Microbes ate my homework
Class practical
Investigate the effects of cellulose-digesting enzymes in microbes on different kinds of paper. This long-term activity allows students to explore the role of microbes in decomposing organic waste and their place in the carbon cycle.
This practical investigates how quickly different kinds of paper decompose under the action of soil microbes. Soil microbes are unusual in the natural world in that they contain cellulases. These are enzymes able to digest cellulose, the fibrous substance which helps to provide plants with a rigid structure. Without these cellulase enzymes in soil microbes, plant material would not decay, and elements such as carbon contained in the material would not be recycled for use by other living things.
Paper is made from woody plants. Cellulose makes up 40-50% of the mature plant cell wall; therefore paper is largely made of cellulose. In this investigation you will find out how microbes in the soil can break down paper over a few weeks. This demonstrates how paper and plant material is broken down in the compost bin.
Cellulase-producing microbes are found in the strangest of places, from termites’ stomachs through to the soil surrounding volcanoes. Scientists are genetically modifying cellulase-producing microbes to get them to produce larger quantities of cellulase. These microbes are being used commercially to produce biofuels from non-food stuff. Currently, most biofuels are made by fermenting edible plant material to produce ethanol. This means using plant products which could be used as food to make fuel instead. If we could produce ethanol from cellulose, we would be able to make use of a huge amount of non-edible plant waste instead, such as stalks from farmland, sawdust and wood chips from forestry operation.
Cellulase-producing bacteria in the guts of herbivores (for example, in the rumen of ruminants and the appendix of rabbits) help those animals to survive by breaking down cellulose so that the animals can use it as a source of energy.
Cellulase-producing microbes therefore play a key role in the carbon cycle, breaking down carbon compounds and releasing methane and carbon dioxide which are of enormous importance in their effects on global climate change.
This practical is based on an investigation called 
Microbes and cellulose (399 KB) published in Practical Microbiology for Secondary Schools © Society for General Microbiology, a booklet compiled by a working party of the Microbiology in Schools Advisory Committee with input from SGM.
Lesson organisation
Each group will set up six tubes containing different paper/ card samples with nutrient broth containing soil microbes. The practical work in the ‘set-up’ session will take around 30 minutes, and each review session will be about 20 minutes. You need to leave at least one week (and up to 3 weeks) between set-up and first review, and a further 1-3 weeks for each subsequent review. Each review will take only 10-15 minutes.
Apparatus and Chemicals
For each group of students:
Sterile 5 cm3 graduated pipette and filler, 1
Clean test tubes with aluminium foil caps or cotton wool plugs, 6
Clean conical flask, 250 cm3, 1
Test tube rack, 1 (to support the tubes for up to 6 weeks)
Sterile nutrient broth, 50 cm3
Soil, 5 g
Samples of different types of paper in 1 cm x 2 cm strips (for example, filter paper, tissue paper, unprinted newspaper, heavily printed newspaper, glossy magazine covers, thin cardboard)
Marker pen, 1
For the class – set up by technician / teacher:
Sterile nutrient broth (50 cm3 per group)
(Note 2)
Soil
Paper samples
Health & Safety and Technical notes
Carry out a full risk assessment before starting any microbiology work (see Note 1 for more details).
1 Before embarking on any practical microbiological investigation carry out a full risk assessment. For detailed safety information on the use of micro-organisms in schools and colleges, refer to Basic Practical Microbiology – A Manual (BPM) which is available, free, from the Society for General Microbiology (email This email address is being protected from spambots. You need JavaScript enabled to view it.) or go to the safety area of the SGM website (http://www.microbiologyonline.org.uk/teachers/safety-information), or refer to the CLEAPSS Laboratory Handbook, section 15.
2 Make sterile nutrient broth by rehydrating tablets (more expensive) or powder according to manufacturer’s instructions. Make just enough (with a little extra for mistakes). (See BPM p6 for more details.)
3 Suitable disinfectants include sodium chlorate(I) (hypochlorite) at concentrations providing 1000 ppm available chlorine for general surface cleaning, or 2500 ppm chlorine for discard pots, or VirKon at 1% (follow manufacturer’s instructions).
4 Cultures and contaminated equipment and materials must be autoclaved at 121°C for 15 minutes before disposal. After sterilisation, all materials can be disposed of with normal waste. Take care to package glass to prevent injury.
Procedure
SAFETY: See Basic Practical Microbiology – a Manual for more information about hazards and risk control measures.
Preparation
a Make up sterile nutrient broth (Note 2).
b Collect soil sample (5 g per working group). Avoid areas where cats may have buried faeces.
c Sterilise the pipettes.
d Make cotton wool plugs for the test tubes.
e Set up discard beakers with appropriate disinfectant fluid (Note 3).
