Bacteria are the most abundant organisms in the soil, present in densities of up to billions of cells per gram of soil (Paul and Clark 1996). Typically, the density of bacterial communities decreases with soil depth, and is greatest in the rhizosphere - the area of soil influenced by plant roots (Paul and Clark 1996). The higher density at the rhizosphere is due to the availability of organic compounds released from the plant roots (Bolton Jr et al. 1993). Bacteria are important decomposers and participants in the carbon cycle, and within the rhizosphere are even more important decomposers than fungi (Griffiths et al. 2007). Many species of bacteria are able to fix atmospheric nitrogen, making significant contributions to the nitrogen cycle (Paul and Clark 1996). Bacteria have been shown to promote plant growth by nutrient cycling and by producing antibiotics, insecticides, and plant growth regulators (Asaka and Shoda 1996; Broadbent et al. 1977; Idris et al. 2004; McConnell and Richards 1959; Turner and Backman 1991).
To study the versatility of soil bacteria, a bacterial colony was isolated from agricultural soil. Tests were performed to determine the bacterial isolate's ability to utilize a range of substrates and to grow in varying environmental conditions. Further observations and tests were used to identify the Genus of the bacterial isolate.
Methods (adapted from Robertson and Egger 2010)
An agricultural soil suspension was prepared with 1g of soil in 99mL of sterile deionized water. A Tryptic Soy Agar (TSA) spread plate was inoculated with 0.1mL of the soil suspension spread evenly using a sterile rod and aseptic technique. Unless otherwise noted, all cultures were incubated at 22°C for 48 hours and then stored at 4°C for five days. Plates were inverted to prevent condensation buildup on the colonies, and broths were placed on a shaker. One week later, an isolated bacterial colony was chosen from the spread plate and used to prepare a TSA streak plate sub-culture. The colony was gram stained and examined on an optical microscope at 1000x. Measurements were calibrated against a stage micrometer.
After one week, the macroscopic appearance of the sub-cultured colonies was observed. Numerous sub-cultures were prepared for various biochemical tests: a starch agar streak plate to test for starch hydrolysis using iodine; a SIM (sulfide, indole, and motility) deep to test for H2S production, indole production (using Kovac's reagent), and motility; a peptone broth to test for ammonification using Nessler's reagent; a nitrite broth to test for nitrification (using Trommsdorf's reagent in dilute H2SO4 to test for nitrite and phenylamine in concentrated H2SO4 to test for nitrate); a nitrate broth to test for denitrification (using sulfanilic acid and N,N-dimethyl-1-naphthylamine to test for nitrite ions followed by zinc powder to test for unreduced nitrate); a thioglycollate broth to test oxygen requirements; and a TSA streak plate to test for catalase (using hydrogen peroxide) and oxidase (using p-Aminodimethylaniline oxalate).
Bacterial growth was tested against temperature by preparing four TSA streak plates, incubated for 36 hours at 4°C, 22°C, 37°C, and 50°C respectively. TSB tubes at pH 3, 5, 7, and 9 were used to test growth against variable pH. Growth at various osmotic pressures was tested using TSA streak plates at 0%, 0.5%, 2%, and 5% NaCl. The pH test tubes and osmotic pressure test plates were incubated at 22°C for 36 hours. After incubation, the TSA streak plates and TSB tubes were stored at 4°C for 5.5 days prior to observation. The extent of growth dependant on pH was determined by using a spectrophotometer to measure the absorbance of each broth at 580nm, with higher absorbance indicating a greater extent of growth. The extent of growth for the temperature and osmotic pressure test plates was determined by visual inspection.
Results
The sub-cultured colonies appeared round, with sharply defined, rough edges (Table 1). A representative colony had a diameter of 7mm and a low, slightly convex profile. The colonies were matte, slightly translucent, and cream colored. Observed at 1000x, cells were gram positive straight rods arranged in chains. Cells were approximately 0.8μm x 2μm in size.
Table 1. Macroscopic and microscopic morphology of an unknown bacterial colony isolated from agricultural soil. The colony was sub-cultured from a community grown from a soil inoculum. Cultures were grown on Tryptic Soy Agar (TSA) media at 22°C for 48 hours.
Colony Morphology
Cell Morphology
Shape
Elevation
Margin
Appearance
Diameter
(mm)
Shape and
arrangement
Dimensions (single cell)
Gram stain
Round, convex
Low
Sharply delineated, rough edge
Cream colored, matte, slightly translucent
7mm
Straight rods in chains
0.8μm
x 2μm
Positive
The colony was able to hydrolyze starch, and exhibited ammonification, nitrification, and denitrification (Table 2). The colony exhibited a slight preference for growth in aerobic conditions and tested positive for catalase activity, but also grew successfully in anaerobic conditions. This indicated that the bacteria were facultative anaerobes. An Oxidase test yielded a negative result, as did tests for H2S and indole production. The bacteria did not appear to be motile, as no cultures were observed away from the stab line in the SIM deep.
The bacteria exhibited optimal growth at 22°C, with growth progressively declining at 37°C and 50°C respectively (Table 3). Even at 50°C, moderate growth was still observed. No growth was observed at 4°C. Colony growth was observed at pH 5, 7, and 9, with an optimal pH of 7 and no growth observed at pH 3. The colony grew at all osmotic pressures tested, from 0% to 5% NaCl. Growth was optimal at 0% NaCl, and declined progressively as the salt concentration increased.
