Prelab Reading The Winogradsky Column

INTRODUCTION e prokaryotic bacteria and archaea exhibit an astonishing metabolic diversity, which far exceeds that of animals, plants, fungi and other higher organisms. e prokaryotes literally keep our biological world turning by recycling all the mineral elements necessary for life support. Microbes drive the chemistry of life and aect the local climate. ey make up 60% of the Earth's biomass, yet it is estimated that less than 1% of microbial species have been identied. By studying microbes, solutions to long-standing environmental and medical challenges may emerge. Scientists are also starting to appreciate the role played by microbes in global climate processes. Two famous microbiologists pioneered the study of these processes: Sergius Winogradsky (1856-1953) and Martinus Willem Beijerinck (1851-1931). While considering the role of microbes in the Earth's biosphere Martinus Beijerinck said of bacterial life, "Everything is everywhere and the media selects." When these words were uttered, Charles Darwin was still formulating his ideas On the Origin of Species back in England and Mendels ideas were buried in obscurity, encased in a musty chest in the basement of his monastery in Austria. Beijerinck was interested in the diversity of life he studied on the microbial level. In contrast to the pure culture studies of other pioneer microbiologists such as Louis Pasteur and Robert Koch, Beijerinck and Winogradsky studied the relationships between dierent types of microorganisms in mixed communities. ey were astonished at the success of these creatures and sought to understand their achievements. It is their work we will be replicating over the next two weeks. THE WINOGRADSKY COLUMN A simple laboratory demonstrationthe Winogradsky column illustrates how dierent microorganisms perform their interdependent roles: the activities of one organism enable another to grow, and viceversa. ese columns are complete, self-contained recycling systems, driven only by energy from light. e columns (Fig. A) are easy to set up with a glass tube, such as a graduated cylinder. Mud from the bottom of a lake or river is collected and supplemented with cellulose (e.g., newspaper), sources of sulfur (magnesium sulfate) and calcium (calcium carbonate), and other salts (potassium phosphate). e rest of the tube is lled with water from the lake or river. e tube is then capped and placed near a window or other light source. All the organisms are present initially in low numbers, but when the tubes are incubated for several weeks the dierent types of

microorganism proliferate and occupy distinct zones where the environmental conditions favor their specic activities. Below is a summary of an idealized column. ANAEROBIC ZONE (OXYGEN DEPLETED) e large amount of cellulose in the column initially promotes rapid microbial growth that soon depletes the oxygen in the sediment and in the water column. Only the very top of the column remains aerated because oxygen diuses very slowly through the surface of the water. e only organisms that can grow in anaerobic conditions are those that perform fermentation and those that perform anaerobic respiration. As a review from Biological Principles I, fermentation is a process in which organic compounds are degraded incompletely; for example, yeasts ferment sugars to alcohol. Anaerobic respiration is a separate and distinct process in which organic substrates are degraded completely to CO2, but using a substance other than oxygen as the terminal electron acceptor. Some bacteria respire by using nitrate or sulfate ions, in the same way as we use oxygen as the terminal electron acceptor during respiration. ere are three basic levels that form in the lower level of the column. At the bottom, depending on the source of the mud, a pink layer with gas vesicles will develop due to purple sulfur bacteria. A characteristic species is Amoebobacter. As you move upwards in the column (or as it ages), some cellulose-degrading Clostridium species start to grow when the oxygen is depleted in the sediment. All Clostridium species are strictly anaerobic because their vegetative cells are killed by exposure to oxygen, but they can survive as spores in aerobic conditions. ey degrade the cellulose to glucose and then ferment the glucose to gain energy, producing a range of simple organic compounds (ethanol, acetic acid, succinic acid, etc.) as the fermentation end products. e sulfur-reducing bacteria, such as Desulfovibrio (Fig. B), can metabolize these fermentation products through anaerobic respiration, using either sulfate or other partly oxidized forms of sulfur (e.g., thiosulfate) as the terminal electron acceptor, generating large amounts of H2S by this process. (In our own aerobic respiration, we use O2 and reduce it to H2O.) e H2S will react with any iron in the sediment, producing black ferrous sulde. is is why lake sediments (and our household drains) are frequently black. However, some of the H2S diuses upwards into the water column, where it is used by other organisms. At the top of the anaerobic zone purple sulfur bacteria such as Chromatium, in a red to purple layer, are processing sulfates into sulfur. At another point Gallionella, a stalked bacteria, processes iron to help create the black layer that forms just below. is level is marked by a strong rust/orange color. MICROAEROPHILIC ZONE (OXYGEN SCARCE) In this zone, oxygen diuses down from the surface but is limited in concentration. Sulfur from the lower part of the column has begun to move up as H2S. is diusion of H2S from the sediment into the water column enables anaerobic photosynthetic bacteria to grow. ey are seen usually as two narrow, brightly colored bands immediately above the sediment: a zone of green sulfur bacteria, such as Chlorobium, characterized by a green or olive color indicative of growing

anaerobic conditions, followed by a zone of purple sulfur bacteria, such as Chromatium, which takes on a red, orange, or rust color. e green and purple sulfur bacteria gain energy from light reactions and produce their organic molecules from CO2 in much the same way that plants do. However, there is one essential dierence: they do not generate oxygen during photosynthesis because they do not use water as the reducer; instead, they use H2S. e following simplied equations show the parallel.

