t. 1.1: . mam. flag... .. . . V _. ‘ . .. . , $.25 . a V .A . . a; Ziafifififihgmgfi . t. i 1....74‘... i3 LIBRARY Michigan State University This is to certify that the thesis entitled ANALYSIS OF PAH-DEGRADING BACTERIA ASSOCIATED M.S. WITH PHYTOREMEDIATION presented by ENDANG SUSILAWATI has been accepted towards fulfillment of the requirements for the degree in Crop & Soil Sciences \\ / Major Professor’s Signature ../ Date MSU is an Affirmative Action/Equal Opportunity Institution _. a—-c.---—-I-o-.-a-¢-o—.---n-n—--l_c-l-l-O-O-O-O-O-O-O-t—n—4—-—— , PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE ipfl 15b. £007 6/01 c:lCIRC/DaIeDue.p65-p.15 ANALYSIS OF PAH-DEGRADING BACTERIA ASSOCIATED WITH PHYTOREMEDIATION By Endang Susilawati A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 2003 ABSTRACT ANALYSIS OF PAH-DEGRADING BACTERIA ASSOCIATED WITH PHYTOREMEDIATION By Endang Susilawati Phytoremediation, the use of plants to clean up organic and inorganic pollutants, is an emerging remediation technology considered to be a cost effective, aesthetically pleasing, and more environmentally compatible alternative to engineering-based methods. Plant-microbe rhizosphere interactions are considered the major mechanism for phytoremediation of some organic contaminants, including polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs). This research study is focused on the soil microbial community response in the rhizosphere of different plant species in phytoremediation field trials in PAH contaminated soils. We hypothesized that different plant species have different effects in the abundance and diversity of bacterial PAH degraders. We utilized a direct PAH metabolism technique, the “Spray Plate Assay”, to quantify culturable and PAH-degrading soil bacteria from five different field treatments: planted with Aster novae-angliae, Eupatorium purpureum, Labelia cardinalis, and Spartina pectinata, and an unplanted control. It was shown that A. novae-angliae and S. pectinata increased total soil bacteria relative to the other treatments. A. novae-angliae and E. purpureum were observed to enrich PAH-degradin g bacteria up to 4 times compared to the other treatments. In summary, this study demonstrated that appropriate plant selection is a critical factor for design of an effective phytoremediation treatment. ACKNOWLEDGEMENTS I would like to express my gratitude and appreciation to many people who made this Masters thesis possible. I am deeply indebted to my major advisor Dr. Clayton L. Rugh from the MSU Crop and Soil Sciences Department whose help, stimulating suggestions, and encouragement helped me in all the time of my study and research for writing of this thesis and supporting my Masters study. I want to thank the Department of Crop and Soil Sciences for giving me a chance to study at Michigan State University. I have furthermore to thank Ford Motor Company for partial funding support of my master study. My special thanks to Dr. Alvin J.M. Smucker and Dr. Terence L. Marsh for their opinions, suggestions, and help to my research program. My great thanks to Dr. Sasha Kravchenko who helped me with statistical training and analysis of my data. Also, I extend my great appreciations to Indonesian Oil Palm Research Institute (IOPRI), who allowed me to continue my graduate study in US and partly supports my graduate study. To my former colleagues from Crop and Soil Sciences Department who supported me in my MS, many thanks for all their help, support, interest and valuable hints. I am especially obliged to Rachada Settavongsin, Nathan Stewart, and Andrew Bender who helped me during my research working until very late. My special thanks also to Sarah Kinder for related computer support and her great suggestions for my research. Last but not least, my great appreciation to Theresa Wood, Cindy Wan, Chris Saffron, Susan Redwine, Sarah Marshall, and Sarah Norris for the great time during my work in phytoremediation laboratory. Thanks are also due to my supervisor in Scientific Writing iii Class, Dr. Snider, who helped me editing my thesis. And also for my writing classmates Harry and Hong Bo who gave me ideas for my thesis during the class. Especially, I would like to give my special thanks and love to my husband whose patient love enabled me to complete my study. I am very grateful for the love and supports of my Mom and Dad who have taken care of my three lovely sons Ega Anugerah Prima, Fakhry Pranayanda, and Kevin Triananda. This is all for you guys. iv TABLE OF CONTENTS PAGE LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ........................................................................................................ vii INTRODUCTION ............................................................................................................ 1 CHAPTER 1. REVIEW OF LITERATURE .................................................................. 3 References .......................................................................................................... 20 CHAPTER 2. EVALUATION OF METHODS FOR ENUMERATION OF SOIL PAH-DEGRADING BACTERIA ........................................... 28 Abstract ............................................................................................................. 29 Introduction ....................................................................................................... 30 Materials and Methods ....................................................................................... 32 Results ............................................................................................................... 36 Discussions ........................................................................................................ 46 Summary and Conclusion .................................................................................. 47 References .......................................................................................................... 49 CHAPTER 3. ANALYSIS OF SOIL MICROBES IN PHYTOREMEDIATION FIELD SOILS ....................................................................................... 51 Abstract ............................................................................................................. 52 Introduction ....................................................................................................... 53 Materials and Methods ...................................................................................... 54 Results ............................................................................................................... 61 Discussions ........................................................................................................ 64 Summary and Conclusion ................................................................................. 71 References ......................................................................................................... 73 LIST OF TABLES PAGE Table 2.1. WST-l OD450 values of planted soil microbial extracts ............................... 39 Table 2.2. Net OD450.630 values of planted and unplanted soil microbe extracts ........... 40 Table 2.3a-d. WST-l Microtiter Assay verification analysis ......................................... 42 Table 2.4. Primary Spray Test analysis of 11 treated soils from phytoremediation field trials ........................................................................................................... 44 Table 2.5. Spray Test Assay confirmation analysis for 10 isolates ............................... 45 Table 3.1. Planted soil treatments in the Phytoremediation Field Study plot ............... 57 Table 3.2. Preliminary Spray Test analysis of treated soils from 19 phytoremediation field trials ............................................................................................................ 63 Table 3.3. Relative phenanthrene biodegrader (ZFU) bacteria] density in selected treatments ........................................................................................................... 67 Table 3.4. Spray Test Assay analysis of 1° isolates against various PAH compounds. 68 vi LIST OF FIGURES PAGE Figure 2.1. Indole Conversion Assay ............................................................................. 37 Figure 2.2a. Spray Plate Assay for PAH-contaminated soil extract ............................. 43 Figure 2.2b. Re-plated culture of isolated Spray-positive colony ................................. 43 Figure 3.1. Phytoremediation Field Study Upland Plot layout schematic ..................... 56 Figure 3.2. Preliminary Spray Plate Assay analysis of phytoremediation study soils... 62 Figure 3.3. Colony Forming Units (CFUS) from soils of selected treatments ............... 65 Figure 3.4. Zone Forming Units (ZFUS) from soils of selected treatments ................... 66 Figure 3.5a-c. Spray Test Assay of selected isolates with 3 PAHs ................................ 69 Figure 3.5d. Spray Test Assay of selected isolates with benzo(a)pyrene ...................... 69 vii INTRODUCTION Polycyclic aromatic hydrocarbons (PAHS) are persistent organic pollutants (POPS) that are widely distributed in the ecosphere. PAHs have been found to have carcinogenic and mutagenic properties and tend to biomagnify in the food chain. PAHs strongly sorb to soil organic matter causing them to be resistant to biological degradation. Engineering-based technologies, such as excavation or thermo/chemical treatments, are the most common approaches for soil remediation, though these are typically expensive and environmentally disruptive. Biological remediation applications are being developed as more affordable and ecologically compatible strategies for environmental remediation. Bioremediation typically refers to microbe-based remediation and has been extensively developed as a treatment for soil and water contamination by organic and elemental pollutants. Phytoremediation, the use of plants to clean up environmental contamination, offers some unique advantages over microbial or engineering-based approaches. Plants have been shown to possess utility against a broad range of pollutant classes, while concurrently extracting water to minimize contaminant migration, harvesting sunlight for energy and deciduous biomass, and regenerating soil fertility. In phytoremediation of organic pollutants, the interaction between microbes and specific plants is thought to be the main process for biodegradation. PAH biodegradation rates are dependent on their chemical properties, site conditions, and soil biological activity. Plants release root exudates containing enzymes, sugars, and secondary metabolite products, which have been shown to stimulate microbial contaminant degradation. However, despite extensive PAH phytoremediation research, basic mechanisms of plant-assisted biodegradation is not well understood. This research is one component of a phytoremediation field study in PAH- impacted soils located at the Ford Rouge Manufacturing Complex (Dearborn, MI). In order to rehabilitate this site using phytoremediation, Ford Motor Company and Michigan State University are directing an ongoing research program to utilize 18 different Michigan native plant species to accelerate the soil biodetoxification process. The Phytoremediation Demonstration Facility was built up in September 2000 and located at Allen Park, Claymine (MI). The focus of this research is to enumerate the bacterial PAH- degrader abundance in planted soils during PAH phytoremediation. Our hypothesis is that each plant species affects the abundance of total bacteria and PAH bacterial degrader in different ways, some of which can contribute to enhanced biodegradation processes. The first phase of this program is to survey published procedures for characterization of PAH- degrading bacteria and to tailor the selected protocol for analysis of phytoremediation treated soils. A modified Spray Plate Assay was ultimately developed to identify and quantify PAH-degrading bacteria in rhizosphere soils of a variety of selected plant species. Over the course of this analysis, we observed that plant root systems greatly influence both soil bacterial density and the ratio of PAH-degrading bacteria. Preliminary experiments suggested that plants may also stimulate particular metabolic activities against different PAH compounds. This research will help to identify essential components of the plant-microbe PAH biodegradation process and allow development of more effective strategies for phytoremediation treatment of organic pollutants. CHAPTER 1 REVIEW OF LITERATURE INTRODUCTION Organic and inorganic contaminants have become major environmental problems for human and wildlife ecosystems. Environmental pollution poses severe economic and medical burdens to human populations due to spoilage of natural resources and elevated incidences of disease in impacted communities. Efforts to clean up these environmental hazards are slowed by both the high cost and extreme size and number of impacted areas. In contrast to intensively engineering based technology, biological methods for ecological remediation have been proposed as a less disruptive, less costly, and more environmentally compatible approach. Organic contaminants are described as compounds containing carbon atoms in their chemical structures and include pesticides, herbicides, chlorinated solvents, petroleum hydrocarbons, polycyclic aromatic hydrocarbons (PAHS), and polychlorinated biphenyls (PCBs). Many of these compounds strongly sorb to soil particles causing them to be resistant to degradation and persistent in the environment. Additionally, the class of organic compounds known as Persistent Organic Pollutants (POPS), including PCBs, PAHS, and some pesticides, can biomagnify in animal tissues posing a major health risk to humans and other living creatures. Inorganic contaminants are elemental pollutants, including nutrient minerals (e. g. nitrogen, phosphorous), heavy metals (e. g. lead, mercury), and radionuclides (e. g. cesium, uranium). Pollution by toxic metals and radionuclides in our environment has resulted from industrial, agricultural, and sewage disposal activities. The primary sources of