Who ate my phenathrene? An investigation of hydrocarbonoclastic organisms in pristine and contaminated soils.

Author: Alexandra Schwarz

Schwarz, Alexandra, 2015 Who ate my phenathrene? An investigation of hydrocarbonoclastic organisms in pristine and contaminated soils., Flinders University, School of Biological Sciences

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The microbial community dynamics of two soil types (sand and clay) were assessed using culture‐independent techniques to investigate the difference in microbial dynamics of soils of different type and contamination histories. This was then used to determine if targets for remediation strategies can be predicted based on baseline community structure alone. Two novel nucleic acid extraction techniques were developed for clayey and sandy soils and the profiling technique of Polymerase Chain Reaction (PCR) Temperature Gradient Gel Electrophoresis (TGGE) was optimised. A comparison between the laboratory standard profiling method, denaturing gradient gel electrophoresis (DGGE), and TGGE determined that although there was a difference in banding pattern of communities, there was no statistical difference in the results of the diversity, thus, strengthening the argument for use of TGGE over DGGE, especially due to the greatly reduced experimental run times and requirement of smaller sample volumes. Baseline fungal communities of the two soil types were investigated using TGGE profiling of the internal transcribe regions (ITS) regions of the rRNA gene. In sandy soils, the most contaminated test pit had the highest fungal diversity. The fungal profiles were dominated by species from the class Eurotiomycetes and included the well‐known hydrocarbon degrading species of Aspergillus and Eurotiales. Dominance of these species did not change depending on contamination level, suggesting a level of adaptability to multiple carbon sources. There appeared to be no correlation between fungal species diversity and contaminant level in the clayey soil type. Similar dominant fungal species were identified in the clay communities to those found in the sandy soils, all of which were part of the phylum Ascomycota. The clayey soils had a higher species diversity and range‐weighted richness compared to sandy soils, which may be due to the pore connectivity theory. As a result of low water connectivity in soils the formation of diverse communities is promoted through creation of microhabitats.The prediction of contaminant mineralisation was undertaken on the sandy soils using PCR amplification of 16S rRNA, ITS region profiling and ‘prediction primers’ (PAH‐RHDα GN‐F and GN‐R for Gram‐negative bacteria and PAH‐RHDα GP‐F and GP‐R for Gram‐positive bacteria). Stable isotope probing (SIP) was used to track the active degraders of 13C‐labelled phenanthrene. Baseline profiling indicated that there was very little difference in fungal diversity but a significant difference in bacterial diversity dependent on contamination history. The pristine soil had the highest fungal diversity at baseline, although the contaminated soil had the highest bacterial diversity. Identification of the dominant fungal and bacterial species highlighted the presence of organisms capable of degradation of various petroleum‐based compounds and other anthropogenic compounds regardless of contamination history. Community response after the simulated contamination event ( 14C‐phenanthrene) showed that the microbial community in the deep pristine and shallow contaminated soils were the most able to adapt to the presence of phenanthrene. The similarity in the microbial community structure of the well adapted soils demonstrated that a highly adaptable fungal community in these soils enabled a rapid response to the introduction of a contaminant. Ten fungal and 15 bacterial species were identified as active degraders of phenanthrene. The fungal degraders were dominated by the phylum Basidiomycota and Ascomyota including the genus Crypotococcus and Tremellales. Bacterial degraders include the genus Alcanivorax, Marinobacter and Enterococcus. There was little synergy between dominant baseline microbes, predicted degraders and those that were determined to be actually degrading the contaminant. It can be concluded from this work that a prediction of the bioremediation potential of a soil cannot be made based solely on baseline microbial diversity. Furthermore, the assessment of baseline communities tends to grossly underestimate the ability of a microbial community (by around 80– 90%) or potentially identify inaccurate remediation targets. This work has demonstrated that there are many complex interactions that occur once a soil is exposed to a contaminant and that a simplistic investigation of the microbialcommunity is not sufficient to determine how a soil will respond to a contamination event.

Keywords: Phenanthrene, molecualr microbial ecology, Temperature Gradient Gel Electrophoresis (TGGE), Stable Istopoe Probing (SIP).
Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Biological Sciences
Supervisor: Prof Ian Menz