Winogradsky Columns Fall 2018: Intro Post

Introduction

Most of the diversity of life on our planet comes from microbes. They can thrive in many environments throughout the biosphere and have been around for billions of years. During the 1880s, Sergei Winogradsky developed a method for culturing a large diversity of microorganisms. He did this by placing pond mud and water into a column with a carbon and sulfur source to create an observable environment.
These contraptions, called "Winogradsky Columns," allow for the creation of microbial mats, which are multi-layered sheets of microorganisms composed mostly of bacteria and archaea. We used this method to test the effects of different salinities on the growth of these microbial mats and the diversity of the microorganisms found within them. Our sample was taken from a fresh water source, therefore it is unlikely that there will be many halotolerant bacteria. With that in mind, we predict that as the salinity increases, the diversity of microbial growth will decrease.

Background

Winogradsky Columns are representations of the natural phenomenon of the development of microbial mats. Communities of microbes allowed to grow without predation tend to stratify based on metabolic and tolerance needs. Communities usually involve organisms that require access to oxygen and light and those with resistance to ultraviolet radiation growing in zones closer to the surface with exposure to those factors, while communities buried lower down have access to byproducts from the decay of organic material and thrive in the euxinic atmosphere.
By combining natural sediment and water in a glass jar and allowing it to incubate for an extended time, a Winogradsky Column is expected to stratify independent of further laboratory manipulation. Here, we gathered samples of sediment and pond water from the WSU duck pond and built columns containing increasing concentrations of salt (NaCl) in an effort to understand how the zonation of the Winogradsky Column would be influenced as the microbial communities from the duck pond are cultivated in the varying ionic concentrations and osmotic conditions.

Hypothesis + Experimental Design

We hypothesized that adding salt to our Winogradsky columns would decrease the amount of diversity present in the columns, and that this would be visible in the number of layers each column formed.
In order to test this hypothesis, we set up our experiment so that each column has a different amount of added salt in the water. The concentrations we decided to test were 0%, 4%, 8%, and 12% salt. Other than the salt concentration, the columns were as near to identical as we could make them. The soil and water were gathered from the same location in the Weber duck pond and homogenized before being added to the columns, and the same amount was added to each column (soil up to 12.6 cm, water up to 18.6 cm). In addition, we added a whole egg to the bottom of each column and mashed them thoroughly with spoons to provide additional carbonate and sulfur to the column. Finally, we wrapped the outside of the column with garbage bags to block light from the sides and promote the formation of clear layers, and placed the columns in the window sill of the lab to incubate.

Variables & Controls

• The variable in our experimental design is based on changing the salinity of the water in the columns. This is done to view the effects of salt concentration on microbial mat growth.
• Our control column was set up using water from the same source that did not have any salt added. We did this to observe the natural growth of the microbial mats.

Field Sampling & Measurements

Field Protocol

The field plan included collecting soil and water samples from the campus to construct the columns. The samples were taken from the Weber State University duck pond and surrounding landscape.The water sample was collected by dipping a small bucket into the North East section of the pond; an area near the fountain that is fairly free of algae growth and debris. A soil sample was taken from the nearby landscaped island to the south using a spade by turning the grass aside and gathering fresh soil. A small bucket was used for transport of the soil sample. The water was tested for salinity content using a refractometer. A clean dropper was used to dispense two drops of the pond water onto the refractometer. The temperatures of the water and soil were measured using a laser temperature gun. Ambient temperature was also measured with the laser temperature gun and verified using web based weather data for Ogden, Utah.

Field Measurements

Air Temp: 31.0 °C
Water Temp: 20.8 °C
Weather: Sunny and warm, not very windy. Very few clouds in the sky.
Location: Weber State Duck Pond; Latitude: 41.1927 Longitude: -111.9464
Time: 2:48 PM
Date: September 7th, 2018
Salinity of pond: 0%

Figure 1: Weber State University Duck Pond, the source of the water for the Winogradsky Column experiments.

Procedure for the Winogradsky Columns

1. Place 1 chicken egg in each of the four glass columns and smash egg up with a cooking spoon.
2. Measure 12.6 cm from the bottom (about 80% of 18.6 cm column) of each glass column and mark with a permanent marker.
3. With another cooking spoon, scoop soil from environmental sample and fill each column to previously marked line, 12.6 cm. Pull out any large grass and roots when possible and compact the dirt as you go.
4. In 1000 ml graduated cylinder measure out 340 mls of pond water.
5. Add the 340 mls of pond water to first column and label as your control.
6. Using three separate 1000 ml graduated cylinders, measure out 500 mls of pond water for each of the remaining three columns.
7. In the first 500 ml pond water sample add 20 grams of NaCl to make a 4% salt concentration solution. Stir solution until salt is completely dissolved.
8. In the second 500 ml pond water sample add 40 grams of NaCl to make an 8% salt concentration solution. Stir solution until salt is completely dissolved.
9. In the third 500 ml pond water sample add 60 grams of NaCl to make a 12% salt concentration solution. Stir solution until salt is completely dissolved.
10. For each separate salt concentration, measure 340 mls of solution and pour into one of the three remaining columns. Label columns according to salt concentration.
11. Use spoon to mix water, soil and egg.
12. Cover top of each column with clear plastic wrap
13. Put all four columns in a window with labels facing away from the window. 

