ACTIVITY: Do Microbes Cause Water Pollution?
INTRODUCTION:
Common sources of water pollution include runoff of soluble materials from farmlands, roads, golf
courses as well as accidental spills and leakage of sewage and perhaps industrial waste. When
these substances reach natural waters (in their travels ‘down’ the watershed ) they encourage the
growth of microbes that either use them as food or are simply stimulated to grow because of the
excess nutrient provided. (Remember, N and P and K are not food, they supplement the building
of macromolecules in the cell.) When photosythetic organisms are stimulated in this way, they
pull carbon into the system (CO2) increasing the biomass in the water. When this material dies, it
is used for food by other organisms that do use the carbon as food and require large amounts of
oxygen. This rapid depletion od oxygen (ecostsems are okay with slow cycling) can be deadly for
fish and other animals.
I adapted this exercise to be an inquiry based activity, and it is originally from ‘Microbes and
Water Pollution,’ an activity in Practical Microbiology for Secondary Schools, a publication of the
Society for General Microbiology and that booklet can be ordered from them for about 12.00
(www.microbiologyonline.org.uk). Because I could not obtain it in e-format, I had to retype most of
it here for you. Please do not mistake the ‘word-for-word’ as plagiarism, I fully acknowledge the
source.
In Part I below, I’ll first describe a very simple approach to this question, the mason jar
‘experiment’ or demonstration. Next, the more formal adapted activity.
PART I. THE MASON JAR ACTIVITY: In WISTR, you will recall that we examined some
mason jars with Long Island Sound water , that had been incubated in the sunlight for 1-2 weeks,
one with fertilizer (Miracle Grow Plant Food) added. The observation? The jar with fertilizer had
become quite green, indicating algal and/or bacterial growth. Why? The microorganisms were
already in the wate, and the fertilizer provides them with the additional nutrients they need for
abundant growth (especially N and P).
Important learning opportunities:
1. This is a powerful lesson, as simple as it may seem! First, microbes are invisible, but their
capabilities are vast. Changing the environment may cause predicted or unpredicted
consequences. In this case you simply added a nutrient.
2. All living things need N and P and may not be getting enough of them. By adding more you
stimulate all sorts of growth. Your jar gets green because you put it in the light, stimulating
algae and photosynthetic bacteria (see other handout explaining the difference). Try putting a
jar in the jar, with nutrients. Will other microbes grow better??
3. Biomass arises through phtosynthesis. All that carbon that arises in the jar does NOT come
from the nutrients added. It comes directly from CO 2 in the air, which is abundant.
Remember, photosynthesizers need CO2 and water to build sugars and starch and all the
molecules in the cell. Added micronutrients and macronutrients N, P etc. are also needed
since some molecules contain N and P as part of their building blocks (DNA and protein).
THE NOTION THAT PLANTS GET BIGGER BECAUSE OF NUTRIENTS is one of the most
common misperceptions in our school system. You can correct that. Carbon, which makes up
the bulk of the biomass of living things, comes from carbon (CO 2) , and carbon released by
burning or respiration also becomes CO2 again. The cycle is very real and students need
your help to understand it,. This experiment helps! The fundamental understanding is far
more important than the equations and definitions.
4. All pollution does not kill growth. Some forms of pollution promote too much growth as
evidenced in this study. You are of course familiar with this term, eutrophication. Why is it
harmful? The growth itself is not toxic, bad, or poisonous. Sometimes algal growth is
unpalatable or non-pleasing in swimming or drinking water settings. But the worst problem is
how this changes ecosystems (the algae will crowd out more natural communities) and
worse, algal ‘blooms’ always die off at some point, and their dead bodies must go
somewhere. This serves as food for non-photosynthesizing organisms, who will RESPIRE
the carbon. Yes, the carbon will return to the atmosphere if this happens close to the surface.
But high levels of respiration quickly use up all the oxygen in the vicinity of the rotting matter.
This drop in oxygen has a very negative impact on animals (fish and invertebrates etc.) in the
water, and may kill them off. Not good!
ALL THIS DISCUSSION STIMULATED FROM A FEW JARS OF WATER!!
Of course, discussion can go on before setting up the water, and jars can be set up in an
experimental fashion with students predicting the outcome.
PART II. MICROBES AND WATER POLLUTION (from SGM booklet)
Learning objectives:
 Microbial populations and communities change in natural waters when pollutants are added
(or any major change occurs)
 One of the major sources of pollution in Long Island Sound is overload of nutrients, or
eutrophication.
