Recycling Agricultural By-Products to Grow Sugarcane on Sandy Soils in South Florida

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Recycling Agricultural By-Products to Grow Sugarcane on Sandy Soils in South Florida
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Gomez, Susanna M
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Soil and Water Science
Committee Chair:
Daroub, Samira H
Committee Members:
Wright, Alan Lee
Mccray, James M
Annable, Michael D
Bhadha, Jehangir

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Subjects / Keywords:
amendments -- sugarcane
Soil and Water Science -- Dissertations, Academic -- UF
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Soil and Water Science thesis, M.S.
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Abstract:
Sustainable farming techniques, such as the use of agriculturalby-products as organic fertilizer, have the potential to improve sugarcane (Saccharum spp) yields grown on sandy soilswith low organic matter content. This study was designed to investigate threetypes of agricultural by-products in the Everglades Agricultural Area for theirpotential use as organic soil amendments and substitute inorganic phosphorus(P) fertilizer to grow sugarcane on sandy soils in south Florida. The mesocosm experimentlasted twelve months during which sugarcane was grown in lysimters using millmud, mill ash, a 50/50 mix of mill mud and mill ash, and water lettuce (WL) asa soil amendment at a high, medium, and low application rates. We hypothesizedthat these amendments would add organic matter (OM) and increase microbialactivity, therefore adding nutrients to sandy soils to increase sugarcane yield.All amendments were compared to the control that only received recommendedrates of inorganic fertilizers. Mill mud treatments (18.3 kg cane lysimter-1)and mix treatments (19.9 kg cane lysimter-1) produced the most biomassand were significantly different than the control at 6.55 kg cane lysimter-1.These two treatments showed higher levels of leaf nutrient concentrations andless nutrient imbalances compared to the control according to the criticalnutrient levels (CNL) and the Diagnosis and Recommendation Integrated System(DRIS). In addition these treatments added the most OM to the soil, whichcontributed to significant increases in microbial biomass, phosphatase activity(PA), cation exchange capacity (CEC), and soil nutrients. Reasons for theseincreases in microbial activity and nutrient availability were attributed tothe low C:N ratio of 15.5 for mill mud and 17.7 for the mix material. The millmud and mix treatments showed no significant decreases in soil OM, CEC, totalP, and available-P across the 12-month sampling period, indicating that thesematerials served as successful organic amendments and P fertilizer for at leastan entire year. Mill ash treatments, except the treatment with the highest rateof mill ash, showed no significant differences from the control in any soilphysiochemical and microbial property. These results were reflected in theharvest data, where the mill ash medium and low application treatments showedno significant differences in the sugarcane biomass or sucrose yield. However,the high C:N ratio of 56:1 suggests that mill ash may degrade slowly and beuseful for ratoon crops and long-term benefits. The WL high and mediumtreatments did not allow the sugarcane to enter the formative growth stage,which was attributed to inhibitory allelopathic compounds excreted the WL. TheWL low treatment failed to significantly alter sugarcane biomass and sucroseyields in comparison to the control, proving its inability to act as an organicsoil amendment.  Future research onlong-term benefits in field conditions, environmental impacts, and the economicfeasibility of using mill mud, mill ash, or a mixture of the two as soilamendments will further help scientists and growers understand how to bestmanage these materials.
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by Susanna M Gomez.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Daroub, Samira H.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 RECYCLING AGRICULTURAL BY PRODUCTS TO GROW SUGARCANE ON SANDY SOILS IN SOUTH FLORIDA By SUSANNA M. GOMEZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Susanna M. Gomez

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3 To my parents

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4 ACKNOWLEDGMENTS I would like to express my deepest thanks to my advisor, Dr. Samira Daroub for her guidance and support throughout my graduate studies. Her patience, encouragement, and advice were invaluable to the completion of my thesis. My gratitude is given to Jehangir Bhadha and Timothy Lang for their guidance during my time at the University of Florida. Their help in the design and execution of my experiment was much appreciated. Recognition is extended to my committee members, Dr. Alan Wright for his help wi th my microbial analyses, Dr. Mabry McCray for his expertise in sugarcane agronomy, and Dr. Michael Annable for his review of my thesis and valuable viewpoint. I would also like to thank Viviana Nadal, Irin a Ognevich, and Yigang Lou for their patience and assist ance with all laboratory work. Mihai Giurcanu is acknowledged for his help and assistance towards my statistical design and analyses. A special thanks to Odiney Campos for willingness to help in the field and laboratory and her enco uragement as a fr iend. Thanks are given to Ernest Guillaume for his help with my sugarcane harvest and to Rhogzhong Ye for his help with my sucrose analysis. Acknowledgements go out to the Everglades Research and Education Center for providing the facilities to conduct thi s resea rch. I would also like to thank The Sugar Cane Grower Cooperative of Florida and James Shrine for their support and for providing the materials for this study. I would also like to thank Mike Sisk of student services in the Soil and Water Scien ce De partment for all his help with my paperwork I want to thank all my friends and family. Their support and encouragement made the difficult task of completing my thesis a lot easier to manage knowing I could also count o n them. Special thanks are given to my boy friend Adam Orndorff for

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5 experiencing the high s and lows of this experience with me. I am forever thankful for his endless encouragement and patience. Lastly, but most importantly, I would like to thank my parents and my sisters. I will never be able to fully express how grateful I am for everything they have taught me and everything they have done for me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Background ................................ ................................ ................................ ............. 13 Sugarcane Overview ................................ ................................ ........................ 13 Agricultural By Products ................................ ................................ ................... 15 Literature Review ................................ ................................ ............................. 16 Project Statement and Objectives ................................ ................................ ........... 18 Materials and Methods ................................ ................................ ............................ 19 Materi als ................................ ................................ ................................ ........... 19 Experimental Design ................................ ................................ ........................ 19 Planting and Fertilizer Applications ................................ ................................ .. 20 Experimental Analyses ................................ ................................ ..................... 21 2 SUGARCANE RESPONSES ................................ ................................ .................. 27 Background ................................ ................................ ................................ ............. 27 Hypothesis and Objectives ................................ ................................ ..................... 28 Methods and Materials ................................ ................................ ............................ 29 Leaf N utrient Concentration ................................ ................................ .............. 29 Yield Measurements ................................ ................................ ......................... 30 Statistical Analysis ................................ ................................ ............................ 31 Results ................................ ................................ ................................ .................... 31 Plant Growth ................................ ................................ ................................ ..... 31 Biomass Yields ................................ ................................ ................................ 32 Sucrose Yields ................................ ................................ ................................ 33 Nutrient Leaf Concentrations ................................ ................................ ............ 33 Discussion ................................ ................................ ................................ .............. 36 Plant Growth and Yield Data ................................ ................................ ............ 36 Nutrient Leaf Concentrat ions ................................ ................................ ............ 39 3 SOIL QUALITY ................................ ................................ ................................ ....... 52 Background ................................ ................................ ................................ ............. 52 Physiochemical Properties ................................ ................................ ............... 52

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7 Mi crobial Properties ................................ ................................ .......................... 53 Hypothesis and Objectives ................................ ................................ ..................... 54 Materials and Methods ................................ ................................ ............................ 55 Soil Sampling and Physical Chemical Analysis ................................ ................ 55 Microbial Analysis ................................ ................................ ............................. 56 Statistical Analysis ................................ ................................ ............................ 57 Results ................................ ................................ ................................ .................... 58 Soil physiochemical Analyses ................................ ................................ .......... 58 Soil Microbial Analyses ................................ ................................ ..................... 61 Discussion ................................ ................................ ................................ .............. 63 Effects of Amendments on Physical and Chemical Properties ......................... 63 Effects of Amendments on Soil Microbial Properties ................................ ........ 66 Correlations between Soil Quality Parameters and Sugarcane Yields ............. 71 4 CONCLUSIONS AND FUTURE WORK ................................ ................................ 88 Summar y and Conclusions ................................ ................................ ..................... 88 Future Work ................................ ................................ ................................ ............ 90 APPENDIX A CRITICAL NUTRIENT LEVELS ................................ ................................ .............. 92 B BRIX AND POL VALUES ................................ ................................ ........................ 94 C WEATHER DATA ................................ ................................ ................................ ... 95 LIST OF REFERENCES ................................ ................................ ............................... 96 BIOGRAPHIC AL SKETCH ................................ ................................ .......................... 103

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8 LIST OF TABLES Table page 1 1 Physiochemical characteristics of the amendments and the sandy soil matrix ... 23 1 2 Application rates (tons/ha) of each amendment at the high, medium, and low depths ................................ ................................ ................................ ................. 24 1 3 Treatment lysimeters fertilizer rates ................................ ................................ .... 25 1 4 Control lysimeters fertilizer rates ................................ ................................ ........ 25 1 5 Amount of phosphorus and nitrogen (kg lysimeter 1 and kg ha 1 ) added to each treatment. ................................ ................................ ................................ ... 26 2 1 Biomass and sucrose yield data. ................................ ................................ ........ 44 2 2 July leaf nutrient concentrations. ................................ ................................ ........ 46 2 3 July Critical Nutrient Level (CNL) categories. ................................ ..................... 47 2 4 September leaf nutrient concentrations. ................................ ............................. 48 2 5 Sept. Critical Nutrient Level (CNL) categories. ................................ ................... 49 2 6 Sugarcane leaf Diagnosis and Recommendation I ntegrated System (DRIS) indices ................................ ................................ ................................ ................ 50 2 7 Correlations between leaf nutrient concentrations in July and the harvest data. ................................ ................................ ................................ ................... 51 2 8 Correlations between leaf nutrient concentrations in September and harvest data. ................................ ................................ ................................ ................... 51 3 1 Soil organic matter (OM) for each treatment for each sampling date. ................ 74 3 2 Cation exchange capacity (CEC) for each t reatment for each sampling date ... 75 3 3 pH for each treatment for each sampling date ................................ .................... 76 3 4 Total phosphorus (TP) in soils for each t reatment for each sampling date ......... 76 3 5 Mehlich 3 phosphorus (M3 P) in soils for each treatment for each sampling date. ................................ ................................ ................................ ................... 78 3 6 Total Kjeldahl nitrogen (TKN) for each treatment for each sampling date. ......... 79 3 7 Soil nitrate ( NO 3 N) for each treatment on each sampling date .......................... 80

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9 3 8 Soil ammonium ( NH 4 N) for each treatment on each sampling date. ................. 80 3 9 Microbial Biomass Carbon (MBC) on each sampling date ................................ 81 3 10 Microbial biomass nitrogen (MBN) on each sampling date ................................ 82 3 11 Microbial biomass phosphorus (MBP) on each sampling date. .......................... 84 3 12 Phosphatase activity (PA) on each sampling date measured by the amount of p nitrophenol released. ................................ ................................ ................... 84 3 13 Correlations between soil quality parameters, biomass yield (kg cane lysimeter 1 ) and sucrose yield (kg sucrose lysimeter 1 ). ................................ ...... 86 3 14 Correlations between the microbial properties of the soil and soil physiochemical properties at the beginning of the experiment ........................... 87 A 1 Sugarcane leaf nutrient critical values and optimum ranges (Anderson and Bowen, 1990). ................................ ................................ ................................ .... 92 A 2 Sugarcane leaf nutrient sufficiency ranges for defining nutrient management categories (McCray and Mylavapu, 2010) ................................ ........................ 93 B 1 Brix values (g of soluble solids per kg of juice) and Pol values (unitless value of polarization of the juice) used in the calculations for % yield of sucrose. ....... 94 C 1 Average monthly air temperature and rainfall ................................ ..................... 95

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10 LIST OF FIGURES Figure page 1 1 Map of the Everglades Agricultural Area in south Florida. Source: South Florida Water Management District. ................................ ................................ ... 22 1 2 Relative N, P, and K requirements (%) for sugarcane during different growth stages. (Adapted fro m Bachchhav, 2005 and FAO, 2013). ................................ 23 1 3 Polyethylene lysimeters containing sandy soil, amendments, and three sugarcane plants. ................................ ................................ ............................... 24 2 1 Change in tiller count for each treatment over 6 month period, May October. ... 43 2 2 Change in dewlap height for each treatment over 6 month period, May October. ................................ ................................ ................................ .............. 43 2 3 Biomass yield for each treatment at harvest in terms of kg cane/lysimeter ........ 44 2 4 Number of millable stalks for each treatment at harvest. ................................ .... 45 2 5 Sucrose yield for each treatment at harvest in kg sucrose/ lysimeter. ................ 45 3 1 Soil organic matter over time for each treatment. ................................ ............... 74 3 2 Cation exchange capacity (CEC) in soils over time for each treatment. ............. 75 3 3 pH of soils over time for each treatment. ................................ ............................ 76 3 4 Total phosphorus (TP) in soils over time for each treatment. ............................. 77 3 5 Mehlich 3 phosphorus in soils over time for each treatment. .............................. 78 3 6 Total Kjeldahl nitrogen (TKN) in soil over time for each treatment. ..................... 79 3 7 Soil nitrate over time for each treatment. ................................ ............................ 80 3 8 Soil ammonium over time for each treatment. ................................ .................... 81 3 9 Microbial biomass carbon of soils over time for each treatment. ........................ 8 2 3 10 Microbial biomass nitro gen of soils over time for each treatment. ...................... 83 3 11 Microbial biomass phosphorus of soils over time for each treatment. ................ 84 3 12 Phosphatase activity in soils over time for each treatment. ................................ 85

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RECYCLING AGRICULTUR AL BY PRODUCTS TO GROW SUG ARCANE ON SANDY SOILS IN SOUTH FLORI DA By Susanna M. Gomez August 2013 Chair: Samira Daroub Major: Soil and Water Science Sustainable farming techniques, such as the use of agricul tural by products as organic fertilizer have the potential to improve sugarcane ( Saccharum spp ) yields grown on sandy soils with low organic matter (OM) content This study was designed to investigate three types of agricultural by products in the Everglades Agricultural Area for their potential use as organic soil amendment s and substitute inorganic phosphorus (P) fertilizer to grow sugarcane on sandy soils in south Florida Th e mesocosm experiment lasted twelve month s during which sugarcane was grown in lysimters using mill mud, mill ash, a 50/50 mix of mill mud and mill as h, and water lettuce (WL ; Pistia stratiotes ) as a soil amendment at a high, medium, and low application rate. We hypothesized that these ame ndments w ould add OM and increase microbial activity, therefore adding nutrients to sandy soils to increase sugarcane yield All amendm ents were compared to the control that only received recommended rates of inorganic fertilizers. Mill mud treatments (18.3 kg cane lysimter 1 ) and mix treatments (19.9 kg cane lysimter 1 ) produced the most biomass and were significantly different than the control at 6.55 kg cane lysimter 1 These two treatments showed higher levels of leaf nutrient concentration s and less nutrient imbalances compared to the control according to the

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12 critical nutrient level (CNL) and the Diagnosis and Recommendation Integrat ed System (DRIS) approach In addition t hese treatments added the most OM to the soil, which contributed to significant increases in microbial biomass, phosphatase activity (PA), cation exchange capacity (CEC), and soil nutrients. Reasons for these increases in mic robial activity and nutrient availability were attributed to the low C:N ratio of 15.5 for mill mud and 17.7 for the mix material. The mill mud and mix treatments showed no significant decrease s in soil OM, CEC, total P, and available P across the 12 month sampling period, indicating that th ese material s served as successful organic amendment s and P fertilizer for at least an entire year. Mill ash treatments, except the treatment with the highest rate of mill ash, showed no significant differences from the control in any soil physiochemical and microbial property. These results were reflected in the harvest data, where the mill ash medium and low application treatments showed no significant differences in the sugarcane biomass or sucrose yield. However, the high C:N ratio of 56 :1 suggest s that mill ash may degrade slowly and be useful for long term benefits with ratoon crops The WL high and medium treatments did not allow the sugarcane to enter the formative growth stage, which was attributed to inhibitory a llelopathic compounds excreted by the WL The WL low treatment failed to significantly increase sugarcane biomass and sucrose yields in comparison to the control, proving its inability to act as an organic soil amendment. Future research on long term benefits in field conditions, environmental impacts, and the economic feasibility of using mill mud, mill ash or a mixture of the two as soil amendments will further help scientist s and growers understand how to best manage t hese materials.

