EXERCISE 2: SIMULATING THE Sergei N.

Winogradsky
ENVIRONMENT WITH A WINOGRADSKY ● Designed the Winogradsky Column in 1880 to
study soil microorganisms
COLUMN
● Pioneer in the investigation of microbial
autotrophy (and microbial ecology in general)
II.I. Winogradsky Column
in the late 1800’s and 1900’s
● Excellent tool for the study of microbial activity
● Strong proponent of examining freshly-isolated
in:
organisms rather than domesticated laboratory
o The soil
strains
o Nutrient cycling
● Winogradsky Column was one of the methods
o Microbial succession
he developed for the study of microbial nutrient
o Ecology
cycling in the environment
● Simple and inexpensive to set up (can be set
up at home)
● Can also be set up in an amazing variety of
ways to study the following:
o Sulfur
o Nitrogen
o Carbon
o Phosphorus
o and other nutrients that most often cycle
between the upper zone and the lower
anaerobic zone
● It is like a miniature enclosed ecosystem which
enriches the microbial communities within a
II.II. Set Up of the Winogradsky Column
sediment or a sediment or soil sample
● The column consists of a transparent cylinder,
o Enables many scientists to study many
for example:
of the microorganisms that play a vital
o Graduated cylinder
role over its biogeological chemical
o Pre-calibrated transparent empty 1.5- or
processes without the need to isolate
2.0-L soda bottle
and culture these microorganisms
● The column will be filled with marine or
individually
freshwater mud (i.e., pond) and a few
● These columns serve as a complete,
substrates that would support bacterial growth
self-contained recycling system driven only by
in the consortium
light energy
● This set-up is a remarkable way to study carbon,
nitrogen, sulfur, phosphorus or other nutrients
o As these undergo cycling between the
upper aerobic zone/layer and the lower
anaerobic zone/layer
● The column will be filled with marine or
freshwater mud (i.e., pond) and a few
substrates that would support bacterial growth
in the consortium
o The mud contains a diverse community
of interdependent microbes
o It takes approximately six to eight weeks
to see layers of microbial growth
 o This allows organisms of any oxygen or
redox requirements to grow
● Particular interest is in looking for conspicuous
sulfur-cycling organisms and photosynthesizers

II.IV. Gradient in Terms of Energy Transfer

● In a light trap setup, from light dependent energy
transfer, the system starts to have a microbial
system that are characterized by chemotrophs
moving down the column
● Microbial zonation and its corresponding niche
processes involved in the column:

I. Chemoheterotrophs

● Some cellulose degrading Clostridium species

II.III. Principles in the Winogradsky may start to grow when the oxygen is depleted
Column in the sediment
● Microbes generate sulfide at the bottom of the o All Clostridium species are strictly
column anaerobic as their vegetative cells are
● Cellulose fermentation slowly releases sugar for killed the moment there is an exposure
use by anaerobic organisms to oxygen
o Sulfate (added as calcium sulfate) then ● But in nature, they can survive as spores in
serves as an electron acceptor for aerobic conditions:
sulfate reducers, generating sulfide o They degrade the cellulose to glucose
o Carbonate is available for autotrophic and then ferment the glucose to gain
growth and as a pH buffer energy, producing a range of simple
● Insoluble calcium salts are relatively used so organic compounds (i.e., ethanol, acetic
that they don't create an overly salty acid, succinic acid, etc.) as the
environment fermented end products
● Metal sulfides (typically iron) create a black ● The sulfur reducing bacteria, which can also be
color at the bottom of the column found at the bottom of the column (i.e.,
o ↑ Sulfide diffuses upward into the Desulfovibrio), can utilize these fermentation
column products produced by Clostridium through
o ↓ Oxygen diffuses downward from the anaerobic respiration using sulfate or any other
surface partly oxidized forms of sulfur (i.e., thiosulfate)
● Sulfur oxidizing organisms consume both where as the terminal electron acceptor now
they meet generating large amounts of hydrogen sulfide
o Resulting in stable counterbalancing o The hydrogen sulfide will now react with
sulfide and oxygen gradients any iron in the sediment, producing this
black ferrous sulfide
 o This is why lake sediments (i.e., o This is now a pathway to a sulfur cycle
household drains) are most frequently that typically occurs in some natural
black in color waters
o However, some of the hydrogen sulfide
diffuses upwards into the water column III. Purple Non-Sulfur bacteria
where it is actually utilized by other
microbial systems

