High-salinity Winogradsky column, 2017

Students: Niki Chondrelli, Steven Moschos 

Department of Biological Applications and Technologies, University of Ioannina

Experiment

We constructed two Winogradsky columns with mud and water from the lagoon in Koronisia, in 20th October 2017. We used newspaper as a carbon source and egg as a source of sulfur and carbonate ions. In one column we also added 50gr of salt to check how it would affect the growth of microorganisms. 

 

Hypothesis

We expect a delay in the bacterial growth in the salt-enriched column but similar colour patterns.

Our hypothesis is based on the fact that in every broad taxonomic group there are halophilic species, therefore no significant difference will be observed in the colour patterns. Possibly the diversity of the high-salinity column will be more limited, but this can not be deduced with simple observation, unless a group is completely absent. Also, due to the increased salinity, the number of microorganisms that survive and consequently grow in this column will be lower compared to the standard and therefore the formation of colonies will be delayed.

Results

Our hypothesis was rejected, since we observed the same rates of bacterial growth in both columns. This could be due to the fact that the mud we collected exhibited high salinity (about 80‰) so halophilic species were already abundant in it, and the addition of salt didn't significantly change their growth conditions.

As it can be seen in the pictures that follow, the colour patterns are similar in both columns throughout the experiment but the colour of the water differs (reddish in the standard column and muddy in the salt-enriched one).

Standard, 26/11/17

Salt, 26/11/17

Standard, 23/11/17

Salt, 23/11/17

Standard, 14/12/17

Salt, 14/12/17

Winogradsy column - Light efect Hipothesis

Four Winogradsy columns were constructed. (pictures from the columns can be found here:( https://drive.google.com/drive/folders/1PRv055DWphKSB6fsl9J6ypOXioezNHev?usp=sharing )

Researchers : George Kazantzidis , Giota Kontogeorgiou, Dimitris Papanikos. 

Department of Biological Applications and Technologies

Hypothesis: Using different color filters for each winogradsky column we expect different microorganisms growth depending on the color allowed in each column.

Materials and methods

Place of experiment

Mud was selected from Amvrakikos Lagoon western Greece (39° 0'25.69"Β, 20°55'7.19"Α) at 20/10/2017.

The weather was sunny and the place from which we gathered the mud was wet but not mumbled. The place is known for high eutrophic levels. Also Amvrakikos lagoon is polluted cause many rivers from Western Greece end to it, carrying a lot of fertilizers from crops.

Materials

Mud and water were taken from the lagoon. In the mud was added 2gr of cellulose (crushed paper) and calcium carbonate (one egg, whole).  The mud was mixed in order to homogenize. In each column (plastic bottle 1,5L) was filled 2/3 with mud (1L) and 1/3 with water(0,5L). one column was used as standard and the others for the Hypothesis. Each from the 3 columns was wrapped with plastic color filter membrane. The three membranes used were blue (450nm), red (680nm) and green (520nm). All four columns were placed in sunny place at marine biology lab (University of ioannina).

Results

Our first hypothesis was that in the standard column we will find all kinds of bacterial communities. By contrast, in the green column we expected to find only purple sulfur and non-sulfur bacteria in the middle of the column because they are the only ones that can use the green light for energy production. In the red column we expected to find green photosynthetic bacteria and not purple  bacteria (because purple bacteria does not absorb red light). In the blue column we expected to find all types of bacteria but in less abundance than in the standard column (blue light is absorbed by all kinds of photosynthetic bacteria). We do not expected changes in the non-photosynthetic bacteria communities. 

Two months after the construction of the column we do not observe any difference in our columns. We assume that is too early to have bacterial growth because the membrane filters reflect a large amount of light outside of the column.

One interesting result is at the water bacterial communities which are grown at all columns but with different microorganisms (observed with eyes) as they form different shapes of communities.

Stabilization of the colonies and appearance of organic molecules

Nutrient data:

·         Mud: 30g

·         Cellulose: 0’5g

·         Sugar (glucose): 0’5g

·         Iron sulphate (FeSO₄): 0’1g

·         Agar: 0’11g

·         Calcium carbonate: 0’5g

·         Sodium chloride: 0’54g

·         Water

Third blog post:

In the last posts, we described the possible bacteria colonies present in our Winogradsky column: from sulphate reducing bacteria to cyanobacteria. In our second post, we detailed the progresses in the first three weeks, and in the present entry we will conclude with all our progresses after two months of research.