toxic metals are the burning of fossil fuels, mining, and smelting activities (Vangronsveld et al., 1996). Inorganic pollutants are disposed and transported into atmospheric, aquatic, and terrestrial environments mainly as solutes or particulates, which may accumulate to high concentrations causing risks to public health. Several different techniques can be used in cleaning up our environment from both organic and inorganic contamination. These techniques can be physical, chemical, or biological removal of the contaminants from the impacted sites. Engineering-based physical and chemical techniques are typically very expensive and some of them may be ineffective in permanently detoxifying the contaminants. Alternatively, biological methods such as bioremediation and phytoremediation are considered cost-effective and ecologically compatible tools for environmental cleanup of a wide array of contaminants. Bioremediation is defined as the use of living organisms, typically microbes, to breakdown or to mineralize hazardous pollutants into less harmful or non-toxic compounds. The goal of bioremediation is to stimulate microorganisms with nutrient and other mineral components that will allow them to degrade and/or detoxify the contaminants. Addition of pre-grown cultures of demonstrated microbial degraders to contaminated sites has also been proposed as a method to enhance contaminant biodegradation rates. Extensive research has been conducted for biological degradation of a wide array of organic contaminants, such as dioxin, trichloroethylene (TCE), petroleum byproducts, and PAHs. Bioremediation of organic contaminants may result in complete degradation of the contaminants or at least reduction to levels below regulatory guidelines. Numerous bacterial strains have been shown to utilize low molecular weight PAHs such as naphthalene, phenanthrene, and anthracene as a sole carbon and energy source (Cemiglia, 1992; Kastner, 1994). Biodegradation rates are influenced by the concentration and chemical properties of the specific PAHS, as well as the diversity and density of the PAH-degrading microorganisms. The low water solubility of PAHs increases soil matrix partitioning and limits contaminant bioavailability for degradation. An additional biological limitation to PAH degradation is the low number of PAH- degrading bacteria typically found in contaminated soils (Kastner, 1994). Phytoremediation is the use of plants and the associated rhizosphere microorganisms to remove, transform, or contain toxic chemicals in soils, sediments, water, and the atmosphere. Phytoremediation has been used to clean up widely different contaminants such as petroleum hydrocarbons, chlorinated solvents, pesticides, explosives, and metals. Similar to bioremediation, phytoremediation applications may reduce installation and operating expenses relative to engineering-based approaches. In addition, plants can promote various ancillary benefits during site remediation including enhancement of soil fertility, soil physical structure, soil aeration, and reduced migration of pollutants to ground water by hydraulic utilization and containment (Chang and Corapcioglu, 1998). Plants have been shown to enhance microbial degradative activities, a process known as plant-assisted biodegradation or phytostimulation. Plant roots provide nutrient from secretions and dead or decaying cells for soil microbes such as simple sugars, amino acids, fatty acids, and organic acids. Root exudates also contain plant secondary metabolites, such as phenolics and terpenes, which may influence the metabolic activity of microbes for degradation of soil organic contaminants. The plant compounds may serve as carbon resources for microorganisms that perform co-metabolic biotransformation, or breakdown of the target contaminants that cannot serve as primary carbon or energy sources. Root exudates have been shown to enhance degradation of organic contaminants relative to biodegradation rates in unplanted or unamended soils (Miya and Firestone, 2001; Siciliano et al., 1998; Yoshitomi, 2001). Previous research was conducted to identify plants capable of enhanced PAH remediation (Wan, 2002). In this study, 18 native Michigan plant species were planted in a Phytoremediation Demonstration field experiment located at the Allen Park Claymine (Allen Park, MI). It was shown that most, though not all, of the tested plant species accelerated total PAH (tPAH) reduction compared to Implanted soils within the first year of the study. These results demonstrated that plant species selection is a critical factor for effective phytoremediation. The objective of this research study is to evaluate the influence of different native Michigan plant species on the total number of culturable bacteria and suspected PAH- degrading bacteria in the treated soils during the phytoremediation process. The results of this study will enhance our understanding of plant-microbe interactions during PAH- phytoremediation and allow improvement for future applications of planted systems to treat impacted soils. LITERATURE REVIEW Environmental Pollution The accumulation of organic and inorganic pollutants in the world’s soils, waters, and atmosphere poses a risk to human, wildlife, and ecological health. In general, terrestrial pollutants are divided into two major categories; heavy metals (e.g. Cdz”, Niz“, Hg“) and organic, or carbon-based, chemicals (Schaffner et al., 2002). Metals can exist in soil as discrete particles or associate with soil organic matters and soil minerals. They are relatively immobile. The aqueous solubility of metals is depended on soil characteristics and strongly influenced by soil pH (Chaney et al., 1997). Organic pollutants include aromatic chemicals such as polycyclic aromatic hydrocarbons (PAHS), and polychlorinated biphenyls (PCBS), which are hydrophobic compounds that tend to bind with organic matter or soil, thus limiting their bioavailability to microbial degradation. The solubility and immobility of these pollutants in soils are influenced by soil physical and chemical characteristics such aeration, soil moisture, temperature, soluble organic matter, (Joner et al., 2001; Robinson et al., 2003) and the hydrophobicity of these pollutants. Substantial remediation efforts are focused on a class of contaminants known as “persistent organic pollutants”, or POPS, due to their toxicity, persistence in the environment, and tendency to biomagnify to harmful concentrations. POPS, such as PCBs, PAHS, and chlorinated pesticides, possess low aqueous solubility and strong adsorption to soil matrices, which further makes them resistant to abiotic decomposition and biodegradation (Eduljee, 2001). POP biodegradation rates are dependent upon their bioavailability, the number of aromatic rings (PAHS), and the degree of halogenation (dioxins, PCBs). Prolonged contaminant residence time in soils and sediments allows these compounds to become “weathered” or more tightly bound to the soil matrix and less available for biodegradation (Alexander, 2000; Kelsey and Alexander, 1996). Though aging reduces the bioavailability of organic pollutants such as chlorinated pesticides, PCBs, and PAHS, it does not eliminate their ecotoxicity. Physical disturbances of contaminated soils and sediments distribute and release toxic organic pollutants into the environment to increase biological exposure. Polycyclic aromatic hydrocarbons (PAHS) PAHS are polycyclic aromatic hydrocarbons composed of two or more benzene rings fused together in linear, angular, and cluster arrangements (Wilson and Jones, 1993). PAHS may be introduced into the soil environment by natural biogeochemical processes or human activities (Benlahcen et al., 1997). Open burning of agricultural debris can generate PAHS, mainly those of low molecular weight PAHS (Kakareka and Kukharchyk, 2003). The principal source of PAHS in the environment is incomplete combustion of fossil fuels (Pothuluri and Cerniglia, 1994). PAHS possess low water solubility and high octanol partitioning coefficients (logKow), resulting in increased sorption to the soil matrix and environmental persistence. The US EPA lists sixteen different PAHS as contaminants of concern (Keith and Telliard, 1979). PAHS are an ongoing source of public health concerns because of their toxic, mutagenic, and carcinogenic properties. PAHS with log Kow less than 5.2 (e.g. naphthalene, anthracene, phenanthrene, fluorene, pyrene, and fluoranthene) significantly reduced the survival and reproduction of bioassay microorganisms (Sverdrup et al., 2002). Most higher molecular weight PAHS, including fluoranthene, benzo(a)anthracene, chrysene, and benzo(a)pyrene, have been shown to be carcinogenic (Cemiglia, 1992). Since some PAHS are potent carcinogens in laboratory studies, they are considered as a cancer risk to humans as well (Dipple, 1990). Metabolic conversion of PAHS can produce reactive diol-epoxide intermediates, which bind to critical targets in DNA (Sims and Overcash, 1983). PAHS have been shown to impact seed germination and plant growth. In the absence of PAHS, lettuce (Lepidium sativum) seed germination was more than 95%. However, seed germination was lowered at increasing PAH concentrations to 75% germination at 50 ppm soil PAH and less than 16% seed germination at 1000 ppm PAH (Maila and Cloete, 2002). Pollution Remediation Technologies Engineering-based remediation applications include excavation and landfill disposal, incineration, solidification, carbon adsorption, UV oxidation, chemical precipitation, and soil vapor extraction. (FRTR, 2003) Among these techniques, excavation and landfill disposal is the most commonly used remediation technology for cleaning up contaminated soils. For excavation and landfill disposal, the contaminated material is removed and transported to off-site facilities, though in some cases, pretreatment of the contaminated media is required to meet land disposal restrictions. This method is not considered a permanent solution because it simply moves the contaminants to another area, which may pose a future hazard. Excavation approaches are typically environmentally disruptive to the site and can be extremely expensive. Compared to engineering technologies, biological approaches such as bioremediation and phytoremediation are considered low cost and possibly more effective long-term treatments (EPA, 2001). Bioremediation and phytoremediation are applicable for cleanup of both organic and inorganic pollutants. In bioremediation, either indigenous or added rrricrobes may be utilized for pollutant degradation. For phytoremediation, plants are used to clean up hazardous chemicals. Phytoremediation may rely upon plant-mediated processes or enhanced conditions for microbial growth and activity for contaminant removal and/or detoxification. Over the last 10-12 years, researchers have conducted laboratory, greenhouse, and pilot-scale field experiments to evaluate phytoremediation as an environmental decontamination technology. Phytoremediation is considered less expensive than standard, engineering-based remediation technologies. For example, the estimated costs for cleaning up lead contaminated soils using an excavation-landfilling approach are approximately $150 to $350 per ton of contaminated soils, meanwhile the estimated costs of phytoremediation treatment of lead-contaminated site, including off-site disposal of hazardous biomass wastes, are $20 to $80 per ton (Ensley, 2000). Phytoremediation may also be less expensive than microbe-based bioremediation techniques. The cost to clean up petroleum hydrocarbon-contaminated site using bioremediation techniques in a fully contained facility was estimated at $20,000 to $60,000 per hectare (15 cm depth), though cheaper using in situ bioremediation at $7,500 to $20,000 per hectare. Phytoremediation treatment for this site was estimated to be between $2,500 to $15,000 per hectare, due primarily to reduced management and soil manipulation oneeds (Cunningham and Ow, 1996). Bioremediation Bioremediation is degradation or detoxification of both organic and inorganic contaminants into nontoxic or less toxic substances. Bioremediation is considered as a potential low-cost alternative technology compared to traditional engineering technologies. In bioremediation, indigenous or supplemented microbial species or complex community interactions are utilized for contaminated environment remediation. 