Winogradsky Column. A month later (Group B2)

It's been  a month since we prepared our column. We can see an increasement of the dark zone (as expected) and the horrible smell persists. No other major changes can be appreciated compared to the previous entry.

In the end we haven't been able to compare different kinds of microorganisms, only the bacteria reponsible for the iron reduction and the black color.

                                                                                                                       Inés, Daniel y Andrea

Winogradsky Column. Two weeks later (Group B2)

A week after we prepared our column no major changes had occured so we will talk about the ones that happened over the next week.
The Winogradsky column has grown as  expected. We can notice that the amount of mud has decreased while the FeS amount, which is black colored, has increased. There's still some mud to be used as substrate on the top. However,  we can observe a methane chamber made by the enormous amount of organic material added. As this chamber can be dangerous we shake the column in order to remove it.

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Also we could appreciate a very hideous smell caused by the sulfhydric acid. We needed to pierce the column too so it wouldn't explode.
Finally, we expect the black zone to increase in size consuming more organic material.

Inés, Daniel y Andrea

Winogradsky Column. First week (Group B2)

A Winogradsky Column is a microecosystem which  allows us to study all the possible combinations of microorganisms  that can grow in several types of environments.

To prepare it, first we mix 30 g of mud with the next ingredients to provide substrates to all the microorganisms so they can develop properly:

- Yeast extract: 3,03 g
- Iron sulphate: 2,84 g
- Calcium carbonate: 5,10 g
- Sugar: 1,05 g
- Sodium chloride: 3,4 g
- Cellulose: 0,5 g

Then we add 30 g of sand combined with 10 g of mud, with some water on top, and we cover the column.
As we used a lot of organic material, we expect the column to turn into a dark color due to the  big amount of iron sulphate added. We also think the oxygen will be consumed quickly, leading to a better development of anaerobic microorganisms.

                                                                                                                Inés, Daniel y Andrea

UCA,_B4_1: First Blog Post

Nutrient data:

·         Mud: 30g

·         Cellulose: 0’5g

·         Sugar (glucose): 0’5g

·         Iron (II) sulphate (FeSO₄): 0’1g

·         Agar: 0’11g

·         Calcium carbonate: 0’5g

·         Sodium chloride: 0’54g

·         Water

In this research, we pretended to observe how well microorganisms evolved and how were they able to develop in an environment rich in some nutrients, such as glucose, cellulose and other metabolites, and how did they compete with other bacteria of the surroundings to reach those substances. Microorganism have been able, since the beginning of live on earth, to intelligently survive in a poor-oxygen-atmosphere earth, in which O₂ concentrations were very poor in contrast to carbon dioxide concentration. Some other inorganic molecules existed in that atmosphere, such as methane and nitrogen. In addition to this, high temperatures and radiation index indicated a very different atmosphere to the present-day atmosphere, so only some microorganisms could use those limited resources to correctly develop its metabolism. It is unbelievable how did those primitive bacteria and unicellular algae managed to survive in those conditions and, thereby, guaranteed the perpetuation of that cellular lineage throughout times. Therefore, we can make grow, practically, any kind of microorganism as long as we provide them their requirements. In the end, this is the objective of our investigation: study how can microorganisms develop in a limited space with limited food and oxygen.

Winogradsky column provided us a way to study how could the mud bacteria develop in an environment with nutrient concentration very different to natural mud: we added approximately 60 grams of silt in a column closed in one of his extremes, and opened to the atmosphere on the other. In normal conditions, we cannot distinguish any microorganism layer in mud, as it viscosity keeps it homogenous. However, in a Winogradsky column it is immobile, and its bottom has a poor oxygen concentration, so we can study several kinds of metabolism (aerobic and anaerobic). In addition, we added a few number of metabolites to see if some bacteria of the mud could break them down. Those metabolites are the following:

·         Sodium Chloride: Some bacteria can break down NaCl into its individual ions (chloride and sodium). This kind of bacteria is known as halobacteria (also called halobacteriacea), and can be found in waters with high salt concentration. It is included in archaea domain, rather than bacteria. Is also included in halophile community, as they need the presence of salt to grow and develop.