One month after the last registers of our column, we can observe that the metal sulphides precipitate is much more concentrated than before: this is to say that the column is darker than before. However, purple sulphur bacteria colonies has developed considerably too. Furthermore, we can appreciate the appearance of a small new layer. This layer seems to have the same colour as the mud we used at the start of the experiment. Occam’s razor would say that the obvious answer is that it’s mud. The question here is why did it appear after all this time, when there were metal sulphides there before. Methane going up, as we’ll see later, could have stirred all the components of the column. We could have stirred them while breaking the methane gap. Or, a more complex answer would be that those sulphides are being decomposed. Could be the action of bacterias, either using the sulfides, or altering the equilibrium it might have with the H₂S. As we already said on our last post, purple sulphur bacteria use the H₂S on their metabolism. According to Le Chatelier’s principle, by using H₂S, the equilibrium constant of formation of those sulphides would decrease, and more H₂S would be formed, consuming sulphides.

But the most important new in our Winogradsky column is the creation of a gap which divides the mud in two parts: this gap keeps one piece of mud raised and suspended in the air, as we can observe in this image:

This is due to the production of methane, which is produced by a process called methanogenesis or bio-methanation, carried out by some microorganisms from the domain Archaea. This was something important and new in our research: we finally obtained organic substances, which are an indisputable evidence of the presence of life. Methane (CH₄) is the simplest organic molecule, as it contains only one carbon atom and four hydrogen atoms. The origin of this carbon atom can be cellulose or calcium carbonate; the two carbon sources we added (it is impossible to determinate it qualitatively). Methane is a waste molecule produced by living beings, and its presence is a clear sign of cellular metabolism. Last exoplanetary researches established the presence of a small concentration of methane in the surface of Mars (approximately 0’01ppm); a very small concentration but not insignificant. Another important fact about the significance of the role of this bacteria in the possible appearance of life is its presence in hydrothermal vents, which are one of the cribs of life on earth. The expulsion of gas methane though this chimney evidence the presence of this microorganisms. To sum up, the appearance of this gas gap in our column is a great new in our research, as we finally can demonstrate the formation of organic molecules. Nevertheless, we cannot determinate quantitatively the concentration of methane in this camera, so we can’t confirm that this gas is pure methane: it can coexist with other inorganic molecules, such as CO₂ (coming from the metabolism of some microorganisms) or even H₂O vapour, among other molecules. But we can confirm that most of this gas is methane, as we can smell an unpleasant scent when we extract the gas from this bag.

In relation to the rest of the Winogradsky column, we cannot make any clear distinction respect to our last posts: most of the column stills being dark, but the purple sulphur bacteria colonies have increased in number and size. The methane gas gap has been removed to fix again both parts of the mud, and a new colour layer has appeared. Attending to the evolution trajectory of the colonies, we do not expect any significant news: maybe purple sulphur bacteria colonies can reach a higher size, and we may observe the appearance of new methane gas gaps spread through the bottom of the column. Therefore, this is the final appearance of our Winogradsky column:

Pablo González García &
                                                                               José Manuel Bellido Gutiérrez

                                                                                  Group 4B

Group 6A, after a month

After a month we can appreciate several changes in the different sediments:

• At the top we can see how the green over the brown colour, that because there are more aerobic bacteria.

• In the middle we see that the zone is still black due to the nonsulfur photosynthetic bacterias, but now has appeared some pink zones due to purple non-S-bacteria.
• in the bottom it still black with some bubbles in there due to the fermentative processes of the microorganisms

Winoblog, The last post, group B7.

          
        In response to the last post’s comment, Beggiatoa isn’t growing here. Simply, it was cyanobacteria.
Cyanobacteria grow in a space with oxygen, at the top of the column. This area can present a ligth brown. This is the part of the column richer in oxygen and poorer in sulfur. However, certain quantities of SH₂ arrive to the first stratum by diffusion from the mud of lower areas. This quantities can be used by sulfide oxidisers.

               To emphasise, at the end of our Winogradsky column, we can observe red and orange colours. 

Rhodospirillum and Rhodopseudomonas produce those colorus. Their abundance depends 

on the amount of hydrogen sulphide that has been produced and that has been utilised. Those 

microorganisms are photoorganotrophs.

                In order to conlcude with our experiment, we think that this experiment have an interesting 

ecologic part. The microorganisms that are involved in the column have the ability to regulate 

a medium that was saturated with nutrients and to generate a gradient of gases.

This microorganisms have a specific metabolic pathways that can produce subtances that others can use

to obtain energy. This “chain relation” through bacterias are so interesnting and can be used in

biotechnological process. For example, this microorganisms can be used to regulate an ecosistem 

that it’s saturated of some substances, this knoledge field is called “Bioremediation”,

and its booming science sector. 

               Other example is generating a “chain” trough some microorganisms to obtain some interesting substances

for humans or pharmaceutical industries.

