Identification Of Bacillus Bacteria From Agricultural Soil Biology Essay

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The roles of microbes in the soil environment are complex, diverse, and crucial to the functioning of every ecosystem. The ecosystem services provided by soil microbes, including bacteria, are to develop soil structure for adequate ventilation and drainage of the soil environment. Drainage and ventilation is important for groundwater recharge and filtration. Soil structure is also an important factor in determining nutrient availability to plants, microbes and other soil organisms. Microbes play major roles in nutrient cycling by converting nutrients, such as nitrogen, to bioavailable forms. Specifically, some soil bacteria form symbiotic relationships with plant roots that dramatically enhance plant growth (Brady and Weil 2008). These services are carried out in the soil environment, which is constantly changing.

The structure and function of a soil community is heavily influenced by ventilation, moisture, temperature, acidity, salinity, and nutrient regimes and fluctuations (Anderson et al. 2009). With increasing anthropogenic influence on soil quality, there is greater selective pressure on soil organisms as fluctuations in environmental become more dramatic (Bell et al. 2009). Ecosystem services of soil communities are irreplaceable, so some researchers have utilized the ability of some microbes to reduce anthropogenic damage. For example, Cheng and Li (2009) have researched which species of Bacillus bacteria can reduce Chromium (VI) to Chromium (III) in metal contaminated soils.

The purpose of this experiment was to isolate a species of bacteria and identify it to the genus level by conducting morphological and biochemical tests on the isolated bacteria (Robertson and Egger 2010).

Methods (Robertson and Egger 2010)

The bacteria were isolated from agricultural soil. Six serial dilutions of soil solution were made, starting with 1:10-2 and decreasing the concentration exponentially to 1:10-7soil::water. The appropriate dilutions were cultured in the following TSA media: slants, broths, deeps, streak plates, pour plates, and spread plates. One culture was prepared and incubated in anaerobic conditions using Brewer's anaerobic medium. The TSA cultures were incubated at 22oC for 48 hours.

From these cultures, a discrete bacterial colony was sub-cultured onto TSA streak plates. Colony morphology was described. Cellular morphology was described using gram staining technique and examining the bacteria at 1000X magnification.

Several biochemical tests were performed to help identify the isolated bacteria. Iodine was used to test for starch hydrolysis. SIM deeps were cultured to test for sulfide metabolism, indole production-tested with Kovac's Reagent, and motility. Peptone broth cultures were prepared and tested with Nessler's Reagent to test for ammonification. Three tests were done to test for nitrification. Firstly, ammonium sulfate culture was tested with Nessler's Reagent, Trommsdorf's Reagent, and dilute sulfuric acid. Secondly, nitrite cultures were tested with Trommsdorf Reagent and dilute sulfuric acid. Thirdly, nitrite cultures were tested with diphenylamine and concentrated sulfuric acid. Denitrification was tested with nitrate broth cultures by adding sulfanilic acid and N.N-dimethyl-1-1-naphthylamine. Two tests were done to determine oxygen requirements: cultures were prepared in thioglycollate medium, and hydrogen peroxide (3%) was added to the colonies on a glass slide.

Optimal environmental growth conditions were tested. Temperature was tested by culturing the bacteria on TSA media and incubating them at 4 oC, 22 oC, 37 oC, and 50oC for 36 hours. The bacteria were cultured in TSB tubes at pH of 3,5,7, and 9 to test acidity tolerance. Salinity tolerance was observed by culturing the bacteria on TSA plates of 0%, 0.5%, 2%, and 5% sodium chloride (Robertson and Egger 2010).

Results

The bacteria appeared to be actinomycete bacteria. It was filamentous in form and had a rough, bumpy, raised texture and filiform boundary. It was pinkish to colourless and dull. The size of individual colonies was not determined because the bacteria occupied the entire area wherever it was cultured. Observations of morphological characteristics, nutrient cycling processes, and growth conditions are summarized in Table 1.

Table 1. Summary of test results for bacterial identification.

Test

Observations and comments

Interpretation

Morphology

Cell morphology

1.05µm x 5.25µm. Rods. Formed into chains.