Challenges

There were a few challenges that we faced as a group, the first being agreeing on a singular idea that we could complete. Tempers were flaring and we were on a verge of an all out brawl. Fortunately, Dr. Frantz was there to calm us down. Once we came up with a plan, we created a list of things to do to execute that plan. First we needed to get the soil and the pond water from the designated area. This is where we ran into our next problem, the heat. It was the middle of September and the lab required us to wear long pants and we need to trek from the Science building all the way to the front of the campus, so that was an inconvenience. The last problem we didn't foresee was the weight of the bucket we used to collect samples. The samples were so heavy that arms were being pulled from their sockets and sweat drenched the carriers. The only solace was that elevators and air-conditioning awaited us on the way back to the lab. Tips for future classes performing this experiment; keep it simple, you're not vaccinating the common cold here, be mindful of the weather outside, and lastly, bring a wagon.

WSU Geomicro 2017, where geo and micro nerds come together for the common good.

Winogradsky Columns... What are we testing? The combination of dirt, carbon, sulfur, and water. Oh, and some unintentional duck feces and twigs and such, and maybe some parts of construction workers made it in there too. So again, what are we testing with all this goodness? What microbes are going to grow and how growth differs based on the variables. Exciting ay? We think so.

What are the variables? Well, a handful of things. It used to be more than a handful of things, but then in the middle of the experiment it became apparent a few of the variables were not logical. So, here we are, with only a handful of variables and the oh-so-typical and necessary “controls.”

Controls, Variables, and Procedure

Conditions of mud sample site:

Air temperature: 30.5 C, 20% humidity, sunny, light breeze

Water temperature: 27 C, calm

DPM temperature: 30.9 C, full of sticks and leaves and fowl excrement

DP Water Retrieval

We used a blue bucket to scoop up duck pond water and poured it into a three-gallon water container and returned it to the lab.

DPM Retrieval

We began on the north side of the pond, Serena overcame her germaphobia to scrape the mud from the bottom of the pond for its sediment only to discover the pond is actually concrete... Pretty big kink in the plan, right? But no worries, eventually we found this plot of mud surrounded by ducks and geese who started coming at us—assuming we had food. We did not. Once they realized this they quickly deserted. So, with kitchen spoons, we dug up this mud which smelled of foul avian feces. We collected the mud in a blue bucket and carefully carried it back to the lab.

Mix Sediment Control

Procedure: An egg was smashed into a column. Equal amounts of DPM and CD were added to the 30% line, and the soils and egg were mixed together in a slurry. More soil was added to the 80% line. Then water from the Weber State duck pond was added to the 90% line. The column was covered with saran wrap secured with a rubber band, and the column was placed in the west-facing window of Tracy Hall lab 462.

Mix Darkness

Procedure: The column was set up in the same way as the mix control. The column’s sides were then covered in a black garbage sack and taped and rubber banded in place. The top of the column was left open to the sunlight from the west-facing window of Tracy Hall lab 462.

The Column of Greatest Importance

Now that we have the basic rundown of this entire Winogradsky experiment, let’s narrow this down to which column we (Emily and Serena—the authors of this wicked good blog post) are going to be observing over the course of the semester.

We chose to observe and track the progress of growth on the “Mix Darkness” column, compared to its control “Mix Sediment.”

Week One:

1           2 3 4

These pictures were taken at about 1:00 pm, Thursday, September 14, 2017. The garbage sack around the dark column was carefully removed and pictures 1 (side facing the window) and 2 (side not facing the window) were taken. The control was merely turned to get shots of the sunlit side (4) and the side that does not face the window (3).

We hypothesize that the garbage sack will affect growth due to the lack of sunlight as well as the insulation. The bag will make the column temperature slightly warmer as well as dark. The diversity of growth, we believe, will consist of microbes that hate UV light and microbes which are facultative when it comes to UV light, resulting in a greater visible diversity than the control, which is exposed to light all the way around the column.

That's all for this week, folks! Stay tuned!

--Serena and Emily