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Reconciling pH for Ammonia Biofiltration in a Cucumber/Tilapia Aquaponics System Using a Perlite Medium

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Title:
Reconciling pH for Ammonia Biofiltration in a Cucumber/Tilapia Aquaponics System Using a Perlite Medium
Creator:
TYSON, RICHARD V.
Copyright Date:
2008

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Subjects / Keywords:
Ammonia ( jstor )
Aquaculture ( jstor )
Aquaponics ( jstor )
Bacteria ( jstor )
Hydroponics ( jstor )
Nitrogen ( jstor )
Nutrients ( jstor )
Perlite ( jstor )
pH ( jstor )
Plants ( jstor )
City of Gainesville ( local )

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University of Florida
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University of Florida
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Copyright Richard V. Tyson. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2009
Resource Identifier:
659806785 ( OCLC )

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1 RECONCILING pH FOR AMMONIA BIOFILTRATION IN A CUCUMBER/TILAPIA AQUAPONICS SYSTEM USING A PERLITE MEDIUM By RICHARD V. TYSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Richard V. Tyson

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3 To my beloved wife, Gladys, and to the loving memory of our parents: James and Alyce Tyson and Sandalio and Juaquina Moreno

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4 ACKNOWLEDGMENTS I wish to express my sincere appreciation and gratitude to Dr. Eric Simonne, chair, and Dr. Marion White, co-chair of my graduate advisory committee, for their encouragement, advice, guidance, and patience throughout my Ph.D. pr ogram. I would like to extend my deepest gratitude to the members of my advisory comm ittee, Dr. Frank Chapman, Dr. Megan Davis, and Dr. Danielle Treadwell for their as sistance, technical support, and gui dance. I would also like to thank Dr. Elizabeth Lamb for her tireless re views of my early wo rk on the project. My deepest gratitude also goes to my supe rvisors at the University of Florida and Seminole County Extension: Dr. Freddie Johnson, Dr. Dan Cantliffe, Barbara Hughes, and Suzy Goldman for their understanding and suppor t of my Ph.D. program activities. My sincere thanks is extended to those i ndividuals who assisted in the setup and implementation of my research during the 2006 sa bbatical at the University of Florida: Dr. Amarat Simonne, Mike Alligood, David Studs till, Aparna Gazula, and Wei-Yea Hsu.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Importance of Hydroponic Vegetable Pr oduction, Aquaculture, and Integrated Aquaponic Systems.............................................................................................................11 Statement of the Problem, Rational and Significance............................................................13 2 REVIEW OF LITERATURE.................................................................................................17 Introduction................................................................................................................... ..........17 The Dichotomy of pH in Aquaponics.....................................................................................18 pH Affects Nitrification Activity.....................................................................................20 pH Determines Ammonia Equilibrium...........................................................................22 pH and Nitrite Accumulation..........................................................................................22 Plant Nutrient Considerations and pH.............................................................................23 Balancing Aquaponic System Water pH.........................................................................25 Ammonia Biofiltration.......................................................................................................... ..25 Aquaculture Biofilters.....................................................................................................25 Plants as Biofilters...........................................................................................................29 Hydroponic Systems and Media.............................................................................................30 Overcoming Limiting Fact ors in Plant Nutrition....................................................................32 Aquaponics: the Potential for Sustainability..........................................................................36 Water and Nitrogen Budgets...........................................................................................37 Further Systems Integration............................................................................................39 Conclusion and Objectives.....................................................................................................41 3 RECONCILING WATE R QUALITY PARAMETERS IMPACTING NITRIFICATION IN AQUAPONICS: THE PH LEVELS...................................................46 Introduction................................................................................................................... ..........46 Materials and Methods.......................................................................................................... .48 Results and Discussion......................................................................................................... ..50 Conclusions.................................................................................................................... .........54

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6 4 EFFECT OF NUTRIENT SOLUTION, NO3 --N CONCENTRATION AND PH ON NITRIFICATION RATE IN PERLITE MEDIUM................................................................57 Introduction................................................................................................................... ..........57 Materials and Methods.......................................................................................................... .60 Results and Discussion......................................................................................................... ..63 Conclusion..................................................................................................................... .........66 5 EFFECT OF WATER pH ON YIEL D AND NUTRITIONAL STATUS OF GREENHOUSE CUCUMBER GROWN IN RECIRCULATING HYDROPONICS..........71 Introduction................................................................................................................... ..........71 Materials and Methods.......................................................................................................... .73 Results and Discussion......................................................................................................... ..75 Conclusion..................................................................................................................... .........79 6 WATER QUALITY INLUENCES A MMONIA BIOFILTRATION AND CUCUMBER YIELD IN RE CIRCULATING AQUAPONICS............................................84 Introduction................................................................................................................... ..........84 Materials and Methods.......................................................................................................... .87 Results and Discussion......................................................................................................... ..91 Conclusion..................................................................................................................... .........98 7 CONCLUSIONS............................................................................................................. ......105 REFERENCES..................................................................................................................... .......108 BIOGRAPHICAL SKETCH.......................................................................................................119

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7 LIST OF TABLES Table page 2-1 Fraction of NH3 in an ammonia solution...........................................................................43 2-2 Average concentrations of mineral nutr ients in plant shoot dry matter that are sufficient for adequate plant growth..................................................................................44 2-3 Percent elemental analysis a nd physical propertie s of perlite...........................................45 3-1 Changes in TAN, NO2-N, and NO3-N concentrations in perlite medium trickling biofilters as affected by water pH......................................................................................55 4-1 Total ammonia nitrogen (TAN), nitrite nitrogen (NO2 -N), and nitrate nitrogen (NO3 -N) concentrations in perlite trickli ng biofilter (root growth medium) when exposed to hydroponic nutrient solution............................................................................69 5-1 Initial water analysis for pH and selected nutrients...........................................................81 5-2 Cucumber shoot fresh and dry weight and plant length on 14 DAT stage of growth as influenced by system water pH and foliar spray................................................................81 5-3 Cucumber shoot nutrient content (% DM) 14 DAT as influenced by solution pH and foliar spray................................................................................................................... ......82 5-4 Cucumber shoot nutrient content (mg/kg) 14 DAT as influenced by solution pH and foliar spray................................................................................................................... ......82 5-5 Concentration of NO3 –N and K in cucumber petiole sap and average season nutrient solution NO3 –N and K levels............................................................................................83 5-6 Cucumber fruit yield as influenced by nutrient solution pH and foliar spray...................83 6-1 ‘Fitness’ cucumber fruit yield resp onse to pH and production system..............................99 6-2 Twenty-four hour total ammonia nitr ogen (TAN) and nitrite nitrogen (N02 -N) concentrations in a perlite trickling biofilter after introduc tion of ammonium chloride....................................................................................................................... .....100 6-3 Twenty-four hour TAN loss from recirc ulating system tank water and perlite trickling biofilter after introduc tion of ammonium chloride............................................101 6-4 Estimate of ammonia and nitrate nitrogen mass balance in an aquaponic system with a raised bed perlite me dia trickling biofilter....................................................................103 6-5 Tilapia initial feeding activity and overal l mortality as influenced by system water pH and production method...............................................................................................104

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8 LIST OF FIGURES Figure page 4-1 Effect of pH on ammonia and ni trite oxidation in perlite medium....................................70 6-1 Most probable number (MPN) of Nitrosomonas sp . bacteria in perlite trickling biofilters as influenced by pH and production method....................................................102 6-2 Perlite trickling biofil ter 24-hour TAN loss as influenced by pH and production method......................................................................................................................... .....102

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9 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RECONCILING pH FOR AMMONIA BIOFILTRATION IN A CUCUMBER/TILAPIA AQUAPONICS SYSTEM USING A PERLITE MEDIUM By Richard V. Tyson May 2007 Chair: Eric H. Simonne Cochair: James M. White Major: Horticultural Science Integrated hydroponic and aquaculture (aquapon ic) production requires balancing pH and water quality for the growth of 3 organisms: plan ts, fish, and nitrifying bacteria. To improve systems integration, a series of trials were conducted to 1) determine the optimum pH for nitrification and evaluate performance of perlite as a biofil ter, 2) determine the effect of hydroponic nutrients on nitrification, 3) make predictions about the contribution of plants and nitrifiers to ammonia biofiltra tion, and 4) establish a reconcil ing pH for ammonia biofiltration and cucumber yield in aquaponics. Tota l ammonium nitrogen (TAN) removal and NO2 --N accumulation in a trickling perlite biofilter increa sed as pH increased from 5.5 to 8.5. Aquaponic biofilter TAN removal rates were 19, 31, and 80 g/m3/d for pH 6.0, 7.0 and 8.0, respectively. Nitrification was unaffected by plant nutrients in solution at optimum levels for hydroponic production. Nutrients may be tailored for plant production (with consideration for fish waste contributions) with no adverse impact on nitrifie rs. Most probable number (MPN) sampling of biofilter cores indicated that aquaculture contro l at pH 7.0 with no plants had a higher (0.01% level) number of Nitrosomonas sp. biofilter bacteria compared to treatments containing plants in the biofilter. However, the highest ammonia biofiltration rate was a quaponic production (plant,

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10 fish, bacteria) at pH 8.0. pH was a more important factor than bacteria p opulation in the rate of ammonia biofiltration—most likely due to pH in duced increases in unionized ammonia, the substrate for the nitrification reaction. Ammoni a biofiltration increased 3.7 times at pH 6.0 when bacteria and plants were in the biofilter co mpared to plants alone. The vigor of tilapia ( Oreochromis niloticus ) feeding increased and mortality decreased as water pH increased from 6.0 to 8.0. Early marketable cucumber fruit yiel d decreased linearly as pH increased from pH 5.0 to pH 8.0. However, total marketable yield was unaffected by pH. The reconciling pH for ammonia biofiltration and cucumber yield shoul d be pH 7.5 to 8.0 given the importance of pH and bacteria to the ammonia biofiltra tion rate, differences in fish vigor, and given that no difference in total cucumber fruit yield among treatments was found.

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11 CHAPTER 1 INTRODUCTION Importance of Hydroponic Vegetable Producti on, Aquaculture, and Integrated Aquaponic Systems Hydroponics is a term used to describe the pr oduction of plants wit hout soil. Plant roots grow in a nutrient solu tion with or without an artificial me dium for mechanical support (Jensen, 1997). Greenhouse hydroponic vegetable producti on is expanding rapidly worldwide (Brentlinger, 1999; Cantliffe and VanSickle, 2000; Resh, 2004; Smither-Kopperl and Cantliffe, 2004; Steta, 2004). Recent estimates of productio n (in hectares) for selected countries are: Spain, 60,000; Israel, 12,141; Holland, 4,100; Me xico, 2,000; England, 1,722; Canada, 800; United States, 400. The value of the commerc ial hydroponic industry worldwide is currently estimated at 6 to 8 billion dollars (Hassall, 2001) . Hydroponics is a very young science. It has been used on a commercial basis for only 50 years. However, gr eenhouse hydroponic vegetable production offers the potential for greater yields and more control of practices that can be environmentally sensitive compared to field grown production (Smither-Kopperl and Cantliffe, 2004). Yield increases of 2.3 (lettuce, Lactuca sativa ), 3.1 (pepper, Capsicum annuum ) 4 and 8.7 (cucumber, Cucumis sativus ), and 6 (tomato, Solanum lycopersicum ) times field grown yields have been reported for hydroponic greenhous e production (Resh, 2004; Smither-Kopperl and Cantliffe, 2004). Greenhouse European cu cumber yields were about 37.9 kg/m2 per year during 1999 when spring and fall crops were combined at the Horticultural Research Unit in Gainesville, Florida (Shaw et al., 200 0). This compares to 5.3 kg/m2 of field grown cucumber yields (one crop) in the 1 999 growing season (Rhodes, 2001). Overall yield of hydroponic greenhouse vegetable crops increased an averaged of 5 times over field grown yields (Resh, 2004; Smither-Kopperl and Cantliffe, 2004).

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12 Aquaculture, the commercial farm production of fish, shellfish and plant products, is the fastest growing sector of the world food economy, increasing by more than 10% per year. Nearly a third of the seafood c onsumed in the world today is a product of farmed aquaculture (Timmons et al., 2002). Consumer demand for aquaculture products is increasing as many wild fisheries stocks have reached, or are very close to, their maximu m sustainable limits. The U.S. catfish ( Ictalurus punctatus ) industry has grown by 100 million kil ograms in the last 7 years. The Atlantic salmon ( Salmo salar ) industry has been adding 50 million kilograms of new production each year for the last 10 year s. Worldwide production of tilapia ( Oreochromis sp .) exceeded 2.2 million metric tons in 2002 with 68% of that total coming from farmed aquaculture (Lim and Webster, 2006). Intensive recirculating aquaculture systems reuse relatively small volumes of water by circulating the water through biofilt ers to remove toxic waste produc ts before returning the water to production tanks (Rakocy et al ., 2006). These systems allow produc tion of fish at much higher levels than extensive pond culture with carrying capacities of 60 kg/m3 vs. 0.6 kg/m3, respectively (Losordo et al., 1998). Recirculating aquaculture is an environmentally responsible alternative to fishing and virtually eliminates bycatch waste which occurs in wild fisheries. However, water discharge/replacement requiremen ts of 5% to 10 % of recirculating water volume per day makes these systems subject to di scharge restrictions du e to concerns with environmental waste management (Timmons et al., 2002). Concentrations of organic matter, inorganic nitrogen and phosphorus in the waste water may be high requiring in-system or postdischarge treatment of effluents (Gutie rrez-Wing and Malone, 2006; Shnel, 2002). Aquaponics is an integrated system th at links hydroponic plant production with recirculating aquaculture (Diver, 2006). The ad vantages of linking fish and plant culture are

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13 shared startup, operating, and infrastructure cost s, fish tank waste nutrient and water removal by plants, reduced water usage a nd waste discharge to the envi ronment, and increased profit potential by producing two cash crops (Rakocy, 1999; Timmons et al., 2002). Properly designed and managed hydroponic and aquaculture systems are considered environmentally responsible alternatives to fiel d grown vegetable production and wild caught fisheries (SmitherKopperl and Cantliffe, 2004; Timmons et al., 2002). Statement of the Problem, Rational and Significance Water and nitrogen budgets fo r conventional field grown vegetable crops are often formulated with the knowledge that a portion of these inputs will be lo st to the environment through leaching, runoff (Hochmuth and Hanlon, 1995) , denitrification and/or volatilization (Cockx and Simonne, 2003). Movement of fertiliz er inputs, especially nitrogen, and buildup of phosphorus in the environment, may adversel y impact natural ecosystems and the water resources they depend on (Mitsch and Gosseli nk, 2000). As a result, farmers are under tremendous pressure to reduce or eliminate nutrientladen water discharges to the environment. Despite significant progress in reducing phosphor us discharges in the Lake Apopka basin (Neal et al., 1996), 6,070 hectares of land for vege table production was pu rchased and taken out of production several ye ars after passage by the Florida legislature of the Lake Apopka Restoration Act of 1996 (Tyson et al., 1996). Si nce this area traditionally multiple cropped radishes ( Raphanus sativus ), sweet corn ( Zea mays ), and cole crops (various crops in the Cruciferae or mustard family), this translated into an average loss of 14,164 hectares of vegetable production and $50 million in farm gate va lue per year. Other agricultural areas of Florida are under discharge rest rictions, including the Everglades Agricultural Area (EAA) covering 279,239 hectares in south Florida. Similar phosphorus reductio n efforts are underway in the EAA (EPA, 2003).

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14 Harvested vegetable acreage in Florida d eclined by 29,907 hectares (21%) during the ten year period from 1994 to 2003 (Bronson, 2005) a nd a third of the loss can be attributed directly to government buyouts over concerns with nutrient discharges to the environment. Designing and managing agricultu ral production systems for mini mal discharge of water and nutrients to the environment such as aquaponi cs, protects groundwater quality, makes water permitting easier to obtain, and may help maintain the long term sustainability of agricultural enterprises. These designs will also reduce conc erns about discharge of nutrients into coastal zones that could contribute to reef die-off and harmful algal blooms. Additional land loss of agricultural production ca n be attributed to urban sprawl. Urban expansion in the United States claimed more th an 400,000 hectares of cultivated lands per year between 1960 and 1990 (Heimlich and Anderson, 2001) . Farms in metropolitan areas make up 33 % of all farms, 16 % of cropland, and produce a th ird of the value of U.S. agricultural output. Over 75 % of the land area of Florida is classifi ed as either metropolitan core or metropolitan edge. The highest rates of popul ation growth occur at the ed ges of metropolitan areas in predominantly rural counties. In order for fa rmers to adapt to rising land values and new residents, they need to change operations to emphasize higher value products, more diversification, intensive producti on on less land, and an urban ma rketing orientation (Heimlich and Anderson, 2001). Advances in technology, limitations in water quality and quantity, environmental regulations, and increasing input costs are driving the aquaculture industry towards similar more intensively managed systems (Fitzimmons, 2003; Guitierrez-Wing and Malone, 2006). Hydroponics and recirculating aquaculture ar e intensive production systems that produce high value agricultural products. More diversification, conservati on of resources, and total yield

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15 increases are possible when these systems are integrated into aquaponic production systems. Developing aquaponic systems for minimal discharg e of nutrients and water to the environment would allow sustainable agricultu ral production in and around metropol itan areas. However, this technology is new, and adoption is limited by a lack of basic production information (McMurtry et al., 1997). Combining hydroponic and aquaculture system s requires reconciling water quality parameters for the survival and growth of plants , fish, and nitrifying bacteria. However, there are many unanswered questions regarding the op timum water quality parameters when the organisms present in aquaponics ar e grown together. In particul ar, a dichotomy exists between the optimum pH for plant nutrient availabi lity in hydroponics (pH 5.5.5; Hochmuth, 2001a) and the optimum pH for nitrifying bacteria ac tivity (7.5.0; Hochheimer and Wheaton, 1998). Aquaculture production is recommended to be ma intained between pH 6.5 and 8.5 (Timmons et al., 2002). Aquaponics has the pote ntial to be a sustainable zero agricultural discharge system (ZADS) since the waste by-products of aquacultur e can be used by plants in hydroponic systems (Tyson, 2004). However, with all its promise as a sustainable alternative to conventional food production, there is limited information on how aquaponic system water quality impacts nitrification in perlite growth me dium, little information on the plant/ nitrification interactions in root growth media biofilters, and how this inte raction affects ammonia biofiltration and plant yield. In addition, it would be beneficial to add fertilizer nu trients to aquaponic production system water to optimize plant nutrient levels pr ovided this does not adversely impact the fish and nitrifying bacteria but more science based information is needed before recommendations can be made. Information is lacking on the rela tive contribution of nitrifying bacteria and plants to system water ammonia biofiltration and the re lative importance each has in the overall system

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16 performance and economic yield. The reconcilin g pH for ammonia biof iltration in aquaponic systems will be determined by the amount of a mmonia removed from system water by plants and nitrifying bacteria and th e effect of pH on vegetable fruit yields. Adoption of properly designed aquaponic systems will conserve our natural resources; water and the fossil fuels required to produce nitr ogen and other plant nutrients. However, clear scientifically based recommendations on system ma nagement are required to remove information barriers to adoption of this technol ogy. The overall goal of this re search was to 1) determine the optimum pH for nitrification and evaluate performance of perlite as a biofilter/root growth medium, 2) determine the affect of hydroponic nu trients on nitrification, 3) make predictions about the contribution of plants and nitrifiers to ammonia biofiltration, and 4) establish a reconciling pH for ammonia bi ofiltration and cucumber yi eld in aquaponics.

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17 CHAPTER 2 REVIEW OF LITERATURE Introduction Aquaponics is an integrated system th at links hydroponic plant production with recirculating aquaculture (Diver, 2006; Timmons et al., 2002). The adva ntages of linking plant and fish culture include fish tank waste nutri ent and water removal by plants, reduced water usage and waste discharge to the environment by both systems, and increas ed profit potential by producing two cash crops (Rakocy, 1997; Rakocy et al., 2006; Timmons, et al., 2002). The most common aquaponic systems to date employ either a media-filled raised bed, nutrient-flow technique (NFT), or floating raft system (Adler et al., 1996; Anonymous, 1997, 1998; Diver, 2006; McMurtry et al., 1997; Ra kocy et al., 1997, 2006; Watten and Busch, 1984) for the plant growing area integrated with a recirculating aquaculture tank system (Timmons et al., 2002). Aquaponic system integration is hampered by the lack of information on a reconciling pH between the optimum pH for hydroponic plant production (5.5.5; Hochmuth, 2001a) and for rapid ammonia biofiltration of system wa ter (7.5.0; Hochheimer and Wheaton, 1998). Recommended pH levels for aquaculture produc tion are between 6.5 and 8.5 (Timmons et al., 2002). There is little information on the use of hydroponic media as biofilters of ammonia, especially newer, more commonly used media, such as perlite. In addition, it would be beneficial to add fertilizer nutrients to aquaponic production system water to optimize plant nutrient levels provided this doe s not adversely impact fish and nitrifying bacteria. A more flexible management strategy for these systems would be to supplement with plant nutrients, which would permit less reliance on the fish and nitrification to provide optimal plant nutrient levels. Information is also lacking on the relati ve contribution of nitrifying bacteria and plants to system water ammonia biofiltration and th e importance each has in the overall system

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18 performance. This will need to be determined to establish a reconciling pH for aquaponic system water. This review will address the importance of pH and water quality in successful aquaponic production especially as they affect ammonia bi ofiltration and cucumber production. The review will identify the limits and dangers associated wi th managing pH and water quality with respect to the organisms present in the system – especi ally nitrifying bacteria and plants. It will emphasize nitrification, the bioche mical reaction that converts NH3 to NO3 and ammonia biofiltration (nitrification + plan t removal of TAN). It will di scuss the hydroponic subsystem, especially systems utilizing perlite medium, wh ich may be used as biofilters for aquaponic systems. Further, the review will discuss limiting factors in plant nutrition and how aquaponic systems may be used to overcome these limits in order to improve system integration and sustainability. The Dichotomy of pH in Aquaponics The concept of pH was developed by a Dani sh biochemist named Soren Sorenson in 1909 to simplify working with solution hydrogen ion concentrations (Myers 2003). The pH of a solution is defined as the negative logarith m (base 10) of the hydrogen ion concentration (Campbell and Reese, 2002). A neutral pH in water based solutions has a pH of 7 (-log 10 -7 = (-7) = 7) and occurs when an equal number of hydrogen (H+) and hydroxyl (OH-) ions are present. When the hydrogen i on concentration increases (> 10-7 mol . L -1) from neutrality, the pH decreases and the solution is termed acidic. When the hydrogen ion concentration decreases (< 10-7 mol.L -1) from neutrality, the pH increases and th e solution is termed alkaline. The pH controls a wide variety of solubility, oxi dation–reduction and equi librium reactions in hydroponics and aquaculture systems (Timmons et al., 2002; De Rijck an d Schrevens, 1999).

