aquatic
bacteria?

• 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)

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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
43
Biofilms
44
Biofilms
Biofilms
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
thickness

• 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
  moisture.
• Their development is most rapid in flowing
  systems where adequate nutrients are available

45
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.

46
Types of Biofilms
Some other typical biofilm locations and types
From Am. Soc. Micro
47
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
48
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
49
Biofilms in a water pipe 1
Biofilm Development in a Water Supply Pipe (1)
1
2
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.
50
Biofilms in a water pipe 2
Biofilm Development in a Water Supply Pipe (2)
3
4
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.
51
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
52
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
53
Formation 2

• Free floating or planktonic bacteria encounter
  the conditioned surface and form a reversible,
  sometimes transient attachment often within
  minutes.
• 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
  attachment.

54
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.

55
Biofilms Image
From Costerton Stewart, Scientific American,
July 2001
56
Biofilm - mature
From Costerton Stewart, Scientific American,
July 2001
57
Streamers
Scanning electron micrograph (SEM) of a
Pseudomonas aeruginosa PANO67 biofilm
Enlarged view of A showing details of
streamers
From Am. Soc. Micro
58
images
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
(green).
59
images
60
images
61
Biodegradation
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
62
Groundwater
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
63
Contact lens case contamination
From ASM - Louise McLaughlin-BorlaceDepartment
of PathologyInstitute of OphthalmologyLondon,
England EC1V 9EL, United Kingdomlmclaugh_at_hgmp.mrc
.ac.uk
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.
64
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
microcolony
From ASM Jean BarbeauFaculty of
DentistryUniversity of MontrealMontreal, Quebec
H3C 3J7, Canadabarbeauj_at_medent.umontreal.ca
65
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
66
Biofilms
Biofilms 3-dimensional view Accumulation
Detachment Simulation and Modelling
67
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
layer.
68
Summary

• 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
  pathways
• 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.

69
Reference Sources

• Reference Sources
• Center for Biofilm Engineering, Montana State
  University
• 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
70
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)

71
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
  acids.
• 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
72
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
73
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

74
Phosphorus Cycle in Lake Erie 1
75
Rate of Phosphorus Cycle in Lake Erie 2
Cycling Rates of Phosphorus
76
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
  Simcoe
• 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
  pathways.
• Lake Simcoe
  Conservation Authority Web Site

WebLink
77
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.

78
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.

79
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
  atmosphere.
• Heat input is not often regarded as pollution,
  but can have significant effects near large scale
  heat outputs such as those from hydro generating
  stations.

80
Inorganic Compounds
Inorganic compounds - details
Acid rain
WebLink
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
Pennsylvania
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.
81
Eutrophication
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.
82
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)  
Photosynthesis
Respiration
83
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)  
Photosynthesis
Respiration
A. is the ionic composition of seawater B. is
the empirical formula for algal cells
84
Equation
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
85
N-P levels
In the system described above (in equation 1),
the elements are used in these ratios for
photosynthesis in the photic zone.
86
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?).
87
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
waters.  
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!
88
Lake NP ratios
This figure gives the same plot for a typical
lake.
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.
89
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 .
90
Eutrophication
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
concentration.
91
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.
92
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.
93
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
94
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.  
95
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
  1C
• P cycle as example in Module 1C
• The Great Lakes
• Control of Eutrophication
• Lake Victoria, East Africa
• Key Concepts (Summary)

96
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.
97
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
98
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
99
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).

100
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
  problem."
• There are some very basic and simple ecological
  principles involved in ecosystem control
  measures.
• The background to the process is to consider the
  whol