The presence of any particular species of bacteria is largely dependent on existing soil conditions and the availability of necessary foods for the bacteria to consume. Of all soil organisms, bacteria possess the greatest metabolic capabilities. They are capable of extremely fast growth and under certain ideal circumstances; the populations of some bacteria can double in just minutes.
Active decomposers, soil bacteria and fungi, operate within a certain temperature and pH range. Mesophiles, the most common bacterial type, prefers soil at 25-35°C (Alexander, 1977). Microorganisms thrive at an optimal pH near neutrality, but grow in a wider range of pHs. A neutral pH provides adequate supplies of inorganic nutrients, a balance of air and water-filled pore space (about 50-60% of water holding capacity) and abundant organic substrates (carbon and energy sources). When any one of these parameter get too far beyond the normal range some segment of the population will likely be stressed. For example, aerobic (oxygen requiring) bacteria will be at a disadvantage when a soil becomes waterlogged and available O2 is depleted through respiration of roots, microbes, and soil animals.
Fungi favour more slightly more acidic soil than bacteria. In soil more acid than pH 5.5, the flora is dominated by filamentous fungi (Alexander, 1971).
Compared with bacteria, actinomycetes are less sensitive to environmental conditions such as heat or drought because they produce structures that allow them to survive these adverse conditions. However, actinomycetes are sensitive to pH and are almost absent below pH 5.
Below this pH, very few bacteria and actinomycetes can survive, while many fungi grow very well. Fungi also survive unfavourable environments with specialized structures called sclerotia and conidia. These store food and other materials until conditions are favourable for them to germinate and form a new mycelium or a fruiting body.
Table 3 displays a few factors affecting microbial distribution in soils.
Table 3. Principle environmental factors affecting soil microbes
The most important limiting factor for microbial growth in soil (assuming moisture is adequate) is the abundance of available organic carbon sources. The vast majority of soil microbes are organotrophs, they require organic carbon compounds to oxidize for energy and to build the organic constituents of their cell bodies. Only a few types of soil bacteria get their carbon from CO2 (autotrophs) and they contribute little to the overall organic matter content of a soil.
The natural abilities of soil microbial population to degrade various compounds are the basis of microbial remediation. Bioremediation is the term applied to technologies that accelerate natural processes for degrading harmful chemicals in soil and groundwater. Microbes that exist naturally in soil produce enzymes which break down hydrocarbons into smaller, less toxic materials. Modern biotechnology has selectively adapted naturally occurring microbes for their ability to digest specific hydrocarbon contaminants such as oil and gasoline. When combined with nutrients, pH stabilizers, oxygen, and surfactants, these microbes attack the offending materials at a rapid rate to minimize contamination and reduce or eliminate the environmental hazard (Blaine Metting Jr., 1993).
AIMS
These laboratory sessions should enable the operator:
- To gain an understanding of the application of aseptic methods use in microbiology.
-
To further student's understanding of the process of dilution and plate counting for the enumeration of microbes.
- To determine the number of bacteria, actinomycetes and fungi present in soil.
- To understand and use the plant count technique.
- To demonstrate activities of organisms in the nitrogen cycle.
EXPERIMENTAL PROCEDURES
ALL EXPERIMENTS HAVE BEEN PRODUCED AS DIRECTED BY THE PROTOCOL PROVIDED.
The most widely used and accepted technique in soil microbiology for enumerating soil microorganisms is the dilution plate count technique.
RESULTS
- Experiment 1: Enumeration of soil microorganisms
After incubation at 25 °C for three days, number of colonies has developed on the plates.
Each medium will favourised the growth of a different type of microorganisms:
- SDA: fungi
- GYEA: actinomycetes
- TSA: bacteria
Table 4 to Table 6 displays the count of colonies on the different plates incubated, organised by media. The number of bacteria, actinomycetes and fungi present in 1g of soil was determine by multiplying the number of colonies counted on each plated by the inverse of the dilution factor (example: on the SDA medium, 69 colonies have formed at a dilution of , thus 69*=0.69*cfu/ml, as 1ml of the solution made with 1g of soil has been poured into the petri dish.).
All calculations will be expressed as a power of , in order to compare results.
TMTC: too many to count.
Table 4: Count of colonies on SDA medium, and number of colony
forming unit.
Table 5: Count of colonies on GYEA medium, and number of colony
forming unit.
Table 6: Count of colonies on TSA medium, and number of colony forming unit.
Plates having between 25 and 250 colonies should be taken into account.
