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The diversity of microbial populations in the soil ecosystems are far much more than the eukaryotic organisms. In one gram of soil there may be ten billion microorganisms of numerous different species (de Freitas et al., 2003). When studying both the structure and function of an ecological unit, the evaluation of microbial communities must take into consideration not only the distribution and abundance of species but also the redundancy present in microbial community and functional diversity (Shukurov et al., 2006, Taccari et al., 2011). de Freitas et al., (2003) defined functional diversity as the number of distinct processes (thus functions) that can potentially be performed by a community where as functional redundancy is measured as the number of different species within the functional groups present in a community.
To apprehend the character of microbial communities in different soil environments, according to Shukurov et al., (2006) it is an essence to have knowledge of community function and functional diversity. These implies actual catabolic action expressed by the microorganisms and its potential activity that is the capability of the community to acclimatize catabolism (de Freitas et al., 2003) or the relative composition and size of integral populations to varying abiotic conditions such as microclimate and added substrate (Taccari et al., 2011).
The population dynamics of microorganisms in soil are extremely difficult to assess due to the complex nature of the soil environment (Taccari et al., 2011, Shukurov et al., 2006, dos Santos et al., 2012). Also the diverse nutritional requirements of microorganisms in soil may not be easily estimated hence isolation of the soil microorganisms (Mohanty and Panda, 2011) and studying them in the laboratory as pure or mixed culture cannot be easily attained. In the case the soil microorganisms can be isolated and brought to the laboratory, there may be not in the same physiological and morphological state as in the soil. In the soil microorganisms interact with other abiotic and biotic entities in the soil so in laboratory cultures it is often difficult to manipulate such conditions hence the ecological balance is not attained.
As a result indirect methods have been developed to study the microbial populations and communities in soil. Soil respiration response is an example of such methods that are widely used. Total population is estimated by measuring the total respiration of the population using soil incubated in jars and carbon dioxide traps (Mohanty and Panda, 2011). This procedure is based on the fact that all soil microorganisms respire and during respiration carbon dioxide concentration changes. In the case when soil is altered with a compound, the soil microflora responds by either using the compound as a substrate or if it is toxic they may die from it and in such case they do not tackle the compound (Zhang et al., 2010; Cleveland et al., 2007; Changming and Moncrieff, 2005). When the compound is used as a substrate, the response often results in elevation in the soil population hence increase in soil respiration. But when the compound is toxic, decline in the population is noted by decrease in soil respiration measured by the carbon dioxide released (Fujii et al., 2010). In recent studies soil respiration have been employed to measure the biomass of microorganisms in soil but fewer studies have been conducted on the population dynamics of specific microbial populations, an example being the microorganisms responsible for biodegradation of toxic compounds in soil (Zhang et al., 2010, Taccari et al., 2011). Chemoheterotrophic bacteria for example differ in the specific organic substrates they use as a carbon and energy source for their growth (Hamamura et al., 2008). The considerable diversities in the substrates that are biodegradable and the capability of individual species to catabolize specific substrates have been used for many years to identify and characterise the microorganisms (Cleveland et al., 2007). In this study the effects of different soil amendments on microbial populations were assessed by soil respiration.
Materials and methods
Table 1: Materials and reagents used in the study
-100ml and 50ml beakers
-2L mason jars
-Blank (to measure CO2in the atmosphere)
-Soil only (control)
-Soil + saw dust (2.5g)
-Soil + paper (2.5g)
-Soil + glucose (2g)
-Soil + ammonium nitrate (1g)
-Soil + engine oil (5ml)
-Soil + roundup (xenobiotic compound)(0.02g)
-Soil + chicken manure (2.5g)
200g of fresh soil was measured into the 2L Mason jars and water was added to bring the moisture holding capacity to 60% and mixed well. To each jar the corresponding treatment to the soil was added and mixed well. Then 25ml of 0.5N NaOH was measured using a burette into a 50ml beaker and placed into the Mason jar. Also approximately 5ml of tap water was poured into a test tube and the tube placed into the Mason jar as a way of maintaining relative humidity. The jars were then tightly sealed and incubated at room temperature for a week.
After a week of incubation, the beaker containing NaOH was removed from the jar and to it drops of BaCl2 was added to precipitate the excess carbonate as BaCO3. Then few drops of phenophthanein indicator were added. Using a burette 0.5N HCl was titrated to the unneutralised alkali until an end point was reached (change from pink colour to clear milky white). The amount of the acid used was recorded for each treatment. After titration the beakers were then washed and another fresh 25ml of 0.5N NaOH was added and then the jars were reincubated. The amount of carbon dioxide evolved during the week was then calculated using the formula CO2= (B-V)NE where V is the volume of acid used in titration, B volume of acid used to titrate the blank, N normality of the acid and E is the equivalent weight ( if data is expressed in terms of carbon E is 6 and if expressed as CO2 E is 22)
Following another week of incubation, beakers of NaOH were removed from the jars and then titration was carried out following the same procedure as the past week. The same procedure was also duplicated in reincubation of the jars, the only modification was the introduction of the Rossy cholodyney slides which were buried in the soil according to the procedure of their preparation.
