In soil environments, microorganisms greatly vary not only in their classification but also their functions, their importance and how they are distributed among soil. One factor that affects the distribution in the soil is requirement of oxygen and microorganisms can be placed in one of five categories based on this need for oxygen; obligate aerobes, microaerophiles, obligate anaerobes, aerotolerant aerobes and facultative anaerobes. These soil microorganisms play an important role in "decomposition and the recycling of essential nutrients that are locked in macromolecules of organic material" (Roberston, S. & Egger, K., 2008). Essential nutrients are released using enzymatic hydrolysis reactions and different types of bacteria perform their own unique group of biochemical reactions. This difference in biochemical reactions is one way microorganisms can be identified. Most soil bacteria are heterotrophs because they use existing organic compounds, such as dead plants, as their source of carbon and energy. Starch, sulfur, and nitrogen are all essential for the synthesis of macromolecules and can be obtained by different chemical reactions which the bacterium may or may not be able to complete.
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Microorganisms can only grow in conditions where their cellular components are fully functional. Although as a group, bacteria can grow over a wide variety of environments, genuses have specific temperatures, pHs, and solute concentrations in which they can endure. The heat sensitivity of enzymes, membranes and other cellular components determines the temperature range in which the bacteria can grow. Bacteria can be divided into the following categories based on temperature range; psychrophiles, mesophiles, thermophiles and hyperthermophiles. The pH of the environment also has an effect on growth of bacteria because it affects the activity of biosynthetic enzymes, the availability of nutrients and other compounds that are harmful to the bacteria. The categories in which bacteria are divided based on pH tolerance are acidophiles, neutrophiles and alkalophiles. Solute concentration also effects where bacteria can grow because of osmotic pressure. The concentration of solute on the outside of the plasma membrane that surrounds the bacteria can either cause the bacteria to burst or to shrink. The following experiment is designed to study these important characteristic of different bacteria as well to learn to isolate and classify a specific bacteria from a soil sample.
To begin our study of soil bacteria, we obtained a sample of two different soils and diluted them to 10-2 through to 10-7 soil dilutions. Using the 10-2 dilution, inoculates were made in three different culture tubes; broths, slants and stabs and three different agar plates; spread plates, pour plates and streak plates. After allowing these bacteria colonies to grow, we recorded amount of growth for each condition. Pure cultures were created by sub-culturing the bacteria colonies and new streak plates and slants were created. Colony morphology observations such as form, elevation, margin, appearance, optical property, texture and color were made for each of the four pure cultures. Using a compound microscope, cellular morphology properties such as cell shape, cell arrangement and cell diameter were observed and recorded for each pure culture.
Staining techniques allowed us to differentiate between the organism and the background, compare different cell morphologies and observe structures such as endospores and capsules. In this experiment we used Gram stain, which is a differential staining technique that categorizes bacteria based on the structure of their cell wall. Gram staining procedures were taken from the lab manual (Roberston, S. & Egger, K., 2008) and based on the results of gram staining; one was able to differentiate each of the bacteria samples as gram-positive or gram-negative.
The next step in the experiment was to determine biochemical reactions which take place in each bacterium. Starch hydrolysis was tested by inoculating each bacterium onto a starch agar plate, allowing the bacteria to grow and testing with iodine. To test for hydrogen sulfide, the bacteria were grown in a sulfur-containing compound and iron salts to observe whether black ferric sulphide is produced. Nitrogen is also essential for the synthesis of macromolecules and can be obtained by ammonification, nitrification and/or denitrification. A series of tests, as indicated in the lab manual (Roberston, S. & Egger, K., 2008), were completed to test for these biochemical transformations. Catalase is the enzyme that breaks down hydrogen peroxide into water and free oxygen. Catalase was tested for by adding 3% hydrogen peroxide to determine if any oxygen is released as a result of the hydrogen peroxide breakdown.
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Optimal environmental factors were determined by testing the growth response in a range of temperatures, pHs and solute concentrations. Each bacterium was tested at temperatures of 4Â°, 10-15Â°, 22Â° and 55Â° and at pH 3, 4, 7 and 9. Solute concentration tolerance was determined by placing each bacterium into different concentration of salt; 0, 0.5, 2, and 5% NaCl. Based on observations and results of biochemical testing, cellular and colony observations and optimum growth factors, the possible identification of the each pure bacterium can be determined using the Bergy's Manual of Systematic Bacteriology.
