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Enhanced Degradation of Ddt via Application of Brewery Waste Product

Permanent Link: http://ufdc.ufl.edu/UFE0045492/00001

Material Information

Title: Enhanced Degradation of Ddt via Application of Brewery Waste Product
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Coppenger, Benjamin J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: apopka -- beer -- bioremediation -- ddt -- enzyme -- laccase -- lake -- peroxidase -- trub
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: DDT is an organochlorine pesticide (OCP) that was used extensively until the 1970s when it was banned, except for use in vector disease control. Due to its recalcitrance properties, it still persists in soils today, threatening the health of flora and fauna in the environment. Due to the soil properties of the study site in the North Shore Restoration Area of Lake Apopka, an in situ bioremediation strategy is necessary. Beer trub, a yeast containing sludge left over after the brewing process, was chosen as an additive and applied to contaminated soil to enhance extracellular enzyme production from native fungi in the soil. Microcosm and mesocosm studies analyzed which ratio of beer trub and water was most effective in enhancing the DDT degradative process. Results suggested that a solution of at least 1.8% beer trub solution was most effective. Extracellular enzyme and biomass assays tracked the time and rates of DDT disappearance in the treatment soils thus supporting the hypothesis that the biodegradation was by fungi. Leachate was also analyzed for extracellular enzyme activity, nitrate, and DDx concentrations. The results supported the recycling of the leachate if resources permit to allow for the reuse of enzymes in the leachate, as well as to allow for complete degradation of DDx in leachate. Due to the amount of beer commercially produced in the U.S., supplies of the cost-effective beer trub do not seem to be limited; meaning this method of bioremediation is possible.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Benjamin J Coppenger.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Thomas, John E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045492:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045492/00001

Material Information

Title: Enhanced Degradation of Ddt via Application of Brewery Waste Product
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Coppenger, Benjamin J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: apopka -- beer -- bioremediation -- ddt -- enzyme -- laccase -- lake -- peroxidase -- trub
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: DDT is an organochlorine pesticide (OCP) that was used extensively until the 1970s when it was banned, except for use in vector disease control. Due to its recalcitrance properties, it still persists in soils today, threatening the health of flora and fauna in the environment. Due to the soil properties of the study site in the North Shore Restoration Area of Lake Apopka, an in situ bioremediation strategy is necessary. Beer trub, a yeast containing sludge left over after the brewing process, was chosen as an additive and applied to contaminated soil to enhance extracellular enzyme production from native fungi in the soil. Microcosm and mesocosm studies analyzed which ratio of beer trub and water was most effective in enhancing the DDT degradative process. Results suggested that a solution of at least 1.8% beer trub solution was most effective. Extracellular enzyme and biomass assays tracked the time and rates of DDT disappearance in the treatment soils thus supporting the hypothesis that the biodegradation was by fungi. Leachate was also analyzed for extracellular enzyme activity, nitrate, and DDx concentrations. The results supported the recycling of the leachate if resources permit to allow for the reuse of enzymes in the leachate, as well as to allow for complete degradation of DDx in leachate. Due to the amount of beer commercially produced in the U.S., supplies of the cost-effective beer trub do not seem to be limited; meaning this method of bioremediation is possible.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Benjamin J Coppenger.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Thomas, John E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045492:00001


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1 ENHANCED DEGRADATION OF DDT VIA APPLICATION OF BREWERY WASTE PRODUCT By BENJAMIN J. COPPENGER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGR EE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Benjamin J. Coppenger

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3 To my family, whose love and support I c ould not have done this without

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4 ACKNOWLEDGMENTS First of all, I would like t o thank Dr. John Thomas for his willingness and patience to work with me throughout these last two and a half years. Without his instruction and expertise, this would not be possible. Also, thank you to my committee members Dr. Hochmuth and Dr. Rollins. Wi thout your help, I would not have learned to consider other ideas and to look at the big picture of what is truly happening. I would also like to thank Moshe Doron for his friendship, support, and willingness to listen and understand. I will always remembe r the entrepreneurial conversations and insightful conversations we had. I am ever indebted to my parents for the sacrifices they made to enable me to achieve my goals. They have showed me true love during times I did not deserve it, and they always were t here when I needed an escape from school. I would also like to thank my brother, Sam. Thank you for including me in all the basketball and football games. I will always cherish those special games where we were there to celebrate a Gator victory (win again st Kentucky in the ODome, win against LSU in the swamp). To Nicole, my lovely bride to be, I am so grateful for your help and support throughout this journey. I do not know how I would have made it this far if you had not been there to provide a good meal, a loving home, and a shoulder to rest on when I was weary. Last but certainly not least, to my EVER PRESENT HELP IN TIME OF NEED with whom all things are possible (Psalm 37:4 5).

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION OF DDT AN D THE STUDY SITE IN LAKE APOPKA, FL ........... 12 Definition and History of DDT ................................ ................................ ................. 12 Chemical and Physical Properties that lead to DDT Recalc itrance ......................... 13 Health and Environmental Effects of DDT ................................ .............................. 13 Necessity of Improved Remediation Strategies ................................ ...................... 15 Study Site ................................ ................................ ................................ ............... 17 Research Objectives and Hypotheses ................................ ................................ .... 19 Thesis Overview ................................ ................................ ................................ ..... 20 2 EVALUATION OF INCREMENTAL BEER TRUB AMENDMENTS TO DDX CONTAMINATED SOIL IN MICROCOSM STUDY ................................ ................. 27 Materials and Methods ................................ ................................ ............................ 29 Soil ................................ ................................ ................................ ................... 29 Microcosm ................................ ................................ ................................ ........ 30 DDx Extraction ................................ ................................ ................................ 31 Accelerated Solvent Extraction (ASE) ................................ .............................. 31 Florisil Extraction ................................ ................................ .............................. 32 Gas Chromatography (GC) Conditions ................................ ............................ 32 Laccase Enzyme Assay ................................ ................................ ................... 34 Required solutions and sample preparation ................................ ............... 34 Laccase e nzyme assay spectrophotometer method ................................ .. 34 Peroxidase Enzyme Assay ................................ ................................ ............... 35 Required solutions and sample preparation ................................ ............... 35 Peroxidase enzyme assay spectrophotometer method ............................. 35 Statistical Analysis ................................ ................................ ............................ 36 Resu lts and Discussion ................................ ................................ ........................... 36 Gas Chromatography Analysis of DDx in Microcosm Study ............................. 36 Laccase and Peroxidase Enzyme Activities ................................ ..................... 37 Conclusions ................................ ................................ ................................ ............ 38

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6 3 ANALYSIS OF DDX DEGRADATION, BIOMASS, AND ENZYME ACTIVITY IN MESOCOSM STUDY ................................ ................................ ............................. 43 Materials and Methods ................................ ................................ ............................ 44 Soil ................................ ................................ ................................ ................... 44 Mesocosm ................................ ................................ ................................ ........ 44 DDx Extraction for Soil Samples ................................ ................................ ...... 48 Accelerated Solvent Extraction (ASE) of Soil Samples for Gas Chromatography ................................ ................................ ........................... 48 Flori sil Extraction for Soil Sample Extracts ................................ ....................... 49 Gas Chromatography (GC) Conditions for Soil and Leachate Samples ........... 50 Laccase Enzyme As say ................................ ................................ ................... 51 Peroxidase Enzyme Assay ................................ ................................ ............... 52 Ergosterol Extraction for High Performance Liquid Chromatography (HPLC) Analysis ................................ ................................ ................................ ......... 53 Accelerated Solvent Extraction (ASE) for HPLC Analysis ................................ 54 HPLC Analysis of Ergosterol ................................ ................................ ............ 54 Nitrate Nitrogen Analysis ................................ ................................ .................. 55 Organic Matter Analysis ................................ ................................ ................... 55 Statistical Analysis ................................ ................................ ............................ 55 Results and Discussion ................................ ................................ ........................... 56 Mesocosm DDx Degradation in Soil ................................ ................................ 56 Mesocosm DDx Degradation in Leachat e ................................ ........................ 58 Mesocosm Soil Enzyme Activity ................................ ................................ ....... 60 Mesocosm Leachate Enzyme Activity ................................ .............................. 61 Mesocosm Soil Ergosterol Concentrations ................................ ....................... 63 Nitrate Concentrations in Leachate and Soil ................................ .................... 63 Conclusion ................................ ................................ ................................ .............. 64 4 DEGRADATION OF DDT ................................ ................................ ....................... 79 Materials and Methods ................................ ................................ ............................ 80 DNA Extraction and PCR Amplification of 18s rRNA Genes ............................ 80 DDT Mineralization Study ................................ ................................ ................. 81 Results and Discussi on ................................ ................................ ........................... 83 DNA Extraction and PCR Amplification of 18s rRNA Genes ............................ 83 DDT Mineralization Study ................................ ................................ .......... 83 Conclusion ................................ ................................ ................................ .............. 84 APPENDIX: RECIPES ................................ ................................ ................................ .. 88 REFERENCES ................................ ................................ ................................ .............. 89 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 94

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7 LIST OF TABLES Table page 1 1 Chemical Structure, IUPAC and common names for major DDT metabolites (adapted from ATSDR, 2002) ................................ ................................ ............. 22 1 2 DDx compounds (adapted from ATSDR, 2002) ................................ ................................ ................................ .... 23 1 3 Physical DDx compounds (adapted from ATSDR, 2002) ................................ ................................ ................................ .... 23 1 4 Study Site characteristics ................................ ................................ ................... 23 4 1 Characteri stics of PCR samples ................................ ................................ ......... 87 A 1 Plating Medium for Soil Fungi Rose Bengal Streptomycin Agar (Martin, 1950) ................................ ................................ ................................ .................. 88 A 2 Basal III medium (per liter) (Tien and Kirk, 1988) ................................ ............... 88 A 3 Trace Element Solution (Tien and Kirk, 1988) ................................ .................... 88

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8 LIST OF FIGURES Figure page 1 1 DDT Poster from 1947 by Trimz Co. Inc. ................................ ............................ 24 1 2 Aerial view of the study site. The red star indicates the study site ZSS3027 in field ZSE J ................................ ................................ ................................ ...... 25 1 3 Picture of Allen Plow used to remediate DDT contaminated soils. ..................... 26 2 1 DDT (g/g dry soil) i n selecte d microcosm treatments ................................ ................................ ................................ .......... 39 2 2 DDE concentrations in selected microcosm treatments spanning duration of experiment. ................................ ................................ ....................... 40 2 3 Laccase enzyme activity. ................................ ................................ .................... 41 2 4 Peroxidase enzyme activity ......... 42 3 1 DDT concentrations in soil samples from treatments durin g mesocosm study. ................................ ................................ ................................ .................. 67 3 2 DDE concentrations in soil samples from treatments during mesocosm study.. ................................ ................................ ................................ ................. 68 3 3 DDT concent rations in leachate samples from treatments during mesocosm study.. ................................ ................................ ............................... 69 3 4 DDE concentrations in leachate samples from treatments during mesocosm study. ................................ ................................ ................................ 70 3 5 Laccase activity in soil for treatments during duration of mesocosm study.. ...... 71 3 6 Peroxidase activity in soil for treatments during duration of mesocosm study.. .. 72 3 7 Laccase activity in leachate for treatments during duration of mesocosm study.. ................................ ................................ ................................ ................. 73 3 8 Peroxidase activity in lea chate for treatments during duration of mesocosm study.. ................................ ................................ ................................ ................. 74 3 9 Soil ergosterol concentrations in treatments during duration of mesocosm study.. ................................ ................................ ................................ ................. 75 3 10 Leachate NO 3 concentrations for treatments during duration of mesocosm study, including day 0 of study.. ................................ ................................ .......... 76

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9 3 11 Leachate NO 3 concentrations for treatments during dura tion of mesocosm study, not including day 0 data.. ................................ ................................ ......... 77 3 12 Soil NO x concentrations for treatments during duration of mesocosm study.. .... 78 4 1 Picture of PCR amplicons recovered from soils.. ................................ ................ 85 4 2 DDT levels for DDT mineralization study.. ................................ ................... 86

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10 Abstract of Thesis Presented to the Gra duate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ENHANCED DEGRADATION OF DDT VIA APPLICATION OF BREWERY WASTE PRODUCT By Benjamin J. Coppenger May 2013 Chair: John Thomas Major: Soil and Water Science DDT is an organochlorine pesticide (OCP) that was used extensively until the 1970s when it was banned, except for use in vector disease control. Due to its recalcitrance properties, it still persists in soils today, th reatening the health of flora and fauna in the environment. Due to the soil properties of the study site in the North Shore Restoration Area of Lake Apopka, an in situ bioremediation strategy is necessary. Beer trub, a yeast containing sludge left over aft er the brewing process, was chosen as an additive and applied to contaminated soil to enhance extracellular enzyme production from native fungi in the soil. Microcosm and mesocosm studies analyzed which ratio of beer trub and water was most effective in en hancing the DDT degradative process. Results suggested that a solution of at least 1.8% beer trub solution was most effective. Extracellular enzyme and biomass assays tracked the time and rates of DDT disappearance in the treatment soils thus supporting th e hypothesis that the biodegradation was by fungi. Leachate was also analyzed for extracellular enzyme activity, nitrate, and DDx concentrations. The results supported the recycling of the leachate if resources permit to allow for the reuse of enzymes in t he leachate, as well as to allow for complete degradation of DDx in leachate. Due to the amount of beer

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11 commercially produced in the U.S., supplies of the cost effective beer trub do not seem to be limited; meaning this method of bioremediation is possible