The first two dilutions on each media were insufficient to get a countable number of colonies.
On the SDA medium, the dilution allowed a count of 69 colonies, but the dilution did not allowed enough colonies to form.
On the TSA medium, not enough colonies formed at any dilution factor.
On the GYEA medium, the dilution allowed 116 colonies to form, but the dilution did not get quite enough forming colonies.
- Experiment 2: Demonstration of ammonification in soil
Ten tubes have been incubated at 30 °C for 3 days. Six of them have been inoculated with known bacteria, two with 1g of soil, and two will act as controls.
The production of ammonia has been observed according to the colour reaction obtained when a few drop of Nesslers reagent to 0.1 ml of the resultant solution.
Table 7 displays these results.
Table 7: Colour intensity and colouration after incubation when adding Nesslers reagent.
Both soil tubes (7 and 8) display the darkest colouration, thus, according to the scale providing, the more production of ammonia occurred in those tubes.
Both control tubes (9 and 10) and tube 1 display the lightest colouration, thus, no or very little ammonia production occurred.
-
Experiment 3: Demonstration of denitrification in soil
Four tubes were incubated for three days at 30 °C, two tubes with nitrate broth (one with soil, one with Pseudomonas aeruginosa), and two tubes with nitrate-free broth (one with soil, one with Pseudomonas aeruginosa).
Every tube contained a Durham tube to check if gas was formed during incubation.
One drop of alpha napthylamine and one drop of sulphanilic reagent were added to each tubes, the presence of nitrites would be demonstrated by a formation of a red colour in the tube.
A pinch of powdered zinc was added to tubes that failed to develop a red colour.
The formation of a red colour after addition of powdered zinc demonstrates the presence of nitrate in the tube.
Table 8 display the results obtained.
Table 8: Results of experience 3.
Gas production did not occur in the nitrate-free tube containing Pseudomonas aeruginosa.
Only the nitrate free broth tube containing Pseudomonas aeruginosa has reacted positively (red colour development) to the addition of reagents, indicating the presence of nitrites.
DISCUSSION
Experience 1
Soil microorganisms can be isolated from the soil environment and grown on artificial media. Different media encourage the growth of different types of microorganisms through the use of inhibitors and specialized growth substrates. The numbers of organisms capable of growth on a specific media are referred to as "colony forming units" (CFU).
Tables 4 to 6 show that the number of bacteria is higher than the number of actinomycetes and fungi. To compare numbers, results are all expressed as a function of. Thus, numbers of bacteria range from 14 to 60 (x ), numbers of actinomycetes range from 11.6 to 18 (x), and numbers of fungi range from 0.5 to 0.69 (x) cfu.
As confirmed by this experiment, the most numerous microbes in soil are bacteria (Table 1), then actinomycetes, and fungi in order.
Table 9 also confirm this order of dominance in numbers by those microbes in relation to the depth of the sampled garden soil.
Table 9:Distribution of microorganisms in numbers per gram of typical garden soil at various depths.
(Source: Internet 1)
Bacteria are generally the more numerous because they are the smallest, most diverse group of microorganisms. They are diverse in shape, the methods by which they get energy, in the types of compounds they can decompose and the conditions under which they can live. Some bacteria can live in the absence of oxygen while others can only live in the presence of oxygen, others can live either with or without oxygen. There are bacterial that can grow at low, medium and high temperatures and some can survive and grow in extremes of pH and water availability. Some bacteria need only the simplest medium to live grow and reproduce. They can live using sunlight for energy, carbon dioxide as a sole carbon source and inorganic ions from their surroundings (Blaine Metting Jr., 1993).
However, for this soil sample to be representative of the rest of the garden soil, we have to assume a homogeneous distribution of microbes within the soil.
Indeed, it is generally assumed that microbial cells are uniformly distributed in the soil penetrating all pores by diffusion. Studies show that the distribution of microbial cells in soil is not diffuse but focal. Microbes, especially bacteria and mycobacteria inhabit soils in colonies (Krasil'nikov, 1958). Thus, the sample could have been misrepresentative, as the particular 1g sample of soil tested could have contained a “cluster” of bacteria not existing in another section of the sample.
The number of microbes in a given soil is also factor to parameters such as seasons. Thus, comparison of samples must relate the season of the year, which it has been sampled at. Table 10 displays a summary of winter and summer microbial population in soils of two different sites (Santee and Bluebird in USA).
Source: USDA, 1971.
Table 10: Summer and winter microbial populations in Santee and Bluebird soils.