6 CO2 + 6 H2O C6H12O6 + 6 O2 (plant photosynthesis) 6 CO2 + 6 H2S C6H12O6 + 6 S (bacterial anaerobic photosynthesis)

Purple sulfur bacteria, such as Chromatium, typically have large cells with sulfur granules as deposits inside the cells. e green sulfur bacteria have smaller cells and typically deposit sulfur externally. e sulfur (or sulfate formed from it) produced by the photosynthetic bacteria returns to the sediment where it can be recycled by Desulfovibrio, part of the sulfur cycle in natural waters. Most of the water column above the photosynthetic bacteria is colored bright red by a large population of purple non-sulfur bacteria. ese include species of Rhodopseudomonas, Rhodospirillum and Rhodomicrobium. A mixed culture of them is shown in the bottle in Fig. C. ese bacteria grow in anaerobic conditions, gaining their energy from light reactions but using organic acids as their carbon source for organic molecule synthesis. eir use of light as an energy source means that they are phototrophs (light feeders) and their use of organic molecules (as opposed to gaseous CO2) as carbon sources means that they are heterotrophs. When we take both of these pieces of information together, we refer to them as photoheterotrophs. e organic acids that they use are the fermentation products of other anaerobic bacteria (e.g., Clostridium species). e purple non-sulfur bacteria are intolerant of high H2S concentrations, so they occur above the zone where the green and purple sulfur bacteria are found.

AEROBIC ZONE (OXYGEN RICH) e top of the water column can contain large populations of diverse bacteria. ese are aerobic organisms that are found in organic-rich freshwater habitats such as shallow ponds and polluted streams. ese organisms are generally agellated, allowing migration and colonization of new areas. In addition, there may be a diverse phototrophic fauna as well from the original water and mud source. At the very top of the zone, the mud is characterized by a light brown color. is is the most oxygen rich part of the mud and the most sulfur poor.

ere are three distinctive types of bacteria that are of special interest. First, any H2S that diuses into the aerobic zone can be oxidized to sulfate by the sulfur-oxidizing bacteria, such as Beggiatoa and iobacillus. ese bacteria gain energy from oxidation of H2S, and they synthesize their own organic matter from CO2. So they are termed chemosynthetic organisms, or chemoautotrophs. Similar types of organisms occur in soils, gaining energy from the oxidation of ammonium to nitrate, which then leaches from the soil and can accumulate in water supplies. Photosynthetic cyanobacteria can grow in the upper zones. is area is characterized by a grassgreen color. ese are the only bacteria that have oxygen-producing photosynthesis like that of plants. ere is very strong evidence that the chloroplasts of plants originated as cyanobacteria (or the ancestors of present-day cyanobacteria) that lived as endosymbionts inside the cells of a primitive eukaryote. Similarly, there is equally strong evidence that the mitochondria of presentday eukaryotes were derived from purple bacteria. Once the cyanobacteria start to grow, they can oxygenate much of the water. is happened in Column 2 in Fig. A; the whole water column was dominated by a mass of cyanobacteria composed of spiral laments. e top of the water column can contain large populations of sheathed bacteria (see Fig. A, Column 1). ese aerobic organisms use organic substrates, but are unusual because as they divide, they synthesize a rigid tubular sheath from which individual cells can escape and swim away to establish new colonies. Many empty sheaths are seen in older colonies. ey are made of a complex mixture of protein, polysaccharide and lipid, and are thought to protect the cells from predation by protozoa. e sheaths also can be encrusted with ferric hydroxide, giving a yellow or rusty appearance to the colonies. SUMMARY e Winogradsky column is a classic demonstration of the metabolic diversity of prokaryotes. All life on earth can be categorized based on the organism's carbon and energy source: energy can be obtained from light reactions (phototrophs) or from chemical oxidations (of organic or inorganic substances) (chemotrophs); the carbon for organic molecule synthesis can be obtained from CO2 (autotrophs) or from available organic compounds (heterotrophs). Combining these categories, we get the four basic life strategies: photoautotrophs (e.g., plants), chemoheterotrophs (e.g., animals and fungi), photoheterotrophs and chemoautotrophs. Only in the bacteria - and among the bacteria within a single Winogradsky column - do we nd all four basic life strategies. e Winogradsky column is also a classic demonstration of how microorganisms occupy highly specic microsites according to their environmental tolerances and their carbon and energy requirements. Finally, the column enables us to see how mineral elements are cycled in natural environments. We focused mainly on sulfur, but there are equivalent cycles for nitrogen, carbon and other elements.