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12 CHAPTER 1 INTRODUCTION OF DDT AND THE STUDY SITE IN LAKE APOPKA, FL Definition and History of DDT DDT (1,1,1 trichloro 2,2 di(4 chlorophenyl)ethane) is an organochlorine pesticide that is made by condensing chloral hydrate with chlorobenzene in concent rated sulfuric acid (Gianessi and Puffer, 1992). It was first synthesized by German doctoral student, Othmar Zeidler, in 1874 at the University of Strasbourg in Germany (EPA report, 1975; Metcalf, 1973). However, it was not until 1939 when Swedish chemist Paul Hermann military began to use DDT to combat diseases such as malaria and typhus in World War II (EPA report, 1975). After World War II ended, the use of DDT became more widespread and was heavily utilized for agricultural and commercial purposes. In fact, in 1947, an advertisement poster was published by Trimz Co., Inc. that a dvertised a wall 1). At the peak of its popularity in the U.S. in 1962, DDT was registered for use on 334 agricultural commodities and about 85,000 tons were being produced annually (Metcalf, 1995 ). Production numbers skyrocketed when, in 1966, 27 million pounds of DDT were being used that year in U.S. agriculture (Gianessi and Puffer, 1992). Although sales for DDT were at an all time high in the 1960s, profit made from DDT sales would not continu Silent Spring was published, which called into question the health effects of DDT and its metabolites DDD (1 chloro 4 [2,2 dichloro 1 (4 chlorophenyl)ethyl]benzene) and DDE (1,1 bis (4 chlorophenyl) 2,2 dichloroethene) (collecti vely known as DDx). Chemical structures of the DDx

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13 compounds are shown in Table 1 1. The publishing of Silent Spring launched the beginning of the environmental movement and in 1972 DDT was highly restricted for use in the U.S. (EPA report, 1975). Althoug h DDT was mostly banned in 1972, it is still used for agriculture and vector disease control in some tropical countries. If it was completely banned, certain countries would most likely fall into an endemic and epidemic of malaria (World Health Organizatio n, 1979). Chemical and Physical Properties that lead to DDT Recalcitrance Even though DDT has been banned since the 1970s in the U.S., it is still persistent in soils today. DDT is highly insoluble in water and has a high affinity for organic matter and s oil particles (ATSDR, 2002). The half life for DDT in the soil ranges from 2 15 years (National Pesticide Information Center, 1999). It can also volatilize via handler application or exposure to air at the soil surface and can be transported great distance s by aeolian transport of dust and soil particles that carry the bound DDT its high affinity for organic matter, DDT is not readily bioavailable for degradation via micro bial metabolic processes. Also, DDT is a xenobiotic, thus limiting the number of microbes that can degrade it due to the lack of similar compounds in nature. However, extracellular enzymes with broader specificities such as laccase and peroxidases have bee n found to degrade DDT (Hou et al 2003; Bonnen et al 1994). DDT can also become more difficult to degrade over time due to DDx compounds sequestering with the soil organic matrix in a process known as aging (Alexander, 1995; 1998). Health and Environm ental Effects of DDT Due to the physical and chemical properties of DDT and its ability to bioaccumulate over time (Kunisue, 2003), DDT has been found to negatively affect

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14 animal and human health. In the U.S. Environmental Protection Agency (EPA) review of DDT in 1975, evidence supporting that DDT was harmful to animals was documented. The report states that DDT was present in animals ranging from phytoplankton to reptiles and birds. It also reports finding levels of DDT in humans (EPA report, 1975). Amphib ians have been found to be most susceptible to DDT, followed by mammals and birds (ATSDR, 2002). DDT has been found to be a cause of bird egg shell thinning which leads to crushed eggs before hatching, thus effecting the mortality rates of newborn birds ( EPA report, 1975). One particular example of a DDT affected ecosystem is Lake Apopka, FL where high amounts of organochlorine pesticides (OCPs) have been found. Several studies have argued that the presence of these OCPs in the Lake Apopka ecosystem have led to altered reproductive outcomes and feminization of fish (Gallagher et al., 2001; Kristensen et al., 2007; Toft et al., 2003), turtles (Guillette et al., 1994), alligators (Guillette et al., 1994; 1996), and Florida panthers (Facemire et al., 1995). D DT, as well as other OCPs, has also been found to be endocrine disruptors and have been classified as probable carcinogens (U.S.E.P.A., 2011). An accumulation of issues with these pesticides, as well as others such as heptachlor and mirex, resulted in the 2001 Stockholm Convention development of a list of OCPs that would be eliminated from production. The ban created at the convention went into force on May 17, 2004 (United Nations Environment Programme, 2005). OCPs, such as DDT, bioaccumulate in the fatty tissues of animal species and are able to biomagnify as they are passed up the food chain. This bioaccumulation in the fatty tissues of animals is possible because of the high lipophility of DDT (as indicated

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15 by the high octanol water distribution coeffici ents (K ow ) of the DDx compounds shown in Table 1 2 and 1 3). Once present in the fatty tissue of animals, it can eventually cause er. In insects, DDT works by interfering with the movement of ions through neuronal membranes, eventually causing convulsions, hyperexcitability, and eventually death for the insect (ATSDR, 2002). The recalcitrance of the DDx compounds are also facilitated by the aromatic chlorine structures of the DDx compounds. The more halogen substituents present on the organic compounds, the more energy is needed for microbes to degrade the carbon halogen bonds (Gohil, et al., 2011; Dobbins, 1995). Necessity of Improv ed Remediation Strategies Due to the recalcitrant physical and chemical properties of the DDx compounds, different methods of remediating soils contaminated with OCPs have been studied. Cucurbits such as cucumbers, melons, and squash have been shown to acc umulate OCPs and thus, some farmers have used these crops to remediate their contaminated soil (Ogburn, 2010). This method, however, has issues. First, the farmer loses money and seed by growing the crops to take up the OCPs. The farmer then has time and l abor costs associated with harvesting and disposing of the crop in a safe and efficient way. Another problem is that the cucurbits can only capture OCPs that the roots can reach. If the OCPs have been transported to a depth deeper than the depth of the roo ts, the OCPs will remain in the soil. Therefore, a different method is needed that is more efficient and does not have depth limits. Wood rot fungi have the ability to produce extracellular enzymes that are indiscriminate and can degrade pesticides, such as DDT. These extracellular enzymes

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16 are not limited as much in depth as cucurbit roots are and do not come with the extra cost of seeding, growth, uprooting, and disposal, thus making them a more affordable remediation plan. Recent studies with Pleurotus o streatus and Agaricus bisporus by Dr. John Thomas (Thomas, 2011) have shown that the addition of a spent mushroom substrate (SMS) to the soil did produce the desired enzymatic activities of lacasse and peroxidase, but the enzymes were not able to effective ly degrade DDx. The study suggested that the lacasse and peroxidase produced by the SMS were unable to reach the contaminants in a sufficient quantity. The hypothesis was that the enzymes produced by the SMS caused the native Nectria sp to stop producing its own enzymes. It was also hypothesized that the enzymes that were produced were in low concentrations and that they only traveled down the water columns in the soil and did not come into contact with the contaminants. A cost effective method that enhan ces the production of extracellular enzymes by endemic wood rot fungi, Nectria sp is needed. Beer trub, the waste sludge leftover after the beer brewing process, contains live and dead yeast, as well as other nutrients. The yeast used in the brewery proce ss, Saccharomyces cerevisiae is also capable of producing extracellular enzymes capable of degrading DDT (Jadhav et al., 2007). Since yeast extract, which is dead yeast, has also been found to enhance extracellular enzyme production (Hou et al., 2004), bee r trub and yeast extract will be compared in their abilities to enhance the production of the extracellular enzymes and the amount of DDx degraded in soil. Beer trub was chosen because it is a cheap waste product that is fairly accessible in most parts of the world and is known to contain yeast.

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17 Study Site The site where the DDx contaminated soil was collected for this study is located just south of Hooper Farm Road in the Lake Apopka North Shore Restoration Area (Site ZSS3027 in field ZSE J) (Figure 1 2). Located in central Florida, Orange and Lake counties border Lake Apopka. The contaminated soil was collected on September 9, 2011. The soil type was mapped on the NRCS Web Soil Survey as Canova Muck on September 13, 2011(See Table 1 4 for other soil char acteristics). The UTM coordinates for the sampling site are zone 17 R, 0445726 for the x coordinate and 3169801 for the y coordinate (Lat: 28.654199437278905; Long: 81.55538519914658). The study site was originally owned by Zellwin Farms Company and was u sed for growing crops like sweet corn, carrots, celery, cucumbers, cabbage, radishes, and other vegetables Farming operations have been discontinued since farming practices were halted in July 1998 (Personal communication, Pam Bowen, 2/24/2012). DDT cont amination of the soil occurred in multiple ways. According to the Saint Management Plan (SJRWMD SWIM Plan), Growers would apply DDT and a host of other OCPs such as dieldrin, toxaphene, and chlordane (SJRWMD SWIM Plan, 2003) to their fields. Also, from 1957 to 1981, Tower Chemical Company (TCC) manufactured, produced, and stored a variety of OCPs in the area. TCC contributed to the contamination by discharging their chemical wastewater i nto 0.5 acre unlined percolation/evaporation pond, which was located over a sink hole. This sink hole allowed wastewater to percolate into the Floridian aquifer. In 1980, a spray irrigation field for waste disposal was created due to an overflow of a waste water pond into Gourd Neck, which is a small spring source that feeds into Lake Apopka (SJRWMD SWIM

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18 PLAN, 2003). The spill of wastewater into Gourd Neck and the surrounding area ended TCC spill, changes in vegetation and fish kills were reported in the Gourd Neck area (Rice and Percival, 1996). Eventually, the U.S. EPA designated the area a Superfund site due to the high concentrations of DDT and other pesticides that were present in t he soil (SJRWMD SWIM Plan, 2003). In the 1990s, the St. Johns River Water Management District (SJRWMD) purchased the property of the study site, as well as all other properties east of the Apopka Beauclair Canal. This large purchase by the SJRWMD was an a ttempt to reduce and/or eliminate the amount of phosphorus running off from farms into Lake Apopka. The phosphorus was released from fertilizers that were applied on the farms and washed into the lake when the growers pumped water off of their property. This the plants in the lake as a result of the algal blooms in the lake water. The eutrophication that was caused by the large amounts of phosphorous washing into Lake Apopka from the northern bordering farm fields killed off a large portion of the bass (SJRWMD, 2006; Personal Communication, Pam Bowen, 2/24/2012). One of the main goals o f the Lake Apopka North Shore Restoration Area was to restore a large portion of the land on the north shore of Lake Apopka to wetlands that wetlands, phosphorous and oth er contaminants would be filtered out of the water flow before reaching the river. Other remediation methods included using an Allen Plow

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19 (Figure 1 that is on top and flips it, es sentially burying the contaminated soil 3 to 4 feet below the surface. Since the majority of the OCPs are found in the top 6 inches of the soil, the Allen Plow buries the OCPs, which are large molecules and do not migrate upward easily in soil, thus limiti ng the exposure of the OCPs to the surface. Once the field has been plowed with the Allen Plow, it generally is flooded so that the original wetland habitat is restored. This method offered one of the safest and most affordable methods of remediation. Howe ver, since our particular study site contains a significant amount of sand, this method of flooding for wetland restoration has not been utilized because the sand allows for a significant potential of OCPs release and entry into the fauna food chain via fi sh uptake (Personal Communication, Pam Bowen, 2/24/2012). Due to the paucity of remediation methods previously employed at the study site, other methods of remediation are necessary. Research Objectives and Hypotheses This thesis attempts to demonstrate that the addition of a beer trub solution to the contaminated soil enhances the remediation and biodegradation process of DDT in soil taken from the previously discussed study site. Since yeast extract has been found to enhance extracellular enzyme product ion (Hou et al., 2003), beer trub and yeast extract were compared in their abilities to enhance the production of the extracellular enzymes and the amount of DDx degraded in the soil. Beer trub was chosen because it is a cheap waste product that is fairly accessible in most parts of the world and is known to contain live yeast. The objectives of this research project are as follows: (1) quantify biodegradation of 1, 1, 1 trichloro 2,2 di(4 chlorophenyl)ethane (DDT), 1 chloro 4 [2,2 dichloro 1 (4 chloropheny l)ethyl]benzene (DDD), and 1,1 bis (4 chlorophenyl) 2,2

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20 dichloroethene (DDE) under various treatments in mesocosms and microcosms, (2) conduct enzyme and biomass analyses on soil samples from the treated mesocosms and microcosms, and (3) conduct a mesocosm study to test whether or not nitrate is a limiting factor in the biodegradation process. Based on the general knowledge related to organochlorine pesticides, the hypotheses for these studies are: (1) that extracellular enzymes, laccase and peroxidase, wi ll biodegrade DDT, DDD, DDE. (2) the water soluble amendments, such as beer trub, will increase fungal biomass, which will correlate to an increase in extracellular enzyme production, which will then correlate to an increase in the degradation of DDx, (3) the mescocosm study will show that nitrate inhibits DDx degradation by limiting extracellular enzyme production. Enzyme production is limited by excessive nitrate because the fungi that produce the extracellular enzymes are not in need of the enzymes crea ting more energy sources when a certain level of beer trub is present. (4) the isolation and identification of fungi will show that there is a native species of a wood rot fungus ubiquitous to the site that can degrade DDx compounds. Thesis Overview This t hesis begins with an introduction to DDT in Chapter 1, covering its rise in popularity and use during the 1940s through the 1960s and the banning of DDT in the 1970s due to its harmful health effects on animals and humans. The study site is introduced alon g with a discussion of why a new cost effective alternative to DDT degradation is needed. Objectives and hypotheses are also stated in Chapter 1. In order to test the objectives and hypotheses mentioned in Chapter 1, an initial microcosm study was conduc ted to test the possibility of using beer trub to augment the degradation process of DDT in our study site soil. Chapter 2 discusses this microcosm

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21 study and states parameters used for analysis of DDT degradation via gas chromatography, the methodology app lied to prepare samples for gas chromatography analysis, as well as the methods for the enzyme assays conducted during the microcosm study. The purpose of the microcosm study was to determine if beer trub was effective in enhancing the degradation process of DDT, and if so, how much beer trub was most successful at enriching the degradative process. Once the amount of beer trub that was most effective in enhancing the degradative process was discovered, we increased the scale of our experiments to a study c onducted with 5 gallon bucket filled with our study site soil. This experiment analyzed for DDT degradation in our study site soil with the application of beer trub, enzyme activity, biomass of fungi in the treated soils throughout the duration of the expe riment, as well as nitrate levels in leachate collected during the experiment and in the soil itself. Chapter 3 discusses this mesocosm study, including methodologies used for each study performed, and results and discussions of the findings. To conclude, Chapter 4 describes more of the microbiological aspect of this research. It focuses on the fungi isolation and DNA testing on the isolated fungi that was done to identify the fungal species present in our study site soil. Once the fungi were isolated and identified, a microcosm DDT mineralization study was done to see if our isolated fungi could indeed degrade DDT via enzyme production. This microcosm mineralization study is also included in Chapter 4, along with the general findings of this research and r ecommendations for further studies.

