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Exploring the Sulfur Nutrient Cycle
Using the Winogradsky Column
BRIAN ROGAN
MICHAEL LEMKE
T
o fully understand the workings of the biological world, it is important that students have a fundamental sense of the natural cycles that provide the
nutrients and energy that power life, as well as a
sense of how these systems evolved. Many teachers
cover carbon cycles and emphasize microbial
processes when reviewing the complexities of nitrogen cycling, but often the sulfur cycle, if covered, is
done so briefly. There may be many reasons for this:
time limitations, the element is less prevalent than
others as a biological constituent, or the topic is
thought to be too complex. However, teaching the
sulfur cycle in conjunction with the classic
Winogradsky column exercise presents the opportunity to cover several important topics simultaneously. The exercise links microbial processes, concepts
of biodiversity, inorganic chemistry, biogeochemical
cycling, evolution, microbiology, and microbial ecol-
BRIAN ROGAN teaches at The New Jewish High School,
Waltham, MA 02453. MICHAEL LEMKE is in the Biology
Department, University of Illinois at Springfield, Springfield,
IL 62703-5407. MICHAEL LEVANDOWSKY and THOMAS GORRELL
work at Haskins Laboratory, Pace University, New York, NY
10038-1598.
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THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
M I C H A E L L E VA N D O W S K Y
THOMAS GORRELL
ogy to help meet the many demands and standards
that are part of today’s science classes.
The Winogradsky column is a glass or clear
plastic column, filled with enriched soil or sediment. When developed, it has an anaerobic lower
zone and aerobic upper zone that allow growth of
microorganisms under conditions similar to those
found in sediments and water rich in nutrients
(Sylvia et al., 1998). Often teachers simply convey
the message that different microorganisms exist in
different strata of the column and that some live in
the aerobic and some in anaerobic zones. However,
this is really where the discovery begins rather than
ends! Explaining the complexity that lies within the
depths of the ecosystem allows deeper insights into
the microbial world.
In the laboratory, the Winogradsky column
demonstrates how the metabolic diversity of
prokaryotes transforms sulfur, an essential constituent of living matter and an abundant element in
the Earth’s crust (Stanier et al., 1976), to different
forms with varying redox states, thus supplying
nutrients and/or energy to the organism. The microbial assemblage that develops in the column spatially separates organisms into distinct layers several
centimeters thick even though in the environment
establishment of similar layers of different organisms
would typically exist in a few millimeters of sediment.
The Winogradsky column creates conditions that
expand the volume of natural processes, allowing a
clear view of naturally-occurring phenomena. Soil samples are collected from wetland habitats, amended with
simple inorganic and organic materials, then exposed to
light as an external energy source. The results are a multicolored column of soil and water, each color linked to
a chemical or biological process. The defined zones of
microbes develop form according to concentration gradients of oxygen, sulfur, nutrients, and light. Each functional microorganism group is dependent on other
functional microbial groups for development.
The Winogradsky column was developed and
named after Sergei Winogradsky (1856-1953), a
Russian microbiologist. He studied the complex interactions between environmental conditions and microbial activities using soil enrichment to isolate pure bacterial cultures (Madigan et. al, 2000). Louis Pasteur,
Robert Koch, and other scientists isolated cultures for
study, but Winogradsky’s work was one of the first to
study microorganisms in mixed enrichment cultures.
The fact that the exercise works under a wide range of
circumstances is a testament to the near ubiquity of certain functional groups of microbes.
The Winogradsky column may also be used to
demonstrate aspects of the earliest, sulfur-based life
forms found on Earth. In an article in Nature, Nisbet
(2000) paints a picture of an environment of early
organisms in the Archean period 2.5 to 4 billion years
ago that are analogous to those found in hydrothermal
vents. Hydrothermal vents were first observed by the
submersible vehicle Alvin that explored the Mid-Atlantic
spreading ridge where the North American and
European plates are inexorably moving apart. This
observation marked the discovery of a system that may
have remained intact since its formation as an ancient
biotic system utilizing simple nutrient cycles as an energy source. In a sense, by creating a Winogradsky column, we are modeling ancient environments, though
perhaps at lower temperatures.