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13 CHAPTER 1 INTRODUCTION Backgroun d The Everglades Agric ultural Area (EAA) consists of 2833 km 2 of the original Everglades region of south Florida (Figure 1 1) which was artificially drained to sustain an annual $2 billion agriculture farming industry within the region (Alvarez and Polopolis, 2012) Sugarcane ( Saccharum spp ) is produced on approximately 1600 km 2 (Morgan et al., 2009). Approximately 80% of H istosols) south of Lake Okeechobee in the EAA (Gilbert et al., 2008). These soils are fertile, highly productive and have high water and nut rient holding capacities. Approximately 20% of sugarcane in Florida is grown on sandy soils bordering the EAA (Gilbert et al., 2008). Sugarcane grown on sandy soil typically produces lower yields than sugarcane grown on muck soil due to the poor water hold ing and nutrient retention capacity of the these soils. However, with the depth of organic soil decreasing over time due to oxidation, the amount of sugarcane grown on sandy soil is slowing increasing. This means that more inorganic fertilizer or more inno vative nutrient management techniques will be needed to grow high yield ing crops in the area. Sugarcane Overview Sugarcane is a tall perennial grass of the genus Saccharum In Florida, it is typically planted from late August through January using vegetati ve propagation and harvested from late October to mid April (Baucum and Rice, 2009). At harvest, stalks, also known as millable cane, are counted, weighed, and crushed for sugarcane juice. In Florida, an average sugarcane stalk weighs about 1.4 kg and cont ains 0.14 kg sugar,

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14 which is about 11% sugar by weight (Baucum and Rice, 2009) After a field is harvested a second crop of stalks, called a ratoon, grows from the old plant stubble. On average, 3 annual crops are harvested from one field before the field is replanted (Baucum and Rice, 2009). Sugarcane has four growth stages: the germination stage, the tillering (formative) stage, the grand growth st age, and the ripening stage ( FAO, 2013 ). The germination stage lasts about 30 to 35 days while the tilleri ng stage starts around 40 days after planting and may last up to 120 days ( Steduto et al. 20 12 ). Tillering is a physiological process of repeated branching of the primary shoot of the plant and provides the crop with an appropriate number of millable stal ks. The grand growth stage starts approximately 120 days after pl anting and lasts up to 270 days. The growth stage is followed by the ripening stage which occurs from day 270 to day 360 in a 12 month crop cycle ( Steduto et al., 2012 ). Sugar synthesis and accumulation of sugar takes place during the ripening phase and vegetative growth is reduced (Steduto et al., 2012 ). Understanding the growth cycles is important for maximizing cane and sugar yields. Figure 1 2 shows when nitrogen ( N ) phosphorus ( P ) and potassium ( K ) are primarily consumed by the plant in relation to its growth stages. Nit rogen is most needed during the tillering phase and excess N during the ripening phase can limit sugar accumulation (Rice et al., 2010). Similar to N, the plant consume s the majority of P during the tillering phase, but it is not needed in as large of quantities. Potassium, however, is most rapidly consumed during the grand growth and ripening phase of the sugarcane growth cycle. This information can aid in efficient wat er and nutrient

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15 management strategies, which may help to explain nutrient levels in the plant and soil during certain times of the growing cycle. Agricultural By Products The sugar industry in Florida produces a number of by products including bagasse, mill ash, and mill mud. Bagasse is a fibrous residue left after the juice is extracted and is re used to fuel the mill boilers to generate steam and electricity burne d. Mill m ud, also known as filter mud, filter cake, or pressmud is solid material left after the filtering of cane juice (Qureshi, 2000). Mill mud contains fairly high quantities of plant nutrient s and is high in organic matter (OM) and moisture content Nutrients have been found to range between 0.9 % to 2.24% for P 1.4% to 2.24% for N and 0.11% to 1.9% for K (Chinlo y and Innes ,1953; 1958; Alexander, 1972; Soloaimalai, 2001; Khan,2011). Moisture content has been reported around 75% (Alexander 1972 ; So laimalai et al., 2001 ) and pH of mill mud has been reported between 6.6 and 7.8 ( Kumar and Mishra, 1991 ; Khan et al., 2011). Mill ash has nutrient concentrations between 0.1% to 0.46% for P 0.0% to 0.13% for N and 0.2% to 0.47% for K (Dee et al., 2002; K han et al., 2011). The pH of mill ash has been reported between 7.5 and 9.2 ( Dee et al., 2002; Khan et al., 2011 ) The composition of both mill mud and mill ash will vary depending on locality, variety of cane, mill efficiency, and clarification method (So laimalai et al. 2001). In addition to mill mud and mill ash, water lettuce ( Pistia stratiotes ) is another potential source of an organic amendment which can be found in abundance in the EAA. Water lettuce (WL) is a floating aquatic weed that shows a high nutrient uptake capacity, fast growth rate, and high biomass produc tion (Reddy and Sutton, 1984).

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16 However, Thick mat s of WL on the canal surface impedes light from reaching submerged aquatic vegetation, which has been shown to increase pH from photosynthe tic activity and cause the co precipitation of P with the CaCO 3 bedrock (Otsuki and Wetzel, 1972; Murphy et al., 1983). The abundance of WL in the canals also decreases the dissolved oxygen in the water column, which negatively affects the biodiversity of the canals and downstream ecosystems. It is also considered a nuisance by farmers since it can clog up drainage pumps in the canals. Harvesting and using water lettuce as a soil amendment may alleviate the problems as sociated with WL in EAA farm canals. L iterature Review Mill mud and mill ash could be used as an organic fertilizer on sandy soils in south Florida since they are typically high in OM, P, N, and calcium (Ca) (Gilbert et al, 2008). In Trinidad, Prasad (1976a) inves tigated the effects of mill mu d, N, P, and K fertilizer on sugarcane yield and sucrose content The results showed that P fertilizer was not required when mill mud was applied in quantities of 20 tons ha 1 dry and that mill mud can partly substitute for K fertilizers. For N it was recommended that the standard applic ation rate in Trinidad of 94 kg N ha 1 should be applied with mill mud, particularly in coarser textured soils. Fewer experiments have been performed using mill ash in comparison to mill mud; however, results from Dee et al. (2002) showed that the addition of mill ash on acidic soil could significantly raise the pH and increase P and s i licon (Si) in the soil to improve soil fertility. Since sandy soils have low OM and low nutrient holding capacities, mill mud applications have been shown to increase P, Ca m agnesium ( Mg ) m anganese ( Mn ) and z inc ( Zn ) in soils, which could help prevent the depletion of soil nutrients and

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17 maintain soil fertility (Prasad, 1976b; Bokhitar and Sakuri, 2005b). A study by Kumar et al. (1985) in India found that mill mud application increased water retention and available water in sandy soils. The addition of mill mud reduced the percolation lo sses of water and nutrients, which enhanced the surface irrigation applying efficiency in sandy soil s With the addition of mill mud, they also saw an increase in c ation exchange capacity ( CEC ) from 3.10 to 11.69 cmolc/kg in their sandy soil amended with mill mud (Kumar et al., 1985) The increase in organic C from the addition of mill mud and mill ash and its effects on microbial activity was investigated in South Africa by Dee et al. (2002). By measuring CO 2 production they found that microbial respiration rates increased after addition of both mill mud and mill ash. The addition of either amendment also d oubled the aryl sulphatase activity and increased acid phosphatase activity by about 17%. A decrease in the accumulation of mineral N with increasing rates of addition of each amendment was also found. The decrease in N was associated with an increase in CO 2 evolution and greater microbial immobilization of N. The use of mill mud as an organic fertilizer has shown positive results in terms of yield increase and sugar content (Yaduvanshi and Ya dav, 1990; Bokhtiar and Sakurai 2005a, 2005b; Prasad, 1976a; Gil bert et al., 2008). In Bangladesh, Bokhtiar and Sakurai (2005a, 2005b) reported tha t mill mud at a rate of 15 tons ha 1 increased the tiller count and the number of millable cane stalk s in both plant and ratoon crops. The results from Yaduvanshi and Yadav (1990) showed that sugarcane biomass increased by 13% after mill mud applications; however, combining mill mud and N fertilizer increased biomass yields by 38%. Gilbert et al. (2008) explored the use of mill mud

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18 appli cation at a rate of 224 tons ha 1 on s andy soils in southern Florida. The study showed that additions of mill mud in a cumulative 3 yr crop cycle produced 4.1 ton sucrose ha 1 a 24% increase more than the standard fertilizer rate s alone. Water lettuce has successfully been demonstrated to hav e the potential for removing P from the water column (Reddy and Sutton, 1984; Lu et al., 2010). However, it may not be effective as a long term sink of P due to the high turnover rate which can generate organic labile detrital material that can accumulate at the sediment water interface. Since these plants have high nutrient uptake ability, they may be used as an organic fertilizer when harvested and dried. Claude Boyd (1969) collected water lettuce from Fort Lauderdale canals to determine the fertilizer u nits of water lettuce on a dry b asis and found it contained 2.5 % N, 0. 31 % P, and 3.5% K Project Statement and Objectives The purpose of this project is to investigate three types of agricultural by products in the EAA for their potential use as soil amend ments and substitute for inorganic P fertilizer on sandy soils. Using mill mud, mill ash, and WL, we hypothesize d that these amendments will add OM and increase microbial activity, therefore adding nutrients to sandy soils to increase the biomass yield and sucrose yield of sugarcane. The objectives of this research were to: 1. Design and set up a mesocosm experiment to test the effectiveness of mill mud, mill ash, water lettuce and a 50/ 50 mixture of mill mud and mill ash to grow sugarcane on sandy soil. 2. Eval uate the effects of each amendment on sugarcane plant growth in terms of plant height, number of tillers, nutrient content of the plant, biomass yield, and sucrose content 3. Evaluate the effects of each amendment on the physio chemical cha racteristics of the soil matrix including the effects on the microbial community in terms of microbial biomass and phosphatase activity.

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19 4. Determine if each amendment effectively improves soil quality and sugarcane yields on sandy soil by comparing each trea tment to the control with no organic amendments through statistical analyses. Materials and Methods Materials The mill mud and mill ash were obtained from the Glades Sugar House in Belle Glade, Florida on behalf of the Sugar Cane Growers Cooperative of Flo rida. The materials arrived fresh to the Everglades R esearch and Education Center in Belle Glade, Florida where they were piled on site for one month before use. Water lettuce was collected from local farm canals in Belle Glade, Florida. It was air dried for 14 days before being ground to pass through a 6.45 cm 2 ( 1in 2 ) mesh Oldsmar sand (Sandy, siliceous, hyperthermic Alfic Arenic Alaquods) was obtained from Clewiston, Florida. This soil is representative of the sandy soil used to grow sugarcane, containi ng 2.4 % OM The physio chemical characteristics of the amendments and the sandy soil matrix are described in Table 1 1 Experimental Design A mesocosm experiment was carried out in 0.265 m 3 ( 70 gallon ) polyethylene lysimeters (Figure 1 3) Each lysimeter was filled with the Oldsmar sand until it was either 15.3 cm (6 in), 10.2 cm (4 in), or 5.1 cm (2 in) from the top of the lysimeter The sandy soil was then amended with mill mud, mill ash, a 50/50 mixture of mill mud and mill ash, or dried WL at a high, medium, or low application rate. The high application added 15.3 cm ( 6 in ) of the amendment into the sandy soil matrix, the medium rate added 10.2 cm (4 in ) of the amendment and the low added 5.1 cm ( 2 i n ) of the amendment to the lysimeter The amendments added were then incorporated into the top 30.5 cm (12 in) of the sandy soil matrix The depths added to each lysimeter were

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20 then co nverted into application rates ( Table 1 2) using the dry weight of the amendments added to each bucket and the area of the bucket ( 0.61 m 2 ). The control lysimeter did not include any amendments, but incorporated the recommended rate of 192 kg ha 1 (160 lb ac 1 ) diammonium phosphate fertilizer (DAP) into the sandy soil matrix (Morgan et al., 2009). The design was a randomized b loc k design with three blocks of 13 sugarcane lysimeters, so each treatment along with the control was cond ucted in triplicates totaling 39 sugarcane lysimeters. Planting and Fertilizer Applications Aft er incorporation of amendments, t hree sugarcane seedl ings of variety CP 78 1628 were transplanted in each lysimeter on March 1 st 2012 This variety is the most widely grown cultivar on mineral soils in Florida ( Rice et al., 2011). During the growing season the lysimeters received the recommended rate of fer tilizer application for sugarcane on sandy soils to supplement essential nutrients. Each experimental lysimeter received ammonium nitrate for N potash for K, SulPoMag for S K and Mg, and a Micromix for Zn, B, Cu, and Mn. The rates were applied to meet the recommended nutrient rates of 202 kg N ha 1 (180 lb N ac 1 ), 280 kg K ha 1 (250 lb K ac 1 ), 6.7 kg Mg ha 1 (6 lb Mg ac 1 ), 2.2 kg Zn ha 1 (2 lb Zn ac 1 ), 1.12 kg B ha 1 (1 lb B ac 1 ), 2.2 kg Cu ha 1 (2 lb Cu ac 1 ), and 5.6 kg Mn ha 1 (5 lb Mn ac 1 ) acco rding to Rice et al. (2010) The control pots received these application in addition to recommended rate of 192 kg ha 1 (160 lb ac 1 ) of DAP fertilizer for P to meet the nutritional recommendation of 84 kg P ha 1 (75 lb P ac 1 ) according to Rice et al. (2010 ). DAP is a water soluble fertilizer that was applied as a one time application. Table 1 3 and Table 1 4 show the fertilizer chosen, the amount applied to each bucket, and how often they were applied

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21 fotr the treatment and control lysimeters. Rates of fertilizer application where converted into kg nutrient lysimeter 1 t o compare the amount of nutrients added to each treatment (Table 1 5). Experimental Ana lyses For the plant analyses, plant height and number of tillers were counted T he height was measured to the top visible dewlap (TV D), a diagnostic tissue that is frequently used in the evaluation of the nutritional status and is located on th e blade joint of the plant leaf. Leaf tissues were collected in July and September and the nutrient compos ition s were analyzed fo r N, P, K, Ca, Mg, Si, Zn, Mn, c opper (Cu), and i ron (Fe). Sugarcane was harvested on November 1 st 2012 where m illable stalks, weight of cane, and sucrose yields were measured. Details of these analyses are explained in C hapter 2. T o test the changes in the physio chemical properties of the soil after incorporation of amendments moisture content, OM CEC, total phosphorus (TP), Mehlich 3 phosphorus (M3), total Kjeldahl nitrogen (TKN), soil nitrate, soil ammonium, and soil pH were ana lyzed. Soil samples were collected on three sampling dates: March 7 th 2012, September 30 th 2012, and March 4 th 2013. Details of these analyses are discussed in C hapter 3. Microbial biomass nitrogen (MBN), microbial biomass carbon (MBC), microbial biom ass phosphorus (MBP), and alkaline phosphatase activity (PA) were analyzed to determine the effects of each amendment on microbial properties of the soil. Details of these pr ocedures are also discussed in C hapter 3.

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22 Figure 1 1 Map of the Everglades Agricultural Area in south Florida. Source: South Florida Water Management District.

