The Identifying Factor Of Azotobacter Biology Essay

It is all excessively easy to bury about the dirt micro-organism that are so important to the wellness of the vegetations and zoologies of an ecosystem. When speech production of dirt micro-organisms, this categorization can be farther broken down into three subcategories: Fungi, Protista and bacteriums, with bacteriums doing up the largest part of the micro-organisms ( Boyle et al. , 2007, Pelczar et Al. 1993 ) . These dirt micro-organisms play a cardinal function to the biogeochemical cycling of foods such as, Nitrogen, Phosphorus and Sulphur ; elements which are damaging for the growing and life of beings ( Bastida et al. , 2007 ) . In add-on to their function in alimentary cycling, dirt micro-organisms mostly contribute to dirty constructions by interrupting down and break uping organic affairs, and are besides an of import nutrient beginning for other beings such as Earth worms and ameba ( Bastida et al. , 2007 ) . Sing the impact of dirt micro-organisms on the environment and ecosystem, it is easy to see how dirt micro-organisms besides impact human life style and economic system. In recent times, a inundation of research has been conducted on the importance of micro-organisms on agribusiness, with involvements in the ability of N repairing bacteriums to replace for nitrogen-bearing fertilisers ( Cakmacki et al. 2006 ) .

This lab focused on the bacterial part of dirt micro-organisms, specifically, the isolation and designation of a individual bacteria from a local dirt sample utilizing a figure of sterile research lab techniques and Bergey ‘s Manual of Systemic Bacteriology ( 1984 ) .

Materials and Methods

The undermentioned methods were taken from the Biology 203 Lab Manual ( Robertson 2008 ) . Two dirt samples were taken, the first from forest dirt and the 2nd, a harsh woody dust, from compost. These two samples were serially diluted to do solutions from 10E-2 to 10E-7, and from each sample pour home bases, angles, run home bases and stocks were inoculated utilizing sterile technique. The settlement morphologies were observed and recorded and each home base was enumerated. From these samples 4 bacteriums were chosen and subcultured onto run home bases and angles. After fixing and Gram Staining the slides the cell morphologies of the bacteriums were observed and noted. The settlements were once more subcultured onto run home bases and tested for the ability to hydrolyse amylum by adding Iodine. The settlements were besides cultured into Sulfide, Indole and Motility ( SIM ) deeps to prove for the presences of the Sulfur rhythm and motility. In add-on Peptone stocks were inoculated and the civilizations were tested for ammonification. Ammonium sulfate stocks and nitrite stocks were inoculated and nitrification was tested for every bit good denitrification was tested for by inoculating nitrate stocks. Aerobic respiration was confirmed when catalase trials were carried out by adding H2O2 to a sample of bacteria. Finally civilizations were separately exposed to each of several different temperatures, salts and degrees of pH in order to find their optimum environmental conditions. A individual settlement of bacterium was chosen to be identified: bacteriums 1 from dirt sample 2.

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The settlement morphology can be described as a glistening opaque white colour with a level and irregular form. The growing was smooth and soft. The diameter of the settlement approximated 15mm. Under 1000x magnification it was revealed that the cells were bacilli, vest and had a diameter of about 2?m. The cells stained Gram negative.

Table 1: Summary of Results for Unidentified Bacterium 1 of Soil Sample 2



Starch Hydrolysis


H2S decrease




Aerobic or Anaerobic




Denitrification ( NO3- to NO2- )


Nitrification ( NH3/NH4+ to NO2- )


Nitrification ( NH3/NH4+ to NO3- )




Optimum temperature

22 & A ; deg ; C

Optimal pH


Optimal salt concentration

0 %

The consequences of the balance of the trials – biochemical and environmental – are summarized by Table 1. It was concluded based on the Iodine and starch reaction that this bacteria hydrolyzed amylum as a beginning of Carbon. The Sulfur rhythm did non happen as there was no black precipitate from the combination of Iron and H sulphide found in the SIM deeps. The SIM deeps did uncover that these bacteriums were non-motile, turning merely on the stab line. The proteins in the peptone stock were degraded to ammonia meaning that this bacteria is an ammonifer. Nitrification was besides confirmed with the bacteriums oxidising the NH3 and NH4+ in the stocks to NO2- and NO3. Denitrification nevertheless, did non happen ; NO3 was non reduced. The add-on of H2O2 led to bubbling as it reacted with catalase nowadays in the cells. Optimal environmental conditions were found to be 22 & A ; deg ; C, pH of 5 and 0 % salt. Between the temperatures of 4, 15, 22 and 54 & A ; deg ; C, growing was strongest at 22 & A ; deg ; C, so 15 and weakest at 37 and 4 & A ; deg ; C. Growth at pH was merely somewhat stronger than at pH 7 but well stronger than at pH 3 and 9 ( refer to postpone 2 ) . Growth in salt was best at 0 % and decreased with.05 % , 2 % and 5 % severally.

