Azospirillum Identification Of An Unknown Soil Bacterium Biology Essay

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Microbial diversity in soil is extremely immense and far surpasses that of eukaryotic organisms (Torsvik and Øvreås 2002). The relationship between the functional diversity of soil microbes and their functions is an area of topic that is largely unknown. However, it has been assumed that the biodiversity influences the stability, productivity, and resilience of ecosystems (Torsvik and Øvreås 2002). Soil microbes are capable of carrying out all known biotic processes and they live in discrete microhabitats (Ingham et al. 1985). Microorganisms found in soil can contribute greatly to the trace gases of the atmosphere (Conrad 1996). The microorganisms participate in production and consumption processes that affect the atmospheric cycles of trace gases such as methane, hydrogen, carbon dioxide, and nitrogen (Conrad 1996).

The objective of this lab was to identify an unknown bacterium, cultured from soil, through a variety of means. These means included visual observations, biochemical testing, and environmental testing.

Methods (Robertson & Egger 2010):

A 10-2 dilution of agricultural soil was used to make the streak plate that this bacterium was found on. Once the bacterium was chosen, it was sub-cultured onto a streak plate. The colony morphology was observed and recorded, as well as the cell morphology.

Next, a series of biochemical tests were performed on the unknown bacterium in order to help indentify it. All inoculations and sub-cultures were incubated for 7 days before the results were observed. The bacterium was tested for its ability to hydrolyze starch by being sub-cultured onto a starch agar plate. It was also inoculated into a SIM deep (Sulfide, Indole, and Motility) to test for its hydrogen sulphide production and its motility. Kovac's Reagent was added to the deep to test for the production of indole. The bacterium was inoculated into a peptone broth and later tested for ammonification with Nessler's Reagent. As well, it was inoculated into both an ammonium sulphate broth and a nitrite broth tube to test for nitrification. Nessler's Reagent was used to test for the presence of ammonia, both Trommsdorf's reagent and dilute sulphuric acid were used to test for nitrite, and diphenylamine reagent and concentrated sulphuric acid were used to test for nitrate. The bacterium was tested for denitrification next. It was incubated in nitrate broth and tested for nitrate reduction using Reagents A and B (sulfanic acid and N,N-dimethyl-1-1-naphthylamine). Reagent C (zinc powder) was used if no colour appeared after the first two reagents were added. The unknown bacterium was then tested for its oxygen requirement by observing its growth in a thioglycollate medium. The last biochemical test performed was the addition of a few drops of hydrogen peroxide to the bacterium, which was then observed for foaming or bubbles.

The bacterium was also tested for the effects of environmental changes. It was sub-cultured onto four different agar plates which were incubated in four different temperatures: 4, 22, 37, and 50oC. It was sub-cultured onto four plates with different salt concentrations: 0, 0.5, 2, and 5% concentrations. Lastly, the unknown was tested for its optimal pH and inoculated into four tubes, each with a different pH: 3, 5, 7, and 9.


The bacterium colony was observed to have a smooth, circular, and raised form with an entire margin. It was shiny, opaque and had a pinkish tinge to it. Microscopically, the bacterium appeared to be rod shaped, with a cluster arrangement and 0.5 by 3 micrometers in size. The bacterium had a negative Gram stain as the cells appeared pink under the microscope. These observations, as well as the results collected from the tests performed on the unknown bacterium, are summarized in table 1.

As seen in table 1, there was no starch hydrolysis, no hydrogen sulphide production, and no indole production. A moderate amount of ammonification occurred and the bacterium showed signs of motility. The bacterium did not appear to convert ammonium to nitrite, but the tests did show signs of nitrite being converted to nitrate. The test conducted to detect the conversion of nitrate to nitrite was negative but something interesting happened when zinc powder was added to test for denitrification. When zinc powder (Reagent C) was added to the nitrate broth that was incubated with the bacterium, a strong pink colour appeared indicating the no denitrification took place. However, after the tube sat with the zinc powder for some time, the colour disappeared from the broth, which indicates that denitrification took place and nitrate was converted all the way to ammonium ions or to nitrogen gas.