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23 Figure 1 2. Relative N, P, and K requirements (%) for sugarcane during different growth stages. (Adapted from Bachchhav, 2005 and FAO, 2013 ). Table 1 1. Physio chemical characteristics of the amendments and the sandy soil matrix Material Moisture (%) OM (%) TP (%) TKN (%) NO 3 N (mg/kg) NH 4 N (mg/kg) pH Mill Mud 55.4 75.5 1.02 2.80 0.00 0.12 8.1 Mill Ash 39.3 45.5 0.60 0.47 0.01 0.00 8.8 50/50 Mix 54.3 61.6 1.01 2.00 0.00 0.01 8.5 Water Lettuce 52.9 71.1 0.34 1.90 0.01 0.18 8.9 Sandy Soil 4.3 2.4 0.08 0.12 0.00 0.00 7.7

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24 Figure 1 3. Polyethylene lysimeters containing sandy soil, amendments, and three sugarcane plants. Table 1 2 Application rates (tons/ha) of each amendment at the high, medium, and low depths Amendment High Medium Low Mill Mud 488 317 175 Mill Ash 481 323 171 50/50 Mud/Ash Mix 365 270 132 Water Lettuce 202 125 65

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25 Table 1 3 Treatment l ysimeters f ertilizer r ates NH 4 NO 3 DAP Potash SulPoMag Micromix Fertilizer Rate ( kg ha 1 ) 97.10 0.00 69.20 33.40 4.03 Lysimeter Rate (g lysimeter 1 ) 36.40 0.00 23.99 12.50 2.30 Application 1 (g lysimeter 1 ) 9.10 0.00 4.97 12.50 2.30 Application 2 (g lysimeter 1 ) 9.10 0.00 9.51 0.00 0.00 Application 3 (g lysimeter 1 ) 9.10 0.00 9.51 0.00 0.00 Application 4 (g lysimeter 1 ) 9.10 0.00 0.00 0.00 0.00 Table 1 4 Control l ysimeters f ertilizer r ates NH 4 NO 3 DAP Potash SulPoMag Micromix Fertilizer Rate ( kg ha 1 ) 81.30 29.9 69.20 33.40 40.30 Lysimeter Rate (g lysimeter 1 ) 30.54 11.21 23.99 12.50 2.30 Application 1 (g lysimeter 1 ) 3.24 11.21 4.97 12.50 2.30 Application 2 (g lysimeter 1 ) 9.10 0.00 9.51 0.00 2.30 Application 3 (g lysimeter 1 ) 9.10 0.00 9.51 0.00 0.00 Application 4 (g lysimeter 1 ) 9.10 0.00 0.00 0.00 0.00

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26 Table 1 5. Amount of total phosphorus and nitrogen added to each treatment. Treatment kg P lysimeter 1 kg N lysimeter 1 kg P ha 1 k g N ha 1 Mud High 0.277 0.508 2959 8113 Mud Medium 0.180 0.494 2873 7886 Mud Low 0.099 0.188 1100 3010 Ash High 0.161 0.126 2568 2011 Ash Medium 0.108 0.085 1725 1351 Ash Low 0.057 0.045 913 715 Mix High 0.206 0.406 3286 6482 Mix Medium 0.152 0.301 2425 4802 Mix Low 0.074 0.147 1185 2347 WL High 0.038 0.123 611 1959 WL Medium 0.024 0.076 379 1215 WL Low 0.012 0.040 197 632 Control 0.005 0.012 84 202 Water Lettuce (WL)

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27 CHAPTER 2 SUGARCANE RESPONSES Background Sugarcane growers rely on inorganic fertilizers to improve yields; however, with increasing fertilizer prices new nutrient management strategies need to be investigated to reduce cost and improve soil fertility (Gilbert et al., 2008). This is especially true in South Florida, where approximately 80,000 ac res of sugarcane is grown on mineral soils with low capacity to hold water and nutrients (McCray et al. 2011). Standard fertilization practices on sandy soils in Florida involve splitting recommended rates into 3 to 4 applications (Anderson,1989) ; however positive yield responses with up to 13 applications have been recorded over a three year period (Obreza et al., 1998). Such large numbers for fertilizer applications could diminish profits, so the use of abundant and local organic amendments such as mill mud, mill ash, and water lettuce (WL) could reduce cost and promote sustainability. The benefits of mill mud application will vary with soil type and fertilizer use (Gilbert et al., 2008), although it has been concluded that mill mud application would be most benefi cial in soils with low available P much like the sandy soils in s outh Florida (Alexander 1972 ; Moberly and Meyer, 1978). Chinloy and Innes (1953) reported that mill mud application at 25 t ons ha 1 could replace 18% of superphosphate to obt ain an equal yield of sugarcane yield. Jurwaker et al. (199 3 ) found that application of 20 t ons ha 1 resulted in a saving of recommended inorganic fertilizer on wheat, rice, maize, and sugarcane. Substantial reduction s of N fertilizers after application of mill mud were observed by Yaduvanshi and Yadav (199 0 )

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28 Application of mill mud at rates of 175 200 t ons ha 1 to sugarcane fields provided enough P for the for the ratoon group while increasing the cane yield up to 37.4 t ons ha 1 ( Solaimali et al., 2001 ). Gilbert et al. (2008) explored the use of mill mud application at a rate of 224 t ons ha 1 on sandy soils in southern Florida. The study showed that additions of mill mud in cumulative 3 yr crop cycle produced additional 4.1 ton sucrose per hectare, a 24% increase more than the standard fertilizer rate alone Mill ash has been shown to improve the height, stalk and straw yield with an application of 50 t ons ha 1 to wheat fields (Khan, 2011). Boyd (1969) stated that a gre en manure was a poss ible use for WL since fertilizer units for N P and K were rather high on a dry weight basis. Hypothesis and Objectives Chapter 2 focuses on how the sugarcane plant s responded to each amendment compared to the control. We hypothesize that mill mud, mill as h, and WL will increase sugarcane yields due to an increase in available nutrients which will be noticeable in the leaf nutrient concentrations The objectives of this chapter are to: 1. Evaluate how sugarcane responds to the different amendments in terms of tiller count and dewlap height over time 2. Compare the sugarcane biomass yield and sucrose yield o f each amendment to the control. 3. Evaluate the nutritional value of each amendment through leaf tissue analysis, critical nutrient levels, and the diagnosis and r ecommen dation integrated system (DRIS) 4. Correlate nutrient leaf concentrations to sugarcane biomass yield and sucrose yields

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29 Methods and Materials Sandy soil borde ring the EAA was amended with mill mud, mill ash, a 50/50 mixtu re of mill mud and mill ash, and dried WL at a high, medium, or low application rate in 0.265 m 3 ( 70 gallon ) polyethylene lysimeters (Table 1 2) Each treatment was compared to the control that received th e recommended rate of diammonium phosphate fertilizer (192 kg DAP ha 1 ) instead of an organic amendment. Each lysimter received three transplanted sugarcane seedlings of the CP 78 1628 variety on March 1 st 2012. Starting in May, the sugarcane plants were measured for height and number of tillers, which later in the season become millable stalks. T he height was measured to the top visible dewlap (TV D), a diagnostic tissue that is frequently used in the evaluation of the nutritional status and is located on th e blade joint of the plant leaf. The sugarcane plants were harvested on November 1 st 2012 Each treatment along with the control was conducted in triplicates totaling 39 sugarcane lysimeters. The design was a randomized block design with three blocks of 13 sugarcane lysimeters. Further details of the experimental design are explained in Chapter 1. This experiment was conducted in the EAA at the Everglades Research Education Center in Belle Glade, Florida. Leaf Nutrient Concentration In the middle (July) and the end (September) of the prime growing season the nutrient composition of the plant tissue were analyzed for N, P, K, Ca, Mg, Zn, Mn, Cu, Fe, and Si Ten top visible dewlap leaves were harvested at random from each lysimeter. The leaf samples were separated from midribs, rinsed with DI water, dried for one week at 60C and ground to pass a 1 mm screen before being sent to the UF/IFAS Soil Testing Laboratory in Gainesville, Florida for analysis. The plant tissue was

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30 analyzed for P, K, Ca, Mg, Z n, Mn, Cu, Si, and Fe using a 12.1 M hydrochloric acid digestion after tissue had been ashed for 5 hours at 500 C in a muffle furnace (EPA, 1996, Method 200.7) Digestions were filtered through a Whatman No. 62 filter and analyzed using an inductively c oup led a rgon p lasma (ICP) ( Spectro Arcos, Marlboro, MA ). Total Kjeldahl nitrogen was determined after a sulfuric acid digestion ( Bremner 19 96 ) and analyzed on an Alpkem Flow IV auto analyzer ( O.I. Analytical, College Station, TX ) using the semi a utomated col orimetric analysis. The nutrient lea f c oncentration results were categorized into very deficient (VD ), deficient (D), marginal (M), sufficient (S), and sufficient plus (SP) based on the Critical Nutrient Level (CNL) approach as described in McCray et al. (2010) To evaluate nutrient imbalances, the Diagnosis and Recommen dation Integrated System (DRIS) was used with the Sugarcane DRI S Ca lculator ( http://erec.ifas.ufl.edu/exte nsion/sugarcane_calculator.shtml ) A negative DRIS index value indicates the nutrient is insufficient relative to other nutrient levels and a positive DRIS index value indicates the nutrient is excessive relative to other nutrients. A DRIS index value of zero or near zero indicates the nutrient is in optimum balance with other nutrients. The Nutrient Balance Index (NBI) was calculated as the sum of the absolute value of all DRIS indices for a sample, where the higher the number the more imbalance Yield M easurements Yield measurements were performed on November 1st 2012. Millable stalks from each lysimeter were counted. Plant fresh weights were used to determine individual stalk weight (kg stalk 1 ) and biomass yield was calculated as the product of stalk number and stalk weight. To determine sucrose concentration a 10 stalk harvest

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31 random sample was collected from each lysimeter milled, and the crusher juice analyzed for brix and pol. Brix, which is a measure of percent soluble solids, was measured u sing a refractometer that automatically corrected for temperature. Pol, which is a unit less measure of the polarization of the sugar solution, was measured using a saccharimeter. The Brix and Pol measurements were plugged into the theoretical recoverable sugar method calculator (Legendre, 1992), which determines percent sucrose. The amount of sucrose per lysimeter was calculated as the product of the percent sucrose and the biomass yield divided by the area of the lysimeter (0.61 m 2 ). Statistical Analysis Analyses of variance for nutrient leaf co ncentration biomass yield, and sucrose yield were performed for a randomized complete bloc k design using the generalized l inear mixed mod els procedure (PROC GLIMMIX) on SAS 9.3 (SAS Inc., 2011). All treatment effec ts were considered significant at p < 0.05, and pairwise comparisons were made using the lsmeans statement with the Tukey method. Plant growth was measured as repeated measures over time and analyzed lsmeans between treatments and across the 6 month sampl ing dates Pearson correlations between nutrient leaf co ncentrations and yield data (biomass and sucrose) were analyzed using a 2 tailed significant (p < 0.05) on SPSS 20 (IBM Corp., 2011) Results Plant Growth The WL high and WL medium treatments did not su rvive their conditions in any of the replicates ; therefore, there will only be data available for the WL low treatments The number of tillers counted once a month showed that all treatments had a similar trend across the months of May through October (Figure 2 1) All treatments peaked in

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32 June, declined in July and August and began to level out in September. Mill mud treatments and mix treatments had the highest number of tillers throughout the six month measurement period, while the control had the lo west number of tillers throughout. The WL low treatment began with the lowest tiller count, but showed a lesser decline after its peak in June. The WL treatment also had the largest variation, and by the last two months was not significantly different than any other treatments The height of each plant increased significantly each month from May to August. All mill ash treatments mill mud medium mill mud low treatment, WL low, and the control showed no significant change i n height compared to the control from September to October. The mill mud high treatment and all mix treatments still showed a significant difference in plant height from September to October. In general, the mill mud treatments and the mix treatments had the greatest height each month wh ile the WL low treatment had the shortest height. Figure 2 2 shows the plant growth over time for each treatment. Biomass Yields Biomass yield (kg of cane lysim e ter 1 ) showed a significant treat ment effect ( p<0.001 ). The treatments that produced the highe st biomass were the mill mud treatments (18.34 kg cane lysim e ter 1 1.85) and the mix treatments (19 89 kg cane lysimter 1 2.52). There were no significant differences between the rates of either of these treatments. The control lysimeters had an average of 6.55 kg cane lysimter 1 ( 0.65 ), which was significantly lower than all mill mud treatments, mix treatments, and the mill ash high treatment These results are illustrated in Table 2 1 and Figure 2 3 The number of millable stalks showed a slightly di fferent trend ( Table 2 1, Figure 2 4 ) The control lysimeters were still the lowest with 13.67 (4.62) stalk s, but were not

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33 significantly different from any treatment except the mill mud treatments and the mix high treatment. The WL low treatment had the s econd lowest weight of yield (6.7 kg cane lysimeter 1 1.91), but was not significantly different from any treatment in terms of millable stalks. This suggests that there were many stalks but were ver y thin and light, which is confi rmed in th e stalk weight data in Table 2 1 All the mix treatments and the mill mud high treatment had significantly higher stalk weights than the control. Sucrose Yields The results for sucrose yields can be seen in Table 2 1 and Figure 2 5 Mix treatments produced the most sucrose with an average of 2.4 kg lysimter 1 ( 0. 2 ) followed by mill mud treatments with 2.1 kg lysimter 1 ( 0.4 ) ; however, there was not a significant difference between these two treatments. The control lysimeter had 0.82 kg sucrose lysimter 1 which was significantly lower t han all the mill mud treatments and all the mix treatments. However, the control was significantly higher than the mill mud high, mill mud medium, and WL low treatment for percent sucrose yield, wh ich is the percent sucrose by weight. Nutrient Leaf C oncentrations Lead nutrient concentration s and their nutrient management categories for July and September are shown in Table s 2 2 2 3 2 4 and 2 5 In the July sampling, 8 out of the 10 leaf nutrients, P, K, Ca, Mg, Fe, Mn, Zn, and Cu were placed in the sufficient or sufficient plus category for mill mud treatments The mill mud had marginal N except mill mud low, which had very deficient N and very deficient Si Mix trea tme nts also showed positive result s with 7 out of the 10 nutrients falling in the sufficient or suffi ci ent plus categories: P, K, Ca Mg, Fe, Mn, and Zn. The Cu and N were marginal and the Si was deficient. The WL low treatment had the same results as the mix treatments. The

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34 control lysimeters had 6 out of the 10 nutrients above marginal (K, Ca, Mg, Fe, Mn and Cu). Mill ash treatment, however, only had 5 out of the 10 nutrients (K, Ca, Mg, Fe, and Mn) placed in the sufficient or sufficient plus category. Silicon was very deficient in all 11 treatments and N did not exceed marginal in any treatment except m i x high. The s tatistical analysis showed that every tested nutrient in each treatment significantly (p<0.0001) decreased from July to September. In Sep tember, P dropped to a marginal standing in the mill mud, mix, and th e WL low treatments and P dropped to very deficient in the mill ash treatments and the control Cu dropped to very deficient in all treatments, Zn d ropped to below sufficient in all treat ments except mill mud, and K dropped to below sufficient in all treatments except mud high, mud medium, and WL low. The DRIS index results, which allows for the examin ation of nutrient balance s, are summarized in Table 2 6 In July, the two macronutrients that appear to be most out of balance are N and Ca. N itrogen is low compared to other nutrients with a DRIS i n dex value of less than 10 especially in the mill mud medium and low treatments, the mill ash medium and low treatments, the mix low treatment, the WL low treatment, and the control Ca lcium is extremely out of balance as a surpl us with DRIS indices of greater than 10 for all treatments. The control has the highest value of 46.1. There are also two micronutrients that are out of balance in July, Zn and Cu. All the mill ash treatments and the WL low treatment have a DRIS value of less than 10 for both these nutrients, indicating a deficiency compared to other nutrients. The control and the mix medium treatment also have a DRIS v alue of less than 10 for Zn. The NBI (the s um of the absolute value of all DRIS indices for a sample) indicates that the control, the WL low,

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35 the mill ash medium and low, and the mill mud low treatments are the most imbalanced since the NBI index value was over 80. The mix treatments had the lowest set of NBI values. In September, nutrient imbalances become more severe; all treatments have a NBI value over 80 except the WL low treatment. N itrogen and Zn are no longer below 10 in the DRIS index; however, Ca and Cu values have become more extreme. The mill ash treatments have the worst imbalance s with NBI values over 250. These hi gh values come from high DRIS indices in N, P, K, Ca, and Mg and extremely low va lues for Cu. Correlations between nutrient leaf concentrations and yield data show that there is a significant correlation (p<0.05) between leaf N, Zn, P, Mg, and Cu and biomass yield (kg cane lysimeter 1 ) in July (Table 2 7) Nitrogen had the highest Pe arson correlation of 0.729 followed by Zn ( 0.619), P (0.594) Mg ( 0.477), and Cu (0.408). M agnesium P, N, Fe, Zn were significantly correlated to biomass yield in September (Table 2 8) Magnesium had the highest Pearson correlation of 0.748 followed by P ( 0.576) N ( 0.441 ), Fe (0.392), and Zn ( 0. 362 ). M anganese was the only lea f nutrient to have a negative correlation, although it was not significant at the 0.5 level. Z inc N, P, Cu, and Mn were significantly correlated to sucrose content ( kg sucrose lysimeter 1 ) in July. Z i n c had the highest Pearson c orrelation of 0.546 followed by N (0.523), P (0.485), and Cu (0.442). M anganese was negatively correlated with a Pearson Correlation of 0.367. In September, only Mg was significantly correlated to sucros e content with a Pearson coefficient of 0.646