·         Cellulose:  Cellulose demotion is carried out by cellulolytic microorganisms. There are a lot of microorganisms capable of metabolizing cellulose, so we can’t just say that they are all aerobic or anaerobic. However, we’ll clasify them in groups and name a few examples. Anaerobic bacterias, for example, are the bacteroides like the bacteroides cellulolyticus, the acetivibrio cellulolyticus, clostridium thermocellum and ruminococcus like the ruminococcus flavefaciens. Anaerobic fungi are the piromonas communis and the sphaeromonas communis. Aerobic bacteria are genera like the cellulomonas or the cellvibrio. And anaerobic fungi are, for example, Trichoderma like the viride, reesei and koningii, penicillium pinophilum, and fusarium solani.

·         Glucose: Almost all organisms are capable of metabolizing glucose, so many that glycolysis is considered an almost universal process. We can’t identify the metabolism of a microorganism based only in the fact that they can metabolize glucose.

·         Iron sulphur: FeSO₄ is used by sulfate-reducing bacteria in anaerobic respiration, reducing the SO₄^2- to H₂S, which can often react with metal ions to produce metal sulfides like FeS, which are insoluble and dark, like brown or black. Some orders that include mostly sulfate-reducing bacteria are the desulfobacterales and the desulfovibrionales. Some genera that include only sulfate-reducing bacteria are the desulfotomaculum and the desulfosporosinus.

·         Agar: Despite agar is universally used as a farming environment, as it provides consistency and food to bacteria, it can be broken down by a special kind of microorganism: agarolytic bacteria. They live in seas and oceans, and can feed with algae agar. We pretended to study if some bacteria could demote agar while we offered consistency to the mud. Cytophaga psychrophila is an agarolytic bacteria.

·         Calcium carbonate: We added CaCO₃ to provide a carbon source. Its breakdown produces carbon dioxide (CO₂) and lime (CaO), a white substance.

A month later

In our Winogradsky's column we can differentiate three zones:

A higher zone of green colour where cyanobacterias and algae that liberate oxygen to environment are situated keeping this zone aerobic.

The second zone is of purple colour, we can find sulphate reducing bacterias, this bacterias give purple colour to this zone. Sulphides are liberated by bacterias to oxygenated highes zone creating a gradient where photosynthetic bacterias that use sulphur are developed.

Finally in the lower zone, what is of black colour there are micro-organisms that make anaerobic breathing or they make fermentatives process producing alcohol and fatty acids like subproducts of their metabolism. This products are substrate to growth of sulphate reducing bacterias.

In conclusion we noticed that a gradient of oxygen and an interdependence between zones of column with different micro-organism is established like we thought.

In the Winogradsky's column put in the dark, no micro-organism have been developed since they need light as energy.

The third week:

One month later:

First week, group 1B

We are going to use bacteria from the pond mud to create and study a mini ecosystem called a Winogradsky column.  It is based on the idea of ecological balance what means cooperation between different types of bacteria. Residues produced by ones constitute substrate for others. First of all we mix the mud with several ingredients to provide food and other substrates for the bacteria to use. These ingredients are:

• ·         30 g of mud.
• ·         3’36 g of salt – to develop halophilic bacteria.
• ·         Shredded paper – to provide Carbon source on the form of cellulose.
• ·         0’99g of gypsum (Calcium sulphate – to provide a source of sulphur - and Calcium carbonate – as another source of carbon).
• ·         0’17 g of Sodium bicarbonate - it will provide Carbon too.
• ·         Pond water

Then, we add the mix to the test tube (1/3 of the tube), we add a mix of sediment and mud (2/3) and we seal it. We place the column next to a window for several weeks.

We expect it to develop halophilic bacteria, some anaerobic bacteria and some aerobic bacteria. Some of them should be able to break down the cellulose in order to give glucose.

Finally, we will post more blog entries to analyse what happens in our Winogradsky column week after week.

