Application of bioremediation technologies for the treatment of Australian soils contaminated with aged polycyclic aromatic hydrocarbons

Polyaromatic hydrocarbons (PAHs) are an important class of organic hydrocarbons which originate from both anthropogenic and natural sources. PAHs are of particular importance due to their persistence, abundance and harmful effects and their carcinogenic, mutagenic and lipophilic properties. Around 60 % of Australia's polluted sites contain hydrocarbons, several of which contain PAHs. When PAH contamination in soil occurred some years ago, the ageing process decreases the fraction of PAHs available for biodegradation. Environmental factors such as pH, temperature, and moisture content, in addition to soil properties, may all affect the bioavailability and bioaccessibility of soil PAHs. These factors assert their influences through changing the properties of soil organic matter and the release of PAHs from soil. Therefore, it is very important to achieve reliable risk assessment of contaminated land with PAHs and treat them efficiently. The binding of PAHs to soil organic matter results in the contaminant becoming more resistant to microbial degradation. The potential use of microorganisms for bioremediation of PAH-contaminated soil has been widely investigated due to the potential for microbial breakdown of complex contaminants into less harmful by-products. However, the application of microbial based bioremediation is not yet commonly practiced for aged PAHs in contaminated soils as the effectiveness of the process is influenced by a complexity of factors related to the microorganisms involved, the nature of the PAH mixture and the contaminated environment. Therefore, remediation of aged PAHs contaminated sites remains a challenge for current and future research. The uniqueness of Australian soils and environmental conditions complicate the use of exogenous bacterial isolates that have been previously shown to degrade PAHs in other polluted sites. For instance, contaminated soils in parched locales of Australia generally lack adequate soil nutrients that are required for the degradation of the hydrocarbons by microorganisms. Additionally, the soil moisture content may not be adequate to maintain the degradation of the hydrocarbon contaminants throughout the year as a result of extreme weather conditions. The main aim of this thesis was to investigate the potential use of different bioremediation strategies to reduce concentrations of aged, weathered PAHs in Australian contaminated soils.  This study was based primarily on the hypothesis that the degradation of aged PAHs can be achieved through a combination of two techniques, biostimulation and bioaugmentation. To test this hypothesis, a detailed experimental program of research was undertaken by (1) developing an extraction protocol to allow quantification of (aged) PAHs from soil, (2) isolating and identifying suitable PAH degrading microorganisms from the contaminated environment with subsequent screening of the identified microorganisms for PAHs degradation potential and (3) investigating the potential application of these microorganisms for bioremediation within microcosms experiments. In the first part of the research, the development and optimisation of a simpler and rapid solvent extraction was developed to characterise 16 USEPA priority PAHs in aged contaminated soils, following analyses by GC-MS / MS. From this study, solvent extraction using both acetone : hexane and chloroform : methanol were found to be most effective. Acetone: hexane appears to be the safest type of solvent extraction method considering the greater toxicity associated with chloroform : methanol. The detection limit for the method was calculated to between  2 and 30 ng PAH g−1 of soil, which was low compared to other methods available in published literature. In the second part of the study, through a selective enrichment process and evaluation on Biolog™ MT2 plates, seven bacterial species capable of using PAHs as the primary source of carbon from polluted soils were isolated. They were identified as Oerskovia paurometabola, Achromobacter sp., Arthrobacter equi, Microbacterium maritypicum, Pantoea sp., Rhodococcus sp., and Sejongia sp. using 16S rRNA gene sequence based molecular identification. Enzyme studies confirmed that all the selected isolates exhibited catechol 1,2-dioxygenase activity, involved in the ortho cleavage pathway of PAH biodegradation.  In addition, the metabolic profiles of these seven isolated bacteria were tested on various substrates using Biolog™ Eco plates.  The metabolic properties of seven isolates indicated that all isolates can use a wide variety of organic substrates; Sejongia sp. was capable of utilising 28 out of the 31 substrates used. This study is the first to report the presence and involvement of Sejongia sp., Arthrobacter equi and Oerskovia paurometabola in the remediation of soil contaminated with PAHs. In the third part of the research, the ability of the bacterial isolates (both individually and as a consortium) for the bioremediation of aged PAH-contaminated soil was investigated, with comparison of bioaugumentation and biostimulation in microcosms for 56 days. At the end of the 56 - day experiment, both treatments showed over 99% degradation of PAHs. Bioaugmentation and biostimulation showed similar degradation performance; however, some inhibitory effect was observed at day 14 in mesocosms inoculated by Achromobacter sp. (NH13) and Pantoea sp. (NH15). With respect to the microbial community composition and prevalence of PAH-degrading genes during the degradation of PAHs, the findings of qPCR showed that Gram-positive degraders of PAH were more prevalent in all treatments. 16S rRNA gene sequencing showed that the organisms that were inoculated did not establish themselves as major components within communities in bioaugmentation treatments. During the treatments, however, significant changes were detected in the bacterial populations, indicating that the natural community displayed an effective yet changing community involved in biodegradation at all stages of the degradation process. At the phylum level, Actinobacteria, Proteobacteria, Bacteroidetes and Gemmatimonadetes increased in abundance in all treatments. At the genus level, ' the relative abundance of Sphingomonas at Day 0 was 35 % but progressively became less dominant (< 2 %) in all microcosms except in microcosms inoculated with Pantoea sp. (14 % on Day 14). However, in all treatments, Sphingomonadaceae (genus unclassified Sphingomonadaceae) increased from 3 % (Biostimulation - Day 0) to around 10 %. The relative abundance of Luteimonas sp. on Day 0 was 6 % but increased three-fold in all treatments by Day 14. Gemmatimonadetes (unclassified) became more dominant in all treatments on Day 28 (biostimulation (5 %), consortium (6 %), Achromobacter sp. (6 %) and Pantoea sp. (4 %) when compared to Day 0 (0.4 %)). Compared with other treatments, an increased abundance of Methylobacterium sp. (15 %) was found in microcosms inoculated with Pantoea sp. on Day 28. Promicromonospora was less dominant in all the treatments except in microcosms inoculated with Achromobacter sp. (12 %) and Pantoea sp (8 %) on Day 14. Biostimulation is therefore suggested as the best approach for remediating PAHs in aged, weathered and heavily polluted soils. The outcomes from this research study bridges some of the knowledge gaps existing in the bioremediation of aged, weathered PAH contaminated soils using biostimulation as the main treatment strategy.