Constructing a Winogradsky column from simple
homemade materials is only one of the exercises discussed here. We also present a technique that uses the
respiration of seed germination to allow the reciprocal
process, anaerobic metabolism, to occur in a simple
glass baking dish. The objectives of this article are to:
1. describe the microbial ecosystem of the
Winogradsky column as a tool for studying the
cycling of sulfur
2. explain how use of the column may illustrate some
features of development of early life on Earth
3. discuss how organisms may be isolated and
grown in a homemade anaerobic chamber.
Useful laboratory and educational extensions of the
exercise are also discussed.
Materials & Methods
Materials needed for the construction of the
Winogradsky column are simple and common. They
include:
• a clear glass or plastic container (e.g., a smoothsided, quart plastic water bottle at least 15 cm in
height and 5 cm in diameter. Plastic bottles are
flexible and can be manipulated to allow for
extraction of species for culturing. Very tall containers require longer development periods and
the bacteria may be more difficult to extract.)
• plastic film and a rubber band
• a wooden dowel
• a sulfur source (e.g., calcium sulfate, magnesium
sulfate, or egg yolk added at about 1-2% of the
soil weight)
• an inorganic carbon source (calcium carbonate
[e.g., chalk or limestone] or baking soda may be
added to 1-2% of soil weight)
• hydric soil (e.g., pond mud or shallow river sediment collected near the surface)
• cellulose (e.g., shredded paper towels)
• a 60-75 watt light source
• water from the same source as the sediment
Break up soil clumps and sieve out larger debris so
the column can be packed evenly. The muddy mixture
should be stirred to gain a uniform consistency and
should include the sulfur and inorganic carbon sources.
Place a 2-3 cm layer of the mud mixture in the column,
add the source of cellulose, and stir and pack with the
dowel. Add as much of the mixture as needed, 2-3 cm at
a time, with gentle tamping with the dowel to force out
trapped air, until the tower of mud is about 5 cm from
the top of the container. Your last layer should be 2-3
cm of water. Cover the opening with plastic film and
secure with a rubber band. Place the column next to a
continuously-lit, low heat, moderate intensity light
source, making sure the column does not overheat.
Examine the columns weekly for at least a month,
recording changes in color as they occur. For the
Winogradsky column to be successful, enough time
must be allowed for the cultures to develop. The
columns may show growth in a week, as indicated by
formation of a black color near the bottom and disintegration of the cellulose (paper), but will probably not
fully form and stabilize for four weeks or more. The
SULFUR NUTRIENT CYCLE
349
ideal situation would be for students to investigate the
column over the course of the year studying ecology,
microbiology, biodiversity, evolution, and other biological themes.
Isolation & Culturing of
Organisms from the
Winogradsky Column
Variations
When pigmented patches are visible in the column
(Figure 1), one can attempt to isolate some of the organisms. Sampling can occur at weekly intervals to check
succession or can be done at the end of the project to
see the final flora of bacteria that develops. Sampling
may be done using a standard bacteriological nichrome
wire loop or hypodermic needle (pierced through the
side of the plastic container), however distinction
between the microbes and mud is often difficult. Look
for Beggiatoa (Figure 2A) or Thiobacillus in the watersediment interface. Flagellated organisms from the column, such as Rhodopseudomonas (Figure 2B) or those
with sulfur inclusion bodies, like Chromatium and
Thiospirillium (Figure 2D) are more easily identified if
you carefully adjust the contrast on a standard light
compound microscope or have a phase-contrast microscope. The green sulfur (Figure 2C) and sulfate-reducing (Figure 2E) bacteria are more difficult to see.
Another method is to plunge a microscope slide into the
soil and allow growth of adherent biofilms to form on
the glass, which is then easily examined under magnification (Anderson & Hairston, 2000).
Construction and development of the Winogradsky
column incorporates several variables. With just a few
changes, different columns can be created to compare
growth rates, microbial populations, and ecological
diversity.
• Sulfur Source
To illustrate the importance of the type of sulfur
as a substrate, sodium sulfide or elemental sulfur
can be added in place of a sulfate. This should
reduce the growth of the sulfate-reducing bacteria and alter the composition of the microbial
community.