Strepto bacillus

Gram stain

Purple

Gram positive

Nutrient cycling

Starch hydrolysis

Yellow colour

Positive

H2S reduction

No black precipitate

Negative

Motility

Growth away from inoculation point

Positive

Indole production

No colour change

Negative

Ammonification

(Organic N  NH3)

No colour change

Negative

Nitrification

(NH4+  NO2-)

No colour change

Negative

Nitrification

(NO2- NO3-)

Light purple

Slightly

Denitrification

(NO3- NO2-)

Deep red

Negative

Denitrification

(NO3-  NH4+ or N2)

Not done because of previous negative result

Negative

Catalase

Bubbling

Positive

Growth conditions

Oxygen tolerance

Growth only near top of media

Obligate aerobe

Optimal temperature

Growth at all temperatures, abundant at 22oC and 55oC

Mesophile

Optimal pH

Spectrophotometry. pH 3 = 0, pH 5 = 0.033,

pH 7 = 0.190, and pH 9 = 0.100

Neutrophile

Optimal salt concentration

Abundant growth at NaCl concentrations < 2%,

very little growth at 5% NaCl

Nonhalophile

Discussion

The probable identity of the bacteria isolated from the agricultural soil is of genus Bacillus. The filamentous nature of the bacteria, the colour, and the rod-shaped cells in chains were consistent with the description of Bacillus (Egger 2010).

Bacillus have three major characteristics which are consistent with the tests done on these soil bacteria: a gram positive membrane, can tolerate oxygen, and can form endospores (Todar 2009). Although no endospores were observed microscopically, it could be that since the bacteria had not been subject to environmental stress there were no endospores were present. The ability of these bacteria to grow at wide ranges of temperatures, from 4oC to 50oC (Table 1), supports the hypothesis they can form endospores. This is because the bacteria were only subject to the extreme heat for 36 hours, then refrigerated at 4oC the remainder of the week. Therefore, the endospore could have survived the extreme heat and re-grown at the lower temperature.

These bacteria metabolized starch, which is consistent with the fact that many Bacillus are found near the rhizosphere where they can use plant roots as a source for carbohydrate polymers for carbon and energy. The inability of these bacteria to reduce sulfate to hydrogen sulfide or to carry out nitrogen transformation processes indicates it does not play a major role in making sulfur or nitrogen bioavailable for plants; this consistent with the metabolism of Bacillus bacteria as their main role is to deter pathogenic species from the plant roots (Choudhary and Johri 2009). The isolated bacteria were also found to be obligate aerobes because of the presence of catalase and the growth pattern in the thioglycollate medium (Table 1). This is consistent with the criteria for Bacillus genus (Todar 2009).

Bacillus are well suited to the soil environment because of their ability to cope with varying salt concentrations, water availability, and pH due to their ability to form endospores when growth conditions are unfavorable. Bacillus are commonly symbionts with plants because of their ability to metabolize extracellular chemicals, such as antibiotics, that deter pathogenic microbes and pests. Some of these extracellular chemicals stimulate plant growth hormones or enhance plant defense mechanisms against pathogens. The bacteria utilize the roots as their source of carbon and energy via bio-polymers produced by the plant (Choudhary and Johri 2009).

Further tests could have been performed to further confirm the identity of these bacteria. The simplest test would be to examine bacterial cells with the microscope after they had been subject to environmental stress, to see if any endospores are present. If these bacteria belong to the genus Bacillus, then the type of antibiotics or anti-pathogenic chemicals that may or may not be produced could be determined. More information could be gained about the bacteria if the DNA sequence was examined and compared to other strains of Bacillus spp.

The tests performed to identify the isolated bacteria are limited because bacterial strains are always changing due to genetic recombination (Todar 2009). There are often discrepancies between characteristics of the bacteria described in the literature and the bacteria isolated in the laboratory, there is not often a complete match. The tests performed were very general in that the results did not narrow the possible identities of the bacteria because microbes that share the same environment will carry out similar processes.

Specific sources of error include the gram stain test, which can be variable for two reasons. Errors in procedure cause faulty results because of over-heating when heat-fixing and improper timing, staining, and rinsing with the dyes and ethanol. Results can vary because some bacteria change between gram positive and negative when at different growth phases (Robertson and Egger 2010). The nitrification test often gives falsely positive results. This is because nitrite is toxic to many bacteria; therefore, they detoxify their surroundings by converting nitrite to nitrate. This does necessarily mean the process of nitrification is a part of their nutritional mode.

A bacteria isolated from agricultural soil was deduced to belong to the genus Bacillus according to the morphological and biochemical tests performed in the laboratory.

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