The greatest population change from summer to winter was a reduction of 75 percent in the actinomycetes group. Aerobic bacteria declined 67 percent and anaerobic bacteria decreased 30 percent in both soils. The fungi remained unchanged throughout the year in both soils.
It is also noticed that assumptions are linked to the technique used (dilution plate count technique). The technique is based on the principle that complete detachment and dispersion of cells from the soil will give rise to discrete colonies when incubated on a petri plate containing nutrient media. It is assumes that a complete dispersion of sample will occur, media will be suitable for the growth of organisms, and once on the media, organisms will not interact (Internet 1).
There are many potential inaccuracies when using the dilution plate technique, which result in an underestimate of the total viable population of cells. The factors responsible for the underestimation are:
- clumps of cells remain aggregated or attached to soil particles;
- cells are killed in the dilution medium;
- spores fail to germinate;
- adsorption of cells on pipette walls;
- high selectivity of the plating medium and incubation conditions (Skinner et al., 1952).
All these factors might affect the numbers of microbes forming on the agar plates and it is generally accepted that plate counts account for < 10% of the total population (Internet 3).
Although, two counts, one for fungi and one for bacteria, have been taken into account for the calculations, when they are under the minimum colonies number that should have been counted. Indeed, plates with between 25 and 250 colonies are suitable for counting. A plate with fewer than 25 colonies is inaccurate because a single contaminant could influence the results and, a plate with greater than 250 colonies is extremely difficult to count. Those two plates have however been taken into account to further enhance conclusions, as they do not contradict them.
Experience 2
Ammonification in soil is the conversion of organic forms of nitrogen (for example, nitrogen in proteins in dead plants and soil animals) to ammonium. The ammonification process is carried out by a wide range of soil organisms. Many different types of bacteria and fungi are involved. Ammonification is also known as nitrogen mineralization and occurs when soil dwelling bacteria decompose dead organic matter, which are composed of complex nitrogen containing compounds such as proteins and amino acids, and these bacteria use the nitrogen they obtain to create their own amino acids and proteins and release the excess nitrogen as ammonium which can then be used by plants (Porteous, 2000).
Results show that the lowest rate of ammonification occurred in tubes1, 9 and 10.
Tubes 9 and 10 are controls, thus no ammonification was expected from those tubes, as no microorganisms were inoculated into them. A colour change indicates production of ammonia, and the intensity of that colour change is proportional to the quantity of ammonia produced. Microorganisms inoculated tubes were all coloured, exception of tube 1 (inoculated with Bacillus cereus).
Tube 1 contained Bacillus cereus, the faint yellow colouration of the tube, indicated that Bacillus cereus did not perform a high level of ammonification during incubation.
Although tube 4 (also inoculated with Bacillus cereus) developed a slight orange colouration. Thus, it can be concluded that Bacillus cereus do participate to ammonification processes to a certain extent.
Following this reasoning, it appears that all three microorganisms have an ammonifying power. Although, according to the colouration intensity, Proteus vulgaris has more power than Bacillus cereus and Pseudomonas fluorescens has more power than Proteus vulgaris. Indeed one notable feature of Proteus vulgaris is the ability to degrade urea to ammonia, by production of the enzyme urease. And, Pseudomonas fluorescens is known to be an ammonifier (Conn and Bright, 1919).
The speed with which this ammonia is formed within a soil varies with the physical and chemical composition of the soil together with the number and physiological efficiency of the various organisms taking part in the process (Greaves, 1922). The formation of ammonia in the soil is the result exclusively of the conjoint activity of numerous lower organisms of very widely different, characters. All three microbes inoculated are bacteria, it is therefore not surprising that both soil tubes (9 and 10) displayed a more intense colouration as bacteria and fungi have ammonifying power. Soils contain a mixture of bacteria and fungi, thus ammonifying power of every type of microbes accumulates. Moreover, it is quite likely that the organisms are even more efficient in the soil in the mixed cultures than they are in the pure cultures, because the transforming of protein nitrogen to ammonia is a complex process which must proceed by steps and some organisms must be more efficient than are others in specific phases of the reaction (Greaves, 1922).
Experience 3
Denitrification is the microbial reduction of to gas. It is a step in the nitrogen cycle, which involves the reduction of nitrates into nitrite, nitrous oxide, ammonia, or elemental nitrogen. It is carried out by certain forms of denitrifying bacteria in the soil and serves as an important part of the breakdown of dead organism. It is responsible for the loss of much of the soil's natural and synthetic fertilizers. This process is favoured most in warm, anaerobic conditions.