Table 2. Results of biochemical tests performed against an unknown bacterial colony isolated from agricultural soil. +: positive test result; −: negative test result.
Starch hydrolysis
H2S
production
Motility
Indole production
Ammonification
Nitrification
to NO3-
+
−
−
−
+
+
Denitrification
to NO2-
Denitrification to NH4 or N2
Oxygen use/tolerance
Catalase activity
Oxidase activity
+
−
Facultative anaerobe
+
−
Table 3. Temperature, pH, and osmotic pressure preferences of an unknown bacterial colony isolated from agricultural soil. Temperature variable sub-cultures were incubated at 4°C, 22°C, 37°C, and 50°C; pH variable sub-cultures were grown on TSB plates at pH 3, 5, 7, and 9; and osmotic pressure variable sub-cultures were grown on TSA streak plates at 0%, 0.5%, 2%, and 5% NaCl. *: a small amount of growth was still observed in these conditions, although a marked decline in growth was noted.
Temperature (°C)
pH
Osmotic Pressure (% NaCl)
Minimum
22
5
0%
Optimal
22
7
0%
Maximum
Not reached
9*
5%*
Classification
Mesophile
Neutrophile
Nonhalophile
Discussion
The isolated bacterium is likely a member of Genus Bacillus, characterized by gram positive cell walls, a straight rod shape no more than 2.5μm in diameter, lack of filaments, aerobic or facultative anaerobic respiration, catalase activity, unpigmented colonies, denitrification, and a lack of sulfide production (Sneath 1986). These characters are all demonstrated by the isolated bacteria. On the other hand, Bacillus sp. are typically motile, and often produce both endospores and capsules (Sneath 1986). Motility, endospores, and capsules were not observed in the isolated bacteria, although only motility was directly tested for and is lacking in some Bacillus species and strains (Sneath 1986). Endospores may not be detectable without the use of phase contrast microscopy, or may not form at all unless the growth medium is supplemented with manganous salts (Sneath 1986). Capsules are not produced by all Bacillus species, and may not be detected without specific staining techniques (Sneath 1986). Therefore, the lack of observable motility, endospores, and capsules does not rule out Bacillus sp. as the probable identity of the isolated bacteria. Another possibility is Genus Cellulomonas, however this was considered less likely as the isolated bacteria grew moderately well at 50°C, whereas members of Cellulomonas rarely grow above 43°C (Jones and Collins 1986). Finally, the isolated bacteria formed cream colored colonies approximately 7mm in diameter, whereas Cellulomonas usually forms bright yellow pigmented colonies 1-3mm in diameter (Jones and Collins 1986).
The use of phase contrast microscopy would aid in detecting endospores, which are characteristic of Bacillus (Sneath 1986). India ink may be used as a stain to determine if capsules are present, presenting as a clear region around bacterial cells if capsules are present (Butt et al. 1936). Various staining techniques can also be used to emphasize flagella, if present (Leifson 1951). Determining if endospores, capsules, and flagella are present would aid in differentiating between Bacillus and Cellulomonas.
Bacillus species are common soil organisms, and their nutritional and ecological roles vary widely (Sneath 1986). Some species of Bacillus, such as B. polymyxa, are known to fix nitrogen (Hino and Wilson 1958). Experiments have shown that the growth of plants is promoted when an active Bacillus community is present (Broadbent et al. 1977; Turner and Backman 1991). Bacillus communities may promote plant growth due to their contribution to nutrient cycling (Broadbent et al. 1997); the production of insecticidal toxins such as those produced by B. thuringiensis (McConnell and Richards 1959); the production of phytopathogen antibiotics by species such as B. subtilis (Asaka and Shoda 1996); and the production of plant growth regulators such as gibberellins and auxins (Broadbent et al. 1977; Idris et al. 2004).
When preparing streak plates for testing growth against temperature and osmotic pressure, it was impossible to be sure that the streaks were identical from plate to plate. Some plates may have been inoculated with more bacterial cells than others, and the distribution of the inoculum over the surface may have varied from plate to plate, possibly affecting the extent of growth observed after incubation. This may be mitigated by using spread plates instead. Testing of bacterial growth against temperature was done in fairly coarse increments. No growth was observed at 4°C, and optimal growth was observed at 22°C. As no temperatures between 4°C and 22°C were tested, a minimum temperature of 22°C is likely an inaccurate assumption. When preparing the SIM deep for testing motility, the inoculating needle used was slightly bent. The stab line was quite wide as a result. To avoid confusing the thickness of the stab line with motility, bacteria was not considered motile unless present far away from the stab line. This may have led to a false negative if motile bacteria migrated only a small distance.
In conclusion, the bacterial isolate from agricultural soil was identified as Genus Bacillus to a moderately high degree of certainty. The bacterial isolate was able to hydrolyze starch; exhibited ammonification, nitrification, and denitrification; grew in both aerobic and anaerobic conditions; and grew moderately well even at 50°C. This range of traits is indicative of a fairly versatile organism, and agrees with other studies which have demonstrated the ability of Bacillus to function in both carbon and nitrogen cycling (Sneath 1986).
