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Identification and characterization of soil bacteria are essential in examining microbially mediated functions that are of agronomic and environmental importance (Robertson & Egger 2010).
Soil bacteria contribute to nutrient cycling through the degradation and humification of organic matter and are principal agents of nitrogen fixation through nitrogenous oxidation and reduction processes (Cappuccino 2002, Leboffe & Pierce 2005). Arthrobacter, Cellulomonas, and Actinomyetes genera are major decomposers of organic proteins and carbohydrates, while Nitrosomonas, Pseudomonas, and Staphylococcus genera are principal agents in ammonia oxidation and nitrate reduction (Egger 2010, Holt 1986). Bacterial distribution in soil is governed by physical soil composition and aggregation, as well as environmental factors such as oxygen tolerance, tonicity gradients, and temperature (Halsall & Gibson 1986).
Microbial isolation and taxonomic classification using morphological characteristics and biochemical or environmental reactions are useful in identifying unknown bacteria (Cappuccino 2002). In the present study, bacterial populations of forest and agricultural soils are isolated and subject to biochemical tests and varying environmental conditions in order to identify the bacteria to a single genus.
Aseptic inoculation of serially diluted agriculture and forest soil samples into TSA and TSB media followed by seven days incubation yielded mixed bacterial genera which were sub-cultured and isolated (N=4).
A series of biochemical tests were performed upon each isolate to examine bacterial response including differential Gram staining, the iodine test for starch hydrolysis, motility and hydrogen sulphide detection in SIM media, and indole complexation with Kovacââ‚¬â„¢s reagent. Nesslerââ‚¬â„¢s reagent was added to peptone and ammonium sulphate broths to test for bacterial ammonification, while nitrification was examined both by acidic phenylamine addition to nitrite broth and by acidic Trommsdorfââ‚¬â„¢s addition to nitrite and ammonium sulphate media. Denitrification was tested in nitrate broth supplemented with sulfanilic acid and N,N-dimethyl-1-1-naphthylamine. Growth distribution in thioglycollate media, the oxidase test, and the hydrogen peroxide test for catalase activity indicated bacterial oxygen tolerance.
TSA and TSB bacterial growth response to temperature (4, 22, 37, 50 Â°C) and osmotic pressure (0, 0.5, 2, 5 % NaCl) were qualitatively measured, while bacterial turbidity in acidic and alkaline condition (3, 5, 7, 9 pH) were spetrophotometrically determined.
As summarized in Table 1, colonial morphology of isolates exhibit convex, opaque, erose spheres 0.2-4.0 mm in diameter and yellow-orange in color, while cellular morphology occurs as a mixture of irregular cocci and clustered, short bacilli.
Table 1. Summary of colonial and cellular morphological characteristics of the sub-
cultured soil bacterial isolate
0.2-4.0 mm diameter
Bacilli with interspersed cocci
Bacilli: 3 um x 1 um
Cocci: 1 um diameter
As shown in Table 2, bacteria are predominantly Gram positive although some saffranin counterstaining is present. Some cellular lateral movement is observed, suggesting some cellular motility. A clear zone encircling bacteria in iodine testing indicates amylase exoenzyme presence, while absence of black growth in SIM media indicates hydrogen sulphide absence (Table 2). Bacteria exhibit minor ammonification and indole production, as indicated by faint reagent color change to light yellow and red, respectively (Table 2). Oxidation of ammonium and nitrite is not observed. Bacteria exhibit strong denitrification to nitrite with a rapid red reagent color change, but do not display reduction to ammonium or molecular nitrogen (Table 2). Bacteria are facultatively anaerobic as indicated by catalase-mediated gas evolution with the application of hydrogen peroxide, absence of color change for the oxidase test, and greater growth near thioglycollate media air interfaces (Table 2).
Table 2. Summary of biochemical tests performed on the sub-cultured soil bacterial
Mostly positive; some counterstaining
NO3- to NO2-
NO3- to NH4+ or N2
NH4+ to NO2-
NO2- to NO3-
Facultatively anaerobic distribution
Bacteria are neutrophilic at optimal pH 7, nonhalophilic with optimal growth at 0% [NaCl] and no growth at 5 % [NaCl], and mesophilic with optimal growth near 22 C and no growth at 50 C (Table 3). However, bacteria also express strong growth at pH 9 and 37 C (Table 3).
Table 3. Summary of growth response to environmental conditions of the sub-cultured
forest soil bacterial isolate
Osmotic pressuire (%NaCl)
On the basis of biochemical and morphological data analysis, the bacterial isolate is a likely member of the genus Cellulomonas. The genus is composed of common soil bacteria of at least 10 species of the family Cellulomonadacae, class Actinobacteria (Rainey et al. 1995).
Cellulomonas cells appear as slender, v-shaped rods that appear shorter or coccoid in older cultures due to exhaustion of fermentable carbohydrate from agar media (Rainey et al. 1995). Cellulomonads are Gram positive, are facultatively aerobic, effectively hydrolyse starch through amylytic exoenzymes, and are not major contributors to nitrification or denitrification (Egger 2010; Holt 1986). Species of Cellulomonas are readily decolorized in Gram staining, while a single polar or sparse lateral flagella of some members confers motility (Rainey et al. 1995; Holt 1986). Rhodococcus and Arthrobacter genera also share similar cellular morphological and biochemical characteristics, but Cellulomonas is readily distinguished by its yellow-orange color and oxidative and fermentative glucose metabolism (Bagnara et al. 1985). Cellulomonas are mesophilic, nonhalophilic bacteria with optimal growth and metabolic activity at neutral or slightly alkaline pH which corresponds to the high growth of the bacterial isolate observed at pH 7 and 9 (Rainey et al. 1995; Holt 1986).
Soil, particularly with much vegetation, is the main habitat for cellulomonads (Halsall & Gibson 1986). In addition to amyltic ability, Cellulomonas is instrumental in environmental degradation by its function in generating and secreting cellulase for the lytic decomposition of cellulose (Halsall & Gibson 1986; Holt 1986). Cellulomonas may form association complexes with other genera to decompose organic matter and enable biological processes such as nitrogen fixation (Halsall & Gibson 1986). Commercial use of Cellulomonas includes composting formulas and waste degradation in cotton and fiber industries (Summers & Srinivasan 1979).
Biochemically, urease and glucose tests may be useful in differentiating Cellulomonas from some Actinobacteria genera (Bagnara et al. 1985). Some Cellulomonas species may exhibit endospore or cyst formation (Egger 2010; Holt 1986). Thus, the endospore test using heated malachite green dye may be effective in positive identification of Cellulomonas (Leboffe & Pierce 2005). Isolation of genomic DNA and PCR amplification will likely provide the most effective means of precisely identifying bacterial genus to the molecular level (Rainey et al. 1995).
Methodological weaknesses of the present study include the limited reproducibility of results, as reporting mainly relied on qualitiative analysis through the use of chromatographic indicators and simple numeric rating scales. Other sources of potential bias or error include differences in incubation or storage conditions, impurities in stock culture or media, as well as calibration error in pipettes or the spectrophotometer. In addition, the effect of metabolic and biochemical diversity at different stages of bacterial life cycles are largely ignored.
Nevertheless, study objectives are met, as successful microbial inoculation, separation, and classification techniques were employed to biochemically and morphologically identify the unknown soil bacteria as a likely member of the genus Cellulomonas.