Table 2: Growth of bacteria 1 at assorted pH based on optical density degrees at 580nm


Bacterial Growth










Each of these stairss aided in the possible designation of the bacteria as Azobacteraceae Azotobacter a genera of bacteriums found in dirt, H2O and roots ( Bergey ‘s Manual, 1984 ) . Due to the dilutant bed of peptidoglycan surrounded by a phospholipid outer membrane as opposed to a thick external bed of peptidoglycan this bacteria stained Gram negative ( Prescot, Harley and Klein 2005 ) . Of critical importance for designation was the presence of catalase, an enzyme nowadays in aerophilic bacteriums that breaks down the toxic by-product of negatron conveyance: H2O2 ( Wang et al. 2008 ) . The hunt to place Bacterium 1 began with these two wide standards: Gram negative and aerophilic respiration. This peculiar bacteria was non-motile, the bacteria grew merely along the stab line in the SIM deep instead than distributing throughout the medium. This turned out to be an of import factor while placing every bit it as Azotobacter which contains both motile and non-motile bacteriums ( Bergey ‘s Manual, 1984 ) . These three qualities entirely pointed in the way of Azotobacter ; the biochemical and environmental trials served to corroborate that Bacterium 1 was so Azotobacter by fiting the features of this peculiar genera to the bacteria.

Nitrification was a common feature between the two and was confirmed to happen in Bacterium 1 when the ammonium hydroxide stock was oxidized to nitrite and nitrite was oxidized to nitrate ( Bergey ‘s Manual, 1984 ) . Denitrification nevertheless, did non happen the nitrate was left integral and un-reduced. Ammonification, the dislocation of N incorporating compounds to ammonia, was besides a common feature of Bacterium 1 and Azotobacter ( Bergey ‘s Manual, 1984, Roberts, 2008 ) . Although the trial for motility in the SIM deep was positive, the sulfur rhythm trial in the SIM had a negative consequence. The S incorporating compounds were non reduced by the bacteria to bring forth H2S and, this consequence even as a negative, was an of import factor in designation because Azotobacter besides does non cut down S ( Bergey ‘s Manual, 1984, Roberts, 2008 ) . Bergey ‘s Manual ( 1984 ) classifies Azotobacter as a heterotroph and, likewise Bacterium 1 was identified as a heterotroph when Iodine was added to the run home base incorporating amylum and no colour alteration occurred in the country under and around the settlement. This was an indicant of the bacteria interrupting down and metabolising the amylum. Finally, the optimum environmental conditions of both Bacterium 1 and Azotobactera were found to be really similar. The optimum conditions were stated as: pH of 4.8 – 8.5, temperature of 15 – 37EsC and low salt ( Bergey ‘s Manual, 1984 ) . Bacterium 1 had really similar environmental conditions of: pH of 5, 22EsC and salt of 0 % NaCl.

Sing the nitrifying and ammonifiying qualities of the Azotobacter, this bacteria plays an of import function in the N rhythm by interrupting down proteins and change overing the N into a signifier that can so be used by other beings ( Butenschoen, Marhan and Scheu, 2007, Cakmakci et Al. 2006 ) . Azotobacter, as one of the more common nitrifying dirt bugs, is known to bring forth a great sum of useable Nitrogen, and hence is closely linked to works growing and wellness ( Cakmakci et al. 2006, Prescot, Harley and Klein 2005 ) . Interestingly, although many nitrifying beings hold a symbiotic relationship with works roots, supplying Nitrogen in exchange for foods, Azotobacter, in peculiar, does non ( Prescot, Harley and Klein 2005 ) . Azotobacter besides carries out starch hydrolysis, and hence AIDSs in the decomposition of organic affair in dirt and the mineralization procedure ( Smith and Smith, 2001 ) .

The designation of Bacterium 1 as Azobacteraceae Azotobacter is non definite and several other trials would hold required in order to turn out this statement as true. One trial which would hold been really helpful would hold been a trial for cyst formation ; a cardinal feature of the Azotobacter ( Bergey ‘s Manual, 1984, Prescot, Harley and Klein 2005 ) . The Azotobacter are non rhizobacteria and therefore it would hold been appropriate if there were some manner of detecting the bacteriums in their natural home ground. In add-on, the trials that were carried out had restrictions to the sum of information that could hold been gleaned from the consequences. The nitrification, ammonification, and denitrification trials were based on a simple colour alteration, and there was no manner of stating the procedure by which these maps, if present, occurred, nor were the trials specific to the concentration of the compounds present. All of these trials and techniques could besides hold been subjected to error, for illustration, measurement mistakes under the microscope, mistakes in consecutive dilutions and even, taint of civilizations.

From a simple compost dirt sample it was possible to insulate and subculture a individual bacteria species. Using assorted biochemical trials such as, trials for nitrification, ammonification, denitrification and the sulfur rhythm, it was possible to find the characteristic metabolic maps of the being. These consequences, in add-on to observation of cell and settlement morphology, particularly Gram staining, enabled the designation of the bacteria as Azobacteraceae Azotobacter.


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