10 Bioremediation effectiveness may be dictated by microbial community structure and density, nutrient availability, aerobic or anaerobic conditions, or other soil physical- chemical characteristics, including pH, moisture, and temperature. Therefore, stable maintenance of optimal habitat conditions is essential, though frequently difficult, for sustained bio-treatment. Bioremediation has been developed as a treatment technology for organic contaminants (Comeau et al., 1993; Steffan et al., 1999). Transformation of organic contaminants during microbial degradation occurs via two different metabolic processes, direct metabolism and co-metabolic degradation. In the first, microbes use the target organic contaminants as a carbon and energy source for their growth and reproduction. In the second process, microbes degrade the organic pollutants through co-metabolic transformation, which means that although they can transform or detoxify organic pollutants they do not use them as their grth substrate (Fijalkowska et al., 1998; Hage et al., 2001). Highly chlorinated carbon such as tetrachloroethene (TCE) and PCBS are resistant to aerobic biodegradation; but they can be reductively dechlorinated under anaerobic conditions (Aulenta et al., 2002; Tiedje et al., 1993). The removal of metals by engineering techniques such as excavation/confinement and soil washing are expensive and may not be effective. Recent studies have demonstrated that bacteria can be used to remediate metal contamination by removing metals from contaminated water or waste streams, sequestering metals in soils and sediments, or solubilizing metals (Lovley and Coates, 1997). Metal bioremediation can be accomplished either by biosorption of metals or by enzymatically catalyzed changes in the metal redox state. For example, some bacteria are able to enzymatically reduce Hg2+ ll to volatile Hg° thereby reducing toxicity and microhabitat mercury levels (Barkay et al., 1992). In other bioremediation process, certain microorganisms transform strongly adsorbing metal into more soluble forms for extraction and removal (Ahmann, 1997). In other applications bacterial, algal and fungal biomass has been used to accumulate and extract metals from aqueous media (Barkley, 1991; Cai et al., 1995; Gadd and White, 1993). PAH bioremediation PAHS are degraded by abiotically, via photolysis or chemical oxidation, though microbiological degradation is the major decomposition process (Cemiglia, 1993). PAH biodegradation rates are dependent upon the concentration and chemical properties of the PAH contaminants, environmental conditions, and microbial community structure (Carmichael and Pfaender, 1997; Cemiglia, 1992; Stapleton, 1998). PAH biodegradation rates may be limited if soil conditions are not optimal for microbial activity or PAHS have low bioavailability due to sorption to soil organic matter. The breakdown rates of PAHS in natural soil environments were observed to decrease over time compared to laboratory studies, possibly due to low nutrient levels, low PAH bioavailability, and heterogeneous soil structure (Amellal et al., 2001). Algal, bacterial, and fungal species have been shown to be capable of PAH biodegradation (Kotterman et al., 1998; Sutherland et al., 1995; Warshawsky et al., 1995). Bacterial PAH biodegradation has been well demonstrated for low molecular weight PAHS such as naphthalene and phenanthrene, though high molecular weight PAHS are more resistant to biodegradation (Cemiglia, 1992; Mahro et al., 1994). Numerous bacterial strains have been identified capable in degrading PAHS with fewer 12 than 3-benzene rings. Some microbes that are capable in degradation of PAHS with greater than two-benzene rings are Mycobacterium, Nocardia, and Rhodococcus (Moody et al., 2001). Mycobacterr'um sp. was demonstrated to rnineralize over 60% of 14C-pyrene after 96 hours of incubation. Rhodococcus Sp. W] was shown to use pyrene as a sole carbon and energy source (Walter, 1991). Other bacteria can utilize the four-ringed PAH fluoranthene as a growth substrate (Juhasz et al., 1997; Rehmann et al., 2001; Sepic et al., 1998). The primary steps for PAH degradation are monooxygenase or dioxygenase enzyme activity, involving the single or double insertion of oxygen atoms into the aromatic nucleus (Heitkamp, 1988). Prokaryotes utilize dioxygenase enzymes to attack PAHS to form dihydrodiols, though eukaryotic organisms such as fungi attack PAH molecules by monooxygenase acivity (Cemiglia, 1992). Monooxygenase breakdown of PAHS has been shown to lead to the formation of epoxide intermediates, which have been demonstrated to induce tumorigenesis in in vitra assays (Sims and Overcash, 1983). Phytoremediation Phytoremediation is defined as the use of plant systems and their rhizosphere microbial communities to remove, degrade, or stabilize environmental contaminants (Flathman and Lanza, 1998). Phytoremediation is also referred to bioremediation, botanical bioremediation, or green bioremediation (Chaney et al., 1997). Phytoremediation is an emerging technology for environmental cleanup generating more than 600 research papers reporting the use of plants for treatment of pollutants in from soil, wetland, and hydroponic media in the years 1997 to 2002 (Singer et al., 2003). The term phytoremediation refers to a diverse collection of technologies using either naturally 13 occurring or genetically engineered plants for environmental remediation. Phytoremediation has been used to treat different types of organic contaminants such as petroleum hydrocarbons, chlorinated solvents, pesticides, and explosives as well as inorganic contaminants such as radionuclides and metals. Phytoremediation technologies There are several categories of phytoremediation treatment technologies: phytoextraction, phytovolatilization, phytostabilization, phytodegradation, and phytostimulation (Cunningham and CW, 1996). Phytoextraction is the use of plants to uptake heavy metal contaminants into plant shoot tissues for harvest and disposal (Baker and Brooks, 1989). Phytovolatilization is the use of plants and associated microbial activity to increase the rate of pollutant extraction from contaminated soils and evaporation from aerial plant parts. Phytostabilization is the use of plants as both a groundcover and hydraulic “pump” to minimize migration of contaminants through soil media. Phytodegradation, or phytotransformation, is the use of plants to take up organic contaminants and metabolize them to non-toxic products. Phytostimulation, also called rhizodegradation, is the use of plants and associated soil microbes to biodegrade organic contaminants in the root zone, or rhizosphere. Phytodegradation of organic pollutants Phytodegradation has been studied for a wide variety of organic compounds. Translocated contaminants may be volatilized, metabolized, or mineralized by plant enzymes (Schnoor et al., 1995). However, plant uptake and metabolism of some classes of organic contaminants may be limited by their hydrophobicity, though more lipophilic compounds may be accumulated by oil-rich roots or plant tissues (Briggs et al., 1982). 14 Less hydrophobic compounds may be more readily translocated through plant tissues (T rapp, 2000). Poplar trees have been widely utilized for phytodegradation and hydraulic control of water—soluble organic pollutants, such as trinitrotoluene (TNT), atrazine, and trichloroethylene (TCE). Poplars were observed to degrade 90% of atrazine in an 80 days hydroponic study (Burken and Schnoor, 1997). The widespread groundwater pollutant, TCE, was effectively phytoextracted in experimental test plots of hybrid poplars (Newman et al., 1999). Hybrid poplars were also used to remove the nitroaromatic explosives, TNT and RDX, from aqueous media (Thompson et al., 1999; Thompson et al., 1998). Equally important to phytodegradation is the ability of phreatophytic, or “water-loving plants”, such as hybrid poplar to utilize large quantities of contaminated water and minimize the migration of soluble pollutants (Burken and Schnoor, 1998; Hinckley et al., 1994; Trapp et al., 2001). Rhizosphere microorganisms are thought to be primarily responsible for biodegradation of organic contaminants, though recent genomic and biochemical surveys suggested a wide variety of plant enzymes that may be capable of transformation, conyugation, and translocation of organic pollutants (Schaffner et al., 2002; Schroder et al., 2001; Schuler, 1996). Phytostimulation Plants may enhance contaminant biodegradation and bioremediation by stimulation of microbial proliferation and metabolic activity in the rhizosphere. The rhizosphere is a zone of increased microbial biomass and metabolic activity at the root- soil interface (Anderson et al., 1993). Plant-microbe interactions have been the focus of 15 extensive agricultural research because of the importance of this symbiosis for crop productivity, e.g. (Miethling et al., 2000; Smalla et al., 2001; Wieland et al., 2001). The association of plant roots and microbial communities has been shown to accelerate the degradation of many organic compounds including pesticides, solvents, petroleum products, and recalcitrant organic contaminants. Roots release sloughed cells and secreted exudates, which contain a wide variety of materials including enzymes, amino acids, sugars, and aromatic and aliphatic plant secondary compounds. Plant-microbe associations may enhance biological treatments of inorganic and organic contaminants. Rhizosphere bacteria were observed to enhance phytoextraction of the inorganic pollutants mercury and selenium (de Souza et al., 1999). The rhizosphere microbial community was also found to significantly improve cadmium and zinc uptake by hyperaccumulator plants grown on metal impacted soils (McGrath et al., 2001). Plant- microbe interactions were also found to be superior then microbe- or plant-only treatments for biodegradation of a variety of organic compounds, including TCE (Walton and Anderson, 1990), 2,4-dichlorophenoxyacetic acid (2,4-D) (Boyle and Shann, 1995), and chlorobenzoic acid (CBA) (Siciliano and Germida, 1998). The chemical composition of root-secreted secondary compounds may be the primary component of the phytostimulation process. Root exudates can contain essential carbon substrates for microbial co-metabolism of aromatic and heterocyclic xenobiotic pollutants (Singer et al., 2003). Purified plant phenolics were shown to serve as selective growth substrates for PCB degrading bacteria (Donnelly et al., 1994). Media supplementation experiments with the plant-derived terpenoid compounds, cumene, thymol, and trans-cinnamic acid, were observed to induce the PCB biodegradation 16 pathway of Arthrobacter sp. (Gilbert and Crowley, 1997). Rhizosphere effects on the microbial metabolism and community structure and activity depend on plant species and the stage of plant growth (Hegde and Fletcher, 1996; Smalla et al., 2001). Mulberry root turnover at the end of the growing season was shown to double the production of phenolic compounds demonstrated to stimulate the growth of PCB-degrading bacteria (Leigh et al., 2002). PAH Phytoremediation Direct PAH phytodegradation may be limited by the low aqueous solubility and high logKow of PAH pollutants. Plants grown on PAH-contaminated soils may accumulate low levels of PAHS via leaf adsorption, leaf deposition of contaminated soil particulates, or root translocation to shoots through the transpiration stream (Fismes et al., 2002). High logK0W compounds desorb from soils slowly and may not readily transfer into root tissues (Luthy et al., 1997). However, roots were predicted and later demonstrated to accumulate lipophilic pollutants, such as pesticides and PAHS, in the lipid phase of the root tissues (Briggs et al., 1982; Schwab et al., 1998; Trapp, 2002). Root adsorption or accumulation may be considered the primary step for PAH phytoremediation. Various plant enzymes have been suggested to contribute to proposed PAH phytodegradation processes, including cytochrome P-450 monoxygenases, gluthathione S-transferases, and glycosyltransferases (Schaffner et al., 2002). Phytostimulation is more likely than direct phytodegradation to be instrumental in PAH phytoremediation processes. Plants aerate soils, which would promote dioxygenase activity, and produce root exudates to stimulate microbial growth and activity (Burken and Schnoor, 1996). 17 Numerous field trials have demonstrated an enhancement of planted soils relative to unplanted for PAH biodegradation (Fiorenza et al., 2000; Pradhan et al., 1998). Tall fescue was demonstrated to increase four—ringed PAH degradation compared to unvegetated treatments of contaminated soils (Robinson et al., 2003). PAH degradation in this study was approximately 35% greater at shallow depths (10-15 cm) than that in medium depth zones (15 — 21 cm). In a separate study, a mixture of nine plant species was twice as effective for pyrene degradation relative to unplanted soils (Liste and Alexander, 2000). A mixture of eight prairie grasses were reported to enhance biodegradation of four persistent PAH compounds (benz(a)anthracene, chrysene, benzo(a)pyrene, and dibenzo(a,h)anthracene) in contaminated soils (Aprill and Sims, 1990). A molecular genetic approach was used to quantify microbial naphthalene catabolic gene abundance in PAH phytoremediation treatments (Siciliano et al., 2003). This study demonstrated that tall fescue (F estuca arundinacea) enhanced naphthalene mineralization and increased specific naphthalene catabolic genes, indicating increased PAH microbial degrader cell numbers. SUMMARY AND CONCLUSION Polycyclic aromatic carbons (PAHS) are widely distributed persistent organic pollutants. Current engineering-based technologies are expensive and may not be practical or permanent options for PAH contamination treatment. Biological remediation approaches may be more suitable alternatives to large-scale cleanup of PAH impacted soils and sediments. Plant-microbe associations have been observed to enhance PAH 18 biodegradation treatments. Though PAH phytostimulation has been extensively studied, many basic biochemical, genetic, and physiological components of the process remain poorly understood. In this current research project, we utilized a selected analytical procedure to evaluate the effectiveness of selected native Michigan plant species to stimulate PAH- degrading bacteria in a phytoremediation field trial. In the future, we hope this study may contribute to more effective phytoremediation applications to clean up PAHS and other organic environmental pollutants. l9 REFERENCES Ahmann, D. (1997). Bioremediation of metal-contaminated soil. Society for Industrial Microbiology News 47, 218-233. Alexander, M. (2000). Aging, bioavailability, and overestimation of risk from environmental pollutants. Environmental Science and Technology 34, 4259-4265. Amellal, N., Portal, J. M., and Berthelin, J. (2001). Effect of soil structure on the bioavailability of polycyclic aromatic hydrocarbons within aggregates of a contaminated soil. Applied Geochemistry 16, 1611-1619. Anderson, T. A., Guthrie, E. A., and Walton, B. T. (1993). Bioremediation in the rhizosphere. Environmental Science & Technology 27, 2630-2636. April], W., and Sims, R. C. (1990). Evaluation of the use of prairie grasses for stimulating polycyclic aromatic hydrocarbon treatment in soil. Chemosphere 20, 253-265. Aulenta, F., Majone, M., Verbo, P., and Tandoi, V. (2002). Complete dechlorination of tetrachloroethene to ethene in presence of methanogenesis and acetogenesis by an anaerobic sediment microcosm. Biodegradation 13, 411-424. Baker, A. J. M., and Brooks, R. R. (1989). Terrestrial higher plants which hyper- accumulate metallic elements: A review of their distribution, ecology and phytochemistry. Biorecovery 1, 81-126. Barkay, T., Turner, R., Saouter, E., and Horn, J. (1992). Mercury biotransforrnations and their potential for remediation of mercury contamination. Biodegradation 3, 147- 159. Barkley, N. P. (1991). Extraction of mercury from groundwater using immobilized algae. Journal of the Air & Waste Management Association 41, 1387-1393. Benlahcen, K. T., Chaoui, A., Budzinski, H., BellocQ, J ., and Garrigues, P. H. (1997). Distribution and sources of polycyclic aromatic hydrocarbons in some Mediterranean coastal sediments. Marine Pollutant Bulletin 34, 298-305. Boyle, J . J ., and Shann, J. R. (1995). Biodegradation of Phenol, 2,4-Dcp, 2,4-D, and 2,4,5-T in field- collected rhizosphere and nonrhizosphere soils. Journal of Environmental Quality 24, 7 82-785. Briggs, G. G., Bromilow, R. H., and Evans, A. A. (1982). Relationships between lipophilicity and root uptake and translocation of non-ionised chemicals by barley. Pesticide Science 13, 495-504. 