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19 Integrating hydroponics and recirculating aqu aculture is hampered by differences in optimal pH recommendations for the nutrient so lution (pH 5.5.5) which baths the plant root system (Hochmuth, 2001a) and the recirculating water (pH 7.0.0) of the fish production tanks (Hochheimer and Wheaton, 1998; Timmons et al., 2002). The former is recommended to maximize the availability of nutrients in solution for uptake by plants. The latter is determined by pH ranges conducive for production of fish and the pH range of more optimal activity of nitrifying bacteria in removi ng ammonia waste from recircula ting waters. Recommendations on pH in plant and fish production are well established. However, sc ientific information is lacking in regards to pH in integrated hydroponic and reci rculating aquaculture syst ems, especially as it relates to the biofiltration of am monia by bacteria and plants. Recommended pH ranges for the nutrient solution irrigation water in greenhouse hydroponic production tends to be sligh tly acidic (5.5.0, Hochmuth, 2001b; 5.5.5, Hochmuth, 2001a; 5.8.4, Resh, 2004) to avoid precipitation of Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+ to insoluble and unavailable salts when pH > 7. Aquaponic recirculating water pH is recommended to be maintained in the range of 7.0 to 7.5 (Timmons et al., 2002) to balance the requirements of biofiltration of toxic fish waste ammonia from system wa ter with the nutritional needs of plants. If aquaponic re circulating water pH is maintain ed at levels more optimum for nitrifying bacteria (7.5.0; Ho chheimer and Wheaton, 1998), plant uptake of certain nutrients could become restricted and plan t yield reduced. Reconciling pH optima for production of plants (5.5.5) and the growth of n itrifying bacteria (7.5.0) in aquaponic systems would significantly improve systems integration and sustaina bility. Integration in this context refers to systems that are connected or combined to func tion together as one unit, each dependant on the other in some way. Sustainability refers to th e capacity of the combin ed units to use self

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20 sustaining resources inherent to th e systems in such a way that the resources are not depleted or the system harmed. pH Affects Nitrification Activity One of the most complex and important subsyste ms of recirculating aquaculture systems is the biofiltration and removal of fish waste ammo nia through nitrification to maintain fish tank water quality (Gutierrez-Wing and Malone, 2006; Masser, et al., 1999). Nitrif ication is a biological process performed by nitrifying bacteria that maintains water quality in recirculating aquaculture systems and has been shown to tran sform 93%% of potentially toxic nitrogenous fish wastes (NH3 -N) into relatively non-toxic NO3 --N in biofiltration units (Prinsloo et al., 1999). Nitrification is the biochemical conversion by bacteria of NH3 to NO3 (Hagopian and Riley, 1998; Madigan et al., 2003; Prosser, 1986). It is a two step process: Primarily Nitrosomonas sp . NH3 + 1 O2 NO2 + H2O + H+ + 84 kcal mol-1 (Equation 2-1) Primarily Nitrobacter sp . NO2 + O2 NO3 +17.8 kcal mol-1 (Equation 2-2) This nitrogen transformation eliminates ammonia from the water. Un-ionized ammonia nitrogen is toxic to fish at levels above 0.05 mg/L (F rancis-Floyd and Watson, 1996) and is dependant on pH and temperature of culture water. Nitrate, th e end product of nitrificati on, is not toxic to fish except at very high levels (channel catfish, Ictalurus punctatus , 96-h LC50 > 6,200 mg/L NO3N; Colt and Tchobanoglous, 1976), although some i nvestigations suggest that prolonged exposure to 200 mg/L NO3-N might decrease the immune res ponse of some fish species (Hrubec et al., 1996). Nitrate is the primary source of N for plants in hydroponi c nutrient solutions at concentrations from 50 to 280 mg/L NO3 -N (Resh, 2004). Hence, the understanding and

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21 management of the nitrification process in aquapo nics is important for the maintenance of water quality and the production of nitrat e nitrogen. The pH is one of the most important environmen tal parameters that can affect the activity of nitrifying bacteria (Antoniou et al., 1990). A wide range of pH optima have been reported from research on the effect of pH on the process of nitrification. In substrates from terrestrial forest environments, increasing pH stimulated net nitrification while decreasing pH depressed it (Ste-Marie and Pare, 1999). Nitr ification in aquaculture biofilters was reported to be most efficient at pH levels from 7.5 to 9.0 (Hochhe imer and Wheaton, 1998), and 7.0 to 8.0 (Masser et al., 1999). In a submerged biofilter investigation, a pH increase of one un it within a range of 5.0 to 9.0, produced a 13% increase in nitrification efficiency (Villav erde, et al., 1997). In another investigation with four different biological filters (under gravel, fluidized bed, non-fluidized bed, and gravel bed) nitrification slowed signi ficantly or stopped when pH dropped below 6.0 (Brunty, 1995). In wastewater tr eatment processes, the pH of a pproximately 7.8 (Antoniou et al., 1990) produced the maximum growth rate of nitr ifying bacteria and 8.4 (Peng et al., 2003) the greatest nitrification rate. No scientifically based optimum pH has been reported for aquaponic systems. The causes of varying pH optima may be attributed to differen ces in substrate, alkalin ity, effluent, or species of nitrifying bacteria present in the syst em. The literature would support maintaining recirculating water pH between 7.5 and 8.5 for ma ximum waste ammonia bi ofiltration within the range of plant and fish production recommenda tions (pH 5.5.5) if this were the only consideration for determining pH. However, th is pH range affects water quality parameters which may adversely impact plant production.

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22 pH Determines Ammonia Equilibrium In water, ammonia exists in two forms, wh ich together are called the Total Ammonium Nitrogen (Francis-Floyd and Watson, 1996) or TAN. The equilibrium reaction is (Campbell and Reese, 2002): NH4 + NH3 + H+. Water temperature and pH will affect which form of ammonia is predominate (Table 2-1). To calcu late the amount of unionized ammonia present, the TAN (TAN = NH4 +-N + NH3 -N) of a water sample must be multiplied by the factor (using the pH and temperature of that sample water) sel ected from Table 2-1. Fo r example, at 28C, the percent of NH3 increases by nearly a factor of 10 for each 1.0 increase in pH and is 0.2%, 2% and 18% of the TAN for pH 6.5, 7.5, and 8.5, respectively. Un-ionized ammonia nitrogen (NH3 – N) at concentrations as low as 0.02.07 mg/L have been shown to slow fish growth and cause ti ssue damage (Masser et al., 1999). The 96-h LC50 for un-ionized ammonia on fi ngerling channel catfish ( Ictalurus punctatus ) was 3.8 mg/L (Colt and Tchobanoglous, 1976). The 96–hour LC50’s vary wide ly by fish species from as low as 0.08 mg/L NH3 -N for pink salmon to 2.2 mg/L NH3 -N for common carp (Timmons et al., 2002). The 72 h LC50 of NH3 for tilapia Oreochromis aurea has been reported at 2.35 mg/L (Redner and Stickney, 1979; as referenced in Lim and Webster, 2006; original document not found). Safe pH ranges will depend largely on the species grown. Thus 5 mg/L TAN at pH 7 would be safe for pink salmon and common carp; but the same TAN at pH 8 would kill pink salmon, but not common carp. Within the range of pH 7 to 8, sp ecies sensitive to union ized ammonia should be grown closer to pH 7 to avoid damage. pH and Nitrite Accumulation The intermediate product of nitrification, nitrite (NO2 -), may be toxic to both fish and plants at low levels. Nitrite at concentrations as low as 5 mg/L in nutrient solution damaged tobacco ( Nicotiana tabacum L.) root tips (Hamilton and Lowe, 1981). Nitrite oxidation activity

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23 was suppressed by elevated pH and ammonium concentrations when urea was used in a hydroponic tobacco float system resulting in the a ccumulation of toxic levels (30-70 mg/L) of nitrite (Pearce et al., 1998). Gila trout ( Oncorhynchus gilae ) exposed to nitrite at 10 mg/L or more for 96 h died (Fuller et al ., 2003) and the 96 h LC50 for bass ( Morone sp .) was 12.8 mg/L (Weirich et al., 1993). Nitrite is toxic to fish because it affects the blood hemoglobin’s ability to carry oxygen (Timmons et al., 2002) a nd is called “brown blood disease.” Nitrite accumulation can occur due to the faster growth of a mmonium oxidizers than nitrite oxidizers (most common in startup biofilte r cycles) or due to the inhibition of Nitrobacter by free ammonia (Villaverde et al., 1997), which is more common under steady state conditions and was reported to start at pH above 7.5 and increasing asymptotically to 85% at pH 8.5. To avoid damage to plants and fish from nitrite accumu lation under steady state conditions, a pH at or below 7.5 would seem to be an appropriate recommendation. Plant Nutrient Considerations and pH Plants require 17 elements (Table 2-2) fo r normal growth and development (Marschner, 2003). Carbon (C), hydrogen (H), and oxygen (O), required in the largest amounts, are supplied by air and water. The others come from soil a nd/or fertilizer: nitrog en (N), phosphorus (P), potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), iron (Fe), copper (Cu), manganese (Mn), boron (B), molybdenum (Mo), zinc (Zn), chlorine (Cl) and nick el (Ni). Since hydroponic systems do not use soil, care must be taken to insu re that all the essential elements necessary for plant growth are supplied to the system in some fashion, usually by adding water soluble fertilizer. The recommended amounts of major elements such as nitrogen and potassium vary depending on crop species, especially in regard to specific crops in hydroponic culture where nutrition can be managed (Hochmuth, 2001a; Hoch muth and Hochmuth, 2003). Cl and Ni are required in such small amounts and are available in the environment (soil or water) so that it is

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24 usually not necessary to supply them in fertilizer applicatio ns. Some elements, although not required, are beneficial to the plant. Silicone, for example, is needed for stem support and gives resistance to fungal infection, es pecially in cucumber (Resh, 2 004). Sodium, cobalt, selenium, and aluminum have also been found to be bene ficial for certain plan ts and circumstances (Marschner, 2003). pH affects the solubility of i ons in solution and the ionic form of several elements (Epstein and Bloom, 2005; De Rijck and Schrevens, 1999). Ac idity increases the sol ubility of sulfates and phosphates (Taiz and Zeiger, 2002) . Increasing ion solu bility facilitates th eir availability to roots. Rhizosphere pH may differ from bulk soil pH by up to two pH units (Marschner, 2003), because of the release from plant roots of H+, OH-, or HCO3 and the excretion of organic acids. Root uptake of ions is an electr ically neutral process. Thus ro ot induced changes in pH are the result of imbalances in the cati on/anion uptake ratio resulting in ne t differences in the release of H+, OH-, or HCO3 by roots. Recommended pH ranges for hydroponic production systems tend to be slightly acidic (5.5 to 6.5, Hochmuth, 2001a; 5.8 to 6.4, Resh, 2004) in order to provide a greater relative availability of most nutrients in solution. Precipitation of Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+ to insoluble and unavailable salts can occur in nutrient solu tion culture at water pH levels above 7. Phosphorus deficiency causing yield reduction in hydroponic tomato es (Wallihan et al., 1977) and iron deficiency with dry matte r yield reduction in sorghum ( Sorghum bicolor ) grown in solution culture (Bernardo et al., 1984) occurred when pH levels were above 7. If aquaponic recirculating water pH is maintained at leve ls more optimum for n itrifying bacteria (7.5.0; Hochheimer and Wheaton, 1998), plant uptake of cer tain nutrients may become restricted and thus plant yield may be reduced.

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25 It may be possible to overcome nutrient defi ciency and increase crop yield under system water pH production conditions > 7.0 with the use of foliar application of certain plant nutrients. Foliar applications of Mg, Zn, a nd Mn fertilizers can effectively correct deficiencies of these nutrients in fruit and vegetabl e crops grown on calcareous soils (usually pH 7.4.4) in south Florida (Li, 2001). Overall yiel d increases of 33% occurred wh en strawberry cultivars were sprayed once per week with Fe when grown in calc areous (pH 8.2) soil (Zaiter and Saad, 1993). Balancing Aquaponic System Water pH Where does the balance in pH lie for aquaponic sy stem water? Is it weighted toward the plant component, the fish component, or the biofilt er housing the nitrifying bacteria? How much do plants contribute to the biof iltration of ammonia and is that relevant to the overall system operation? If the plant contributi on to ammonia biofiltration is sign ificant then are the nitrifying bacteria and their special water quality require ments needed? Since unionized ammonia and nitrite are toxic to fish, and bot h increase with increasing pH, then what is the tipping point—the pH that the system should not go above? The adoption of aquaponics is hampered by a lack of scientifically based answers to some of these questions and the need to make sense of these dichotomies in pH. When this is accomplished, reasonable and understandable recommendations for system operation will make system adoption easier and more efficient. Ammonia Biofiltration Aquaculture Biofilters Recirculating systems must incorporate both so lids removal and biol ogical filtration into the water reconditioning process to achieve prop er water quality for fish and plants (Harmon, 2001). Solids removal is accomplished when recirc ulating water passes through a material that intercepts suspended particles. A biofilter is simply a surface on which bacteria grow. Biological filtration can take plac e anywhere in the system where recirculating water comes in

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26 contact with a surface to which nitrifying bacteria are attach ed–this may include tank walls, interior surfaces of pipes and ev en plant roots. However, to provide sufficient biofiltration activity to maintain optimum water quality in in tensive recirculating a quaculture, where (TAN) loading can be high, separate biofilter equipment is currently required. Biofilters used in recirculating aquaculture are of two main types: fixed film (attached growth) and suspended growth where microorgani sms are maintained in suspension (GutierrezWing and Malone, 2006). Suspended growth systems are not common because of their high level of management and reputation for instab ility. Thus, most of the biofiltration in recirculating systems are aerobic, fixed film biofilters. These biofilters basically consist of a porous solid phase on which nitrifying bacteria grow and extract nutrien ts from water passing over this solid phase (Wheaton, 1993). Water ma y enter the biofilter fr om the top, side or bottom and exit from the bottom, side or top, depending on design location relative to the water level of the fish tank. There are four basic types of biofilter designs : submerged bed, rotating disc, fluidized bed, and trickli ng (Tetzlaff and Heidinger, 1990). Submerged bed biofilters are characterized by tank water being pumped through a medium that is consta ntly underwater. The rotating disc biofilter consists of a series of parallel circular plates mounted on a shaft and rotating as a round drum with th e lower part submerged and the upper part above water. The plates are the substrate the bacteria grow on. In a fluidized bed biofilter, water enters the bottom of a medium containing cylinder under high pressure and exits out the top after being acted on by the filter. In trickling filter s water enters the top and flows down through the medium and keeps the bacteria wet but never completely submerged. Trickling filters are maintained above the water level of the fish tank.

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27 Of all the water quality parameters which aff ect fish, ammonia is th e most important after oxygen (Francis-Floyd and Watson, 1996). Ammonia is the main excretion product from fish and uneaten feed. It can quickly become a concern because of the buildup of un-ionized ammonia and nitrite, both of which can be toxi c to fish at very lo w levels (Harmon, 2001; McGee and Cichra, 2000) as disc ussed previously. Ammonia is usually not a problem if the biological filters are properly si zed for the loading rate and carry ing capacity and if adequate water flow is maintained (Fowler, et al., 1994). Hockheimer and Wheaton (1998) recommend that the system water move th rough the biological filter at le ast 2 times per hour. However, Rakocy et al. (1997) were unable to detect any difference in tilapi a growth rate, total weight, or survival between water exchange rates of 0.55 and 1.25 times per hour. Perhaps both of these flow rates were too low to detect a difference. McGee and Cichra (1999) recommend a 3:1 fish tank to biofilter volume ratio as being a more than sufficient design for biofilters. The ammonia generation load is based on the fish feeding rate and could be assumed to be 10% of the protein in the feed becomes the amm onia-N generation rate (Timmons et al., 2002). The size of the biofilter depends on the amount of ammonia added to the system which is closely related to the feeding ra te and efficiency of food utilization (Tetzlaff and Heidinger, 1990). Van Gorder (2000) indicated that feed levels change with fish size as fingerlings consume a much higher percentage of their body weight (5% %) than harvestable size fish (0.75%%). Another way to determine ammonia – N load is to consider that generally 2.2 to 6.6 kg of ammonia are produced for each 220 kg of feed. Thus, 220 kg of fish being fed 6.6 kg of fish feed per day (3% of body wt/d) produce 0.1 kg of ammonia per day. Chapman (2000) puts these feed levels at 6 to 15% of body weight for young fish (<25 g) and 1% to 3% of body weight for older fish (>25 g).

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28 Trickling biofilters provide nitr ification, aeration, and some carbon dioxide removal in one unit (Losordo, et al., 1999). The main disadvantage of trickling filters is that they are relatively large and biofilter media are expensive. The quantity of bacteria available to oxidize ammonia is limited by the surface area of the biofilter medium t hus an important factor in biofilter design is to get the maximum amount of surface area in to a given volume (Harmon, 2001). However, when particle size is reduced, filter clogging may increase and the ability of oxygenated water to mix well within the filter decreases. Clogging of the medium may occur if the solids are not prefiltered. Volumetric nitrifi cation rates of about 90 g to tal ammonia nitrogen (TAN)/m3 per day can be expected with trickling filters (Losordo et al., 1999). When designing these filters into a recirculating system for nitrif ication (assuming 2.5 percent of th e feed becomes TAN), a design criteria of 3.6 kg feed/day/m3 of trickling medium should be us ed. Based on these numbers, and a feeding rate of 3% of fish body weight per day, 1 m3 of trickling medium biofilter should support 120 kg of fish. With a carry ing capacity at harvest of 60kg/m3, 1 m3 of trickling medium biofilter would be required for ev ery 2000 L of fish tank water, a 1 to 2 ratio. Increased removal of ammonia by a trickling biof ilter was found with increasing c oncentrations of ammonia in pond water (Rijn and Rivera 1990) and removal ra te was considered substrate-limited with respect to ammonia. Research on freshwater recirculating a quaculture biofilters should focus on cost competitiveness, low head and low energy use op eration in support of large scale facilities (Gutierrez-Wing and Malone, 2006). The efficiency of biofilters need not be associated with use of expensive commercial biofiltration devices (Prinsloo, et al., 1999). When two types of trickling filters were compared, one containi ng PVC shavings (surface contact area of 1,220 m2), the other a more sophisticated commercially av ailable biofilter made up of Siporax porous

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29 sintered glass cylinders (surface water contact area of 32,000 m2), the PVC biofilter was more efficient at breaking down nitrogenous wastes (efficiency 96% and 93%, respectively). Efficiency of biofilter media consisting of hydroponic media such as perlite has not been scientifically established. This should be accomplished in order to more effectively integrate sustainable hydroponic and aquaculture systems. Plants as Biofilters Plant uptake is one of the most widely r ecognized biological processes for contaminant removal in wastewater treatment wetlands (Debusk, 1999; Mitsch and Gosselink, 2000). Ammonium nitrogen removal effi ciencies of 86% to 98% were reported from a constructed wetlands system receiving aquaculture wastewat er (Lin et al., 2002). In hydroponic greenhouse plant production systems receivi ng aquaculture wastewater, Adle r (1996) found that differences in nutrient removal rates of nitrate nitrogen and phosphorus were dependant on plant numbers and effluent flow rate. If plant numbers are increased sufficiently, nutrient concentration can decrease to levels that may be too low to sustain plant growth. A quaponic wastewater cleanup cost abatement alone can be a major factor in integrating hydroponic a nd aquaculture systems (Adler, 2001; Adler, et al., 2000). Plant uptake was insufficient to remove cont aminants (Prinsloo et al., 1999) in one aquaponic trial due to a high ratio of fish to plants. Rakocy et al. (1997) were able to establish a balanced system by maintaining a large plant gr owing area relative to fish production area in a commercial scale aquaponics system . Rakocy (1999) indicated that su fficient nitrification occurs in lettuce floating raft systems when correct ra tios of fish feed to plant growing area are maintained. In a tilapia/floating romaine lettuce aquaponic system, each square meter of hydroponic growing area removed 0.56 g of ammoni a-nitrogen, 0.62 g of nitrite-nitrogen, 0.83 g of total nitrogen, and 0.17 g of total phosphorus per day.

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30 Plant roots were found to be more comp etitive for ammonium than the ammoniumoxidizing bacterial species Nitrosomonas europaea (Verhagen et al., 1994). There may be a possibility for less reliance on nitrification in aquaculture biofilters for ammonia removal when sufficient plants are present in aquaponic system s. The optimum ratio of nitrate to ammonium nitrogen in hydroponic nutrient solutions is 75:25 (Cockx and Simonne, 2003; Simonne et al., 1992). Consequently, a source of nitrate-nitr ogen would be needed for plant uptake in aquaponics either through nitrification or supplemental fertil ization for optimum plant growth. In addition, certain plant nutrients can fall belo w sufficiency standards in aquaponics (McMurtry et al., 1990; Rakocy et al., 1997; Se awright et al., 1998) without s upplemental fertilization. Thus methods to make up this deficit without adversel y impacting fish and nitrifying bacteria need further investigation. Hydroponic Systems and Media Hydroponics is the term used to describe the production of plan ts without soil. Plant roots grow in a nutrient solu tion with or without an artificial me dium for mechanical support (Jensen, 1997). Most fruits and vegetables are grown in field soil. Soil serv es two basic purposes: it acts as a reservoir for essential elements and wate r and it provides physical support for the plant (Johnson et al., 1985). Soilless cultu re (hydroponics) is an artificia l means of providing plants with support and a reservoir for nutrients and water. The growing medium can be perlite, vermiculite, rockwool, peatmoss, coir, composted pine bark, sawdust, sand or gravel. Water only systems such as the nutrient flow technique and the floating raft system utilize artificial means of support for the plant. Many hydroponic systems have been developed and the technology is rapidly changing (Resh, 2004, Tyson, et al., 1999, 2001), but they have only been used commercially for the last 50 years. Most systems are housed inside a greenhouse (considered Controlled Environment Agriculture or CES) but they can be used outdoors.

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31 The most common hydroponic systems in Florida today use some form of perlite medium which provides anchorage for the plant roots (Tyson, 2002). A nutrient solution is pumped through the medium and mineral elements are taken up by the roots. Other systems in use include the nutrient flow techni que (NFT) where the nutrient solu tion trickles down a plastic or PVC trough, rockwool culture where slabs of fibrous rockwool anchor plants as nutrient solution drips through them, and floating hydroponics, where a raft containi ng plants floats in a nutrient solution. Commercial production of leafy salad crops in greenhouses co ntaining floating raft systems have been used in Florida and Canada since the 1980’s (Resh, 2004, Spillane, 2001). Perlite is a generic term for naturally occurr ing volcanic glass or rock (Reed, 1996). When this material is heated, it pops like popcorn and expands from four to tw enty times its original volume. The expansion process cr eates a white angular pearl like pe bble that is light weight (32 kg/m3) and adaptable for numerous applications su ch as low to high temperature and acoustic insulation, as fillers, as adsorp tion carriers, and light weight aggregate construction, among other uses (Anonymous, 2007). Table 2-3 describes a typical elemental an alysis and physical properties of perlite. It also contains fluoride (17 mg/L, Reed, 1996). Certain ornamental plants such as dracaena ( Dracaena marginata ) have been shown to be sensi tive to fluoride. Perlite has a high water holding capacity a nd high aeration properties (Hoc hmuth and Hochmuth, 2003). Medium to course grade horti cultural perlite is recommended for use in hydroponic vegetable production. Perlite has become th e most commonly used plant gr owing medium in the Florida greenhouse vegetable industry (Tyson, 2002, Tyson et al., 2001). Perlite has also been used successfully as a filter to remove gaseous ammonia and othe r waste gases (Flanagan, et al., 2002; Joshi, et al., 2000; Wri ght and Raper, 1998).

