Katie Clark, Willis W. Colson, Analisa Brown, and Terrell Bright
Microbiology Lab 3100
Dr. Jenifer Turco
November 25, 2008
Abstract
A Winogradsky column is a wonderful tool by which an otherwise complex environment can be simulated and explored in laboratory. Three Winogradsky columns were constructed identically except for a single aspect. One column contained calicium sulfate, one contained calcium carbonate, and the control contain both. The columns were monitored over approximately a ten-week period. We hypothesized that growth would be greater in the periphyton region than the surface microlayer biofilm within the column. This did not hold true however as our observation would lead us to surmise that other factors played critical roles in the growth of both biofilm as well as periphyton communities.
Introduction
In 1888 Sergi Winogradsky devised a selective enrichment technique for phototrophic bacteria; the “Winogradsky Column”. The Winogradsky column is an example of a mixed culture method. Its purpose is to help determine and demonstrate the growth of microorganisms, while monitoring their activity over a period of time. The column is compiled with mud from a freshwater source; in this particular experiment the water and organic material was obtained from Big Brown lake located in Lake Park GA. A source of carbon is then added as well as (CaCO3) and a source of sulfur (CaSO4). These materials will help to promote the growth of different microorganisms pending their nutritional requirements.
In conducting the following experiment numerous biofilms were formed. Biofilms are microorganisms that are held together by a matrix of extracellular polymeric substances. Adhesion to the liquid surface is believed to occur through proteins and polysaccharides by their hydrophobic regions. Biofilms can include a variety of microorganisms including but not limited to; fungi, algae, protozoa, and bacteria. Surface microlayer biofilms in particular are biofilms that loosely adhere to the surface of static or dynamic liquid mediums (i.e. Lakes, ponds, rivers, and streams). Individual (planktonic) bacterial cells have the ability to adhere to surfaces. Other planktonic bacteria can then attach to the adhered bacteria. This process of continued adhesion eventually leads to multilayers of bacteria on the surface. A large amount of extra cellular polymeric substances (EPS) accompany the bacterial cells, creating a matrix throughout the biofilm. Surface microlayer biofilms are made up of phototropic aerobic organisms and are considered to be generally weaker in composition than biofilms that are in a more mature stage of development.
Periphyton communities by contrast are sessile organisms that live attached to surfaces projecting from the bottom of freshwater aquatic environments. Periphyton communities are known to exhibit rapid response to environmental conditions and nutritional environments. An excess of periphyton communities in agricultural and waste management facilities has been known to be problematic in controlling aquatic environments necessary for these industries to function effectively. Periphyton communities are aerobic and tend to have greater a degree of growth pending exposure to UV rays.
Due to the nature of attachment mechanisms and substrate properties for these two ecological niches it is believed that the organismal diversity will be greater in the periphyton communities. Considering the metabolic processes of biofilm formation it is unlikely that organismal growth is going to be divergent. In this particular experiment three columns were created. The first column assembled conforming to the standard procedure for creation of a Winogradsky column. A column was then created that lacked CaCO3. Finally a column was assembled that lacked CaSO4. Considering all metabolic and nutritional factors the control column is likely to show the most organismal diversity for both niches. Calcium carbonate is commonly used to neutralize acidic conditions in industrial runoff so therefore a much lower number of acidophiles present in both niches. Calcium Carbonate is also beneficial in the formation of lime deposits hence logically the periphyton niche would stand a greater chance for adhesion and therefore greater diversity. Calcium sulfate is necessary for the nutritional needs of sulfur-oxidizing lithotrophic bacteria. The absence of the Calcium Sulfate would logically lead to a decrease in lithotrophic organisms and an increase in organotrophs in both niches.
Materials and Methods
The mud and water for the columns were collected from “Big Brown Lake” a part of Sunset Hunting and Fishing Club located near Lake Park, GA on August 30, 2008. The exact location of extraction is marked in Fig. 1. The mud was collected as follows. At a depth of one foot, the researcher used a flat bladed shovel to remove the upper layer from the lake bottom. After the detritus was removed the researcher collected the mud in Tupperware containers. Care was taken to make sure that no allochtonous material (i.e. sticks and leaves) was collected with the mud. The water was collected in Tupperware containers as well. The mud and water were kept at room temperature for three days until the columns were assembled.
