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Seminario 1-Documento2 PDF
Seminario 1-Documento2 PDF
348 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
centimeters thick even though in the environment 3. discuss how organisms may be isolated and
establishment of similar layers of different organisms grown in a homemade anaerobic chamber.
would typically exist in a few millimeters of sediment.
Useful laboratory and educational extensions of the
The Winogradsky column creates conditions that
exercise are also discussed.
expand the volume of natural processes, allowing a
clear view of naturally-occurring phenomena. Soil sam-
ples are collected from wetland habitats, amended with Materials & Methods
simple inorganic and organic materials, then exposed to Materials needed for the construction of the
light as an external energy source. The results are a mul- Winogradsky column are simple and common. They
ticolored column of soil and water, each color linked to include:
a chemical or biological process. The defined zones of
microbes develop form according to concentration gra- a clear glass or plastic container (e.g., a smooth-
dients of oxygen, sulfur, nutrients, and light. Each func- sided, quart plastic water bottle at least 15 cm in
tional microorganism group is dependent on other height and 5 cm in diameter. Plastic bottles are
functional microbial groups for development. flexible and can be manipulated to allow for
extraction of species for culturing. Very tall con-
The Winogradsky column was developed and tainers require longer development periods and
named after Sergei Winogradsky (1856-1953), a the bacteria may be more difficult to extract.)
Russian microbiologist. He studied the complex inter-
actions between environmental conditions and micro- plastic film and a rubber band
bial activities using soil enrichment to isolate pure bac- a wooden dowel
terial cultures (Madigan et. al, 2000). Louis Pasteur, a sulfur source (e.g., calcium sulfate, magnesium
Robert Koch, and other scientists isolated cultures for sulfate, or egg yolk added at about 1-2% of the
study, but Winogradskys work was one of the first to soil weight)
study microorganisms in mixed enrichment cultures.
The fact that the exercise works under a wide range of an inorganic carbon source (calcium carbonate
circumstances is a testament to the near ubiquity of cer- [e.g., chalk or limestone] or baking soda may be
tain functional groups of microbes. added to 1-2% of soil weight)
hydric soil (e.g., pond mud or shallow river sedi-
The Winogradsky column may also be used to
ment collected near the surface)
demonstrate aspects of the earliest, sulfur-based life
forms found on Earth. In an article in Nature, Nisbet cellulose (e.g., shredded paper towels)
(2000) paints a picture of an environment of early a 60-75 watt light source
organisms in the Archean period 2.5 to 4 billion years
ago that are analogous to those found in hydrothermal water from the same source as the sediment
vents. Hydrothermal vents were first observed by the Break up soil clumps and sieve out larger debris so
submersible vehicle Alvin that explored the Mid-Atlantic the column can be packed evenly. The muddy mixture
spreading ridge where the North American and should be stirred to gain a uniform consistency and
European plates are inexorably moving apart. This should include the sulfur and inorganic carbon sources.
observation marked the discovery of a system that may Place a 2-3 cm layer of the mud mixture in the column,
have remained intact since its formation as an ancient add the source of cellulose, and stir and pack with the
biotic system utilizing simple nutrient cycles as an ener- dowel. Add as much of the mixture as needed, 2-3 cm at
gy source. In a sense, by creating a Winogradsky col- a time, with gentle tamping with the dowel to force out
umn, we are modeling ancient environments, though trapped air, until the tower of mud is about 5 cm from
perhaps at lower temperatures. the top of the container. Your last layer should be 2-3
Constructing a Winogradsky column from simple cm of water. Cover the opening with plastic film and
homemade materials is only one of the exercises dis- secure with a rubber band. Place the column next to a
cussed here. We also present a technique that uses the continuously-lit, low heat, moderate intensity light
respiration of seed germination to allow the reciprocal source, making sure the column does not overheat.
process, anaerobic metabolism, to occur in a simple Examine the columns weekly for at least a month,
glass baking dish. The objectives of this article are to: recording changes in color as they occur. For the
1. describe the microbial ecosystem of the Winogradsky column to be successful, enough time
Winogradsky column as a tool for studying the must be allowed for the cultures to develop. The
cycling of sulfur columns may show growth in a week, as indicated by
formation of a black color near the bottom and disinte-
2. explain how use of the column may illustrate some gration of the cellulose (paper), but will probably not
features of development of early life on Earth fully form and stabilize for four weeks or more. The
350 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
BIOLOGY
Aerobic
heterotrophic colonies N/A
Phillic
Thiobacillus chemolithotrophic rods negative
Water-Soil Interface
Purple non-sulfur Rhodospirilium photoheterotrophic vibrio-spiral negative
Bacteria Rhodopseudomonas photoheterotrophic rods negative
Microaerophilic
Micro-Aero
Red-Violet Zone
Purple sulfur Chromatium photoheterotrophic ovals or rods negative
Bacteria
Anaerobic
High Black Zone Non-sulfur Clostridium fermentative rods with positive
Obligate Anaerobic endospores
Bacteria
Figure 1.
