Most soils are home to a diverse community of microorganisms, including fungi and many different types of both Gram-positive and Gram-negative bacteria (Robertson and Egger 2008). The types of bacteria that thrive depend, like with any other growth medium, on the particular soil's environmental factors and nutrient availability and how this matches up with the bacteria's requirements for survival (Robertson and Egger 2008). Most bacteria are facultative anaerobes and can survive with or without oxygen, but others can be obligate aerobes or anaerobes, microaerophiles or aerotolerant (Robertson and Egger 2008). Different types of bacteria are able to perform different metabolic functions related to decomposition and recycling of nutrients found in organic soil matter. They are essential to the breakdown of carbon, sulfur and nitrogen compounds to forms that plants are able to use (Robertson and Egger 2008). Microorganisms also have a specific range of environmental conditions - including temperature, acidity, and solute concentration - in which they can grow. For temperature, most soil bacteria would likely fit into the category of mesophiles (moderate) or in colder climates, psychrophiles. They are commonly neutrophilic, preferring a pH in the 5.5-8 range (Robertson and Egger 2008).
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By performing biochemical tests to evaluate ability to metabolize specific compounds and analyzing growth at different temperatures, pHs and salt concentrations it is possible to determine the identity of an unknown bacterial isolate.
Methods: Soil samples were serially diluted from 10-2 to 10-7 and cultured aseptically. From these cultures, individual bacterial colonies were sub-cultured and grown on Tryptic Soy Agar (TSA) growth medium. Once the individual bacteria species had been isolated, a number of tests were performed for identification purposes.
Colonies were examined for form, elevation, margin, appearance, optical property, colour, texture and diameter. Gram stained slides were prepared to determine whether the bacteria was Gram-positive or Gram-negative, and cells were observed under a microscope at 1000x power to determine the cell shape and arrangement. Biochemical tests were performed to determine whether the bacterial isolate was motile, able to reduce H2S and hydrolyze starch, contained catalase, and carry out ammonification, nitrification and denitrification (Robertson and Egger 2008). Additionally, the bacterial isolate was sub-cultured and grown at temperatures of 4, 10-15, 22, and 55°C; pH of 3, 5, 7, and 9; and salt concentrations of 0%, 0.5%, 2% and 5% to determine optimal environmental conditions.
Results: Gram stain was positive, and the cells were rod-shaped and grouped together. Individual colonies were circular and pale yellow in colour, with an average diameter of about 1mm. The bacterial isolate did not respond to many of the biochemical tests, but was positive for nitrification of NH3/ NH4+ to NO3-, and denitrification of NO3- to NO2- (Table 1). A positive reaction was also found for the catalase reaction with H2O2, and the bacteria grew beyond the stab line in the SIM deep indicating motility. The optimal growing conditions were found to be a temperature of 22°C, pH of 7 and 0.5% salt concentration.
Table 1. Results of tests performed on unknown bacterial isolate.
optical property: opaque
colour: pale yellow
cell shape: bacillus
cell arrangement: clustered
(NO3- to NO2-)
(NO3- to NH3 or N2)
(NH3/NH4+ to NO2-)
(NH3/ NH4+ to NO3-)
Optimal salt concentration
Discussion: Based on the information contained in Bergey's Manual (2nd edition), the unknown bacterium is most likely of the genus Caryophanon. This genus contains Gram-positive, rod-shaped aerobic bacteria. Caryophanon have a characteristic pale yellow colour and are motile, but are inactive to most standard biochemical tests. The optimal pH is around 8, optimal temperature 25°C, very similar to the unknown bacterium's optimal pH of 7 and optimal temperature 22°C. Caryophanon latum is found most often in cow dung (Weeks and Kelly 1957; Dean 1963) and has been found in compost (Ryckeboer et all 2003).
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Due to its inactivity to most standard tests, little is known about Caryophanon and only two species have been identified, C. latum and Caryophanon tenue. The two species are very similar other than a slight difference in size (Bergey's Manual 1986). Though Bergey's Manual classifies Caryophanon as aerobic, it has been suggested that it is actually microaerophilic and is thus able to survive passage through the rumen of a cow, where conditions are mostly anaerobic (Dean 1963).
Since there is little information available about Caryophanon other than its inactivity to most common tests, if the unknown is indeed of this genus more testing may not be of any help in confirming the classification however inactivity to more biochemical tests could be considered affirmative to the potential identification.
The pH testing results may have been affected by the inability to break up large chunks of bacterial growth that could have resulted in higher absorbance readings.