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Simulating the Environment With A Winogradsky Column

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A Formal Report

Submitted to

Sonia N. Javier, M.Sc. & Jonathan Carlo A. Briones, M.Sc.

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Department of Biological Sciences

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College of Science

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University of Santo Tomas

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By

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4 Bio 5 Group 2

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Ysabel Aira E. Azucena

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Judy Ann G. Bacud

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Shannon Cake M. Baquiran

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Timothy John L. Bautista

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March 2016

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Abstract

30Introduction. The Winogradsky column is a tool that was designed by Sergie N.

31Winogradsky to study microbial activity and succession and nutrient cycling of
32carbon, nitrogen, sulfur, phosphorus, and other nutrients. The objective of the
33experiment was to create a model microbial ecosystem that would show
34microbial activity and nutrient cycles of carbon and sulfur.
35Method. An empty plastic container was filled with a mixture of pounded egg
36yolk, pulverized egg shell, shredded newspaper, soil, and pond water and
37covered with plastic wrap. The column was observed for four weeks for microbial
38activity and nutrient cycling
39Result. The bacterial growth responds in patterns or in distinct zones reflecting
40the substrate and aerobic concentrations. Different types of microorganisms such
41as such as Cyanobacteria and Clostridium proliferated and occupied distinct
42zones where the environmental conditions favour their specific activities.
43Conclusion. The Winogradsky column demonstrates metabolic diversity of
44microorganisms and illustrates how different microorganisms perform their
45interdependent roles wherein the activities of one organism enable another to
46grow.
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48Keywords: Winogradsky column, microbial activity, Nutrient cycling
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50Introduction
51The Winogradsky column is a tool that was designed by Sergie N. Winogradsky
52to study microbial activity and succession and nutrient cycling of carbon,
53nitrogen, sulfur, phosphorus, and other nutrients. The set-up is ideal for studying
54growth of microorganisms because the materials needed to construct a
55Winogradsky column are inexpensive and easy to obtain. The column consists of

56a graduated cylinder or pre-calibrated empty soda bottle, filled with water, soil,
57and other substrates that would supplement nutrients and allow bacterial growth.
58The column is covered with saran wrap and rubber band to hinder evaporation of
59water. It is exposed to illumination to also allow phototrophic microorganisms to
60thrive. Shredded newspaper is used as primary source of carbon while hard61boiled egg is the primary source of sulfur. Two zones compose the Winogradsky
62column, the upper aerobic zone and lower anaerobic zone. The presence of an
63aerobic liquid air biofilm or pellicle may be seen on the surface. The column
64becomes more anaerobic at the lower layers. The objective of the experiment is
65to create a model microbial ecosystem that would show microbial activity and
66nutrient cycles of carbon and sulfur.
67Materials and Methods
68A 2-L clear, empty soda bottle was cut and marked from 0.0 to 100.0-mL with
69intervals of 5.0-mL. Egg yolk from a hard-boiled egg was pounded and the egg
70shell was pulverized. Shredded newspaper and soil/mud were mixed with the
71egg yolk and egg shell. The mixture was placed inside the calibrated soda bottle
72that will serve as the column. The bottle was filled with pond water until the
73surface of the mixture is covered by 3.0 to 4.0 cm. The mixture was stirred to
74release air bubbles and was left undisturbed for five minutes. More pond water
75was then added until the 500.0-mL was reached. The Winogradsky column was
76covered with plastic wrap and rubber band. Observations were done for four
77weeks and the while making sure that the water level was maintained by adding
78more pond water.
79Results and Discussion
80The end of the experiment left the column with few distinct layers: mold growing
81on top with black, white and red/ purple film, and green and white colored crust; a
82dark red/ brown layer; and a dark purple layer.
83The uppermost layer is consists of photosynthetic organisms including algae and
84cyanobacteria which, in presence of sunlight, these organisms photosynthesize

85to prepare their own food and oxygen is evolved in the reaction. The white film
86within and right below the water surface is the Microaerophillic zone where, even
87though one side is exposed to oxygen, very little O 2 diffuses slowly through water.
88This layer consists of bacteria such as Beggiatoa which oxidize the remaining
89Hydrogen-Sulfide into Sulfuric acid. The energy from which is then used to
90process other organic molecules. The upper portion of sand column is reddish
91brown

with

the

growth

of

non-sulfur

anaerobic

photoheterotrophs

92like Rhodospirillum. Below the red-brown zone, the red-purple zone indicates the
93growth of purple sulfur bacteria, Chromatium spp. and Ectothiorhodospirillum
94spp., which are photosynthetic organism. These organisms oxidize H2S into
95elemental sulfur (Shrestha, 2015).
96Decomposition and sulfate reduction activity of bacteria occur at the base of the
97column. The breaking down of the materials that make up the substrates
98contributed to the production of different substances needed by the bacteria at
99different levels of stratification. The different chemical demands each bacteria
100define the layer where they can thrive.

Anaerobic microorganisms cause the

101release of sugar by cellulose fermentation. Reduction of sulfate causes the

102release of hydrogen sulfide gas at the lower layers and decreases at the higher
103layers. The formation of a gradient allows a great range of habitats for various
104microorganisms. Carbonate in the column also functions as pH buffer and is
105important of the autotrophs. Lower portions of the Winogradsky column are often
106colonized by green photoautotrophic and purple sulfur bacteria. The aerobic layer
107is often occupied by oxygenic cyanobacteria. Heterotrophic microorganisms also
108thrive inside the column. Examples of these are obligate anaerobes like clostridia
109and methanogenic bacteria as well. Metal sulfides like iron usually contribute to
110the black color at the bottom of the column. Sulfur oxidizing organisms consume
111oxygen that diffuse downward and sulfide that diffuse upward. This reaction
112results to a stable counterbalance of sulfide and oxygen and ultimately allows the
113growth of organisms with either oxygen or redox requirements.
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Table 1.0 Weekly observation of the Winogradsky Column

Observations

Week 0
Rotten

Odors

egg

Color of the soil

odor
Gray
None

Condensation on
plastic cover
Crust forming in
the Bottle

Film on Surface
of the water

None

None

Week 1

Week 2

Week 3

Week 4

Rancid odor

Rancid odor

Rancid odor

Gray

Gray

Gray

Gray

colorless

colorless

colorless

Colorless

Green and

Green and White

White Crust

crust

Rancid odor

Green and
white crust

Green Crust
Green, white

Green,

Green and

and

red/brown,

Black, White and

white film

red/brown

and white

red/ purple film.

film

film

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Figure 1.0 Week 1

Figure 2.0 Week 2

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Figure 3.0 Week 3

Figure 4.0 Week 4

120Conclusion
121The Winogradsky column demonstrates metabolic diversity of microorganisms
122that grow in response to different environmental conditions. Different types of
123microorganisms

proliferated

and

occupied

distinct

zones

where

the

124environmental conditions favour their specific activities such as Cyanobacteria

125and Clostridium. Decomposition of different substrates contributed to the diffusion
126of hydrogen sulfide, oxygen, and other substances that create the stratification of
127microbial growth.
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129References
130Brown,
J.
(2013).
Winogradsky
Columns.
Retrieved
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http://www.mbio.ncsu.edu/mb452/Winogradsky_columns/wc.html

from

132
133Shrestha,
T.
(2015).
The
Sufur
Cycle.
Retrieved
134
http://upendrats.blogspot.com/2015_03_01_archive.html

from

135
136Winogradsky
Columns.
(n.d.).
Retrieved
from
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http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/winogradsky.html
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