Identification Of Sporosarcina Bacteria Isolated From Forest Soil Biology Essay

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Microbial communities are an immensely important part of soil structure and function. Microbes cycle nutrients, such as carbon and nitrogen, in the soil, making them available for use by plants (Rogers and Tate III 2001, Wu et al. 2008), and they affect the physical structure of soil by forming microaggregates of soil particles (Wu et al. 2008). The microbial community is also influenced by the soil; the soil type and its properties (Wu et al. 2008) and the litter type (Rogers and Tate III 2001) can affect the size and activity of the microbes. The flora growing in the soil can also have a major impact on microbial communities; forest and agricultural soils having very different community compositions (Macdonald 2009).

There are several ways to identify the microbes composing a soil community, including soil enzyme activity, microbial community enumeration, and metabolic diversity patterns (Rogers and Tate III 2001). In this study, bacteria were cultivated from agricultural and forest soils, and a single bacteria was identified by characteristics such as cell morphology and biochemical cycling and metabolism.

Methods

The methods were adapted from Roberston and Egger (2010). Cultures were prepared from 10-2 dilutions of agricultural and forest soils in slants, broths, deeps, streak plates, spread plates, and anaerobic spread plates, and from dilutions of 10-4-10-7 pour plates were prepared. From these, four distinct colonies were subcultured on streak plates and slants, and gram staining was applied to the original cultures. The subcultures were then cultured in TSA plates, starch agar plates, SIM (Sulfide, Indole, and Motility) deeps, peptone, ammonium sulfate, nitrite, nitrate, and thioglycollate broths to be tested for nutrient cycling. TSA subcultures were tested with H2O2 and p-Aminodimethylaniline oxalate, an additional test not included in Robertson and Egger (2010), starch plates tested with iodine, peptone and ammonium sulfate broths with Nessler's reagent, ammonium sulfate and nitrite broths with Trommsdorf's reagent and dilute H2SO4, nitrite broths with diphenylamine and concentrated H2SO4, nitrate broths with Reagents A,B, and C, SIM deeps observed for motility, H2S production, and tested with Kovac's reagent, and thioglycollate broths observed for oxygen preference. The subcultures were then cultured at 4, 22, 37, and 50°C on TSA plates to test for temperature preference, in broths at pH 3, 5, 7, and 9, and in NaCl concentrations of 0, 0.5, 2, and 5%. The temperature and NaCl plates were observed for growth extent, and the absorbance at 580nm was measured for the pH tubes. From the information collected, a single bacterial genus from the forest soil was identified.

Results

The isolated bacteria was a gram-positive coccus of diameter 1.46um with cells that did not aggregate together and were found to be motile (Table 1). Colonies were round and shiny, with a pale pink to orange cream color ranging to non-pigmented (Table 1). The bacteria did not hydrolyze starch, reduce hydrogen sulfide, or produce indole (Table 1). Nitrate was not denitrified, but tests indicated that the bacteria did nitrify nitrite to nitrate, and produced ammonia from nitrogen compounds (Table 1). Tests for oxygen tolerance indicated an aerobic or microaerophilic bacteria, although there was no oxidase or catalase activity (Table 1). Growth occurred between 4 and 22°C, optimally at neutral pH, and in up to 2% NaCl (Table 1). From the observations and test results, it was determined that the bacteria was most likely a member of the genus Sporosarcina (Egger 2010).

Table 1. Summary of the characteristics observed and tested for to identify the forest soil bacteria of genus Sporosarcina.

Test

Results

Colony Morphology

Circular, convex, shiny, opaque, smooth, slightly pink/orange/no pigment, entire margin

Cell Morphology

Single coccus, 1.46um

Gram Stain

Positive

Starch Hydrolysis

None

H2S Reduction

None

Motility

Motile

Ammonification

Strong ammonifier

Indole production

None

Denitrification (NO3-to NO2-)

None

Denitrification (NO3-to NH4+to NO2-)

None

Nitrification (NH4+to NO2-)

None

Nitrification (NO2-to NO3-)

Yes

Catalase / Oxidase

No catalase activity / No oxidase activity

Oxygen Tolerance

Aerobic/Microaerophilic

Optimal Temperature

Growth at 4-22°C (Psychrotroph)

Optimal pH

Optimal growth at pH 7 (Neutrophile)

Optimal Salt Concentration

Growth up to 2% NaCl (Nonhalophile)

Discussion

Using colony morphology, cell morphology and gram-stain, the possible identity of the bacteria was first narrowed to the genera Micrococcus, Sporosarcina, Staphylococcus, Sarcina, and Arthrobacter (Egger 2010). Motility, ammonification, colony morphology, and cell morphology indicated that of these, Sporosarcina was the most probable identity (Egger 2010). Lack of denitrification, starch hydrolysis, indole production, and H2S production also supported the identity, as did oxygen tolerance (Egger 2010). These characteristics were further supported by Claus and Fahmy (1986), Kocur and Martinec (1963), and Yoon et al. (2001). Although Egger (2010) indicated that Sporosarcina was both oxidase and catalase positive, some species in the genus are oxidase negative (Kocur and Martinec 1963). Egger (2010) also indicated that Sporosarcina cells aggregated as diplococci or in tetrads, but Kocur and Martinec (1963) and Claus and Fahmy (1986) have shown that there are species in the genus occuring as single coccus, as the isolated bacteria did. Several species of Sporosarcina are psychrotrophic or psychrophlic (Yoon et al. 2001, Reddy et al. 2003) as the isolated bacteria was. The isolated bacteria did not reduce nitrates to nitrites as some species in the genus do, but it has been shown that this trait can be variable (Kocur and Martinec 1963, Yoon et al. 2001).

