This experiment is courtesy of
Wilson Middle School
Lorraine Holowach, Ph.D.
Rohm and Haas, Company
Spring House, PA
This project provides students with an
understanding of what the term "biodegradable" means and
provides them with the awareness that man-made products just
don't go away when they discard them. Students will also
develop an understanding of the difference between synthetic
(man-made) and natural materials. They should be able to
reason that man tries to imitate nature in terms of what is
produced but that imitation fails in terms of
- To observe the degradation or lack
of degradation of certain materials by soil
- To conduct a controlled scientific
- To understand the meaning of
- To recognize the stability of
man-made polymers and their lack of
- To appreciate the persistence of
products in the environment.
- To develop an understanding of how
slowly changes may occur to materials when they are
- To recognize the responsibility we
all have to reduce, reuse and recycle our "trash."
- To evaluate the need to produce
products that are biodegradable.
We are members of a disposable
society. We dump or landfill what we no longer need. Just
what happens to discarded trash after we have no need for
it? Contrary to what you believe, there is no "trash fairy."
What happens then to that trash? The problem is, maybe
nothing at all. That's a dilemma. Our trash may remain in a
stable state for years, decades, or even millennia. Maybe
even forever. That's frightening.
The real problem lies with those
products that we label disposable. It is estimated that
about 100,000,000 tons of packaging is discarded worldwide
per year. This includes sheets, strapping, shrink wraps,
trash bags, bottles, and beverage rings. The United States
alone generates 160,000 tons per year of solid waste; 18% of
this volume is plastic, 38% is paper, and 2% is glass. Most
of these products are inherently stable and do not
Just recently the word biodegradable
has appeared on the labels of some trash bags, diapers, and
various other plastics. Even more recently manufacturers
have been asked to remove the word biodegradable from their
labels because these products are not truly biodegradable,
rather they disintegrate into smaller units of the whole,
but the original product is still there.
Making products that degrade
adequately into environmentally safe byproducts should be
the concern of every manufacturer in the world. We must
remember that manufacturing is consumer-driven. We have to
consider what kinds of things we'd be willing to give up to
clean up! If we don't approach this problem very soon our
children are destined to live in a world of trash piles and
pollution. Recycling is a partial solution to waste
reduction, but it's not a solution to the long range problem
of cleaning up the environment. It is important that
students be aware that the trash they create may be around
for a very long time. How can they help as individuals? They
can reduce, reuse, and recycle and be aware.
Many of us believe that all of our
trash degrades by some natural mechanism after it has been
taken to the landfill or enters the natural environment.
It's true that many things that we dispose of are recycled
back into the environment. Some materials deteriorate and
some degrade. There are three ways materials may degrade.
Photodegradation is stimulated by sunlight, usually
ultraviolet radiation. Biodegradation is stimulated by the
enzymatic action of microorganisms. Chemical degradation
occurs when the bonds between molecules break due to
inherent instability of the material. These processes aid in
breaking down (degrading) materials so that they can be
recycled by natural processes.
Biodegradation, the focus of this
lesson, is accomplished by microorganisms. Microorganisms
are capable of utilizing many types of carbon-containing
molecules as substrates for growth, metabolism and cell
division. The major elements required for microbial growth
in oxygen-containing environments are carbon, nitrogen,
hydrogen and phosphorous. Elements that are also essential
but required in much smaller quantities include sodium,
potassium, calcium, magnesium, sulfur, iodine, iron, boron,
copper, cobalt, molybdenum and zinc. This project focuses
upon the ability of soil microorganisms to use natural and
man-made materials as carbon sources. Complete degradation
would result in a product being degraded to carbon dioxide,
water and minerals and could be accomplished by one or more
of the processes of chemical, photo- or microbial
degradation. Some products do not detectably degrade at all
(styrofoam and many plastics, for example) or are only
partially degraded (plastics, paper products). In some
instances, intermediates formed during degradation may be
more toxic than the starting material.
The students should be introduced to
the term "polymer." There are many familiar examples of
naturally-occurring polymers, including cellulose, DNA,
proteins, starch. Naturally-occurring polymers are
biodegradable &emdash; they are synthesized and degraded by
enzymatic processes. Natural products degrade at very
different rates depending upon chemical composition;
chemical or structural modifications can alter the rate of
degradation. Some natural products, such as lignin from
trees, degrade only very slowly (consider how long it takes
a fallen tree to decompose in the forest). An important
reason why these naturally-occurring polymers are not
incorporated into consumer products is that they do not
generally possess the physical characteristics that make
chemically synthesized polymers indispensable to our
life-style, or the expense of using naturally-occurring
materials is too high.