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32 The most common recirculating aquaponic system s to date employ either a media-filled raised bed, nutrient-flow technique (NFT), or floating raft system (Adler et al., 1996; Anonymous, 1997, 1998; Diver, 2006; McMurtry et al., 1997; Rakocy et al., 2006, 1997; Watten and Busch, 1984) for the plant growing area. Of those systems, the media filled bed has potential for providing for solids removal, biolog ical filtration, and root zone space for plant production. Perlite is the mo st common plant growing medi um used in hydroponic plant production in Florida (Tyson et al., 2001). It has also been investigated as a soilless culture alternative to soil fumigation with methyl bromide in field grown tomato and pepper ( Capsicum annuum L) production (Hochmuth et al., 2002). The type of soilless media in which plants grow has been shown to significantly affect nitrifying bacteria counts (Lang and Elliott, 1997). However, perlite medium has not been investigat ed with respect to the activity of nitrifying bacteria in an aquaponic biofilter. Overcoming Limiting Factors in Plant Nutrition Yields of most crops in the Un ited States have increased an average of 3% per year for the past 30 years (Wallace and Wallace, 1993). This has been accomplished in part by breeding and in part by improving the efficiency of inputs and overcoming limiting production factors such as plant nutrient and water stress. The production and use of fertilizers has pl ayed a large role in this increase in productivity. For example, a doubling of agricu ltural food production over the last 35 years was accompanied by a 7-fold increase in nitrogen fertilization (Tillman, 1999). Nitrogen is an essential nutrien t element and the fertilizer nutrient required in largest amounts by plants. It can accumulate to levels up to 5% of plant dry matter (Marschner, 2003). Nitrogen is an essential constituen t of proteins and amino acids. It is assimilated into plants primarily in the form of nitrate (NO3 -) and ammonium (NH4 +). The manufacture of nitrogen fertilizer is expensive, requi ring large amounts of non-renewable energy resources. In addition,

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33 the uptake and assimilation of nitr ogen in the plant is energy c onsuming for the plant, requiring 15 moles of ATP for the reduction of one mole of nitrate and 5 moles of ATP for the assimilation of one mole of ammonia. Fluctuating nutrient environments require that organisms continually adapt with specific responses to either cope w ith nutrient limitations or adju st to oversupply (Grossman and Takahashi, 2001). Nitrogen limitation can in fluence the physiology and morphology of plants reducing cell division rates and metabolic activi ties. For example, a feedback control of photosynthesis is related to the ca rbon to nitrogen balance (Paul and Pellny, 2002). An adequate supply of building materials (C-skeletons) and energy is necessary to carry out many plant functions. Recent developments in addressing the problem of limiting nutrients focus on manipulating gene expression to improve yield by improving absorption and nutrient efficiency (Good et al., 2004; Grotz and Guerin ot, 2002). However, for the mo re traditional horticulturist, there may be ways of improving nutrient efficien cy in the short run by manipulating production system design. In an integrated hydroponic and a quaculture system, nitrogen in the nutrient solution was reduced 3.5 times compared to tradit ional solution concentrations to produce lettuce in a 2.5 year continuous multiple cropping pilot pr oject (Rakocy et al., 1997). It was suggested that this was the result of the nutrient solution constantly bathi ng the plants roots compared to intermittent applications, which may be the case in more trad itional hydroponic applications. This was also suggested by much earlier work (Olson, 1950). Olson (1950) was able to esta blish that nutri ents were absorbed at a constant rate regardless of concentration, as long as the overa ll proportion and concentrat ion of nutrients in solution remained nearly the same, and that th e nutrient solution was thoroughly mixed. More

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34 recent work (Schon and Compton, 1997b) illustrate s the importance of irrigation frequency on the effects of nutrient solution concentration. Irrigation frequency for greenhouse hydroponic cu cumbers is usually determined by the use of the weighing lysimeter system (Sc hon and Compton, 1997b). The length of each irrigation event is determined by the amount of time needed to obtain some leaching fraction (LF) that can range from 15% to 40% of th e applied irrigation (Schon and Compton, 1997a). The LF is defined as the volume of nutrient solution leached, divided by the total volume of solution delivered. This leaching process ensures the replenishment of nutrients in the media to recommended levels. The frequency of the irrigation event will be determined by the loss of weight (due to water uptake) of the bag, pot, or slab containing the growing media. When the water loss from the substrate reaches a level that may result in loss of plant turgor, irrigation is initiated. In this way the plant receives sufficien t water to prevent deficit stress. Under this regime, with a 20% LF in perlite media pots, ir rigation frequency increased from twice per day (for 3 min. each) early in the growth season, to 10 times dail y (for 10 min. each) during the cucumber harvest season (Cha verria et al., 2005). However, despite regimes of commercial ly recognized, commonly used irrigation frequencies described above for greenhouse cucumb ers, nitrate-N concentrations in rockwool media slabs can drop below the depletion level (< 10 mg/L N) just prior to harvest (Schon and Compton, 1997b). This N depleti on from substrate occurred even when the nutrient solution concentrations ranged from 90 to 175 mg/L N; this low level can reduce cr op yields. Increased N concentrations were recommended (between 225 and 275 mg/L N) for the nutrient solution so that N did not drop below depl etion level in the substrate between irrigation events.

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35 In soils, nutrients move to the surface of roots by diffusion and bulk flow of the soil solution resulting from transpiration (Taiz and Ze iger, 2002). Concentration gradients can form in the soil solution as nutrients are taken up by the roots and the c oncentration of nutrients at the root surface is lowered compared to the surrounding area. This can result in a nutrient depletion zone near the root surf ace. The capacity for continuous growth by roots however, extends this region of nutrient uptake beyond the depletion zone. Thus, optimum nutrient acquisition by plants depends on the capacity of their root system s not only to absorb nutrients, but also to grow into fresh soil. In hydroponic production, the media volume is finite and nutrient depletion can be recovered only in the next ir rigation event. The results described above, from Schon and Compton (1997b), indicate that N depleti on does occur at lower N nutrient solution concentrations, and that irrigation frequencie s adequate to prevent water stress are not necessarily adequate to prevent nutrient depletion except at high N nutrient solution concentrations. Therefore, it seems logical to propose that more frequent flushing of the media with lower concentrations of N would obviate N de pletion between irrigati on events. If this flushing was continuous, there would be no appreciable depletion of nutrients in the root zone. This reasoning could be applied to all nutrien ts in the nutrient solu tion and may provide an avenue for production of plants at pH levels > 7.0 . The constant recirculation of the nutrient solution across the root zone woul d obviate low nutrient concentrations of ions with potential for precipitation (Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+) to insoluble and unavailable salts at pH levels > 7.0. These data suggest that optimum plant yiel ds may be maintained when lower nutrient solution concentrations are consta ntly provided to the root syst em. Further investigation is

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36 needed to establish the reasons why this occurs. This reduction in nutrient solution concentration requirement may be due to plant conservation of resources since the constant water and nutrient supply negates the need for the production of expl oratory roots or it may be due to the increased turgor pressure (pushing growth) associated with a constant supply of wa ter to the transpiration stream, or both. In addition, the relationship between nitrogen, sucrose, and carbon supply, and the matrix of processes mediating plant growth that utilize those reso urces may be simplified, when a constant – sufficient and not excessive or restrictive supply of N and other nutrients are available to the plant. Since N is the major mineral nutrient assimilated by plants, considerable savings could be obtained by cha nging practices to insure optimum yield with reduced N input. The end result should be reduced nutrient solution concentrations required for optimum yield and significant saving to growers, consumers, and society. Aquaponics: the Potential for Sustainability Aquaponics fits closely into the definition of sustainable agriculture as defined by the 1990 Farm Bill, Title XVI, Subtitle A, Sec. 1603. Aquaponi cs is 1) “an integrated system of plant and animal production practices” using vegetables wi th aquaculture species, 2) “having a sitespecific application” in greenhouse production units. It will 3) “over the long term satisfy human food needs” and “enhance environmental quali ty” by producing crops using environmentally friendly practices that minimize water and nutri ent waste discharges to the environment. Aquaponics will 4) “make the most use of nonrenewable resources” by conserving nitrogen fertilizer, produced from non-renewable fuels, and wa ter. It will 5) “integrate natural biological cycles” by using nitrifying bacteria in the process of nitrifica tion to convert harmful ammonia fish waste to usable, safe, nitr ate nitrogen for plants. Aquaponics will 6) “sustain the economic viability of farm operations” and “enhance the quality of life for farmersand society as a

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37 whole” by producing food in a sustainable ag ricultural production method and in an environmentally bio-rational manner without wa steful discharge to the environment. In hydroponic systems, nutrients are precisely controlled and spoon fed into the plant growing area as needed and recommended from rese arch trials. Optimum yields are obtained by adjusting nutrient amounts depending on the crop vari ety and stage of growth of the plant. In aquaponic systems, most nutrients are part of the aquaculture waste stream and dependent on daily feed amounts based on fish weight and de nsity. However, the nutrients available in wastewater alone are insufficien t to maintain maximum plant productivity (Rakocy et al., 1997; Seawright et al., 1998) which is an important factor when producing in expensive greenhouse facilities. More research into plant nutrient management in aquaponics is needed before widespread system adoption can be realized. Ther e is no single ratio of plant biomass to fish biomass that results in equilibrium concentratio ns of most of the essential plant nutrients (Seawright et al., 1998). Thus op timum plant yield in aquaponics requires nutrient management and supplementation. However, aquaponics does si gnificantly reduce the need for fertilizer inputs, especially applied nitr ogen (Rakocy et al., 1997). Nitr ogen is an essential nutrient element and the fertilizer nutrient required in largest amounts by plants (Marschner, 2003). The manufacture of nitrogen fertiliz er is expensive, requiring larg e amounts of non-renewable energy resources. Understanding and management of the nitrification process in aquaponics is critical for the maintenance of water qu ality, the production of nitrate n itrogen, and the management of nutrients for plant production in these integrated systems. Water and Nitrogen Budgets Designing agricultural production systems for mini mal discharge of water and nutrients to the environment protects groundwater quality and makes water permitting easier to obtain. It also would protect coastal waters from harm ful algal blooms. A zero-discharge tilapia

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38 recirculating system has been successfully eval uated (Shnel, 2002) and used a combination of a trickling biofilter, fluidized bed reactor, and a sedimentation/digestion basin to maintain system water quality during a 331-day grow -out period. The ideal scenario would be a zero agricultural discharge system (ZADS) where inputs become a harvestable product with no discharge to the environment. One potential ZADS produc tion arrangement combines hydroponic plant production and recirculating aqu aculture systems into what is known as aquaponics. The potential for plants to use the by-products of a quaculture and keep recirculating water clean have been documented (Adler, 1996; Adle r et al., 2000; Lin et al., 2002). Two major components of both hydroponic a nd aquaculture systems are water and nitrogen. Most recirculating aquaculture system s replace 5% to 10% of system water daily to prevent the buildup of toxic levels of ammoni a and other fish by-products and provide makeup water for evaporation and for backwashing fi lters (Masser et al., 1999). The irrigation requirement for a field grown wate rmelon crop in southwest Florida is about 4 liters of water per meter squared of growing area per day (Kovach , 1984). Greenhouse crops require as much as 1.9 liters of water per plant pe r day (Hochmuth, 2001a). Given recommended greenhouse plant densities (Marr, 1995), water use would be about 4.5 liters per meter squa red per day. A single plant can use between 0.5 to 5 liters of water per day dependi ng on its size, maturity, and the growing season or temperature. If we assume an average of 3 liters of water use per plant per day, 100 plants could satisfy the water replacement requirements of a recirculating aquaculture tank containing 3000 (at 10%) or 6,000 liters (at 5% replacement). The main applied nutrient in plant production – nitrogen – could be supplied by fish in an aquaponic system (Rakocy et al.,1997). Sufficient nitrification to convert 75% of the ammonia to nitrate would be preferred since the recommended NO3 to NH4 + ratio in hydroponics is 75:25

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39 (Cockx and Simonne, 2003; Simonne et al., 1992). One hundred kilograms of fish could produce an average of 90 g of ammonia nitrogen pe r day being fed at 3% body weight and 3% of feed becoming ammonia-N (Losordo et al ., 1998). Nitrogen requirements of hydroponic vegetable plants can range from 1 to 20 g per plant per season (Hochmuth, personal communication, 2005) depending on the crop specie s (lettuce, cucumber, pepper or tomato – ranked low to high nitrogen requirement). A fi sh production rate of 90 g of N per day would support 4,050 lettuce plants (45 day crop and 1 g of N requirement per plant) or 1,080 cucumber plants (120 day crop and 10 g of N requirement) or 1,215 pepper or tomato plants (270 day crop and 20 g of N requirement). Rakocy (1997) was able to obtain a 1.5% system volume daily water replacement with rain water as the sole source of wa ter for a tilapia and lettuce a quaponic system. Also, potassium, calcium, or iron concentrations may fall below sufficiency standards in aquaponics without supplemental fertilization (M cMurtry et al., 1990; Rakocy et al.,1997). Plant nutrient applications in aquaponics as we ll as the goal of zero discharge to the environment of water and nutrients need further inve stigation in order to improve systems in tegration and sustainability. Further Systems Integration The difficulty in finding a median environment between plant, fish, a nd nitrifying bacteria culture in aquaponics has resulted in less integration of the sy stems than would be ideal for maximizing space and infrastructure thus reduc ing the potential overall profitability of aquaponics. Most serious aquaponi c trials to date actually use two systems, one hydroponic, one aquaculture, connected by pipes with water reci rculating through both (Adler et al., 1996; Diver, 2006; Jones, 2001; Rakocy et al., 1997; Rakoc y et al., 2006; Timmons et al., 2002). The addition of a hydroponic system does usually eliminat e the need for a separa te biofilter for the aquaculture component (Rakocy, 1999, Anonymous , 1997) since the plant growing area can

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40 provide biofiltration. However, there are conf licting recommendations on the ratio of plant growing area to fish rearing surface area to main tain a balanced system—from 1:1 to 10:1. This may be due to the different hydroponic subsystems us ed with gravel (1:1) and float (2:1 to 10:1) being examples. Gravel media probably contai ns more space per meter squared for nitrifying bacterial growth to occur compared to floating sy stems. The use of gravel culture is no longer very common (Tyson et al., 2001) and has been re placed by lighter media such as perlite. The most common hydroponic systems in Florida today use some form of perlite medium which provides anchorage for the plant roots (T yson, 2002). Perlite is light weight and thus would facilitate plant production above aquacult ure tanks. It should ha ve the characteristics necessary to make a good biofilte r (low density and high porosity), but this has not been scientifically shown. If hydroponic and aquaculture systems were integrated vertically in space, higher returns per unit area of space would be po ssible, which is important when producing inside expensive greenhouse stru ctures. Gross monetary re turns for hydroponic tomatoes of $47/m2 of greenhouse space can be e xpected (Smith et al., 2003) during a ten month production period. Similar gross returns of multip le cropped hydroponic greenhouse cucumbers are possible. Despite the advantages of high yield a nd potential gross returns, the cost of producing hydroponic vegetables on a per kilogr am basis is usually higher than the cost of the field grown product and thus requires a greate r return in the marketplace to be profitable (Hochmuth and Belibasis, 1991; Olson et al., 2006 ). Tilapia production harves t densities of 60 g/L (Rakocy, 1999) can be expected during a similar (ten month) period. A value of $1.65/kg for whole tilapia, with gro ss returns of $44/m2 of tank surface area is possible using rectangular recirculating tanks filled with wa ter to a level of 0.61 m. Using th e vertical space above the fish tanks for growing plants would significantly increase returns per square meter of greenhouse

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41 space. Assuming yields are similar to when these crops are grown alone, ($47 tomatoes or cucumbers + $44 for tilapia (assume 50% space util ization in the greenhouse for the fish tanks with walkways between) then $91 per square meter of greenhouse space per year may be possible for a tomato/tilapia or cu cumber/tilapia combination. Smith (2003) indicates that small positive changes in price and yield can significantly improve cash flows and gross margins for tomato greenhouse enterprises. The addition of another cash crop with the cost savings of increased systems integration (sharing equipmen t and structures) should improve profitability by reducing cost of production. Conclusion and Objectives In summary, water quality parameters affected by operating pH of aquaponic system water can provide potentially toxic conditions for fish a nd plants, and affect ammonia biofiltration rates of system water. No scien tifically based information is av ailable on pH effects on ammonia biofiltration rate in perlite medium or on the effect of hydroponic nutrien t solution concentration on nitrification. Reconciling the pH optima for ammonia biof iltration and plant yield will increase aquaponic system inte gration and sustainability. The goal of this project was to establish a reconciling pH for ammonia biofiltration and cucumber ( Cucumis sativus ) yield in an aquaponic system cont aining a perlite trickling biofilter / root growth medium. In addition, based on trial re sults, perlite will be eval uated as a medium for aquaponic biofilters. The relative contribution of pl ants and nitrifiers to the biofiltration of ammonia will also be assessed. Specific objectives were to: 1. Determine the optimum pH for nitrification in a trickling biofilter containing perlite medium within the range of recommended pH’s for hydroponic (5.5 to 6.5) and recirculating aquaculture (6.5 to 8.5) systems (Chapter 3). 2. Determine the nitrification rate respons e in the designed biofilter to hydroponic nutrient solution, NO3 --N concentrations, and to pH levels near optimal for plants (6.5) and nitrificati on (8.5) (Chapter 4).

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42 3. Establish a pH range for optimum pr oduction of greenhouse cucumber in the designed biofilter system and determine if foliar applied nutrients could provide plant rescue of nutrient deficien cy at high pH (Chapter 5). 4. Determine the ammonia biofiltration rate and evaluate a perlite trickling biofilter/root growth medium in aquaponic production (Chapter 6). 5. Make predictions about the relative contribution of plants and nitrifiers to the biofiltration of amm onia (Chapter 6). 6. Establish the reconciling pH for ammonia biofiltration and cucumber yield in recirculating aquaponi cs (Chapter 6).

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43 Table 2-1. Fraction of NH3 in an ammonia solution.z Temperature pH (C) 6.5 7.0 7.5 8.0 8.5 20 0.0013 0.0039 0.0124 0.0381 0.1112 21 0.0013 0.0042 0.0133 0.0408 0.1186 22 0.0015 0.0046 0.0143 0.0438 0.1264 23 0.0016 0.0049 0.0153 0.0469 0.1356 24 0.0017 0.0053 0.0164 0.0502 0.1431 25 0.0018 0.0056 0.0176 0.0537 0.1521 26 0.0019 0.0060 0.0189 0.0574 0.1614 27 0.0021 0.0065 0.0202 0.0613 0.1711 28 0.0022 0.0069 0.0216 0.0654 0.1812 29 0.0024 0.0074 0.0232 0.0697 0.1916 30 0.0025 0.0080 0.0248 0.0743 0.2025 31 0.0027 0.0085 0.0265 0.0791 0.2137 32 0.0029 0.0091 0.0283 0.0842 0.2253 zFrom Lim, C. and C.D. Webster. 2006. T ilapia: Biology, culture, and nutrition. The Food Products Press, Binghamton, N.Y.

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44 Table 2-2. Average concentratio ns of mineral nutrients in pl ant shoot dry matter that are sufficient for adequate plant growth.z mol/g mg/kg Relative number Element Abbreviation dry wt (ppm) % of atoms Molybdenum Mo 0.001 0.1 1 Nickel Ni ~0.001 ~0.1 1 Copper Cu 0.01 6 100 Zinc Zn 0.30 20 300 Manganese Mn 1.0 50 1 000 Iron Fe 2.0 100 2 000 Boron B 2.0 20 2 000 Chlorine Cl 3.0 100 3 000 Sulfur S 30 0.1 30 000 Phosphorus P 60 0.2 60 000 Magnesium Mg 80 0.2 80 000 Calcium Ca 125 0.5 125 000 Potassium K 250 1.0 250 000 Nitrogen N 1000 1.5 1 000 000 z From Marschner, H. 2003. Mineral nutrition of higher plants. Academic Press, Elsevier Science Ltd.

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45 Table 2-3. Percent elemental analysis and physical prope rties of perlite.z Typical percent elemental analysis y Silicon 33.8 Aluminum 7.2 Potassium 3.5 Sodium 3.4 Iron 0.6 Calcium 0.6 Magnesium 0.2 Trace 0.2 Bound water 3.0 Oxygen (by difference) 47.5 Typical physical properties Color White Refractive index 1.5 Free moisture, maximum 0.5% pH (of water slurry) 6.5-8.0 Specific gravity 2.2-2.4 Bulk density (loose weight) As desired but usually 32-400 kg/m3 Mesh size As desired 4 mesh and finer z From Anonymous. 2007. Basic facts about perlite. The Perlite Institute, Inc., Harrisburg, PA. Retrieved April 1, 2007, from http://www.perlite.org/ y All analysis are shown in elemental form even though the actual forms present are unavailable and bound in mixed glassy silicates. Free silica may be present in small amounts.