The Winogradsky columns for this experiment were constructed of 1 liter clear plastic bottles (recycled Dasani bottles). The bottles were cut just above the converging ridge of the bottle in order to maintain the rigidity of the bottles. Approximately 4 cups of mud and 3 cups of water were collected for each column constructed. Several sheets of newsprint were cross-shredded to provide a carbon source for the column. One shredded sheet was used per column (20 x 25 cm). Plastic beakers (500 ml) were used to measure and mix mud with shredded paper, calcium carbonate, and calcium sulfate before adding the ingredients to the bottles. The mud and the shredded paper were mixed thoroughly within the beaker before being added to the column bottles. All bottles were equipped with a 1” X 12” inch strip of aluminum foil so that light would be shielded from a portion of the side of each column. The strip of foil was attached with transparent tape in such a way that it could be detached and reattached upon each observation.
To the control column 1 teaspoon of calcium sulfate (CaSO4), and 1 teaspoon of calcium carbonate (CaCO3) was added. Two test columns were created, each lacking either calcium sulfate (CaSO4) or calcium carbonate (CaCO3). All three columns were labeled with a sharpie (control, Ø CaSO4, or Ø CaCO3). After placing the respective mixtures in their corresponding bottles extra mud was added to bring the mud level to 5 cm from the opening. The mud was then packed firmly to remove any trapped gases and open spaces. The columns were then filled with water collected from the source. Glass slides were inserted to approximately halfway point into the mud. Each column possessed a glass slide that was inserted on the day of construction and was not removed until the last day of observation. To prevent evaporation cellophane was placed over the opening and secured with a rubber band. Between monitoring session the columns were placed side by side on a west facing windowsill on the second floor of the Bailey Science Center on the campus of Valdosta State University. The columns were positioned such that the aluminum strip was as close as possible to the window without making contact.
The columns were constructed on September 2, 2008 and were monitored roughly once a week until November 13, 2008 (9 observations). Observations consisted of written descriptions being made on appearance of the water, the mud, the covering, the smell of the column, the slides placed into the mud, and well as wet mounts. The observations can be found in appendix A. Upon each observation photographs were taken using a Canon Power Shot SX 100 IS. The photographs can be found in appendix B.
Results
When the data was examined collectively (i.e. macroscopic observation, documentation, and microscopic photographic data; Table 1 and 2 Figures 1-15) the following was found in the Control column. Over the course of the experiment the soil had no discernable change in color remaining dark ebony throughout. There was no change in the aphotic zone throughout the entirety of the experiment. A sulfur smell was noted on occasional observation but was not a pungent odor. The macroscopic examination of the water was noted to become progressively murky with clearing occurring upon the fifth examination of the column. From the fifth examination to the final examination the water again became murky in appearance. The formation of the surface micro-layer was initially slight green with small biomass that increased in thickness until the eighth examination. The color of the biofilm was initially green with brown introduced upon the third examination of the column and remaining a mixture of brown and green throughout the course of the experiment (Fig.2). Incidentally, a green substance was found to be growing on the soil surface. Microscopic examination of previous green substance was performed (Fig.3). The differentiation of soil layers was insignificant with only slight change noted upon the eighth examination. Upon microscopic examination the control column possessed a myriad of microbiological growth over the observational period. Figure 4 is but one example of the Euglenid that was observed consistently throughout the observation period. Complex groups of organisms were adhering to the slide inserted in the substrate as soon as week three. Figure 5 includes several different members from the cyanobacteria genus Anabaena. Among the Anabaena are multiple Closterium, nonflagellated algae. Over time the Anabaena and the Closterium that had been prevalent on the slides that were inserted in the media became less obvious (Fig. 6).
In the column that was absent of CaSO4 the soil progressively became lighter with a final result of dark brown upon final examination. Again there was no change in the aphotic zone throughout the duration of the experiment. There was no smell noted with this column at any point of the experiment. When the water was examined macroscopically it became slightly murky on first examination progressing to an almost opaque appearance with a brown tint by the conclusion of the experiment. The surface microlayer biofilm was not noted on initial examination of the column; however, on the second examination a red/orange biofilm was noted. The red/orange film continued to increase in surface area and biomass throughout the duration of the experiment eventually covering the exposed sides of the column (Fig. 7). There was no differentiation of soil layers noted. Under microscopic examination in week three the slides pulled from the ØCaSO4 column possessed Coelastrum as well as some Anabaena (Fig. 8). Closterium remained absent from the slides until week 8 (Fig. 9). Ref. Fig. 10 show that Arthrospira were present toward the end of the observational period. Of the slides examined the ØCaSO4 slides showed the least density of microbial growth.
