Soil microbial communities are very important to their surrounding ecosystems. Through metabolic processes microorganisms decompose organic material and cycle nutrients; some are even able to degrade pollutants (Nie et al. 2009). In agricultural environments nitrogen can be exhausted in the soil unless plants, like Faba beans, that form symbiotic relationships with nitrogen fixing bacteria are rotated in as a crop (Köpke and Nemecek, 2010). Microbial relationships are very important for sustainable farming. According to Köpke and Nemecek (2010) these nitrifying bacteria produce H2, which is used as an energy source by other bacteria and therefore increases biomass of many other microbial organisms. The conditions in which the microbes live greatly affect their populations and communities. It is found that populations of microbes are greater in the rhizosphere than in bulk soil (Nie et al. 2009). Also, the communities of bulk soils tend to be more greatly affected by salt concentration and water availability than those in the rhizosphere (Nie et al. 2009). Microbial biomass promotes vegetation growth and fertilizers can affect microbial biomass especially since nutrients like phosphorus are usually limiting in plant and microbial growth (Heinze et al. 2009). Heinze et al. (2009) found that higher pH levels promoted growth in microbial communities. Manure, which is often used as an organic fertilizer, increases the soil pH and therefore the growth of microbial communities and vegetation (Heinze et al. 2009). In this study a bacterial isolate from agricultural soil was isolated and tested to determine its identity and role in the environment.
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MATERIALS AND METHODS: (Robertson and Egger, 2010)
I cultured a Tryptic Soy Agar (TSA) streak plate with a 10-2 dilution of Agricultural soil that I made and incubated it for 48 hours at room temperature (about 22oC). I sub-cultured four individual bacterial colonies on a TSA streak plates and TSA slants. Using the same four colonies I conducted gram stains and observed the bacteria under 1000X magnification on a calibrated microscope.
Colonies from the sub-cultured TSA streak plates were incubated then kept at 4oC and tested the following week. Bacteria were cultured on a starch agar plates and tested for starch hydrolysis by adding iodine. I cultured SIM deeps and looked for motility, production, and, after adding Kovac's reagent, Indole production. I inoculated Peptone tubes and I added a loopful to a drop of Nessler's reagent to test for ammonification. I inoculated ammonium sulfate tubes and a nitrite tubes and added a loopful of each to separate wells containing 3 drops of Trommsdorf's reagent and a drop of dilute to test for nitrification. I also added a loopful of ammonium sulfate broth to a drop of Nessler's reagent to test for nitrification. Nitrate tubes were inoculated and I added sulfanilic acid and N,N-dimthyl-1-1-naphthylamine to test for denitrification. I inoculated tubes of thioglycollate to test oxygen tolerance. TSA plates were inoculated and then I added 3% to a section (test for catalase) and P-aminodimethylaniline oxalate to another section (test for Oxidation).
Bacteria were then tested in differing environmental conditions. Some TSA plates were inoculated and incubated at varying temperatures (4, 22, 37, and 50oC) to test optimum temperature and tolerance while others were inoculated using different concentrations of salt (0, 0.5, 2, and 5% NaCl) to test optimum salt concentrations and tolerance. I inoculated TSB tubes at varying pH`s (pH 3, 5, 7, and 9) and measured bacterial growth using a spectrophotometer set at 580nm.
In table 1 the physical characteristics of the unknown bacteria and their colonies are noted.
Table 1. Observable Physical characteristics of Unknown Bacterial Colony and Individuals.
Colony morphology Slightly raised, bumpy and smooth, shiny, opaque,
and a beige/orange colour.
Cell morphology Bacillus (1.024.08μm) and variable, clumped
Motility No movement away from stab line (-)
Bacilli were present but they weren't the only cell type, instead cell type seemed to vary, having some cocci forms and filaments as well (Table 1). Table 2 below is shows results of many biochemical tests on the colonies.
Table 2. Biochemically tested characteristics of Unknown Bacterial Colony.
Gram Strain +
Starch Hydrolysis +
Indole production -
Denitrification ( to ) +
Denitrification ( to or ) -
Nitrification ( to) -
Always on Time
Marked to Standard
Nitrification ( to ) +
The colony tested positive in the gram test and therefore has peptidoglycan (table 2). Ammonification occurred (Table 2) but not very much as the peptone broth with the Nessler's reagent only turned light yellow. The reaction of Trommsdorf's reagent and sulfuric acid with the cultured nitrate broth gave a dark brown colour, which was still considered positive (Table 2) for nitrification although a positive reaction is usually indicated by blue/black. Starch hydrolysis was positive (Table 2) because no blue/black colour developed around the colony, but no purple colour occurred anywhere on the entire plate, even in areas not colonized by any of the 4 bacterial cultures. Table 3 below is the result of culturing bacteria in several different media.