• Acidity
Acid affects the biotic component of our environment and alters its function. Changing the pH
can affect which species grow and dominate.
Many of the standard sulfur reducers are adapted
to pH 6-8 (Madigan et al., 2000). Creating a more
acidic or alkaline environment shifts the community composition and alters sulfur cycling.
• Heat
Thermophilic bacteria are adapted to higher heat
than most (i.e., mesophilic) bacteria. Mud from
some sources (e.g., hot springs) may harbor thermo-tolerant or even thermophilic bacteria. If you
put the column close to a light source that produces heat, these may grow. This could lead to a
classroom discussion about thermal vent biological communities.
• Osmotic Stress or Marine Stimulation
Columns with different salt concentration can
illustrate several principles. If you begin with a
freshwater or wetland source, salt can be a stressing agent favoring halophilic and halotolerant
bacteria. On the other hand, you can show that
nutrient cycles also occur normally in marine
environments by simply collecting muds and
water from a marine source and letting the column develop as described.
• Type of Light (Wavelength)
Fluorescent lights, incandescent lights, or light
filters (i.e., colored cellophane) that remove part
of the spectrum from a light source could be
used to select for organisms with different
absorptive pigments.
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THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
Growing microorganisms isolated from the diverse
conditions of the Winogradsky column will be challenging, yet students interested in the complexity of the
mechanisms underlying the fundamental processes of
nature will want to explore this process. Several methods for culturing microorganisms from the column have
been described and include pipetting mud from each
colored zone to individual tubes for incubation with
light (Benson, 2002) or separating the mud, layer by
layer, and drawing off the liquid just above the layer for
culturing or microscope observation (Atlas & Bartha,
1998). If the latter method is part of your plan, constructing a Winogradsky column out of a clear, smoothsided cylinder is very helpful. After removing the plastic
wrap or plugs from the ends, the mud can be pushed
out in a single unit and sampling can be easily done. In
addition, specific microbiological media recipes (e.g.,
Atlas, 1995; DIFCO Laboratories, 1984) are available to
establish enrichment cultures of some of the organisms.
Although one can buy jars (e.g., GasPak system) and
other materials (e.g., Brewer’s Petri plate, Wright’s Tube;
Harley & Prescott, 1999) designed specifically for anaerobic culture, we present here a method for culturing
anaerobic organisms that can work well under classroom conditions using simple household items. The
method is also intrinsically interesting for students
because of the irony of pitting one biological process
(i.e., seed respiration) to provide conditions for another
SULFUR NUTRIENT CYCLE
351
Hydrogen Sulfide Concentration
Black Zone
Green Zone
Red-Violet Zone
Water-Soil Interface
Water
Air
Winogradsky Column
Beggiatoa
Sulfur-oxidizing
Bacteria
Non-sulfur
Obligate Anaerobic
Bacteria
Clostridium
Desulfovibrio
Chlorobium
Green sulfur
Bacteria
Sulfur-reducing
Bacteria
Chromatium
Rhodospirilium
Rhodopseudomonas
Purple sulfur
Bacteria
Purple non-sulfur
Bacteria
various
various
various
Diatoms
Cyanobacteria
Protists
Thiobacillus
R E P R E S E N TAT I V E
GENUS
ORGANISMS
BIOLOGY
fermentative
chemoheterotrophic
photoheterotrophic
photoheterotrophic
photoheterotrophic
photoheterotrophic
non-photosynthetic
chemolithotrophic
chemolithotrophic
photosynthetic
photosynthetic
photosynthetic or
heterotrophic
M E TA B O L I S M
rods with
endospores
vibrio
straight or curved
rods
ovals or rods
vibrio-spiral
rods
rods
filamentous
silica frustule
singular or filament
singular or simple
colonies
MORPHOLOGY
positive
negative
negative
negative
negative
negative
negative
negative
N/A
N/A
N/A
GRAM
S TA I N
Diagram of a typical Winogradsky column showing zones of growth that correspond to oxygen and sulfide gradients. Organisms frequently found in the different microhabitats and their metabolic
and other characteristics are shown in register with column zones.
