Microorganisms, such as bacteria, fungi, algae, protozoa and other organisms, within mixed communities can be found in soil, and they interact with each other in various ways (Singleton 1992). Microorganisms play an essential part in biochemical processes that recycle sulfur, nitrogen, and carbon and in maintaining soil productivity. They break down organic compounds to inorganic compounds, which can be used as nutrients by plants, thus serving as link between animals and plants (Singleton 1992). Bacteria compose the majority of microbial population in soil; species of Bacillus, Clostridium, Arthrobacter, Pseudomonas, Rhizobium, Azotobacter, and Nitrobacter are generally present (Pelczar 1993). Other microorganisms such as fungi exist abundantly near the soil surface where oxygen is readily available (Pelczar 1993). Due to their contribution to soil structure formation; decomposition of organic matter; toxin removal; and the biochemical cycling of carbon, nitrogen, phosphorous, and sulfur, microorganisms are crucial in maintaining the soil function, and besides, their extent of diversity is critical to the maintenance of soil health and quality (Pelczar 1993). The number and kinds of microorganisms are affected by the presence of roots and the extent of the root system in soil. Our objective is to isolate and identify a pure culture of soil bacterium from a mixed population of soil microorganisms (Pelczar 1993).
Methods
To start with, we cultured the mixed populations of aerobic and anaerobic microorganisms from an agricultural soil sample. First, a series of serial dilution were prepared from an agricultural soil sample and they are 10-2, 10-3, 10-4, 10-5,10 -6,10 -7. Four pour plates were prepared by pipetting inoculum from the 10-4,1 0-5, 10-6, 10-7 dilutions of the agricultural soil sample, and were incubated at 22 oC for 48 hours, and were then placed in the fridge (4oC). In order to obtain the pure culture of the soil bacteria from the mixed populations, the streak plate subculture was prepared from one of the colonies in the pour plate. And gram-staining was performed for the bacterium. Bacterial colony morphology was recorded and cellular arrangement was examined using compound microscope. Biochemical activities of the soil bacterial isolate in the cycling of carbon, nitrogen and sulfur were tested; those activities include starch hydrolysis, H2S production, motility, ammonification, nitrification, denitrification, oxygen tolerance and catalase production. To examine the environmental factors, such as temperature, PH and osmotic pressure, on the growth of the bacterial isolate, the isolate was tested at 4, 22, 37 and 50 oC; at pH 3, 5, 7 and 9; and at different concentrations of salt (0, 0.5, 2,and 5% NaCL) under incubation for one week (Robertson and Egger 2010).
RESULT
The strain we studied was isolated from the pour plates incubated at 22 oC. The isolate was Gram positive, facultative anaerobic, catalase positive, motile rods with flagellated organisms. Our strain produced flat, filamentous, rough, rhizoid colonies with a diameter of 15mm, and they are dull, opaque and white. The cells are rod-shaped and are arranged as a single bacillus with a dimension of 4um*1um (table 1). It was tested positive for starch hydrolysis, ammonification, denitrification (NO3- to NO2-), nitrification (NH4+ to NO2-) and catalase production (table 1). It showed a wide growth temperature range from 4 to 50oC, with an optimal temperature of 37 oC, and it had a PH growth range from PH 3 to PH 9 with an optimal Ph of 7, but there was no growth at PH5 (table 1). It tolerated salt concentration from 0% to 5%, and grew best at 0.5% and was inhibited by high concentration of salt in the medium (table 1).
Table 1. Main characteristics of the strain we studied.
Characterictics
Temperature range
4-50 oC
Optimal temperature
37 oC
PH range
3-9
% NaCL range
0%-5%
Cell dimensions (um)
4*1um
Chains of cells
_
Colony diameter (mm)
15 mm
Gram stain
+
Starch hydrolysis
+
H2S reduction
_
Motility
+
Ammonification
+
Indole production
_
Denitrification (NO3- to NO2-)
+
Denitrification (NH4+ to N2)
_
Nitrification (NH4+ to NO2-)
+
Nitrification (NO2- to NO3-)
_
Discussion
Our isolate was identified as strains of Bacillus species. Their colonial and microscopic morphologies and biochemical test profiles were typical for these species. The cells of genus Bacillus are rod-shaped and may occur singly or in chains (Goodfellow et al, 1998), compared to our observation that the cells were rod-shaped and were arranged as a single bacillus. The rods may be quite small (0.5*1.2um) or rather large (2.5*10 um) (Goodfellow et al, 1998), and our measured dimension of the rods is about 1*4 um, which is within the above range. Our result indicates the production of catalase, which matches the profile of genus Bacillus that most species produce catalase (Goodfellow et al, 1998). Most Bacillus species are motile by means of flagella (Egger and Robertson 2010) and our test showed the indication of motility. The cells can be gram-positive or negative or positive only in the early stage of growth (Goodfellow et al, 1998), and our test suggests that the cell is gram-positive. Cell morphology of Bacillus strains varies greatly with environmental factors, including composition of medium, temperature of inoculation, humidity; however, our strain has as the same form of rhizoid as Bacillus mycoides (Goodfellow et al, 1998). Colonies of pure culture can be translucent or opaque, whitish or cream-colored in a single plate, related to the density of endospores within the colonies (Goodfellow et al, 1998), while our strain is opaque and white. Strains of Our isolated bacteria were facultively anaerobic while the published data suggest that they can be aerobic or facultively anaerobic. Endospores are formed by Bacillus species in unfavorable environment (Goodfellow et al, 1998). The impervious coat that surrounds the endospore can prevent it from adverse environment such as excessive heat, freezing and desiccation, so they can be dormant in nature for a long period of time
