The Identifying Factor Of Azotobacter
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It is all too easy to forget about the soil microorganisms that are so crucial to the health of the flora and fauna of an ecosystem. When speaking of soil microorganisms, this classification can be further broken down into three subcategories: fungi, protista and bacteria, with bacteria making up the largest portion of the microorganisms (Boyle et al., 2007, Pelczar et al. 1993). These soil microorganisms play a central role to the biogeochemical cycling of nutrients such as, Nitrogen, Phosphorus and Sulphur; elements which are detrimental for the growth and life of organisms (Bastida et al., 2007). In addition to their role in nutrient cycling, soil microorganisms largely contribute to soil structures by breaking down and decomposing organic matters, and are also an important food source for other organisms such as earth worms and amoebae (Bastida et al., 2007). Considering the impact of soil microorganisms on the environment and ecosystem, it is easy to see how soil microorganisms also impact human lifestyle and economy. In recent times, a flood of research has been conducted on the importance of microorganisms on agriculture, with interests in the ability of nitrogen fixing bacteria to substitute for nitrogenous fertilizers (Cakmacki et al. 2006).
This lab focused on the bacterial portion of soil microorganisms, specifically, the isolation and identification of a single bacterium from a local soil sample using a number of aseptic laboratory techniques and Bergey's Manual of Systemic Bacteriology (1984).
Materials and Methods
The following methods were taken from the Biology 203 Lab Manual (Robertson 2008). Two soil samples were taken, the first from forest soil and the second, a coarse woody debris, from compost. These two samples were serially diluted to make solutions from 10E-2 to 10E-7, and from each sample pour plates, slants, streak plates and broths were inoculated using aseptic technique. The colony morphologies were observed and recorded and each plate was enumerated. From these samples 4 bacteria were chosen and subcultured onto streak plates and slants. After preparing and Gram Staining the slides the cell morphologies of the bacteria were observed and noted. The colonies were again subcultured onto streak plates and tested for the ability to hydrolyze starch by adding Iodine. The colonies were also cultured into Sulfide, Indole and Motility (SIM) deeps to test for the presences of the Sulfur cycle and motility. In addition Peptone broths were inoculated and the cultures were tested for ammonification. Ammonium sulfate broths and nitrite broths were inoculated and nitrification was tested for as well denitrification was tested for by inoculating nitrate broths. Aerobic respiration was confirmed when catalase tests were carried out by adding H2O2 to a sample of bacterium. Finally cultures were individually exposed to each of several different temperatures, salinities and levels of pH in order to determine their optimal environmental conditions. A single colony of bacteria was chosen to be identified: bacteria 1 from soil sample 2.
The colony morphology can be described as a glistening opaque white color with a flat and irregular shape. The growth was smooth and soft. The diameter of the colony approximated 15mm. Under 1000x magnification it was revealed that the cells were bacilli, singlet and had a diameter of approximately 2μm. The cells stained Gram negative.
Table 1: Summary of Results for Unidentified Bacterium 1 of Soil Sample 2
Aerobic or Anaerobic
Denitrification (NO3- to NO2-)
Nitrification (NH3/NH4+ to NO2-)
Nitrification (NH3/NH4+ to NO3-)
Optimal salt concentration
The results of the remainder of the tests - biochemical and environmental - are summarized by Table 1. It was concluded based on the Iodine and starch reaction that this bacterium hydrolyzed starch as a source of Carbon. The Sulfur cycle did not occur as there was no black precipitate from the combination of Iron and hydrogen sulfide found in the SIM deeps. The SIM deeps did reveal that these bacteria were non-motile, growing only on the stab line. The proteins in the peptone broth were degraded to ammonia signifying that this bacterium is an ammonifer. Nitrification was also confirmed with the bacteria oxidizing the NH3 and NH4+ in the broths to NO2- and NO3. Denitrification however, did not occur; NO3 was not reduced. The addition of H2O2 led to bubbling as it reacted with catalase present in the cells. Optimal environmental conditions were found to be 22°C, pH of 5 and 0% salinity. Between the temperatures of 4, 15, 22 and 54°C, growth was strongest at 22°C, then 15 and weakest at 37 and 4°C . Growth at pH was only slightly stronger than at pH 7 but substantially stronger than at pH 3 and 9 (refer to table 2). Growth in salinity was best at 0% and decreased with .05%, 2% and 5% respectively.
