Using a Winogradsky Column to enrich microbes as they are by simulating various conditions and to predict Microcosm Biofilm Patterns using time lapse tracing and regression analysis

Abstract

Of all the microbes known most are uncultured because of their fastidious nature . Most part of microbial interactions are still unexplored. Herein we use an age old tool, Winogradsky column to enrich, simulate the microbes as they are and predict microcosm biofilm patterns. We have worked on many simulation parameters to better the Winogradsky column in every way. We describe methods to bring the interactions of the microbes in biofilms at a mathematical level. We also have explored the various practical applications possible out of winogradsky column like using it as a universal enrichment medium for all microbes to grow as they are and also to isolate and evolve purpose based microbes for degradation studies, harnessing the redox potential of microbial succession.

Keywords

Winogradsky Column/ Microcosm/ Biofilm/ microbial succession/ simulation/ degradation/ nutrient cycle/ regression

Introduction

The Winogradsky column is a glass or clear plastic column, filled with enriched soil or sedi- ment. When developed, it has an anaerobic lower zone and aerobic upper zone that allow growth of microorganisms under conditions similar to those found in sediments and water rich in nutrients (Sylvia et al., 1998).

Often teachers simply convey the message that different microorganisms exist in different strata of the column and that some live in the aerobic and some in anaerobic zones. However, this is really where the discovery begins rather than ends! Explaining the complexity that lies within the depths of the ecosystem allows deeper insights into the microbial world (Rogan et al., 2005).

Construction and development of Winogradsky column incorporates several variables. With just a few changes, different columns can be created to compare growth rates, microbial populations, and ecological diversity (Rogan et al., 2005)

In each experiment, set up one winogradsky column to serve as a control. This column will be the standard and each of the other columns should vary in only one aspect, such as concentration of a given pesticide from the control column. The results in the column may be obtained by observing the colour changes within the columns. Each change represents the activity of a different group of microorganisms. The sequence of events in the winogradsky column may take as little as six weeks to two months under warm conditions. Under cooler conditions, the reactions will occur more slowly. The succession observed by Kobyashy and Okuda (Hattori 1973) in a column containing soil and water from a rice paddy required two and a half months and occured in this order: 1)green algae (green appeared in about one week); 2) sulphate reducing bacteria (black) were evident at four weeks; 3) photosynthetic purple bacteria (red) appeared five to six weeks later; and, 4) the purple non sulphur bacteria (green) were seen within the next few weeks. The winogradsky column can be a useful tool to demonstrate succession and microbial interdependence with relative ease and simplicity. Also we can follow the chemical reactions and recycling of material within the column to gain an understanding of how they occur in nature (Pigage et al., 1985)

The Winogradsky column was developed and named after Sergei Winogradsky (1856-1953), a Russian microbiologist. He studied the complex interactions between environmental conditions and microbial activities using soil enrichment to isolate pure bacterial cultures (Madigan et al., 2000)

Over one hundred years ago, Sergei Winogradsky studied the microbial organisms inhabiting sulphide-rich black mud ecosystems and pioneered our understanding of chemolithotrophy through his experiments with sulphate and nitrate reducing organisms. Although many of the microbes from black mud environments (which are often used to inoculate so-called "Winogradsky columns") have been studied for decades, the vast majority of the microorganisms present in natural black mud ecosystems remain unknown to microbiologists. While the physiology and biochemistry of microorganisms are best studied in pure cultures, this may prove very tedious in the case of black mud microbes, because the complex community has co-evolved for millions of years. Syntrophy -- the requirement of a microbe to associate with another microbial species for metabolites -- is probably critical to the survival of most of the species within this complex environment (Tanner et al., 2000)

An excellent but perhaps overlooked tool for the study of microbial activity in the soil, nutrient cycling, microbial succession and ecology is the Winogradsky column (Pigage et al., 1985)

What we have done is a time lapse trace of biofilm patterns in the Winogradsky column, taking equivalent weights of each biofilm patterns by cutting the traces of biofilms and weighing them individually and then using regression equations to estimate the biomass with time. Thus over a period of time we were able to give a regression coefficient to each of the coloured pattern in the column and hence give a simulation and prediction tool.

Materials and Methods

Sample Collection

Pond soil and water were collected from the water soil interphase of ponds at Government Botanical Gardens, Ooty. This place was selected because these gardens have been undisturbed natural biosphere (Figure 2).

Procedure of making a Winogradsky column (Anderson et al 1999) the soil sample was cleaned of debris, stones, pebbles, grass clippings, leaves and moving insects. This is used as the control column and standard reference (Figure 3).

1. Fill one fourth of a 250ml glass measuring cylinder with the soil.
2. Mix the soil with 2g of cellulose, 2g of calcium carbonate, and 2g of calcium sulphate.
3. Cover upto three fourth of the column with soil slurry.
4. Let the soil set for five minutes to release trapped air bubbles.
5. Add water leaving a 2cm gap at top.
6. Incubate the column where it will receive daylight or artificial light.
7. Observe the column over the next several weeks for development of layers, smell, colours, and zones.

