Winogradsky column lab page!


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Welcome to the Winogradsky column lab page! Students from the Departments of Biological Applications and Technology, University of Ioannina and Icthyology and Aquatic Environment, University of Thessaly, Greece and the Microbiology course, Faculty of Sciences, University of Cádiz, Spain, discuss their findings on Winogradsky columns they constructed!

If you want to add a post, please feel free to contact the blog administrators (Hera Karayanni, Sokratis Papaspyrou or Kostas Kormas)!



Καλωσορίσατε στη σελίδα των Winobloggers! Διαδικτυακός τόπος συνάντησης φοιτητών, φοιτητριών και διδασκόντων δύο Τμημάτων από την Ελλάδα: Tμήμα Βιολογικών Εφαρμογών και Τεχνολογιών, Παν/μιο Ιωαννίνων και Τμήμα Γεωπονίας, Ιχθυολογίας και Υδάτινου Περιβάλλοντος, Παν/μιο Θεσσαλίας και ενός από την Ισπανία: Σχολή Θετικών Επιστημών, Πανεπιστήμιο του Cadiz. Παρακολουθούμε, σχολιάζουμε, ρωτάμε, απαντάμε σχετικά με τα πειράματά μας, τις στήλες Winogradsky!


Bienvenidos a la pagina web de los Winobloggers! Aquí los estudiantes y profesores de dos departamentos griegos, el Departamento de Aplicaciones y Tecnologías Biológicas de la Universidad de Ioannina y el Departmento de Agricultura, Ictiología y Sistemas Acuáticos de la Universidad de Thessalia, junto con los estudiantes de Microbiología de la Facultad de Ciencias en la Universidad de Cádiz, se reúnen para observar, comentar, preguntar y responder a preguntas relacionadas con nuestro experimento, la columna Winogradsky.


Winogradksy columns

Winogradksy columns
'In the field of observation, chance only favors the prepared mind' Pasteur 1854

Blog posts

Tuesday, 16 January 2018

Winogradsky Columns

Acidic column (column A) and column with an alternative source of sulfur (column B)

Team; Cheiladakis Emmanouel, Grizi Olga,Varoucha Effrosyni  

Sampling
Sampling was carried out at Koronisia, Amvrakikos Bay (Greece) on 20/10/2017. Three columns were made; one as the control, one with low pH (pH=3), and the last one containing an alternative source of sulfur (rice). The nutrients used are presented below:


Control column
Column Α
Column Β
Egg (sulfur source)
25 g
25g
-
Newspaper (Carbon source)
1 g
1 g
1 g
Mud
1/3 of the bottle
1/3 of the bottle
1/3 of the bottle
Mud with added nutrients
1/3 of the bottle
1/3 of the bottle
1/3 of the bottle
Lagoon Water (pH=8.5)
350 ml
350 ml
350 ml
Lemon Juice (pH=3)
-
100 ml
-
Rice (sulfur source)
-
-
150 g

.The rest of the bottle stayed empty, in order for aerobic bacteria to grow             

After the sampling process, the columns were placed in an accessible by light spot, at room temperature. The duration of the experiment was approximately 3 months. Through this period the columns were compared by the appearance of colour layers.

Pic.1; Typical colour zones due to bacterial growth in a Control Winogradsky column.
Hypotheses
  1. In column A, pH value was stabilized at 3, where fermentation is not expected to occur in the lower layers as the fermentation bacteria (e.g Clostridium) cannot survive. As a result there is no carbon acids (which are produced by Clostridium) for sulfate-reducing bacteria to grow. This prevents or reduces the appearance of the upper layers. Also, due to the acidic environment, neither cyanobacteria will grow. In this case, no colour pattern is expected.
  1. The same amount of sulfur was added in column B, using rice as a source, and if bacteria use it in the same way, we do not expect significant differences in the colour pattern between the control and the column B. 


  • Pic.2; Hypothesis A- What is expected to happen in the acidic column
Results
A.Hypothesis accepted; there was no zone observation because acidic pH prevented the growth of the expected micro-organisms.

B.Hypothesis rejected; there was also no zone observation, so it was proven that bacteria don’t use the sulfur from the rice in the same way they use it from the egg.

It needs to be clarified, though, that there is a possibility low pH or rice as a sulfur source just delayed the growth process and not completely prevents it. So in order to be absolutely sure of the result, observation for a longer period is essential.

Phases of the experiment
ü   In every photo column B is at left,control column is in the middle and column A is at right.

Pic.3; Winogradsky columns- Day 1



Pic. 4; Winogradsky columns- After 2 months



Pic.5; Winogradsky Columns- Final Phase



Wednesday, 3 January 2018

Yeast enriched and water bath Winogradsky Columns

Students: Bakratsas Georgios , Nikolaou Tasos  
Department of Biological Applications and Technologies, University of Ioannina

Experimental course
Sampling for the construction of the Winogradsky column took place on 20/10/2017 in the Koronissia region on the Gulf of Amvrakikos. After collecting the necessary amount of sludge (for 3 Winogradsky columns), it was enriched with the following nutrients:
• 3 g CaCO3
• 3 g CaSO4
• 3/4 of the newspaper page
• 3 eggs
• 12 g of onion-garlic mixture.
It is noted that a small amount of sludge was not enriched with nutrients to form the upper layer of the Winogradsky columns.
Our team constructed 3 columns, which were filled with 3/5 sludge, 1/5 water from the lagoon and 1/5 air. From the three columns one was the control and was placed at room temperature near a window (access to solar radiation ), the second identical to the control was placed in a water bath at a temperature of 50-55 ° C while the third was enriched with 8 g of activated yeast in the sludge layer and placed at room temperature near a window.
The second and third columns will be compared to the control.

Experimental hypotheses

1. The column placed in the water bath is expected to have less microbial variation due to the effect of temperature, which only allows the development of heat-resistant micro-organisms.

2. Larger microbial diversity is expected in the yeast column since, with the introduction of yeast, the production of alcohol which is a nutrient of the sulfur-reducing bacteria is accelerated. Therefore, the sulfur cycle is enhanced by increasing the populations of micro-organisms affected by it.

Results

At the time being, the experiment is not in a stage in which we can gather sufficient data about the bacteria in order to conclude a specific result.
Over the course of two months, we only observed a small bacterial growth in the control Winogradsky column in the upper sludge layer. In particular, the developed bacteria had a reddish color which lead us to the assumption that they belonged to the purple non-sulfur bacteria. Apart from the bacteria, there was a development of a white Fungi in the upper layer. Regarding the second and the third column, there was no observable bacterial development.
 As far as the hypotheses are concerned, both of them were rejected since neither the addition of yeast did accelerate the bacterial growth (more bacterial growth in the control) nor the amount of time for the processing of the experiment was enough for the development of heat-resistant micro-organisms (no comparison with the control is available).

Picture 1 Winogradsky columns first day
  
















 Picture 2 Purple bacteria and white Fungi in Control


  













Saturday, 30 December 2017

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

Sunday, 17 December 2017

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.







Friday, 23 June 2017

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