20 Burken, J. G., and Schnoor, J. L. (1998). Predictive relationships for uptake of organic contaminants by hybrid poplar trees. Environmental Science & Technology 32, 3379-3385. Burken, J. G., and Schnoor, J. L. (1997). Uptake and metabolism of atrazine by poplar trees. Environmental Science & Technology 31, 1399-1406. Burken, J. G., and Schnoor, L. (1996). Phytorrnediation: Plant uptake of atrazine and role of root exudates. Journal of Environmental Engineering 122, 958-963. Cai, X. H., Traina, S. T., Logan, T. J ., Gustafson, T., and Sayre, R. T. (1995). Applications of eukaryotic algae for the removal of heavy metals from water. Molecular Marine Biology and Biotechnology 4, 338-344. Carmichael, L. M., and Pfaender, F. K. (1997). Polynuclear aromatic hydrocarbon metabolism in soils: relationship to soil characteristics and preexposure. Environmental Toxicology and Chemistry 16, 666-675. Cemiglia, C. E. (1992). Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351-368. Cemiglia, C. E. (1993). Biodegradation of polycyclic aromatic hydrocarbons. Current Opinion in Biotechnology 4, 331-338. Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S., and Baker, A. J. M. (1997). Phytoremediation of soil metals. Current Opinion in Biotechnology 8, 279-284. Chang, Y. Y., and Corapcioglu, M. Y. (1998). Plant-enhanced subsurface bioremediation of nonvolatile hydrocarbons. Journal of Environmental Engineering-Asce 124, 162-169. Comeau, Y., Greer, C. W., and Samson, R. (1993). Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation. Applied Microbiology and Biotechnology 38, 681-687. Cunningham, S. D., and Ow, D.‘ W. (1996). Promises and prospects of phytoremediation. Plant Physiology 110, 715-719. de Souza, M. P., Huang, C. P. A., Chee, N ., and Terry, N. (1999). Rhizosphere bacteria enhance the accumulation of selenium and mercury in wetland plants. Planta 209, 259-263. Dipple, A., Cheng, S.C., Bigger, AH. (1990). Polycyclic aromatic hydrocarbon carcinogens. In Mutagens and Carcinogens in the Diet, M. W. Pariza, 21 Aeschbacher, H.U., Felton, J .S., and Sato, 8., ed. (New York: Wiley-Liss Inc.), pp. 109-127. Donnelly, P. K., Hegde, R. S., and Fletcher, J. S. (1994). Growth of PCB-degrading bacteria on compounds from photosynthetic plants. Chemosphere 28, 981-988. Eduljee, G. H. (2001). Budget and Source Inventories: Issues and Challenges. In Persistent Organic Pollutants: Environmental Behaviour and Pathways for Human Exposure, S. Harrad, ed. (Boston: Kluwer Academic Publishers), pp. 1-28. Ensley, B. D. (2000). Rationale for use of phytoremediation. In Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment, 1. Raskin and B. D. Ensley, eds. (New York: John Wiley & Sons, Inc.), pp. 3-11. EPA. (2001). Brownfields Technology Primer: Selecting and Using Phytoremediation for Site Clean Up. In http://www.epa.gov/tio/download/remed/phytoremprimerpdf. Fijalkowska, S., Lisowska, K., and Dlugonski, J. (1998). Bacterial elimination of polycyclic aromatic hydrocarbons and heavy metals. Journal of Basic Microbiology 38, 361-369. Fiorenza, S., Oubre, C. L., and Ward, C. H. (2000). Phytoremediation of Hydrocarbon- Contaminated Soil (Boca Raton, FL: Lewis Publishers), pp. 164. Fismes, J ., Ganier, C. P., Bissonet, P. E., and Morel, J. L. (2002). Soil-to-root transfer and translocation of polycyclic aromatic hydrocarbons by vegetables grown on industrial contaminated soils. Journal of Environmental Quality 31, 1649-1656. Flathman, P. E., and Lanza, G. R. (1998). Phytoremediation: Current views on an emerging green technology. Journal of Soil Contamination 7, 415-432. FRTR. (2003). The remediation technology screening matrix. In http://www.frtr.gov/matrix2/section3/tableIL2.html. Gadd, G. M., and White, C. (1993). Microbial treatment of metal pollution - a working biotechnology? Trends in Biotechnology 11, 353-359. Gilbert, E. S., and Crowley, D. E. (1997). Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter Sp. strain B1B. Applied and Environmental Microbiology 63, 1933-1938. Hage, J. C., Kiestra, F. D. G., and Hartmans, S. (2001). Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp strain DCAl. Applied Microbiology and Biotechnology 57, 548-554. 22 Hegde, R. S., and Fletcher, J. S. (1996). Influence of plant growth stage and season on the release of root phenolics by mulberry as related to development of phytoremediation technology. Chemosphere 32, 2471-2479. Heitkamp, M. A., J. P. Freeman, D. W. Miller, C. E. Cemiglia (1988). Pyrene degradation by a Mycobacterium sp.: Identification of ring oxidation and ring fission products. Applied and Environmental Microbiology 54, 2556-2565. Hinckley, T. M., Brooks, J. R., Cermak, J., Ceulemans, R., Kucera, J., Meinzer, F. C., and Roberts, D. A. (1994). Water flux in a hybrid poplar stand. Tree Physiology 14, 1005-1018. Joner, E. J ., Johansen, A., Loibner, A. P., Dela Cruz, M. A., Szolar, O. H. J ., Portal, J. M., and Leyval, C. (2001). Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHS) in spiked soil. Environmental Science & Technology 35, 2773-2777. J uhasz, A. L., Britz, M. L., and Stanley, G. A. (1997). Degradation of fluoranthene, pyrene, benz(a)anthracene, and dibendz(a,h)anthracene by Burkholderia cepacia. Journal of Applied Microbiology 83, 189-198. Kakareka, S. V., and Kukharchyk, T. I. (2003). PAH emission from the open burning of agricultural debris. The Science of The Total Environment 308, 257-261. Kastner, M., M. B. Jammali, B. Mahro (1994). Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH). Appl. Microbiol. Biotechnology 41, 267-273. Keith, L. H., and Telliard, W. A. (1979). Priority pollutants: A perspective review. Environmental Science and Technology 13. Kelsey, J. W., and Alexander, M. (1996). Declining bioavailability and inapproriate estimation of risk of persistent compounds. Environmental Toxicology and Chemistry 16, 582-585. Kotterman, M. J. J., Rietberg, H. J., Hage, A., and Field, J. A. (1998). Polycyclic aromatic hydrocarbons oxidation by the white-rot fungus Bjerkandera sp. strain B0855 in the presence of nonionic surfactants. Biotechnology and Bioengineering 5 7, 220-227. Leigh, M. B., Fletcher, J. 8., Eu, X. 0., and Schmitz, F. J. (2002). Root turnover: An important source of microbial substrates in rhizosphere remediation of recalcitrant contaminants. Environmental Science & Technology 36, 1579-1583. 23 Liste, H. H., and Alexander, M. (2000). Plant-promoted pyrene degradation in soil. Chemosphere 40, 7-10. Lovley, D. R., and Coates, J. D. (1997). Bioremediation of metal contamination. Current Opinion in Biotechnology 8, 285-289. Luthy, R. G., Aiken, G. R., Brusseau, M. L., Cunningham, S. D., Gschwend, P. M., Pignatello, J. J ., Reinhard, M., Traina, S. J ., Weber, W. J ., and Westall, J. C. (1997). Sequestration of hydrophobic organic contaminants by geosorbents. Environmental Science & Technology 31, 3341-3347. Mahro, B., Schaefer, G., and Kastner, M. (1994). Pathways of microbial degradation of polycyclic aromatic hydrocarbons in soil. In Bioremediation of Chlorinated and PAH Compounds, pp. 203-217. Maila, M. P., and Cloete, T. E. (2002). Germination of Lepidium sativum as a method to evaluate polycyclic aromatic hydrocarbons (PAHS) removal from contaminated soil. International Biodeterioration & Biodegradation 50, 107-113. McGrath, S. P., Zhao, F. J ., and Lombi, E. (2001). Plant and rhizosphere processes involved in phytoremediation of metal-contaminated soils. Plant and Soil 232, 207—214. Miethling, R., Wieland, G., Backhaus, H., and Tebbe, C. C. (2000). Variation of microbial rhizosphere communities in response to crop species, soil origin, and inoculation with Sinorhizobium meliloti L33. Microbial Ecology 40, 43-56. Miya, R. K., and Firestone, M. K. (2001). Enhanced phenanthrene biodegradation in soil by slender oat root exudates and root debris. Journal of Environmental Quality 30, 191 1-1918. Moody, J. D., Freeman, J. P., Doerge, D. R., and Cemiglia, C. E. (2001). Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-l. Applied and Environmental Microbiology 67, 1476-1483. Newman, L. A., Wang, X. P., Muiznieks, I. A., Ekuan, G., Ruszaj, M., Cortellucci, R., Domroes, D., Karscig, G., Newman, T., Crarnpton, R. S., Hashmonay, R. A., Yost, M. G., Heilman, P. E., Duffy, J., Gordon, M. P., and Strand, 8. E. (1999). Remediation of trichloroethylene in an artificial aquifer with trees: A controlled field study. Environmental Science & Technology 33, 2257-2265. Pothuluri, J. V., and Cemiglia, C. E. (1994). Microbial Metabolism of Polycyclic Aromatic Hydrocarbons. In Biological Degradation and Bioremediation of Toxic Chemicals, G. R. Chaudhry, ed. (Portland: Dioscorides Press.), pp. 92-124. 24 Pradhan, S. P., Conrad, J. R., Paterek, J. R., and Srivastava, V. J. (1998). Potential of phytoremediation for treatment of PAHS in soil at MGP sites. Journal of Soil Contamination 7, 467-480. Rehmann, K., Hertkom, N ., and Kettrup, A. A. (2001). Fluoranthene metabolism in Mycobacterium sp. strain KR20: identity of pathway intermediates during degradation and growth. Microbiology 147, 2783-2794. Robinson, B., Green, 8., Mills, T., Clothier, B., van der Velde, M., Laplane, R., Fung, L., Deurer, M., Hurst, S., and Thayalakumaran, T. (2003). Phytoremediation: using plants as biopums to improve degraded environments. Australian Journal of Soil Research 41. Robinson, S. L., Novak, J. T., Widdowson, M. A., Crosswell, S. B., and Fetterolf, G. J. (2003). Fileds and laboratory evaluation of the impact of Tall Fescue on polycyclic aromatic hydrocarbon degradation in an aged creosote-contaminated surface soil. Journal of Environmental Engineering 129, 232-240. Schaffner, A., Messner, B., Langebartels, C., and Sandermann, H. (2002). Genes and enzymes for In-Planta phytoremediation of air, water, and soil. Acta Biotechnologica 22, 141-152. Schnoor, J. L., Licht, L. A., McCutcheon, S. C., Wolfe, N. L., and Carreira, L. H. (1995). Phytoremediation of organic and nutrient contaminants. Environmental Science & Technology 29, A318-A323. Schroder, P., Scheer, C., and Belford, E. J. D. (2001). Metabolism of organic xenobiotics in plants: conjugating enzymes and metabolic end points. Minerva Biotechnologica 13, 85-91. Schuler, M. A. (1996). Plant Cytochrome P450 Monooxygenase. Critical Reviews in Plant Sciences 15, 235-284. Schwab, A. P., A] Assi, A. A., and Banks, M. K. (1998). Adsorption of naphthalene onto plant roots. Journal of Environmental Quality 27, 220-224. Sepic, E., Bricelj, M., and Leskovsek, H. (1998). Degradation of fluoranthene by Pasteurella sp. [FA and Mycobacterium sp. PYR-l: Isolation and identification of metabolites. Journal of Applied Microbiology 85, 746-754. Siciliano, S. D., and Germida, J. J. (1998). Degradation of chlorinated benzoic acid mixtures by plant-bacteria associations. Environmental Toxicology and Chemistry I 7, 728-733. Siciliano, S. D., Germida, J. J., Banks, K., and Greer, C. W. (2003). Changes in microbial community composition and function during a polyaromatic hydrocarbon 25 phytoremediation field trial. Applied and Environmental Microbiology 69, 483- 489. Siciliano, S. D., Goldie, H., and Germida, J. J. (1998). Enzymatic activity in root exudates of Dahurian wild rye (Elymus dauricus) that degrades 2-chlorobenzoic acid. Journal of Agricultural and Food Chemistry 46, 5-7. Sims, R. C., and Overcash, M. R. (1983). Fate of polynuclear aromatic compounds (PNAS) in soil-plant systems. Residue Reviews 88, 1-68. Singer, C. A., Crowley, D. E., and Thompson, 1. P. (2003). Secondary plant metabolites in phytoremediation and biotransformation. TRENDS in Biotechnology 21, 123- 1 130. Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H., and Berg, G. (2001). Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: Plant-dependent enrichment and seasonal shifts revealed. Applied and Environmental Microbiology 67, 4742- 475 1. Stapleton, R. D., G. S. Sayler (1998). Assessment of the microbiological potential for the natural attenuation of petroleum hydrocarbons in a shallow aquifer system. Microbial Ecology 36, 349-361. Steffan, R. J ., Sperry, K. L., Walsh, M. T., Vainberg, S., and Condee, C. W. (1999). Field-scale evaluation of in situ bioaugmentation for remediation of chlorinated solvents in groundwater. Environmental Science & Technology 33, 2771-2781. Sutherland, J. B., Rafii, F., Khan, A. A., and Cemiglia, C. E. (1995). Mechanisms of polycyclic aromatic hydrocarbon degradation. In Microbial Transformation and Degradation of Toxic Organic Chemicals, L. Y. Young and C. E. Cemiglia, eds. (New York: John Wiley & Sons, Inc.). Sverdrup, L. E., Nielsen, T., and Krogh, P. H. (2002). Soil ecotoxicity of polycyclic aromatic hydrocarbons in relation to soil sorption, lipophilicity, and water solubility. Environmental of Science and Technology 36, 2429-2435. Thompson, P. L., Ramer, L. A., and Schnoor, J. L. (1999). Hexahydro-l,3,5-trinitro— 1,3,5-triazine translocation in poplar trees. Environmental Toxicology and Chemistry 18, 279-284. Thompson, P. L., Ramer, L. A., and Schnoor, J. L. (1998). Uptake and transformation of TNT by hybrid poplar trees. Environmental Science & Technology 32, 975-980. Tiedje, J. M., Quensen III, I. F., Chee-Sanford, J., and Schimel, J. P. (1993). Microbial reductive dechlorination of PCBS. Biodegradation 4, 231-240. 