An erroneous characteristic the indication that the isolated bacteria nitrified ammonium ions to nitrate. This is typically a characteristic of heterotrophic nitrifying bacteria like Nitrosomonas, Nitrosococcus and Nitrobacter, aerobic gram-negative bacteria (Egger 2010). The indicative test results were odd, as Nessler's reagent indicated no ammonia and Trommsdorf's reagent indicated no nitrite in the ammonium sulfate broth, and diphenylamine reagent indicated the presence of nitrate in the nitrite broth (Table 1). The test of the nitrite broth with Trommsdorf's reagent, however, produced a brown product for all four of the bacteria tested; a negative result should have produced no reaction, leaving the liquid clear, and a positive result should have yielded a blue-black colour (Robertson and Egger 2010). No explanation was found for this phenomenon.

The negative result of the catalase test is also problematic. All indications are that Sporosarcina are catalase positive (Kocur and Martinec 1963, Claus and Fahmy 1986, Yoon et al. 2001, Reddy et al. 2003, Egger 2010). The only catalase negative bacteria provided by Egger (2010) were Sarcina, Actinomycetes, and Nitrobacter, all of which did not match the other characteristics of the bacteria. No explanation was found for this indication, though Busse et al. (1996) stated that test for catalase can be difficult to standardize and interpret.

The reliability and success of the tests used must be taken into consideration when identifying a bacteria such as Sporosarcina. Many of the tests used to identify Sporosarcina indicated the presence of biochemical pathways and phenotypes which can be variable between species of the same genus (Busse et al. 1996). For example, Egger (2010) and Kocur and Martinec (1963) show that Sporosarcina are oxidase negative, while Clause and Fahmy (1986) indicate that the genus is oxidase positive. Some results for these tests, such as the nitrite broth reaction with Trommsdorf's reagent, cannot be explained if they do not give one of the expected results. Busse et al. (1996) also point out that many tests, for example those for indole, catalase, and oxidase, are difficult to standardize and interpret, which may explain the negative results for the catalase test. They also point out that newly isolated strains of a culture may react differently than those which have been stored or subcultured over a longer period of time and that many identification systems were designed for specific taxa and may not work the same way with other organisms (Busse et al. 1996). It must also be considered that some techniques and tests used, such as gram-staining, take practice to be done properly and well. All the tests undertaken in the lab were being performed for the first time by someone with no previous experience. In order to ensure accurate results, it is suggested that multiple cultures be made in test media and the tests themselves be performed in duplicate.

There are also a variety of other tests that could be used to confirm the identity of Sporosarcina. As Sporosarcina are endospore forming bacteria (Kocur and Martinec 1963, Claus and Fahmy 1986, Yoon et al. 2001), testing for endospore formation using terbium diplocolinate photoluminescence (Pellegrino et al. 1998) or total luminescence spectroscopy (Smith et al. 2004) could substantiate the identification. Some species have the ability to decompose casein, gelatin, and tyrosin (Claus and Fahmy 1986), so testing for removal of these substance from medium may indicate Sporosarcina. It has also been found that Sporosarcina possess menaquinone systems (Claus and Fahmy 1986) so identification of the these could contribute to a positive identification. 16S rRNA analysis is an extremely useful tool for identifying phylogenetic relationships between bacteria (Busse et al. 1996) which has been successfully used to distinguish species by their similarities to Sporosarcina sequences in Antarctic soil (Aislabie et al. 2006, Aislabie et al. 2008), and so could be used identify the isolated bacteria as a member of the genus.

Like the bacteria in Antarctica, Sporosarcina are predominantly found in soil (Claus et al. 1986, Acha et al. 2009), as well as some species such as S. macmurdoensis found in ponds. One of the large roles of Sporosarcina in the soil is the production of urease, which catalyzes the production of CO2 and ammonia, lowering the pH of surrounding soils and causing the precipitation of mineral ions (Acha et al. 2009). The addition of urea to the soil can have a detrimental effect on germinating seedlings and young plants, as well as populations of microorganisms (Omar and Ismail 1999), and species like S. ureae play an active role in the decomposition of urea (Claus and Fahmy 1986).

To the extent possible with the given biochemical and nutrient tests and observations, the bacteria isolated from the forest soil was identified as a member of the genus Sporosarcina. Not all tests, such as the catalase test, matched to Sporosarcina, but the similarities between Sporosarcina and the isolated bacteria were enough to provide an identification as a member of the genus. Without more biochemical testing, or even DNA or RNA analysis, it cannot be said for certain that the bacteria is a member of Sporosarcina but the study was successful in providing a very probable identity for the bacteria as Sporosarcina.

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