Winogradsky Column- Report and Analysis

MICROBIAL GROWTH IN AREAS OF VARYING A EROBIC AND SUBSTRATE CONDITIONS DAVID ROSENBERG, SARAH CHOMALI, T OMERM ADAR, AND NELLIE RODRIGUEZ DECEMBER, 2010The Winogradsky column is a method to bring otherwise inaccessible communities into the lab. It re-creates the natural environments for bacterial growth, creating an ideal environment to research various nutrient cycles. Saturated, enriched dirt is taken and supplemented with sulfur and carbon and sealed off. The bacterial growth responds in patterns, or zones , reflecting the substrate and aerobic concentrations. In our experiment, an exorbitant amount of Sulfur was used, creating atypically large zones of Sulfur-reducing bacteria such as Chlorobium and Chromatium. The research of microbial evolution also has great impact on Science and Medicine.

IntroductionMicrobiology, the study of organisms not visible to the unassisted eye1, has emerged as a subject of vital importance to the scientific community. Microorganisms, more commonly called microbes, are involved in a wide variety of life processes such as nitrogen fixation, mineral decomposition, and even the preparation of many foods through fermentation (Society for General Microbiology). They are also brilliantly adapted to their environments. Some microbes, commonly called Extremophiles, have been recorded to live in temperatures as high as 130rC2 or in environments acidic as pH 03. The study of Microbiology began with Antoni van Leeuwenhoek and his discovery of microorganisms in 1675 with the microscope of his own design (Payne, 1970). This discovery1

This field was originally thought of as the study of Prokaryotes exclusively. However, recent discoveries have shown some visible Prokaryotes and some microscopic Eukaryotes. 2 Strain 121 Recorded life at 121rC 3 Picrophilus has been recorded to live at pH -0.06.

Page

I

Microbial Growth in Areas of Varying Aerobic and Substrate Concentrations

put forth the evidence needed to end the idea of spontaneous generation can originate from inorganic materials

the idea that life

simultaneously laying the groundwork for biogenesis

life can only originate from other living sources . Perhaps the most important event in the evolution of microbes is the acquisition of Photosynthesis using H2O as a reducer by cyanobacteria. This process allowed O2 to enter and form the atmosphere we have today earth4 (Beatty et. al, 2005). Of course one cannot talk about Microbiology without mentioning the Russian scientist Sergei Winogradsky. He studied the complex interactions between environmental conditions and microbial activity by isolating various bacterial cultures. In his research he discovered the idea of Chemosynthesis living organisms can live completely off of inorganic compounds (Ackert, irrevocably changing the way life would evolve on

2006). This discovery gave Science an answer as to how bacteria were able to survive prephotosynthesis (though Winogradsky probably never realized the ramifications of this discovery himself). The obvious obstacle in studying microbial communities is their size. Also, the habitats they are naturally found in can be very hard to research5. The solution to this problem can also be accredited to Winogradsky- in what is called the Winogradsky Column. (Rogan et. al, 2005) The design of the column is simple. Soil or other enriched dirt (such as sand) is taken from the bottom of a pond, river, or ocean. The soil is then supplemented with water (ideally) from the4 5

An oxygenic atmosphere allowed for the development of terrestrial life. Hydrothermal vents or the ocean floor for example.

Page

II


same source, as well as sources of carbon and sulfur, the container is then sealed off. Through various biological processes an O2/ H2S concentration gradient is formed. This gradient allows the various floras to culture, bringing researchable data in vitro. Law of Conservation, Biological and Geochemical Cycles In an enclosed environment6, such as our planet, we maintain what is known as the Law of Conservation of Mass. This rule states that the overall chemical mass in said environment must remain constant. In simpler terms, if no new substances enter the environment, the overall concentrations of any given atom will remain constant, even if it is in different form. (Campbell et. al, 2009) In response to this law, many organisms evolved to attain nutrients via chemosynthesis creating what we call nutrient cycles. In these cycles, consumers and producers work

together to maintain homeostatic concentrations at each part of the cycle. At each level, the different organisms process the same substances in different forms; each byproduct is then subsequently used by a consumer at the next level. This eventually leads to the original chemical or gas being restored to the atmosphere in its usable form7. In most research, we see four main cycles; the carbon cycle, phosphorus cycle, nitrogen cycle, and the water cycle. However, in the Winogradsky Column, since we are dealing with an anaerobic environment we mostly examine the sulfur cycle.