FINAL SCORE

As we can observed, the areas are more  distinct  than the previous time we saw the column As noted, at the bottom we can still notice a dark area due to anaerobic microorganisms and at the top we can also notice a green area due to aerobic microorganisms.The black coloured area at the bottom of the column is due to the presence of  iron sulfide which degrades organic matter thus we can notice that black colour . In addition, a small pick coloured area can be observed in the middle region of the column and that is due to a substance produced by purple sulfur bacteria which are responsible for that pink colour.

A MONTH LATER

Over time, different colours can be noticed in our column . As expected we can  distinguish a darker area  at the bottom of our column and that is due to  the content of substances such as sulfates ( sodium and calcium sulfates) and organic matter(Carbon source) we added at the beginning of the process where only  anaerobic microorganisms can be found. On the contrary, at the top of our column there is a green coloured area that shows the presence of  photosynthetic microorganisms.

Winogradsky Column Evolution (Group A)

Winogradsky Column After three weeks (Group A)

The column that has been in contact with sunlight has created two levels inside the column. The upper level has been pushed and raised to the top because of the gases that the microorganisms of the lower level have caused. The upper level has a clearer color and contrasts with the other level. The color is grayish brown, probably due to the death of microorganisms at this level. On the other hand, a layer of gases has been created, as we have already said. This layer with the small amount of water in the upper level causes oxygen to pass to the lower level. For this reason, microorganisms that function without oxygen can not be developed.

We have lowered the upper level and removed the gas layer, we also introduced some water.

We see in the inferior level diverse colors, purple, brown, orange and the characteristic black that has been intensified enormously.

Let's talk about the column that has been kept in the dark. No major changes are seen in the column. The colors have been maintained. The column has been compacted a little by the top, the cracks have disappeared. The same amount of water is maintained at the top and from time to time there are bubbles there are bubbles that rise from the end of the column, gases generated by microorganisms.

Winogradsky Column After one week (Group A)

In the picture we can not see to many differences between both columns.

We think that the black color in both columns are from the combination of iron and sulfur.

On the other hand, we think that they look similar because the column that is in the sunlight doesn’t need it as energy source. 

 

The left picture is the column in darkness and the right picture is the column exposed to the light.

Winogradsky Column First Week (Group A)

Winogradsky column is a mechanism that allows the crop of microorganisms. The objetive is to be able to differenciate some communities of microorganisms. For that, we are going to preparate two columns, one of them will be in the darkness and the other one will be in the sunlight.

Our columns have 20.15 g of mud, some of CaCO3 (this is for enrich living organism autotrophs), CaSO4 (this produces energy), glucose (it is a food and energy source), and agar (this can resists to some microorganisms, that’s why not every microorganisms are going to use it). Finally, we are going to put some salt water, mix it and distribute our mixture into two columns.

Then we are going to add more salt water and we will put one of them in the darkness and the other one in the sunlight. 

Final post: ''Results of a Winogradsky column''. Group A4.

In this post we are going to discuss the evolution of the column in the fifth (the last) week,
The surface is more green compairing with the fourth week, due to the growth of microalgae and cianobacteria (aerobic photosinthetic). Furthermore in this part of the column there are more bubbles because there is more oxygen than the previous week.
In the middle of the column the pink is more intense because the anaerobic photosintetic bacteria have been producing more sulphure. For that reason when we hit the column in the table the smell is worse than the last week.
The part of the column that is reached by the sunlight has a brown colour while the otherside is black, due to the presence of heterotrophic bacteria which oxidate iron, in the brown part.
To sum up, comparing with the prediction in the first post we can conclude thet,
1. Halophilic bacteria haven't appeared apparently.
2.Cianobacteria (autotrophic bacteria) have grown in the surface of our column, as we expected.
3.Heterotrophic microorganisms have appeared too (they oxidate iron).
4.Microorganisms which can assimilate the sulfur have appeared.
        