If denitrification has occurred in the incubated tubes, gas formation should be observable in the Durham tubes, or a colour change should developed when reagents are added.
Pseudomonas aeruginosa is well known amongst microbiologist for its denitrification abilities and physiological versatility. Pseudomonas aeruginosa is a denitrifying bacterium, a microorganism whose action results in the conversion of nitrates in soil to free atmospheric nitrogen. It is commonly found in terrestrial soil, and can grow in a variety of low-nutrient conditions (Doudoroff and Palleroni, 1974).
Pseudomonas aeruginosa is a representative of a wide range of other denitrifying bacteria. Indeed, microbiologists have even though of this microbe for introducing atmospheric nitrogen in Mars atmosphere (Hart et al., 2000)
Results will be discussed tube by tube:
-
Tube 1: nitrate broth, Pseudomonas aeruginosa
Tube 1 displayed a gas bubble in the Durham tube, from this, conclusion could be made that di-nitrogen gas has been produced by Pseudomonas aeruginosa.
Moreover, there was no colour change when reagents were added, this indicated that no nitrites were present; and after powdered zinc addition, no colour change occurred either, indicating that no nitrates were present. Before incubation, nitrates were present from the nitrate broth, thus nitrates have been fully reduced to di-nitrogen gas, as there is no nitrites present and no nitrates left.
- Tube 2: nitrate broth, soil
Tube 2 displayed a gas bubble in the Durham tube, from this, conclusion could be made that the microbial population investigated in experience 1 has produced di-nitrogen gas. Moreover, there was no colour change when reagents were added, this indicated that no nitrites were present; and after powdered zinc addition, no colour change occurred, indicating that no nitrates were present. Before incubation, nitrates were present from the nitrate broth, thus nitrates have been fully reduced to di-nitrogen gas as there is no nitrite present and no nitrate left.
-
Tube 3: nitrate-free broth, Pseudomonas aeruginosa
Tube 3 do not displayed gas production in its Durham tube. If denitrification had occurred, then the nitrate was not yet reduced to di-nitrogen gas. After addition of reagents, a colour change occurred. This indicated the presence of nitrite in the tube.
Theoretically, no nitrate should be present in the tube, as the broth was nitrate free, thus no di-nitrogen gas should be produced. However, presence of nitrites has been in evidence by the red colour development. The exact content of the broth is unknown, and this content might have some type of nitrogenous compounds which Pseudomonas aeruginosa might have been able to reduce to nitrite. Another reason for the presence of nitrites might be from contamination of the tube.
- Tube 4: nitrate-free broth, soil
Tube 4 displayed a gas bubble in the Durham tube, from this conclusion could be made that the microbial population investigated in experience 1 has produced di-nitrogen gas. No dominant colour change occurred when reagents were added, this indicated that no nitrites were present (although a little bit of red colour developed on the side of the tube), and after powdered zinc addition, no colour change occurred, indicating that no nitrate was present. Although no nitrate was added to the broth, production of gas occurred but no nitrite or nitrate were present after incubation, which might indicate that microorganisms in the soil have reduced residual nitrate or nitrite that were present within the soil itself. This conclusion could be demonstrated by testing the soil before incubation for presence of nitrate and nitrite. In this case, the denetrification process has been fully undertaken as no nitrite or nitrate was present but only di-nitrogen gas.
Scope for bioremediation and soil quality
The metabolic diversity and capacity of the soil microflora has allowed the development of methods for the bioremediation of soils contaminated by hazardous wastes or spilled petroleum products on both land and sea. Bioremediation may be defined as the controlled use of microorganisms for the destruction of chemical pollutants.
In biostimulation, the environment into which the material has been spilled or otherwise introduced is made favourable for the rapid development of microbes. Typically, this process involves adding sufficient nitrogen and phosphorus fertilizer to overcome nutrient limitations to microbial growth and providing some mechanism for increased aeration of the system. These practices encourage development of the indigenous microbial population, which usually contains microbes able to degrade the compounds of interest.
Bioaugmentation is the addition of an external microbial population in order to speed up the degradation process. Numerous microbes have been developed for such purposes. However, the full measure of the usefulness of such microbial products is not yet known (Internet 4).
Studying soil microbes is also important to gain knowledge about the health of soils.