Many chemically-synthesized polymers
are very rich in carbon, for example, polyethylene,
polystyrene and poly(vinyl chloride); however, organisms do
not possess enzymes that degrade these materials, and they
are generally very stable to chemical or photolytic
degradation. Thus, these materials persist for very long
times in landfills and the open environment.
In this lesson, students will develop
an awareness of the persistence of man-made polymers; those
various plastic products that participate in every aspect of
our lives. They include six pack beverage rings, diapers,
styrofoam cups, milk and juice jugs, food wraps, detergents
to list a few. The efficacy of these products is remarkable,
but what of their final destination?
Degradation – a chemical reaction leading to the
breaking of bonds in molecules such that new molecules may
be formed as opposed to deterioration. This process uses
carbon, nitrogen, moisture and microbes.
Biodegradation – a degradation process in which a
living organism, like bacterium or fungus, metabolizes or
breaks down a material through an enzymatic process.
Recycle – to use again; pass through a series
of changes or treatments in order to regain materials for
Photodegradation – A degradation process initiated by
light, such as sunlight or a UV source.
Deterioration – The
fragmentation of an article in which the individual
fragments retain the properties of the original article and
which is caused by environmental or physical forces.
Polymer – A large molecule built by the
repetition of small, simple chemical units (they are the
basis for all plastics).
The following are three separate
procedures that can be followed in order for students to
observe the process of biodegradation or the lack of it in
certain materials. It would be advisable to begin these
procedures at the start of the school year so that students
may follow their results for as long as possible, even the
entire year. It must also be noted that bacteria and or
fungi may grow prolifically, and should be grown in closed
containers, and should also be sterilized in an autoclave
when the procedure is completed. These microorganisms should not be handled by
students. Before beginning experiments, be sure to read the
article on "Safety in Microbiology Experiments" available
from Sister Helen M. Burke, Ph.D., Chemistry Department,
Chestnut Hill College, Philadelphia, PA 19118, (215)
Observing Degradation Using Soil in
"Zipper" plastic bags, quart
Compost or rich garden soil (compost
may be made or obtained at your local county recycling
center for free)
Variety of materials to be tested for
biodegradability (cellulose filter paper, chewing gum and
packaging, toilet and facial tissue, paper bags, newspaper,
styrofoam, aluminum foil, leaves, fruit, grass
clippings). Do Not Use Animal
Products. They Will Putrefy!
- Give each student a plastic bag
partially filled with three cups of uniformly moistened
- Let each student choose one item
to be placed in the bag to observe for
- Thoroughly wet the items with
water, blot excess water away from the surface, place the
item inside the bag on the soil so that the item is in
good contact with the soil and may be easily observed
through the bag.
- Insert a plastic straw at one edge
of the bag, zip the bag closed so that the straw extends
out of one side of the bag to allow some air into the
bag. Do not insert the end of the straw into the
- Label each bag with student's
name, date, item added, soil type, or other treatment ,
- Place the bags in a drawer or box
and leave in a warm location away from drafts and
temperature fluctuations. Bags should not be left in
- Observe and record results weekly
or as appropriate in a journal. Have students compare and
contrast their variables, using information about the
chemical and physical composition and natural or
synthetic origin when making their observations. Handle
the bags with care so that the material remains in good
contact with the soil and also remains visible.
- Have students make a hypothesis
about how their material will look at the end of the
- Disposal: Remove straws from bags,
and destroy living microorganisms by heat sterilization
or addition of chemical disinfectant to the bags. For
chemical disinfection, prepare a solution of Lysol�,
Clorox� or alcohol according to the recommendations
of Pogosian in the article "Safety in Microbiology
Experiments". Allow the bags to sit overnight, pour off
liquid, seal bags and dispose of bags and soil in the
- Try using different types of soil
(sterile, garden, worm, mushroom, etc.)
- Try setting up other variables
such as temperature, humidity, radiation.
- Try heating the soil to kill
organisms before adding item.
- What conditions could you devise
that would speed up degradation? (Will a gum wrapper on
top of a table degrade as fast as a wrapper in
- Try adding fertilizer, such as
plant food in water solution, to speed the rate of
degradation (see supplement for explanation).
- Before beginning the experiment,
what did you think your variable would look like over
time? Should it degrade or not?
- What kind of changes took place in
- Is your material biodegradable?
How do you know?