26 Trapp, S. (2002). Dynamic root uptake model for neutral lipophilic organics. Environmental Toxicology and Chemistry 21, 203-206. Trapp, S. (2000). Modeling uptake into roots and subsequent translocation of neutral and ionisable organic compounds. Pest Management Science 56, 767-778. Trapp, S., Kohler, A., Larsen, L. C., Zambrano, K. C., and Karlson, U. (2001). Phytotoxicity of fresh and weathered diesel and gasoline to willow and poplar trees. Journal of Soils & Sediments 1, 71-76. Vangronsveld, J., Colpaert, J. V., and VanTichelen, K. K. (1996). Reclamation of a bare industrial area contaminated by non- ferrous metals: Physicochemical and biological evaluation of the durability of soil treatment and revegetation. Environmental Pollution 94, 131-140. Walter, U., M. Beyer, J. Klein, H. J. Rehm (1991). Degradation of pyrene by Rhodococcus sp. UWl. Appl. Microbiol. Biotechnology 34, 671-676. Walton, B. T., and Anderson, T. A. (1990). Microbial degradation of trichloroethylene in the rhizosphere - potential application to biological remediation of waste sites. Applied and Environmental Microbiology 56, 1012-1016. Wan, C. S. M. (2002). Phytoremediation of polycyclic aromatic hydrocarbon contaminated soil using native Michigan plant species. In Crop and Soil Science (East Lansing: Michigan State University), pp. 122. Warshawsky, D., Schneider, J., Reilman, R., and LaDow, K. (1995). Detection of polycyclic aromaticshydrocarbons metabolites in aquatic organisms. Bioindicator of environmental health, 1-12. Wieland, G., Neumann, R., and Backhaus, H. (2001). Variation of microbial communities in soil, rhizosphere, and rhizoplane in response to crop species, soil type, and crop development. Applied and Environmental Microbiology 67, 5849-5854. Wilson, 8. C., and Jones, K. C. (1993). Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHS): a review. Environmental Pollution 81, 229-249. Yoshitomi, K. J., and Shann, JR. (2001). Corn (Zea mays L.) root exudates and their impact on 14C-pyrene mineralization. Soil Biology & Biochemistry 33, 1769- 1776. 27 CHAPTER 2 EVALUATION OF METHODS FOR ENUMERATION OF PAH-DEGRADIN G SOIL BACTERIA 28 ABSTRACT Evaluation of methods for enumeration of PAH-degrading soil bacteria Plant-microbe interactions are important in both organic and inorganic contaminant remediation. To maximize bioremediation effectiveness, optimum conditions for microbial growth and activities need to be maintained. Plants provide microbial growth substrates and improve soil physical and chemical properties such as aeration and hydrological aspects and releasing organic and inorganic substrates for biological remediation, a process known as phytostimulation. However, the mechanisms for plant-enhanced polyaromatic hydrocarbon (PAH) biodegradation are not completely understood. In this research study, we evaluated different published protocols for quantifying PAH-degrading bacteria from treated and untreated contaminated soils. Three different in vitro plating methods were testing for PAH-biodegrading bacteria identification and enumeration, including the Indole Conversion Assay, the Microtiter Plate Metabolism Assay, and the Spray Plate Assay. Each of these procedures are limited by culture-based bacterial bias, though provide relatively easy methods for bacterial community analysis. The Indole Assay is less specific for PAHS, instead screening for aromatic bacterial dioxygenase activity, though the Microtiter and Spray Plate Assays are designed to directly analyze PAH metabolism. Based on this survey, the Spray Plate Assay was found more reliable, reproducible, sensitive, and specific for PAH-degrading soil bacteria identification, isolation, and quantification compared to the other two techniques. The Spray Plate Assay offers a relatively rapid and dependable tool for further study of root-microbe interactions during PAH phytoremediation. 29 INTRODUCTION Persistent organic pollutants (POPS) such as polychlorinated biphenyls (PCB) and polycyclic aromatic hydrocarbons (PAHS) have become widespread environmental problem. POPS are typically hydrophobic, strongly soil-sorbed, and possess relatively low bioavailability. Microbial biodegradation of organic contaminant has been shown to be an important process for pollutant dissipation in the soil environment (Kastner, 1994). However, endogenous microbial activity may not be sufficient for site decontamination. Bioremediation, usually defined as the addition selected microbes or stimulation of microbial metabolic processes, has been explored as a cleanup strategy, though may be restricted by various factors, including limited nutrient supply (e. g. carbon and energy sources), anoxic conditions (for aerobic degradation), or suboptimal environmental conditions (e. g. temperature, moisture). The use of plants has been shown to enhance organic contaminant biodegradation by improving physical and chemical properties of soil (Schwitzguebel, 2002). Root- microbe interactions have been shown to be an important mechanism for plant-enhanced biodegradation, also called phytostimulation. Various studies have shown that the root zone, or rhizosphere, increased microbial growth and metabolic activities in soils impacted by various contaminants, including PCBS (Leigh et al., 2002), PAHS (Joner et al., 2001), and chlorinated solvents (Jordahleta1., 1997). However, the precise mechanisms of the rhizosphere-mediated interaction between plants and microbes are still poorly understood. To develop additional tools for analysis of the PAH phytostimulation process, this study evaluates different methods to characterize the bacterial biodegradin g fraction of the soil microbial community in a PAH phytoremediation field trial. 30 A variety of metabolic assays are available for analysis of PAH degrading soil bacteria, including the Indole Conversion Assay (Indole Assay), the Microtiter PAH Metabolism Assay (Microtiter Assay), and the Spray Plate Assay. The Indole Assay is a general test for expression of dioxygenase enzymes and has been shown to screen for expression of enzymatic activity for oxidative-cleavage of toluene, PAH compounds, or biphenyl (Ensley, 1983). The principal bacterial mechanism for aerobic PAH degradation is oxidative attack of the carbon rings by the multi- component dioxygenase enzyme (Sutherland et al., 1995). The Indole Assay is based on alteration of colorless indole vapor to dark blue indigo precipitate in cells expressing dioxygenase activity. The Indole Assay is simple to perform and could serve as a useful means of primary screening of soil bacteria for PAH biodegraders. The Microtiter Assay is a potentially hi gh-throughput method to screen native soil communities or bacterial isolates for specific carbon, i.e. PAH, metabolism. This assay uses 96-well microtiter plates to culture microbial samples in defined media, e. g. PAH substrates, followed by spectrophotometric quantification of cell growth and culture density (J ohnsen et al., 2002). The microtiter cultures are quantified by relative dehydrogenase activity using Water Soluble Tetrazolium (W ST-l) as a colorimetric indicator. In the presence of living cells, the WST-l reagent is reduced by dehydrogenases in viable cells to form a water-soluble forrnazan (~450 nm, yellow- orange color range). The Microtiter Assay could be used to screen for and possibly isolate PAH-degraders in treated and/or contaminated soil extracts. The Spray Plate Assay is also a Petri dish culture assay, though is more specific for PAH metabolizing bacteria than Indole Assay. In the Spray Plate Assay, PAHS are 31 applied as volatile solutions onto plated, pre-grown colonies with the solvent allowed to evaporate leaving a thin, cloudy residue of the compound of interest. PAH-degrading bacteria are distinguished by formation of clear areas surrounding the colony due to PAH uptake and utilization (Kiyohara, 1982). The mechanism of clear zone formation during the Spray Plate Assay may be due to bacterial secretion of degradative enzymes or production of biosurfactants to increase the solubility of the PAH. The Spray Plate Assay has been used in several studies of PAH degrading bacteria (Ahn et al., 1999; Kastner, 1994; Kiyohara, 1982; Samantha et al., 1999). We evaluated these three PAH-biodegrader identification and enumeration protocols for their effectiveness, reproducibility, and reliability for quantifying PAH- degrading bacteria from different PAH impacted soils. The objective of this study is to determine the most reproducible and consistent PAH metabolism assay to enumerate PAH-degradin g bacteria. The selected bacterial PAH degrader assay from this survey will be used in further studies of phytoremediation field trials. MATERIALS AND METHODS Three different bacterial plating methods were examined for enumeration of bacterial PAH degrader density in untreated Rouge Complex Coke Oven area soils, greenhouse tested soils, and soils from the Phytoremediation Field Study site. Indole Conversion Assay To perform the Indole Conversion Assay (“Indole Assay”), soil microbial extracts or pure bacterial cultures were plated to achieve a final colony density of less than 300 CFU per plate on YEPG agar medium {per L: 0.05g yeast extract, 0.05g ammonium 32 nitrate, 0.5g Bacto Peptone, 0.25g Glucose, 15.0g Bacto Agar (Sigma; St Louis, MO), adjusted to pH 8.0}. After 3-5 days incubation at 25°C small amount (~0.2g) of indole crystal (Aldrich Chemicals Co.; Milwaukee, WI) was spread on the inside of the lid of the inverted, pre-grown plates and then incubated in a vented incubator for 1-3 days. A positive result was scored if the bacterial colony turned blue due to indigo formation from the colorless indole vapor (Ensley, 1983). Indole-positive bacteria were isolated on YEPG agar plates. Indole-positive isolate were reanalyzed for survival, reproducibility of indigo conversion, and secondary analysis using the Spray Plate PAH-degradation assay. PAH Metabolism Microtiter Assay Soil Microbial Extract M icrotiter Assay Analysis For analysis of the PAH Metabolism Microtiter Assay protocol (“Microtiter Assay”) (Johnsen et al., 2002), we used soil bacterial extracts from planted and unplanted soils treatments. Microtiter plates (96-well, 12 x 8) were prepared by addition of 20uL of phenanthrene (PHEN) solution (5mg/mL hexane) per well and allowing the hexane to evaporate. N o carbon-substrate control plates (IBEX) were prepared by application and evaporation of only 20uL hexane in the wells. Planted and unplanted soils were extracted with SPP (10g sin 90 SPP). A 4-fold dilution series of soil suspension was prepared by suspending lmL of the first soil extraction into 3mL of phosphate salt media (PSM). A 2001.11 aliquot of 1/160-fold dilution of each soil extract suspension was added to 6 replicate wells in the first row of the microtiter plates with 20010 of sequential 4-fold dilutions added to each successive row. The plates were wrapped with plastic film and incubated dark without shaking at room temperature (~22-25°C) in a fume hood cabinet 33 for 3 weeks. The microtiter plate cultures were analyzed for cell growth and viability by addition of 50].rL of mixed carbon medium (MCM) containing glucose, pyruvic acid, and succinic acid (Sigma; St Louis, MO) each at 16.6mM plus IOILL of the respiration- indicator reagent, Water Soluble Tetrazolium (W ST-l; Roche Diagnostics GmbH; Indianapolis, IN). The plates were secured to a shaker incubator platform and shaken at 300rpm at 25°C for 4-5 hours. Bacterial cell density was determined based on spectrophotometric intensity of WST-l color change (A450) minus the background interference (A630). Phenanthrene-degrader bacterial cell density was calculated by subtracting the HEX-plate A450-A630 value from the PHEN-plate A450-A63o value. WST-I Microtiter Assay Calibration Analysis To evaluate the consistency and versatility of the Microtiter Assay, we used pure cultures of suspected PAH degrader isolates, the Pseudomonas putida G7 strain (positive control), and Agrobacterium tumefaciens LBA4404 (negative control). Bacterial isolates were grown in Luria Bertani medium (LB: 5g/ L NaCl (EM Science; Gibbstown, NJ), 5g] L yeast extract, and 10g/ L tryptone (Becton Dickinson and Co.; Sparks, MD)} to a culture OD630 of ~0.4 — 0.5. Serial dilutions of the bacterial lines were plated to determine the CFU/ unit OD630 for each isolate. For the Microtiter Assay trial, bacterial cells were washed twice with pelleting by centrifugation (5000rpm, 1.5 minutes) and resuspended in phosphate salt mineral (PSM) medium to OD630 ~ 0.4. Serial dilutions were performed in a HEX-only treated microtiter plate using a multi-pipetter to sequentially transfer and mixlOOuL of inocula from each row into 200p.L of PSM medium of the adjacent wells for a 3X 3-fold dilution series. One column of microtiter plate wells was used as an uninoculated control containing only 200uL PSM medium. The standard MCM-WSTl 34 reagent mixture (60111) was added to each well, the plates shaken at 300rpm at 25°C for 90 minutes, and then analyzed on the spectrophotometer at A450 and A630. The plates were then centrifuged at 4000rpm for 10 minutes at 15°C to pellet the bacteria and cell debris. 