Table 1: A summary of the results of the tests performed to identify the unknown bacterium



Colony morphology

Circular form, raised, entire margin, shiny, opaque, pinkish/cream colour, smooth texture, 2.5 mm diameter

Cell morphology

Rod, cluster, 3 x 0.5 um

Gram Stain


Starch Hydrolysis


Hydrogen Sulfide production




Indole production



Moderate amount

Nitrification (NH4+ to NO2-)


Nitrification (NO2- to NO3-)


Denitrification (NO3- to NO2-)


Denitrification (NO3- to NH4+ or N2)

There was none at first, but the colour disappeared which indicated a positive result

Oxygen tolerance

Not at the top or the bottom of the tube: Microaerophile



Optimal Temperature

22oC (Mesophile)

Optimal pH

7 (Neutrophile)

Optimal salt concentration

0% NaCl (Nonhalophile)

The bacterium showed signs of being a microaerophile as there was no growth evident neither at the bottom nor directly at the top of the tube. The bacterium showed positivity as a catalase. The bacterium's apparent optimal temperature was 22oC, which would be a mesophile. A pH of seven was optimal for the unknown, as well as a salt concentration of zero percent. This would make the bacterium a neutrophile and a nonhalophile.


The unknown bacterium is potentially identified as belonging to the genus Azospirillum. Azospirillum has been identified in the Bergey's Manual of Systematic Bacteriology (Garrity et al. 2005) as a bacterium that occurs free-living in soil. As well, Coninck et al. (1988) studied Azospirillum that originated from agricultural soil, as this particular unknown bacterium did. Azospirillum generally have light to dark pink colonies, an optimal temperature of 33-41oC, a pH of 5.5 to 7.4, are motile, and can be aerobic or microaerobic (Garrity et al. 2005). Our sample had a light pink colony, an optimal temperature of 22oC, an optimal pH of 7, and showed microaerophilic tendencies. As well, our sample had motility and was nonhalophilic, which were both stated as qualities of Azospirillum in the Bergey's Manual (2005). Both Azospirillum and the unknown bacterium do not hydrolyze starch and do not produce indole, but they are both positive for catalase activity and ammonification (Egger 2010). The cell morphology of the unknown generally matches up with that of the Azospirillum. The unknown had a rod shape and was 3 by 0.5 micrometers in size. The Azospirillum can be a slightly curved or a straight rod with dimensions of 1 by 2 to 4 micrometers (Egger 2010). Both are Gram negative as well.

Both Azospirillum and the unknown are negative for the nitrification of ammonium to nitrite. However, the other nitrification test and both the denitrification tests are opposite in the unknown and Azospirillum. The unknown tested positive for the conversion of nitrite to nitrate, but Azospirillum does not do this (Egger 2010). Our unknown tested negative for conversion of nitrate to nitrite as well as from nitrate to ammonium or nitrogen gas. Azospirillum does both these conversions (Egger 2010). However, our unknown did something unusual. The colour that was present, which indicated there was no denitrification from nitrate to ammonium or nitrogen gas, disappeared over the course of an hour or so. The lack of colour indicates that denitrification did indeed take place. In Bergey's Manual (Garrity et al. 2005), it states that Azospirillum may dissimilate nitrate to nitrite or to nitrogen gas under severe oxygen limitations. This is maybe what the unknown did.

Azospirillum can grow in mircoaerophilic conditions and can be used as a crop inoculant in agricultural soils to promote plant growth (Coninck et al. 1988). It has the ability to fix nitrogen gas, interact with plants, and produce phytohormones (Michiels et al. 1989). Increased yields of cereal and forage grasses can result from Azospirillum being added to the soil. It acts to improve root development, increase the rate of water and mineral uptake, and biologically fixing nitrogen (Okon 1985).

It might have been helpful to do an oxidase test on our unknown to further identify it. Because of the uncertainty of the nitrification and denitrification tests and of the mis-match between the unknown and Azospirillum for these tests, it would be beneficial to do these tests again to get a more certain result. Contamination may have affected our results.

We utilized a number of biochemical tests and techniques to identify an unknown bacterium of the soil nature. It was possibly identified as belonging to the genus Azospirillum, but more tests should be completed to confirm.