Due to their strong soil sorption capacity, molecular stability, and hydrophobicity, PAHs interact with nonpolar soil domains such as organic matter and become highly resistant to soil microbial degradation that is the primary removal pathway in contaminated soils . Several  bio-remediation-enhancement technologies have been applied to increase PAH bio-availability and/or soil microbial abundance and activity such as surfactant amendment, bio-augmentation of known PAH-degrading microorganisms, and phytoremediation, to overcome the bio-availability limitations and environmental stability of PAHs. However, bio-remediation is a complex process involving numerous abiotic and biotic interactions that can result in the inhibition of soil microbial activity and lead to ineffective PAH degradation. Therefore, the effects of various  bio-remediation techniques that are commonly used for PAH-contaminated soils must be systematically investigated to address these issues and explore effective PAH remediation options. Because microbial degradation plays a vital role in PAH dissipation, the effects of remediation treatments, individually or in combination, on the soil microbial community must also be understood to ensure the protection of the environment and human health. This dissertation is compiled into five chapters. Chapter 1 is a review of the literature to-date that is relevant to the specific objectives of the dissertation. Chapters 2, 3, and 4 are each dedicated to one of the specific objectives. The first manuscript and second manuscript were published in Environmental Pollution and Chemosphere, respectively , while the third manuscript is being prepared for submission to Science of the Total Environment. Chapter 5 provides conclusions and recommendations for future research. Polycyclic aromatic hydrocarbons are aromatic hydrocarbons with two or more fused benzene rings and are ubiquitous environmental contaminants that are formed during the thermal decomposition of organic molecules.

Approximately 700 sites on the United States Environmental Protection Agency National Priorities List are polluted with PAHs,hydroponic barley fodder system of which more than 100 are PAH-contaminated soil sites . Bio-remediation, the use of microorganisms to transform hazardous compounds to nonhazardous forms, has been recognized as an efficient, cost-effective, and versatile approach to clean up PAH-contaminated soils . However, due to their hydrophobicity, PAHs are strongly associated with nonpolar soil domains such as soil organic matter or soil micropores, and therefore may not be bio-available to degrading microorganisms . Synthetic surfactants at concentrations both above and below their critical micelle concentration have been used to enhance the availability of PAHs to the degrading microbes and increase PAH biodegradation . At greater concentrations, a surfactant may become toxic to soil microbes by forming mixed micelles with the phospholipid bilayer of the cell membrane and solubilizing the cell membranes, inducing enzymatic disorders, cell penetration and even cell apoptosis . Because of the detrimental effects of synthetic surfactants, biosurfactants, notably rhamnolipid biosurfactants produced by Pseudomonas aeruginosa, have become known as environmentally benign alternatives to synthetic surfactants . Biosurfactants have similar properties to synthetic surfactants, including high surface-active properties and micelle formation; however, biosurfactants possess distinct advantages over synthetic surfactants. For example, rhamnolipid biosurfactants typically have better biocompatibility with cell membranes, resulting in lower toxicity to microorganisms, stability under extreme environmental conditions such as temperature, pH, and salinity, and relatively low soil-sorption tendency . The production of surface-active compounds by microorganisms is a critical microbial process that affects the bioavailability of PAHs and other hydrophobic organic contaminants in soil. For example, Mycobacterium vanbaalenii PYR-1, an isolate from an oil-contaminated estuary near the Gulf of Mexico, has been shown to produce trehalose-containing glycolipids, a type of surface-active biosurfactant, to enhance PAH solubility and degradation.Environmental stressors such as chemical toxicity have the potential to inhibit microbial activity that can lead to ineffective PAH biodegradation .