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36 Discussion Plant Growth and Yield Data Results indicated that mill mud, mill ash, and WL can have a significant effect on sugarcane growth and yield. The mill mud treatments produced on average of 180% more bi omass than the control. The mill ash treatment produced an average of 69% more biomass than the control, while t he WL treatment only produced 2.3% more than the control. With no significant differences between the WL and the control, the resources it takes to harvest, dry, and grind the WL may not be worth the benefits. The 50/50 mix of mill mud and mill ash produced the most with 204% more biomass than the control. This study was carried out i n lysim e ters with an area of 0.61 m 2 so it is unlikely that a large field study would see such drastic increases. Prasad (1976 a ) showed that applications of mill mud at 34 t ons ha 1 increased biomass yields by only 9.9%. Srivastava et al. (2012) showed a 30% biomass increase after application of 10 t ons ha 1 of mill mud. Yaduvansh et al. (1990) showed an increase of up to 65% with mill mud applications at 30 t ons ha 1 combined with 50 kg N ; however, we used much larger rates (Table 1 2) Gilbert et al. (2008) applied 224 t ons mill mud ha 1 a similar rate to those used in our experiment on sandy soil from south Florida and found only an increase in sugarcane biomass (tons cane ha 1 ) of 26%. However, he did find large increases in biomass compared to the controls of only inorganic fertilizer s during the first ratoon (a 58%increase) and the s econd ratoon (a 167% increase), which may indicate the potential of mill mud to be used as a slow release amendment. The lysimeters amended with WL at high (202 tons ha 1 ) and medium (125 tons ha 1 ) rates failed to support the growth of sugarcane in our lysimeters. The release of

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37 inhibitory allelopathic compounds may be responsible. Alliotta et al. (1991) and Gross (2003) found that WL contains lipophili c algicidal compounds, such as steroid derivatives and various fatty acids t hat were especially inhibitory. The release of allelopathic compounds interfere with settlement and/or growth of competitors in their vicinity (Gross, 2003), so these compounds may have inhibited the growth of the sugarcane plant. The WL low treatm ent, with 65 tons ha 1 was able to sustain growth through the 12 month experiment, but showed no significant increase in biomass or sucrose yield compared to the control (Table 2 1). Our results showed a significant increase in sucrose content after the a ddition of the mill mud, mill ash, and a 50/50 mix The mill mud treatments produced a n average of 160% more sucrose than the control. The mill ash treatment produced an average of 69% more sucrose than the control. The 50/50 mix of mill mud and mill ash p roduced 195% more sucrose than the control. However, the WL treatment produced 12.5 % less sucrose than the control, but was not significantly different. Although the mix and mud treatments produced the most sucrose in terms of kg sucrose lysimeter 1 the y had the lowest percent sucrose by weight, also known as percent yield of sucrose (Table 2 1) The control and mill ash treatments had the highest percent yield of sucrose. The reduced percent sucrose yield with higher mill mud rates is often observed ( Pr asad, 1976a; Bokhtiar and Sakurai, 2005a ), but is often a trade off for having increased biomass or cane yield. This may have happened because certain elements such as N influence how much sucrose the plant will store during the ri pening phase. Sugarcane w ill store a higher percent of sucrose when N is

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38 limited 6 to 8 weeks prior to harvest (Rice et al., 2010), which was shown in the leaf nutrients results for the control and the mill ash treatments. The dewlap height measurements and tiller counts showed t hat the type of amendment influences the scale of the measurements, but not the overall pattern of tiller count or dewlap height. It is possible that the height of the plant may have eventually reached a plateau, and that the type of amendment could influence when this would occur. This idea stemmed from the fact that during the last two months of measurements the mill mud high treatment and all mix treatments showed a signif icant difference in plant height, indica ting that it may not be near a plateau in height. However, all the other treatments showed no significant change in plant height during the last two months of measurement before harvest. The increase in number of m illable stalks of our treatments compared to the control was consistent with Bokhitar and Sakurai (2005 a ), Chatterjee et al. (1979), and Gilbert et al. (2008). The increased tiller population might be because the combination of chemical fertilizers and or ganic sources are more effectively used than chemical source alone. Matin et al. (1989) showed that tiller survivability increased from 35% to 60% for treatments receiving both chemical fertilizers and organic amendments versus those only receiving N, P, a nd K fertilizers. A study by Bokhitar and Sakurai (2005b) showed an increasing trend in production of millable canes where a mixture of organic amendments and chemical fertilizer was applied and increased up to 86.3% over control. The higher number of millable canes found in their organic amended plots was attributed to a decrease in tiller mortality due to higher nutrient uptake.

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39 There were no significant differences (p<0.05) for application rates within each treatment for number of millable stalks, k g cane per lysimeter, and kg sucrose per lysimeter. All rates could be considered high as they were all over 100 tons ha 1 whereas other studies used 10 tons of mill mud ha 1 (Srivastava et al., 2012), 15 tons of mill mud ha 1 (Bokhtiar and Sakurai, 2005a ,b), and 50 tons mill ash ha 1 (Khan, 2011). However, large of applications of 175 200 tons of mill mud ha 1 ( Soloaimalai, 2001 ) and 224 tons of mill mud ha 1 (Gilbert et al., 2008) have been applied to sugarcane plots with positive results. Large applica tion rates may be useful to growers in hopes to extend the longevity of the amendments in future ratoon cycles, especially since mill mud has been shown to increase yields in ratoon crops (Prasad 1976a; Bokhtiar and Sakuraia, 2005a ; Gilbert et al., 2008). The longevity of the amendments is expected since they serve a slow release fertilizer, while the DAP is a water soluble and readily available source of P. In addition a large, one time application may be more economical than multiple applications to susta in ratoon crops since transport cost are substantial due to the high moisture content of organic ame ndments (Qureshi et al., 2000). The increase in sugarcane biomass yield and sucrose yield compared to the control show s positive results for mill mud, mill ash, and a mixture of mill mud and mill ash as an organic amendment. There are many reasons why these amendments affected the harvest results in a favorable manner including the possible addition of organic matter, increased microbial activity, or an incre ase in available nutrients. These possible explanation s will be evaluated further in C hapter 3 Nutrient Leaf C oncentrations The CNL approach is a simplistic method that compares the leaf nutrient concentrations with a set of reference standards that incl udes established critical values

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40 (Miles, 2010). Values above or below the optimum range indicate a deficiency or toxicity that may limit growth. However, the CNL approach is very time sensitive because reference standards are calibrated for nutrient concen trations at a specific stage in the growth cycle (McCray et al., 2009a). A study by McCray et al. (2009b) confirmed that the leaf concentration critical values for Florida sugarcane developed by Anderson and Bowen (1990) and McCray et al. ( 2009a ) are most reliable for leaf samples collected during June and July. Therefore, our analysis will be more focused on the values obtained during the July sampling, rather than the leaf samples collected in September. Our CNL analysis from July indicates that N, Si, and Cu are marginal to deficient for every treatment including the control. These results are similar to the results by McCray et al. (2009 b ) which found that nutrients with the highest incidence of deficiency in mineral soils in south Florida were Si, N, Mg, and Fe. In addition, they showed that the sugarcane variety used in this study, CP 78 1628, generally had higher incidences of N, P, Si, and Fe deficiencies than any other cultivar. However, our study showed no deficiencies in P, Mg, and Fe, so it is possible the mill mud, mill ash, and WL supplemented these nutrients. The N deficiencies found by this study may be a cause for concern since N plays a pivotal role in yield optimization because it interacts and drives the uptake of many other nutrients (Miles, 2010). All the mill ash treatments and the control were categorized as very deficient with N concentrations below 1.6%. This may have occurred because N has the greatest influence on cane ripening of all the nutrient elemen ts and the cane will actually store a higher percent of sucrose when N is limited 6 to 8 weeks prior to harvest (Rice et al., 2010). This severe N deficiency in mill ash

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41 may have influenced the deficiencies in P, Cu, and Zn since Gosnell and Long (1971) re ported that N deficiency caused a noticeable reduction in uptake of P, K, Ca, Mg, Mn, Cu and Zn. The mill mud, mix, and WL treatments, however, had marginal N and showed that the other nutrient concentrations were still in the sufficient or sufficient plus category. Another possible reason the mill ash treatments have lower leaf nutrient concentrations is the high pH of 8.8. Zinc Mn, and Fe usually decrease in availability due to less microbial activity and because P complexes with Ca and Mg at a pH greate r than 8 (Brady and Weil, 2008). In addition to the deficiencies in Si and N in all treatments, the results of the leaf analysi s showed there was also an excess of P, K, and Mg in the mill mud and mix treatments. Barry et al. (2001) also noted increases in phosphorus concentrations above plant requirements from applications of mill mud to sugarcane fields in Australia. To determine if either the excess of nutrients or deficiencies caused nutrient imbalances in the plants the DRIS approach was implemented. Ca lcium was a large cause of nutrient imbalances with DRIS indices above 10 for all treatments and a high DRIS index of 46.1 in the control. The Ca imbalance may be a cause for concern since nutrient interactions occur in the plant and in adsorption site s in the soil for ions with the same valances and similar diameter s such as Mg and Mn (Wilkinson et al., 2000). However, the Mg and Mn indices for all treatments are close to zero, indicating no nutrient imbalances. The DRIS i n dex of less than 10 for N in the mill mud medium, mill mud low, ash medium, ash low, mix low, WL low, and the control reemphasized the need for optimum levels of N. Many studies have shown that mill mud applied along

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42 with N fertilizers greatly enhanced the yield (Yaduvanshi et. al, 1 990, Gilbert et. al, 2008; Srivastava et al., 2013). Although the WL treatment only had Si and Cu deficient in CNL, it had the highest NBI of 120.5 These large nutrient imbalances may have caused the low biomass and sucrose yield The mill ash medium and low treatments also had large NBI values of over 80. The main cause of the imbalance for mill ash came from Ca N, Zn and Cu. However, the N imbalance in the mill ash treatments was not as severe as in the mill mud treatments The mix of mill mud and mill ash has the lowest NBI indicating that it receives the best attributes of mill mud and mill ash without causing imbalances. All treatments except WL low, mill ash medium, and mill ash low have a lower nutrient balance index than the control. Combining t hese results with our biomass yield and sucrose yield results suggest that mill mud and a mix of mill mud and mill ash have a strong potential for its use as a P fertilizer and organic amendment. In addition, the DRIS analysis showed no imbalances of P in any treatment. To correct the imbalances shown in the results, additional supplements of N would be recommended. A study by McCray et al. (2010) showed that sugarcane responses to DRIS recommended fertilizer application during the summer was too late to result in substantial yield improvements. Therefore, the resu lts of our nutrient leaf analys i s would be most beneficial and cost effective to take the recommendations and optimize fertilizer applications the following year.

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43 Figure 2 1 Change in tiller c ount for each treatment over 6 month period, May October. Figure 2 2. Change in d ewla p h eight for each treatment over 6 month period, May October

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44 Table 2 1 Biomass and sucrose yield data. Biomass Yield Data Sucrose Yield Data Tr eatment Millable Stalks Stalk Wt. (kg) Kg cane % Sucrose Yield Kg sucrose Mud High 29* 0.71 20.5 10.5* 2.1 Mud Medium 30* 0.56 17.0 10.7* 1.8 Mud Low 26* 0.67 17.6 12.8 2.3 Ash High 18 0.66 11.9 12.8 1.5 Ash Medium 16 0.66 10.3 12.6 1.3 Ash Low 16 0.58 9.5 12.8 1.2 Mix High 29* 0.74 21.1 10.9 2.3 Mix Medium 25* 0.82 20.5 12.0 2. 5 Mix Low 26* 0.71 18.1 12. 9 2. 3* WL Low 19 0.42 6.7 9.6* 0.7 Control 14 0.50 6.6 12.5 0.8 Water Lettuce (WL) Weight (kg) on per lysim e ter basis Treatments with an asterisk are significantly different (p<0.05) from the control Figure 2 3. Biomass yield for each treatment at harvest in terms of kg cane/lysimeter

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45 Figure 2 4 Number of millable stalks for each treatment at harvest. Figure 2 5. Sucrose yield for each treatment at harvest in kg sucrose/ lysimeter.

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46 Table 2 2 July leaf nutrient concentrations. Water Lettuce (WL) Concentrations are significantly different (p<0.05) from the control N (%) P (%) K (%) Ca (%) Mg (%) Si (%) Fe (mg/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) Mud High 1.96 0.26 1.45 0.49 0.25 0.06 77.4 29.4 18.5 3.24 Mud Medium 1.91 0.30 1.57 0.62 0.29 0.06 83.1 32.1 21.4 4.38 Mud Low 1.55 0.28 1.24 0.67 0.26 0.06 73.9 34.3 20.7 4.32 Ash High 1.53 0.18 1.16 0.34 0.19 0.07 60.5 20.6 12.0 2.15 Ash Medium 1.38 0.21 1.25 0.41 0.23 0.08 60.78 20.9 11.1 1.74 Ash Low 1.37 0.21 1.21 0.44 0.19 0.07 58.1 24.4 11.5 1.84 Mix High 2.15 0.27 1.41 0.44 0.29 0.07 74.9 23.4 17.5 3.17 Mix Medium 1.94 0.28 1.47 0.49 0.31 0.07 76.3 26.2 17.3 3.07 Mix Low 1.61 0.25 1.38 0.42 0.25 0.06 63.3 17.2 18.0 3.11 WL Low 1.62 0.24 1.43 0.54 0.28 0.06 87.7 60.0 16.3 2.31 Control 1.36 0.20 1.17 0.59 0.18 0.05 63.2 30.4 14.3 3.94

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47 Table 2 3 July Critical Nutrient Level ( CNL ) categories Water Lettuce (WL) Sufficient Plus(SP), Sufficient (S), Marginal (M), Deficient (D), and Very Deficient (VD). N (%) P (%) K (%) Ca (%) Mg (%) Si (%) Fe (mg/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) Mud High M S SP SP SP VD S S S M Mud Medium M SP SP SP SP VD SP S S S Mud Low VD SP S SP SP VD S S S S Ash High VD D S S S VD S S VD D Ash Medium VD M S S S VD S S VD VD Ash Low VD M S S S VD S S VD VD Mix High S SP SP S SP VD S S S M Mix Medium M SP SP SP SP VD S S S M Mix Low M SP SP S SP VD S M S M WL Low M S SP S SP VD SP SP S D Control VD M S S S VD S S D S

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48 Table 2 4 September leaf nutrient concentrations Water Lettuce (WL) *Concentrations are significantly different (p<0.05) from the control. N (%) P (%) K (%) Ca (%) Mg (%) Si (%) Fe (mg/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) Mud High 1 .54 0.19 1.12 0.4 0 0.21 0.04 48.7 27. 1 18.3 1.24 Mud Medium 1.83 0.21 1.07 0.46 0.24 0.04 49.2 26.8 19.5 1.44 Mud Low 1.37 0.18 0.88 0.54 0.21 0.04 40.2 27.1 18.0 1.55 Ash High 1.15 0.13 0.83 0.31 0.18 0.06 28. 8 16.0 9.2 0.37 Ash Medium 1.11 0.14 0.85 0.31 0.15 0.05 29.1 15.0 9.3 0.38 Ash Low 1.05 0.13 0.87 0.35 0.12 0.05 28.5 16.6 9.9 0.32 Mix High 1.65 0.18 0.99 0.34 0.22 0.06 42.4 18.9 15.2 0.77 Mix Medium 1.39 0.17 0.88 0.4 0 0.21 0.06 38.3 19.1 13.0 0.69 Mix Low 1.19 0.16 0.89 0.41 0.17 0.04 36.1 17.1 13.1 0.66 WL Low 1.47 0.17 1.21 0.34 0.15 0.03 42.7 21.6 16.4 2.8 Control 1.1 0.14 0.85 0.55 0.13 0.04 38. 5 22 .0 13.1 1.07

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49 Table 2 5 Sept. Critical Nutrient Level (CNL) categories Water Lettuce (WL) Sufficient Plus (SP), Sufficient (S), Marginal (M), Deficient (D), and Very Deficient (VD). N (%) P (%) K (%) Ca (%) Mg (%) Si (%) Fe (mg/kg) Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) Mud High VD M S S S VD D S S VD Mud Medium M M S SF S VD D S S VD Mud Low VD M D SF S VD D S S VD Ash High VD VD D S S VD VD M VD VD Ash Medium VD VD D S S VD VD D VD VD Ash Low VD VD D S M VD VD M VD VD Mix High S M M S S VD D M M VD Mix Medium VD M D S S VD VD M D VD Mix Low VD VD D S S VD VD M D VD WL Low VD M S S S VD D S M D Control VD VD D SP M VD VD S D VD