Table 2: Growth of bacterium 1 at various pH based on absorbance levels at 580nm
Each of these steps aided in the possible identification of the bacterium as Azobacteraceae Azotobacter a genera of bacteria found in soil, water and roots (Bergey's Manual, 1984). Due to the thinner layer of peptidoglycan surrounded by a phospholipid outer membrane as opposed to a thick external layer of peptidoglycan this bacterium stained Gram negative (Prescot, Harley and Klein 2005). Of vital importance for identification was the presence of catalase, an enzyme present in aerobic bacteria that breaks down the toxic byproduct of electron transport: H2O2 (Wang et al. 2008). The search to identify Bacterium 1 began with these two broad criteria: Gram negative and aerobic respiration. This particular bacterium was non-motile, the bacterium grew only along the stab line in the SIM deep rather than spreading throughout the medium. This turned out to be an important factor while identifying as it as Azotobacter which contains both motile and non-motile bacteria (Bergey's Manual, 1984). These three qualities alone pointed in the direction of Azotobacter; the biochemical and environmental tests served to confirm that Bacterium 1 was indeed Azotobacter by matching the characteristics of this particular genera to the bacterium.
Nitrification was a common characteristic between the two and was confirmed to occur in Bacterium 1 when the ammonia broth was oxidized to nitrite and nitrite was oxidized to nitrate (Bergey's Manual, 1984). Denitrification however, did not occur the nitrate was left intact and un-reduced. Ammonification, the breakdown of nitrogen containing compounds to ammonia, was also a common characteristic of Bacterium 1 and Azotobacter (Bergey's Manual, 1984, Roberts, 2008). Although the test for motility in the SIM deep was positive, the sulfur cycle test in the SIM had a negative result. The sulfur containing compounds were not reduced by the bacterium to produce H2S and, this result even as a negative, was an important factor in identification because Azotobacter also does not reduce sulfur (Bergey's Manual, 1984, Roberts, 2008). Bergey's Manual (1984) classifies Azotobacter as a heterotroph and, similarly Bacterium 1 was identified as a heterotroph when Iodine was added to the streak plate containing starch and no color change occurred in the area under and around the colony. This was an indication of the bacterium breaking down and metabolizing the starch. Finally, the optimal environmental conditions of both Bacterium 1 and Azotobactera were found to be very similar. The optimal conditions were stated as: pH of 4.8 - 8.5, temperature of 15 - 37ËšC and low salinity (Bergey's Manual, 1984). Bacterium 1 had very similar environmental conditions of: pH of 5, 22ËšC and salinity of 0% NaCl.
Considering the nitrifying and ammonifiying qualities of the Azotobacter, this bacterium plays an important role in the nitrogen cycle by breaking down proteins and converting the nitrogen into a form that can then be used by other organisms (Butenschoen, Marhan and Scheu, 2007, Cakmakci et al. 2006). Azotobacter, as one of the more common nitrifying soil microbes, is known to produce a great amount of usable Nitrogen, and therefore is closely linked to plant growth and health (Cakmakci et al. 2006, Prescot, Harley and Klein 2005). Interestingly, although many nitrifying organisms hold a symbiotic relationship with plant roots, providing Nitrogen in exchange for nutrients, Azotobacter, in particular, does not (Prescot, Harley and Klein 2005). Azotobacter also carries out starch hydrolysis, and therefore aids in the decomposition of organic matter in soil and the mineralization process (Smith and Smith, 2001).
The identification of Bacterium 1 as Azobacteraceae Azotobacter is not definite and several other tests would have required in order to prove this statement as true. One test which would have been very helpful would have been a test for cyst formation; a key characteristic of the Azotobacter (Bergey's Manual, 1984, Prescot, Harley and Klein 2005). The Azotobacter are not rhizobacteria and therefore it would have been appropriate if there were some way of observing the bacteria in their natural habitat. In addition, the tests that were carried out had limitations to the amount of information that could have been gleaned from the results. The nitrification, ammonification, and denitrification tests were based on a simple color change, and there was no way of telling the process by which these functions, if present, occurred, nor were the tests specific to the concentration of the compounds present. All of these tests and techniques could also have been subjected to error, for example, measurement errors under the microscope, errors in serial dilutions and even, contamination of cultures.
From a simple compost soil sample it was possible to isolate and subculture a single bacterium species. Using various biochemical tests such as, tests for nitrification, ammonification, denitrification and the sulfur cycle, it was possible to determine the characteristic metabolic functions of the organism. These results, in addition to observation of cell and colony morphology, especially Gram staining, enabled the identification of the bacterium as Azobacteraceae Azotobacter.
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