100uL of supernatant were pipetted to a new microtiter plate in a 1:1 dilution in 100uL of fresh PSM medium and analyzed again on the spectrophotometer at A450 and A630. Spray Plate Assay To directly quantify PAH metabolizing colonies on culture plates, we utilized the Spray Plate Assay (Ahn et al., 1999). Bacteria were extracted from soil samples by suspending 1.0g of soil with modified SPP buffer: 0.1% tetrasodium pyrophosphate (Sigma; St Louis, MO), 0.1mM EGTA (Sigma; St Louis, MO), 0.4mM Tween20 (Bio- Rad Laboratories, CA), 0.01% Bacto Peptone (Becton Dickinson and Co.; Sparks, MD), and 0.007% yeast extract (Becton Dickinson and Co.; Sparks, MD). The soil suspension was extracted in 10mL SPP using a vortex mixer set at maximum speed for 303, paused for 308, and re-vortexed for 308. Serial dilutions were prepared in SPP and plated at concentrations intended to generate a consistent, easily countable number of bacteria (~100 CFU) on YEPG agar plates with pH adjusted to pH 8.0 to match the Coke Oven soil pH. Inoculated plates were incubated for 5 days at 25°C and visible colonies counted to calculate the total colony forming units (CFU). Pre-grown plates were sprayed in a fume hood cabinet with 1% phenanthrene (Sigma, St Louis, MO) acetone solution using a Thin Layer Chromatography (TLC) sprayer connected to the exhaust port (~20 psi) of a vacuum pump. Phenanthrene sprayed plates were incubated for 5 days at 25°C before scoring. The formation of clear zones around colonies (ZFUS) was scored as positive responses for PAH-degrading bacteria (Kiyohara, 1982). Soil extract CFU and ZFU 35 values were obtained by multiplying plated colony counts by the extract dilution factor and soil moisture correction factor to convert values to per g soil dry weight (e. g. CFU/ g DW). For secondary screening of Spray Assay positives, approximately 2100 spray- assayed, phenanthrene-degrading bacterial isolates were transferred to YPEG plates at ~25 isolates/plate, except for fast growing colonies (~5 isolates/plate). Bacterial isolates were replica patch-plated on multiple plates using inoculated toothpicks and incubated at 25°C for 3 days to allow colony development. The pre-grown patch plates were tested via the Spray Plate Assay using 1% phenanthrene, incubated at 28°C, and scored for clear zone development after 5 days incubation. RESULTS Indole Conversion Assay Indole Assayed colonies were scored positive by conversion of the colorless indole vapor to dark blue indigo precipitate on the colony (Figure 2.1). The Indole Assay was applied to untreated Rouge soils collected from 12 different locations at Ford Rouge Manufacturing Complex. Approximately 3.916 >3.916 1.847 1/3 0.062 0.963 2.162 1.032 0.456 1/9 0.018 0.096 0.571 0.240 0.119 2.3d. Post-centrifugation Cell Respiration (OD450) (corrected for dilution) Dilution P. putida Agrobacter UPD-02 UPD-03 UPD-07 undiluted 0.804 2.822 3.888 4.112 1.350 1/3 0.024 0.680 1.612 0.706 0.330 1/9 0.012 0.040 0.450 0.1 18 0.086 42 Figure 2.2a. Spray Plate Assay for PAH-contaminated soil extract. The plate was sprayed with phenanthrene and analyzed after approximately 4-5 days incubation. Several clear- zone forming colonies (“Spray-positive”) are marked with numbers. Images in this thesis are presented in color. Figure 2.2b. Re-plated culture of isolated Spray-positive colony. A single putative PAH- degrading colony was grown and re-tested with 2° Spray Plate Assay for Phenanthrene. Images in this thesis are presented in color. 43 Table 2.4. Primary Spray Test analysis of treated soils from 11 phytoremediation field trials. Soil microbial extracts were of selected planted treatments collected July 2002 from the phytoremediation field trial site and Spray Assayed using phenanthrene. Shown are average (AVG) visible colonies (CFU) and Spray-positives (ZFU) (per g soil DW) per plate. Planted Treatment C1233: G 21:31:; G (ZFU 72:52 100) Joe Pye Weed 106 9.4 8.9 Ninebark 116 7.8 6.7 Meadowsweet 109 6.9 6.3 Prairie Cordgrass 110 5.9 5.5 Unplanted Control 98 4.9 5.0 New England Aster 107 5.4 5.0 Leadplant 125 5.5 4.4 Pasture Thistle 90 3.5 3.9 Big Bluestem 90 2.4 2.7 Cardinal Flower 94 2.2 2.6 Little Bluestem 97 1.7 1.8 Table 2.5. Spray Test Assay confirmation analysis for 1° isolates. PhenD = phenanthrene degrader. The % 2° PhenD is the percentage of re-tested 1° PhenD isolates displaying Spray-positive phenotype on patch plates (2° PhenD). 1° PhenD % - Planted Treatments Tested 2° Phen" 2° Phen” Ninebark 108 100 92.6 Meadowsweet 1 17 107 91.5 Joe Pye Weed 112 101 90.2 Cardinal Flower 101 91 90.1 Lead Plant 99 88 88.9 Pasture Thistle 107 94 87.9 Prairie Cordgrass 110 94 85.5 New England Aster 106 87 82.1 Big Bluestem 104 85 81.7 Unplanted Control 107 86 80.4 Little Bluestem 100 77 77.0 Total 2138 1869 87.4 45 DISCUSSION In this research study, various methods were evaluated for determination of the number and metabolic activity of PAH-degrading bacteria. The Indole Assay is a general test for dioxygenase activity in bacterial cells under aerobic conditions. However, Indole Assay screening of PAH-degrading bacteria did not produce consistently reproducible indicator results and also caused mortality in treated bacterial colonies. Given the need to re-culture PAH-biodegrader isolates, the Indole Assay was not found to be an effective method for soil microbe analysis. The Microtiter Assay was designed to quantify the proliferation of PAH bacterial degraders based on their respiration activity using the WST-1 indicator reagent. Microtiter culture of soil microbial extracts using phenanthrene as a carbon source gave highly variable results, particularly with respect to the extract dilution series. It is possible that soil coextractants served as a heterogeneous carbon substrate that inconsistently distributed across the microtiter wells. Soil “contaminants”, such as humics, phenols, etc., could have confounded the phenanthrene sole-carbon design of the assay leading to the apparently random results. WST-1 reaction analysis using pure bacterial strains showed highly different responses to the reagent among the tested strains. This observation creates concern that reliance upon the WST-1 colorimetric reading alone could overlook the effects of different bacterial species dominance during soil microbial community analysis. Centrifugation was shown to remove most cell particulates (OD630), though not evenly for all bacterial strains, e. g. UPD-02. It was concluded that more detailed study of the Microtiter Assay would be required to consider it useful for soil PAH-biodegrader analysis. In the future research, the effect different 46 factors such as carbon sources used in this study and the application of centrifugation on the WST-1 experiment need to be studied further. The Spray Plate Assay was the most dependable of the methods tested. This method was highly reproducible, relatively easy to perform, and specific for bacterial PAH metabolism. Unlike the Indole Assay, the Spray Plate treatment did not result in colony mortality and allowed isolation of biodegraders for further analysis. Secondary screening of 1° isolates from mixed soil extracts was essential for confirmation of Spray- positive scoring. The 1° Spray Test false positives (~10-15%) were likely due to difficulty isolating individual colonies on crowded, densely grown treatment plates, though true positives were easily resolved with follow-up analysis. Subsequently, the Spray Plate Assay was determined to be an effective method for soil community PAH degrader analysis in our ongoing phytoremediation research program. SUMMARY AND CONCLUSION Phytoremediation, the use of plants to clean up or neutralize organic contaminants such as PAHS is considered an unobtrusive and cost effective method to clean up contaminated soil, groundwater, and sediments. Bacterial culturing methods are convenient and effective procedures to study microbial metabolic activities associated with phytoremediation. Based on this study of various published protocols for PAH biodegrader analysis, the Spray Plate Assay was found to be the most reliable and manageable method for analysis of PAH-degrading bacteria from contaminated soils. Though this study used phenanthrene as the tested PAH, this procedure should be effective for analysis of other PAH compounds. It is also possible that the “standard” 47 Spray Plate Assay as described in this study could be modified to use multiple PAHS or bacterial pre-treatments with putative plant-derived compounds as co-metabolites. The Spray Plate Assay may also be applied to other organic contaminants with similar chemical properties to PAHS. Environmental microbial sources could be tested against PCBS, nitroaromatics, and other hydrocarbon pollutants using the Spray Plate Assay methodology. The ability to directly isolate biodegrader colonies provides additional opportunities for specific analysis of plant-stimulated processes. For example, the isolated degraders could now be tested against additional PAHS, root derived substances, or under varied treatment conditions. Development of an effective method for analysis of PAH-degrading bacteria is a critical step for studying phytostimulation processes. 48 REFERENCES Ahn, Y., Sanseverino, J ., and Sayler, G. (1999). Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation 10, 149-157. Ensley, B. D., B. J. Ratzkin, T. D. Osslund., M. J. Simon (1983). Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222, 167-169. Johnsen, A. R., Bendixen, K., and Karlson, U. (2002). Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1. Applied and Environmental Microbiology 68, 2683-2689. Joner, E. J ., Johansen, A., Loibner, A. P., Dela Cruz, M. A., Szolar, O. H. J ., Portal, J. M., and Leyval, C. (2001). Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHS) in spiked soil. Environmental Science & Technology 35, 2773-2777. J ordahl, J. L., Foster, L., Schnoor, J. L., and Alvarez, P. J. J. (1997). Effect of hybrid poplar trees on microbial populations important to hazardous waste bioremediation. Environmental Toxicology and Chemistry 16, 1318-1321. Kastner, M., M. B. J ammali, B. Mahro (1994). Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH). Appl. Microbiol. Biotechnology 41, 267-273. Kiyohara, H., K. Nagao, K. Yana (1982). Rapid screen for bacteria degrading water- insoluble, solid hydrocarbons on agar plates. Applied and Environmental Microbiology 43, 454-457. Leigh, M. B., Fletcher, J. 8., Fu, X. 0., and Schmitz, F. J. (2002). Root turnover: An important source of microbial substrates in rhizosphere remediation of recalcitrant contaminants. Environmental Science & Technology 36, 1579-1583. Samantha, S. K., Chakraborti, A. K., and Jain, R. K. (1999). Degradation of phenanthrene by different bacteria: Evidence for novel transformation sequences involving the formation of l-naphthol. Applied Microbiology and Biotechnology 53, 98-107. Schwitzguebel (2002). Hype or hope: The potential of phytoremediation as an emerging green technology. Federal Facilities Environmental Journal 11, 109-125. Sutherland, J. B., Rafii, F., Khan, A. A., and Cemiglia, C. E. (1995). Mechanisms of polycyclic aromatic hydrocarbon degradation. In: Microbial Transformation and 49 Degradation of Toxic Organic Chemicals, L. Y. Young and C. E. Cemiglia, eds. (New York: John Wiley & Sons, Inc.). 50 CHAPTER 3 ANALYSIS OF PAH-DEGRADING SOIL BACTERIA IN PHYTOREMEDIATION FIELD TRIALS 51 ABSTRACT Analysis of PAH-Degrading Soil Bacteria in Phytoremediation Field Trials Rhizosphere systems have been extensively researched as a remedial tool for cleanup of polycyclic aromatic hydrocarbon (PAH) pollution. Root-microbe interactions are an important component of PAH-phytoremediation, since microbes have been shown to be the primary mechanism of environmental PAH degradation or detoxification. Further understanding of these processes is essential for advancement of PAH bioremediation technologies. In this research, we conducted preliminary screening for PAH degrading bacteria abundance in contarrrinated soils treated with 18 different native Michigan plant species. Phenanthrene metabolism assays suggested that each plant had a different effect on total soil bacteria, total PAH degrading bacteria, and the relative abundance of PAH biodegraders. These differences were studied in greater detail by more extensive analysis of four selected plant species: Aster novae-angliae, Eupatorium purpureum, Spartina pectinata, and Lobelia cardinalis. Most of the planted treatments were observed to enhance overall bacterial density, though only two species, Aster novae- angliae and Eupatorium purpureum, were shown to consistently increase soil bacterial PAH degrader cell density. In addition, these two species’ enhancement of PAH biodegrader numbers was observed to be most pronounced at the end of the growing season, due possibly to root senescence and nutrient release into the rhizosphere. Further study of >2000 phenanthrene degrading bacterial isolates showed that many strains could degrade multiple PAH compounds. A more detailed analysis of this capability with regard to particular plant rhizosphere influence would greatly contribute for design and management of more effective PAH phytoremediative treatments. 52 INTRODUCTION Polyaromatic hydrocarbons (PAHS) are a class of hydrophobic contaminants with low aqueous solubility and a strong affinity for soil organic matter causing them to be environmentally persistent pollutants. Some PAH compounds are lipophilic with a tendency to bioaccumulate to hazardous concentrations at higher trophic levels, so are considered high cleanup priority pollutants. Extensive research has been conducted on microbial PAH degradation at the molecular, biochemical, and ecological levels (Cemiglia, 1993; Harayama, 1997; Wilson and Jones, 1993). Natural PAH biodegradation rates are highly variable depending on the particular PAH compound chemistry, geochemical site conditions, and microbial community structure (Shuttleworth and Cemiglia, 1995). The use of microbial biodegradation of PAHS has been proposed as an attractive method for PAH-contaminated site remediation. Phytoremediation, the use of plants to remove and/or detoxify environmental pollutants, is an alternative emerging technology. Plants may not directly uptake hydrophobic pollutants, such as PAHS (Briggs et al., 1982), though have been suggested to contain or secrete enzymes potentially capable of transformation of some aromatic pollutants (Schaffner et al., 2002). Soil microbial metabolism is considered the primary mechanism for environmental PAH degradation. Plants have been demonstrated to accelerate endogenous biodegradation rates for various organic pollutants, including polychlorinated biphenyls and PAHS (Donnelly et al., 1994; Muratova et al., 2003). It has been observed that vegetated treatments decreased PAHS level much faster than nonvegetated controls (Aprill and Sims, 1990; Liste and Alexander, 2000). Laboratory studies showed that plant root secreted nutrients and organic compounds may facilitate 53' microbial growth and PAH biodegradation (Daane et al., 2001; Reilley et al., 1996). Despite these important observations, there remains a limited understanding of the basic plant-microbe mechanisms of PAH-phytoremediation (Ortega-Calvo et al., 2003). The goal of this research study was to quantify the effect of planted systems on the number of total bacteria and PAH bacterial degrader during PAH phytoremediation. The phytoremediation field