Research Goals: The primary goals of this research are: a) to gain an understanding of the transport and degradation mechanisms involved in soil bioremediation systems for a variety of PAHs; 2) to define the role of biofilms, in terms of growth, structure and chemistry in PAH biodegradation, and 3) to understand the mass transport of nutrients and contaminants to biofilm microorganisms. A porous sand media in a glass column is used to simulate a soil contaminated by PAHs; samples ports as well as flowcells are located along the column, so that PAH removal profiles, physical and chemical structure s and growth patterns of the biofilm can be determined.

Overview: Polycyclic aromatic hydrocarbons (PAHs) are polynuclear chemicals containing at least two condensed rings. Their unique characteristics make them of interest for new research and investigation: high densities, low solubilities, high hydrophobicities low vapor pressures and therefore, low biodegradabilities. They are produced by the incomplete combustion of coal, oil and gas, garbage and other organic substances; there is also knowledge about the use of these chemicals in pharmaceutical and dying industries, for pesticides, plastics and wood preservation.

In-situ bioremediation has been proposed as a new alternative for PAH degradation in soils, showing excellent results. Factors like low water solubility, lack of appropriate microorganisms, lack of needed nutrients or the need for oxygen often limit the effectiveness of in-situ bioremediation of PAHs, though.

Assuming black-box approaches, different studies have been conducted on soil pore water, giving a poor description of the actual rate of disappearance of the PAHs. However, no research has been reported on the biofilm surrounding the soil particles, which is the primary site where biodegradation occurs. The characterization of the soil biofilm will be performed in this study by:

• Determination of biofilm physical structure by 1) confocal scanning laser microscopy (CSLM), which will help with the measurement of the biofilm thickness, heterogeneity and growth patterns, and 2) micro-slicing techniques, which will help with the measurement of the biofilm density and porosity.

• Determination of biofilm chemical characteristics by using microelectrodes: pH, redox potential, dissolved oxygen, nitrates, ammonia.

• Determination of firmly and weakly attached biomass (by measuring lipid phosphates) and of polysaccharide content of the exopolymer matrix (by carbohydrate and protein contents and volatile solids)

• Determination of the solid phase PAHs (SPAH) by analyzing sand samples. This will give the total PAH content of sand and biofilm

• Determination of biofilm cell count and content by using standard plate counting procedures to give a general description of the number and type of biological community structures.

• Determination of specific PAH degraders within the biofilm by using Fluorescence In – Situ Hybridization (FISH)

Progress to Date: Glass columns and flow cells have been designed and manufactured. Columns and flow cells have been packed with a mixture of sand and mixed liquor (from an aeration tank of a municipal wastewater treatment plant) and biofilms have been developed. Low and high molecular weight PAHs, such as naphthalene,phenanthrene and pyrene, have been used as sole-substrate systems and in binary and tertiary mixtures to examine substrate interactions of cometabolism and competitive inhibition on porous media biofilms. It was shown that phenanthrene and pyrene could not be degraded as sole carbon sources in the system, but binary systems of the 3and 4-ring PAHs with acetate and napthalene stimulated their degradation, up to 87.9% and 70.1% removal efficiencies respectively. However, in the tertiary systems the presence of phenanthrene inhibited pyrene degradation. Absorption of PAHs to sand media was measured to negligible biofilm growth, development and changes in composition were analyzed over time, showing increases in both firmly and loosely attached viable biomass, as well as extracellular polymeric substance production to form complex matrix.Heterogeneous surface films and variety of biological aggregate structures and growth pattern were observed by confocal microscopy.

Application of Knowledge: Bioremediation of contaminated soil and groundwater, biofilm processes in wastewater treatment, PAH biodegradation.

Future Directions: The use of nonionic surfactants is being investigated as a method to improve the solubility and transport of PAHs in biofilm systems. Both positive and negative effects have been frequently observed and widely reported in the literature, but no research had been done yet with surfactants in porous media biofilms.

Biofilm physical structure will be quantitatively described from 3-dimensional confocal laser scanning microscopy to monitor biofilm development and growth, and quantify environmental factors affecting biofilm heterogeneity, size and morphology.

Techniques Incorporated: PAHs: gas chromatography; biofilm physical structure: confocal scanning laser microscopy, micro-slicing; biofilm chemical composition: microelectrodes (ORP, pH, nitrates, ammonia, DO); viable biomass: lipid phosphates; extracellular polymeric substances: carbohydrates and protein.

Keywords:
PAH biodegradation
Biofilm
Bioremediation
Microelectrodes
Confocal microscopy