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

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.

2. 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

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

  

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

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.