Winogradsky Column Experiment

Winogradsky Column Experiment 

Winogradsky Column Experiment is a simple way of cultivating diverse microorganisms and study how they interact with each other.

Nutrients: Lentils as source of Ferrum

                  Two Eggs as source of Sulfur

                  Sugar as source of Carbon

Sampling Procedure: On October 16th we went to the Kalodiki marsh (Photo 1) in order to collect mud and water and each student had to create at least one Winogradsky column. Bottles of 1.5lt volume were used as columns. The two-thirds of my column were filled with mud mixed with nutrients and the other one-third was filled with water. Firstly, I collected the mud and mixed it with all nutrients. Then, I filled with the enriched mud the two-thirds of my bottle and added the water which originated from the same marshland. The bottle was covered with a black plastic bag and was left to the hydrobiology lab to incubate in room temperature.

Hypothesis: The Ferrum would help the Photosynthetic microorganisms to grow but the black plastic bag would be an obstacle in this precdure. So I supposed that finally I would't see the green photosynthetic bacteria in the top of my column.

Complications: In October 18th I went to relieve the gas pressure in my bottle but it exploded . I suppose that the reason of this explosion was the production of large amounts of gases and especially of Ηydrogen Sulfide from the sulfur bacteria.

Due to this incident, I decided to create a new colum:

The two-thirds of the new column were filled with mud from the University campus and the rest was filled with water from the eutrophic lake of Ioannina, Pamvotis. The first one-third was filled with mud mixed with one egg as source of sulfur and shredded paper as source of carbon, the second one-third had mud without any nutrient and the last one-third had was filled with lake water. The bottle was left to incubate in the lab at room temperature.

Results: After almost two months of incubation, in the end of the semester I did not notice any sign of growth in my new column(Photo 2). These results, compared to the ones of my fellow students that used mud from the marshland, may indicate that the mud from the University campus was not so rich in microorganisms as the one collected from Kalodiki wetland. 

          

Photo 1: The Kalodiki marsh                          Photo 2: The new column after

                                                                          two months of incubation

Winogradsky Column

Vragkalis Georgios

Department of Biological Applications and Technology

University of Ioannina

Hypothesis: We expect to notice green and purple sulfur bacteria in the mud and cyanobacteria in the water.

Sampling Areas: Mud was selected from Kalodiki wetland, while water was selected from Pamvotis lake. Both systems are eutrophic. The weather was sunny and warm without any rain for a long period of time, which caused the wetland to be dried up on some of its regions.

The Column: Mud was selected from the wetland. 2/3 of mud were used to fill the column (1,5L plastic bottle). 1/3 of it was mixed with a whole egg and shredded paper and put at the bottom of the column. Nothing was put on the rest of the mud. Finally, the column was filled with 0,5L of water from Pamvotis lake. The column was maintained in room temperature and exposed to light.

Results: After a week, cyanobacteria were noticed in the mud and in the water. On day 19, purple and green sulfur bacteria were noticed in the anaerobic zone of the column and became more visible on day 26. On day 40, we can see some spots of black pigment at the bottom of the column. Those are sedimentations of iron sulfide II. Though we can’t see them macroscopically, we could also say that sulfate reducing bacteria exist at the bottom of the column.

Below you can see some pictures of the column:

Στήλη Winogradsky by Daphne Fessa

Εισαγωγή

Σκοπός του πειράματος ήταν η δημιουργία στήλης Winogradsky με χώμα και νερό από το έλος Καλοδικίου. Στην τοποθεσία αυτή, την ημερομηνία άφιξης (.....) επικρατούσαν συνθήκες ξηρασίας. Το έλος ήταν αποξηραμένο και υπήρχαν τοπικές λίμνες σε σημεία στην έκτασή της. 

Υλικά/Μέθοδοι

• Ως πηγή θείου χρησιμοποιήθηκε ορισμένη ποσότητα σκόρδου σε μορφή σκόνης (~2g)
• Ως πηγή άνθρακα χρησιμοποιήθηκε εφημερίδα
• Ακολουθήθηκε ως έχει η υπόλοιπη πειραματική πορεία
• Η στήλη αναπτύχθηκε σε εξωτερικό περιβάλλον, παρουσία φωτός στα Ιωάννινα

Πραγματοποιήθηκε ολική ανάδευση λόγω πτώσης του μπουκαλιού την τρίτη ημέρα μετά τη δημιουργία της στήλης.

