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Soil is a habitat for a variety of organisms, including bacteria, fungi, protozoa, insects, algae, nematodes, worms and many other animals (Prescott et al., 2006). These microorganisms are responsible for the formation, maintenance, and in some situations, the degradation and disappearance of soils (Prescott et al., 2006). Soil is a complex of microbial communities all contributing to the biological importance of this ecosystem. These microbes are responsible for the formation of minerals, the degradation of hydrocarbons, pesticides and the decomposition of organic matter (Prescott et al., 2006) through enzymatic hydrolysis (Robertson, 2009). The microbial community also influences plants in many direct and indirect ways, such as symbiotic relations with specific nitrogen fixing bacteria and plant nodules. Soil microorganisms also have interesting interaction with the atmosphere through their activities in biochemical cycling of the carbon, nitrogen, iron and manganese cycles (Prescott et al., 2006).
Soil microorganisms are maintained by nutrient sources such as materials leaching from the surface, decomposition of buried plant remains and the methane synthesis (Prescott et al., 2006). "Microorganisms can only grow in environments where conditions allow their cellular components to function properly (Robertson, 2009)". Soil provides an environment for microorganisms that are in close physical contact with oxygen. Most soil bacteria are located on the surface of soil particles where the bacterial population can approach 10Ë†8 and 10Ë†9 cells per gram dry weight of soil (Prescott et al., 2006). In Garden soil nitrogen is largely responsible for the health of the plants. Nitrogen is made available to plants by nitrogen fixing bacteria which convert nitrogen into nitrates, a form plants can use. Soil is generally evaluated on fertility and texture. Fertility is a combination of essential nutrients and a pH that makes these nutrients available to the plants. (Iannotti, About.com ) The identification of a bacterial colony based on biochemical tests and appearance was performed on bacterial isolates from garden soil.
The procedures were taken for Egger and Robertson, Lab Manual (2009). The bacteria transfer for all tests was carried out aseptically. 1.05g of garden soil was weighed and dissolved in 99ml of sterile deionised water. The solution was transferred onto a TSA streak plate and incubated at 25 ËšC for ~ 48 hours then placed in a fridge at 4ËšC for 5 days. The colony morphology tests were performed on the original colony, including a Gram stain. The colonies were then sub cultured. The sub cultures were used to record the cellular morphology. In week four the biochemical tests were performed in the isolated bacterial colonies. The colonies were tested for Starch Hydrolysis by streaking the bacterium onto the starch agar plates, and incubating them for 48 hours at 25ËšC. In week five iodine was added to the edge of the colonies. H2S and Motility was tested using SIM (Sulfide, Indole, and Motility) deeps. Ammonification was tested using peptone broths, Nitrification test were performed using ammonium sulfate and nitrite broth and Denitrification tests were carried out using nitrate broth. The broths were incubated at 25ËšC for seven days. The Ammonification test was completed by adding 1 drop of Nessler's reagentto a loop full of broth. Nitrificaiton tests were completed using two tests, in the first test one drop of Nessler's reagent was added to a loop full of ammonium sulfate broth. In the second test 3 drops of Trommsdorf's reagent and one drop of H2SO4 was added to a loop full of the nitrite broth. If this test was negative for nitrite, one drop of phenylamine and 2 drops of concentrated H2SO4 was added to a loop full of nitrite broth. The Denitrification test was concluded by adding reagents A and B to the nitrate broth. If there was no color change then reagent C was added. The Catalase test was done with 3% hydrogen peroxide and a loop full of bacterium. The environmental factors that influence bacterial growth were tested using TSA plates incubated at 13-15ËšC, 26ËšC, 37ËšC, and 50ËšC. Furthermore, TSB tubes at pH 3, pH 5, pH 7, and pH 9 were used. Also, TSA plates with 0%, 0.5%, 2% and 5% NaCl concentrations. The extent of bacteria growth was visually measured by comparing the growth for the different temperatures and osmotic pressures. The pH's were measured using a spectrophotometer.