Table 3. Effect of Environmental Factors on Bacterial Colony Growth
Factor Observation (and Classification)
Oxygen tolerance Most growth at surface ( rich, therefore Aerobe)
Optimum temperature 4-22oC (Psychrophile - Mesophile)
Optimal pH 3 (Acidophile)
Optimal salt concentration 0-0.5 NaCl (Growth at 5% NaCl, Halotolerant)
The bacteria grew very well at both 4 and 22oC (Table 3) and seemed to be able to grow over a wide temperature range. The bacteria were able to survive in all salt concentrations (Table 3) but did not grow as well at high concentrations and lost coloration. The bacterial growth at pH 3 was over double the growth at all other pH levels, therefore the bacteria were acidophiles (Table 3).
The unknown microbe's suspected genus is Rhodococcus. This suspicion is gleaned from characteristics shared by the unknown and Rhodococcus. Williams (1984) tells us that Rhodococcus is positive for gram stain, catalase, ammonification, and denitrification, which are all consistant with the unknown bacterial traits (Table 2). Williams (1984) also gives a mesophilic growth range of 15-40oC for Rhodococcus while my tests indicate a temperature range that encompasses, and is greater than, Rhodococcus' range with an optimum temperature at the lower end of this range (Table 3). However Rhodococcus has been shown to have enzymes which allow it to function in -1.8-2.0oC temperatures of Antarctic seas (Pini et al. 2007). This coincides with my results of high growth at 4oC, but also introduces a source of error because this means the bacteria would be able to grow while they were in the fridge at 4oC. The unknown showed rod shaped bacteria of 1.024.08μm (Table 1). This agreed with the diameter range of 0.8-1.0μm (Egger, 2010) for Rhodococcus. The cell morphology seemed confusing as there was more than just the bacilli type present, but this is explained by Rhodococcus' rod-coccus life cycle which involves filaments and clumping (Willaims, 1984) which were also seen in the unknown (Table 1). We also find that Rhodococcus can live in salt concentrations up to 5% which is the same as the unknown (Williams, 1984). The colony morphology is described by Williams (1984) as rough, smooth or mucoid with a cream, yellow, orange, or red colour which coincides with the unknown (Table 1). The unknown tests positive for Starch hydrolysis, but negative for oxidation, production (Table 2), and motility (Table 1) as does the Rhodococcus (Egger, 2010). The starch hydrolysis test was a bit concerning because there was no indication of any starch left on the plate at all, even in spots where no bacterial colonies grew. To remove this possible source of error additional starch hydrolysis tests could be conducted using only one unknown bacteria on the TSA plate rather than four. Rhodococcus is aerobic (Egger, 2010) and the unknown's growth is mostly near the surface of the thioglycollate medium, however there were a few colonies growing on the sides of the test tube along its length which makes it unclear if the bacteria are obligately or facultatively aerobic.
More tests could be conducted for identification, many of which are based on metabolic processes of the bacteria or their composition. To be sure of the genus Rhodococcus I would like to examine the unknown's amino acid and sugar composition, and using thin layer chromatography I would test for amino acids and mycolic acid (Williams, 1984). Genus Rhodococcus did not match in all areas though because the unknown was positive for nitrification ( to ) as seen in table 2, but according to Egger (2010) Rhodococcus is negative for nitrification. Egger (2010) does warn against basing the identity too heavily on the nitrification. There is also a chance that results for the unknown are affected by other bacterial colonies, especially because for many of the tests multiple colonies were grown and tested on the same TSA plate. One of the four colonies seemed to be invasive and infiltrated other colonies on a few occasions, thus making testing more difficult and less accurate.
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The unknown bacteria has an important role in soil ecology and the nitrogen cycle as it is able to nitrify to which enables the nitrogen to be taken up by plants (Krome et al. 2009). Rhodococcus erythropolis (a potential identity of the unknown) can clean up petroleum pollutants from soil environments through bioremediation (Lin et al. 2010). Further tests on the unknown could involve adding petroleum to soil and bioaugmenting some samples with the unknown bacteria and leaving other samples as negative controls. The petroleum could then be extracted from the soil using dichloromethane (Lin et al. 2010) to see if a there is significant difference in the remaining amounts of petroleum. One of the roles of the unknown microbe in the environment, nitrification, was detected by biochemical testing. The exact identity of the bacteria remains unknown but there is strong evidence to support that it belongs to the genus Rhodococcus.
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Robertson, S. and Egger, K. 2010. BIOL 203 Microbiology Laboratory Manual. UNBC.
Williams, S.T. 1984. Bergey's Systematic Bacteriology Vol. 4, Lippincott William and Wilkins 351 West Camden St. Baltimore, MD 21201-2436 USA, pages 2362-2367.