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22 Table 1 1. Chemical Structure, IUPAC and common names for major DDT metabolites (adapted from ATSDR, 2002) IUPAC name Common Name Chemical Structure 1,1,1 trichloro 2,2 bis (p chlorophenyl)ethane DDT 1,1, 1 trichloro 2(o chlorophenyl) 2 (p chlorophenyl)ethane DDT 1,1 dichloro2,2 bis(p chlorophenyl) ethylene DDE 1,1 dichloro 2(o chlorophenyl) 2 (p chlorophenyl) ethylene DDE 1,1 dichloro 2,2bis(p chlorophenyl)ethane DDD 1,1 dichloro 2 (o chlorophenyl)2 (p chlorophenyl)ethane DDD

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23 Table 1 DDx compounds (adapted from ATSDR, 2002) Property DDT DDD DDE Solubility in Water (mg/L at 25C) 0.025 0.90 0.12 Log K ow 6.91 6.02 6.51 Log K oc 5.18 5.18 4.70 K ow = octanol water partition coefficient; K oc = organic carbon coefficients Table 1 DDx compounds (adapted from ATSDR, 2002) Property o DDT DDD DDE Solubility in Water (mg/L at 25C) 0.085 0.14 0.1 Log K ow 6.79 5.87 6.00 Log K oc 5.35 5.19 5.19 K ow = octanol water partition coefficient; K oc = organic carbon coefficients Table 1 4. Study Site characteristics NRCS Mapp ed Soil Type Canova Muck % Sand 60.2* %Silt 14.6* %Clay 17.5* % other (Organic Matter, Water) 7.7* pH 5.5** DDE 391 ug/kg (dryweight)*** DDT 339 ug/kg (dry weight)*** DDD 190 ug/kg (dry weight)*** signifies values taken from Natio nal Resources Conservation Service (NRCS) Online Web Soil Survey on January 15, 2013. / ** Signifies pH value determined in the lab that conducted this thesis study on January 15, 2013. / ***Signifies concentration values of pesticides in question at the s tudy site which were measured by Pace Analytical Service, Inc. from a grab sample taken Nov. 4, 2010

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2 4 Figure 1 1. DDT Poster from 1947 by Trimz Co. Inc.

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25 Figure 1 2. Aerial view of the study site. The red star indicates the study site ZSS3027 i n field ZSE J

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26 Figure 1 3. Picture of Allen Plow used to remediate DDT contaminated soils.

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27 CHAPTER 2 EVALUATION OF INCREMENTAL BEER TRUB AMENDMENTS TO DDX CONTAMINATED SOIL IN MICROCOSM STUDY When the Saint Johns River Water Manag ement District (SJRWMD) began the Lake Apopka North Shore Restoration project, one of the main goals was to eventually convert the entire restoration area to natural wetlands in the expectation that the wetlands would filter out contaminants in surface run off flowing southward into Lake Apopka. As discussed in the previous chapter, our particular study site was deemed inappropriate for wetland conversion because of the threat of the persistent DDT in the mineral soil being transported through the soil profi le into the groundwater and eventually into the lake via water flow transportation. Therefore, to achieve the acceptable levels. Many different methods of remediating DD T contaminated sites have been studied, including cultivation of cucurbits, like melons and squash, to phytoremediate DDT (Ogburn, 2010) and the use of nano scale zero valent iron applications to clean up DDT contaminated soil (El Temsah et al., 2012). The premise behind the use of metals such as iron to degrade DDT is that the metals react with the organic contaminant causing a reductive dehalogenation, or more specifically, a reductive dechlorination, transformation to occur. This transformation results i n a loss of the outer DDT chlorines allowing for chemical degradation (Hanna, 2012). Other remediation strategies include contaminated soil excavation that utilizes ex situ treatments such as incineration (El Temsah, 2012). Although there has been some suc cess with these various treatments, there are still major flaws with these different remediation strategies. The use of metals to enhance DDT degradation pose serious environmental concerns (El Temsah, 2012)

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28 and the use of cucurbits to phytoremediate DDT i s limited by plant root depth. Ex situ remediation strategies are logistically difficult to manage and at times pose further environmental exposure to the contaminant. The main issue with all of these strategies, however, is the limitation imposed by the h igh cost of these remediation methods. It is important to remember that the remediation strategy chosen has to make economic sense and allow the landowner to retain capital to redevelop the restored land, or afford to flood the land and lose the potential agricultural revenue, as is the case of the aforementioned Lake Apopka study site. The use of metals, the transportation of contaminated soil, and use of heavy machinery make the remediation prohibitively expensive. Therefore a more cost effective method o f remediation is required. One possible cost effective approach to degradation of DDT is enhancing the production of extracellular enzymes such as lacasse and peroxidase from wood rot fungi. Lacasse and peroxidase enzymes have been shown to degrade D DT (H ou et al., 2003; Bonnen et al 1994) due to their nondiscriminatory catalytic characteristics. These enzymes that are capable of degrading DDx are not limited by depth of root and do not produce high volumes of waste material. However, due to laccase and peroxidase being oxidase enzymes, they are dependent upon the presence of oxygen for catalysis (Baldrian, 2005). Furthermore, wood rot fungi are naturally occurring in Florida soil, which greatly reduces the cost of remediation. Recent studies with two typ es of wood rot fungi Pleurotus ostreatus and Agaricus bisporus by Dr. John Thomas (Thomas, 2011) have shown that the addition of a spent mushroom substrate (SMS) to the soil did produce the desired lacasse and peroxidase activities, but in low quantities. This lack of enzyme production makes sense, especially for laccase production,

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29 because extracellular laccase enzymes are produced via secondary metabolic processes in basidiomycete fungi (Hou et al., 2003). Also, it was discovered that a native wood rot fu ngal species were present in the study soil. It was hypothesized that the enzymes produced by the SMS in low concentrations caused the native Nectria sp. to stop producing lacasse and peroxidase enzymes, and as a result, the degradation of DDx was slowed. It was also hypothesized that the enzymes that were produced were in low concentrations and only traveled down the water columns in the soil and may not have come into contact with the contaminants. Therefore, a cost effective method that enhances the pro duction of extracellular enzymes by the native wood rot fungi is needed. Since yeast extract has been found to enhance extracel lular enzyme production (Hou et al., 2004), beer trub (spent brewery grain slurry) and yeast extract were compared in their abili ties to enhance the production of the extracellular enzymes and the subsequent degradation of DDx in the soil. Beer trub was chosen because it is a cheap waste product that is fairly accessible in most parts of the world and is known to contain live yeast and some nutrients. Though we expected beer trub to enhance lacasse and peroxidase production and thus increase DDT degradation, the most suitable amount of beer trub to be added was unknown. To test this, we added various amounts of beer trub to mason jar s filled with our DDx contaminated soil and measured degradation rate of the isomers of DDT, DDE, and DDD along with the laccase and peroxidase activity in the soil. Materials and Methods Soil The soil that was used in this study was collected from the Nor th Shore Restoration Area (NSRA) at Lake Apopka, FL. The contaminated soil was collected on

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30 September 9, 2011. The soil type was mapped on the NRCS Web Soil Survey as Canova Muck on September 13, 2011. The soil was transported back to the University of Flo rida (UF) in Gainesville, FL by truck and was stored in a greenhouse managed by the faculty and graduate assistants of the Soil and Water Science Department at UF in Gainesville. The soil was sieved through a 2 millimeter mesh sieve and homogenized by mixi ng before being added to the 32 oz. (946 ml) mason jars (Ball Corporation, Broomfield, CO) was 5.5 using 1:2 soil: water mixture as measured with a Fisher Scientific/ Denver Instrument Com pany (Bohemia, New York) Accumet pH meter 50 electrode. Microcosm The microcosm study was constructed using the sieved homogenized soil from the NSRA of Lake Apopka. A total of 34 mason jars (32 oz.; 946 ml) were filled about full with the sample (wet) soil. Each jar was weighed once after the soil and beer trub treatments were added, so that the moisture content in each jar could be maintained during the duration of the experiment. 17 treatments were analyzed, and each treatment was duplicated (totalin g 34 mason jars). Treatments 1a and 1b were treated with 3% (w/w) water only as a lower control. Beer trub was then increased by 0.2% to the remaining 32 mason jars (i.e. Treatment 2a/2b = 0.2% trub, 3a/3b = 0.4% trub, 4a/4b = 0.6% trub, etc.). Treatment 1 7a and 17b were treated with 3.0% yeast extract (Fisher Scientific Inc., Atlanta, GA) as a control. The mason jars were clear glass and were kept covered by tin screw cap lids to limit light and moisture exposure when stored in an incubator at a constant t emperature of 25C. They were removed weekly and adjusted for evaporative loss of water. Samples were collected on a biweekly basis over a 4 month period The beer trub was obtained from Swamp Head Brewery in

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31 Gainesville, Fl. on 10/6/2011. The beer trub co Saccharomyces cerevisiae ) Labs Inc., Boulder, Co, USA). The yeast cultures we re characterized with the following properties: 73 80% attenuation, medium flocculation, optimum fermentation temperature of 68 73F, and a high alcohol tolerance (Wyeast Laboratories, Inc. 2012. Odell, OR, USA; White Labs Inc., Boulder, Co, USA). DDx Ex traction To analyze soils for DDx concentrations, soil samples had to undergo an accelerated solvent extraction (ASE) and a florisil cleanup. Both extraction methods were modified as indicated below from the procedures set forth in USEPA method #3545 (U.S. E.P.A., 2000) and in a previous study for the Dionex Accelerated Solvent Extractor 100 (Thomas et al., 2008). Accelerated Solvent Extraction (ASE) A 30 mm diameter cellulose filter with 45 m pores (Dionex Corporation, P/N 056780) was placed at the bott om of the 34 mL stainless steel extraction cell. About 1/3 of the cell was filled with clean Ottawa sand (20/30 mesh size). Next, 1.00 g Hydromatrix (Agilent Technologies, Santa Clara, CA, USA), a bulking and drying agent comprised of pelletized diatomaceo us earth, was added to the cell. This was followed by approximately 2 grams of wet soil from the NSRA of Lake Apopka that had been sieved through a 2 mm mesh screen after the exact weight to two decimal places was recorded. Lastly, enough clean Ottawa sand to fill the extraction cell was added and the capsule was closed to form a tight seal. The sample was extracted on a Dionex 100 Accelerated Solvent Extractor at 100 C and 1500 psi using 4:1 methylene chloride:

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32 acetone. Once the sample was extracted and f lushed into a clean ASE glass sample bottle, the samples were transferred to clean, labeled test tubes and stored at 4 C. Florisil Extraction Three mL of hexane: acetone (9:1 v/v) were pipetted into pre packed florisil rticle size) (Varian Inc., Palo Alto, CA) for column conditioning. The florisil clean meniscus touched the top of the Florisil column packing to keep column moist. Next, 2 mL of ASE extract sample was adde d to the florisil clean up column. ASE extract sample was then eluted with 9 mL of hexane: acetone (9:1 v/v) until the column was completely dry. Eight mL of the cleaned extract were removed, pipetted into cleaned and burned glass tubes. The 8 mL of cleane d extract was then air dried with a gentle flow of air so as not to splash the extract sample out of the glassware. Complete dryness must be achieved to remove acetone and residual methylene chloride. The dried residual was re dissolved with 0.5 mL hexane. It was necessary to remove all methylene chloride and acetone due to matrix response enhancement by these compounds (Schmeck and Wenclawiak, 2005). The re dissolved 0.5 mL solution was then pipetted into a 2 mL amber glass gas chromatograph (GC) vial (Fis her Scientific Inc., Atlanta, GA) and then crimped with a 12x32mm aluminum crimp seal with pre fitted PTFE lined septa (Fisher Scientific Inc., Atlanta, GA). The crimped GC vial containing the ASE and florisil extracted sample was then analyzed by GC. Gas Chromatography (GC) Conditions A Perkin Elmer Autosystem Gas Chromatograph (GC) equipped with an electron capture detector (ECD) and an autosampler were utilized in the analysis of the extracted samples. Our laboratory has established percent recovery lim its of 102.8 for

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33 o,p DDE, 88.7 for p,p DDE, 89.3 for o,p DDD, 74.6 for p,p DDD, 74.9 for o,p DDT, and 84.9 for p,p DDT. The following parameters were used in the GC analysis based on this less than 15% of error in consecutive injections were obtained): the GC column was a RTX CLPesticide (Restek, Inc. Bellefonte, PA ) column measuring 30 m x 0.32 mm ID Column conditions were: helium gas as the carrier at flow rate of 1.33 mL/min, nitrogen as the make up gas with a flow rate of 50 mL/min, electron capture detector (ECD) at 330 C, and injector temperature at 200 C in splitles volume in slow injection mode. The GC temperature program was: initial temperature 140 C for 1.0 minutes, ramp at 30 C/min to 240 C, then hold for 2.0 minutes. The DDE, 10.31 minut DDE, 10.56 DDT, and DDT. Under these conditions, the detection limit can be mL for o,p DDE, DDD, 0.039 DDT (Hubaux and Vos, 1970). Hence, the standard deviation. A four standard set was run in triplicate at the beginning and end of each GC run, with one standard set run after approximately 20 samples for quality control. An R 2 value of no less than 0.998 was achieved for every DDx compound stand ard for all three standard runs before and after sample analysis was conducted on the GC.