- What is your material made
- What do you think will happen to
your material over the next 10 years?
- As a class make a list of products
that will stay stable in the environment and not degrade
for a very long time.
- Make a list of things that you
would not want to degrade (e.g. paints, clothing)
- What did people do before plastics
and disposables were available?
- How could we minimize the use of
disposables by reusing products and still maintain food
safety, hygiene, freshness, etc.?
- Make a list of products that will
outlive their usefulness.
Observing Degradation In A Minimal
Minimal liquid media (prepare or
Distilled water (if media is to be
made by teacher or students, can be purchased at grocery
store or pharmacy)
125-milliliter flasks, baby food jars,
other clear containers (disposable plastic flasks can be
purchased from suppliers such as Fisher Scientific or
Carolina Biological Supply)
Soil (garden soil, sterile potting
Cellulose filter papers or chewing gum
and wrappers, or other materials to test
Preparation of Minimal Liquid
Although bacterial media and culture
containers are normally sterilized before use, this
experiment may be successfully conducted using unsterilized
media and containers, since only minimal media is used and
very few microorganisms survive under these conditions.
Appropriate controls (media with no substrate) should be set
up to observe whether unsterilized media supports
microorganism growth. If appropriate facilities are
available, the media and glassware may be sterilized before
To prepare 1000 milliliters of minimal
1. In a 2-Liter container, add 800
milliliters of distilled water
2. In the water, dissolve the
grams K2HPO4 (dibasic
3. After salts are dissolved, bring
the volume to 1000 milliliters with distilled water.
4. Check pH using pH paper or pH
meter; pH should be between 6.5 and 7.5 and does not need
- Add 30 milliliters minimal liquid
media to each container.
- Add 2 grams of soil to appropriate
containers (should have at least one control lacking
- Wet the item to be tested with
water or minimal liquid media.
- Place the item to be tested in the
container with media. You may want to use a plastic straw
to support the item so that it does not rest against the
sides of the container (if the item absorbs water, water
will wick up the side of the container and observations
will not be as clear as if water surface is undisturbed
around the item being tested).
- Close the container with a lid or
a cotton plug. Do not seal tightly, as air must be able
to enter the container during the experiment.
- Incubate the containers at room
temperature. Keep away from drafts and sunlight.
- Observe and record results once a
week as long as possible over the school year.
- Dispose of containers after
decontamination by heat sterilization or chemical
disinfection following the instructions in Pogosian's
article "Safety in Microbiology Experiments".
Observing Degradation On Agar Media In
Minimal liquid media (see Experiment
2, prepare or purchase)
Bacteriological Agar (Difco)
Parafilm (American Can Co)
Disposable plastic petri plates
Soil (compost, garden soils)
Materials to be tested for biodegradability
- Prepare minimal liquid media as in
Experiment #2. Add 12 grams agar per liter.
- Heat media in an open vessel,
preferably a large beaker, at least twice the volume of
- Heat solution gently until agar
dissolves. The liquid need not boil but should be watched
carefully so that the agar does not burn. The solution
should be yellowish but completely clear when agar is
- Allow agar to cool to about 45 to
50 degrees C, then dispense into petri plates. Pour agar
media into a smaller vessel (500-mL Erlenmeyer flask or
beaker) to make dispensing easier. Open each plate only
long enough to dispense media, avoid unnecessary exposure
of media to the open air.
- Allow agar to solidify, about one
hour. Plates should be poured at least one day in advance
of the experiment.
- Wet the material to be tested, and
place the item firmly in contact with the medium using
forceps. Do not touch agar with fingers (although this is
not a sterile system, any extraneous contamination should
be avoided). Place a small scoop of thoroughly moistened
soil at the edge of the material, in contact with the
- Close the plate and seal edges
with parafilm. Label plates on the bottom (lids can be
switched, bottoms cannot!).
- Observe and record results at
least once weekly for as long as possible during the
- Disposal- Dispose of containers
after decontamination by heat sterilization or chemical
disinfection following the instructions in Pogosian's
article "Safety in Microbiology Experiments".
In this series of experiments, the
students will study whether familiar materials, both natural
and man-made can be utilized as carbon sources by consortia
of microorganisms obtained from soil samples. The students
will prepare a minimal bacterial growth medium which
supplies nitrogen in the form of ammonium salt, oxygen from
the air, essential microelements from tap water, a buffer to
maintain physiological pH, and an object that will be tested
as a source of carbon. Suggestions for biodegradable
materials include filter paper (pure cellulose), different
types of processed papers (cellulose plus other additives),
plant matter (grass clippings, leaves, fruit peels.)