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46 CHAPTER 3 RECONCILING WATE R QUALITY PARAMETERS IMPACTING NITRIFICATION IN AQUAPONICS: THE PH LEVELS Introduction Aquaponics is an integrated system th at links hydroponic plant production with recirculating aquaculture (Diver, 2006). The advantages of linking fish and plant culture together are shared startup, opera ting and infrastructure costs, fish waste nutrient removal by plants, reduced water usage, and increased prof it potential by producing two cash crops (Rakocy, 1999; Timmons, et al., 2002). The potential of plan ts and fish for production in aquaponics has been investigated (Adler et al., 1996; Anonymo us, 1997, 1998; McMurtry et al., 1997; Rakocy et al., 2006, 1997; Watten and Busch, 1984). One of the most complex and important subsys tems of recirculating aquaculture is the biofiltration and removal of fish waste. Reci rculating systems must incorporate both solids removal and ammonia biofiltration into the wate r reconditioning process to achieve proper water quality for fish and plants (Harmon, 2001). Amm onia is the main excretion product from fish. Both un-ionized ammonia and nitrite can be toxi c to fish at very lo w levels (Harmon, 2001; McGee and Cichra, 2000). In the process of nitrification, certain autotrophic bacteria (primarily Nitrosomonas ) oxidize ammonia to nitrite and others (primarily Nitrobacter ) oxidize nitrite to nitrate. The overall reaction of nitrification can be writte n as (Hagopian and Riley, 1998): Nitrosomonas NH3 + 1.5O2 NO2 + H2O + H+ + 84 kcal mol-1 (Equation 3-1) Nitrobacter NO2 + 0.5 O2 NO3 +17.8 kcal mol-1 (Equation 3-1) This nitrogen transformation elimin ates ammonia from the water. Nitrate is generally not toxic to fish except at very high levels (96-h LC50 > 1000mg/L NO3-N; Colt and Tchobanoglous,

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47 1976) and is the primary source of nitrogen fo r plants in hydroponic systems (Hochmuth, 2001a; Resh, 2004). Nitrate and ammonium (NO3 and NH4 +) are the most common form s of nitrogen taken up by vegetable crops (Cockx and Simonne, 2003). However, they should be regarded as two different nutrients because they affect plant me tabolism differently. Plant nutrient uptake is a process that is electricall y neutral. Uptake of NH4 + may depress uptake of the essential cations (K+, Ca2-, Mg2+). The optimum nitrate to ammonium ratio for vegetables grown in hydroponics is 75:25 (Simonne et al., 1992). When ammonium is the dominant form of n itrogen available for plant uptake, a smaller plant will result. Thus where the nitrogen source in aquaponics comes primarily from the fish, the nitrification process is important for nitrate uptake by plants. The fish, the plants, and the nitrifyi ng bacteria rely on the same recirculating water for optimum growth hence water quality parameters have to be favorable for all three organisms in a selfsustaining aquaponic system. The effects of water quality on nitrifying b acteria have not been investigated from the standpoint of conditions that can be pres ent in aquaponic systems. The pH is one of the most important environmen tal parameters that can affect the activity of nitrifying bacteria (Prosser, 1986). R ecommended pH ranges for hydroponic systems are between 5.5 and 6.5 (Hochmuth, 2001a) and for aquaculture systems are between 6.5 and 8.5 (Timmons et al., 2002). A wide range of pH optima have been reported from research on the effect of pH on nitrifica tion rate. In substrates from terrest rial forest environments, increasing pH stimulated net nitrification while decreasi ng pH depressed it (Ste-Marie and Pare, 1999). Nitrification in aquacult ure biofilters was reported to be most efficient at pH levels from about 7.5 to 9.0 (Hochheimer and Wheaton, 1998), and 7.0 to 8.0 (Masser et al., 1999). In a submerged biofilter investigation, a pH increas e of one unit within a range of 5.0 to 9.0,

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48 produced a 13% increase in nitrification efficien cy (Villaverde, et al., 1997). In another investigation with four different biological filters (under gravel, fluidized bed, non-fluidized bed, and gravel bed) nitrification slowed signi ficantly or stopped when pH dropped below 6.0 (Brunty, 1995). The pH of approximately 7.8 pro duced the maximum growth rate of nitrifying bacteria for wastewater treatment processes (Antoniou et al., 1990) . The causes of varying pH optima may be attributed to differe nces in substrate, effluent, alka linity, or species of nitrifying bacteria present in the system. The most common recirculating aquaponic system s to date employ either a media-filled raised bed, nutrient-flow technique (NFT), or floating raft system (Adler et al., 1996; Anonymous, 1997, 1998; Diver, 2006; McMurtry et al., 1997; Rakocy et al., 2006,1997; Watten and Busch, 1984) for growing plants. Of those systems, the media filled bed has potential for providing for solids removal, biological filtratio n, and root zone space for plant production. Perlite is the most common plant growing medium used in hydroponic plant production in Florida (Tyson et al., 2001). It ha s also been investigated as a so illess culture alternative to soil fumigation with methyl bromide in field grown tomato and pepper produc tion (Hochmuth et al., 2002). However, perlite medium has not been in vestigated with respec t to the activity of nitrifying bacteria in an aquaponic biofilter. The type of soi lless media in which plants grow has been shown to significantly affect nitrifying bacteria counts (Lang and Elliott, 1997). The purpose of this investigation wa s to determine the nitrificatio n activity response to pH ranging from 5.5 to 8.5 in a trickling biological filt ration system containing perlite medium. Materials and Methods Two experiments were conducted in 2004 in a Dutch-style glass greenhouse with pad and fan cooling system at the Seminole Community College Horticultural Unit, Sanford, Fla. Sixteen perlite medium trickling biofilters were set up in a randomized block design with four

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49 treatments (pH 5.5, 6.5, 7.5, 8.5). Twenty liters of tap water were added to the 80-L plastic biofilter boxes which were kept closed during the experiment. Air vents in the upper section of the boxes allowed for natural ventilation and gas exchange. Screen colanders were placed above the water on plastic stools in each box and filled w ith 6.5 L of horticultural grade coarse perlite. Water was recirculated through the perlite with an aquarium pump at the average rate of 1.9 L/min. Sodium bicarbonate and potassium hydroxi de (Plant Food Systems, Zellwood, FL) were added to raise pH during experiment 1 and pot assium hydroxide was used to raise pH in experiment 2. Phosphoric acid (Plant Food Sy stems) was added to lower pH and sodium bicarbonate was added to increase alkalinity as needed during both trials. Experiment 1 biofilter setup began on 20 Jan. wi th water and perlite added to the tanks and recirculating pumps installed. On 21 Jan., “P roline” Aqua-Coat (Dech lorinator/Substrate Conditioner; Aquatic Eco-Systems, Apopka, FL ) was added at 1.3 ml per tank. Ammonium chloride was added at 25 mg/L resulting in 5.0 mg/L total ammonium n itrogen concentration in the recirculating solution. “P roline” Bio-Booster nutrient soluti on was added at 0.3 ml per tank. ‘Proline’ Freshwater Nitrifying B acteria (Aquatic Eco-Systems) wa s added to the perlite at the rate of 2.5 ml/L of tank wate r. The “Proline” products are proprietary blends of water conditioner, nutrients, and nitrifying bacteria recommended for use when beginning new biofilter startup cycles in recirculating aquaculture. On 27 Jan., another 1.5 ml/L of nitrifying bacteria was added to each tank in an effort to speed up the nitrification process. Total ammonia nitrogen (TAN = NH4 +-N plus NH3-N), nitrite nitroge n, nitrate nitrogen, pH, dissolved oxygen, soluble salts, salinity, and temperature measurements were taken every 4 d beginning on 21 Jan.. Ammonium chloride (0.125 g) was added to the 8.5 pH treatment on 1 Feb., and to the other treatments on 9 Feb. On e week after setup, aquarium heaters were

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50 installed in the boxes to maintain recirculati ng water temperatures be tween 26C and 31C. Upon completion of experiment 1, boxes and equipm ent were disassembled, triple rinsed, and dried prior to assembly for experiment 2. Experiment 2 biofilter setup began on 3 Mar., with water and fresh perlite added to the tanks and recirculating pumps installed. Aqua rium heaters were reinstalled. On 10 Mar., “Proline” Aqua-Coat (Dechlorinator/Substrate Conditioner) was added at 1.3 ml per tank. Ammonium chloride was added at 25mg/L. “Pro line” Bio-Booster nutrient solution was added at 0.3 ml per tank. On 11 Mar., “Proline” Freshwater Nitrifying Bacteria was added to the perlite at the rate of 10 ml/L of tank water. Total ammonium nitrogen, nitrite nitrogen, nitrate nitrogen, and pH measurements were taken every 4 d wh ile dissolved oxygen, solubl e salts, salinity, and temperature water quality data were taken every 8 d beginning on 11 Mar. Total ammonia nitrogen (range 1.0 to 8.0 mg/L), nitrite (l ow range, 0.1 to 0.8 mg/L), chlorine, and alkalinity were measured with La Motte Test Kits. Nitrit e (high range, 0 to 150 mg/L) was measured using a Hanna Ion Specific Me ter. Nitrate was measured using a Cardy Ion Specific Meter (0 to 9,900 mg/L). Dissolved oxygen, specific conductivity, temperature, and salinity were measured using a YSI Model 85 mete r. Both experiments used a randomized block design with four replications. Data were analyzed using a Statistical Analysis System (SAS) software and Duncan’s Multiple Range Test us ing a P value of <0.05. The pH data were measured using a Fisher Scientific AR15 Accumet Research pH meter. Results and Discussion These experiments are based on typical startup characteristics for bringing a new biological filter system up to full capacity (Tetzlaff and Heidinger, 1990; Timmons et al., 2002). Relative nitrification activity is measured based on the time it ta kes after introducti on of nitrifying

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51 bacteria to convert ammonia to nitrate. A significant experiment by pH interaction was present in enough data sets to warrant discussion by experiment. Total ammonia nitrogen (TAN) decreased from 5 mg/L to zero, 12 d after the introduction of nitrifying bacteria to the biof ilters maintained at a target pH of 8.5 (Table 3-1). A similar reduction in TAN for the target pH of 7.5 t ook 20 d and for pH 6.5 took 20 (Exp.1) and 24 (Exp. 2) d. TAN did decline an average of 44 % in 28 days during the trials at pH 5.5 but nitrite accumulation was not detected. Nitr ite began to be measured in the biofilter water 8 (pH 8.5), 16 (pH 7.5), and 16 (pH 6.5) d after in troduction of nitrifying bacter ia. No nitrite was measured in the biofilters maintained at a pH of 5.5. Nitrate readings were inconsistent but did indicate a trend towards increased nitrate buildup over time which would be consistent with the oxidation of ammonia to nitrate. The inconsistency ma y be due to the wide range of the Cardy Ion Specific Meter (0 to 9,900 ppm) and the low range of the nitrate measured. There was conservation of nitrogen through th e nitrification process from am monia to nitrate. Overall, results indicate nitrifying bacteria activity in pe rlite medium trickling biofilters increased as pH increased and was greatest at pH 8.5. Average water quality parameters during e xperiments 1 and 2 respectively were 7.4 and 7.0 mg/L dissolved oxygen, 521 and 493 S/cm sp ecific conductivity, 0.25 and 0.24 ppt (parts per thousand) salinity, and 28.1 and 29.8 C temperat ure. The use of sodium bicarbonate to raise pH in experiment 1 resulted in higher specific conductivity compared w ith experiment 2 where potassium hydroxide was used. Seasonally averag e greenhouse temperatures were higher during experiment 2 compared to experiment 1. Season pH values during experiment 1 ranged from 5.2 to 5.7, 6.1 to 6.4, 6.7 to 7.5, 8.5 to 8.6, and during experiment 2 ranged from 5.5 to 5.7, 6.1 to 6.5, 7.1 to 7.7, and 8.0 to 8.6 for pH treatments 5. 5, 6.5, 7.5, and 8.5, respectively. Nitrification

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52 is an acid producing process requ iring adjustment of recirculati ng water to maintain target pH levels. Actual pH values were within the target pH range for the treatments. Nitrite accumulation in the tria ls (Table 3-1) averaged 4.9 mg/L or 98 % of the applied TAN at pH 8.5. The only source of nitrite in the aerobic biofilter system was from oxidation of ammonia by the applied Nitrosomonas bacteria. Therefore, TAN loss from other sources, i.e. ammonia volatilization, were minimized below 2% at pH 8.5 in the biofilter boxes and at least 98% of the observed TAN loss there was from n itrification. Since ammonia volatilization increases with increasing pH, we can assume TAN losses at the lower pH’s were also primarily due to nitrification. Therefore, the loss of T AN from the biofilters was primarily the result of oxidation of ammonia by the applied nitrifying bacteria a nd these losses occurred at a faster rate as pH increased from 5.5 to 8.5. The lack of accumulation of nitrite at pH 5.5 was likely due to the slow rate of nitrificati on occurring (Alleman, 1985). This accumulation is more evident at high pH than at low pH. Nitrification is a dynamic process and as nitrite accumulates, it is simultaneously oxidized to nitrate. Reconciling water quality parameters: The pH recommendations for aquaculture systems range between 6.5 and 8.5 (Timmons et al., 2002). For a pH range between 2.0 and 7.0, ammonia in solution is completely present as NH4 + (De Rijck and Schrevens 1999). However, as pH increases above 7.0, there is an increase in the un-ionized NH3 form of ammonia and a decrease in the ionic NH4 + form. Un-ionized ammonia is the mo st toxic form for fish with 96-h LC50 varying by species from 0.08 mg/L NH3-N for pink salmon ( Oncorhynchus gorbuscha ) to 2.2 mg/L for common carp ( Cyprinus carpio ) (Timmons et al., 2002). The pH tolerances of plants can range from 5.0 to 7.6 depending on the species (Maynard and Hochmuth, 1997). However, recommended pH ranges for hydroponic nutri ent solutions tend to be slightly acidic

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53 (5.5 to 6.5–Hochmuth, 2001a; 5.8 to 6.4–Resh, 2004) due to problems with plant nutrient solubility. At pH levels above 7.0 there can be reduced micronutrient a nd phosphorus solubility. If aquaponic recirculating water pH is maintained at levels optimu m for nitrifying bacteria (8.5), plant uptake of certain nutrients may become restricted and un-ionized ammonia levels may become toxic to the fish. Plant uptake is one of the most widely r ecognized biological processes for contaminant removal in wastewater treatment wetlands (Debusk, 1999). Ammonium nitrogen removal efficiencies of 86% to 98% were reported from a constructed wetlands system receiving aquaculture wastewater (Lin et al., 2002). In hydroponic gr eenhouse plant production systems receiving aquaculture wa stewater, Adler (1996) found that differences in nutrient removal rates of nitrate nitrogen and phosphorus were dependant on plant numbers and effluent flow rate. If plant numbers are increased suffici ently, nutrient concentration can decrease to levels that may be too low to sustain plant growth. Plant roots were found to be more competitive for ammonium than the ammonium -oxidizing bacterial species Nitrosomonas europaea (Verhagen et al., 1994). There may be less reliance on nitr ification for ammonia removal when sufficient plants are present in aquaponic systems. Howe ver, since the optimum ratio of nitrate to ammonium nitrogen in hydroponic nutrient solutions is 75:25 (Cockx and Simonne, 2003; Simonne et al., 1992), a source of nitrate-nitrogen would be needed for plant uptake either through nitrification or supplementa l fertilization for optimum plan t growth. Since certain plant nutrients can fall below sufficiency standards in aquaponics (McMurtr y et al., 1990) without supplemental fertilization, methods to make up this deficit without adversely impacting fish and nitrifying bacteria need furt her investigation.

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54 Conclusions The loss of total ammonia nitrogen from perlite medium trickling biofilters increased as pH increased from 5.5 to 8.5 and these losses we re primarily the result of nitrification. The recommended pH for aquaculture systems is from 6.5 to 8.5 and for hydroponic systems is between 5.5 and 6.5. However, pH extremes s hould be avoided when reconciling pH between fish, plants, and bacteria sin ce high alkaline conditions reduce th e solubility of certain plant nutrients and increase the presence of the un-ionized (more toxic to fish) form of ammonia. It should be possible to maintain aquaponic wate r at a pH range of 6.5 to 7.0, levels more conducive to hydroponic plant nutrie nt uptake and reduced un-ionized ammonia levels, without a significant buildup of ammonia in the recirculatin g water provided there are a sufficient number of plants present for uptake and reduction of nutri ent loads in the system water and water flow rate through the root zone is adequate. Even tho ugh nitrification is slower at pH 6.5 than at pH 8.5, the increased uptake and uti lization of ammonia by plants should make up for the reduced nitrifying activity. Plant nutrient availability could be enhanced by supplemental fertilization of the plant growing medium or by foliar application of specific elements. Reconciling differences in optimum water qualit y for plants, fish, and nitrifying bacteria will be necessary to successfully integrate hydroponic and aquaculture systems. More information is needed on aquaponic systems c ontaining soilless media such as perlite and vermiculite. Also, the affects of pH and hydroponi c nutrient concentration of the system water, as well as methods of plant nutri ent application on nitr ifying bacteria activity and growth and yield of plants and aquatic organisms n eed to be investigated more fully.

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55 Table 3-1. Changes in TAN, NO2-N, and NO3-N concentrations in pe rlite medium trickling biofilters as affected by water pH. Target pH Day 0z Day 4 Day 8 Day 12 Day 16 Day 20 Day 24 Day 28 Day 32 Experiment 1 Total ammonia nitrogen (mg/L) 8.5 5.0 a y 4.0 b 1.1 c 0 c 0 c 0 b 0 b 0 b 7.5 5.0 a 5.0 a 3.9 b 4.5 b 1.3 bc 0 b 0 b 0 b 6.5 5.0 a 5.0 a 4.4 a 4.9 a 2.5 b 0 b 0 b 0 b 5.5 5.0 a 5.0 a 4.5 a 4.9 a 4.6 a 4.1 a 3.6 a 3.0 a L** L** L** L** L** L** L** Significance x Q** Q** Q** Q** Q** Q** Experiment 2 8.5 5.0 a 3.3 b 2.0 b 0 b 0 c 0 b 0 b 0 b 0 b 7.5 5.0 a 5.0 a 5.0 a 4.4 a 2.8 b 0 b 0 b 0 b 0 b 6.5 5.0 a 5.0 a 5.0 a 4.3 a 3.5 a 1.8 a 0.5 b 0 b 0 b 5.5 5.0 a 5.0 a 5.0 a 4.6 a 3.8 a 3.0 a 2.8 a 2.6 a 2.4 a L** L** L** L** L** L** L** L** Significance Q** Q** Q** Q** Q** Q** Exp x pH P-valuew 0.01 0.09 0.14 0.21 0.01 0.11 0.83 Experiment 1 Nitrite nitrogen (mg/L) 8.5 0 a 0 a 0.9a 5.3 a 3.7 a 1.2 a 0 b 0 a 7.5 0 a 0 a 0 b 0.3 b 2.6 b 1.6 a 0.9 a 0 a 6.5 0 a 0 a 0 b 0.1 b 2.1 b 1.5 a 0.1 b 0 a 5.5 0 a 0 a 0 b 0 b 0 c 0 c 0 b 0 a L** L** L** L** Q** Significance Q** Q** Experiment 2 8.5 0 a 0 a 0.6 a 4.5 a 2.0 ab 0.2 b 0 b 0 a 0 a 7.5 0 a 0 a 0 b 0.5 b 2.9 a 4.3 a 2.0 ab 0 a 0 a 6.5 0 a 0 a 0 b 0 b 0.2 b 1.2 ab 2.8 a 3.3 a 0.3 a 5.5 0 a 0 a 0 b 0 b 0 b 0 b 0 b 0 a 0 a L** L** L* Q* Q* Significance Q** Q** Exp x pH P-value 0.71 0.78 0.29 0.17 0.07 0.07

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56 Table 3-1. Continued Experiment 1 Nitrate nitrogen (mg/L) 8.5 3.8 a 3.0 a 1.5 a 5.0 a 2.0 a 8.3 a 7.8 a 8.8 a 7.5 2.0 b 2.0 b 0.3 b 3.0 b 1.3 b 4.3 b 3.0 b 5.0 b 6.5 2.0 b 2.0 b 0.3 b 3.0 b 0.3 c 3.8 b 3.0 b 4.0 c 5.5 2.0 b 2.0 b 0.5 b 3.0 b 0 c 2.8 c 1.0 c 2.5 d L** L** L** L** L** L** L** L** Signific. Q** Q** Q* Q* Q* Q** Q** Q** Experiment 2 8.5 1.5 a 0 a 3.8 a 1.0 a 5.5 a 3.8 a 4.5 a 5.3 b 5.8 b 7.5 1.0 b 0 a 3.8 a 0.5 ab 4.5 b 3.0 ab 4.5 a 6.5 a 6.8 a 6.5 0 c 0 a 3.0 b 0.3 b 4.0 b 2.5 bc 2.8 b 5.0 b 6.5 ab 5.5 0 c 0 a 3.0 b 0 b 4.0 b 2.0 c 2.0 c 3.5 c 4.0 c L** L** L** L** L** L** L** L** Significance Q** Q** Exp x pH P-value 0.01 0.01 0.32 0.01 0.27 0.01 0.01 0.01 zNitrifying bacteria introduced to the biofilters. yWithin columns, means followed by different letters are significantly different; four replicates xLinear and Quadratic effects were signifi cant at the 5% (*) or 1% (**) level. wP values for experiment x pH interaction

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57 CHAPTER 4 EFFECT OF NUTRIENT SOLUTION, NO3 --N CONCENTRATION AND PH ON NITRIFICATION RATE IN PERLITE MEDIUM Introduction Aquaponics is an integrated system th at links hydroponic plant production with recirculating aquaculture (Diver, 2006). The most common aquaponic systems to date employ either a media-filled raised bed, nutrient-flow tec hnique (NFT), or floating raft system (Adler et al., 1996; Anonymous, 1997, 1998; Diver, 2006; McMurtry et al., 1997; Rakocy et al., 2006,1997; Watten and Busch, 1984) for the plant grow ing area integrated w ith a recirculating aquaculture tank system (Timmons et al., 2002). The advantages of linking plant and fish culture include fish tank waste nutrient and water removal by plants, reduced water usage and waste discharge to the environment by both systems, and increased profit potential by producing two cash crops (Rakocy et al., 2006; Rakocy, 1997; Timmons , et al., 2002). The media-filled raised bed system has potential for providing biological filtration and a root zone space for plant production. The type of media in which plants grow has been shown to significantly affect nitrifying bacteria counts (soilless potting media; Lang and Elliott, 1997) and nitrification rate (various soil types; Prosser, 1986), but no inform ation was found on nitrifi cation rate in perlite medium. Perlite was chosen as the medium for this trial because it was the most common root growth medium used in hydroponic plant producti on in Florida during 2001 (Tyson et al., 2001). Nitrification is a biological process that maintains water quality in recirculating aquaculture systems and has been shown to tran sform 93%-96% of nitrogenous fish wastes (NH3 into NO3 -) in biofiltration units (Prinsloo et al ., 1999). Un-ionized ammonia nitrogen (NH3 -N) at concentrations as low as 0.02 -0.07 mg/L reduced fish gr owth and cause tissue damage (Masser et al., 1999). The 96–h LC50 for un-ioni zed ammonia on fingerling channel catfish

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58 ( Ictalurus punctatus ) was 3.8 mg/L (Colt and Tchobanoglous, 1976). The reactions involved in nitrification may be summari zed as (Madigan et al., 2003): Nitrosifying bacteria (primarily Nitrosomonas ) NH3 + 1 O2 NO2 + H+ + H2O G0 = -275 kJ/reaction (Equation 4-1) Nitrifying bacteria (primarily Nitrobacter ) NO2 + O2 NO3 G0 = -74.1 kJ/reaction (Equation 4-2) The intermediate product of nitrification, nitrite (NO2 -), may be toxic to both fish and plants at low levels. Nitrite at concentrations as low as 5 mg/L in nutrient solution damaged tobacco ( Nicotiana tabacum L.) root tips (Hamilton and Lowe, 1981). Nitrite oxidation activity was suppressed by elevated pH and ammonium concentrations when urea was used in a hydroponic tobacco float system resulting in the accumulation of toxic levels (30 mg/L) of nitrite (Pearce et al., 1998). Gila trout ( Oncorhynchus gilae ) exposed to nitrite at 10 mg/L or more for 96 h died (Fuller et al ., 2003) and the 96 h LC50 for bass ( Morone sp .) was 12.8 mg/L (Weirich et al., 1993). Nitrate, the end product of nitrification, is not toxic to fish except at concentrations much greater than those typically used in nutrient solu tion for plant production (catfish 96–h LC50,200 mg/L NO3 -N; Colt and Tchobanoglous, 1976) although some investigations suggest that prolonged exposure to 200 mg/L NO3 - – N might decrease the immune response of some fish species (Hrubec et al., 1996). Nitrate is the primary source of N for plants in hydroponic nutrien t solutions at concentra tions from 50 to 280 mg/L NO3 -N (Resh, 2004). Hence, the understanding and management of the nitrification pr ocess in aquaponics is important for the maintenance of water qua lity and the production of nitrate nitrogen. There is no single ratio of plant biomass to fish biomass that results in equilibrium concentrations of most of the essential plant nutrients (Seawright et al., 1998). The levels of

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59 most nutrients over 2 year s in a commercial scale aquaponic tilapia/lettuce ( Lactuca sativa ) system remained well below the initial concentrations of nutri ents in hydroponic formulations (62-779 vs. 1200-1900 mg/L total dissolved solutes) , but normal lettuce growth was obtained with fertilizer supplementation of K, Ca and Fe (Rakocy et al., 1997). Levels of NO3 -N averaged 36 mg/L during the 2.5 years, well below those recommended for hydroponic lettuce nutrient formulations (115 mg/L NO3 -N; Resh, 2004). Aquaponic systems that rely solely on fish waste to supply nutrients for plants ha ve reported low levels of phosphorus, potassium, iron and manganese (Adler et al., 1996) and phos phorus, sulfur, potassium and iron (Seawright et al., 1998) in recirculating water. It would be beneficial to supplement aquaponic water with hydroponic fertilizer to optimize nutri ent levels for plants ; however, research on nitrification in aquaponic systems under these conditions is lacking. The pH is one of the most important water qualit y parameters that can affect the activity of nitrifying bacteria in aquaculture biofilters (H ochheimer and Wheaton, 1998; Villaverde et al., 1997). The pH recommendations for aquaculture systems range between 6.5 and 8.5 (Timmons et al., 2002), whereas, the pH to lerances of plants can range from 5.0 to 7.6 depending on the species (Maynard and Hochmuth, 1997). Ho wever, recommended pH ranges for hydroponic production systems tend to be slightly acidic ( 5.5 to 6.5 by Hochmuth, 2001a; 5.8 to 6.4 by Resh, 2004). Precipitation of Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+ to insoluble and unavailable salts can occur in nutrient soluti on culture at water pH levels above 7. If aquaponic recirculating water pH is maintained at levels more optimum for nitrifying bacteria (7.5.0; Hochheimer and Wheaton, 1998), plant uptake of certain nutrients may become restricted and thus plant yield may be reduced.