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34 Laccase Enzyme Assay Required solutions and sample p reparation The laccase enzyme assay was adapted from Elisashvili et al. (2008). It required a 50 mM Na Acetate s olution with pH adjusted to a pH of 4.0 with glacial acetic acid, an 2,2' azino bis(3 ethylbenzothiazoline 6 sulphonic acid) (ABTS) solution, and a 1:1 enzyme solution. The 1:1 enzyme solution was made with approximately 2 grams of soil and 2 mL of deioniz ed water. The solution was shaken for 5 minutes and then the liquid solution was poured into 2 mL centrifuge tubes. The tubes were centrifuged on an Eppendorf Centrifuge 5415 C (Eppendorf AG, Hamburg, Germany) for 8 minutes at 14rpm achieve maximum separat ion of enzyme solution and soil. Once centrifuged, the liquid enzyme solution was pipetted into cleaned and burned glass tubes and stored on ice. Laccase enzyme assay spectrophotometer m ethod The laccase enzyme assay was conducted with a Hitachi model # U 2000 Double Beam UV/Vis Spectrophotometer with a 5 cell changer autosampler (Hitachi Instruments, Inc., Danbury, CT). First, to a spectrophotometer cuvette, 0.650 mL of Na Acetate solution (50 mM) was added. Next, 100 L of the ABTS solution and 250 L o f the prepared enzyme solution prepared as above were added to the cuvette. The top of the cuvette was covered with parafilm and rotated vertically 5 times to ensure proper mixing. The cuvette with the mixed sample was placed in the Hitachi U 2000 Double B eam UV/Vis Spectrophotometer autosampler holder and sliding cover on the spectrophotometer was closed. Absorbance was measured at 418 nm every minute for 5 minutes at room temperature. The two absorbance readings with the greatest positive difference were used for enzyme activity calculations. A molar extinction coefficient of

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35 36,000 M 1 cm 1 was used in the enzyme activity calculations (Alcade and Butler, 2003). Calculations of laccase enzyme activity were also adapted from Alcade and Butler (2003). Perox idase Enzyme Assay Required solutions and sample p reparation The peroxidase enzyme assay was adapted from Tien and Kirk (1988). It required a 10 mM veratryl alcohol (3,4 Dimethoxybenzyl alcohol) solution, a 0.25 M d Tartaric Acid (pH 2.5) solution, a 10 mM H 2 O 2 solution, and the same 1:1 enzyme solution that was prepared for the laccase enzyme solution. Peroxidase enzyme assay spectrophotometer m ethod The peroxidase enzyme assays were performed on the same Hitachi model#U 2000 Double Beam UV/Vis Spectroph otometer with a 5 cell changer autosampler (Hitachi Instruments, Inc., Danbury, CT) that the laccase enzyme assays were conducted with. First, to a spectrophotometer cuvette, 200 L of the 10 mM veratryl alcohol solution was added. Then, 40 L of the 10 mM H 2 O 2 200 L of the 0.25 M d Tartaric Acid, 600 L of deionized water, and 250 L of enzyme solution were added to the cuvette. The top of the cuvette was covered with parafilm and rotated vertically 5 times to ensure proper mixing. Next, the cuvette with the mixed sample was placed in the Hitachi U 2000 Double Beam UV/Vis Spectrophotometer autosampler holder and the sliding cover on the spectrophotometer was closed. Absorbance was measured at 310 nm every minute for 5 minutes at room temperature. The two absorbance readings with the greatest positive difference were used for enzyme activity calculations. Differing from the molar extinction coefficient value of 36,000 M 1 cm 1 used in the enzyme activity calculations for the laccase enzyme assay method, a m olar extinction coefficient

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36 of 9,300 M 1 cm 1 was used in the peroxidase enzyme activity calculations (Tien and Kirk, 1988). Calculations of peroxidase enzyme activity were also adapted from Tien and Kirk (1988). Statistical Analysis Statistical compari sons were conducted using one way ANOVA analysis on SAS Range (HSD) Test for yield. Values with p<0.05 were considered significantly different and variables in the graphs not notated by the same letter are significantly different. Results and Discussion Based on results produced by previous experiments on substrate addition to DDx contaminated soil to enhance DDx degradation (Thomas, 2011), this research project tested the de gradative effects of adding beer trub to contaminated soil. Beer trub was chosen because it is a cost effective source of micronutrients and yeast, which has been found to enhance extracel lular enzyme production (Hou et al., 2004). Though beer trub was hyp othesized to enhance the production of extracellular enzymes, and, therefore, enhance degradation of DDx, it was unknown what amount of beer trub would be most effective in enhancing the degradative process. Gas Chromatography Analysis of DDx in Microcosm Study The microcosm study was designed to test 16 incremental concentrations of beer trub added to each 32 oz. mason jar containing contaminated soil, plus one additional treatment consisting of 3.00% (vol/weight) yeast extract. The first treatment out of the 16 treatments was a negative control consisting of only tap water. Each treatment was duplicated. The data from this study suggested that from 0.00% to 1.8% of beer trub, DDT levels decreased. To verify this decrease, statistical analysis was conducte d using

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37 statistical tests showed that 1.8 % trub was significantly different from treatments 0.00% trub to 1.4% trub. However, beyond 1.8% trub, DDT levels were not reduced as effec tively (Figure 2 1). This suggested that there was some inhibitory factor that prohibited continued DDT degradation past 1.8% beer trub. Based on other work, it was hypothesized that one factor that may have inhibited continued degradation of DDT was an in crease in nitrate from the increase in beer trub (Wang and Broadbent, 1973; Smith, 1992). On day zero of the experiment, the highest DDE concentrations were 0.06 0.08 g/g dry soil, and these values were found in treatments 1.2 1.8% beer trub. At the end of the experiment, the majority of the treatments showed an increase in DDE concentrations (Figure 2 2). This was expected due to DDE being a degradation product of DDT (ATSDR, 2002). The generally elevated concentration values of DDE on the final day of t he experiment suggest that DDT degradation did occur, and that a longer testing period would be required if complete DDx degradation was desired. The treatment of 1.8 % beer trub, however, showed almost complete degradation of DDT and DDE, providing furthe r evidence that 1.8% beer trub was more effective in enhancing the degradative process of DDx. Laccase and Peroxidase Enzyme Activities Laccase and peroxidase enzyme assays were conducted throughout the experiment to monitor enzyme activities in the soil. The hypothesis was that the addition of beer trub would enhance enzyme production, thus enhancing the degradation of DDx. The laccase data shows a general trend of laccase activity (units of enzyme per L of enzyme solution needed to convert one Mol in on e minute) increasing over time.

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38 The most notable increase can be found in the 1.8% beer trub treatment, where the Day 12 laccase activity is zero, the Day 44 activity shows an increase, and then the enzyme activity had greatly increased by Day 58. This obs DDT degradation aspect of the microcosm study in that the 1.8% beer trub treatment DDT degradation. As stated earlier, the extracellular enzymes (laccase and peroxidase) are capable of degra ding DDT, and therefore a higher level of enzyme activity, especially laccase activity, should be found in the treatment most suited to enhance DDT degradation. The peroxidase results show similar trends with a consistent increase in peroxidase enzyme acti vity not observed until treatment 10, which is the 1.8% beer trub treatment (Figure 2 4). Conclusions significant organic matter content over the past decades. Multiple remediati on strategies have been studied in hopes of finding an efficient means of degrading DDT and restoring contaminated soil. This research tested the feasibility of adding beer trub to DDx contaminated soil to enhance DDx degradation via enhancement of extrace llular enzyme production from native fungi, which have of the capacity to degrade DDT (Hou et al ., 2004; Bonnen et al 1994). This study found that 1.8% beer trub was most effective in enhancing enzyme production as well as enhancing the degradative proce ss of DDx in a microcosm setting. It also found that past 1.8% beer trub addition, DDT levels were not as effectively lowered, suggesting the presence of an inhibiting factor that prevented efficient DDT degradation. Further studies will be conducted to te st if nitrate is the possible inhibitory factor in these treatments, as is expected from reviewing literature on this issue (Wang and Broadbent, 1973).

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39 Figure 2 DDT (g/g dry soil) i n selected microcosm treatments. Letters represent statistical differences b etween treatments. Treatments represented by the same letter denotes statistically similar res ults, whereas treatments represented by different letters denotes statistical differe nces.

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40 Figure 2 DDE concentrations in selected microcosm treatments sp anning duration of experiment.

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41 Figure 2 3. Laccase enzyme activity (units enzyme per L enzym e solu tion needed to convert 1 Mol in one minute) for all treatments over duration of micr ocosm experiment. Each consecutive treatment represents a 0.2% increase in beer trub, with treatment 1 containing 0% beer trub and treatment treatment 16 containing the most amount of beer trub at 3%. Treatment 17 represents a 3% yeast extract t reatment. Percentages of treatment added are based on vol/weight ratio.

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42 Figure 2 4. Peroxidase enzyme activity (units enzyme per L enzyme solution needed to convert 1 Mol in one minute) for all treatments over duration of mi crocosm experiment. Each consecutive treatment represents a 0.2% increase in beer trub, w ith treatment 1 containing 0% beer trub and treatment treatment 16 conta ining the most amoun t of beer trub at 3%. Treatment 17 represents a 3% yeast extra ct treatment. Percentages of treatment added are based on vol/weight ratio.

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43 CHAPTER 3 ANALYSIS OF DDX DEGRADATION, BIOMASS, AND ENZYME ACTIVITY IN MESOCOSM STUDY Exist ing literature discussing bioremediation of DDT points out that the main flaw with bioremediation methods is that there are no naturally occurring organisms that can effectively degrade DDT (Mitra et al., 2001). However, as discovered by previous experimen ts from our lab, naturally occurring wood rot fungi, which produce enzymes capable of DDT degradation, do exist in the soil from our study site at the Lake Apopka North Shore Restoration Area. As indicated by Hou et al. (2003), however, the enzymes that ar e involved in the natural degradation of DDT, specifically laccase enzymes, are produced via secondary metabolic processes in basidiomycete fungi which greatly limit the amount of enzymes produced. Therefore, this research seeks to find a cost effective wa y of enhancing enzyme production from naturally occurring organisms in the soil. The previous microcosm study found that beer trub, the leftover yeast sludge from the beer brewing process, was a possible source of enhancement of the aforementioned extrace llular enzymes needed in the DDT degradation process. As is with the case of all lab experiments, there is a lack of certainty in whether or not the researched remediation method is applicable to a larger scale scenario. Financial cost of implementing the remediation method as well as the expected and unexpected environmental impacts are important factors to keep in mind when carrying out large scale experiments. The previous microcosm study showed that more than a 1.8% application of beer trub, produced a diminishing efficiency in DDT breakdown, which supports the idea of a inhibitory factor being present in the degradation process. Literature supports this idea in that nitrate has been found to be a limiting factor in the degradation of DDT (Wang et al., 1 973). As far as financial costs are concerned, the

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44 approximately 4 gallons of beer trub that was collected for this research was free, which greatly reduces application costs. Since beer trub is a brewery waste product, other landowners seeking to utilize this remediation method could pick up the beer trub free of charge as well. In an effort to study the potential environmental impacts of the application of beer trub to a contaminated land, leachate studies were also conducted. Materials and Methods Soil The soil that was used in this study was collected from the same plot of land used in the previous microcosm study (the North Shore Restoration Area (NSRA) at Lake Apopka, FL). The soil for this experiment was collected on July 20, 2012. The soil collected for this time was handled exactly like the handling of the collected soil for the microcosm experiment. For further information regarding the soil type and preparation sec tion. Mesocosm The mesocosm study was assembled in a greenhouse at the University of Florida in Gainesville, FL. The experimental design consisted of filling nine 5 gallon buckets with approximately nine centimeters of small stones and gravel, and then o n top of the rocks, adding approximately 23 centimeters of DDT contaminated soil from the NSRA of Lake Apopka sample site. This amount of soil and rocks was based on bulk density measurements taken at the NSRA study site. Bulk density was determined by tak ing four soil cores at the study site. The tool used to take the soil cores had a predetermined volume and the soil that was collected with the coring tool was taken sealed in plastic ziplock bags and taken back to our lab at the University of Florida in