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50 Table 2 6 Sugarcane leaf Diagnosis and Recommen dation Integrated System (DRIS) indices. A DRIS index value near zero indicates the nutrient is in optimum balance with other nutrients. DRI S index values of 10 are in bold. Nutrient balance Indices (NBI) greater than 80 are in bold. Sample ID N P K Ca Mg Fe Mn Zn Cu NBI Mill Mud High July 5.4 0.4 2.3 19.1 0.5 2.5 0.0 3.7 4.3 38.1 Mill Mud Medium July 15.7 0.9 1.6 26.1 0.7 2.0 0.0 4.5 2.6 54.0 Mill Mud Low July 24.6 3.3 6.7 39.3 1.3 2.9 0.0 4.0 3.1 85.1 Mill Ash High July 0.9 1.3 3.5 16.7 2.5 3.0 0.0 15.5 12.4 55.8 Mill Ash Medium July 12.1 8.3 8.9 27.5 9.1 5.6 0.0 23.7 23.5 118.8 Mill Ash Low July 10.2 6.1 6.7 33.1 1.9 3.0 0.0 20.5 20.1 101.5 Mix High July 0.2 3.4 1.8 11.7 4.2 2.0 0.0 9.5 5.8 38.4 Mix Medium July 8.4 5.2 2.2 17.8 7.0 0.6 0.0 11.2 8.8 61.2 Mix Low July 11.5 1.7 1.9 15.2 3.2 2.9 1.3 2.7 3.7 44.1 WL Low July 22.3 2.1 2.9 29.7 6.9 10.4 10.3 13.1 22.8 120.5 Control July 18.3 3.6 3.3 46.1 6.1 1.6 0.0 10.2 2.9 92.1 Mill Mud High Sept 0.7 2.5 4.0 27.8 6.2 2.2 1.6 3.7 44.2 92.8 Mill Mud Medium Sept 3.5 2.7 0.7 31.2 8.2 10.5 0.0 3.7 38.1 98.6 Mill Mud Low Sept 8.5 0.6 7.8 53.2 6.0 17.5 0.0 4.1 28.8 126.5 Mill Ash High Sept 16.1 15.5 24.0 55.9 32.0 9.3 4.4 2.4 136.1 295.8 Mill Ash Medium Sept 15.1 17.5 24.0 55.2 20.7 8.3 3.8 1.8 126.2 272.6 Mill Ash Low Sept 11.4 16.3 28.9 75.6 14.3 6.2 5.5 9.5 155.4 323.2 Mix High Sept 11.5 8.8 6.9 27.7 17.9 4.7 1.9 5.2 75.2 159.8 Mix Medium Sept 5.5 8.7 4.5 47.3 19.6 7.4 2.3 1.6 82.2 179.2 Mix Low Sept 0.4 8.8 8.8 54.8 8.8 7.5 2.1 3.8 79.1 174.0 WL Low Sept 3.4 4.7 7.2 17.0 3.1 9.3 0.0 1.9 1.8 48.4 Control Sept 10.2 6.2 3.0 79.0 6.5 8.3 1.4 4.3 42.0 160.7 Water Lettuce (WL) DRIS indices that are 10 units away from zero indicate critical imbalances compared to other nutrients. NBI values ( s um of the absolute value of all DRIS indices for a sample) above 80 indicate critical nutrient imbalances in the system.

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51 Table 2 7 Correlations between l eaf nutrient concentrations in July and the harvest data. Soil Quality Parameter Kg Cane Lysimeter 1 Kg Sucrose Lysimeter 1 Pearson Correlation Significance (2 tailed) Pearson Correlation Significance (2 tailed) Nitrogen 0. 729** 0.000 0.523** 0.002 Phosphorus 0.590** 0.000 0.485** 0.004 Potassium 0.322 0.059 0.229 0.200 Calcium 0.003 0.986 0.002 0.993 Magnesium 0.477** 0.005 0.338 0.005 Zinc 0.619** 0.000 0.546** 0.001 Manganese 0.329 0.061 0.367* 0.036 Copper 0.408* 0.018 0.442** 0.010 Iron 0.189 0.292 0.025 0.889 Silicon 0.045 0.803 0.010 0.955 ** Correlation is significant at the 0.01 level (2 tailed). Correlation is significant at the 0.05 level (2 tailed). Table 2 8 Correlations between l eaf nutrient concentrations in September and harvest data. Soil Quality Parameter Kg Cane Lysimeter 1 Kg Sucrose Lysimeter 1 Pearson Correlation Significance (2 tailed) Pearson Correlation Significance (2 tailed) Nitrogen 0.441* 0.010 0.198 0.270 Phosphorus 0.576** 0.000 0.310 0.070 Potassium 0.013 0.942 0.211 0.238 Calcium 0.092 0.611 0.221 0.217 Magnesium 0.748** 0.000 0.66** 0.000 Zinc 0.362* 0.039 0.166 0.357 Manganese 0.273 0.124 0.238 0.182 Copper 0.198 0.268 0.286 0.107 Iron 0.392* 0.024 0.157 0.384 Silicon 0.214 0.233 0.247 0.166 ** Correlation is significant at the 0.01 level (2 tailed). Correlation is significant at the 0.05 level (2 tailed).

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52 CHAPTER 3 SOIL QUALITY Background Soil is a medium for plant growth and supports diverse microbial communities that play important roles in ecosystem processes (Ye at al., 2007). Soil provides nutrients, water, and anchorage to the growing plants. Sugarcane can be grown on organic soils or mineral soils; however, sugarcane gown on mineral soils require more maintenance of proper physical, chemical and biological conditions. The addition of organic amendments such as mill mud and mill ash, can alter the physical, chemical and biological conditions of the soil, which may be necessa ry for realizing higher growth yield and quality of sugarcane. Maintaining or increasing organic matter (OM) in so ils through the incorporation of amendments can i mprove drainage, infiltration, and tilth (Rosen and Allan, 2007) The added OM also provides a source of energy for soil microbes, which results in the release of plant available nutrients (Rosen and Allan, 2007). Poor sugarcane production in Florida sandy soils appears to be a function of several soil chemical attributes ( Hanlon et al, 2011 ) Soil pH, OM content, and soil extractable calcium (Ca) magnesium (Mg) and potassium (K) were all lower in Florida sandy soils ( Muchovej et. al, ). Mill mud has been shown to increase K levels by 2.2 times and extractable phosphorus ( P ) levels up to 8.4 times higher than sugarcane plots without mill mud on Florida sandy soils (Morris et al., 2007) Physiochemical Properties Mill mud, mill ash, and water lettuce ( WL ) hav e the potential to alter the physical, chemical, and biological properties of sandy soils to improve soil quality. The a ddition of

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53 mill mud has been shown to improve soil ti l th, aeration, and water holding capacity (Solaimali et al., 2001). Mill mud applic ations ha ve been shown to increase soil nitrogen (N) extractable P, K, and Ca on sandy soils (Golden 1975; Prasad, 1974; Prasad, 1976 b ). Clarson (1983) recorded an increase in cation exchange capacity ( CEC ) values from 0.85 to 8.13 cmol c kg 1 and Kumar et al. (1985) saw an increase in CEC from 3.10 to 11.69 cmol c kg 1 in sandy soil amended with mill mud. A study by Khan (2011) showed that mill mud at an application rate of 20 t ons ha 1 decrease d bulk density increased soil porosity increased available K from 172 to 293 mg kg 1 increase d total N from 300 to 1070 mg kg 1 and increased available P from 7.5 to 290 mg kg 1 The same studies showed that mill ash at an application rate of 50 t ons ha 1 also decreased bulk density increased total po rosity, increased available K from 172 to 210 mg kg 1 and increased available P from 7.5 to 44 mg kg 1 Microbial Properties Microbial communities play important roles in soil processes such as decomposition of OM and nutrient cycling (Ye et al., 2007). The composition and activity of these communities are related to the efficiency of nutrient cycling and ecosystem function (Yao et al., 2000). Organic material added to soil supplies energy during decomposi tion, th us stimulating microbial activity and increasing the multiplication of their cells. This means that if mill mud, mill ash, and WL influence soil physical and chemical properties such as pH, moisture, OM content, and nutrient availability, then they will al so have effects on the microbial community composition and activity. This study investigated the effects of each amendment on microbial biomass nitrogen, carbon, and phosphorus (MBN, MBC, MBP) as well as alkaline phosphatase activity.

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54 P hosphatase s are a g roup of enzymes that catalyzes the hydrolysis of H 3 PO 4 removing phosphate groups from many types of m olecules, including nucleotides and proteins (Tabat abai, 1994 ). The activities of these enzymes are critical for the degradation of soil OM and plant detri tus and are regulated by many environmental factors (Wright and Reddy, 2001). It is important to estimate the phosphatase activity because it leads to the release of orthophosphate from the organic bound P fraction, which is assimilated by microorganisms a nd plants (Xie et al., 2011). A study by Dee et al. (2002) found microbial respiration rates increased after addition of both mill mud and mill ash and increased acid phosphatase acti vity by about 17%, showing evidence that the addition of organic amendmen ts may increase phosphatase activity. However, W right and Reddy (2001) found that an increase in phosphorus concentrations may inhibit alkaline phosphatase activity in areas that are naturally low in P. Since our sandy soil matrix has only 0.08% TP, it is possible that the amendments may decrease activity due to an increase in P concentrations. Hypothesis and Objectives Chapter 3 focuses on how the amendments affect soil quality and how each one differs from the control. Soil quality is the ca pacity of a soil to function for specific land uses or within ecosystem boundaries (USDA, 1995). Our hypothesis states that mill mud, mill ash, and WL will improve soil quality through the addition of OM which will increase microbial activity and lead to an increase i n available nutrients. It is hypothesized that these increases in available nutrients will be significantly correlated with sugarcane biomass and sucrose yields. The objectives of this chapter are to: 1. Evaluate how each amendment changes the physiochemical properties of the soil compared to the control.

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55 2. Compare how each amendment affects the microbial biomass and phosphatase activity of the soil with respect to the control. 3. Assess how the physiochemical and microbial properties change during the 12 month ev aluation period for each amendment and the control. 4. Correlate soil properties to sugarcane biomass and sucrose yields Materials and Methods Sandy soil bordering the Everglades Agricultural Area (EAA) was amended with mill mud, mill ash, a 50/50 mixt ure of mill mud and mill ash, and dried WL at a high, medium, or low application rate (Table 1 2) in 0.265 m 3 ( 70 gallon ) polyethylene lysimeters Each treatment was compared to the control that received the recommended rate ( 192 kg ha 1 ) of diammonium phospha te fertilizer (DAP) instead of an organic amendment. Each lysimter received three transplanted sugarcane seedlings of the CP 78 1628 variety on March 1 st 2012 and were harvested on November 1 st 2012. Each treatment along with the control was conducted i n triplicates totaling 39 sugarcane lysimeters. The design was a randomized block design with three blocks of 13 sugarcane lysimeters. Further details of the experimental design are explained in Chapter 1. This experiment was conducted in the EAA at the Ev erglades Research Education Center in Belle Glade, Florida. Soil Sampling and Physical Chemical Analysis So il samples from each lysimeter were collected at the beginning of the experiment, at 6 months, and at 12 months up to 15 cm The samples were h omogen ized and sieved to 2mm and stored at 4C until use. Moisture content was measured as the mass loss after drying for 24 hours at 105C. Soil OM content was measured by the loss on ignition methods after ashing the samples for 16 hours at

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56 550C (Anderson 19 76) .The pH was analyzed using a 1: 1 water to soil mixture (Thomas, 1996). The CEC was estimated using the ammonium acetate (pH 7) method (Sumner and Miller 1996) and analyzed for ammonium on a Lachat analyzer (Hach Company, Loveland, CO) Total Kjeldahl nitrogen (TKN) was analyzed using a sulfuric acid digestion (Bremner 19 96 ) and analyzed on a Lachat analyzer (Hach Company, Loveland, CO) Soil nitrate and ammonium were measured using a 2.0 M KCL extraction at a 1:10 soil: KCl ratio. Soil nitrate was analyzed using the cadmium co lumn reduction method (EPA, 1 83a, Method 353.3) on an AQ2+ discrete analyzer (Seal Analytical Inc, Mequan, WI) and soil ammonium was measured on a spectrometer using the colorimetric sodium salicylate method (Mulvaney, 1996). T otal P was analyzed using dried and ashed soil following the ascorbic acid method ( EPA, 1983b, Method 365.4) and measured on a Alpkem Auto Analyzer (O.I Analytical, Glattburgg, Zurich) Extractable P was measured using the Mehlich 3 (M3) extraction (Mehlic h, 1984). Microbial Analysis Microbial biomass C (MBC), N (MBN) and P (MBP) were analyzed using the chloroform Fumigation Extraction method (Horwath and Paul, 1994). MBC and MBN were extracted with 2.0 M KCl, while MBP was extracted using 0.5 M NaHCO 3 Th e amount of KCl extract C was determined with a total organic carbon analyzer (Schimadzu, Narcross, GA). The MBC was calculated from the difference in extractable C between fumigated and unfumigated samples using a conversion factor of 0.35 (Horwath and P aul, 1994). The amount of KCl extract N was analyzed for nitrate using the cadmium column reduction method (EPA, 183a, Mathod 353.3) on an AQ2+ discrete analyzer (Seal Analytical Inc., Mequan, WI) Soil ammonium was measured on a spectrometer using the col orimetric sodium salicylate method (Mulvaney, 1996). The

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57 following formula was used to determine the MBN with a correction factor of 0.68 (Horwath and Paul, 1994): Microbial Biomass N = [(NO 3fumigated +NH 4fumigated ) (NO 3unfumigated +NH 4unfumigated )] / 0 .68. The total P content of the NaHCO 3 extracts used for MBP was analyzed on a Alpkem Auto Analyzer (O.I. Analytical, Glattbrugg, Zurich) using the ascorbic acid method ( EPA, 1983b, Method 365.4). The MBP was determined as the difference in total P of NaHC O 3 extracts from fumigated and unfumigated samples Alkaline phosphate activity (PA) was measured based on the colorimetric estimation of p nitrophenol released by microbial activity when the soil was incubated with a pH 11 buffered p nirtrophenyl phosphate solution (Tabatabai and Bremner 1969). Statistical Analysis Analyses of variance for OM, TP, M3 P, NO 3 N NH 4 N TKN, CEC, pH, MBN, MBC, MBP, and PA was performed for a randomized complete block design using the generalized linear mixed models procedure (PROC GLIMMIX) of SAS 9.3 (SAS Inc., 2011). All treatment effects were considered significant at p < 0.05, and pairwise comparisons were made using the lsmeans statement with the Tukey method. Each soil quality parameter was analyzed as a repeate d measure over time using lsmeans at each sampling date. Pearson correlations between soil properties at the first sampling date on March 7 th 2012 and biomass yield data were analyzed using a 2 tailed significance (p<0.05) on SPSS 20 (IBM Corp., 2011) Pe arson correlations between soil properti es at the second sampling date o n September 30 th 2012 and biomass yield data were analyzed using a 2 tailed significance (p<0.05) on SPSS 20 (IBM Corp., 2011 ). Sucrose yields were analyzed using the second date beca use the soil was

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58 sampled values because approximately one month before sugarcane harvest and sucrose yields are more dependent of soil pro perties during the ripening growth stag e. Results Soil physiochemical Analyses The mill mud high and ash high treatment s consistently had the highest percentage of OM at each sampling date and were always significantly higher than the control (Table 3 1 ). At the beginning of the experiment, March o f 2012, the mill mud high (25% OM) and mill ash high (23% OM) treatm ent had about 10 times the amou nt of OM than the control (2.3 %). The mill mud medium, mill mud low, mix high, and mix medium had 4 times to 7 times higher OM content than the control.; however the lack of significant differences in due to large standard de viations between the replicates. The mill ash low, mix low, an d WL low treatments showed 2 3 times higher OM than the control. The difference between each treatment and the control remained consistent at each sampling date (Figure 3 1) so the OM did not s ignificantly differ across the sampling dates except for the mill mud high treatment where in dro pped significantly in September, 2012 For CEC the mill mud high and medium consistently had the highest levels, followed by the mix high and mix medium tre atments (Table 3 2 ). During the first sampling date, March of 2012, only the mill mud high (28. 1 cmol c kg 1 soil) and the mill mud medium treatments (20. 6 cmol c kg 1 soil soils) were significantly d ifferent than the control (4.5 cmol c kg 1 soil). By the th ird sampling in March of 2013, all mill mud treatments, mill ash high and the mix high and medium treatments were significantly different than the control. Although the CEC had a decr easing trend in most treatments

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59 (Figure 3 2) there was no significant d ifference between samplin g dates for any treatment. At the beginning of the experiment, the pH of the soil was above 8.0 for all treatments including the control (Table 3 3 ) All treatments, except the mix treatments, significantly increased the pH. The control had a pH o f 8.02, which increased up to 8. 93 with the addition of mill ash at 481 t ons ha 1 The mill ash treatments consistently had the highest pH on each sampling date and the mill ash high was always signifi cantly higher than the control. The pH in all mud treatments, all ash treatments, and the WL low treatment dropped significantly from the first sampling date (March, 2012) to the second date six months later (September, 2012) as illustrated in Table 3 3 and Figure 3 3 However, the contr ol and the mix treatment s showed no significant differences over the sampling dates. Total phosphorus (TP) showed no significant differences over the sampling dates for any treatment (Table 3 4 Figure 3 4 ). The mill ash high an d mill mud high treatments consistently had the highest TP values and were significantly higher than the control in March, 2012 However, only the mill ash high treatment was significantly different than the control (about 5 times higher) at each sampling date. All other treatments, except the WL low treatment, had at least double the amount of TP than the control; however, they were not significantly different due to large standard deviations. The high levels of TP in the mill ash high treatment is surprising since the physiochemica l characteristics of the amendments in Table 1 1 shows that mill ash has a lower percentage of TP than mill mud and the 50/50 mix of mill mud and mill ash.