Η επιστημονική υπόθεση που έγινε στην αρχή του πειράματος ήταν η εξής: 

1. Αναμένεται γρηγορότερη ανάπτυξη των βακτηρίων που έχουν απαίτηση σε θείο στην περίπτωση που η περιεκτικότητα θείου στο σκόρδο είναι μεγαλύτερη από αυτή των κρόκων αυγών.
2. Αναμένεται καθυστερημένη ανάπτυξη βακτηρίων που έχουν απαίτηση σε θείο, εφόσον η περιεκτικότητα θείου στο σκόρδο είναι μικρότερη από αυτή των κρόκων αυγών.
3. Κανονική ανάπτυξη αερόβιων οργανισμών.

Ο έλεγχος της επιστημονικής υπόθεσης πραγματοποιήθηκε με οπτική παρατήρηση των διάφορων χρωμάτων (πράσινο, κόκκινο) που αντιπροσωπεύουν την ανάπτυξη των αντίστοιχων οργανισμών.

Αποτελέσματα

Παρατηρήθηκε έντονη ανάπτυξη κυανοβακτήρια και φυτοπλαγκτού, δηλαδή των αερόβιων φωτοσυνθετικών οργανισμών, επομένως η υπόθεση υπ' αριθμό "3" επιβεβαιώθηκε. Το ίζημα της στήλης παρέμεινε καφέ, δεν εμφάνισε αποχρώσεις πράσινου ή κόκκινου. Το γεγονός αυτό δείχνει ότι τα βακτήρια που εξαρτώνται από θείο, τα πορφυρά και πράσινα θειοαναγωγικά και θειοοξειδωτικά δεν αναπτύχθηκαν και συνεπώς πιθανόν ισχύει η υπόθεση "2" έναντι της "1", ενώ η λάσπη κατά βάση αποτελείται από θειικά και ανθρακικά άλατα σε συνδυασμό με κυτταρίνη ή άλλα οργανικά.

Η στήλη παρουσίασε αντίθετα αποτελέσματα, όσον αφορά την ανταλλαγή αερίων με το περιβάλλον. Αναμενόμενη ήταν η εκτόνωση αερίων από το εσωτερικό της στήλης, ωστόσο το μπουκάλι αντλούσε αέρια και συμπιέστηκε αρκετές φορές, ως αποτέλεσμα της μεγάλης ανάγκης που είχε το εσωτερικό του.

Συζήτηση

Η ανάπτυξη των αερόβιων μικροοργανισμών (κυανοβακτηρίων και φυτοπλαγκτό) ήταν τόσο έντονη με αποτέλεσμα τη δημιουργία bloom στη στήλη. Από την άλλη, η περιεκτικότητα του σκόρδου σε θείο δεν φάνηκε ικανοποιητική, προκειμένου να αποδώσει την ανάπτυξη και άρα τα χρώματα των θειοοξειδωτικών και αναγωγικών βακτηρίων.

Winogradsky column-Too many added nutrients

Collected mud and lake water from lake Pamvotis, Ioannina, were mixed with a whole egg (70gr), confectioner’s sugar (15gr), shredded paper (5gr). The first bottle was filled with the mixture and then lake water, while the second one had the mixture on the bottom and then just mud and lake water. Hypothetically, the first bottle was supposed to grow and develop the zones faster than the bottle 2. The bottles (1,5L each) were kept for 70 days outside on indirect sunlight. 
The first 5 days the temperature was high, and the nutrients too many, so the bottles needed decontamination every day. In bottle 1 especially, there was a gas overproduction by bacteria, so all the water was removed, so the bottle could deflate. After that, the experiment continued with the bottle 2, wich was not opened again. No changes were found in the mud of bottle 2. The bottle, from day 2, had trapped gases throughout the mud that was produced by bacteria. The trapped gases possibly created a non-friendly environment, so bacteria couldn’t grow further. The only change found, was a discolouration of the water. It turned brown-red because some cyanobacteria that produce those photosynthetic pigments developed successfully in there. They even developed some darker colonies on the top of the water. Photos are from days 2, 14, 29, 50 of the experiment.

How does a blue transparent bottle affect the growth of micro-organisms?

Hi everyone,

This is my final report about my Winogradsky Columns.

Materials

1. Transparent plastic bottle (1.5 L)
2. Mud from Lake Pamvotis (2/3 of the bottle)
3. Water from Lake Pamvotis (1/3 of the bottle)
4. Carbon source (5 g newspaper in small pieces )
5. Source of sulfur (1 whole boiled egg 70g)

Hypothesis

Since bottle (2) contained 1/3 of the nutritions compared to bottle (1), it would have a slower growth rate (of the organisms it contained) as its metabolic processes would be slowed down .

Bottle (1)                                                     Bottle(2) 
After 60 days 

Summary of Results

The experimental hypothesis was not confirmed as bottle (2) with limited enrichment materials contained greater abundance of microorganisms than bottle (1) with evenly distributed enrichment materials throughout.