Nondegradable materials include synthetic polymers
(polyethylene, polystyrene), or metals (aluminum
A stick of gum in its wrappers is a
good substrate to test, as it is a familiar object composed
of parts that are very different in their compositions, and
therefore their biodegradability. The outer paper wrapper is
composed of cellulose, dyes, polymeric binders that hold the
paper together and possibly some wax. Over time, microbial
growth should develop on paper surfaces, and the paper
should discolor and deteriorate. The inner aluminum foil
wrapper is a paper-backed sheet of metal. Inorganic aluminum
will not support microbial growth, as it does not contain
carbon. Degradation of the backing may be observed. Gum
sticks are usually coated on the outside with some kind of
sugar and flavoring agents. Sucrose, fructose or glucose
coat "sugared" gums, while sugar alcohols (sorbitol,
xylitol) are used to sweeten many "sugarless" gums. These
sugars are excellent carbon sources for microbial growth,
and very shortly after the experiment is initiated,
microbial growth will coat the gum stick surface. The gum
itself, however, is a nonbiodegradable petroleum products
which will not support microbial growth, and therefore, will
not degrade. The stick may swell as it becomes
In addition to gum, cellulose filter
papers can be used as substrates for soil microorganisms in
liquid media or on agar in petri plates.
While this experiment can be carried
out with any of the media described in Experiments 1, 2, or
3, it is best suited to testing on agar plates. The
substrate, or carbon source, used in this experiment is
cellulose filter paper, which can be purchased from any
scientific supply source. Cellulose filter paper is uniform
in composition and is not treated with any preservatives
that may interfere with microbial activity (many consumer
products are treated with preservatives to inhibit microbial
Controls are incorporated into this
experiment which should provide students with the
opportunity to observe and discuss the roles of the various
components in the experiment: microorganisms are necessary,
but they require carbon (cellulose) and nitrogen (ammonium
ions in media).
Supplement to Experimental
Selection and Containment of
Teachers are strongly advised
to read the article "Safety In Microbiology Experiments"
before planning student experiments. Microbiological
experiments can be conducted safely, however proper
precautions, such as judicious choice of bacterial sources,
proper containment and disposal practices must be
Preparation of Media For Flask and
Petri Dish Experiments:
Many schools lack equipment
and facilities to prepare the media for the experiments
which call for liquid and agar media. Custom-made, sterile
liquid or agar media may be obtained from Carolina
Biological Supply, Burlington, North Carolina. Orders for
custom-preparation of the media suggested in this lesson,
may be placed by contacting Ms. Juliana Hauser, Head-
Microbiology Department, Carolina Biological
Supply, Burlington, NC (919)-584-0381.
Prices will be approximately $3.00 per 100 mL of media
(Petri plates required approximately 15 milliliters each of
agar media). In addition, Carolina Biological Supply offers
a wealth of live and prepared specimens, hands-on kits with
full instructions, equipment and supplies useful for
biology, chemistry, physics, environmental science study
appropriate for students from kindergarten to high school.
Teachers may obtain a free copy of Carolina's catalog by
Disposal of Samples Containing Live
Bacterial and Fungal Cultures:
Proper disposal of live
cultures after experiments are terminated can be
accomplished by pressurized heat sterilization (autoclaving)
or by chemical disinfection. Instructions on how to
chemically disinfect cultures using alcohol or commercial
disinfectants, such as Lysol� or Clorox� are
included in the article "Safety In Microbiology Experiments"
by Barbara Pogosian.
Additional Suggestion for "Zipper"
The Exxon Valdez oil spill off
the Coast of Prince Edward Sound in 1990, while an
environmental disaster of epic proportions, provided a
fascinating example of the ability of endemic populations of
microorganisms to cleanse the environment, provided that
adequate supplies of nutrients are available. While many
people spent months hosing and wiping oil from the beaches,
scientists studying bioremediation as an alternative to
physical cleanup found that when the beach was sprayed with
fertilizer (plenty of Nitrogen and a balance of other
essential nutrients), the surface of the beach was cleaned
of oil (carbon!!) in a remarkably short time. This study is
a dramatic example of the adaptability of endemic microbial
populations to using novel carbon compounds when the
environment is rich in essential nutrients. A similar
situation could be stimulated in the soil bags by adding
fertilizer, for example a solution of Miracle Grow� or
Peter's plant food, to the soil in the bags.
This experiment is courtesy of