Figure 1.
High
Anaerobic
Low
Aerobic
Chemistry
Microaerophilic
Micro-Aero
Phillic
A.
process (i.e., anaerobic respiration). This gives the teacher yet
another chance to test the critical thinking of the student.
B.
C.
D.
E.
Figure 2.
Drawings and photomicrographs of some representative organisms found in a typical Winogradsky
column.Photomicrographs shown in E used with permission from N. Pfennig.
A. Drawings by S.Winogradsky of the sulfur-oxidizing bacteria Beggiatoa.
Fig. 1 shows drawings of the tip of a Beggiatoa alba filament that becomes depleted in sulfur
globules (dots inside filament) over time: a) B. alba grown with sulfides, b) same species
grown in low-sulfide water for 24 h, c) B. alba in low sulfide after 48 hours.Fig. 2: Beggiatoa
media bacterial filament. Fig. 3: tip of B. minima. Fig. 4: degenerated B. alba lacking sulfur
(Winogradsky, 1949).
B. Non-sulfur purple bacteria Rhodopseudomonas (left) and Rhodospirilum (right) (Stanier et al.,
1976).
C. Green sulfur bacteria Chlorobium limicola (Stanier et al., 1976).
D. Purple sulfur bacteria Chromatium (left) and Thiospirillium (right) (Stanier et al., 1976).
E. Sulfate-reducing bacteria Desulfovibrio desulfuricans (left) and Desulfonema limicola (right)
(Madigan et al., 2000).
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THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
The medium has the following components: 0.01%
NaS•9H2O (i.e., 0.01 g/100 ml
water), 0.05% yeast extract,
0.05% sodium malate, 0.05%
L-cysteine, and 1.5% agar in
solution. This is most easily
prepared by adding 0.01 g
NaS•9H2O, 0.05 g yeast
extract, 0.05 g sodium malate,
0.05 g L-cysteine, and 1.5 g
agar to 100 ml water. Adjust
the pH to 7.3 using dilute acid
or base (i.e., < 0.1 M NaOH or
HCl) and bring to boil, then
dispense ingredients in clean
Petri dishes. Isolate mud from
the red or green pigmented
anaerobic or microaerophilic
areas of the column and
innoculate each to different
plates by streaking.
Place 1-2 cm layer of live
oats or other seeds, such as
grass seed, in a glass casserole
dish, add enough water to
moisten well, and cover with
moist paper towels. Place the
inoculated (streaked) Petri
dishes on this layer. Cover the
whole tray with a glass or plastic plate sealed around the
edges with Vaseline to make an
oxygen seal. Add a light source
above the chamber to give light
for photosynthesis (Figure 3).
As the seeds germinate, they
respire, and oxygen is depleted
in the sealed incubation tray,
and carbon dioxide is produced, making an appropriate
atmosphere for anaerobic photosynthesizers.
The media and chamber
work as follows: the presence
of sodium sulfide and cysteine (an amino acid with an
exposed sulfhydryl group)
helps maintain reducing conditions; it is also a key component for anoxygenic photo-
synthesis in these microbes (i.e., a source of reduced
sulfur or electron donor, for some of the green sulfur
bacteria). Certain red anaerobic photosynthesizers can
use organic compounds, such as malic acid, as electron
donors, so this medium serves double duty for this
type of culture enrichment. The generalized anaerobic
process is:
CO + 2H X ____> (CH O) + 2X + H O
2
2
2
2
where X stands for some reducing agent (or none at
all—some anaerobic bacteria can simply use H2 by itself).
This metabolic reaction is thought to be ancestral in the
sense of biochemical evolution to the familiar photosynthetic process in modern oxygenic photosynthesis,
in which X has become oxygen.