It is possible that the color of bottle (1) (transparent blue) affected the light that penetrated it and thus the growth of photosynthetic organisms.

In fact, visible light consists of wavelengths of 400-700 nm. Blue light corresponds to wavelength (λ)= 450 nm, and therefore this λ could not be absorbed by the micro-organisms as it is reflected by the bottle.

In the diagram below you can see the absorption spectra of three basic dyes in photosynthesis:chlorophyll a, chlorophyll b, β-carotene.

All three pigments show a peak for λ ~ 450 nm

Source: khanacademy.org

In particular, all micro-organisms that could be cultured in Winogradsky colums show a peak in their absorption of wavelength λ ~ 450 nm through their pigments.

Source:

Colorful niches of phototrophic microorganisms shaped by vibrations of the water molecule

The ISME Journal 1 p.271-282 (2007) - M. Stomp et. al.

volum1, pageM. Stomp et. 

Especially, purple sulfur bacteria,acorrding to a study (Photosynthetic development of purple sulfur bacteria during illumination with green light-Osnitskaia LK, Chudina VI-1977),use short wavelengths of natural light, blue and green, for CO2 assimilation and biosynthesis of biomass, proteins, and pigments.

Which means that the photosynthetic ability of the micro-organisms in bottle (1) was lowered because their pigments didn't absorb wavelength λ ~ 450 nm. That way, the growth of photosynthetic organisms in bottle (1) was slowed down and in the course of the same time showed lower abundance of micro-organisms than bottle (2).

Christina Diamantopoulou

Department of Biological Applications and Technologies

University of Ioannina-January 2019

UCA_A1, A2: results

Our column was saturated with a lot of nutrients that may have done a bad effect on some microorganisms. We have thought the hypothesis that these microorganism can´t grow because of this saturation that have made microorganisms produce second metabolites which have changed pH's environment and maybe that is the reason why some microorganisms died. Also, de high concentration of glucose inhibited some reactions that could be necessary to the microorganisms. On the other hand, microorganisms that were able to live in this condition are the ones which we mentioned in the last post. These are anaerobe microorganisms who produce sulfhydryl acid which precipitates as iron sulphide II (black) and the acid mentioned is the bubble we see on the top.

On the other hand, the column A1 presents the variety of microorganism that we expect. We have to differentiate two zones, the anaerobic and the aerobic:

In the aerobic zone we can see many different types of colonies. These microorganisms usually inhabit in aquatic zone as we can relate in the photos. If they are phototroph they should have a Green coloration, that´s because they have chlorophyll pigments in their chloroplasts. As we can relate in the picture our Winograd sky column also present purple/Brown colonies of phototrophic bacteria’s, they have that colour because they don´t present any chlorophyll pigments.

In the anaerobic zone we discover the presence of sulphate reductors, that kind of microorganism have black pigmentation and are named chemotrophic. We can´t see any other relevant colonies, our theory is that the capacity of reproduction in that organism is better than the others one.

In conclusion the theories that we propose on the first days of class, was not confirmed. We talk about the presence of lactic oxidators but instead we can only observe the most habitual microorganism.

Apart from that we we´re observing the increase of many microorganism likewise we also see the difference between the strata that appears in the column and how they distribute making a microecosystem.

UCA_C3,C4_day90

Few changes have been observed.
-C4
Under strictly anaerobic conditions after a few weeks, and using the load of cellulose provided by the remains of paper incorporated in the sediment as the primary source for its metabolism, appear the bacteria of the genus Clostridium,and that's why an orange color is appreciated.

C3

-Sulfur-reducing bacteria are displayed as a deep black layer and are represented by Desulfovibrio, using sulfate to produce SH2, which will react with any iron present in the sediment, producing ferrous sulfide, which gives color Black. 

UCA_7B,8B_1: Day 89

Winogradsky column- Day 89

Group 7B (Lucas Garín Ortega and Alba Mejías Gallardo)

Group 8B (Laura Lucena del Amo and Noelia Moares Fernández)

The last time we visited our columns, the one made by group 8B, wich was kept in the light, had the following microorganisms:

·        At the bottom of the column, there was a black layer wich means that there are anaerobic microorganisms that breath with sulphate. The bubbles in thar área, are from H₂S, and they are dur to thet breathing.

·        Also, there was a few orange layers wich means that photoeterooganotrophs had been proliferated.

The main diference between the last time we visited our column and this is that there are more orange layers and they are bigger.

After a few weeks we visited our column, the one made by group 7B, for the last time and observed the following:

·   The water that remains on the surface is still clear, so aerobic microorganisms

    have not proliferated

·   The clear layer previously found in the middle of our column has decreased.

    The bottom of the column has become a lighter color, and around this change,

    numerous cracks are found due to gases. These are due to the respiration of the

    microorganisms that are proliferating.