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60 Waste from fish rarely supplies plant nutrients in adequate amounts without supplementation (Adler et al., 1996; Rakocy et al ., 1997; Seawright et al ., 1998), thus providing a nutrient solution to optimize plant production is ju stifiable. However, nutrient solution effects on nitrification in aquaponic syst ems are untested. Testing indivi dual plant nutrients for effect on system nitrification was deemed time and resource consuming; hence, a commercial hydroponic fertilizer blend was chosen as nutrient source for the expe riment. If no adverse affect on nitrification is observed with these nutrien ts, then a large gap between research and application for nutrient effects c ould be quickly filled at once. The fish, the plants, and the nitrifying bacter ia rely on the same recirculating water for growth hence water quality parameters have to be acceptable for all three organisms in a selfsustaining aquaponic system. The objective of this research was to dete rmine the nitrification response in a perlite trickli ng biofilter (root growth medi um) exposed to hydroponic nutrient solution, varying NO3 --N concentrations, and to pH leve ls optimum for pl ants (6.5) and nitrification (8.5). These will be used to ma ke predictions about water quality effects, nitrification / bi ofiltration, and plant yiel d in aquaponic systems. Materials and Methods Two similar experiments were conducted in Sanford, FL: the first (30 June Aug., 2004) in a laboratory facility at the Horticultural Unit at Semi nole Community College, and the second (21 Jan. Feb. 2005) in a nearby garage facility due to hurricane damage at the Horticultural Unit. Aquarium heaters were installed in th e biofilter boxes during experiment 2 since that facility was not heated in the winter. Twenty liters of tap water were added to 80-L plastic biofilter boxes which were kept covered (nitrifying bacteria ar e adversely sensitive to UV light ) during the experiments except during data collection. Air vent s in the upper side section of the boxes allowed for natural

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61 ventilation and gas exchange. Screen colanders were placed above the water on plastic stools in each box and filled with 7 L of horticultural grad e coarse perlite. Water was re-circulated through the perlite with a quarium pumps at the average rate of 1.9 L/min. Biofilter tank setup began one week before the start of the experiments with water and nutrients added followed by addition of 1 ml per tank of “Proline Aqua-Coat” (Dechlorinator/Substrate Conditi oner), and 0.5 ml per tank of “P roline Bio-Booster” solution. Perlite and recirculating pumps were added to the tanks prior to adjustment of tank water pH to treatment levels. Potassium hydroxide was added to raise pH and phosphoric acid was added to lower pH as needed during both trials (Plant Fo od Systems, Zellwood, FL). The pH was the last water quality adjustment made prior to inocula tion of the tanks with nitrifying bacteria. Experiments began with the addition of “Pro line Freshwater Nitrifying Bacteria” (active cultures of Nitrosomonas and Nitrobacter bacteria product number 239211) to the perlite at the rate of 150 ml per tank on 30 June, 2004 and 21 Ja n., 2005 for experiments 1 and 2, respectively. The “Proline” products are propriet ary blends of water conditione r, nutrients, and nitrifying bacteria recommended by the manufacturer for use when beginning new biofilter startup cycles in recirculating aquaculture (Aqua tic Eco-Systems, Apopka, FL). The pH treatments were 6.5 and 8.5 (established as described above). Hydroponic nutrient solution treatments were 1) tap water, no added nut rients, 2) tap water, 100 mg/L nitrate nitrogen plus complete hydroponic plant nutr ient solution or 3) tap water, 200 mg/L nitrate nitrogen plus complete nutrient solution. The complete hydro ponic nutrient solution co nsisted of 600 mg/L NFT Vegetable Formula (hydroponic fertilizer blend), 600mg/L calcium nitrate for the 100+ nitrate nitrogen treatments and 600 mg/L NFT Vegetable Formula, 600 mg/L calcium nitrate, and 600mg/L potassium nitrate for the 200+ nitrate nitrogen treatments. Fe rtilizer was obtained

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62 from Grower’s Supply Center, Lynn Haven, FL . The NFT vegetable formula as applied consisted of 18 mg/L nitrate nitrogen, 39 mg/L phosphorus, 134.5 mg/L potassium, 32 mg/L magnesium, 2.7 mg/L iron, 0.2 mg/L zinc, 0.4 mg /L manganese, 0.07 mg/L copper, 1.0 mg/L boron, and 0.07 mg/L molybdenum. The nutrient sources were deri ved from potassium sulfate, monopotassium phosphate, magnesium sulfate, pot assium nitrate, iron EDTA, zinc EDTA, manganese EDTA, copper EDTA, sodium borate and sodium molybdate. pH was measured using an AR15 Accumet Research pH meter (Fisher Scientific International, Inc., Hampton, NH). Total ammoni a nitrogen (range 1.0 to 8.0 mg/L), nitrite (low range, 0.1 to 0.8 mg/L) and alkalinity were meas ured with test kits (LaMotte Company, Chestertown, MD). Nitrite (high range, 0 to 150 mg/L) was measured using an ion specific meter (Hanna Instruments USA, Woonsocket, RI). Nitrate was measured using an ion specific electrode cardy meter (Spectrum Technologies, Inc ., Plainfield, IL). Dissolved oxygen, specific conductivity (EC), temperature, and salinity were measured us ing a YSI Model 85 meter (YSI Inc., Yellow Springs, OH). Total ammonia nitrogen (TAN = NH4 +-N + NH3-N), nitrite nitrogen (NO2 -N), nitrate nitrogen (NO3 -N) and pH measurements were take n on 30 June and 21 Jan., just prior to inoculation with nitrifying bact eria (for experiment 1 and 2, re spectively) and every 5 d after inoculation in each experiment. Dissolved oxyge n, soluble salts, salinity, and temperature water quality data were taken just prio r to inoculation on 30 June and 21 Jan. and every 10 d thereafter. Ammonia oxidation rate was calcula ted based on the time in days after inoculat ion (DAI) it took for TAN concentrations to reach a measured valu e of 0. Nitrite oxidation rate was determined by assuming conservation of nitrogen through the reaction (8 mg/L TAN become 8 mg/L NO2 --N) and the time interval from first measurement of NO2 -N concentration until it was resolved to 0.

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63 Sodium bicarbonate (NaHCO3) was added when necessary to maintain recirculating water alkalinity above 50 mg/L CaCO3. Alkalinity is defined as the total amount of titratable bases in water expressed as mg/L equivalent calcium carbonate. Sodium bica rbonate is 83g/eq and 1meq/L = 50 mg/L as CaCO3 (Timmons et al., 2002). A randomized block design with four replicatio ns was used in both experiments. Data were analyzed using Statistical Analysis System (SAS) software and Du ncan’s Multiple Range Test using a P value of <0.05. Results and Discussion Average water quality parameters during expe riments 1 and 2, respectively, were 7.0 and 6.1 mg/L dissolved oxygen (more than adequate for the ammonia oxidation reaction to proceed in Eq. 4-1), and 29.1 and 30C. Electrical conductivity (EC) averaged 447 and 719 S/cm for the no nutrient solution treatment, 1415 and 1934 S/cm for the 100 mg/L nitrate plus complete nutrient solution, and 2241 and 2812 S/cm for the 200 mg/L nitrate plus complete nutrient solution treatments. Salinity in parts per thousand averaged 0.23 and 0.35 for the no nutrient solution treatment, 0.71 and 0.98 for the 100 mg/L nitrate plus complete nutrient solution, and 1.14 and 1.46 for the 200 mg/L nitrate plus comp lete nutrient soluti on treatments. The differences in EC and salinity between Experime nts 1 and 2 were most likely due to different public water sources for each experiment. The wa ter source for Experiment 2 had higher initial soluble salts for the make up water. Season pH values during experiment 1 ranged from 5.9 to 7.3 and 8.2 to 8.7 and during Experiment 2 from 6.1 to 7.0 and 8.3 to 8.6, for pH treatments 6.5 and 8.5, respectively. Ni trification is an acid producing process (ammonia oxidation produces protons H+ in Eq.1) requiring adjustment of recirculating water to main tain target pH levels. The measured pH was within the targ et pH range for the treatments.

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64 This experiment was based on typical startup characteristics for bri nging a new biological filter up to full capacity in aqu aculture systems (Timmons et al ., 2002). Relative nitrification activity was measured based on the number of days after inoculation (DAI) it takes after introduction of nitrifying b acteria to convert TAN to NO3 -N. The end point for the oxidation reactions was the date on which NH3 -N and NO2 -N concentrations reached 0 mg/L (Madigan et al., 2003). The complete nitr ification reaction occurred in 45 days (10 sampling dates) for experiment 1 and in 25 days (6 sa mpling dates) for experiment 2. This was most likely the result of a more active batch of starter bacteria for experiment 2. As a result of the differences in the speed of the oxidation reactions, data will be presented separately for each experiment. In addition, no significant interacti on occurred between nutrient/NO3 -N concentration and pH for ammonia and nitrite oxidation. Therefore, data are presented as the main effects of nutrient/NO3 -N concentration and the main eff ects of pH on nitr ification rate. No significant difference was observed in nitr ification rate when system water contained no nutrient solution versus a complete hydroponi c nutrient solution (Table 4-1). Since NO3 -N is the end product of nitrite oxidation in the nitrifi cation reaction (Eq. 4-2), NO3 -N concentrations of 100 and 200 mg/L NO3 -N were compared to test for a possible feedback inhibition effect on the reaction (Eq. 4-2), which did not occur. No significant difference in nitrification rate with NO3 -N concentrations of 0, 100, or 200 mg /L where observed (Table 4-1). Results indicate that hydroponic plant nutrient solutions at co ncentrations found in plant production systems are unlikely to reduce nitrific ation rate in perlite medium. Thus plant nutrients in aquaponic systems may be tailor ed for optimum production of the plant (with consideration of the contribution by fish wa ste) without concern for adverse impacts on nitrifying bacteria.

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65 Nitrification was significantly affected by sy stem water pH (Fig. 4-1). Total ammonia nitrogen concentration was cl osest to 0 on 35 and 25 DAI at pH 6.5, and 20 and 15 DAI at pH 8.5, for Experiment 1 and 2, respectively. Th e ammonia oxidation rate s were 231 and 300 g L-1 d-1 at pH 6.5 and 400 and 540 g L-1 d-1 at pH 8.5, in Experiments 1 and 2, respectively. The ammonia oxidation rates proceeded 1.75 times faster at pH 8.5 compared to pH 6.5. This was most likely due to the increase in substrate for ox idation because ammonia in water exists as two compounds: ionized NH4 + and un-ionized NH3 ammonia. Ammonia oxidation involves the uncharged ammonia NH3 (Prosser, 1986) and the concentra tion of uncharged ammonia in water increases almost 10 fold as pH increase s from 7.0 to 8.0 (Masser et al., 1999). Nitrite oxidation occurred at the rates of 231 and 375 g L-1 d-1 for pH 6.5 and 267 and 540 g L-1 d-1 for pH 8.5 for Experiments 1 and 2, respec tively. Nitrite oxidation rates proceeded 1.2 and 1.4 times faster at pH 8.5 compared to pH 6 .5, in Experiments 1 and 2, respectively. Nitrite nitrogen concentra tion reached a peak of 4.2 and 3.8 mg NO2 --N /L for Experiments 1 and 2, respectively (Fig.4-1). Nitrifi cation is a dynamic process and as nitrite nitrogen accumulates, it is simultaneously oxidized to nitrate nitrogen. However, since the minimum doubling times for ammonia oxidizers are about 7 h compared to 13 h for nitrite oxidizers (Prosser, 1986), the potential for nitrite buildup exists. In batch tests with sewages, mi nimal nitrite was present at pH 6, but at pH values of 8.4 and 9.2 the rate of ammonia oxidation surpassed that of nitrite oxidation causing an accumulation of nitrite (Alleman, 1985). The current investigation indicated similar results as ammonia oxidation pr oceeded at a higher rate compared to nitrite oxidation (1.75 vs. 1.3 times faster) at pH 8.5 comp ared to pH 6.5, resulting in increased nitrite accumulation (Fig. 4-1). Nitrite at concentrations as low as 5 mg/L in nutrient solution damaged tobacco root tips (Hamilton and Lowe, 1981) and G ila trout exposed to nitrite at 10 mg/L or

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66 more for 96 h died (Fuller et al., 2003). Ca ution should be exercised in aquaponic system management to avoid practices that may pr oduce surges in TAN and subsequent nitrite accumulation. Hence, nitrite should be monitored routinely. The advantages of increasing th e rate of nitrification would be to allow greater stocking density for fish and increased nutrient loads for plants thereby increasi ng productivity potential. The nitrification efficiency in this perlite medium biofilter startup cycle increased 19% and 26% at pH 8.5 compared to pH 6.5, for Experiments 1 a nd 2, respectively (Fig. 41). In a submerged biofilter investigation, a pH in crease of one unit within a ra nge of 5.0 to 9.0, produced a 13% increase in nitrification rate (Villaverde et al., 1997) similar to the current study results. However, even though nitrification proceeds faster at pH 8.5 compared to pH 6.5, the potential for increased nitrite accumulation at the higher pH exists, with subsequent danger for the fish (brown blood disease; Masser et al., 1999) and plants (root damage; Hamilton and Lowe, 1981; Pearce et al., 1998). Also, increasing pH from 7. 0 to 8.0 results in a near exponential increase in un-ionized ammonia concentration in system water, which can be toxic to fish (Colt and Tchobanoglous, 1976; Masser et al., 1999) . In addition, nutrient avai lability for plant uptake at pH above 7 may be restricted due to precipitation of Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+ to insoluble and unavailable salts (Resh, 2004). The a dvantages of increased n itrification efficiency at the higher pH weighed against th e potential increased water quality risks to fish and plants are therefore not justified. Management of pH in aquaponic systems is currently maintained near 7 to compromise between plant and nitrifying bacter ia preferences (Rakocy et al., 1997) and the current work supports this compromise. Conclusion Reconciling water quality parameters in su stainable aquaponic (i ntegrated hydroponic and recirculating aquaculture) systems requires balanc ing nutrients and pH for the optimal growth of

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67 3 organisms: the plant, the fish, and the nitrifying bacteria. Nitr ifying bacteria convert fish waste into NO3 -N that may be used by the plants. Fish waste rarely supplies nutrients in adequate amounts for plants without supplementation. Increas ing nitrification rate and efficiency would allow greater stocking density for fish and increa sed nutrient loads for plants. The objective of this research was to determine th e nitrification rate re sponse in a perlite tric kling biofilter (root growth medium) exposed to hydropo nic nutrient solution, varying NO3 --N concentrations, and to pH levels optimum for plants ( 6.5) and nitrificat ion (8.5). The experiment used recirculating tank batch culture and was based on typical startup characteristics for bringing biological filters up to full capacity in aquaculture systems. No significant difference (P value < 0.05) in ni trification rate was f ound when recirculating system water contained no nutrient solution ve rsus a complete hydroponic nutrient solution or NO3 -N concentrations of 0, 100, or 200 mg/L. Th ese results indicate that hydroponic plant nutrient supplementation to concentrations f ound in plant production systems do not significantly affect nitrification rate in perlite medium. N itrification was significantly impacted by water pH. Ammonia oxidation of initial to tal ammonia nitrogen (TAN = NH4 +-N + NH3 -N = 8 mg/L) occurred at the rates of 231 and 300 g/L/d at pH 6.5 and 400 and 540 g/L/d at pH 8.5, for experiments 1 and 2, respectively. The rates proc eeded 1.75 times faster at pH 8.5 than at pH 6.5. Nitrite oxidation occurred at the rates of 231 and 375 g/L/d for pH 6.5 and 267 and 540 g/L/d for pH 8.5 and proceeded 1.2 and 1.4 times faster, respectively. The increased ammonia oxidation rate (1.75) compared to nitrite oxidat ion rate (1.3) at pH 8.5 resulted in accumulation of NO2 - – N to levels near those harmful to plants and fish (observed peaks of 4.2 and 3.8 mg/L NO2 -N, respectively). The potential for increas ed levels of un-ionized ammonia which are toxic to fish and reduced plant nutrient uptak e from micronutrient precipitation are additional

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68 problems associated with pH 8.5. The advantages of increased nitrification efficiency, which averaged 23% in the current trials at the highe r pH, when weighed against the potential increased water quality risks to the fish and plant, justify a compromise between pH optima for nitrification and plant production to pH 7 for aquaponic system water. A more flexible management strategy for these systems would be to supplement with pl ant nutrients, which would permit less reliance on the fish and nitrification to provi de optimal plant nutrient levels.

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69 Table 4-1. Total ammonia nitr ogen (TAN), nitrite nitrogen (NO2 -N), and nitrate nitrogen (NO3 -N) concentrations in perlit e trickling biofilter (root growth medium) when exposed to hydroponic nutrient solution. Nutrient Days after Inoculation Solution 0 z 5 10 15 20 25 30 35 40 45 Experiment 1 Total ammonia nitrogen (mg/L) 0 y 8.0a x 7.1a 6.1a 3.1a 1.6a 1.1a 0.6a 0.0a 0.0a 0.0a 100+ 8.1a 6.7b 6.0a 3.5a 1.5a 1.4a 0.0a 0.0a 0.0a 0.0a 200+ 8.1a 7.0ab 6.5a 3.9a 1.6a 1.4a 0.6a 0.1a 0.0a 0.0a Experiment 2 0 7.5b 6.8a 3.4a 1.7a 0.1a 0.0a 0.0a 100+ 7.7b 6.8a 3.3a 1.8a 0.9a 0.1a 0.0a 200+ 8.1a 7.4a 3.4a 2.1a 0.6a 0. 1a 0.0a Experiment 1 Nitrite nitrogen (mg/L) 0 0.0a 0.0a 0.0a 1.6a 2.5a 1.8a 1.1b 1.6a 0.3a 0.0a 100+ 0.0a 0.0a 0.0a 0.9a 2.4a 3.5a 3.0a 2.0a 0.6a 0.4a 200+ 0.0a 0.0a 0.0a 0.9a 2.4a 2.4a 1.1b 0.8a 1.2a 0.6a Experiment 2 0 0.0a 0.1a 1.7b 2.4a 1.0a 0.6a 0.0a 100+ 0.0a 0.5a 2.4ab 2.1a 2.0a 0. 0a 0.0a 200+ 0.0a 0.3a 2.9a 3.2a 1.2a 0. 4a 0.0a Experiment 1 Nitrate nitrogen (mg/L) 0 3c 2c 3c 2c 2c 3c 4c 5c 6c 5c 100+ 104b 94b 106b 102b 102b 104b 106b 115b 119b 114b 200+ 201a 180a 194a 194a 200a 204a 211a 220a 229a 221a Experiment 2 0 2c 2c 3c 2c 4c 5c 4c 100+ 110b 114b 119b 103b 119b 129b 116b 200+ 205a 199a 216a 190a 216a 239a 213a ______________________________________________________________________________ z Nitrifying bacteria introduced to the biofilters. y Hydroponic nutrient solution 0 (tap water only), 100mg/L nitrate pl us complete plant nutrient solution or 200 mg/L plus complete nutrient solution. x Within columns, means followed by di fferent letters are significantly different at the 0.05 level; four replicates.

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70 A0 1 2 3 4 5 6 7 8 9 051015202530354045 Days After InoculationTAN or NO2 -N mg/L pH 8.5 TAN pH 8.5 NO2-N pH 6.5 TAN pH 6.5 NO2-N B0 1 2 3 4 5 6 7 8 9 051015202530 Days After InoculationTAN or NO2 -N mg/L pH 8.5 TAN pH 8.5 NO2-N pH 6.5 TAN pH 6.5 NO2-N Note: Error bars represent SE (n=4). Figure 4-1. Effect of pH on amm onia and nitrite oxidation in perl ite medium. A) Experiment 1 B) Experiment 2.