While the use of synthetic surfactants for bio-remediation of PAHcontaminated soils is well documented, albeit with inconsistent results, the application of biosurfactants in bio-remediation is relatively limited. In particular, few researchers have compared the performance of synthetic and biosurfactants in PAH biodegradation in native or bioaugmented soils, leading to inclusive views on their respective advantages and limitations. The objective of this study was to evaluate the effect of a commonly used synthetic surfactant, Brij-35, and rhamnolipid biosurfactant, at three amendment rates on PAH biodegradation in two soils. Biodegradation was determined in both native soils as well as soils augmented with a known PAH degrader, M. vanbaalenii PYR-1. 14C-Pyrene was used as a model PAH compound and mineralization to 14CO2 was measured as an endpoint of biodegradation.A San Emigdio fine sandy loam soil and Perry clay soil with substantial differences in organic matter and soil texture and initially free of PAH contamination were collected from the 0–30 cm depth at the South Coast Research and Extension Center in Irvine, California and Crossett, Arkansas, respectively. The soils were sieved through a 2-mm mesh before use. Soil particle-size distribution and textural classification were determined using the 12-h hydrometer method . The sand, silt, and clay percentages of the clay soil and sandy loam were 3%, 19%, 78% and 66%, 17%, and 17%, respectively. The soil total organic carbon was analyzed by hightemperature combustion using an Elementar Vario MAX C/N instrument after the addition of HCl for carbonate removal . The clay soil had a TOC of 2.42% and the sandy loam soil had a TOC of 0.66%. The CMC of the two surfactants was determined using a Du Noüy ringtensiometer . Briefly, the force required to raise a platinum wire ring from the surface of serial dilutions of surfactant stock was measured and the CMC was determined from the inflection point plotting the surface tension and logarithm of surfactant concentration. The CMC for the rhamnolipid biosurfactant was determined to be 70 mg L−1 , which was similar to previous studies using rhamnolipid biosurfactants . The emulsification activity of the surfactants was determined by mixing 2 mL surfactant with 2 mL n-hexadecane and vortexed for 2 min. The emulsion stability and the emulsification index were determined according to Youssef et al. .

An emulsion was considered stable if E24 was ≥50% ; both Brij-35 and rhamnolipid biosurfactant exhibited good emulsion stability. The 14C-pyrene mineralization assays were conducted using 250-mL Erlenmeyer flask respirometers, each consisting of a rubber stopper covered with aluminum foil and a 7 mL glass scintillation vial containing 1 mL 1.0 M NaOH suspended from the stopper. The NaOH acted as a trap for 14CO2 and hence offered a measurement of mineralization of 14Cpyrene . For each sample, 2 g soil was weighed into each respirometer and spiked with 10 mg kg−1 non-labeled pyrene and 1800 Bq of 14C-pyrene in 1.0 mL acetone. The treated flask was closed for 1 h to allow the chemical to disperse throughout the soil sample and then opened to allow the solvent to evaporate overnight . An additional 8 g soil was added and mixed with a stainless steel spatula over a period of 5 d. Following the chemical treatment, 10 mL sterilized MBS solution was added to the native soil treatment, followed by the addition of 10 mL surfactant solution at 0.1X, 1.0X, or 10X CMC, resulting in initial amendment rates of 21.6, 216, and 2160 μg surfactant g-dry soil−1 for Brij-35 and 14, 140, and 1400 μg surfactant g-dry soil−1 for rhamnolipid biosurfactant, respectively. For the bioaugmented treatment, 5 mL of sterilized MBS solution and 5 mL M. vanbaalenii PYR-1-MBS solution were added to yield approximately 107 cells g−1 soil,livestock fodder system and followed by the addition of 10 mL surfactant solution at 0.1X, 1.0X, or 10X CMC. All treatments contained soil slurries in a 1:2 soil:water ratio as Doick and Semple determined that this soil:water ratio resulted in greater overall mineralization and improved reproducibility as compared to other solidwater ratios. After spiking, the respirometers were sealed and placed in an incubator at 23 °C and mixed at 100 rpm. The 14CO2 traps were changed every 12 h for the first 2 d, and then daily for 2–7 d, followed by sampling approximately every 3 d thereafter. The trapped 14C-activity was measured after the addition of 6 mL Ultima Gold XR scintillation cocktail and subsequent liquid scintillation counting on a Beckman LS 5000TD Liquid Scintillation Counter . Respirometers were opened briefly on a weekly basis for aeration. Following the 50-d incubation period, the total 14C activity was determined by sample oxidation on an OX-500 Biological Oxidizer to determine 14C-activity remaining in the soil. Briefly, an aliquot of soil sample was air-dried in a fume hood and combusted at 900 °C for 4 min. The evolved 14CO2 was trapped in 15 mL Ultima Gold XR scintillation cocktail, followed by quantification on LSC . Blank soil samples were spiked with a known amount of 14C-pyrene in preliminary experiments and combusted to test the 14C recovery of the combustion method. The mean recovery was ≥90%. The mass balance of 14C-pyrene activity was greater than 80% among all soil treatments. Both untreated respirometers and mercuric-chloride-sterilized controls were analyzed as analytical blanks and indicated no abiotic losses of 14C-activity to the 14CO2 trap. Triplicate samples for each treatment were used in both surfactant toxicity evaluation and 14C-pyrene mineralization experiments.

