LIBRARY M'Chigan State ' University PLACE IN RETURN BOX to roman this checkout from your «card. TO AVOID FINES return on or baton data duo. DATE DUE DATE DUE DATE DUE MSU Is An Affirmattvo Adm/Ema! Opportunity Institution Wm1 ABSTRACT THE EFFECT OF COMPOSTING PROCESS PARAMETERS ON THE MINERALIZATION OF ATRAZINE By Nishant Rao Composting offers a relatively inexpensive and environmentally safe method for the potential bioremediation of pesticide-laden rinsewater. The purpose of this study was to evaluate whether co-composting of lignocellulosic substrates and pesticide would be a viable option for the disposal of the pesticide. Atrazine was chosen because it is the most extensively used pesticide in the US. Composting was carried out in 2-liter laboratory scale composters. Degradation and mineralization of atrazine were followed by the use of MC-radiolabeled atrazine. The effects of composting process parameters such as temperature, type of substrate, and inoculum on the mineralization of atrazine were studied. Mineralization of atrazine during composting with poplar wood was investigated at three temperatures: 25°C, 37°C, and 55°C. Maximal mineralization of poplar wood carbon to CO; was observed at 37°C, with 10% mineralization of the poplar wood at the end of 84 days of incubation. Mineralization of atrazine was minimal in all cases. The enhancement of degradation and mineralization of atrazine by composting with pretreated lignocellulosic materials as compared to untreated lignocellulosics was also evaluated. Wood that was subjected to steam explosion (STEX wood) or ammonia explosion (AFEX wood), untreated wood (native), and shredded newspaper were selected as composting substrates. The results showed that there was no significant enhancement in atrazine mineralization when composted with the pretreated woods (AFEX wood and STEX wood) as compared to that observed with the native wood. Thus, pretreatment of the wood, which was hypothesized to lead to increased substrate and atrazine mineralization, was seen to have no added effect on atrazine mineralization. Finally, the effect of an exogenous inoculum of the white-rot fungus Phanerochaete chrysosporium on atrazine mineralization during composting of poplar wood was investigated. The addition of an aqueous conidial suspension of P. chrysosporium inoculum (Strain BKM-F—l767) significantly enhanced mineralization, resulting in a 14% mineralization of atrazine in 94 days of composting compared to 1% mineralization observed in controls without the P. chrysosporium inoculum. Preliminary modeling and process design suggested the feasibility of co-composting atrazine and poplar wood with an inoculum of P. chrysosporium as a viable option for the disposal of atrazine- contaminated rinsewater. ACKNOWLEDGMENTS First and foremost I would like to thank my family and friends for all their support and help during the bad times and good. I would like to thank Dr. Hans E. Grethlein for his guidance during the course of my work. I also would like to express my gratitude to Dr. C. A. Reddy whose help was invaluable during the difficult times of my research. I also would like to thank Dr. Ramani Narayan, Dr. Larry Forney, and Dr. Robert Hickey for their suggestions and help during the course of my dissertation. iv TABLE OF CONTENTS List of Tables ............................................. List of Figures ............................................ Chapter I: Introduction ............................. . ...... Chapter II: Literature Review ............................... l. Composting ........................................ l. Composting Process ............................... 2. Composting Applications ............................ 3. Factors Influencing the Composting Process .............. 1. Temperature ................................... 2. Moisture ...................................... 3. Aeration and Oxygen Supply ....................... 4. Other Parameters ................................ 2. Pesticides ......................................... l. Atrazine ........................................ 2. Fate of Pesticides in the Environment ................... 3. Degradation of Atrazine ............................ I. Abiotic Degradation .............................. 2. Pure Culture Degradation .......................... 3. In situ Degradation ............................... 4. Disposal Options for Pesticide Contaminated Sources ....... 5. Bioremediation of Pesticides Using Exogenous Inocula ..... 3. Pesticide Degradation During Composting ................. 1. Pesticide Degradation During Composting of Lignocellulosics 4. Research Objectives .................................. References ........................................ Chapter 111: Effect of Temperature on the Mineralization of Atrazine during Composting with Poplar Wood ............ ABSTRACT ........................................ 50 51 51 52 Pesticides ......................................... Compost Substrates ................................. Composting System ................................. Procedures for CO; Evolution and Fractionation of Compost RESULTS AND DISCUSSION .......................... Effect of temperature on substrate mineralization ............ Effect of temperature on atrazine mineralization ............ Effect of corn cobs as composting substrate ................ Effect of corn cobs on atrazine mineralization .............. Atrazine degradation during composting .................. CONCLUSIONS ..................................... Chapter IV: Mineralization of Atrazine During Composting With Untreated and Pretreated Lignocellulosic Substrates ........ ABSTRACT ........................................ INTRODUCTION .................................... MATERIALS AND METHODS ......................... Pesticides ......................................... Compost Substrates ................................. Composting System ................................. Compost Analysis ................................... Substrate Composition ............................ Enzymatic Hydrolysis .............................. C02 Evolution ................................. Compost Extraction Procedure ..................... Thin Layer Chromatography ...................... RESULTS AND DISCUSSION ......................... Substrate Composition ............................... Enzymatic Hydrolysis ................................ Total C02 Evolution ................................ Atrazine Mineralization and Degradation ................. CONCLUSIONS ..................................... REFERENCES ...................................... Chapter V: Mineralization of Atrazine During Temperature Controlled Composting of Poplar Wood With and Without an Exogenous Inoculum of Phanerochaete chrysosporium ABSTRACT ........................................ INTRODUCTION .................................... 52 52 53 53 54 54 55 55 55 55 56 57 65 66 69 69 69 70 71 71 71 71 71 72 72 72 73 73 74 76 76 87 87 MATERIALS AND METHODS ........................ 89 Pesticides ......................................... 89 Compost Substrates ................................. . 89 Compost Inoculum ................................. 9O Composting System ................................. 90 C02 Evolution ..................................... 91 RESULTS AND DISCUSSION ......................... 91 Substrate mineralization .............................. 91 Atrazine mineralization ............................... 92 Effect of corn amendment on substrate mineralization ....... 93 Effect of corn amendment on atrazine mineralization ........ 93 REFERENCES ...................................... 95 Chapter VI: Preliminary Process Design and Modelling 104 Introduction 104 Process Design 104 Process Modelling 110 Chapter VII: Overall Conclusions and Future Directions .......... 117 Appendices .............................................. 119 Appendix A: Effect of UN Ratio and Moisture Content on the Compostm' g of Poplar Wood ........................... 119 SUMMARY ........................................ 120 INTRODUCTION ................................... 120 MATERIALS AND METHODS ......................... 121 Compost Substrate .................................. 121 Compost Inoculum .................................. 121 Composting System ................................. 121 RESULTS AND DISCUSSION .......................... 122 REFERENCES ........................... ' ........... 123 vii LIST OF TABLES Table title page 2.1 Annual Usage Estimates of Pesticides in the US (Aspelin et al., 1991) ....................................... 17 2.2 s-Triazine herbicides and transformation products ....... 18 2.3 Summary of atrazine degradation research in pure cultures . . 27 2.4 Summary of atrazine (in situ) degradation research ....... 30 4.1 Composition of the lignocellulosic substrates used for composting .................................... 80 viii Figure 2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4 LIST OF FIGURES title Conceptual composting process. From Gray et al. (19713) Typical temperature and pH patterns in windrow composting. From Biddlestone and Gray (1985) ..................... Interdependence of composting process parameters. From Campbell et al. (1990b) ............................. a) General sructure of s-triazines (See Table 2.2 for substituents b) Structure of atrazine and common metabolites ......... Conversion of poplar wood to C02 at three temperatures. Values presented are means :1: half range for duplicate composters. ...................................... Mineralization of ”C-ring labeled atrazine to 1“co, during the composting of poplar wood at three different temperatures. Values presented are means 1 half range for duplicate composters. ...................................... Conversion of substrate carbon to CO; during composting at 55°C. Values presented are means :1: half range for duplicate composters. ...................................... Mineralization of l4c-ring labeled atrazine to ”co, during composting at 55°C. Values presented are means :1: half range for duplicate composters ............................ ix page 14 19 6O 61 62 63 3.5 4.1 4.2 4.3 4.4 4.5 5.1 5.2 Distribution of 14C from compost samples at different time periods in various extraction solvents. Extraction procedures used are described in Materials and Methods. ‘NaOH’ in the legend box refers to the fraction of 14C radiolabel extracted into the N aOH+Na4P207 solution. ‘Bound’ in the legend box refers to the unextracted fraction of 1°C radiolabel. ............. Glucose yields from the enzymatic hydrolysis of untreated poplar wood (Native), ammonia exploded (AFEX) wood, steam exploded (STEX) wood, and newspaper. Each substrate was treated with cellulase and B-glucosidase at 80 U/g dry substrate each as previously described (Thompson et al., 1992). Values presented are means :1: one standard deviation. ...... Fraction of substrate carbon converted to C02, Values presented are means+one standard deviation ............... Mineralization of l‘iC-Atrazine to 14C02 during composting with different lignocellulosic substrates. Values presented are means + one standard deviation. ....................... Comparison between total C02 production (substrate mineralization) and 14CO; production (atrazine mineralization) for each of the substrates. ............................ Distribution of 14C from compost samples at different time periods in various extraction solvents. Extraction procedures used are described in Materials and Methods. ‘NaOH’ in the legend box refers to the fraction of 1°C radiolabel extracted into the NaOH+Na4P207 solution. ‘Bound’ in the legend box refers to the unextracted fraction of 1°C radiolabel. ............. Effect of addition of a spore inoculum of P. chrysosporium (PC) to poplar wood. Composting was carried out at 37°C. Values presented are means :1: half range for duplicate composters. ...................................... Effect of addition of an exogenous inoculum of P. chrysosporium (PC) in the form of blended mycelia to poplar wood. Composting was carried out at 37°C. Values presented are means a: half range for duplicate composters. .......... 81 82 83 84 85 99 100 5.3 5.4 5.5 6.1 6.2 6.3 6.4 A.1 A2 A3 A.4 Mineralization of ”C-ring labeled atrazine to 14CO2 during the composting of poplar wood with and without the addition of an exogenous inoculum of P. chrysosporium (PC). Values presented are means :1: half range for duplicate composters. . . Fraction of initial substrate carbon converted to C02 during the composting of poplar wood with and without an amendment of corn at 37°C. Values presented are means :|: half range for duplicate composters. .............................. Mineralization of l“c-nng labeled atrazine to l“co. during the composting of poplar wood with and without an amendment of corn. Values presented are means :l: half range for duplicate composters. ...................................... Conceptual co-composting process .................... Mineralization of atrazine and wood during composting with an inoculum of P. chrysosporium ........................ Model prediction for substrate mineralization ............. Model prediction for atrazine mineralization .............. Conversion of initial carbon to CO; during the composting of poplar wood at an initial C/N ratio of 10:1 and varying moisture contents. ................................. Conversion of initial carbon to C02 during the composting of poplar wood at an initial C/N ratio of 30:1 and varying moisture contents. ................................. Conversion of initial carbon to C02 during the composting of poplar wood at an initial C/N ratio of 50:1 and varying moisture contents. ................................. Conversion of initial carbon to C02 during the composting of poplar wood at an initial moisture content of 70% and varying C/N ratios. ....................................... xi 101 102 103 105 111 115 116 125 126 127 128 Chapter I: INTRODUCTION Many pesticides commonly used in agricultural and lawn care applications pose a potential threat to public health and environmental quality. There is increasing recognition that soils at many agrichemical facilities and farms have been contaminated with high concentrations of pesticides through accidental spills or improper rinsing and discharge procedures (Long, 1989). High concentrations of ordinarily biodegradable pesticides can be persistent in soils partly because they inhibit microbial activity (W interlin et al., 1989; Dzantor and Felsot, 1991). High concentrations of pesticides have also been found to be more mobile than low concentrations (Davidson et al., 1980). The combination of persistence and greater mobility increases the risk of surface and groundwater contamination and emphasizes the need for expeditious cleanup. Current disposal options for these contaminated wastes include excavation and subsequent landfilling or incineration of solid wastes. Discharging of liquid wastes into drainage systems has been known to occur, with possible contamination of natural water systems (Seiber, 1991). Such options are being phased out with the passage of stricter laws aimed at curbing environmental and health problems. Disposal methods such as landfilling and incineration are expensive and do not always address the problem of contaminant detoxification. As more contaminated sites are discovered, it is becoming increasingly important to seek cleanup technologies that can be easily adapted to a variety of situations. Examples of disposal options that would fit the category of environmentally friendly technologies include in-situ remediation, landfarming (also known as land application or land treatment) and biological treatments such as bioremediation, bioaugmentation, composting, and anaerobic digestion. Composting, a prime example of biological treatment, is a relatively inexpensive and easily manageable alternative and has been widely used in the disposal of municipal solid wastes (Gray et al., 1973) and yard wastes (Michel et al., 1993). Composting has also been shown to be effective in the bioremediation of a variety of xenobiotics (Williams et al., 1992; Lemon and Pylypiw, 1992; Vogtmann, 1984). The potential for bioremdiation of contaminated sources such as pesticide rinsewater and contaminated soils, using composting is promising primarily because of the intensity of the microbial activity within a composting matrix. The overall transformation potential for contaminants within a composting mass is worth considering over other decontamination methods for a variety of reasons. First, elevated temperatures facilitate a higher reaction rate. Second, the opportunity for cometabolism (degradation of a recalcitrant compound or contaminant while a microorganism obtains its carbon and energy from other easily utilizable compounds) is enhanced due to the range of alternative substrates present and the high level of microbial activity. Finally, the changing physical and chemical microenvironments within a composting mass result in a diversity of microbial communities and metabolic activity, thus increasing the number and type of microorganisms to which the contaminant is exposed. Composting could be used with lignocellulosic substances as substrates, since previous studies have shown that lignocellulose degrading enzymes may also be important for the degradation of pesticides and xenobiotics (Hammel, 1992; Boominathan and Reddy, 1992). Lignocellulosic materials have also been shown to concentrate pesticides from wastewater sources with good sorption characteristics (Toller and Flaim, 1988; Hetzel et al., 1989; Mullins et al., 1993) suggesting that co-composting of pesticides may be a disposal option. In the case of the pesticide-laden rinse water, a model lignocellulosic material such as wood could be used as the adsorption agent and the composting substrate to optimize the rates and extent of mineralization of the pesticide. The data gathered from the work could then be extended to more readily available lignocellulosics such as corn stover, corn cobs, and other agricultural byproducts. Although research has been done in the area of pesticide degradation in soils, very little has been done in elucidating the co—composting of pesticides with lignocellulosic substrates. Research needs to be conducted in order to better understand the factors controlling the simultaneous metabolism of the lignocellulosic substrate and the various amendments in the form of pesticides or contaminated soils. The overall objective of this study was to verify whether gratuitous mineralization of pesticides was possible during the the composting of lignocellulosic substrates. This would enable us to evaluate co- composting of pesticides with various lignocellulosic materials as a viable method for disposal of the pesticide. Atrazine was chosen as the pesticide to be studied due to its widespread use in the US. (it accounts for about 12% of all pesticides used in this country), and due to its recalcitrance to degradation in the environment. This dissertation is divided into seven chapters, including this first chapter (Introduction) followed by a review of relevant literature in Chapter 11. Chapters III through V constitute the research papers from this dissertation. Process design and modelling are discussed in Chapter VI. Overall conclusions and future directions are presented in Chapter VII. Appendix A presents preliminary data on the effect of substrate moisture content and C/N ratio on conversion of substrate to C02. Chapter 11: Literature Review Research in the fields of composting and pesticide degradation has been very diverse, encompassing a wide variety of areas such as composting substrates, parameters, and systems, fate of pesticides in the environment, and degradation of pesticides by physical, chemical, and microbiological means. Because of the diversity of research in the areas of composting and pesticides, the literature review presented here focuses on areas that have relevance to this study. This chapter presents a summary of research in the fields of composting, fate of pesticides in the environment, degradation (pure culture and in situ) of atrazine, disposal options for pesticide contaminated sources, bioremediation of pesticides using added microbial inocula, and finally, pesticide degradation during composting. l. Composting 1.1 Composting Process Composting is one of the many paths in nature which contribute to the closure of the carbon cycle by recycling carbon that has been used in the synthesis of various organic compounds. Composting involves the biodegradation of organic compounds by a mixed microbial consortia to carbon dioxide and water and leading to the formation of a stable humus-like product, which has been used as a soil conditioner. As a biological process it is subject to the constraints of all biological processes, namely, limitations imposed by microbial population and genetic traits, and process parameters such as temperature, moisture, and nutrients. Composting is a method of solid waste management where the organic component of the solid waste stream is biologically decomposed to a state in which it can be handled, stored, and/or put to some end use. A variety of composting operations are in use at present that include windrow composting (passive aeration and forced aeration), and a host of in vessel systems that include rotary drums and agitated bins (Hang, 1993). A brief description of the general succession of events that occur during a common composting process (windrow composting), is presented in the next few paragraphs. A conceptual diagram of the processes that occur during composting is shown in Figure 2.1. The composting process begins with preparing the material for composting by adjusting moisture content and/or addition of a nitrogen source. Depending on the composting material this initial step is followed by the addition of amendments such as sawdust, manure, and yard wastes, and/or bulking agents such as wood chips, to make the composting material more amenable to handling and to provide structural support and maintain air spaces in the compost matrix. The conditioned material is then placed into piles or windrows. Microbial activity in the presence of oxygen and moisture generates heat during the degradation of the organic fraction of the composting material. Temperature is a good process indicator, since the release of heat is an outcome of metabolic activity. As easily degradable compounds get utilized by the microbial species, temperature rapidly increases in the compost matrix. These thermophilic temperatures can be maintained for several weeks as seen in Figure 2.2 which represents general temperature trends that occur during windrow composting. With a decline in the amount of easily degradable substrates, microbial metabolism switches to the hydrolysis and assimilation of the polymeric materials in the compost matrix which is a relatively slower [hi--——___.. 