The colony morphology for the bacteria isolate from garden soil had a circular form, convex elevation, and entire margin, and a smooth texture. The colonies appearance was shinny with a translucent, orange color. The diameter of the colony was approximately 1.5mm. The cellular morphology was streptobacillus, with a dimension of about 23.76um. The test results are included in Table 1. The test results were negative for motility, H2S reduction and ammonificaiton. All other test results were positive. Results are not available for Detnitrification to N2 and Nitrification to NO3Ë‰ because previous detnitrification and nitrification test were positive, therefore further tests did not need to be done. The bacterium colony stained purple with the counter stain safranin die therefore, the colony was Gram positive. When iodine was added to the edges of the colonies the agar remained clear consequently, the bacteria hydrolysed starch. There was no black precipitate in the SIM therefore, no H2S was produced. Furthermore the bacteria cells did not migrate away for the stab line in the SIM therefore the bacterium is not motile. Since there was no color change when Nessler's reagent was added to the peptone broth the bacterium cannot carry out ammonification. The bacterium was positive for nitrification since both the ammonium sulfate and nitrite broths experienced color changes when the reagents were added to them. Finally the addition of reagents A and B caused the nitrate broth to go pink immediately, therefore the bacteria was also positive for nitrification.
Table 1. Results for Biochemical Tests of Unknown Bacterium
Denitrification (NO3Ë‰ to NO2Ë‰)
Denitrification (NO3Ë‰ to NH3 to N2)
Nitrification (NH3/ NH4+ to NO2)
Nitrification (NH3/ NH4+ to NO3Ë‰)
Optimal Salt Concentration
(0% - 0.5%)
Aureobacterium was the bacteria genus isolated from the garden soil. The unique biochemical tests, environmental preferences and colony/cellular morphology led to the positive identification. Aereobacterium is Gram-positive, aerobic, neutrophilic and rod shaped bacterium (Wu et al., 2008). A defining characteristic ao Aureobacterium is that is grows well between 25ËšC-30ËšC although, its full range of growth is between 10ËšC-40ËšC (Krieg and Holt, 1984). Some species are molite, while other are not. All species are catalase-positive, and many species hydrolys starch. Furtheremore many species reduce nitrate to nitrate (Krieg and Holt, 1984). Other characters that identified the bacterium were the colonies morphology. Aureobacterium are convex, circular, shinny and have entire edges (Krieg and Holt, 1984).
Auerebacterium are found in soil and other enviromental areas, such as the sea, depending on the specific species. The bacterial comunity in soil is associated with plant roots. Plant roots are a common place for Auerebacterium to be found (Siciliano and Germinda, 1999). Aurobacterium have the ability to use picric acid and other nitrogen substituted phenols as a sole carbon source and to completely degrade picric acid to the level where no aromatic degradation products can be detected. Past methods of disposing wastes containing picric acid have included dumping at specified land-fill areas, dumping in deep water at sea and incineration (World Intellectual Property Orginization, 1994). All of these methods carry some potential for harm to the environment. Therfore degrading picric acid compounds completely can be an effective way to remediate picric acid contaminated environments (World Intellectual Property Orginization, 1994).
Some other tests that could be performed the further identify the bacterium are an endospore stain and an urease test. Aureobacterium is negative for both of these tests (Krieg and Holt, 1984). The spore stain is a diferential stain used to detect the presence and location of spores in the bacterial cells (Leboffe and Pierce, 2005). The urease test is used to differentiate organisms based on their ability to hydrolize urea with the enyme urease (Leboffe and Pierce, 2005).
Errors in the identification of Aureobacterium may have occurred due to inexperience in the aseptic and culturing techniques. There may have been insufficient biochemical tests to accurately identify the bacterium. This problem could be solved by performing additional tests. Human error also occurs via each individual reading the results with their own perception.
The bacterial isolate from garden soil was positively identified as belonging to the Genus Aureobacterium.