The column lacking the calcium carbonate had soil of an ebony appearance that remained uniform throughout the experiment. There was no visible change in the aphotic zone noted. In contrast to the other columns examined, this column had a sulfur smell that increased in intensity over the duration of the experiment. Upon macroscopic examination of the water, it was murky with a green tint on first examination with increasing opacity throughout becoming completely opaque (green) by the fifth examination (Fig. 11). The formation of the surface microlayer biofilm was noted upon first examination as a virulent with an increase in biomass and surface area throughout the duration of the experiment, eventually adhering to the column walls (Fig.12). Again there was no differentiation of soil layers noted. Upon the week four observation of the ØCaCO3 a diverse microbiological growth was formed upon an inserted slide. Anabaena and Anacystis dominate Fig. 13. Later observation showed a rapid decline of these organisms (Fig. 14). Not until the slide that had been placed into the column on day one was viewed were any notable biofilms witnessed (Fig. 15).
Discussion
As noted in the control column an environment containing both the Calcium Sulfate and the Calcium carbonate, the diversity of the microorganisms remained static. There were a large number of euglenids noted throughout the observations, which can be partially explained by the phagocytic nature of certain types of freshwater euglenids who thrive on an environment rich in both bacteria and microflagellets. On final examination of the column neither the periphyton niche nor the surface microlayer biofilm was noted to have a substantial biomass or propensity for adhesion. Anabaena and the Closterium that had been prevalent on the slides showed a marked decrease in both niches by the end of the experiment. Anabaena a known diazotroph can be assumed to have lost its supply of nitrogen. This can be attributed to the Column being covered with cellophane therefore severely inhibiting the ability to obtain atmospheric N2.
In the column that was lacking the calcium sulfate noted large quantities of Coelastrum a eukaryote that is planktonic in nature. Given Coelastrums planktonic nature it was noted to be prevalent in the surface microlayer biofilm. Some Anabaena was again noted with a great decrease in density at the conclusion of the experiment. Arthrospira a cyanobacteria was noted to appear at the closing stages of the experiment. This is significant in that while the diazotrophs were decreasing in number because of the lack of N2 Arthospira increased in number due to its proclivity for carbonate rich environments. It should be noted that the surface microlayer biofilm is thought to consist of red planktonic cells and had substantial propensity for adhesion. The periphyton niche examined when the full- time slide was removed showed crystalline structures but no evidence of motile organisms even when examined under high objective (Fig. 16).
The final column lacking Calcium carbonate Anacystis, a genus of blue-green algae in the class Cyanophycea was noted in large quantities. Anabaena and Anacystis are both cyanobacteria, which correlate to the abundance in the aerobic zone of the column. Again, the decrease in density of these organisms can be directly contributed to the unavailability of atmospheric nitrogen. The periphtyon region showed little growth throughout the experiment with only substantial organisms noted on examination of the full-time slide. However, it should be noted that the surface microlayer biofilm had the most substantial biofilm and the greatest adhesion.
In conclusion it would seem that the influence of nutritional factors presents direct outcome on diversity and establishment of periphyton regions as well as surface microlayer biofilms. While the initial hypothesis inferred that growth would be greater in the periphyton region than the surface microlayer biofilm that did not hold true. This can surmise that the effect of biomineralization, a key factor in establishing adhesion and propagation of both surface and submerged bioflim formation, responds better to a highly oxygenated environment. Perhaps, if the experiment had gone on for a longer duration, the temperature of the column had been altered, or if UV exposure had been constant then a greater microbial density and diversity would be apparent in both niches.
Literature Cited
Barton, L. (1995). Sulfate-Reducing Bacteria, Biotechnology Handbooks 8. Plenum Press, N.Y.
Brown, Alfred E. Benson's Microbiological Applications: Laboratory Manual in General Microbiology (Complete Version. 11th ed. Boston: McGraw-Hill: Higher Education, 2009.
Chapman, V.J. (1968) The Algae. St. Martins Press.
Herrero A. and Flores E. (editor). (2008). The Cyanobacteria: Molecular Biology, Genomics and Evolution, 1st ed., Caister Academic Press.
Madigan, Michael T., John M. Martinko, Paul V. Dunlap, and David P. Clark. Brock: Biology of Microorganisms. 12th ed. San Francisco: Pearson Education, 2009.
Patrick, K., Jjemba (2004). Environmental Microbiology Principles and Applications. Science Publishers Inc
Van den Hoek, C., Mann, D.G., & Jahns, M.M. (1995). Algae: an introduction to phycology, Cambridge University Press
Wells CL, Wilkins TD (1996). Botulism and Clostridium botulinum in: Baron's Medical Microbiology (Baron S et al, eds.), 4th ed., Univ of Texas Medical Branch
Whitton, B. A. and M. Potts (eds.) 2000. Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht, Boston.
1 comment:
Since Winogradsky's column favors an anerobic enviornment. Is there any bypass to overcome this deficit. Frankline, Research Scholar, India.
Post a Comment