To sum up, after that experiment we understand better the relations that can be established in our "Winogradsky column”. What`s more, now we understand the real biotechnological potential of bacteria. This experiment does not finish here, it’s only the beginning 

Figure 1: Cyanobacteria

Figure 2: Rhodospirillum and Rhodopseudomonas 

Figure 3:  Photo taken 26th May

UCA_8A_3: The aerobic zone shrinks.

After several weeks, the aerobic zone of our column has nearly disappeared and all of our column is mostly black, indicating the presence of anaerobic bacteria. We can confirm this from the fact that almost all the aerobic algae that were at the top of the column have died and disappeared.

The reason behind this is that by putting a big sulfate source, anaerobic bacteria have proliferated. They produce H₂S, which is toxic to aerobic bacteria, so we can assume this H₂S has reached the top of the column, causing the algae to die.

We can assume this aerobic zone will disappear completely in a few days and the only thing left in our column will be anaerobic bacteria.

Group 6A, After a week

After a week we only can aprecciate three deposited sediments in function of the microorganism:

• At the top of the column it turned to a brown colour and a bit green, it can be due to aerobic bacterias growing on it.
• In the middle we can se a black zone, it can be because of nonsulfur photosynthetic bacteria.
• At the bottom we can observe a gray zone where a big bubble it can be produced by microorgasim which have a big fermentative capacity.

Group 6A, first day

The Winogradsky column is a micrography of an acuatic ecosystem which is exposed to a permantent solar source, it allows the growth of several kind of microorganism.
In our case, we added:

1. Cellulose: 0,51 g.
2. NaCl: 2..1 g.
3. Na2SO4: 1.2 g.
4. Calcium Carbonate:0.65 g.
5. Calcium Sulfate: 0.81 g.
6. Glucose: 0.59 g.

After adding this enrichment material, we expect microorganism to grow in there. 

• Halo bacterias because we added NaCl
• Bacterias capable of metabolizing cellulose.
• CaCO3 and glucose as carbon source for organism.
• Na2SO4 and calcium sulfate like energy source doing fermentatives process.

<We didn´t take any photo of the winogradsky column>

Last week (4 C)

 Three weeks later

Three weeks later the main black color has disappeared. Now we can see that several organisms has grown. For example, there are seaweeds occupying the majority of our Winogradsky column. These seaweeds are the same ones that grow in Río San Pedro's sediment. We can also appreciate the development of the sulfur fixative bacteria in a reddish tone at the bottom of the column, where there is no oxygen. We have achieve our obtective: creating a growth medium with a gradient of oxygen. At the bottom, only sulfur fixative bacteria have grown, which doesn't need oxygen. At the top, there are photosynthetic seaweeds, which use oxygen in their metabolism.

Week 2 of our Winogradsky column (4 C)

Second week:

After one week, we can see that the colour of our Winogradski column has turned motsly into black. At the top of the column there is a thin layer of turbid water. The Río San Pedro's mud and sediment contained several microorganisms and depending on the nutrients we added, different forms of organisms will have developed. In the mood under the water we can see a reddish tone and a small seaweed. This reddish tone appears because of sulfur fixative bacteria that has grown thaks to the calcium sulfate as a sulfur source for them.

The reason of the black colour is the big amount of organic matter that has been formed because of the compounds we added at the beggining of the experiment such as sugar (an important carbon source).  

UCA_8A_2: Day 14- Why, Winogradsky, why?

We added 20g of mud from Rio San Pedro, 60g of sand and some water, 0.2g of sugar, 0.2g of cellulose, 0.2g of plaster and 0.2g of sodium sulphate.

Today, our Winogradsky column appeared mostly black, mainly in the middle.

Anaerobic bacteria, which are in the deep zone of the column, have proliferate too much because of adding a lot of sulfate sources. They use these sulfate sources in their metabolism, producing H₂S that has been accumulated at the bottom of the column (that is why depths are grey). That excess of H₂S, which is toxic, prevent the growth of other bacteria so none of them can live on the surroundings.

What happened at the top? This part is still brown and looks like some kind of algae have grown due to they were further away from the source of H₂S. But it will not last too much until the H₂S reaches the top, killing them.

Survival of the fittest, life is too hard inside our winogradsky column.

UCA_8A_1: First Day!

The target of this practice is to create an aquatic ecosystem. It would let us learn about how are the bacteria from the mud of a river. In this case, our mud is from the Rio of San Pedro.

We added 20g of mud, 60g of sand and some water. 

We wanted the bacteria to have a good culture medium, so we also added the following nutrients:

-          -As a source of carbon, 0.2g of sugar and 0.2g of cellulose.

-          -As a source of sulfur, 0.2g of plaster and 0.2g of sodium sulphate.

