Winogradsky Column Formal Report Final 1 PDF

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Ecology Lab Report

Simulating the Environment with a Winogradsky Column

Biology

203L EXPT 2 PAGE 18

Louise Manangan, Katrina Pangilinan, Edward Paz

Olivas,

Trisha

Pagtakhan,

Marian

Department of Biological Sciences, College of Science

Abstract The Winogradsky column is a method to bring otherwise inaccessible communities into the lab. It re-creates the natural environments for bacterial growth, creating an ideal environment to research various nutrient cycles. Saturated, enriched dirt is taken and supplemented by the egg yolks and sealed off. The bacterial growth responds in patterns, or “zones”, reflecting the substrate and aerobic concentrations. The prokaryotic bacteria and archaea exhibit an astonishing metabolic diversity, which far exceeds that of animals, plants, fungi and other higher organisms. The prokaryotes literally keep our biological world turning by recycling all the mineral elements necessary for life support. The Winogradsky column - illustrates how different microorganisms perform their interdependent roles: the activities of one organism enable another to grow, and vice-versa.

Introduction Microbial communities are found in pond mud, and these organisms are capable of producing metabolic by-products that are required for the survival of other organisms within the environment. By using the Winogradsky column, these products can be studied, and the interdependent relationship of the microorganisms can be observed. This column can act as a replica of the microbial environment. The Winogradsky column is a miniature, self-contained ecosystem which models ecological conditions in varying ways. It was invented by the Russian bacteriologist Sergei N. Winogradsky in 1880. The column is composed of a transparent cylindrical container which is filled with a few substrates (ex. Soil/mud) and marine or freshwater. The column is usually covered to prevent evaporation. Illumination (sunlight) is provided to promote the growth of

Ecology Lab Report microscopic organisms (ex. phototrophs). This composition will provide the information needed to study sulfur, nitrogen, carbon, phosphorus, and other nutrients, which undergo cycling between the aerobic zone (upper layer) and the anaerobic zone (lower layer). Moreover, these gradients (light gradient, temperature gradient, nutrient, O 2 and H2S concentration gradients) result in a complex interaction of microbes with their environment and with one another resulting in a series of community successions and, ultimately, stratification of microbial populations in the water column. Different microbes live according to different concentrations of H 2S and O2, and this gives the column a zonal appearance after 6-8 weeks of microbial growth. Winogradsky columns demonstrate the interdependence of diverse microorganisms in complex communities for survival and growth. In this exercise, the students aim 1) to create a microcosm in which complex microbial community processes affect the surrounding environment, 2) to gain an appreciation for the diversity of methods microorganisms use to gain energy from oxygenproducing photosynthesis and bacterial photosynthesis, and 3) to diagram the carbon and sulfur cycles as it occurs in a Winogradsky column.

Materials and Methodology The materials utilized for this exercise are the following:             

Clear 1.5-liter soda bottle 5 cups soil from garden 5 cups water from Fountain of Wisdom 1 empty 1.0 pint plastic container 1 large spoon or spatula 10.0 grams shredded newspaper or 5.0 grams calcium carbonate 1 hard boiled egg yolk or 5.0 grams calcium sulfate Plastic wrap Rubber bands Permanent markers Ruler Pair of scissors/cutter Light source (i.e. natural or artificial)

Figure 1. Materials used in exercise

A. Collection of mud/sediments/soil samples In preparation for this exercise, the group first gathered soil from a garden. Then, water samples were also collected from the aquatic environment where the soil was gathered. In this exercise, the group collected pond water samples from the Fountain of Wisdom located inside the campus of University of Santo Tomas. Lastly, the gathered soil were cleared of with organic debris (i.e. plant materials like roots, grass clippings, leaves, and dead/live invertebrates, pebbles, rocks, etc.).

Ecology Lab Report B. Construction of the Winogradsky Column To take the appearance of a column, the neck of the 1.5-liter clear soda bottle was cut with a pair of scissors/cutter. Then, the plastic column was marked from about 0.0 to 100.0-ml with intervals of 5.0-ml.

Figure 2. Pond water sample and calibrated plastic column

Next, the egg yolk was separated from the egg and was pounded until the lumps disappeared. The egg shells were also removed from the egg. The egg shells were then pounded until almost pulverized. Using the large spoon/spatula, the pounded egg yolk and egg shells were homogeneously mixed inside the plastic container along with 5.0 grams of shredded newspaper and cleaned 30.0 grams of soil sample.

Figure 3. Preparation of homogenous soil mixture

Then, the mixture was transferred inside the clear plastic column and it was made sure that none of the sample mixture was left on the side. Then, enough pond water was added to cover

Ecology Lab Report the surface of the soil mixture by 3.0- to 4.0-cm. The mixture was stirred to release any trapped air bubbles in the soil and was let sit for 5.0-mins. Additional pond water was poured until soil remained under water by a depth of about 500.0-ml. Initial readings of the soil sample (Ssm 0) and water column (Hclm0) was recorded. The improvised plastic column was then covered with cling/plastic wrap and fastened by rubber bands while making sure that air was not allowed in. The Winogradsky Column set-up was then exposed at room temperature and placed in a sunny window for 7 weeks. The height of the water column was maintained after every reading for the past 7 weeks.