6

An enclosed environment is one where no new resources are entering. For example, H2O is released by the atmosphere in liquid form, where it can be processed by producers and consumers, and then it sent back into the atmosphere in gaseous form.7

Page

III


In a typical Winogradsky column, we can expect that via chemosynthesis, different zones of Bacteria flourish at different levels, the most prominent of which is usually the non-sulfur photosynthetic bacteria such as Rhodomicrobium and Rhodopseudomona.

Our ExperimentOur goal was to observe how different microbial communities would develop in a controlled environment with varying aerobic and substrate concentrations. The hypothesis we were testing was if more sulfur than carbon were to be added to the substrate the zones that form would be reflective of that change; i.e., the sulfur-synthesizing bacteria would be more prominent. To test this, ~2L of sand was taken from Brighton Beach in Brooklyn. The sand was then saturated with tap water from the lab. It was then supplemented with 3 eggs8 and 2.5 slices of American cheese as sulfur sources, as well as 2.5 eggshells as a carbon source. The column was then sealed off and left to culture under sunlight for a period of six weeks. After two weeks, the column showed the beginning stages of layer formation, with a darker layer at the bottom, with the layers getting lighter as you move upward. Most of the water was gathered at the top, above the sand layers, showing an opaque white color. These first weeks showed rapid microbial growth above the water, which leveled out thereafter. This growth occurred mostly around the edge of the column, along the bottle.

8

One boiled

Page

IV


Therewere also small masses of orange-colored bacteria (very similar to Rhodospirillium but I cannot say so with certainty) that were floating in the water. Upon uncovering the column (which in hindsight, may have compromised our experiments validity), it exhibited a very strong sulfuric odor (which began to wear off in later weeks). After another week, a new layer began to form. This layer was a dark purple color, in contrast with the other light green layers. The water mass became taller, and the mold mass became more condensed. The orange masses migrated towards the edges of the column, joining the mold masses.. After another week, the orange masses once floating in the water virtually disappeared. However, a green mucus-like mass appeared in its stead. Under the mold, a black layer began to form, almost as if it was a base to the mold (which was steadily decreasing in mass). Week 4 also showed the appearance of a new layer, this one a slightly lighter shade of purple atop the existing one. After another week, the layers were continually becoming more defined from each other. There is now a clear distinction between the green (2), purple (2), and opaque layers. Also, sprouts were starting to come up from the black layer, through the continually decaying mold mass. At the end of the experiment the layers became even more distinct from each other. The higher green layer and the lower purple layer (the S-reducing bacteria) took up the largest combined

Page

V

portion of the column.


DiscussionThe end of the experiment left the column with many distinct layers: A light, very textured green layer; a darker, more solid layer; a dark purple layer; a slightly lighter purple layer; a white layer; a green-tinged water layer; and mold growing on top. Obligatory anaerobic bacteria at the bottom of the column such as Clostridium break down the cellulose into its glucose bases, further breaking them down, by means of fermentation, into ethanol and other organic molecules. Other bacteria such as Desulfovibrio respire using these compounds to reduce the sulfate from the eggs and cheese. These processes quickly deplete any remaining O2 at the bottom of the column. Desulfovibrio release Hydrogen-Sulfide as a byproduct of said sulfate reduction. This causes a concentration gradient in the column between O2 and H2S (Higher O2 at top). The Hydrogen-Sulfide is then picked up by two layers of photosynthetic bacteria that use S as a reducer Chlorobium (Green)and Chromatium (Purple). These floras, as mentioned before, were very successful and took up a large portion of the column. The position of these layers (Chlorobium on top) is determined by each Bacterium s S tolerance. Atop these two layers, a layer of non-sulfur photosynthetic bacteria (Rhodomicrobium) cultured. These bacteria use Ethanol (remaining from Chlostridium) as a photosynthetic reducer. The white layer that lies just underneath the water is called the microaerophilic region. Even though one side is exposed to oxygen, it is very little as O2 diffuses very slowly through water.