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.
352 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
synthesis in these microbes (i.e., a source of reduced lished themselves as symbionts inside the cells of a
sulfur or electron donor, for some of the green sulfur primitive eukaryote.
bacteria). Certain red anaerobic photosynthesizers can
A little deeper in the column, hydrogen sulfide gas
use organic compounds, such as malic acid, as electron
(H2S) is diffusing upward into the aerobic zone. Part of
donors, so this medium serves double duty for this
the sulfur cycle is evident here. The H2S gas has been
type of culture enrichment. The generalized anaerobic
produced by anaerobic microorganisms near the bot-
process is:
tom of the column. These organisms reduced the sulfate
CO + 2H X ____> (CH O) + 2X + H O
2 2 2 2 originally mixed into the soil. Near the top of the col-
umn, the H2S can be oxidized back to sulfate by the sul-
where X stands for some reducing agent (or none at
fur-oxidizing bacteria, such as the genera Beggiatoa and
allsome anaerobic bacteria can simply use H2 by itself).
Thiobacillus (Atlas & Bartha, 1998). These bacteria are
This metabolic reaction is thought to be ancestral in the
chemoautotrophs and gain energy from the oxidation
sense of biochemical evolution to the familiar photo-
of reduced sulfur to elemental sulfur or to sulfate and
synthetic process in modern oxygenic photosynthesis,
they can also synthesize organic compounds autotroph-
in which X has become oxygen.
ically from CO2. Thiobacillus oxidizes sulfur while
Beggiatoa is sulfide-oxidizing.
Discussion
Microaerophilic Zone
Relating Microbiology to Nutrient The diffusion of H2S from the sediment below
Cycling in the Column enables anaerobic photosynthetic bacteria (which typ-
ically appear in brightly colored bands) to grow. From
After a month to six weeks, the column should sta- bottom to top, green sulfur bacteria (GSB), such as
bilize into three distinct zones and develop communi- Chlorobium, create an olive-green color zone. Purple
ties of bacteria specific to their environmental require- non-sulfur bacteria (PNSB), such as Rhodospirilum and
ments (Figure 1). Rhodopseudomonas, usually require a small amount of
oxygen and are located nearer to the top of column than
Aerobic Zone are the GSB. Growth of these organisms results in a dark
red-rust color.
The top of the water column can contain large pop-
ulations of diverse bacteria and protists. These are aer- The metabolism of both GSB and PNSB provides an
obic organisms found in organic-rich freshwater excellent opportunity to draw comparisons between oxy-
habitats such as shallow ponds and polluted streams. genic photosynthesis (oxygen producing, like green
The bacteria are often flagellated, allowing them to plants) and anoxygenic photosynthesis (non-oxygen pro-
migrate and establish themselves in new areas ducing organism that pre-dated green plants). GSB and
(Madigan et al., 2000). In addition, there may be larger PNSB gain energy from light reactions and metabolize
protozoa and invertebrates CO2 in the same way as plants
from the original water and do. Yet, because they use H2S
mud source. At the very top instead of water as the source
of the zone the mud is the of hydrogen (reducing power),
most oxygen-rich part of the they produce a more oxidized
column, often colored a light- sulfur product (Atlas &
brown from iron-oxide pre- Bartha, 1998). Consider plant
cipitate. photosynthesis (i.e., 6CO2 +
6H2O => C6H12O6 + 6O2)
Oxygen-producing expressed as CO2 + H2O ___>
organisms, such as the photo- [CH2O] + O2. Then it is easy
synthetic cyanobacteria, show the parallel processes
often grow above the mud, between photosynthesis and
forming a green zone. These hydrogen sulfide oxidation
are the only bacteria that (Atlas & Bartha, 1998) as:
have photosynthesis like that
of plants. In fact, there is very CO + H O __> [CH O] + O
2 2 2 2
354 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
Figure 4.
The sulfur cycle emphasizing the transition in oxidation state of the different sulfur compounds (after Fenchel et al., 1998) and the bacteria-
mediated processes that occur in aerobic or anaerobic habitats.
356 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005