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60 Available P measured as Mehlich 3 phosphorus (M3 P), had a much stronger treatment affect than TP where all treatments except mill ash low, mi x low, and WL low, w ere significantly higher than the control at the beginning and the end of the experiment (Table 3 5) The mill mud high, ash high, mix high, and mix medium had the strongest affect compared to the control. Each had at least 12 times more M3 P than the control at each sampling date All treatments, except mill mud high and medium, showed no significant difference in M3 P concentrations over time. The mill mud high treatment increased significa ntly after six months, while the mill mud medium showed a significant drop in M3 P after 6 months, but then increased again at the 12 month sampling date (Table 3 5, Figure 3 5) For TKN which measures organic N and ammonium, the mill mud treatments cons istently had the highest concentrations. However, the mill mud high treatment was the only treatment that was significantly different than the control at each sampling date (Table 3 6). Mill mud high ha d a TKN concentration of 0.97% TKN in March of 2012 0 .37% in September of 2012 and 0.90% in March of 21013, while the control had 0.10%, 0.06%, and 0.09% TKN in each sampling date All other treatments had higher levels of TKN but were not significantly different from the control except for mill mud medium (5 times higher) and mix medium (4 times higher) in March of 2013 All treatments, except mill mud high, mill mud medium, and mix medium showed no significant difference in TKN concentrations over time. The mud high, mill mud medium, and mix medium treatment showed a drop during the second sampling date before increasing significantly again at the third sampling date (Table 3 6, Figure 3 6)

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61 Soil nitrate (NO 3 N) was highest in March of 2012, but decline significantly by Septem ber of 2012 in all treatments except the WL low (Table 3 7, Figure 3 7 ). The decline in soil nitrate could be due to plant uptake and leaching. The WL low treatment consistently had the lowest nitrate concentration and showed no significant difference over time. During the first sampling date only the mix high (110.8 mg kg 1 ) and the mix medium (103.3 mg kg 1 ) were significantly higher than the control (12.34 mg kg 1 ). During the next two sampling dates, no treatments were significantly different than the c ontrol. Soil ammonium (NH 4 N ) significantly decreased over time in the WL low, mill mud m edium, mix high, and mix medium, which was most likely due to conversion to soil nitrate (Table 3 8 Figure 3 8 ). At the first sampling date in March of 2012, WL had the highest NH 4 + concentrations at 21. 0 mg kg 1 followed by mill mud medium (16. 2 mg kg 1 ), and mill mud high(15.7 mg kg 1 ). Despite the large difference s i n concentrations compared to the control (4.2 mg kg 1 ), these values were not significantly different. The mill mud high and mill mud medium continued to retain higher concentrations of ammonium over time and were significantly higher than the control in the second and third sampling date, except for mill mu d medium in March of 2013. Soil Microbial Analyses At the first sampling date in March of 2012, the mill mud high (3.61 g C kg 1 soil) and mill mud medium treatment (2.79 g C kg 1 soil) had significantly higher levels of soil MBC than the control (0.08 g C kg 1 soil) as shown in Table 3 9 By the second sampling date, only mill mud medium was significantly higher than the control and by the third date no treatments were significantly different th an the control. T reatment s showed different trends between the first and second sampling date but by March,

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62 2013 they all converge d to similar val ues. The mill mud treatments started out the highest, but declined significantly over time, while mill ash treatments and the contro l showed a significant increase in M BC between March of 2012 and March of 2013 (Table 3 9, Figure 3 9) The mix high and mix medium treatment showed no significant difference over time. The mill mud high and mill mud medium treatments showed the greatest impact on soil MBN because they are significantly higher than the control during the first and last sampling date s (Table 3 10). During the first sampling date, the mix high treatment had about 10 times more MBN than the control; however, it significantly decreases after the first sampling d ate and no longer remained significantly different than the control. All other treatments had higher MB N, but not statistically significant MBN values compared to the control. The control, mill ash low, mix low, and WL low treatments showed no significant change across the sampling dates, while the mill ash high, mill ash medium, and mill mud medium decreased over time. The mill mud high, mill mud medium, mix high, and mix medium showed a decrease after 6 months, followed by an increase after another 6 mont hs which parallels the trend for these treatments for TK N (Table 3 10. Figure 3 10) The trends for soil MBP over time showed similar results to MBN in that the mill mud treatments, mix treatments, and WL treatment significantly decreased from the first t o second sampling date and increased significantly from the second to third sampling date (Table 3 11, Figure 3 11 ). The mill ash treatments and the control decreased significantly after the first 6 months, but did not show a significant increase the foll owing 6 m onths like the other treatments The mill mud high and medium, the mix high and

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63 medium, and the ash high showed the greatest impact on MBP since they were significantly higher than the control at each sampling date (Table 3 11). Phosphatase activity (PA) was measured as the amount of p nitrophenol released by microbial activity during the decomposition of a given substrat e. The mill mud high and mill mud medium treatments consistently had the highest PA and were significantly high er than the control with at least 12 t imes more PA each sampling date (Table 3 12). The mill mud low, the mix high, and mix medium were also significantly higher than the control during the first and third sampling date s The ash treatments and the WL trea tments seemed to have little effect on PA since they were never significantly higher than the control. The mill mud treatments declined significantly from the first sampling date to the last sampling date, while all other treatments showed no significant change of time (Table 3 12. Figure 3 12) Discussion Effects of Amendments on Physical and Chemical Properties All treatments, except the WL low, mill ash low, and mix low treatment, showed an increase in available M3 P compared to the control throughout the 12 month measurement period, which show the benefits of these amendments as phosphorus fertilizer since they increased available P. The mill mud high treatment had an average of 885 mg P kg 1 soil, the mix high treatment had 699 mg P kg 1 soil, and the mix high had 673 mg P kg 1 soil. All these are significantly higher than the control which had 29.3 mg P kg 1 soil. All treatments showed either an increasing trend in available P or no significant change in available P across the sampling period, showing that these treatments may be a long term, slow release P fertilizer (Figure 3 5) These results can explain the sufficient or sufficient plus P shown in the leaf nutrient concentration

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64 analysis in Chapter 2. Both the nutrient leaf P and the M3 P was signi ficantly and positively correlated at the 0.01 level to the sugarcane biomass yield data (Table 2 7 Table 3 13 ). Mehlich 3 P was also significantly correlated at the 0.01 level to the sucrose yield data. Soil nitrate, ammonium, and TKN showed a decrease from the first sample date in March to the second sample date in September. It is assumed that conversion of ammonium to nitrate and plant uptake was responsible for this decrease since sugarcane plants consume most N during the first few months of growth ( Bachchhav, 2005 ; Rice e t al., 2010). This is because the N requirement of sugarcane is greatest during the tillering phase of the sugarcane growth cycle, which usually commences 40 days after planting and may last up to 120 days ( Bachchhav, 2005 ). Leachi ng of nitrate may have also been responsible fo r the decline in N over the 12 month period T otal Kjeldhal N, soil nitrate and ammonium all had large variability in the replicates; however the mud and mix treatments generally added the most N to the soil The increase in TKN, nitrate, and ammonium in these show that these amendments are more effective than t he control because they either add additional nitrogen into the soil, are effective at retaining nutrients added by nitrogen fertilizer through the addition of OM or a combination of the two. The mill ash treatments showed negligible increases in soil ammonium and nitrate compared to the control. This may be attributed to the high C:N ratio of mill ash of 56 :1 This large C:N ratio implies that the mill ash mater ial would cause immobilization of inorganic N into microbial biomass. Microbial biomass N C:N ratios, a nd microbial activity will be discussed in further detail in the Effects of Amendments on Soil Microbial Properties section

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65 The amendments also affected the physical and chemical properties of the soil through the addition of OM The OM in the soils was significantly correlated (p<0.01) with TP, M3 P, TKN, CEC, MBC, MBN, MBP, and phosphatase activity, which re emphasizes the importance and benefits of the OM added from the amendments (Table 3 13 ). Organic matter has an especially pronounced effect on the water holding capacity of sandy soils (Brady and Weil, 2008). A study b y Hudson (1994) showed that as OM content increased in sandy soils from 0.5 to 3%, available water holding capacity of the soil more than doubled. Although water holding capacity was n ot measured, it is likely that the mill mud, mill ash, and mix treatments increased water holding capacity since they all more than tripled the percent of OM in the soil compared to the control. For each treatment, except mill mud high, t he OM did not sign ificantly change over time showing that the all the amendments may be a good, stable source of OM that could provide benefits to ratoon cycles. The CEC of the soil increased after the incorporation of the amendments, especially in the mill mud and mix tre atments. The relationship between OM and CEC may have also contributed to the increase in available nutrients for the sugarcane plant. Both OM and CEC was significantly correlated (p<0.05) to sugarcane biomass yield and suga rcane sucrose yields (Table 3 13 ). The CEC of the soil also showed no significant changes across sampling dates for any of the amendments. This shows promise for these amendments for the long term, especially the treatments with the highest levels of CEC such as the mill mud and mix tre atments. The pH of the soil was significantly and negatively correlated to sugarcane biomass yields (Table 3 13), which was expected since nutrient availability become s

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66 limited at a pH greater than 8. All treatments, including the control had an average p H of above 8.0 at the first sampling date The increase in pH after the addition of mill mud and mill ash was also seen in other studies (Barry et al., 2001 ; Morris et al., 2007 ; Khan, 2011). Gilbert et al. (2008) used mill mud obtained from the same mill used in our experiment and found that the Ca concentration was at 7.8%, which provided a liming effect on the soil. The high Ca content in mill mud may come from the underlying limestone bedrock in the EAA (Gilbert et. al, 2008). The soil matrix used in our experiment, before incorporation of the amendments, had a pH of 7.7. I t is possible that the mill mud and mill ash may show increased nutrient availability in soil with lower pH values. The pH of sandy soil in Florida is variable; o ther experiments use d to grow sugarcane on sandy soils in south Florida reported soil pH around 5.0 (Morris et al., 2007; Gilbert et al., 2008). The results showed that mill mud significantly increased OM CEC, M3 P, TKN, and NH 4 N compared to the control. Mix treatments showed promise with increases in all physiochemical properties, but large variabil ity in the replicates, so only increases in CEC, M3 P, and NO 3 N were significantly higher compared to the control. The mill ash trea tments increased the pH significantly compared to the control, which may have limited the availability of other nutrients. Only the mill ash high treatment significantly increased the percent TP in the soil compared to the control. The WL treatments were u nsuccessful as they did not improve soil quality and did not significantly alter any physiochemical properties the soil. Effects of Amendments on Soil Microbial Properties M icrobial biomass was affected by the addition of organic amendments, which was exp ected since microbial communities are in close contact with microenvironments

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67 and are easily subjected to change following alteration of soil (Corstanje et al. 2007). The mill mud and mix treatments were especially influential compared to the control since they provide t he necessary energy and nutrients to support an increase in microbial biomass. This is extremely critical because it has been demonstrated that nutrient availability, such as P and N, greatly influences soil microbial activity and function ( Wright and Reddy 2001; Corstanje et al. 2007; Ye et al. 2009). Our experiment highlights th is point since TKN, soil nitrate, and M3 P are all significantly correlated with MBC, MBN, and MBP a t the 0.01 level (Table 3 14 ) Mill ash treatments and the control showed deficient N and marginal P according the critical nutrient levels discussed in Chapter 2, which may be due to their lower microbial biomass and activity. Mill ash treatments also had highest soil pH with an average of 8.8, which may have li mited microbial activity. The mill mud and mix treatments had the greatest impacts on microbial biomass and activity. The mud treatments had significantly higher MBC during the first two sampling dates, higher MBN during the first and third sampling date, higher MBP during a ll sampling dates, and increases in PA during all sampling dates in comparison to the control. The mix treatments had an increase in MBP and PA compared to the control. Higher levels of OM for microbial degradation is one reason for t he higher values than the control since OM improves soil aeration and water retention, which have also been correlated to increases in microbial activity and microbial biomass (He et al., 1997). The range in MBC levels (0.08 g C kg 1 3.61 g C kg 1 ), MB N levels (2.0 mg N kg 1 175 mg N kg 1 ), and MBP levels (0.10 mg P kg 1 204 mg P kg 1 ) are similar rates to other studies that used the chloroform fumigation method to test microbial biomass

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68 on agricultural field amended with organic amendments (Schnure r et al., 1985; He et al., 1997; Paul and Solaiman, 2004). Our study showed the largest MBN and MBP values after the first sampling in March, which was taken one week after the amendments were added, before seeing a decline in MBN and MBP by our next sampl e date 6 months later. This is also consistent with microbial biomass studies by Friedel et al. (2000) and Paul and Solaiman ( 2004 ) which showed a peak in microbial biomass after seven days. The decline in biomass by our 6 month sampling date followed by the increase at the 12 month sampling date may be attributed to the decrease in nutrient availability due to plant uptake followed by an increase in nutrient availability after the sugarcane was harvested in between the 6 month and 12 month sampling date. A seasonal study in the UK by He et al. (1997) showed a decrease in MBP from March to September followed by an increase in MBP from October to Decembe r due to the moisture content of the soil. This is consistent with the trends in our experiment for MBP a nd MBN; therefore, r ainfall accumu lation and moisture content may also be attributed to the decrease in microbial activity observed in the September soil samples. August and September of 2012 received 51.74 cm (20.37 in ) of rainfall, which included Tropica l Storm Isaac in late August (Table C 1) The large accumulation and intensity of rainfall result ed in prolonged periods of flooding in all lysimeters possibly decreasing the micro bial activity and N content of the soil due to denitrification. To alleviate the effects on the flooding, each lysimeters were artificially drained two times during the month of August, which may ha ve resulted in nitrate leaching. For MBC, each amendment behaved differently over time. Mill ash treatments and the control treatment increased significantly over time, while the mill mud

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69 treatments decreased significantly over time. T he mix treatments remained approximately the same. This may be attributed to the quality of the O M added from each amendment. The C:N and C:P ratios are important to determine the likelihood that a material will be mineralized or immobilized and how efficiently the OM will be utilized by the microbial community (Ye et al., 2007). Using the physiochemi cal propert ies of the amendments (Table 1 1 ) and assuming OM is 58% carbon, we can calculate the C:P and C:N ratios of the amendments. Materials with C:P ratios under 200 :1 exhibit net mineralization and release dissolved inorganic P, while C:P ratios abo ve 300 show net immobilization and a net decrease of dissolved inorganic P ( Reddy and DeLaune, 2008 ). The C:P ratios of our amendments were calculated to be 42.0 for mill mud, 43.7 for mill ash, 35.3 for the mix, and 123 for WL All amendments have a C:P ratio under 200:1 so all should release dissolved inorganic P from the degradation of OM which shows potential for these amendments applied to substitute inorganic P fertilizers However, the C:N ratios are also needed to determine the quality of OM and h ow rapidly it will be degraded. Materials with C:N ratio s below 20 will show a net mineralization and release inorganic N while a C:N ratio above 20 would show net immob ilization ( Reddy and DeLaune, 200 8 ). We calculated the C:N ratios of our amendments to be 15.5 for mill mud, 17.7 for the mix, 24.5 for WL, and 56.2 for mill ash. The low C:N ratio of the mill mud amendments would explain the high availability of nutrients immediately after incorporation into the soil matrix and the high amount of MBC and MBN The decrease in MBC over time may have occurred since the OM was being degraded rapidly causing a shift in the C:N ratio. The opposite may be said about the mill ash treatments, which had the highest C:N ratio of 56.2. The material would be