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45 G ainesville, FL where the soil was dried overnight in an oven set to 65 C and then weighed. The bulk density equated to 1.23 g/cc. Before the soil was placed in each experiment bucket, the soil had been sieved and homogenized. Prior to filling the buckets with rocks and soil, each treatment bucket had holes drilled into the bottom of it using a 15/64 inch (0.6 cm) drill bit. The holes were spaced 0.5 inches (1.27 cm) apart, and this pattern of drilling the holes extended over the entire surface of the bott om of each bucket. This allowed leachate to percolate through the bucket and prevented the soil conditions in each treatment bucket from becoming anaerobic. Once each treatment bucket was filled with rocks and the study site soil as described above, each t reatment bucket was placed inside another 5 gallon bucket that served as a leachate collection container. Every leachate collection bucket had a half inch diameter hole drilled into the bottom of it, to allow leachate to be collected from the bottom of the bucket. When leachate collection was not being conducted, the holes at the bottom of each leachate bucket were closed with a rubber stopper to prevent the leachate from escaping the catchment containers. O f the nine buckets, three served as negative contr ols and were treated with 3.0 % (v/w) tap water, based on wet weight of soil in the buckets. The 3.0 % value was determined by calculating the average percent moisture of our soil, which was 10.79 %. Next, the weighted average of the available water capaci ty was determined to be 13 % ( websoilsurvey.nrcs.usda.gov ) The difference of the weighted average of the available water capacity and the average percent moisture of our soil was then calculated, giving us the amount of liquid solution we were able to add to the soil before field capacity was reached. Another three buckets were treated with a beer trub/water solution totaling 3.0

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46 % of wet weight of soil in buckets. Of that 3.0 % beer trub/water solution, 1.8% of it was beer trub, and the remaining 1.2% of it was water. The remaining three buckets were treated with a 3.0% (v/w) beer trub only solution based on wet weight of soil in the buckets. The treatments were labeled A 3.0% beer trub treatment. Each replicate for the respective treatment was numbered 1 3; i.e. treatment B 2 signified the second replicate of the 1.8% beer trub treatment. A third number at the end of the sampl e label signified the collection date, i.e. C 1 4 denoted a sample from the first replicate from the treatment with 3.0% beer trub, taken on the fourth sampling day. Each treatment was mixed in a cement mixer for five minutes, with periodic scraping of the inside walls of the cement mixer to ensure the added liquid solutions were completely mixed in with the soil. All sampling began on August 6, 2012. Soil samples were collected bi weekly and leachate samples were collected on a daily basis up until Septemb er 11, 2012, when the leachate collection schedule changed to collecting every other day due to a decrease in the flow rate of the water through the buckets from soil compaction and possible uptake by increased biomass in the soil. The leachate was weighed each day of collection in order to determine the volume of water that was passing through the soil profile. The value for the volume of water leached through the soil profile was used in determining the concentration of nitrate that was percolating throug h the soil. After each time the leachate was collected from the catchment buckets, new water was applied to the soil. In order to maintain as close to the same level of soil moisture as possible in the buckets, a ratio of approximately 19.28 grams of wet s oil per 1 ml of water was

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47 continued throughout the duration of the project. This ratio was determined in the beginning of the experiment when 1 liter of water was applied to each bucket, which contained approximately 19,277.68 grams of soil. Due to limited resources and manpower, leachate was only analyzed on a weekly basis. Leachate samples were analyzed for nitrate concentrations, enzyme activity, and DDx concentrations in the water. The purpose of the leachate analysis for these three factors was three f old. First, the nitrate level in the leachate was analyzed to see if the beer trub would cause a large amount of nitrate to leach through the soil profile and potentially end up in the ground water. This is a concern because of the eutrophication problems that Lake Apopka has experienced. The second reason for analyzing the leachate was to determine if a substantial amount of the enzymes that were involved in the DDx degradation process were leaching through the soil profile. If this was found to be so, the n the leachate could serve as a source for these valued enzymes and could be recycled. Lastly, the leachate was analyzed for any DDx compounds that may have eluted from the soil. It was hypothesized that there would not be a significant amount of DDx compo unds in the contain appreciable concentrations of the DDx compounds, it would support the argument that the leachate should be recycled through the soil profile so that the DDx compounds could continue their degradation process. Soil temperature readings taken at depths of approximately 15 cm were recorded on every sample day. The beer trub used in this experiment was donated by Swamp Head Brewery in Gainesville, Fl. o

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48 following properties: 73 80% attenuation, medium flocculation, optimum fermentation temperature of 68 73F, and a high alcohol tolerance (Wyeast Laboratories, Inc. 2012. Odell, OR, USA; White Labs Inc., Boulder, Co, USA). The trub had a pH of 4.9 and approximately 14 % dry soli ds. DDx Extraction for Soil Samples Mirroring DDx extraction procedures from the previous chapter, soil samples underwent an accelerated solvent extraction (ASE) method as well as a florisil cleanup method. The ASE method as well as the florisil cleanup me thod was modified from the procedures set forth in USEPA method #3545 (U.S.E.P.A., 2000) and in a previous study for the Dionex Accelerated Solvent Extractor 100 (Thomas et al., 2008). Accelerated Solvent Extraction (ASE) of Soil Samples for Gas Chromato graphy ASE method. The procedure began by applying a 45 m pore 30mm diameter filter (Dionex Corporation, P/N 056780) into a 34 mL stainless steel extraction cell, so that the 45 m filter is completely flush with the bottom of the extraction cell. Clean Ottawa sand is poured into the extraction cell, filling about 1/3 of the cell. Next, 1.00 g of a bulking and drying agent comprised of pelletized diatomaceous earth called Hydromat rix (Agilent Technologies, Santa Clara, CA, USA) was added on top of the Ottawa sand in the extraction cell. Following the addition of the Hydromatrix, approximately 2 g of wet soil from the study site in Lake Apopka was added to the cell. Before the soil was added to the cell, the soil was weighed and recorded. The Hydromatrix and the soil were mixed so as to enable as much soil to come in contact with the Hydromatrix as possible. The rest of the extraction cell was then filled with

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49 more Ottawa sand, and t hen the lid of the cell was screwed on tight to ensure pressure would be retained in the cell during extraction. The cell was placed on a Dionex 100 Accelerated Solvent Extractor at 100 C and 1500 psi using 4:1(v:v) methylene chloride: acetone. Once the s ample was extracted and flushed into a clean ASE glass sample bottle, the sample was transferred to a clean test tube, labeled, and placed at 4 C for storage. Florisil Extraction for Soil Sample Extracts As stated above, the soil samples were extracted i n a 4:1(v:v) methylene chloride: acetone solution. In order to avoid matrix response enhancement with the samples on the gas chromatograph (Schmeck and Wenclawiak, 2005), the samples had to be flushed through a pre (200 m particle size) (Varian Inc., Palo Alto, CA). To ensure the compounds in question that were in the ASE samples were transferred through the florisil column, 3 mL of hexane: acetone (9:1 v/v) was first pipetted into the florisil column and allowed to percolate through the column until the meniscus of the hexane: acetone (9:1 v/v) reached the base of the column. 2 mL of the ASE sample was then added to the column. The added sample was then flushed with a total of 9 mL hexane: acetone (9:1 v/v) to ensur e all of the compounds in question had been flushed through the florisil column. Once the samples had been filtered through the florisil columns, 8 mL of the filtered sample was pipetted into a 20 mL test tube and air dried to concentrate the sample for GC analysis. Once the sample was air dried, 500 L of hexane was added to the tube. The hexane was rinsed throughout the test tube containing the dried sample to ensure all the DDx that remained on the glass of the test tube was mixed into the hexane. The sa mple solution was then transferred into a 2 mL amber glass gas

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50 chromatograph (GC) vial (Fisher Scientific Inc., Atlanta, GA) and then crimped with a 12x32mm aluminum crimp seal with pre fitted PTFE lined septa (Fisher Scientific Inc., Atlanta, GA). The sea led GC vial containing the sample was then placed on the GC autosampler rack and analyzed by the GC. Liquid leachate samples were cleaned and treated the exact same way, except that they did not need to undergo the ASE extraction step and methylene chlori de was used instead of hexane:acetone for florisil cleanup per USEPA method #3545 (U.S.E.P.A., 2000) Gas Chromatography (GC) Conditions for Soil and Leachate Samples The prepared samples were placed on the autosampler wrack on the GC and an electron captur e dector (ECD) was used to analyze the samples on the GC. Percent recovery limits of 102.8 for o,p DDE, 88.7 for p,p DDE, 89.3 for o,p DDD, 74.6 for p,p DDD, 74.9 for o,p DDT, and 84.9 for p,p DDT had been predetermined by our lab. A RTX CLPesticide (Reste k, Inc.) column was used for this analysis since this type of was helium, flowing at a rate of 1.33 mL/min, and nitrogen was the make up gas with a flow rate of 50 mL/min. The ECD temperature was set at 330 C, and the injector injection mode. The GC temperature p rogram was set so that for the first minute, the initial temperature was 140 C. Then, the temperature was ramped up at a rate of 30 C/min until a temperature of 240 C was reached. That temperature of 240 C was held for 2.0 minutes. The retention times DDE, DDD, DDT. In calculating the

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51 detection limit, we determined using a 90% con fidence level that the detection limit for DDT DDT (Hubaux and Vos, 1970). Th erefore, the average detection limit value equated to be is 0.29 with a 0.10 standard deviation for DDx in of standards were run before and after the samples were run, with a stand set run approximately every 20 samples for quality control. Each sample was run in triplicate in order to increase accuracy of the results. An R 2 value of no less than 0.998 was achieved for every DDx compound standard for all three standard runs before and after sample analysis was conducted on the GC. Laccase Enzyme Assay As discussed in the previous chapter, the laccase enzyme assay was modified from an assay developed by Elisashvili et al. (2008). The components of this enzyme assay were a 50 mM Na Acetate solution with pH adjusted to a pH of 4.0 with glacial acetic acid, an 2,2' azino bis(3 ethylbenzothiazoline 6 sulphonic acid) (ABTS) solution, and a 1:1 enzyme solution. The 1:1 enzyme solution was made by mixing approximately 2 grams of soil and 2 mL of deionized water in a 20 mL test tube. The test tube was shaken for 5 minutes. Once the solution had been shaken adequately, the liquid solution was poured into 2 mL centrifuge tubes. The 2 mL centrifuge tubes containing the enzyme solutio n were then placed on an Eppendorf Centrifuge 5415 C (Eppendorf AG, Hamburg, Germany) for 8 minutes 14 rpm. Once centrifuging was complete, the enzyme solution was pipetted to cleaned and burned test tubes and stored on ice to

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52 activity to discontinue before the solution could be analyzed. Once the samples were ready to be analyzed, 0.650 mL of Na Acetate solution (50 mM) was added to the spectrophotometer cuvette. Next, 100 L of the ABTS solution and 250 L of the prepared en zyme solution mentioned above were added to the cuvette. Parafilm was then placed on the top of the cuvette to prevent the sample to spill, as the cuvette was rotated vertically five times to allow the sample and the reagents to properly mix for analysis. The sample containing cuvette was then placed in compartment was closed to prevent light infiltration. The spectrophotometer that was used for this analysis was a Hitachi U 2000 Doubl e Beam UV/Vis Spectrophotometer with a 5 cell changer autosampler (Hitachi Instruments, Inc., Danbury, CT). Samples were analyzed in the spectrophotometer at an absorbance level of 418 nm every minute for 5 minutes at room temperature. The two absorbance r eadings with the greatest positive difference were used for enzyme activity calculations. Adhering to Alcade and Butler (2003), the molar extinction coefficient value of 36,000 M 1 cm 1 was used to determine laccase enzyme activity. Peroxidase Enzyme Assa y The second enzyme assayed was peroxidase. For the peroxidase assay, the method of Tien and Kirk (1988) was modified. We used a 10 mM veratryl alcohol (3,4 Dimethoxybenzyl alcohol) solution, a 0.25 M d Tartaric Acid (pH 2.5) solution, a 10 mM H 2 O 2 soluti on, and the same 1:1 enzyme solution that was prepared for the laccase enzyme solution. The recipe for making the peroxidase sample is as follows: add 200 L of the 10 mM veratryl alcohol solution, 40 L of the 10 mM H 2 O 2 200 L of the 0.25

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53 M d tartaric acid, 600 L of deionized water, and 250 L of enzyme solution to the spectrophotometer cuvette. Similarly to the laccase method, once the cuvette is filled with the proper ingredients, the cuvette was covered with parafilm and the cuvette was vertically r otated five times to ensure proper mixing. The cuvette with the mixed sample was then placed in the autosampler compartment and the compartment sliding door was closed. The peroxidase assay was conducted on the same Hitachi U 2000 Double Beam UV/Vis Spectr ophotometer with a 5 cell changer autosampler (Hitachi Instruments, Inc., Danbury, CT) that the laccase enzyme assay was conducted on. The sample was analyzed in the spectrophotometer with an absorbance value of 310 nm every minute for 5 minutes at room te mperature. Mirroring the laccase assay method, the calculation of the peroxidase enzyme activity involved using the two absorbance readings with the greatest positive difference for enzyme activity calculations. Differing from the laccase enzyme activity c alculation method however, a molar extinction coefficient of 9,300 M 1 cm 1 was used in the peroxidase enzyme activity calculations (Tien and Kirk, 1988). Ergosterol Extraction for High Performance Liquid Chromatography (HPLC) Analysis One hypotheses of th is experiment is that with an increase rate in beer trub application to contaminated soil, fungal biomass will increase. In order to measure biomass, ergosterol concentrations in samples were analyzed. Ergosterol is a fungal sterol that only occurs in fung al species and some microalgae, and can be analyzed to determine relative fungal biomass (Djajakirana, et al., 1996). The method used for measuring biomass in this study was derived from the HPLC method in Djajakirana, et al. (1996). To quantify ergosterol concentration in the soil samples, samples needed to

