WINOGRADSKY COLUMN.pdf

UST College of Science

Department of Biological Sciences

UNIVERSITY OF SANTO TOMAS COLLEGE OF SCIENCE

 Activity 2 Winogradsky Column Ecology Laboratory BIO 203L

4 Biology 2 Group 1

Celina Joyce Aniceto Tricia Anne Barot Eleazar John Cruz Rafaella Beatriz Kraft

18 April 2017

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UST College of Science

Department of Biological Sciences

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 ABSTRACT

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The Winogradsky Column is an inexpensive and indispensable tool in microbial ecology

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that illustrates the interdependent roles that prokaryotes play in sustaining life. In this

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activity, the experimental set-up was composed of a mixture of newspaper, egg shell, egg

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yolk, soil, and pond water in a transparent plastic bottle. Two set-ups were made, one

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exposed to sunlight and the other covered with aluminium foil, to determine the function

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of sunlight on the growth of microorganisms and different activities happening within the

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column. The study was conducted for two months; wherein the set-ups were

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photographed and observed twice every week. After two months, both set-ups showed

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changes in color and odor, and exhibited various layers which indicated the sep aration of

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the aerobic and anaerobic microorganisms into distinct zones that favour their specific

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metabolic activities. Nutrient cycling was evident because of the obvious proliferation of

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different microbes that required nutrients produced by other bacteria. This activity enabled

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our group to create a microcosm in which complex microbial community processes affect

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the surrounding environment. Also, it provided knowledge regarding the different

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processes of how the carbon and sulfur cycles occur within a Winogradsky Column.

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Keywords: Winogradsky Column, nutrient cycling, soil microbial ecology

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Department of Biological Sciences

INTRODUCTION

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Life is sustained by numerous cycles that are dependent on the taxonomic and

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metabolic diversity of microorganisms. For instance, the metabolic diversity of

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prokaryotes enables the sulfur cycle, an essential constituent of life, by transforming it

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into various forms that can be used by other organisms. One such way to illustrate this in

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the laboratory is with the use of the Winogradsky Column which was developed by two

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microbiologists, Sergius Winogradsky and Martinus Willem Beijerinck, who wanted to

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understand the interdependent roles that various microorganisms play in order to sustain

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life (Anderson & Hairston, 1999; Ackert, 2007).

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The Winogradsky Column is a complete, self-contained recycling system that is

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driven by light. Using inexpensive materials, it creates conditions that demonstrate the

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natural processes in which nutrients are cycled in the biosphere. Also, the Winogradsky

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column is used to show the different aspects of how life was possible in early Earth which

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was dominated by sulfur-based, anaerobic microbes (Rogan et al., 2005).

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In this activity, the ability to create a microcosm in which complex microbial

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community processes affect the surrounding environment was cultivated. Also, the

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processes of how the carbon and sulfur cycles occur within a Winogradsky Column were

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understood.

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MATERIALS AND METHODS

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Sample collection.  Approximately 300g of garden soil and 500mL of pond water were

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collected from four different sites [(1) Bacoor, Cavite; (2) Holy Family Residence; (3)

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Calauan, Laguna; (4) Tarlac] then combined to obtain integrated soil and water samples.

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Winogradsky column. The neck of two 2L transparent soda bottles were cut. The yolk

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of one hardboiled egg was separated from the egg white then made into small crumbs.

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The eggshells were pulverized into a fine powder. The egg yolk crumbs and eggshell

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powder were mixed along with shredded newspaper and the integrated soil sa mple. The

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soil mixture was divided into two and placed in the two cut soda bottles. The integrated

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pond water sample was added to each soil mixture until it filled approximately ¾ of the

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soda bottle. The open end of each soda bottle was covered with cling wrap. One column

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was exposed to sunlight while the other was covered with aluminium foil. The columns

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were observed and photographed twice a week for two months.