Page

VI


This layer consists of bacteria such as Beggiatoa which oxidize the remaining Hydrogen-Sulfide into Sulfuric acid. The energy from which is then used to process other organic molecules. In the water, Aerobic bacteria such as Algae and Cyanobacteria carry out standard photosynthesis. Both of these processes (Aerobic Photosynthesis, and processing of organic molecules) emit oxygen as a byproduct, contributing to the concentration gradient. In many experiments, there is a presumed superiority of fungus to bacteria in the breakdown of cellulose (Hunt, Stewart, and Cole, 1986); this lead to the rapid microbial growth in the beginning weeks. However, due to its mass, the mold rose to the top of the water, separating it from its nutrients; this lead to the rapid decay of the mold.

ConclusionIn the Winogradsky column, various bacterial floras grow in response to different environmental conditions. In our experiment, where there was a higher-than-normal concentration of Sulfur, the growth of Sulfur-reducing photosynthetic bacteria was positively correlated as a result. The two layers consisting of Chlorobium and Chromatium showed prominence in the column as opposed to the non-sulfur bacteria such as Rhodomicrobium. Fungus was also shown to grow rapidly, but was unable to sustain that growth, quickly decaying after two weeks.

Microbes and their Value to Science

Page

VII

The Evolution of Microbial Photosynthesis


The first evidence of life, as indicated by the fossil record, emerged on earth approximately 3.7 billion years ago. These cells (the first microorganisms) lived underwater, mostly near hydrothermal vents. These organisms adapted to survive via chemosynthesis, living on inorganic compounds such as sulfur among others. Roughly 800 million years later, bacteria very similar to today s cyanobacteria were the first organisms to learn to use water as a photosynthetic reducer. This new form of respiration introduced gaseous oxygen into the planet s atmosphere, forever changing the future of life as we now know it (Beatty et. al, 2005). Through various levels of endosymbiosis, eukaryotes were evolved approximately a half billion years later. There is substantial evidence that many of the traits that ensure the survival of eukaryotes are derivatives of endosymbiosis, including plastids and mitochondria (Campbell et. al, 2009). This pattern of endosymbiosis even continues today. The Sea-Slug Elysiachlorotica may be a perfect example of this. These mollusks, as well as related species have ingested the chloroplasts of the various algae surrounding it. This has not only given E. chlorotica its green color (an advantageous evolutionary trait), chloroplasts also have given it the ability to carry out photosynthesis in times of starvation. There is much debate as to whether this can be called endosymbiosis as it is only an organelle, but important nonetheless; and may be indicative of what is to come. (Rumpho, Summer, and Manhart, 2000) (Pfitzenmeyer, 1960) Life on Mars The evolutionary patterns of various microorganisms have ramifications in many other areas of science as well, including the possibility of life on Mars. Certain elements of Mars s atmosphere

Page

VIII


have very similar, if not identical characteristics to what lab data shows, was existent on Earth in the earlier stages of evolution. (Beatty et. al, 2005) (Grady and Wright, 2006) (Tung, Bramall, and Price, 2005) Horizontal Gene Transfer The evolutionary progression, aided by the fossil record as well as genome decoding, also gives us a lot of information about horizontal gene transfer9. As mentioned before, most of today s organisms have evolved based on that genetic transfer. In fact, eukaryotes first arose from gene transfer between Bacteria and Archea. Further transfer lead to the development of complex, multicellular organisms. (Campbell et. al, 2009) (Raymond et. al, 2003)

Evolution and Disease ControlThe evolution of microorganisms does not only have an effect on the larger scale; some microorganisms10 have shown rapid evolution in very short periods of time, sometimes with disastrous effects. HIV is a pathogen which has a very fast rate of replication, as well as a high mutation rate. Most of these mutations usually do not have significant consequences on the effects of the virus, but some actually give it more resistance to various drugs, this causes the world of Science, Virology and Medicine in particular, a lot of grief. Shortly after promising treatments for the disease were implemented, the virus evolved a new, resistant strain making the disease even harder to treat.

9

Horizontal transfer is the transfer of genetic traits and information between different species. Although viruses are not yet considered living, the generally are catagorized in the field of Microbiology. I am using the term microorganism to be more inclusive.10

Page

IX


This phenomenon is not exclusive to HIV. It is a common factor in the treatment of manydiseases such as MRSA among others. (Hutchinson, 2001) The study of the evolution of microorganisms is important to science, not only as to understand our existence, but also to ensure the continued viability of that existence. Of course with the treatment of disease, many argue that we are hindering natural selection, and thereby slowing evolutionary progression, but there is not much we can do about that.

