Soil Microbial Diversity: Influence of Geographic Location and Hydro-carbon Pollutants

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Chapter 2 EVALUATION OF MICROBIAL DIVERSITY OF DIFFERENT SOIL LAYERS AT A CONTAMINATED DIESEL SITE

Abstract

In this study, we evaluated the hydrocarbon removal efficiency and microbial diversity of different soil layers. The soil layers with high counts of recoverable hydrocarbon degrading bacteria had the highest hydrocarbon removal rate compared to soil layers with low counts of hydrocarbon degrading bacteria. Removal efficiency was 48% in the topsoil compared to 31% and 11% in the 1.5 m and 1 m respectively. There was no significant difference between the Total Petroleum hydrocarbon (TPH) removal in the nutrient amended treatments and the controls at 1 m and 1.5 m soil layers. The respiration rate reflected the difference in the number of bacteria in each soil layer and the availability of nutrients. The high O2 consumption rate corresponded positively with the high TPH removal rate. Analysis of the microbial diversity in the different soil layers using functional diversity (community level physiological profile using Biolog) and genetic diversity using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of 16 SrDNA revealed differences in substrate utilisation patterns and DGGE profiles of 16 SrDNA fragments respectively. The microbial diversity as revealed by DNA fragments was reduced in the highly contaminated soil layer (1.5 m) compared to the topsoil and the soil layer at 1 m.

Introduction

The effect of hydrocarbon contamination on soil microbial communities has been studied (Atlas et al., 1991; Wünsche et al., 1995; Lindstrom et al., 1999; MacNaughton et al., 1999; Stephen et al., 1999; Juck et al., 2000; Bundy et al., 2002). However, these studies only investigated the influence of hydrocarbons using mainly the topsoil. Information about the microbial diversity of different soil layers at a given site is lacking. Because oil contamination normally penetrates deeper than the top layer, it is important to understand the distribution of degrading populations with soil depth and how the distribution patterns influence the efficiency of biodegradation. The subsurface soil environment, though devoid of sufficient nutrients, oxygen and other factors, harbors an array of soil microorganisms that plays an important role in decomposition and the recycling of nutrients (Krumholz, 1998). It is widely presumed that the number of heterotrophic bacteria changes with increasing depth. This can be attributed to spatial and resources factors which can influence the microbial diversity of the soil (Zhou et al., 2002). Shallow subsurface micro-flora appears to be predominantly prokaryotic, appears to be specially adapted for growth and survival in nutrient poor conditions, includes strains that can function throughout a wide range of nutrient concentrations and may sometimes exert significant effect on groundwater chemistry (Ghiorse and Balkwill, 1983; Balkwill and Ghiorse, 1985; Bone and Balkwill, 1988; Ghiorse and Wilson, 1988; Balkwill et al., 1989). The availability of hydrocarbons in the vadose zone can alter the diversity of the heterotrophic community due to an increase in the carbon substrate. According to Atlas (1981), Leahy & Colwell (1990), the number of hydrocarbon bacteria and their relative abundance in the bacterial communities increases significantly in the presence of readily available hydrocarbons. Also the changes in hydrocarbon content in soil results in characteristic shifts of the substrate utilisation patterns by the microorganisms and that the altered pattern of substrate utilisation corresponds with similar changes in abundance of hydrocarbons in the soils (Wünsche et al., 1995). This is not surprising,and in accordance with the theories about gene accumulation and selection pressures, we can predict lower abundance of hydrocarbon degraders with depth as selection pressure and growth conditions in general lowers with depth. In this study, we investigated the hydrocarbon removal capacity and the microbial diversity of different soil layers after diesel contamination. The capacity of the soil layers in removing hydrocarbons was evaluated (using simple microbial assays), while microbial diversity was evaluated using functional diversity (community level physiological profiles using Biolog micro plates) and genetic diversity (PCR-Denaturing Gradient Gel Electrophoresis of 16SrRNA).

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Materials and Methods

Soil: The contaminated soil layers were collected in sterile bags from a dieselcontaminated site at Coalsbrook, in the Free State Province, South Africa. The soil collected was a loam soil with depth to ground water, 2m. The organic carbon of the uncontaminated topsoil was 0.9%. The electron acceptors were not measured. The soil layers were collected one month after contamination by a leaking diesel pipeline. Direct push drilling to 2 m was used to sample the contaminated soil layers at a depth of 1 m (CS1m) and 1.5 m (CS1.5m). The contaminated topsoil layer (CTS) was collected within 10 cm of the soil surface. Uncontaminated topsoil (UCTS) was also collected from the same site. Samples were kept at 4°C until analysis, which was completed within 24 h.
Microbiological analysis: 100 mℓ of 0.2% tetra-sodium pyrophosphate was added to 250 mℓ Erlenmeyer flask containing 10 g of the soil from each sample. The flasks were placed on a shaker (140 rpm) for 45 min. The mixtures in the flasks were allowed to settle for 5 min after mixing. Serial dilutions (with saline solution) were done using the samples before inoculating both the agar plate and the Biolog GN plates. The Total Recoverable Heterotrophs (TRHs) were enumerated by spread plate technique using nutrient agar (Biolab Diagnostics). The hydrocarbon-degrading bacteria were isolated as described by Margesin and Schinner (1999a) with diesel being the only source of carbon and energy. Both agar plates were incubated at 28°C and counted after 24 h and 7 d respectively. The bacterial counts were not corrected for the dry mass of the soil. Bacterial counts were done in triplicates. Analysis of Variance (ANOVA) was used to determine the difference between the treatments.
Carbon source utilization pattern determination: Sample dilutions were done as described above. Heterotrophic plate count data were used to adjust the samples to similar cell density for Gram Negative Biolog plate inoculation. 100 µℓ of the each sample was added to each well. The Biolog plates were read at 600 nm using Bio-Tek Elx800 microreader (Bio-Tek Instruments Inc) before incubation at 28°C. The plates were further read after 24, 48 and 72 h. Readings of the micro plates were made in triplicate. Statistical analyses were done using STATISTICA for Windows release 5.1.

Chapter 1 : General Introduction
Chapter 2 : Evaluation of Microbial Diversity of Different Soil Layers at a Contaminated Diesel Site
Chapter 3 : Evaluation of Microbial Communities Colonizing Stone Ballasts at Diesel Depots
Chapter 4 : Soil Microbial Diversity: Influence of Geographic Location and Hydro-carbon Pollutants
Chapter 5 : Multiplanted and Monoculture Rhizoremediation of Polycyclic Aromatic Hydrocarbons (PAHS) from the Soil
Chapter 6 : Germination of Lepidiun Sativum as a Method of Evaluating the Removal of Polyaromatic Hydrocarbons (PAHS) from Contaminated Soil
Chapter 7 : The Use of Biological Activities to Monitor the Removal of Fuel Contaminants: Perspective for Monitoring Hydrocarbon Contamination.
Chapter 8 : Bioremediation of Petroleum Hydrocarbons through Landfarming: Are Simplicity and Cost-Effectiveness the Only Advantages?
Chapter 9 : Conclusions and Perspectives