BS/LBS159H Spring Semester 2004:

Preparation of a Winogradsky Column

Introduction
Prokaryotes (Bacteria and Archea) collectively encompass a taxonomic and a metabolic diversity that far exceeds that of plants and animals (the so-called higher animals). This week we will set up a model microbial ecosystem (a microcosm) called a Winogradsky column that will simply and elegantly demonstrate a cross section of that diversity. Sergei Winogradsky (1856-1953) who, along with Martinus Willem Beijerinck (18511931), developed the technique, was a pioneering russian microbiologist who worked in the late 1800s and early 1900s, when microbiology was still a fledgling science. At a time when microbiological methods barely existed and most microbiologists focused on the germ-theory of disease, the isolation of individual microbes and the characterization of domesticated laboratory strains, Winogradsky focused on environmental microbiology and developed techniques for cultivating and studying complex microbial communities (such as the Winogradsky column). In so doing, he made great advances in our understanding of environmental microbiology, microbial diversity, microbial physiology, microbial autotrophy (the ability to obtain energy from non-organic substances) and environmental nutrient cycling (particularly sulfur and nitrogen cycling). He is considered one of the founding fathers of microbial ecology. The Winogradsky column is a miniature, self-contained ecosystem that in many ways models ecological conditions found in a typical Michigan lake in late summer. Spring thaw results in a mixing of lake water so that the water is more or less uniform at different depths. As summer progresses gradients begin to form in the water column (light gradient, temperature gradient, nutrient, O2 and H2S concentration gradients). These gradients result in a complex interaction of microbes with their environment and with one another resulting in a series of community successions and, ultimately, stratification of microbial populations in the water column. Similarly, the Winogradsky column begins as a uniform slurry of soil, mud or lake sediment and water (preferably from some outdoor source) supplemented with nutrients (carbon and sulfur). The ingredients are mixed into a uniform slurry, poured into a tall, transparant vessicle such as a test tube, graduated cylinder or soda bottle and exposed to sunlight to provide energy for the system and to enrich for both aerobic and anoxic photosynthetic bacteria. The sediment, soil and water serve as inocula of diverse microbial populations with diverse metabolic processes. A series of gradients (e.g. oxygen, H2S) soon develop in the column as a result of bacterial metabolic processes. For example, aerobic photosynthetic organisms and exposure to the air will produce O2 near the top of the column which can then be used by other microbes for aereobic respiration. Similarly, sulfate reducers in the anoxic region

near the bottom of the column will produce H2S, which can be used by H2S utilizers and anoxic phototrophs. Different organisms soon gain a competative advantage in different regions of the column causing a stratification and continual regional succession of organisms. Because this culture system more closely resembles a natural environment than do traditional culture methods, a wider variety of physiological types can be observed than by the more standard culture methods. Also, different microbes will be concentrated (enriched) in specific regions of the column where local conditions are optimal for their particular growth (e.g areobes at the top in the oxic zone and anaerobes at the bottom, anoxic zone). Winogradsky columns demonstrate the interdependence of diverse microorganisms in complex communities for survival and growth. As such, variations in Winogradsky column ingredients and manipulations are as infinite as your imagination will allow. Virtually any microbial process could be studied with the correct design and initial inocula.
EXAMPLE: IF YOU WERE AN ENVIRONMENTAL ENGINEER INTERESTED IN DISCOVERING MICROORGANISMS TO CLEAN UP A SITE CONTAMINATED WITH (SAY) PCBs, YOU MIGHT ADD PCBs TO THE COLUMN. ORGANISMS IN YOUR SEDIMENT CAPABLE OF DEGRADING PCBS WOULD HAVE A COMPETATIVE ADVANTANGE (YOU WOULD BE ENRICHING FOR PCB DEGRADERS). AS STRATA BEGIN TO FORM, THE COLUMN COULD BE SAMPLED OVER TIME AT DIFFERENT DEPTHS TO TEST FOR PCB DEGRADATION AND TO CHARACTERIZE THE MICROBIAL POPULATIONS ASSOCIATED WITH THAT DEGRADATION.

There is a wealth of information about Winogradsky columns on the web. A quick Google search will help you find them (see #4 of Results, below).

Exercise Objectives
1. To create a microcosm (a model microbial ecosystem) in which complex microbial communities are cultivated. 2. To gain an appreciation for the diversity of methods microorganisms use to gain energy and how those metabolic processes affect the surrounding environment and, thus, determine microbial community composition and succession.

Materials
250 ml Beaker Wooden applicators Test tubes 20 ml beaker (to cover the column) Lake sediment (mud) and dry soil from outdoor source (sources of organisms and nutrients) Water from one of various sources (solvent and an additional source of organisms)

Calcium sulfate (CaSO4a pH buffer and an electron acceptor for anaerobic respiration) Rice, shredded newspaper or sawdust (carbon sources) Microscope slides (optional)

Method
1. In the 250 ml beaker, prepare a slurry consisting of the following ingredients in the following order: Four parts lake sediment One part dry soil One part CaSO4 add a small amount of water and mix to make slurry. Use more soil or water to adjust the consistancy. The slurry should be pourable without being too runny (The TAs will help you determine the correct consistancy). 2. Add a few kernels of rice and a few bits of newspaper and or some sawdust to the bottom of a test tube. Pour the slurry you prepared in step 1 into the tube until its about 75-80% full (avoid trapping air bubbles). Add additional water to fill the tube to about 1 cm from the top. 3. Cover the tube with the 20 ml beaker and place in the rack near the window. Incubate at room temperature. Always maintain a water layer on top of the column. 4. The instructor and TAs will set up similar columns which will be kept in the dark (NEGATIVE CONTROLS). 5. (Optional) If you wish, you can set up more than one column using different ingredients to compare the effects on community development AND/OR microscope slides can be inserted into the columns to monitor community development at the microscopic level.

Results
1. Observe your column at least once a week and record any changes that occur (ie, formation of gas pockets, blackening, development of coloration, etc.) in your laboratory notebook. It usually takes 2 to 4 weeks for chromogenic, anaerobic photosynthetic bacteria to become obvious but incubation of the columns can continue long beyond that to follow long-term community development You may want to sample the overlying water and record microscopic observations. 2. Compare your column to the columns of other teams and those that have been kept in the dark. 3. Include the observations you record in your lab notebooks with the carbons that you turn in weekly. 4. Do an on-line search for more information about the potential, microbial processes taking place in your Winogradsky column. List at two URLs that you have found in your lab notebook.

References
Pigage, Helen K. "The Winogradsky Column: A Miniature Pond Bottom." American Biology Teacher (47/4), April, 1985. Pp. 239-240. Sagan, Dorion, and Margulis, Lynn. Garden of Microbial Delights. Boston: Harcourt, Brace, Jovanovich. 1988