Statistical analyses including analysis of variance and post-hoc Tukey’s range test were performed using SAS® 9.3 . Least square means for significant effects were compared using a protected least significant difference procedure at α = 0.05. The addition of Brij-35 at all concentrations in the native clay soil treatments resulted in the shortest lag period before PAH mineralization commenced as compared to the unamended clay soil treatment . After 25 d of incubation, the addition of Brij-35 surfactant at all rates in the native clay soil resulted in greater 14C-pyrene mineralization than the unamended native clay soil treatment . In the native sandy loam soil, the addition of Brij-35 at the low or high rate resulted in a longer lag period compared to the unamended control , while amendment of the Brij-35 surfactant at the medium rate shortened the lag period to 21 d . However, after 50 d of incubation, all Brij-35 surfactant treatments had greater 14C-pyrene mineralization as compared to the unamended native sandy loam soil, and the enhancement may be attributed to increased bio-availability of 14C-pyrene to the native soil microorganisms .Several studies showed that the addition of Brij-35 surfactant and rhamnolipid biosurfactant enhanced PAH desorption in PAH-contaminated soil systems, making the contaminants more bio-available. Bueno-Montes et al. used Tenax extraction to measure the bioaccessible fractions of PAHs in aged, creosote-polluted and MGP soils and showed that the addition of Brij-35 at 25X CMC resulted in significant PAH solubilization in both soils. Congiu and Ortega-Calvo also used Tenax extraction and 14C-pyrene to evaluate the effect of rhamnolipid biosurfactant on PAH solubilization and concluded that the rhamnolipid biosurfactant significantly increased the solubilization of pyrene that was initially sorbed to soil. Additionally, Adrion et al. examined the effects of four nonionic surfactants and rhamnolipid biosurfactant below their respective CMCs on PAH desorption in PAH-contaminated soil from a former MGP site. They observed that all surfactants increased PAH desorption; however, all surfactants except the rhamnolipid biosurfactant significantly increased PAH biodegradation, possibly due to rhamnolipid biosurfactant having the least effect on PAH desorption. In this study, the addition of rhamnolipid biosurfactant at the medium and high rates resulted in significant inhibition of 14C-pyrene mineralization as compared to the unamended soils . The application of rhamnolipid biosurfactant at the medium and high rates resulted in an increased lag period in all native and bioaugmented soil treatments, as well as smaller biodegradation rates in the bioaugmented soil treatments as compared to the unamended treatments . Because of its biological origin, it is likely that the rhamnolipid biosurfactant is more amendable as asubstrate for soil microorganisms. In this case, the rhamnolipid biosurfactant likely acted as a more favorable carbon source compared to pyrene in the soil system, and the native or inoculated biodegraders exhausted the rhamnolipid biosurfactant as a carbon and energy source first, before resorting to pyrene as a substrate. This biphasic growth and sequential use of two different carbon substrates, where the easier carbon source supporting faster microbial growth is mineralized first, is referred to as “diauxie” . This possibility was supported by the eventual increased 14C-pyrene mineralization in the bioaugmented soils amended with rhamnolipid biosurfactant . Several previous studies also showed preferential degradation of the externally added rhamnolipid biosurfactant instead of the target compound. Vipulanandan and Ren observed the preferential degradation by a Pseudomonas sp. of rhamnolipid biosurfactant over solubilized naphthalene in biosurfactant and Triton X-100 solutions. The addition of rhamnolipid biosurfactant increased the time for complete naphthalene degradation to 40 d, as compared to only 100 h for Triton X-100, even though the presence of rhamnolipid biosurfactant clearly increased the solubility of naphthalene by 30 times its aqueous solubility.