UCA_3C, 4C_1: Day 1. Column’s preparation.

Materials:

- 20 g of Rio San Pedro’s sediment.

- Destilled water/sea water (group 4) and sea water (group 3).

- 0,3 grammes of filter paper (both groups)

- 0,1 grammes of CaSO4 and 0,3 grammes of SO4- (group 3)

- 100 grammes of sand.

Objectives.

The objective of this experiment is to recognise the different microorganisms depending on their metabolism, nutrition, etc. For that, we choose the nutrients and environment of the column depending on the microorganisms that we want to benefit.

Preparation:

We add 20 grammes of Rio san pedro’s sediment in a plastic recipient. With the purpose of getting it viscous, we mix it with some water. Each group add their chosen products (paper, sulfate…), and we keep mixing and adding water until necessary. We achieve a thick mass that we can get into the tube with the help of a funnel. Then we hit it softly so the sediment will go down on the tube, and the trapped bubbles go away. We mark the tube to indicate the level of the preparation, so we know where the nutrients are.

We prepare another mix with 20 grammes of sediment, 100 grammes of sand and some water, and we put it into the tube. On the top of the column, we add some more water (2 cm).

Hypothesis

We think that , in the column with only filter paper, are going to grow the chemoorganoheterotroph organisms, and, in the other hand, in the column with sulfate, are going to grow the chemolithoheterotroph organisms.

UCA_8B,7B_Day 8

Winnogradsky column - Day 8

Group B8(Laura Lucena Del Amo, Noelia Moares Fernández)
GroupB7(Pablo Lucas Ezequiel Garin Ortega, Alba Mejías Gallardo)

We are back one week later to discuss about the changes that we have observed in both columns, the column exposed to sunlight (B8) and the column kept in darkness in which we added glucose (B7).

In the column exposed to sunlight (B8), which had Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4, we find these characteristics:

• The column is darker in the bottom, with black sediments. This probably is caused by the FeS precipitate, produced by anaerobic microorganisms from Fe (provided by the Rio San Pedro sediment) and SO4 (from the CaSO4). 
• The top of the tube is much lighter than the bottom, due to the proliferation of photoautotroph microorganisms. These organisms produce O2, which helps keeping a gradient of oxygen.
• Because of this gradient of oxygen aerobic microorganisms will grow close to the top of the column and anaerobic microorganisms will grow in the bottom.
• There are a few of little bubbles in the column, they may be bubbles of N2 (from the sediment) as well as bubbles of H2S (mostly in the bottom).
• The main difference observed this week is that we can perceive two different sides of the tube in the middle:

1. One side lighter ( with an orange hue), this is the side exposed to sunlight. The explanation of this phenomenon is based in the fact that there might have proliferated photoorganoheterotrophic bacteria in the medium.
2. The other side is darker, the same that we observed last week. There might be chemoorganoheterotroph microorganisms.

While in the B7 tube, kept in darkness (absence of light) and in which we added Rio San Pedro sediment + 0.3 g paper + 0.1 g CaSO4 + glucose, we find these changes:

• This week's column is way darker than the last one due to the breathing of the H₂S reacting with Fe²⁺ and obtaining HFe, which gives the black tone. This reaction is produced by anaerobic microorganisms. The Fe²⁺ comes from the sediment of San Pedro river, and the H₂S is caused by the fermentation of present sugar in the column.
• Many small bubbles appear, we think that these bubbles are formed by CO2 and CH4 probably due to the fermentation of the chemo-heterotrophs microorganisms.
• The main difference observed this week is:

1. There aren´t accumulation of fumes and it has been generated a layer of dirty water on the top. In this water seems to have been suspended micro-organisms, although it may also be sediment of San Pedro river.

UCA_GRUPOA1_ENTRADA1

In the A2 group's winogradsky column we added agar, yeast extract and skimmed milk powder. All this apart from the basic components: mud, sand, celullose and CaCO4.
We put this column to light. We have to see the differences between this column and the A1 group's column which have different components and is put to light too.

About column A2 we expect the appereance of  microorganisms able to demean lactose.

UCA_8B,7B_Day 7

Winogradsky column – Day 7

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

One week after the preparation of the Winogradsky column, both groups have observed significant changes in our columns.

The changes of the group B7 (column in darkness) are described with details below:

• We observed a huge amount of black sediments spread all over tube. This is caused by the reaction of H2S with Fe²⁺ resulting in HFe that presents a black color. This reaction is produced by anaerobic microorganisms. The Fe²⁺ comes from the sediment of San Pedro river, and the H2S is caused by the fermentation of present sugar in the column.
• Many small bubbles appear, but there is a big bubble of gas that has separated in two the column. We think that these bubbles are formed by CO2 and CH4 probably due to the fermentation of the chemo-heterotrophs microorganisms.
• we think that in the zones with lighter colors, the microorganisms chemo-heterotrophs haven't proliferated.

After the observation of the tube, we have provoked the exit of the gas bubble to prevent the spillage.

Now, we will explain closely the changes observed in the tube exposed to sunlight of group B8:

• In the bottom of the column, we can observe an accumulation of black sediments. This indicates the proliferation of microorganisms able to breathe SO4^- and make the reactions commented previously in this zone.
• Also, we see bubbles in the middle of the colums. This gas may be N2 or H2S. We think that a part from these bubbles are formed by the sugar fermentation of micoorganisms chemoheterotrophs.
• In the top of the column, we see a fine laver of lighter color. It can be provoked by photoautrophic microorganisms (anaerobic). They provide oxygen to the rest of the tube, creating a gradient of oxygen. Due to this fact, we will find aerobic organisms close to the top and anaerobic microoorganisms in the bottom.

Finally, we return the columns to their previous places and we'll wait for new changes in next week.

Winogadsky column. Day 7. UCA B1 B2

Hello everyone, we´re back a week later to check the progress of  our columns.

As we can see in the image, there are significant differences between both columns:
    - The column that had the teaspoon of salt didsfavoured the growth of microorganisms, maybe because of the osmosis.
    - In the column that hadn´t the salt we could appreciate a larger dark area at the bottom which is caused by microorganisms so we could say that they grew better in this column.


As we mentioned in the last blog  entry, both of our columns were exposed to sunlight so that it wasn´t a variable. It was done in this way so we could see how the salt affects the growth of microorganisms. 

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