Azospirillum Identification Of An Unknown Soil Bacterium Biology Essay

Microbial diverseness in dirt is highly huge and far surpasses that of eucaryotic beings ( Torsvik and Ovreas 2002 ) . The relationship between the functional diverseness of dirt bugs and their maps is an country of subject that is mostly unknown. However, it has been assumed that the biodiversity influences the stableness, productiveness, and resiliency of ecosystems ( Torsvik and Ovreas 2002 ) . Soil bugs are capable of transporting out all known biotic procedures and they live in distinct microhabitats ( Ingham et al. 1985 ) . Microorganisms found in dirt can lend greatly to the hint gases of the ambiance ( Conrad 1996 ) . The microorganisms participate in production and ingestion processes that affect the atmospheric rhythms of hint gases such as methane, H, C dioxide, and N ( Conrad 1996 ) .

The aim of this lab was to place an unknown bacteria, cultured from dirt, through a assortment of agencies. These agencies included ocular observations, biochemical testing, and environmental testing.

Methods ( Robertson & A ; Egger 2010 ) :

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A 10-2 dilution of agricultural dirt was used to do the run home base that this bacteria was found on. Once the bacteria was chosen, it was sub-cultured onto a streak home base. The settlement morphology was observed and recorded, every bit good as the cell morphology.

Next, a series of biochemical trials were performed on the unknown bacteria in order to assist indentify it. All vaccinations and sub-cultures were incubated for 7 yearss before the consequences were observed. The bacteria was tested for its ability to hydrolyse amylum by being sub-cultured onto a amylum agar home base. It was besides inoculated into a SIM deep ( Sulfide, Indole, and Motility ) to prove for its H sulfide production and its motility. Kovac ‘s Reagent was added to the deep to prove for the production of indole. The bacteria was inoculated into a peptone stock and subsequently tested for ammonification with Nessler ‘s Reagent. As good, it was inoculated into both an ammonium sulfate stock and a nitrite stock tubing to prove for nitrification. Nessler ‘s Reagent was used to prove for the presence of ammonium hydroxide, both Trommsdorf ‘s reagent and dilute sulfuric acid were used to prove for nitrite, and diphenylamine reagent and concentrated sulfuric acid were used to prove for nitrate. The bacteria was tested for denitrification next. It was incubated in nitrate stock and tested for nitrate decrease utilizing Reagents A and B ( sulfanic acid and N, N-dimethyl-1-1-naphthylamine ) . Reagent C ( zinc pulverization ) was used if no coloring material appeared after the first two reagents were added. The unknown bacteria was so tested for its O demand by detecting its growing in a thioglycollate medium. The last biochemical trial performed was the add-on of a few beads of H peroxide to the bacteria, which was so observed for frothing or bubbles.

The bacteria was besides tested for the effects of environmental alterations. It was sub-cultured onto four different agar home bases which were incubated in four different temperatures: 4, 22, 37, and 50oC. It was sub-cultured onto four home bases with different salt concentrations: 0, 0.5, 2, and 5 % concentrations. Last, the terra incognita was tested for its optimum pH and inoculated into four tubings, each with a different pH: 3, 5, 7, and 9.


The bacteria settlement was observed to hold a smooth, round, and raised signifier with an full border. It was glistening, opaque and had a pinkish touch to it. Microscopically, the bacteria appeared to be rod shaped, with a bunch agreement and 0.5 by 3 microns in size. The bacteria had a negative Gram discoloration as the cells appeared pink under the microscope. These observations, every bit good as the consequences collected from the trials performed on the unknown bacteria, are summarized in table 1.

As seen in table 1, there was no amylum hydrolysis, no H sulfide production, and no indole production. A moderate sum of ammonification occurred and the bacteria showed marks of motility. The bacteria did non look to change over ammonium to nitrite, but the trials did demo marks of nitrite being converted to nitrate. The trial conducted to observe the transition of nitrate to nitrite was negative but something interesting happened when Zn pulverization was added to prove for denitrification. When Zn pulverization ( Reagent C ) was added to the nitrate stock that was incubated with the bacteria, a strong pink coloring material appeared bespeaking the no denitrification took topographic point. However, after the tubing sat with the Zn pulverization for some clip, the coloring material disappeared from the stock, which indicates that denitrification took topographic point and nitrate was converted all the manner to ammonium ions or to nitrogen gas.

