Winogradsky column. Day 0. UCA B1 B2

Hello, we are groups B1 and B2 and we are going to explain how we made a Winogradsky column experiment.

       First of all, we added 20g of mud in our test tube. Later we mixed the mud with a little bit of San Pedro´s river water trying to get a viscous texture. The next step was adding 0,3g of paper and 0,1g of CaCO3. We prepared a 100g of sand and 20g of mud mix and we cleaned the test tube edges. In this moment wee got a 2 cm layer of water.

Hypothesis: The aim of this experiment is checking how the concentration of salt affects to microorganisms. For this, one group prepared a Wingardsky column without salt and the other group added a teaspoon of salt. Both columns were exposed to the sunlight.

Winogradsky column. Day 0. UCA C2 C1

Helo! We are Rafael Pardo Velasco, Jaime Pérez Leiva, Blanca Ruiz Alonso, María Oliva Pareja González, Covadonga Muñoz Raya and Lorena Rodríguez Rivero! We will explain how we have made our Winogradsky column!

We have made our Winogradsky colum with:

Rio San Pedro sediment, 0,5g of CaCO₃ and filter paper.

That colum has been made for this way:

- We put 20g of sediment and much water in a plastic container. When all the compounds became viscous, we put it in a test tube, covering ⅓ of it.

- We put slowly 0,5g of CaCO₃ and much filter paper inside the tube. Before we had mixed them without introducing air bubbles.

- We carefuly hit the tube to send the sediment to the botton of the tube and to catch all the air bubbles.

-We washed the plastic container.

- We mixed 100g of sand and 20g of sediment without enriching.

- We repeated the third step and cleaned the tube borders.

- We waited for until all the compounds proned.

- A water layer was formed on the top of the tube.

Observations:

Because of the addition of the filter paper and the CaCO3, we have created an environment that is enriched in CO2, furthermore, mixing all the compounds of our column, have ensured a fast diffusion of nutrients and gases inside the tube, so gradients are more widespread.

We made two Winogradsky columns for the same way to study the influence of light on them, putting one of our columns in light and the other in darkness. We supose that, in the column that we have put in darkness, it will inhibit the growth of photoautotrophic microorganisms. So, that column will only depend on quimioautotrophic microoorganisms, that pick up its energy of carbonated and sulfurated compounds. The other column will depend on light too.

UCA_4A-3A_DAY 7

RIO SAN PEDRO'S SEDIMENT + RIO SAN PEDRO'S WATER + RIO SAN PEDRO'S SAND + 0.5g NaCO3 + Filter paper + 0.25g NaCl 

Hello lovers of microbiology! 

We are going to explain how we made our Winogradsky column experiment. 

First of all, we mixed in a bucket river “San Pedro”’s sediments and river water.  Then, we added 0,5g of NaCO3 and a piece of paper (cellulose). Finally, we mixed river’s sand we the previous mix (if it is necesary we add a little bit o water, but trying to not make de mix a liquid).

Then, we introduced the mix in a big test tube, in our case, we added 0,25g of NaCl for encourage the development of halophilous bacteriums.  For making it funnier we did two test tubes with the same composition, but one of them will be in the oscurity and we put the other in the light so we expect that in the second tube we will find more fotosintetic bacteriums so we can apreciate the differences between both tests tubes. 

What will happen the next time we observe them?

On the right we can see first a photo of the column that goes on the dark in day 0 and above we can see a photo of the column that goes on the light in day 0. 

On the right, we see the same columns but after 7 days. 

UCA_5A-6A-7A_1: Day 0 – Starting Winogradsky Column’s project!

Winogradsky Column Materials:

All groups included 40g Rio San Pedro sediment, 100g sand and sea water.

5A group’s column: 0,31g tryptone; 0,62g powdered milk; 0,1g CaSO₄.

6A group’s column: 0,74g agar, 0,74g CaCO₃.

7A group’s column: 0,1g CaSO₄, 0,3g paper (cellulose).

Procedure:

1º-. We took off the solids residues from the sediment. Then, we mixed 20g of Rio San Pedro sediment with sea water, and after that we included the previous materials mentioned for each group.

2º-. Once the mixture was homogenized, we put it into a test tube. Secondly, we mixed 100g of sand and 20g Rio San Pedro sediment, with sea water. Both mixtures’ texture should be like milkshake’s texture. 