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54 undergo an Accelerated Solvent Extraction (ASE) ergosterol extraction method for High Performance Liquid Chromatography (HPLC) analysis. The ASE method was modified from the procedures set forth in USEP A method #3545 (U.S.E.P.A., 2000). Accelerated Solvent Extraction (ASE) for HPLC Analysis First, a 45 m pore 30mm diameter filter (Dionex Corporation, P/N 056780) was placed at the bottom of the 34 mL stainless steel extraction cell. About 1/3 of the ce ll was then filled with clean Ottawa sand (Fisher Scientific Inc., Atlanta, GA). Next, 1.00 g Hydromatrix (a bulking and drying agent comprised of pelletized diatomaceous earth) was added to the cell. It was followed by approximately 2 grams of wet soil fr om the NSRA of Lake Apopka that had been sieved through a 2 mm mesh screen after the exact weight to two significant figures was recorded. Lastly, enough clean Ottawa sand to fill the extraction cell was added and the capsule was closed to form a tight sea l. The sample was extracted on a Dionex 100 Accelerated Solvent Extractor at 100 C and 1500 psi using methanol. Once the sample was extracted and flushed into a clean ASE glass sample bottle, the samples were transferred to clean, labeled test tubes and s tored at 4 C. HPLC Analysis of Ergosterol Following ASE extraction, the samples were run through HPLC for quantification of ergosterol concentration in the soil samples. HPLC parameters were main column 12.5 cm Spherisorb ODS II S5 (Knauer Vertex 12 cm m ain column,0.5 cm pre column) with mobile phase 91 % methanol and 9% water (v/v) at a flow rate 1.0 ml per min. with UV/ Visible wavelength detector set at 282 nm. A constant room temperature of 25 C was maintained. Retention time of ergosterol was around 6.3 min. The detection limit for ergosterol for this system was 0.37 g/ml (Hubaux, 1970; Lyman, 1977). A 1,000 g/ml

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55 stock standard solution of ergosterol (Sigma E 6510) was prepared in bidistilled ethanol (96%, denaturized). Nitrate Nitrogen Analysis On ce soil and leachate samples were collected, they were stored in a 4 C refrigerator for no more than 4 weeks before nitrate analysis was conducted. Denitrification may have occurred due to lag time between sample collection and analysis. Soil and leachat e samples were sent to the UF/IFAS Analytical Services Laboratories Soil Testing Lab at the University of Florida in Gainesville, FL for nitrate nitrogen analysis. The Soil Testing Lab follows EPA method 353.2 for nitrate analysis (U.S.E.P.A., 1993). Orga nic Matter Analysis A few samples representative of the study site were sent to the UF/IFAS Analytical Services Laboratories Soil Testing Lab at the University of Florida in Gainesville, FL for organic matter analysis via Loss on Ignition (ASTM, 2000). Statistical Analysis In an effort to determine statistically significance and differences in the results Studentized Range (HSD) Test for Yield was used as control treatments w ere tested against the variable treatments, as values of p<0.05 were considered significantly different. Treatments presented in the graphs depicting results that are represented by the same alphabetical letter denote statistical similarity, while treatmen ts that are represented by different letters signify treatments that are statistically different.

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56 Results and Discussion Mesocosm DDx Degradation in Soil DDT concentrations were similar, reporting concentr ations close to 1.5 g/g dry soil. As the mesocosm experiment continued, the following observations were made. As expected, the Water Control treatment which consisted of 3 % water only (volume/weight) remained nearly constant DDT concentr ation. The 1.8 % beer trub treatment, which was the most DDT DDT degradation, beginning noticeably around day 49 of DDT degradation con tinued gradually until reaching a concentration of approximately 0.2 g/g dry soil. The 3 % beer trub treatment reported a DDT concentration between the 65 day and the 80 day mark, DDT concentration of approximat ely 1.3 g/g dry soil around day 65 and DDT of close to 0.5 g/g dry soil by about day 80 (Figure 3 1). However, due to the lack of precision in analyzing for DDx compounds, none of the treatments are statistically different. Though there are no statistical differences, it still appears that there are decreases in DDT concentrations in th e various treatments with time. DDT concentration in the soil during the mesocosm study, a few deduct ions can be made. The first supports the findings from the previous microcosm study. The 1.8 % beer trub treatment does appear to have DDT. However, this mesocosm study showed that the 3 % beer trub treatment also influenced p, DDT degradation. The difference between

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57 DDT degradation began. The 1.8 % beer trub treatment seemed to effect degradation rates earlier, approximately around the 30 day mark whereas the 3 % beer trub treatment took longer to effect degradation rates by not enhancing degradation until past the 65 day mark. However, once degradation occured in the 3 % beer trub treatment, degradation rates were higher than the 1.8% trub treatm ent. DDT in DDE concentrations for the DDE is a byproduct of p DDE around the time that p,p DDE concentration in the Water Control remained essentially constant. The 1.8 % beer trub treatme nt exhibited p,p DDE concentrations at a constant level until around day 35 where they increased from around 0.7 g/g dry soil to approximately 1 g/g dry soil. DDE can be seen in Figure 3 2 to decrease steadily until it reaches a concen tration of about 0.1 g/g dry soil by day 92. Statistically speaking, the 1.8% trub treatment is not completely different from the Water Control treatment and the 3% trub treatment. The 3 % beer trub treatment reported similar degradation patterns for p,p DDE as it did for the p,p DDT. The p,p DDE concentrations remained around 0.9 g/g dry soil DDE concentrations were found to be around 0.4 g/g dry soil. DDE was undetectable in the soil

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58 by day 93 (Figure 3 2). The 3% trub treatment was statistically less than the Water DDE was degraded in the 3% treatment than in the Water Control. Similar to p,p DDT degradation rates the 1.8 % beer trub treatment and the 3 % beer trub treatment both showed the capability of enhancing p,p DDT degradation, although the 3 % beer trub took longer to effect degradation rates. Though the 3 % beer trub treatment took longer, it showed a mor DDE than the DDE is assumed to be due to more extracellular enzymes acting upon the DDE as a result of more fungal growth production in the 3% trub treatments. With more nutrients available from the added beer trub, fungal growth was able to increase, thus increasing the amount of extracellular enzymes produced once the need for enzyme production was realized. Since the beer trub also has been found to secrete extrace llular enzymes, it is also likely that complete DDE degradation was due to the enzyme production of the trub (Jadhav et al., 2007). The soil from the study site contained approximately 8.52 % organic matter. Mesocosm DDx Degradation in Leachate The mesocos m study was not only expanded in scale from the microcosm study, but also in the number of medias that were studied. The leachate that resulted from the beer trub/water mixture applications had to be analyzed to determine if the leachate posed a potential DDT DDT that were leached through the soil in the beginning of the study were low, ranging in concentrations from 0.00 2 to 0.007 g/ml. However, leachate that was analyzed for day DDT concentrations in the

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59 DDT in the leachate, with a concentratio n around 0.04 g/ml. The 1.8 % beer trub treatment DDT concentration of approximately 0.006 g/ml and the 3 % beer trub treatment indicated a concentration of 0.01 g/ml (Figure 3 3). Day 106 leachate DDT levels had decreased to around the 0.006 g/ml value. DDT concentrations in the leachate, the 3% trub treatment reported statistically less DDT in the leachate on the first and last sampling event. This is expected to be due to t he increased beer trub causing a more tortuous flow path for the first sampling day, thus limiting the time the DDT can leach out, and more degradation of DDT in the 3% treatment over time, thus causing less DDT to be able to leach through the soil. The o DDE in the leachate was the water control DDE concentrations occurred on day 49 (Figure 3 4). Although, statistically speaking, the concentrations reported on day 49 are not statistica lly different from the other sampling times due to the large error bar. However, DDE in the water control treatment, it is reasonable because DDE is more water sol DDT (Keil et al., 1969) and there was a lack of enzymes being produced around day 49 (Figure 3 5 and Figure 3 6) to completely degrade the small amount of DDT that had been naturally degraded in the water control treatment (Figure 3 1 and Figure 3 DDE was therefore free to wash through the soil profile DDT degradation.

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60 Mesocosm Soil Enzyme Activity The purpose of the addition of the beer trub was not solely to increase fun gal biomass by providing nutrients for fungal growth, but the trub also was expected to increase enzyme activity itself (Jadhav et al., 2007) and, consequentially, have a positive effect on the degradation of DDT. To provide further evidence for this study that the beer trub does augment fungal enzyme production, laccase and peroxidase enzyme assays were conducted. The laccase assays conducted during the mesocosm experiment reported low levels of enzyme activity in the beginning of the experiment. Then, ar ound day 52, enzyme activity levels significantly increase in the treatments, reporting values of up to 1.5, 2.0, and approximately 3.25 for the water control, 1.8 % beer trub treatment, and 3 % beer trub treatment respectively. After day 52, enzyme activi ty levels did not fall below 1.00 for the rest of the experiment. On day 52, the 3 % beer trub treatment resulted in the highest amount of laccase enzyme activity out of the three treatments. By day 65, the 1.8 % beer trub treatment indicated the highest a mount of laccase enzyme activity, and the remaining two sampling days reported paralleled enzyme activity values (Figure 3 5). These reported values of laccase enzyme activity generally align with the DDx degradation that occurred during the same time of t he experiment (Figure 3 1 and Figure 3 2). The peroxidase assays reported similar findings where data showed a significant spike of enzyme activity on day 52. Three things to note about the peroxidase assay data (Figure 3 DDT around day 52 of the study (Figure 3 DDE

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61 (Figure 3 DDT c oncentration value of the 1.8% beer trub towards the end of the study in relation to the other treatments (Figure 3 1). First, more peroxidase activity was reported in the 1.8 % beer trub treatment around day 52 compared to the others. Around day 50 is al DDT degradation began to be consistently observed during the study. Secondly, the peroxidase significantly spiked in the 3 % beer trub treatment around day 62, which is when the 3 % beer trub treatments DDT degradation. Thirdly, at the end of the study the peroxidase activity is either equal or greater in the 1.8% beer trub treatment compared to the other treatments, showing that there is still identifiable peroxidase activity occurring in the beer trub treat DDT concentration is smaller in the 1.8% beer trub treatment than the other treatments. It can be deduced from this that the 1.8% beer trub treatment contained more laccase and peroxidase activity towards DDT to continue to be degraded at the end of the study. Mesocosm Leachate Enzyme Activity Another aspect of analyzing the leachate collected during the mesocosm study is to discern whether or not the leachate contai ns enough active extracellular enzymes to be recycled back through the soil profile so that the extracellular enzymes in the leachate can continue degrading the remaining DDx in the soil, being that complete degradation of DDx has not already occurred. Lac case and peroxidase were analyzed in the leachate because, as stated previously, they have been found to be capable of degrading DDT (Hou et al., 2004). The laccase activity in the leachate on day 7 for the 3 % beer trub treatment was approximately 0.3. T he other treatments did not show any enzyme activity. Laccase

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62 activity was identified in the leachate for both the beer trub samples on day 32 and for the remainder of the experiment. The highest value of laccase activity reported by any of the treatments is approximately 0.4, compared to the highest laccase enzyme activity value of approximately 3.3 for the soil samples. The highest value for laccase enzyme activity of 0.4 falls around the value of the laccase enzyme activity in the soil samples before p,p DDT degradation had begun consistently (Figure 3 7 and Figure 3 5). However, none of the treatments were statistically different, thus showing a lack of significant increase in laccase activity in the leachate throughout the experiment. Therefore, since the leachate laccase enzyme activity value was not at a level found to DDT degradation, the leachate does not need to be recycled solely for the laccase activity in the leachate. The peroxidase enzyme ac tivity in the leachate reveals a different finding. The enzyme activity values reported at the end of the study for all the treatments range from 0.8 to approximately 1 enzyme activity unit (Figure 3 8). These values of leachate resemble peroxidase enzyme activity values found in the soil samples during the time DDT degradation began to occur more consistently. For example, around day DDT degradation was being observed, peroxidase enzyme activity in soil samples were reporting activit y values of 0.8. Peroxidase assays reported a significant increase in peroxidase activity around day 53 and then again on the last three sampling events. This shows that there was a significant increase in peroxidase activity in the leachate as time went o n. Due to this finding, it could be reasonable for leachate to be recycled through the soil profile, if only for the sake of reemploying the peroxidase DDT in the soil.