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71 CHAPTER 5 EFFECT OF WATER PH ON YIELD AND NUTRITIONAL STATUS OF GREENHOUSE CUCUMBER GROWN IN RECIRCULATING HYDROPONICS Introduction Fresh cucumber ( Cucumis sativus ) was grown on 23,136 hectares with a market value of 235 million dollars in the United States during 2 005 (National Agricultural Statistics Service, 2006). It is also an import ant vegetable in greenhouse produc tion systems (Tyson et al., 2001) and has potential for production in integrated hydroponic and a quaculture systems (aquaponics) (Timmons et al., 2002). Aquaponic production requi res balancing water qua lity and pH for the optimal growth of three groups of organisms: plants, fish, and nitrifying bacteria. One of the most important water quality mana gement requirements of aquaculture systems is to prevent the buildup of ammonia in system wa ter. In water, ammonia exists in two forms, which together are called the Total Ammonium Nitrogen (Fra ncis-Floyd and Watson, 1996) or TAN (TAN = NH4 +N + NH3 – N). The equilibrium reaction is NH4 + NH3 + H+ (Campbell and Reese, 2002). Water temperature and pH a ffect which form of ammonia predominates. NH4 +-N is predominate below pH 7.0. As pH in creases from 7.0 to 8.0 there is a ten-fold increase in NH3-N. Unionized ammonia (NH3) is toxic to fish at concentrations above 0.05 mg/L (Francis-Floyd and Watson, 1996), producing mortality at 0.08 mg/L NH3-N for pink salmon and 2.2 mg/L NH3-N for common carp (Timmons et al., 2002). Thus ammonia biofiltration (nitrification) of system water by nitrifying bacter ia is needed for maintenance of water quality by conversion of fish waste ammonia to NO3 -N which is relatively nontoxic to fish and may be used by plants. Recommended water pH for greenhouse hydroponic production is 5.5.0 (Hochmuth, 2001b), 5.5.5 (Hochmuth, 2001a) and 5.8.4 (Resh, 2004). This slightly acid pH helps reduce precipitation of Fe2+, Mn2+, PO4 3-, Ca2+ and Mg2+ into insoluble and unavailable salts

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72 which may occur at water pH levels > 7.0. Aqua ponic recirculating water pH is recommended to be maintained between 7.0 and 7.5 (Timmons et al., 2002) to balance pH for ammonia biofiltration with nutritional requir ements of the plant. Aquacu lture biofilter nitrification was reported to be most efficient at pH 7.5.0 (Hochheimer and Wheaton, 1998) and 7.0.0 (Masser et al., 1999). Nitrificati on efficiency increased 13% with each unit increase in pH from 5.0 to 9.0 (Villaverde et al., 1997) in submerged biofilters. In a nother investigation with four different biological filters (unde r gravel, fluidized bed, non-flui dized bed, and gravel bed) nitrification slowed signifi cantly or stopped when pH dropped below 6.0 (Brunty, 1995). A reconciling pH between the requirements of rapid ammonia biofiltration and the nutritional requirements of crops in hydroponi c production has not been scientifically established. It may be possibl e to overcome nutrient deficien cy and maintain crop yield under system water pH production conditions > 7.0 with th e use of foliar application of micronutrients. Foliar applications of Mg, Zn, and Mn can effectiv ely correct deficiencies in fruit and vegetable crops grown on calcareous soils with a pH 7.4 to 8.4 (Li, 2001). Overall yield increases of 33% occurred when strawberry ( Fragaria ananassa ) cultivars were sprayed once per week with Fe (1.0 kg Fe/ha) when grown in calcareous soil at pH 8.2 (Zaiter and Saad, 1993). Fish stocking density in inte nsively managed recirculating aq uaculture systems is directly related to the capacity of the biofilter to process and preven t the buildup of toxic ammonia since 10% of the protein in fish feed becomes ammoni a nitrogen in the system water (Timmons et al., 2002). If greenhouse crops could be grown at pH 7.0.0 without a redu ction in yield, then ammonia biofiltration rates may be improved in integrated aquaponic systems. This would allow greater fish stocking densities producing more plant nutrients fr om fish waste thus conserving applied fertilizer and thereby improving aqua ponic system integration and sustainability.

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73 The most common recirculating aquaponic syst ems employ either a media-filled raised bed, nutrient-flow technique (NFT), or floati ng raft system (Adler et al., 1996; Anonymous, 1997; Diver, 2006; McMurtry et al., 1997; Rakocy et al., 200 6,1997; Watten and Busch, 1984) for the plant growing area. Of those systems, the media filled bed has potential for providing ammonia biofiltration and a root zone sp ace for plant production. Using a continuous recirculating system perlite media bed with poten tial for use in aquaponics, the purpose of this investigation was to 1) determ ine the effect of pH on greenhous e cucumber yield at water pH between 5.0 and 8.0 and 2) assess the possibili ty of restoring nutri tion and yield by foliar fertilization at pH 7.0 and 8.0. Materials and Methods The experiment was conducted in a passi vely ventilated gr eenhouse at the Polk Correctional Prison Farm in Sanford, FL. Si x treatments were arranged in a randomized complete block design with three replications. All treatments had recirculating water maintained for a range of pH values as follows: 1) pH 5.0, 2) pH 6.0, 3) pH 7.0, 4) pH 8.0, 5) pH 7.0 with foliar applied nutrients (7-fs), a nd 6) pH 8.0 with foliar applied nutrients (8-fs). Plastic, 80-L rectangular tanks were filled with 40 L of ta p water on 11 Aug., 2005. The plastic recirculating tanks were placed 90 cm apart in a single row parallel to the length of the house. A complete hydroponic nutrient solution consisting of 600 mg/L NFT Vegetable Formula (hydroponic fertilizer blend Grower’s Suppl y Center, Lynn Haven, FL), 600 mg/L calcium nitrate (Ca(NO3)2) and 300 mg/L magnesium sulfate (MgSO4) were added to each tank. The NFT vegetable formula as applied cons isted of 18 mg/L nitrate nitrogen ( NO3 -N), 39 mg/L phosphorus (P), 134.5 mg/L potassium (K), 32 mg /L magnesium Mg), 2.7 mg/L iron (Fe), 0.2 mg/L zinc (Zn), 0.4 mg/L manganese (Mn), 0.07 mg/L copper (Cu), 1.0 mg/L boron (B), and 0.07 mg/L molybdenum (Mo), using potassium sulfate, monopotassium phosphate, magnesium

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74 sulfate, potassium nitrate, iron EDTA, zinc EDTA, manganese EDTA, copper EDTA, sodium borate and sodium molybdate as nutrient sources . Phosphoric acid was used to lower pH and potassium hydroxide was used to raise pH during the trial as needed to maintain pH at treatment levels. Composite water samples for each treat ment (75ml/tank) were collected on 15 Aug. and frozen for specific elemental analysis of starti ng solution (Table 5-1). Electrical conductivity (EC) was maintained between 1 and 2 ds/cm dur ing the experiment by periodically adding the above fertilizer at the same ratios when required. Germination trays with drainage slits in the bottom were placed above the water on plastic stools in each tank on 15 Aug. Natural burlap wa s double layered in the trays, horticultural grade course perlite was added, a nd water distribution plates were placed on top of the perlite. Water was pumped to the plates and re-circu lated through the perlite with aquarium pumps (model no. SP800, Aquatic Eco-systems, Apopk a, FL) at the rate of 1.9 L/min. Two ‘Millagon’ (De Ruiter S eeds, Inc., Lakewood, CO) European cucumber seedlings were planted into the perlite of each plot on 16 Aug (0 DAT). This variety has powdery mildew tolerance and excellent fruit uniformity (Hochmuth et al., 1996). Two additional transplants were placed on the distribution plates of each co ntainer on 16 Aug. and their roots were bathed with constant recirculating nutrient solution for 14 d. These additional plants were included to obtain early season shoot tissue elemental content. A foliar nutrient application was made once weekly beginning 7 DAT at the rate of 20ml/L INP 3500 chelated nutritional complex (Plant Food Systems, Zellwood, FL) with 6g/L potassium nitrate to treatments 7-fs and 8-fs. Nutritional content of foliar spray was 780, 2,640, 0.4, 1.0, 0.9, 0.2, 0.2, 0.04, and 0.0008 mg/L of N, K, Mg, S, Fe, Mn, Zn, B, and Mo, respecti vely. Plants in the distribution plates were harvested 14 DAT and shoot tissue processed us ing the dry ash digestion method (Mills and

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75 Jones, 1996). Tissue analysis and composite wa ter sampling were analyzed by Agro Services International, Inc., Orange City, FL. usi ng a segmented stream autoanalyzer for N, spectrophotometer colorimetry for P, atomic abso rption for K, Ca, Mg, Mn, Fe, Zn, and Cu, and the curcumin method for B (Plank, 1992). Cucumbers were trellised to overhead wires and pruned to a single leader stem. Fruit were harvested every 2 days between 34 to 55 DAT. Fruits showing poor tip fill and angled fruit approaching 45% were considered non-marketable and were pruned from the plants as soon as defects were detected. Those with marketable potential were allowed to grow to commercial size. Early yield was calculated fr om fruits in the first three harvests and total yield from all ten harvests. Nitrate nitrogen concentrations in petiole sa p were measured using ion specific electrode meters (Cardy Spectrum Technologies, Inc., Plainf ield, IL) on 22 and 45 DAT. The cardy meter was also used to measure NO3 -N and K concentrations in th e nutrient solution during the experiment. Water pH and EC were measured using an Accumet Research pH meter (model no. AR15, Fisher Scientific Interna tional, Inc., Hampton, NH) and a YSI Model 85 meter (YSI Inc., Yellow Springs, OH), respectively. Data were analyzed using ANOVA (SAS, 2001) and Duncan’s Multiple Range Test using a P value of 0.05. Data were analyzed for significant linear and quadratic trends over the nonrepeating equal distant pH units. Results and Discussion Season pH values ranged from 3.8 to 5.9, 5.4 to 6.9, 6.4 to 7.6, 7.2 to 8.5, 6.3 to 7.7, and 7.2 to 8.5, respectively, for treatments pH 5.0, 6.0, 7.0, 8.0, 7-fs, and 8-fs. The measured pH was within the target pH ranges of the treatments for the trial. Composite samples from the nutrient solutions of treatments were analyzed for selected nutrient concentrations after fertilizer addition

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76 and pH adjustment (four days after tank setup) to determine potential availability for uptake by the plant (Table 5-1). The concentrations of Ca , P, Fe, and Mn in the nutrient solution declined as pH increased, but magnesium was unaffected by pH. These nutrients were identified as potentially restricted at high pH due to pr ecipitation to in soluble salts (R esh, 2004). Shoot fresh and dry weight, and length of young cucumber plants on14 DAT were similar among pH 5.0, 6.0, or 7.0 treatments, but were signifi cantly reduced in the pH 8 treatment (Table 5-2). Shoot fresh and dry weight and length declined linearly as the pH increased from 5.0 to 8.0. Differences between pH 7.0 and 7.0-fs and 8.0 and 8.0-fs were not signi ficantly different. Foliar spray at one week after pl anting had no effect on ea rly growth. This is most significant at pH 8.0 since plants where alrea dy smaller at this stage of gr owth compared to the other treatments (Table 5-2). This difference in ea rly biomass production most likely contributed to the significant difference in early marketable yi eld observed between pH 5.0 and 8.0 (Table 5-6). Results of shoot tissue analysis from the samp les above indicate that Mg content increased as pH increased from 6.0 to 8.0 (Table 5-3). This was most likely due to the competition for binding and transport sites on the plasma me mbrane (Marshner, 2003) between cations Mn2+ and Mg2+ and the declining concentration of Mn2+ in the nutrient solution as pH increased (Table 51). Nitrogen and phosphorus content were si gnificantly reduced at pH 8.0 compared to 5.0, 6.0, or 7.0. This may be a result of reduced P in the nu trient solution as pH in creased (Table 5-1) but not for N since NO3 -N increased as pH increased (Table 5-6). However, these results suggest that (1) lower concentrations in recirculating solutions may be adequate and (2) reduced P in solution is affected primarily by water only at pH 8.0. Although foliar spray samples were not washed prior to analysis, no significant increas e in N, K, Ca, Mg, Cu, or B were observed compared to unsprayed treatments (pH 7.0 versus 7-fs and 8.0 and 8-fs). Consequently, the

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77 increase in Fe, Mn, and Zn, were most likely actual tissue content increases and not residual spray on the leaves (Table 5-4). This agrees with earlier work (Li, 2001; Zaiter and Saad, 1993) for overcoming nutrient deficiency at high pH for Fe, Mn, and Zn. Visual observation of cucumber foliage on 24 DAT showed pH 5.0 with dark green leaf color, pH 6.0 with medium green leaf color, pH 7.0 with slightly mottled yellow leaf color, pH 8.0 with pronounced mottled yellow leaves, pH 7fs with noticeably less mottled leaf appearance compared to pH 7, and pH 8-fs with slightly less mottled leaf color than pH 8.0. At this point in the experiment three foliar sprays had been made and indicate that visu al symptoms of nutrient deficiency were less pronounced with foliar nutriti onal sprays compared to unsprayed treatments. The concentrations of NO3 -N in petiole sap were unaffected by pH (Table 5-5). Sap leaf petiole NO3 -N levels were higher than recomm ended for cucumber production (800-1000 mg/L NO3 -N first flower stage, 400-600 mg/L first ha rvest; Hochmuth, 2003), thus nutrient solution concentrations during the tria l were kept below recommended levels of 113-275 mg/L N (Chaverria et al., 2005; Hochmuth, 2001a; Scho n and Compton, 1997b) and averaged between 52 and 84 mg/L (Table 5-5). Tissue concentrations of all nutrients tested were within adequate to high ranges (Table 5-3 and 5-4) with the exception of manganese, which was low < 30 mg/kg at pH 8.0 (Olson et al., 2006). However, manganese was restored to an adequate level by foliar spray. Even though Fe dropped to the same level (T able 5-1) as the source water at pH 8 (0.03 mg/L), the plant was able to accumulate 70 mg/kg Fe in the shoot tissue, we ll within an adequate range for the plant (Table54). This indicates that the analyzed nutritional content of the cucumber plants appeared adequate at all pH levels (except manganese at pH 8.0) even though certain nutrient levels were lower than reco mmended (Hochmuth, 2001a;Olson, et al., 2006) in the nutrient solution for non-circulat ing systems (Table 5-1 and 5-5). This may be due to the

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78 continuous recirculation of the nut rient solution through the media, bathing the roots of the plant, resulting in no depletion of nutrien ts in the root zone despite lo w nutrient concentrations in the solution. Two studies (Olson, 1950; Rakocy et al., 1997) indicate that optimum plant yields may be maintained at lower nutrient so lution concentrations if roots are constantly exposed to the solution rather than receiving it intermittently. In soils, nutrients move to the surface of roots by diffusion and bulk flow of the soil solution resul ting from transpiration (Taiz and Zeiger, 2002). Concentration gradients can form in the soil so lution as nutrients are taken up by the roots and the concentration of nutrients at the root surf ace is lowered compared to the surrounding area. This can result in a nutrient depletion zone near the root surface. The capacity for continuous growth by roots however, extends this region of nutrient uptake beyond the depletion zone. Thus, optimum nutrient acquisition by plants depends on the capacit y of their root systems not only to absorb nutrients, but al so, to grow into fresh soil. In hydroponic production, the media volume is finite and nutrient depletion can be recovered only in the next irrigation event. N depletion can occur at lower N (90-175 mg/L) nutrient solutio n concentrations (Schon and Compton (1997b) under commercially recognized intermittent fertigation of cucumber in hydroponic media. Irrigation frequencies adequate to prevent water stress are not necessarily adequate to prevent nutrient depletion except at high N (225-275 mg/L) nutrient solution concentrations. Therefore, it seems logical to propose that more frequent flushing of the media with lower concentrations of N would obviate N de pletion between irrigati on events. If this flushing was continuous, there would be no appreciable depletion of nutrients in the root zone. This reasoning could apply to all nutrients in the solution. Thus precipitation of certain nutrients at pH 8.0, suggested in Table 5-1, may not limit the overall nutritional status of the plant

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79 provided continuous recirculation through the roo t/media zone occurs to eliminate nutrient depletion. Early marketable cucumber fruit yield was si gnificantly higher at pH 5.0 compared to pH 8.0 (Table 5-6). Foliar sprays during the grow ing season did not increas e yields compared to unsprayed treatments. Total marketable and cu ll cucumber fruit yields were not significantly different among treatments. Early marketable yiel d response to pH was si gnificantly linear at the 5.2% level. Results indicate an early yield advantage to keep ing nutrient solution pH between 5.0 and 7.0 but not an advantage for total yield. This would indicate that maintaining the recirculating pH at 8.0 to accommoda te nitrifying bacteria activity would be detrimental from an economic standpoint if the grower were producing for an early market but would not adversely affect the overall production during middle to late portions of a multiple cropped season. Conclusion Shoot fresh, dry weight, and length of cucu mber plants harvested on 14 DAT decreased linearly as pH increased from 5.0 to 8.0. Early ma rketable cucumber fruit yield was higher at pH 5 compared to pH 8 but total yield was unaffect ed by pH treatment. Foliar nutritional sprays during the season reduced visual deficiency symptoms at pH 7.0 and 8.0, but did not significantly increase yield. Fo liar spray rescue treatments did not work. However, results suggest that nutrient con centrations in recirculating systems can be maintained lower than in non-circulating systems overcoming potential limits to production which may occur from selected nutrients prone to precipitation at pH > 7.0. This study indicates that aquaponic systems utilizing cucumber in recircul ating aquaponic culture may be ma intained at pH levels more optimum for nitrifying bacteria 7.5.0 with no reduction in tota l yield except during production for early season markets where pH 7.0 would be recommended. Increased system ammonia biofiltration through nitrification will allow higher fish stocking densities producing more plant

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80 nutrients from fish waste thus conserving a pplied fertilizer and th ereby improving aquaponic system integration and sustainability.

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81 Table 5-1. Initial water analysis for pH and selected nutrients. Composite Actual Solution elem ental concentration (mg/L) Sample pH Ca Mg P Fe Mn H2O 7.6 31.2 7.6 0.3 0.03 0.03 pH 5.0 5.4 102.8 45.0 66.4 0.16 0.24 pH 6.0 6.1 87.3 40.4 34.4 0.06 0.16 pH 7.0 7.0 80.1 43.8 15.7 0.08 0.07 pH 8.0 8.3 73.5 42.9 2.8 0.03 0.04 Recom.z level 5.5-6.5 130.0 50.0 62.0 2.5 0.62 zRecommended nutrient solution level for non-circulating hydroponic cucumbers (Hochmuth, 2001b) Table 5-2. Cucumber shoot fresh and dry weight and plant length on 14 DAT stage of growth as influenced by system water pH and foliar spray. Target pH Shoot fresh wt. Shoot dry wt. Shoot length g g cm 5.0 70.5 az 10.2 a 57 a 6.0 68.2 a 10.0 a 56 a 7.0 68.5 a 9.3 a 55 a 8.0 42.0 b 6.5 b 40 b Contrast L*y L** L* 7.0-fsx 65.2 a 8.8 a 50 ab 8.0-fs 41.3 b 6.3 b 40 b zWithin columns, means followed by different letters are significantly different; three replicates. yLinear contrast were significant at the 5% (*) or 1% (**) level. xfs = Foliar nutritional spray once per week.

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82 Table 5-3. Cucumber shoot nut rient content (% DM) 14 DAT as influenced by solution pH and foliar spray. Target pH N P K Ca Mg % % % % % 5.0 5.2 a 0.78 a 4.4 ab 2.9 ab 0.83 c 6.0 5.2 a 0.74 a 5.4 a 2.2 c 0.80 c 7.0 5.1 a 0.71 a 4.7 ab 2.5 bc 1.10 b 8.0 4.5 b 0.39 b 4.5 ab 3.1 a 1.43 a Contrast L** Q* L** Q* Q* L** 7.0-fsx 5.3 a 0.69 a 5.1 ab 2.3 bc 1.17 b 8.0-fs 4.7 b 0.36 b 4.2 b 2.5 bc 1.17 b Sufficiency Rangew 2.5-5.0 0.25-0.6 1.6-3.0 1.0-3.5 0.3-0.6 ________________________________________________________________________ zWithin columns, means followed by different letters are significantly different; three replicates. yLinear and quadratic contrast s were significant at the 5% (*) or 1% (**) level. xfs = Foliar nutritional spray once per week. wPlant tissue analysis at early bloom st age for cucumber, dry weight basis. (Olson et al., 2006) Table 5-4. Cucumber shoot nutri ent content (mg/kg) 14 DAT as influenced by solution pH and foliar spray. Target Shoot nutrient concentration pH Fe Mn Zn Cu B mg/kg mg/kg mg/kg mg/kg mg/kg 5.0 120 bz 143 a 71 b 10 a 96 a 6.0 84 b 57 bc 61 b 7 b 84 a 7.0 82 b 31 c 63 b 10 ab 87 a 8.0 70 b 24 c 66 b 11 a 91 a Contrast L** Q** y L** Q* 7.0-fsx 367 a 68 b 109 a 10 ab 87 a 8.0-fs 363 a 79 b 120 a 11 a 98 a Sufficiency Rangew 40-100 30-100 20-50 5-10 20-60 ________________________________________________________________________ zWithin columns, means followed by different letter s are significantly different; three replicates. yLinear and quadratic effect s were significant at the 5% (*) or 1% (**) level. xfs = Foliar nutritional spray once per week. wPlant tissue analysis at early bloom stage for cucumber, dry weight ba sis (Olson et al., 2006).

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83 Table 5-5. Concentration of NO3 –N and K in cucumber petiole sap and average season nutrient solution NO3 –N and K levels. Petiole sap Avg. season nutrient Target First flower Sixth harvest solution concentration _____ pH NO3 –N NO3 –N K mg/L mg/L 5.0 1150 az 1233 a 52 c 71 ab 6.0 1100 a 1053 a 61 bc 54 b 7.0 1123 a 1163 a 72 ab 60 b 8.0 1133 a 1083 a 78 a 84 a Contrast L**y Q* 7.0-fs x 1167 a 983 a 73 ab 56 b 8.0-fs 1233 a 1267 a 84 a 67 ab Recom. Levelsw 800-1000 400-600v 133 150 zWithin columns, means followed by different letter s are significantly different; three replicates. yLinear and quadratic effect s were significant at the 5% (*) or 1% (**) level. xfs = Foliar nutritional spray once per week. w Recommended for petiole sap (Olson et al., 2006) and N and K in non-circulating hydroponics (Hochmuth, 2001b). v First harvest recommendation. Table 5-6. Cucumber fruit yi eld as influenced by nutrien t solution pH and foliar spray. Target pH Early marketablez Total marketable Total cull kg/plant no/plant kg/pl ant no/plant kg/ plant no/plant 5.0 1.38 ay 4.2 a 2.81 a 6.83 a 0.24 a 0.7 a 6.0 1.04 ab 2.8 ab 3.18 a 7.17 a 0.61 a 0.7 a 7.0 0.73 ab 2.3 ab 2.93 a 7.00 a 0.42 a 0.5 a 8.0 0.62 b 2.0 b 2.83 a 7.17 a 0.03 a 0.2 a Contrast 0.052x 7.0-fsw 1.08 ab 3.2 ab 3.16 a 7.83 a 0.13 a 0.2 a 8.0-fs 0.64 b 2.2 b 2.58 a 6.67 a 0.25 a 0.3 a ________________________________________________________________________ zEarly = first three harvests, Total = all ten harvests, Marketable = 34-42 cm in length, less than 45 fruit angle, few blemishes, Cull = greater than 45 fruit angle, poor tip fill, frequent blemishes. yWithin columns, means followed by different lette rs are significantly different; three replicates. xSignificant linear contrast trend between pH 5.0 through 8.0 (p = 0.052) level for early marketable fruit. wfs = Foliar nutritional spray once per week.

