The contamination of environmental media by total petroleum hydrocarbons (TPH) is a concern in many parts of the world; particularly as most petroleum components like polycyclic aromatic hydrocarbons (PAHs) are either toxic or carcinogens. In South Africa, the sale of major petroleum products by the South African Petroleum Industry Association (SAPIA) reveals that about 21 billion litres of petroleum products are sold per year. These products include bitumen, diesel, fuel oil, illum paraffin, jet fuel and petrol. In addition, 19.5 million tonnes of crude oil are brought into South Africa annually to feed the country’s four refineries. The production of oily sludges at refineries, transportation, storage, and handling of petroleum products by end users, results in environmental contamination. The soil environment is particularly vulnerable to hydrocarbon contamination as most of the accidental spillages by trucks, rail locomotives and pipelines have a direct impact on the soil medium. As most of the petroleum compounds are either toxic or carcinogenic, their removal from the soil is necessary. The literature reveals that biological treatment of hydrocarbons is cost effective compared to other treatment options. However, in order to improve the efficiency of biological treatments, there is a need to understand the microbial diversity of TPH stressed environments and how simple biomonitoring ‘instruments’ can be used to evaluate the removal of hydrocarbons from the soil. The message from the literature indicates some potential solutions to the existing problems associated with soil microbial diversity and biotreatment of hydrocarbon contaminated soil, which must be investigated. The main aim of this work was to evaluate the microbial diversity of the different soil environments disturbed by Total Petroleum Hydrocarbons (TPHs) and the potential use of plants and microorganisms in monitoring and removing hydrocarbons from the soil. In addition, the potential of the culture-independent methods in complementing, the culture-dependent methods when evaluating soil microbial diversity were also evaluated. The polyphasic approach was successfully used in evaluating microbial diversity in both hydrocarbon-contaminated and uncontaminated soils. The approach involved the use of community level physiological profiles (CLPP) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to evaluate the effects of hydrocarbons on the soil microbial communities of both the contaminated and non-contaminated soil layers at a diesel contaminated site. Because of the ability of the molecular methods (PCR-DGGE) to complement the CLPP, the polyphasic approach is recommended when evaluating soil microbial diversity and the effect of pollutants on microbial community structure as the approach appears to compensate for the limitations of each of the methods of evaluating microbial diversity. However, further work is needed to improve the recovery of bacteria from the soil, particularly where the interest is to evaluate the availability of the indigenous microbial populations for bioremediation. The substrate utilisation pattern and 16S DNA fragments of the soil microbial communites in different soil layers at a diesel contaminated site were different. The substrate utilisation pattern of the topsoil was different from the substrate utilisation pattern of the soil layers below 1m. In addition, the substrate utilisation pattern of the contaminated and uncontaminated soil layers were different. 16S DNA fragments of the different soil layers were also different. While the metabolic activities of different samples as reflected by CLPP does not necessarily imply the difference in community structure of the samples, PCR-DGGE revealed differences in 16S DNA fragments and this complemented the results of the culture based methods. The results suggest that the use of functional and genetic approaches (in combination) have a better chance of revealing a ‘clearer’ picture of soil microbial diversity. The distribution of hydrocarbon-utilising bacteria and the efficiency of biodegradation of hydrocarbons vary with soil depth. The biodegradation rate of hydrocarbon was highest in the topsoil compared to other soil layers and this was supported by the high number of hydrocarbon-degrading bacteria in the topsoil compared to soil layers at and below 1m. The results suggest that the biological removal of hydrocarbons varies in different soil layers and that microbial diversity as measured by CLPP and PCR-DGGE varies with depth in hydrocarbon-contaminated soil. The information about metabolic activities of different soil layers is important when assessing the footprints of degradation processes during monitored natural attenuation (MNA). However, further studies are required to understand the effect of (not only) other pollutants, but the influence of soil components (pore volume, level of adsorbents and other environmental factors) on the microbial diversity of different soil layers in both ‘shallow’ and deep aquifers. The microbial diversity of different environments contaminated by hydrocarbons has different community level physiological profiles. At diesel depots where similar hydrocarbons are used for maintenance of locomotives, the number of bacteria (both total culturable heterotrophic bacteria and hydrocarbon-degrading bacteria) was proportional to the level of hydrocarbon contamination. However, there was no significant difference in the level of total culturable heterotrophs (TCHs) and the hydrocarbon degrading bacteria. In addition, the biological activities as evaluated by CO2 production were higher in nutrient amended treatments in which high numbers of TCHs were present. Microbial diversity of polluted surfaces needs to be studied further to investigate the concentration or the thickness of the hydrocarbons layer on the rock surfaces that encourages the attachment or colonization of the TCHs and the hydrocarbon-degrading bacteria. The hydrocarbons rather than the geographical origin of the soil sample appear to be more important in determining functional or species diversity within the bacterial communities. The samples from different locations were as different as samples from the same location but from contaminated versus uncontaminated soil. The results of the soils from different locations artificially contaminated by different hydrocarbons also reached the same conclusion. However, further work is required to investigate the importance of soil heterogeneity in community studies of soil environments contaminated by similar hydrocarbons. The removal of Polycyclic Aromatic Hydrocarbons (PAHs) in multi-planted soil microcosm was higher compared to PAHs removal in monoculture soil microcosms. In addition, the PAH removal was higher in the vegetated soil microcosms compared to the non-vegetated microcosms. There was however, no significant difference in the PAH removal in the soil microcosms planted with Branchiaria serrata and the microcosm with Eulisine corocana. The Principle Component Analysis (PCA) and Cluster analysis used to analyse the functional diversity of the different treatments revealed differences in the metabolic fingerprints of the PAH contaminated and non-contaminated soils. However the differences in metabolic diversity between the multi-planted and mono-planted treatments were not clearly revealed. The results suggest that multi-plant rhizoremediation using tolerant plant species rather than monoculture rhizoremediation have the potential to enhance pollutant removal in moderately contaminated soils. Lepidium sativum, a plant with short germination period, was successfully used to monitor, the removal of Polycyclic Aromatic Hydrocarbons (PAHs) from the soil. The sensitivity of L. sativum eased with increasing concentration of the polycyclic aromatic hydrocarbons in the artificially contaminated soil while no germination occurred in the historically polluted soil. When used during phytoremediation of PAH, the germination level of L. sativum was inhibited during the first weeks, after which germination increased, possibly due to PAH dissipation from the soil. The methodology based on the sensitivity of L. sativum to PAH can be used as a monitoring tool in bioremediation of soil contaminated with PAH. However, the methodology should be developed further to gain more knowledge on aspects of bioavailability of PAH in both the aged as well as the freshly spiked soil. Also critical is the sensitivity of the seeds to other pollutants (e.g. heavy metals), which are most likely to occur in the presence of the PAHs. Although the biological activities have the potential to monitor the removal of hydrocarbons from the soil, the methodologies have not been developed sufficiently to cater for the heterogeneity of the soil and to differentiate toxicity by the parent compound and the metabolites. At present, it is best that they be used to complement existing conventional monitoring instruments. Finally, the biological removal of hydrocarbons is cost-effective compared to other treatments. However, inherent physical, chemical and biological limitation hampers the efficient utilisation of the bioremediation technologies. Biostimulation approaches involving the stimulation of indigenous pollutant-degrading bacteria should be preferred ahead of bioaugumentation. The latter approach should be considered when the contaminated site does not have the indigenous pollutant-degrading bacteria. Even in this case, the aim should be to ‘seed’ the biodegradation knowledge to the indigenous microbial populations due to poor survival of the added strains.
Thesis (PhD (Biotechnology))--University of Pretoria, 2007.