1: '6 OXYGEN MICRO- f—‘ oacAmsus * MOISTURE - ————— 1 ‘} PROTEINS A AMINO-ACIDS are: A - L‘ Hvoaxres —" v .CELLULOSE .- l I LIGNIN ‘ 1 ASH . INTERMEDIATE F_j,__ METABOLITES moasxmc u “ mraoceu —j CYCLE ’ new 1 JEATH "'"'"'ORGANISMS : 1, I HUHUS °" :1} COHPOST Figure 2.1: Conceptual composting process. From Gray et al. (1971a). O O N El Temperature 1°C ) Temperature peak - point of stability Spore-forming 70 _. bacteria and octinomycetes / \ \ 8W I Breakdown " ° ’ t l rners 60 sohbles ° poy Fungi Fungi / killed ‘ re-estoblisn 50 - Ternperoture / 40- , Curve 30 - Ammonia d 9 evolution Curve 20 pH .. 7- Soil animals move in / .. '5 Formation of humic oods Acid generation - a c 1 l 1 ’11; . J Meso- Tnerrno- Cooling down Motoring philic phllic stage stage Time Figure 2.2: Typical temperature and pH patterns in windrow composting. From Biddlestone and Gray (1985). ' A'kOllne Acidic O I process. This results in a decrease in heat generation, and temperatures gradually drop till the compost matrix reaches ambient temperatures. This characteristic temperature pattern over time reflects changes in the rate and type of decomposition taking place as composting proceeds. The final stage of composting, a comparatively slow process compared to the previous stages, involves maturing of the composting material into a stable product. This takes place at ambient temperatures with the action of predominantly mesophilic organisms giving rise to humus and humic acids via polymerization and condensation reactions (Gray et al., 1971a). Humic acids result from condensation of plant lignin residues with bacterial protein and can also be produced by microorganisms by synthesizing carbohydrates to polycyclic compounds in the presence of nitrogen. These processes that occur during composting are brought about by the activities of microbial communities, each of which is suited to an environment of relatively limited duration. The main classes of microorganisms encountered are bacteria and fungi, with bacteria being responsible for the initial breakdown of the organic material and for a large part of the initial heat released into the composting mass (Finstein and Morris, 1975). The initial metabolism of the easily degradable organic fraction by bacteria leads to the subsequent colonization of the compost by actinomycetes and fungi which can utilize a relatively wider array of substrates. A general drop in microbial activity is seen with the increase in temperature, with little or no activity at the peak temperatures reached in the windrow (Strom, 1985; McKinley and Vestal, 1985; McKinley and Vestal, 1984). This leads to a drop in the temperatures with recolonization of the compost by actinomycetes and fungi (Gray et al., 1971a). This period of decline of temperature to the ambient temperature is referred to as the curing or maturing of the compost and is accompanied by a slower rate of composting and lower oxygen consumption rates. This general description of windrow composting is generally valid for most materials undergoing large-scale composting (Gray et al., 197la,b). 1.2 Composting applications Although a major emphasis of composting applications has been for the disposal of municipal and domestic solid wastes (Haug, 1993), composting has also been proposed as the answer to the disposal of other types of wastes. Vallini et. al. (1984) found composting to be a viable alternative for the disposal of food factory, fruit, vegetable, cork and tannery wastes. They found that tannery wastes, a highly polluting and biologically toxic substance, could be transformed quickly and inexpensively by composting, into an organic fertilizer for crops. Lopez-Real (1984) highlighted the use of high-temperature composting as a resource recovery system for agro-industrial wastes, while Biddlestone and Gray (1991) suggested composting as a source for the production of a peat alternative. The composting of a variety of substrates including tree bark, sewage sludge, municipal refuse, and newsprint has also been investigated (Ashbolt and Line, 1982; Atchley and Clark, 1979; Campbell et al., 1990a,b; DeNobili and Petrussi, 1988; Ferrari, 1987; Hang, 1979; Jeris and Regan, 1973a,b,c). Composting has also been studied as an alternate means of disposal for xenobiotics and industrial wastes (W interlin et al., 1986; Vogtmann et al., 1984; Fogarty and Tuovinen, 1991). 10 1.3 Factors influencing the composting process 1.3.1 Temperature Temperature plays an important rolein the composting process as described in section 1.1. Composting essentially takes place within two ranges of temperature (see Figure I) referred to as mesophilic (IO-45°C) and thermophilic (>45°C) though the cut off between the two ranges is ill-defined. Mesophilic temperatures allow effective composting, as observed by McKinley and Vestal (1985, 1984) who reported the greatest microbial activity in compost samples taken from lower temperature areas (35°C-45°C) of sewage sludge windrow composts. Similar findings were reported by Jeris and Regan ( 1973a) who investigated the bench-scale composting of newsprint and observed the highest CO; production rate at 48°C. They also reported that the composting of stabilized municipal refuse was found to have an optimum at 40°C in shake flask experiments. The optimum temperature during the lab-scale composting of ground garbage was found to be 45°C (Snell, 1957), while that for the solid state fermentation of straw by Chaeromium cellulolyticum was found to be 37°C (Abdullah et al., 1985), reflective of the lower growth temperatures for fungi. Hogan et al. (1989a) evaluated the composting of sludge amended with aliphatic and polyaromatic hydrocarbons at two temperatures (35°C and 50°C). They noted that volatile solids and hydrocarbon losses were greater at the lower temperature, keeping with the notion that higher microbial activity is generally observed at mesophilic temperatures. Most commercial operations, however, maintain thermophilic conditions since pathogens, weed seeds, and fly larvae are destroyed at those temperatures (Composting Handbook, 1992). Microbial metabolism during composting releases large amounts of 11 energy as heat, resulting in an increase in the temperature of the compost due to the self- insulating qualities of the composting material (Composting Handbook, 1992). Heat accumulation can push temperatures well above 60°C which affects the compost microorganisms, leading to a slowing of the process. Temperatures can continue to rise above 70°C at which point most of the microorganisms either die or become dormant. At this stage the compost process effectively stops and does not resume until the microbial population recovers after a drop in temperature. Researchers have also shown composting to be efficient at thermophilic temperatures. Schulze (1962) observed that temperature was directly related to microbial oxygen uptake rate between 30°C and 70°C during the small-scale composting of garbage. Suler and Finstein (1977) observed that optimum composting was achieved at 55 to 60°C during the lab-scale composting of table scrap. Strom (1985) reported that bacterial species diversity decreased markedly during the lab-scale composting of table scraps and newspaper above a temperature of 60°C and concluded that the maximum desirable temperature for composting was thus 60°C. Cathcart et al. (1986) concluded that the optimum temperature for composting unshredded and shredded crab scrap was 63°C and 56°C respectively. Attempts have also been successfully made to control the temperature at a desired set-point for optimal composting by the control of aeration to the compost matrix (Hogan et al., 1989a,b; Ryoo et al., 1991; Finstein et al., 1992). To summarize, increase in temperature leads to an increase in the rates of degradation of the organics in the compost materials, thus increasing the rate of composting. On the other hand, the outcome of excessive increase in compost temperature is the decrease in the microbial activity in the compost, resulting in a decrease in the rate 12 of composting. Depending on the substrate and the composting system being used, an optimum temperature exists, at which a balance between the two opposite effects of temperature can be achieved. 1.3.2 Moisture Since composting is a microbially mediated process, moisture plays a critical role in the composting process. Water provides the medium for chemical reactions, transportation of nutrients and enzymes, and allows for microbial movement. Desirable moisture content is linked to particle size and porosity of the composting material. In theory, optimal microbial activity is achieved at saturated conditions, with cessation of activity below a 15% moisture content. However, aeration is adversely affected at saturated conditions, possibly leading to anaerobic conditions. Thus, in practice, a range of 50-90% moisture, depending on the composting substrate, is recommended (Biddlestone and Gray, 1985). Jeris and Regan (1973b) investigated the composting of refuse containing 60-70% paper and noted that the highest oxygen consumption rate was at a moisture content of 67%. Suler and Finstein (1977) reported 60% moisture to be optimum for the lab-scale composting of table scrap. The optimum moisture content was found to lie in a range of 52 to 58% during the lab-scale composting of ground garbage (Snell, 1957). The optimum moisture content for the lab-scale solid state fermentation of straw by Chaetomium cellulolyticum was found to be 80% (Abdullah et al., 1985). Cathcart et al. (1986) concluded that the optimum moisture content for composting unshredded and shredded crab scrap was 67% and 55% respectively and attributed the higher optimum moisture content for unshredded scrap to larger particle size and increased porosity. 13 1.3.3 Aeration and Oxygen supply Aeration serves to provide oxygen to the composting material, as well as removal of heat, water vapor, and gases that are generated during composting. Limiting the supply of oxygen slows the compost process and creates anaerobic conditions in the compost. Suler and Finstein (1977) reported that higher C02 evolution (indicating higher mineralization of organic carbon) was observed when the exhaust air from lab-scale composters had oxygen concentrations greater than 10%. They observed that air flow rates that left 2% Oz in the exhaust resulted in lower CO; generation, emphasizing the importance of oxygen supply. This was also shown by Kaneko and Fujita (1992) who conducted lab-scale composting of newsprint and dog food at 50°C, and concluded that the efficiency of composting could be ensured by controlling the oxygen concentration in the exhaust gas to about 10%. McKinley and Vestal (1985b) observed significant improvements in the rates of microbial metabolism and growth when aeration was used to keep the temperature below 58°C during the small-scale composting of municipal sewage sludge, compared with piles composted simultaneously at higher temperatures (60 to 84°C). This highlights the interdependence of temperature, moisture content and aeration in obtaining optimal composting conditions as presented in Figure 2.3. 1.3.4 Other parameters The composting process is also affected by other factors such as UN ratio, pH, and particle size of the composting substrate. Carbon and nitrogen are the primary nutrients required by microorganisms. Microorganisms use carbon for energy and growth while nitrogen is essential for protein synthesis. Since microorganisms contain approximately 50% carbon and 5% nitrogen on a 14 breakdown f > I Particle size I ( aurtace area [Substrate I ”may )| Free air space lfi I a l a water activ'ay t [Decomposition l‘ I M ture content I A ois metabolic water} A nutrient : i r i a evaporation J g g Aeration ( 2 _ .. .. __J L v coolin L 9 >l Temperature I rate at reaction. sobbility at 02 and nutrients Figure 2.3: Interdependence of composting process parameters. From Campbell et al. ( 1990b). 15 dry weight basis, and approximately 20-40% of the carbon present intially in the compost material is converted to microbial biomass (Gray et al., 1971), the requirement of nitrogen in the feed is 24 parts/ 100 parts of initial carbon. Thus, raw materials with a CM ratio of about 25:1 to 50:1 are ideal for active composting although higher ratios are also acceptable depending on the amount of available carbon (Biddlestone and Gray, 1985; Kayhanian and Tchobanoglous, 1992). Due to the variety of microorganisms involved, the composting process is relatively insensitive to pH, especially within the range of values generally seen with composting materials. One instance where pH is a factor is in the case of materials with a high nitrogen content, where a process pH of >8.5 can lead to the formation of ammonia, creating odor problems (Composting Handbook, 1992). This can be easily controlled by adjusting the ON ratio of the feed above 25: 1. Since microbial decomposition occurs on particle surfaces, degradability can be improved by reducing the particle size (which increases the available surface area), as long as porosity is not affected (Composting Handbook, 1992). 2. Pesticides Pesticide usage in the US. has been relatively stable at about 1.1 billion pounds of active ingredient during recent years (Aspelin et al., 1991). An estimated 45 billion dollars worth of agricultural pesticides, corresponding to about two thirds of the total pesticide sales, are sold in the United States every year. This includes about 21,000 pesticides registered under the Federal Pesticide Law. Failure to use proper procedures at pesticide mixing and handling sites and improper disposal of pesticide laden rinsewater can result in the contamination of soil, surface water and ground water (Myrik, 1990, N orwood, 1990, 16 Toller and Flaim, 1988). One of the most important issues facing the agrichemical industry is the cleanup and prevention of site contamination at retail dealerships. Atrazine, alachlor, cyanazine, and metolachlor were the pesticides most frequently found in contaminated soil sites (Buzicky et al., 1992), a finding directly related to the amount of use of these pesticides (Table 2.1). A report stated that the ground water of more than half the states in the US. contained agricultural pesticides (Chemistry and Engineering News, 68:26-40, 1990). The findings from another report (EPA, 1988) showed the presence of 74 different pesticides in the ground waters of 38 states, more than half of which originated from agricultural uses. These reports highlight the seriousness of the extent of contamination, due to pesticide usage, and emphasize the urgent need to find cost effective solutions for the disposal or remediation of contaminated sources. However, many of the current treatment options are very expensive as observed by Myrick (1992), who estimated a cleanup cost of between three to five million dollars for just a single dealership site. He also foresaw a potential savings in the billions of dollars to the agrichemical industry contingent on the development of inexpensive site remediation technologies for contaminated dealership sites. 2.1 Atrazine The s-triazine group of herbicides which include atrazine, sirnazine, and cyanizine (Figure 2.4, Table 2.2) are widely used in agriculture in the US. for the control of annual grasses and broad leaf weeds in corn (Cook, 1987; Aspelin et al., 1991). In addition atrazine is used extensively as a preemergent herbicide for other crops including sorghum, sugar cane, macadamia nut, and pineapple, as well as for weed control on rangeland and along railroads and highways (Herbicide Handbook, 1979). Atrazine alone accounts for 17 Table 2.1: Annual Usage Estimates of Pesticides in the US. (Aspelin et al., 1991) Pesticide Usage in Million Pounds Active Ingredient Atrazine 70-90 Alachlor 60-75 2,4-D 40-65 Metolachlor 40-55 1,3-D 35-45 Trifluralin 30-40 Cyanazine 20-30 Carbaryl 10-15 Chlorpyrifos 8-16 Maneb/Mancozeb 8-12 Methyl Parathion 8-12 18 Table 2.2: s-Triazine herbicides and transformation productsa Substituent Chemical Formula Common Name Abbreviationb R1 R2 R; C] Csz C3H7i 2-Chloro-4-ethylamino-6- Atrazine CIET isopropylamino-s-triazine C1 H C3H7i 2-Chloro-4-amino—6- Deethylatrazine CIAT isopropylamino-s—triazine Cl H Csz 2-Chloro-4»ethylamino—6- Deisopropyl CEAT amino-s-triazine atrazine Cl H H Chloro-diarnino-s-triazine Dealkylatrazine CAAT Cl C2H5 C2H5 2-Chloro-4,6-bis(ethylamino)- Simazine CEET s-triazine Cl C3H7i C3H7i 2-Chloro-4,6- Propazine CIIT bis(isopropylamino)es—triazine Cl C2H5 C3116(CN) 2-[(4-Chloro-6-Ethylamino-s Cyanazine triazine-2-yl)amino]-2- methylpropionitrile NH; H H 2,4,6-Triamino-s-triazine Melamine AAAT OH €sz C3H7i 2-Hydroxy-4wethylamino-6- Hydroxyatrazine OIET isopropylamino s-triazine OH H C3H7i 2-Hydroxy-4-amino-6- Deethyl OIAT isopropylamino s-triazine hydroxyatrazine OH CH H 2-Hydroxy-4—ethylamino—6— Deisopropyl OEAT amino s-triazine hydroxyatrazine OH H H 2-Hydroxy-4,6-diamino s- Ammeline OAAT triazine a - Refer to Figure 2.4(a) for general structure of s-triazines b - From the system developed by Cook (1987) N R3HN * N k a) Cl N X N C,H,iHN JR N k NHC2H, Atrazine Cl N A N HIN A N k NHC2H, Deisopropylatrazine b) OH A N N 1N1 Hydroxyatrazine C,H,iHN NHCZH, C1 A N’ N Jen/km, Deethylatrazine C,H,iHN Figure 2.4: a) General structure of s—triazines (See Table 2.2 for substituents) h) Structure of atrazine and common metabolites 20 about 12% of all the pesticides used in the US. and is most heavily used in the midwest. Over 36 million kilograms of atrazine were applied nationwide in 1990 (Periera and Rostad, 1990). Atrazine and its metabolites, deethylatrazine and deisopropylatrazine (Figure 2.4b) are the most frequently found pesticides in surface waters of the rnidwestem U.S. (Thurman et al., 1991). Atrazine has been detected in lakes and streams at levels ranging from 0.1 to 30 jig/L with peak concentrations up to 1000 ug/L known to occur in surface runoff from agricultural fields adjacent to bodies of water during times of application (Day, 1991). These concentrations generally exceed the maximum contaminant level of 3 jig/L that took effect in 1992 (EPA, 1991). 2.2. Fate of pesticides in the environment Degradation and sorption are two of the most important processes governing the fate of pesticides in the environment. Processes involved in the degradation of pesticides can be classified under three main categories: physical, chemical, and microbiological. The two primary physical processes involved in the degradation are light and heat. Photolysis of pesticide residues is extremely significant on vegetation, on the soil surface, and in water (Coats, 1991). Thermal decomposition often occurs in conjunction with the photodegradative reactions. Solar radiation is therefore responsible for the degradation by both, photolysis and thermal decomposition. Chemical degradation occurs as a result of the various reactive agents in the pesticide formulations and in the environment. Water is responsible for considerable breakdown of pesticides in solution, especially in conjunction with pH for pH sensitive compounds. In most environments, oxidative reactions involving oxygen, ozone and 21 peroxides are the most frequent degradative pathways observed (Hapeman-Somich, 1992). Biological agents are also significant in the degradation of pesticides. Microorganisms are the most important group of degraders based on their prevalence in the environment. The major strategies exhibited by the microbes include catabolism, co» metabolism, and extracellular enzymatic activity in the presence or absence of microbes (Coats, 1991). The influence of sorption on the biodegradation of organic contaminants has been recognized as an important issue in environmental science (Alexander, 1991). Factors cited as reducing the availability of pollutants for biodegradation included sorption to soils, presence in a physically inaccessible state, and binding of the pesticides in a manner as to prevent their transformation by microbial activity (Alexander, 1991). Sorption has been generally considered to hinder or limit the rate and extent of pesticide mineralization, as indicated by Ogram et al. (1985) and Weissenfels et al. (1992). But Guerin and Boyd (1992) showed that the availability of sorbed naphthalene was different for two different bacterial species, and that generalizations regarding the bioavailability of sorbed organic contaminants and pesticides were inappropriate. Similar to sorption, the contact time between pesticidesiand soils, referred to as pesticide aging is another important factor influencing the bioavailability of pesticides. Steinberg et al. (1987) compared the biodegradation of residual ethylene dibromide in field weathered soils to those of fieshly added ethylene dibromide. Dramatic differences were observed showing aged ethylene dibromide to be persistent, whereas fi‘eshly added radiolabeled ethylene dibromide was readily degraded within the same soil sample. 22 2.3. Degradation of atrazine Atrazine, along with the others in the s-triazine group, are relatively, persistent in the environment, with the most heavily substituted and chlorinated s-triazine analogs being the least biodegradable. However, both bacteria and fungi have been shown to mineralize atrazine to varying degrees (Cook, 1987), though atrazine uptake and metabolism was very limited in algae (Jones et al., 1984; Butler et al., 1975). Atrazine can be degraded by either biotic or abiotic processes. (Mandelbaum et al., 1993b). N-dealkylation, dechlorination and hydroxylation at the 2-position, deamination, and ring cleavage are the major degradative processes for atrazine. Biotic degradation of atrazine generally results in the production of desethylatrazine or desisopropylatrazine (Levanon, 1993) with the mineralization of the alkyl-amino side chains mainly due to fungal activity. Research has shown deethylatrazine to be the more stable and dominant initial biotic degradation product. The abiotic degradation of atrazine results in hydrolysis with the initial formation of hydroxyatrazine. Deethylatrazine is almost as phytotoxic as atrazine while deisopropylatrazine is five times less phytotoxic. Dealkylatrazine (2-chloro-4,6-diamino-s- triazine) and hydroxyatrazine are non-phytotoxic (W inkelmann and Klaine, 1991). 