II. Anaerobic Photosynthetic Bacteria

● Most of the water columns above the
photosynthetic bacteria will be colored bright red
by large populations of Purple Non-Sulfur
bacteria
o Include species of Rhodopseudomonas,
● The diffusion of these hydrogen sulfide from the
Rhodospirillum, and Rhodomicrobium
sediment into the water column now enables
o Grow in anaerobic conditions, gaining
these anaerobic, photosynthetic bacteria to grow
their energy from light reactions BUT
o Purple Sulfur bacteria (Chromatium)
while using organic acid as their
o Green Sulfur bacteria (Chlorobium)
carbon source for cellular synthesis
*Appears usually as two narrow brightly colored
▪ This is why they are termed
bands just immediately above the sediment
“photoheterotrophs”
● Both Green and Purple Sulfur bacteria gain
● The organic acid that they use are the
energy from the light reaction and produce their
fermentation products of other anaerobic
cellular materials from carbon dioxide (in much
bacteria (usually Clostridium)
the same way plants do)
o However the Purple Non-Sulfur bacteria
are intolerant when there is a high
Plant photosynthesis vs. Bacterial anaerobic
amount of hydrogen sulfide
photosynthesis (with regards to the equation)
▪ Reason why they occur
immediately above the zone
where the Green and Purple
● Only essential difference: Bacterial anaerobic Sulfur bacteria are found
photosynthesis does not generate oxygen during
photosynthesis, IV. Remaining Oxygenated Zone
o As they do not use water as the ● Many microorganisms can also grow in the
reductant. Instead, they use hydrogen remaining oxygenated zone (top of the water
sulfide column)

Purple vs. Green Sulfur Bacteria Three distinctive types are of special interest:

1. Sulfur Oxidizing bacteria (Beggiatoa)

DIFFERENCES

PURPLE GREEN

Cell Size Larger Smaller ● Diffuses or accepts hydrogen sulfide that

moves to the aerobic zone
Sulfur Deposits inside Deposits externally ● Any of these hydrogen sulfide that diffuses into
Deposit the cell the aerobic zone can be oxidized to sulfate by
the sulfur oxidizing bacteria
● Sulfate form of the Green Sulfur bacteria ● Gains energy from the oxidation of hydrogen
(produced by the photosynthetic bacteria) will sulfide and synthesize their own organic
return to the sediment: matter from carbon dioxide
o Where it can be recycled by o Reason why they are termed
Desulfovibrio (or any sulfur-reducing “chemoautotrophs”
microorganism
● Similar types of organism occur in soils, gaining
energy from the oxidation of ammonium to Summary
nitrate, which then leeches and sips from the soil ● The Winogradsky column is a classic
and can accumulate in most of the water supply demonstration of the metabolic diversity of
o Particularly in some of our agricultural prokaryotes
lands o All life on Earth can be categorized in
terms of the organisms’ carbon and
2. Photosynthetic Cyanobacteria energy source
● Energy can be obtained from light reactions
o If you obtain it from a light reaction, you
are a phototroph
o If from chemical oxidation of organic
substances, you become chemotrophs
● Colored in green, and can be seen and can
o And the carbon for cellular synthesis
grow in the upper zones
can be obtained from carbon dioxide, in
● Only bacteria that have oxygen evolving
which you become autotrophs
photosynthesis, similar with plants
o Or if from free-form organic compounds,
o There is strong evidence that the
you become heterotrophs
chloroplasts of plants have evolutionarily
● Combining these categories, these are the four
originated from the cyanobacteria (or
basic life strategies:
the ancestor of present-day
o Photoautotrophs - Represented by the
cyanobacteria) that at least lived as a
environment by plants
symbiont inside the cell of a primitive
o Chemoheterotrophs - Like humans
eukaryote
and other animals and fungi
o Similarly, there is equally strong
o Photoheterotrophs
evidence that the mitochondria of
o Chemoautotrophs
present day eukaryotes were derived
● Only in the bacteria and among the bacteria
from the mitochondria of Purple bacteria
within a single Winogradsky column can be
● Once the cyanobacteria starts to grow, they can
found all the four basic life strategies
now oxygenate much of the water zones of the
● The Winogradsky column is also a classic
column
demonstration of how microorganisms occupy
highly specific microcytes according to their
3. Sheathed Biofilms
environmental tolerances and their carbon and
energy requirements
● Finally, the column enables us to see how
mineral elements are cycled in the natural
environment
● Aerobic organisms
o Use organic substrates but are unusual
because as the bacterial cells divide,
they synthesize a rigid, tubular sheet
from which individual cells can escape
and swim away to establish new
colonies
● Many empty sheets are seen in older colonies
o They are made up of a complex mixture
of protein, polysaccharides, and lipids
o Brought to protect the cells from the
predations of some protozoa
● The sheet can usually be observed encrusted
with ferric oxide often giving a thin layer of a
yellow or crusty appearance in the colonies just
above of the photosynthetic cyanobacteria