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Soil microorganisms are important in cycling carbon and nitrogen, and with water movement (Tolli and King 2005). The variety and metabolic diversity of bacteria in soil is great, containing nitrogen-fixers, hydrogen-oxidizers, sulfur-oxidizers and nitrifyers (Beffa et al. 1995). Composting by bacteria is a significant process required by humans to manage organic wastes such as landfills, sewage, and in the production of agricultural soil (Beffa et al. 1995). Generally, fewer bacteria are found in agricultural soil than in forest soil, but, in both, diversity increases with depth (Tolli and King 2005). All soil microorganisms vary depending on environmental factors such as temperature, water content, biological variables as well as differences in soil type and cultivation practices (Tolli and King 2005).
A bacterium will be isolated through sub-culturing from a forest soil sample. Biochemical tests will be performed, and effects on growth due to environmental factors will be observed for the isolated bacterium. The characteristics of known bacteria types will be compared with the isolated bacterium to determine its identity.
The following tests were performed as described by Robertson and Egger (2010). Serial dilutions of 10-7 were prepared beginning with 1 g of a forest soil sample in 99 mL of deionized water as the 10-2 dilution. Using the aseptic technique and the 10-2 dilution, a broth, a slant and a deep were inoculated. Also using the 10-2 dilution, a streak plate and two spread plates (one on Brewer's anaerobic medium) were prepared. Four pour plates were made using the 10-4 to 10-7 dilutions of the soil sample. One week later a distinct colony was selected from the aerobic streak plate and sub-cultured aseptically on to a new TSA plate and TSA slant. A gram-stain was done for the selected bacteria colony. Observations were recorded all throughout this procedure, and the colony and cellular morphology recorded by sight and light microscope, respectfully. After the colonies were successfully sub-cultured, biochemical tests were performed, including tests for starch hydrolysis, hydrogen sulfide production and motility, ammonification, nitrification, dentrification (nitrate reduction), oxygen tolerance and presence of catalase and oxidase. The catalase test was performed by sub-culturing the bacterium on a TSA plate, and one-week later adding hydrogen peroxide, watching for bubbles indicating catalase activity. We looked for a colour change to purple with the addition of para-Aminodimethylaniline oxalate to indicate the presence of oxidase. Bacterial growth depending on environmental factors of varying temperature, pH and osmotic pressure were tested and observed. Finally, the observations and results were combined and a potential identity for the isolated forest soil bacterium was determined.
After sub-culturing, macroscopic colony morphology was highly distinct. The colony had a round form with convex elevation, a smooth margin, and was an opaque cream colour. It appeared shiny and very slimy.
Cellular morphology was discovered with the use of a light microscope at 1000X magnification. The isolated bacterium had a streptobacillus form, approximately 0.06 µm by 2 µm. No endospores, cysts, or capsules were visible. The bacterium was found to be gram-positive.
Biochemical tests displayed characteristics useful for determining the bacterium's genera (Table 1). The isolated bacterium did not hydrolyze starch, produce hydrogen sulfide or ammonify (Table 1). The colony was found to be somewhat motile. It was able to denitrify NO3- to NO2-, but not NO3- to NH4+ or N2 (Table 1). The isolate nitrified NO2- to NO3-, but not NH4+ to NO2- (Table 1). The bacterium did not produce indole or oxidase, but it did produce catalase, and was determined to be a facultative anaerobe (Table 1).
Table 1. Biochemical Tests Results of Unknown Isolated Bacterium from Forest Soil Sample
Denitrification (NO3- to NO2-)
Dentrification (NO3- to NH4+ or N2)
Nitrification (NH4+ to NO2-)
Nitrification (NO2- to NO3-)
The effects of environmental factors on bacterial growth were observed (Table 2). The bacterium grew best at incubation temperatures of 37°C downwards to 4°C (Table 2). Using a spectrophotometer to measure growth it was found this bacterium preferred a pH of 7, but grew at pH 5 (Table 2). Growth of this bacterium decreased as the percentage of NaCl increased, and it grew well only at osmotic pressures of 0% and 0.5% NaCl (Table 2).
Table 2. Preference of the Isolated Bacterium for Growth as Effected by Environmental Factors
37°C to 4°C
5 to 7
The isolated bacterium from the forest soil sample is assumed to be from the genus Xanthobacter. The isolated bacterium shares many characteristics with Xanthobacter, although there are some minor variations between the two.
In appearance, the isolated bacterium was very similar to Xanthobacter in that it had a round, high form with an entire margin and was shiny in appearance (Holt and Krieg 1984). Xanthobacter often has a yellow colour that is caused by the pigment zeaxanthin dirhamnoside (Hertzberg et al. 1976). The isolated bacterium had more of a cream colour than yellow. However, white Xanthobacter species do exist; they just have low levels of this pigment (Hertzberg et al. 1976). As is characteristic of Xanthobacter, the isolated bacterium produced copious amounts of slime (van Ginkel and de Bont 1986).