Table 1: A sum-up of the consequences of the trials performed to place the unknown bacteria



Colony morphology

Round signifier, raised, full border, shiny, opaque, pinkish/cream coloring material, smooth texture, 2.5 millimeter diameter

Cell morphology

Rod, bunch, 3 ten 0.5 um

Gram Stain


Starch Hydrolysis


Hydrogen Sulfide production




Indole production



Moderate sum

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 coloring material disappeared which indicated a positive consequence

Oxygen tolerance

Not at the top or the underside of the tubing: Microaerophile



Optimum Temperature

22oC ( Mesophile )

Optimal pH

7 ( Neutrophile )

Optimal salt concentration

0 % NaCl ( Nonhalophile )

The bacteria showed marks of being a microaerophile as there was no growing evident neither at the bottom nor straight at the top of the tubing. The bacteria showed positiveness as a catalase. The bacteria ‘s evident optimum temperature was 22oC, which would be a mesophile. A pH of seven was optimum for the unknown, every bit good as a salt concentration of zero per centum. This would do the bacteria a neutrophile and a nonhalophile.


The unknown bacteria 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 bacteria that occurs free-living in dirt. As good, Coninck et Al. ( 1988 ) studied Azospirillum that originated from agricultural dirt, as this peculiar unknown bacteria did. Azospirillum by and large have light to dark pink settlements, an optimum temperature of 33-41oC, a pH of 5.5 to 7.4, are motile, and can be aerophilic or microaerobic ( Garrity et al. 2005 ) . Our sample had a light pink settlement, an optimum temperature of 22oC, an optimum pH of 7, and showed microaerophilic inclinations. As good, 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 bacteria do non hydrolyse amylum and do non bring forth indole, but they are both positive for catalase activity and ammonification ( Egger 2010 ) . The cell morphology of the unknown by and large matches up with that of the Azospirillum. The unknown had a rod form and was 3 by 0.5 microns in size. The Azospirillum can be a somewhat curved or a consecutive rod with dimensions of 1 by 2 to 4 microns ( Egger 2010 ) . Both are Gram negative every bit good.

Both Azospirillum and the unknown are negative for the nitrification of ammonium to nitrite. However, the other nitrification trial and both the denitrification trials are opposite in the unknown and Azospirillum. The unknown tested positive for the transition of nitrite to nitrate, but Azospirillum does non make this ( Egger 2010 ) . Our unknown tested negative for transition of nitrate to nitrite every bit good as from nitrate to ammonium or nitrogen gas. Azospirillum does both these transitions ( Egger 2010 ) . However, our unknown did something unusual. The coloring material that was present, which indicated there was no denitrification from nitrate to ammonium or nitrogen gas, disappeared over the class of an hr or so. The deficiency of coloring material indicates that denitrification did so take topographic point. In Bergey ‘s Manual ( Garrity et al. 2005 ) , it states that Azospirillum may dissimilate nitrate to nitrite or to nitrogen gas under terrible O restrictions. This is possibly what the unknown did.

Azospirillum can turn in mircoaerophilic conditions and can be used as a harvest inoculum in agricultural dirts to advance works growing ( Coninck et al. 1988 ) . It has the ability to repair N gas, interact with workss, and bring forth plant hormones ( Michiels et al. 1989 ) . Increased outputs of cereal and eatage grasses can ensue from Azospirillum being added to the dirt. It acts to better root development, increase the rate of H2O and mineral consumption, and biologically repairing N ( Okon 1985 ) .

It might hold been helpful to make an oxidase trial on our unknown to farther place it. Because of the uncertainness of the nitrification and denitrification trials and of the mis-match between the unknown and Azospirillum for these trials, it would be good to make these trials once more to acquire a more certain consequence. Contamination may hold affected our consequences.

We utilized a figure of biochemical trials and techniques to place an unknown bacteria of the dirt nature. It was perchance identified as belonging to the genus Azospirillum, but more trials should be completed to corroborate.


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