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63 Mesocosm Soil Ergosterol C oncentrations One of the indicators that the beer trub had increased fungal growth in the soil, and subsequently enhanced extracellular enzyme production is the presence of ergosterol, a fungal sterol that can indicate fungal biomass, in the soil (Djajakir ana, et al., 1996). Therefore, an increase in fungal biomass theoretically should have been DDT. The data shows low levels of ergosterol during the beginning of the experiment until day 63, where the ergosterol conten t in the treatments significantly increases especially in the 3% trub treatment (Figure 3 9). As time went on, all treatments showed a significant increase in ergosterol concentrations. Nitrate Concentrations in Leachate and Soil From the results of the microcosm study, we hypothesized that the low DDT degredation activity of the 3 % beer trub treatment was due to some inhibitory factor. Based on literature, it was assumed that nitrate was the inhibitory factor (Wang et al., 1973) and that because the 1.8 % beer trub treatment did not contain as much nitrate it DDT degradation data shows different results and suggests that microbiological processes played a larger role in the lag time of DDT degradation in the 3 % beer trub treatment. The nitrate data for the mesocosm study (Figure 3 10, Figure 3 11, and 3 12) suggest other hypotheses. Figure 3 10 shows a significantly higher amount of nitrate in the leachate at the first samp ling time. This is thought to either be high nitrate levels due to the combination of beer trub washing through the soil profile and the formation of nitrate via nitrification. Figure 3 11 shows statistically lower nitrate levels in the beginning of the mesocosm experiment, with nitrate levels in the leachate significantly increasing around

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64 day 53. More specifically, the 1.8% beer trub treatment showed higher nitrate values than the other two treatments. This seems questionable at first because it would s eem that the treatment with the highest concentration of beer trub would contain the highest nitrate concentration. However, the ammonification of proteins produced by the isolated fungus in the soil (to be discussed in more detail in Ch. 4) is capable of indirectly producing nitrates in the soil. Since the beer trub treatments reports significant increases in fungal biomass towards the end of the experiment, it is reasonable that there would be an increase in proteins produced by the increase in fungi, and therefore an increase in nitrate caused by the nitrification of ammonia in the soil. Paralleling this increase in leached nitrate towards the end of the experiment, it would stand to reason that with an increase in water soluble nitrate towards the end of the experiment, there would be a decrease in nitrate levels in the soil at the end of the study. As shown in Figure 3 12, this is indeed what occurred. The nitrate levels in the soil significantly decreased around the same time (around day 50) that nitrat e levels in the leachate were reported. Also, it is expected that a large portion of the nitrate in the soil was taken up by the fungal growth which had increased in the soil around the same time. Conclusion The first microcosm study showed that beer trub had a positive effect on enhancing DDT degradation and that the 1.8 % beer trub treatment was the most effective at doing so. The data from the microcosm study as well as literature suggested that there might be an inhibitory factor preventing treatments with greater than 1.8 % beer trub from effectively degrading DDT. This mesocosm study purposed to study these hypotheses further, as well as analyze for enzyme activity, biomass levels, and

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65 leachate to see if the leachate would pose a possible threat to th e environment as a whole if the beer trub treatments were applied in a larger scale scenario. The DDx analysis found that both the 1.8 % and the 3 % beer trub treatments enhanced DDx degradation, but that there is a difference in the initiation of DDx deg radation between the 1.8 % and 3 % beer trub treatment. It also found that although the 3 % beer trub treatment took longer to begin to effect DDx degradation, once degradation began, the rates appeared faster and minimal concentration levels were achieved more completely. The data also inferred that the 3 % beer trub treatment was DDE due to the increased fungal growth producing more extracellular enzymes The enzyme data, ergosterol data, and nitrate da ta supported the use of beer trub as an additive to augment the DDx degradation process as well. Laccase enzyme activity was significantly increased by the addition of the beer trub treatments, and the times of the increases in activity coincided with the time that DDx degradation consistently began, as well was the case for the peroxidase assays. The leachate enzyme assays supported the recycling of the leachate mostly for the reuse of peroxidase enzyme activity, as the laccase enzyme activity in the leach ate did not appear to be enough to enhance DDx degradation. The ergosterol data showed that with the addition of the beer trub, fungal biomass significantly increases as time goes on Instead of suggesting it was an inhibitory factor, the nitrate data sugg ested that nitrate helped spur on fungal growth towards the end of the experiment. With the NO x levels in the soil significantly decreasing around the same time the ergosterol

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66 concentrations in the soil were increasing, it stands to reason that nitrate was being taken up by the fungi in the soil which used the nitrate as a food source. The significant increase in nitrate levels in the leachate around the same time suggest that the nitrate that was not used by the fungi was leached through the soil. Though t here was a large spike in nitrate levels on Day 1 in the leachate, in a real world scenario, the majority of the nitrate leached would not pose an environmental threat, as it would denitrify in subsurface anaerobic conditions. Therefore, nitrate is not exp ected to be a potential source of pollution, but further study is needed to confirm this hypothesis. Further studies on the role and impact of nitrate on the system are certainly necessary for a bution to the system as a whole, as well as its contribution of other nutrients. For a real world application example, if the 1.8 % beer trub treatment was to be applied to an acre of land for the purpose of intensifying DDT degradation, approximately 13, 245 kg of beer trub would be required. In order to produce this amount of beer trub, approximately 175,200 liters (46,283 gallons) of beer must be produced. Seeing how commercial breweries such as Anheuser Busch produce a few billion gallons of beer each y ear (answerparty.com), 46,282 gallons should not be an issue for the amount of trub that is needed in a 1.8 % solution. Due to the commercial availability of potential sources of beer trub, the idea of using an approximately 2 % beer trub solution to help remediate DDT contaminated soil is realistically viable

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67 Figure 3 DDT concentrations in soil samples from treatments during mesocosm study. All error bars represent one standard deviation. Similar l etters den ote no statistical significance on day of treatment.

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68 Figure 3 DDE concentrations in soil samples from treatments during mesocosm study. Al l error bars represent one standard deviation. Similar l etter s denote no statistical significance on day of treatment.

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69 Figure 3 DDT concentrations in leachate samples from treatments during mesocosm study. Error bars represent one standard deviation. Simila r l etters denote no statistical significance on day of treatment.

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70 Figure 3 DDE concentrations in leachate samples from tre atments during mesocosm study. Error bars represent one standard deviation Similar l etters denote no statistical significance on day of treatment.

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71 Figure 3 5. Laccase activity in soil for treatments during duration of mesocosm study. Enzyme activity = units of enzyme per L of enz yme solution needed to convert 1 Mol in one minute. Letters represent statistical significance in enzyme activity in clusters of treatments throughout the study. Different letters r epresent statistically different results.

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72 Figure 3 6. Peroxidase activity in soil for treatments during durat ion of mesocosm study. Enzyme activity = units of enzyme per L of enzyme solutio n needed to convert 1 Mol in one minute. Letters represent statistical significance in enzyme activity in clusters of treatments throughout the study. Different letters represen t statistically different results.

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73 Figure 3 7. Laccase activity in leachate for treatments during dur ation of mesocosm study. En zyme activity = units of enzyme per L of enzyme solut ion needed to convert 1 Mol in one minute. Letters represent statistical significan ce in enzyme activity in clusters of treatments throughout the study. Different letters represent statistically differ ent results.

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74 Figure 3 8. Peroxidase activity in leachate for treatments during dur ation of mesocosm study. Enzyme Activity = units of enzyme per L of enzyme solu tion needed to convert 1 Mol in one minute. Lett ers represent statistical significan ce in enzyme activity in clusters of treatments throughout the study. Different letters represent statistically different results.

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75 Figure 3 9. Soil ergosterol concentrations in treatments during du ration of mesocosm study. Ergosterol concentrations indicate fungal biomass levels i n soil. Error bars represent one standard deviation Different letters represent statistical differences between clusters of treatments throughout th e study.

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76 Figure 3 10. Leachate NO 3 concentrations for treatments during durat ion of mesocosm study, including day 0 of study. Error bars represent one standard deviation. Different letters represent statis tical differences between clusters of treatments throughout the study.

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77 Figure 3 11. Leachate NO 3 concentrations for treatments during duratio n of mesocosm study, not including da y 0 data. Error bars represent one standard deviation. Different letters represent statistical differences between clusters of treatments throughout the study.

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78 Figure 3 12. Soil NO 3 concentrations for treatments during duration of mesocosm s tudy. Error bars represent one standard deviation. Different letters represent statistical differences between clusters of treatments throughout the study.

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79 CHAPTER 4 DDT In previous e xperiments conducted by this laboratory, control treatments were found to contain naturally occurring fungi suspected to be wood rot fungi due to DDT concentrations decreasing overtime. However, DDT concentrations did not decrease significantly enough for the desired level of DDT remediation. This finding led to the hypothesis that the naturally occurring fungi in the soil were low in population or required extra nutrients to produce enzymes levels sufficient to degrade the DDT that was in the study sit e soil. As stated previously, some varieties of wood rot fungi have been found to degrade DDT via the release of extracellular enzymes with catalytic capacity to breakdown DDT. To produce these enzymes in sufficient quantities requires enhanced metabolism by the fungi. For the required nutrients, beer trub was chosen because it is a cost effective waste product that contains the living yeast Saccharomyces cerevisiae, which has been found to enhance extracel lular enzyme production (Hou et al., 2004; Jadhav et al., 2007) thus enabling it to contribute directly to DDT degradation (Kallman et al., 1963). The beer trub was applied to the soil, as described in previous chapters, to augment the growth of the ubiquitous wood rot fungi in the soil. This first microc osm experiment results supported the hypothesis that the beer trub could indeed enhance fungal growth, and subsequently increased enzyme productivity. The next experiment was larger in scale and purposed to verify the microcosm study, as well as test addit ional parameters, such as nitrate and biomass levels. As the mesocosm study was underway and similar trends in DDT degradation that were reported in the microcosm study were being

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80 observed, identification of one of the fungal species as well as its capabil ity to degrade the DDT in our study site soil were evaluated. Materials and Methods DNA Extraction and PCR Amplification of 18s rRNA Genes DNA was extracted from soil samples taken from the Lake Apopka North Shore Restoration study site using the PowerSoi l DNA Isolation kit (Mobio Laboratories that the naturally occurring fungi in our sample soil were wood rot fungi, and therefore one of our primer sets was specific to bas idiomycetes, the phyla containing the majority of wood rot fungi. However, a more general fungal primer set was used as well in order to identify other possible strains of fungi. The first primer set consisted of ITS1 F and ITS 4B, with the ITS 4B being t he basidiomycetes specific primer. The second primer set that was used was not specific to any phyla of fungi, and consisted of the primers ITS1 F and ITS4 R. The sequences of these primers are as follows: ITSl CTTGGT CAT TTA GAG GAA GTA A TCC TCCGCT TAT TGA TAT GC CAG GAG ACT TGT ACA CGG TCC AG (Prewitt et al., 2008). In order to optimize PCR analysis, various concentrations of the primer sets and isolated DNA were run in the PCR reactions (Table 4.1). The PCR am plification program comprised of an initial denaturing period set at 94C for 1.5 minutes, followed by 40 cycles with a melting temperature set at 95C for 35 seconds, an annealing temperature of 55C for 55 seconds, followed by an extension temperature of 72C for one minute. A final extension temperature of 72C was held for 10 minutes (Jasalavich et al., 2000). To verify the PCR amplifications were suitable to send off for DNA identification, the amplification products were run separated

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81 by gel electroph oresis on a 1% agarose gel made in trisacetate EDTA buffer and stained using ethidium bromide. The gels were then observed under UV light (Figure 4 1). Once the gels were observed and an optimum sample had been chosen for DNA identification, the sample was sent off to the Genome Sequencing Laboratory at the University of Florida. DDT Mineralization Study In order to verify that the fungus native to our study site soil was capable of degrading DDT, an isolated fungal DDT mineralization study was conducted. F irst, the fungus in the study site soil had to be isolated. For isolation, 10 grams of sifted (2mm) soil were added to 100 mL of a sterile Basal III medium solution (Appendix A). Next, 10 mL of a Trace Element Solution (TES) (Appendix A) was added via syri nge filter to the 100 mL Basal III medium solution. Approximately 2 grams of yeast extract was then added to the solution. (Tien and Kirk, 1988). The mixed solution was then placed in an oscillating incubator set at room temperature. Serial dilutions were done on the fungi in the Tien and Kirk (1988) solutions until no soil was left in the beaker. Next, the fungi was streaked on a Rose Bengal plate (Appendix A) (Martin et al., 1950; Martin, 1950) so that a single fungus could be isolated for study. The crit eria for choosing fungal hyphae from the Rose Bengal plate is that the fungus had to have decolorized the Rose Bengal plate. This decolorization of the Rose Bengal indicates the presence of extracellular enzymes (Arora et al., 2001; Hou et al., 2004). Onc e the fungus was isolated and devoid of any soil particulate matter, the DDT mineralization experiment was constructed. The mineralization study consisted of a total of three treatments, each treatment being replicated three times. The first treatment cons isted of the 100 mL of sterile Basal III medium, 10 mL of the TES, 0.2 grams yeast

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82 extract, five Teflon strips (12mm wide by 25 mm long) were spiked with 20 L of 1000 The second treatment was made up of the 100 mL of sterile Basal III medium, 10 mL of the TES, 0.2 grams yeast extract, and five Teflon strips that were spiked with 20 L of e added, a fungal transfer taken from fungal growth grown in the 100 ml Tien and Kirk broth (1988) as described previously was made to the 100 mL Basal III medium and autoclaved. After it was autoclaved, the TES, yeast extract, and Teflon strips were added This treatment served as the positive control, as it contained a sterile form of the fungal transfer. The last treatment consisted of the same ingredients as the first two treatments, except that it had a live fungal growth transfer taken from a similiar fungal that when sampling was done, a single Teflon strip was taken at a time, leaving the remaining strips in the solution for further sampling. The premise of this design is that not leach into the water based solution in the beaker due to DDT being highly insoluble in water. Sampling was conducted on a weekly basis. The sampling method was modified from Gohil (2011). It involved the removal of the Teflon strip from the solution and placing the strip in a clean glass test tube. 10 mL of methanol were added to the tube, and the tube would be shaken for 15 minutes to allow the DDT on the Teflon strip to transfer into the methanol. After shaking, 500 L of the 10 mL methanol solution were pipetted into an amber GC vile and analyzed via GC with an ECD detector. The same

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83 GC conditions as stated in previous chapters were used for analysis Preliminary experiments with this method produced a % recovery of 85.5. Results and Discussion DNA Extraction and PCR Amplification of 18s rRNA Genes After the PCR amplicons were sent to the Genome Sequencing Laboratory at the University of Florida, the DNA sequence of the PCR products was searched for on the Basic Local Alignment Search Tool (BLAST). The results showed a 99% Max identity for multiple strains of Penicillium chrysogenum Other strains that were listed included other species of Penicillium as well as a strain of Hypocrea lixii although the Penicillium chrysogenum strains were more prevalent in the results. These results from BLAST aligned with the PCR results, which showed that the basidiomycetes specific primer sets did not have any bands for our isolated fungal DNA, though the general fungi primer sets did produce a visible band on the gel (Figure 4 1). Penicillium is in the phyla ascomycetes, which is the second phyla in the subkingdom Dikarya, with basdiomycetes being the other phyla. P enicillium is most known for producing Penicillin, which is an antibiotic (Paul and Thomas, 1996; Bruggink et al., 1998). Penicillium chrysogenum has been found to produce laccase enzymes, which as noted in previous chapters and in this chapter, is an extr acellular enzyme that is capable of degrading DDT (El Shora et al., 2008; Rodriguez et al., 1996; Hou et al ., 2004; Bonnen et al 1994). DDT Mineralization Study In order to provide further evidence that the isolated Penicillium spp. fungus was capa DDT, a mineralization study was carried out. The results after 3 weeks of sampling do not show any statistical

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84 treatme nts in terms of DDT concentrations for the treatments (Figure 4 2). A longer time span of sampling and testing may be required for the degradation potential of the isolated fungus to be determined. Conclusion In order to learn more about the system that w as present in the microcosm and DDT mineralization study were necessary. After running PCR and having the DNA of the fungus sequenced, it seems probable that the fungus present in our soil is so me strain of Penicillium This is noteworthy because Penicillium spp. have been reported to produce enzymatic activities that degrade DDT. Penicillium is also capable of secreting amino acids, which after they have undergone ammonification and nitrificatio n, can produce nitrate, which may explain the nitrate levels found in treatments in the mesocosm study. Although the DDT mineralization study did not show significant evidence of DDT degradation with the isolated fungus treatments after three weeks of sam pling, it is still possible that this Penicillium spp., being adapted to a soil ecosystem and with the potential to metabolize DDT, could be a contributor to DDT in the soil if a proper temporal and spatial environment is established.