Xanthobacter had common cellular morphology to the isolated bacterium as it was observed under the microscope. The isolated bacterium was found to be streptobacillus with dimensions of 0.6 µm by 2 µm, which is in line with that of Xanthobacter, characterized by rods of varying sizes of 0.4 µm - 1 µm to 0.8 µm - 6 µm (Holt and Krieg 1984). The gram stain procedure showed the isolated bacteria to be gram-positive. Xanthobacter is gram-variable but it usually stains gram-positive (Holt and Krieg 1984). However, because it contains lipopolysaccharides it belongs to the gram-negative bacterial group (Holt and Krieg 1984). Neither endospores, cysts, or capsules were observed; Xanthobacter only sometimes produces capsules (van Ginkel and de Bont 1986)).
In the biochemical tests it was found this bacterium does not hydrolyze starch, produce hydrogen sulfide or undergo ammonification (Table 1), just as Xanthobacter does not (Holt and Krieg 1984). The isolated bacterium displayed slight motility. Xanthobacter species can be either motile or nonmotile (van Ginkel and de Bont 1986).
This bacterium completed denitrification from NO3- to NO2-, but did not denitrify NO3- to NH4+ or N2 (Table 1), the same as Xanthobacter (Egger 2010). This bacterium was found to complete nitrification (both NH4+ to NO2- and NO2- to NO3-) (Table 1) but Xanthobacter does not nitrify (Holt and Krieg 1984). However, there are heterotrophic nitrifyers commonly present in soil that may be giving this positive result for nitrification in this sample (Tolli and King 2005). In addition, the slime produced by Xanthobacter commonly traps contaminants which could include these heterotrophic nitrifyers (Holt and Krieg 1984).
The isolated bacterium did not produce indole or oxidase, but it did produce catalase (Table 1). Xanthobacter does not produce indole, but produces both oxidase and catalase (Egger 2010). As a presumed species of Xanthobacter the isolated bacterium would be expected to produce catalase. Through observations of bacterial growth in the thiogycollate medium the isolated bacterium was determined to be a facultative anaerobe (Table 1) where as Xanthobacter are classified as obligate aerobes (Holt and Krieg 1984). This is a minor difference, and was determined for the isolated bacterium by observed growth in thioglycollate medium.
The effects on growth of the isolated bacterium with varying environmental factors gave further evidence that it belongs to the genus Xanthobacter. The optimal temperature for the isolated bacterium ranged from 37°C to 4°C (Table 2) characterizing it between a psychrotroph and mesophile, like Xanthobacter which has an optimal temperature of 28°C to 31°C (Padden et al. 1997). Xanthobacter species are neutrophiles or alkalophiles, adding alkali to adjust the acidic pH of the slime, and have an optimal pH of 5.8 to 9.0 (Holt and Krieg 1984). The isolated bacterium was found to prefer a pH between 5 and 7 (Table 2). Growth was highest with a lower osmotic pressure and salt concentration (Table 2) indicating the isolate to be a nonhalophile like the nonhalophile/halotolerant Xanthobacter (Holt and Krieg 1984).
There are many other tests that could help confirm the identity of this isolated bacterium as a member of the genus Xanthobacter. As their zeaxanthin dirhamnoside is what separates them from other yellow-pigmented lithotrophs and nitrogen fixers, isolating this pigment with ethanol as described by Hertzberg et al. (1976) would assist in confirmation. To confirm the results of the Gram-reaction the bacteria should be tested for the presence of lipopolysaccharides (Holt and Krieg 1984). Xanthobacter stains gram-positive or variable, but because it contains lipopolysaccharides it belongs to the gram-negative bacterial group (Holt and Krieg 1984).
Although the cellular morphology characteristics between the isolated bacterium and Xanthobacter were very similar it should be noted that according to Holt and Krieg (1984) shape, gram-stain and motility are the main causes of error in identity of bacteria. In addition, selecting a single colony does not ensure purity, especially when it has not been sub-cultured many times (Holt and Krieg 1984).
Xanthobacter is found free-living in soil with decaying organic material, or in water (Holt and Krieg 1984). In an experiment by Padden et al. (1997) Xanthobacter was commonly found around the roots of marigold plants (Tagetes patula and Tagetes erecta) where it could grow on the many released organic nutrients such as carbon and sulfur. It was suggested the production of extracellular slime is for attachment to the root surface.
A bacterium was successfully separated from a forest soil sample. After colony and cellular morphology observations, biochemical tests, and observations on the effects of varying environmental factors on growth, the isolated bacterium was assumed to belong to the genus Xanthobacter based on the numerous shared characteristics.