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