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