The numbers, biomass, activity, and community structure of the organisms, which comprise the soil food web, can be used as indicators of ecosystem health because these organisms perform critical processes and functions. Soil decomposers (bacteria, fungi and possibly certain arthropods) are responsible for nutrient retention in soil. If nutrients are not retained within an ecosystem, future productivity of the ecosystem will be reduced as well as cause problems for systems into which those nutrients move, especially aquatic portions of the landscape (Hendrix et al, 1986).
CONCLUSION
These laboratory sessions have enabled the operator:
- To appreciate the importance of applying aseptic techniques when handling microorganisms potentially pathogenic.
- To gain knowledge and handling experience of microbial enumeration technique such as dilution plate counting.
- To identify microbes present in garden soil, and calculate their abundance.
- To understand the role of microbes within soils, and especially their role in the nitrogen cycle.
- To understand the importance of bacteria as a remedial agent in contaminated soils, and the relationship between microbial communities and health of soils.
REFERENCES
- Alexander, M. 1971. Microbial Ecology. John Wiley and Sons. Chichester.
- Alexander, M. 1977. Introduction to Soil Microbiology, 2nd. Ed. Krieger Publ. Co., Melbourne, FL.
- Blaine Metting Jr, F. 1993. Soil microbial ecology. Applications in agricultural and environmental management. Mracel Dekker, Inc.
- Conn, H. J., Bright, J. W. 1919. The Journal of Agricultural Research. January 6, - March 31, 1919.
- Dindal, D. 1990. Soil Biology Guide. John Wiley and Sons. 1349 pp.
-
Doudoroff, M. and Palleroni, N. J., Part 7: Gram-negative Aerobic Rods and Cocci. Family I: Pseudomonaceae. Genus I: Pseudomonas in Bergeys. Manual of Determinative Microbiology, eds. R. E. Buchanan and N. E. Gibbons, Williams and Wilkins, Baltimore, pp. 217-222, 1974.
-
Graham, J.H., and D.J. Mitchell. 1997. Biological control of soilborne plant pathogens and nematodes. In: D.M. Sylvia, J.J. Fuhrman, P.G. Hartel and D.A. Zuberer (eds.), Principles and applications of soil microbiology. Prentice-Hall, Englewood Cliffs, N.J.
- Greaves, J. E. 1922. Agricultural Bacteriology. Constable and Company, Ltd.
- Hart, S. D., Currier, P. A., and Thomas, D. J. 2000. Denitrification by Pseudomonas aeruginosa Under Simulated Engineered Martian Conditions.
- Hendrix, P.F., R.W. Parmelee, D.A. Crossley, Jr., D.C. Coleman, E.P. Odum, and P.M. Groffman. 1986. Detritus foodwebs in conventional and no-tillage agroecosystems. Bioscience 36:374-380.
- Krasil'nikov, N. A. 1958. Soil and microorganisms and higher plants. Academy of Sciences of the USSR Institute of Microbiology. Published
for THE NATIONAL SCIENCE FOUNDATION, WASHINGTON, D.C.
and THE DEPARTMENT OF AGRICULTURE, USA by THE ISRAEL PROGRAM FOR SCIENTIFIC TRANSLATIONS 1961.
- Nelson, E.B. 1997. Microbiology of turfgrass soils. Grounds Maintenance. March, 1997.
-
Paul, E. A., Clark, F. E. 1996. Soil microbiology and biochemistry. 2nd. Ed. Academic Press.
- Porteous, A. 2000. Dictionary of Environmental Science and Technology. (Third Editon). John Wiley & Sons, Ltd.
-
Sylvia, D., Fuhrmann, J., Hartel, P., and Zuberer, D. 1997. Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, N.J.
-
Skinner, F. A., and P. C. T. Jones, and J. E. Mollison. 1952. A comparison of a direct and plate counting technique for quantitative estimation of soil microorganisms. J. Gen Microbiol. 6:261-271. Scholes, R.J. and Scholes, M.C. September 1993. The Role of Nonliving Organic Matter in the Earth’s Carbon Cycle. Zepp, R. G. and Sonntag, Ch., ed. The Effect of Land Use on Nonliving Organic Matter in the Soil. p. 209-226.
- USDA. Forest Service Research Note - I50 February 1971. Microbial populations in two swamp soils of south Carolina.
- Winfield, A. 1995. Environmental Chemistry. Cambridge University Press.
-
Internet 1: Soil Productivity - Plate Count Method
- Internet 2: The soil food web
- Internet 3: Enumeration of microbes in laboratory
- Internet 4: Soil biological properties