The following the Rossy cholodyney slides were removed. Heat fixation was carried out and the slides were stained with crystal violet and methylene blue and kept for microscopic observation. The NaOH containing beakers were also removed from the jars and titration with the acid was carried out as before. Reincubation was also done but now the slides were not included.
Finally after the last incubation titration was carried out. The stored Rossy cholodyney slides were then observed using the microscope. On a final step all glassware were cleaned and the soil treatments were disposed off in plastic bag. Results obtained were then analysed by ANOVA.
Results and analysis
Table 1: ANOVA comparison of carbon dioxide evolved in different soil treatments
Soil only 1
Soil only 2
Chicken manure 1
Chicken manure 2
Source of Variation
There is a significant statistical difference on the carbon dioxide evolved in the different soil treatments. The F value calculated from the carbon dioxide evolved from different soil treatments far exceeds the F critical value; F value calculated is 4.086703 while the F critical value is 1.880175 hence a significant statistical difference. This is a clear depiction that soil respiration in treated soils differed between treatments.
Figure 1: Carbon dioxide evolved by respiring microorganisms in different soil treatments in a period of four weeks
In all the soil treatments the level of carbon dioxide evolved decreased over the four week period. Substrates added that were readily utilized by the soil microorganisms lead to the high carbon dioxide evolved hence high rate of soil respiration. As the substrate added in the soil depleted as the microorganisms utilized them the carbon dioxide evolved decreased. Lower levels of carbon dioxide evolved from soil treatment such as pesticide treated soil indicated that the pesticide was utilized by the soil microorganisms but this substrate had adverse effect on the soil microorganisms as the respiration rate of them decreased hence there was a reduction in the microbial population in that particular soil.
Figure 2: Microorganisms isolated from the soil treatments by Rossy cholodyney slides
Soil microbial populations respond differently to substrate added as a treatment to the soil. Table 1 showed a statistically significant result when comparing the soil respiration of microbial populations in the different soil treatments. Soil treatment affected the microbial populations in soil, microorganisms that can utilize a substrate added were able to increase in population hence their respiration rate increased as the level of carbon dioxide evolved was more when compared to other soil treatments that had lower levels of carbon dioxide evolved. Glucose treated soil had a higher carbon dioxide evolved but as the time passed the level of carbon dioxide decreased; this is due to the fact that the concentration of glucose added to the soil decreased as the microorganisms used it as a source of energy and growth. Glucose is the primary carbohydrate source of energy; it is a compound of highest priority to microorganisms as an energy source so when it is added to the soil it will be utilised quickly hence respiration rate increases as microbial biomass increases as a result of glucose utilization.
The most critical component in the microbial degradation of compounds is the carbon to nitrogen ratio in the substrate. Compounds with low carbon to nitrogen ratio are easily degraded by soil microorganisms and those that have a relatively high carbon to nitrogen ratio are easily degraded when there is an external supply of nitrogen to the microorganisms. Organic compounds also are easily degraded by soil microorganisms since the microorganisms have evolved side by side with such compounds hence they have developed metabolic pathways that can tackle the compound easily and obtain nutrients and energy from the degradation of such compounds. In figure 1, compounds such as glucose, chicken manure and paper had a higher respiration rate compared to oil, pesticide and NH4NO3 which had a lower respiration rate. Glucose, chicken manure and paper are organic compounds while pesticide and NH4NO3 are inorganic compounds but oil is an organic compound from the lithosphere with a high carbon to nitrogen ratio.
One important factor is that chicken manure raises the pH of soil to the range 6.3 to 7.4 which is optimal for the growth of known oil-utilizing bacteria.
Figure 2 shows that the soil which have been treated with glucose have the highest carbon dioxide followed by the soil which were treated with chicken manure, paper, oil, NH4NO3, untreated soil and pesticides respectively. Based on the literature review from…………… high amount of CO2 implies that the soil respiration is high since CO2 is a product of respiration. Glucose, Chicken manure, and paper have a lower carbon to nitrogen ratio hence it is easy for the microorganisms to degrade them by the process of respiration. Oil and NH4NO3 require a high carbon to nitrogen ratio and hence it is difficult for them to be degraded, on top of that NH4 is toxic to microorganisms therefore it will reduce the microbial population reducing the amount of soil respiration and this also implies to the pesticides.
In fig 1 the gradual decrease in the amount of carbon dioxide produced per week might have resulted due to the reduction of nutrients in the soil and that fluctuations in the amount of CO2 produced between the weeks on the same treatment might be due to the fact that when introducing different treatments, microorganisms take them as foreign substances and hence they take time to adopt to them