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RESULTS

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Table 1. Winogradsky column observations Date Light

Dark

24 Jan

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31 Jan

3 Feb

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7 Feb

10 Feb

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Department of Biological Sciences

14 Feb

21 Feb

28 Feb

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Department of Biological Sciences

7 Mar

21 Mar

28 Mar

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Department of Biological Sciences

31 Mar

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Table 2. Weekly observations of the Winogradsky column (light set-up) Observations

Week 1 (Jan 24 & 31)

Week 2 (Feb 3 & 10)

Week 3 (Feb 14 & 21)

Week 4 (Feb 28)

Odor

None

None

Foul

Foul

Foul

Foul

Foul

Color of soil

Brown

Brown

Black

Black

Black

Black

Black

Present

Present

Present

Present

Orange

Orange

Orange

Thin film

ring

ring

ring

(light

present

present

present

colored)

at the

at the

at the

surface

surface

surface

Condensation on plastic

 Absent Absent Present

cover

Crust forming None

None

None

in the bottle

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Department of Biological Sciences

Green

Green

Green

Green

Green

moss-

moss-

moss-

moss-

moss-

like

like

like

like

like

band

band

band

band

band

Film on the surface of the

None

None

water

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Table 2 cont. Observations

Week 5 (Mar 7 & 10)

Week 6 (Mar 17 & 21)

Week 7 (Mar 28 & 31)

Odor

None

None

Foul

Foul

Foul

Foul

Color of soil

Black

Black

Black

Black

Black

Black

Present

Present

Present

Present

Present

Present

Orange

Orange

Orange

Orange

Orange

Orange

ring

ring

ring

ring

ring

ring

present

present

present

present

present

present

at the

at the

at the

at the

at the

at the

surface

surface

surface

surface

surface

surface

Green

Green

Green

Green Green

Green

moss-like

moss-like

band

band

Condensation on plastic cover

Crust forming in the bottle

Film on the moss-

moss-

moss-

moss-

surface of the like

like

like

like

water band

band

band

band

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Department of Biological Sciences

Table 3. Weekly observations of the Winogradsky column (dark set-up) Observations Odor

Week 1 (Jan 24 & 31)

Week 2 (Feb 3 & 10)

Week 3 (Feb 14 & 21)

Week 4 (Feb 28)

None

None

Foul

Foul

Foul

Foul

Foul

Brown

Brown

Brown

Brown

Brown

Black

Black

 Absent Absent Absent

Absent

Absent

Present

Present

Color of soil Condensation on plastic cover Thin Crust

Thin film orange

forming in

None

None

None

None

None

(light crust on

the bottle

orange) sides Mold-like

Mold-like

White

White

film on

film on

mold-like

mold-like

top with

top with

film on

film on

bubbles

bubbles

top

top

Film on the surface of

None

None

None

the water

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Table 3 cont. Observations

Week 5 (Mar 7 & 10)

Week 6 (Mar 17 & 21)

Week 7 (Mar 28 & 31)

Odor

None

None

Foul

Foul

Foul

Foul

Color of soil

Black

Black

Black

Black

Black

Black

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Condensation on

Present

Present

Present

Thin

Thin

Thin

Present

Present

Present

Orange

Orange

Orange

crust on

crust on

crust on

sides

sides

sides

plastic cover

Crust orange

orange

orange

forming in crust on

crust on

crust on

the bottle sides

sides

sides

White

White

White

White

White

White

mold-like

mold-like

mold-like

mold-like

mold-like

mold-like

film on

film on

film on

film on

film on

film on

top

top

top

top

top

top

Film on the surface of the water

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DISCUSSION

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The Winogradsky Column is an inexpensive device used to study the different

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functions of various microorganisms in nutrient cycling and sustaining life. As oxygen

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diffuses downward from the surface, fermentation products and microbial metabolites

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diffuse upward. The cycling of nutrients within the column creates various chemical

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gradients that are necessary for the growth of certain organisms, enabling their

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proliferation in distinct zones and creating a vertical distribution of microbes similar to

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those present in natural ecosystems (Anderson & Hairston, 1999). In order for a

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Winogradsky column to work, it initially requires a sul fur, inorganic carbon, and cellulose

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source. In the experimental set-up, the egg yolk crumbs served as the sulfur source, the

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Department of Biological Sciences

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eggshell powder served as the inorganic carbon source, and the shredded newspaper

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served as the cellulose source (Rogan et al., 2005).

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Growth was observed in the columns one week after it was prepared (Table 1).

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Both set-ups turned into a dark colored mixture with visible green growths at the top. The

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dark color of the mixture is attributed to rapid microbial growth promoted by the presence

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of cellulose. The green growths observed at the top of the mixture indicate the growth of

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green, photosynthetic microorganisms such as cyanobacteria and algae. The rapid

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growth of microorganisms depletes the oxygen present at the bottom of the set-up,

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creating an anoxic environment. At such conditions only anaerobic bacteria, such as

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Clostridium, can survive. Anaerobes degrade cellulose and produce fermentation

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products, such as organic acids and alcohols, which diffuse upward. These fermentation

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products together with the sulfur and inorganic carbon sources added into the mixture are

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utilized by anaerobic, sulfur-reducing organisms, such as Desulfovibrio, to produce

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hydrogen sulfide.