UCA_C2_2 - The process of our column

 A week after developing our Winogradsky column, we can start seeing important and different changes. First of all, fermenting cellulose bacterias have grown (corresponding to the black colour of the background). These bacterias might be the Clostridium genre. We can also see some grey parts due to sulphate reducting bacterias.
In the aerobic area, the top of the column, seaweeds may have grown so that they could produce oxygen after doing photosynthesis with the light the column receive everyday (the column is next to a window). This fact could be an explication to the bubbles we see at the top.
In the middle of the column there is no remarkable change. Here you have some photos to see all these changes and to follow better the evolution of our column.

Irene, Raúl and Nieves

UCA_6B_1: Day 1 - OUR LITTLE TOWN OF MICROORGANISMS

UCA_6B_1: Day 1 -  OUR LITTLE TOWN OF MICROORGANISMS

Hi! We are Sara and Kenia.

We are biotechnology students at Cadiz Science university. We are going to explain how our Winogradsky column develops. Our goal is to create two gradients (one of them is H2S and the another one is of O2) where different microorganisms will grow.

We have added 30,1 g of mud from Rio San Pedro to our column and we have mixed it with 0,1 g of CaSO4. This mix is TO crEate a gradient of SO4.

After that, we have added 0,1 g of NaHCO3 thaT produces CO2 : if the microorganisms are autotroph, they will make their own nutrients from inorganic carbon.

We have also added 0,1 g of cellulose so these microorganisms can feed themselves, because cellulose is a polymer of glucose .

 we have put all of them in a cristal tube.

Finally, we have put our column near the window, so the microorganisms can catch the sunlight and take advantage of it.

We will see the evolution of the column during a few months, so we will show you photos of our winogradsky column along the time.

UCA_1C_Week 6

                                                         SECOND POST


Hello! It´s been six weeks since we prepared our column and now it´s time to talk about the changes we have seen. Here we have some photos.

As we can see, at the bottom of the column it has appeared a black colour joined with a pink colour. We suppose that in this area there is not oxygen, so we can find microorganisms which are able to make fermentative processes.


The waste of these microorganims is used by bacteria that reduce the sulphate. The result of this process is the liberation of sulphides. It goes to the top of the column which is oxygenated by cyanobacteria and algae.

Also, there are photosynthetic bacteria that use the sulphur we can find in the column due to the sulphide gradient.
                                                                                          Victoria, Andrea, Alessia.

UCA_E7-8_2: 6th week.

WINOGRADSKY COLUMN (6th week)

Rio San Pedro sediment + 0,3 g NaCl + 0,3 K3PO4 + 0,4 g sugar + 0,4 g CaSO4

Several weeks after the Winogradsky column was made, many appreciable changes were observed in each column. 

In this image, you can see the different areas of each column after about six weeks of its completion.

In the column on the left, which was exposed to sunlight, there are four areas where different microorganisms have intervened, whereas in the right column, which was devoid of light, has three distinct areas. These changes are due to the action of different bacteria in each area of the column.

Column exposed to light.

This column is characterized by the presence of light, so the bacteria are photosynthetic.

The lower zone is characterized by being anaerobic and fermenting, but without oxygen. This fermentation transforms the organic matter into sulfur as a gas (H2S normally). This gas decreases as we move through the column to higher areas. In this zone, the bacteria transform the sulfur present in CaSO2 and transform it into H2S. This area usually presents a black color and occupies much of the column.

As we rise in the column, the hydrogen sulphide from the bottom is consumed by anaerobic bacteria that reduces the present sulfur. This area has a black color and also occupies a large area of the column.

The brown area has the presence of sulfur and oxygen. This zone is aerobic because diverse bacteria oxidize the sulfur coming from inferior zones. Sulphates appear in this zone.

At the top of the column is the aerobic zone, where there is a higher concentration of oxygen. For this reason, bacteria that inhabits aquatic environments appear. This area is rich in organic matter and is the smallest area of the column.

Column not exposed to light.

This column presents bacteria that do not use sunlight for their evolution, that is, they are chemosynthetic. In the areas of the column there are many similarities to those mentioned above in the previous column.

The lower zone is also similar, where the organic matter is transformed by the bacteria into sulfur, obtaining a great amount of that element.