(Robertson and Egger 2010). Endospore structure can be confirmed by subjecting it to temperature of 70-80C for 10min and cultivate it under appropriate condition (Goodfellow et al, 1998). The form of endospores is characteristic features of Bacillus species; it is usually cylindrical, or ellipsoidal or oval or round (Goodfellow et al, 1998). However, it should be noticed that endospores can not be formed under all cultural conditions, and thus nonsporulating Bacillus species may be misidentified for other genera. Biochemical reactions such as denitrification, nitrification, ammonification and starch hydrolysis were detected (Goodfellow et al, 1998). Denitrification is common among Bacillus species, during which nitrate is used as a terminal electron acceptor during respiration and nitrite is generated (Goodfellow et al, 1998).Our result confirmed reaction because nitrate was reduced to nitrite with no further reduction. Bacillus species are ammonifiers, capable of transforming N containing organic compounds, such as proteins, to ammonia (Egger and Robertson 2010). Our isolate carried out ammonification since ammonia was detected. Our result shows that starch was consumed by our bacteria, using starch as their carbon source, and it matches McSpadden’s result (2004). All the strains of Bacillus species tested in McSpadden’s experiment gave negative results in indole and H2S production tests, corresponding to our results that Indole and H2S were not produced (2004). Bacillus species can grow at both low and high temperature (Goodfellow et al, 1998); our result shows that our bacteria can grow at temperature ranging between 4 and 50 oC, with an optimal temperature of 37 oC. Their ability to withstand high temperature can be attributed to the growth of endospores when returned to an optimal temperature (Goodfellow et al, 1998). Our bacteria can grow at PH from 3 to 9, with an optimal PH of 7, but can not grow at 5, compared to the published data which suggest that Bacillus species can grow at low and high PHs (Goodfellow et al, 1998). The fact that there was no growth at PH 5 can be attributed to errors made during our experiment. When we were using the aseptic technique to inoculate the agar plates; cells could be killed before incubation due to the killing of inoculum by the heated loop. Some strains of Bacillus species are salt tolerant, some can only grow under solution with certain salt concentration (Goodfellow et al, 1998).Our bacteria have wide growth range of salt concentration,with an optimal concentration of 5.
In order to determine the range of growth temperatures, growth of culture was observed at 50 oC and 65 oC after 24h of incubation, at 37 oC after 2 days, and at 15oC after 14 days (Llarch et al. 1996). Instead of observing the growth of the culture one week after incubation, we can improve our experiment by observing cultures that grow at different temperatures after different period time of incubation.
Utilization of organic acids such as citrate and propionate can be tested by inoculating slants of citrate and propionate utilization medium, and incubating them for 14 days. A red color indicates utilization of organic acids. Production of dihydroxyacetone can be tested by inoculating plates of tyrosine agar with one streak of inoculum and incubate them, clearing of the tyrosine crystals around and below the growth gives a positive result (Goodfellow et al, 1998).
Bacillus species play a vital role in the biological cycling of carbon and nitrogen due to their ability to degrade a series of biopolymers (Goodfellow et al, 1998). Besides, Bacillus species can promote crop health and productivity by suppressing plant pathogens and pests, and their uses in agriculture are being exploited. Some strains of Bacillus species have been developed as biological fungicides, insecticides, and nematicides or generic plant growth promoters (McSpadden 2004). Our objectives were achieved as we isolated our bacteria and identified them as Bacillus species.
LITERATURE CITED
Goodfellow, M., Williams, S.T., Mordarski M.. 1998. Bergey’s Manual of Systematic Bacteriology, Volumn 2. Springer, NY, pp. 1104-25.
Llarch, A., Logan, N.A., Castellvi, J., Prieto, M.J., Guinea, J.. 1996. Isolation and Characterization of Thermophilic Bacillus spp.from Geothermal Environments on Deception Island, South Shetland Archipelago.Microb Ecol. 34:58-65.
McSpadden, G.B.. 2004. Ecology of Bacillus and Paenibacillus
spp. in agricultural systems. Phytopathology 94:1252-1258.
Pelczar, M.J.. 1993. Microbiology--concepts and applications. McGraw-Hill, NY, pp. 77-83.
Robertson, S and Egger, K. 2010. BIOL 203 Microbiology Laboratory Manual. UNBC.
Singleton, Paul. 1992. Introduction to bacteria : for students of biology, biotechnology & medicine / Paul Singleton. Chichester, NY, pp. 144-9.
(Robertson and Egger 2010)