Simulation of varied environmental factors

By building variations of Winogradsky column one factor at a time- other conditions were kept unchanged.

Variations of nutrients

Column 1 contains carbon source only: 2 g cellulose

Column 2 contains sulphur source only: 2g CaSO4

Column 3 contains carbonate source only: 2g of CaCO3 Column 4 has no added nutrients at all

Variations of pH

pH values were maintained by adding buffer tablets to the water of the column. Column 5 pH3

Column 6 pH5 Column 7 pH7 Column 8 pH9

Variations of light

In a water body, the visible light ranges from red to blue from top to bottom. To simulate the depth (with which the prevalent wavelength changes), the following colour papers were used to cover the

Column.

Column 9 black

Column 10 blue

Column 11 red

Column 12 orange

Variations of temperature

Temperatures possible in lab were used to match the environmental temperature. Column 13 AC temperature of 25 degrees

Column 14 room temperature of around 27 degrees

Column 15 incubation temperature of 37 degrees

Column 16 outdoor temperature of 30 degrees

Variations of salinity

Winogradsky column was watered with the following values of salinity of water. It decides the osmotic pressure.

Column 17: 1% salt concentration

Column 18 : 2.5% salt concentration

Column 19 : 5% salt concentration

Column 20 : 10% salt concentration

Variations of texture

The texture of the soil decides the porosity.

Column 21 red garden soil

Column 22 sponge

Column 23 sand Column 24 clay

Variations of hard substrates

To make degradation possible, the columns were made to contain the hard substrates as a sole source of carbon by starvation.

Column 25 coir

Column 26 dye

Column 27 naphthalein

Column 28 urea

Column 29 paraffin

Column 30 ash

Column 31 chitin

Standard control column

Column 32 contains standard winogradsky column used as control for all the other variations.

Electrochemical gradient potential

In column 33, the electrochemical gradient potential between the top and the bottom of the standard column was monitored by inserting multimeter probes (Anderson, 1999)

Pressure

Pressure increases with depth of water column. A Winogradsky set up was designed (no 34) to create pressure more than the atmospheric pressure. Using one way walves to the outlet and inlet, the pressure inside the closed column was increased by closing the outlet and suppling air through pumping using the pasteur pipette bulb attached to the inlet.

Wind and waves

A Winogradsky tank (no 35) was designed to simulate the wind through a nozzle of air pump and waves by a pedalling motor.

Isolation of Methylotrophs

Purpose: We may use 13C methanol (NMR sensitive) to grow methylotrophs and later use the lysed culture for growing our cells that express protein of interest. Idea was to substitute 13C methanol for glucose Cost effective. Sole source of carbon is methanol (column number 36).

Incubation

Incubation was done at room temperature and under artificial lighting in algal growth chambers for three months. Readings were taken at the end of each month.

Tracking biofilm pattern

The changing biofilm patterns of the Winogradsky columns were kept track by tracing the outlines of the biofilm patterns with colour markers on a rectangular piece of polythene sheet around the column (NASA quest)

Quantitative data of biofilm patterns

The biofilm patterns out on polythene sheets were cut according to the color markers and weighed on a microbalance. Thus the equivalent weights of biofilm patterns were got (NASA quest).

Biofilm pattern prediction formulae

Statistical tool chosen for prediction of past and future patterns of biofilms was done by using regression equations. This equation also fills the gaps in the scattered data and completes the regression curve.

The first step taken towards this is to establish a standard set of values observed over a period of time in a given set of simulation conditions. And then once the biomass values are got over a considerable period of time, then this data can be used to get the other parameters.

The formulae used are

a value denotes biofilm equivalent weight in grams e value denotes time in days

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Results

Effects of environmental factor variation on biofilm pattern

The biofilm patterns obtained in the columns were traced out on polythene sheets resulting in winogradsky map. Each pattern was cut and weighed periodically (Figure 17a, b).

Simulation of microbial succession

Microbial populations grew in succession in the winogradsky column kept as standard (Figure 19) . A white biofilm at the bottom at the bottom followed by black coloration of soil followed by purple and green biofilms appeared in the soil layer with green algal biofilms in the water layer (Figure 1)

Variants of the winogradsky column gave different biofilm patterns indicating that different pond microcosms can be simulated by manipulating the environmental factors. So it is worth designing a grand column where we can manipulate all factors precisely.

All results of column variations were interpreted using table. The change of patterns and the appearance and disappearance of patterns indicates the changes occuring in the microenvironments created by gradients developed according to the factors manipulated.

Nutrients

Even in limiting conditions of nutrients, green, rust, and black biofilm patterns appeared indicating their ability to survive in such conditions. Which may also mean that soil by itself has enough carbon substrates to be degraded anaerobically and start the microbial succession (Figure 4).

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