Next is the largest area of this column, coloured black, where bacteria that uses all the sulfur from the bottom appears. Consequently, there is no intermediate zone in which the sulfur is oxygenated. This area is anaerobic.

The top is aerobic and it is where bacteria transform oxygen. This area is very small compared to the other two.

Moving forward in time, more changes will be seen in the column, as well as bacteria continuing to grow.

Ana García Ramos, Carlota Borne Bernal, Ernesto Segundo Mendoza, Maria del Carmen Espinosa Corona and Pablo Carrasco Ercilla.

UCA_E7-8_1

WINOGRADSKY COLUMN (first day)

Rio San Pedro sediment + 0,3 g NaCl + 0,3 K3PO4 + 0,4 g sugar + 0,4 g CaSO4

The purpose of this practice is to observe the color change and the increase of microorganisms over time in a medium created by ourselves with the reactives elements mentioned above.

We introduce the same mixture of the same quantity into two different tubes. One of them will be exposed to light and the other will not.

Then, we will explain why we have added each of the aforementioned compounds:

• The sucrose is added to bring carbon to the medium as every living thing needs a source from which it can obtain it.
• CaSO4 provides sulfur to the medium. It will allow energy-capturing bacteria to grow through the reduction of this.
• With the addition of K3PO4 we are contributing with phosphorus to the medium, which is used by microorganisms for their metabolism and it is an essential macronutrient for the growth and development of living beings.
• We added NaCl to have a more saline medium than we had originally, and thus favor the growth of halophilic bacteria.

          

In this image we can observe the state of our tubes immediately after the deposition of the mixture in them and therefore they present a homogeneous and compact color throughout the column.

The tube that is in contact with the sun has a different development than the one that is not.

Over time, we will see different colors and changes in our column due to the growth of microorganisms. These variations can be seen because each bacteria will grow under the conditions that are most favorable to them along the column.

Ana García Ramos, Carlota Borne Bernal, Ernesto Segundo Mendoza, Maria del Carmen Espinosa Corona and Pablo Carrasco Ercilla

Second post: The evolution of our Winogradsky column. Group A4.

In this post we will discuss the progress of our column during the last four weeks.

First week:
Four parts are distinguished:
At the bottom of the tube we can see bubbles which expands to the Surface . It seems like a volcano.
Due to the presence of an acumulation of organic material, the microorganisms make methanogenesis, althought this is one of the less efficient source of energy. This process produce the grey colour .
Above the grey colour, we can observe a black area where oxigen can´t access ,so the microorganisms who live there are anaerobic and instead of using this molecule they take the sulfate and release  sulfuric acid and carbon dioxide. The sulfuric acid reacts with iron producing iron sulfate, which is a black precipitate.
In the third zone, the orange and black colours are mixed . This part can be reached by oxigen , so microorganisms are aerobic and organoheterothophs. The orange colour is a consequence of the photosinthetic pigments.
In the last part, at the top of the column, there is some orange water without any algae but in the surface there is an oily liquid due to the bacteria.

                      
Second week:
This time, we can distinguish three parts:
The bottom of the tube has stayed grey, due to the methanogenesis, as the last week.
The orange liquid of the surface has not change neither.
The principal changes have occured in the black zone of the tube (at the middle). It has expanded, because of the acumulation of organic material.
At the surface of the  mud, a green colour has appeared which indicates presence of photosinthetic microalgae.
                                         
Third week:
The black zone of the bottom of the tube has not changed. However, in the middle of the tube a light pink colour has appeared, due to the presence of a special photosintetic bacteria. It assimilates sulphur and produce sulfate instead of oxigen.
At the surface, the orange colour is mixed with green and brown. As we said, the green colour has appeared because of the growth of photosinthetic microalgae.

                   

Fourth week:
The major change is that the pink colour has become more intense and dark, because the bacteria has increased the concentration of sulfate.
At the same time, it has appeared bubbles at the surface because of the presence of oxigen produced by photosinthetic microalgae and cianobacteria.
The gas produced in the methanogenesis has created some breaks which we eliminated by hitting the table with the column.
The column was warm due to the biological activity and the solar heat.
In general, we have noticed a disgusting smell every week because of the excess of sulphur.