3º-. After that, we added the second mixture to test tube until we filled up to 2/3 of the test tube. We left the mixture stand a little bit. 

4º-. Finally, we added 2-3 cm of sea water on the top. 

Hypothesis

5A: When the project has finished, we won't find aerobic bacterias owing to the plenty sources of N and S. In our column, two predominant colors will be found. In the lowest part of the test tube, there will be a black color which is caused by bacterias with a more anaerobic metabolism (respiration S₂O₃^- -> H₂S). It will take up about 1/3 of the solid part of the column. Above it, we will find a grey color, which is caused by other anaerobic bacterias (respiration NO₃^- -> NO₂^- -> N₂).

6A: In conclusion, after having thrown to the column CaCO₃ (calcium carbonate) and Agar the sample will keep the color it has because it does not have sulfur and although this in the dark it will not turn blue which would be the color it must acquire when the bacteria feed on sulfur.

7A: We thought that our column will have aerobic and anaerobic bacterias, due to the two feeding sources, the cellulose from the paper and the CaSO₄. We also thought that our column will change its color because we will have anaerobic bacterias producing H₂S from SO₄^-. Then the H₂S will react with the iron in the mud to produce a dark-blue color. Besides, our column is exposed to sunlight. 

We will report more about our columns in the next entry coming soon.

 A5 Winogradksy Column.

 A6 Winogradsky Column

 A7 Winogradsky Column

UCA_4B_3B_day7

San Pedro's River sediment + 0.3 g paper + 0.1 g CaSO4

Group 3B: Marcelo Gómez Herrera, Alvaro Lucero Garófano and José Luis Hernández Fernández.

Group 4B: Paula Gilabert Prieto and Pilar Grosso Rodríguez.

Hello everyone!

Today we will explane you how we have done ours Winogradsky's columns.

Firstly, we added San Pedro's river mud (1/3 of the length of the column), exactly 20 grammes.

We must remove big leaves and sticks.

Then we dilute the mud with a little bit of water from the river.

We didn't used distilled water due to the fact that the difference of concentrations will lead to lysis.

We added 0.1 grammes of CaSO4 and 0,3 grammes of paper(celulose). This is for create an atmosphere rich in sulphire and CO2.

Secondly we took another 20 mud grammes and we mixed it with sand.

Finally filled the column with mud until reaching to the top.

Hypothesis:

We have prepared 2 columns exactly with the same compositions. (Explaned in a few lines before)

The unique variable that can change the column is the light.

So we will keep one of them in contact with sunlight, and the other one will be kept in the darkness.

Because of that, the column kept in the light will grow photosyntetic organism, meanwhile in the darknesses will grow chemosyntetic organisms.

UCA_5B, 6B_1: Day 6

Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4 + (4 g sugar)

We made two Winogradsky columns with:

• 40 g Rio San Pedro sediment and 100 g soil.
• 0.3 g paper, whose cellulose will serve as a source of organic carbon for our microorganisms.
• 0.1 g CaSO₄, as a sulfur source.
• Rio San Pedro water -note that not any water would do the job, for the osmotic shock would kill our microorganisms- to saturate the mud.
• 4 g sugar in our second column.

Depending on the metabolism of the different microorganisms found in Rio San Pedro, we could expect diverse results by the addition of sugar to our Winogradsky column:

•  In case there are yeasts -such as Saccharomyces cerevisiae-, some of the sugar we added will undergo alcoholic fermentation, and it’ll be turned to ethanol and CO₂. If this happens, it’s likely that the ethanol will be found at the bottom of the column, as this is where -according to the O₂ gradient- anaerobic processes can take place.
• On the contrary, if there are other kinds of fermentative microorganisms, such as lactic acid bacteria, it won’t be ethanol but lactic acid what we’ll find -and maybe this could be checked by using spectrophotometric methods-.
•  However, another possibility could be the entire degradation and oxidation of the sugar by heterotrophic bacteria, in which case no other metabolic products will be found.

In the column with no sugar, the source of carbon will be much scarcer, and therefore, we forecast a slower growth rate.

Since the columns will be kept in the dark, we are certain that no phototrophic bacteria will be able to survive; though we do expect, due to the presence of a sulfur source, sulfate-reducing bacteria, producers of hydrogen sulfide (H₂S). 