Results: Table 1. Summary of Results for Unknown Bacteria
Form: irregular, Elevation: flat, Margin: undulate, Appearance: dull, Optical Property: slightly translucent, Color: pale yellow, Texture: smooth, Diameter: 4mm
Cell Shape: bacillus, Cell arrangement: majority single, Dimensions: 5 x 1
Denitrification (NO3- to NO2-)
Denitrification (NO3- to NH3 or N2)
Nitrification (NH3/NH4+ to NO2-)
Nitrification (NH3/NH4+ to NO3-)
Optimal salt concentration
The isolated bacteria had an irregular, flat and undulate shape with a dull, translucent pale yellow color as shown in row one of Table 1. The surface of the colony was smooth and four millimetres in diameter. Under the microscope, the bacteria of choice mainly appeared as single bacillus with a diameter of approximately one micrometer. In the Gram staining of the bacteria, it was determined that the bacterium was gram-positive because it stained purple. The bacterium of interest tested slightly positive for starch hydrolysis due to color change to yellow although not to the black precipitate present if the bacteria cannot hydrolyze starch at all. H2S reduction and motility characteristics showed in row five and six of Table 1. were tested using SIM deeps. When sulfur is reduced it will combine with the iron salts to form a visible black ferric sulfide but our bacterium's color remained unchanged and there was lack of bacterial growth from the original stab line, therefore both H2S reduction and motility were negative.
The nitrogen cycle transforms N-containing organic compounds and elemental N2 into inorganic forms using ammonification, nitrification, denitrification and N fixation and these results are shown in rows seven to eleven in Table 1. In the test for ammonification the bacteria under investigation turned a yellow and therefore ammonification did occur and there were moderate amounts of ammonia present. In testing for nitrification, the bacterium of interest presented a yellow-orange color, allowing us to determine that ammonia was present. When Trommosdorf's reagent and H2SO4 was added to the broth, a clear color was produced meaning that there was no nitrite present. When the bacterium was grown in a nitrite broth, this test showed the presence of both nitrite and nitrate, meaning that some nitrification did occur. Denitrification was tested and the isolated bacteria showed that although nitrate was not reduced to nitrate, some denitrification occurred due to presence of ammonium ions or nitrogen gas. The presence of the enzyme catalase was also tested which appears on the last row of Table 1. Since bubbles were observed during the test of our bacteria, we can confirm our bacterium tested positive for catalase activity.
The possible identity of the isolated bacterium was Arthrobacter and more specifically matched to the species, aurescens. Arthrobacter are sometimes known to represent the most common single bacterial group of specimens from soil and the isolated bacteria came from the forest soil sample (Funke et al. 1996). Arthrobacter are known to have colonies about 3-5mm in diameter and usually a cream or buff color although some species are a shade of yellow (Keddie et al., 1986).The isolated bacteria was found to be a pale yellow color with a diameter of 4mm. Arthrobacter found by Irlinger et al. (2005) were found to be yellow, smooth and convex, which is similar to the isolated bacteria only it appeared to be flat instead of convex. Although Arthrobacter have a marked rod-coccus growth cycle, in our bacteria we only observed bacillus shape cells (Keddie et al., 1986). The species of the genus Arthrobacter are gram-positive, catalase-positive and aerobic which matches the observations found for the bacterium of interest (Reedy et al., 2000). Another general characteristic of the genus Arthrobacter is non-motile which was also observed from the bacteria of interest. The optimum temperature of Arthrobacter is 25-30Â°C and not surviving at 63Â°C which correlates to the data for the chosen bacterium which had optimum growth at 22Â° but no growth at 55Â° (Keddie et al., 1986). The bacteria of interest also grew best at pH of 5-7, which resembles Arthrobacter who grow at near neutral pH (Keddie et al., 1986).
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Arthrobacter is found to be increasingly important due increase use harmful chemicals in soils because they are found to reduce hexavalent chromium which is known to degrade agricultural pesticides. Arthrobacter is found to not only be able to reduce chromium but also have high resistance to Cr(V1) (Megharaj et al, 2003).
Other test may have been preformed to help determine the identity such as the presence of spores. A common characteristic of Arthrobacter is they are non-spore forming, which can be determined by staining techniques and then observation under a microscope. Another characteristic is oxidase activity which can be determined using the modified oxidase test of Faller & Schleifer such as used by Irlinger et al. (2005). In Funke et al. (1996), all strains were found to complete hydrolysis of gelatin which was detected by immersing film strips. Some of the tests that were preformed were limited not only by resources but time available to perform the tests. Some of the test preformed involved a lot of comparison with the other bacteria being tested because determining whether the test was positive or negative was not very distinct or always clear.
The main source of error that may have caused incorrect results is inexperience with aseptic techniques. This lack of experience may have lead to contaminations throughout the experiment which would affect the overall results. Other sources of error may include incorrect results or recording of biochemical testing.