2.3.1. Abiotic Degradation As mentioned earlier in section 2.2, abiotic degradation of pesticides by physical or chemical processes has been shown to contribute to the overall degradation of the pesticide. There have been quite a few studies on the effect of abiotic factors on the degradation and possible mineralization of atrazine. Hapeman-Somich et al. (1992) reported that the aqueous ozonation of atrazine at a concentration of 0.153 uM (33 ppm) gave rise to a variety of products including CIAT, CDIT, CEAT, and CAAT. They also 23 found that the s-triazine ring remained intact and that the chorine atom was not removed. Similar results were obtained by Adams and Randke (1992) with the exception of recovery of hydroxy metabolites of atrazine. A study by Hance (1979) on the effect of soil pH on the degradation of atrazine showed that degradation was relatively insensitive to soil pH ranging from 5.1 to 8.2. On the other hand, Best and Weber (1974) reported that atrazine degradation in a soil at pH 5.5 was greater than that observed in the same soil adjusted to pH 7.5. Moyer and Blackshaw (1993) found that atrazine dissipation was related to the amount of rainfall received (and thus the soil moisture content), with greater amounts of rainfall leading to a correspondingly greater dissipation of atrazine. A modified Fenton system (Pratap and Lemley, 1994) consisting of electrochemical generation of iron in the presence of hydrogen peroxide was shown to be effective in degrading 90% of an aqueous solution of atrazine in about 2 hours. Another process that was studied was the vacuum- ultraviolet photolysis of atrazine in water (Gonzalez et al., 1994). Up to 50% of the initial atrazine was found to be degraded to cyanuric acid though the extent of mineralization to C02 was minimal. Ro et al. (1995) found that sodium azide could chemically transform atrazine to 3- ethylamino, 5-isopropylamino-s-triazyl azide and 3-ethylamino, 5-isopropylamino—s- triazinone. A complete dissipation of 10 mg/L of atrazine was observed with a 1% sodium azide solution, within 21 days of anaerobic incubation. Koskinen et al. (1994) observed a 50% degradation of analytical grade and formulated atrazine in 330 and 2772 minutes respectively during ultrasonication of aqueous solutions of the pesticide. No mineralization of atrazine was observed. Widmer et al. (1993) found no significant loss in atrazine concentration to occur during storage in well water and deionized water for 19 weeks. 24 Almost all the systems described above used very dilute aqueous solutions of atrazine (up to 33 ppm) and were not shown to mineralize atrazine. These methods showed increased rates of degradation of atrazine to its metabolites but suffered the drawback of being unable to effect atrazine mineralization. Thus the use of such systems in atrazine disposal strategies would necessitate additional steps to further eliminate the metabolites created. 2.3.2. Pure Culture Degradation Donnelly et al. (1993) studied the degradation of 2,4-D and atrazine by nine different fungal species. The pesticides were used at two concentrations, 1 and 4 mM, corresponding to 215 and 860 ppm of atrazine, and at three nitrogen levels: 0, 1, and 10 mM. They found that there was no significant mineralization of the l4C-ring labeled atrazine at the end of 8 weeks, though the cultures grew at both atrazine concentrations. This was in agreement with the findings of Kaufman and Blake (1970) who found no mineralization of 1°C-ring labeled atrazine by soil fungi, though mineralization by N- dealkylation was observed when l4C-chain labeled atrazine was used. In contrast, enrichment cultures containing 14C-ring labeled atrazine at a concentration of 100 ppm were shown to mineralize 80% or more of atrazine to 14C02 in 3 days (Mandelbaum et al., 1993a). This was attributed to the use of citrate and sucrose as mixed carbon sources and atrazine as the sole nitrogen source. The differences in the findings of these groups could be attributed to the fact that atrazine degradation and mineralization might be mediated by a mixed microbial consortia. This hypothesis is supported by the finding of Mandelbaum et al. (1993a) who noticed that more than 200 pure cultures isolated from the enrichment 25 cultures failed to utilize atrazine, whereas mixing these pure cultures restored atrazine mineralization. Behki and Khan (1986) isolated three species of Pseudomonas capable of utilizing atrazine as a sole source of carbon, from soil with a long history of atrazine application. Atrazine was metabolized via N-dealkylation with a preferential formation of deisopropylatrazine over deethylatrazine. These researchers also isolated a Rhodococcus strain (B-30) which rapidly degraded atrazine to its mono and di dealkylated analogs in 72 hours (Bth and Khan, 1994). The authors indicated that the presence of both alkyl groups and the presence of chlorine at the 2-position was necessary for the N -dealkylation of either alkyl group by the bacterial strain. This meant that the metabolites of atrazine could not be degraded further by the bacteria. Thus, atrazine metabolism has been observed to mainly occur by N-dealkylation and hydrolysis, with minimal degradation by ring cleavage, although recent efforts at isolating organisms capable of extensive mineralization of atrazine by ring cleavage have been successful. Radosevich et al. (1995) isolated a bacterial species which mineralized 40% of the initial ring labeled atrazine (22 ppm) in about 100 hours, while Yanze- Kontchou and Gschwind (1994) reported the isolation of a Pseudomonas strain (DSM 93- 99) which was capable of mineralizing 50% of a 30 ppm atrazine solution in 50 days. Atrazine metabolites detected at the end of the 50 days were cyanuric acid and hydroxyatrazine. Mandelbaum et al. (1995) reported isolating a Pseudomonas species that could mineralize 80% of a 100 ppm atrazine solution to 14C02 in 150 hours. This bacterium was also shown to degrade atrazine at concentrations of 1000 ppm in agar plates by the appearance of clearing zones on indicator agar plates. 26 A summary of the research on the degradation of atrazine by pure cultures of organisms is presented in Table 2.3. 2.3.3. In situ Degradation Numerous studies have been conducted to date on the degradation and mineralization of atrazine by microorganisms in situ in soils and other habitats (Erickson and Lee, 1989; Cook 1987). Kolpin and Kalkhoff (1993) observed a substantial decrease in atrazine concentration in a 11.2 km stretch of water in a creek in Iowa. They found that the concentrations of two biotic atrazine degradation products (CIAT and CEAT) were constant or decreasing downstream, suggesting an abiotic degradation process, possibly photolytic. Assaf and Turco (1994) found that atrazine degradation in soils amended with carbon as mannitol, and with nitrogen as urea at levels of 10, 30, 50, or 80 mg/kg was similar to degradation in unamended soils. They observed that 39% of applied atrazine was mineralized after 326 days regardless of the initial carbon or niU'ogen treatment. The major metabolite recovered was hydroxyatrazine with lesser quantities of the dealkylated metabolites also detected. Hance (1973) showed that the addition of inorganic nutrients and straw to soils doubled the rate of degradation of atrazine and decreased the half life of atrazine by 50%, though the effect of amendments on atrazine mineralization was not investigated. Winkelmann and Klaine (1991) reported that 59% of the ring labeled dealkylatrazine and 21% of the ring labeled hydroxyatrazine were mineralized by soil microcosms after 180 days of incubation. They also reported an exponential decrease in 27 Table 2.3: Summary of atrazine degradation research in pure cultures Researcher(s) Amount of Microorganism(s) Findingsa Atrazine (ppm) Mandelbaum et al. 100 Mixed bacterial culture 80% in 3 days (1993a) Mandelbaum et al. 100 Pseudomonas sp. 80% in 150 hours (1995) Radosevich et al. 22 Bacterial sp. 40% in 100 hours ( 1995) Yanze-Kontchou 30 Pseudomonas sp. 50% in 50 days and Gschwind (‘94) Donnelly et al. 215, 860 Nine different fungi (b) (1993) Kaufman and Blake 5 Twelve soil fungi (b) (1970) Behki and Khan 50 Pseudomonas sp. (c) ( 1986) Behki and Khan 15 Rhodococcus sp. (c) ( 1994) Mougin et al. 0.43 P. chrysosporium (b), (c) (1994) Selim and Wang 0.2 Granular activated 99% decrease in (1994) carbon bed atrazine concentration a - Percentages refer to amount of mineralization of MC-ring-labeled atrazine to ”C02 b - No mineralization by ring cleavage c - Mineralization of atrazine by N-dealkylation only 28 atrazine concentration over a period of 180 days, with > 90% disappearance in the first 60 days. Bacterial mixed cultures which had been previously reported to mineralize atrazine in liquid growth medium (Mandelbaum et al., 1993a), were further used in a study to determine whether they could metabolize atrazine in soil (Mandelbaum et al., 1993b). More than 90% of both atrazine and hydroxyatrazine were degraded after 24 hours of inoculation with the bacterial cultures, though the mineralization of atrazine obtained in pure cultures was not observed in soils. Wolf and Martin (1975) studied the degradation of ring labeled atrazine, cyanuric acid, and dealkylatrazine in soils and in pure culture. They observed an 18% mineralization of atrazine to 14C02 after 550 days of incubation as compared to 40% mineralization of dealkylatrazine in 192 days, and 87% mineralization of cyanuric acid in 16 days. R0 and Chung (1995), investigating the aerobic biotransformation of atrazine in wetland sediment, found that atrazine concentration dropped from 10 mg/L to less than 10 jig/L within three weeks of incubation at room temperature. In a related study, Chung et al. (1995) observed a much slower rate of degradation of atrazine under anaerobic conditions, with atrazine (10 ppm initial concentration) dropping to below detectable levels after 38 weeks of incubation. Solid-state fermentation of atrazine using bioreactors containing nutrient enriched peat moss resulted in an 86% disappearance of atrazine at the end of 26 weeks (Mullins et al., 1993), while the use of bioreactors containing steam exploded wood was shown to decrease solvent extractability of atrazine by 80% within 320 days (Berry et al., 1992) On the other hand, researchers have reported atrazine to be recalcitrant to ring cleavage in various environments including alluvial-aquifer sediments (McMahon et al., 29 1992), natural aquifers (Agertved et al., 1992), soils inoculated with Phanerochaete chrysosporium (Hickey et al., 1994), and soils (Nair and Schnoor, 1992; Dao et al., 1979; Skipper and Volk, 1972; Skipper et al., 1967; Goswarni and Green, 1971). The ultimate fate of atrazine in soils was investigated by Schiavon (1988) who found 49 to 67% of the initial l4C radiolabeled atrazine ill bound residues in the 0 to 6 cm level of soil columns incubated under field conditions for a year. Bidealkylated atrazine (CAAT) was found to form the highest amounts of bound residues followed by the monoalkylated metabolites and last of all by hydroxyatrazine. Khan (1991) reported that 54% of the initial l4C-atrazine (25 ppm) was found in bound residues after an incubation period of a year. Analysis of the residues revealed the presence of atrazine (3.7 ppm), and its metabolites hydroxyatrazine (1.5 ppm), deethylatrazine (2.1 ppm) and deisopropylatrazine (1.1 ppm). Similar results were observed by Winkelmann and Klaine (1991) who showed that, after 180 days incubation with soil microcosms, soil bound residues of atrazine and its metabolites accounted for as much as 60% of the initial radioactivity applied (as atrazine) to the microcosms. Blumhorst and Weber (1994) conducted experiments to investigate the relationship between chemical and microbial degradation of atrazine in soils ranging in pH from 5.3 to 8.1. They found atrazine degradation to be dominated by chemical processes at moderately acidic pH and by microbial processes at neutral pH. The research discussed in this section has been summarized in Table 2.4. 30 Table 2.4: Summary of atrazine (in situ) degradation research Researcher(s) Amount of Environment Findings atrazm’ 6 (ppm) Assaf and Turco 10 Soils 39% mineralization in (1994) 326 days Levanon (1993) 1 Soils 29% mineralization in 32 days Wolf and Martin 2.5 Soil 18% mineralization in (1975) 550 days Nair and Schnoor 0.37 Soil microcosms 1.5% mineralization in (1992) 100 days Skipper and Volk 2-3 Soil 0.1% mineralization in (1972) 2 weeks R0 and Chung 10 Wetland sediment 99% degradation in 3 (1995) weeks Winkelmann and 2 Soil 90% degradation in 60 Klaine (1991) days Hance (1973) 10 Soils + amendments Degradation rate (N utrients/Straw) doubled with amendment addition Hickey et al. (1994) 25 Soil amended with P. (a) chrysosporium Goswarni and Green 10-20 Submerged soils (a) ( 197 1) Dao et al. 1.5 Soil (a) ( 1979) Agertved et al. 0.4 Natural aquifer N o degradation ( 1992) (a) - No mineralization of atrazine by ring cleavage observed 31 2.4. Disposal Options for Pesticide Contaminated Sources The two main options currently available on a commercial basis for the disposal of pesticide contaminated water are evaporation/dc gradation and filtration technologies (Seiber, 1991). Evaporation based approaches include the pouring of the pesticide laden water on a bed of soil or some other matrix and allowing for the concentrating of the pesticide on the matrix by natural evaporation. The next step would be the degradation of the concentrated pesticide by the action of sunlight, microorganisms, chemical reactions, etc. The filtration process employs a similar strategy with activated charcoal as the matrix for adsorption/concentration of the pesticide. Though these processes address the issue of pesticide removal from waste streams, they still face the problem of disposal of the pesticide laden matrices. Options for disposal include composting, microbial treatment, incineration, and encasement. Research has been conducted with different matrices as adsorbents for pesticide laden wastes. Mullins et al., (1989) found peat moss to be an effective adsorbent for the pesticide diazinon, and observed a drop in diazinon concentration from initial values of 4000 to 32000 mg/kg to about 1 to 7 mg/kg after 18 weeks. Removal efficiencies of up to 98% were observed for the pesticides paraquat, diquat, and amitrole, using chemically modified peat as the adsorbent (MacCarthy and Djebbar, 1986). Extractive liquid membrane technology was used to assess the feasibility of extracting pesticides from rinsewaters typical of those arising at dealership sites (Norwood, 1992). Results showed a removal of 85.4 and 92.9% of 2,4-D and atrazine respectively after 15-20 minutes of mixing time. 32 Dennis and Kobylinski (1983), studied the effect of adsorption of seven different pesticides by a granular activated carbon system. They observed that, at pesticide loading rates of up to 100 ppm, absorption efficiencies of 95% and greater were achieved in 21 hours by 45 lb of granular carbon. A simple activated charcoal filtration system, with a startup cost of about $1200, was investigated by Massey et al. (1992). They observed a drop in pesticide concentrations from initial values of 300 to 1000 ppm to less than 10 ppm after 120 minutes of filtration through, and adsorption by the activated charcoal. Incubation of alachlor treated charcoal with a mixed culture of microorganisms resulted in approximately a 50% loss of alachlor after 50 days. Hunter (1992) showed supercritical extraction using C02 to be a viable alternative for the removal of pesticides from contaminated soils, with >95% removal of atrazine, bentazon, alachlor, and permethrin. The advantages of using C02 were that CO2 is non- polluting, inexpensive, and easily recycled. The disadvantages were the high cost, high operating pressure (3000 psi), and the fact that different contaminants could require different extracting conditions. Martinez-Inigo and Alrnendros (1992) found the addition of composted evergreen oak to soils significantly enhanced the sorption of atrazine as compared to composted evergreen alone. 2.5. Bioremediation of Pesticides Using Added Microbial Inocula Dzantor and Felsot (1991) found that introducing an alachlor cometabolizing fungus into contaminated soil at 0.015% w/w caused a marginal increase in the degradation of 100 ppm of alachlor. Increasing the amount of inoculum to 0.045% w/w showed a slightly higher rate of alachlor loss (50% of the initial alachlor at the end of 55 ya-.. 33 days, as compared to 40% with 0.015% inoculum and 30% with no inoculum). They also found that amending the soils with 2% corn residue completely masked the effects of fungal inoculation. Addition of a pentachlorophenol utilizing Arthrobacter to soil enhanced the removal of the pesticide (Edgehill and Firm, 1983). Similar results were observed by inoculation of contaminated soil with a pentachlorophenol degrading Flavobacterium species, however, several inoculations were required for substantial removal of the pesticide (Crawford and Mohn, 1985). The reason for the rapid decline of the F lavobacrerium species was thought to be the inability to compete with the native microorganisms. Repeated applications of Pseudomonas cepacia strain AC1100 were also needed to maintain biodegradation of 2,4,5-T in soil, and the strain rapidly died in the absence of 2,4,5-T (Kilbane et al., 1983). Successful field scale bioremediation of soils contaminated with up to 9000 mg of chlorophenols per kg of dry soil by composting has been demonstrated (Valo et al., 1986). Bioremediation has its basis in the physiology and the ecology of the microorganisms. The survival and activity of externally introduced microorganisms in any environment is an important factor in attempts to use them in field applications. Cleanup methods thus have to be designed around the capabilities of the microorganisms, which requires detailed knowledge of the biodegradation pathways and the requirements and behavior of the microorganisms. 3. Pesticide Degradation During Composting Composting has proved to be an effective technology for the bioremediation of nitrated aromatics and nitrated triazine explosives contaminated soils (Williams et al., 34 1992; Williams et al., 1990; Doyle et al., 1986). In addition, a wide variety of pesticides including atrazine, 2,4-D, diazinon, parathion, chlorpyrifos, pendimethialin and benomyl have been shown to degrade/disappear during composting (Vogtmann, 1984; Lemmon and Pylypiw, 1992). Harrad et al. (1991) found municipal yard waste composting to concentrate and increase the amounts of chlorophenols, chlorobenzenes, PCDDs, and PCDFs. Compost amendments were found to enhance the degradation of 2,4-D, MCPA, and benthiocarb in soils with complete degradation of the herbicides in 8 days as opposed to 13 days without the amendment (Duah-Yentumi and Kuwatsuka, 1982). These researchers also studied the effects of anaerobic (reductive-flooded) and aerobic (oxidative-flooded) conditions on the degradation of the same herbicides and found that compost amendments promoted the degradation of the pesticides under aerobic conditions, whereas under anaerobic conditions the degradation was very slow, with compost amendments having no effect on the process (Duah-Yentumi and Kuwatsuka, 1980). Rose and Mercer (1964) explored the potential of composting for decomposing diazinon, parathion, DDT, and dieldrin. They found that composting lowered the diazinon concentration by 98% in 42 days, while a 50% degradation of parathion was noticed in 12 days. They found that composting had a minimal effect on DDT and dieldrin. Research by others (Wilson et al., 1983; Geunzi and Beard, 1968) showed that these recalcitrant compounds (DDT and dieldrin) could be degraded using a combination of techniques such as composting followed by anaerobic digestion. The behavior of five pesticide residues was studied during aerobic and semi- anaerobic composting of cotton gin wastes (Winterlin et al., 1986). Propargite, 35 methidathion, and chlorate residues declined significantly during both composting treatments, while DEF and paraquat were stable to composting. Vogtrnann et. al. (1984) demonstrated that greater than 80% degradability could be obtained on average for a variety of pesticides. However, degradation of the pesticides in both studies was based on the disappearance of the pesticides and not on complete mineralization. Thus there is ample evidence of the ability of compost matrices to degrade a broad range of pesticides. Despite the publication of guidelines for the use of composting in the treatment of hazardous materials and industrial wastes (Becker et al., 1985; Kaplan and Kaplan, 1982; Willson et al., 1982; Muller and Korte, 1976) there is a lack of information regarding the processes involved and the ultimate fate of xenobiotics and pesticides in composting systems (Fogarty and Tuovinen, 1991). 3.1 Pesticide Degradation during Composting of Lignocellulosics One avenue of research that has not been well investigated is the co-metabolism of pesticides during the composting of lignocellulosic substrates. Lignocellulosic materials are an ideal choice as a composting substrate because they have been shown to concentrate pesticides from wastewater sources owing to their high sorption characteristics (Mullins et al., 1993; Hetzel et al., 1989; Toller and Flaim, 1988). Furthermore, enzyme systems of certain lignocellulose degrading organisms might also gratuitously degrade xenobiotics due to their non-specificity, and structural similarity of the xenobiotics to portions of the lignocellulose substructure. The non-specificity arises from the fact that these enzyme systems are oxidative in nature (rather than hydrolytic), using free radical intermediates to accomplish the task of degrading the substrates (Reddy, 1995; Boominathan and Reddy, 1992; Harnmel, 1992; Bumpus et al., 1985). 36 The main class of organisms exhibiting this phenomenon are the lignin degrading white rot fungi, of which P. chrysosporium has been the most widely studied (Boominathan and Reddy, 1992; Crawford and Crawford, 1980). 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Bianchin, A. Pera, and M. DeBertoldi (1984). Composting agro- industrial byproducts. Biocycle, May/June : 4346,51. Valo, R. and M. Salkinoja-Salonen (1986). Bioreclamation of chlorophenol-contaminated soil by composting. Appl. Microbial. Biotechnol. 25, 68-75. Vogtmann, H., P. v. Fragstein, and P. Draeger (1984). The degradation of agrochemicals during composting. In Proceedings of the Second International Symposium Peat in Agriculture and Horticulture. K. M. Schallinger (ed.). pp. 357-378. Weissenfels, W.D., H.J. Klewer, and J. Langhoff (1992). Adsorption of polycyclic aromatic hydrocarbons (PAHs) by soil particles: influence on biodegradability and biotoxicity. Appl. Microbial. Technol. 36, 689-696. Widmer, S. K., J. M. Olson, and W. C. Koskinen (1993). Kinetics of atrazine hydrolysis in water. J. Environ. Sci. Health B28(1), 19-28. Williams, RT. and CA. Myler (1990). Bioremediation using composting. Biocycle November, 78-80, 82. Williams, R.T., P.S. Zeigenfuss, and W.B. Sisk (1992). Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. J. Ind. Microbial. 9, 137-144. Willson, G.B., J .F. Parr, J .M. Taylor, and L.J. Sikora (1982). Land treatment of industrial wastes : Principles and practices. Biocycle. Jan./Feb., 37-42. 49 Willson, G.B., L.J. Sikora, and J.F. Parr (1983). Composting of chemical industrial wastes prior to land application. In Land treatment of hazardous wastes, Noyes Data Corp., Park Ridge, New Jersey. Winkelmann, DA. and SJ. Klaine (1991). Atrazine metabolite behavior in soil-core microcosms. In L. Somasundararn and JR. Coates (eds.), Pesticide transformation products: Fate and significance in the environment. ACS Symposium Series 459, American Chemical Society, Washington, DC. Winterlin, W.L., M.M. McChesney, S.R. Schoen, and J.N. Seiber (1986). Chemical residues during screening, composting and soil incorporation of cotton gin waste. J. Environ. Sci. Health. B21(6), 507-528. Wolf, D. C. and J. P. Martin (1975). Microbial decomposition of ring-”C atrazine, cyanuric acid, and 2-chloro-4,6-diamino-s-triazine. J. Environ. Qual. 4, 134-139. Yanze-Kontchou, C. and N. Gschwind (1994). Mineralization of the herbicide atrazine as a carbon source by a Pseudomonas strain. Appl. Environ. Microbiol. 60, 4297-4302. Chapter 111 Effect of Temperature on the Transformation of Atrazine During the Composting of Poplar Wood and Corn Cobs (Manuscript) 50 51 ABSTRACT Mineralization of atrazine during the composting of poplar wood was investigated at three different temperatures: 25°C, 37°C, and 55°C. In addition, the effect of poplar wood as the composting substrate versus corn cobs as the substrate on atrazine mineralization was investigated at 55°C. Maximal mineralization of poplar wood carbon to CO; was observed at 37°C, with 10% mineralization of the poplar wood at the end of 84 days of incubation. Also, the extent of mineralization of corn cobs was higher at 55°C (>15%) than that of poplar wood as substrate at 55°C (<6%). Mineralization of atrazine was minimal in all cases. INTRODUCTION Composting is an easily manageable, environmentally safe, and relatively inexpensive alternative for the disposal of municipal solid wastes and yard wastes (Fogarty and Tuovinen, 1991; Michel et al., 1993). Lignocellulosic materials are an ideal choice as a substrate for the disposal of pesticides via composting, because lignocellulose degrading enzymes may be important for the degradation of pesticides and xenobiotics (Reddy, 1995; Boonrinathan and Reddy, 1992; Hammel, 1992; Fogarty and Tuovinen, 1991; Bumpus and Aust, 1987). Furthermore, they have been shown to concentrate pesticides from wastewater sources owing to their high sorption characteristics (Mullins et al., 1993; Hetzel et al., 1989). Atrazine degradation and mineralization in soils and by microorganisms in vitro has been studied extensively at mesophilic temperatures (Erikson and Lee, 1989; Cook, 1987). Kaufman and Blake (1970) reported that no l“co, was evolved from ”c-ting labeled 52 atrazine in pure culture solutions of soil fungi maintained at 24°C. Nair and Schnoor (1992) found about 1% mineralization of atrazine in 125 days in soils incubated at 25°C. McCormick and Hiltbold (1966) investigated the microbial degradation of atrazine in soils at different temperatures. They concluded that the rate of degradation approximately doubles with each 10 degree increase from 10°C to 30°C. However, there is no information on the relative rate and extent of mineralization of atrazine when it was composted with lignocellulosic substrates at different temperatures. The rate and extent of mineralization of atrazine during composting of lignocellulosic substrates at three different temperatures, 25°C, 37°C, and 55°C are presented in this paper. MATERIALS AND METHODS Pesticides [U-t-ing-“clAtrazine (specific activity: 7.8 mCi/mmol; purity>98%) was obtained from Sigma Chemicals, St. Louis, MO. AAtrex 4L, a commercially available sprayable atrazine emulsion (50% active ingredient), was obtained from Ciba-Geigy (Greensboro, NC). Analytical grade atrazine was obtained from Chem Service (West Chester, PA). C ampast Substrates The poplar wood used in this study was provided by the NSF Center for Microbial Ecology at Michigan State University, and is a hybrid between Papulus nigra and Papulus deltoides and has been designated Papulus x euramericana cv. Eugenei. The wood was ground in a Wiley mill through a #10 screen (1.7 mm mesh size) and adjusted to 70% moisture (g/g wet wt.) by adding distilled water. Corn cobs obtained from a local store (Soldan’s Pet Supplies, Lansing, M1) were also adjusted to 70% moisture and used as a 53 substrate in another experiment. Each substrate was loaded into duplicate composters to give 100 g dry substrate per composter. The composters were amended with [U-ring- ”C]Atrazine (5.6 uC per composter) and AAtrex (500 mg atrazine/kg dry substrate). The inoculum (10% w/w) used for composting was obtained from 10-week-old wood compost piles operated by a large scale composting facility (Hollandia Gardens, Holland, MI). Composting System Composting was carried out in a laboratory scale composting system recently described by Michel et al. (1993). The effect of temperature on atrazine mineralization was studied under laboratory conditions by running the composters at three different temperatures: 25°C, 37°C, and 55°C. In the last case, the composters were started out at room temperature in a temperature programmable incubator, the temperature ramped to rise at a rate of 5°C a day up to a temperature of 55°C, and then held constant at 55°C for the duration of the experiment to simulate temperature patterns generally observed in windrow composting using yard trimmings as the substrate (Michel et al., 1993). The second set of composters were placed in a temperature controlled room maintained at 37°C, while the third set of composters were incubated at ambient temperature (~25°C). Praceduresfar measuring C02 Evolution and F ractianatian of the Compost. Total CO; as well as the amount of MC02 evolved during composting was measured as described by Michel et al. (1993). Extraction of atrazine and its metabolites were carried out using the procedure described by Rao et al. (1995). Unextractable radiolabel was measured by combusting 100 mg samples of extracted compost as described by Michel et al. (1995). 54 RESULTS AND DISCUSSION Effect of temperature on substrate mineralization. Mineralization of substrate carbon to CO; was most effective at 37°C (Figure 3.1). A 10% conversion of biomass to CO; was achieved at the end of 84 days of composting at 37°C as compared to a 5.9% conversion at 55°C and 4.6% conversion at 25°C. Statistical analysis showed that the amounts of C02 produced at the three temperatures were statistically different. The higher conversion at 37°C suggests higher microbial activity at that temperature. McKinley and Vestal (1985, 1984) reported the greatest microbial activity in compost samples taken from lower temperature areas (25°C-45°C) of sewage sludge windrow composts. Snell (1957) investigated the effect of temperature on the lab-scale composting of ground garbage and reported an optimum temperature of 45°C based on the oxygen uptake rates in the composters. On the other hand, Schulze (1962) observed that temperature was directly related to microbial oxygen uptake rate between 30°C and 70°C during the small-scale composting of garbage. Other researchers have reported composting temperature optima around 55-60°C based on the amount of substrate converted to C02, using substrates such as sewage sludge (MacGregor et al., 1981), table scrap (Suler and Finstein, 1977), and municipal refuse (Jeris and Regan, 1973). However, interpretation of these results is diffith due to the variations in the compost substrates and differences in the composting systems. Since the compost substrate used in this study was poplar wood, a slow degrading substrate principally susceptible to fungal attack (Boominathan and Reddy, 1992), the higher conversion seen at 37°C could be an indicator of fungal degradation rather than bacterial degradation. 55 Effect of temperature on atrazine mineralization. Mineralization of atrazine during the composting of poplar wood as the substrate was minimal at the three different temperatures (Figure 3.2), with an observed mineralization of only about 1%, and did not reflect the differences seen in the conversion of total substrate carbon to C02. Also, the radiochemical purity of 1“C labeled atrazine was reported as only >98%. Thus there could be a possibility that the 14C02 evolved might not be from 14C labeled atrazine. These results indicate a lack of direct correlation between the mineralization of poplar wood and that of atrazine. Similar results were reported by other researchers (Nair and Schnoor, 1992; Kaufman and Blake, 1970) who observed minimal mineralization of l“c-ring labeled atrazine to "co: in soils at 25°C. Effect of an amendment on substrate mineralization. The rate and extent of conversion of corn cobs to C02 at 55°C was about 18% after 84 days of composting, whereas in otherwise identical composters, but with poplar wood as the substrate, <6% mineralization was observed (Figure 3.3). Effect of amendment on atrazine mineralization. Mineralization of atrazine was minimal either with corn cobs or poplar wood as the substrate (Figure 3.4). For example, about 1% mineralization of atrazine was observed with wood as the substrate but only about half that much mineralization was observed with corn cobs as the substrate. Thus, there was no apparent correlation between the rate and extent of atrazine mineralization and mineralization of corn cobs to C02. Atrazine degradation during composting. Radioactivity distribution data (Figure 3.5) showed that on day zero, 1"C was primarily in the chloroform fraction. Samples taken on 56 day 60, on the other hand, showed a change in the distribution with a greater amount of the 14C in the NaOH+Na4P201 fraction, which extracted polar metabolites and hunric components. For example, 95% of the extracted 1“C in the day zero sample from wood (in composters at 55°C), was in the chloroform fraction which extracted atrazine, with 4% in the methanol fraction which extracted non-polar metabolites of atrazine. On the other hand, only 14% was extractable into the chloroform fraction at the end of 60 days of composting, whereas 40% was extractable into the methanol fraction and 15% into the NaOH+Na4P207 fraction with 28% in bound (unextractable) residues. This general trend towards lesser amounts of radioactivity in the chloroform fraction and greater amounts of radioactivity in the methanol and NaOH+Na4P207 fractions was seen in the other composts as well. These results suggest that atrazine is being transformed into more polar metabolites and/or is complexing with hunric components, as shown recently in the case of 2,4-D composting by Michel et al. (1995), who showed that about 23% of 2,4-D carbon is present in the high molecular weight humate fraction during the composting of 2,4-D with yard trimmings. Our results are further supported by the findings of Winkelmann and Klaine (1991) who showed that, after 180 days incubation with soil microcosms, soil bound residues of atrazine and its metabolites accounted for as much as 60% of the initial radioactivity applied (as atrazine) to the microcosms. CONCLUSIONS Mineralization of atrazine during the composting of wood was evaluated at three different temperatures: 25°C, 37°C, and 55°C. Conversion of wood was higher at 37°C than at 25°C or 55°C. Mineralization of atrazine was minimal at all three temperatures. A 57 comparison of wood as the substrate at 55°C to corn cobs (also at 55°C) showed that a higher percentage (18%) of the carbon from corn cobs was mineralized to CO; as compared to that from poplar wood as substrate; however, mineralization of atrazine to 1‘co. was minimal (<2%) with either of the substrates. REFERENCES Boominathan, K., and C. A. Reddy (1992). Fungal degradation of lignin: biotechnological applications, p. 763-822. In D. K Arora, R. P. Elander, and K. G. Mukerji (ed.), Handbook of applied mycology, vol. 4. Fungal biotechnology. Marcel Dekker, Inc., New York. Bumpus, J. A. and S. D. Aust (1987). Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysosporium: involvement of the lignin-degrading system. Biaassays 6, 116-200. Cook, A. M. (1987). Biodegradation of s-triazines. FEMS Microbial. Rev. 46, 93-116. Erickson, L. E. and K. H. Lee (1989). Degradation of atrazine and related s-triazines. Critical Rev. Environ. Control 19, 1-14. Fogarty, A. M. and O. H. Tuovinen (1991). Microbiological degradation of pesticides in yard waste composting. Microbiol. Rev. 55, 225-233. Hammel, K. E. (1992). Oxidation of aromatic pollutants by lignin-degrading fungi and their extracellular peroxidases, p. 41-60. In H. Sigel and A. Sigel (ed.), Metal ions in biological systems, vol. 28. Degradation of environmental pollutants by rrricroorganisms and their metalloenzymes. Marcel Dekker, Inc., New York. Hetzel, G. H., D. E. Mullins, R. W. Young, and J. M. Simmonds (1989). Disposal of dilute and concentrated agricultural pesticides using absorption and chemical and microbial degradation. p. 239-248. In: D. L. Weigmann (ed.), Pesticides in terrestrial and aquatic environments, Proc. Natl. Res. Conf. Virginia Water Resourc. Res. Center, Virginia Polytech. Inst. and State Univ. Blacksburg, VA. Hickey, W. J ., D. J. Fuster, and R. T. Lamar (1994). Transformation of atrazine in soil by Phanerochaete chrysasparium. Sail Biol. Biochem. 26, 1665-1671. Jeris, J. S., and R. W. Regan (1973). Controlling environmental parameters for optimal composting. 1. Experimental procedures and temperature. Compost Sci. 14, 10-15. 58 Kaufman, D. D. and J. Blake (1970). Degradation of atrazine by soil fungi. Soil Biol. Biochem. 2, 73-80. MacGregor, S. T., F. C. Miller, K. M. Psarianos, and M. S. Finstein (1981). Composting process comtrol based on interaction between microbial heat output and temperature. Appl. Environ. Microbial. 41, 1321-1330. McCormick, L. L. and A. E. Hiltbold (1966). Microbiological decomposition of atrazine and diuron in soils. Weeds 14, 77-82. McKinley, V. L., and J. R. Vestal (1985). Physical and chemical correlates of microbial activity and biomass in composting municipal sewage sludge. Appl. Environ. Microbial. 50, 1395-1403. McKinley, V. L., and J. R. Vestal (1984). Biokinetic analyses of adaptation and succession: microbial activity in composting municipal sewage sludge. Appl. Environ. Microbiol. 47, 933-941. Michel Jr., F. C., C. A. Reddy, L. J.Forney (1995). Microbial degradation and humification of the lawn care pesticide 2,4-Dichlorophenoxyacetic acid during the composting of yard trimmings. Appl. Environ. Microbial. 61, 2566-2571. Michel Jr., F. C., C. A. Reddy, and L. J. Forney (1993). Yard waste composting: Studies using different mixes of leaves and grass in a laboratory scale system. Compost Sci. Util. 1, 85-96. Mullins, D. E., R. W. Young, D. F. Berry 9 J.-D. Gu and G. H. Hetzel (1993). Biologically based sorbents and their potential use in pesticide waste disposal during composting. In: K. D. Racke and A. R. Leslie (eds.), Pesticides in Urban Environments: Fate and significance. ACS Symposium Series No.522, American Chemical Society, Washington, DC, 113-126. Nair D. R. and J. L Schnoor (1992). Effect of two electron acceptors on au'azine mineralization rates in soil. Environ. Sci. Technol. 26(11), 2298-2300. Rao, N., H. E. Grethlein, and C. A. Reddy (1995). Mineralization of atrazine during the composting of untreated and pretreated lignocellulosic materials. Accepted for publication in Compost Sci. Util. Reddy, C. A. (1995). The potential for white-rot fungi in the treatment of pollutants. Curr. Opin. Biatechnal. 6, 320-328. Schulze, K. L. (1962). Continuous thermophilic composting. Appl. Microbial. 10, 108- 122. 59 Snell, J. R (1957). Some engineering aspects of high-rate composting. J. Sanit. Eng. Div. Proc. Am. Soc. Civ. Eng. 83, Paper 1178, 1-36. Suler, D. J., and M. S. Finstein (1977). Effect of temperature, aeration, and moisture on C0; formation in bench-scale, continuously thermophililc composting of soild waste. Appl. Environ. Microbial. 33, 345-350. Winkelmann, DA. and SJ. Klaine (1991). Atrazine metabolite behavior in soil-core microcosms. In L. Somasundararn and LR. Coates (ed.), Pesticide transformation products: Fate and significance in the environment. ACS Symposium Series 459, American Chemical Society, Washington, DC, 75-92. Yadav, J. S. and C. A. Reddy (1993). Degradation of benzene, toluene, ethylbenzene, and xylene (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysasparium. Appl. Environ. Microbial. 59, 756-762. «0.16 8 —0— Wood 37°C 2 0.14 — —0— Wood 55°C '8 —A— Wood 25°C t g 0.12 '- C 8 g 0.10 - o .n . ° :5 o a 0.08 —' . E 006 .4 . . a . g I I a ' - - o I A ‘ 5’3 0.04 — . . ' ' ‘ . 0 II ‘ g A c ' A . g 0.02 “ . "/"’= A E ,A 0-00 ‘9’ I I l I 0 20 40 60 80 Days 100 Figure 3.1: Conversion of poplar wood to CO; at three temperatures. Values presented are means :1: half range for duplicate composters. 61 3 —0— Wood 37°C —0— Wood 55°C + Wood 25°C 8 2 'fi 2 - E -_ ‘6 0.— ° 5 c . .2 I AO‘IA “ A ii . its-2.3.3": . -- la 1 _ ‘i‘JiAE'ig‘..f I I a; _/- in... .E E O I l 60 80 100 Days Figure 3.2: Mineralization of l“C-ring labeled atrazine to 14C02 during the composting of poplar wood at three different temperatures. Values presented are means :1: half range for duplicate composters. 62 0.20 0.18 - 0.16 A 0.14 - 0.12 - 0.10 - 0.08 - 0.06 - Fraction of Substrate Carbon converted to CO2 -0— Wood 55°C --0— Cobs 55°C 40 60 80 Days 100 Figure 3.3: Conversion of substrate carbon to C02 during composting at 55°C. Values presented are means :1: half range for duplicate composters. 63 N l Mineralization of atrazine (%) -0— Wood 55°C —0- Cobs 55°C a... 1- ...oo°. .. . III-I... I I II "— I'.’ .o 0'9 I l I F 0 20 40 60 80 Days 100 Figure 3.4: Mineralization of l“c-ring labeled atrazine to 1“co, during composting at 55°C. Values presented are means :t: half range for duplicate composters. A. Wood 25 B. Wood 37 Distribution of "C aayo Dayao oayeo Dayo Day30 Dayso I CHCI3 El MeOH I NaOH I H20 I 002 El Bound Figure 3.5 Distribution of 14C from compost samples at different time periods in various extraction solvents. Extraction procedures used are described in Materials and Methods. ‘NaOH’ in the legend box refers to the fraction of 14C radiolabel extracted into the NaOH+Na4P207 solution. ‘Bound’ in the legend box refers to the unextracted fraction of ”c radiolabel. Chapter IV Mineralization of Atrazine During Composting with Untreated and Pretreated Lignocellulosic Materials Published in: Compost Science and Utilization 3:38-46 (1995). 65 66 Abstract Composting offers a relatively inexpensive and environmentally safe method for the potential bioremediation of pesticide-laden rinsewater. The purpose of this study was to evaluate whether degradation and mineralization of atrazine, a pesticide used extensively in the US, can be enhanced by composting with pretreated lignocellulosic materials as compared to untreated lignocellulosics. Wood that was subjected to steam explosion (STEX wood) or ammonia explosion (AFEX wood), untreated wood (native), and shredded newspaper were selected as the composting substrates. These substrates which differed in composition as determined by the quantitative saccharification and enzymatic hydrolysis techniques, were amended with [U-ring-“C]Atrazine (500 ppm, 5.6 uCi per composter) and composted in 2-liter lab-scale composters. The results showed that the highest rate and extent of total organic matter mineralization to CO; was observed with AFEX wood as the substrate, but atrazine mineralization was relatively higher (11%) with paper as the substrate. There was no significant enhancement in atrazine mineralization when composted with the pretreated woods (AFEX wood and STEX wood) as compared to that observed with the native wood. Thus, pretreatment of the wood, which was hypothesised to lead to increased substrate and atrazine mineralization, was seen to have no added effect on atrazine mineralization. Introduction A variety of pesticides in common use in agricultural and lawn care applications represents a potential threat to public health and environmental quality. Failure to use proper procedures at pesticide mixing and handling sites and improper disposal of 67 pesticide laden rinsewater, which can contain from 30 to 2000 mg/L pesticide, can result in contamination of soil, surface water, and groundwater (Myrik, 1990; Norwood, 1990; Toller and Flaim, 1988). Atrazine is one of the most widely used pesticides in the U.S. accounting for about 12% of all the pesticides used in this country (Aspelin et al., 1991). Over 36 million kg of atrazine were applied nationwide in 1990 (Periera and Rostad, 1990). Atrazine and its metabolites are the most frequently detected pesticides in surface waters of the midwestern US (Thurman et a1, 1991). Composting is a relatively inexpensive, easily manageable, and environmentally safe alternative that is increasingly being used in the disposal of municipal solid wastes and yard wastes (Fogarty and Tuovinen, 1991; Michel et al., 1993). Lignocellulosic materials are an ideal choice as a composting substrate because they have been shown to concentrate pesticides from wastewater sources owing to their high sorption characteristics (Toller and Flaim, 1988; Hetzel et al., 1989; Mullins et al., 1993). Furthermore, lignocellulose degrading enzymes may be important for the degradation of pesticides and xenobiotics (Hammel, 1992; Boorrrinathan and Reddy, 1992; Fogarty and Tuovinen, 1991; Bumpus and Aust, 1987). Mullins et al. (1993) proposed that pesticides sorbed on lignocellulosic materials such as steam exploded wood, newspaper, peanut hulls, and peat moss could be degraded to nontoxic products via composting and that this system represents a cost effective and technically uncomplicated approach to treat pesticide laden wastewater. These investigators found peat moss and steam exploded wood to be excellent sorption materials for the pesticides Chlorpyrifos and metolachlor, with a nearly complete removal of the pesticide from solution. 68 Solid-state fermentation of atrazine using bioreactors containing steam exploded wood was shown to decrease solvent extractability of atrazine by 80% within 320 days (Berry et al., 1993). Solid-state fermentation of atrazine using bioreactors containing nutrient enriched peat moss resulted in an 86% disappearance of atrazine at the end of 26 weeks (Mullins et al., 1993). However, the extent of mineralization of atrazine and its fate (mineralization, degradation to polar metabolites, and humification) during composting was not investigated. Also, there is no information on the relative rate and extent of mineralization of atrazine when it was composted with different lignocellulosic substrates. The rate and extent of mineralization of atrazine during composting of lignocellulosic substrates with differing physical and chemical characteristics are presented in this paper. The pretreated substrates were chosen based on the increase in surface area due to the pretreatment (Thompson et al., 1992) and due to the increase in pore size of the substrates leading to better substrate accessibility to enzymes (Grous et al., 1986; Dale and Moreira, 1982). The hypothesis was that pretreatment of the substrate would result in faster conversion of substrate to C02 and would also lead to increased mineralization of atrazine in the compost matrix. Also, steam explosion and ammonia explosion are two of the more widely used pretreatment procedures for increasing the accessibility of the carbohydrate polymers of wood to cellulases and herrricellulases (Grous et al., 1986; Dale and Moreira, 1982). 69 Materials and Methods Pesticides [U-ring-"clAtrazine (specific activity: 7.8 mCi/mmol; purity>98%) was obtained from Sigma Chemicals, St. Louis, MO. Analytical grade atrazine was obtained from Chem Service (West Chester, PA). AAtrex 4L, a commercially available sprayable atrazine emulsion (50% active ingredient), was obtained from Ciba-Geigy (Greensboro, NC). Compost Substrates Four substrates were used in this study: untreated poplar wood (Native), ammonia exploded (AFEX) wood, steam exploded (STEX) wood, and shredded newspaper, a lignocellulosic substrate derived from chemi-mechanical treatment of wood. The poplar used in this study was a hybrid grown at the Kellogg Biological Station and was provided by the NSF Center for Microbial Ecology at Michigan State University. The clone is a hybrid between Papulus nigra and Papulus deltaides and has been designated Papulus x euramericana var. eugenei. The poplar wood was ground in a Wiley mill through a #10 screen (1.68 mm mesh size) and was used in this form or was subjected to the following pretreatrnents: 1. steam explosion at 350 psi for 5 minutes (Grous et a1, 1986); and 2. ammonia explosion in which the native wood (at 30% moisture, g/g dry wood) was treated for 30 min with anhydrous ammonia (3 kg per kg dry wood) at 50°C and a conventional AFEX explosion was used (Dale and Moreira, 1982). The AFEX wood was provided to us by Dr. Bruce Dale, Texas A&M University, College Station, TX. Newspaper shredded to 3/8” strips, was obtained from Applegate Insulation, Lansing, MI. 70 Water was added to each lignocellulosic substrate to 70% moisture (g/g wet wt.) and each substrate was loaded into three replicate composters. The composters were amended with [U -ring-”C]Atrazine (5.6 uCi per composter) and AAtrex (500 ppm atrazine/g dry substrate). The inoculum (10%) used for composting was obtained from 10- week-old wood compost piles operated by a large scale composting facility (Hollandia Gardens, Holland, MI). Composting System Composting was carried out in the laboratory scale composting system recently described by Michel et al. (1993). In brief, the system consisted of a rubber stoppered 2- liter, wide mouth glass jar with two plastic screens (1cm and 1mm mesh opening) forming a false bottom. Aeration was provided through a hole just below the level of the two screens. C02-free, humidified air for the composters was obtained by passing the air through a flask containing 5 N NaOH to remove C02 and then through a 5 gallon carboy containing 2.5 gallons of distilled water. The air flow to each of the composters was set at 100 ml/min by means of a needle valve placed just upstream of the composters. The exhaust gas from each composter passed through a polyethylene tube containing polyurethane foam plugs to trap any volatiles and then into two 5N NaOH containers to trap C02 present in the exhaust gas. To ensure that the 1“C in the NaOH traps was due to MC02 and not due to volatilized l°C-atrazine or other volatile organics derived from 14C- atrazine, a barium chloride precipitation step was included as previously described (Yadav and Reddy, 1993). The entire system was placed in a temperature controlled room which was maintained at 37°C. 71 Compost Analysis Substrate Composition. The quantitative saccharification technique developed by Saeman et al. (1945) was used to determine the cellulose, hemicellulose, and lignin contents of the initial compost substrates. The procedure consisted of hydrolysis of the samples with 72% sulfuric acid for an hour, dilution to a 4% acid solution, and then autoclaving at 121°C for an hour. Sugar concentrations in the hydrolysate were measured using high pressure liquid chromatography (Converse et al., 1989). Glucose obtained from sample hydrolysis was assumed to originate from cellulose and five carbon sugars and mannose were assumed to originate from henricellulose (Grous et al., 1986; Thompson et al., 1992). Enzymatic Hydrolysis. Enzymatic hydrolysis of each lignocellulosic substrate used for composting was carried out with cellulase and B-glucosidase using the method of Thompson et al. (1992). Cellulase (Novo CCN 3000, a cellulase from Trichoderma reesei with a cellulase activity of 52.1 FPU/ml) and B-glucosidase (Novozym TN 188, isolated from Aspergillus niger and having an activity of 588 U/ml) were purchased from Novo Industries (Copenhagen, Denmark). The enzymes were used at a level of 80 units/g dry substrate. Glucose yield from the hydrolysis was calculated as a percentage of the theoretical maximum glucose yield based on the amount of cellulose in the original sample. CO; Evolution. Total C02 as well as the amount of 1“C02 trapped in the NaOH trap was measured as described by Michel et al. (1993). Compost Extraction Procedure. Since atrazine and its metabolites exhibit a wide range of polarities and solubilities (Judge et al., 1993), a modified extraction procedure based on 72 the methods of Smith (1981), FDA (1982), and McCall (1981) was used to determine the distribution of radioactivity in the composts. The extraction procedure was based on the fact that solubility of atrazine is 52000 ppm in chloroform, 18000 ppm in methanol, and 33 ppm in a polar solvent such as water. Samples were periodically taken from the composters and refrigerated until analyzed. The samples were successively extracted with chloroform and methanol (to extract atrazine and its non-polar metabolites), 0.1M NaOH+Na4P207 solution (to extract polar metabolites and the humic components), and water. Each extraction was carried out with a given solvent till no further 1“C was extracted. l"C extracted in each step was calculated by mixing an aliquot of the sample with liquid scintillation cocktail and counting as previously described (Michel et al., 1993). Thin Layer Chromatography. The chloroform and methanol extracts were concentrated to about 30 [11 each and 10 ul was spotted on Silica gel 60, F254 TLC plates (Merck, EM Industries, Gibbstown, NJ) to detect the presence of residual 1°C-atrazine. The plates were developed using a chloroform-acetone (3:2, v/v) mobile phase (Judge et al., 1993). Radioactive spots on the plate were scanned using a Bioscan System 200 Imaging scanner (Bioscan Inc., Washington, DC). Results and Discussion Substrate Composition. The composition of the lignocellulosic materials, obtained by the quantitative saccharification technique, is shown in Table 4.1. The composition of the native wood and AFEX wood were very similar. The STEX wood on the other hand showed a reduced henricellulose content (i.e. xylan, mannan, arabinan, and galactan) of 8.16%, as compared to 16.17% in native wood, and an increase in the glucan content. The 73 composition of the shredded newspaper was similar to that of native wood though there was a qualitative difference in the hemicellulose content. Enzymatic Hydrolysis. Enzymatic hydrolysis data (Figure 4.1) showed that the rate of glucose release as well as the total yield were the greatest with STEX wood (91.5% of the theoretical maximum) which was attributed to the removal of the hemicellulose making cellulose more accessible to cellulase and B-glucosidase (Grous et al., 1986). AFEX wood and newspaper gave similar results with about 50% of the theoretical glucose yield at the end of 48 hours of hydrolysis. Native wood gave the slowest rate and yield of glucose (21.8%). These results agree with previous studies which showed an increase in glucose yield from pretreated wood (Grous et al., 1986). Total CO; Evolution. AFEX wood showed the highest rate and extent of total C02 evolution ("co2 as well as unlabeled co.) with 57.7% of the substrate carbon converted to CO; at the end of 160 days of composting (Figure 4.2). The total extent of C02 production from native wood was only a fraction of that produced by the AFEX wood. Paper and STEX wood showed a relatively long lag period of about 40 days after which there was a substantial increase in the rate of C02 evolution. At the end of 160 days of composting, the amounts of C02 produced fi'om paper, native wood, and STEX wood were not statistically different, while the amount of C02 produced from AFEX wood was statistically different compared to the other three substrates. The higher mineralization observed during the composting of AFEX wood could be due to a higher niu'ogen content in the AFEX wood than that in the other substrates. For example, AFEX wood had a UN ratio of 140:1 while native wood, STEX wood, and paper had C/N ratios of 360:1, 410:1, 74 and 900:1 respectively. Thus, even though the pretreatments resulted in increased glucose yields as seen from the enzymatic hydrolysis data, these results were not reflected during the composting of these substrates. Atrazine mineralization and degradation. Mineralization data for l“(z-atrazine during composting (Figure 4.3) showed that newspaper supports about 7% mineralization (range: 4 to 11%) after 160 days of composting. By comparison, the AFEX wood, STEX wood, and native wood supported 4.3 %, 1.8%, and 3.3% mineralization, respectively, in the same period. These results are comparable with those of earlier investigators who showed that under aerobic conditions 1.5 % of the s-triazine ring of atrazine was mineralized after 100 days of incubation in soil microcosms receiving 0.37 ppm of atrazine (Nair and Schnoor, 1992). Statistical analysis using the student’s t-test (p=0.05) showed that there was no difference in the cumulative l"C02 output at the end of 160 days of composting between any of the substrates. The size of the error bars for atrazine mineralization were much higher in composters with paper as the substrate because activity dropped in one of the three replicate composters from day 50 onwards. Also, the rate and extent of atrazine mineralization, and mineralization of total carbon to CO; were different except in the case of STEX wood (Figure 4.4). This could mean that the mechanisms controlling the conversion of substrate carbon to CO; were different from those responsible for the mineralization of atrazine. An observation of the trends in atrazine mineralization (Figures 4.3 and 4.4) showed that atrazine mineralization followed biphasic kinetics with a slower mineralization rate during the first 70 days of composting and a faster rate after this initial period. This could be attributed to a change in 75 the microbial population during that period or to the triggering of some enzyme system(s) in populations already present in the compost matrix. Radioactivity distribution data (Figure 4.5) showed that on day zero, 1°C was primarily in the chloroform fiaction. Samples taken on day 160, on the other hand, showed a change in the distribution with a substantial fraction of the 1“C in the NaOH+Na4P207 fraction. For example, 95% of the extracted 1°C in the day zero sample of the AFEX wood compost was in the chloroform fraction, whereas only 0.3% of the 1“C was extractable into this fraction in day 160 samples. This general trend towards decreasing amounts of radioactivity in the chloroform fraction and increasing amounts of radioactivity in the N aOH + Na4P207 fraction was seen in the other three composts as well. These results suggest the transformation of atrazine carbon to more polar metabolites and/or its complexing with humic components as has been recently shown in the case of 2,4-D composting with yard wastes (Michel et al., 1994). Also, the amount of unextractable (bound) radiolabel increased in the compost samples as composting progressed. For example, about 2% of the radiolabel was unextractable in the day zero sample of AFEX wood, whereas 39% was unextractable in day 160 samples. Our results are supported by the findings of Winkelmann and Klaine (1991) who showed that, after 180 days incubation with soil microcosms, soil bound residues of atrazine and its metabolites accounted for as much as 60% of the initial radioactivity applied (as atrazine) to the microcosms. In support of the above data, the TLC results showed the presence of atrazine on plates spotted with chloroform and methanol extracts from day 0 samples, but not on plates spotted with similar extracts from day 160 samples (data not shown). These results 76 showed that atrazine disappears completely during composting. Winkelmann and Klaine (1991) also showed that atrazine concentrations decrease exponentially over a period of 180 days in soil microcosms. Conclusions The results of this study show complete disappearance of atrazine during 160 days of composting. Up to 11% mineralization of atrazine was observed when it was composted with selected lignocellulosic substrates. No significant differences in atrazine mineralization were observed with untreated wood or pretreated woods as the compost substrates. The time course profiles of organic carbon mineralization to C02 generally paralleled atrazine mineralization except with AFEX wood as the substrate. The extent of total organic carbon mineralization observed with a given substrate did not always reflect in the extent atrazine mineralization observed with that particular substrate. ACKNOWLEDGMENTS We wish to acknowledge Dr. Fred Michel for reviewing this manuscript and Michigan Biotechnology Institute for the use of some of the analytical instrumentation. We thank Dr. Bruce Dale for providing us the AFEX wood. This research was supported in part by The Cooperative State Research Service, US Department of Agriculture under agreement no 94341890067. References Agertved, J ., K. Rugge, and J .F. Barker (1992). Transformation of the herbicides MCPP and atrazine under natural aquifer conditions. Ground Water. 30, 500-506. Aspelin, A.L., A.H. Grube, and V. Kibler (1991). Pesticide industry sales and usage: 1989 market estimates. Document #H-7503W, Office of pesticide programs, US EPA, Washington, DC. 77 Berry, D.F., R. A. Tomkinson, G. H. Hetzel, D.E. Mullins, and RE. Young (1993). Evaluation of solid-state fermentation techniques to dispose of atrazine and carbofuran. J. Environ. Qual. 22, 366-374. Boominathan, K., and C. A. Reddy (1992). Fungal degradation of lignin: biotechnological applications, p. 763-822. In D.K. Arora, R.P. Elander, and K.G. Mukerji (ed.), Handbook of applied mycology, vol. 4. Fungal biotechnology. Marcel Dekker, Inc., New York. Bumpus, J .A. and SD. Aust (1987). Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysasparium: involvement of the lignin-degrading system. Biaassays. 6, 116-200. Converse, A.O., K. Kwarteng, B.E. Grethlein, and H. Ooshima (1989). Kinetics of thermochemical pretreatment of lignocellulosic materials. Appl. Biochem. Biotechnol. 20, 63-7 8. Dale, B.E. and M.J. Moreira (1982). A freeze-explosion technique for increasing cellulose hydrolysis. Biatechnol. and Biaeng. Symp. 12, 3143. FDA (1982). The determination of chlorotriazine residues in plant material, animal tissues, and water using the ultraviolet method. FDA Pesticide Analytical Manual, Volume II. US Food and Drug Administration, Washington, DC. Fogarty, A.M. and 0.11. Tuovinen (1991). Microbiological degradation of pesticides in yard waste composting. Microbiol. Rev. 55, 225-233. Grous, W.R., A.O. Converse, and B.E. Grethlein (1986). Effect of steam explosion pretreatment on pore size and enzymatic hydrolysis of poplar. Enzyme Microb. Technol. vol. 8, 274-280. Hammel, KE. (1992). Oxidation of aromatic pollutants by lignin-degrading fungi and their extracellular peroxidases, p. 41-60. In H. Sigel and A. Sigel (ed.), Metal ions in biological system, vol. 28. Degradation of environmental pollutants by microorganisms and their metalloenzymes. Marcel Dekker, Inc., New York. Hetzel, G.H., B.E. Mullins, R.W. Young, and J .M. Simmonds (1989). Disposal of dilute and concentrated agricultural pesticides using absorption and chemical and microbial degradation. p. 239-248. In: D.L. Weigmann (ed.), Pesticides in terrestrial and aquatic environments, Proc. Natl. Res. Conf. Virginia Water Resourc. Res. Center, Virginia Polytech. Inst. and State Univ. Blacksburg, VA. Judge, D.N., B.E. Mullins, and RE. Young (1993). High performance thin layer chromatography of several pesticides and their major environmental by-products. J. Planar Chromatogr. 6, 300-306. 78 McCall, PJ., S.A. Vrona, and 8.8. Kelly (1981). Fate of uniformly carbon-14 ring labeled 2,4,5-trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid. J. Agric. Food Chem. 29, 100-107. McMahon, P.B., F.H. Chapelle, and M.L. J agucki (1992). Atrazine mineralization potential of alluvial-aquifer sediments under aerobic conditions. Environ. Sci. Technol. 26, 1556-1559. Michel Jr., F.C., C.A. Reddy, and L. J. F orney (1993). Yard waste composting: Studies using different mixes of leaves and grass in a laboratory scale system. Compost Sci. Util. 1, 85-96. Michel J r., F.C., C.A. Reddy, L.J.Forney (1994). Fate of the lawn care pesticides 2,4— D and Diazinon during yard waste composting. In: Proc. 87 th Annual Meeting, Air and Waste Management Association, Cincinnati, OH. Paper # 94-TP45B.06, Vol., 14 A, 10 pp. Mullins, B.E., R.W. Young, D.F. Berry , J.-D. Gu and G.H. Hetzel (1993). Biologically based sorbents and their potential use in pesticide waste disposal during composting. In: K.D. Racke and AR. Leslie (eds.), Pesticides in Urban Environments: Fate and significance. ACS Symposium Series No.522, American Chemical Society, Washington, DC. Myrik, C. (1990). AgriChemical dealership site assessment and remediation. Proc. Infor. Exchgn. Meet, Memphis, TN. Nat. AgriChem. Retail. Assn. Nair, D.R. and J .L Schnoor (1992). Effect of two electron acceptors on atrazine mineralization rates in soil. Environ. Sci. Technol. 26, 2298-2300. Norwood, V.M. (1990). A literature review of waste treatment technologies which may be applicable to wastes generated at fertilizer/agrichemical dealer sites. Tennessee Valley Authority bulletin Y-214. Periera, W.E. and CE. Rostad (1990). Occurrence, distribution, and transport of herbicides and their degradation products in the lower Mississippi river and its tributaries. Environ.Sci. Technol. 24, 1400-1406. Saeman, J .F., J.L. Bubl, and EB. Harris (1945). Quantitative saccharification of wood and cellulose. Industrial Engineering and Chemistry. 17, 35-37. Smith, A.E. (1981). Comparison of solvent systems for the extraction of atrazine, benzoylprop, flamprop, and trifluralin from weathered field soils. J. Agric. F aad Chem, 29, 1 1 1-115. 79 Thompson, D.N., H.-C. Chen, and HE. Grethlein (1992). Comparison of pretreatment methods on the basis of available surface area. Biaresaurce Technol. 39, 155-163. Thurman, E.M., D.A. Goolsby, M.T. Meyer, D.W. K0plin (1991). Herbicides in surface waters of the midwestern United States: The effect of Spring flush. Environ. Sci. Technol. 25, 1794-1796. Teller, G. and G. M. Flaim (1988). A filtering unit for removal of pesticide residues from aqueous solutions. Water Res. 22, 657-661. Yadav, J .S. and C.A. Reddy (1993). Degradation of benzene, toluene, ethylbenzene, and xylene (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysasparium. Appl. Environ. Microbial. 59, 756-762. Winkelmann, D.A. and SJ. Klaine (1991). Atrazine metabolite behavior in soil-core microcosms. In L. Somasundaram and J .R. Coates (ed.), Pesticide transformation products: Fate and significance in the environment. ACS Symposium Series 459, American Chemical Society, Washington, DC. 80 Table 4.1: Composition of the lignocellulosic substrates used for compostingal Substrate Lignin Cilucanb Xylan° Galactan° Arabinan° Mannanc Total Native 24.37 43.11 14.12 0.29 0.35 1.41 83.65 AFEX 24.15 42.61 13.09 0.18 0.42 1.65 82.10 STEX 27.88 54.51 6.92 0.24 0.02 0.98 90.55 Paper 28.43 43.68 4.68 1.50 0.96 8.42 87.67 'Numbers refer to percentage composition by dry weight of each composting substrate. Native refers to untreated poplar wood; AFEX and STEX refer to ammonia exploded and steam exploded wood, respectively; and paper refers to newspaper. ° Derived from cellulose. ° Derived from hemicellulose. 81 100 g 80 - —9'— Native g —o— AFEX "é —v— STEX 0 —v— Pa er 5 60 - p ‘6 e: 2 o '5. 4O — O U) o 0 2 (5 20 —,v,( “e o l I l l 0 10 20 30 40 Hours Figure 4.1: Glucose yields from the enzymatic hydrolysis of untreated poplar wood (Native), ammonia exploded (AFEX) wood, steam exploded (STEX) wood, and newspaper. Each substrate was treated with cellulase and B-glucosidase at 80 U/g dry substrate each as previously described (Thompson et al., 1992). Values presented are means :1: one standard deviation. 82 0.7 . r l o" —9— Native 0 + AFEX 3 0.6 — —v— STEX '8 —v— Paper 5 g 0.5 — 8 fl 3 04 — it . __ -- O .. .. a . ‘5 0.3 — .- ei 7r .r: a “-1 G :1 .2 *5 23 in Figure 4.2: Fraction of substrate carbon converted to C02, Values presented are means+one standard deviation. 200 83 8 .. —9— Native 7 _ + AFEX —e— STEX __ —v-— Paper 6 _. 01 1 Mlnerallzatlon of atrazlne (%) (D .h l L N l k \ l l l l O 20 4O 60 80 100 120 140 160 180 Days Figure 4.3: Mineralization of l“C-Atrazine to 1“C02 during composting with different lignocellulosic substrates. Values presented are means + one standard deviation. c Native AFEX g 0.35 0.8 8 0.30 - —0— Total 002 0.7 - —0— Total CO2 " -l:l- “CO *01 05 - -l:l— 14CO *0.1 g 0.25 - 2 - - 2 .5 020 - 0'5 q .— 0.4 - 0 0.15 ~ g 0 10 0.3 .1 g - 0.2 - § 0.05 - 0.1 _ a 0.00 CT I I I I I 0.0 0- 0 30 60 90 120150180 0 30 60 90120150180 Days Days t: STEX Paper g 0.20 0.8 8 —0— Total 002 0.7 - —0— Total 002 — 0-16 1 -t:l—“co *0.1 E 2 9." 5 0.12 - '6 O 0.08 - 5’ g 0.04 - B 0.00 I I T I I 0- 0 30 60 90 120150180 Days Days Figure 4.4:.Comparison between total C02 production (substrate mineralization) and l‘COz production (atrazine mineralization) for each of the substrates. 85 A. Native c. STEX i D. PAPER Distribution of "C 00110 W40 0817160 peyo Day40 Day160 I ChC|3 El MeOH I NaOH I H20 I 002 E] Bound Figure 4.5: Distribution of 1“C from compost samples at different time periods in various extraction solvents. Extraction procedures used are described in Materials and Methods. ‘NaOH’ in the legend box refers to the fraction of 14C radiolabel extracted into the NaOH+Na4P207 solution. ‘Bound’ in the legend box refers to the unextracted fraction of 1“c radiolabel. Chapter V Mineralization of Atrazine During Temperature Controlled Composting of Poplar Wood and Corn with and without an Exogenous Inoculum of Phanerochaete chrysasparium (Manuscript) 86 87 ABSTRACT The effect of an exogenous inoculum of the white-rot fungus Phanerochaete chrysasparium on atrazine mineralization during the composting of poplar wood was investigated using 2-liter lab-scale composters. [U-ring-“C]Atrazine was added at 500 [lg/g dry substrate (10 uCi) to each composter. P. chrysasparium (Strain BKM-F-1767) inoculum was added as an aqueous conidial suspension (9.6 x 10’ spores/g dry substrate). The addition of P. chrysasparium inoculum significantly enhanced mineralization of atrazine, resulting in a 14% mineralization of atrazine in 94 days of composting compared to 1% mineralization observed in the controls without the P. chrysasparium inoculum. INTRODUCTION Atrazine is one of the most widely used pesticides in the US accounting for about 12% of all the pesticides used in this country (1). Over 36 million kg of atrazine were applied nationwide to agricultural land for the control of annual grasses and broad leaf weeds in 1990 (20). Atrazine and its metabolites are the most frequently detected pesticides in surface waters of the midwestem US (24). Atrazine, along with the others in the s-triazine group, is relatively persistent in the environment, with the most heavily substituted and chlorinated s-triazine analogs being the least biodegradable (7). Numerous studies have been conducted to date on the degradation and mineralization of atrazine by microorganisms in pure culture and in situ in soils and other habitats (7,9). 88 Biodegradation of atrazine has been attributed mainly to fungi (12,13) although recent studies have shown atrazine degradation and mineralization by bacterial _ species also (3,14,21). Mullins et al. (18) investigated the use of lignocellulosic materials such as steam exploded wood and peat moss as absorbents, for a cost effective approach to treat pesticide laden wastewater. These investigators found peat moss and steam exploded wood to be excellent sorption materials for atrazine, with a nearly complete removal of the pesticide from solution (19). Solid-state fermentation of atrazine using bioreactors, containing nutrient enriched peat moss resulted in an 86% disappearance of atrazine at the end of 26 weeks (18). In a recent study, Rao et al. (22) reported upto 11% mineralization of atrazine in 160 days during the composting of lignocellulosic substrates. White rot fungi, as exemplified by Phanerochaete chrysasparium, were shown to be efficient degraders of lignin (a complex, heterogeneous aromatic polymer) in wood. Lignin modifying enzymes of these organisms are relatively non-specific and were shown to fortuitously degrade a variety of chloroaromatic environmental pollutants such as polychlorinated biphenyls and dioxins (5,6,8,23). Hickey et al. (11) recently investigated the potential of P. chrysasparium for degrading atrazine in contaminated soils as well as in liquid cultures in the laboratory, and reported insignificant mineralization of ring labeled atrazine to 14C02. Mougin et al. (17) observed a 48% decrease of the initial atrazine (0.43 ppm) after 4 days of incubation with P. chrysasparium but they also failed to show significant mineralization of “c-ring labeled atrazine to 1“co. However, the potential of P. chrysasparium to nrineralize atrazine during the composting of lignocellulosic materials such as wood has not been investigated before. In this study, we investigated the 89 mineralization of atrazine during the composting of poplar wood with and without an exogenous inoculum of P. chrysasparium. MATERIALS AND METHODS Pesticides. [U-ling-‘°C]Atrazine (specific activity: 25 mCi/mmol; purity>98%) was obtained from Sigma Chemicals, St. Louis, MO. AAtrex 4L, a commercially available sprayable atrazine emulsion (50% active ingredient), was obtained from Ciba-Geigy (Greensboro, NC). Analytical grade atrazine was obtained from Chem Service (West Chester, PA). Compost Substrates. The poplar wood used in this study was provided by the NSF Center for Microbial Ecology at Michigan State University, and is a hybrid between Papulus nigra and Papulus deltaides and has been designated Papulus x euramericana cv. Eugenei. The poplar wood was ground in a Wiley mill through a #10 screen (1.7 nrrn mesh size) and adjusted to 70% moisture (g/g wet wt.) by adding distilled water. Coarsely ground corn, obtained froma local pet supply store (Soldan’s Pet Supplies, Lansing, MI), was adjusted to 50% moisture and used as an amendment to the wood (1:1 w/w) in one experiment. Wood and wood amended with corn were each loaded into two sets of duplicate composters to give 100 g dry substrate per composter. All the composters were amended with [U-ring-"c1Atrazine (10 uCi per composter) and AAtrex (500 pg atrazine/g dry substrate). Initial carbon content of the substrates was determined using a Leco Carbon Analyzer (Model #598-550, Leco Inc., St. Joseph, MI) by the Michigan State University Soil Testing Laboratory. These values were also verified using the 90 quantitative saccharification procedure described elsewhere (22). Compost Inoculum. Inoculum (10% w/w) for all composters came from 10-week- old compost piles operated by a large scale composting facility (Hollandia Gardens, Holland, MI). In addition, P. chrysasparium (Strain BKM-F-l767) was added as conidial spores at the rate of 9.6 x 105 spores/g dry substrate to one set of composters with wood alone and another set of composters with wood and corn. Conidial suspensions were prepared as previously described (4). Composting System. Composting was carried out in the laboratory scale composting system recently described by Michel et al. (16). In brief, the system consisted of a rubber stoppered 2- liter, wide mouth glass jar with two plastic screens (1cm and 1mm mesh opening) forming a false floor. Aeration was provided through a hole just below the level of the two screens. C02-free, humidified air for the composters was obtained by passing the air through a flask containing 5 N NaOH to remove C02 and then through a 5 gallon carboy containing 2.5 gallons of distilled water. The air flow to each of the composters was set at 100 ml/min by means of a needle valve placed just upstream of the composters. The exhaust gas from each composter passed through polyurethane foam plugs to trap any volatiles and then into two 5N NaOH containers to trap C02 present in the exhaust gas. To ensure that the 14C in the NaOH traps was due to 1“C02 and not due to volatilized l“C- atrazine or other volatile organics derived from l“C-atrazine, a barium chloride precipitation step was included as previously described (27). The entire system was placed in a temperature controlled room which was maintained at 37°C since P. chrysasparium is known to grow optimally at 37°C-40°C (5,6,8). 91 CO2 Evolution. Total C02 trapped in the N aOH traps during composting was measured by titrating an aliquot of the NaOH solution against standardized HCl. The procedure involved removal of a 0.5 ml aliquot of the NaOH solution, dilution with 1 ml of water, and precipitation of Na2CO3 with 3 ml of a 70 g/l solution of BaCl2. 0.5 ml of this diluted solution was then titrated against standardized HCl. The titration value obtained was used to calculate the amount of CO2 trapped in the test tubes. The amount of 1“co. trapped was measured by mixing 0.2 ml of the NaOH solution with 0.8 ml water and 15 ml of scintillation cocktail (Safety-Solve, Research Products International Corp., Mount Prospect, IL) in 20 ml scintillation vials. These were then counted using a scintillation counter (Tri-Carb 1500, Packard Instrument Co., Downers Grove, IL) after letting the samples sit overnight to eliminate chemiluminescence effects. RESULTS AND DISCUSSION Substrate mineralization. The evolution of CO2 from the composters with wood as substrate with and without P. chrysasparium inoculum, is shown in Figure 5.1. The rate of evolution of CO2 was much higher in the composters inoculated with P. chrysasparium, as compared to the composters without the P. chrysasparium inoculum. For example there was 61% conversion of the initial substrate carbon to CO2 at the end of 94 days of composting in composters with P. chrysasparium as compared to a 10% conversion observed in the controls. The increased rate of mineralization observed in the composters inoculated with P. chrysasparium (after the first 15 days of composting) was also 92 accompanied by a visible colonization of the substrate by P. chrysasparium. It is of interest that in experiments in which P. chrysasparium was added to poplar wood as a mycelia] blend (Figure 5.2), rather than as a conidial suspension (Figure 5.1), no appreciable difference in the conversion of biomass to CO2 was seen in the composters with and without the P. chrysasparium inoculum. Atrazine mineralization. Mineralization patterns of atrazine (Figure 5.3) were similar to the mineralization patterns of wood seen in Figure 5.1. l“C-atrazine mineralization to 1“CO2 was 14% at the end of 94 days of composting in composters with wood and P. chrysasparium as compared to a 1% mineralization observed in the controls with wood alone. These results are contrary to the findings of Hickey et al. (11) who investigated the potential of P. chrysasparium to bioremediate atrazine-contaminated soils in laboratory studies. They found that l“co. production from soils with l4c-ring labeled atrazine was insignificant. They also reported that atrazine was not metabolized by P. chrysasparium grown in liquid cultures. 0n the other hand, Mougin et al. (17) observed a 48% decrease of the initial atrazine (0.43 ppm) in liquid cultures of P. chrysasparium within the first 4 days of incubation. However, they found only mineralization of the ethyl side chain of atrazine but no mineralization of the atrazine ring. Kaufman and Blake (12) also reported mineralization of the ethyl side chain but no mineralization of the atrazine ring in their study on degradation of atrazine by soil fungi. The evolution of 1“C02 from ”C-ring labeled atrazine observed in our study clearly indicates mineralization of atrazine by ring cleavage, a phenomenon not observed by these researchers. The use of wood, the natural subsu'ate for P. chrysasparium, may account for the observed mineralization of 93 atrazine by ring cleavage in our study. Other researchers have reported mineralization of atrazine by ling cleavage though the amounts of atrazine loaded initially were very low. Assaf and Turco (2) found a 17% mineralization of atrazine (10 ppm initial concentration) in soils after 90 days of incubation. Mineralization levelled out at 39% at the end of 326 days of incubation. Levanon (13) reported a 29% conversion of ring labeled atrazine (1 ppm) to 1“co. in 32 days of incubation in soils. This amounts to atrazine conversion to CO2 of 3.9 ppm observed by Assaf and Turco (2) and 0.29 ppm by Levanon (13). In comparison, our results show mineralization of 70 ppm of the initially loaded atrazine to C02 after 94 days of incubation. Effect of corn amendment on substrate mineralization. The effect of addition of corn as an amendment to the wood is presented in Figures 5.4 and 5.5. The high rate and extent of conversion of biomass to CO2 might be attributable to the fact that com (due to its starch content) is a readily utilizable substrate (Figure 5.4). The addition of P. chrysasparium to composters with wood and corn did not result in an increase in mineralization of the substrate carbon to C02. The conversion of substrate to CO2 in the composters with an added inoculum of P. chrysasparium (57%) was statistically different compared to that in composters without the inoculum(64%). Effect of corn amendment on atrazine mineralization. Mineralization of atrazine at the end of 94 days of composting in reactors containing wood + corn and receiving a P. chrysasparium inoculum, was not statistically different (p = 0.05) from that observed ill identical reactors not receiving the P. chrysasparium inoculum. Thus, there was no 94 apparent correlation between the rate and extent of atrazine mineralization and mineralization of total carbon to CO2. Previous research has been inconclusive on the effect of energy sources and/or nutrients on the degradation of atrazine. Assaf and Turco (2) reported that atrazine degradation in soils amended with mannitol as a carbon source, and with urea as a nitrogen source at levels of 10-80 mg/kg, was similar to the degradation observed ill unamended soils. On the other hand, McCormick and Hiltbold (15) observed that the rate of atrazine decomposition was increased by the addition of glucose to soils. Wagner and Chahal (26) also found that atrazine degradation was accelerated by the presence of glucose. Hance (10) reported that the addition of straw, inorganic salts, or a combination of both approximately doubled the rate of atrazine degradation in soils. Our results showed much lower mineralization of atrazine in composters containing wood + corn and inoculated with P. chrysasparium as compared to that observed in composters containing wood alone and inoculated with P. chrysasparium. This could be due to the fact that the composters amended with corn were colonized rapidly by a Mucor species, a starch utilizing fungus, in the first week of composting (data not shown). Thus the addition of corn as amendment to the wood served to select for a Mucor sp., a starch utilizing fungus, while growth of the wood degrading fungus, P. chrysasparium was inhibited in these composters. The difference in atrazine mineralization seen in the composters containing wood and those containing wood + corn could be attributed to the difference in the enzyme systems of the fungi colonizing the compost substrates. In conclusion, the results of this study show that the addition of an exogenous 95 inoculum of P. chrysasparium to composters with poplar wood as the substrate resulted in mineralization of 14% of the initial atrazine in 94 days of composting as compared to 1% mineralization observed in the controls. Mineralization of atrazine was considerably lower (3.8% mineralization in 94 days) in composters receiving wood, corn and P. chrysasparium inoculum. ACKNOWLEDGMENTS We acknowedge Drs. Donald Penner and Muralee Nair for the use of the scintillation counter, and Dr. C. Srinivasan for providing P. chrysasparium. This research was supported in part by The Cooperative State Research Service, US Department of Agriculture under agreement number 94341890067. REFERENCES l. Aspelin, A. L., A. H. Grube, and V. Kibler. 1991. Pesticide industry sales and usage: 1989 market estimates. Document #H-7503W, Office of pesticide programs, US EPA, Washington, DC. 2. Assaf, N. A. and R. F. Turco. 1994. Influence of carbon and nitrogen application on the mineralization of atrazine and its metabolites in soil. Pesticide Sci. 41:41-47. 3. Behki, R. M. and S. U. Khan. 1994. Degradation of atrazine, propazine, and Simazine by Rhadacoccus strain B-30. J. Agric. Food Chem. 42: 1237-1241. 4. Boominathan, K., S. B. Dass, T. A. Randall, R. L. Kelly, and C. A. Reddy. 1990. Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysasparium. J. Bacteriol. 172:260-265. 5. Boominathan, K. and C. A. Reddy. 1992. Fungal degradation of lignin: Biotechnological applications. pp. 763-822. In D.K. Arora, R.P. Elander, and K.G. 10. ll. 12. 13. 14. 15. 16. 17. 96 Mukerji (eds.) Handbook of Applied Mycology. Vol. 4: Fungal Biotechnology. Marcel Dekker. New York. Bumpus, J. A., M. Tien, D. Wright, and s. D. Aust. 1985. Oxidation of persistent environmental pollutants by a white rot fungus. Science 228:1434-1436. Cook, A. M. 1987. Biodegradation of s-tliazines. FEMS Microbiol. Rev. 46:93-116. Crawford, D. L. and R. C. Crawford. 1980. Microbial degradation of lignin. Enzyme Microb. Technol. 2: 1 1-22. Erickson, L. E. and K. H. Lee. 1989. Degradation of atrazine and related s- triazines. Critical Reviews in Environmental Control 19: 1- 14. Hance, R. J. 1973. The effect of nutrients on the decomposition of the herbicides atrazine and linuron incubated with soil. Pesticide Sci. 4:817-822. Hickey, W. J ., D. J. Fuster, and R. T. Lamar. 1994. Transformation of atrazine ill soil by Phanerochaete chrysasparium. Soil Biol. Biochem. 26: 1665-1671. Kaufman, D. D. and J. Blake. 1970. Degradation of atrazine by soil fungi. Soil Biol. Biochem. 2:73-80. Levanon, D. 1993. Roles of fungi and bacteria in the mineralization of the pesticides atrazine, alachlor, malathion, and carbofuran in soil. Soil Biol. Biochem. 25:1097- 1105. Mandelbaum, R. T., D. L. Allan, and L. P. Wackett. 1995. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Appl. Environ. Microbiol. 61: 1451-1457. McCormick, L. L. and A. E. Hiltbold. 1966. Microbiological decomposition of atrazine and diuron in soils. Weeds 14:77-82. Michel Jr., F. C., C. A. Reddy, and L. Forney. 1993. Yard waste composting: Studies using different mixes of leaves and grass in a laboratory scale system. Compost Sci. Util. 1:85-96. Mougin, C., C. Laugero, M. Asther, J. Dubroca, P. Frasse, and M. Asther. 1994. Biotransformation of the herbicide atrazine by the white rot fungus Phanerochaete chrysasparium. Appl. Environ. Microbiol. 60:705-708. 18. 19. 21. 97 Mullins, D. E., R. W. Young, D. F. Berry , J.- D. Gu and G. H. Hetzel (1993). Biologically based sorbents and their potential use in pesticide waste disposal during composting, pp. 113-126. In: K.D. Racke and AR. Leslie (eds.), Pesticides in Urban Environments: Fate and significance. ACS Symposium Series No.522, American Chemical Society, Washington, DC. Mullins, D. E., R. W. Young, G. H. Hetzel, and D. F. Berry (1992). Pesticide wastewater cleanup using demulsification, sorption, and filtration followed by chemical and biological degradation, pp. 166-176. In: J. B. Bourke, A. S. Felsot, T. J. Gilding, J. K. Jensen, and J. N. Seiber (eds.), Pesticide Waste Management. ACS Symposium Series No. 510, American Chemical Society, Washington, DC. Periera, W. E. and C. E. Rostad. 1990. Occurrence, distribution, and transport of herbicides and their degradation products in the lower Mississippi river and its tributaries. Environ.Sci. Technol. 24: 1400-1406. Radosevich, M., S. J. Traina, Y.-L. Hao, and O. H. Tuovinen. 1995. Degradation and mineralization of atrazine by a soil bacterial isolate. Appl. Environ. Microbiol. 61:297-302. 22. Rao, N., H. E. Grethlein, and C. A. Reddy. (1995). Mineralization of atrazine during the composting of untreated and pretreated lignocellulosic substrates. Compost Sci. Util. 3:38:46. Reddy, C. A. 1995. The potential for white-rot fungi in the treatment of pollutants. Curr. Opinion Biotechnol. 6:320-328. Thurman, E. M., D. A. Goolsby, M. T. Meyer, D. W. Koplin. 1991. Herbicides in surface waters of the midwestern United States: The effect of Spring flush. Environ. Sci. Technol. 25:1794-1796. . Tien, M. and T. K. Kirk. 1988. Lignin peroxidase of Phanerochaete chrysasparium. Meth. Enzymol. 161:38-249. Wagner, G. H. and K. S. Chahal. 1966. Decomposition of carbon-14 labelled atrazine in soil samples from Sanbom field. Soil Sci. Soc. Amer. Proc. 30:752-754. 18. 19. 21. 97 Mullins, D. E., R. W. Young, D. F. Berry , J.- D. Gu and G. H. Hetzel (1993). Biologically based sorbents and their potential use in pesticide waste disposal during composting, pp. 113-126. In: K.D. Racke and AR. Leslie (eds.), Pesticides in Urban Environments: Fate and significance. ACS Symposium Series No.522, American Chemical Society, Washington, DC. Mullins, D. E., R. W. Young, G. H. Hetzel, and D. F. Berry (1992). Pesticide wastewater cleanup using demulsification, sorption, and filtration followed by chemical and biological degradation, pp. 166-176. In: J. B. Bourke, A. S. Felsot, T. J. Gilding, J. K. Jensen, and J. N. Seiber (eds.), Pesticide Waste Management. ACS Symposium Series No. 510, American Chemical Society, Washington, DC. Periera, W. E. and C. E. Rostad. 1990. Occurrence, distribution, and transport of herbicides and their degradation products in the lower Mississippi river and its tributaries. Environ.Sci. Technol. 24: 1400-1406. Radosevich, M., S. J. Traina, Y.-L. Hao, and O. H. Tuovinen. 1995. Degradation and mineralization of atrazine by a soil bacterial isolate. Appl. Environ. Microbiol. 61:297-302. 22. Rao, N., H. E. Grethlein, and C. A. Reddy. (1995). Mineralization of atrazine during the composting of untreated and pretreated lignocellulosic substrates. Compost Sci. Util. 3:38:46. Reddy, C. A. 1995. The potential for white-rot fungi in the treatment of pollutants. Curr. Opinion Biotechnol. 6:320—328. Thurman, E. M., D. A. Goolsby, M. T. Meyer, D. W. Koplin. 1991. Herbicides in surface waters of the midwestem United States: The effect of Spring flush. Environ. Sci. Technol. 25: 1794-1796. Tien, M. and T. K. Kirk. 1988. Lignin peroxidase of Phanerochaete chrysasparium. Meth. Enzymol. 161:38-249. Wagner, G. H. and K. S. Chahal. 1966. Decomposition of carbon-14 labelled atrazine in soil samples from Sanbom field. Soil Sci. Soc. Amer. Proc. 30:752-754. 98 27. Yadav, J. S. and C. A. Reddy. 1993. Degradation of benzene, toluene, ethylbenzene, and xylene (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysasparium. Appl. Environ. Microbiol. 59:756-762. 99 N 8 0.7 - —0— Wood 3 —Cl— Wood + PC g 0.6 “ I I I I > _! ' g . o 0.5 _ [i- : “I! 3 1." U _' C, I fl g 0.3 - _ 3 I" .1 t?) 0.2 — . h I O u 5 -" . . e- 0.1 — ,I' . . . O C 6 .I "I. ‘ ° . . E 15:: - ° 0-0 ‘l r I T l 0 20 40 60 80 100 Days Figure 5.1: Effect of addition of a spore inoculum of P. chrysasparium (PC) to poplar wood. Composting was carried out at 37°C. Values presented are means :1: half range for duplicate composters. 100 0.7 N O .2 —D— Wood + PC 8 g 0.5 - C 8 C 0.4 — E G O .9 0.3 .. .8 .‘o" I 5 0.2 — . - '6 . ' e ' g I I . . C _ e g 0.1 " .(Q'l. & . ”.13:- ./"’ 0.0 | .l I l 0 20 40 60 80 100 Figure 5.2: Effect of addition of an exogenous inoculum of P. chrysasparium (PC) in the form of blended mycelia to poplar wood. Composting was carried out at 37°C. Values presented are means :1: half range for duplicate composters. 101 .L a) —O— Wood —D— Wood + PC .4 .5 l _L N 1 tion of atrazine (%) E3 l 8 T / _ T .1' ,ll -- = 6 a q” H z. a u- a .f 1" .5 4 - I .:*- E l I' ‘L 2 _ _.-I.|..- ..l-' . e 0 I'-- e 0 9 . . . .I!'5“' ° ' 0 '" r l l l 0 20 40 60 80 Days 100 Figure 5.3 Mineralization of l4C-ring labeled atrazine to 1“CO2 during the composting of poplar wood with and without the addition of an exogenous inoculum of P. chrysasparium (PC). Values presented are means :1: half range for duplicate composters. 102 N 0.7 8 —0— Wood 0 —l:l— Wood + Corn _ . E 05“ —A-Wood+Com+PC _.-' I... A .I ‘ A E) I... A‘ A1 = 0.5 - III-I'- AAAAA 8 an... A. : ‘PI ‘A .8 0.4 "‘ I"... AAA 3 I. AAAA o 5!; 2 0.3 - ,5“ s :- ‘e'o‘ '3 r m 0.2 — u .- l/ O ‘1" c - g 0.1 “ a" . . . . o O O E! 5/ . e ' . .’ ° IL . 0-0 ' l l l T 0 20 40 60 80 Days 100 Figure 5.4: Fraction of initial substrate carbon converted to CO2 during the composting of poplar wood with and without an amendment of corn at 37°C. Values presented are means :1: half range for duplicate composters. 103 16 14 .1 —0— W000 —D— Wood + Corn 3 —-Ar- Wood + Corn + PC L 12 - 0 .E N E 10 d ‘5 '6 r: 8 - .9 «one .8 6 — E . . a I 4- -- if I. I A “H'- !'. 2 - "-!1.A‘:‘~"‘5‘ ' ,, _ II-Jlg-iA'A! !_A .9 - . . . .Aeeaeew'mew . - 0 Ah“ I l I l 0 20 40 60 80 100 Figure 5.5: Mineralization of l4C-ring labeled atrazine to 14CO2 during the composting of poplar wood with and without an amendment of corn. Values presented are means 1 half range for duplicate composters. Chapter VI: Preliminary Process Design and Modelling Introduction: The objective of this study was to evaluate a scheme for the disposal of pesticide contaminated water and pesticide rinsewater. From the results of the previous chapter it is evident that co-composting the pesticide with lignocellulosic materials is an effective method for the disposal of the pesticide. This chapter considers the commercial implementation of a co-composting process for the disposal of the entrained pesticide. A theoretical model was also developed to explain the mineralization of poplar wood and atrazine during composting with an inoculum of P. chrysasparium. Process Design: A schematic diagram of the conceptual process for the disposal of pesticide- contaminated water by co-composting with lignocellulosic materials is shown in Figure 6.1. The entire process from the point of generating/receiving the pesticide-contaminated water to the co-composting of the pesticide with the lignocellulosic substrate is a very simple one with respect to equipment considerations. Mixing of the contaminated water and the composting substrate is very easily achieved by spraying the water on the substrate in the required amounts to achieve the desired pesticide concentration and moisture content in the composting substrate. The composting process itself requires a minimum of equipment for the completion of the process, and includes equipment that control the temperature of the compost windrow to the desired set point. In our case the desired set point would be the optimum temperature for the growth of Phanerochaete chrysasparium. 104 105 Inoculum Contaminated Rinsewater , V Composting “”ng ‘ Process Absorbent (Subsuate) Figure 6.1: Conceptual co-composting process Temperature control of the compost windrow can be achieved in a couple of ways. One would be by controlling the windrow configuration (windrow length, width, and height), thus allowing for the regulation of conductive heat loss from the compost pile. This option is not feasible in our case since conduction accounts for only 2.4% of all heat loss in field scale composters, the other 97.6% occuring due to evaporative cooling and sensible heat rise of air in the pile (Hogan et al., 1989). Thus a better alternative to controlling the pile temperature would be by the control of aeration through the windrow with the objective of removal of excess heat (above the desired set point) by evaporation of moisture from the composter. 106 Assuming that composting follows first order reaction kinetics, we get the following equation from a mass balance of the composting process: dm E—Hm, —me) (1) where: m. = mass of composting material at time t me = amount of uncomposted material at end of composting process k = decompostion rate The theoretical air flow rate required to maintain a constant temperature is then: =(A H...) '68ch where: Ahc = heat of combustion AH.“ = enthalpy difference of inlet and outlet air kgc = amount of compostable material Total air flow at time t can be calculated from the following equation: (3) 107 where: Qt = total air flow, m3/day p. = density of air, kg.5./rn3 Fan power required to maintain this air flow: _ Q. A p (m3/sXPascals) P . e efi‘icrency where: P = power (watts) Ap = pressure drop across the compost pile 8 = efficiency of the fan and pressure drop is given by the equation (Higgins, 1982): Ap=a-(%) -d (mmH2O) where: A = floor area of compost pile (m2) (1 = depth of compost pile (m) 3,11 = parameters (4) (5) 108 We can use the following values to calculate the fan power required: Parameter Value Source k 0.026 clay'l Experimental data .1111,; 20 MJ/kg Keener et al. (1993) Air enthalpy (inlet)° 62 KJ/kg Perry (1984) Air enthalpy (outlet)b 184 KJ/kg Perry (1984) m0 3000 kg Assumption in. 1000 kg Experimental Data Air density 1.2928 leg/m3 Perry (1984) Floor area 20 m2 Assumption Windrow depth 2 m Assumption a 1.23x10" Keener et al. (1993) n 1.55 Keener et al. (1993) a - assuming inlet air at 21°C and 50% relative humidity b - assuming outlet air at 40°C and 100% relative humidity Using the values listed above: q = (0.026)(20,000)/(184-62) = 4.262 kgmlkg/day 109 Initial air flow required would be: Qo = (3000-1000)(4.262)/1.2928 = 6593.9 m3/day = 0.0763 m3/s Pressure drop across the compost pile would be: Ap = (1.23x10")(2)(6593.9/20)‘-” = 0.1968 mm H2O = 1.93 Pascals Initial fan power required: Fan power = (0.0763)(1.93)/0.6 = 0.245 Watts = 1.83 x 10“ hp This would be the maximum power requirement since the amount of compostable material would decrease with time and also assuming that the maximum rate of decomposition would be in the initial stages of composting. Assuming cost of electricity to be 6 cents/kWhr, the power requirement cost for the process would be quite minimal (about lclmonth). 110 Process Model: The mineralization of wood and amine during composting with an , inoculum of P. chrysasparium observed in the experiments of Chapter V (see Figure 6.2) were modelled as follows. Growth of biomass was modelled using the Verlhurst and Pearl equation (Bailey and 01118, 1986). dx 3; - loco-ix) (6) The second order term in the equation was introduced to explain the lag observed in the mineralization of both the substrates (wood and atrazine), which could be attributed to the addition of P. chrysasparium in the form of spores rather than as mycelia. Integrating Equation 6 gives: dx —— = dt I loc(1 - Y x) I (7) =5 _d_x_+_y_ dx Jdt ’01: k l-yr= (8) =>— n—-——ln———=kt (9) lll 0.7 .. 0.6-1 o 0 .2 0.5- i 0 > 8 4- o 0. 8 g 3 0.8- 3 a: '5 g 0.2-1 *6 E “- 0.1- 0.0 -= 0 —O— Biomass mineralization —Ci— Atrazine mineralization 100 Figure 6.2 Mineralization of atrazine and wood during composting with an inoculum of P. chrysasparium. 112 x l-‘yx (10) x0 l-‘Yxo =, il"_7x_o = en (11) x0 l-yx Upon rearranging this gives: kt x0e (12) x=1 kl ’Yxo +7xoe This is the solution to Equation 6 and is a logistic curve that gives: (13) x5: 1 r where: x, is the biomass at the end of the experiment. The product formation kinetics (mineralization of wood and atrazine to CO2) were modelled using the Ludeking-Piret equation (Bailey and Ollis, 1986) which combines growth associated and non-growth associated contributions. dp dx (14) Integrating Equation 14 gives: jdp=otjdx+pjrdr (15) 113 Substituting the value of x from Equation 12 into Equation 14: kt x0e dt :5 — =0l x-x + (16) P Po ( 0) BIl-Yxo‘VYxoeu .. 2 ., .7 =>p-po—a(x-xo)+yk ln(l-'Yxo+‘yxoe ) ( ) Substituting Equations 12 and 13 in Equation 17: kt 3pm, =0”, e -1 +&m[1—fl+fle“] (18) l-.§£.+£9.ekt k x3 x3 x x Equation 18 was used to fit the experimental data obtained in Figure 6.2 using a commercially available software (PeakFit, Jandel Sientific). The results obtained from the process are as follows: For biomass mineralization: 0.085: P " P0 = (18.5X0.1{1_ 0 036+ 0 0380.085! - I] (r2 = 0996) (19) For atrazine mineralization: €0.05: _ = 5.84 0.1 —1 r2=0.999 20 p p° ( X 11-0.03+0.03e°-°" ] ( ) ( ) 114 From the correlation coefficients obtained we see that the theoretical model explains the experimental data very well. The model predictions for Equations 19 and 20 are shown in Figures 6.3 and 6.4 respectively. It is of interest to note that the model predicts all product formation to be growth associated with no non- grth associated contribution. The model also predicts a decrease in the lag with increase in the initial amount of biomass. The preliminary process design and model predictions indicate the fesasibility of a large scale composting process for the disposal of pesticide-contaminated water. References: Bailey, J. E. and D. F. Ollis (1986). “Biochemical Engineering Fundamentals”. 2nd Edition. McGraw-Hill Book Co. Hogan, J. A., F.C. Miller, and M. S. Finstein (1989). Physical modelling of the composting ecosystem. Applied and Environmental Microbiology 55: 1082-1092. Keener, H. M., C. Marugg, R. C. Hansen, and H. A. J. Hoitink (1993). Optimizing the efficiency of the composting process. In: H. A. J. Hoitink and H. M. Keener (eds.), Science and engineering of composting: Design, environmental, microbiological, and utilization aspects. Renaissance Publications, Worthington, Ohio. Perry (1984). “Perry’s Chemical Engineers’ Handbook”. 6th Edition. R. H. Perry and D. W. Green (eds.). McGraw-Hill Book Co. 115 70 0 Experimental data — Model prediction 60-1 Percentage of substrate Carbon converted to CO2 0 20 40 60 80 100 Figure 6.3 Model prediction for substrate (wood) mineralization. 116 15 O" 0 Experimental data C; —— Model prediction i. t o > C 3 10 — C o e a O 2 g a: .n a s — '5 e or .8 r: o 2 o a. 0 -€ 0 100 Figure 6.4 Model prediction for atrazine mineralization. Chapter VII: Overall Conclusions and Future Directions 7.1 Overall Conclusions The objective of this study was to investigate the potential for the degradation and mineralization of atrazine by gratuitous metabolism during the composting of lignocellulosic materials. Preliminary results indicated that a moisture content of 70% and higher C/N ratios resulted in a greater conversion of the p0plar wood substrate to CO2. Results from a study on the effect of temperature on the mineralization of poplar wood and atrazine indicated that substrate conversion during lab-scale composting was optimal at a mesophilic temperature of 37°C, though atrazine mineralization was minimal at all temperatures. Subsequent composting experiments conducted at 37°C with untreated and pretreated lignocellulosic substrates showed that pretreating the substrates had little effect on the mineralization of atrazine. Mineralization of atrazine was significantly enhanced when P. chrysasparium, a wood degrading white-rot fungus, was added as an inoculum to the composters. In summary, under the lab—scale composting conditions maintained in our study, optimum mineralization of atrazine was achieved at a moisture content of 70%, composting temperature of 37°C, and with the addition of an exogenous inoculum of P. chrysasparium. This study represents a significant step forward in the field of composting as a disposal option for pesticide contaminated sources, since research has not been conducted previously to investigate the co-metabolism of pesticides during the composting of poplar wood. This is also the first direct evidence of atrazine mineralization during the composting of lignocellulosic substrates with P. chrysasparium. This seems to suggest the 117 118 atrazine by ring cleavage, since earlier investigators reported no mineralization of atrazine (by ring cleavage) by P. chrysasparium in pure cultures (Mougin et al., 1994) and in soils amended with P. chrysasparium (Hickey et al., 1994). 7.2 Future Directions A pilot scale composting operation to confirm the results obtained in the laboratory scale experiments would be a logical extension to this study. The pilot scale operation would also confirm the results of the preliminary process design and modelling. An elucidation of the role of lignocellulosic materials in the mineralization of atrazine by P. chrysasparium, including a clarification of the enzymatic processes that lead to the mineralization of atrazine would be another area that could be explored. This could be carried out by using shake flask cultures of P. chrysasparium with atrazine and wood as substrates, since isolating the enzymes responsible for atrazine mineralization in a solid state fermentation environment might be difficult. Another area that can be investigated is the effect of different types of wood or lignocellulosics on the mineralization of atrazine by P. chrysasparium, based on the evidence that different types of wood have been shown to induce the expression of different enzymes and/or different levels of enzymes. Also, the effect of pesticide loading rates on pesticide mineralization is another area that bears investigation. Appendix A Effect of C/N Ratio and Moisture Content on the Composting of Poplar Wood Published in: Biotechnology Letters 17: 889-892 (1995). 119 120 SUMMARY Milled poplar wood (1.7 mm mesh size) was composted in lab-scale reactors. Initial C/N ratios were adjusted to 10:1, 30: 1, and 50:1 using urea as the nitrogen source. At each C/N ratio, three moisture levels (30, 50, and 70%) were tested. C/N ratios of 50:1 or 30:1 and moisture content of 70% favored more effective composting as indicated by higher levels of mineralization of the poplar wood to CO2. INTRODUCTION Composting is fast becoming the primary disposal option for solid wastes such as leaves, grass, and woody materials, because of the proposed ban on the land filling and incineration of these wastes in many states in the US. Compared to the composting of yard wastes (Michel et al., 1993) there have been relatively few studies to date on the composting of woody materials such as wood pallets, wood processing residues, and Christmas trees. Furthermore, substantial variations in results were reported by different investigators. For example, Poincelot and Day (1973) showed that changing the ON ratio from 41 :1 to 35:1 nearly doubled the amount of degradation of leaf cellulose. Campbell and Tripepi (1991) recommended a relatively broad target C/N ratio of 40:1 to 70:1 for effective composting of wood wastes. Darbyshire et al. (1989) reported that a C/N ratio of 39:1 and a moisture content of 60% are effective for the decomposition of milled spruce bark, while Haug (1980) on the other hand indicated that high moisture content hinders 121 aeration and induces undesirable anaerobic conditions during composting. In this study, we used a laboratory-scale composting system recently designed by us (Michel et al., 1993) to investigate how the mineralization of milled poplar wood is affected in composters with varying moisture contents (30, 50, or 70%) and initial C/N ratios of 10:1, 30:1, and 50:1. MATERIALS AND METHODS Compost substrate. The poplar wood used in this study was a hybrid between Papulus nigra and Papulus deltaides and has been designated Papulus x euramericana eugenei. and was grown at the Kellogg Biological Station and was provided by the NSF Center for Microbial Ecology at Michigan State University. The poplar wood was ground in a Wiley mill through a #10 screen (1.7 mm mesh size). The substrate was amended with urea to give C/N ratios of 10:1, 30: 1, and 50: 1, and enough sterile distilled water was added as a spray to give moisture contents of 30%, 50%, or 70%. Compost inoculum. The inoculum used for composting was obtained from 10- week-old wood compost piles operated by a large scale composting facility (Hollandia Gardens, Holland, MI). A 10% w/w inoculum was used for all composters. Composting System. Composting was carried out in a laboratory scale composting system described by Michel et al. (1993). In brief, the system consisted of rubber-st0ppered 2-liter, wide mouth glass jars with two plastic screens (1cm and 1mm mesh opening) forming a false bottom. Aeration was provided through a hole just below 122 the level of the two screens. CO2-free, humidified air for the composters was provided by passing the air through a flask containing 5 N NaOH to remove CO2 and then through a 5 gallon carboy containing 2.5 gallons of distilled water. The air flow to each of the composters was set at 100 mllmin by means of a needle valve placed just upstream of the composters. The exhaust gas from each composter passed through two 5N N aOH containers to trap CO2 present in the exhaust gas. The amount of C02 trapped was measured as described by Michel et al. (1993). The entire system was maintained at 37°C. RESULTS AND DISCUSSION Milled poplar was used in this study since reduction in particle size has been shown to increase the decomposition and humiflcation rates during composting (N ’Dayegamiye and Isfan, 1991; Poincelot, 1974). Mineralization of biomass as measured by the C02 released from the composters allowed comparison of the effect of moisture content and UN ratio on the composting of poplar wood. The time course of organic matter mineralization at an initial C/N ratio of 10:1 and varying moisture content is shown in Figure A.1. Mineralization of biomass was rather minimal (<3%) at 30% and 50% moisture as compared to 15% mineralization observed at 70% moisture. CO2 production profiles from the composters with an initial C/N ratio of 30:1 and varying moisture contents are shown in Figure A2. The extent of mineralization in composters with 30 and 50% moisture was roughly comparable in the initial stages and 123 was virtually identical at the end of 108 days. In comparison, the rate and extent of CO2 generation was much greater at 70% moisture, with no detectable lag. Mineralization trends in composters with initial C/N ratio of 50:1 (Figure A.3) were similar to those observed in composters with an initial C/N ratio of 30: 1. For example, the extent of conversion of substrate carbon to C02 observed in composters with 30 and 50% moisture content was relatively low at C/N ratios of both 30:1 and 50:1. Also, the behavior of the composters with 70% moisture content and C/N ratios of 30:1 and 50:1 were nearly identical with about 15.8% conversion of biomass to CO2 (Figure A.4). This suggests that wood composting is effective even at a relatively high C/N ratio of 50:1. The results presented in Figure A.4 also show that even the control run without the addition of nitrogen (C/N ratio of 820:1) showed mineralization of substrate carbon comparable to that observed in composters with C/N ratio of 30:1 and moisture contents of 30 and 50%. The results clearly indicate that mineralization of the substrate occurs optimally with a C/N ratio of 30:1 to 50:1 and a moisture content of 70% and further suggest that lower C/N ratios tend to be inhibitory to composting. Our results are in agreement with those of Zadrazil (1980) who showed that low concentrations of nitrogen in the form of ammonium nitrate (0.25%) increased decomposition rates of straw while high levels of ammonium nitrate (0.75 and 1.25%) hindered decomposition. REFERENCES Bono, J. J ., N Chalaux, and B. Chabbert (1992). Biaresaurce Technology 40, 119-124. Campbell, A. G. and R. R. Tripepi (1991). F arest Products Journal 41, 55-57. 124 Darbyshire, J. F., M. S. Davison, G. J. Gaskin, and C. D. Campbell (1989). Biol. Wastes 30. 275-287. Haug, R. T. (1980). Compost Engineering: Principles and Practice. Ann Arbor Science, Ann Arbor. Michel Jr., F. C., C. A. Reddy, and L. J. Fomey (1993). Compost Sci. Util. 1, 85-96. N’Dayegamiye, A. and D. Isfan (1991). Can. J. Soil Sci. 71, 475-484. Poincelot, R. P. (1974). Compost Science 15, 24-31. Poincelot, R. P. and P. R. Day (1973). Compost Science 13, 23-25. Zadrazil, F. (1980). European J. Appl. Microbial. Biotechnol. 9, 31-35. 125 CM Ratio 10:1 0.16 —O— 30% Moisture —Cl— 50% Moisture —A— 70% Moisture p _o —L —L to .1:- l l 0.10 - 0.08 -1 0.06 e 0.04 - Fraction of initial Carbon converted to C02 0.02 -1 0.00 ' 0 20 40 60 80 100 120 Figure A.l: Conversion of initial carbon to CO2 during the composting of poplar wood at an initial C/N ratio of 10:1 and varying moisture contents. 126 CIN Ratio 30:1 0.18 —O— 30% Moisture 0.16 — —D— 50% Moisture 8" —A— 70% Moisture A 3 0.14 -1 U A ‘ a c 0: 0.12 -1 > A C 8 . ‘ c 0.10 - AAA 0 -A 8 0 0.08 - :g E 0.06 - O 8 23 0.04 - E LL 0.02 - 0.00 " 0 20 40 60 80 100 120 Figure A.2 Conversion of initial carbon to CO2 during the composting of poplar wood at an initial CIN ratio of 30:1 and varying moisture contents. 127 CIN Ratio 50:1 0.18 0 16 _ —O— 30% Moisture or ' —Cl— 50% Moisture . ‘ 8 —A— 70% Moisture A 9, 0.14 - “ 'c A ‘ A 9:) a ' g) 0.12 - ‘ C A O A .I O A .0 8 0.10 - 2‘ ' .3 l 0 lg /“ I .0 0 0.08 - .A . ' ' E A I . :E 0.06 - A- _. - , ' g ‘ I'. . '3 0 04 "I 0 ' — A‘ l. e “ .Ll 0 u- ‘ L. O. 0.02 ‘ A I. . ‘ I. 0.. 0.00 .. (.7 .l(..(. (.01 I I 0 _ 20 40 60 80 100 Days 120 Figure A.3 Conversion of initial carbon to CO2 during the composting of poplar wood at an initial CIN ratio of 50:1 and varying moisture contents. 128 70% Moisture 0.18 0.16 _ —O— C/N Ratio 10.1 A O“ —D— C/N Ratio 30:1 ”/5- 0 —A— UN Ratio 50:1 “/5 .. 2 0-14 " —v— No added N ./-' 8 4"" ' t I g 0.12 a ‘/ 00 8 A" A4' 0 v g 0.10 -‘ 'v‘ AIAI- v." E 71‘ ‘- V V v . 0 0.08 - g ' ’ '5 o :43 o E 0.06 — o 8 =5 0.04 d E u. 0.02 - 0-00 ‘ . I I I I I O 20 40 60 80 100 Days 120 Figure A.4: Conversion of initial carbon to C02 during the composting of poplar wood at an initial moisture content of 70% and varying CIN ratios. 129 APPENDIX B Tabulated Data Table title page B.1 Data for Figure 3.1 ............................... 131 B2 Data for Figure 3.2 ............................... 132 B3 Data for Figure 3.3 ............................... 133 3.4 Data for Figure 3.4 ............................... 134 B5 Data for Figure 3.5 ............................... 135 8.6 Data for Figure 4.1 ............................... 136 B.7 Data for Figure 4.2 ............................... 137 B.8 Data for Figure 4.3 ............................... 138 8.9 Data for Figure 4.4 ............................... 139 B.10 Data for Figure 4.5 ............................... 140 B.11 Data for Figure 5.1 ............................... 141 B.12 Data for Figure 5.2 ............................... 142 B.13 Data for Figure 5.3 ............................... 143 B.14 Data for Figure 5.4 ............................... 144 B.15 Data for Figure 5.5 ............................... 145 3.16 Data for Figure 6.2 ............................... 146 3.17 B.18 B.19 8.20 B21 B22 130 Data for Figure 6.3 ............................... Data for Figure 6.4 ............................... Data for Figure A.1 ............................... Data for Figure A.2 ............................... Data for Figure A.3 ............................... Data for Figure A.4 ............................... 147 148 149 150 151 152 Table 13.1: Data for Figure 3.1 131 9 < 333388$$853888338o Wood 55°C Wood 25°C Wood 37°C Averag Half rang: Averag Half range Day Average Half rang 0.0000 0.0000 0.0000 0.0000 0 0.0000 0.0000 0.01 10 0.001 1 0.0099 0.0004 6 0.0182 0.0022 0.0153 0.0013 0.0143 0.0000 10 0.0284 0.0056 0.0210 0.0018 0.0169 0.0000 14 0.0375 0.0066 0.0269 0.0001 0.0210 0.0000 18 0.0450 0.0059 0.0307 0.0016 0.0241 0.0001 24 0.0516 0.0064 0.0338 0.0007 0.0261 0.0001 28 0.0576 0.0041 0.0369 0.0009 0.0299 0.0001 33 0.0651 0.0046 0.0416 0.0007 0.0324 0.0004 38 0.0697 0.0054 0.0422 0.0001 0.0334 0.0006 42 0.0749 0.0042 0.0453 0.0000 0.0352 0.0010 49 0.0824 0.0045 0.0476 0.0006 0.0376 0.0012 54 0.0870 0.0044 0.0501 0.0010 0.0397 0.0008 60 0.091 1 0.0032 0.0532 0.0005 0.0431 0.0009 65 0.0927 0.0025 0.0550 0.0003 0.0439 0.0012 74 0.0973 0.0020 0.0573 0.0012 0.0452 0.0014 79 0.0992 0.0027 0.0577 0.0012 0.0461 0.0012 88 0.1051 0.0057 0.0590 0.0014 0.0465 0.0012 Table 3.2: Data for Figure 3.2 132 Day $3338$88863888838o Wood 55°C Wood 25°C Wood 37°C Averag Half rang Average Halt range Day Averag Half rang 0.0000 0.0000 0.0000 0.0000 0 0.0000 0.0000 0.1044 0.0174 0.0963 0.0093 6 0.1389 0.0365 0.1492 0.0146 0.1962 0.0035 10 0.3286 0.0333 0.2570 0.0031 0.3923 0.0068 14 0.4383 0.0331 0.4768 0.0241 0.541 1 0.0656 18 0.4522 0.0423 0.7942 0.0772 0.7633 0.0958 24 0.5677 0.0665 0.8165 0.0583 0.8423 0.0905 28 0.5814 0.0792 0.8953 0.0471 0.9949 0.0912 33 0.6814 0.0646 0.9206 0.0550 0.9949 0.0912 38 0.7378 0.0661 0.9664 0.0666 1.031 1 0.0926 42 0.7895 0.0639 0.9997 0.0468 1.1345 0.0649 49 0.8766 0.0509 1.0056 0.0410 1.1757 0.0718 54 0.9565 0.0486 1.0377 0.0568 1.1939 0.0821 60 1.0663 0.0544 1 .0643 0.0478 1 .2242 0.0772 65 1 .1275 0.0709 1.1023 0.0602 1.2691 0.0872 74 1.2250 0.1610 1.1133 0.0712 1.2984 0.0752 79 1.3160 0.2428 1 .1 176 0.0756 1.3281 0.0855 88 1 .4680 0.3719 1 .1609 0.0753 1 .3516 0.0754 Table 13.3: Data for Figure 3.3 133 Wood 55°C Day Averag Half range 0 0.0000 0.0000 10 0.0110 0.0011 14 0.0153 0.0013 20 0.0210 0.0018 26 0.0269 0.0001 32 0.0307 0.0016 35 0.0338 0.0007 41 0.0369 0.0009 45 0.0416 0.0007 49 0.0422 0.0001 53 0.0453 0.0000 59 0.0476 0.0006 63 0.0501 0.0010 68 0.0532 0.0005 73 0.0550 0.0003 77 0.0573 0.0012 81 0.0577 0.0012 84 0.0590 0.0014 Cobs 55°C AveraE Half range 0.0000 0.0000 0.0319 0.0002 0.0455 0.0009 0.0580 0.0008 0.0754 0.0010 0.0819 0.0005 0.0909 0.0002 0.1010 0.0045 0.1082 0.0075 0.1123 0.0095 0.1220 0.0132 0.1325 0.01 1 1 0.1375 0.0107 0.1454 0.0099 0.1511 0.0095 0.1544 0.0096 0.1547 0.0099 0.1591 0.0114 Table BA: Data for Figure 3.4 134 Day $3338888353888838o Wood 55°C Cobs 55°C Averag Half range Averag Half rang 0.0000 0.0000 0.0000 0.0000 0.1044 0.0174 0.2843 0.0159 0.1492 0.0146 0.3077 0.0191 0.2570 0.0031 0.3284 0.0265 0.4768 0.0241 0.3682 0.0421 0.7942 0.0772 0.3871 0.0330 0.8165 0.0583 0.3950 0.0410 0.8953 0.0471 0.4197 0.0348 0.9206 0.0550 0.4263 0.0394 0.9664 0.0666 0.4453 0.0356 0.9997 0.0468 0.4570 0.0279 1 .0058 0.0410 0.4897 0.0291 1.0377 0.0568 0.5094 0.0313 1 .0643 0.0478 0.5325 0.0347 1.1023 0.0602 0.5799 0.0449 1.1 133 0.0712 0.5824 0.0424 1.1 176 0.0756 0.5962 0.0286 1.1609 0.0753 0.6213 0.0356 Table B.5: Data for Figure 3.5 135 ChCls MeOH NaOH H20 C02 Bound Wood 25°C Day 0 100491.3 4021.4 36.75 191.5 0 1090.059 Day 30 8732782 3246580 1 565572 1880705 1 34334.5 1 645452 Day 60 7623253 2782227 1505333 2299621 206916 2350058 Wood 37°C Day 0 1004913 4021 .4 36.75 191 .5 0 1090.059 Day 30 2977954 7031470 2895231 229583.6 1513835 2583450 Day 60 1399850 7701231 3062916 1598403 4516595 2671062 Wood 55°C Day 0 100491.3 4021.4 36.75 191.5 0 1090.059 Day 30 21 96903 6563652 2176271 325801 .4 1397745 3681 661 Day 60 2038878 60091 32 2182022 2581 63.5 1 76984 4229462 Cobs 55°C Day 0 1 14153 2784.4 737.75 74.2 0 1225.44 Day 30 1375303 4191373 9444908 377663.1 68125 1214582 Day 60 1298248 3785900 8526447 373885 86179 1315579 Table B.6: Data for Figure 4.1 136 Native STEX AFEX Hours Average Std.Dev. Average Std.Dev. Average Std.Dev. Average Std.Dev. #ankN-e-‘O 8 0.000 7.192 8.219 9.818 1 1.873 15.069 15.412 19.864 21 .804 0.000 0.1 14 0.000 0.000 0.000 0.000 0.1 14 0.000 0.342 0.000 28.422 38.358 51 .760 67.126 78.448 80.990 90.1 17 91 .504 0.000 0.231 0.231 0.462 2.1 95 1 .502 1 .733 3.004 3.466 0.000 1 6.096 1 8.380 22.71 8 28.768 35.21 8 37.844 46.349 51 .524 0.000 0.255 0.471 0.731 0.666 0.797 0.987 0.323 0.21 5 Newspaper 0.000 0.000 16.240 0.187 21 .958 0.323 26.914 0.656 33.166 0.494 38.275 0.755 41 .401 0.988 46.204 1 .347 48.339 1 .029 Table B.7: Data for Figure 4.2 137 Native AF EX STEX Paper Day Average Std. Dev. Average Std. Dev. Aveggg Std. Dev. Averagi Std. Dev. 0 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 6 0.0182 0.0022 0.0735 0.0158 0.0043 0.0008 0.0076 0.0014 10 0.0284 0.0056 0.0931 0.0092 0.0059 0.0009 0.01 10 0.0015 14 0.0375 0.0066 0.1155 0.0059 0.0073 0.0011 0.0146 0.0023 18 0.0450 0.0059 0.1329 0.0035 0.0077 0.0012 0.0152 0.0023 24 0.0516 0.0084 0.1480 0.0080 0.0097 0.0025 0.0187 0.0025 28 0.0576 0.0041 0.1699 0.0107 0.01 18 0.0032 0.0216 0.0019 33 0.0651 0.0046 0.2045 0.0272 0.0141 0.0036 0.0239 0.0020 38 0.0697 0.0054 0.2484 0.0267 0.0157 0.0050 0.0269 0.0020 42 0.0749 0.0042 0.2870 0.0404 0.0185 0.0059 0.0353 0.0078 49 0.0824 0.0045 0.3248 0.0493 0.0250 0.0108 0.0553 0.0284 54 0.0870 0.0044 0.3484 0.0547 0.0390 0.0154 0.0734 0.0491 60 0.0911 0.0032 0.3672 0.0575 0.0495 0.0190 0.0881 0.0672 65 0.0927 0.0025 0.3791 0.0556 0.0534 0.0194 0.0994 0.0783 74 0.0973 0.0020 0.4033 0.0552 0.0723 0.0238 0.1085 0.0840 79 0.0992 0.0027 0.4129 0.0559 0.0829 0.0217 0.1108 0.0853 88 0.1051 0.0057 0.4388 0.0607 0.1009 0.0217 0.1233 0.0965 95 0.1079 0.0055 0.4538 0.0631 0.1087 0.0236 0.1357 0.1 123 102 0.11 19 0.0048 0.4654 0.0847 0.1 167 0.0238 0.1461 0.1240 108 0.1 159 0.0051 0.4768 0.0680 0.1218 0.0242 0.1574 0.1352 112 0.1220 0.0046 0.4867 0.0703 0.1268 0.0244 0.1629 0.1374 124 0.1279 0.0045 0.5067 0.0754 0.1384 0.0247 0.1799 0.1569 130 0.1301 0.0053 0.5197 0.0786 0.1423 0.0232 0.1868 0.1641 138 0.1358 0.0044 0.5303 0.0781 0.1503 0.0198 0.1928 0.1676 143 0.1363 0.0041 0.5338 0.0781 0.1521 0.0198 0.1971 0.1717 151 0.1406 0.0048 0.5437 0.0809 0.1557 0.0201 0.2032 0.1769 180 0.1474 0.0051 0.5574 0.0844 0.1642 0.0173 0.2102 0.1805 Table B.8: Data for Figure 4.3 138 Day Native AFEX STEX Paper Average Std. Dev. Average Std. Dev. Averag Std. Dev. Average Std. Dev. 0 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 6 0.1389 0.0365 0.1957 0.0078 0.0194 0.0031 0.4755 0.0008 10 0.3286 0.0333 0.2654 0.0302 0.0305 0.0045 0.6437 0.0202 14 0.4363 0.0331 0.2893 0.0253 0.0367 0.0060 0.7737 0.0260 18 0.4522 0.0423 0.2923 0.0279 0.0547 0.0005 0.8646 0.0102 24 0.5677 0.0665 0.3296 0.01 14 0.0691 0.0047 0.9671 0.0212 28 0.5814 0.0792 0.3490 0.0088 0.0828 0.0063 1 .1497 0.0373 33 0.6814 0.0646 0.3566 0.0152 0.1029 0.0060 1.2381 0.0325 38 0.7378 0.0661 0.3566 0.0152 0.1094 0.0031 1.2530 0.0475 42 0.7895 0.0639 0.3579 0.0140 0.1415 0.0316 1.3493 0.0775 49 0.8766 0.0509 0.4134 0.0027 0.1558 0.0173 1.4846 0.1354 54 0.9565 0.0486 0.4731 0.0234 0.1897 0.0140 1.6385 0.2285 60 1.0663 0.0544 0.5921 0.0751 0.2367 0.0056 1.6886 0.2131 65 1.1275 0.0709 0.7018 0.1848 0.2637 0.0049 1.7074 0.1958 74 1.2250 0.1610 0.9104 0.2358 0.8429 0.0606 1.8623 0.2515 79 1.3160 0.2428 0.9823 0.2200 0.9552 0.0382 1.9123 0.2640 88 1.4680 0.3719 1.2872 0.3573 1.0261 0.0042 2.2399 0.5024 95 1.7074 0.3780 1.6159 0.4024 1.2272 0.0504 2.6979 0.9117 102 1.8083 0.3399 1.7900 0.3881 1.2363 0.0510 3.0564 1.2171 108 1.8906 0.2645 2.0031 0.4560 1 .3220 0.0152 3.3931 1 .5035 112 2.0476 0.3268 2.1164 0.4173 1.3627 0.0412 3.7414 1.7806 124 2.2618 0.2577 2.5502 0.4315 1.4571 0.0574 4.3262 2.2565 130 2.4391 0.1949 2.7931 0.5249 1.4975 0.0691 5.0510 2.8466 138 2.9131 0.0650 3.6687 0.6052 1.6441 0.0912 5.4196 2.9923 143 2.9424 0.0878 3.7422 0.5904 1.6627 0.0899 5.8632 3.3973 151 3.0985 0.0018 4.0361 0.5345 1.7368 0.0791 6.5909 3.9778 160 3.3014 0.0745 4.3016 0.4518 1.7736 0.0968 6.9527 4.3037 Table B.9: Data for Figure 4.4 139 Day Native AF EX STEX Paper Total C02 “(302 Total C02 “002 Total CO; “(302 Total C02 “(302 0 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 6 0.0182 0.0139 0.0735 0.0196 0.0043 0.0019 0.0076 0.0476 10 0.0284 0.0329 0.0931 0.0265 0.0059 0.0031 0.01 10 0.0644 14 0.0375 0.0436 0.1 155 0.0289 0.0073 0.0037 0.0146 0.0774 18 0.0450 0.0452 0.1329 0.0292 0.0077 0.0055 0.0152 0.0865 24 0.0516 0.0568 0.1480 0.0330 0.0097 0.0069 0.0187 0.0967 28 0.0576 0.0581 0.1699 0.0349 0.01 18 0.0083 0.0216 0.1 150 33 0.0651 0.0681 0.2045 0.0357 0.0141 0.0103 0.0239 0.1238 38 0.0697 0.0738 0.2484 0.0357 0.0157 0.0109 0.0269 0.1253 42 0.0749 0.0790 0.2870 0.0358 0.0185 0.0142 0.0353 0.1349 49 0.0824 0.0877 0.3248 0.0413 0.0250 0.0156 0.0553 0.1485 54 0.0870 0.0957 0.3484 0.0473 0.0390 0.0190 0.0734 0.1638 60 0.091 1 0.1066 0.3672 0.0592 0.0495 0.0237 0.0881 0.1689 65 0.0927 0.1 127 0.3791 0.0702 0.0534 0.0264 0.0994 0.1707 74 0.0973 0.1225 0.4033 0.0910 0.0723 0.0843 0.1085 0.1862 79 0.0992 0.1316 0.4129 0.0982 0.0829 0.0955 0.1 108 0.1912 88 0.1051 0.1468 0.4388 0.1287 0.1009 0.1026 0.1233 0.2240 95 0.1079 0.1707 0.4538 0.1616 0.1087 0.1227 0.1357 0.2698 102 0.1119 0.1808 0.4654 0.1790 0.1167 0.1236 0.1461 0.3056 108 0.1 159 0.1891 0.4768 0.2003 0.1218 0.1322 0.1574 0.3393 112 0.1220 0.2048 0.4867 0.21 16 0.1268 0.1363 0.1629 0.3741 124 0.1279 0.2262 0.5067 0.2550 0.1364 0.1457 0.1799 0.4326 130 0.1301 0.2439 0.5197 0.2793 0.1423 0.1497 0.1868 0.5051 138 0.1358 0.2913 0.5303 0.3669 0.1503 0.1644 0.1928 0.5420 143 0.1363 0.2942 0.5338 0.3742 0.1521 0.1663 0.1971 0.5863 151 0.1406 0.3098 0.5437 0.4036 0.1557 0.1737 0.2032 0.6591 160 0.1474 0.3301 0.5574 0.4302 0.1642 0.1774 0.2102 0.6953 Table 13.10: Data for Figure 4.5 140 ChCl3 MeOH NaOH H20 CO; Bound Native Day 0 100491.3 4021.4 36.75 _ 191.5 0 1090.059 Day 40 1331614 1937492 1784633 59265.5 91639.0 3142859 Day 160 1349651 2427741 2261819 3248113 410071.4 3551869 AFEX Day 0 114153 2784.4 737.75 74.2 0 2920.203 Day 40 2123439 2183782 3097970 77457.6 44299.2 2948196 Day 160 32787.68 1007718 4124086 4589238 5343062 3962125 STEX Day 0 125399.6 5717.5 423.5 100 0 2262.97 Day 40 1225898 4390178 2701964 65810.1 13588.05 1378757 Day 160 79210.48 4110693 3514895 2836122 220307.4 1700660 Paper Day 0 1959401 3583 415.25 31 1.6 0 421 1.914 Day 40 240017.8 1935721 2515004 32041.1 84537.5 3839674 Day 160 80540.05 1588553 1838069 295513 413118.6 4576381 141 Table B.11: Data for Figure 5.1 Wood Wood+PC Day AveraE Halt rang Day Averag Halt rang; 0 0.0000 0.0000 0 0.0000 0.0000 6 0.0182 0.0022 3 0.0102 0.0017 10 0.0284 0.0056 6 0.0260 0.0026 14 0.0375 0.0066 8 0.0401 0.0011 18 0.0450 0.0059 10 0.0473 0.0004 24 0.0516 0.0064 12 0.0544 0.0024 28 0.0576 0.0041 14 0.0611 0.0046 33 0.0651 0.0046 16 0.0682 0.0058 38 0.0697 0.0054 18 0.0757 0.0098 42 0.0749 0.0042 20 0.0809 0.0117 49 0.0824 0.0045 22 0.0965 0.0183 54 0.0870 0.0044 24 0.1128 0.0262 60 0.0911 0.0032 26 0.1297 0.0323 65 0.0927 0.0025 28 0.1473 0.0353 74 0.0973 0.0020 30 0.1700 0.0362 79 0.0992 0.0027 32 0.1924 0.0359 88 0.1051 0.0057 34 0.2161 0.0356 95 0.1079 0.0055 36 0.2430 0.0344 38 0.2640 0.0336 40 0.2855 0.0343 43 0.3241 0.0318 45 0.3453 0.0311 47 0.3651 0.0323 49 0.3881 0.0292 51 0.4088 0.0256 53 0.4269 0.0212 55 0.4427 0.0208 57 0.4621 0.0155 59 0.4757 0.0106 61 0.4903 0.0064 63 0.5077 0.0035 67 0.5311 0.0071 69 0.5455 0.0121 73 0.5585 0.0205 76 0.5708 0.0266 81 0.5832 0.0316 85 0.5956 0.0354 89 0.6016 0.0393 94 0.6095 0.0411 Table B.12: Data for Figure 5.2 142 Wood Wood + PC Day Average Halt raga Day Average Halt rang 0 0.0000 0.0000 0 0.0000 0.0000 16 0.0387 0.0001 8 0.0284 0.0003 24 0.0478 0.0035 16 0.0377 0.0003 31 0.0632 0.0088 23 0.0509 0.0016 33 0.0681 0.01 14 25 0.0561 0.0026 40 0.0974 0.0200 32 0.0855 0.0086 49 0.1051 0.0240 41 0.1065 0.0189 56 0.1221 0.0265 48 0.1382 0.0107 59 0.1261 0.0273 51 0.1468 0.0089 64 0.1406 0.0222 56 0.1604 0.0077 68 0.1458 0.0258 60 0.1695 0.0064 76 0.1596 0.0286 68 0.1876 0.0057 83 0.1700 0.0314 75 0.2025 0.0049 82 0.2222 0.0044 143 Table B.13: Data for Figure 5.3 Day Wood + PC Day Wood Average Halt rang Average Halt rang 0 0.0000 0.0000 0 0.0000 0.0000 3 0.0142 0.0076 6 0.1389 0.0365 6 0.2417 0.0135 10 0.3286 0.0333 8 0.3719 0.0330 14 0.4363 0.0331 10 0.4430 0.0328 18 0.4522 0.0423 12 0.5364 0.0370 24 0.5677 0.0665 14 0.6024 0.0324 28 0.5814 0.0792 16 0.7054 0.0382 33 0.6814 0.0646 18 0.7835 0.0457 38 0.7378 0.0661 20 0.8762 0.0700 42 0.7895 0.0639 22 1 .0268 0.1326 49 0.8766 0.0509 24 1.1825 0.2028 54 0.9565 0.0486 26 1.3864 0.271 1 60 1.0663 0.0544 28 1 .5496 0.3164 65 1 .1275 0.0709 30 1.7862 0.3712 74 1.2250 0.1610 32 2.0093 0.4109 79 1.3160 0.2428 34 2.2396 0.4774 88 1.4680 0.3719 36 2.4821 0.5681 38 2.6765 0.6305 40 2.8923 0.7181 43 3.3072 0.8414 45 3.5863 0.9480 47 3.9340 1.1 143 49 4.3576 1.1970 51 4.7891 1.3415 53 5.1962 1.4619 55 5.6148 1.6369 57 6.1718 1.7890 59 6.6537 1.8758 61 7.1023 1.9403 63 7.6064 1.9169 67 8.6895 1.9257 69 9.1997 1.8705 73 10.2577 1.6905 76 10.9807 1.51 17 81 12.1437 1 .1 134 85 12.7868 0.8258 89 13.4585 0.5679 94 14.2570 0.3134 144 Table B.14: Data for Figure 5.4 Wood+Corn Wood+Com+PC Wood Day Averag Halt rang Averae Halt range DayI Average Halt range 0 0.0000 0.0000 0.0000 0.0000 0 0.0000 0.0000 3 0.0455 0.0012 0.0507 0.0010 6 0.0182 0.0022 6 0.0836 0.0068 0.1013 0.0004 10 0.0284 0.0056 8 0.1136 0.0092 0.1412 0.0111 14 0.0375 0.0066 10 0.1452 0.0042 0.1603 0.0201 18 0.0450 0.0059 12 0.1919 0.0010 0.2026 0.0213 24 0.0516 0.0064 14 0.2374 0.0001 0.2376 0.0104 28 0.0576 0.0041 16 0.2813 0.0021 0.2694 0.0025 33 0.0651 0.0046 18 0.3192 0.0027 0.2927 0.0105 38 0.0697 0.0054 20 0.3414 0.0025 0.3064 0.0155 42 0.0749 0.0042 22 0.3656 0.0060 0.3212 0.0200 49 0.0824 0.0045 24 0.3849 0.0099 0.3331 0.0215 54 0.0870 0.0044 26 0.4024 0.0116 0.3444 0.0251 60 0.0911 0.0032 28 0.4195 0.0137 0.3542 0.0262 65 0.0927 0.0025 30 0.4352 0.0157 0.3638 0.0284 74 0.0973 0.0020 32 0.4492 0.0172 0.3709 0.0303 79 0.0992 0.0027 34 0.4599 0.0161 0.3791 0.0317 88 0.1051 0.0057 36 0.4729 0.0178 0.3853 0.0327 95 0.1079 0.0055 38 0.4816 0.0183 0.3929 0.0349 40 0.4928 0.0187 0.4066 0.0332 43 0.5103 0.0203 0.4286 0.0291 45 0.5156 0.0206 0.4362 0.0270 47 0.5231 0.0206 0.4466 0.0239 49 0.5328 0.0217 0.4581 0.0221 51 0.5396 0.0219 0.4663 0.0203 53 0.5454 0.0228 0.4733 0.0181 55 0.5544 0.0225 0.4808 0.0195 57 0.5604 0.0225 0.4883 0.0184 59 0.5651 0.0227 0.4932 0.0176 61 0.5702 0.0233 0.4993 0.0169 63 0.5781 0.0232 0.5062 0.0159 67 0.5907 0.0232 0.5177 0.0142 69 0.5990 0.0230 0.5270 0.0139 73 0.6029 0.0233 0.5345 0.01 15 76 0.6099 0.0223 0.5418 0.01 16 81 0.6219 0.0217 0.5531 0.01 14 85 0.6316 0.0201 0.5576 0.01 13 89 0.6407 0.0169 0.5640 0.01 17 94 0.6452 0.0124 0.5739 0.0135 Table B.15: Data for Figure 5.5 145 DAY 88833383838388383888888388883833358mmuo Wood+Corn 0.0000 0.2228 0.2423 0.3030 0.3731 0.4330 0.4809 0.5336 0.5658 0.5900 0.6307 0.691 4 0.7641 0.8463 0.9548 1 .0763 1 .21 07 1 .4433 1 .61 54 1 .7683 2.01 75 2.1 800 2.3653 2.5508 2.7305 2.8708 3.1 220 3.2823 3.4052 3.5482 3.6696 3.9290 4.0377 4.2441 4.4320 4.6749 4.9641 5.1 142 5.2146 0.0000 0.0155 0.0146 0.0271 0.0448 0.0640 0.0636 0.0653 0.0626 0.0617 0.0361 0.0009 0.0519 0.0985 0.1669 0.2402 0.3309 0.4435 0.5289 0.6159 0.7370 0.8447 0.9274 1 .0268 1 .1214 1 .1918 1 .3562 1 .4160 1 .4704 1 .5315 1 .5722 1 .6965 1 .7288 1 .8218 1 .8451 1 .9067 1 .8413 1 .8425 1 .7670 Wood+Com+PC 0.0000 0.2962 0.3405 0.3525 0.3757 0.4145 0.5012 0.571 6 0.6123 0.6567 0.7038 0.7496 0.7699 0.7946 0.81 39 0.8288 0.8602 0.8941 0.9068 0.9320 1 .1 314 1 .2638 1 .4045 1 .5374 1 .6588 1 .761 0 1 .8793 2.0710 2.1 689 2.2768 2.3944 2.6476 2.7467 2.9491 3.1 005 3.3074 3.461 9 3.61 90 3.8012 Averag Halt rang Averag Halt range 0.0000 0.0507 0.0689 0.0642 0.0441 0.0210 0.0470 0.0612 0.0525 0.0664 0.0862 0.1 148 0.1236 0.1278 0.1325 0.1403 0.1386 0.1479 0.1523 0.1491 0.0032 0.0805 0.1474 0.1847 0.2146 0.2386 0.2514 0.3487 0.3642 0.3933 0.4223 0.5067 0.5408 0.6247 0.6828 0.7345 0.7883 0.8457 0.8844 Wood Day Average Halt range 0 6 10 14 18 24 §8838$888 79 0.0000 0.1 389 0.3286 0.4363 0.4522 0.5677 0.5814 0.6814 0.7378 0.7895 0.8766 0.9565 1 .0663 1 .1275 1 .2250 1 .31 60 1 .4680 0.0000 0.0365 0.0333 0.0331 0.0423 0.0665 0.0792 0.0646 0.0661 0.0639 0.0509 0.0486 0.0544 0.0709 0.1 61 0 0.2428 0.371 9 Table 3.16: Data for Figure 6.2 146 Wood+PC Day Total 002 “(:02 0 0.0000 0.0000 3 0.0102 0.0001 6 0.0260 0.0024 8 0.0401 0.0037 10 0.0473 0.0044 12 0.0544 0.0054 14 0.0611 0.0060 16 0.0682 0.0071 18 0.0757 0.0078 20 0.0809 0.0088 22 0.0965 0.0103 24 0.1128 0.0118 26 0.1297 0.0139 28 0.1473 0.0155 30 0.1700 0.0179 32 0.1924 0.0201 34 0.2161 0.0224 36 0.2430 0.0248 38 0.2640 0.0268 40 0.2855 0.0289 43 0.3241 0.0331 45 0.3453 0.0359 47 0.3651 0.0393 49 0.3881 0.0436 51 0.4088 0.0479 53 0.4269 0.0520 55 0.4427 0.0561 57 0.4621 0.0617 59 0.4757 0.0665 61 0.4903 0.0710 63 0.5077 0.0761 67 0.531 1 0.0869 69 0.5455 0.0920 73 0.5585 0.1026 76 0.5708 0.1098 81 0.5832 0.1214 85 0.5956 0.1279 89 0.6016 0.1346 94 0.6095 0.1426 Table B.17: Data for Figure 6.3 147 Total 002 data Day Actual Predicted 0 0.0000 0.0000 3 1.0235 0.5193 6 2.6010 1.1770 8 4.0067 1.7081 10 4.7290 2.3257 12 5.4416 3.0415 14 6.1121 3.8678 16 6.8247 4.8174 18 7.5696 5.9028 20 8.0911 7.1360 22 9.6459 8.5275 24 11.2783 10.0858 26 12.9724 11.8157 28 14.7344 13.7179 30 17.0017 15.7875 32 19.2367 18.0137 34 21.6109 20.3790 36 24.2960 22.8595 38 26.3982 25.4253 40 28.5457 28.0421 43 32.4066 31.9808 45 34.5282 34.5606 47 36.5105 37.0621 49 38.8134 39.4543 51 40.8799 41.7120 53 42.6906 43.8164 55 44.2745 45.7550 57 46.2146 47.5220 59 47.5686 49.1168 61 49.0294 50.5435 63 50.7720 51.8098 67 53.1106 53.9035 69 54.5487 54.7552 73 55.8475 56.1314 76 57.0816 56.9245 81 58.3222 57.8973 85 59.5595 58.4395 89 60.1555 58.8308 94 60.9523 59.1689 Table B.18: Data for Figure 6.4 148 “002 data Day Actual Predicted 0 0.0000 0.0000 3 0.0142 0.0913 6 0.2417 0.1963 8 0.3719 0.2747 10 0.4430 0.3607 12 0.5364 0.4549 14 0.6024 0.5578 16 0.7054 0.6702 18 0.7835 0.7929 20 0.8762 0.9266 22 1.0268 1.0721 24 1.1825 1.2302 26 1.3864 1.4017 28 1.5496 1.5874 30 1.7862 1.7881 32 2.0093 2.0046 34 2.2396 2.2376 36 2.4821 2.4877 38 2.6765 2.7556 40 2.8923 3.0417 43 3.3072 3.5056 45 3.5863 3.8383 47 3.9340 4.1897 49 4.3576 4.5594 51 4.7891 4.9470 53 5.1962 5.3516 55 5.6148 5.7724 57 6.1718 6.2080 59 6.6537 6.6570 61 7.1023 7.1176 63 7.6064 7.5878 67 8.6895 8.5489 69 9.1997 9.0350 73 10.2577 10.0066 76 10.9807 10.7250 81 12.1437 11.8805 85 12.—7868 12.7498 89 13.4585 13.5571 94 14.2570 14.4673 Table B.19: Data for Figure A.l 149 CIN Ratio 10:1 pg 30% Moisture 50% Moisture 70% Moisture 0 0.0000 0.0000 0.0000 4 0.0012 0.0007 0.0013 6 0.0012 0.0010 0.0029 10 0.0016 0.0012 0.0110 11 0.0018 0.0023 0.0119 13 0.0018 0.0023 0.0153 14 0.0020 0.0027 0.0166 18 0.0020 0.0028 0.0216 21 0.0025 0.0031 0.0239 24 0.0028 0.0032 0.0296 25 0.0035 0.0033 0.0325 28 0.0045 0.0046 0.0370 31 0.0045 0.0046 0.0388 32 0.0045 0.0047 0.0397 35 0.0060 0.0063 0.0454 36 0.0060 0.0063 0.0479 39 0.0061 0.0063 0.0508 41 0.0064 0.0070 0.0558 42 0.0071 0.0075 0.0581 45 0.0071 0.0075 0.0595 47 0.0078 0.0076 0.0610 49 0.0085 0.0083 0.0627 52 0.0097 0.0088 0.0653 55 0.0114 0.0091 0.0676 59 0.0126 0.0095 0.0731 68 0.0172 0.0139 0.0819 71 0.0182 0.0169 0.0879 76 0.0189 0.0213 0.0975 80 0.0206 0.0237 0.1055 87 0.0226 0.0293 0.1202 90 0.0226 0.0300 0.1210 96 0.0272 0.0348 0.1323 105 0.0289 0.0392 0.1414 108 0.0304 0.0419 0.1429 Table 3.20: Data for Figure A.2 150 CIN Ratio 30:1 E 30% Moisture 50% Moisture 70% Moisture 0 0.0000 0.0000 0.0000 4 0.0012 0.0010 0.0153 6 0.0012 0.0012 0.0268 8 0.0304 10 0.0020 0.0023 0.0375 11 0.0020 0.0023 0.0399 13 0.0020 0.0024 0.0445 14 0.0025 0.0032 0.0470 18 0.0025 0.0069 0.0555 21 0.0028 0.01 1 1 0.0605 24 0.0029 0.0157 0.0668 25 0.0031 0.0178 0.0690 28 0.0044 0.0210 0.0744 31 0.0044 0.0226 0.0770 32 0.0045 0.0247 0.0800 35 0.0058 0.0283 0.0855 36 0.0058 0.0288 0.0866 39 0.0058 0.0303 0.0906 41 0.0065 0.0319 0.0932 42 0.0072 0.0329 0.0949 45 0.0072 0.0338 0.0977 47 0.0084 0.0350 0.0999 49 0.0096 0.0363 0.1024 52 0.0161 0.0385 0.1062 55 0.0217 0.0401 0.1088 59 0.0257 0.0421 0.1132 68 0.0331 0.0491 0.1237 71 0.0348 0.0515 0.1265 76 0.0369 0.0540 0.1300 80 0.0399 0.0569 0.1333 87 0.0515 0.0623 0.1390 90 0.0548 0.0636 0.1404 96 0.0647 0.0704 0.1474 105 0.0737 0.0771 0.1532 108 0.0759 0.0795 0.1558 Table 3.21: Data for Figure A.3 151 CIN Ratio 50:1 Day 30% Moisture 50% Moisture 70% Moisture 0 0.0000 0.0000 0.0000 4 0.0007 0.0006 0.0101 6 0.0010 0.001 1 0.0169 8 0.0227 10 0.0013 0.0051 0.0302 11 0.0013 0.0060 0.0326 13 0.0013 0.0086 0.0367 14 0.0018 0.01 13 0.0396 18 0.0018 0.0184 0.0498 21 0.0021 0.021 1 0.0567 24 0.0021 0.0239 0.0654 25 0.0025 0.0249 0.0672 28 0.0038 0.0300 0.0734 31 0.0038 0.0328 0.0776 32 ' 0.0041 0.0342 0.0798 35 0.0056 0.0387 0.0852 36 0.0058 0.0395 0.0859 39 0.0061 0.0427 0.0899 41 0.0061 0.0451 0.0930 42 0.0087 0.0464 0.0945 45 0.0108 0.0487 0.0975 47 0.0137 0.0505 0.1014 49 0.0166 0.0524 0.1043 52 0.0252 0.0562 0.1086 55 0.0296 0.0583 0.1 1 12 59 0.0347 0.0616 0.1 164 68 0.0539 0.0743 0.1269 71 0.0568 0.0775 0.1297 76 0.0648 0.0818 0.1327 80 0.0701 0.0844 0.1357 87 0.0807 0.0918 0.1410 90 0.0842 0.0941 0.1419 96 0.0946 0.1020 0.1482 105 0.1036 0.1079 0.1547 108 0.1053 0.1101 0.1571 152 Table 13.22: Data for Figure A.4 70 % Moisture Day CIN 10:1 CIN 30:1 CIN 50:1 Day No added N 0 0.0000 0.0000 0.0000 0 0.0000 4 0.0013 0.0153 0.0101 6 0.0182 6 0.0029 0.0268 0.0169 10 0.0284 8 0.0304 0.0227 14 0.0375 10 0.01 10 0.0375 0.0302 18 0.0450 11 0.0119 0.0399 0.0326 24 0.0516 13 0.0153 0.0445 0.0367 28 0.0576 14 0.0166 0.0470 0.0396 33 0.0651 18 0.0216 0.0555 0.0498 38 0.0697 21 0.0239 0.0605 0.0567 42 0.0749 24 0.0296 0.0668 0.0654 49 0.0824 25 0.0325 0.0690 0.0672 54 0.0870 28 0.0370 0.0744 0.0734 60 0.091 1 31 0.0388 0.0770 0.0776 65 0.0927 32 0.0397 0.0800 0.0798 74 0.0973 35 0.0454 0.0855 0.0852 79 0.0992 36 0.0479 0.0866 0.0859 88 0.1051 39 0.0508 0.0906 0.0899 41 0.0558 0.0932 0.0930 42 0.0581 0.0949 0.0945 45 0.0595 0.0977 0.0975 47 0.0610 0.0999 0.1014 49 0.0627 0.1024 0.1043 52 0.0653 0.1062 0.1086 55 0.0676 0.1088 0.1112 59 0.0731 0.1132 0.1164 68 0.0819 0.1237 0.1269 71 0.0879 0.1265 0.1297 76 0.0975 0.1300 0.1327 80 0.1055 0.1333 0.1357 87 0.1202 0.1390 0.1410 90 0.1210 0.1404 0.1419 96 0.1323 0.1474 0.1482 105 0.1414 0.1532 0.1547 108 0.1429 0.1558 0.1571 "‘illlllllllll’llllllllli