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84 CHAPTER 6 WATER QUALITY INLUENCES AMMONIA BI OFILTRATION AND CUCUMBER YIELD IN RECIRCULATING AQUAPONICS Introduction Aquaponics is the integrated production of hydroponic and aquaculture systems. Cucumber ( Cucumis sativus ) is an important hydroponic gr eenhouse crop (Tyson et al., 2001) with potential for production in aquaponic systems (Timmons et al., 2002). Worldwide production of tilapia ( Oreochromis sp .) exceeded 2.2 million metric tons in 2002 with 68% of that total coming from farmed aquaculture (Lim and Webster, 2006). Properly designed and managed hydroponic and aquaculture systems ar e considered environmentally responsible alternatives to field grown vegetable production and wild caught fisheries (Smither-Kopperl and Cantliffe, 2004; Timmons et al., 2002). Aquaponics fits closely into the definition of sustainable agriculture in the 1990 Farm Bill, Title XVI, Subtitle A, Sec. 1603. Aquaponics is “an integrated system of plant and animal production practices” using vegetables with a quaculture species, “having a site-specific application” in greenhouse production units. It will “over the long te rm satisfy human food needs” and “enhance environmental quality” by producing crops using practices that minimize water and nutrient waste discharges to the enviro nment. Aquaponics will “make the most use of nonrenewable resources” by conser ving fertilizer nitrogen derive d from fossil fuels and reducing water use. It will “integrate na tural biological cycles” by using nitrifying bacteria in the process of nitrification to convert harmful ammonia fish waste to usable, nitrate nitrogen for plants. Aquaponics will “sustain the economic viability of farm operations” and “enhance the quality of life for farmersand society as a whole” by pro ducing food in a sustaina ble bio-rational manner without wasteful discharge to the environment. However, with all its promise, there is no information in the literature on how aquaponic syst em water quality impacts nitrification in a

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85 perlite biofilter/root growth medium, little information on the plant root/nitrifying bacteria interaction affects on ammonia bi ofiltration, and how th is interaction aff ects plant yield and nitrifying bacter ia activity. Aquaponic production requires balancing nutrient concentrations and pH for the optimal growth of 3 organisms: plants, fish, and nitrifying bacteria. Recommended pH for aquaculture is 6.5.5 (Timmons et al., 2002) and for greenho use cucumber is 5.5.0 (Hochmuth, 2001a). Aquaculture biofilter nitr ification was reported to be most efficient at 7.5–9.0 (Hochheimer and Wheaton, 1998). The reactions involved in nitrifica tion may be summarized as (Madigan et al., 2003): Nitrosifying bacteria (primarily Nitrosomonas ) NH3 + 1 O2 NO2 + H+ + H2O G0 = -275 kJ/reaction (Equation 6-1) Nitrifying bacteria (primarily Nitrobacter ) NO2 + O2 NO3 G0 = -74.1 kJ/reaction (Equation 6-2) Unionized ammonia is the substrate ion used for the nitrification reacti on (Prosser, 1986). In water, ammonia exists in two forms, which t ogether are called the To tal Ammonium Nitrogen (Francis-Floyd and Watson, 1996) or TAN (TAN = NH4 +N + NH3 – N). The equilibrium reaction is as follows (Campbell and Reese, 2002): NH4 + NH3 + H+. Water temperature and pH will affect which form of ammonia is pre dominate. For example, at 22C the unionized ammonia fraction of TAN is 0.46% and 4.4% for pH 7.0 and 8.0, respectively (Francis-Floyd and Watson, 1996). This represents nearly a ten fold increase unionized ammonia substrate for the nitrification reaction. A c oncentration versus activity plot of a biofilter (Hagopian and Riley, 1998) will often demonstrate a first order response (a ctivity increases with increase in substrate concentration). Increased removal of ammonia by a trickling biofilter wa s found with increasing

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86 concentrations of ammonia in pond water (Rij n and Rivera 1990) and removal rate was considered substrate-limited with respect to ammonia. These reported effects and pH range differences require reconciling water quality parameters affec ting ammonia biofiltration and cucumber in aquaponics to improve systems integration and sustainability. Recommended “consensus” pH levels have not been sc ientifically establis hed in aquaponics. The Most Probable Number (MPN) technique is a method of estimating the numbers of microorganisms in foods, wastewater, enrichment cu ltures, or natural samp les of water or soil (Madigan et al., 2002). A selective culture medium is prepared to target the growth of specific organisms or groups of organisms such as nitr ifying bacteria. The MPN method has been used to enumerate nitrifying bacteria in sediments of aquatic envi ronments (Feray et al., 1999; Smorczewski and Schmidt, 1991), and terrestrial so ils (Prosser, 1986; Papen and Berg, 1998). It has also been used in a hydroponic system (Sch warz et al., 1999) and in soilless potting media (Lang and Elliott, 1997). MPN bacterial cell num bers can be used to compare the relative production environment effect on b acterial reproduction provided in itial cell inoculation numbers are the same among treatments. Maintaining aquaponic system water above pH 7.0 should increase system ammonia biofiltration by nitrification, ther eby allowing higher fish stoc king densities, producing more plant nutrients from fish waste. This will conserve applied fertilizer and thereby improve aquaponic systems integration and sustainability. The purpose of this investigation was to 1) determine the ammonia biofiltration rate of a perl ite trickling biofilter/root growth medium in aquaponic production, 2) make predicti ons about the relative contribu tion of plants and nitrifiers to the biofiltration of ammonia and 3) establish the reconciling pH for a mmonia biofiltration and cucumber yield in recirculating aquaponics.

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87 Materials and Methods The experiment was conducted in a pad and fan greenhouse with polye thylene cover at the University of Florida Horticultural Sciences De partment Teaching Park, Gainesville, FL, from 6 July to 12 Oct., 2006. The 5 treatments were 1) aquaponic pH 6.0, 2) aquaponic pH 7.0, 3) aquaponic pH 8.0, 4) hydroponic control pH 6.0, and 5) aquaculture control pH 7.0, with four replicates. Aquaponic treatments consisted of cucumber, tilapia, and nitrifying bacteria. Hydroponic system contained cucumber plants on ly and aquaculture control system contained fish and nitrifying bacteria. Recirculating ta nks were placed 75 cm on center down a single row in the greenhouse running parallel to the north/south sidewalls. E ach circular tank (180-L, model DM52, Aquatic Eco-systems, Apopka, FL) was filled with 100 L of well water on 6 July, 2006 Phosphoric acid was used to lower pH and pot assium hydroxide was used to raise pH to treatment levels (target 6.0, 7.0, and 8.0) during the experiment. Sodium bicarbonate (NaHCO3) and CaCO3 were added to tank water during the trial to maintain alkalinity (a measure of the capacity of water to neutralize acids, also known as the buffering capacity, due primarily to the presence of available bicarbonate, carbonate, and hydroxide ions). Alkalinity during the experiment averaged 35, 42, 84, 39 and 41 mg/L, re spectively, for treatments described above. Biofilters consisting of rectangular plastic milk carrying cases lined with natural burlap and filled with 20 L of horticulturalgrade perlite were placed on top of the recirculating tanks on 14 July. One aquarium pump (model HX2500, Aquatic Ecosystems, Apopka, FL), was placed in the bottom of each tank and water was re-circulated to distribution plat es on top of the perlite in the biofilter and allowed to trickle dow n through the perlite back to the tanks at the average rate of 100 L/hr. An additional pump (model SP800, Aqua tic Eco-systems, Apopka, FL) was added on 30 Aug. to each tank to increase water turnover rate s through the biofilter from 1 to 3 turnovers of tank water per hour.

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88 An ammonia surge test was conducted to de termine ammonia volatilization rates from tanks at pH 6.0, 7.0 and 8.0 prior to inoculation of bacteria and introducti on of fish. Ammonium chloride (2.5 g/tank) was added on 12 July to create a TAN concentration of 6 mg/L. TAN measurements were taken on 13 July (all tanks reading 6 mg/L TAN) and 20 July. Ammonia volatilization loss in mg/L/d was determined by subtracting beginning and ending TAN and dividing by 7. A complete hydroponic nutrient solution consisting of 200 mg/L NFT Vegetable Formula (hydroponic fertilizer blend, Grower’s Supply Ce nter, Lynn Haven, FL), was added to each tank on 21 July. The NFT vegetable formula cons isted of 6 mg/L nitrate nitrogen, 13 mg/L phosphorus, 45 mg/L potassium, 11 mg/L magnesi um, 0.9 mg/L iron, 0.07 mg/L zinc, 0.1 mg/L manganese, 0.02 mg/L copper, 0.3 mg/L boron, a nd 0.02 mg/L molybdenum, using potassium sulfate, monopotassium phosphate, magnesium su lfate, potassium nitrate, iron EDTA, zinc EDTA, manganese EDTA, copper EDTA, sodium borate and sodium molybdate as nutrient sources. In addition to the Vegeta ble Formula, other nutrients [Ca(NO3)2, KNO3, and MgSO4] were added during the season base d on water analysis and visual observations, to maintain plant nutrition and similar soluble salt concentrations among treatments. Plant nutritional status was monitored by measuring leaf petiole sap NO3 -N and K levels beginning on 26 Aug. and every two weeks thereafter. One leaf petiole per plot from the most recently fully expanded leaf (from the 6th to 8th leaf below the growing point) wa s measured. Leaf petiole sap NO3 -N and K measurements were made using ion specific el ectrode meters (Cardy Spectrum Technologies, Inc., Plainfield, IL ). In addition, NO3 -N, NO2 -N, NH4 + -N, and K+ levels in recirculating tank water were measured weekly. Tank water tota l ammonia nitrogen (low range 1.0 to 8.0 mg/L), nitrite nitrogen (low range, 0.1 to 0.8 mg/L) and alkalinity were meas ured with test kits (LaMotte

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89 Company, Chestertown, MD). TAN (high range 1.0 to 50.0 mg/L), nitrite nitrogen (high range, 0 to 150 mg/L) were measured using an ion specific meter (Hanna Instruments USA, Woonsocket, RI). Nitrate nitrogen was measured using an ion specific electrode (Cardy meter, range 0 to 9,900 mg/L, Spectrum Tec hnologies, Inc., Plainfield, IL). Ammonium chloride (2 g/tank, 23 July a nd 0.5 g/tank, 30 July) was added to provide adequate ammonia for the nitrification reaction (Eq. 6-1). Nitrifying bacteria were added to the biofilters (except the hydroponic control) on 26 July (170 ml, product no. 239211, Proline Freshwater Nitrifying Bacteria). The b acteria solution cont ained a mix of 50% Nitrosomonas sp . and 50% Nitrobacter sp . with a count of 6.7 x 104 cells per ml according the supplier (Aquatic Eco-systems, Apopka, FL). Another 50 ml per ta nk of Proline Freshwater Nitrifying Bacteria was added on 14, 15, and 16 Aug. to control an ammonia spike after introduction of fish. Fish (Nile tilapia, Oreochromis niloticus from Harbor Branch Oceanographic Institution, Fort Pierce, FL) were stocked into treatment tanks (except hydroponic co ntrol) on 1-2 Aug. at similar density of 15 fish averagi ng 32.4 g/fish per tank). Care was taken to insure that an equal proportion of large and small fish were in each tank. The fish population size was variable ranging from a low of 8 g to a high of 122 g at in itial stocking. At the end of the trial it was noticed that several fish had sp awned with live fry in their m ouths indicating a mixed population of male and female fish. Based on previous greenhouse trials (Hochm uth et al., 1996) ‘Fitness’ (Asgrow Seed Company, St. Louis, MO) European cucumber seeds were planted into the pe rlite of the biofilter of each plot except the aquacultu re control treatment on 4 Aug, four seeds on each side of the distribution plate. Plant germination was comp lete in all plots on 7 Aug. (= 0 DAG). Starter fertilizer (400 ml of 2g/L veg mix) was a dded to each tank biofilter around the young seedlings

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90 to stimulate growth on 2 DAG. Plants were thinned to 4 plants per biofilter on 7 DAG. Cucumbers vines were trellised to overhead wires and pruned to a si ngle leader stem. Fruit were harvested 36 DAG and each week thereafter for f our weeks. An ammonia surge test was conducted after the last cucumber harvest to determine ammonia biofiltration rates from treatment ta nks. Ammonium chloride (3 g/tank for all treatments except hydroponic contro l which received 1g/tank due to presence of residual TAN from applied fertilizer) was added at 11:30 am on 10 Oct. TAN measurements were taken at noon on 10 Oct. and every 6 hrs. thereafter for 24 hrs. Ammonia biofiltr ation rates in mg/L/d were determined by subtracting beginning and ending TAN except for aquaponic treatment pH 8.0 which was determined by subtracting beginning TAN from the first 6 hour TAN measurement and multiplying by 4. End of season MPN estimates of the Nitrosomonas sp . bacterial cell populations in the biofilters were made using the three tube seri al dilution method (Feng, 2001). Core samples of 200 ml perlite were taken from the middle of each biofilter on 12 Oct. Samples were mixed into a composite for each treatment and refrigerated at 5C. Nitrifier culture medium for Nitrosomonas sp . was prepared and autoclaved as descri bed (ATCC, 2006) on 17 Oct. Ninety grow-out and ten inoculation test tubes were prepared for each of the 5 treatments. The ATCC liquid medium in the test tubes measured 0 mg/L for NO2 -N and 5 mg/L for NO3 -N prior to inoculation with nitrifying bacteria core samples. Grow-out test t ubes were replicated three times with 30 test tubes per replicate per treatment. On 21 Oct., 200 ml sub-samples of perlite from each treatment were mixed with 200 ml D.I. water and blended in a Hamilton Beech blender for 30 s. One ml of this blended solution was used to inoculate MPN test tubes. Tubes were shaken 8 hrs. per day for 30 days and then measured for nitrifier activity. A measurement 0.1 mg/L

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91 NO2 -N or 10 mg/L NO3 -N was considered positive for Nitrosomonas bacteria activity (Eq. 6-1 and Eq. 6-2). NO2 -N (low range, 0.1 to 0.8 mg/L) was m easured with a test kit (LaMotte Company, Chestertown, MD) and NO3 -N was measured using an ion specific electrode (Cardy meter, range 0 to 9,900 mg/L). One measurem ent for each replicate (3 measurements per treatment) were recorded based on statistical tables (Feng, 2001) and used to determine data significance by ANOVA (SAS, 2001) and Duncan’s Mu ltiple Range Test at the 0.05 level. Water pH was measured 2 to 6 times per w eek (avg. of 4 measurements) using pH meter model WD-35624-86 (Oakton Instruments, Vernon H ills, IL). Water dissolved oxygen, specific conductivity (EC), temperature, and salinity were measured on ce every two weeks using a YSI Model 85 meter (YSI Inc., Yellow Springs, OH). The experimental design was a randomized comp lete block design with 4 replications. Data were analyzed using ANOVA (SAS, 2001) a nd Duncan’s Multiple Range Test at the 0.05 level. Significant linear and quadratic trends over the non-repeating e qual interval pH units (excluding the hydroponic and aquaculture control treatments) were analyzed using the method of orthogonal polynomials (Gomez and Gomez, 1984). Results and Discussion Average high and low greenhouse air te mperatures were 35.3 and 24.4, 38.1 and 24.1, 31.7 and 23.5, and 29.4 and 19.4C for the months of July, August, September, and October, respectively. Average tank water temp eratures were 27.0, 29.3, 25.9, and 23.3C and oxygen levels were 5.5, 4.6, 3.9, and 4.4 mg/L for the same four months. Average season tank water TAN was 2.1, 0.7, 0.3, 3.0, and 0.7 and NO2 --N was 0.2, 1.8, 1.4, 0, and 2.1, respectively, for the five treatments as listed above. Season pH values ranged from 5.5 to 6.7, 6.5 to 7.5, 7.1 to 8.3, 5.5 to 6.8, and 6.5 to 7.4, respectively, for treatmen ts 1) aquaponic pH 6.0, 2) aquaponic pH 7.0, 3) aquaponic pH 8.0, 4) hydroponic control pH 6.0, and 5) aquaculture control pH 7.0.

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92 Nitrification is an acid producing pro cess (ammonia oxidation produces protons H+ in Eq.6-1) requiring adjustment of recirculating water to mainta in target pH levels. Actual pH values were close to the target pH ranges for the treatments. Visual nutrient deficiency symptoms (mottle d yellow middle to lower leaves) were most notable early in the growing season on pH 8.0 plan ts. The plants grown at pH 7.0 showed light mottled yellow leaf symptoms. These symptoms were significantly reduced at pH 8.0 and disappeared at pH 7.0 when ferti lizer applications were increase d prior to fruit set. Early marketable cucumber fruit yield (weight and numb er) decreased linearly as pH increased from 6.0 to 8.0 (Table 6-1). Total marketable and cull cucumber fruit yields were not significantly different among treatments. Results indicate an early yield advantag e to keeping nutrient solution pH between 6.0 and 7.0, but not an advantage for total yield. This would indicate that keeping the recirculating pH at 7.5.0 to accommoda te nitrifying bacteria activity would be detrimental from an economic standpoint only if the grower were producing for an early market window but would not adversely affect the overall pr oduction for the year during normal multiple cropped middle and late season peri ods. Early market windows can occur when seasonal crop production shifts fr om one region to another and are important in order to target temporary spikes in price from supply interrupt ion or to extend seasonal product availability from the incoming startup production region. Nitrifying activity is commonly determ ined by measuring ammonia oxidation, intermediate and end product production such as nitrite or nitrate accumulation, and/or oxygen uptake (Hagopian and Riley, 1998; Prosser, 1986). Ammonia biofiltration rate of the perlite trickling biofilters in aquaponic production was determined by measuring ammonia loss during a 24–hour period after introduction of ammonia to the system water (Table 6-2). This ammonia

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93 surge test was conducted after the last cucumber fruit harvest. Ammonia loss from the system increased linearly as the pH in creased from 6.0 to 8.0. The linear increase in ammonia loss could be the result of an increased unionized ammonia (NH3 Eq. 6-1) concentration in the system water due to increasing pH (Francis-Floyd and Watson, 1996). Increased biofilter activity occurs with an increase in substrate (NH3) concentration (Hagopian and R iley, 1998). Linear trends in nitrite buildup (6 h and 12 h samples, Table 6-2) , confirm that ammonia oxidation increased at the 0.05 level as pH increased from 6.0 to 8.0. Nitr ite accumulation in a st eady state biofilter is low compared to a biofilter in startup cycle due to mature populations of nitr ifiers able to process nitrite even though inhibition of Nitrobacter sp. (Eq. 6-2) at high pH slows the conversion of NO2 -N to NO3 -N somewhat (Prosser, 1986). For the system as designed (20 L of tric kling perlite biofilter medium for 100 L of recirculating water – 1:5 ratio), ammonia lo ss from system water was 3.8, 6.1, 16, 1.3, and 5.9 mg/L/day (Table 6-3), for treatments pH 6.0-aqpon, 7.0-aqpon, and 8.0-aqpon, 6.0-hc, and 7.0ac, respectively. During a similar ammonia surge test at the beginning of the experiment after system setup but before inocula tion with bacteria, the maximum volatilization loss of ammonia from these tanks was 0.39, 0.53, and 0.63 mg/L/d fo r pH 6.0, 7.0, and 8.0, respectively. Thus the difference between these numbers is the mini mum ammonia biofiltration occurring by the process of nitrificati on (7.0-ac, aquaculture control), pl ant uptake of ammonia (6.0-hc, hydroponic control) and nitrifi cation and plant uptake (6.0 -aqpon, 7.0-aqpon, and 8.0-aqpon). Since average greenhouse temperatures were 6C cooler during the second ammonia surge test and volatilization decrea ses with decreasing temperatures it is likely that the actual ammonia biofiltration is very near the measured amm onia loss in Table 6-2 for each treatment. Considering TAN loss volumetrically, as a functi on of biofilter volume, 16 mg/L ammonia loss

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94 from 100 L of tank water per day using a 20 L perl ite trickling biofilter would be equivalent to 80g TAN removal /m3 of biofilter/d, which is near th e amount recommended for trickling biofilters in aquaculture production–90g TAN/m3/d (Losordo, et al., 1999). For all treatments, the volumetric (TAN removal per biofilter media volume) results were 19, 31, 80, 6, and 29g TAN /m3/d, respectively, for the above treatm ents (Figure 6-3). Comparing 6 g/m3/d (TAN removal with cucumber plants only), and 19 g/m3/d (with plants and nitr ifying bacteria) at pH 6.0, indicates that nitrifiers were 3.2 times more efficient as plants in removing ammonia from aquaponic system water under the conditions of this experiment. More work should be done to test the nitrification/plant biofiltration relati onship during very active st ages of plant growth, since the current test was done after the last ha rvest when plants were mature but not actively growing. Recirculating aquaculture systems are us ually intensively managed with maximum carrying capacities of 60 g fish per liter of water for systems with oxygen injection and 30 g/L for natural air aerated systems (Masser et al., 199 9; Megan Davis, personal communication). If tilapia grown at maximum carrying capacity were fed at 1.5% of body wei ght per day with 30% protein in the feed (10% of pr otein in feed becomes the ammo nia generation – Timmons et al., 2002) then ammonia generation from feed would be producing 13.5 and 27 mg/L/day of ammonia nitrogen for the naturally aerated and oxygen injected systems, respectively. The ammonia removal rate of the designed biofilter in this experiment (perlite volume to tank water volume) of 1/5 was sufficient to oxidize ammoni a from a naturally aerated system at maximum carrying capacity with production water at pH 8.0, but system biofilter volume relative to tank water volume would have to increase to matc h the ammonia generation rate based on the stocking/feeding scenarios for wate r held at pH 7.0 or 6.0. Thus biofilter volumes need to be

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95 adjusted to compensate for differences in am monia removal rates caused by production pH. Recirculating systems with high densities of fish may need to be managed with water quality parameters closer to those favoring nitrificati on (7.5.0) in order to e fficiently convert waste ammonia. Results of Most Probable Number (MPN) bacterial cell counts from biofilter core sampling indicate that the aquaculture control (pH 7.0aqpon) with no plants in the biofilter had a significantly higher (0.01% level) number of Nitrosomonas sp . bacteria compared to treatments containing plants in the biofilter, which were not significantly different among themselves (Figure 6-1). In other work, numbers of nitr ifying bacteria were reduced 200-fold in the presence of plants than without them as r oots were more competitive for ammonium than Nitrosomonas europaea (Verhagen et al., 1994). However, in the current trial, aquaponic (plants, fish and bacteria) pH 8.0 had the best a mmonia biofiltration rate of all treatments (Table 6-2). MPN bacteria counts (Figure 6-1) were not a good indicator of biofilter performance (Figure 6-2). This indicates that pH of system water is a more important factor in determining biofilter activity than bacterial population and is most likel y due to pH induced increases in unionized ammonia available for th e nitrification reaction (Eq. 1) as the pH increases. The hydroponic control was not inoculated with nitrifying bacteria but the MPN test indicated a low level of bacteria cells /ml of perlite. Therefore, some unintentional or low level natural inoculation occurred duri ng the growing season. Two hundred g of fish feed (41% protein wi th 10% of the protein becoming the ammonia generation amount (Timmons et al., 2002) were used during th e trial resulting in 8.2 g of ammonia nitrogen being released into the system water from the fish feed (Table 6-4). Fertilizer was added to tank water during the season to mainta in similar overall soluble salt levels in all