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85 Figure 4 1. P icture of PCR amplicons recovered from soils following various treatments containing incremental amounts of extracted DNA and different primer sets Amplicons were separated by 1.5 % agarose gel electrophoresis. Marker 1000 Second primers set DNA concentration 1/10 1/100 0

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86 Figure 4 DDT levels in live fungus, no fungus, and autoclave d fungus treatments for DDT mineralization study. Error bars represent one standard deviation.

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87 Table 4 1. Characteristics of PCR samples Sample # Vol. of Go Taq Green Master Mix (Promega Corporation) Primer used Vol. of Primer Vol. of nuclease free water Vol. of DNA 1 25 L Basidiomycetes specific 1 L of undiluted primer 22 L 1 L of diluted 1/10 2 25 L Basidiomycetes specific 1 L of undiluted primer 22 L 1 L of diluted 1 /100 3 25 L Basidiomycetes specific 1 L of diluted 1/10 primer 22 L 1 L of diluted 1/10 4 25 L Basidiomycetes specific 1 L of diluted 1/10 primer 22 L 1 L of diluted 1/100 5 25 L General Fungi 1 L of undiluted primer 22 L 1 L of diluted 1/10 6 25 L General Fungi 1 L of undiluted primer 22 L 1 L of diluted 1/100 7 25 L General Fungi 1 L of diluted 1/10 primer 22 L 1 L of diluted 1/10 8 25 L General Fungi 1 L of diluted 1/10 primer 22 L 1 L of diluted 1/100 9 25 L Basidiomycete s specific 1L of diluted 1/10 primer 13 L 10 L of undiluted DNA 10 25 L Basidiomycetes specific 1 L of undiluted Primer 23 L No DNA

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88 APPENDIX A RECIPES Table A 1. Plating Medium for Soil Fungi Rose Bengal Streptomycin Agar (Martin, 1950) Ingredients Amount Glucose 10 g Peptone 5 g KH 2 PO 4 1 g MgSO 4 7 H 2 O 0.5 g Agar 18 g Distilled H 2 0 1 L Rose Bengal 33 mg Streptomycin 30 mg pH 5.5 Table A 2. Basal III medium (per liter) (Tien and Kirk, 1988) Ingredients Amount KH 2 PO 4 20 g Mg SO 4 5 g CaCl 2 1 g Table A 3. Trace Element Solution (Tien and Kirk, 1988) Ingredients Amount MgSO 4 3 g MnSO 4 0.5 g NaCl 1 g FeSO 4 7H 2 0 0.1 g CoCl 2 0.1 g ZnSO 4 7H 2 0 0.1 g CuSO 4 0.1 g AlK(SO 4 ) 2 12 H 2 0 10 mg H 3 BO 3 10 mg Na 2 MoO 4 2H 2 0 10 m g Nitrilotriacetate 1.5 g Deionized Water 1 L

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89 REFERENCES Alcade M, Butler T (2003) Colorimetric assays for screening laccases. Methods in Molecular Biology, Directed Enzyme Evolution: Screening and Selection Methods 230:1531 1538 Alexander M (1995 ) How toxic are toxic chemicals in soil? Environ. Sci. Technol 29:2713 2717 Alexander M, Robertson B (1998) Sequestration of DDT and dieldrin in soil: Disappearance of acute toxicity but not the compounds. Environmental Toxicology and Chemistry 17:1034 1 038 Arora DS, Gill PK (2001) Comparison of two assay procedures for lignin peroxidase. Enzyme Microbiol Tech 28:602 605 ASTM (2000) Standard test methods for moisture, ash, and organic matter of peat and other organic soils. Method D 2974 00. American Soc iety for Testing and Materials. West Conshohocken, PA. (ATSDR) Agency for Toxic Substances and Disease Registry (2002) Toxicological profile for DDT, DDE, and DDD. US Department of Health and Human Services. Public Health Service. Division of Toxicology/To xicology Information Branch. 1600 Clifton Road NE, E 29 Atlanta, Georgia 30333 Bonnen A, Anton L, Orth A (1994) Lignin degrading enzymes of the commercial button mushroom, Agaricus bisporus. Applied and Environmental Microbiology 60:960 965 Bruggink A, Ro os EC, de Vroom E (1998) Penicillin acylase in the industrial production of Lactam antibiotics. Organic Process Research & Development 2:128 133 Djajakirana G, Joergensen RG, Meyer B (1996) Ergosterol and microbial biomass relationship in soil. Biology a nd Fertility of Soils 22:299 304 Dobbin P, Powell AK, McEwan AG, Richardson DJ (1995) The influence of chelating agents on the dissimilatory recution of iron(III) by Shewanella putrefaciens. Biometals 8:163 173 El Shora HM, Youssef MM, Khalaf SA (2008) In ducers and inhibitors of laccase from Penicillium. Biotechnology 7:35 42 Elisashvili V, Kachlishvili E, Penninckx M (2008) Effect of growth substrate, method of fermentation, and nitrogen source on lignocellulose degrading enzyme production by white rot ba sidiomycetes. Journal of Industrial Microbiology and Biotechnology 35:1531 1538.

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90 Facemire CF, Gross TS, Guillette Jr. LJ (1995) Reproductive impairment in the Florida panther: nature or nurture? Environmental Health Perspectives 103:79 86 Gallagher EP, G ross TS, Sheehy KM (2001) Decreased glutathione S transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameiurus nebulosus). Aquat Toxicol 55:223 237 Gianessi LP, Puffer CA (1992) Insecticide use in U.S. crop producti on. Washington, DC: Resources for the Future. U.S. EPA Cooperative Agreement CR 813719 Gohil, H (2011) Optimization of anaerobic degradation of 1,1,1 trichloro 2,2 di(4 chlorophenyl)ethane(DDT), 1 chloro 4 [2,2 dichloro 1 (4 chlorophenyl)ethyl]benzene(DDD) and 1,1 bis (4 chlorophenyl) 2,2 dichloroethene(DDE) in organic muck soils of Lake Apopka. University of Florida Guillette Jr. LJ, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR (1994) Developmental abnormalities of the gonad and abnormal sex ho rmone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environmental Health Perspectives 102:680 688 Guillette Jr. LJ, Pickford DB, Crain DA, Rooney AA, Percival HF (1996) Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment. Gen Comp Endocrinol 101:32 42 Hou H, Zhou J, Wang J, Du C, Yan B (2003) Enhancement of laccase production by pleurotus ostreatus and its use for the decolorization of anthraquinon e dye. Process Biochemistry 39: 1415 1419 Hubaux A, Vos G (1970) Decision and detection limits for linear calibration curves. Analytical Chemistry 42:849 855 Jadhav JP, Parshetti GK, Kalme SD, Govindwar SP (2007) Chemosphere 68:394 4 00 Jasalavich, CA, Ostrofsky A, Jellison J (2000) Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA. Applied and Environmental Microbiology 66:4725 4734 Kallma n BJ, Andrews AK (1963) Reductive dechlorination of DDT to DDD by yeast.Science 141:1050 1051 Kristensen T, Edwards TM, Kohno S, Baatrup E, Guillette Jr. LJ (2007) Fecundity, 17 beta estradiol concentrations and expression of vitellogenin and estrogen rece ptor genes throughout the ovarian cycle in female Eastern mosquitofish from three lakes in Florida. Aquat Toxicol 81:245 255.

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91 Kunisue T, Watanbe M, Subramanian A, Sethuraman A, Titenko AM, Qui V, Prudente M, Tanabe S (2003) Accumulation features of persist ent organochlorines in resident and migratory birds from Asia. Environmental Pollution 125:157 172 Lyman O (1977) An introduction to statistical methods and data analysis. Wadsworth Publishing Co., Belmont, California, p.659 Martin JP (1950) Use of acid, R ose Bengal, and streptomycin in the plate method for estimating soil fungi. Soil Science 69:215 232 Martin JP, Harding RB (1950) Comparitive effects of 2 bacterial growth preventatives, acid (pH 4) and Rose Bengal plus streptomycin, on the nature of soil f ungi developing on dilution plates. Soil Science Society of America Proceedings 15:159 162 Metcalf RL (1995) Insect control technology. Kirk Othmer Encyclopedia of Chemical Technology 14:524 602 Metcalf RL (1973) A Century of DDT. Journal of Agriculture an d Food Chemistry 21:511 519 National Pesticide Information Center (1999) DDT General Fact Sheet. Oregon State University, 310 Weniger Hall, Corvallis, Oregon 97331 Ogburn P (2010) Would you like some DDT with that organic cucumber? The Goat Blog, High Cou ntry News. ( http://www.hcn.org/blogs/goat/would you like some ddt with that organic cucumber ). Accessed July 9 th 2011. Paul GC, Thomas CR (1996) A structured model for hyphal differentiation and Penicillium production using Penicillium chrysogenum. Biotechnology and Bioengineering 51:558 572. Prewitt ML, Diehl SV, McElroy TC, Diehl WJ (2008) Comparison of general fungal and basidiomycete specific ITS primers fo r identification of wood decay fungi. Forest Products Journal 58:66 71 Rodriguez A, Falcon MA, Carnicero A, Perestelo F, De la Fuente G, Trojanowski J (1996) Laccase activities of Penicillium chrysogenum in relation to lignin degradation. Appl Microbiol Bi otechnol 45:399 403 Rice KG, Percival HF (1996) Effects of environmental contaminants on the Technical report 53. Fla. Coop. Fish and Wildlife Res. Unit, University of Florida, Gaine sville.

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92 Schmeck T, Wenclawiak B (2005) Sediment matrix induced response enhancement in gas chromatograph mass spectrometric quantification of insecticides in four different solvent extracts from ultrasonic and soxhlet extraction. Chromatographia 62:159 165 Smith NA (1992) Nitrate reduction and ATNC formation by brewery wild yeasts. J. Insl. Brew 98:415 420 St. Johns River Water Management District (SJRWMD) (2003) SJRWMD SWIM plan. St. Johns River Water Management District. St. Johns River Water Management District (SJRWMD) (2006) Lake Apopka restoration area land management plan. St. Johns River Water Management District. Thomas J (2011) Bioremediation of DDE, DDD, and DDT in sandy soil from Site ZSS3027 in Field ZSE J in the Lake Apopka North Shore Res toration Area: Contract #26703;Deliverable 4 Spent Mushroom Substrate Mesocosm Study Draft Report.St. Johns River Water Management District. Lake Apopka, Florida Thomas J, Ogram A, Ou L (2008) Bioremediation of DDE, DDD, and DDR in soil from the Lake Ap opka North Shore Restoration Area Contract #SK91314 Deliverable 5 Microcosm Report. St. Johns River Water Management District. Palatka, Florida. Tien M, Kirk T (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods of Enzymology 161:238 249 Toft G, Edwards TM, Baatrup E, Guillette Jr. LJ (2003) Disturbed sexual characteristics in male mosquitofish (Gambusia holbrooki) from a lake contaminated with endocrine disruptors. Environmental Health Perspectives 111:695 701 United Nat ions Environment Programme (2005) Ridding the world of pops: A guide to the stockholm convention on persistent organic pollutants. Secretariat of the Stockholm Convention on Persistent Organic Pollutants.United Nations Environment Programme (UNEP) Chemical s. International Environment House U.S. Environmental Protection Agency (1975) DDT: A review of scientific and economic aspects of the decision to ban its use as a pesticide. Criteria and Evaluation Division. Office of Pesticide Programs. U.S. Environment al Protection Agency. 401 N. Street. SW Washington, D.C. 20460 U.S. Environmental Protection Agency (2007) Test methods for evaluating solid waste 846. 5th edition. Office of Solid Waste and Emergency Response., Washington, D.C.

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93 U.S. Environmental Protection Agency (1993) Method 353.2: Determination of nitrate nitrite nitrogen by automated colorimetry Revision 2.0. Inorganic Chemistry Branch Chemistry Research Division. Environmental monitoring systems laboratory office of research an d development. U.S. Environmental Protection Agency. Cincinnati, Ohio 45268 Wang CH, Broadbent FE (1973) Effect of soil treatments on losses of two Chloronitrobenzene fungicides. J. Environ. Quality 2:511 514

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94 BIOGRAPHICAL SKETCH Benjamin J. Coppenger was born in Pensacola, FL and studied Maritime Studies with an Environmental Science minor at the University of West Florida While at the University of West Florida, he developed a strong passion for environmental remediation. This passion led him to the Uni versity of Florida where he learned about soil and water remediation research under the tutelage of Dr. John Thomas. He hopes to apply the skills and knowledge he gained at the University of Florida to help other global communities gain better access to cl ean food and clean drinking water.