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70 more stabl e in the soil and degrade more slowly over time, which may be beneficial for ratoon crops and long terms goals. The increase in MBC in the ash treatments and in the control may also be due to changes in C:N and C:P ratios over time or from the crop residue s left after the harvest of sugarcane between the second and third sampling date. Paul and Solaiman (2004) found that MBC and MBN formation was highest in amendments that had the highest C:N ratios. These finding are inconsistent with our results since mil l mud had significantly higher MBC and MBN and both mill mud and mix treatments had significantly higher MBP values than mill ash and control treatments. Paul and Solaiman (2004 ) attributed the correlation between high C:N ratios to high MBC and MBN values to increases in immobilization. Our results may have differed because although the mill ash treatment ha d the highest C:N ratio other factors such as limited nutrient availability and high pH may have decreased microbial populations and activity. Microb ial properties, including enzyme activities such as phosphatase activity (PA), are critical to the first step in the degradation of OM and can be used as indicators for soil quality since it is af fected by nutrient availability (W right and Reddy, 2001). M i ll mud treatments followed by the mix treatments exhibited significantly higher PA than the control. Significant correlations (p<0.001) between PA and TP, M3 P, MBP, TKN, and MBN imply that that enzyme activity is related to both soil P parameters and oth er e nvironmental factors (Table 3 14 ). The positive correlation between soil P parameters and PA differs from finding s in other studies. W right and Reddy (2001) and Ye et al (2007) found no correlations between PA and soil P parameters and found that PA is

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71 often repressed by high dissolved P concentrations as feedback inhibition. On the contrary our results show that the treatments with high available P concentrations (mill mud and mix treatments) have significantly higher levels of PA than the control. However, relationships between microbial parameters, extracellular enzymes, and soil properties are often contradictory since they are not clearly observ ed in the field and have a wide range of factors that influence enzyme activity including redox conditions and nutrient substrates (Marsden and Gray, 1986; W right and Reddy, 2001). In our case, higher PA seems intuitive since these mill mud and mix amendm ents are rapidly degraded due to their low C:N and C:P ratios. Overall our lysimeter study showed that the microbial activity and biomass were dependent on soil attributes such as nutrient availability and the OM quality of the amendments. Mill mud treatm ents had significantly higher MBC and MBN and both mill mud and mix treatments had significantly higher MBP values and PA than the control However, the mill ash amendments may be more stable and last longer in the soil due to a high C:N ratio, which may be beneficial in the long run. The WL treatment failed to improve soil quality through the increase in microbial activity since it was not significantly different from the control in MBC, MBN, MBP, or PA. Correlations between Soil Quality Parameters and S ugarcane Yields This chapter focused on how each amendment affected soil quality. Soil quality was measured through OM, CEC, pH, TP, M3, TKN, NO 3 N, NH 4 N, MBC, MBN, MBN, and PA. However, by definition s oil quality is the capacity of a soil to function fo r specific land use, which in this case was to grow sugarcane. Tables 3 13 illustrate that PA, MBC, CEC, M3 P, MBN, TKN, and NO 3 N are significantly correlated with sugarcane biomass yield at the 0.01 level and OM is positively correlated at the 0.05 leve l. Since

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72 these amendments were added to the lysimeters as an inorganic phosphorus fertilizer substitute, the correlation between yield and available P (M3 P) and PA shows the importance of the amendments to provide P to the plant. The mill mud, mill ash, a nd mix materials were all successful in providing available P in the soil compared to the control (Table 3 5). The CEC was also highly significantly correlated to biomass yield, which means that the amendments were able to hold cations such as Mg 2+ and Ca 2+ for plant consumption more readily in the soil compared to the control. These results are reflected in the lea f nutrient concentration data (Table 2 3) where the mill mud and mix treatments had sufficient plus Mg 2+ and Ca 2+ and also had the highest CEC values (Table 3 2). The pH of the soil was negatively and significantly correlated to biomass yield at the 0.05 level with a Pearson Correlation of 0.431 (Table 3 13) This confirms prior thought the high pH of the mill ash amendment of 8.8 (Table 1 1), negatively affected growth through the inhibition of microbial activity and immobilization of nutrients. The sucrose yield was significantly and positively correlated with MBN TKN, M3 P, TP, and OM at the 0.0l level and with CEC at the 0.05 level (Table 3 1 3 ) These correlations are taken at the second sampling date because that date was approximately one month before harvest and sucrose yield are more dependent on soil properties during the ripening stage right before harvest Our correlation results in dicate the importance of nutrient availability to the production of sucrose; h owever, sucrose yield has typically been known to be correlated with low temperatures, low N levels, and restricted water supply (FAO, 2013).

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73 Based on the se significant correlat ions between biomass yields sucrose yield s, and soil properties such as OM, TP, M 3, TKN, CEC, MBC, MBN, MBP, PA, and pH, it appears that sugarcane production is a function of several soils chemical and microbial attributes.

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74 Table 3 1. Soil organic matte r ( OM ) for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 OM ( % ) OM ( % ) OM ( % ) Mud High 25.0 a A 16. 8 a B 19.6 ba BA Mud Medium 17.3 bac A 14.8 ba A 16.5 bac A Mud Low 10.3 bac A 7.0 ba A 10.4 bdac A Ash High 22.6 ba A 22. 2 a A 21.6 a A Ash Medium 6.2 bc A 8. 9 ba A 7.9 bdc A Ash Low 7. 2 bc A 5.8 ba A 6.1 dc A Mix High 13. 8 bac A 13.6 ba A 13.6 bdac A Mix Medium 11.8 bac A 16.5 ba A 13.6 bdac A Mix Low 4.1 c A 4.3 b A 4.9 dc A Water Lettuce Low 4.3 c A 3.2 b A 2.8 d A Control 2.3 c A 2.1 b A 2.2 d A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 1. Soil organic matter over time for each treatment.

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75 Table 3 2. Cation exchange capacity (CEC) for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 CEC (cmol c /kg) CEC (cmol c /kg) CEC (cmol c /kg) Mud High 28.1 a A 21.9 a A 18.7 a A Mud Medium 20. 6 ba A 17.4 ba A 14.5 b a A Mud Low 11. 7 bc A 11.1 ebdac A 14. 5 b a A Ash High 12.9 bc A 15.2 bdac A 15.2 a A Ash Medium 4.9 c A 9.4 ebdc A 8.3 b a c A Ash Low 6.46 c A 7.1 ebdc A 9.4 b a c A Mix High 13.1 bc A 14.7 ebdac A 12.3 a A Mix Medium 14.3 bc A 16.2 bac A 17.3 a A Mix Low 6.13 c A 5.8 edc A 8.0 b a c A Water Lettuce Low 5.2 c A 4. 8 ed A 5.7 bac A Control 4.5 c A 4.0 e A 3.5 c A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 2. Cation exchange capacity (CEC) in soils over time for each treatment.

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76 Table 3 3 pH for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 pH pH pH Mud High 8.34 dc A 7.96 bc B 7.63 b B Mud Medium 8.34 dc A 7.86 bc B 7.61 b B Mud Low 8.31 dc A 7.84 c B 7.73 ba B Ash High 8.93 a A 8.49 a B 8.30 ba B Ash Medium 8.74 ba A 8.36 ba B 8.10 ba B Ash Low 8.69 ba A 7.94 bc B 8.17 a B Mix High 8.04 fe A 8.35 ba A 7.97 ba A Mix Medium 8.01 f A 8.24 bac A 7.92 ba A Mix Low 8.12 dfe A 7.99 bac BA 7.77 ba B Water Lettuce Low 8.47 bc A 8.11 bac B 8.05 ba B Control 8.02 f A 7.90 bc A 7.79 ba A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 3. pH of soils over time for each treatment.

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77 Table 3 4 Total phosphorus (TP) in soils for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 TP ( % ) TP ( % ) TP ( % ) Mud High 0. 4 4 ba A 0.38 ba A 0. 5 3 a A Mud Medium 0.30 bac A 0.25 ba A 0.30 bac A Mud Low 0.17 bac A 0.1 3 ba A 0.19 bc A Ash High 0.50 a A 0.59 a A 0.49 ba A Ash Medium 0.10 bc A 0.21 ba A 0.17 bc A Ash Low 0.11 bc A 0.12 ba A 0.10 c A Mix High 0.25 bac A 0.28 ba A 0.29 bac A Mix Medium 0.23 bac A 0.40 ba A 0.50 bac A Mix Low 0.1 8 bac A 0.07 ba A 0.09 c A Water Lettuce Low 0.08 c A 0.04 b A 0.04 c A Control 0.11 c A 0.03 b A 0.04 c A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 4. Total phosphorus (TP) in soils over time for each treatment.

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78 Table 3 5. Mehlich 3 phosphorus (M3 P) in soils for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 M3 P (mg/kg) M3 P (mg/kg) M3 P (mg/kg) Mud High 671.3 ba B 981. 7 a A 1002.3 a A Mud Medium 677.0 ba A 315. 7 bdc B 848. 7 ba A Mud Low 520.0 ba A 379.0 bdc A 449. 7 edc A Ash High 765.3 a A 707. 7 ba A 546.0 bdc A Ash Medium 474. 7 bac A 327.3 bdc A 474. 0 bdc A Ash Low 285.3 bdc A 219.3 bdc A 267.0 edf A Mix High 670.0 ba A 662.3 bac A 764. 7 bac A Mix Medium 587. 7 ba A 673. 7 bac A 692.3 bac A Mix Low 107. 7 d A 165.3 dc A 173.0 edf A Water Lettuce Low 122. 7 dc A 72.0 d A 61. 7 ef A Control 47. 7 d A 19.0 d A 21.3 f A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 5. Mehlich 3 phosphorus in soils over time for each treatment.

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79 Table 3 6 Total Kjeldahl nitrogen (TKN) for each treatment for each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 TKN (%) TKN (%) TKN (%) Mud High 0.97 a A 0.37 a B 0.90 a A Mud Medium 0.58 ba BA 0.33 ba B 0.69 a A Mud Low 0.37 b A 0.25 ba A 0.38 bdc A Ash High 0.24 b A 0.15 ba A 0.20 dc A Ash Medium 0.14 b A 0.11 ba A 0.15 dc A Ash Low 0.16 b A 0.06 b A 0.16 dc A Mix High 0.36 b A 0.25 ba A 0.41 bdc A Mix Medium 0.34 b BA 0.30 ba B 0.57 bac A Mix Low 0.17 b A 0.10 ba A 0.18 dc A Water Lettuce Low 0.17 b A 0.10 ba A 0.12 dc A Control 0.10 b A 0.06 b A 0.09 d A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 6. Total Kjeldahl nitrogen (TKN) in soil over time for each treatment.

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80 Table 3 7 Soil nitrate ( NO 3 N) for each treatment on each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 NO 3 N (mg/kg) NO 3 N (mg/kg) NO 3 N (mg/kg) Mud High 29. 2 b A 6.6 a B 2.3 a B Mud Medium 41.4 b A 7.1 a B 3.9 a B Mud Low 19.8 b A 6.1 a B 3.9 a B Ash High 17.1 b A 2.3 a B 3.9 a B Ash Medium 34.7 b A 2.4 a B 2.7 a B Ash Low 12.4 b A 1.0 a B 2.9 a B Mix High 110. 8 a A 4.8 a B 3.8 a B Mix Medium 103.3 a A 6.7 a B 4.3 a B Mix Low 23.9 b A 1.7 a B 2.3 a B Water Lettuce Low 3.3 b A 1.4 a A 2.7 a A Control 12.3 b A 1.4 a B 3.1 a B Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 7. Soil nitrate over time for each treatment.

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81 Table 3 8. Soil ammonium ( NH 4 N) for each treatment on each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 NH 4 N (mg/kg) NH 4 N (mg/kg) NH 4 N (mg/kg) Mud High 15.7 a A 12.0 a A 7.5 a A Mud Medium 16.2 a A 11.4 a A 1. 9 b B Mud Low 6.6 a A 5.0 ba A 1.0 b A Ash High 2.9 a A 3.4 b A 1.0 b A Ash Medium 2.7 a A 2.6 b A 0. 68 b A Ash Low 3. 7 a A 2.1 b A 0.6 3 b A Mix High 7.2 a A 7.4 ba A 1.6 b B Mix Medium 8.5 a A 7.6 ba A 1.3 b B Mix Low 4.0 a A 3.4 b A 0.7 3 b A Water Lettuce Low 21.0 a A 6.1 ba B 0.6 1 b C Control 4.2 a A 2.2 b A 0. 46 b A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 8. Soil ammonium over time for each treatment.

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82 Table 3 9 Microbial Biomass Carbon (MBC) on each sampling date LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 MBC (g/kg) MBC (g/kg) MBC (g/kg) Mud High 3.6 1 a A 1.5 0 ba B 1.6 1 a B Mud Medium 2. 79 ba A 2. 17 a A 1. 19 a B Mud Low 1. 55 b a c A 1.6 2 ba A 1.7 1 a A Ash High 0.11 c B 0.7 9 ba B A 1.2 3 a A Ash Medium 0.06 b c B 0.64 ba B A 1.8 4 a A Ash Low 0.20 c B 0.8 2 ba B A 1. 36 a A Mix High 1.7 2 b a c A 1. 68 ba A 1.8 4 a A Mix Medium 1.5 2 bac A 1.3 2 ba A 1. 35 a A Mix Low 0.21 c B 1. 1 0 ba BA 1. 85 a A Water Lettuce Low 0.5 1 c A 0.42 b BA 1.4 3 a A Control 0.0 8 c B 1.06 ba BA 1. 69 a A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 9. Microbial biomass carbon of soils over time for each treatment.

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83 Table 3 10 Microbial biomass nitrogen (MBN) on each sampling date LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 MBN (mg/kg) MBN (mg/kg) MBN (mg/kg) Mud High 175.4 a A 31. 6 a B 60.6 a BA Mud Medium 107. 2 a A 25.2 a B 48.6 ba B Mud Low 56. 1 ba A 9.0 a B 36. 3 bac BA Ash High 50.2 b a A 18.0 a B 18.6 bc B Ash Medium 96. 9 ba A 5.7 a B 16. 7 bc B Ash Low 16.4 b A 8.8 a A 12. 8 bc A Mix High 201.2 a A 14.9 a C 38. 1 bac B Mix Medium 79.2 ba A 21. 8 a B 34. 8 bac BA Mix Low 48. 4 ba A 10.3 a A 21. 6 bc A Water Lettuce Low 19.9 b A 9. 7 a A 14. 7 bc A Control 22.8 b A 5.6 a A 10. 8 c A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 10. Microbial biomass nitrogen of soils over time for each treatment.

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84 Table 3 11 Microbial biomass phosphorus (MBP) on each sampling date. LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 MBP (mg/kg) MBP (mg/kg) MBP (mg/kg) Mud High 167.7 a A 38.4 a B 8 2.0 ba BA Mud Medium 204.2 a A 29.6 a B 139.7 a A Mud Low 86.5 ba A 11. 6 b a B 38. 8 ba BA Ash High 185.4 a A 17. 9 a B 49.2 ba B Ash Medium 56.3 b a A 12. 6 b a B 20. 2 b c B Ash Low 63. 7 b a A 2.3 b B 4.5 b c B Mix High 134. 6 b a A 17.9 a B 88. 3 ba BA Mix Medium 113. 2 b a A 23. 8 a B 58.2 ba BA Mix Low 17. 9 b A 4.5 b A 20.5 b c A Water Lettuce Low 22.1 b A 1.6 b B 5. 7 b c BA Control 22. 9 b A 1.0 b B 0.1 0 c B Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 11. Microbial biomass phosphorus of soils over time for each treatment.

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85 Table 3 12 Phosphatase activity (PA) on each sampling date measured by the amount of p nitrophenol released LS means with the same letter are not significantly different (p<0.05) March, 2012 September, 2012 March, 2013 PA (mg/kg) NH 4 (mg/kg) NH 4 (mg/kg) Mud High 276.5 a A 188.1 a BA 131.3 a B Mud Medium 241.5 ba A 143.2 a B 122.9 a B Mud Low 144.9 bc A 37.6 b B 82.2 bac BA Ash High 57.5 ecd A 90.7 ba A 27.8 bdc A Ash Medium 32.5 ecd A 24.3 b A 28.3 bdc A Ash Low 25.2 ed A 16.4 b A 26.9 b d c A Mix High 136.3 bcd A 75.2 ba A 91.8 ba A Mix Medium 79.5 dc A 112.7 ba A 103.1 a A Mix Low 16.7 e A 10.2 b A 13.0 dc A Water Lettuce Low 31.7 ed A 16.5 b A 13.9 dc A Control 17.1 e A 12.3 b A 9.8 d A Lower case letters represent differences between treatments Upper case letters represent differences between sampling dates Figure 3 12. Phosphatase activity in soils over time for each treatment.