References Exploring the Sulfur Nutrient Cycle Using the Winogradsky Column Author(s): Brian Rogan, Michael Lemke, Michael Levandowsky, Thomas Gorrell Source: The American Biology Teacher, Vol. 67, No. 6 (Aug., 2005), pp. 348-356 Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis Author(s): Mary E. Rumpho, Elizabeth J. Summer, James R. Manhart Source: Plant Physiology, Vol. 123, No. 1 (May, 2000), pp. 29-38 Notes on the Nudibranch, Elysiachlorotica, from Chesapeake Bay, Maryland Author(s): Hayes T. Pfitzenmeyer Source: Chesapeake Science, Vol. 1, No. 2 (Jun., 1960), pp. 114-115 The Role of Microbes in Agriculture: Sergei Vinogradskii's Discovery and Investigation of Chemosynthesis, 1880-1910 Author(s): Lloyd T. Ackert Jr. Source: Journal of the History of Biology, Vol. 39, No. 2, Biology and Agriculture (Summer,2006), pp. 373-406 http://www.youblisher.com/p/12754-The-Winogradsky-Column/ http://serc.carleton.edu/microbelife/topics/special_collections/winogradsky.html http://www.sumanasinc.com/webcontent/animations/content/winogradsky.html Early Stage Microbial Growth in Winogradsky Plates - an Alternative to Using Columns Journal of Honors Lab Investigations 2(1): 25 - 30. 2002 Julia Slaughter, Sarah Slaughter, Jan Weaver, John Burkhardt people.wcsu.edu/gyurer/files/Winogradsky.doc Concepts of Sulfur, Carbon, and Nitrogen Transformations in Soil: Evaluation by Simulation Modeling Author(s): H. W. Hunt, J. W. B. Stewart, C. V. Cole Source: Biogeochemistry, Vol. 2, No. 2 (1986), pp. 163-177 An Obligately Photosynthetic Bacterial Anaerobe from a Deep-Sea Hydrothermal Vent

Page

X


Author(s): J. Thomas Beatty, JrgOvermann, Michael T. Lince, Ann K. Manske, Andrew S.Lang, Robert E. Blankenship, Cindy L. Van Dover, Tracey A. Martinson, F. Gerald Plumley,Bob B. Buchanan Source: Proceedings of the National Academy of Sciences of the United States of America,Vol. 102, No. 26 (Jun. 28, 2005), pp. 9306-9310 Evolution of Photosynthetic Prokaryotes: A Maximum-Likelihood Mapping Approach Author(s): Jason Raymond, Olga Zhaxybayeva, J. Peter Gogarten, Robert E. Blankenship Source: Philosophical Transactions: Biological Sciences, Vol. 358, No. 1429, Chloroplasts and Mitochondria: Functional Genomics and Evolution (Jan. 29, 2003), pp. 223-230 The Carbon Cycle on Early Earth: And on Mars? Author(s): Monica M. Grady and Ian Wright Source: Philosophical Transactions: Biological Sciences, Vol. 361, No. 1474, Conditions for the Emergence of Life on the Early Earth (Oct. 29, 2006), pp. 1703-1713 The Ecological Role of Water-Column Microbes in the Sea F. Azam, T. Fenche, J. G. Field, J. S. Gra, L. A. Meyer-Rei and F. Thingstad MARINE ECOLOGY - PROGRESS SERIES Mar. Ecol. Prog. Ser. Vol. 10: 257-263, 198 The Biology and Evolution of HIV Author(s): Janis Faye Hutchinson Source: Annual Review of Anthropology, Vol. 30, (2001), pp. 85-108 Microbial Origin of Excess Methane in Glacial Ice and Implications for Life on Mars Author(s): H. C. Tung; N. E. Bramall; P. B. Price Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 51, Enzymatic Rescue of Myelination (Dec. 20, 2005), pp. 18292-18296 http://www.microbiologyonline.org.uk/about-microbiology The Cleere Observer: A biography of Antoni van Leeuwenhoek A.S. Payne Macmillan, 1970

Page

XI


Appendix

The various bacterial layers and their respective reaction formulas

Page

XII


Source: Hunt, Stewart, and Cole, 1986 A depiction of the order in which Organic materials get broken down.

Page

XIII


Source: Rogan et. al, 2005 A description of the bacteria generally found in the Winogradsky column, as well as the concentration gradients.

Page

XIV


Page

XV

Documents

Winogradsky Column- Report and Analysis