UCA_8B,7B_1:Day 1

Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4

Group B8 (Laura Lucena Del Amo and Noelia Moares Fernández) 
Group B7(Pablo Lucas Ezequiel Garín Ortega and Alba Mejías Gallardo)

Objective:

Our main goal in this experiment is to realize the variety of different microorganisms that we can find in a generic sample with the proper envrioment (such as light diponibility, oxygen difusion...). We are also looking forward to understand the complex variety of metabolic capacities of microrganisms and how the waste products from their metabolism  can be the metabolic requirement of other group of microorganism.
Through this experiment we can verify our main hypothesis.


Labware

• Transparent bottle
• Test tube
• Sediment/soil
• Water in situ
• Funnels
• Enrichment materials: paper (celulose) and CaSO4
• Electronic balance
• Parafilm
• Wash-bottle with water in situ
• Tupper
• Agitator
• Spatulas
• Tube and syringe
• Shovel

Experimental protocol

1. We add 20 g of mud (from Rio San Pedro) in a tupper, until we fill a third part of it.
2. We mix the mud with a moderate amount of water, until it has the appearance of a milkshake. 
3. Mix the enrichment substratum with the hydrated mud. (We added 0,3g of paper/celulosa and 0,1g of CaSO4).
4. Transfer the sediment that we have mixed to the test tube little by little with the aid of a funnel. We must tap it while transfering until it doesn't have any bubble).
5. Clean the material used.
6. Prepare another mix of sediment, this time with 100g of sand and 20g of mud (from Rio San Pedro).
7. Repeat step 4, adding a little bit of water until everything is hydrated (not in excess) and tranfered.
8. Clean the top of the test tube with paper.
9. Let the sediment rest for some minutes.
10. Add 2cm of water above the sediment. (We must let an air layer on the top of the tube, at least of 2-3cm).
11. Cover with parafilm.
12. Label the test tube, being careful not to cover the light source.

Initial hypotesis

We prepared two different test tubes to observe the proliferation of microorganisms in the winogradsky column, one was prepared by us (group B8) and the second one was prepared by  group B7. 

In the first one we added celulose and CaSO4, this tube is going to receive a natural light source,

meanwhile the second tube is going to be kept in darkness and has a glucose supply.

Our hypotesis is that in the first tube will grow up photosynthetic (autotroph) microorganisms on the top, there will also be heterotrophic microorganisms (chemoorganotroph) at the bottom. This is due to the fact that the organisms on the top receive a bigger amount of light and the organisms at the bottom get the nutrients from the photosynthetic organisms. These organisms are probably going to be mostly aerobic since they have an oxygen supply from the air. The only microorganisms that will not be aerobic are the ones that we find at the bottom, because they don't get enough oxygen.

In the second tube we suppose that will proliferate chemorganotroph microorganisms as well as chemoinorganotroph microorganisms (autotroph), there will be aerobic organisms on the top and anaerobic organisms at the bottom.

a)

b)    

Some pictures of our test tubes once that are filled with sediment and ready to let microorganisms proliferate inside of it. We hope we get good results that verify our hypothesis.

a) tube exposed to light, with Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4

b) tube not exposed to light, with Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4 and a supply glucose

UCA_5C_1:26/02/2018_0,5g of dextrose+ Río San Pedro sediment. UCA_6C_1:26/02/2018_0,25g CaSO4+ 0,25g paper + Río San Pedro sediment. UCA_7C_1:26/02/2018_0,5g of NaCl+ Río San Pedro sediment.

Hi everyone!

We are all students of 1º year of Biotechnology. In this post, we are going to describe our Winogradsky column experiment.

In first place, we weighted 20 g of sediment and mixed it with salt water so that it had a liquid texture like a smoothie. After doing that, we tipped out the mixture into the test tube being careful with the air bubbles.

Secondly, we weighted and mixed 20 g of sediment with 100g of soil until they become an homogeneous mix. Later, we added the blend to the test tube avoiding the formation of air bubbles. We eliminated the bubbles formed bumping the test tube against the workplace so that they could come out of the column.

We also had to add 2-3 ml of water above the semisolid mash and we had to leave 2 cm empty at the top of the column so that we could close the test tube. We repeat this process three times. In each column, we added differents substances:

C5: 0,5g dextrose.

C6: 0,25g CaSO₄+ 0,25g paper.

C5: 0,5g of NaCl.

The column was placed in a luminous area.