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86 Table 3 13. Correlations between soil quality parameters, biomass yield (kg cane lysimeter 1 ) and sucrose yield (kg sucrose lysimeter 1 ). Soil Quality Parameter Kg Cane Lysimeter 1 Kg Sucrose Lysimeter 1 Pearson Correlation Significance (2 tailed) Pearson Correlation Significance (2 tailed) Organic Matter 0.386* 0.027 0.422** 0.010 Total P 0.284 0.109 0.448** 0.009 Available P 0.509 0.003 0.467** 0.297 Soil Nitrate 0.469** 0.006 0.187 0.207 Soil Ammonium 0.026 0.886 0.226 0.002 Total Kjeldahl N 0.470 0.006 0.511** 0.147 pH 0.431* 0.012 0.258 0.018 Cation Exchange Capacity 0.530** 0.002 0.410* 0.001 Microbial Biomass N 0.475** 0.006 0.538** 0.001 Microbial Biomass C 0.584** 0.000 0.101 0.576 Microbial Biomass P 0.158 0.380 0.278 0.117 Phosphatase Activity 0.578** 0.000 0.241 0.176 ** Correlation is significant at the 0.01 level (2 tailed). Correlation is significant at the 0.05 level (2 tailed)

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87 Table 3 1 4 Correlations between the microbial properties of the soil and soil physiochemical properties at the beginning of the experiment OM TP M3 NO 3 N NH 4 N TKN pH CEC MBN Pearson Correlation .421 ** .300 .582 ** .442 ** .017 .505 ** .326 .514 ** Significance .009 067 .000 .006 .917 .001 .046 .001 MBC Pearson Correlation .490 ** .226 .352 .006 .299 .758 ** .168 .673 ** Significance .002 .167 .030 .973 .064 .000 .308 .000 MBP Pearson Correlation .605 ** .480 ** .533 ** .032 .087 .466 ** .173 .561 ** Significance .000 .002 .001 .844 .596 .003 .293 .000 PA Pearson Correlation .631 ** .412 ** .597 ** .056 .116 .739 ** .170 .789 ** Significance 000 .009 .000 .735 .481 .000 .300 .000 OM Pearson Correlation .885 ** .798 ** .117 .107 .786 ** .196 .894 ** Significance .000 .000 .477 .517 .000 .231 .000 ** Correlation is significant at the 0.01 level (2 tailed). Correlation is significant at the 0.05 level (2 tailed).

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88 CHAPTER 4 CONCLUSIONS AND FUTURE WORK Summary and Conclusions Based on this research, mill mud and a 50/50 mix of mill mud and mill ash are potential candidates for organic soil amendments and substitutes for inorganic P fertilizers to grow sugarcane on sandy soils in south Florida. Mill mud and the mix treatments added the most organic matter (OM) to the soil, which contributed to the si gnificant increases in microbial biomass, phosphatase activity (PA) cation exchange capacity (CEC) and nutrient availability as seen in the nutrient leaf concentrations and soil quality data. As predicted in our hypothesis, these factors significantly co rrelated with sugarcane biomass yields and these treatments produced significantly more cane and sucrose than the control, which received only recommended rates of inorganic fertilizers. The mill mud and mix treatments showed no significant decrease in so il OM CEC, TP, available P and PA across the 12 month sampling period, indicating that this material served as a successful organic amendment and P fertilizer for at least an entire year. These results from the lysimeter study show that when mill mud is applied with a rate of at least 175 t ha 1 and a 50/50 mix of mill mud and mill ash is applied at rates of at least 132 t ha 1 then no additional P fertilizers are necessary during the first crop cycle. It is expected that these materials would serve as a slow release fertilizer and last into ratoon crops and out perform conventional fertilizer applications, which are water soluble and a readily available source of nutrients. However, verification on the field level is necessary.

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89 According to the and the Diagnosis and Recommendation Integrated System (DRIS) for the analysis of nutrient leaf concentrations, the mill mud and mix treatments also have the fewer nutrient imbalances compared to the contro l mill ash treatments, and the water lettuce (WL) low tre atment during the prime growing season. Leaf nutrient concentrations for N in all treatments were lower than sufficient; however, the mill mud and mix treatments had at least marginal N concentrations. Considering the major role N plays in the growth of su garcane and the synthesis of sugar, N management in conjunction with these amendments is critical to optimize sugarcane biomass and sucrose yields. Mill ash treatments failed to significantly increase biomass and sucrose yields compared to the control. Th ese result are attributed to the high nutrient imbalances shown in the DRIS analysis, insignificant increases in soil nutrients compared to the control, and the high C:N ratios that limit microbial degradation of the material. However, the slow degradation of the mill ash amendment may be useful for ratoon crops and long term benefits. Therefore, it may be recommended to use a mixture of mill mud and mill ash to combine the mud material with a low C:N ratio with the more recalcitrant ash material. Water le ttuce also failed to significantly alter sugarcane biomass and sucrose yields in comparison to the control. The WL high and medium treatments did not allow the sugarcane to enter the formative growth stage, proving its inability to act as an organic soil a mendment and P fertilizer. Water lettuce has been shown to release allelopathic chemicals that may have inhibited the growth of sugarcane into the formative stage. The remaining WL low treatments showed high nutrient imbalances in

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90 the DRIS analyses and wer e not significantly different from the control in biomass yield, sucrose yields, any physiochemical soil properties, or any microbial properties. The difference between the high, medium, and low rates for each treatment did not affect the sugarcane biomas s or sucrose yield during the 12 month crop cycle. However, the higher application rate is needed to supply ratoon crops with nutrients and OM and to limit subsequent applications. For several soil properties, including TP, M3 P, TKN, and PA the high appl ication rate resulted in significantly higher content of these nutrients than the low applications showing promising results in terms of extending the longevi ty of the amendments for future ratoon cycles. Future Work The research presented in this thesis o pens the door for many new research possibilities involving the use of mill mud and mill ash as organic amendments in Florida. Field studies need to be implemented to gain a better perspective of how the amendments will affect yields at a larger scale. In addition, experiments are needed to monitor the effect of each amendment over a longer period of time for ratoon crops. Studies on the positive and negative environmental impacts such as leaching, runoff, or nutrient retention or studies on the effects of these amen dments on muck soils in the Everglades Agricultural Area could also be important follow up projects. An economic analysis of recycling these sugar by products in the south Florida region would also be extremely beneficial. When considering the economic feasibility of organic amendments to soil, the unit cost of the material, the recommended application ra tes, be considered (Hanlon et al., 2012). Overall, the cost of mill mud and mill ash

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91 a pplication, analyses of the nutritional values and benefits compared to in organic fertilizer need to be reviewed further

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92 APPENDIX A CRITICAL NUTRIENT LEVELS Table A 1. Sugarcane leaf nutrient cri tical values and optimum ranges ( Anderson and Bowen 1990). Nutrient Critical Value Optimum Range ----------------------------% ---------------------------Nitrogen (N) 1.80 2.00 2.60 Phosphorus (P) 0.19 0.22 0.30 Potassium (K) 0.90 1.00 1.60 Calcium (Ca) 0.20 0.20 0.45 Magnesium (Mg) 0.12 0.15 0.32 Silicon (Si) 0.50 > 0.70 ---------------------------mg/kg ------------------------Iron (Fe) ----50 105 Manganese (Mn) ----12 100 Zinc (Zn) 15 16 32 Copper (Cu) 3 4 8

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93 Table A 2 Sugarcane leaf nutrient sufficiency ranges for defining nutrient management categories (McCray and Mylavapu, 2010) Sufficiency Category N P K Mg Si Fe Mn Zn Cu -----------------------------------% ---------------------------------------------------------mg/kg ----------------------Very Deficient <1.6 <0.17 <0.80 <0.11 <0.20 <40 <12 <13 <2.0 Deficient 1.60 1.79 0.17 0.18 0.80 0.89 0.11 0.12 0.20 0.49 40 49 12 15 13 14 2.0 2.9 Marginal 1.80 1.99 0.19 0.21 0.90 0.99 0.13 0.14 0.50 0.59 50 54 16 19 15 16 3.0 3.9 Sufficient 2.00 2.30 0.22 0.26 1.00 1.30 0.15 0.24 0.60 0.80 55 80 20 60 17 25 4.0 6.0 Sufficient Plus 2.31 2.60 0.27 0.30 1.31 1.60 0.25 0.32 0.81 1.00 81 105 61 100 26 32 6.1 8.0 High >2.60 >0.30 >1.60 >0.32 >1.00 >105 >100 >32 >8.0 Leaf nutrient concentrations are for top visible dewlap blades (without midrib). Suggested sampling period is June July Very Deficient: Estimated production losses > 25% Deficient: Estimated production losses 6 25% Marginal: Estimated production losses 1 10%

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94 APPENDIX B BRIX AND POL VALUES Table B 1. Brix values (g of soluble solids per kg of juice) and Pol values (unitless value of polarization of the juice) used in the calculations for % yield of sucrose. Treatment Brix Pol Mud High 17.9 63. 2 Mud Medium 17.7 64.1 Mud Low 19.8 75.6 Ash High 20.0 76.0 Ash Medium 20.1 75.4 Ash Low 19.9 75.9 Mix High 18.1 65. 3 Mix Medium 19.3 71.5 Mix Low 20.0 76.0 FAV Low 16.7 58.3 Control 19.4 73.9

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95 APPENDIX C WEATHER DATA Table C 1. Average monthly air temperature and rainfall Month Year Average Temperature C (F) Total Rainfall cm (in) Feb 12 19.8 (67.6) 1.8 ( 0.69 ) Mar 12 20.8 (69.4) 2.1 ( 0.81 ) Apr 12 25.9 (78.7) 6.4 ( 2.5 0) May 12 29.5 (85.1) 10.0 ( 3.95 ) Jun 12 25.4 (77.7) 15.6 ( 6.16 ) Jul 12 26.0 (78.8) 15.4 ( 6.06 ) Aug 12 25.9 (78.7) 34.8 ( 13.69 ) Sep 12 25.0 (77.0) 17.0 ( 6.68 ) Oct 12 22.8 (73.0) 9.9 ( 3.9 0) Nov 12 17.6 (63.0) 0.4 ( 0.15 ) Dec 12 18.6 (65.5) 9.1 ( 3.6 0) Jan 13 19.3 (66.7) 0.4 ( 0.16 ) Feb 13 18.3 (65.0) 6.5 ( 2.56 ) Mar 13 16.7 (62.0) 5.2 ( 2.05 ) Data from Everglades Research and Education Center (EREC) weather s tation

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96 LIST OF REFERENCES Alexander, K.E.F. 1972. Filter cake. S. Afr. Sugar J. 56:71 77. Aliotta, G., P. Monaco, G. Pinto, A. Pollio, and L. Previtera 1991. P otential alleloch emicals from pistia stratiotes J Chem Ecol. 17: 2223 2234. Alvarez, J. and C. Polopolus, 2012. The Florida Sugar Industry. EDIS document SC 042 Department of Food and Resource Economics, Florida Cooperative Extension Service, Institute of Food and Agricultural S ciences, University of Florida. Anderson, J.M. 1976. An ignition method for determination of phosphorus in lake sediments. Water Res 10: 329 331 Anderson, D.L. 1989. A review: Soils, nutrition, and fertility practices of the Florida sugarcane industry. Proc. Soil Crop Sci. Soc. Fla. 49:78 87. Anderson, D.L. and J.E. Bowen. 1990. Sugarcane nutrition. Atlanta, Georgia: Potash and Phosphatas e Institute. Bachchhav, S. 2005. Fertigation technology for increasing sugarcane production. Indian J fertilsers. 1: 85 89. Barry, G.A., G.E Rayment A.J. Jeffrey and A.M. Price 2001.Changes in cane soil properties from applications of sugarmill by pr oducts. J. Australian Proceedings of Sugar Cane Technologists. 23:185 191 Baucum, L.E. and R.W. Rice 2009. An Overview of Florida Sugarcane. EDIS document SS AGR 232, Department of Agronomy, Florida Cooperative Extension Service, Institute of Food and Agr icultural S ciences, University of Florida. Brady, N.C. and R.R. Weil. 2008. The Nature and Properties of Soils. Prentice Hall, Upper Saddle River, New Jersey. Bre mner, J. M. 19 96 Nitrogen Total. In Sparks (ed) Methods of soil analysis, part 3 : Chemical Properties SSSA Book Ser ies no. 5. Madison, Wisconsin. pp 1085 1121. Bokhtiar, S.M. and K. Sakurai. 2005a. Effects of organic manure and chemical fertilizer on soil fertility and productivity of plant and ratoon crops of sugarcane. Arch. Agron. Soil Sci. 51:325 344. Bokhtiar, S.M. and K. Sakurai. 2005b. Integrated use of organic manure and chemical fertilizer on growth, yield, and quality of sugarcane in high Ganges river floodplain soils of Bangladesh. Commun. Soil Sci. Plant Anal. 36:1823 1837

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99 Lu, Q., Z.L. He, D.A. Graetz, P.J. Stofella, and X. Yang. 2010. Phytorem ediation toremove nutrients and improve eutrophic stormwaters using water lettuce (Pistia stratiotes L.). Environ Sci Pollut R 17:84 96. Marsden, W.L., and P.P. Gray. 1986. Enzymatic hydrolysis of cellulose lignocellulose materials. Crit. Rev. Biotechnol. 3:235 276 Matin, M.A., M.A. H ossain, and M.A.S Miah. 198 9. Tiller dynamics in sugarcane variety isd 16. Bangladesh J Sugarcane. 11: 1 6. McCray, J.M., R.W., Rice, I.V. Ezenwa, T.A. Lang, and L. Baucum 2009a. Sugarcane Plant Nutrient Diagnosis. EDIS docume nt SS AGR 128, one of a series of the Agronomy Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida McCray, J. M., S. Ji, G. Powell, G. Montes, R. Perdomo, and Y. Luo. 2009b Seasonal concentrations of leaf nutrients in Florida sugarcane. Sugar Cane Int 27:17 24. McCray, J. M., S. Ji, G. Powell, G. Montes, and R. Perdomo. 2010. Sugarcane Response to DRIS Based Fertilizer Supplements in Florida. J Agron Cop Sci. 196: 66 75. MC r ay, J.M., L. Baucum, K. Morgan, and S, Ji 2011. Sugarcane yield responses to mill mud and compost application on mineral soils in Florida. Proceedings from the 2011 joint annual American Society of Sugar Cane Technologist conference. Int Sugar J. 113: 744 745. Meh lich, A. 1984. Mehlich 3 soil test extractant: a modification of Mehlich 2 extractant. Communn So il Sci Plant Anal. 15 : 1409 1416. Miles, N. 2010. Challenges and opportunities in leaf nutrient data interpretation. Proc S Afr Sug Technol Ass. 83: 205 21 5. Moberly, P.K. and J.H. Meyer. 1978. Filter cake: A field and glasshouse evaluation. Proc. S. Afr. Sugar Technol. Assoc. 52:131 136. Morgan, K.T., J.M. McCray, R.W. Rice, and L.E. Baucum 2009. Review of current sugarcane fertilizer recommendations: A repor t from the UF/IFAS sugarcane fe rtilizer standards task force. EDIS document SL 295. Soil and Water Science Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida Morris, D.R., R.A. Gilbert C. R. Rainbolt, R.E. Perdomo, G. Powell, B. Eiland, and G. Montes. 2007. Soil organic matter and macronutrient levels after mill mud application to sugarcane growing in a sand soil. Proceeding of the 2007 American Society of Sugar Cane Technologist joint annu al conference. Sugar Cane Int. 24: 35 37

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103 BIOGRAPHICAL SKETCH Susanna Gomez was born in Chesapeake degree in e nvironmental s cience with a minor in w etland s ciences from Virginia Tech in the spring of 2011 She joined the Soil and Water Science Department at the University of Florida in the fall of 2011 with Dr. Samira Daroub. Susanna completed her program at UF with a Master of Science with a minor in e nvironmental e ngineering and an interdisciplinary wetland certificate in the summer of 2013.