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By the fourth week, the soil-pond water mixture has a distinct odour similar to that

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of rotten eggs because of the sulfur by-products of certain microorganisms. The columns

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also showed different zones with distinct colors, starting from the bottom, the colors were

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opaque black, green, red, and dark green with sparse orange-brown areas for the light

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set-up, orange-brown with sparse dark green areas for the dark set-up. The different

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colors at distinct zones present in the column indicate the type of microorganism that has

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accumulated in that area. The development of Clostridium and Desulfovibrio appear as

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blackened areas, due to the formation of ferrous sulfide, in the lower portion of the column

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where conditions are anaerobic. The sulfide products of Desulfovibrio are then used by

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anaerobic photosynthetic bacteria such as Chlorobium to serve as its final electron

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acceptor, yielding elemental sulfur and water; its g rowth was indicated by the green zone

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directly above the blackened area at the lower portion of the column. Above this zone,

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was a red colored band which can be ascribed to the accumulation of non-sulfur bacteria

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that are able to grow in microaerophilic conditions such as Rhodospirillum  and

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Rhodopseudomonas . These organisms are photoheterotrophs that trap light energy and

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use organic molecules as both electron and carbon sources. The presence of this zone

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in the dark set-up is attributed to fact that the foil covering the experimental set-up was

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lost. Lastly, the combination of dark green and orange-brown areas at the topmost layer

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in both the light and dark set-ups indicate the growth of photosynthetic cyanobacteria,

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green algae, and sulfur-oxidizing organisms such as Thiobacillus. Sulfur-oxidizing

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microbes are chemoautotrophs that oxidize hydroge n sulfide to sulfate to gain energy for

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the synthesis of organic matter. The sulfate produced cycles back to the anaerobic

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sediment of the column to be used by Clostridium, completing the sulfur cycle within the

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closed system.

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The key factor that sustains life within the Winogradsky column is nutrient cycling.

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In the column, carbon, hydrogen, and oxygen are cycled through aerobic respiration and

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photosynthesis. On the other hand, sulfur, which is an important nutritional requirement

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for most life, is cycled within the column through aerobic and anaerobic respiration. The

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sulfur cycle is dependent on the element’s chemical variability. Changes in the oxidation

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states of sulfur are mediated by microbial metabolisms, which is perfectly illustrated within

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the Winogradsky column. For instance, anaerobic species use elemental sulfur as the

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terminal electron acceptor in respiration which reduces it into hydrogen sulfide. While

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others can use thiosulfate or sulfate as an electron receptor. Also, algae and many

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heterotrophic microorganisms may utilize sulfate by incorporating it into proteins

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(Anderson & Hairston, 1999; Deacon, 2003; Rogan et al., 2005).

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CONCLUSION

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The Winogradsky column is an inexpensive device used to illustrate complex

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cycles that occur within the biosphere. The interplay of various organisms in the cycling

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of nutrients prove the importance of microbial metabolic diversity. This activity enabled

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our group to create a microcosm in which complex microbial community processes affect

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the surrounding environment. Also, it provided knowledge regarding the different

124

processes of how the carbon and sulfur cycles occur within a Winogradsky Column.

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REFERENCES

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 Ackert, L. T. (2007). The “cycle of life” in ecology: Sergei Vinogradskii’s soil microbiology,

1885-1940. Journal of the History of Biology, 40, 109-145.

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 Anderson, D. C. & Hairston, R. V. (1999). The Winogradsky column & biofilms: models

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for teaching nutrient cycling & succession i n an ecosystem. The American Biology

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Teacher, 61(6), 453-459.

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Deacon, J. (2003). The Microbial World: Winogradsky Column: perpetual life in a tube.

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Retrieved

on

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April

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http://archive.bio.ed.ac.uk/jdeacon/microbes/winograd.htm

2017,

from:

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Rogan, B., Lemke, M., Levandowsky, M., & Gorrell, T. (2005). Exploring the sulfur nutrient

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cycle using the Winogradsky column. The American Biology Teacher, 67 (6), 348-

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356.

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