Discussion
A little deeper in the column, hydrogen sulfide gas
(H2S) is diffusing upward into the aerobic zone. Part of
the sulfur cycle is evident here. The H2S gas has been
produced by anaerobic microorganisms near the bottom of the column. These organisms reduced the sulfate
originally mixed into the soil. Near the top of the column, the H2S can be oxidized back to sulfate by the sulfur-oxidizing bacteria, such as the genera Beggiatoa and
Thiobacillus (Atlas & Bartha, 1998). These bacteria are
chemoautotrophs and gain energy from the oxidation
of reduced sulfur to elemental sulfur or to sulfate and
they can also synthesize organic compounds autotrophically from CO2. Thiobacillus oxidizes sulfur while
Beggiatoa is sulfide-oxidizing.
Microaerophilic Zone
Relating Microbiology to Nutrient
Cycling in the Column
After a month to six weeks, the column should stabilize into three distinct zones and develop communities of bacteria specific to their environmental requirements (Figure 1).
Aerobic Zone
The top of the water column can contain large populations of diverse bacteria and protists. These are aerobic organisms found in organic-rich freshwater
habitats such as shallow ponds and polluted streams.
The bacteria are often flagellated, allowing them to
migrate and establish themselves in new areas
(Madigan et al., 2000). In addition, there may be larger
protozoa and invertebrates
from the original water and
mud source. At the very top
of the zone the mud is the
most oxygen-rich part of the
column, often colored a lightbrown from iron-oxide precipitate.
Oxygen-producing
organisms, such as the photosynthetic
cyanobacteria,
often grow above the mud,
forming a green zone. These
are the only bacteria that
have photosynthesis like that
of plants. In fact, there is very
strong evidence that the
chloroplasts of plants originated
from
ancestral
cyanobacteria that estab-
lished themselves as symbionts inside the cells of a
primitive eukaryote.
The diffusion of H2S from the sediment below
enables anaerobic photosynthetic bacteria (which typically appear in brightly colored bands) to grow. From
bottom to top, green sulfur bacteria (GSB), such as
Chlorobium, create an olive-green color zone. Purple
non-sulfur bacteria (PNSB), such as Rhodospirilum and
Rhodopseudomonas, usually require a small amount of
oxygen and are located nearer to the top of column than
are the GSB. Growth of these organisms results in a dark
red-rust color.
The metabolism of both GSB and PNSB provides an
excellent opportunity to draw comparisons between oxygenic photosynthesis (oxygen producing, like green
plants) and anoxygenic photosynthesis (non-oxygen producing organism that pre-dated green plants). GSB and
PNSB gain energy from light reactions and metabolize
CO2 in the same way as plants
do. Yet, because they use H2S
instead of water as the source
of hydrogen (reducing power),
they produce a more oxidized
sulfur product (Atlas &
Bartha, 1998). Consider plant
photosynthesis (i.e., 6CO2 +
6H2O => C6H12O6 + 6O2)
expressed as CO2 + H2O ___>
[CH2O] + O2. Then it is easy
show the parallel processes
between photosynthesis and
hydrogen sulfide oxidation
(Atlas & Bartha, 1998) as:
CO + H O __> [CH O] + O
2
Figure 3.
A simple, yet effective, anaerobic chamber can be constructed
from common household items as depicted in this diagram.
2
2
2
(plant photosynthesis)
CO + H S __> [CH O] + S
2
2
2
(bacterial anoxygenic photosynthesis)
SULFUR NUTRIENT CYCLE
353
Anaerobic Zone
The Sulfur Cycle
Organisms that grow in anaerobic conditions ferment organic matter or perform anaerobic respiration.
Fermentation is a process in which organic compounds
are degraded incompletely; for example, yeasts ferment
sugars to alcohol. Anaerobic respiration is a process in
which a substance other than oxygen is the terminal
electron acceptor.
Sulfur is an assimilable, nutritional requirement for
most life, and yet represents a energy conduit, or dissimilatory pathway, for some microorganisms. As a
nutrient, it is a component of several amino acids
required for protein synthesis, and a number of other
important biochemical components of the cell. Its
chemical versatility, or ability to exist in several oxidation states (Figure 4), makes it a significant part of the
energy cycles of many organisms. Because of this range
of oxidation states, sulfur compounds can act as electron acceptors and donors. As electrons move from one
molecule to another they also lose or gain energy. Thus
the many microbial transformations of sulfur compounds form a basic part of energy metabolism.
