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One handful of soil can have an uncountable number of microorganisms, each contributing to the functioning of the ecosystem in their own way (Torsvik and Ovreas 2002).
There is immense structural and functional diversity among the soil microorganisms, and still many microorganisms have not yet been observed under the microscope (Torsvik and Ovreas 2002). Microorganisms inhabiting soil can belong to a wide range of groups, from the less common Archaea to the more common types of aerobic bacteria, such as gram negative or gram positive bacteria (Willey et al. 2008). The more common strains of gram positive or negative bacteria are usually coccus-shaped, bacillus-shaped, or spiral in shape and include genii, such as Bacillus and Staphylococcus (Robertson and Egger 2008).
Bacterial species also have a diverse range of activities and allow biogeochemical processes, such as cycling of important elements, to occur (Willey et al. 2008). Bacteria have the capability to undergo common ecological processes such as decomposition, mineralization, and hummification, which all transform reduced organic matter into inorganic substances and nutrients (Torsvik and Ovreas 2002). Examples of this are the gram positive and negative bacteria, which can undergo many ecologically important processes such as ammonification, a process that converts organic nitrogen into the inorganic nitrogenous species, ammonia (Robertson and Egger 2008). These metabolic processes create the perfect habitat for larger eukaryotic organisms, supplying them with the inorganic substances in the soil required for growth (Willey et al. 2008).
The composition of the microbial communities is determined by varying environmental factors such as temperature, acidity, light, oxygen content, salinity, soil texture, and availability
of required nutrients (Wakelin et al. 2007). These factors change across time and space and favor certain microorganisms over others (Wakelin et al. 2007). Most microbes have unique metabolic processes that allow them to survive in conditions where other microbes would perish (Wakelin et al. 2007). Most microbes growing in soil are facultative anaerobes (Robertson and Egger 2008). Soil bacteria are also mesophilic, adapted to temperatures between 25oC and 45oC (Robertson and Egger 2008). Compost soil can be composed of thermophilic bacteria, which can grow between 45oC and 70oC (Robertson and Egger 2008). Soil bacteria are accustomed to an optimum pH between 5.5 and 8, and therefore are called neutrophiles (Robertson and Egger 2008). Soil bacteria also grow optimally in low to moderate salt conditions (Wakelin et al. 2007).
Identifying microorganisms based on their characteristics is key to industries, such as medicine and agriculture. One key method in identification is Gram stain, where gram-negative and positive bacteria can be differentiated based on the composition of their cell wall (Robertson and Egger 2008). The types of biochemical processes, such as starch hydrolysis, hydrogen sulfide production, nitrification, ammonification, and denitrification, the soil microbe is capable of undergoing also serves as a tool for their identification (Robertson and Egger 2008). All these processes require enzymes to carry out their functions, and the presence of these enzymes can be determined by biochemical tests (Robertson and Egger 2008). For example, Nessler's reagent can determine the presence of ammonia in the culture, and therefore can determine whether the microorganism has the enzymes capable of carrying out the process of ammonification (Robertson and Egger 2008). The objectives of this study was to isolate an aerobic bacterial species from forest soil, and by culturing it and running a series of biochemical tests, to determine the bacterial isolates characteristics, optimum growth conditions and thus its identity.
Materials and Methods
A 10-2 serial dilution was prepared from one gram of forest soil, according to the procedures described in Biology 203 Lab Manual (Robertson and Egger 2008). A further 10-4 dilution was prepared and the bacteria in this soil sample were cultured by preparing Tryptic Soy Agar (TSA) pour plates. Out of all of the colonies growing on the TSA plate, one distinct cream-colored colony was isolated and subcultured onto a streak TSA plate. Using the original colony on the pour plate, gram staining was conducted (Robertson and Egger 2008). The bacteria were then examined under a microscope and their dimensions and characteristics were recorded. The iodine test was conducted by inoculating the bacterial isolate onto a starch agar plate for 48 hours at 25oC, after which iodine was added to the plate to test for starch hydrolysis (Robertson and Egger 2008). To test for H2S production and motility, Sulfide Indole and Motility deeps were inoculated and incubated for 48 hours at 25oC (Robertson and Egger 2008). Ammonification, nitrification, and denitrification ability was tested by culturing the unknown bacteria in peptone, ammonium sulfate, nitrite, and nitrate broth tubes, respectively, for 7 days at 25oC, and by using reagents and procedures listed in Biology 203 Lab Manual (Robertson and Egger 2008). Catalase presence was determined by pouring 3% H2O2 onto a slide containing the bacteria. To determine optimal temperature, new subcultured TSA plates were incubated at either 4oC, 10oC, 15oC, 22oC, or 50oC for 36 hours and then examined. Four subcultured TSB tubes, each of a different pH of either 3,5,7 or 9, were incubated for 36 hours at 25oC. To determine the optimum pH, a spectrophotometer set at wavelength of 580nm was used to measure the extent of turbidity or growth in each tube (Robertson and Egger 2008). Four TSA plates, each containing a different salt concentration of either 0%, 0.5%, 2%, or 5% NaCl were inoculated and incubated for 36 hours at 25oC. This allowed the optimal NaCl concentration to be determined for growth of the unknown bacteria.