We mixed all those substances with the mud and the sand, that’s how  we created our column. From the part of top of the column to the deepest part, the concentration of oxygen decrease, so that’s how we know that the bacteria at the top of the column are aerobic and the ones that are at the deepest part are anaerobic. There’s also a gradient of sulfide. It comes from a very low concentration at the top of the column to a higher concentration at the bottom. 

There's a photo from the 7th day. We can apreciate that the colour of the bottom is darker, the first day the colour was equal in the whole column.

UCA_3A_3:Day 98 - This is not the end!

Finally, the Winogradsky column illustrates how different microorganisms perform their interdependent roles: the activities of one organism enable another to grow, and vice-versa. These columns are complete, self-contained recycling systems, driven only by energy from light.

We added a lot of salt in our column because we wanted halophilic bateria to grow but they didn`t.

Instead of that, they have grown cyanobacteria (indicated by the Green color) and photoheterotroph bacteria (indicated by the orange color produced by the iron oxide).

The white color is due to the production of hydrogen sulphide precipitate produced by purple sulfur bacteria, which use H₂S instead of H₂O as reducing agent so they don’t produce O₂ (they do anoxigenic photosynthesis).

Due to the initial conditions, we suppose that halophilic bacteria will grow because of the amount of sat we added.

But this is not the end, a lot of microorganisms can grow in our column in summer!

First day group 2A UCA

Nutrient data:

·         Mud: 30g

·         Cellulose: 0’5g

·         Sugar (glucose): 0’5g

·         Iron sulphate (FeSO₄): 0’1g

·         Agar: 0’11g

·         Calcium carbonate: 0’5g

·         Sodium chloride: 0’54g

·         Water

Second blog post:

With this research, we intended to demonstrate how could some microorganisms of the natural environment of Cádiz marsh develop in a Winogradsky column, thanks to the addition of some kinds of different metabolites, such as calcium carbonate, sodium chloride, iron sulphate, cellulose or glucose, among others. We wanted to study how could they grow in an environment with different concentrations of oxygen (while the column gets deeper, the concentration of oxygen decreases) and sulphate (while we get near to the surface, the concentration of sulphate is lower).

The very first days, we appreciated the apparition of bacteria colonies. This was demonstrated because of the colour layers:

        

As we described, the surface is richer in oxygen, so the microorganism that grow in this zone are aerobic, microaerophiles or facultative anaerobic. Some weeks later, we will observe the presence of cyanobacteria; a kind of photosynthetic archaea. It is known that this microorganism was the responsible of the appearance of the present day’s oxidant atmosphere 2.400 million of years ago. Immediately below, there is an orange-yellow-brown liquid. We have four hypotheses for this:

·         Option A: Iron Chloride (FeCl₃). In this molecule, iron is in its oxidized form (Fe³⁺). Iron sulphate is dissociated in Fe²⁺ and SO₄^2-. Fe²⁺ can be oxidized with water molecules (or simply with other molecules present in the column) to Fe³⁺, and this cation can react with chloride ions coming from NaCl, forming iron chloride. This reaction is not very likely because of the high solubility of this molecule. In addition, this reaction is not spontaneous even in standard conditions (ΔG=37’9KJ/mol).

·         Option B: Iron sulphate can be dissociated in Fe²⁺ (which has a yellow colour) and SO₄^2-. Part of the ferrous ions can be oxidized into Fe³⁺ (which has an orange colour), and the solution of the top of the column will be a mixture of both ions.

·         Option C: Iron oxides. Iron sulphate can be dissociated in dissolution, resulting in two different ions. It can also react with water to form iron monoxide (FeO) and sulphuric acid (H₂SO₄). Iron monoxide, when diluted, acquires an orange-red colour too. Also, iron (Fe) can be oxidised because of the action of water to form Fe₂O₃, another orange-red compound.

In conclusion, the orange solution can be a result of a mixture of these substances.

According to the rest of the column, which has a black coloration with some red tones, we can obtain some conclusions. First, we must talk about sulphate reducing bacteria (there are several kinds of bacteria which are based on this metabolism). They transform iron sulphate into hydrogen sulphide (H₂S), which, in natural conditions, can react with several kinds of metals to form metal sulphides such as FeS, with a black-brown coloration (which gives the shown coloration of the column) and a rotten egg odour. The purple coloration is due to the purple sulphur bacteria, which take advantage of the hydrogen sulphide obtained in the degradation of iron sulphate to participate in the photosynthesis. These bacteria are microaerophilic or anaerobic: some of the bacteria included in this group can tolerate oxygen (but they do not use it in their metabolism) or they must not be around oxygen (it is considered a poison for them). Thus, they can live in zones poor in oxygen (as it is the bottom of the column) and carry out photosynthesis, using non-organic-carbon metabolites. In the following pictures, we can see this first bacteria: