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Carbon and nitrogen pools and development of alkali extractable organic matter over time, in hog manure… Smith, Susan Lois 1994

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CARBON AND NITROGEN POOLS AND DEVELOPMENT OF ALKALI EXTRACTABLE ORGANIC MATTER OVER TIME, IN HOG MANURE SOLIDS COMPOSTED IN-VESSEL by SUSAN LOIS SMITH B.Sc, The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Soil Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1994 © Susan Lois Smith, 1994 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of \fh / ^dl&fKJ. The University of British Columbia Vancouver, Canada Date \^LM4 < ' Vs , / ^ / Abstract In three separate experiments, separated hog manure solids and hemlock sawdust were composted in-vessel for a period of 46 to 55 days. The effects of temperature and aeration on monitored carbon and nitrogen pools were examined. Composting temperatures were monitored throughout and samples were taken periodically in order to study carbon and nitrogen pools in humus fractions (humic acid (HA) and fractions of fulvic acid (FA)); in overall compost fractions (polysaccharides, total carbon and nitrogen); and in the water-soluble phase (water-soluble carbon). This was done in order to examine how these nutrient pools were affected by the frequency of aeration; and to obtain information on the processes involved in the development of the alkaline extract over time. For experiments 1 and 2, a bucket-scale apparatus was used for composting. In experiment 3 smaller scale apparatus and controlled temperature waterbaths were employed. For experiments 1 and 2, the effects of aeration were examined on hog manure solids composted with hemlock sawdust for 51 to 55 days in 90 litre buckets. Aeration was carried out by mixing the compost with a metal auger attached to a hand-held drill. The aeration treatments varied in intensity from once every 14 days to once every 2 to 3 days. In the third experiment, composting of separated hog manure solids and hemlock sawdust was carried out using smaller 2 litre vessels submerged in controlled temperature waterbaths, with mixing occurring by shaking manually every other day throughout. Over a period of 46 days, the effects of high and low temperatures on carbon and nitrogen pools and the development of the alkaline extract were examined. Significant increases in utilization of FA carbon (CF), carbohydrate-rich FA carbon (Ca) and the ratio of carbon to nitrogen in carbohydrate-rich FA carbon (C/Nca) were noted under aeration treatment 'A3'. This was the only aeration treatment in the bucket-scale composting to impact the metabolically accelerated thermophilic phase, increasing the oxygen content and optimizing the environment for FA carbon degraders. Overall, in terms of C/N ratios, the most consistent trends were found in the carbohydrate-rich component of FA (FAca). With the exception of experiment 3-' low temperature' compost, C/Nc3 values decreased over time as a result of decreasing Ca. Due to design flaws in the bench-scale waterbath compost system, it was not possible to control moisture loss, making it difficult to definitely attribute chemical changes to the influence of controlled temperatures. Regardless of temperature, utilization of total polysaccharides was substantially lower in bench-scale controlled temperature waterbath composting. This suggests that the activity of organisms which degrade complex carbohydrates may be negatively influenced by the homogeneous environment characteristic of controlled temperature composting. i i Table of Contents Page I X X I X Abstract Table of Contents i i i List of Figures vii List of Tables Acknowledgements xiii Table of Abbreviations xiv Chapter 1 - Introduction, Literature Review and Thesis Objectives 1.1 Introduction l 1.2 Literature Review 3 1.2.1 Chemical, Physical and Biological Aspects of Composting Processes 3 1.2.1a Substrate: 3 1.2.1b Microbial Activity and Metabolic Heat: 4 1.2.1c Microbial Activity and Moisture: 7 1.2. Id Microbial Utilization of Carbon and Nitrogen: 8 1.2.1e Carbohydrates: 11 1.2.2 Humus 14 1.2.2a The Nature of Humus Substances: 14 1.2.2b The Formation of Humus Substances: 16 1.2.2c Compost Humus Studies: 18 1.2.3 Soil and Compost Matrices 21 1.2.4 Chemical Methods Theory 22 1.2.4a Total Carbon: 22 1.2.4b Kjeldahl Nitrogen: 23 1.2.4c Polysaccharides: 24 1.2.4d Humus Extraction, Fractionation and Analysis: 24 1.3 Preliminary Static Pile Composting Trials 25 1.4 Thesis Objectives 27 1.5 Description of Manure Management System 28 1.6 Bulking Agents 29 i i i Table of Contents (continued) Page 1.7 In-Vessel Composting 29 Chapter 2 - Materials and Methods 2.1 Composting Materials and Set-Up 30 2.1.1 Experiments 1 and 2 - Bucket-Scale Composting 30 2.1.2 Experiment 3 - Bench-Scale Composting 32 2.1.2a "High Temperature' Composting: 33 2.1.2b * Compost Simulation' Composting: 33 2.1.2c "Low Temperature' Composting: 34 2.2 Chemical Analyses 35 2.2.1 Procedures For Fresh Sample Analysis 35 2.2.1a Total Moisture (MoisT): 35 2.2.1b Water-Soluble Carbon (WSC): 36 2.2.2 Procedures For Air-Dried Ground Sample Analysis 37 2.2.2a Total Carbon (CT): 37 2.2.2b Nitrogen (NT): 37 2.2.2c Polysaccharides: 37 2.2.2d Humus Extraction, Fractionation and Analysis For Total Carbon and Nitrogen: 38 2.3 Statistics 38 2.3.1 Experiment 1 - Statistical Analysis 39 2.3.2 Experiment 2 - Statistical Analysis 39 2.3.3 Experiment 3 - Statistical Analysis 40 Chapter 3 - Results and Discussion 3.1 Experiment 1: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Hog Manure Solids Compost Under Aeration Treatments Aj'andA2' 41 3.1.1 Temperature 41 3.1.2 Moisture Gradient 50 3.1.3 Sampling 52 3.1.4 Changes in Chemical Properties 52 3.1.4a Carbon and Polysaccharide Pools: 53 3.1.4b Nitrogen Pools: 63 I V Table of Contents (continued) Page 3.1.4c Carbon to Nitrogen Ratios (C/N): 67 3.1.4d Total Moisture (MoisT): 70 3.2 Experiment 2: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Hog Manure Solids Compost Under Aeration Treatments 'A2' and 'A3' 72 3.2.1 Temperature 72 3.2.2 Sampling 80 3.2.3 Changes in Chemical Properties 80 3.2.3a Carbon and Polysaccharide Pools: 81 3.2.3b Nitrogen Pools: 89 3.2.3c Carbon to Nitrogen Ratios (C/N): 94 3.2.3d Total Moisture (MoisT): 97 3.3 Experiment 3: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Bench-Scale Hog Manure Solids Compost Under Controlled Temperature Regimes 99 3.3.1 Temperature 99 3.3.2 Sampling 101 3.3.3 Moisture 101 3.3.4 Comparison of Controlled Temperature ' Compost Simulation' Compost (WB2) With Bucket-Scale Compost 103 3.3.5 Evaluation of Controlled Temperature Composting 106 3.3.5a Components ofHumic Acid: 107 3.3.5b Components of Carbohydrate-Rich (Non PVP-Adsorbed) Fulvic Acid (FA J: 108 3.3.5c Polysaccharides: 110 3.3.5d Alkali Extractable Carbon (CJ: 112 3.3.5e Water-Soluble Carbon (WSC): 113 3.3.5f Ratio of Total Carbon to Nitrogen (C/NT): 114 Chapter 4 - Summary Discussion 116 4.1 Temperature 117 4.1.1 Effect of Increased Aeration on the Thermophilic Phase 117 v Table of Contents (continued) Page 4.1.2 Effect of Increased Aeration on the Cooling TransitionBetween Thermophilic and Maturation Phases 119 4.1.3 Temporal Variability in Temperature 120 4.2 Chemical Results 121 4.2.1 Effects of Aeration on Carbon and Nitrogen Pools 122 4.2.2 Ratio of Manure to Sawdust 123 4.2.3 Total Moisture (MoisT) 124 4.2.4 Carbon 125 4.2.5 Carbon to Nitrogen Ratios 126 4.2.6 Polysaccharides 128 Chapter 5 - Conclusions 130 Chapter 6 - Bibliography 135 Appendix A - Experiment 1 - Analysis of Variance 140 Appendix B - Experiment 2 - Analysis of Variance 147 Appendix C - Experiment 3 - Analysis of Variance 154 Appendix D - Raw Data 170 V I List of Figures Chapter 2 - Materials and Methods Page Figure 2.1: Bucket Showing Thermocouple Positions - Experiments 1 and 2 31 Figure 22: Temperature Profile of "Compost Simulation' Waterbath 2 - Experiment 3 34 Chapter 3 - Results and Discussion Figure 3.1: Bucket Showing Thermocouple Positions - Experiments 1 and 2 41 Figure 32: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A,' - Experiment 1, Bucket 1 42 Figure 3.3: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A,' - Experiment 1, Bucket 2 42 Figure 3.4: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A,' - Experiment 1, Bucket 3 43 Figure 3.5: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment \ V - Experiment 1, Bucket 4 43 Figure 3.6: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment SA2' - Experiment 1, Bucket 5 44 Figure 3.7: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment A2' - Experiment 1, Bucket 6 44 Figure 3.8: Temporal Variation in Temperature As Measured By Thermocouple 1 (Uppermost) - Experiment 1, Buckets 1-6 47 Figure 3.9: Temporal Variation in Temperature As Measured By Thermocouple 2 - Experiment 1, Buckets 1-6 47 Figure 3.10: Temporal Variation in Temperature As Measured By Thermocouple 3 (Middle) - Experiment 1, Buckets 1-6 48 Figure 3.11: Temporal Variation in Temperature As Measured By Thermocouple 4 - Experiment 1, Buckets 1-6 48 Figure 3.12: Temporal Variation in Temperature As Measured By Thermocouple 5 (Bottom) - Experiment 1, Buckets 1-6 49 Figure 3.13: Moisture Gradient From Day 7 to Day 23 in Composted Hog Manure Solids Experiment 1, Aeration Treatment'A,' - Bucket Averages (1 to 3) 50 Figure 3.14: Moisture Gradient From Day 7 to Day 23 in Composted Hog Manure Solids Experiment 1, Aeration Treatment SA2' - Bucket Averages (4 to 6) 50 Figure 3.15: Change in Labile Polysaccharides (LPSS) Over Time, in a Hog Manure Solids Compost - Experiment 1 54 Figure 3.16: Change in Total Polysaccharides (TPSS) Over Time, in a Hog Manure Solids Compost - Experiment 1 54 vxx List of Figures (cont'd) Page Figure 3.17: Change in Water-Soluble Carbon (WSC) Over Time, in a Hog Manure Solids Compost - Experiment 1 56 Figure 3.18: Change in Non-PVP Adsorbed Carbohydrate-Rich Fulvic Acid Carbon (Cca) Over Time, in a Hog Manure Solids Compost - Experiment 1 56 Figure 3.19: Effect of Aeration Treatments (At and Aj) on Total Carbon (CT) Over Time, in a Hog Manure Solids Compost - Experiment 1 57 Figure 3.20: Effect of Interaction Between Aeration Treatments (A, and A )^ and Time on Fulvic Acid Carbon (CF) in a Hog Manure Solids Compost - Experiment 1 59 Figure 3.21: Change Humic Acid Carbon (CH) Over Time, in a Hog Manure Solids Compost - Experiment 1 60 Figure 3.22: Change in the Ratio of Humic Acid Carbon (CH) to Fulvic Acid Carbon (CF) "CH/C^ Over Time, in a Hog Manure Solids Compost - Experiment 1 62 Figure 3.23: Change in Total Nitrogen (NT) Over Time, in a Hog Manure Solids Compost - Experiment 1 64 Figure 3.24: Change in Humic Acid Nitrogen (N„) Over Time, in a Hog Manure Solids Compost - Experiment 1 65 Figure 3.25: Change in the Ratio of Carbon to Nitrogen in the Fulvic Acid (C/NF) Over Time, in a Hog Manure Solids Compost - Experiment 1 67 Figure 3.26: Effect of Aeration Treatments (A, and A,) On The Ratio of Carbon to Nitrogen in the Carbohydrate-Rich Fraction of Fulvic Acid (C/Nca) Over Time, in a Hog Manure Solids Compost - Experiment 1 68 Figure 3.27: Effect of Interaction Between Time and Aeration Treatments (At and Aj) On Carbon/Nitrogen in Humic Acid (C/NH) in a Hog Manure Solids Compost - Experiment 1 69 Figure 3.28: Effect of Interaction Between Time and Aeration Treatments (A, and Aj) On Carbon/Nitrogen (C/NT) in a Hog Manure Solids Compost - Experiment 1 69 Figure 3.29: Change in Total Moisture (MoisT) Over Time, in a Hog Manure Solids Compost - Experiment 1 70 Figure 3.30: Bucket Showing Thermocouple Positions - Experiments 1 and 2 72 Figure 3.31: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment A2' - Experiment 2, Bucket 1 73 Figure 3.32: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 2, Bucket 2 73 Figure 3.33: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment A2' - Experiment 2, Bucket 3 74 v i i i List of Figures (cont'd) Page Figure 3.34: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment XA3' - Experiment 2, Bucket 4 74 Figure 3.35: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A3' - Experiment 2, Bucket 5 75 Figure 3.36: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment "A-,1 - Experiment 2, Bucket 6 75 Figure 3.37: Temporal Variation in Temperature As Measured By Thermocouple 1 (Uppermost) - Experiment 2, Buckets 1-6 77 Figure 3.38: Temporal Variation in Temperature As Measured By Thermocouple 2 - Experiment 2, Buckets 1-6 78 Figure 3.39: Temporal Variation in Temperature As Measured By Thermocouple 3 (Middle) - Experiment 2, Buckets 1-6 78 Figure 3.40: Temporal Variation in Temperature As Measured By Thermocouple 4 - Experiment 2, Buckets 1-6 79 Figure 3.41: Temporal Variation in Temperature As Measured By Thermocouple 5 (Bottom) - Experiment 2, Buckets 1-6 79 Figure 3.42: Effect of Aeration Treatments A2' and "A3( Over Time, on Total Carbon (CT) in a Hog Manure Solids Compost - Experiment 2 82 Figure 3.43: Effect of Aeration Treatments "A2' and 'A3' on Carbohydrate-Rich Fulvic Acid Carbon (Cca) Over Time, in a Hog Manure Solids Compost - Experiment 2 83 Figure 3.44: Effect of Aeration Treatments "A2 and 'A3' on Fulvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 2 83 Figure 3.45: Change in Phenolic-Rich Fulvic Acid Carbon (CA) Over Time, in a Hog Manure Solids Compost - Experiment 2 85 Figure 3.46: Change in the Ratio of Phenolic-Rich Fulvic Acid Carbon (CA) to Fulvic Acid Carbon (CF), *CJCf' Over Time, in a Hog Manure Solids Compost - Experiment 2 86 Figure 3.47: Change in Total Extractable Carbon (CJ Over Time, in a Hog Manure Solids Compost - Experiment 2 89 Figure 3.48: Change in Carbohydrate-Rich Fulvic Acid Nitrogen (Nca) Over Time, in a Hog Manure Solids Compost - Experiment 2 90 Figure 3.49: Effect of Aeration Treatments A2' and "A3' Over Time, on Total Nitrogen (NT) in a Hog Manure Solids Compost - Experiment 2 91 Figure 3.50: Effect of Aeration Treatments "A2( and A3' Over Time, on Humic Acid Nitrogen (Nn) in a Hog Manure Solids Compost - Experiment 2 91 xx List of Figures (cont'd) Page Figure 3.51: Effect of Aeration Treatments A2' and "A3' Over Time, in the Ratio of Carbon to Nitrogen in Carbohydrate-Rich Fulvic Acid (C/Nca) in a Hog Manure Solids Compost - Experiment 2 94 Figure 3.52: Change in the Ratio of Total Carbon to Nitrogen (C/NT) Over Time, in a Hog Manure Solids Compost - Experiment 2 95 Figure 3.53: Change in the Ratio of Carbon to Nitrogen of Humic Acid (C/NH) Over Time, in a Hog Manure Solids Compost - Experiment 2 96 Figure 3.54: The Effect of Interaction Between Aeration Treatments (A2 and A3) and Time on Total Moisture (MoiSr) in a Hog Manure Solids Compost - Experiment 2 97 Figure 3.55: Temperature Profile Over Time of Hog Manure Solids Composted in a Bench-Scale High Temperature' Waterbath (WB1) - Experiment 3 99 Figure 3.56: Temperature Profile Over Time of Hog Manure Solids Composted in a Bench-Scale "Compost Simulation' Waterbath (WB2) - Experiment 3 100 Figure 3.57: Temperature Profile Over Time of Hog Manure Solids Composted in a Bench-Scale Low Temperature' Waterbath (WB3) - Experiment 3 100 Figure 3.58: Change in the Ratio of Carbon to Nitrogen in Humic Acid (C/NH) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 107 Figure 3.59: Change in Carbon/Nitrogen in Carbohydrate-Rich Fulvic Acid (C/Nca) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 109 Figure 3.60: Change in Carbon in Carbohydrate-Rich Fulvic Acid (Cca) in Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 110 Figure 3.61: Change in Labile Polysaccharides (LPSS) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 111 Figure 3.62: Change in Total Polysaccharides (TPSS) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 112 Figure 3.63: Change in Extractable Carbon (Ce) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 113 Figure 3.64: Change in Water-Soluble Carbon (WSC) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 114 x List of Figures (cont'd) Page Figure 3.65: Change in Carbon/Nitrogen (C/NT) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 115 x i List of Tables Page Chapter 3 - Results and Discussion Table 3.1: Summary of Changes in Humic Acid Carbon (CH) and Fulvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 1 (Buckets 1-6) Table 3.2: Changing Carbon and Nitrogen Pools in a Hog Manure Solids Compost - Experiment 1, Buckets 1 to 6 Table 3.3: Summary of Changes in Phenolic-Rich Fulvic Acid Carbon (CA) and Fulvic Acid Carbon Over Time, in a Hog Manure Solids Compost - Experiment 2 Table 3.4: Summary of Changes in Humic Acid Carbon (CH) and Fulvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 2 Table 3.5: Changing Carbon and Nitrogen Pools in a Hog Manure Solids Compost - Experiment 2 Table 3.6: Sterile Water Added (in grams) To Hog Manure Solids Composted in Bench-Scale Waterbaths - Experiment 3 Table 3.7: Chemical Characteristics of Hog Manure Solids Compost Over Time: Bucket-Scale (Experiments 1 and 2) and Bench-Scale (Experiment 3) Table 3.8: Changing Ratios of Carbon and Nitrogen Pools in Hog Manure Solids Compost: Bucket-Scale (Experiments 1 and 2) and Bench-Scale (Experiments 3) Table 3.9: Summary of Changes in C„ and NH Over Time in a Hog Manure Solids Compost Under Three Controlled Temperature Regimes - Experiment 3 Table 3.10: Summary of Changes in Cca and Nca Over Time in a Hog Manure Solids Compost Under Three Controlled Temperature Regimes - Experiment 3 Chapter 4 - Summary Discussion Table 4.1: Summary of Chemical Changes Over Time in Experiments 1, 2 and 3 - Hog Manure Solids Composting 121 Chapter 5 - Conclusions Table 5.1: Summary of Chemical Changes Over Time in Experiments 1, 2 and 3 - Hog Manure Solids Composting 130 x i i 63 66 87 88 93 102 104 105 108 109 Acknowledgements Funding for this project was made possible through a grant from Science Council, B.C./B.C. Environment : Environment Research. I would like to thank members of my committee: Dick Beames, Les Lavkulich, Lawrence Lowe and Bill Ramey for advice, encouragement and support (technical and moral) throughout. Special thanks need to go to my main supervisor, Lawrence Lowe, for use of his lab, financial support (beyond the funding of the grant) and guidance; and also to Les Lavkulich for (among other things) help with final thesis revisions. Thanks to Art Bomke for exercising his signing powers when necessary during the period after Lawrence's retirement. I am very grateful to Peter Hill and the contribution of his hogs to the project. Thanks also to Ruth McDougal and J.C. Yu of The Sustainable Hog Producers Association of B.C. I want to express my appreciation for the faculty members and students of the Department of Soil Science at U.B.C. Their support, assistance and friendship has been invaluable over the past three years. Technical assistance in the lab from Carole Dyck, Huang Quian (Jane) and Bernie Von Spindler is much appreciated. Thanks need to go Andy Black and Rick Ketder in Biometeorology, for advice and assistance on the datalogger and its operation. Thanks to Tim Ballard for coming to the rescue every single time the distillation unit decided that it was shower time. Thanks also to Martin Hilmer and Roza Leyderman. Thanks to Donna Dean, Joan Esau, Rhian Evans, Angela Griffiths, Aweis Issa, Michael Shum and Sandy Traichel for lots of laughs. Thanks for letting me hang out with you guys. Aside from the Department of Soil Science, contributions from several departments at U.B.C. made this research possible. They include the departments of Animal Science, Bioresources Engineering and Microbiology. Thanks to Ted Cathcart, the manager of the South Campus Animal Science Research Farm, for providing space to conduct the bucket-scale composting experiments. Thanks to Bill Ramey of the Department of Microbiology, for providing lab space and equipment in the teaching lab of the Microbiology building for the bench-scale waterbath composting experiment. Thanks to Nicole Albrecht and Pearl Chan of the Department of Microbiology for valuable assistance in the lab. Thanks to N. Jackson and J. Pehlke of the Department of Bioresources Engineering, for modifying and donating the auger to the project. Thanks to Alex Dumitrescu for assistance throughout. Lasdy and most importantly, I would like to thank my parents and Mike Nekic without whom this would never have been possible. x i i i Table of Abbreviations1 Terms Mixed at day 0 and day 7, and then mixed every 14 days Mixed every 7 days Mixed every 2-3 days, until day 21, then mixed every 7 days Total moisture (%) Water-soluble carbon (%) Total carbon (%) Total nitrogen (%) Total carbon/total nitrogen Humic acid Total carbon in the HA (%) Nitrogen in the HA (%) Total carbon in the HA/nitrogen in the HA Fulvic acid Total carbon in the FA (%) Nitrogen in the FA (%) Total carbon in the FA/nitrogen in the FA Polyvinyl polypyrrolidone Non-PVP-adsorbed fulvic acid Total carbon in the non-PVP-adsorbed fulvic acid (%) Nitrogen in the non-PVP-adsorbed fulvic acid (%) Total carbon in the FAca/nitrogen in the FAca PVP-adsorbed fulvic acid Total carbon in the PVP-adsorbed fulvic acid (%) Text A, A2 A3 MoiSj wsc Cx NT C/NT HA CH N„ C/NH FA c F NF C/Np PVP FAca cca Nca C/Nca FAA c A Figures Al A2 A3 Mois(T) WSC C(T) N(T) C/N(T) HA C(H) N(H) QN(H) FA C(F) N(F) C/N(F) PVP FA(ca) C(ca) N(ca) C/N(ca) FA (A) C(A) xiv Terms Nitrogen in the PVP-adsorbed fulvic acid (%) Total carbon in the humic acid/total carbon in the fulvic acid Total carbon in the PVP-adsorbed fulvic acid/total carbon in the fulvic acid Total Extractable Carbon Per cent glucose equivalents Labile polysaccharides (%glucose equivalents) Total polysaccharides (%glucose equivalents) Thermocouple Waterbath Values expressed on a wet mass basis Values expressed on an oven-dried mass basis Bucket 1 Text NA CH/CF cycp Ce %gluc. equiv. LPSS TPSS T WB W.M. o.d. B Figures N(A) C(H)/C(F) C(A)/C(F) C(e) %gluc. equiv. LPSS TPSS T WB W.M. o.d. B 'all percentages based on oven-dried mass except Moisj- which is determined on a wet sample basis. xv Chapter 1 - Introduction, Literature Review and Thesis Objectives 1.1 Introduction The trend, in agriculture, away from small individual farms to intensively managed operations has created dramatic increases and concentrations of waste. A case in point is the hog industry. By 1980, it was common to find separate farrowing and finishing herds in operations containing 1,000 to 10,000 animals, as well as a few 10,000 to 50,000-hog operations (Loehr, 1984). For the future, it is believed that the size of hog farms in the U.S. is on the increase (Pond, 1991). Farm productivity has increased since 1940, while farm labour has declined drastically and mechanization has increased. This trend toward mechanization was an attempt to offset the effects of rising inflation and a decreasing or constant farm income. Mechanization has popularized the use of slatted floors in hog and dairy barns and the resultant handling of manure as a water-based slurry. Slatted floors have become a labour-saving design approach in large confinement buildings. Now, a large part of a farmer's working day (35-40%) can be freed up from cleaning if animals are housed on slatted floors and their waste is dropped through to a water-filled collection pit (Loehr, 1974). A by-product of mechanization is a high-moisture organic waste that is unsuitable for discharge because of its high biochemical oxygen demand. A typical 200 sow (farrow to finish) hog farm produces, annually, 8.4 x 105 litres of separated solids containing 20% dry matter (Lowe et al, 1993). Agricultural wastes are a problem because they are a potential source of nitrates and other toxins which can contaminate drinking water and cause eutrophication. These wastes can contain pesticides, metals and pathogens. The feces of livestock consist, chiefly, of food that has escaped digestion. This is predominantly cellulose fibre, but also includes protein, excess nitrogen in the form of urea (for animals), and potassium. Feces also contain residue from digestive fluids, waste mineral 1 matter, worn-out cells from intestinal linings, mucus, bacteria, and foreign matter such as dirt consumed along with food. Calcium, magnesium, iron, and phosphorus are also voided, chiefly, in the feces. For hogs, manure production ranges from 6-8% of body weight per day (Loehr, 1974). Hog producers are faced with problems of small land-bases for waste disposal, compounded with severe nuisance odours. Excessive nutrient pollution from lands used for waste disposal or storage causes imbalances in natural ecological systems, contamination of groundwater, and increased eutrophication. Depletion of oxygen in surface waters can cause fish kills and septic conditions. Microorganisms in waste discharges impair the use of surface waters for recreational use (Loehr, 1974; Lo et al., 1990). On the other hand, agricultural wastes also contain nutrients and organic matter which can be recycled to enhance growth media for plants. Composting of solids is a means of detoxifying waste and reducing its volume. During this process, biological decomposition and stabilization of organic substrates occurs, under conditions which allow development of thermophilic temperatures as a result of biologically-produced heat (Haug, 1980; Miller, 1992). Ideally, the final product is sufficiently stable for storage and application to the land, without adverse environmental effects. Composting has a few major applications in terms of the process and the final product. It is a means of treating organic wastes such as leaves, sludge or hazardous wastes. The final product can be used in a number of ways. Compost is used for agricultural or horticultural purposes, or as a substrate for the cultivation of mushrooms. Depending on the application, the desired moisture content of compost will vary. For mushroom composting, a very high moisture content of 68 to 75% is desired in the finished compost because of the great moismre demands of the subsequent mushroom crop. In waste treatment composting, achieving a drier final compost is usually desirable because of its decreased bulk and weight, and improved handling in terms of storage and transport (Miller, 1992). 2 Accompanied by the microbial decomposition of unstable organics in composting is the development of humus, a component of the alkali soluble fraction of organic matter. This highly polymerized organic product is less affected by immediate biodegradation and may be compared with soil humus. While the nature, origin and properties of humus is not fully agreed upon, this review attempts to summarize what is presently understood about the formation and chemical nature of humus extracted from natural and controlled environments. The compost substrate provides the chemical and physical environment for the biological oxidation of organic matter by a quick succession of bacterial and fungal populations. Reduced carbon compounds are the energy source used by these aerobic decomposers as they metabolize available nitrogen. One of the aims of this study was to analyse changing carbon and nitrogen pools in order to determine their relative importance under different environmental conditions. Information gained may lead to a better understanding of composting processes. 1.2 Literature Review 1.2.1 Chemical, Physical and Biological Aspects of Composting Processes 1.2.1a Substrate: In spite of the fact that there is a wide variety of starting materials to choose from when composting, there are some common initial chemical conditions which are important to the process. A starting mixture of plant and animal residues, generally, has an abundance of readily available reduced carbon compounds such as short-chained volatile fatty acids, simple sugars and polysaccharides, as well as nitrogen-containing compounds such as complex proteins and amino acids (De Bertoldi et al, 1989; Golueke, 1977; McKinley et al., 1989). These readily available carbon 3 compounds are the main initial energy sources for microorganisms as they metabolize available nitrogen (Golueke, 1977). The key to successful composting is to provide optimal starting-conditions for aerobic decomposers. Some important factors which affect conditions are moisture-content, aeration, the ratio of carbon to nitrogen (C/N), pH and salinity. In general, the starting-moisture content should be 45-60% (Loehr, 1974; Dalzell et. al., 1987; de Bertoldi et al., 1989; Weller et. al., 1977; Schonborn, 1981) the C/N should be 25-35:1 (Dalzell et. al., 1987; Schonborn, 1981; Sussman, 1982); the pH should be 5.5-8.0 (near-neutral) (de Bertoldi, 1987; Schonborn, 1981); and the salinity should not exceed 2g NaCl/1 (de Bertoldi et. al., 1987). 1.2.1b Microbial Activity and Metabolic Heat: The composting process is an exothermic biological oxidation of organic matter carried out by a dynamic and quick succession of populations of aerobic microorganisms (Iglesias et al., 1988). Most of the heat generated in a compost results from aerobic respiration, while other forms of metabolism such as fermentation and anaerobic respiration play minor roles (Miller, 1992; The Staff ofBiocycle, 1989). The three main classes of microorganisms encountered in composting are bacteria, actinomycetes and fungi (Golueke, 1977). Bacteria are largely responsible for the initial decomposition of organic material and for a large part of the heat energy released into the composting mass (Golueke, 1977; Miller, 1992). Both actinomycetes and fungi use organic acids, sugars, starches, hemicelluloses, cellulose, proteins, polypeptides, amino acids and a large number of other organic compounds. Actinomycetes appear to be more important as decomposers of the more resistant cellulose and, to some extent, ligneous compounds (Golueke, 1977). Fungi are very important decomposers of cellulose (Dalzell et al., 1987; Miller, 1992). 4 The composting process can be separated into distinct stages: initially mesophilic, then thermophilic and finally maturation. Because the starting temperature generally reflects ambient conditions of about 20° C to 30° C, it is referred to as mesophilic. Given adequate starting conditions, the compost temperature rises quickly to the thermophilic range of 45° C to 65° C where it is typically maintained for three to five days (the Staff of Biocycle, 1989). Temperature increases are a function of metabolic heat evolution and heat conservation. Thermophilic temperatures are achieved because metabolic heat production exceeds losses. The extent of the thermophilic phase depends on the substrate and on processing strategy and can be extended for weeks under certain conditions (Miller, 1992). After this period of peak activity, temperatures decline to ambient conditions and the lengthy maturation phase begins. Golueke (1977) reports that fungi and actinomycetes do not appear in appreciable numbers until the process has advanced. De Bertoldi et al. (1982), however, have noted that mesophilic and thermophilic fungi are fairly active in the initial mesophilic period and in the early portion of the thermophilic phase. Both tend to disappear at peak temperatures of 70° C and then resume their biological activity when the temperature begins to drop (Miller, 1992). The elevated temperatures, characteristic of the thermophilic phase, substantially contribute to the high rates of decomposition possible during composting. For a given enzyme, activity rates generally double with a 10° C temperature rise, until the inactivation temperature is achieved. Optimal decomposition rates occur in the mid- to upper-50°C range (Miller, 1992). McKinley et al. (1977) observed lower rates of microbial metabolism in high temperature zones of an aerated static pile compost. When zones between 55° and 60° C were compared with 35° to 50° C areas, the lower temperature zone exhibited the greatest microbial activity. Unless, however, moisture, aeration and other nutrient variables are controlled adequately, as was not the case here, one cannot conclude that temperature is the sole factor controlling microbial activity. 5 Composting activity rates decrease at temperatures above 60° C. This may be caused by heat accumulation and resultant high temperatures which severely inhibit further microbial activity (Miller, 1992). Golueke (1977) and Finstein et al. (1984) agree that the upper level of the optimum range of thermophiles is between 55° C and 60° C. When the temperature exceeds 60° C, spore-forming organisms begin to enter the spore or resistant stage. Temperatures of 65°C to 70°C probably exceed the thermal deathpoints of most representatives of fungi and actinomycetes (Golueke, 1977). The nature of the thermophilic stage appears to be affected by many things working in combination. Increased metabolic activity is accompanied by changes in environmental factors causing rapid changes in populations (Miller, 1992). The eventual decrease in the thermophilic temperature may be due to an exhaustion of easily degradable nutrients (Loehr, 1974). Finstein (1982) could not find a cause and effect relationship between temperature and microbial activity as measured by oxygen consumption, and suggested that perhaps microbial activity is influenced by changing substrate, over time, as well as by temperature. It is thought that the initial rapid straight-line rise in temperature is made possible by the availability of easily decomposable starches, sugars, proteins and amino acids. As the temperature increase of the composting mass tapers off, only the more resistant compounds remain and the temperature begins a final and inevitable drop (Golueke, 1977). De Bertoldi et al. (1982) report that during the phase of temperature decline, transformation of cellulose and pectin is at its peak. 6 1.2.1c Microbial Activity and Moisture: Water is required for physiological needs and solution of substrate and salts. It acts as a medium for bacterial colonization and is a determinant of gas exchange. Optimum metabolic rates can be achieved in compost by reaching the maximum water content that still does not restrict 02 transfer or utilization (Miller, 1992). The compost reaction is a biochemical transformation of raw organic wastes by microorganisms whose metabolism occurs in the water-soluble phase. Most microorganisms, in solid phase systems, are attached to surfaces and are active at the water-solids interface. These microflora metabolize substances from the solids through enzymatic activity. The solubilized substances are utilized to support growth, or they accumulate in the liquid phase (Hirai, 1984; Inbar et al., 1990). Excessive moisture slows the accumulation of metabolic heat for a number of reasons. Heat storage is mostly determined by water because of its high specific heat and because water normally constitutes approximately two-thirds of the composting mass. An increase in water content decreases porosity and gas diffusivity and, thereby, interferes with aeration necessary for aerobic metabolism (the Staff of Biocycle, 1989). In materials with a large amount of lipid which turns liquid at composting temperatures; it has been recommended that moisture and lipid be considered together in determining the total liquid content (Miller, 1992). Limitations caused by lack of moisture are more complex than those caused by excess water. As matric potentials become more negative than -70kPa, colonization by bacteria is strongly inhibited. Large bacterial cells such as Bacillus spp. which are preponderant during the highly active thermophilic stage, require thicker water films to function. Fungi and actinomycetes, because of their mycelial nature, are thought to be less affected by the matric potential of compost (Miller, 1992). 7 An important moisture consideration is metabolic production of H20. Mass balance studies of a variety of composts have reported the metabolic production of 0.50-0.63g H20 per gram of material composted (Miller, 1992). 1.2. Id Microbial Utilization of Carbon and Nitrogen: Bacteria, actinomycetes and fungi assimilate carbon and nitrogen differendy. Bacteria use less nitrogen to degrade a unit of carbon, than do actinomycetes or fungi. The growth of bacteria is, therefore, initially favoured in waste substrates rich in nitrogenous compounds. Later, fungi are better adapted to consume the remaining complex carbohydrates such as cellulose, non-cellulosic polysaccharides and lignin. The early thermophilic attack by bacteria on the initial proteinaceous substrate frees nitrogen through ammonification and makes it available for subsequent populations (Miller, 1992). Because reduced carbon compounds are the energy source used by microbes as they metabolize available nitrogen, the ratio of carbon to nitrogen (C/N) in a substrate is often measured in order to characterize its nutrient status (Dalzell et. al., 1987; Morel et. al., 1985). In order to prevent nitrogen starvation in plants, a C/N ratio of 20:1 is recommended for finished compost intended for soil amendment (Hirai et al., 1984). During the initial stages of decomposition, readily degradable water-soluble materials are used more rapidly than resistant ligno-cellulose tissues (Whitely et al., 1994). At this time, the breakdown of organic substrates for energy and incorporation into the microbial biomass is the crucial mechanism (Libmont et al., 1993). The C/N values of finished composted materials can fall within the range of 5:1 to 20:1, depending upon the type of raw materials (Hirai et al , 1984). N'Dayegamiye et al. (1991) studied changes in a variety of composted materials, and found that the C/N ratios of all materials decreased 8 over time. After 36 months of composting, the C/N ratio of a mix of two parts cattle manure to one part wood shavings decreased from 43:1 to 17:1. The C/N ratio of a mixture of composting cattle manure and peat moss decreased from 48:1 to 35:1 in the same time. Yoshida et al. (1979) composted various organic raw wastes which ranged in starting total carbon values from 47.94%(o.d.)' in rice bran to 23.15%(o.d.) in sewage sludge. Nitrogen values of these same raw materials ranged from 2.39%(o.d.) for rice bran to 2.59%(o.d.) for sewage sludge. After either laboratory scale or pilot scale composting for four months, the C/N ratio of rice bran had decreased from 20.0:1 to 15.8:1. The nitrogen increased by about 20%(o.d.) while the carbon decreased by less than l%(o.d.). For sewage sludge, the C/N ratio started at 8.9:1 and finished at 9.6:1. In this case there was an increase in nitrogen of almost 30%(o.d.), and a much more noticeable decrease in carbon of almost 25%(o.d.). The degradability of the carbon and the relative abundance of nitrogen are two factors which may have caused this drastic disappearance of carbon in the composted sewage sludge. The rice bran raw material used for the study likely contained higher amounts of relatively more inert cellulose and ligneous material compared to the sewage sludge. Hirai et. al (1984) found that the water extracts of well-matured composts had a ratio of organic carbon to organic nitrogen in the range of 4:1 to 7:1, while overall C/N ratios of composted materials varied from about 8:1 to 18:1. Some researchers have proposed that the ratio of organic carbon to organic nitrogen in the water extract could serve as a reliable indicator of compost maturity. Others argue that this does not yield a consistent relationship, when composts of various sources are analyzed (Inbar et al, 1990). Morel et al. (1985) have proposed that it may be preferable to interpret C/N ratios according to the initial characteristics of compost. In a review of data from several compost studies, however, Iglesias et al. (1988) found that there appears to be no correlation between o.d.=values reported on an oven-dried mass basis 9 the ratio of the final C/N value to its initial C/N value (final C/N:initial C/N) and the final maturity of a material. Because the nature of microbial activity changes as composts mature, characteristics of the water extract are useful indicators of compost maturity (Inbar et al., 1990). Others suggest that because water-soluble components include degradation products of many components of the compost, the significance of any changes is not easily assessed (Iglesias et al., 1988; Mathur et al , 1993). Initially, water extracts from compost contain amino acids, aliphatic acids, peptides, sugars and their polymers (proteins and polysaccharides). As composting progresses, the easily metabolizable and carbon-rich aliphatic groups and amino acids decline while nitrogen-rich peptides increase (Mathur et al., 1993). Saviozzi et al. (1987) found that, in the initial stage of composting, water-soluble organic carbon and nitrogen increased. It was suggested that the increase of these water-soluble nutrients was due to the increasing rate of decomposition of complex substances by microorganisms. The study also found that after approximately one week of composting, a gradual decrease of soluble organic components occurred. The authors suggest that, early in the composting process, hydrolysis and solubilization of complex substances was predominating over mineralization and immobilization processes. Chanyasak et al. (1981) examined water extracts from various composted materials. After shaking a fresh sample in water for thirty minutes at 60° C, the filtered (0.45 yum) extracts were analysed for total organic carbon (T.O.C.) using an autoanalyser. Total organic carbon in the water extract (water-soluble organic carbon) ranged from 0.50%(o.d.) in composted chicken manure to 0.01%(o.d.) in composted leaves. The water extract of sewage sludge and rice husks measured 0.33%(o.d.) T.O.C, and one from a mixture of cow and hog manure composted with straw contained 0.13%(o.d.) T.O.C. Inorganic carbon was found to be present in negligible amounts. 10 Garcia et al. (1990) measured total water-soluble carbon (WSC) from the water extract of various composts using an autoanalyser. A variety of mixtures of city refuse, aerobic sewage sludge, peat residue and grape debris were composted for about seven months. Starting WSC values ranged from 4.56% to 1.27%(o.d.) and over a period of 210 days decreased to a range of 0.24% to 0.61%(o.d.). It was found that when compared with other carbon pools studied such as total carbon and alkali extractable carbon, WSC showed the greatest decreases in all compost mixtures. The authors suggest that, in the composting environment, WSC was relatively easier for microorganisms to degrade. In a mixture of city refuse and peat residue composted for 210 days, Garcia et al. (1990) report that the C/N ratio decreased from 33.90:1 to 11.80:1, total carbon decreased from 37.30% to 15.30%(o.d.), total nitrogen increased from 1.10% to 1.29%(o.d.) and WSC decreased from 2.25% to 0.61%(o.d.). 1.2.1e Carbohydrates In soil, polysaccharides such as cellulose, hemicellulose and starch are probably the most important readily available energy substrates for microorganisms. Cellulose occurs mainly as plant cell wall material and is relatively resistant to decomposition. Hemicellulose does not refer to any specific group of polysaccharide, but is defined as plant cell-wall polysaccharide normally found in association with cellulose. Hemicellulose is relatively less resistant to degradation than cellulose. Starch is a readily degradable polysaccharide relative to hemicellulose and cellulose. In addition to these three types of polysaccharides, it is probable that polysaccharides of microbial origin are present in soil (Greenland et al., 1975). In soil, the rate of disappearance of hemicellulose is initially faster than that of cellulose. Eventually however, the degradation of hemicellulose becomes slower than that of cellulose. This less 11 degradable hemicellulose may be newly synthesized polysaccharide produced by microorganisms decomposing plant material (Greenland et al., 1975). Compost organic material is a mixture of proteins, lignin, minerals and reducing sugars which include hemicellulose, cellulose and starch, all present in a wide range of concentrations (Dalzell et. al., 1987). Inoko et al. (1979) recommend that the C/N ratio of a compost intended for soil amendment should be below 20:1; the total nitrogen should be above 2% (o.d.); and the ratio of carbon in the reducing sugars to total carbon should be below 35:100. These factors are important in order to prevent rapid decomposition which results when a high C/N ratio compost with a low nitrogen content and concentrated amounts of reducing sugars is introduced to a natural soil system. The result of this rapid decomposition is generally nitrogen-starvation for the plants as the microorganisms tie up native soil nitrogen in their oxidation of available carbohydrate (Inoko et al., 1979). As the carbon level of compost decreases over time; cellulose, hemicellulose, lignin, soluble organic components, amino acids and simple sugars are affected. Not all polysaccharides decrease with equal rapidity, and polysaccharides are also synthesized by some composting microorganisms (Mathur et al., 1993). Simple polysaccharides are progressively decomposed by the microflora which develop during the thermophilic and maturation phases. Water-soluble polysaccharides have been noted to decrease much faster than both starch and cellulose (Golueke, 1977; Morel et al., 1985). At the end of the composting period, the quantity of more stable polysaccharide such as cellulose is found to be relatively high, and this seems to correspond with newly synthesized, stable microbial components rather than with those fractions which are not yet decomposed (Morel et al., 1985). Inoko et al. (1979) measured hemicellulose and cellulose in a variety of municipal refuse composts. These materials were composted in-vessel and received continuous ventilation and frequent mixing for nine days before being piled for forty-eight days. Hemicellulose and cellulose were determined via a micro-copper titrimetric determination of hydrolysates. Starting hemicellulose ranged 12 from 3% to 16%(o.d.) and cellulose ranged from 8% to 35%(o.d.). During composting, hemicellulose and cellulose increased for the first seven days of composting and showed a rapid decrease for the next seven days. This was followed by a gradual decrease until the end of the sixty days of composting. On average, the finished composts contained 5.4%(o.d.) hemicellulose and 14.1%(o.d.) cellulose. In soils, Oades (1966) examined carbohydrate contents of soils determined by acid hydrolysis, charcoal clarification and anthrone colorimetry. 0.30% (glucose equivalents) carbohydrate was found in a Podzol. In agricultural soils, carbohydrate values ranged from 0.06 to 0.12% glucose equivalents. Using gel chromatography, Hirai et. al (1984) found that as the compost degradation processes advanced, there was a shift towards higher molecular weight peptides and polysaccharides in the water extract. Similar patterns have been observed in various types of raw materials composted. Chanyasak et al. (1980) composted a mixture of sewage sludge and rice husks for approximately 550 hours and analysed water-soluble polysaccharide via gel chromatography. It was found that the amount of polysaccharide increased from 0.126% to 0.161%(o.d.) with composting. After composting for 550 hours, the study showed that high molecular weight compounds contributed to a larger portion of the polysaccharides content. It was suggested that these high molecular weight compounds were intermediate products of biological degradation. 1.2.2 Humus 1.2.2a The Nature of Humus Substances: Accompanied by the microbial decomposition of unstable organics in composting is the development of humus, a component of the alkali soluble fraction of organic matter. During composting, organic carbon and easily degradable components decrease while highly polymerized 13 organic products increase. These new organic products are less affected by immediate biodegradation and may be compared with soil humus (Gonzalez-Vila et al., 1985; Inbar et al. 1989; Morel et. al., 1985). Humus substances have not been adequately related to any of the existing groups identified as organic compounds. The nature, origin and properties of these substances are not fully understood. They do not exhibit specific chemical characteristics such as a sharp melting point, exact refractive index, exact chemical composition or a definite infra-red spectra (Schnitzer, 1978). These humus substances are however, regularly isolated from soils, decomposing plant residues and peats (Kononova, 1966). They arise from the chemical and biological degradation of plant and animal residues and from synthetic activities of microorganisms (Schnitzer, 1978). In developed soils, humus forms a large part of the total reserve of organic matter (Aiken et al., 1985; Kononova, 1966; Schnitzer, 1978). Humus is a dark-coloured, acidic, predominantly aromatic, hydrophilic, chemically complex, polyelectrolyte-like material (Schnitzer, 1978). It is composed of an amorphous combination of aliphatic and polyphenols carbon structures. It includes not only strictly humus substances, but also products from advanced decomposition of organic residues and products resynthesized by microorganisms. These may include protein-like substances, carbohydrates, lignins, waxes, resins, fats and tannins. Humus substances are usually partitioned into three fractions. Two fractions are soluble in strong alkali: humic acid (HA) and fulvic acid (FA). The third fraction is referred to as is humin and is insoluble in alkali or acid (Aiken et al., 1985; Hayes, 1985; Kononova, 1966; Schnitzer, 1978). Humic acids are a fraction of substances extracted from the soil by alkali which form amorphous precipitates with acids. From the accumulated infra-red spectra and partition paper-chromatography investigations on humic acids of different soils, certain common features about their composition and structure emerge. Humic acids are regarded as high molecular weight compounds 14 with a complex structure. They are thought to be formed during the condensation of two or three compounds. Two main components which are considered important are compounds of phenolic or quinoid nature and nitrogen-containing amino-acids and peptides. As well, a third carbohydrate component is thought to be significant. This carbohydrate component is considered to be a part of humic acid and not a contaminant. It is generally accepted that products of the resynthesis of microbial plasma participate in the formation of humic acid. As carbohydrates of specific nature are a common component of bacterial slime, this could account for the presence of carbohydrate residues in humic acid (Kononova, 1966). Fulvic acid is soluble in water at any pH (Aiken et al., 1985). It is also defined, operationally, as the acid-soluble portion of the alkaline extract (Schnitzer, 1975). Fulvic acid is lower in molecular weight and less aromatic than its humic acid counterpart. If oxygen is considered, important differences between humic acid and fulvic acid are noticed. In fulvic acid, oxygen can be accounted for mainly in carboxyl (COOH), hydroxyl (OH) and carbonyl (CO) functional groups (Schnitzer, 1975; Stevenson, 1985). In humic acid, a high proportion of the oxygen occurs as a structural component of the nucleus in the form of ether (C-O-C) or ester linkages (Stevenson, 1985). Infra-red spectra and elemental composition analyses show that, relative to humic acids, fulvic acids are weakly aromatic structures with a predominance of aliphatic side chains (Kononova, 1966). Humin is thought to represent the least oxidized fraction of organic matter. A lower number of acidic functional groups may be the reason it remains insoluble in strong alkali (Hayes, 1985). 1.2.2b The Formation of Humus Substances: A completely satisfactory scheme for explaining the nature and occurrence of humic and fulvic acids in diverse environments has not yet been developed. Several pathways are thought to exist for the formation of humus substances during the decay of plant and animal residues in soil. 15 For many years, lignin was viewed as the source of humic substances. Waksman (1938) concluded that the nitrogen contained in humic acids resulted from the condensation of modified lignin with microbially synthesized protein. According to this theory, lignin is incompletely utilized by microorganisms and the residuum becomes part of soil humus. Modifications include loss of methoxyl (0-CH3) groups, generation of o-hydroxyphenols, and the oxidation of aliphatic side chains to form carboxyl groups. The modified polymeric material is subject to further unknown changes to yield first humic acids and through further fragmentation and oxidation, fulvic acids. Structural and behavioral similarities between humic acid and lignin, as well as the observation that humic acids have properties similar to oxidized lignins strongly supported Waksman's lignin theory of humic acid formation. Present-day investigators now however, feel that the contribution of lignin to humus formation has been exaggerated (Stevenson, 1982; Waksman, 1938). Kononova (1966) considered humus formation to be a complex two-stage process in which plant and animal residues undergo profound transformations. The process involves the decomposition and conversion by microbes of plant and animal tissues into simpler chemical compounds along with some mineralization. The simpler chemical compounds are used to synthesize organic compounds, forming high-molecular weight humus substances of specific nature, such as humic acids and fulvic acids. As previously stated, investigators feel that the contribution of lignin to humus formation has been exaggerated, and now favour a mechanism which involves quinones. Instead of the residuum of lignin degradation being the contribution to humus, it is theorized that phenolic aldehyde building units are released from lignin during microbiological attack which then undergo enzymatic conversion to quinones. These quinones polymerize in the presence or absence of amino compounds to form humic-like macromolecules. As well, a similar pathway is thought to be present where polyphenols are synthesized by microorganisms from non-lignin carbon sources such as cellulose. The polyphenols 16 are then enzymatically oxidized to quinones and converted to dark-coloured humic substances in the same manner as lignin-derived quinones (Stevenson, 1982). After isolating humic and fulvic acids from a Chernozem, a number of fractions were isolated through dialysis and examined in terms of their elementary composition, exchange capacity and behaviour towards electrolytes. A gradual change in the nature of the humic and fulvic fractions indicated that humic acids and fulvic acids are not necessarily independent groups of humus substances, but may be a single sequence of groups linked by a chain of interconversions (Kononova, 1966). Contradictory theories have been proposed as to whether high molecular weight humic acids are more mature than their lower molecular weight fulvic acid counterparts. On the basis of observations that humic acids from a Podzol appear closer in nature to their fulvic acid counterparts than do humic acids from a relatively more developed Chernozem, Kononova (1966) has suggested that fulvic acids are the least mature representatives when compared with humic acid. Schnitzer (1975), however, suggests that the more complex, high-molecular weight humic materials are formed first and that these are then degraded, most likely oxidatively, into lower molecular weight materials. Thus, over time in soil, the sequence of events appears to be a gradual conversion from humic acid to fulvic acid (Schnitzer, 1975). 1.2.2c Compost Humus Studies: In general, it is felt that there is a shift from fulvic acid to humic acid during composting, as well as an increase in aromaticity of HA coupled with incorporation of proteinaceous substances. Garcia et al. (1990) measured total carbon, alkali extractable carbon (HA-carbon and FA-carbon) and water-soluble carbon (WSC) in various mixtures of materials composted over seven months. The study found that while total carbon and WSC decreased throughout, extractable carbon 17 decreased from day 0 of composting to day 91 and then increased slightly from day 91 to day 210 which was considered to be the maturation phase. This suggests that humification products were resynfhesized in the latter maturation phase of composting (Garcia et al., 1990). Inbar et al. (1989,1990) studied humic substances extracted from a cattle manure solids compost. CPMAS I3C-NMR spectra of humic acids showed a decrease in the polysaccharide region of the spectra and an increase in aromaticity after 147 days of composting. In addition, carboxyl, carbonyl, phenolic, methoxyl and alkyl groups increased. This agreed with the crude fibre analysis which indicated the disappearance by 33%(o.d.) of acid-hydrolysed sugars in the humic acid. In spite of this decrease in polysaccharide concentration, the peaks indicating polysaccharides dominated in all the spectra. Fourier Transform Infrared (FTIR) spectra agreed with the 13C-NMR data. With composting, FTIR spectra of the HA revealed an increase in carboxylate ions (COO), a decrease in alkyl carbon (CH3), and a decrease in aliphatic carbon and carbohydrates (C-O). In addition to an increase in aromaticity and a decrease in polysaccharide concentration, this data indicate that a decrease in HA aliphatic chains had occurred during composting. 13C-NMR spectra and crude fibre analysis also indicated that the lignin content of the HA of the fresh cattle manure solids was 20%(o.d.) while that of the HA of the composted material was 32%(o.d.). It was suggested that higher levels of modified lignin in composted HA may explain the increase in aromaticity and functional groups such as phenolic hydroxyls and carboxyls (Inbar et al. 1989, 1990). It is believed that, during composting, low molecular-weight humic substances (FA) produced initially, are progressively polymerized into less soluble macromolecular substances. Observed 18 increases in compost HA relative to FA over time, have supported this conclusion. An observed increase in EJE62 ratios of HA's however is contradictory, and suggests a decrease in molecular weight of HA's during composting (Mathur et al., 1993). Inbar et al. (1990) fractionated the alkaline extract of a cattle manure solids compost into acid-insoluble humic acid and acid-soluble fulvic acid. Both fractions were found to increase over 147 days of composting. The ratio of carbon in the HA (CH) to carbon in the FA (CF) was reported to increase from 1.0 to 1.8. These values were similar to those reported for soils such as peat and grasslands, which contain high levels of humic acid and characteristically exhibit CyCp ratios greater than 1.0. This trend of an increase in CyCp from fresh to composted waste has been reported by other researchers. It indicates that raw materials, in general, contain high levels of FA and low levels of HA. As decomposition proceeds, the FA either decreases or remains unchanged while HA increases (Inbar et al, 1990). Again, in the same study, Inbar et al. (1990) isolated a non-humic fraction (FAca) from the FA using an XAD-8 resin. The resin separates out the FAM by adsorbing the phenolic-rich fraction of FA (FAA) and allows the FAca to pass through. The study found that FAca increased during composting, while its phenolic-rich counterpart (FAA) remained constant. Finally, in the same study, Inbar et al. (1990) analysed cattle manure solids compost HA for elemental composition and functional groups. These results were compared to a "model HA' which represented a large number of soils formed under widely differing conditions. In the compost HA, carbon and hydrogen contents were found to be higher than in the "model HA', but agreed with contents reported in HA's from Chernozems, humus allophanes, peat bogs and peats. Oxygen contents in the compost HA were lower than that of the "model HA', suggesting a low degree of oxidation in the compost HA. When compared with soil HA's , lower ratios of hydrogen to carbon in compost HA 2E4/E6=absorbance at 465nm /absorbance at 665nm 19 were found, indicating a high degree of aromaticity as found with peat. Compared with the " model * HA', compost HA contained appreciably less oxygen-containing functional groups. This was similar to the contents of oxygen-containing functional groups reported for HA from decomposing plant residues and various organic wastes. The composition of cattle manure solids compost HA was found to have changed little over the 147 day composting period, in spite of the fact that the concentration of alkali extractable organic matter increased significantly during the same time. The authors concluded that this evidence, and the 13C-NMR and FTIR spectra indicate that after 147 days of composting, the HA's of cattle manure solids compost were similar to poorly oxidized soil humic acids (Inbar et al., 1990). Riffaldi et al. (1983) found that the percentage of HA and FA which occurs in different fresh organic wastes varied considerably from one waste type to another. The study found that the humus of cattle manure had a high content of HA, while straw, sludge and poultry manure humus were high in FA. The same study also found that fresh organic waste HA's showed lower C/N ratios than their FA counterparts. It was suggested that the lower C/N ratio seen in HA indicates a higher degree of stabilization and oxidation relative to the FA. The study also showed that organic waste HA's exhibited higher contents of total acidity than their FA counterparts. This is unlike the situation seen for soil humus (Riffaldi et al., 1983). Riffaldi et al. (1983) found that most of the fresh organic waste HA's and FA's in their study showed much higher nitrogen contents than did soil humic compounds. Gonzalez-Vila et al. (1985) found similar results when examining HA's extracted from municipal refuse compost. Riffaldi et al. (1983) attributed their findings to incomplete hydrolysis of proteinaceous constituents contained in the raw waste. Gonzalez-Vila et al. (1985) suggest that proteinaceous materials present in the refuse became incorporated in the HA fraction during the slow maturation process of composting. 20 Finally, Riffaldi et al. (1983) compared the contents of acidic functional groups and EJE6 ratios found in organic waste HA's and FA's, with that of soil humus. Organic waste HA's and FA's contained appreciably less acidic functional groups. This was particularly apparent in organic waste FA's These relatively mild states of oxidation suggest that the organic wastes contained young forms of humic acids (Riffaldi et al., 1983). EJE6 values for raw organic waste FLAs and FA's were lower than values corresponding for soil HA's and FA's. Since low EJE6 ratios are associated with relatively large molecular size or high molecular weight, Riffaldi et al. (1983) suggest that organic waste HA's and FA's are of higher molecular weight than soil HA's and FA's. It was concluded that organic waste humic substances, once applied to the soil, gradually evolve and become closer in their properties to natural soil humic substances. The findings of Riffaldi et al. (1983) were contradicted by those of Gonzalez-Vila et al. (1985) and Inbar et al. (19901). They reported EJE6 values of HA's extracted from compost which were relatively higher than soil HA's. These findings indicate that compost HA's were lower in molecular weight than soil HA's (Gonzalez-Vila et al., 1985; Inbar et al , 19901). 1.2.3 Soil and Compost Matrices In studying the alkali extractable organic matter of a compost it is important to be aware of the similarities as well as the differences between compost and soil. Both are heterogeneous media that possess both liquid and solid phases. In soils, physical factors such as temperature, moisture content or bulk density are largely imposed externally, whereas in composting systems these physical qualities are a consequence of internal events (Miller, 1992). Most importandy for this study, both matrices have a biological component which affect humus transformations. 21 In contrast to a mineral soil, an important distinction of compost is the fact that it is primarily an organic matrix containing a very high density of organic substrate. Consequently, the rate of metabolic activity per unit volume is much greater in compost than in soil. The very slowly changing and structurally stable soil matrices contrast sharply with the accelerated biological activity, typical of compost, which profoundly alters its own physical environment (Miller, 1992). With maturity though, the capacity for a mature compost to support microbial acdvity is eventually much more similar to that of native soil humus (Mathur et al., 1993). In spite of the differences in metabolic rates of reactions which occur in soil and compost, both matrices share common attributes. Despite attempts to homogenize compost, gradients between highly oxidizing and highly reducing conditions exist, as they do in soil. For both types of matrices these aerobic and anaerobic microenvironments are able to coexist within close proximity of one another (Miller, 1992). 1.2.4 Chemical Methods Theory This secdon briefly explains the methods used in the study. Most of the methods have been developed for use in soil and not on compost. Since some composts appear to have slightly different chemical characteristics when compared to soil organic matter, it was felt that a discussion of the methods was important in order to assess their applicability. 1.2.4a Total Carbon: Total carbon, in an aqueous extract, can be determined with an automatic carbon analyzer (Astro Model 1850). An aliquot of a sample is mixed with O, and Na2S208 (a powerful oxidizing agent) and pumped past an ultra violet (U.V.) source. A U.V. catalyzed oxidation of reduced carbon-compounds occurs and an infrared determination is made on the resulting C02. This method is fast, accurate and repeatable. Total carbon in an air-dried ground sample can be determined using a dry combustion method in which the sample is burned in a stream of purified 02. In a LECO induction furnace, high frequency electromagnetic (e.m.) radiation is used to heat the sample. Since soil (and compost) cannot be directly heated this way, Sn and Fe chips must be mixed with the sample prior to combustion. These metal accelerators, absorb e.m. radiation and act as indirect sources of heat for the sample which is in close contact. Total carbon, in the sample is oxidized to C02 which is measured, as a volume, in a gas burette. Again, this method is fast, accurate, and repeatable (Nelson et al., 1982). 1.2.4b Kjeldahl Nitrogen: The Kjeldahl method can be used to determine total nitrogen, except for N03, N02 and N-N linked compounds. The sample is digested in concentrated H2S04 in order to oxidize reduced carbon and to convert any organic and ammonium nitrogen to NH4S04. H2S04 + 2R-NH3 - (NH4)2S04 In a semi-micro Kjeldahl distillation unit, the resulting digest is then made alkaline in order to convert NH4S04 to gaseous NH3. This N-compound is, then, trapped in a boric acid trap through steam distillation: NH3 + H3BO3 - NH4+ + H2B03 The resulting ammonium borate is titrated with standard acid in order to determine the nitrogen-content: HC1!STO) + (NH4)H2B03 - H3B03 + (NH4)C1 23 This is an accurate method which involves the conversion of organic nitrogen to NH4+-N (Bremner et al., 1982). 1.2.4c Polysaccharides: It is possible to determine two polysaccharide pools through acid digestion: total and labile. The method of Lowe (1993) is based on the release of saccharide monomers by hydrolysis with sulfuric acid, followed by colorimetric estimation of total sugar content in the hydrolysates, using the "phenol- sulfuric acid" reagent. Labile polysaccharides (LPSS) refer to the more easily degradable carbohydrate fraction and include hemicellulose and other non-cellulosic cell wall material. LPSS can be determined by incubating the sample with dilute acid: 0.5M H2S04. The recovery of more resistant cellulose in the total polysaccharide pool (TPSS) can be achieved through pretreatment of the sample with relatively more concentrated 12M H2S04 prior to hydrolysis with 0.5M H2S04 (Lowe, 1993). These polysaccharide procedures do not give an accurate absolute measure of polysaccharides because of variations in colour produced by different sugar monomers, and because sugar yields vary with variation in hydrolysis procedures and sample types (i.e. soil type). The procedure is useful, however, in assessing changes in polysaccharides over time, or in comparative studies (Lowe, 1993). 1.2.4d Humus Extraction and Fractionation: Part of humus is thought to contain relatively more hydrophilic functional groups that are not repulsed by strong alkali; and it is, therefore, soluble in a solvent such as NaOH. In a high pH environment, the acidic functional groups of humus, become charged. Repulsion of these charged groups causes the polymer to expand into a randomly coiled structure which associates with water and remains dissolved (Hayes, 1985). 24 Humin is the fraction of humus substances that cannot be extracted from soil by dilute base or acid. According to Schnitzer (1978), its insolubility in soil seems to arise from its being firmly adsorbed on or bonded to inorganic soil and sediment constituents. As well, it seems likely that a relative lack of oxidized functional groups renders humin less hydrophilic than its alkali soluble counterpart (Hayes, 1985). The alkali extract can be further fractionated into humic acid (HA) and fulvic acid (FA). Humic acid is insoluble in acid. This is pardy explained by its aromatic character. The more aromatic a compound the less hydrophilic or acid-soluble it tends to be (Kononova, 1966). As well, in a low pH environment, H+ ions associate with the acidic functional groups. The undissociated acids become involved in inter- and intra-molecular H-bonding processes. As a result, the humic acid molecules shrink, water is excluded from the matrix and precipitation takes place (Hayes, 1985). Fulvic acid is soluble at any pH because of its lower molecular weight and higher contents of acidic functional groups. These characteristics make salt complexes of fulvic acids more soluble than those of humic acids. As well, in spite of the fact that fulvic acids possess acidic functional groups, they are thought to lack the regularity in the arrangements of these groups to allow regular and close sequencing of inter- and intra-molecular H-bonding (Hayes, 1985). Fulvic acid can be further fractionated by passing it through polyvinyl polypyrrolidone (PVP). This adsorbs a fraction of FA which is relatively richer in phenolics (FAA). The fraction which passes through PVP (FAca) is carbohydrate rich, and has a relatively higher molecular weight than PVP-adsorbed fulvic acid (FAA) (Lowe, 1975). 25 1.3 Preliminary Static Pile Composting Trials Hazardous wastes and their potential as a valuable resource have been identified as an issue by the Sustainable Development Research Institute (SDRI), at the University of British Columbia. Agricultural wastes such as hog manure and the feasibility of composting as a management alternative is an aspect worthy of exploration. SDRI began its composting project in May 1991. Preliminary pilot-scale composting studies on hog manure solids were carried out by Dr. Victor Lo in collaboration with Dr. L.E. Lowe, using one cubic metre aerated static piles. Forced-air aeration was provided via perforated piping located at the bottom of the piles. The main objectives of the preliminary trials were to assess variability within the compost pile, and to evaluate sampling methods and analytical procedures for monitoring changes in chemical properties. The focus of the chemical analysis was on decomposition of starting materials and accumulation of products such as humus materials. An attempt was made to simultaneously monitor chemical transformations, moisture and temperature conditions, and microbial features at various stages of composting. This created some design problems because of the rather different requirements for sampling, preservation and analysis needed for chemical versus microbiological studies. In terms of temperature, very high values were found in the first week, with thermophilic temperatures continuing to the end of 28 days of composting. Temperatures significantly above ambient persisted until the end of the experiment, on day 58. With depth in the piles, the presence of gradients was noted: increasing for moisture and decreasing for temperature. Lowe attributed the moisture gradient mainly to gravity (Lowe et al, 1993). Higher moisture contents and low 26 temperatures at the lower level suggest the occurrence of anaerobic zones which are associated with excessive moisture and low rates of metabolic heat production. In addition, lower temperature zones with depth may indicate excessive cooling as a result of too much forced-air aeration from the bottom. Overall, decreasing trends over time were found for total carbon (Cj-), total polysaccharides (TPSS) and the ratio of total carbon to total nitrogen (C/NT). An increasing trend over time was found for total nitrogen (NT). Lowe et al. (1993) reported substantial increases in humic acid carbon (CH), and slight increases in fulvic acid carbon (CF) and carbohydrate-rich fulvic acid carbon (C^), over 58 days of composting. Slight decreases were reported for phenolic-rich fulvic acid carbon (CA). Values in CH ranged from 2.92%(o.d.) at day 0 to 5.66% at day 58. CF ranged from 4.74%(o.d.) to 5.05%, and Cca ranged from 3.65% to 4.19%. CA ranged from 1.09%(o.d.) to 0.86%. These results indicate that accumulation of humus substances occurred over time. Changes were most apparent for humic acid carbon. Increases were found in Qi/Cp from 0.62 to 1.12 from day 0 to day 58, indicating a general shift from CF to CH over time. Phenolic acids are major decomposition products of lignin and other plant polyphenols and have been implicated in some cases of phytotoxicity. Lowe et al. (1993) found significant levels of extractable phenolic acids in uncomposted swine waste solids and sawdust. Levels were substantially lower by day 3 and day 42 of composting, consistent with active microbial populations preventing significant accumulation of such compounds. 1.4 Thesis Objectives Three composting experiments were performed on hog manure solids mixed with hemlock sawdust. In order to characterize hog manure compost, the issue examined in this thesis is humus fractionation and chemical properties of humus. The main purpose of the study was to determine the 27 effects of time and aeration frequency on the temporal development of the alkaline extract of composting hog manure. The term alkaline extract refers specifically to humic acid and fulvic acid which are two components found in humus. This was accomplished through the analysis of carbohydrates and carbon and nitrogen pools under controlled experimental conditions. An ancillary purpose of the study was to assess the effect of externally controlled temperature on the proposed chemical parameters. The first two experiments utilized a bucket-scale apparatus where the effects of time and frequency of aeration could be examined. The third experiment was a smaller bench-scale study to assess the effect of externally controlled temperature on the compost. 1.5 Description of Source Manure Management System Hog manure solids were obtained for the composting project from the Lower Fraser Valley, B.C. hog farm of Peter Hill (18504 20th Ave., Surrey), where a belt press separator is employed. The separator consists of cylindrical rubber rollers and a conveyor belt. The hog bam was designed such that the pigs stand on slats over top of an angled collection pit which is about one foot deep. Periodically the pit is flushed with water. The flush water or slurry, after passing through the bam, collects in a storage tank outside the bam. After allowing some solids to settle, this high moisture slurry (>90% moisture) is pumped to the separator. The slurry is carried along a conveyor belt and passed through two rubber rollers which press out the water. Solids containing approximately 80% total moisture are dropped through a screen onto a collection pile outside the bam. 28 1.6 Bulking Agents In the Lower Fraser Valley, B.C., hog manure solids have been composted successfully with or without bulking agents depending on the starting moisture of the waste (Lo, 1993). In high moisture sewage sludge or animal wastes, a bulking agent such as wood chips, straw or bark is often used to increase porosity (Miller, 1992). For this thesis, hemlock sawdust was used as a bulking agent in order to decrease the starting moisture to approximately 70%. In sawdust used to compost hog manure solids, Lowe et al. (1993) reported C/NT values ranging from 2840:1 to 671:1. In the same sawdust, average carbon values of 48.69%(o.d.)± 1.36 were reported along with NT values of 0.0499%+0.0237. This data suggests that the variability in C/NT for sawdust was a result of variability in the very low NT values. 1.7 In-Vessel Composting Smaller scale (~90 litres) bucket composting was used in experiments 1 and 2 of this thesis to allow regular and effective mixing in order to reduce the variability in temperature and moisture which was encountered in the preliminary static pile trials. For experiment 3, composting was carried out in even smaller vessels (~2 litres) using controlled-tempera ture waterbafhs to better homogenize the temperature environment of the substrate. 29 Chapter 2 - Materials and Methods: 2.1 Composting Materials and Set-up 2.1.1 Experiments 1 and 2 - Bucket-Scale Composting Hog manure solids (79.4% W.M.1) were obtained from the farm of Peter Hill (18504 20th Ave., Surrey), where a belt-press liquid-solid separator was employed. Untreated hemlock sawdust, from Westcoast Cellufibre, was used as a bulking agent. In experiments 1 and 2, composting was performed using 90.0 litre (20 gallon) plastic buckets (Rubbermaid-Roughneck), wrapped with fibreglass insulation. Compost temperatures were monitored with copper-constantin thermocouples attached to a datalogger (Campbell Scientific - Model CR10). Each thermocouple was approximately ten cm. long and was attached to a lead measuring 5 to 7 metres in length. Temperature was measured in five positions per bucket, such that the top, middle and bottom parts of the compost were monitored (Figure 2.1). 'W.M.=value determined on a wet mass basis 30 Figure 2.1: Bucket Showing Thermocouple Positions - Experiments 1 and 2 12 cm 0T1 12 cm 0T3 12 cm 6 cm 18 cm GT2 12 cm ^ T 4 The thermocouples were inserted through holes in the sides of the buckets, such that the sensors penetrated to the centre of the compost. Measurements were made every sixty seconds and half-hour averages were sent to final storage for each thermocouple. For experiment 1, six insulation wrapped buckets were filled with a mixture of five parts hog manure solids and one part hemlock sawdust (measured by volume). On composting day 0, these 5:1 ratios of manure to sawdust were mixed with an auger (60 cm. in length) which was attached to a hand-held drill (Makita). The mixed buckets were placed on top of hollow plastic flooring (~3.5 cm wide) for the duration of the experiment. Three buckets, numbered from 1 to 3, were aerated using the auger and drill on the seventh day of composting and once every fourteen days, thereafter, until the experiment was terminated on day 51. This is referred to as aeration treatment "A/. The other buckets, numbered 4 to 6, were aerated more frequently. Buckets 4 to 6 were mixed, using the same method as with buckets 1 to 3, every seven days from day 0 until the end of the experiment. This aeration regime is referred to as 'A-,'. Before mixing on days 7 and 23, samples weighing 31 approximately ten grams were taken from the bucket holes, at each thermocouple location. After mixing, composite samples weighing approximately two hundred and fifty grams were taken from each bucket, on days 0, 7, 23 and 51. The same bucket-scale composting method was used in experiment 2, with four additional modifications concerning aeration, sampling and starting moisture. While aeration treatment " A2' was again used for three buckets, numbered from 1 to 3; aeration treatment "A/ was abandoned for a more frequent regime (A3). Three buckets, numbered from 4 to 6, were mixed every two to three days, for the first twenty-one days of composting. After twenty-one days, all buckets (1-6) were mixed every seven days, until the end of the trial. This is referred to as aeration treatment "A3'. In addition, sampling periods were increased from four to seven. Composite samples weighing approximately two hundred and fifty grams were taken from each bucket after mixing, on days 0, 7, 14, 21, 28, 42, and 55. In experiment 2, two minor changes were made in an attempt to obtain a drier starting mix. The manure was drained for seventy-two hours, after collection from the source, and the ratio of manure to sawdust was decreased from 5:1 to 4:1. 2.1.2 Experiment 3 - Bench-Scale Composting In experiment 3, bench-scale composting was performed using 2 litre buckets (Nalgene). Five parts hog manure solids (Peter Hill) and one part screened hemlock sawdust {Westcoast Cellufibre) were mixed together and composted in controlled temperature waterbaths (Thelco "Model 83') for 46 days. Previous to filling the buckets, the 5:1 combination of manure to sawdust was thoroughly mixed, using an auger and drill as in experiments 1 and 2. Three vessels were placed in each of three 32 waterbaths. In an attempt to minimize losses of moisture through evaporation, film wrap seals were placed over the tops of the composting vessels. A composite sample weighing approximately one kilogram was obtained on day 0 of composting. Each vessel had a thermocouple inserted, from the top, through the plastic film lid into the compost. Temperatures were measured every sixty seconds and hourly averages were sent to final-storage of a datalogger (Campbell Scientific - Model CR10). Every other day, throughout the experiment, each bucket was removed from the waterbath and mixed. This was achieved by placing a screw-top lid on a vessel and shaking it vigorously for two minutes. Each vessel was mixed and replaced back into its waterbath within approximately ten to fifteen minutes. Sampling occurred after mixing, on days 0, 12 and 46 of the experiment. On day 12, compost samples weighing approximately two hundred and fifty grams were removed from each vessel. On day 46, final samples ranging in size from one hundred and fifty to two hundred grams were obtained. 2.7.2a "High Temperature' Composting: Buckets 1 to 3 were placed in a "high temperature' waterbath (WB1) which was maintained at a temperature of 60° C, for 46 days. 2.1.2b "Compost Simulation' Composting: Buckets 4 to 6 were placed in a "compost simulation' waterbath (WB2) which was adjusted periodically, such that it imitated the temperature profile observed for bucket 6 of experiment 2 (Figure 2.2). 33 Figure 2.2: Temperature Profile of Compost Simulation1 Waterbath 2 - Experiment 3 7 0 . 0 — * -i / AOP -j-*-—V u if^tf^v 5 d . « Oj 4 0 . 0 i-i I § 3 0 . 0 •Va 1 ^ -"-*!-IK) . . .a?. _3n_ a? ?*g«? OBSB lO l O 3 0 4 0 C o m p a n t t a B *I"ii*x«5 ( d a y s ) The temperatures in both the 'high temperature' waterbath and the 'compost simulation' waterbath were gradually adjusted upwards to the thermophilic range within the first 24 hours of composting. 2.1.2c "Low Temperature' Composting: Buckets 7 to 9 were placed in a low temperature' waterbath (WB3) which was controlled such that the temperature ranged from 26° C to 28° C, for the duration of the experiment. 34 2.2 Chemical Analyses Determinations were made for total moisture (Moisr), total carbon (Q.), water-soluble carbon (WSC), total nitrogen (NT), labile and total polysaccharides (LPSS and TPSS); and total carbon and nitrogen in the humic acid (CH and NH), the fulvic acid (CF and NF), and the non-PVP adsorbed carbohydrate-rich portion of the fulvic acid (Cca and Nca). Carbon and nitrogen in the PVP adsorbed phenolic-rich portion of the fulvic acid (CA and NA) was calculated by difference (/. e. CF - Cca = CA). In addition, total extractable carbon (Ce) as a fraction of CT was determined. With the exception of determinations for WSC and MoisT, all analyses were carried out on air-dried, ground compost. A Tecator sample mill (Cyclotec Model 1093) grinder was used to grind air-dried samples to 0.5 mm. Fresh compost samples were immediately placed in air-tight containers until preparations of samples for water extracts and total moisture determinations were started. This always occurred within three hours of sampling. After samples for moisture and the water extracts were obtained, the remainders of the samples were immediately unsealed in order to air-dry at room temperature. Mass Balance - Although it is recognized that a mass balance approach is useful, because of constraints in experimental designs it was not attempted. All values were determined as percentages of the total sample. 2.2.1 Procedures for Fresh Sample Analysis 2.2.1a Total Moisture (MoisT): 5 gram compost samples (fresh) were oven-dried at 105° C for 16 hours. 35 2.2.1b Water-soluble Carbon (WSC): Total carbon was determined on prepared water extracts using an Astro 1850 Carbon Analyzer. Values were compared against a series of working potassium acid phthalate standards. In an attempt to progressively decrease colloidal carbon, a different method for obtaining the water extract was used for each experiment. Experiment 1 - Water Extract: A 10 gram sample of fresh compost was diluted with 100 ml distilled H20 and shaken for 16 to 20 hours. Solids were separated from the supernatant by passing through a 270 mesh screen (100 /urn), and centrifuging (Sorvall RC2-B) for twenty minutes at 5860 x g. After diluting to 250 ml with distilled H20, the supernatant was centrifuged (Beckman J2-21M/E) for twenty minutes at 4720 x g, filtered through Whatman #1 filter-paper, and stored overnight at 4°C. Experiment 2 - Water Extract: The same procedure from experiment 1 was used for experiment 2, with the exception that modifications were made in centrifuge speeds used. After passing through a 270 mesh screen the supernatant was centrifuged (Beckman J2-21M/E) for ten minutes at 9630 x g. After diluting to 250 ml, the supernatant was centrifuged (Beckman J2-21M/E) for ten minutes at 19,800 x g. Experiment 3 - Wrater Extract: A 10 gram sample of fresh compost was diluted with 100 ml distilled H20 and shaken for 16 to 20 hours. No further dilution of the sample occurred. Solids were separated from the supernatant by passing through a filterless buchner funnel (under suction), and by refrigerating at 4°C overnight, to allow settiing of solids. After centrifuging (Beckman J2-21M/E) at 19,800 x g for 20 minutes, the supernatant was sequentially filtered through Whatman #1 and Whatman #42 filter papers. Finally, the supernatant was passed through a 0.2um disposable 36 nylon millipore filter (Alltech 2045) via a 5 ml. syringe (Hamilton 1005TLL) and stored at 4°C, overnight. Millipore filtration was performed to ensure that colloidal carbon (>0.2um) was excluded from the analysis. 2.2.2 Procedures for Air-dried, Ground Sample Analysis 2.2.2a Total Carbon (CT): 0.05 gram compost samples were mixed with Fe and Sn accelerator chips (LECO) and analysed for total carbon using a LECO high-temperature induction furnace and carbon analyser (Furnace Model 521 and Analyzer Model 572-200), by the method of Nelson et al. (1982). 2.2.2b Nitrogen (NT): 0.075 gram compost samples were analysed for nitrogen, using the method of Bremner et al. (1982). Under a condenser, samples were acid-digested with 1.9 grams of KjSOVCuSO^Se catalyst; and then steam-distilled with a strong alkali (ION NaOH), using a semi-micro-Kjeldahl apparatus. The resulting NH3-N was trapped in 5% boric acid, and titrated using 0.02N Standard HC1 (with bromocresol green/methyl red indicator). 2.2.2c Polysaccharides: 0.05 g samples of compost were acid-hydrolysed and analysed for labile polysaccharides (LPSS) and total polysaccharides (TPSS), using the method of Lowe (1993). A Fisher Sterilizer (Model 750) was used to autoclave samples. After autoclaving, cooled samples were filtered through 37 Whatman #1 paper and diluted to 250 ml. A Spectronic 20 spectrophotometer was used to read absorbances off "phenol-sulfuric-hydrolysate" mixtures, at 480 nm. Results were determined as % glucose equivalents (%gluc. equiv.) of either LPSS or TPSS. 2.2.2d Humus Extraction, Fractionation and Analysis For Total Carbon and Nitrogen: The method of Lowe (1980) was used for extraction and fractionation of humic acid (HA), fulvic acid (FA) and non-PVP-adsorbed carbohydrate-rich FA (FAca) from 2 gram samples of compost. Prepared solutions of HA, FA and FAca were stored at 4°C for further carbon and nitrogen analysis. Total Carbon in HA, FA, FAca and FAA (CH, CF, Cca, CA ): An Astro 1850 carbon analyser was used to measure total carbon. Total carbon in the PVP-adsorbed phenolic-rich portion of FA (CA) was calculated by the difference between CF and Cca. Total extractable carbon (Ce ) was determined as the fraction of Q- which consists of alkali extractable carbon (CH + CF). The calculation used was: CJC„+CJ*IOO Nitrogen in HA, FA, FAca FAA (N0, NF, Nca and NA): 25 ml aqueous samples were analysed for nitrogen, using the semi-micro Kjeldahl method. After boiling off the water, samples were acid-digested, using a K2S04/CuS04/Se catalyst; steam-distilled with a strong alkali into a boric acid trap and titrated using standard acid as previously described. Nitrogen in the PVP-adsorbed phenolic-rich portion of FA (NA) was calculated by the difference between NF and NCa. 38 2.3 Statistics 2.3.1 Experiment 1 - Statistical Analysis Experiment 1 was a 2x4 factorial, with two aeration treatments and four sampling times as the treatment factors. There were three replications (buckets) per aeration treatment. Aeration was considered to be fixed and time was random (Zar, 1984). A two-way analysis of variance was performed, using Systat-V.4, in order to determine significant effects of time or aeration on the chemical parameters measured at a critical level of 0.05. Multiple contrast of means was the post-hoc test used to determine which time treatment means were significantly different with an overall critical protection level of 0.05 (Wilkinson, 1989). 2.3.2 Experiment 2 - Statistical Analysis Experiment 2 was a 2x7 factorial, with two aeration treatments and seven sampling times as the treatment factors. There were three replications (buckets) per aeration treatment. Aeration was considered to be fixed and time was random (Zar, 1984). A two-way analysis of variance was performed, using Systat-V.4, in order to determine significant effects of time or aeration on the chemical parameters measured at a critical level of 0.05. Multiple contrast of means was the post-hoc test used to determine which time treatment means were significantly different with an overall critical protection level of 0.05 (Wilkinson, 1989). 39 2.3.3 Experiment 3 Statistical Analysis Each waterbath was a 1x3 factorial, with three sampling times as the treatment. A one-way analysis of variance was performed, using Systat-V.4 to determine significant effects of time on the chemical parameters measured at a critical level of 0.05. Multiple contrast of means was the post-hoc test used to determine which time treatment means were significantiy different with an overall critical protection level of 0.05 (Wilkinson, 1989). 40 Chapter 3 - Results and Discussion 3.1 Experiment 1: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Hog Manure Solids Compost Under Aeration Treatments A/ and A2' 3.1.1 Temperature In graphic representations of the temperature profiles of each bucket (Figures 3.1-3.7), it can be noted that all bucket temperatures rose quickly and reached or exceeded 55° C within twenty-four hours. Thereafter, the temperatures decreased, notably by day 7. This suggests that, for experiment 1, the thermophilic phase was essentially complete before application of the first aeration treatment. Figure 3.1: Bucket Showing Thermocouple Positions - Experiments 1 and 2 41 Figure 3.2: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'Aj' - Experiment 1, Bucket 1 u I r O 7 14 2 1 2 S 3 3 4 2 4 9 C o m p o s t i n g T i m e ( d « 7 i ) • o f a n i B B U r a a o t d v d at 2 3 3 0 h o u n - B l - T l B i - r a B 1 - T 3 B 1 - T 4 - B l - T S - -A.mt>ienx J^IT Figure 3.3: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A,' - Experiment 1, Bucket 2 u 2 D H io.o a 1 14 21 2« 35 4 2 4» C o m p o a t l n g T l m » Cdmywy * M v « i u r » n » l i r»«or4*d ai 3 3 3 0 t tovr* 42 Figure 3.4: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A,' - Experiment 1, Bucket 3 u 43 4 0 . 0 cL so.o s la.o O 7 14 21 2 * 35 42 * » C o m p o i t t n g T i m e C<l»y»> * M « « i u r « m » n t i t i o o r d t d at 2 3 3 0 h o n r i Figure 3.5: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment A2' - Experiment 1, Bucket 4 B 4 - T 1 - B 4 - T 2 B 4 - T 3 B 4 - T 4 - B 4 T 5 Ambient Air lo.e —f-a 7 1* 21 Z» 35 41 +• 3S C o m p o s t i o g T i m e (c lays) * M « a » t a m a n l l raDvrdtd »t 2330 b o a » 43 Figure 3.6: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 1, Bucket 5 u ! -HB- B 5 - T 1 - * - B 5 - T 2 — B 5 - T 3 —O- B S - T 4 —o— B S - T 5 A t a b i o n t A U ; ° 7 * M « * t u r e A e a t i 14 2 1 2 8 3 5 4 2 4 » C o m p o s t i n g T i m e ( d a y s ) r e v o t d o d *t 2 3 9 * h o u r i 3 6 Figure 3.7: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 1, Bucket 6 u I-E - B 4 - T 1 - B 6 - T 2 - B S - T 3 - M - T 4 - B « - T J IO.0 « 7 14 3 1 1 » 3 5 4 2 4 9 J « C o m p o s t i n g T i m e ( d a y s ) ' M s l B v n m a a t i r v e a r d e d at 2 3 3 0 h o u r s 44 Aeration treatment "A/ was applied to buckets 1 to 3. For this compost, the temperature of the upper thermocouples increased rapidly to the thermophilic range in the first twenty-four hours, peaking between day 2 and 3 before declining (Figures 3.2-3.5). After the day 7 aeration treatment, there was a brief increase back to the thermophilic range for two buckets (1 and 2) which had fallen below 55° C, before declining toward ambient conditions. After the day 21 aeration treatment, bucket temperatures increased to 45° to 50° C, peaking between day 29 to day 34. The increase in temperatures for all buckets after three weeks of composting suggests that the day 21 aeration treatment may have extended the transition from the thermophilic stage to the maturation stage by supplying a limiting nutrient: oxygen. After day 42, all bucket temperatures had declined once again to ambient conditions, showing no further increases in temperature. This suggests that some factor other than oxygen was limiting microbial activity at this stage. The more frequent aeration treatment ' A2' was applied to buckets 4 to 6 (Figures 3.6 to 3.8). Their temperature profiles behaved similarly but not as uniformly as with the Aj-compost (Figures 3.2 to 3.4). After a rapid increase to the thermophilic range, the temperatures peaked between day 2 and 4 and then began to decline. For the A2-compost, there was a brief increase in bucket temperatures following the day 7 aeration treatment, and then a steep decline toward ambient temperatures. After the day 14 aeration treatment, the temperature of bucket 4 was the only bucket to briefly increase back to the thermophilic range before continuing its decline in temperature. After day 21, the temperature profile of bucket 6 increased dramatically to about 45° C, similar to the Aj-compost. Buckets 4 and 5 showed much weaker temperature increases to about 27° to 30° C, after the day 21 aeration treatment. Similar to the A,-compost, the temperatures of the A2-compost showed no further increases from about day 30, declining steadily to ambient conditions. With the exception of bucket 4 (A2), the lengths of the thermophilic and transition cooling phases to ambient temperatures do not appear to have been different for aeration treatments in 45 experiment 1. Overall, thermophilic temperatures were maintained for 4 to 6 days (8 days for bucket 4). By day 42, the decline of all compost temperatures had levelled off to slightly above ambient conditions. Figures 3.1 to 3.8 show that for both the 'A/- and A2-compost, the upper parts of the compost, which are represented by positions T i ' and 'T2', initially reached the highest temperatures until approximately day 7. At this point, these upper positions quickly became the lowest temperatures relative to the other thermocouples. This probably arose because of considerable loss in volume of the compost which was observed but not quantified. As the compost volume decreased, "IT- and T2-position thermocouples became progressively more exposed to the upper outer surface, and their temperatures became relatively more influenced by ambient temperature conditions. In spite of this, individual thermocouple temperatures indicate that early in the process, the lower portions of the material were much cooler than the upper portions. In fact, the deepest thermocouple positions (T4 and T5) never exceeded 50 ° C during the experiment. Figures 3.8 to 3.12 describe the temporal variation in thermocouple temperatures between buckets 1 to 6. 46 Figure 3.8: Temporal Variation in Temperature As Measured By Thermocouple 1 (Uppermost) - Experiment 1, Buckets 1-6 u S 3 0 . 0 2 Q . o —j r T 1 4 2 1 2 8 C o m p o i t i n g 1 4 2 4 9 Figure 3.9: Temporal Variation in Temperature As Measured By Thermocouple 2 Experiment 1, Buckets 1-6 u 2 «5 c o 1 5 . 0 O -7 t-* 2 1 2 « 3 3 -*2 •*» C o m p o i t i n g X l m o C c i j l y B ) ' M < u v » n « n l l r a o o r d a d a t 2 3 3 0 h o u r a 47 Figure 3.10: Temporal Variation in Temperature As Measured By Thermocouple 3 (Middle) Experiment 1, Buckets 1-6 u 1 S o H 2 3 3 Q h o o t l Figure 3.11: Temporal Variation in Temperature As Measured By Thermocouple 4 Experiment 1, Buckets 1-6 u g. i s I y 3 O . 0 ! H T* j \ i i ___ f \ AY T ) i i r ! i 1. r j J_ XT i ' ' I ' k M o a * t i f e n e a c i r 14- 2 1 2 a 3 S 4 2 * » 5 « C o m i > o m l a g T I t n o C « * * y « > o a o r d « d * t 3 J 3 0 h o o » » 48 Figure 3.12: Temporal Variation in Temperature As Measured By Thermocouple 5 (Bottom) Experiment 1, Buckets 1-6 u 1 c o 2 0 . 0 1 , , , • , 1 , r O tO 2 0 SO <»0 SO C n m p o a t i n g T"lxn.e ( d m y > ) The error bars in Figures 3.8 to 3.12 indicate the sample standard deviation of temperature measured at each of the five thermocouple positions (Figure 3.1). It can be seen that the spread of the measurements was not homogeneous throughout the experiment. The variability between measurements narrowed as the temperature approached ambient conditions, and increased during periods in which the temperatures were raised above ambient conditions. The variable influence of biological activity which drives temperature increases may have increased variability at temperatures above ambient conditions. The relatively larger deviation of the observations at day 28 is a reflection that not all buckets responded with an increase in temperature after the day 21 aeration. A review of the temporal variation in temperature within the separate aeration treatments (At and A2) showed similar increases in variability at temperatures above ambient conditions (data not shown). 49 3.1.2 Moisture Gradient Figures 3.13 to 3.14 show the average values of day 7 and day 23 moisture determinations of samples taken at individual thermocouple positions for compost under aeration treatments " Aj' and "A2'. Figure 3.13: Moisture Gradient From Day 7 to Day 23 in Composted Hog Manure Solids Experiment 1, Aeration Treatment 'A/ - Bucket Averages (1 to 3) &$ J O . O s -50 Figure 3.14: Moisture Gradient From Day 7 to Day 23 in Composted Hog Manure Solids Experiment 1, Aeration Treatment 'A2' - Bucket Averages (4 to 6) 7 C O rtO-O g_ so.o The error bars in Figures 3.13 to 3.14 indicate the sample standard deviation of moisture values (Moisj) at each of the five thermocouple positions. Day 7 and day 23 moisture determinations from individual thermocouple samples, indicate the presence of moisture gradients which increased with depth regardless of aeration frequency. Moisture gradients were probably due to gravity and drying from the upper compost surface. Variability in Moisj for the A,-compost was greatest at day 7 and narrowed substantially by day 23 for both aeration treatments (Figure 3.13). This was particularly apparent at positions "T2', 'T3' and 'T4'. Differences in variability between sample days were not as notable in the A2-compost (Figure 3.14), suggesting that it was mixed more thoroughly than the Arcompost.. 51 3.1.3 Sampling Sampling occurred on days 0, 7, 23 and 51 of experiment 1. The initial rapid temperature build-up and decline which occurred before the application of any aeration treatment was not expected. Since this most accelerated metabolic phase of composting had passed without any additional aeration occurring after the day 0-mixing, the sampling frequency was less intensive than had originally been intended. Instead of sampling every fourteen days after day 7, sampling only occurred twice past day 7. 3.1.4 Changes in Chemical Properties Twenty-one chemical parameters which are representative of important nutrient pools for composting organisms were measured (directiy or indirecdy) over time in experiment 1. A tabulated form of the two-way analysis of variance results on the effects of time and aeration is located in Appendix A. Of the twenty-one parameters studied, time had a significant effect on twelve at a 5% level of significance. These twelve were Moisr, WSC, CT, NT, CH, NH, C/NF, Cca, C/N^, CH/CF, LPSS and TPSS (Appendix A). Two of these parameters also showed apparently significant effects of aeration treatments 'A{ and "A2': CT and C/Nca. A review of the plotted data, however, indicates increased variability in day 0-values for these two parameters, causing the appearance of segregated populations of 'A,'- and A2-values. A review of the data past day 0 for CT and C/N^ did not show that two separate populations of values existed as a result of the imposed aeration regimes. A decrease in variability for Cr and C/Nca with mixing suggests that the compost became more homogeneous with time. Ce, NF, Nca and NA did not change significantiy in experiment 1. Significant interactions occurred between time and aeration treatments for C/NT, C/NH, CF, CA and CJCf. A 52 review of these data suggests that heterogeneity in the starting mix may have caused the appearance of interactions. After day 0, plots of the data showed no clear evidence of interaction effects for C/NT, C/NH, CF, CA and Q/Cp. The results are presented in a series of figures plotted against time. For parameters in which the analysis of variance showed significant effects of aeration treatments or interactions, the data was graphed as 'A/- and A2-populations. Using Cricketgraph (v. 1.3.1), best-fit lines were interpolated through the data. All MoiSj- values are reported on a per cent wet mass basis (W.M.). The rest are reported on a per cent oven-dried mass basis (o.d.). In addition, a tabulated form of all data is located in Appendix D, Part 1. 3.1.4a Carbon and Polysaccharide Pools: LPSS and TPSS decreased from day 0 through day 51 of the experiment (Figures 3.15-3*16). 53 Figure 3.15: Change in Labile Polysaccharides (LPSS) Over Time, in a Hog Manure Solids Compost - Experiment 1 . 5 SO.O — ! -g 4 0 . 0 Amy -X* 2 0 . 0 —(-O T 1 4 2 1 2 » 3 3 4 1 4 « 3 « C u m p u t t l n j T i m e ( d » y » > Figure 3.16: Change in Total Polysaccharides (TPSS) Over Time, in a Hog Manure Solids Compost - Experiment 1 Sjo Amy O 'S 31 8 0 . 0 —| g p a so.o —i -•to.o — j - r = - ^ l -d « r 5 1 3 0 . 0 O T 1 4 2 1 3 « 3 5 4 2 4 « 3 « C o m p o a i i n g T i m e ( d a y * ) • V « l u » « d a t v r m l n a d o n » n o v « n - d r l * d t > « a l a 54 At day 0, LPSS values averaged 47.5% (glue, equiv.) and decreased to 28.0% by day 51. Average TPSS values started at 67.7% (glue, equiv.) and decreased to 40.9%. These values are considerably higher than values reported by Inoko et al. (1979) for municipal refuse compost. This could be attributed to a different method of hydrolysis and measurement, and perhaps character differences between the composting substrates. Compared with mineral soils, the polysaccharide contents found in this experiment were, at the very least, 100 times greater (Oades, 1966). The steady decrease of LPSS and TPSS in experiment 1 suggests that, initially, microbial populations existed which may have been capable of utilizing both resistant and relatively more biodegradable carbohydrates. This type of utilization may have occurred throughout the study period regardless of temperature fluctuations. On the other hand, the magnitude of LPSS values relative to TPSS, indicates that TPSS was dominated throughout by relatively more degradable carbohydrate than cellulose. WSC, Cca and Cr did not begin to decrease significantiy until after day 7 of composting (Figures 3.17 to 3.19). 55 Figure 3.17: Change in Water-Soluble Carbon (WSC) Over Time in a Hog Manure Solids Compost - Experiment 1 2 . 2 0 2 . 0 0 l . B O * -£* - -(SB "mt "»r i 1 I I 1.40 —+-- a g s i OS d.y 2 3 -^3S &'' m m l.oo —t-~T~ -33W-\ 1-* 21 2 8 35 -*2 -*9 SO C o m p o i t l a $ T i m e C<i*y»> ""Vailwm de t0rmi&0tf o » aa. o v o a - i i r i o 4 basia n - 4 » Figure 3.18: Change in Non-PVP Adsorbed Carbohydrate-Rich Fulvic Acid Carbon (Cca) Over Time, in a Hog Manure Solids Compost - Experiment 1 ms ao i S T - - . , IB m IB - J 3 D -3.00 —| , 1 : , ; ; : , . ; , , , p~ O 7 14 2 1 2H 33 -*2 -»« C o m p o s t i n g T i m o ( d s y a ) ' V t f o # i d « t » r m i o * d o « • » o v « n - d r U d b a i t s n - 2 5 56 Figure 3.19: Effect of Aeration Treatments (A1 and Aj) on Total Carbon (CT) Over Time, in a Hog Manure Solids Compost - Experiment 1 o 4tf.O 4 5 . 0 4 4 . 0 4 3 . 0 t 4 -K A.1 S J ^ * 1 ' —-A 2 " ! < | : 1 * SI aa ' i ( i i * CCT> A l BB CCT) A 2 O "7 I* 21 2 8 3 5 4 2 -•» 3 « C o m p o t t l a i T i m e C<J»y«) * D a t d r n i i i « t i o f t i T a p o t t s d o o o v f l t t - d t i s d b t a t i WSC started out averaging 1.71%(o.d.) and decreased significantiy from day 7 to day 51 of composting. Final values averaged 1.01%(o.d.). These determinations agreed with WSC values reported by Garcia et al. (1990), but were much higher than those extracted and measured by Chanyasak et al. (1981). Differences in the literature could be a reflection of variability in methodology and material characteristics. The lack of change until day 7 for WSC suggests that an expanding population of organisms was making significant water-soluble carbohydrate contributions. The decrease in WSC, after day 7 through day 51, reflects the activity of an established microbial population utilizing readily available nutrients and producing relatively lower amounts of water-soluble carbohydrate-like by-products (Figure 3.17). Cca decreased from day 7 to day 23 and then levelled off through day 51 (Figure 3.18). At day 0, values averaged 4.28%(o.d.) and decreased to 3.60% by day 51. Although these values are similar in magnitude to those reported by Lowe et al. (1993), their decreasing trend contradicts the 57 slightly increasing trend in Cca, which was found by Lowe et al. (1993). One would expect this carbohydrate-rich material to be more available to organisms and for it to behave in a pattern similar to that of relatively more available nutrients like LPSS and WSC. A levelling off of the decrease in Cca may not necessarily indicate a decrease in utilization. Negative enrichment of C a may have occurred due to preferential oxidation losses of other reduced carbonaceous material in the substrate. A two-way analysis of variance for Cr showed significant effects of aeration treatments "A/ and "A2' (Figure 3.19). A review of the data, however, did not show that two separate populations existed as a result of imposed aeration regimes past day 0. A relatively larger spread in day 0 values appears to have narrowed by day 7. Variation in Q. determinations may have acted as a sensitive indicator that homogeneity was not sufficiendy achieved after the initial mix of two substances differing widely in carbon-contents: sawdust and manure. This segregation of populations at day 0 could possibly have been caused by differences between the manure that was rationed into buckets 1 to 3 (A,) and manure that went into buckets 4 to 6 (A2) at the time of the day 0-mix. At the time of mixing, the manure was first rationed to A,-buckets, and then to A2-buckets. During the interval between rationing for the two sets of buckets, some settiing of water-soluble nutrients in the manure due to gravity may have occurred which could have affected homogeneity of mixing. CT remained unchanged until day 7, decreasing significandy until day 23 (Figure 3.19). Starting values of Cj averaged 44.9%(o.d.) and decreased to 43.2% by day 51. These values and the slightiy decreasing trend resemble that of rice bran compost which was reported by Yoshida et al. (1979). As with WSC, the early lack of decrease in C^ may be a reflection of carbohydrate contributions from an expanding population of organisms. The decrease in CT from day 7 to day 23 suggests utilization of reduced carbon compounds and the release of C02 through oxidation, during this period. 58 A two way analysis of variance indicated interactions between time and aeration treatments for CF, CA and Q/Cp determinations. Figure 3.20 shows that for CF, a large spread in the day 0 values occurred. Figure 3.20: Effect of Interaction Between Aeration Treatments (Ai and AJ and Time on Fulvic Acid Carbon (CF) in a Hog Manure Solids Compost - Experiment 1 «.oo -gy-! SB CC3F) A 3 u 4 . 0 0 I O T 14 2 1 2 » 3 3 4 2 •*& 5 6 C a m p o i t t a g T i m e C<*&ys> " D * t * r m L t i a t l o x i » r v p o r t a d O H o * e B - d r l t d b a t i k n - 2 5 This variability of day 0-CF values suggests segregation of "A,'- and A2-populations, which should not have been the case since all six buckets were treated identically at this point. As with Q-, there may have been a lack of homogeneity in the starting mix, to which CF was a relatively sensitive indicator. Similar interactions were also found upon examination of CA and CJCP data (not shown). For both CF and CA, the values appear to have decreased over time. Average values for CF ranged from 5.24% (o.d.) at day 0 to 4.29% by day 51. CA values averaged from 0.96% (o.d.) to 0.70% during the same time period. These ranges in CF and CA were similar in magnitude to that reported by Lowe et al. (1993). 59 In general, it appears that all fractions of fulvic acid carbon decreased at some point during composting. This agrees with the findings of Inbar et al. (1990) on CF from cattle manure solids compost, but it disagrees with the findings of both Inbar et al. (1990) and Lowe et al. (1993) who report increases in Cca and no change in CA during composting. The previously mentioned interaction which occurred between time and aeration treatments for CJCp was not unexpected, as this ratio would carry influences from both CA and CF trends. Average values for CJCf ranged from 0.18 at day 0, to 0.16 by day 51. CH increased from day 0 to day 7 of composting and then levelled off through day 51 (Figure 3.21). Figure 3.21: Change in Humic Acid Carbon (CH) Over Time, in a Hog Manure Solids Compost - Experiment 1 3-i, as,. 3J O -7 14 2 1 2 S 3 5 4 3 +9 C o f f l p c i H [ i i i | i T f w o ( d a y s ) » V « l u » » < i * t « r m l c t * « i o n « n o v a n - d r t w d %>«•!• At day 0, average CH values were 4.14%(o.d.) and had increased to 5.29% by day 51. Although Lowe et al. (1993) reported a larger overall increase in CH, the final Q, values found here 60 agree with their findings. The increase in CH from day 0 to day 7 was surprising because one would expect formation of humic acid to occur largely in the latter part of composting. There could possibly have been contamination of non-humic substances such as lignin into the humic acid fraction which could explain an initial enrichment followed by no change. The early increase in CH may also have been a result of negative enrichment because of the preferential loss of carbonaceous material through oxidation. The absence of change from day 7 through day 51 suggests steady state conditions for CH during the post-thermophilic phases of composting. Non-humic substances like lignin may have been enriching the humic acid carbon pool while oxidation and condensation of HA carbonaceous structures was occurring. It is possible that non-humic lignin compounds became microbially transformed into polyphenolic humic structures, while more easily oxidizable external aliphatic chains and polysaccharides were oxidized to C02 and removed. This suggests that structures represented by CH may not have changed much in composition during the post-thermophilic phases of composting, but may have become more humic and less lignaceous in character. CH/CF values increased significantly from day 0 to day 23 of composting (Figure 3.22). 61 Figure 3.22: Change in The Ratio of Humic Acid Carbon (CH) to Fulvic Acid Carbon (CF) 'CH/CF' Over Time, in a Hog Manure Solids Compost - Experiment 1 u O . S O SB/ * - m •3}^~-~ amy o 0.70 - t i l O.tSO I O 7 1* 2 1 2 8 3 3 4 3 •*» C o m p o i t i n ) T l m o ( d » y « ) *"V»li»«»» d B t B r f f l t n a d o n m.n o v a n - d r l e d b a s f i Day 0 values for CH/CF averaged 0.79 and increased to 1.23 by day 51. The magnitude and trend agree with the findings of Lowe et al. (1993). An increase in CH/CF has also been reported by Inbar et al. (1990). Table 3.1 summarizes changes in humic acid and fulvic acid carbon over time for experiment 1. 62 Table 3.1: Summary of Changes in Humic Acid Carbon (C„) and Futvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 1 (Buckets 1-6) Day 0 to 7 Day 7 to 23 Day 23 to 51 CH at bnc nc cF cint int int C„/CK T t i nc upvalues increased significantly b=no significant change occurred c=significant interactions occurred between time and aeration treatments The early increase in CH accounts partly for the increase in CyCp (Table 3.1). Changes in CF were difficult to draw conclusions about due to interactions between aeration and time treatments. Increases in CH/CF from day 7 to day 14 however, suggest that CF decreased during this time while CH remained unchanged. Ce did not change significantly during experiment 1 (data not shown). Average values ranged from 21.4%(o.d.) at day 0 to 22.2% by day 51. Steady state conditions for Ce contradict findings of Garcia et al. (1990), where the Ce of various compost mixes decreased over time. 3.1.4b Nitrogen Pools: Aeration treatments "A/ and "A2' did not significandy affect nitrogen pools. They either increased over time at some point, or remained unchanged. NT increased from day 0 through day 51, probably reflecting the preferential oxidation of carbonaceous material coupled with the retention of nitrogen in the solid residue (Figure 3.23). 63 Figure 3.23: Change in Total Nitrogen (NT) Over Time, in a Hog Manure Solids Compost - Experiment 1 s§ m ~-sB a "¥" l . O O —i : ; ; . 1 ; ; ; ; 1 , , . ; ; . J [ O 7 1 4 2 1 2 6 3 5 - 4 2 49 5<S ! C o m p o s t i l i K T i m e C d a y i ) j 32.-4.7 l • v « t u # » d » t » r m i A « d o n a n D v * a - d t t * d ba» ia i i Day 0-NT values averaged 1.19%(o.d.) and increased to 2.29% by day 51. This increase was more drastic than that observed by Yoshida et al. (1979) in various composts differing widely in carbon contents. NH increased significantly during experiment 1 (Figures 3.24). 64 Figure 3.24: Change in Humic Acid Nitrogen (NH) Over Time in a Hog Manure Solids Compost - Experiment 1 s§ 0 . 4 0 —f-0 . 3 0 •>!•-'-day 2.3 _ 33 -'"i 2) i ^ - ' j W* V M 1 U B M d e t i 1 14 2 1 2 a 3 5 4 2 4 » C o m p c m i n g T i m e C<3*y«> » i a « d o n a a o v e n - d r i e d b»*isi» NH remained unchanged until day 7 and then increased through day 51. This suggests that incorporation of nitrogen into humic acid, in the form of amino structures, occurred in the post-thermophilic stages of composting. Day 0 values for NH averaged 0.297%(o.d.) and increased to 0.481% by day 51. NF, Nca and NA did not change significantly during composting (data not shown). Average values for NF ranged from 0.352% (o.d.) at day 0 to 0.384% by day 51. During the same period, average Nca ranged from 0.366% (o.d.) to 0.389%. Measurements for NA were negligible, averaging 0.002% throughout. Since NA was determined by the difference between NF and Na , its negligible and sometimes negative values could be a reflection of contamination of nitrogen into the Nca fraction from degrading PVP. The likelihood of this occurring was minimized by avoiding the use of aging PVP. For this experiment, PVP was used for no longer than six weeks after it was acid washed and 65 rinsed with distilled H20. Negligible amounts of NA may suggest that development and incorporation of nitrogen into phenolic-rich FAA structures was minimal. Table 3.2 summarizes changes found in carbon and nitrogen pools over time for experiment 1. Table 3.2: Changing Carbon and Nitrogen Pools in a Hog Manure Solids Compost Experiment 1, Buckets 1 to 6 \^-r NT C/NT CH N„ C/NH cF NF C/NF c Nca C/Nca cA NA cc Composting Time (days) 0 t o 7 *nc CT dint T nc int int nc i nc nc i int nc nc 7 to 23 bl t i int nc T int int nc nc ! nc i 4-int nc nc 23 to 51 nc i int nc i int int nc nc nc nc nc int nc nc a=no significant ciwnge occurred b=values decreased significantly c=values increased significantly d=significant interaction occurred between time and aeration treatments 66 3.1.4c Carbon to Nitrogen Ratios (C/N): C/Np decreased from day 0 to day 7, and then remained unchanged through day 51 (Figure 3.25). Figure 3.25: Change in the Ratio of Carbon to Nitrogen in the Fulvic Acid (C/NF) Over Time in a Hog Manure Solids Compost - Experiment 1 l O . O X9.0 i a . o 17 . o us.o I S O 1 4 . 0 1 3 . 0 1 2 . O l l . O l O . O » .o - V a l u e s U _ z i | J -j ! 1 . . . . ..... i Am.y o iv._. ..... .... . ! t 1 ! ^ --N. IS -• **'' r csr--.. tm 1 •3 9~ ~ d « y 23 J I j fci ; i33 1 r 1 4 2 1 2 8 3 3 4 2 -49 S < C o m p o H t i n g T i m e C*3*y"B>> t a r t n l n s d o n «.» o v » n - d r i « t c i t » H » i s n - 2 7 C/Np decreased from an average value of 15.1 at day 0, to 11.2 by day 51. As NF remained unchanged throughout, an early decrease in CF appears to have controlled the C/NF ratio (Table 3.2). A two-way analysis of variance showed that aeration had a significant effect on C/Nca. As with CT, a review of the data past day 0, however, did not show that two separate populations existed as a result of imposed aeration regimes (Figure 3.26). 67 Figure 3.26: Effect of Aeration Treatments (Al and Aj) On The Ratio of Carbon to Nitrogen in the Carbohydrate-Rich Fraction of the Fulvic Acid (C/Nca) Over Time, in a Hog Manure Solids Compost - Experiment 1 + -:*-' * t •• -^ j '•-.. ] J j "I j A . 1 aK 3 } ^ 3D *T W ~~-——^ A.3. 1 i ! I ! 1 ~~ B i •m \ i * C / N ( o « ) A l ss C/N<o*5 A.2 * 0 « l e r m i n t t i o n 7 1 4 2 1 2 f 3 5 H2 -4» C o m p o s t i n g T i m e ( d n y i ) r e p o r t e d o n . o v t t n - c l r i a d h a a i i n - 2 7 As with Cr, CF and CA, a large spread in the C/Nca day 0 values occurred. The possible reasons for this variability have been discussed earlier. C/Nca values decreased significandy from day 0 to day 23, average values ranging from 11.8 at day 0, to 9.2 by day 51. Interactions occurred between time and aeration treatments for C/NH and C/NT, making their trends difficult to comment on (Figures 3.27 to 3.28). 68 Figure 3.27: Effect of Interaction Between Time and Aeration Treatments (Aj and Aj) On Carbon/Nitrogen in Humic Acid (C/NH) in a Hog Manure Solids Compost - Experiment 1 2 ! S3 ! 1 A 2 ! j / i \ 1 A .1 -^  x^ " + '• 3 ) ©'-,. '•--.. —._. i 1 j 3! i 1 i 1 C / N C H } A l lO.O O 7 l*t 2 1 2 B 3 3 4 a 4S» 5 6 C o m p o s t i n g T i m e ( d a y s ) " D e l B r m l i i i t l D i i i t « p o t t o d o n o v o Q - d T l B d b a s i s Figure 3.28: Effect of Interaction Between Time and Aeration Treatments (A, and A^ On Carbon/Nitrogen (C/NT) in a Hog Manure Solids Compost - Experiment 1 - C / N ( T ) A l co C/NCT) A 2 u • D e t o r t n i a a l l c ~ 1 ~" ' ~ " — ; — - — j — T -—-r r — - t — — 7 14 2 1 2 S 3 3 4 2 4-3 C a m p o A t i n g T i m e ( d t y s ) raade o a o v o n - d r i o d h n i i 69 Average C/NH values ranged from 13.9 at day 0 to 11.0 by day 51. C/NT average values ranged from 37.3 to 18.9 during the same period. Similar C/NT ranges have been reported by N'Dayegamiye et al. (1991) in a cattle manure solids and wood shavings compost. 3.1.4.d Total Moisture (MoisT): Moist remained unchanged until day 23 and then it increased through day 51 (Figure 3.29). Figure 3.29: Change in Total Moisture (Mois^ Over Time, in a Hog Manure Solids Compost - Experiment 1 ss o 2 53 Q3 E3 H a h m -^r J i -j LS3 a » y o 3 J S I 3 3 m : ^ 1 HI - " S I " ^ ' 1 - 5 -=23 33 S3 -31 d . y —^—~~ 2 J S 1 —"" *a G 3 r l l2 ] r E l 1 ! ! 1 * V i l u o § d e t e r m i n e d 1 4 2 1 2 8 3 3 4 2 -*5» o i l e n w o t - m « « » t > « « i s tt-55 At day 0, MoisT values averaged 71.57%(W.M.) and increased to 72.57% by day 51. In spite of the fact that the evolution of steam was observed during mixing from day 7 to day 21 of composting, this removal of water from the system as a result of metabolic heat did not decrease the moisture content of the compost. This suggests that negative enrichment of water occurred due to 70 preferential oxidation of carbonaceous material. As microbial heat production and evaporation had declined after day 23, the observed late increase in moisture suggests that in addition to negative enrichment of water, metabolic contributions of water were significantly increasing the moisture content of the composting environment. The initial steady-state conditions of Moisx agree with that found by Lo et al. (1993), in the composting of hog manure solids and sawdust for 14 days by the aerated static pile method. 71 3.2 Experiment 2: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Hog Manure Solids Compost Under Aeration Treatments A2' and A3' 3.2.1 Temperature In graphic representations of the temperature profiles of each bucket (Figures 3.30-3.36), it can be noted that all bucket temperatures rose quickly and reached or exceeded 55° C within thirty-six hours. Thereafter, the temperatures decreased, notably by day 14. It is evident that the application of the more frequent aeration treatment 'A3' occurred throughout the thermophilic phase of composting, which lasted from day 2 or 3 until day 10 to 13. Figure 3.30: Bucket Showing Thermocouple Positions - Experiments 1 and 2 72 Figure 3.31: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 2, Bucket 1 6o.o —r^a .ay —OB- B l - T l —<*- B 1 T 2 — — B 1 - T 3 — a - B 1 - T 4 —J— B l - T J A . z n t > l e t i l A l l 1 0 . 0 , , - ^ - | . , , ; [ _ _ _ , . O 7 t-4 2 1 2fl 3 5 -*2 4 9 S « C o m p o s t i n g H m e ( d a y a ) w f v £ * « s a r * m « i L t a r « c o ( d * d * t 2 3 3 0 b o a r s Figure 3.32: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 2, Bucket 2 - B 2 - T 1 - B 2 - T 2 B 2 - T 3 - B 2 - T * - B 2 - T 3 - A m b i e n t Jt.lt I O . O 0 7 1* 21 28 35 42 * » 56 C o t n p o i t l n g T i m e ( d » y s ) ' M a a a n t t m e n l t r n a o r d e d at 2 J 3 0 h o u n 73 Figure 3.33: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A2' - Experiment 2, Bucket 3 u £ : —so— B 3 - T 1 •—»— B 3 - T 2 — B 3 - T 3 I —a— B3-T4-; —o— B 3 - T 5 [ - A m b i e n t Air m i x *o.o - » % — « -lO.O —r-O 7 14 2 1 2» 35 4 3 4 9 C o m p o s t i n g T in i f l (d*.y»3 l « k t u r * m * A t a r « c o r d » d at 2 3 3 0 h o u r s Figure 3.34: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A3' - Experiment 2, Bucket 4 - B 4 - T I - B - 4 - T 2 B 4 . - T 3 - B 4 - T 4 - B 4 - T 5 - A m b i e n t A i r I lO.O —{-' M M i v r a m a n t a r 14 21 2« 35 4 1 4 9 C a m p o s t i a g T t n t e Cdny*) • a o r d a d «t 2 3 3 0 h o u r ! 74 Figure 3.35: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment A3' - Experiment 2, Bucket 5 CO. en —®- B 5 - T 1 1 —«— B 5 - T 2 ! — ^ - B S - T 3 i —O— B 5 - T 4 1 —3— B 5 - T 5 J A a b l v a i i 1 1 1 Air J m f - m i x O 7 14 2 1 Za 3 5 4 2 *9 SB C o m p O B t J n g T i m e C d * y « > * M * a s v r » t z x * n t * r * c o r 4 * d »T 2 9 3 0 h o u r s Figure 3.36: Temperature Profile in a Hog Manure Solids Compost Under Aeration Treatment 'A3' - Experiment 2, Bucket 6 B 6 - T 1 B S - T 2 H « - T 3 B 6 - T 4 B 6 - T 5 A m b i e n t A i r O 7 * M e a a u t »tn B a n 14 2 1 2 8 3 5 4 2 4 3 5 6 C o m p o s t i n g T i m e ( d a y s ) B o a r d e d i t 2 3 3 0 h o u r i 75 Buckets 1 to 3 had aeration treatment 'A2' applied to them (Figures 3.31-3.33). Their upper thermocouple temperatures rose quickly from mixing at day 0 and reached the thermophilic range by about day 2 to 3 before declining. For the most part, compost temperatures remained at or above 55° C until day 7, increasing briefly following the aeration at day 7. By day 14, temperatures had declined below the thermophilic range and increased for a brief period back up to 55° C, after the day 14-aeration treatment. Temperatures declined smoothly after the day 21 aeration treatment, reaching ambient conditions at about day 39 to 41. This smooth decline suggests that some factor other than oxygen was limiting metabolic activity and the subsequent heat production in the A2-compost, at this stage. Buckets 4 to 6 had the more frequent aeration treatment "A/ applied to them (Figures 3.34-3.36). Upper thermocouple temperatures rose quickly and reached the thermophilic range by about day 3 to 5, and remained at or above 55° C until day 12. Temperatures declined smoothly after the day 12 aeration, reaching ambient conditions by about day 32 to 34. Following aeration treatments, the small sharp increases in temperature observed in the A2-compost were not notable in the A3-compost. There appears to be no difference between the lengths of the thermophilic phases for aeration treatments in experiment 2. Overall, thermophilic temperatures were maintained for 9 to 10 days, for both 'A2'- and A3-compost. The transition cooling phase of the A3-compost was shorter than that observed for the A2-compost, ending about 7 days earlier. From Figures 3.31 to 3.36 it can be seen that initially, as with experiment 1, the lower portions of the material (T4 and T5) were much cooler than the upper portions early in the process. After about day 14, however, the uppermost "TT and 'T2' positions became the lowest temperatures relative to the other thermocouple positions. As with experiment 1, this was probably caused by a 76 considerable decrease in volume which occurred over time, gradually exposing the upper thermocouples to cooler ambient temperatures. Figures 3.37 to 3.41 describe the temporal variation in thermocouple temperatures between buckets 1 to 6. Figure 3.37: Temporal Variation in Temperature As Measured By Thermocouple 1 (Uppermost) - Experiment 2, Buckets 1-6 o 3 4 D . O S 3 0 . 0 o "T -/ \ _ y ——f— Nc-t ? 1 X ! -"N^X ^"*—-~ " l_^ J L1J O 7 14- 2 1 2 » 3 5 4 3 - * 0 C o m p o s t i n g T i m e ( d a y s ) * K-S o a a u r a IT> 0 n t m r o o o i - d w d « £ 2 3 3 0 h o u r m 77 Figure 3.38: Temporal Variation in Temperature As Measured By Thermocouple 2 Experiment 2, Buckets 1-6 14 2 1 2 S 3 5 4 3 4 9 C o m p o s t i n g T i r c o C<JBy») k M e a t u r » f f l 0 f t t s r n c o i d n d At 2 3 3 0 tic Figure 3.39: Temporal Variation in Temperature As Measured By Thermocouple 3 (Middle) Experiment 2, Buckets 1-6 u a o 78 Figure 3.40: Temporal Variation in Temperature As Measured By Thermocouple 4 Experiment 2, Buckets 1-6 u ! 43 3 ] «1 ! <u & Tern 3 0 . 0 2 0 . 0 O l O 2 0 3 0 - » 0 S O C o m p o s t i n f T i m e ( d a y s ) • u r a m * n t a r « u a r d « t i J a t 2 3 3 0 h o u m Figure 3.41: Temporal Variation in Temperature As Measured By Thermocouple 5 (Bottom) Experiment 2, Buckets 1-6 1 eraturc (°C) Temp ! S O - t o 3 0 2 0 o o o o ! 1 L - r 'fC ~T~ 1 _ Y - • - \ "^•s. . T --- • ^ 5 O 7 1 4 2 1 2 8 3 3 -*-2 4 » C o m p a a t t l i i T l m o ( d « y » ) * 9 * C o « s u r o m o i x t * e « « o r d o i i « t 2 3 3 0 h o u r s 79 The error bars in Figures 3.37 to 3.41 indicate the sample standard deviation of temperatures at a given thermocouple position between buckets over time. As with experiment 1, it can be seen that the spread of the measurements was not homogeneous throughout experiment 2. The error tended to narrow as the temperature approached ambient conditions, and increased at temperatures above ambient conditions. A review of variability in temperatures within aeration treatments (A2 and A3) showed similar increases in variability at temperatures above ambient conditions (not shown). As for experiment 1, this may be attributed to the variable influence of biological activity. Compared with experiment 1, the spread of experiment 2 temperature measurements appears to be narrower throughout. This could be a result of improved substrate homogeneity as a result of more thorough and frequent mixing. 3.2.2 Sampling Sampling occurred on days 0, 7, 14, 21, 28, 42 and 55. It was more intensive for experiment 2 because at least one aeration treatment (A3) was impacting the important thermophilic metabolic phase. It was felt that more information could be obtained about the effect of aeration on the utilization of nutrient pools if sampling was more intensive than in experiment 1. 3.2.3 Changes in Chemical Properties Twenty-one chemical parameters which are representative of important nutrient pools for metabolizing organisms were measured (directly and indirectly) throughout experiment 2. A tabulated form of the two-way analysis of variance results on the effects of time and aeration is located in Appendix B. Of the twenty-one parameters studied, time had a significant effect on thirteen at a 5% 80 level of significance. These were Q, NT, C/NT, CH, NH, C/NH) CF, NF) Cca, Nca, C/NM, CA, Ce and CjCp. (Appendix B). In addition, six of these parameters also showed apparent significant effects of aeration treatments "A2' and 'A3' at a 5% level of significance. These were Cj., NT, NH, CF, C ,^, and C/Nca. A review of the data for these six parameters showed two separate populations of values as a result of the imposed aeration regimes for all except Cj. Relatively larger variability in day 0 data for Cp may have caused the appearance of two separate populations of values at this point. As for experiment 1, a review of the Q- data past day 0 shows a decrease in variability, and also does not show further segregation of populations as a result of imposed aeration regimes. NA did not change significantly in experiment 2. Significant interactions occurred between time and aeration treatments for Moi^, WSC, CH, C/NF) (yCp, LPSS and TPSS. The results are presented in a series of figures plotted against time. For parameters in which the analysis of variance showed significant effects of aeration treatments or interactions, the data was graphed as separate "A2' and "A3' populations. MoisT values are reported on a per cent wet mass basis (W.M.). The rest are reported on a per cent oven-dried mass basis (o.d.). Using Cricketgraph (v. 1.3.1), best-fit lines were interpolated through the data. A tabulated form of all data is located in Appendix D, Part 2. 3.2.3a Carbon and Polysaccharide Pools: CT decreased with time and increased frequency of aeration (Figure 3.42). 81 Figure 3.42: Effect of Aeration Treatments A2' and A3' Over Time, on Total Carbon (CT) in a Hog Manure Solids Compost 1 - Experiment 2 (_) 4 * . 0 j *• 1 2S j •^j _ v ^ - -A--2 r») * \ \ « ' \LCS 2 S I ---"" _— & :*5i ^ t— -— ~m -• 2S"'"'" 5S _--—--. 1 A . 3 j ^ i "~nST' """ '* "" ^g^-""' *• | ; ""1 '* ! J a \ i HE CCT) A 2 I » C ( T ) A 3 I •*2.0 O 7 1* 2 1 2B 3 5 4 3 4 9 5 6 C o m p o i t i n g T l m o C<i«y«) ( A 2 ) n - 1 9 ( A 3 ) n - 2 5 As with experiment 1, a two-way analysis of variance showed significant effects of aeration treatments for CT. Similarly, a review of the data does not show that two separate Q- populations existed past day 0 as a result of imposed aeration regimes. A large spread in these day 0 values occurred and was possibly due to an initial incomplete mixing of manure and sawdust. With mixing, Figure 3.42 shows a narrowing in the spread in values by day 7. Cr values decreased significandy from day 0 to day 7, and from day 28 to day 42. At day 0, values averaged 45.5%(o.d) and decreased to 44.5% by day 51. Compared with experiment 1, CT values were slightly higher and did not decrease as steadily in experiment 2. This was probably the result of the narrower ratio of manure to sawdust used in experiment 2. CF and Cca decreased with both time and increased aeration frequency (Figures 3.43-3.44). 82 Figure 3.43: Effect of Aeration Treatments A2' and A3' on Carbohydrate-Rich Fulvic Acid Carbon (CCJ Over Time, in a Hog Manure Solids Compost - Experiment 2 - k - - \ - - . j » -2 . 0 0 J&--f^-~r~ -m ao CC<J«} A 2 • C ( c » ) A 3 ¥ - ! 1 i ! 1 ] — - r — ! — - ! 1 r — r - — r p -o -7 i» a i as 35 *a *s» s« C o m p o a t l n g T i m e C<3*ir*) ' V m l u i i d e t t r m i i e d am u o r a a - d t U d b a s i s ( A 2 > n - 2 1 ( A 3 ) n - 2 3 Figure 3.44: Effect of Aeration Treatments A2' and A3' on Fulvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 2 C ( F ) A 2 i C ( F ) A 3 I * V « t u » * d e l -.SH-OO - r — r -o o r n i i «*« 14 2 1 2 8 3 5 4 2 C o m p o s t i n g X i m e ( t l s y s ) 49 sa ( A 2 ) n - 2 1 ( A 3 ) a - 2 3 83 At day 0, Cca averaged 3.81%(o.d.), and by day 55, Cca values under aeration regime 'A,' decreased to 3.32%. During this time, values under aeration regime " A3' decreased more dramatically to 3.05%. Aj-C^ remained unchanged until day 7, decreased significandy to day 14 and then levelled off. This follows similar experiment 1 trends in which carbohydrate-rich carbon pools were utilized quickly after day 7 by an established population of organisms. A rCc a decreased significandy from day 0 to day 7 and from day 28 to day 42 (Figure 3.43). This decreasing trend over time agrees with that found in experiment 1. For CF, day 0-values averaged 5.12%(o.d.). A2-CF decreased significandy from day 0 to day 14 and levelled off to finish at 3.76% by day 55 (Figure 3.44). Average A3-CF values decreased more dramatically to 3.47%. Under aeration treatment "A/, CF decreased significandy not only from day 0 to day 14, but also from day 28 to day 42. Experiment 2-Cca and -CF were similar in magnitude to those found in experiment 1. The decreasing trends found in experiment 2-Cca and -CF values, however, contradict the slightly increasing trend found in static pile composting by Lowe et al. (1993). In this experiment, aeration through mixing may have provided more optimal conditions for organisms to degrade fulvic acid carbon compounds. Mixing may have inoculated relatively nondegraded microenvironments in the substrate with organisms capable of degrading CF and Cca, promoting utilization. CA decreased over time (Figure 3.45). 84 Figure 3.45: Change in Phenolic-Rich Fulvic Acid Carbon (CA) Over Time, in a Hog Manure Solids Compost - Experiment 2 < i . e o 1 . 3 0 l . - t O l . a o 1 . 2 0 1 . 1 0 l . O O o.&o o . s o 0 . 7 0 0 . 6 0 O . S O O. -40 0 . 3 0 " V a t u w a d a » t * r x n i n a » d -f h i. i <, "E B 3 *? i * « y O ffi "\ J - ^ C —- * " T 7 S \ J ~ -* «J»y 3- 1 _J J " d « \ til ^ . ^ '* a ^ ss d " y :t) ^*S~~^~ 2 • 4-my ' • — --4-2 1 -_SB aa i d»J" a » j ~""*--- -* ' ---433 ! .. . ffi.._ J L20 ! 1 4 2 1 2 9 3 3 4 2 4 9 C o m p o n t i n ^ T i m e ( d A y s ) i • *» o v » n - t i r l « d b a i i n At day 0, CA averaged 1.31%(o.d.) and decreased to 0.44% by day 55. Values remained unchanged until day 7, decreased to day 14 and levelled off until day 42. CA then decreased through day 55. The magnitudes and decreasing trend found in CA are similar to that reported by Lowe et al. (1993). The absence of a decrease of CA from day 14 to day 42, regardless of aeration treatment, may be a result of negative enrichment due to losses from polysaccharide pools. CJCp remained unchanged until day 7, decreased to day 14 and then levelled off until day 42. It then decreased through day 55 (Figure 3.46). 85 Figure 3.46: Change in the Ratio of Phenolic-Rich Fulvic Acid Carbon (CA) to Fulvic Acid Carbon (CF), 'CJC¥' Over Time, in a Hog Manure Solids Compost - Experiment 2 j ! o i 1 . 1 0 * OS m ,—m .*. • * « r •7 ! ; i o.ao —f-f * » y 1+ 'sa_ as * V « l u c » d e t o t m i a e d o n • a . _ , , , P , , . . . , j _ 1+ 2 1 2 8 3 5 42 •*» C o m p o M t i n g T i m e : ( d a y s ) o v e o - d r l e d b » * i v Average C^Cp values declined from 0.26 at day 0 to 0.12 by day 55. Values found here are similar in magnitude to those found in experiment 1. Table 3.3 summarizes changes in phenolic-rich fulvic acid carbon (CA) and fulvic acid carbon (CF) found in experiment 2. 86 Table 3.3: Summary of Changes in Phenolic-Rich Futvic Acid Carbon (Q) and Fidvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 2 Day 0 to 7 Day 7 to 14 Day 14 to 21 Day 21 to 28 Day 28 to 42 Day 42 to 55 c A *nc bi nc nc nc " A2-CF 1 -nc nc nc nc A3-CF i 1 nc nc 1 nc cjc¥ nc I nc nc nc i a=no significant cliange occurred b=values decreased significantly Since both CA and CF declined during early and late periods, the accompanying decrease in CyCp suggests that utilization of CA was a dominant factor (Table 3.3.). Interactions between time and aeration treatments occurred for LPSS, TPSS, WSC, CH and CyCp making their trends difficult to interpret (data not shown). Average LPSS values ranged from 46.0% (glue, equiv.) to 30.9% from day 0 to day 55. During the same period, average TPSS values ranged from 63.7% (glue, equiv.) to 47.4%. These values were similar to those found in experiment 1. Average WSC values ranged from 1.01%(o.d.) at day 0 to 0.54% by day 55. Utilization of LPSS and TPSS may have contributed by-products of metabolism to the water-soluble phase measured as WSC. Starting WSC values were substantially lower than those found in experiment 1. This difference may be a result of the lower ratio of manure to sawdust in the starting mix, or it may be a result of using increased centrifugation speeds in obtaining the water extract (Chapter 2, section 2.2. lb). The method for preparation of the water extract used in experiment 2 may have been a more efficient one for excluding colloidal carbon from the analysis of WSC, resulting in lower WSC values. 87 Average CH values range from 3.26%(o.d.) at day 0 to 4.75% by day 55. Average CyCp values ranged from 0.64 at day 0 to 1.32 by day 55. Table 3.4 summarizes changes found in humic acid carbon and fulvic acid carbon in experiment 2. Table 3.4: Summary of Changes in Humic Acid Carbon (CH) and Fulvic Acid Carbon (CF) Over Time, in a Hog Manure Solids Compost - Experiment 2 Day 0 to 7 Day 7 to 14 Day 14 to 21 Day 21 to 28 Day 28 to 42 Day 42 to 55 CH •int int int int int int A r C F bi 1 cnc nc nc nc A3-CF I i nc nc I nc t-'H/^'F int int int int int int a=significant interaction occurred between time and aeration treatments b=values decreased significantly c=no significant change occurred Ce fluctuated over time (Figure 3.47). 88 Figure 3.47: Change in Total Extractable Carbon (Cc) Over Time, in a Hog Manure Solids Compost - Experiment 2 6 32 \ S I * v » l « « t d * t * r a [ l l-» 2 1 2 8 3 3 -»2 -*9 C o m p o s t i n g X i m e C<J»y""} Mn o r « n - d f l * d b * a i a — [ r 5 6 At day 0, Ce values averaged 18.0%(o.d.)- Figure 3.47 shows that values increased and decreased over time, finishing at an average value of 18.7% by day 55. 3.2.3b Nitrogen Pools: Unlike experiment 1 where Nca and NF did not change significandy over time, Nca and NF fluctuated throughout experiment 2. 89 Figure 3.48: Change in Carbohydrate-Rich Fulvic Acid Nitrogen (Nca) Over Time, in a Hog Manure Solids Compost - Experiment 2 zf IS) i 1^ <i»rr T Am.y 1 4 d » T 3 3 ^a ^ V B I U B I d e t e r m I n o d o n 14 2 1 5 * 3 5 4 2 4ft C o m p o s t i n g T i r n « <cJ*y»> o v » n - d r t » d fc»*»i» Average Nca values started at 0.303%(o.d.) and peaked at 0.366% by day 7, before declining until day 14 and then levelling off (Figure 3.48). At day 55, average Nca values were 0.310%(o.d.). Like Nca, NF increased significandy from 0.309%(o.d.) at day 0 to 0.388% by day 7 (data not shown). Values then declined until day 14, and then levelled off through day 55 to finish at 0.311%. These early increases suggest that incorporation of nitrogen into fulvic acid, in the form of amino structures, was occurring as the microbial population was initially expanding and increasing its level of metabolic activity. For both Nca and NF, starting values were relatively low compared with experiment 1. This may be a reflection of the narrower manure to sawdust ratio in the starting mix of experiment 2. In spite of these initial differences, final concentrations were more similar to those found in experiment 1. NT and N„ increased with both time and increased aeration frequency (Figures 3.49-3.50). 90 Figure 3.49: Effect of Aeration Treatments A2' and A3' Over Time, on Total Nitrogen (NT) in a Hog Manure Solids Compost - Experiment 2 2 . 0 0 f-2B N ( T ) A . 2 •> KTCT> A . 3 "=W o.oo O 7 1 4 2 1 2 8 3 5 4 2 49 C o m p o i t l n g T i m e ( d * y a > ' V i l u a i d * t « r m l n » d o n « n o v e n - d r l s d B a a l * CA.23 n - 2 4 ( A 3 ) n - 2 4 Figure 3.50: Effect of Aeration Treatments 'A,' and A3' Over Time, on Humic Acid Nitrogen (NH) in a Hog Manure Solids Compost - Experiment 2 fa M < H ) A 2 a a> 6 2f —ET m ~w ' * V H . J U » M < J > l > r r o ( n « d o n 1 4 3 1 3 * 3 5 4 3 4 9 3 6 C o m p o a i t l n g T i m e idmymy o v a n - d r l a d h > a « t a ( A 2 ) t i - 2 1 ( A 3 ) n - 2 3 91 At day 0, NT values averaged 1.12%(o.d.). By day 55, A2-NT increased to 2.03% and A3-NT increased more dramatically to 2.23% (Figure 3.49). As with experiment 1, the increase in NT can probably be attributed to preferential loss of carbonaceous material coupled with nitrogen retention in the accumulating solid residue. This increase was extended over a longer period under the more frequent aeration treatment "A3', where day 0-NT increased to day 28 and then levelled off. Under aeration treatment "A2', NT remained unchanged until day 14 and increased until day 21. Regardless of aeration treatments, NH did not change significantly until day 7. For both aeration treatments (A2 and A3) it increased until day 21, and then levelled off through day 55 (Figure 3.50). This suggests that the enrichment of humic acid with nitrogen was occurring during the thermophilic and transition cooling stages of composting. At day 0, NH values averaged 0.297%(o.d.). By day 55, A2-NH increased to 0.370%, and A3-NH increased more dramatically to 0.413%. Enhanced activity of microorganisms in the A3-compost due to increased oxygen may have increased NH through incorporation of nitrogen into humic acid structures. The trends over time and magnitudes of NT and NH values were similar to those of experiment 1. As with experiment 1, NA-values were negligible and did not change significantiy during composting. At day 0, values averaged 0.012%(o.d.) and by day 55 values averaged 0.003% (data not shown). Table 3.5 summarizes carbon and nitrogen pool changes which occurred over time in experiment 2. 92 Table 3.5: Changing Carbon and Nitrogen Pools in a Hog Manure Solids Compost Experiment 2 * *r NT C / N T CH NH C/N H cF NF C / N F c Nca C / N c a cA NA ce Composting Time (days) 0 t o 7 H A 2 -nc A3-T i d in t A 2 - n c A 3 - n c -+• A 2 - i A 3 - i I int A 2 -nc A r i t A 2 -nc A34 nc nc 7 to 14 bnc A 2 -nc A3-T i int A2-T A3-T nc A24 A3-I | int A24 A 3 -nc 1 A 2 -nc A 3 -nc i nc 1 14 to 21 nc A2-CT A34 l int A , - : A 3 - : nc A 2 -nc A 3 -nc nc int A 2 -nc yV3-nc nc A 2 -nc nc nc nc -21 to 28 nc A2-nc A34 nc int A24" A34 nc A2-nc A3-nc nc int A2-nc A3-nc nc A2-nc nc nc nc nc 28 to 42 i A2-nc A3-nc nc int A2-nc A3-nc nc A2-nc A34 nc int A2-nc A34 nc A2-nc nc nc nc nc 42 to 55 nc A2-nc A3-nc nc int A2-nc A3-nc nc A2-nc A3-nc nc int A2-nc A3-nc nc A2-nc nc { nc -a=values decreased significantly b=no significant change occurred c=values increased significantly d=significant interaction occurred between aeration and time treatments 93 Compared with experiment 1, the increases in NT and NH were more restricted to the early and middle time periods of experiment 2 (Table 3.5). These two nitrogen pools do not appear to have been affected during maturation. Table 3.5 shows that under aeration treatment 'A3', trends in CA and CF were similar to that of CA and Cj. A lag in utilization, lasting from about week 1 or 2 to day 28 or 42, was a common factor with these parameters. 3.2.3c Carbon to Nitrogen Ratios (C/N): The analysis of variance and mean contrasts showed that C/Nca values under aeration treatment ' A2' did not change significandy over time. C/Nca values under aeration treatment " A3' did, however, decrease from day 0 to day 7, levelling off through day 55 (Figure 3.51). Figure 3.51: Effect of Aeration Treatments A2' and 'A3' Over Time, in the Ratio of Carbon to Nitrogen in Carbohydrate-Rich Fulvic Acid (C/NCJ in a Hog Manure Solids Compost Experiment 2 00 C V N < o « > A . 2 • C / N ( . « ) A. a _ 1 — , — , — I — , — , — , — , — , — , — r a 7 1* xt 3 * 3S *i +* s « C u t n p c l i o g T i m s C<>«y»J * V « l u « « i l » t » r m t a e d am •&. a v M a . 4 r t B d b » t a ( A 3 ) n - 2 1 ( A 3 ) n - 2 7 94 Day 0 C/Nca values averaged 12.4 and finished at 10.6 by day 55, for aeration treatment * A2'. A3-C/Nca values averaged 10.0 by day 55. The decreasing trend over time and magnitudes of experiment 2-C/Nca were similar to that found in experiment 1. The early decrease in A3-C/Nca values illustrate the influence of the early decrease in Cca and increase in Nra on C/Nca (Table 3.5). C/NT decreased from day 0 to day 21 of composting (Figure 3.52). Figure 3.52: Change in the Ratio of Total Carbon to Total Nitrogen (C/NT) Over Time, in a Hog Manure Solids Compost Experiment 2 CJ 3 0 . 0 s i a "-..SB A»y +-Z 14 2 1 2 8 3 5 4 2 4 9 J « C o m p o « t l n B T i m e ( d » y » ) n mwx o v e n - d r !•><! b a i l s * " V « l t i » B d o t n r m l n e d «> Day 0 C/NT values averaged 42.8 and decreased to 20.0 by day 55. The steady decrease in C/NT was likely controlled mainly by NT which increased from day 0 to day 21 (Table 3.5). Although there is evidence of the disappearance of several other carbon pools such as CF, Cca, and CA, from day 0 to day 21, this was not reflected in the CT trend. This suggests that a dominant fraction of the total 95 carbon pool was very resistant to degradation. Woody material such as sawdust contains cellulose and lignin compounds which are degraded very slowly. Compared with experiment 1, slightly higher starting C/NT values were found in experiment 2. This was likely a result of the narrower manure to sawdust ratio used in experiment 2. In spite of these initial differences, the final experiment 2-C/NT values were similar to those found in experiment 1. An interaction occurred between time and aeration treatments for C/NF making trends difficult to interpret. Average C/NF values ranged from 17.0 at day 0 to 11.6 by day 55 (data not shown). The magnitude of these values was similar to that of experiment 1. C/NH increased from day 0 to day 7 (Figure 3.53). Figure 3.53: Change in the Ratio of Carbon to Nitrogen of Humic Acid (C/NH) Over Time, in a Hog Manure Solids Compost - Experiment 2 i ; „. ! z~ ; u i - V " . 1 5 . 0 1 . 4 . 0 1 3 . O 1 2 . 0 l l . O l O . O 9 . 0 s .o 7 . 0 i : 1 / «> ,' i; '^--. "1 ! -! _L_ s ; ! o U * A c i * t * r t » j -^ d a y •"^SS V d t y 1-4 d » y O 1 '• ; y i t CZam n a d . o n « n -r .TD*,-—Cral •• s . . g [§ S3 d . j - 2 1 d * r " lol 3 3 2 1 3t« 3 J S 4 2 4 0 p o f l t i n K T i m e < c 3 « . y » > o v « n - d r i « d b a i l s « ! 3 3 ' , | 5 6 i 1 1 1 | j j | i n— <4-8 j i 96 At day 0, C/NH values averaged 8.3 and increased to 12.4 by day 55. The increase in C/NH reflects a day 0 increase in CH while NH remained unchanged. After day 7, CH continued increasing along with NH until day 21. This resulted in C/NH values levelling off after day 7 (Table 3.5). Compared with experiment 1, starting C/NH values were substantially lower for experiment 2. Final values, however, were similar to those of experiment 1. Lower day 0-C/NH values resulted from substantially lower starting CH. This may reflect the narrower ratio of manure to sawdust used in the experiment 2 starting mix. 3.2.3.d Total Moisture (MoisT): An interaction occurred between time and aeration treatments for Moisx (Figure 3.54). Figure 3.54: The Effect of Interaction Between Aeration Treatments (A2 and A3) and Time on Total Moisture (MoisT) in a Hog Manure Solids Compost - Experiment 2 M o U ( T ) M o I « ( T ) A 2 I A 3 ! e? 05 7 3 . 0 0 - « S -o 2 -Ski '88 gj IT -70 .00 ~+-• V i l u . . d O f 1* 2 1 2.» 3 5 * 2 4 » C o m p o a t i n g T i r a o < d « y « > • t a r m l n x t o n w * t - m « K B 1>«MI» ( A 2 ) n - 6 3 ( A 3 ) n - < 5 2 97 Average MoisT values ranged from 72.54%(W.M.) at day 0 to 73.54% by day 55. In spite of the increase in bulking agent, Moisr values did not decrease below 70%(W.M.) and were very similar to values found in experiment 1. 98 3.3 Experiment 3: Changes in Carbon and Nitrogen Pools and Development of Alkali Extractable Organic Matter Over Time, in a Bench-Scale Hog Manure Solids Compost Under Controlled Temperature Regimes 3.3.1 Temperature Rgures 3.55 to 3.57 show the temperature profiles over time for waterbaths 1 to 3 and that of their associated 2 litre-buckets of composting hog manure solids. Figure 3.55: Temperature Profile Over Time of Hog Manure Solids Composted in a Bench-Scale High Temperature' Waterbath (WB1) - Experiment 3 r u s - B u c k e t 2 - H i . k . t 3 • W a t a r b a t h 1 20.0 —J r——i r— p o to 2 0 so *o C o m p o s t i n g T i m s ( d a y » ) apavataraa x»«a*««>d at 3SOO feoasa 99 Figure 3.56: Temperature Profile Over Time of Hog Manure Solids Composted in a Bench-Scale Compost Simulation' Waterbath (WB2) - Experiment 3 u ! 2 so.a C3 o 1 . . . ^ f e l , f '"\ f "1 ' 1 ' ' ' 1 ; I ' " 1 B u o k a t A • B n o k s t 3 - B u a k « t tf • W a t e r b a t l l 2 l O JO SO 4 0 C o m p o « t i o t T i m e Cd»y») * T « a B e - * * * l * * » « * « a A * a « « A* 13(H) • » « * Figure 3.57: Temperature Profile of Hog Manure Solids Composted in a Bench-Scale Low Temperature' Waterbath (WB3) - Experiment 3 o o O Urn 3 H. s & B v o k e t 1 -D««k«t S - B n i t k a t 9 • W » t . r b » t h 3 bT»ot|*«r««mx*B r*««x-4*«l «t 2 SO 9 h « « i 100 After sampling on day 12, it can be seen from Figures 3.55 to 3.57 that the bucket temperatures tended to remain below the temperatures of the waterbaths. This was particularly so for the buckets in the 'high temperature' waterbath (WB1). This probably occurred because on day 12 , approximately half of the compost was removed from each bucket for sampling, resulting in the bucket thermocouples being incompletely buried in the remaining compost. The lower bucket temperatures probably resulted from the influence of the cooler air temperatures surrounding the compost. 3.3.2 Sampling Sampling occurred on days 0, 12 and 46. Because of substantial loss in volumes which occurred as a result of both sampling and the composting process, it was not possible to sample more frequently. 3.3.3 Moisture After about day 24 of composting, it became necessary to add sterile water regularly to the compost in waterbaths 1 and 2. Based on observations from a short preliminary waterbath experiment, water was added in small increments until the surface particles of the compost were "just glistening". This state of "just glistening" particles was used as an indication that Moisx was approximately 70%. Table 3.6 shows the amounts of sterile water added to each composting bucket throughout experiment 3. 101 Table 3.6: Sterile Water Added (in grams) To Hog Manure Solids Composted in Bench-Scale Waterbaths - Experiment 3 Time (days) 24 26 28 30 32 34 36 38 40 42 44 1 46 'High Temperature' Waterbath 1 (WBl) Bl 12 24 45 30 30 25 25 45 45 25 B2 18 15 24 45 30 30 25 25 45 45 25 B3 15 30 30 24 45 25 30 35 45 45 45 25 'Compost Simulation' Waterbath 2 (WB2) B4 9 12 15 8 B5 15 15 8 B6 6 15 15 8 'Low Temperature' Waterbath 3 (WB3) B7 15 B8 15 B9 15 As can be seen from Table 3.6, it was necessary to add water to the 'high temperature' compost (WBl) every 2 days after day 26 of composting. The 'compost simulation' compost (WB2) also required additions of water for a brief period during the transition phase from thermophilic to ambient temperatures. By day 38, the 'low temperature' compost (WB3) began to dry noticeably at the surface and required minor additions of water on day 42 of composting. Estimation of Moisj- by visual observation of the 'WBl'- and WB2-compost started to become difficult after day 21 because the compost became very dark, sticky and pasty. This made it very difficult to determine "just glistening" particles. Consequendy, final Mois,- values of 102 the compost from these two waterbaths fell well below 70%. On day 46, approximately 150 to 200 grams of compost remained in each bucket for final sampling. Judging from the amounts of water added to the compost after day 24 (Table 3.6), and the remaining amount of compost on day 46; it is estimated that MoisT for the 'high temperature' compost (WB1) fluctuated from about 60% to 10% every 2 days, during the last 22 days of the experiment. 3.3.4 Comparison of Controlled Temperature "Compost Simulation' Compost (WB2) With Bucket-Scale Compost It would be appropriate to precede any results and discussion of the effects of low and high temperature composting with an evaluation of the microscale controlled temperature composting method used in experiment 3. Overall, the chemical character of WB2-compost more closely resembled that of experiment 1-compost than experiment 2-compost. The main differences between the experiment 3 and experiments 1 and 2 were found upon examination of Moisp and TPSS values. Tables 3.7 to 3.8 show similarities and differences in some important nutrient pools between composts from experiments 1, 2 and the " compost simulation' compost (WB2) of experiment 3. Moisx is reported an a per cent wet mass basis (W.M.). The rest of the parameters are reported on an oven-dried mass basis (o.d.) Values reported in Tables 3.7 and 3.8 are averages of all determinations made at each specified time period. 103 Table 3.7: Chemical Characteristics of Hog Manure Solids Compost Over Time: Bucket-Scale (Experiments 1 and 2) and Bench-Scale (Experiment 3) Experiment 1 - Day 0 (Bucket-Scale) Experiment 2 - Day 0 (Bucket-Scale) WB2 - Day 0 (Bench-Scale) Experiment 2 - Day 14 WB2 - Day 12 Experiment 1 - Day 51 Experiment 2 - Day 55 WB2 - Day 46 MoiSr (%) 71.57 72.54 73.26 72.44 72.64 72.57 73.54 57.98 LPSS (% glue, equiv.) 47.5 46.0 47.4 38.8 38.9 28.0 30.9 35.2 TPSS (% glue, equiv.) 67.7 63.7 68.5 56.3 65.5 40.9 47.4 55.8 C/NT 37.3/1 42.8/1 37.2/1 27.6/1 30.3/1 18.9/1 20.8/1 22.0/1 The chemical characteristics of WB2-compost at day 0 most closely resembled experiment 1 starting values. This may reflect the fact the same 5:1 ratios of manure to sawdust were used for both experiments 1 and 3, while a 4:1 ratio was used for experiment 2. At day 12, with the exception of TPSS, other chemical parameters measured in the WB2-compost were similar to day 14 experiment 2 values. By day 46, \VB2-Moisj. had decreased substantially compared with experiments 1 and 2, and TPSS remained higher. From Table 3.7 it can be seen that the final MoiSj for WB2-compost was substantially lower than that found in experiments 1 and 2. This arose as a result of previously mentioned changes in physical characteristics of the compost which made moisture estimation and adjustment difficult. 104 Total polysaccharides (TPSS) remained high for WB2-compost compared with experiments 1- and 2-compost This could be an indication that some factor was inhibiting the activity of cellulose-degraders. Early in the process, this may have been due to an absence of a variety of temperature microenvironments. Later in the experiment, low Moisp could have been contributing to inhibition of cellulose utilization. Table 3.8: Changing Ratios of Carbon and Nitrogen Pools in Hog Manure Solids Compost: Bucket-Scale (Experiments 1 and 2) and Bench-Scale (Experiment 3) Experiment 1 - Day 0 (Bucket-Scale) Experiment 2 - Day 0 (Bucket-Scale) WB2 - Day 0 (Bench-Scale) Experiment 2 - Day 14 WB2 - Day 12 Experiment 1 - Day 51 Experiment 2 - Day 55 WB2 - Day 46 C/NT 37.3/1 42.8/1 37.2/1 27.6/1 30.3/1 18.9/1 20.8/1 22.0/1 C/NH 13.9/1 8.3/1 13.4/1 13.1/1 13.7/1 11.0/1 12.4/1 10.3/1 C/NF 15.1/1 17.0/1 15.1/1 11.4/1 10.7/1 11.2/1 11.6/1 9.3/1 C/Nca 11.8/1 12.4/1 12.8/1 9.9/1 9.9/1 9.2/1 10.3/1 8.5/1 v-tj/Cp 0.79 0.64 0.39 1.08 0.83 1.23 1.32 1.55 CJCF 0.18 0.26 0.19 0.18 0.11 0.16 0.12 0.14 Table 3.8 shows that starting WE^-CyCf values were relatively low when compared with experiments 1 and 2. This resulted from a low starting concentration of WB2-CH (Appendix D, Part 3). In spite of this, the starting C/N ratio of humic acid (C/NH) found in WB2-compost is similar to that found in experiment 1. 105 3.3.5 Evaluation of Controlled Temperature Bench-Scale Composting Despite the use of plastic film covers and frequent additions of water, moisture content was found to be an out of control variable in bench-scale composting. By day 24, ' compost simulation' compost (WB2) and " low temperature' compost (WB3) appeared very wet and had formed large anaerobic-looking balls. At this point, the plastic film lids were removed from the buckets in an attempt to dry out the compost. Later on, the film lids were replaced when the compost appeared to be drying out excessively. In contrast, the 'high temperature' compost (WB1) dried out very quickly despite attempts to maintain adequate moisture levels by adding water at frequent intervals (Table 3.9). Because of fluctuations in compost moisture contents between waterbaths, conclusions as to the effects of controlled temperature on the chemical parameters studied were difficult to make. Twenty-one chemical parameters were measured (direcdy or indirectiy) as was done with experiments 1 and 2. A tabulated form of the one-way analysis of variance results on the effect of time is located in Appendix C. This discussion will only point out some interesting changes which occurred in the "high temperature' (WB1) and 'low temperature' (WB3) waterbaths. For this purpose, some of the parameters measured were averaged and presented graphically over time. Moisj- values are reported on a per cent wet mass basis (W.M.). The rest of the chemical parameters were determined on an oven-dried mass basis (o.d.) and are reported as percentages. In addition, a tabulated form of all data is presented in Appendix D, Part 3. 106 Table 3.9 summarizes changes in HA components examined. Table 3.9: Summary of Changes in C„ and Nn Over Time in a Hog Manure Solids Compost Under Three Controlled Temperature Regimes - Experiment 3 Day 0 to 12 Day 12 to 46 'High Temperature' (WBl) c H »t T (%) i T C/NH bnc ci 'Compost Simulation' (WB2) c H (%) t T NH (%) » -C/NH nc i 'Low Temperature* (WB3) CH T T NH (%) T r C/NH i nc a=valu£s increased significantly b=no significant change occurred c=values decreased significantly Trends in C/NH values suggest that narrowing of this ratio during composting occurred regardless of temperature. 3.3.5b Components of Carbohydrate-Rich (Non PVP-Adsorbed) Fulvic Acid (FA<J: In contrast with components of humic acid, changes in FAca differed between high and low temperature waterbaths. C/Nca values of composts from "high temperature' compost (WBl) and "compost simulation' compost (WB2) increased significandy from day 0 to day 46. Values for 'low temperature' compost (WB3) increased from day 0 to day 12 and remained unchanged through day 46 (Figure 3.59). 108 Figure 3.59: Change in Carbon/Nitrogen in Carbohydrate-Rich Fulvic Acid (C/Nca) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 1S.O 1 4 . O 1 3 . 0 11-O « l l . O 0" 10-° 9 . 0 i T.O | A further examination of data showed that for all three waterbaths Cca and Nca decreased over time to affect the decreases in C/Nca. Table 3.10 summarizes changes which occurred in Cc and Nca in experiment 3. Table 3.10: Summary of Changes in Cca and Nca Over Time in a Hog Manure Solids Compost Under Three Controlled Temperature Regimes - Experiment 3 Day 0 to 12 Day 12 to 46 'High Temperature' (WB1) (%) a j i (%) i l C/Nca '-* 'Compost Simulation1 (WB2) * i Nca (%) ^ bnc C/Ncl i I 'Low Temperature' (WB3) r v- c a (%) * nc (%) a nc C/Nca CT nc a=va/uei decreased significantly b=no significant change occurred c=values increased significantly 109 — a — w » l - H o t — • • — W B 2 - C o n p o i t S i a a , - - — W B 1 - C o l d O lO ZO 3 0 4 0 SO C o i a p o > t i i t g T l x a e C d « y » ) The trends in carbon and nitrogen shown in Table 3.10 suggest that decreases in C^ from WBl- and WB2-compost exceeded decreases in N^ in order for C/Nca to decrease throughout. From day 0 to day 12, the increase in C/N^ in the "low temperature' compost (WB3) indicates that Nca decreases exceeded losses in Cca. Figure 3.60 shows that the concentration of CM changed the least in the "low temperature' compost (WB3). This could possibly be due to cold temperature-inhibition of Cca-utilizing organisms. Figure 3.60: Change in Carbon in the Carbohydrate-Rich Fraction of Fulvic Acid (Cca) in Hog Manure Solids Composted Under Three Controlled Temperatures - Experiment 3 u 3.0O —!-2 .00 W B l - Hot WB2 Compo. l Sin WBS - Cold O 10 10 30 4 0 C o m p o f t i f l g T i m e <d«ys ) *V*luos d0totml&ed on an o-ren-dfittd lammm baaia 3.3.5c Polysaccharides: LPSS values found in the "low temperature' compost (WB3) did not begin to decrease significandy until day 12. In contrast, LPSS values found in the "hot temperature' compost (WBl) and the 'compost simulation' compost (WB2) decreased significandy from day 0 to day 12 and then levelled off through day 46 (Figure 3.61). 110 Figure 3.61: Change in Labile Polysaccharides (LPSS) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 4 * o .3 • • — W B l - Mot -«— WBJ - C o n p o . l aim.. - — - W » » - Cold 3a O lO 2 0 SO * 0 C o m p o a t l a g T L t n a C<&*y*> * v « I v « * r # p o r t t n t om « » o v e a - 4 s i o 4 m a * a b « » l « In experiment 3, TPSS changed significantly only for the Mow temperature' compost (WB3). Values decreased significantly from day 12 to 46, similar to its LPSS counterpart. For the 'high temperature' compost (WBl) and the 'compost simulation' compost (WB2), TPSS did not change significantly over time (Figure 3.62). Ill Figure 3.62: Change in Total Polysaccharides (TPSS) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 g* 3 •a S5 so - f -— w » t 1 —«— WB2 ) - - - - - WBl - Hot - Conp«ftt - C o ] * l l n . i ' i ' I ! 1 O 10 20 30 40 Campo«tifig Tim** (days) iralaei r«port*d on. •» oven-iirUi nmu bftkla 3.3.5J 4^/Jba/i Extractable Carbon (CJ: Ct contents found in the Mow temperature' compost (WB3) did not change significandy in experiment 3. Ce values from WBl- and WB2-compost were found to decrease from day 0 to day 12, levelling off through day 46 (Figure 3.63). 112 Figure 3.63: Change in Extractable Carbon (Ce) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 ao.s —f-m W B l - • — W B l - - - - WB3 - Hot - Compost t i n . - Cold O lO 2 0 3 0 4 0 C o x n p o s t i u f T t n m ( d a y i ) * V * l o # i d t « t « r n L a * f i o n l a ovf»m~«lri«<i tn.mmm b u i t These trends in Ce suggest that increases in extractable carbon were inhibited by low temperatures. 3.3.5e Water-Soluble Carbon (WSC): WSC values determined from WBl-compost did not change significantiy until day 12 of composting and then decreased through day 46. Values for WBl- and WB2-composts increased from day 0 to day 12 and then decreased significantly through day 46 (Figure 3.64). 113 Figure 3.64: Change in Water-Soluble Carbon (WSC) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 •WBl - Hot W M - c « r o n aim. WM - Cold O lO 2 0 30 4 « Conpaal<n( Time (dsya) »v«ls«« rep«rtDd on. •» ar»a-iltL«4 u«»a b*»ia Early increases in WSC seen in the "compost simulation' compost (WB2) and 'low temperature' composts (WB3) suggest that initially, contributions to this most available carbon pool exceeded metabolic losses. High temperatures in the WBl-compost may have inhibited utilization. WSC utilization in the latter part of composting appeared to be independent of temperature. 3.3.5f Ratio of Toted Carbon to Nitrogen (C/NT): C/NT decreased significantly for composts under all three temperature treatments (Figure 3.65). 114 Figure 3.65: Change in Carbon/Nitrogen (C/NT) of Hog Manure Solids Composted Under Three Controlled Temperature Regimes - Experiment 3 Z SO.O u • & . W I I - H o t «— W » 2 - C o w o H « U -«-- W B * - C o l d * V « l u e * d « l « r < n l a * d a n C/NT values for WB1- and WB2-compost decreased significandy from day 0 to day 46. Values for the 'low temperature' compost (WB3) did not begin decreasing until day 12. Change in C/NT, in all cases, was due to increases in NT, as CT did not change significandy for any of the temperature treatments in experiment 3. 115 Chapter 4 Summary Discussion In Chapter 3, the results of the three experiments were discussed individually. In this summary chapter, an overview of the set of experiments will be presented by comparing and contrasting the findings of the separate experiments. Conclusions based on comparisons between experiments must be made cautiously because of differences which existed between the starting compositions of compost used for experiments 1 and 2. In addition, moisture (Moisj) fluctuated much more extremely in experiment 3 than in either experiments 1 and 2. In this thesis, environmental factors affecting the utilization of various carbon and nitrogen pools were examined. It has been well established that composting is a dynamic process carried out under controlled conditions, in which reduced carbon compounds provide energy for microorganisms which utilize available nitrogen to produce tissue protein in the expansion of their populations (Golueke, 1977; Iglesias et al., 1988; Miller, 1992). The changing rates of metabolic activity which occur throughout the process depend on many environmental conditions such as aeration, temperature and nutrient status of the substrate (deBertoldi et al., 1989; Golueke, 1977; Loehr, 1974; Miller, 1992). In this thesis the changing nutrient status over time was examined in order to determine if different rates of aeration and extreme environmental temperatures had positive or negative effects on the composting process. 116 4.1 Temperature 4.1.1 Effect of Increased Aeration on the Thermophilic Phase The high temperature thermophilic phase is considered to be the most metabolically important phase of composting (Miller, 1992). In fact, it differentiates composting from other biological processes in which metabolism is controlled by the C/N status of a heterogeneous substrate such as soil. In an aerobic environment, the presence of concentrated, highly reduced, easily degradable carbonaceous material in organic wastes such as manure provide optimal conditions for accelerated metabolic activity and heat production (Golueke, 1977; Miller, 1992; The Staff of Biocycle, 1989). At increased temperatures, rates of enzyme production and activity increase and promote oxidation of organic material (Miller, 1992). This is desirable when one is attempting to reduce the polluting potential of a waste such as manure. In composting, unless fresh waste is continuously added to the system, the length of the thermophilic phase is generally very short (Miller, 1992). It is not understood fully whether the length of the thermophilic phase is affected more by the changing nutrient status of the substrate over time or by the high temperature which results from increased metabolic activity (Finstein, 1982; Loehr, 1974; Miller, 1992). Since the high rates of metabolism which support such high temperatures characteristic of the thermophilic phase are for the most part aerobic in nature, it is likely that oxygen is an important nutrient which is utilized quickly. The temperature profiles for experiments 1 and 2 indicate that the thermophilic range was maintained for a minimum of three days. A comparison of the effect of aeration treatments on the length of the thermophilic phase must be limited to an examination of temperature profiles of buckets under aeration regimes 'A2' and 'A,' in experiment 2. This is because neither of the 117 aeration treatments used in experiment 1 (Aj and A2) were frequent enough to impact the thermophilic stage (Figures 3.2 to 3.7). In experiment 2, Figures 3.31 to 3.33 show that brief increases in temperature occurred in the A2-compost after the day 7- and day 14-aeration treatments, slightly interrupting the gradual decline from the thermophilic range which was occurring at this time. The declines in the A3-compost temperatures were smoother (Figures 3.34 to 3.36). This suggests that the less frequently aerated A2-compost system was not supporting maximum rates of metabolic activity before the day 7 and day 14 mixing events. The brief increases in A2-compost temperatures after the day 7- and day 14-aeration treatments suggest that mixing served to supply some limiting nutrient which briefly optimized and consequendy increased metabolic rates of activity. Further study of actual differences in oxygen consumption occurring with increased rates of aeration would further support the conclusion that these brief increases in temperature are associated with increases in nutrient utilization (oxidation) which might affect the quality of the final product. A sufficiendy aerated system such as with the A3-compost may be more beneficial to the whole composting process, in terms of increasing nutrient utilization and stabilization of unstable organic wastes. The churning method of mixing used in experiments 1 and 2, not only provides aeration but it also homogenized the substrate. Beneficial effects to metabolism could have occurred through inoculation of nutrient-rich parts of the substrate with immobile organisms. There was no difference in the length of the thermophilic phase for aeration treatments within experiments 1 or 2. There was, however, a difference in the length of the thermophilic phases between experiments 1 and 2. In experiment 1, under aeration treatments "A/ and "A2', the thermophilic phase lasted from 4 to 6 days for all but one bucket. In contrast, the A2- and A3-compost of experiment 2, maintained thermophilic temperatures for 9 to 10 days. This may reflect 118 the different ratios of manure to sawdust used for experiments 1 and 2. The narrower ratio used for experiment 2 may have optimized metabolic conditions for thermophilic activity by increasing the starting ratio of total carbon to nitrogen. 4.1.2 Effect of Aeration on the Cooling Transition Between Thermophilic and Maturation Phases At the end of the thermophilic phase, declines in metabolism and heat production cause temperatures to decline (Golueke, 1977; Miller, 1992). If oxygen is a limiting nutrient and its depletion causes termination of the thermophilic phase, then increased aeration during the cooling transition phase may have beneficial effects on metabolic activity causing an extension of the cooling transition period before lower temperatures of the maturation phase are reached. An extension of the cooling transition phase may be beneficial to a process where oxygen is limiting during the thermophilic phase. An extended cooling transition may indicate that maximum nutrient utilization did not occur during the thermophilic phase and rich enough nutrient conditions still existed during the immediate post-fhermophilic period such that metabolic activity was maintaining higher temperatures, delaying the low temperature maturation phase. An examination of experiment 1 temperature profiles showed that the main factor differentiating Arcompost from A2-compost was a strong increase in temperature seen in the Arcompost which interrupted the decline to ambient conditions, after the day 21 aeration (Figures 3.2 to 3.7). In spite of this, there did not appear to be any differences in the length of the cooling transition period for aeration treatments in experiment 1. By day 42, the decline in temperatures of both A,- and A2-composts had levelled off slightly above ambient conditions. 119 In experiment 2, the transition cooling phase for the A3-compost was shorter than that observed for the A2-compost, ending about 7 days earlier. This suggests that nutrients were exhausted by metabolizing organisms more quickly in the more frequendy aerated A3-compost, possibly due to an increased supply of oxygen. 4.1.3 Temporal Variability in Temperature The increase in temporal variability of temperature above ambient conditions observed in experiments 1 and 2, suggests that microenvironments varying in conditions existed in the bucket-scale composting regardless of aeration treatments imposed (Figures 3.8 to 3.12, and 3.37 to 3.41). The presence of microenvironments were likely associated with thoroughness of mixing, and observed temperature and moisture gradients which occurred with depth. The statistical analysis of experiment 1 data showed significant interactions between time and aeration treatments for C/NT, C/NH, CF, CA and CyCf. Plots of the data over time showed segregation of populations due to aeration treatments (Aj and A2) at day 0. This was probably due to variability in day 0 data suggesting that the starting compost was not a homogeneous enough mix of sawdust and manure. This separation of day 0 values also occurred for CT and C/Nca. The statistical analysis showed significant effects of aeration treatments which were not apparent in plots of the data past the day 0 sampling period. This phenomenon of increased variability in day 0 data was not a problem in experiment 2 for any parameters except CT. This suggests that thoroughness of mixing at day 0 increased in experiment 2, improving the homogeneity of the starting compost. In spite of this, similar temperature gradients with depth were found in experiment 2. 120 4.2 Chemical Results Table 4.1 summarizes changes in nutrient pools over time in hog manure solids compost for experiments 1, 2 and 3. MoiSx is reported on a wet mass basis (W.M.). The rest of the values are reported on an oven-dried mass basis (o.d.). Values in Table 4.1 represent averages of all determinations. Table 4.1: Summary of Chemical Changes Over Time in Experiments 1,2 and 3 Hog Manure Solids Composting MoisT {%) CT (%) NT (%) C/NT WSC (%) LPSS (% glue, equiv.) TPSS (% glue, equiv.) C„ (%) NH (%) Experiment 1 Day 0 71.57 44.9 1.19 37.3 1.71 47.5 67.7 4.14 0.297 Day 51 72.57 43.2 2.29 18.9 1.01 28.0 40.9 5.29 0.481 Experiment 2 Day 0 72.54 45.5 1.12 42.8 1.01 46.0 63.7 3.26 0.297 Day 55 73.54 44.6 A2 2.03 A3 2.23 20.8 0.54 30.9 47.4 4.75 A2 0.370 A3 0.413 Experiment 3 Day 0 73.26 45.1 1.21 37.2 1.20 47.4 68.5 2.38 0.178 High Temp. (WBl) Day 46 44.24 46.3 1.84 21.8 0.89 33.1 58.1 4.35 0.366 Comp. Sim. (WB2) Day 46 57.98 45.9 2.09 22.0 1.09 35.2 55.8 4.70 0.455 Low Temp. (WB3) Day 46 58.58 46.1 2.13 25.2 0.69 38.6 54.2 4.93 0.509 Table 4.1: (Continued) C/NH CF (%) NF(%) C/NF Cca {%) Nca (%) C/Ncl CA (%) NA (%) Ce (%) Cn/Cp eye, Experiment 1 Day 0 13.9 5.24 0.352 15.1 4.28 0.366 11.8 0.96 0.002 21.4 0.79 0.18 Day 51 11.0 4.29 0.384 11.2 3.60 0.389 9.2 0.70 0.002 22.2 1.23 0.16 Experiment 2 Day 0 8.3 5.12 0.303 17.0 3.81 0.309 12.4 1.31 0.13 18.0 0.64 0.26 Day 55 12.4 A2 3.76 A3 3.47 0.311 11.6 A2 3.32 A3 3.05 0.310 A2 10.6 A3 10.0 0.44 0.003 18.7 0.26 0.12 Experiment 3 Day 0 13.4 6.16 0.408 15.1 5.00 0.390 12.8 1.16 0.018 18.9 0.39 0.19 High Temp. (WBl) Day 46 11.9 2.90 0.368 7.9 2.48 0.364 6.8 0.42 0.004 15.7 1.50 0.15 Comp. Sim. (WB2) Day 46 10.3 3.04 0.333 9.3 2.60 0.313 8.5 0.43 0.021 16.8 1.55 0.14 Low Temp. (WB3) Day 46 9.7 4.35 0.298 14.6 3.52 0.262 13.4 0.83 0.036 20.1 1.14 0.19 4.2.1 Effects of Aeration on Carbon and Nitrogen Pools There were no significant effects of aeration treatments in experiment 1 on carbon and nitrogen pools in monitored humus fractions (HA and FA); in overall compost fractions (polysaccharides, total carbon and nitrogen) and in the water-soluble phase (water-soluble carbon). 12 - 7 This could be attributed to the fact that neither of the aeration treatments (Al and A2) impacted the most metabolically active thermophilic phase in experiment 1. In experiment 2, there were significant differences between aeration treatments "A2' and "A3' in utilization of several carbon and nitrogen pools. Under the more frequent aeration treatment (A3), there were increases in concentrations of NT, and NH; and decreases in CF and Ca and C/Nca (Table 4.1). Differences seen in nutrient utilization in the A3-compost might be attributed to the fact that aeration occurred throughout the metabolically accelerated thermophilic phase where oxygen requirements would be relatively high. 4.2.2 Ratio of Manure to Sawdust From Table 4.1 it can be seen that decreasing the manure to sawdust ratio from 5:1 to 4:1 in experiment 2 did not produce the desired effect of reducing day 0-Moisx. It did cause a slight increase in day 0-C/NT for experiment 2 relative to experiments 1 and 3. Differences between experiments 1 and 2 in terms of the magnitude and chemical trends over time were apparent for C/NH. For experiments 1 and 3, C/NH values started relatively higher and narrowed during the composting process (Table 4.1). Starting C/NH values for experiment 2 were relatively low but eventually increased to values similar to that of experiment 1. An examination of CH and NH values show that low day 0-CH values were the reason starting C/NH values were low for experiment 2. This may have been caused by the narrower ratio of manure to sawdust used for the experiment 2 compost. 123 4.2.3 Total Moisture (MoisT) Although one may want to achieve a drier final product when composting, the oxidation processes characteristic of the process cause metabolic water to be produced in substantial quantities (Miller, 1992). Moisx tended to increase significantly over time in the bucket scale composting experiments. This was not surprising given that the period during which the temperature was high enough to remove excess moisture occurred during only a fraction of the entire composting period. The overall increase in total moisture could be attributed to metabolic production of water exceeding the rate of evaporation of water. This very likely occurred after the high temperature cooling phase had ended. It is likely that the oxidation and consequent losses of carbonaceous material in the form C02 also had the effect of negatively enriching Moisx. Moisx decreased substantially in the bench-scale waterbath composting experiments regardless of the imposed temperature. It is possible that metabolic production of water was inhibited in these systems due to lack of microenvironments. A substrate which possesses temperature and moisture gradients such as were present for experiments 1 and 2 may have provided more optimal conditions for composting microorganisms. In a substrate such is this which would contain a wide variety of carbon sources, a wide variety of environments may be necessary in order to support a variety of organisms. On the other hand, greater moisture losses may have occurred in the bench-scale composting as a result of the smaller size of the apparatus. The bench-scale apparatus may not have allowed for enough substrate to trap metabolically produced water. This may particularly have become a factor for experiment 3, after approximately half of the substrate was removed at the time of the day 12 sampling. 124 4.2.4 Carbon During the initial stages of decomposition, the breakdown of organic substrates for energy and incorporation into the microbial biomass is believed to be the crucial mechanism driving the composting process (Libmont et al, 1993). It is also thought that water-soluble nutrients such as carbon and nitrogen may represent not only progressively decreasing nutrient pools but also intermediate products of metabolism (Mathur et al., 1993). For experiment 1, decreases in carbon pools such as WSC, Cca and CT were not significant until after day 7 of composting. These early steady-state conditions suggest an expanding microbial population which is utilizing and contributing to these carbon pools through oxidation of carbonaceous material and consequent production of bacterial slime and by-products of incomplete oxidation. For experiment 2, trends in carbon pools indicate a lag in utilization between day 14 to about day 28 for CT, CF, Cca and CA. After day 28, relatively more degradable carbon pools (Cca and CF) from compost under aeration treatment 'A3' began to decrease after an initial increase from day 7 to day 14 and then a levelling off. During the cooling transition phase between the thermophilic and maturation stages of composting, demand for oxygen was probably decreasing as metabolic activity and heat production was declining. Aeration after day 14 may have cooled the compost, creating a suboptimal environment for oxidation of reduced carbon compounds. After day 28, the subsequent reduction in frequency of aeration for treatment "A3' may have allowed the temperature to build up sufficiently for utilization of CF and CM to continue. For experiment 2, the same lag in utilization was observed for CA and Q regardless of aeration treatments. Steady-state conditions of CA from day 14 to 42 may not indicate a lag in utilization, but may be a result of negative enrichment due to losses from the polysaccharide pools. 125 Cj- decreased very little in experiments 1 and 2, and not at all in experiment 3. This suggests the presence of a substantial amount of relatively inert carbonaceous material such as lignin compounds and cellulose. Given its more highly oxidized and less carbohydrate-like nature, one would expect humic acid to remain relatively stable throughout the composting process, compared to fulvic acid carbon. For experiment 1, CH increased briefly from day 0 to day 7. Metabolic utilization of more available carbon material in the fulvic fraction and carbohydrate pools may have caused a negative enrichment of HA carbon. This increase in CH and then steady-state conditions for the duration of the experiment could be partly explained by contamination of the alkali extract with non-humic substances like lignin. Early enrichment of the humic acid carbon pool, coupled with oxidation and condensation of HA carbonaceous structures, may be possible given the accelerated nature of metabolic processes during this high temperature period. This transformation within HA may have been the case particularly during the post-thermophilic stages where the concentration of CH remained unchanged. It is possible that lignin compounds were transformed to become more humic in nature. NH increased significantly during the post-thermophilic period suggesting incorporation of nitrogen into humic acid in the form of amino structures. The trend in HA carbon for experiment 2 was less clear as interactions occurred between time and aeration treatments for CH and CJCp. 4.2.5 Carbon to Nitrogen Ratios In spite of the fact that CT values changed very little or not at all during the composting process, the ratios of carbon to nitrogen in several fractions examined narrowed quite substantially. 126 These results agree with the findings of other researchers studying compost containing a relatively inert carbon source such as sawdust (Yoshida et al., 1979). In bucket-scale composting, interactions between aeration and time treatments occurred for C/NT, C/Np and CF. The interactions were inconsistent from experiment 1 to experiment 2. Table 4.1 shows that, in general, C/NT narrowed over time for all experiments. In general, C/NF and C/Nca narrowed over time for all composts except the 'low temperature' compost in experiment 3. Examinations of individual carbon and nitrogen pools showed that different processes occurred to affect the narrowing of C/N ratios for humic and fulvic acid. For HA, the narrowing of the ratio was affected by increases in both CH and NH. In general, narrowing of the C/N in FA and FA^ was associated with decreasing carbon and very little change in nitrogen. For experiment 1, C/NH narrowed over time, with both CH and NH increasing during composting. For experiment 3, the results were similar, regardless of temperature treatment. The trends in components of FAca are consistent for all three experiments. With the exception of the Tow temperature' compost (WB3), C/Nca values decreased over time as a result of decreasing C^. The phases during which Cca was utilized varied from one experiment to another. In contrast, C/NM found in the Tow temperature' compost (WB3) increased briefly due to decreases in both Cca and Nca. A review of the data indicates that overall, C^ was utilized much less in the Tow temperature' system. It has been suggested by researchers (Iglesias et al., 1988; Mathur et al., 1993) that the use of C/N ratios as indexes of maturity or quality is problematic in composting. The presence of high amounts of urea or inorganic nitrogen compounds in an unstable compost could cause the ratio of carbon to nitrogen (total or water-soluble) to be low (Mathur et al., 1993). This could mislead one into assuming a compost is mature and biostable, when in fact these inorganic forms of nitrogen make the compost readily susceptible to further decomposition given the appropriate 127 environmental conditions. In addition, inorganic forms of nitrogen such as water-soluble nitrate and ammonium would be a source of pollution. The narrowing of the C/N ratio of fulvic acid may be a more reliable indication of stabilization since nitrogen in the FA is by definition free of inorganic forms of nitrogen. 4.2.6 Polysaccharides During the initial stages of decomposition, readily degradable water-soluble materials are used more rapidly than resistant ligno-cellulose tissues (Whitely et al., 1994). LPSS and TPSS are representative of carbohydrates which vary greatiy in degradability. In experiment 1, LPSS and TPSS were utilized throughout, regardless of the composting phase. The magnitude of the LPSS values relative to TPSS, indicates that TPSS was dominated throughout by relatively more degradable carbohydrate rather than cellulose for all experiments. It is difficult to determine the extent of cellulose utilization based on the data. However, since cellulose degraders are not generally active during the thermophilic phase (Golueke, 1977; Miller, 1992), the decrease in both LPSS and TPSS at this stage may represent utilization of carbohydrate other than cellulose. For experiment 2, interactions occurred between time and aeration treatments for LPSS and TPSS. The values measured and decreasing trends over time appeared similar to those found in experiment 1 composting. For experiment 3, the overall utilization of TPSS was low relative to experiments 1 and 2. The reason for this may have been due to the homogeneous nature of the substrate in this controlled temperature system. The initial thermophilic temperatures imposed on waterbaths 1-and 2-compost probably created conditions suboptimal for cellulose-degraders such as fungi. In 128 the case of the "compost simulation' compost (WB2), because there were no low temperature microenvironments for cellulose-degraders to survive in, they may have been unable to recolonize and utilize cellulose once cooling of the substrate occurred. The Mow' temperature compost (WB3) may have provided conditions where the temperature was too low for any substantial decomposition of TPSS to occur over the course of 46 days. This could have been due to a temperature environment which was suboptimal for enzyme activation (Miller, 1992). 129 Chapter 5 - Conclusions Conclusions based on comparisons between experiments must be made cautiously because of differences which existed between the starting compositions of compost used for bucket-scale composting in experiments 1 and 2. In addition, MoisT fluctuated much more extremely in the bench-scale waterbath compost than in either of the bucket-scale experiments. Table 5.1 summarizes changes in nutrient pools over time in hog manure solids compost for experiments 1, 2 and 3. Moisj- is reported on a wet mass basis (W.M.). The rest of the values are reported on an oven-dried mass basis (o.d.). Values in Table 5.1 represent averages of all determinations. Table 5.1: Summary of Chemical Changes Over Time in Experiments 1,2 and 3 Hog Manure Solids Composting MoiSj (%) CT (%) NT (%) C/NT WSC (%) LPSS (% glue, equiv.) Experiment 1 Day 0 71.57 44.9 1.19 37.3 1.71 47.5 Day 51 72.57 43.2 2.29 18.9 1.01 28.0 Experiment 2 Day 0 72.54 45.5 1.12 42.8 1.01 46.0 Day 55 73.54 44.6 A2 2.03 A3 2.23 20.8 0.54 30.9 Experiment 3 Day 0 73.26 45.1 1.21 37.2 1.20 47.4 High Temp. (WBl) Day 46 44.24 46.3 1.84 21.8 0.89 33.1 Comp. Sim. (WB2) Day 46 57.98 45.9 2.09 22.0 1.09 35.2 Low Temp. (WB3) Day 46 58.58 46.1 2.13 25.2 0.69 38.6 130 Table 5.1: (Continued) CH (%) NH (%) C/NH CF (%) NF (%) C/NF Cca (%) Nca (%) C/Nca CA (%) NA (%) C, (%) Cu/Cp cjc. Experiment 1 Day 0 4.14 0.297 13.9 5.24 0.352 15.1 4.28 0.366 11.8 0.96 0.002 21.4 0.79 0.18 Day 51 5.29 0.481 11.0 4.29 0.384 11.2 3.60 0.389 9.2 0.70 0.002 22.2 1.23 0.16 Experiment 2 Day 0 3.26 0.297 8.3 5.12 0.303 17.0 3.81 0.309 12.4 1.31 0.13 18.0 0.64 0.26 Day 55 4.75 A2 0.370 A3 0.413 12.4 A2 3.76 A3 3.47 0.311 11.6 A2 3.32 A3 3.05 0.310 A2 10.6 A3 10.0 0.44 0.003 18.7 0.26 0.12 Experiment 3 Day 0 2.38 0.178 13.4 6.16 0.408 15.1 5.00 0.390 12.8 1.16 0.018 18.9 0.39 0.19 High Temp. (WBl) Day 46 4.35 0.366 11.9 2.90 0.368 7.9 2.48 0.364 6.8 0.42 0.004 15.7 1.50 0.15 Comp. Sim. (WB2) Day 46 4.70 0.455 10.3 3.04 0.333 9.3 2.60 0.313 8.5 0.43 0.021 16.8 1.55 0.14 Low Temp. (WB3) Day 46 4.93 0.509 9.7 4.35 0.298 14.6 3.52 0.262 13.4 0.83 0.036 20.1 1.14 0.19 131 Composting in 90 litre vessels (bucket-scale) proved useful in characterizing a developing hog manure solids compost in terms of carbon and nitrogen pools in humus fractions (HA and FA); in overall compost fractions (polysaccharides, total carbon and nitrogen) and in the water-soluble phase (water-soluble carbon). In experiment 1, the chemical effects of aeration treatments A! and A2 were studied over time on a bucket-scale compost composed of 5 parts separated hog manure solids and 1 part hemlock sawdust. Initially, WSC, Cp and Cca remained constant before decreasing, suggesting that during the stage of thermophilic expansion microorganisms were both utilizing and making contributions to these carbon pools. CH increased briefly until the end of the thermophilic phase and then remained constant throughout. Further study of changes into the oxidation characteristics and the nature of the carbon structures are necessary to determine whether conversion of lignin plays an important role in humus transformation in composting. Increases in NT and NH probably reflect negative enrichment due to oxidation losses of carbonaceous material as well as concentration of organic nitrogen structures in humic acid. The trend in HA carbon for experiment 2 was less clear as interactions occurred between time and aeration treatments for CH and CJCp. Aeration treatments Ai and A2 did not impact the thermophilic phase and were not shown to have significant effects on the chemical parameters monitored. In experiment 2, the chemical effects of aeration treatments A2 and A3 were studied over time on a bucket-scale compost composed of 4 parts separated hog manure solids and 1 part hemlock sawdust. Trends in carbon pools indicate a lag in utilization after the thermophilic phase, between day 14 to about day 28 for Cr, CF, Cra and CA. This lag in utilization was the case for Q. and CA trends, regardless of aeration treatments. For relatively more available pools (CF and Cca), the lag in utilization was evident only under the more frequent aeration treatment (A3), suggesting 132 aeration every 2 to 3 days is possibly inhibitive to oxidation processes when applied during the transition cooling phase between thermophilic and maturation stages of composting. In experiment 2, there were significant differences between aeration treatments "A2' and "A3' in utilization of several carbon and nitrogen pools. Under the more frequent aeration treatment (A3), there were increases in concentrations of NT and NH, and decreases in CF, Cca and C/Nca. Differences seen in nutrient utilization could possibly be attributed to the fact that aeration in the A3-compost occurred during the metabolically accelerated thermophilic phase where oxygen requirements would be relatively high. Problems in control of moisture losses and thoroughness of mixing were encountered when hog manure solids were composted under controlled temperatures in 2 litre vessels. Tightly sealed vessels which are aerated periodically through the provision of forced air or by a churning mechanism such as a hand mixer might better prevent excessive moisture loss and the formation of anaerobic balls in the compost. In experiment 3, the chemical effects of extreme temperatures on bench-scale hog manure solids compost were studied. Because of design flaws in the system, moisture was not a constant factor when comparisons were made between "high temperature', Mow temperature' and "compost simulation' systems. Moisture fluctuations were the most extreme in the "high temperature' system, but were also a problem in the "compost simulation' compost. Although an analysis of variance on the effects of controlled temperature showed significant chemical changes for "high' and "low' temperature composts, it is difficult to definitely attribute any chemical differences to the influence of controlled temperatures. The fluctuation in moisture values likely had an effect on utilization processes as well. In general, the overall utilization of TPSS was substantially lower for bench-scale waterbath composting, relative to bucket-scale composting, regardless of moisture. This suggests 133 that the homogeneous nature of the controlled temperature systems may have inhibited cellulose-decomposers such as fungi. Overall, the ratios of carbon to nitrogen in several fractions narrowed quite substantially in bucket-scale and bench-scale controlled temperature composting. 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Prentice Ha., Englewood Cliffs, New Jersey 07632. 139 Appendix A: Experiment 1 - Analysis of Variance1 Table al: Experiment 1 - Dependent Variable: Total Moisture (MoisT) N=55 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 11.6 30.4 3.74 129.7 R=0.52 DF 1 3 3 47 Mean Square 11.6 10.1 1.25 2.76 R2=0.27 F-Value 9.3 3.67 0.45 P 0.06 0.02 0.72 Table al: Experiment 1 - Dependent Variable: Water-Soluble Carbon (WSC) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.001 3.33 0.23 1.13 R=0.87 DF 1 3 3 40 Mean Square 0.01 1.11 0.08 0.03 R2=0.76 F-Vahie 0.13 39.3 2.69 P 0.74 0.000 0.06 Table a3: Experiment 1 - Dependent Variable: Total Carbon (CT) N=30 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 2.76 17.4 0.44 10.9 R=0.80 DF 1 3 3 22 Mean Square 2.76 5.81 0.14 0.50 R2=0.64 F-Vahte 18.6 11.7 0.30 P 0.02 0.000 0.83 140 Table a4: Experiment 1 - Dependent Variable: Total Nitrogen (NT) N=45 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.14 7.53 0.05 0.40 R=0.98 DF 1 3 3 37 Mean Square 0.14 2.51 0.02 0.01 R2=0.95 F-Value 7.99 229.4 1.65 P 0.07 0.000 0.20 Table a5: Experiment 1 - Dependent Variable: Total Carbon/Nitrogen (C/Nj) N=56 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 71.4 2782.9 16.2 72.2 R=0.99 DF 1 3 3 48 Mean Square 71.4 927.6 5.41 1.50 R2=0.98 F-Value 13.2 617.0 3.60 P 0.04 0.000 0.02 Table a6: Experiment 1 - Dependent Variable: Total Carbon in the HA (CH) N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.003 4.72 0.42 3.19 R=0.79 DF 1 3 3 17 Mean Square 0.003 1.58 0.14 0.19 R2=0.62 F-Value 0.02 8.39 0.74 P 0.90 0.001 0.54 141 Table a7: Experiment 1 - Dependent Variable: Nitrogen in HA (NjJ N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.000003 0.12 0.002 0.01 R=0.95 DF 1 3 3 17 Mean Square 0.000003 0.04 0.001 0.001 R2=0.90 F-Value 0.004 51.6 0.99 P 0.96 0.000 0.42 Table a8: Experiment 1 - Dependent Variable: C/N in the HA (C/NB) N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.0001 52.3 7.18 11.5 R=0.92 DF 1 3 3 19 Mean Square 0.0001 17.4 2.39 0.60 R2=0.84 F-Value 0.00004 28.8 3.95 P 1.00 0.000 0.02 Table a9: Experiment 1 - Dependent Variable: Total Carbon in the FA (CF) N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.16 4.50 1.25 1.94 R=0.87 DF 1 3 3 17 Mean Square 0.16 1.50 0.42 0.11 R2=0.75 F-Value 0.39 13.16 3.66 P 0.58 0.000 0.03 142 Table alO: Experiment 1 - Dep. Var.: Nitrogen in FA (NF) N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.002 0.01 0.001 0.037 R=0.52 DF 1 3 3 17 Mean Square 0.002 0.004 0.0004 0.002 R2=0.28 F-VaJue 4.57 1.68 0.19 P 0.12 0.21 0.90 Table all: Experiment 1 - Dependent Variable: C/N in the FA (C/NF) N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.009 57.3 3.65 32.7 R-0.81 DF 1 3 3 19 Mean Square 0.009 19.1 1.22 1.72 R2=0.65 F-Value 0.007 11.1 0.71 P 0.94 0.000 0.56 Table all: Experiment 1 - Dep. Var.: Total Carbon in FAca (C,J N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.02 2.05 0.26 1.21 R=0.81 DF I 3 3 17 Mean Square 0.02 0.68 0.08 0.07 R2=0.66 F-Value 0.21 9.62 1.20 P 0.64 0.001 0.34 143 Table a!3: Experiment 1 - Dep. Var.: Nitrogen in the FAea (NJ) N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.007 0.005 0.004 0.02 R=0.67 DF 1 3 3 17 Mean Square 0.007 0.002 0.001 0.001 R2=0.45 F-Value 5.90 1.34 1.11 P 0.09 0.30 0.37 Table al4: Experiment 1 - Dependent Variable: C/N in the K4M (C/NJ N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 3.61 23.6 0.58 7.49 R=0.88 DF 1 3 3 19 Mean Square 3.61 7.86 0.19 0.39 R2=0.78 F-Value 18.7 19.9 0.49 P 0.02 0.000 0.69 Table al5: Experiment 1 - Dependent Variable: Carbon in the FAA (Q) N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.06 0.49 0.40 0.18 R=0.91 DF 1 3 3 17 Mean Square 0.06 0.16 0.13 0.01 R2=0.84 F-Value 0.49 15.1 12.2 P 0.54 0.000 0.000 144 Table al6: Experiment 1 - Dependent Variable: Nitrogen in the FAA (NJ N=25 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.0001 0.0003 0.0002 0.001 R=0.72 DF 1 3 3 17 Mean Square 0.0001 0.0001 0.00007 0.00004 R2=0.52 F-Value 1.48 3.02 2.03 P 0.31 0.06 0.15 Table al7: Experiment 1 - Dependent Variable: Cj/Cp N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.005 0.75 0.007 0.13 R=0.93 DF 1 3 3 19 Mean Square 0.005 0.25 0.002 0.007 R2=0.86 F-Value 2.19 37.6 0.36 P 0.24 0.000 0.78 Table aI8: Experiment 1 - Dependent Variable: C/CF) N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.001 0.005 0.007 0.004 R=0.88 DF 1 3 3 19 Mean Square 0.001 0.002 0.002 0.0002 R2=0.78 F-Value 0.58 8.51 12.1 P 0.50 0.001 0.000 145 Table al9: Experiment 1 - Dependent Variable: Labile Polysaccharides (LPSS) N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.69 1383.7 55.9 163.4 R=0.95 DF 1 3 3 19 Mean Square 0.69 461.2 18.6 8.60 R2=0.90 F-Value 0.04 53.6 2.16 P 0.86 0.000 0.13 Table a20: Experiment 1 - Dependent Variable: Total Polysaccharides (TPSS) N=27 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 6.05 2900.8 65.8 150.7 R=0.98 DF 1 3 3 19 Mean Square 6.05 966.9 22.9 7.93 R2=0.95 F-Value 0.28 121.9 2.76 P 0.64 0.000 0.07 Table all: Experiment 1 - Dependent Variable: Total Extractable Carbon (CJ N=24 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 2.27 12.6 13.3 36.5 R=0.66 DF 1 3 3 16 Mean Square 2.27 4.18 4.45 2.28 R2=0.44 F-Value 0.51 1.83 1.95 P 0.53 0.18 0.16 146 Appendix B: Experiment 2 - Analysis of Variance1 Table bl: Experiment 2 - Dependent Variable: Total Moisture (MoisT) N=125 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 19.3 70.0 13.4 67.0 R=0.78 DF 1 6 6 111 Mean Square 19.3 11.7 2.24 0.60 R2=0.60 F-Value 8.64 19.3 3.70 P 0.03 0.000 0.002 Table b2: Experiment 2 - Dependent Variable: Water-Soluble Carbon (WSC) N=78 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.15 10.2 0.17 0.12 R=0.99 DF 1 6 6 64 Mean Square 0.15 1.71 0.03 0.002 R2=0.99 F-Value 5.15 900.6 14.9 P 0.06 0.000 0.000 Table b3: Experiment 2 - Dependent Variable: Total Carbon (C?) N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 5.02 15.5 2.81 26.3 R=0.66 DF 1 6 6 30 Mean Square 5.02 2.59 0.47 0.88 R2=0.44 F-Value 10.7 2.96 0.54 P 0.02 0.02 0.78 147 Table b4: Experiment 2 - Dependent Variable: Total Nitrogen (Nj) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.21 6.48 0.14 0.53 R=0.96 DF 1 6 6 34 Mean Square 0.21 1.08 0.02 0.02 R2=0.93 F-Vahte 9.29 69.6 1.48 P 0.02 0.000 0.21 Table b5: Experiment 2 - Dependent Variable: Total C/N (C/Nj) N=50 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 30.8 2221.7 84.3 281.1 R=0.95 DF 1 6 6 36 Mean Square 30.8 370.3 14.0 7.80 R2=0.91 F-Value 2.20 47.4 1.80 P 0.19 0.000 0.13 Table b6: Experiment 2 - Dependent Variable: Total Carbon in the HA (CH) N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.14 13.4 0.41 0.50 R=0.98 DF 1 6 6 30 Mean Square 0.14 2.24 0.07 0.02 R2=0.96 F-Value 2.05 132.4 4.06 P 0.20 0.000 0.004 148 Table b7: Experiment 2 - Dependent Variable: Nitrogen in the HA (Ng) N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.01 0.07 0.003 0.008 R=96 DF 1 6 6 30 Mean Square 0.01 0.01 0.0005 0.0003 R2=91 F-Vahte 21.1 39.6 1.74 P 0.004 0.000 0.15 Table b8: Experiment 2 - Dependent Variable: C/N in the HA (C/N„) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 4.81 56.4 7.52 21.6 R=0.87 DF 1 6 6 34 Mean Square 4.81 9.40 1.25 0.63 R2=0.76 F-Value 3.84 14.8 1.98 P 0.10 0.000 0.10 Table b9: Experiment 2 - Dependent Variable: Total Carbon in the FA (CF) N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 1.59 11.3 0.18 0.85 R=0.97 DF 1 6 6 30 Mean Square 1.59 1.89 0.03 0.028 R2=0.94 F-Value 54.4 67.4 1.02 P 0.000 0.000 0.43 149 Table blO: Experiment 2 - Dependent Variable: Nitrogen in the FA (NF) N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.001 0.04 0.003 0.01 R=0.87 DF 1 6 6 30 Mean Square 0.001 0.006 0.0005 0.0004 R2=0.75 F-Vaute 1.30 13.4 1.12 P 0.30 0.000 0.38 Table bll: Experiment 2 - Dependent Variable: C/N in the FA (C/N) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 6.12 143.1 7.93 8.42 R=0.98 DF 1 6 6 34 Mean Square 6.12 23.8 1.32 0.25 R2=0.95 F-Vahie 4.63 96.3 5.34 P 0.08 0.000 0.001 Table bl2: Experiment 2 - Dependent Variable: Total Carbon in the FA^ ( C ^ N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.88 2.29 0.11 0.65 R=0.92 DF 1 6 6 30 Mean Square 0.88 0.38 0.02 0.02 R2=0.84 F-Vahte 46.8 17.5 0.87 P 0.000 0.000 0.53 150 Table bl3: Experiment 2 - Dependent Variable: Nitrogen in the FAa (N<J N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.000 0.02 0.001 0.03 R=0.62 DF 1 6 6 30 Mean Square 0.0001 0.003 0.0002 0.001 R2=0.39 F-Value 0.43 2.87 0.25 P 0.54 0.02 0.96 Table bl4: Experiment 2 - Dependent Variable: C/N in the FAa {C/NJ) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 11.3 32.6 3.85 43.1 R=0.73 DF 1 6 6 34 Mean Square 11.3 5.43 0.64 1.27 R2=0.53 F-Value 17.6 4.29 0.51 P 0.006 0.003 0.80 Table bl5: Experiment 2 - Dependent Variable: Carbon in the FAA (CJ N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.10 3.82 0.11 0.32 R=0.96 DF 1 6 6 30 Mean Square 0.10 0.64 0.02 0.01 R2=0.93 F-Value 5.56 60.5 1.75 P 0.06 0.000 0.14 151 Table b!6: Experiment 2 - Dependent Variable: Nitrogen in the FAA (NJ N=44 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.0003 0.005 0.005 0.02 R=0.61 DF 1 6 6 30 Mean Square 0.0003 0.001 0.001 0.001 R2=0.37 F-Vahte 0.39 1.39 1.31 P 0.55 0.25 0.28 Table bl7: Experiment 2 - Dependent Variable: C^CF N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.21 2.47 0.04 0.05 R=0.99 DF 1 6 6 34 Mean Square 0.21 0.41 0.007 0.001 R2=0.98 F~Vaaie 29.4 293.8 5.00 P 0.002 0.000 0.001 Table b!8: Experiment 2 - Dependent Variable: C/CF N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.0003 0.09 0.005 0.02 R=0.92 DF 1 6 6 34 Mean Square 0.0003 0.02 0.001 0.001 R2=0.84 F-Vahie 0.36 27.7 1.42 P 0.57 0.000 0.24 152 Table b!9: Experiment 2 - Dependent Variable: Labile Polysaccharides (LPSS) N=48 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 19.9 1347.7 38.7 60.6 R=0.98 DF 1 6 6 34 Mean Square 19.9 224.6 6.45 1.78 R2=0.96 F-Vaute 3.08 126.1 3.62 P 0.13 0.000 0.007 Table b20: Experiment 2 - Dependent Variable: Total Polysaccharides (TPSS) N=46 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 48.4 1975.6 122.1 91.6 R=0.98 DF 1 6 6 32 Mean Square 48.4 329.3 20.4 2.86 R2=0.96 F-Vaaie 16.9 115.0 7.11 P 0.000 0.000 0.000 Table b2l: Experiment 2 - Dependent Variable: Total Extractable Carbon (Ce) N=40 Sources of Error Aeration Sample Day Aeration * Sample Day Experimental Error Sum of Squares 0.75 20.3 5.80 12.3 R=0.83 DF 1 6 6 26 Mean Square 0.75 3.39 0.97 0.47 R2=0.69 F-Vabie 0.77 7.15 2.04 P 0.41 0.0001 0.10 153 Appendix C: Experiment 3 - Analysis of Variance1 Part 1: 'Hot' Waterbath 1 Table cl: Experiment 3(WB1) - Dependent Variable: Total Moisture (MoisT) N=17 Sources of Error Sample Day Experimental Error Sum of Squares 2067.5 72.0 R=0.98 DF 2 14 Mean Square 1033.7 5.14 R2=0.97 F-Vahte 201.1 P 0.000 Table c2: Experiment 3(WB1) - Dependent Variable: Water-Soluble Carbon (WSC) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.35 0.12 R=0.86 DF 2 9 Mean Square 0.18 0.01 R2=0.74 F-Vahie 13.1 P 0.002 Table c3: Experiment 3(WB1) - Dependent Variable: Total Carbon (CT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 1.61 6.83 R=0.44 DF 2 5 Mean Square 0.80 1.37 R2=0.19 F-Vahie 0.59 P 0.59 Table c4: Experiment 3(WB1) - Dependent Variable: Total Nitrogen (NT) N=7 Sources of Error Sample Day Experimental Error Sum of Squares 0.94 0.01 R=0.99 DF 2 4 Mean Square 0.47 0.003 R2=0.99 F-Vaute 181.5 P 0.000 154 Table c5: Experiment 3(WB1) - Dependent Variable: Total C/N (C/NT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 304.8 9.83 R=0.98 DF 2 5 Mean Square 152.4 1.97 R2=0.97 F-Vahte 77.5 P 0.0002 Table c6: Experiment 3(WB1) - Dependent Variable: Total Carbon in the HA (Cg) N=14 Sources of Error Sample Day Experimental Error Sum of Squares 9.40 0.66 R=0.97 DF 2 11 Mean Square 4.70 0.06 R2=0.93 F-Vahie 78.2 P 0.000 Table c7: Experiment 3(WB1) - Dependent Variable: Nitrogen in the HA (Njj) N=14 Sources of Error Sample Day Experimental Error Sum of Squares 0.09 0.01 R=0.96 DF 2 11 Mean Square 0.04 0.001 R2=0.93 F-Value 73.7 P 0.000 Table c8: Experiment 3(WB1) - Dependent Variable: C/N in the HA (C/N^ N=17 Sources of Error Sample Day Experimental Error Sum of Squares 13.3 4.98 R=0.85 DF 2 14 Mean Square 6.65 0.36 R2=0.73 F-Value 18.7 P 0.0001 155 Table c9: Experiment 3(WB1) - Dependent Variable: Total Carbon in the FA (CF) N=14 Sources of Error Sample Day Experimental Error Sum of Squares 30.1 0.43 R=0.99 DF 2 11 Mean Square 15.0 0.04 R2=0.99 F-Vaute 388.7 P 0.000 Table clO: Experiment 3(WB1) - Dependent Variable: Nitrogen in the FA (NF) N=14 Sources of Error Sample Day Experimental Error Sum of Squares 0.01 0.004 R=0.80 DF 2 11 Mean Square 0.004 0.000 R2=0.64 F-Value 9.95 P 0.003 Table ell: Experiment 3(WB1) - Dependent Variable: C/N in the FA (C/NF) N=17 Sources of Error Sample Day Experimental Error Sum of Squares 149.9 5.89 R=0.98 DF 2 14 Mean Square 74.9 0.42 R2=0.96 F-Vahie 178.2 P 0.000 Table c!2: Experiment 3(WB1) - Dependent Variable: Total Carbon in the FA^ (C^ N=14 Sources of Error Sample Day Experimental Error Sum of Squares 16.6 1.42 R=0.96 DF 2 11 Mean Square 8.28 0.13 R2=0.92 F-Vahie 64.0 P 0.000 156 Table c!3: Experiment 3(WB1) - Dependent Variable: Nitrogen in the FAca (NJ) N=14 Sources of Error Sample Day Experimental Error Sum of Squares 0.004 0.004 R=0.68 DF 2 11 Mean Square 0.002 0.000 R2=0.47 F-Value 4.84 P 0.03 Table cl4: Experiment 3(WB1) - Dependent Variable: C/Ninthe FA^ (C/NJ N=17 Sources of Error Sample Day Experimental Error Sum of Squares 101.7 4.10 R=0.98 DF 2 14 Mean Square 50.8 0.29 R2=0.96 F-Value 173.5 P 0.000 Table cl5: Experiment 3(WB1) - Dependent Variable: Total Carbon in the FAA (CJ N=14 Sources of Error Sample Day Experimental Error Sum of Squares 1.90 0.67 R=0.86 DF 2 11 Mean Square 0.95 0.06 R2=0.74 F-Value 15.4 P 0.001 Table cl6: Experiment 3(WB1) - Dependent Variable: Nitrogen in the FAA (N^J N=14 Sources of Error Sample Day Experimental Error Sum of Squares 0.000 0.001 R=0.62 DF 2 11 Mean Square 0.000 0.000 R2=0.38 F-Value 3.37 P 0.07 157 Table cl7: Experiment 3(WB1) - Cj/CF N=17 Sources of Error Sample Day Experimental Error Sum of Squares 3.37 0.07 R=0.99 DF 2 14 Mean Square 1.68 0.005 R2=0.98 F-Vabte 331.2 P 0.000 Table c!8: Experiment 3(WB1) - C/CF N=16 Sources of Error Sample Day Experimental Error Sum of Squares 0.004 0.02 R=0.39 DF 2 13 Mean Square 0.002 0.002 R2=0.15 F-Value 1.16 P 0.34 Table c!9: Experiment 3(WB1) - Labile Polysaccharides (LPSS) N=9 Sources of Error Sample Day Experimental Error Sum of Squares 357.8 117.6 R=0.87 DF 2 6 Mean Square 178.9 19.6 R2=0.75 F-Value 9.13 P 0.02 Table c20: Experiment 3(WB1) - Total Polysaccharides (TPSS) N=13 Sources of Error Sample Day Experimental Error Sum of Squares 2167.5 6658.9 R=0.50 DF 2 10 Mean Square 1083.8 665.9 R2=0.25 F-Vatue 1.63 P 0.24 158 Table c21: Experiment 3(WB1) - Total Extractable Carbon (CJ N=14 Sources of Error Sample Day Experimental Error Sum of Squares 44.4 4.18 R=0.96 DF 2 11 Mean Square 22.2 0.38 R2=0.91 F-Value 58.5 P 0.0000 Part 2: 'Compost Simulation' Waterbath 2 Table c22: Experiment 3(WB2) - Dependent Variable: Total Moisture (MoisT) N=20 Sources of Error Sample Day Experimental Error Sum of Squares 930.7 19.9 R=0.99 DF 2 17 Mean Square 465.3 1.17 R2=0.98 F-Value 397.9 P 0.000 Table c23: Experiment 3(WB2) - Dependent Variable: Water-Soluble Carbon (WSC) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.23 0.10 R=0.83 DF 2 9 Mean Square 0.11 0.01 R2=0.68 F-Value 9.78 P 0.006 Table c24: Experiment 3(WB2) - Dependent Variable: Total Carbon (CT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 0.94 7.86 R=0.33 DF 2 5 Mean Square 0.47 1.57 R2=0.11 F-Value 0.30 P 0.75 159 Table c25: Experiment 3(WB2) - Dependent Variable: Total Nitrogen (NT) N=7 Sources of Error Sample Day Experimental Error Sum of Squares 0.83 0.05 R=0.97 DF 2 4 Mean Square 0.42 0.01 R2=0.95 F-Vabte 34.9 P 0.003 Table c26: Experiment 3(WB2) - Dependent Variable: Total C/N (C/NT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 286.1 13.3 R=0.98 DF 2 5 Mean Square 143.0 2.67 R2=0.96 F-Value 53.6 P 0.000 Table c27: Experiment 3(WB2) - Dependent Variable: Total Carbon in the HA (Cg) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 10.8 2.05 R=0.92 DF 2 9 Mean Square 5.38 0.23 R2=0.84 F-Value 23.6 P 0.000 Table c28: Experiment 3(WB2) - Dependent Variable: Nitrogen in the HA (N„) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.16 0.01 R=0.96 DF 2 9 Mean Square 0.08 0.001 R2=0.92 F-Vahie 53.1 P 0.000 160 Table c29: Experiment 3(WB2) - Dependent Variable: C/NintheHA (C/Ng) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 23.3 3.14 R=0.94 DF 2 9 Mean Square 11.6 0.35 R2=0.88 F-Value 33.3 P 0.000 Table c30: Experiment 3(WB2) - Dependent Variable: Total Carbon in the FA (CF) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 22 A 0.54 R=0.99 DF 2 9 Mean Square 11.0 0.06 R2=0.98 F-Value 182.7 P 0.000 Table c31: Experiment 3(WB2) - Dependent Variable: Nitrogen in the FA (NF) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.01 0.01 R=0.69 DF 2 9 Mean Square 0.01 0.001 R2=0.48 F-Value 4.08 P 0.06 Table c32: Experiment 3(WB2) - Dependent Variable: C/NintheFA (C/NF) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 81.4 6.91 R=0.96 DF 2 9 Mean Square 40.7 0.77 R2=0.92 F-Value 53.0 P 0.000 161 Table c33: Experiment 3(WB2) - Dependent Variable: Total Carbon in the FAa (C^ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 12.3 1.21 R=0.95 DF 2 9 Mean Square 6.16 0.14 R2=0.91 F-Value 45.7 P 0.000 Table c34: Experiment 3(WB2) - Dependent Variable: Nitrogen in the FA^ (N^ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.01 0.01 R=0.72 DF 2 9 Mean Square 0.01 0.001 R2=0.52 F-Value 4.83 P 0.04 Table c35: Experiment 3(WB2) - Dependent Variable: C/N in the FACtt (C/NJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 41.9 5.08 R=0.94 DF 2 9 Mean Square 20.9 0.56 R2=0.89 F-Value 37.1 P 0.000 Table c36: Experiment 3(WB2) - Dependent Variable: Total Carbon in the FAA (CJ N=14 Sources of Error Sample Day Experimental Error Sum of Squares 1.90 0.67 RO.86 DF 2 11 Mean Square 0.95 0.06 R2=0.74 F-Value 15.4 P 0.001 162 Table c37: Experiment 3(WB2) - Dependent Variable: Nitrogen in the FAA (NJ N= Sources of Error Sample Day Experimental Error Sum of Squares R= DF Mean Square R2= F-Vahte P TabU c38: Experiment 3(WB2) - CJC? N=12 Sources of Error Sample Day Experimental Error Sum of Squares 2.70 0.20 R=0.96 DF 2 9 Mean Square 1.35 0.02 R2=0.93 F-Value 61.6 P 0.000 Table c39: Experiment 3(WB2) - C/CF N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.01 0.01 R=0.76 DF 2 9 Mean Square 0.01 0.001 R2=0.57 F-Vauie 6.05 P 0.02 Table c40: Experiment 3(WB2) - Labile Polysaccharides (LPSS) N=9 Sources of Error Sample Day Experimental Error Sum of Squares 236.2 78.7 R=0.87 DF 2 6 Mean Square 118.1 13.1 R2=0.75 F-Vaaie 9.00 P 0.02 163 Table c41: Experiment 3(WB2) - Total Polysaccharides (TPSS) N=13 Sources of Error Sample Day Experimental Error Sum of Squares 262.8 165.0 R=0.78 DF 2 6 Mean Square 131.4 27.5 R2=0.61 F-Value 4.78 P 0.06 Table c42: Experiment 3(WB2) - Total Extractable Carbon (CJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 18.7 165.0 R=0.90 DF 2 6 Mean Square 9.34 27.5 R2=0.80 F-Value 18.5 P 0.001 Part 3: 'Cold' Waterbath 3 Table c43: Experiment 3(WB3) - Dependent Variable: Total Moisture (MoisT) N=20 Sources of Error Sample Day Experimental Error Sum of Squares 1017.5 64.8 R=0.97 DF 2 17 Mean Square 508.8 3.81 R2=0.94 F-Value 133.5 P 0.000 Table c44: Experiment 3(WB3) - Dependent Variable: Water-Soluble Carbon (WSC) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.52 0.06 R=0.94 DF 2 9 Mean Square 0.26 0.01 R2=0.89 F-Value 37.3 P 0.000 164 Table c45: Experiment 3(WB3) - Dependent Variable: Total Carbon (CT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 1.91 7.26 R=0.46 DF 2 5 Mean Square 0.95 1.45 R2=0.21 F-Vahte 0.66 P 0.56 Table c46: Experiment 3(WB3) - Dependent Variable: Total Nitrogen (NT) N=7 Sources of Error Sample Day Experimental Error Sum of Squares 0.46 0.02 R=0.98 DF 2 4 Mean Square 0..23 0.01 R2=0.95 F-Value 38.9 P 0.002 Table c47: Experiment 3(WB3) - Dependent Variable: Total C/N (C/NT) N=8 Sources of Error Sample Day Experimental Error Sum of Squares 191.7 16.0 R=0.96 DF 2 5 Mean Square 95.9 3.20 R2=0.92 F-Value 30.1 P 0.002 Table c48: Experiment 3(WB3) - Dependent Variable: Total Carbon in the HA (Cg) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 14.7 0.30 R=0.99 DF 2 9 Mean Square 7.36 0.03 R2=0.98 F-Value 217.2 P 0.000 165 Table c49: Experiment 3(WB3) - Dependent Variable: Nitrogen in the HA (Ns) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.26 0.004 R=0.99 DF 2 9 Mean Square 0.13 0.000 R2=0.98 F-Vabte 282.2 P 0.000 Table c50: Experiment 3(WB3) - Dependent Variable: C/N in the HA (C/Ng) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 40.1 1.51 R=0.98 DF 2 9 Mean Square 20.0 0.17 R2=0.96 F-Value 119.3 P 0.000 Table c51: Experiment 3(WB3) - Dependent Variable: Total Carbon in the FA (CP) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 8.21 0.22 R=0.99 DF 2 9 Mean Square 4.11 0.02 R2=0.97 F-Value 166.4 P 0.000 Table c52: Experiment 3(WB3) - Dependent Variable: Nitrogen in the FA (NF) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.04 0.002 R=0.97 DF 2 9 Mean Square 0.02 0.000 R2=0.94 F-Value 75.9 P 0.000 166 Table c53: Experiment 3(WB3) - Dependent Variable: C/N in the FA (C/NF) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 5.30 2.71 R=0.81 DF 2 9 Mean Square 2.65 0.30 R2=0.66 F-Value 8.79 P 0.01 Table c54: Experiment 3(WB3) - Dependent Variable: Total Carbon in the FAm (C„) N=12 Sources of Error Sample Day Experimental Error Sum of Squares 5.33 0.97 R=0.92 DF 2 9 Mean Square 2.66 0.11 R2=0.85 F-Value 24.6 P 0.000 Table c55: Experiment 3(WB3) - Dependent Variable: Nitrogen in the FA„, (NCJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.05 0.004 R=0.96 DF 2 9 Mean Square 0.02 0.000 R2=0.92 F-Value 53.3 P 0.000 Table c56: Experiment 3(WB3) - Dependent Variable: C/N inthe FAea (C/NJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 7.30 5.02 R=0.77 DF 2 9 Mean Square 3.65 0.56 R2=0.59 F-Value 6.54 P 0.02 167 Table c57: Experiment 3(WB3) - Dependent Variable: Total Carbon in the FAA (CJ N=14 Sources of Error Sample Day Experimental Error Sum of Squares 0.32 0.34 R=0.69 DF 2 9 Mean Square 0.16 0.04 R2=0.48 F-Vatue 4.19 P 0.05 Table c58: Experiment 3(WB3) - Dependent Variable: Nitrogen in the FAA (NJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.001 0.001 R=0.71 DF 2 9 Mean Square 0.000 0.000 R2=0.50 F-Value 4.52 P 0.04 Table c59: Experiment 3(WB3) - C,/CF N=12 Sources of Error Sample Day Experimental Error Sum of Squares 1.24 0.01 R=0.995 DF 2 9 Mean Square 0.62 0.001 R2=0.99 F-Vatue 414.6 P 0.000 Table c60: Experiment 3(WB3) - CJCP N=12 Sources of Error Sample Day Experimental Error Sum of Squares 0.000 0.01 R=0.17 DF 2 9 Mean Square 0.000 0.001 R2=0.03 F-Value 0.14 P 0.87 168 Table c61: Experiment 3(WB3) - Labile Polysaccharides (LPSS) N=10 Sources of Error Sample Day Experimental Error Sum of Squares 210.9 32.2 R=0.93 DF 2 7 Mean Square 105.5 4.60 R2=0.87 F-Vahie 22.9 P 0.001 Table c62: Experiment 3(WB3) - Total Polysaccharides (TPSS) N=10 Sources of Error Sample Day Experimental Error Sum of Squares 1185.0 319.2 R=0.89 DF 2 7 Mean Square 592.5 45.6 R2=0.79 F-Value 13.0 P 0.004 Table c63: Experiment 3(WB3) - Total Extractable Carbon (CJ N=12 Sources of Error Sample Day Experimental Error Sum of Squares 2.86 3.51 R=0.67 DF 2 9 Mean Square 1.43 0.39 R2=0.45 F-Value 3.68 P 0.07 1 coefficient of determination. It is the ratio of the treatment sum of squares to the total sum of squares. It indicates how much of the total variation in an analysis of variance is explained by the treatments. 169 Appendix D: Raw Data Moisj- is reported on a per cent wet mass basis. The rest of the parameters are expressed on a per cent oven-dried mass basis. LPSS and TPSS are reported as glucose equivalents. Part 1: Experiment 1 - Bucket-scale 1 I) Experiment 1:Aeration Treatment 'Ax' Average ; 1 !i I Average 1 1 i i Average ] Average Bucket 1 1 2 2 3 3 1 1 i 2 3 3 1 1 2 2 3 1 I 1 2 i ~> 3 3 3 Sample Day 0 0 0 0 0 0 7 7 7 7 7 7 23 23 23 23 23 23 51 51 51 51 51 51 51 51 51 MoisT (%) 73.36 72.35 70.16 69.31 70.26 69.49 70.82 71.63 70.87 69.30 69.71 70.81 71.16 70.58 71.55 70.62 71.35 70.23 72.97 72.43 71.52 73.82 74.65 75.26 70.66 71.74 70.89 69.80 70.76 70.42 72.00 CT (%) 45.0 46.1 45.4 45.5 44.4 45.1 44.9 44.8 43.8 43.3 43.4 43.5 42.3 42.9 45.6 43.6 NT (%) 1.16 1.19 1.17 1.16 1.13 1.06 1.15 1.44 1.45 1.59 1.64 1.41 1.49 1.50 1.78 1.83 1.92 1.88 1.83 1.80 1.84 2.23 2.28 2.29 i 22 2.39 2.28 C/NT 38.8 37.9 39.4 39.7 40.2 42.6 39.8 30.9 30.8 28.5 27.6 31.9 30.2 30.0 24.5 23.9 22.5 23.0 23.7 24.1 23.6 18.9 18.5 18.8 20.5 19.0 19.2 wsc LPSS (%) (%gluc. 1.69 1.82 1.73 1.70 1.30 1.33 1.60 1.45 1.46 1.71 1.64 1.51 1.48 1.54 1.48 1.41 1.57 1.54 1.33 1.40 1.45 0.95 0.98 1.11 0.99 1.10 1.13 1.04 equiv.) 48.1 46.2 42.1 45.5 46.7 42.0 43.3 44.0 36.1 34.8 36.1 37.3 36.1 27.6 26.7 28.3 28.2 27.7 TPSS (% glucj equiv.) 61.3 64.8 70.8 65.7 63.9 61.8 66.7 64.1 51.4 51.8 54.8 54.2 53. lj 40.2 39.7 41.1 42.7 40.91 170 JO © o ' l o — (N <Nj<Ni — — — — — . — . o o ; ©' i o o c © o o r-!t^- *n *o;-oil O •=2 U oo oo r~ o t/1 *C r*l Ov ON OO o o O i? r^  m IT, \or<> o t~-irs o o © © -<© o ©' o vo oo o o o o 00 1 f", O <N <N O © O O " O i O O O I O u s? 00 (N CO o —'• — o t o o o 2: ; ON <N O fO O *fi 00 00 ;—^  o-i c4 <N|—- o d o v-> i/~, o o ON d d O —• • ON m fN liTi O , C*- ON ON j ON z 3? C-J Tf ON :t/~. <r~, oo = 0 c~, 04 r ^ i o o^ (N mioo c j \ m ! C">, r j - r*-, (N ^ i n »n oo oe Ci m c-\ r- tr~ i <; ON O c i i c-, i m c-i " i d o o 0 £ OO Ov Tf TT I f- -O </-> — <N <N ! C^ | r ^ NO OC Tf Tf j Tf [ r o c~> CN p~.: CN d C V 5 0 J O — CN Tf i Tf f S CN : Tf Tf «/•> Tf ! c", r\j c"i NO f S M I T , Tf O N I O O I O N OO ON !vo;i z s? m M ( > ,<r. i n oo ; c i r n <N Tf ! — j 00 Tf l/~, O ! — ! Vi ON ON c l C*! c*t S c i m Tf i y s? !S © rr * n f i <S~, I "/"> r r I T in- r r T I T i I z 0 Z J! U S? ;r- -— r- | <N |Tf rf —< <ri Tf ON ON (N O r~- 50 i Tf \ c-> Tf r ^ c"i Tf r*^  0O O NC V> — — O ON Tf c~, ON lo Tf Tf fNj r - oo ON C I CI c , o d d m r-- — ! ^ i vO 1 " ! 00 ! 00 O CN ON CN | Tf i / i Tf -rf d; d d d : d 00 : — CN. - i t fN! un! tri v-j ONSNCI 'O iT j - t v-i 1 *y-)| i — C\| CN | ~ C-l C", ; - N IN (N (N I i> CO n L. f\ > ! aj SB n L. 9 •» <] j< i c/^ SB H ; ' t . , 5»-> • \<< i J w • S I L. J* > < f"-f( ^Experiment 1: Aeration Treatment 'Ai' j i ! i ! j j 1; : Average | j j! ;j jl |] ii i \ Average Average ! 1 | j | j Average_ Bucket 4 4 4 4 > 5 5 5 6 6 6 6 4 4 5 5 5 s 6 6 6 6 4 4 > > 6 6 4 4 4 5 \ 5 6 6 6 Sample Day 0 0 0 0 0 0 0 0 0 0 0 0 7 7 7 7 7 7 7 7 7 7 23 23 23 23 23 23 51 51 51 51 51 51 51 51 51 MOIST (%) 72.49 73.44 71.95 72.43 70.83 69.12 75.26 72.22 71.90 73.51 71.65 70.92 68.95 68.65 70.93 74.09 73,67 71.91 72.38 69.72 70.22 72.00 74.44 75,56 74.58 73,79 74.53 73.73 72.11 71.23 71.54 73.50 Cr (%) 45.2 44.3 45.3 44.0 44.1 45.0 44.7 44.8 44.5 43.4 44.1 44.5 44.0 44.2 43.7 43.0 43.0 43.3 42.4 43.2 42.5 42.7 NT (%) 1.27 1.25 1.22 1.19 1.22 1.26 1.24 1.68 1.62 1.69 1.58 1.58 1.63 2.23 2.28 1.89 1.96 1.97 2.00 2.06 2.32 2.41 2.02 2.20 2.57 2.30 CfSr 35.7 36.1 35.0 35.4 37.1 38.0 36.0 36.8 36.0 34.9 36.7 35.6 36.1 26.6 26.4 26.9 25.7 27.3 26.1 28.1 28.2 27.8 27.9 27.1 19.6 19.2 1"} -7 22.0 21.9 21.5 21.1 18.3 17.6 21.5 19.7 16.5 18.7 vvsc (•/.) ( 2.01 2.03 1.76 1.69 1.80 1.68 1.83 1.90 1.94 1.46 1.46 1.47 1.44 1.61 1.57 1.59 1.14 1.13 1.26 1.28 1.33 0.90 0.91 0.80 1.29 1.00 0.98 0.98 LPSS % glue equlv.) 46.4 47.6 57.4 44.8 49.0 36.0 39.6 42.8 39.5 33.9 35.0 35.9 34.9 28.8 30.3 26.5 28.5 TPSS (% glue equlv.) 69.0 69.8 73.0 65.1 69.2 i 59.2 62.4 62.5 61.4 46.0 | 50.11 i 48.91 48.4 43.3 43.2 j j| Jl 36.41| I  II 4y=J o o o OO i O N ; */*> C~- i/~> i NO o o l d o o o oo NO t— o o © O iON —• : 0 —• — —• m ^ 5? ; i r , C. 'O M - " , ' 0 | ' A t O \ O O ^ - O O T r v O ^ t N T t ^ O O v M h- o -n ! C O O o o © ! o j r - ON O O I O ; 0 0 I O N z Z i? z u Z i? u s? r^ —. I r^ 00 rr — C-J r*~> OO m m O N — —• © O O O •* —It-; r^ (N <N —' © C I O © O NO C-, — (^N l^> —• T J 1 </-> iv-; §y aji B Part 2: Experiment 2 - Bucket-scale Experiment 2: Aeration Treatment 'Az' -'1 ;; :i i Average I j j i Average 1 ! i 1 Average Average Bucket 1 1 1 2 2 2 3 3 3 ] 1 1 ~! 2 2 3 3 3 1 1 1 "> "t 2 3 3 3 I I 1 2 2 2 3 3 3 Sample Day 0 0 0 0 0 0 0 0 o 7 7 7 7 7 7 7 7 7 14 14 14 14 14 14 14 14 14 21 21 21 21 21 21 21 21 21 Molsr (•/.) 73.02 72.51 72.60 70.66 72.75 72.41 72.61 72.46 72.44 72.38 72.15 71.24 71.25 71.53 72.07 72.10 71.76 69.73 70.97 71.42 72.16 70.72 72.11 72.24 71.90 72.65 71.95 72.37 72.74 72.09 72.49 72.89 72.77 73.54 72.30 71.82 72.63 72.37 73.63 72.72 Day 0 to Day 21 CT (%) («>42.0 46.7 46.3 46.5 44.3 45.2 42.3 43.9 44.6 43,2 44.9 44.2 44.9 45.5 44.1 44.8 NT (%) 1.10 1.24 1.09 1.14 1.32 1.41 1.35 1.36 1.63 1.13 1.66 1.48 1.82 1.86 1.S3 1.84 C/NT 37.7 42.6 40.2 33.5 31.9 31.3 32.3 27.3 38.2 27.1 30.8 24.6 24.4 24.1 24.4 VVSC (%) ( 1.08 1.00 1.04 1.04 1.53 1.57 1.44 1.36 1.42 1.42 1.46 1.42 1.34 1.32 1.41 1.34 1.31 1.36 1.22 1.14 1.16 1.12 1.14 1.14 1.15 LPSS % glue equlv.) 43.3 46.2 47.3 45.6 40.0 41.9 41.4 41.1 41.5 39.3 40.0 36.5 39.3 37.2 38.6 36.8 37.7 37.6 TPSS (% glue) equlv.j 64.2 67.1 64.6 65.3 57.5 59.3 59.6 58.8 57.7 57.4 57.3 54.0 56.6 57.6 j 56.61 ! 39,7 51.31 a 'value has been rejected as extraneous on the basis of a Q-test (Dixon, 1986) 174 Experiment 2: Aeration Treatment 'Ai' - Day 28 to Day 55 Sample Bucket Day 28 28 28 28 28 28 28 28 28 M O I S T (%) 69.59 70.94 71.23 74.57 73.57 73.47 72.07 72.90 71.67 CT (%) 44.7 45.3 46.2 NT (%) 2.08 1.93 1.97 C/NT WSC LPSS (%) (%gluc. equiv.) TPSS (% glucj equiv.' 21.5 23.5 23.5 0.82 0.80 0.83 0.79 0.85 0.88 34.3 33.9 34.3 50.2! 49.0 52.2 Average 72.22 45.4 .99 22.8 0.83 34.2 50.4 42 42 42 42 42 42 42 42 42 73.39 73.82 72.82 73.83 74.17 73.90 73.64 76.43 74.19 43.3 44.1 44.9 2.16 1.99 .09 20.1 22.2 21.5 0.58 0.62 0.57 0.63 0.69 0.69 32.5 32. 31.3 45.4 48.2 43.8 Average 74.02 44. 2.08 21.3 0.63 31.9 45.8 55 55 55 55 55 55 55 55 55 73.46 73.81 73.52 74.11 73.46 73.43 73.23 73.02 72.76 45.0 (a)49. 45.1 2.18 1.79 2.12 1.81 2.16 2.10 20.7 25.2 20.9 21.5 0.52 0.49 0.50 0.48 0.54 0.55 31.8 33.3 29.6 47.3 49.8 47.0 Average^ 73.42 45. ,.03 22.1 0.51 31.6 48.0 a-wlue has been rejected as extraneous on the basis of a Q-test (Dixon, 19S6) 175 \n I! Experiment 2: Aeration Treatment 'Ai' - Day 0 to Day 21 Sample Bucket Day Moisr CT NT C/NT WSC LPSS TPSS (%) (%) (%) (%) (%glue. (•/. glue ; I I Average 1 i Average ! ! ! ! i i ! Average 1 I Average 4 4 4 5 s 5 6 6 6 4 4 4 5 5 5 6 6 6 4 4 4 5 5 5 6 6 6 4 4 4 5 5 s 5 6 6 6 0 0 0 0 0 0 0 0 0 7 7 7 7 7 ~7 7 7 7 14 14 14 14 14 14 14 14 14 21 21 21 21 21 21 21 21 21 21 73.26 73.42 72.96 72.77 71.71 71.71 72.29 72.77 73.28 72.69 71.82 71.69 71.65 72.22 71.49 71.64 72.55 72.80 72.39 72.03 72.20 72.22 72.37 73.47 73.68 73.93 72.14 72.56 72.65 72.80 74.75 73.96 74.06 74.86 74.51 73.91 74.20 73.20 73.60 74,12 43.6 47.4 44.7 44.1 44.9 44.6 43.8 43.7 44.1 45.5 43.8 44.1 43.0 44.1 44.4 44.8 43.5 44,2 1.22 1.16 0.89 1.09 1.36 1.45 1.43 1.42 1.75 1,77 1.84 1.79 2.05 2.01 1.98 2.01 35.8 40.7 50.5 49.8 44.2 32.8 30.2 30.5 31.2 26.0 24.7 23.9 23.3 24.5 21.7 22,2 22.0 22.0 0.94 1.02 0.98 0.98 1.55 1.53 1.50 1.48 1.50 1.48 1.51 1.58 1.58 1,65 1,72 1.62 1.65 1.63 1.17 1.24 1.27 1.30 1.28 1.34 1,27 equiv.) 47.1 44.5 45.1 48.3 46.2 42.4 42.2 43.3 42.7 40.4 37.8 37.0 38.4 33.2 34.6 32.9 33.6 equiv.) 62.l1 63.21 1 63.2 61.5 62.5 62.1 61.8 63.5 | 62.4 57.8 56.8 j 53.3 j 56J9I 48.91 I 48.01 I j 1 46.7! 1 i 47JH 177 Experiment 2. i i ll i i! Average | | ; Average Average Bucket 4 4 4 5 5 5 6 6 6 4 4 4 4 5 5 5 6 6 6 4 4 4 5 5 5 6 6 6 6 Aeration Treatment 'A3' - Day 28 to Day Sample Dav 28 28 28 28 28 28 28 28 28 42 42 42 42 42 42 42 42 42 42 55 55 55 55 55 55 55 55 55 55 MoisT (%) 73.88 74.30 74.37 75.46 75.53 73.92 73.45 73.69 74.33 73.91 74.58 72.55 74.19 75.22 75.41 73.82 73.84 74.11 74.18 73.50 73.26 73.51 75.32 73.68 73.70 72.97 73.54 73.38 73.65 CT (%) 44.5 43.9 45.0 44.1 44.4 43.9 43.0 42.8 43.2 44.8 45.2 44.0 43.3 44.3 NT (%) 2.22 2.22 2.12 2.19 2.14 2.17 2.14 2.15 2.35 2.23 2.37 2.20 2.15 2.09 2.23 55 C/NT 20.0 19.8 20.3 20.8 20.2 20.5 19.8 20.0 20.1 19.1 20.1 19.1 20.5 20.5 20.7 21.0 20.2 20.1 wsc 1.02 1.02 0.92 0.98 0.97 0.99 0.98 0.65 0.64 0.67 0.65 0.65 0.65 0.65 0.56 0.54 0.59 0.59 0.57 0.61 0.58 LPSS (% glue, equiv.) 30.8 30.7 30.3 32.3 31.0 29.2 29.4 29.7 31.4 29.9 29.7 29.3 30.7 32.0 30.4 TPSS (% glue, equiv.) 45.1 50.3 44.3! 47.4 46.8 44.6 44.4 44.9 45.6 44.9 47.4 44.21 46.7! 49.6 47.0 178 I N (N M Id © d 1 y_ ! ^ i i m O rn •o r- '-o o d d ON (N O oo CN r-^  r-^  r-' O od i n \ ON 0s- r ^ M M (N IN o d d d zs? ;3SS ION 04 oo rXJ m O r~-rs O ' « </i CN — — o d d oo r^ r~ r- r-~ oo|r-~ d © oo oo - ~ O O r*i c~ —* —< < N c-i - * —< - -d d d d d d d o d d <>- i n o —' OO 0 \ ON Cv' Z S? • a 5 z i ? us? ;o oc o^ l _ vo r-i J— ON oo 11" i O O O O IT) OO Tf od od od oo C\ — <0 oo co r*l r*i - r s ^ i M i> co oc od • — iri TT ON od ON m — O J ON m ON O ON ON VO OO od od © ^ 1-j <o • ON ON OO CO > — r^i S O O O O o o o o d d © o d d o VI V) m ON' ON ON" — <N — (N <N d d d d c — o f - i r*1 rr, \0 \Q ON ON \ 0 - H i n - O ^ ' O ON ON ON ON i—" ON ON ' d d < < N ! C N —; O r n <s w r o O * n | o est <N ^ | < N NO 00 CO r*"> c*"> n-i oo k i TT oo oc ! r n r o r*"i — m m ON — O (M m m ;<n ICN — c s C*N r*"i —i CN O ON O ON ' d « d - c ' —- NO r r OO —< fS <N — - H W (N| N o d d W M m ON —« NO d -^ -^ r*i c*i <~n OO TT — •f*"t m r*~i • ^O CO r- *n — O I v s ! ON — NC — *n ^ O ! m j r ^ —. o CS — ( S i — ' t n m r*~> T O IO O (N (N N f s r i r i r s —- ON m f^ n <N <N ^O r f CM <n ^ r r r pS-r*~i r o m I m i • o ^O (N CS *-» CN m *T oc m m m , r ^ n * r r r f i ^ f | T f TT TT t t f T l T f l — — — — — w j _ _ _ _ _ _ !(N (N fS N M Ol j in. in, i n '•O | r r i n i n vO I «N f S M f S N M T ' t T fl- t T f I T T TT * r i n NC i >< 179 Part 3: Experiment 3 - Microscale NO I •8 5 § C/3 1/3 C/5 !/3 -o a "3D x ^ cJ s "5b x > s a> ^ - v > S <u Z x U ^  5« — 83 C/3 ! ! >-Q w <u - 4 u 3 CO ^ O — T NO NO in r--CNj in NO od oo in -* •* TT ;inj<N • 00 I r-' NO I NO rr |CN ^ticN co CN ^^  oo O C\l co <— — CN CN *~~ ^ — ^ - < i i CM CN ~^ O CN »—-f CN T-* NO oo oo in co co NO co Tf NO NO CN O ON CN INi rf TT CN co co co co r- t-~ t-» r- r— CN ;co • i> 'CN co co in !NO NO NO NO ON m m CO CN| CO CN in NO ON 00 CO in ON CN in in NOION r - N O N O O N O < N o o o o C N I O N t - ^ o o O S c O T f r f i n r O co i C N co in CN C N co <-* C N co r--!r- r~-r--r-~r--r-r~-r~-t--O O O O O O ; ( N ( N ( N M ( N N i N M ( N ) r-; oo co in ON NO r^  od <n in in in Tf CN NO co o in CO CO CO NO r- r^ r- o oo © ~-i o o NO r-~ CN —«' --<" CN CN CN ON TT rf O *—_ —; CN CN CN q Tf vi NO NO NO' r^ TT rr a> [ S£! 93! - i « l >| <\ — — CN CN C N CO CO CO 4) s i S3 1 -i <W I > < oo in CO CO ON OO CO ON 0O NO —< CO CO O ON r^ in co NO NO NO NO t t r^ ^ f — CN CO CO I 180 i ^~ o ON r - tn c-t i ON ] r-- o i r^l CN —; *-; <N — . j ~ - : — r ^ d d o d d d ' d j d o | J ; M t « t d i d d d d r-J v~i r*1 O fN -— O N ! r- T T rr O N I r*~,' — «~i i-o ^ i C rr ro rr rr rr r*^  j r^ * CT\ O N O r^ ' O 1 [ rr NC rr ^ j 1 ^ ! d d d d d didlc: d —• d i d i — — —'• — j s b U v? z s? u S 5 Z J? 0 S z D z s? z 0 Z i? u £ o tt -«f o r^  -o oo r-" oo o c> o\ o I —; | -rf- in r e Tf m \ m I m *o v . in O — ON CN m — > 00 I O CN fN O fN (N O I —• ! O o o o o o o ! o i© O O O © © ©' i O I o O m | < n ! CN O O m O - j O ! " O O O o o | o : o o o o o c j o I o © © o TT c e r f \D •— O I ^C j tN ^O O r e I © ! 00 M h - -(N <rs ~-* O v i r-- i —.; ^o r^ o <N ! Tf j r e Tf r f r f r _ ,_ __ ,_ _ Q ; ,_; , -^J 0 * 0 " Q I Q I Q 0 ' 0 ' o •oo i r i o q © Tt i n i o O | r - © '•o >/"i! TT ' r e TT CO r -c s <N (N r e r j r e i c s [ oo cr\ o Cv! <> i r^ MS* vo l-0 t--^ r^  r^ „ <n ° r--rr 00 r T rr 00 o ON rr rr u-1 oo ON o LTi Wl 0 0 r T ° rr rr "V^ l 0 0 I O r r jr*~i o «^i CN u~) O o "Tl t n 00 rr o r^i r'^ o — 00 IN o rr O N rvj „ _, ° rr; fr~~ oc O m j m i^-ON o o r e O i n (N oc 00 CI o oc C4 c^ t ^ DC v. o c-4 rs oc 1 o rr ("Nt oo r^ rr O 00 rr (N ON r~ 0C r<i O r -j O \ - - 0> M C1. rn -^ T m ^1- m -rr i r f cc I—• m oo co u-j! CNI ^ t VD o\ 0 ! m rn vo •ocliri i 'O oo un ^D m r^-< ro m § ro f re re re re ! o o o !rj* c* TJ- *-> - O M N ^ q T , oo r e r e r f r e r-j O^O IO o o o o i i \ o ! < J o o r -- ' ^ r - — r -00! r e o CN ^C O tr-, ' O O^  '00 'OC o .^ ^ ; r e r e r e r e i r e ! <N r^ c-j r ^ j (N t i ; p o Tf Tt O1. i r e ! oo r-^  o ^ j cv i r e ' r e r e in •^"Trri'— —• r-j r^ I —• t~- i n -rr ^ o r e [ oc j o O in o l ^ ! — r e rr, r—1 co »n oo oo GN i n | r - - ! v c >n — 0 ' r e : i n o *r-, mi'O _ „ ^_ ^_ _ _ J _« ; (SJ c^, r , , r ^ j 0 j j r r . ( ^ j . rr.t ( ^ [ o o o o o o , o o c l o l r - ^c 'CO -— ue r s : o o i ^ oo r^ t-- jce j Tf <N - ^ (Nlv--, l^f — in tn <je o t r e i tn T r e Ovlre;-— r - r s r e j r e I r s c s r s r s r s e - i l r s i r e r e r e r - j i r e i r t r r TJ- -rrlTf 13 C/} r s CJ e t rN | . vo vo O ^C' i — _ _ — _ [ T j - r r - ^ T f I • — - r ) f ^ i l ; — OJ r e rr-, t \< Ki 38T <•< !'« 195 lire l < ire : | S 9 i \> >< ! 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