Investigation
a Label 6 test tubes A–F, together with your name and date.
b Use a graduated pipette and filler to place 5 cm3 of nutrient broth in tube A. Carefully drop in a 1 cm x 2 cm sample of filter paper.
c Place 5 g of soil and 30 cm3 of nutrient broth in the conical flask. Swirl the contents to form a suspension. Allow this to settle for a minute to avoid blocking the pipette.
d Pipette 5 cm3 of the supernatant of the nutrient broth / soil suspension into each of the five remaining tubes. Put the pipette into a discard beaker.
e Into tube B, carefully drop in a 1 cm x 2 cm sample of filter paper.
f Put a 1 cm x 2 cm sample of a different kind of paper or card into each of the other four tubes C–F.
g Stopper each of the tubes with either cotton wool or loosely cover with aluminium foil.
h Record the contents of each of the six tubes in a table.
| Tube | Treatment | Appearance after …… weeks | Appearance after …… weeks |
|---|---|---|---|
| A |
Nutrient broth (sterile) + filter paper |
|
|
| B |
Nutrient broth + soil + filter paper |
|
|
| C |
Nutrient broth + soil + |
|
|
| D |
Nutrient broth + soil + |
|
|
| E |
Nutrient broth + soil + |
|
|
| F |
Nutrient broth + soil + |
|
|
 |
 |
i Leave the tubes at room temperature for at least a week.
j Before reviewing the tubes, give each a tap with your finger. Carefully observe what happens to the paper strip. Do not take out the cotton wool stoppers.
k Record your results in the table.
l Dispose of the soil suspension immediately after the first lesson, and the contents of the tubes safely at the end of the investigation (Note 4).
Teaching notes
Cellulose is a fibrous substance that helps to provide plants with a rigid structure. It makes up 40-50% of the mature plant cell wall, and is the most abundant carbohydrate. The molecules are very large and very long and contain carbon, hydrogen and oxygen. In wood, forest and agricultural wastes, and in waste paper, cellulose occurs in a complex mixture with lignin (another plant polymer) called lignocellulose.
The microbes that can decompose and thus recycle it are extremely important in maintaining the turnover of organic matter in the carbon cycle. On land, the major decomposers of cellulose are fungi aided by a few bacteria. Cellulolytic bacteria include species of Cellulomonas, Pseudomonas and Ruminococcus. Cellulolytic fungi include Chaetonium, Fusarium, Myrothecium and Trichoderma. Ask students to locate the position of soil microbes on a diagram of the carbon cycle.
Cellulose is not soluble in water, so microbes cannot absorb it into their cells. They secrete cellulase enzymes which partly digest cellulose and break it down to soluble sugar molecules that can be absorbed and used. Higher organisms do not make cellulases; this means that herbivores cannot digest cellulose themselves. They depend on cellulolytic bacteria in their intestinal tracts to do the job for them. This can be a complicated process – involving regurgitation, chewing and swallowing in ruminants, or re-ingestion of faecal pellets in rabbits. This is an interesting opportunity for students to carry out some research into different methods of digesting cellulose. Humans cannot digest cellulose at all, so all the cellulose we eat passes through our digestive system unchanged. This is called ‘dietary fibre’.
Students are often very aware of issues associated with recycling. They may be interested to think about the length of time paper of different sorts would sit in landfill before rotting if it is not recycled. This may also introduce discussion about how different paper treatments make them harder to recycle.
If you have access to a compost bin or wormery, you could compare the results of this investigation with the rate (and manner) of decomposition of similar paper samples in the compost bin and wormery. Be aware of the risks posed by mould spores in compost. If you choose to work with material in a domestic compost bin, make sure no cooked food or meat products go into the compost, and that there is no evidence of rodent activity around the bin. Practise good personal hygiene after handling compost.
There is scope for higher level investigations using this and related techniques such as:
- exploring the effect of temperature on the activity of cellulolytic microbes or cellulases (from school science suppliers)
- exploring the cellulolytic activity of microbes from different soils
- exploring the effect on cellulolytic activity of adding nutrients to the soil samples
- exploring whether fungi or bacteria are more important in terms of cellulolytic activity in particular types of soil.
Health & Safety checked, November 2008
Downloads
Download the student sheet 
Microbes ate my homework (73 KB) with questions and answers.
Download the original protocol from SGM Microbes and cellulose (399 KB)
Web links
www.microbiologyonline.org.uk
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms. This practical is adapted from Practical Microbiology for Secondary Schools. These booklets are available free of charge.
www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/pracbiotech.html
The NCBE is a rich source of up-to-date protocols and practical equipment for biotechnology practicals in schools. These notes (from 1993) show two more protocols for assessing cellulase activity – by digesting cellulose in an agar plate (similar methodology to the starch and protein methods on the Practical Biology site) and by measuring the change in viscosity of wallpaper paste in syringe barrels!
(Websites accessed October 2011)