Three primary strata form in the lower level of the
column. The uppermost anaerobic layer often contains
Gallionella and other iron-oxidizers. Enrich for these by
adding a source of iron (i.e., a nail or piece of steel
wool). If you isolate Gallionella and look after closing
down the condenser iris (or using a phase-contrast
scope), you will see cells and stalks that appear as twisted threads. These organisms oxidize iron and produce a
rust-colored iron oxide layer.
Moving deeper into the column, purple sulfur bacteria, such as Chromatium, may be found and these bacteria produce a red-to-purple layer in the soil. Purple sulfur bacteria reduce sulfates to sulfur; a type of metabolism that emerged on Earth early in the planet’s history.
From an evolutionary standpoint, there is strong evidence that the mitochondria of present-day eukaryotes
were derived from the purple bacteria (Margulis et al.,
1986).
In the deepest layers are obligate anaerobes that
scavenge and metabolize sulfur and carbon in ways we
do not often discuss in the science classroom. Sulfurreducing bacteria like Desulfovibrio utilize fermentation
products (see below) in anaerobic respiration, using
either sulfate or other partly-oxidized forms of sulfur
(e.g., thiosulfate) to produce the H2S gas that diffuses
through the column. The H2S spontaneously complexes
with iron to form a black ferrous sulfide (FeS). This is
why lake sediments (and our household drains) are frequently black.
Some of these organisms are anaerobic cellulosedegraders, such as Clostridium, that grow when oxygen
is depleted in the sediment. Though Clostridia cells are
killed by exposure to oxygen, these organisms produce
spores that can survive aerobic conditions (Madigan et
al., 2000). They degrade the cellulose to glucose and
then ferment the glucose to gain energy, producing a
range of simple organic compounds (e.g., ethanol, acetic
acid) as the fermentation end products. Sometimes, at
the very bottom of the column, depending on the
source of the mud, a pink layer will develop due to purple sulfur bacteria with gas vesicles. A characteristic
species is Amoebobacter, which also photsynthesizes
using H2S (Atlas & Bartha, 1998).
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THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
A number of species can use elemental sulfur anaerobically as a terminal electron acceptor (the usual role of
oxygen in aerobic respiration), reducing sulfur to hydrogen sulfide (H2S). Others can use thiosulfate or sulfate as
an electron receptor. A number of sulfur pathways exist
in the column (Figure 4). There are numerous species in
this environment beyond those mentioned, and each
has its own unique contribution to the sulfur cycle.
Additional Questions
Other areas can be investigated as an activity with
students. The column is, in fact, almost limitless as a
source of questions and projects
1. If iron is increased, how might that affect growth
of the organisms in the column?
2. Are there methanogens (i.e., microorganisms that
produce combustible methane) in the column?
How would they be detected? (Hint: Use a lighted match to check the head space or gas of an
“old” column).
3. What would be the effects of manipulating the
pH? Adding salt? Adding organic nutrients?
Manipulating the temperature? Manipulating the
spectral influx in the light source with filters?
Conclusions
The Winogradsky column is a complex system and
an excellent example of an investigation that can span
the level from guided inquiry all the way to open-ended
projects that can occupy students’ imaginations and
studies for months. It is also a window into the biodiversity of our world. The Winogradsky column is an excellent way to show students that not all bacteria are
pathogens and they have an important role in the geochemical cycling of the biosphere, one they have been fulfilling since life first began nearly four billion years ago.
Figure 4.
The sulfur cycle emphasizing the transition in oxidation state of the different sulfur compounds (after Fenchel et al., 1998) and the bacteriamediated processes that occur in aerobic or anaerobic habitats.
SULFUR NUTRIENT CYCLE
355
Acknowledgments
We wish to extend a special thank you to Dr.
Norbert Pfennig for his permission to use his photomicrographs in this publication and to Sherry Hutson
(UIS) for graphic design assistance. We wish to thank
the 2000 Woodrow Wilson National Fellowship
Foundation Summer Leadership Institute for Teachers
that focused on biodiversity for creating the forum for
learning and interaction; one of the many end products
being this article.
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