The unidentified bacterial colony isolated and cultured has a variety of distinguishable features (Table 1). The shiny, smooth, and flat colony has an irregular form with undulated margins (Table 1). It is cream in color and individual colonies are 9mm in diameter (Table 1). The colony consists of 0.5umx3um singular, rod-shaped, gram positive bacteria, which do not hydrolyze starch or reduce hydrogen sulfide (Table 1). Mobility was noticed in deep tubes (Table 1). The bacterial isolate is an ammonifier, and degrades organic nitrogen into ammonia, but does not undergo nitrification (Table 1). Bacterial cultures are capable of undergoing denitrification, by reducing nitrate to ammonium ions or nitrogen gas (Table 1). The unknown bacterial colony is composed of aerobic bacteria that degrade hydrogen peroxide via the enzyme catalase (Table 1). The bacteria are mesophilic and neutrophilic, and grow at an optimum temperature of 22oC, and at an optimum pH of seven. The optimal salt concentration is 2% NaCl (Table 1).
By using the Bergey's Manual of Systematic Bacteriology (Holt 1986), the bacterial isolate was identified to be a member of the genus Bacillus. Some of the identifying characteristics were that that the unknown bacteria were gram positive, rod-shaped, and catalase-positive. Other identifying characteristics were that the bacteria could not hydrolyze starch or produce hydrogen sulfide, but could undergo ammonification and denitrification. Colony morphology also helped in determining the bacterial isolate from other rod-shaped, gram positive bacteria.
Bacillus consists of a wide variety of species, and consequently, they have a wide variety of roles in nature. Bacillus is widely distributed across the world and occur in almost any habitat, due to the resistance of their endospores, which are highly resistant to heat, drying and many disinfectants (Holt 1986). Species are usually aerobic or facultative anaerobes (Holt 1986).
Table 1. Summary of Results for Isolated Unknown Bacterium.
Optical property: opaque
Dimensions: 0.5um x 3um
(NO3 - to NO2 -)
(NO3 - to NH3 or N2)
(NH3/NH4 + to NO2 -)
(NH3/NH4 + to NO3 -)
Optimal salt concentration
One of their most ecologically important activities of decomposing organic materials and transforming them into inorganic nutrients for plants depends on their ability to undergo ammonification and denitrification (Holt 1986). The ammonia or nitrogen gas produced from these processes can be transformed into other inorganic nitrogenous species by other microorganisms, making nitrogen available to larger eukaryotic organisms (Willey et al. 2008). Some species of Bacillus are also pathogenic, and infect insects, plants, and humans (Holt 1986).
This makes them excellent pesticide or biological control agents, used in agriculture to kill off unwanted insects and fungi (Gao et al. 2007). One example is the use of Bacillus subtilis as a pesticide to kill the green mold pathogen infecting citrus fruit (Leelasuphakul et al. 2007). Taking advantage of their abilities to decompose certain chemicals such as esters and aromatic compounds, they have also been used in bioremediation, in an effort to take pollutants and anthropogenic chemicals out of the earth (Lakshmi et al. 2007).
There were limitations to the tests that were performed. For example, the results of gram staining depend on the user's technique; if a poor technique is used then false results could be obtained (Leboffe and Pierce 2005). This was one of the sources of error that could have affected our results. Gram staining depends on the age of the culture, where older cultures give different results (Leboffe and Pierce 2005). Also, it wasn't possible to determine if the bacteria denitrified nitrate to ammonia or to nitrogen gas, which could have helped in our identification. Other sources of error could have occurred due to the imperfect techniques used to culture and test the bacterial isolate.
Another test that could have helped in identifying the bacteria was the Gelatinase test, that determines the bacteria's ability to produce gelatinases (Leboffe and Pierce 2005). In addition, the capsule stain could have determined the ability of the bacterial isolate to produce a capsule, and the endospore stain could have determined the presence of endospores (Leboffe and Pierce 2005). Bacilllus is known to be positive for these tests. Also, a test determining the pH after the bacteria was cultured on glucose could have helped in identifying the bacterial isolate, since marked acidity on glucose is a characteristic of Bacillus (Holt 1986).
In conclusion, our objectives were reached and a bacterial isolate was successfully isolated from forest soil and cultured. The characteristics and optimum growth conditions were found and it was identified to the genus Bacillus.