 Eutrophication causes overgrowth of photosynthetic organisms which adds biomass to an
aquatic system (Biomass = total amount of carbon).
 Extra biomass, when ‘burned’ in respiration, can seriously deplete oxygen levels.
MATERIALS:
 River, Lake, Pond, or Seawater, collected fresh, several liters for several groups of students
(be careful not to collect where there is sewage effluent. If suspected, use gloves when
sampling and handling)
 Hay or soil or mud
 1-5g nutrient broth powder
 1g potassium nitrate
 1g potassium phosphate
 6 flasks (500ml size) or jars
 measuring container
 cheese cloth and rubber bands
 Filter paper, funnels
 balance
 marking pens etc.
INQUIRY BASED PREPARATION:
Pose questions: What factors in water contribute to algal growth? What conditions favor such
growth? What conditions favor non-photosynthetic microbial growth? What would be the evidence
of such growth? Different groups of students might pose different questions.
Propose Hypotheses: Ask the students to formulate hypotheses to address these questions. For
example: Increased input of Nitrogen will enhance algal growth when sunlight is present.
Design and Test: How will students test their hypothesis? What will be their controls? For
example, in the above example, controls would include samples without added N, samples
incubated in the dark, etc.
Determine Method of Measurement: Please address – what will be measured as evidence of
enhanced growth??? Whereas there are techniques for extracting and measuring chlorophyll, a
simpler approach would be to measure biomass. Filter a known volume of water. Filter the
sample. Dry the filter. Weight the filter. If the flask was GREEN and no carbon was added to the
flask, one can assume this biomass is a result of photosynthesis. This cannot be assumed if you
add carbon to flask (nutrient broth powder) and/or sample is incubated in the dark.
NOTE: Cloudiness can also be measured as an indicator of bacterial growth, using a
spectrophotometer, or by comparing to known ‘cloudiness’ standards. A simple test of placing the
cloudy solution in a tube and trying to ‘read’ fonts of different sizes through the tube can be a
rough way of quantifying cloudiness, if you do not have a spec. I add this because a cloudy nongreen solution without much biomass may be tough to weigh on filter paper. The paper will weigh
so much more, that it will be hard to get an accurate value for the small amount of biomass added
to it, even though the appearance of cloudiness clearly tells you that it is there. Rule of thumb,
filter paper works well for algal biomass, cloudiness measurement is best for non green growth.
Of course, students may choose to tabulate their results as simply EITHER/OR, YES/NO for the
presence or absence of growth, or the presence or absence of photosynthetic growth.
Predictions: After designing and setting a plan for the experiment, students should make an exact
prediction about their hypothesis, for the experiment that they will be doing. What experimental
results would clearly demonstrate support for their hypothesis?? What results would clearly not
support the hypothesis, suggesting some alternate explanation?? Please, please, stay away from
using the words PROVE or DISPROVE with your students. The results of experiments like these
do not prove or disprove anything,. They simply lend or do not lend support.
PROTOCOL:
1. Label the flasks according to the type of treatment desired. (If you have enough materials,
replicates are a good approach. This means, more than one flask for each treatment, 3 being
a good number).
2. Amounts that can be used for treatments: For P treatment, add .1g potassium phosphate, for
N treatment .1g potassium nitrate, for nutrient broth treatment (Carbon and nutrients) add 1 g
nutrient broth powder; for soil/sediment/hay/grass, add about 1T. This treatment is used if
students postulate that material must be added to flask to provide microbes as opposed to
microbes already being present in the water.
NOTE: Please keep in mind that nutrient broth consists of proteins and nutrients, don’t let the
name mislead you, it is a very different addition than N or P which are strictly nutrients. This
treatment adds a lot of carbon to the system)
3. Measure 100ml water into each flask. Place clean squares of cheescloth (3-4 layers) or top,
and fasten with rubber band, need not be tight. Cotton plugs will work too if you have them.
4. Incubate under appropriate conditions. Light would of course require a windowsill or plant
light. Be sure that the flasks will not heat up more than their non-light counterparts.
Sometimes we wrap flasks in foil and place them under the same lights for the dark
treatment, so temp is about the same.
5. Over the next 2-4 weeks, many changes will occur in the flasks. The observations will
stimulate much discussion and can be recorded on a report sheet or in a notebook.
HOWEVER, be sure to decide on some stopping point at which the results must be
measured and recorded and a decision made about support of the hypothesis.