experiment was conducted at the Phytoremediation Demonstration Facility (Dearbom, MI). PAH-impacted soils were planted with a variety of single plant species and monitored for PAH contaminant fate under vegetated and non- vegetated treatments (W an, 2002). This paper describes our analysis of the influence of selected plant rhizospheres on the soil bacterial PAH-biodegrader community. Characterization of the soil microbe response to planted treatment will allow us to better understand the phytostimulation process and ultimately design more effective phytoremediation systems for restoration of PAH, as well as for other organic pollutant, contaminated habitats. MATERIALS AND METHODS Phytoremediation Field Site Design The Phytoremediation Field Demonstration site was constructed at the Allen Park Claymine (Allen Park, MI) by URS Corporation (Willow Grove, PA) in the summer 2000. Polyaromatic Hydrocarbon (PAH)-contaminated soils were obtained from the grounds surrounding the Coke Oven Facility at the Ford Rouge Manufacturing Complex (Dearbom, MI). The Coke Oven soils were amended with composted yard litter (10% v/v; Ypsilanti County, MI) and composted poultry manure (5% v/v; Herbruck’s Poultry 54 Farm, Saranac, MI) to a final volume of approximately 200 cubic yards of Rouge Soil Mix. The amended Rouge Soil Mix was distributed into an excavated pit, lined with 40 mil high-density polyethylene (HDPE), to form the “Upland Plot” (20’ x 100’ x ~24” depth). The Upland Plot was overlaid by a grid of walk boards to divide the plot into ~5’ x 5’ sub-plots, or “cells”, for planting. The Upland Plot contains 60 cells, of which 3 cells were not used due to the presence of utility fixtures, 3 cells were left unplanted as treatment controls, and 54 planted cells (Figure 3.1a). Each vegetated cell was planted in September 2000 with 12 plugs of a single species with 3 replicate cells for each of the 18 different, selected plant species (Table 3.1). The field plots were fertilized with an N-P-K solution (20-20-20) at a concentration 475 ppm weekly for the first year and twice yearly for the 2002 and 2003 growing seasons. The reduction of PAH compounds in the Upland soils was observed to be ~25-40% 1 year after planting (Wan, 2002). Soil Sample Collection Soil samples were collected from the Upland Plot of the Phytoremediation Field Site (Dearbom, Michigan). Soils were collected in July 2002, July 2003 and October 2003. A 2” diameter soil probe was used to obtain 2 cores (Jul 02 and Jul 03) and 3 cores (Oct 03) from each cell. Samples were obtained from 2-10” depth. The soil transferred to 150mL amber jars with Teflon-lined caps after removing was rocks and mulch debris. During the Oct 03 sampling event, rhizosphere soils were collected from excavated root systems from each of the planted treatments in the Upland Plot. Rhizosphere soils were defined as soil obtained from root cluster obtained only after aggressive shaking by hand. Soil samples were transferred to amber vials and kept on ice in coolers in the field for transfer to a 4°C walk-in cold room at the MSU Phytoremediation Laboratory for further 55 aux—ace 95.558 .3583 3:33 208 SC 3828 2:5 E2585 9353:: use 328% E v3.83 2: Duo .28 20:82 :8 8E ecu—m3 o5 o5 Esocm .mEoEEob coquoEouoism Boon—om .£.m Bsmfi .8353 so: 98 3:33 Bo: Bo; 2x: an 53» e832: mzoo A23 Ev :EEE... @232 one .58 e835 65:8 @8535 o5. .m=o_§>o5pe .36on Ema no a: a how mm 29d. 9 Bowen 2:253: BEEN—ac: Lo 928% Eocotfi M: “8 some 8% moumozmoc 0 382a hm Co 25 wEEeEoo bmé x .m.vrv :3 a $5852 Egg comm ”$5632“ =< .36 253% 6.8828 Bob: SE @533 roam EoE seafloofiouoicm ._.m oSwE 2: 3o >0: So 9:. new 92 an 95 new 96 no. 0 m < :N a 3 S 3 m~ 3 Nfi 3 A: a w h v m N g £6 25mm E 2: Se do“ ooa So :ao >0: cwo Sm m6 oom com how £a So cow 5 am com now 9:. £> 3 new 32 En an 025 use to new use new Em no. £> sow So can D Em m6 be. >0: Bow fie E «E oom x cow be com smo oEa 8m 2e :8 Sm oEa Em to 6m “we can am new 9:. gm £> 6w ecu Em moo w»; to 08m 95 moo m Sm oow Com :ao oEm am am ago he x h: Em So am L8 >0: 2: Bow Com x 96 ecu ago 25 moo $3 so Em 6w :m m? no. so .6 “ma 9.: new 95 < :N a M: E 3 mm 3 m— 2 2 3 a w b e m e. m N m .26. SEE 56 Table 3.1. Planted soil treatments in the Phytoremediation Field Study plot. Treatment # Scientific Name Common Name Plant ID 1 Amorpha canescens Leadplant AMOCAN 2 Andropogon gerardii Big Bluestem ANDGER 3 Andropogon scoparius Little Bluestem ANDSCO 4 Aster novae-angliae* New England Aster ASTNOV 5 Carex sprengelii Sprengel Sedge CARSPR 6 Ceanothus americanus New Jersey Tea CEAAME 7 C irsium discolor Pasture Thistle CIRDIS 8 Eupatorium perfoliatum Boneset EUPPER 9 Eupatorium purpureum* Joe Pye Weed EUPPUR 10 Geum triflorum Prairie Smoke GEUTRI 11 Hystrix patula Bottlebrush Grass HYSPAT 12 Lobelia cardinalis* Cardinal Flower LOBCAR l3 Physocarpus opulifolius Ninebark PHYOPU 14 Scirpus atrovirens Green Bulrush SCIATR 15 Silfhiu’". Prairie-dock SILTER tertbmthmaceum 16 Spartina pectinata* Prairie Cordgrass SPAPEC 17 Spirea alba Meadowsweet SPIALB 18 Viburnum dentatum Arrowwood Viburnum VIBDEN Control Treatments 19 Unplanted Control* Unplanted Field Cell UNPLNT *These plant treatments were used in the primary study processing. Soil samples were prepared for analysis by sieving with a stainless steel 8- inch diameter, 2.36mm sieve (Gilson Co.; Worthington, OH) to remove the remaining soil debris and homogenize the sample. Sieved samples were stored dark in amber jars at 4°C until use in experiments. Soil Bacteria PAH Degrader Analysis Preliminary bacterial analyses were performed using the Spray Plate Assay of phytoremediation study soils collected in Jun02 after approximately 9 months storage at 4°C. Equivalent quantities of soils from each of the 3 replicate treatments were mixed to create a composite soil sample for each of 19 different soil treatments (18 plant species and l unplanted) (Table 3.1 and Figure 3.1a). In this preliminary soil microbial Spray Test trial, we utilized the Spray Plate Assay (Ahn etal., 1999). Soil bacteria were extracted mixing 1.0g of soil with modified SPP buffer". 0.1% tetrasodium pyrophosphate (Sigma; St Louis, MO), 0.1mM EGTA (Sigma; St Louis, MO), 0.4mM Tween20 (Bio- Rad Laboratories, CA), 0.01% Bacto Peptone (Becton Dickinson; Sparks, MD), and 0.007% yeast extract (Becton Dickinson; Sparks, MD). The soil suspension was extracted in 10mL SPP using a vortex mixer set at maximum speed for 30s, paused for 308, and re- vortexed for 30s. Serial dilutions were prepared in SPP and plated at dilutions to achieve ~120 CFU on YEPG agar plates adjusted to pH 8.0 to match the Coke Oven soil pH. Twenty plates were inoculated with 0.1ILL extract suspension for each treatment soil and incubated for 5 days at 25°C and visible colonies counted to calculate the total colony forming units (CFU). Fifteen pre-grown plates (~100-120 colonies each) were sprayed in a fume hood cabinet with 1% phenanthrene solution (Sigma, St Louis, MO) in acetone (w/v) using a Thin Layer Chromatography (TLC) sprayer connected to the exhaust port 58 (~20 psi) of a vacuum pump. Phenanthrene sprayed plates were incubated for 5 days at 25°C before scoring. The formation of clear rings around colonies, or “zone forming units’ (ZFUS), were scored as PAH-degrading bacteria (Kiyohara, 1982). Soil extract CFU and ZFU values were obtained by multiplying the plated colony counts by the extract dilution factor and soil moisture correction factor to convert values to per g soil dry weight (e.g. CFU / g DW). There are several reasons why phenanthrene was used in this experiment: a) generates easily observable cloudy residue on the plates during spray application, so clear zones are easy to detect; b) unlike naphthalene, phenanthrene has a relatively low vapor pressure (0.018 Pa at 25°C) and does not evaporate too quickly at room temperature (Marlowe et al., 2002); c) phenanthrene serves as a signature PAH compound because it contains both bay- and K-regions, similar with most carcinogenic PAHS (Samantha et al., 1999). Phenanthrene is often used as a model substrate for carcinogenic PAHS such as benzo(a)pyrene, benzo(a)anthracene, and chrysene (Samantha et al., 1999). For analysis of the Jul 03 bulk soil, Oct 03 bulk soil, and Oct 03 rhizosphere soil samples, a modified Spray Test Assay procedure was used. Soil bacterial extracts were prepared by suspending 4.0g soils in 36mL SPP buffer in 50mL conical tubes (Corning Inc.; Corning, NJ) with homogenization using a vortex mixer at maximum speed for 1min, paused 10s, and re-vortexed for 1min. In this experiment, four selected planted treatments were used in the spray plate study: Aster novae-angliae (New England Aster), Eupatorium purpureum (Joe Pye Weed), Lobelia cardinalis (Cardinal Flower), and Spartina pectinata (Prarie Cordgrass) (Figure 3.1b). These four plant species were selected for more extensive analysis due to two factors: distinct soil CFU, ZFU, and 59 ZFU/CFU values in previous trials and the observation that these species’ subplots contained very minimal or no weed interference. Unplanted cells were used as non- vegetated control treatments. The various replicate cells were widely and randomly distributed around the 2000 sq ft Upland Plot study area. Ten-fold serial dilutions of treatment soil extracts were prepared in SPP buffer and plated on YEPG agar with incubation at 25°C for 5 days. The phenanthrene Spray Test Assay was performed on 15 pre-grown plates (~150-200 colonies per plate) for each of the 3 replicate field samples of each soil treatment with analysis of soil bacterial extract CFUs and phenanthrene-ZFUS as before. For secondary confirmation and broad-spectrum PAH screening, approximately 2100 Spray-positive, phenanthrene-degrading bacterial isolates from the preliminary trial experiment were transferred to YPEG plates at ~25 isolates / plate, except for fast growing colonies (~5 isolates/plate). Bacterial isolates were replica patch-plated on multiple plates using sterile toothpicks and incubated at 25°C for 3 days to allow colony development. The pre-grown plates were tested via the Spray Plate Assay for 6 different PAHs: 1% phenanthrene (Sigma; St Louis, MO), 1% anthracene (Sigma; St Louis, MO), 1% fluoranthene (Aldrich Chemical Co.; Milwaukee, WI), 0.5% pyrene (Aldrich Chemical Co.; Milwaukee, WI), 0.5% chrysene (Aldrich Chemical Co.; Milwaukee, WI), and 0.5% of benzo(a)pyrene (Fluka Chemica; Steinheim, Switzerland). Each plate was sprayed with a single acetone-solubilized PAH compound and incubated at 28°C. Phenanthrene sprayed plates were scored for clear zone development after 5 days incubation, while other PAH-sprayed plates were observed after 1, 2, or 4 weeks. CFU and ZFU calculations were conducted as for the previous trials. 60 RESULTS Soil Microbial Analysis Preliminary Spray Plate Assay survey A preliminary survey of soil microbes from the 18 different plant species treatment in the phytoremediation field study displayed varied numbers of both total heterotrophic bacteria (CFU) and PAH-degrading bacteria (ZFU). In general, most planted treatments displayed apparently higher numbers of CFUs and ZFUs relative to the unplanted treatment (Figure 3.2). The unplanted ZFU to CFU ratio was near the mean ratio value for all of the treatments (Table 3.2). This preliminary experiment is not statistically resolvable due to the absence of replicates. Bacterial PAH-degrader analysis of phytoremediation field study soils Five selected phytoremediation field study treatments (Aster novae-angliae, Eupatorium purpureum, Lobelia cardinalis, Spartina pectinata, and an unplanted control) were examined for evidence of phytostimulation via the Spray Plate Assay for phenanthrene degrading bacteria. This analysis showed augmentation of both total soil bacteria (Figure 3.3) and PAH degrader bacteria (Figure 3.4) by most of the plant species at both sample dates. For the July 2003 soils, three out of four planted treatments, Spartina, Aster, and Eupatorium, have significantly higher soil CFU numbers compared to the unplanted (p value = 0.0161). All planted treatments had higher biodegrader counts (ZFU) than the unplanted treatment (p value = 0.064). For the October 2003 soils samples, all vegetated treatments had higher total bacterial density compared to the unplanted treatment, though only two planted treatments, Aster and Eupatorium, displayed significantly higher biodegrader numbers than the unplanted treatment (p value 61 I I I I I I I I I I I I I I I CFU per g soil DW (x106) 8 I I I T I I I I I ZFU per g soil DW (x10°) fieéféyé‘w c." “6‘ ESQ-.3 Figure 3.2. Preliminary Spray Plate Assay analysis of phytoremediation study soils. The upper chart Shows the Colony Forming Units (CFU) and lower chart is for the Zone Forming Units (ZFU). Y-axis is in 10° colonies per g soil DW for both data. The bars of both charts are aligned with treatment names at the bottom of the lower chart: l8 planted treatments (clear bars) and one unplanted treatment (black bar). There are no error bars, since these data are from one soil extraction per treatment (no replicates). 