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96 treatments. Since there was no plant uptake, the aquaculture control received much less NO3 -N. Makeup water from the source well had a NO3 -N concentration of 4 mg /L. Perlite contains 0.06% N and a dry weight of 117 g/L. Since this fraction of the filter could not be separated from the ending filter measurements containing r oots and bacteria it wa s included in the input section. The burlap used to line the biofilter contai ners was made of jute and hemp plants which together had an N content of 1.6%. The burlap was not recovered separately from the biofilter because of disintegration by the e nd of the season. The estimate of ammonia and nitrate nitrogen mass balance in the aquaponic treatments (6-,7,8-aqpon) indicate an average of 51 g total nitrogen input to the system w ith 16 g being withdrawn for the plant stem biomass and 16 grams for the fruit biomass. Of this nitrogen input total, 38 g consisted of fertilizer nitrogen and 8.2 g from fish feed. The ammonia and nitrite spike in system water which occurred one week after stocking the tanks with fish resulted in large wa ter changes which would normally not have to be made under steady state conditions. Thus the losses of nitrogen from the system observed in the discharge water (Table 6-4) is mo st likely higher than would be e xpected. Slightly acidic soils (pH 5.5.5) generally favors root growth (Taiz and Zeiger, 2002). The roots growing out of the bottom of the biofilters were recovered. Dr y weight and subsequent nitrogen recovered decreased as pH increased. The difference between the input and output nitrogen which averaged 3 g per treatment could be attributed to ammonia volat ilization from the system water which was not recoverable. Although no differences in tilapia growth were expected among treatments due to their adaptation to wide ranges of water quality conditions (Chapm an, 2000; Lim and Webster, 2006; Watanabe et al., 1997) there were significant differences in in itial feeding activity and fish mortality by treatment (Table 6-5). Initial feed ing activity (average data from 5 days), or the

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97 response of fish when feed is first thrown in to the re-circulating ta nks, increased and fish mortality decreased as pH incr eased from 6.0 to 8.0. The vigor of fish feeding activity is a reflection of the general health and stress level of the fish (L im and Webster, 2006) and results imply that fish were healthier, under less environm ental stress, and more likely to survive as pH increased from 6.0 to 8.0. There are several possible reasons for this difference among treatments that could have occurred in combination. First, there was an ammonia and nitrite spik e in the system water seven days after stocking which resulted in gill damage. This damage was diagnosed at the Fish Health Lab at the University of Florida’s Fish eries and Aquatic Sciences Department and it was suggested that the fish could recover within three to four weeks provided water ammonia was kept low and adequate aeration maintained. Consequently, water changes were made and aeration added with tank water recirculation rates increased from one to three revolutions through the biofilter per hour. A se cond possible reason to explain th e difference is that the fish population size was variable ranging from a low of 8 g to a high of 122 g at initial stocking. Care was taken at initial stocki ng and during the season so that the same relative proportion of small and large fish were in each tank to en sure biomass consistency among tanks. However, these size differences contribute to a social hierar chy with more aggressive (male or larger fish) affecting the feeding activity of subordinate fi sh (Lim and Webster, 2006) especially at low densities, ie, < 100 fish per m3. At the end of the trial it wa s noticed that several fish had spawned with live fry in their mouths indicati ng a mixed population of male and female fish. Third, the available fish population was limited so that fish were moved between tanks (no more than one pH difference) during the experiment to maintain a similar density among treatment tanks. This handling probably al so increased stress on the fish. Another reference (Chen et al.,

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98 2001) found an interaction between lethal disso lved oxygen levels and pH. Lethal DO for O. mossambica tilapia was 7.14, 4.02, 3.36, 0.84 and 3.20 mg/L at pH 4.0, 5.0, 6.0, 8.3, and 9.6, respectively. Conclusion Biofilter removal of total ammonia nitrogen (T AN) increased linearly in a perlite trickling biofilter/root growth medium and occurred at the rate of 3.8, 6.1, and 16 mg/L/d of system water for aquaponic treatments pH 6.0, 7.0, and 8.0, re spectively. Maximum volumetric ammonia biofiltration rate for the biofilters was 80 g/m3/d for aquaponic production at pH 8.0. MPN analysis of Nitrosomonas sp. bacteria populations indicated a significantly higher population of bacteria in biofilters without plant roots. Howeve r, the highest ammonia biofiltration rate in the trial occurred in aquaponic plots produced at pH 8.0. Thus pH appeared more important than bacteria population in removing ammonia from biof ilters. Fish vigor increased as pH increased from 6.0 to 8.0. Early marketable cucumber fru it yield in re-circulating integrated hydroponic and aquaculture (aquaponic) production decreased linearly as pH increased from 6.0 to 8.0. However, there were no differences in total marketable yield among treatments. Results indicate that given the importance of pH in biof ilter activity and that total cucumber yields are unaffected by pH in the range of 6.0 to 8.0, then aquaponic systems may be maintained at pH levels more optimum for nitrifying bacteria (7.5.0) except during prod uction for early season markets where pH 7.0 would be recommended.

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99 Table 6-1. ‘Fitness’ cucumb er fruit yield response to pH and production system. Target pH & prod. Early marketablez Total marketable Total cull method kg/plant no/plant kg/plant no/plant kg/plant no/plant 6.0-aqpony 1.52 ax 3.3 a 3.64 a 8.3 a 0.44 a 3.3 a 7.0-aqpon 1.32 a 2.9 a 4.12 a 9.7 a 0.33 a 3.0 a 8.0-aqpon 0.67 b 1.8 b 3.54 a 8.8 a 0.33 a 3.0 a Contrastw L** L* 6.0-hc 1.57 a 3.3 a 3.63 a 8.4 a 0.53 a 3.9 a zEarly = first harvest 36 DAG, Total = four harvests – 36, 43, 50, 58 DAG, Marketable = 34-42 cm in length, less than 45 fruit angle, few blemishe s, Cull = greater than 45 fruit angle, poor tip fill, frequent blemishes. Average of 4 plants per plot; 4 reps. y6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fi sh and nitrifying ba cteria, 6.0-hc = pH hydroponic control. xWithin columns, means followed by different letter s are significantly diff erent at the 0.05 level; four replicates. wLinear contrasts were significant at the 5% (*) or 1% (**) level.

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100 Table 6-2. Twenty-four hour total ammonia nitrogen (TAN) and nitrite nitrogen (N02 -N) concentrations in a perlite trickling biofilte r after introduction of ammonium chloride. Target pH & Hours after introduction of ammonia prod. method 0z 6 12 18 24 Total ammonia nitrogen (mg/L) 6.0-aqpon y 6.1 ab x 4.9 a 4.0 a 2.9 b 2.4 b 7.0-aqpon 6.4 a 3.4 b 2.1 b 1.3 c 0.3 c 8.0-aqpon 6.0 ab 2.0 c 0.0 c 0.0 d 0.0 c Contrast w L** L** L** L**Q** 6.0-hc 5.4 b 4.8 ab 4.5 a 4.4 a 4.4 a 7.0-ac 6.5 a 4.5 ab 2.1 b 1.4 c 0.6 c Nitrite nitrogen (mg/L) 6.0-aqpon 0.0 a 0.0 b 0.0 c 0.0 a 0.0 a 7.0-aqpon 0.0 a 0.3 b 0.3 cb 0.1 a 0.1 a 8.0-aqpon 0.0 a 1.4 a 1.1 a 0.2 a 0.0 a Contrast L** L** 6.0-hc 0.0 a 0.0 b 0.0 c 0.0 a 0.0 a 7.0-ac 0.0 a 0.3 b 0.4 b 0.0 a 0.1 a zAmmonium chloride introdu ced into the biofilters. y6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fi sh and nitrifying ba cteria, 6.0-hc = pH hydroponic control with plants , 7.0-ac = pH aquaculture control with fish and nitrifying bacteria. xWithin columns, means followed by different letter s are significantly diff erent at the 0.05 level; four replicates. wLinear and quadratic effect s were significant at the 5% (*) or 1% (**) level.

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101 Table 6-3. Twenty-four hour TAN loss from recircul ating system tank water and perlite trickling biofilter after introduction of ammonium chloride. Target pH & Prod. Method TAN lossz TAN lossy mg/L/d g/m3/d 6-aqponx 3.8 cw 19 c 7-aqpon 6.1 b 31 b 8-aqpon 16.0 a 80 a Contrastv L**Q** L**Q** 6-hc 1.3 d 6 d 7-ac 5.9 b 29 b zLoss of TAN from recirculating tank water. yLoss of TAN converted to biofilter volume. x6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fi sh and nitrifying ba cteria, 6.0-hc = pH hydroponic control with plants , 7.0-ac = pH aquaculture control with fish and nitrifying bacteria. wWithin columns, means followed by different letters are significantly different at th e 0.05 level; four replicates. vLinear effects were signif icant at 1% (**) level.

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102 0200040006000800010000 6-aqpon 7-aqpon 8-aqpon 6-hc 7-acpH & Production MethodMPN x 1000 Note: 6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fish and n itrifying bacteria, 6.0-hc = pH hydroponic control with plants , 7.0-ac = pH aquaculture control with fish and nitrifying bacteria. Error bars represent SE (n=3). Figure 6-1. Most probable number (MPN) of Nitrosomonas sp . bacteria in perlite trickling biofilters as influenced by pH and production method. 020406080100 6-aqpon 7-aqpon 8-aqpon 6-hc 7-acpH & Production Methodg/m3/d Note: 6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fish and n itrifying bacteria, 6.0-hc = pH hydroponic control with plants , 7.0-ac = pH aquaculture control with fish and nitrifying bacteria. Error bars represent SE (n=4). Figure 6-2. Perlite trickling biofilter 24-hour TAN loss as influenced by pH and production method.

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103 Table 6-4. Estimate of ammonia and nitrate nitrogen mass balance in an aquaponic system with a raised bed perlite media trickling biofilter. Nitrogen pH and production method z Source 6-aqpon 7-aqpon 8-aqpon 6-hc 7-ac Nitrogen input – g/tank Fish: Stocking 12.3 12.0 13.0 12.5 Feed NH4 + -N 8.2 8.2 8.2 8.2 Fertilizer: NO3 -N 35.6 35.6 35.6 34.9 7.2 NH4 + -N 2.2 2.2 2.3 3.0 2.2 Makeup water: y NO3 -N 2.9 3.0 2.9 2.1 2.2 Biofilter: Perlite 1.4 1.4 1.4 1.4 1.4 Burlap 0.5 0.5 0.5 0.5 0.5 Total nitrogen 63.1 62.9 63.9 41.9 34.2 Nitrogen output – g/tank Plant: Shoot 16.7 15.7 15.7 15.7 Root 0.12 0.02 0.01 0.30 Fruit 15.1 16.6 16.8 15.0 Fish: Net loss 8.4 6.2 4.4 4.6 Harvest 5.7 6.2 6.1 5.5 Filters: Perlite 4.4 5.4 4.3 2.9 4.4 Solids 0.24 0.17 0.24 0.01 0.11 Discharge water: NO3 -N 8.8 9.0 12.4 4.1 15.0 NO2 -N 0.1 1.0 0.8 0.0 1.1 NH4 + -N 1.8 0.04 0.0 1.3 0.04 Total nitrogen 61.4 60.3 60.7 39.3 30.7 z6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plan ts, fish and nitrifying bacteria, 6.0-hc = pH hydroponic control with plants , 7.0-ac = pH aqua culture control with fish and nitrifying bacteria. yWell water contained 4 mg/L NO3 -N.

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104 Table 6-5. Tilapia initial feeding activity and overall mortality as influenced by system water pH and production method. Target pH & Initial feedingz Fishy prod. Method activity mortality 6.0-aqponx 1.6 bw 18.5 a 7.0-aqpon 2.3 ab 8.0 b 8.0-aqpon 4.0 a 3.4 b Contrastv L** L** 7.0-ac 3.0 ab 8.8 b zInitial feeding activity rating: 1 = don’t come to feed, 3 = half strike feed, and 5 = all strike feed. yAvg. no. per tank dead during growing season. x6.0-aqpon, 7.0-aqpon, 8.0-aqon = pH with plants, fish and nitrifying bacteria, 6.0-hc = pH hydroponic cont rol with plants, 7.0-ac = pH aquaculture control with fish and nitrifying bacteria. wWithin columns, means followed by di fferent letters are significantly different at the 0.05 le vel; four replicates. vLinear effects were signif icant at 1% (**) level.

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105 CHAPTER 7 CONCLUSIONS Combining hydroponic plant and aquaculture fish production systems (aquaponics) requires reconciling water quality parameters for the survival and growth of plants, fish, and nitrifying bacteria. Aquaponics ha s the potential to be a sustaina ble minimum discharge or zero agricultural discharg e system since the waste by-products of aquaculture can be used by plants in hydroponic systems. However, with all its promis e as a sustainable alternative to conventional food production, many unanswered questions must be resolved regarding optimum water quality parameters when the organisms present in aquaponics are grown together. Aquaculture production water pH is recommen ded to be between 6.5 and 8.5. However, there is a dichotomy between th e optimum pH for plan t nutrient availability in hydroponics (pH 5.5.5; Hochmuth, 2001a) and pH maintained at le vels more optimum for nitrifying bacteria activity (7.5.0; Hochheimer and Wheaton, 1998). There is no information in the literature on how aquaponic system water quality im pacts nitrification when perlite growth medium is used as the biofilter medium, little information on the plan t/nitrification interactions in root growth media biofilters, and how this interaction affects ammonia biofiltration and plant yield. In addition, systems management would be improved with the addition of fe rtilizer nutrients to aquaponic system water to optimize plant nutrient levels but science based information is needed before recommendations can be made. The re conciling pH in aquaponic systems for plant production and nitrification will be affected by their relative impor tance as biological filters and the pH effects on plant yields and nitrification. In order to improve the integration of sustainable aquaponic systems, a series of trials were conducted to 1) determine the optimum pH for nitrification and evaluate perlite as an aquaponic biofilter/root growth medium 2) determine the affect of hydroponic nutrients on ni trification, 3) make predictions about the relative contribution

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106 of plants and nitrifiers to the biofiltration of ammonia, and 4) establish a reconciling pH for ammonia biofiltration and cucumber yiel d in an aquaponic production system. Nitrification activity in a perlite medium tric kling biofilter, as evidenced by ammonia loss and nitrite accumulation, increased linearly as pH increased from 6.5 to 8.5. Biofilter removal of TAN was 19, 31, and 80 g/m3/d for aquaponic perlite biofilters operating at pH 6.0, 7.0, and 8.0, respectively, at average tank wa ter temperatures of 22.2C. Optimum performance for tricking biofilters in aquaculture have been reported as 90 g/m3/d. Thus perlite, in addition to being a common medium for plant growth, provides adeq uate TAN removal rates when used as a biofilter/root zone media in aquaponic production at pH 8.0. No difference in nitrification rate was found when recirculating system water contained no nutrient solution versus a complete hydroponic nutrient solution at nitrate nitrogen concentrations of 100 or 200 mg/L. Thus fertil izer nutrients, at leve ls commonly used in hydroponics, may be added to aqua ponic systems if needed to provide optimum plant nutrition with no significant adverse impact on nitrifying b acteria. The concentration of certain elements (Ca, Fe, and Mn) in the nutrien t solution declined as pH incr eased from 5.0 to 8.0. Nutrient depletion of the root zone can occur with low nutrient solution levels and intermittent irrigation applications. However, since the nutrient solu tion was continuously recirculating, cucumber shoot uptake was within or n ear the sufficiency range. Ammo nia biofiltration was 3.7 times higher for aquaponic treatments (plants, fish a nd nitrifying bacteria) at pH 6.0 compared to a hydroponic control (plants only) at pH 6.0 indicating that nitrif ication activity contributes significantly more to TAN loss from system water than plant removal of ammonia. The presence of roots in the perlite biof ilter reduces the most probable number (MPN) cell count of Nitrosomonas sp. bacteria compared to biofilters wit hout plants. However, pH was a more

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107 significant factor affecting amm onia biofiltration than bacteria population with removal rates maximized at aquaponic treatmen t pH 8.0. Even though early marketable cucumber fruit yield decreased linearly as pH increased from 5.0 to 8.0 and 6.0 to 8.0, total marketable yield was unaffected. For the recirculating aquaponic system under study—perlite trickli ng biofilter / root growth medium with cucumber and tilapia—the greatest ammonia biofiltr ation (nitrification and plant removal of TAN) was 80 g/m3/d and occurred at pH 8.0. This was four times the amount of TAN removal at pH 6.0. Keeping pH at more optimum levels for nitr ifying bacteria activity will allow increased fish stocking density. This will provide additional plant nutrients from the waste stream and reduced need for fertilizer supp lementation, thus increasing systems integration and sustainability. In add ition, systems management may be improved by the addition of fertilizer to system water when needed without harm to nitrifying bacteria. The reconciling pH for ammonia biofiltration and cucumber yield in this recirculating aquaponic system should be pH 7.5.0 given the importance of pH to the ammonia biofiltra tion rate and given that no difference in total cucumber fruit yield among treatments was found.

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108 REFERENCES Adler, P. 2001. Overview of economic ev aluation of phosphorus removal by plants. Aquaponics J., 5:15. Adler, P.R., J.K. Harper, F. Takeda, E.D. Wade and S.T. Summerfelt. 2000. Economic evaluation of hydroponics and other treatm ent options for phosphorus removal in aquaculture effluent. Ho rtScience, 35(6):993. Adler, P.R., F. Takeda, D.M. Glenn and S.T. Summerfelt. 1996. Utilizing byproducts to enhance aquaculture sustainabili ty. World Aquaculture, 27(2):24. Alleman, J.E. 1985. Elevated nitrate occurr ence in biological waste-water treatment systems. Water Sci. Technol . 17:407. Anonymous. 2007. Basic facts about perlite. The Perlite Institute, Inc., Harrisburg, PA. Retrieved April 1, 2007, from http://www.perlite.org/ Anonymous. 1998. Linking hydroponics to an 8 80 gallon recycle fish rearing system. The Conservation Fund’s Freshwater Institute, Shepherdstown, WV, 17 pgs. Anonymous. 1997. The Freshwater Institute na tural gas powered aquaponic system design manual. The Conservation Fund Freshwat er Institute, Shepherdstown, WV, 37 pgs. Antoniou, P., J. Hamilton, B. Koopman, R. Ja in, B. Holloway, G. Lyberatos and S.A. Svoronos. 1990. Effect of temperature and ph on the effective maximum specific growth rate of nitrifying bacteria . Water Research, 24(1):97. ATCC. 2006. ATCC 221 Nitrosomonas, Ameri can Type Culture Collection, Manassas, VA. Retrieved October 2, 2006 from http://www.bibliographics .com/LAB/PCR/NITRIFIER%20CULTURE%20MEDIA.htm Bernardo, L.M., R.B. Clark and J.W. Maranvill e. 1984. Nitrate/ammonium ratio effects on nutrient solution pH, dry matter yield, and nitr ogen uptake of sorghum. J. Plant Nut. 7(10):1389. Brentlinger, D. 1999. Status of the commercial hydroponic in dustry in the United States. Proc. Intl. Symp. Gr owing Media Hydroponics . Acta Hort. 481:731. Bronson, C.H. 2005. Florida Agricultura l Statistical Directory 2005. Florida Department of Agriculture and Consumer Services, Florida Agriculture Statistics Services, Tallahassee, FL. Brunty, J.L. 1995. Biological filtration fo r ornamental fish production and factors affecting total ammonia nitrogen and nitrite removal rates. University of Florida, Dept. of Agriculture & Biological Engineering, M.S. Thesis:143 pgs.

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118 Wheaton, F.W. 1993. Aquacultural engine ering. Krieger Publishing Company, Malabar, FL: 708 pgs. Wright, P.C., and J.A. Raper. 1998. Investig ation into the viabil ity of a liquid-film three-phase spouted bed biofilter. J. Chemical Technology and Biotechnology, 73 (3):281-291. Zaiter, H.Z. and I. Saad. 1993. Yield of iron-sprayed and non-sprayed strawberry cultivars grown on high pH calcareous soil. J. Plant Nut. 16:281-296.

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119 BIOGRAPHICAL SKETCH Richard V. Tyson was born on November 30, 1950 in West Palm Beach, Florida. The youngest of three children, he grew up in Pa hokee, Florida, graduating from Pahokee High School in 1968. Richard attended Florida Stat e University during th e 1968 school year and played offensive guard on the freshman foot ball team. During 1969, he attended Palm Beach Community College and then served in the United States Army Medical Corp from 1970 as a Clinical Specialist. During the next several years, Richard worked with a landscape company, was co-owner of a retail plant store, and worked for the United States Sugar Corporatio n as a clarifier operator and juice chemist. He earned his Associate of Arts degree from Palm Beach Community College in 1977 and entered the University of Florida Ho rticultural Sciences Department in 1978. He received his Bachelor of Scien ce degree in 1980, majoring in plant science, and a Certificate in Tropical Agriculture the same year. He entere d graduate school in the same department and received a Master of Science in 198 3 with a vegetable crop emphasis. Richard began serving as a commercial ve getable Extension Agent in Dade County, Florida in the fall of 1982 where he served for fi ve years. He then took a position with Collier Farms, Immokalee, FL, a subsidiary of Collier Enterprises, as tomato division manager from 1987 to 1994. During that time, he managed the production and harvesting of an average of 1,000 acres of tomatoes each year with double cr opped production of watermelons, sweet corn, cucumbers, yellow squash, and zucchini. Richard became a science instructor at Im mokalee High School in 1994. He taught classes in marine biology, honors physics, and physical scie nces. In 1995, he moved to central Florida to take the position of multicounty commercial vegetable Extension Agent in five Florida counties: Seminole, Volusia, Orange, Lake, a nd Sumter. He was housed with the Seminole

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120 County Extension Service in Sanford, FL. In 1999, the position changed to include 50% commercial turfgrass in three c ounties and landscape maintenance responsibility in one county, in addition to the commercial vegetable duties. Richard received several awards as an Extension Agent includ ing the Sadler Distinguished Extension Professional and Enhancement Aw ard and the Marshall and Mildred Watkins Professional Improvement Award. He was also a national winner in the National Association of County Agricultural Agents, Search for Excellence in Crop Production program. On a personal level, Richard was married to Gladys Moreno in June of 1981. Their daughter Natalie Lissette was born in October of 1984 and their son Alexander James was born in August of 1987. He has served in various leadership roles in the United Methodist Church in the communities he has lived including chairman of the Leadership Council of the First United Methodist Church in Sanford for three year s. Richard and Gladys celebrated their 25th wedding anniversary in 2006. Richard entered the Universi ty of Florida’s Employee Education Program in 2002 to pursue the PhD degree. He passed the qualifying exam and was admitted to candidacy in the spring of 2005, with a successful defense of the dissertation in March of 2007. He received his PhD degree in Horticultural Science in May of 2007.