Joint Bacterial-Fungal Consortia for the In-Situ Bioremediation of Polycyclic Aromatic Hydrocarbons in Estuarine Sediments

Polycyclic aromatic hydrocarbons (PAHs) are a class of over 100 chemicals found at various EPA Superfund sites across the United States formed through the incomplete combustion of organic compounds. These environmental contaminants are of concern due to their carcinogenicity, mutagenicity, and teratogenicity. Bioremediation using microorganisms is an economically efficient and environmentally sustainable process for PAH transformation in the environment. However, bacterial bioremediation schemes are limited in their ability to transform high molecular weight (HMW) PAHs. Through exploiting non-specific extracellular fungal enzymes in a mixed fungal-bacterial consortia, HMW PAHs have the ability to become bioavailable to bacteria, and ultimately be transformed. This approach is not widely used in the field of bioremediation, as there is limited understanding around interkingdom relationships between bacteria and fungi. There is limited knowledge of the array of extracellular enzymes that may assist with the transformation of PAHs, and whether these enzymes can be biostimulated or antagonized to increase the removal of PAHs in the environment. Additionally, a knowledge gap exists with regards to the survivability of introduced fungal and bacterial isolates, and whether a mixed fungal-bacteria consortia will provide an advantage in soil communities. This dissertation work will focus on developing a joint fungal-bacterial consortia that will exploit the cross-kingdom interactions of fungi and bacteria to assist in the removal and transformation of PAHs. This will be examined through isolating and identifying fungal isolates from creosote contaminated soil and assessing them for their ability to be biostimulated to produce extracellular enzymes that can be analogized for PAH transformation. Once determined, fungal and bacterial isolates will be assessed together for their ability to produce a joint biofilm. Lastly, fungal, and bacterial isolates will be assessed for their transformation capability and survivability amongst the microflora community in PAH soils.

This dissertation's first research objective was to identify and characterize indigenous fungi for a biostimulation/bioaugmentation scheme targeting PAHs. The goal of this objective was to find promising indigenous fungal isolates for further investigation in the transformation of PAHs. First, creosote contaminated soil was diluted and plated onto an array of growth agars to provide a representative overview of the fungal soil community. Fungi were isolated, cultured, and screened for the enzymatic production of laccase (Lac), manganese peroxidase (MnP), and tannase, along with the ability to degrade cellulose and starch. Fungal isolates were then incubated alongside complex amendments and Lac production will be measured through absorbance. Fungal isolates and complex amendments were lastly incubated alongside PAHs to measure transformation. From this objective, it was found that wide variety of fungal isolates native to creosote contaminated sediment were found to be able to produce extracellular enzymes that may assist in the biotransformation of PAHs. It was also found that complex amendments may also be used to increase extracellular enzymatic production to assist with PAH transformation. The complex grasshopper amendment is shown to be the most promising complex amendment to use in a future scheme.

This dissertation's second objective was to observe and characterize intra- and interkingdom behaviors in a mixed fungal-bacterial consortium for the improved transformation of PAHs. This research goal aimed to investigate the interactions between fungi and bacteria that could aid in the transformation and degradation of PAHs, as well as understand the potential for antagonistic and mutualistic interactions within soil communities. In this objective, a series of fungal permutations were performed from a select group of previously isolated indigenous fungi. The fungal permutations were visually assessed for unique growth patterns and assessed for changes in enzymatic functions. Permutations were then examined in the presence of a complex amendment to compare enzymatic production of fungal consortia to monoculture isolates. A select group of fungal isolates were then incubated alongside PAH degrading bacteria and examined for biofilm formation using optical density. Finally, fungal-bacterial mixed consortia and select model PAHs were incubated with the model complex amendment and measured for transformation capability. It was found that fungi will develop different morphological responses in the presence of other isolates. From this objective it was found that a mixed consortia between Novosphingobium pentaromativorans and filamentous fungi such as Penicilium.513 and Trichoderma.508 were deemed to be more advantageous for bacterial cell adhesion than yeast and yeast-like fungi such as Scheffersomyces.502 and Aureobasidium.509. When placed against model PAHs: FLA, PHE, PYR, and BaP, it was found that nearly all the fungal isolates saw improvements in their ability to degrade when placed alongside the bacteria. Additionally, when co-cultures were provided with a grasshopper amendment, increases in PAH degradation were observed.

The final objective of this dissertation work was to assess the performance of a mixed fungal bacterial consortia in a PAH-spiked soil microflora community. The purpose of this research objective is to assess the viability of a mixed fungal bacterial consortia in a soil reactor that mimics field conditions. Reactors were developed to assess community changes in PAH-spiked soil and non-spiked soil. Reactors were also inoculated with either monocultures or cocultures to explore how each variation affects the microbial community over time. Reactors were also biostimulated with a complex grasshopper amendment. Over a 60-day period, reactors were sacrificed at various points. RNA was extracted out of the soil at each time point and used to analyze the bacterial and fungal communities. Furthermore, soil samples were evaluated for PAH degradation at every time interval. No correlation between mixed fungal-bacterial consortia and PAH degradation was observed based on this objective. In comparison to their naturally attenuated counterparts, soil reactors that were subjected to biostimulation, bioaugmentation, and biostimulation-bioaugmentation analyses did not demonstrate appreciable variations in community structure.

This dissertation work will help engineers and researchers create efficient and sustainable PAH remediation strategies. This dissertation addresses long-standing knowledge gaps in mixed consortia fungal behavior and fungal-bacterial interactions. This work provides a framework for future investigations into cross-kingdom interaction, improved bioaugmentation methods, and optimized biostimulation for target organisms.