62 Table 3.2. Preliminary Spray Test analysis of treated soils from 19 phytoremediation field trials. Soil microbial extracts were of selected planted treatments collected July 2002 from the phytoremediation field trial site and Spray Assayed using phenanthrene. Shown are average visible colonies (CFU) and Spray-positives (ZFU) (per g soil DW), relative abundance of CFU per plate of 15 plated sub-samples and percentages of PAH bacterial degrader. CFU per g soil ZFU per g soil Treatment nw (x10°) nw (xro‘) %ZFU HYSPAT 116.3 10.8 9.3 EUPPUR 106.3 9.4 8.9 VIBDEN 163.4 13.7 8.4 GEUTRI 147.9 11.9 8.0 SILTER 96.3 7.4 7.7 CEAAME 109.2 7.9 7.3 PHYOPU 115.8 7.8 6.7 SPIALB 109.3 6.9 6.3 SPAPEC 109.7 5.9 5.4 EUPPER 118.5 5.9 5.0 UNPLNT 97.7 4.9 5.0 ASTNOV 106.8 5.1 4.8 AMOCAN 125.0 5.5 4.4 CIRDIS 89.9 3.5 3.9 SCIATR 93.0 3.6 3.8 ANDGER 89.7 2.4 2.7 LOBCAR 93.9 2.2 2.4 CARSPR 117.8 2.6 2.2 ANDSCO 96.5 1.7 1.8 63 0.0696). Only total heterotropic bacteria were enhanced in rhizosphere soil samples (Oct03), with no difference for ZFUS relative to the unplanted (Figure 3.3 and Figure 3.4). The percentages of PAH bacterial degrader to the total bacteria for each treatment were shown on Table 3.3. On the study of the ability of isolated Phenanthrene degrader screened on the preliminary experiment to utilize different type of PAHS, it was shown that some phenanthrene degrader isolates were able to degrade fluoranthene, pyrene, and anthracene. However, only a few of them could metabolized chrysene and benzo(a) pyrene (Table 3.3). Figure 3.5a and 3.5b showed phenanthrene degrading screened on anthracene, fluoranthene, and benzo(a)pyrene sprayed plates. DISCUSSION In this research study, the soil bacterial PAH degrader consortia was enumerated using the Spray Plate Assay. Phenanthrene, a three-ring angular PAH, was applied in the Spray Plate analyses to observe the abundance and diversity of PAH-degrading bacteria during phytoremediation. Two spray plate assay experiments were conducted for soil microbe analysis of the phytoremediation study. The preliminary spray plate experiment demonstrated that each plant species appears to affect abundance and diversity of soil bacterial communities to a different extent. However, the lack of replications in this trial experiment limits the interpretation of this data. In addition, these tested soil samples had been stored at 4°C for ~9 months prior to analysis, which may have permitted changes in soil microbial community structure. For the more extensive soil microbe analysis of the 4 planted and 1 unplanted phytoremediation field study treatments collected from, we Total CFU per 9 soil DW (x10°) 0 - L_1 ASTNOV EUPPUR LOBCAR SPAPEC UNPLNT ' “ 1, = Bulk Jul 03 soils - = Rhizosvhere Oct 03 soils = Bulk Oct 03 soils Note: a: significantly different at a = 0.05 compared to bulk Jul 03 UNPLNT treatment b: significantly different at a = 0.05 compared to bulk Oct 03 UNPLNT treatment c: significantly different at a = 0.1 compared to bulk Oct 03 UNPLNT treatment Figure 3.3. Colony Forming Units (CFUs) from soils of selected treatments. CFU values are given as per g soil dry weight for bulk soils (Jul 03 and Oct 03) and rhizosphere soils (Oct 03) from 5 different selected treatments (four planted treatments and one unplanted treatment). Bars represent mean values and error bars are standard error of means. N = 3 replicates. 65 b {a 5 '9 I 5 T D 3 3 l a: a a 8 r a . a . » - I II u. , ,, ASTNOV EUPPUR LOBCAR SPAPEC UNPLNT ' = JUI)’ 2003 SOIIS - = Rhizosphere October 2003 soils = October 2003 soils Note: a: significantly different at a = 0.1 compared to Jul 03 UNPLNT treatment b: significantly different at 01 = 0.1 compared to Oct 03 UNPLNT treatment Figure 3.4. Zone Forming Units (ZFUS) from soils of selected treatments. ZFU values are given as per g soil dry weight for bulk soils (Jul 03 and Oct 03) and rhizosphere soils (Oct 03) from 5 different selected treatments (four planted treatments and one unplanted treatment). Bars represent mean values and error bars are standard error of means. N = 3 replicates. 66 Table 3.3. Relative phenanthrene biodegrader (ZFU) bacterial density in selected treatments. Shown is average relative abundance of PAH bacterial degrader to total bacteria (ZFU / CFU x 100%) for soil samples collected July 2003 (bulk soil) and October 2003 (bulk soil and rhizosphere soil) from 5 different treated soils from phytoremediation field trials. (ZFU / CFU) % Treatmems Bulk Soil Bulk Soil Rhizosphere Soil Jul 03 Oct 03 Oct 03 ASTNOV 1.9 2.2 0-6 EUPPUR 2.1 2.1 0.5 LOBCAR 2.2 1.1 1-0 SPAPEC 1.3 0.9 1-0 UNPLNT 1.1 1.4 - 67 Table 3.4. Spray Test Assay analysis of 1° isolates against various PAH compounds. Abbreviation key: PhenD = phenanthrene degrader; AnthD = anthracene degrader; FluoD = fluoranthene degrader; PyreD = pyrene degrader; ChryD = chrysene degrader; BapyD = benzo(a)pyrene degrader. The % for 2° PhenD was calculated from total 1° PhenD Tested. The Spray-postive %’s for the remaining PAHS were calculated from total 2° PhenD positives. Note: All 2° PhenD negative colonies were also Spray-negative for the other tested PAHS. 1° PhenD 2° Planted Treatments Tested PhenD AnthD Fluo” PyreD ChryD BapyD Leadplant 99 88 6 6 4 0 0 Little Bluestem 100 77 15 24 23 0 0 Big Bluestem 104 85 11 33 28 0 1 New England Aster 106 87 9 36 28 0 0 Sprengel Sedge 106 96 15 33 31 0 0 New Jersey Tea 109 91 20 27 23 0 0 Pasture Thistle 107 94 14 28 30 0 0 Boneset 106 94 1 1 26 24 0 0 Joe Pye Weed 112 101 14 13 17 0 0 Prairie Smoke 110 102 5 6 6 0 0 Bottlebrush Grass 108 103 7 15 13 0 0 Cardinal Flower 101 91 27 42 39 0 0 Ninebark 108 100 15 15 14 0 0 Green Bulrush 108 91 16 18 17 0 0 Prairie Dock 107 98 11 23 19 0 0 Prairie Cordgrass 1 10 94 6 l 1 20 0 0 Meadowsweet 1 17 107 29 24 14 0 0 Arrowwood Viburnum 105 94 7 15 12 1 0 Unplanted Controls Unplanted Soil 107 86 5 16 15 1 0 Untreated Rouge Soil 108 90 2 2 3 0 0 Total 2138 1869 245 413 380 2 l % Positive 87.4% 13.1 % 22.1 % 20.3% 0.1 % 0.1 % 68 Figure 3.5a-c. Spray Test Assay of selected isolates with 3 PAHS. Plated isolates were tested with (a) phenanthrene; (b) anthracene; or (c) pyrene (5 days incubation). Images in this thesis are presented in color. Figure 3.5d. Spray Test Assay of selected isolates with benzo(a)pyrene. Sprayed plates were incubated for 4wk. Images in this thesis are presented in color. 69 independently analyzed each of the replicate sub-plots. In addition to this more statistically representative analysis, we increased the soil mass extracted from 1.0g to 4.0g to minimize microbial heterogeneity bias in the soil matrix. These measures gave, in our estimation, a more representative understanding of the plant contribution to the microbial community structure. It has been reported that microbial degradation is a major factor affecting PAH degradation (Heitkamp, 1988; Reilley et al., 1996). Plant secondary metabolites such as phenolic and terpenoid compounds have structural similarities to PAHS and may facilitate co-metabolic induction of degradation activity toward organic contaminants through co- metabolic processes (Fletcher and Hegde, 1995; Miya and Firestone, 2001; Singer et al., 2003). Vegetated enhancement of soil microbial density has also been reported in numerous studies (Nichols, 1997; Reilley et al., 1996; Siciliano et al., 2003). It was expected that the higher number of bacterial degraders in soil, the higher increased of PAH degradation. However, even though Spartina pectinata and Lobelia cardinalis displayed significant higher of CFU, but they did not increase ZFU compared to the unplanted treatment. Plants are species specific for root exudate quantity and chemical composition, as well as variable under different growing conditions and over the course of the growing season. Chemical compounds in root exudates have also been demonstrated to inhibit the growth of microbial degraders. It has been reported that some root exudate compounds may have negative effects on xenobiotic degradation by repressing catabolic pathways or by increasing the sorption capacity of the soil to make contaminants less bioavailable for microbial degradation (J ordahl et al., 1997). 70 This experiment showed that Aster novae-angliae and Eupatorium purpureum may have particular utility for phytoremediation applications due to their enrichment of both total soil microbes and bacterial degraders. The idea of phytostimulation by Aster and Eupatorium was supported by previous soil PAH concentration analyses performed in our laboratory. Soil PAH concentrations were observed to be significantly reduced by A. novae-angliae and E. purpureum, as well as A. gerardii and S. teribinthinaceum, in the phytoremediation field study (Wan, 2002). Unlike the bulk soil samples, rhizosphere soils collected in Oct 03 showed little difference from the unplanted treatment. CFU and ZFU values of rhizosphere soils were both significantly lower than bulk soil cores taken in October 2003. This result was not anticipated because the rhizosphere is considered the most metabolically active zone in the soil for microbial activity (Curl and Truelove, 1986). It is possible that the expected rhizosphere microbial enrichment was not observed for the Oct 03 sampling event due to the lateness in the calendar year, typically near or already in the dormant phase for Michigan vegetation. More frequent sampling may be necessary to observe dynamic growing season effects for rhizosphere soils. Future studies need to be performed to observe directly the effect of different plant Species on the number of PAH-degradin g bacteria using several different type of PAH compounds as substrates. SUMMARY AND CONCLUSION In this study, selected native Michigan plant species were used in an experimental phytoremediation study for rehabilitation of industrially impacted soils. Based on analysis of four planted and one unplanted treatment in this study, we observed that 71 different plant species had distinctive influences on the number of total soil bacteria and the number of PAH-degrading bacteria. Aster novae-angliae and Eupatorium purpureum were shown to consistently stimulate biodegrader numbers in treated soils. Though plants may increase total and PAH-degradin g bacteria compared to unplanted treatments, each plant species influences microbial growth and activity differently. Some plant species may actually inhibit contaminant degradation by production of antimicrobial compounds or selection for non-biodegrading bacterial populations. Therefore, plant species selection is critical for phytoremediation of persistent organic contaminants such as PAHS. Further study of the beneficial interactions between plants and microbes for organic contaminant degradation will lead to more advanced strategies for effective phytoremediation. 72 REFERENCES Ahn, Y., Sanseverino, J ., and Sayler, G. (1999). Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation 10, 149-157. April], W., and Sims, R. C. (1990). Evaluation of the use of prairie grasses for stimulating polycyclic aromatic hydrocarbon treatment in soil. Chemosphere 20, 253-265. Briggs, G. G., Bromilow, R. H., and Evans, A. A. (1982). Relationships between lipophilicity and root uptake and translocation of non-ionized chemicals by barley. Pesticide Science 13, 495-504. Cemiglia, C. E. (1993). Biodegradation of polycyclic aromatic hydrocarbons. Current Opinion in Biotechnology 4, 331-338. Curl, E. A., and Truelove, B. (1986). The Rhizosphere (New York: Springer-Verlag Berlin Heidelberg). Daane, L. L., Harjono, I., Zylstra, G. J ., and Haggblom, M. M. (2001). Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants. Applied and Environmental Microbiology 67, 2683-2691. Donnelly, P. K., Hegde, R. S., and Fletcher, J. S. (1994). Growth of PCB-degrading bacteria on compounds from photosynthetic plants. Chemosphere 28, 981-988. Fletcher, J. S., and Hegde, R. S. (1995). Release of phenols by perennial plant roots and their potential importance in bioremediation. Chemosphere 31 , 3009-3016. Harayama, S. (1997). Polycyclic aromatic hydrocarbon bioremediation design. Current Opinion in Biotechnology 8, 268-273. Heitkamp, M. A., W. Franklin, C. E. Cemiglia (1988). Microbial metabolism of polycyclic aromatic hydrocarbons: Isolation and characterization of a pyrene- degrading bacterium. Applied and Environmental Microbiology 54, 2549-2555. Jordahl, J. L., Foster, L., Schnoor, J. L., and Alvarez, P. J. J. (1997). Effect of hybrid poplar trees on microbial populations important to hazardous waste bioremediation. Environmental Toxicology and Chemistry 16, 1318-1321. Kiyohara, H., K. Nagao, K. Yana (1982). Rapid screen for bacteria degrading water- insoluble, solid hydrocarbons on agar plates. Applied and Environmental Microbiology 43, 454-457. 73 Liste, H. H., and Alexander, M. (2000). Plant-promoted pyrene degradation in soil. Chemosphere 40, 7-10. Marlowe, E. M., Wang, J. M., Pepper, 1. L., and Maier, R. M. (2002). Application of a reverse transcription-PCR assay to monitor regulation of the catabolic nahAc gene during phenanthrene degradation. Biodegradation 13, 251-260. Miya, R. K., and Firestone, M. K. (2001). Enhanced phenanthrene biodegradation in soil by slender oat root exudates and root debris. Journal of Environmental Quality 30, 191 1-1918. Muratova, A., T. Hubner, S., Tischer, O., Turkovskaya, M., Moder, M., and Kuschk, P. (2003). Plant-rhizosphere-microflora association during phytoremediation of PAH-contaminated soil. International Journal of Phytoremediation 5, 137-151. Nichols, D. C. W., H. B. Rogers, C. A. Beyrouty, C. M. Reynolds (1997). Rhizosphere microbial populations in contaminated soils. Water, Air, and Soil Pollution 95, 165-178. Ortega-Calvo, J. J ., Marchenko, A. I., Vorobyov, A. V., and Borovick, R. V. (2003). Chemotaxis in polycyclic aromatic hydrocarbon-degrading bacteria isolated from coal-tar and oil-polluted rhizosphere. FEMS Microbiology Ecology 44, 373-381. Reilley, K. A., Banks, M. K., and Schwab, A. P. (1996). Dissipation of polycyclic aromatic hydrocarbons in the rhizosphere. Journal of Environmental Quality 25, 212-219. Samantha, S. K., Chakraborti, A. K., and Jain, R. K. (1999). Degradation of phenanthrene by different bacteriazevidence for novel transformation sequences involving the formation of 1 -naphthol. Appl Microbiol Biotechn0153, 98-107. Schaffner, A., Messner, B., Langebartels, C., and Sandermann, H. (2002). Genes and enzymes for In-Planta phytoremediation of air, water, and soil. Acta Biotechnologica 22, 141-152. Shuttleworth, K. L., and Cemiglia, C. E. (1995). Environmental aspects of PAH biodegradtion. Applied Biochemistry and Biotechnology 54, 291-302. Siciliano, S. D., Germida, J. J., Banks, K., and Greer, C. W. (2003). Changes in microbial community composition and function during a polyaromatic hydrocarbon phytoremediation field trial. Applied and Environmental Microbiology 69, 483- 489. Singer, C. A., Crowley, D. E., and Thompson, 1. P. (2003). Secondary plant metabolites in phytoremediation and biotransformation. Trends in Biotechnology 21, 123- 1 130. 74 Wan, C. S. M. (2002). Phytoremediation of polycyclic aromatic hydrocarbon contaminated soil using native Michigan plant Species. In Crop and Soil Science (East Lansing: Michigan State University), pp. 122. Wilson, S. C., and Jones, K. C. (1993). Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHS): a review. Environmental Pollution 81, 229-249. 75