                    

Winogradsky Column Day 0. Group A5

We added into the column:

- Pond mud: 24.7 g.
- Paper (5 pieces): 0.5 g aprox.
- Yeast: 0.4 g.
- Calcium carbonate: 0.3 g.
- Sea water.

The mud will be the medium of the column where the microorganism will grow. Each component will apport some nutrient for each kind of microorganism.

The paper will be used as the source of glucose by the chemotrophic microorganism but, at the same time, it will only permit to grow the microorganism that can digest the cellulose. The yeast offers a lot of nutrients due to its amount of amino acids. Litotrophic microorganism will grow using the calcium carbonate as the carbon source. It is a saline medium because of the sea water so only will grow microorganism that resist those conditions.

At the end, there will appear different layers. Each colour that appears in the column will mean the growth of different microorganism. At the bottom of the column, will grow anaerobic or facultative anaerobic microorganism. Also, the sun light will be used by autotrophic microorgnanism that synthesize glucose by photosynthesis (cianobacteria).

WINOGRADSKY COLUMN DAY 0: HYPOTHESIS (GROUP 6C)

Pond mud→ 20,81g

Yeast extract→ 0,1g

Sodium sulfate→ 0,4g

Calcium sulfate → 0,1g

Pieces of paper→ 0,1g

For our Winogradsky column we use pond mud as a culture medium. The yeast extract can  be used for microorganisms as a nutrient source because  it is high in  B vitamin, and it contains amino acids and other factors  that are ideal for their growth.In addition,  we have used sodium sulfate and calcium sulfate as a source of sulfur that will be used for the anaerobic microorganisms that grow at the bottom of our column in order to being able to accomplish their breathing process.Finally,we have added little pieces of paper that can be  used as a carbon source (cellulose) so the organisms can obtain  energy from the synthesis of glucose which is  required for their biological processes.

As a result of all this, we predict that there will be able to notice  a few layers.We will be able to find aerobic microorganisms as there is enough oxygen for them to use. Facultative anaerobic organisms will be found in the layers that are in between, and anaerobic organisms will be found in the deepest layer of our column.

Winoblog, Third post, group B7.

          This is the third post which we make about our “Winogradsky column”.

       By this time, the column has changed a lot. For example, microalgae have appeared in our column. To be more accurate, they have appeared in a water’s zone with oxygen.

        

           On the one hand, some gas bubbles have appeared in our column. That bubbles 
as a sign of metabolic activitiy. Cianobacteria produces oxygen using another componets of our “widnogradsky column”.

        On the other hand, stratification can be better appreciated. For example, under the water, there are a large area of ​​greenish gray. This is because microorganisms are growing up there, more specifically Beggiatoa is growing there.

         To sum up, we can se how the bio-system that we have created is becoming stable. Besides, we can appreciate differents strata that a couple of days before there werent there. 

Figure 1: Microalgae.

Figure 2: Photo taken 17th April.

UCA_3A_2: Day 48 - Our column in process.

In the first week, we can see a black colour at the bottom of the column. This is a result of sulphydric production. Besides, there is water in the top of the column, where we can find an orangey brown colour (this is the zone provided with oxygen).  Moreover, consequently to have added a lot of mud and just a bit of sand, there is a part in the middle of the column that is completely grey and homogenous, where gases can’t pass through it. Due to this point, we can guess that there will be two separated communities.

In the second week, we observe that the homogenous part has decreased. Besides, we see a black colour in the top of the mud (there are bacteria that feed of sulphate and produce sulphydric acid). In another hand, the brown community are bacteria that are able to use oxygen.

After two weeks, we focus on our column again. As a result of have added a lot of mud, the development of the microorganisms  is very slow. The continuous black colour indicate us that bacteria keep producing H₂S. Now, there is an orange colour that is a signal of the existence of photoheterotrophs microorganisms producers of iron.