·   Finally, the orange layer appreciated on the surface has increased.

UCA_A4_A3_day85

-COLUMN THAT WAS IN THE DARK:
We can see that all the column is darker, this is because have increased the number of anaerobic microorganisms, since they have carried out a respiration with sulphate, releasing H2S, which precipitates with iron, producing a black FeS compound.
Also, previously we had a gas bubble in the middle of the column, and now we haven´t it, this may be due to the weight of the land has elevated it to the top of the column. •

-COLUMN THAT WAS IN THE LIGHT:

In this case, the column has more than one colour, the column has more than one colour, we can see that there are areas that predominate the black colour by the same phenomenon that we explained before, due to the precipitation of FeS produced by the anaerobic respiration of some microorganisms. 

There are small areas of the column that have an orange colour, produced by photoheteroorganotrophic microorganisms such as Rhodospirillum. 

Finally, comment that there is a small gas bubble, this can be methane, carbon dioxide or even sulfuric acid. 

UCA_C5_C6_C7 day 90

C6: Ana Valdivia Aceituno, Curro Polo Castellano y Sergio Valiente Vélez.

C5: David Ruiz Romero y Carlos Pinto Perea.

C7: Noemí Toro Barrios, Nerea Rojas Sanz, Jose Manuel Pozo Suárez.

Hi everyone!

Here we are going to compare de progress of our Winogradsky columns from the day 44 till the day 94 after beginning this experiment. As we can see, the columns have suffered too many changes. The matter that we agree at the beginning has already been consumed.

Figures 2: 90 days

Figures 1: 40 days

C6: 0,25 g paper +0,25g CaSO4 + Sun light

C6 column has experimented a differentiation of the organization of microorganism with the pass of the time:

On top, from 40 days till today: we can find a green colour that indicates the presence of cyanobacteria; they need to be in the surface to catch gasses like O₂ because they perform oxygenic photosynthesis. This colour can also indicate the presence of algae.

Bellow this, we can see an orange colour that indicates the presence of photoheteroorganotrophs organisms (for example: Rhodospirillum). They use sunlight as a principal source of energy and organic matter from outside as a source of carbon.

This organic matter should come from cyanobacteria of above because they produce secondary metabolites that are sent out when they don’t need them anymore.

Below the orange area of the column, a dark region is placed. Its black and grey colours are due to the existence of anaerobial breath which produces a precipitate of FeS or Fe.

There are also some gas bubbles whose composition can be methane, carbon dioxide or hydrogen sulphide.

The pink region of the column, which is placed under the grey and black one, is probably where Chromatium are placed. As we can compare, this region is bigger whit the time.

Finally, in the very bottom of the column there is a dark green region associated to Chlorobium as they are green bacteria which are anaerobial and phototrophs.

Now, let’s talk about C5 column: The column is still dark on the 20 of April.

Figure 1:40 days

On the one hand, one face of the column is less dark than the other face because it has been exposed to the sun.

On the other hand, in the centre we can see a brown spot that maybe due to pollution. Eventually, there is water in the top.

Along the column it can be observe the presence of purple sulphur bacteria, anaerobic photoautotroph anoxygenic organisms that live at the bottom of the column.

It can also remake the fact that all the initial glucose has been converted to hydrogen sulphide.

Then, on June 1 our Winogradsky column hasn’t changed.

Figure 2:90 days

The dark area covers practically the entire column. This black zone is compound of anaerobic respiration with sulphate, producing hydrogen sulphide which precipitates iron with abiotic form as black iron sulphide.

One side of the column is less dark than the other because it has been exposed to the sun.

Continuing with the observation, in the centre we can see brown spots that may be due to contamination. Sometimes, there is water in the upper part of the column and dry remains of gases that have emanated from the surface liquid (methane, hydrogen sulphide, carbon dioxide ...).

Throughout the column, the presence of purple sulphur bacteria can be observed; anoxygenic photoautotrophic organisms (of the Chromatium type) that live in the lower part of the column. It may also be because all the initial glucose has been converted to hydrogen sulphide.

By the end, the column C7 has been changing a lot throughout these days:

Figure 1: 40 days

Figure 2: 90 days

 First; it shows a brown colour. Some crevices can be seen in the sediment, as well as, dark grey spots. These crevices may show up because of gas production such as CO2, CH4, H2S... Furthermore, orange spots can be seen too, which indicate the presence of photoautotrophic organisms.

It has divided in two parts: a lighter one and a darker one. It must be because the sunlight only lights up in the lighter one. So, we think that in the lighter one there will be more photoautotrophic organisms while in the other will be more chemoautotrophic organisms. The darker one should be formed by organisms that reduces sulphur because the colour black indicates us that it contains sulphur.