We have an hypothesis of what can occur a week after preparing the column. We think that:

In C5 (0,5g dextrose):

Although now the C5 ´s column Winogradsky   is the darkest (due to the fact that it presents in his composition organic matter), in the future it  will have a clearer color. We base on two reasons:

It will be exposed to the solar light, that’s why the  photosynthetic organisms will predominate in the column.

·         It is highly probable that the quantity of initial glucose is close to ending. This means that  the heterotrophs organisms aren´t going to have food source. In the other hand, autotrophs organisms generate more organic matter.

In C6 (0,25g CaSO₄+ 0,25g paper):

·         At the superior surface of the column there will be aerobial phototrophs, because hera there will be more oxigen than on the bottom.

·         Around the rest of the column there will be anaerobial phototrophs.

·         In the interior of the column there will be heterotrophs.

·         In the inferior region of our column there will be organotrophs.

·         At the top, there will be lithotrophs.

In C7 (0,5g of NaCl):

Our hypothesis is that the most of microorganisms are autotrophs because we have improved the conditions for growing autotrophy’s microorganisms. We think that in the surface of the test tube will grow photoautotrophs’ microorganisms because our Winogradksy column is near to the sun light. At the bottom,  almost all the microorganisms will be anaerobics because there will not be oxygen enough for aerobic breath.

Ilustración 1:C5

 Ilustración 2: C6

Ilustración 3:C7

Interesting reading material...

Petri's dish vs. Winogradsky's column –

shifting boundaries between purity and mixture of microbes

Winogradsky Column - Sediment depth hypothesis

Students: Nikolopoulou Ioanna, Lamprou Andriana

Department of Biological Applications and Technologies, University of Ioannina

Hypothesis

The microbial diversity correlates to the depth of the sediment in an inversely proportional way, with photosynthetic microorganisms being more prominent closer to the surface of the sediment.

Material and methods

The samples were collected from the Logarou lagoon, Koronisia, Greece at 20/10/2017. Sediment from the surface (0-2 cm) and from greater depth (18-25 cm), 700ml each, was enriched with 10 gr white sugar and 1 gr Ca2SO4 .Each sample was then placed in a transparent plastic bottle of 1,5L capacity and then 700ml of ultrapure water were added. The Winogadsky columns were stored at room temperature next to a window and were observed on a weekly basis.

Results

 On the first day of the experiment a difference was observed in the color of the two sediments. The superficial sediment had a dark green color, whereas the deep sediment appeared slightly brown. With the progression of time there was an increase of the sediment's volume as well as the appearance of small gaps in it, due to the production of gas, in both columns. This is mostly due to the presence of sulfate- and sulfur-reducing bacteria that produce H2S, which had a very distinct odor. The column with the superficial sediment presented a change in the color of the water as it obtained a green hue, while there was a slime-like formation in the surface of the water, possibly due to the presence of photosynthetic microorganisms (photo 2). At the same time there was no change in the appearance of the water in the other column.

 Approximately two months later (10/01/2018) an interesting observation was made. Regarding the deep sediment, a distinct stratification occured which was the most significant change in the appearance of this column. Specifically, a big part of the sediment turned from brown to black, while the water also obtained a blackish tint. This is due to the presence of black ferrous sulphide which is produced after the chemical reaction of H2S with any iron that is present in the sediment, due to the strong presence of sulfur- and sulfate-reducing bacteria. At this point the consistency of the deep sediment resembles the natural sratification that was observed in a larger scale in the field during the sampling process. It is important to note that in this column during the experiment, the water did not turn green, whereas in the column with the superficial sediment the water turned greener with time. However, no stratification was observed in the superficial sediment during the experiment. 

The presence of green color in the water of the column with the superficial sediment indicates the presence of photosynthetic microorganisms, which seem to be absent from the other column. Due to the use of ultrapure water, any microorganisms that are present in the column must have originated from the sediment, hence only the superficial sediment seems to have photosynthetic mircoorganisms. The production of gas in both columns is a strong indication that sulfur- and sulfate-reducing bacteria are present in both columns and therefore at both depths. 

 We conclude that the superficial sediment has a greater diversity in microorganisms, specifically more that are involved in photosynthesis. However, we can not estimate accurately the diversity in both samples through optical observation alone, so molecular assessment should be implemented. Furthermore, it is important to note that the sediment can act as a microbial sink, which can provide microorganisms to the ecosystem.

Image 1:Winogradsky columns at 20/10/2017(1),13/11/2017(2), 10/01/2018(3)  Left column: deep sediment. Right column: superficial sediment