Figure 4. Winogradsky Column setup at Week 0

C. Observations of the Winogradsky Column The set-up was checked and the observations were recorded every week for the past seven weeks. Detailed observations on the appearance of the column, from bottom to top, and from the light to dark sides were noted. While keeping the cover of the plastic column, the appearance of the column was recorded as any changes observed in color patterns or growth occurring in the soil and soil-water interface over time. The following observations were documented as: Table 1. Observation with cover on

OBSERVATIONS Odors Color of the soil Condensation on plastic cover Crust forming in the bottle Film on the

WEEK 0

WEEK 1

WEEK 2

WEEK 3

WEEK 4

WEEK 5

WEEK 6

WEEK 7

Ecology Lab Report surface of the water Table 2. Observation with cover off

OBSERVATIONS

WEEK 0

WEEK 1

WEEK 2

WEEK 3

WEEK 4

WEEK 5

WEEK 6

WEEK 7

Odors Film on the surface of the water Crust build up in the column Macroscopic organisms

Key to potential observations: a) Aerobic colors  Green – eukaryal algae or cyanobacteria  Red/brown – cyanobacteria or thiobacilli  Red/purple – purple non-sulfur Bacteria  White – sulfur oxidizing Bacteria b) Anaerobic colors  Red/purple – purple sulfur Bacteria  Green – green sulfur Bacteria  Black – sulfate reducers c) Gas  In the water column is probably O2 from oxygenic photosynthesis  In the aerobic zone is probably CO2 from respiration  In the anaerobic zone is probably CH4 from methanogenesis  Tracks in the upper layers of the sediment are formed by “worms”  Small specks swimming in the water column are crustaceans, e.g. Daphnia & Cyclops

Results & Discussion Over time gradients of different nutrients should have formed in the Winogradsky columns. These gradients affect where different microbes grow within the columns. For example, over time there is more oxygen at the top of a column than at the bottom, and this means that microbes that can tolerate or produce oxygen will be found at the top. Microbes that cannot tolerate free oxygen (called anaerobic bacteria) will be further down. Similarly, microbes that need light to make energy (via photosynthesis or a similar process) will need to live where they can get light in the column.

Ecology Lab Report After about one to two weeks, depending on how much light the columns receive, some green coloring should appear in the columns receiving light on the illuminated sides. This is mostly due to cyanobacteria and algae, which needs light. The column in the dark should remain dark brown. In the column that had egg yolk, areas of darker green, purple, and/or black coloring may have developed over time near the bottom— these colorings could be groups of certain anaerobic bacteria: green sulfur bacteria, purple sulfur bacteria, and sulfate-reducing bacteria, respectively. Sulfate-reducing bacteria actually eat sulfur and make hydrogen sulfide gas, which is eaten by the green and purple sulfur bacteria. In the column that had newspaper, some areas of brown, orange, red or purple may be evident near the middle— these colorings could be groups of purple non-sulfur bacteria, which need a carbon source to thrive. In addition, worms, snails, shrimp or other small organisms in the water, but probably not many (if any) in the bottle with the egg yolk, because hydrogen sulfide is toxic to most organisms OBSERVATIONS Odors

WEEK 0 None

WEEK 1

Sewer/sulfuric Color of the soil

Condensation on plastic cover Crust forming in the bottle

Light gray

Mixture of gray and light brown

WEEK 3

Smells like horse poop

Intense horse poop; "burak"

Colorless None

WEEK 4

Light gray

Light gray

Colorless Thicker than last week reddish brown crust

Colorless

Light orange

Cloudlike appearance

Lesser diameter of black and white crust

WEEK 6 Faint stench

Faint stench

Brown Red to black

None

Formation of air bubbles, lard-like appearance, combination of black and white film

WEEK 5 Faint stench

Gray

None

Thin reddish brown Film on the surface of the water

WEEK 2

Mixture of black, brown, and white film w/ bubbles

Brown

Mixture of gray and brown Brown

Red to brown/black

Red to brown

Thick crust, mixture of black, brown, gray, white w/ bubbles

Thick crust, mixture of black, brown, gray, white w/ bubbles

Mixture of gray and brown

Figure 1. Observations with Cover On OBSERVATIONS

WEEK 0

WEEK 1

WEEK 2

WEEK 3

WEEK 4

WEEK 5

WEEK 6

Odors

None

Very strong stench; Creek/sewage

Sewage with horse poop

Strong foul stench; "burak"

Less intense odor

Film on the surface of the water

None

Dry white crust over film Darker, thicker crust

Vomit-like appearance with bubbles; mixture of black, white, and orange film Orange layer

Less intense odor Thick crust, mixture of black, brown, gray, white w/ bubbles s Dark red with

Less intense odor Thick crust, mixture of black, brown, gray, white w/ bubbles

Crust build up in

None

Wet tissue-like appearance with mold-like residues Light orange layer

Mixture of black, brown, and white film w/ bubbles Red to black

Mixture of white,

Ecology Lab Report than last week

the column Macroscopic organisms

None Molds

Appearance of molds

Molds

Molds

white particles

dark red, brown

Molds

Molds

Figure 2. Observations with Cover Off

a) Week 0

1

d) Week 3

b) b) b) e) Week 4

Week

c) c) c) Week 2 f) Week 5 Figure 3. Winogrdadsky model of Weeks 1-5

Ecology Lab Report

g) Week 6 Figure 4. Winogrdadsky model of Week 6 Conclusion The Winogradsky column demonstrates metabolic diversity of microorganisms that grow in response to different environmental conditions. In this experimnent, where the column was exposed at room temperature and placed in a sunny window for 6 weeks, different types of microorganisms proliferated and occupied distinct zones where the environmental conditions favour their specific activities such as Cyanobacteria (upper zone; aerobic) and Clostridium (lower zone; anaerobic).

References Deacon, J (n.d.) The microbial world: Winogradsky column: Perpetual life in a tube. Retrieved from: http://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm Perry, et al. (2002). Microbial life(1st Ed.). Sunderland, MA: Sinauer Associates http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/winogradsky.html https://www.msu.edu/course/lbs/159h/winogradsky04.pdf http://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm...