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Potential Bioremediation System for Nitrate Removal from Plant Nursery Runoff Water

Permanent Link: http://ufdc.ufl.edu/UFE0021368/00001

Material Information

Title: Potential Bioremediation System for Nitrate Removal from Plant Nursery Runoff Water
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Mozdzen, Miguel Angel
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: denitrification, fertigation, fertilizers, leaching, nitrate, nitrogen, nursery, nutrients, reduction, runoff
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: High levels of nitrate (NO3) in ornamental plant nursery runoff water can contribute to euthrophication of water bodies, and to human health issues such as baby blue syndrome. The objectives of these studies were to characterize the range of nitrate concentrations and loadings in surface runoff water from two typical ornamental plant nurseries, and to evaluate the potential use of a common aquaculture biofiltration media for removing nitrate from nursery generated runoff water. A foliage plant nursery and a bedding plant nursery were used to evaluate NO3-N losses. Losses of NO3-N at the foliage nursery during a typical fertigation cycle ranged from 0.8 to 1.2 kg per acre, with concentrations ranging from 70 to 274 mg/L. Losses at the bedding plant nursery ranged from 186 to 405 g per acre with concentrations ranging from 0.7 to 26.3 mg/L. The bioremediation experiments indicated that pulsing NO3-N considerably increased the lag phase for induction of optimal NO3-N removal when dissolved organic carbon (DOC) was not limited. Results also indicated that pulsing DOC once optimal nitrate removal was achieved reduced NO3-N removal rates significantly. Using sucrose as the source of carbon, optimal nitrate removal rates of 5.97 mg and 3.06 mg of NO3-N/hour were achieved after 7 and 16 days in separate studies. Performing total water exchanges did not appear to affect the rate of NO3-N removal once the optimal rate was achieved.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Miguel Angel Mozdzen.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Wilson, Patrick C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021368:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021368/00001

Material Information

Title: Potential Bioremediation System for Nitrate Removal from Plant Nursery Runoff Water
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Mozdzen, Miguel Angel
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: denitrification, fertigation, fertilizers, leaching, nitrate, nitrogen, nursery, nutrients, reduction, runoff
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: High levels of nitrate (NO3) in ornamental plant nursery runoff water can contribute to euthrophication of water bodies, and to human health issues such as baby blue syndrome. The objectives of these studies were to characterize the range of nitrate concentrations and loadings in surface runoff water from two typical ornamental plant nurseries, and to evaluate the potential use of a common aquaculture biofiltration media for removing nitrate from nursery generated runoff water. A foliage plant nursery and a bedding plant nursery were used to evaluate NO3-N losses. Losses of NO3-N at the foliage nursery during a typical fertigation cycle ranged from 0.8 to 1.2 kg per acre, with concentrations ranging from 70 to 274 mg/L. Losses at the bedding plant nursery ranged from 186 to 405 g per acre with concentrations ranging from 0.7 to 26.3 mg/L. The bioremediation experiments indicated that pulsing NO3-N considerably increased the lag phase for induction of optimal NO3-N removal when dissolved organic carbon (DOC) was not limited. Results also indicated that pulsing DOC once optimal nitrate removal was achieved reduced NO3-N removal rates significantly. Using sucrose as the source of carbon, optimal nitrate removal rates of 5.97 mg and 3.06 mg of NO3-N/hour were achieved after 7 and 16 days in separate studies. Performing total water exchanges did not appear to affect the rate of NO3-N removal once the optimal rate was achieved.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Miguel Angel Mozdzen.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Wilson, Patrick C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021368:00001


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de95b51f035f875e401c100c4fde2e14e76cd8ff







POTENTIAL BIOREMEDIATION SYSTEM FOR NITRATE REMOVAL FROM PLANT
NURSERY RUNOFF WATER




















By

MIGUEL ANGEL MOZDZEN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007
































O 2007 Miguel Angel Mozdzen


































To my deceased father Walter Joseph Mozdzen, dad, mentor and friend; and to my daughter and
wife who were my principal driving force throughout.









ACKNOWLEDGMENTS

This research was supported by USDA/ARS proj ects 6618-13000-002-00D and 6618-

13000-002-04S. Proj ect -04S is supported by the USDA-ARS Floriculture and Nursery

Research Initiative. I would like to thank the Floriculture and Nursery Research Initiative

(FNRI), USDA-ARS, and the Horticulture Research Institute (HRI) for funding this research. I

would also like to thank the members of my graduate committee: Patrick Wilson, Thomas

Yeager, Joseph Albano, and Andrew Ogram for allowing me to work on this fascinating proj ect

and for their support, patience, and guidance during my graduate program. I would like to

specially thank my chair: Patrick Wilson, for always keeping his door open for me to discuss and

correct any aspects of my thesis proj ect, and I truly appreciate all of the time he dedicated to me.

He always pushed me to excel, and encouraged me during the rough times. I would also like to

thank Patrick Wilson' s and Joseph Albano' s laboratory staff for their assistance and support.

I would like to give special thanks to my wife Sonia who supported me, especially when I

was up late at night, and gone during weekends. Her love, patience, and support allowed me to

succeed in my masters program. Also, I would like to dedicate this proj ect to my parents who

always encouraged, and supported me during my scholarly pursuits.












TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............7............ ....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ..... ._ ................. ............_........9


CHAPTER


1 INTRODUCTION ................. ...............10.......... ......


Background ................. ...............10.................
Obj ectives ................. ...............12.......... .....

2 CHARACTERIZING NITRATE CONCENTRATIONS AND LOADINGS IN
SURFACE RUNOFF WATERS FROM TWO ORNAMENTAL PLANT NURSERIES....13


Introducti on ................. ...............13.................
M materials and M ethods .............. ...............14....
Site Description ................ ...............14.................
Discharge Measurements ................. ...............15........... ....
Sampling ................. ...............16.................
A analysis .............. .... ........... ...... ....... .. .. ... ...........1
Staged surface runoff events: foliage plant production nursery ............... ..............18
Staged surface runoff events: bedding plant production nursery ................... ..........19
Results & Discussion................ ..............1
Foliage Plant Production Nursery .............. ...............19....
Bedding Plant Production Nursery ................. ......... ...............20. ....
Conclusions............... ..............2


3 CHARACTERIZATION OF THE POTENTIAL BIOFILTRATION SYSTEM............._._.28


Introducti on ........._._ ...... .. ...............28...

Obj ectives .............. ... ...............3 0...._.._.....
Materials and Methods ............... ...... ... .............3
Capture of Native Denitrifying Microflora .............. ...............30....
Experimental Units ............ ..... .._ ............... 1....
Nitrate and Carbon Analysis .............. ...............32....
Physical/Chemical Measurements ....__ ......_____ .......___ .............3
Experim ental Design ..................... ........ ....... ...............3
Effects of NO3-N availability (pulsing) on NO3-N removal potential .....................33
Effects of carbon availability (pulsing) on NO3-N removal potential .....................34











Confirmation ofNO3-N removal rates under optimal NO3-N and DOC dosing
scenarios .............. .. ....... ......... ... ..... .......3
Effect of total water exchange on NO3-N removal potential ................. ................3 5
Data Analysis............... ...............36
Results and Discussion ................. .......... ............ ...............3
Effects of NO3-N Availability (Pulsing) on NO3-N Removal Potential .........................36
Physical Conditions..................... ............... ... .............. ..... ..........3
Effect of Carbon Availability (Pulsing) on NO3-N Removal Potential ..........................38
Confirmation ofNO3 Removal Rates under Optimal NO3 and DOC Dosing
Scenarios ............... ... ........... ............ ..... .... .. ....... .......3
Effect of Total Water Exchange on NO3-N Removal Potential .................. ...............39
Conclusions............... ..............4

APPENDIX

A FERTIGATION EVENT WATER VOLUME INPUT AT THE FOLIAGE NURSERY
FLOW STUDIES. 1) SUMMER. 2) FALL................. ...............49

B NO3-N LOADINGS APPLIED TO CROPS DURING THE FLOW STUDIES AT THE
FOLIAGE NURSERY. 1) SUMMER. 2) FALL................. ...............50

C NO3-N RUNOFF CONCENTRATIONS AT THE FOLIAGE NURSERY. 1)
SUMMER. 2) FALL................. ...............51

D NO3-N LOADINGS APPLIED TO CROPS DURING THE FLOW STUDIES AT THE
BEDDING NURSERY. 1) SPRING. 2) SUMMER .............. ...............53....

E NO3-N RUNOFF CONCENTRATIONS AT THE BEDDING NURSERY. 1) SPRING.
2) SUMMER ................. ...............54.................

F SUMMARY TABLE FOR WATER AND NO3-N LOADS FOR ALL FLOW
S TUDIE S ................. ...............56.......... ......

G DISSOLVED ORGANIC CARBON (DOC) RESULTS DURING THE SPRING 2005
NO3 -N REMOVAL RATES STUDIES UNDER OPTIMAL DO SING SCENARIO S........5 7

H REGRESSIONS OBTAINED DURING THE CALIBRATIONS OF THE V-NOTCH
WEIRS AT THE FOLIAGE NURSERY. A) PIPE 1. B) PIPE 2. C) PIPE 3 ........................58

I REGRESSIONS OBTAINED DURING THE CALIBRATIONS OF THE V-NOTCH
WEIRS AT THE BEDDING NURSERY ................. ...............60........... ...

LIST OF REFERENCES ................. ...............61................

BIOGRAPHICAL SKETCH .............. ...............63....










LIST OF TABLES


Table Page

2-1 Exponential equations describing the discharge at the foliage nursery .............................23

2-2 Exponential equations describing the discharge at the bedding nursery ...........................23

3-1 Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load
for the daily NO3-dosing. ............. ...............41.....

3-2 Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load
for the daily NO3-dosing during the pulsed carbon study ................. ................. ...._41

3-3 Predicted time intervals (h) for removing 25%, 50%, 75%, and 90% of the NO3-N
load for the daily NO3-dosing treatment for the spring 2005 study ................. ................42

3-4 Linear regression coefficients, DOC consumption, and C:N ratios during optimal
NO3-N removal period (Day 7 to 17). ............. ...............42.....










LIST OF FIGURES


Figure Page

2-1. Fertigation event cumulative water volume runoff for the flow studies at the foliage
nursery. A) Summer. B) Fall............... ...............24..

2-2. Cumulative NO3-N load for each discharge pipe during the flow studies at the foliage
nursery. A) Summer. B) Fall............... ...............25..

2-3 Cumulative runoff volume for each discharge pipe during the flow studies at the
bedding nursery. A) Spring. B) Summer. ............. ...............26.....

2-4 Cumulative NO3-N loads for each discharge pipe during the flow studies at the
bedding nursery. A) Spring. B) Summer. ............. ...............27.....

3-1 NO3-pulSing experimental design ...._ ................. ...............43.....

3-2 DOC-pulsing experimental design............... ...............43.

3-3 Percentage of NO3 TemOVal during the summer 2004 studies. A) 8 h and B) 24 h after
dosing with 25 mg/L of NO3-N and DOC constantly saturated. ............. ....................44

3-4 Summary of A) dissolved oxygen concentrations. B) Redox potential. C) pH
measurements during the summer 2004 studies. ............. ...............45.....

3-5 NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N
during the summer 2004 studies with DOC saturated .............. ..... ............... 4

3-6 NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N
during the summer 2004 studies with DOC depleted ................ ................ ........ .46

3-7 Time required for removing 90% of 25 mg/L NO3-N during the spring 2005
confirmation study under optimal NO3 and DOC scenarios ................. ............. .......47

3-8. Native microflora harvesting apparatus. .............. ...............48....









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

POTENTIAL BIOREMEDIATION SYSTEM FOR NITRATE REMOVAL FROM PLANT
NURSERY RUNOFF WATER

By

Miguel Angel Mozdzen

August 2007

Chair: Chris Wilson
Major: Soil and Water Science

High levels of nitrate (NO3) in Ornamental plant nursery runoff water can contribute to

euthrophication of water bodies, and to human health issues such as baby blue syndrome. The

obj ectives of these studies were to characterize the range of nitrate concentrations and loadings

in surface runoff water from two typical ornamental plant nurseries, and to evaluate the potential

use of a common aquaculture biofiltration media for removing nitrate from nursery generated

runoff water. A foliage plant nursery and a bedding plant nursery were used to evaluate NO3-N

losses. Losses of NO3-N at the foliage nursery during a typical fertigation cycle ranged from 0.8

to 1.2 kg per acre, with concentrations ranging from 70 to 274 mg/L. Losses at the bedding plant

nursery ranged from 186 to 405 g per acre with concentrations ranging from 0.7 to 26.3 mg/L.

The bioremediation experiments indicated that pulsing NO3-N considerably increased the lag

phase for induction of optimal NO3-N removal when dissolved organic carbon (DOC) was not

limited. Results also indicated that pulsing DOC once optimal nitrate removal was achieved

reduced NO3-N removal rates significantly. Using sucrose as the source of carbon, optimal

nitrate removal rates of 5.97 mg and 3.06 mg of NO3-N/h were achieved after 7 and 16 days in

separate studies. Performing total water exchanges did not appear to affect the rate of NO3-N

removal once the optimal rate was achieved.









CHAPTER 1
INTTRODUCTION

Background

The ornamental plant production industry is among the fastest growing segments of

American agriculture. Much of the plant material marketed in the United States is produced in

containers in nurseries located in the Southern and Pacific coast states (Alexander 1993). These

nursery operations may use large amounts of nutrients and water, which can lead to nutrient

leaching and losses in runoff water. High levels of NO3 in HUTSery leachate and runoff waters can

contribute to environmental problems related to euthrophication of water bodies. NO3 is also

known to cause methemoglobinemia (blue baby syndrome) in infants (Ziebarth 1991).

Plant nurseries use diverse irrigation and fertilization systems. Overhead and

microirrigation are the most common irrigation systems used today. The most common

fertilization strategies include incorporation in the substrate, top-dressing, and application

through the irrigation system. Fertilizer formulations range from controlled-release to water-

soluble. Regardless of the fertilizer type, nitrate leaching and losses in surface water can occur.

Even controlled-release fertilizers (CRF) have a high potential for nitrate leaching under

conditions of high temperatures and excessive moisture. Also, mishandling CRF may cause

cracking of the granule coatings and result in a quick release of nitrate (Dumroese 1995).

According to Mancino (1990), nitrate-nitrogen (NO3-N) is the predominant form of

nitrogen leached from plant containers containing at least 20% of an organic amendment such as

sphagnum peat. The high leaching potential of NO3 is due to its negative charge. Most substrates

used for ornamental plant production have a very high cation exchange capacity and a very low

anion exchange capacity, resulting in little absorptive capacity of the negatively charged NO3

ion, and a very high absorptive capacity for the positively charged ammonium ion (NH4). Due to









the substrates low anion holding capacity, the NO3 mOlecules can move freely through the

substrates, into surface waters, and/or ground waters.

Alexander (1993) reported that NO3-N concentrations in runoff waters from plant

nurseries in the Southern United States ranged from 1.6 55.0 mg/L. Studies have also shown

NO3-Nlevels in the upper 3 feet of soil under some benches and near retention ponds to exceed

2,245 kg/ha (2000 Lbs/acre), in nurseries that have been operating for over 10 years (Amos

1993).

Best management practices (BMPs) can reduce NO3 leaching and losses in runoff, and

may include the use of retention structures and water recycling. Retention structures are designed

to retain a fraction of runoff water on site, allowing opportunity for natural processes to remove

nutrients and sediments. Alexander (1993) reported that a lined retention structures used alone

was only 20% effective in removing nutrients, while a system that included a lined retention

structures, water treatment, and water recycling was 80% effective in removing nitrogen.

However, once filled to capacity, retention structures may discharge surface runoff water before

natural processes are able to remove NO3. In addition, NO3 leaching can also occur in areas with

high water tables where liners for retention structures cannot be used, such as in many areas of

South and Central Florida. Further more, as water is recycled, salts and some nutrients may

increase with each cycle causing a significant reduction in production, and other problems

associated with over fertilization, including weed and nuisance algae growth on production

surfaces and in water collection structures (Alexander 1993). Treatment of recirculated water

may alleviate some of these problems.

Due to the recent increase in nursery/greenhouse operations in the Southern U.S., and the

high potential for NO3 leaching and runoff, federal and state governments are evaluating NO3










sources in nursery operations (Amos 1993). The management of nitrogen to prevent surface and

ground water contamination may affect many nursery operations in the future. Many BMP's are

now available that can help reduce NO3 leaching and runoff (Yeager et al. 1997). However, NO3

leaching and runoff losses can occur even when using appropriate BMP's. In this event, other

strategies must be considered to help reduce NO3 l0ads in runoff waters.

One possible strategy is the use of biofiltration systems originally developed for the

aquaculture industry. These bioailtration systems have a relatively small footprint, use relatively

low cost infrastructure and operating costs, and are designed to remove large quantities of NH4-

N through nitrifieation processes. Because these systems are designed to treat relatively large

volumes of water within a short period of time, a similar system might me useful for in-situ NO3-

N removal from runoff water near production areas. However, NO3-N and NH4-N removal by

bioniltration occurs under very different conditions. In order for NO3-N removal to occur by

bioniltration, appropriate redox conditions must be met, and a source of carbon must be provided

at a minimum of a 2: 1 ratio C: N (Aesoy 1998). The following studies were designed to address

the following obj ectives:

Obj ectives

1. Characterize the range of NO3-N concentrations and loadings likely in surface runoff
water from ornamental plant nurseries.

2. Capture native denitrifying microflora from surface water drainage systems using
bioniltration substrate.

3. Evaluate the effect of NO3-N availability (pulsing) on NO3-N removal potential using
captured native denitrifying microflora.

4. Evaluate the effect of carbon availability (pulsing) on NO3-N removal potential using
captured native denitrifying microflora.

5. Evaluate the effect of total water exchange on NO3-N removal potential using captured
native denitrifying microflora.









CHAPTER 2
CHARACTERIZING NITRATE CONCENTRATIONS AND LOADINGS IN SURFACE
RUNOFF WATERS FROM TWO ORNAMENTAL PLANT NURSERIES

Introduction

One overall obj ective of this program was to design a biofiltration system for use in

ornamental plant production areas. In order to accomplish this, some knowledge regarding NO3-

N concentrations and loadings is needed. NO3 is an important source of nitrogen, because it is

the main form that most plants assimilate directly. NO3 is commonly used by the ornamental

plant industry, and is applied through either soluble (such as ammonium nitrate (NH4NO3) and

calcium nitrate (CaNO3)), Or controlled-release fertilizers (CRF) (such as Osmocote and

Nutricote). The most common soluble fertilizer-containing NO3-N is NH4NO3. It is widely used

by the industry because of its high solubility and ease of application through irrigation systems.

NO3 mOlecules have a high potential for leaching in nursery substrates due to their

negative charge, and the absence of anion holding capacity in most substrates used by the

industry. High amounts of NO3 leaching from plant nurseries can result in serious environmental

problems when it enters natural aquatic ecosystems in high concentrations, resulting in

euthrophication. Euthrophication often results in hypoxia, which can lead to fish kills and a rapid

degradation of more nutrient-poor, aquatic ecosystems. Health concerns can also arise when

infants drink ground water with high levels of NO3 CauSing "Blue Baby Syndrome" (Ziebarth

1991).

Fertilizer Formulations vary in their susceptibility to leaching. The use of CRF can reduce

NO3 leaching by regulating its release (Dumroese 1995). Escobar et al. (2004) investigated

nitrogen leaching from olive trees grown in plastic pots in which total nitrogen losses were

higher when NH4NO3 and CaNO3 were applied, and lower with controlled-release fertilizers.

However, high temperatures and moisture can increase the release rate of nitrate from CRF's,










making them more susceptible to leaching. Mishandling CRF's can also cause the fertilizer' s

granule coating to crack, resulting in a quick release of NO3 (Mancino 1990).

The amount of NO3 that leaches and is lost in runoff water from plant nursery operations

may be influenced by several factors including irrigation and nutrition management practices.

Many nursery operations use inefficient overhead irrigation systems, which can result in the

delivery of up to 80% of the irrigation water to non-target areas (Alexander 1993). To

compensate for the inefficiencies in water placement, growers may increase the volume of

irrigation water applied, increasing the risk of NO3 losses by leaching and runoff. Other

operations use microirrigation systems with soluble fertilizers for fertigation. Improperly

managed, these systems can result in leaching losses of more than 72% of the water applied per

container (Dumroese 1995), and up to 95% of the NO3 applied (Bigelow 2001). According to

Mancino (1990), NO3-N is the predominant form of nitrogen leached from plant containers

containing at least 20% of an organic amendment such as sphagnum peat. Other leaching studies

found similar results where substrates incorporated with inorganic and organic amendments such

as sphagnum peat leached more than 95% of the NO3 applied, while NH4 leaching was negligible

(Bigelow 2001).

* Hypothesis: Nitrate loss occurs in surface runoff water from container ornamental plant
nurseries.

* Objective: To characterize nitrate losses in surface runoff water from two container
nurseries.

Materials and Methods

Site Description

Two nurseries were selected for investigation of NO3 losses in surface runoff/drainage

water. Both nurseries were located in South Florida (St. Lucie and Martin Counties). The

nursery located in St. Lucie County produces high quality foliage plants using fertigation through









a drip irrigation system. This nursery will be referred to in this study as the "Foliage Nursery"

(FN). This nursery employs a water recycling system, and the water used to irrigate/fertigate is

pumped from a non-lined water reservoir. The total area of the drained production area studied at

this nursery was I hectare (ha) (2.5 acres or 108,900 ft2). This area was drained through three

discrete discharge pipes.

The nursery located in Martin County produces high quality bedding plants using an

overhead irrigation/fertigation micro-sprinkler system, coupled with the addition of controlled-

release fertilizers in the container substrate. This nursery will be referred to in this study as the

"Bedding Nursery" (BN). This nursery does not recycle water. Water used to irrigate/fertigate is

pumped from an onsite well. The nursery collects its runoff water in a small non-lined pond.

During storm events runoff water leaves the nursery site once the pond reaches capacity. The

total nursery area studied at this location was 0. 112 ha (0.28 acres or 12,000 ft2), and was drained

through two discrete drainage pipes.

Discharge Measurements

V-notch weirs were constructed and used at both nursery sites to estimate the instantaneous

and cumulative flow volumes of runoff water during monitored events. The respective weirs

were constructed of PVC sewer pipe by cutting a v-notch into the pipe at angles varying from 30

to 600. Plexiglass panels with a thickness of 5 mm were marked at 1-cm intervals, and glued with

clear silicon to each side of the v-notch. The constructed v-notch weirs were attached to 900

elbows, which were attached to the downstream end of each discharge pipe evaluated.

Three 25.4 cm (10-in) discharge pipes drained the FN study area, while a 10 and 15 cm (4

and 6-in) discharge pipe drained the BN study area. In order to estimate discharge volumes

during monitored events, a flow rate versus depth of flow (through each weir) relationship was

derived by regression analysis. Each weir was manually calibrated at 1 cm depth increments










throughout the useable range. Regression equations used for each nursery are shown in Table

2.1.

Water depth readings were taken every 5 min, and the flow (L/min) was calculated using

the regression equation determined during each weir's calibration. The weir calibrations were

checked during each irrigation/fertigation event to confirm accuracy. The total amount of water

discharged through each pipe during the irrigation/fertigation event was calculated by averaging

the flows every 5 min. The average flow was then multiplied by the time (5 min) to determine

the water volume discharged during each 5-min interval. Water discharge volumes for each 5-

min interval during the entire duration of the runoff event were summed to estimate total

discharge per drainage pipe. Total discharge from the production area was estimated by summing

the total volume for each pipe.

Sampling

To determine the amount of water and NO3 l0ads applied during an irrigation/fertigation

event, sampling containers were randomly placed throughout the different irrigation zones within

the production area monitored. For the FN, five gallon buckets were used as sampling containers

to collect irrigation/fertigation water. Two buckets were randomly placed inside each zone

within the area being studied, and one microirrigation emitter was placed inside each bucket.

There were a total of twelve zones in the production area with two buckets per zone for a total of

24 buckets. At the end of the irrigation/fertigation event the total volume of water per bucket was

measured, the average water volume applied per emitter within each zone was determined, and

then multiplied by the total number of functional emitters present in each respective zone during

the irrigation/fertigation event. The total water volume applied per zone was summed to

determine the total volume of water applied during the event at the production area being

evaluated.









For the BN, plastic cups were used as sampling containers to collect irrigation/fertigation

water. Two cups were randomly placed inside each zone within the area being studied to directly

collect the water applied by the overhead micro sprinkler irrigation system. There were a total of

six (6) zones in the area with two (2) cups per zone for a total of 12 cups. The total volume of

water applied for all zones during the irrigation/fertigation event was determined using a flow

meter at the well. Flow measurements were taken every 5 min, and the flow was multiplied by

the time or duration the pumping occurred in order to determine the total water volume applied.

To determine the NO3-N loads applied during the irrigation/fertigation event, a water

sample was collected from each bucket or cup. Samples were preserved by adding 2 drops of 11

N sulfuric acid to lower the pH below 2, and were immediately cooled (4oC) with ice. Samples

were refrigerated until analysis. The average of the NO3-N concentrations per zone was

determined, and then multiplied by the total volume of water applied per zone to determine the

total load applied per zone. The total NO3-N load applied during an irrigation/fertigation event

was determined by adding the loads of all zones within the production area.

The total NO3-N load discharged during the irrigation/fertigation event was estimated by

sampling for NO3-N at 10-min intervals from the water discharged at the v-notch weirs. The

total water volume in liters discharged at the weir during each 10-min interval was multiplied by

the NO3-N concentration (mg/L) sampled at each particular interval to estimate the total load in

milligrams of NO3-N. Finally, the total NO3-N load lost during the irrigation/fertigation event

was calculated by adding all of the 10-min interval loads for the entire runoff event.

Analysis

NO3-N samples collected from the nurseries runoff events were centrifuged for 5 min at

3000 revolutions per minute (rpm) to remove any suspended solids. Then, 100 CIL of 1 N

hydrochloric acid (HC1) was added to 5 ml of each centrifuged sample prior to









spectrophotometric analysis using a Cary model 300 Bio UV-visible spectrophotometer at 220

nanometers (nm) (Walnut Creek, CA). All NO3-N samples were analyzed using the NO3-N

ultraviolet spectrophotometric screening method described under Standard Methods for the

Examination of Water and Wastewater (Clesceri et al. 1998). In order to account for possible

interference from dissolved organic materials, light absorbance was also measured at 275 nm.

The corrected UV-light absorbance (Abs) of NO3-N in the sample was calculated using the

following equation:

Abs (corr) = Abs (220) 2 x Abs (275)

Results from NO3-N screening method were confirmed using a Westco auto-analyzer

(model: Smartchem) and USEPA method 353.1 for colorimetric determination of NO3-N.

Results were also confirmed using a Dionex ion chromatograph model: ICS-1000 (Dionex

Corporation Sunnyvale, CA) and operating under conditions outlined in USEPA method 300.6.

Staged surface runoff events: foliage plant production nursery

Runoff sampling events were staged on two separate occasions at the FN.

Irrigation/fertigation was applied through emitters at a rate of 3.875 L/h Each

irrigation/fertigation cycle was 0.5 h in duration. During the summer event, two cycles were

performed for each of the 12 zones of the production area, resulting in a total application of 3.8 L

(1gal) per emitter. The total water volume applied during the summer 2004 irrigation/fertigation

event was 57,853 L (15,285 gal) (appendix A). During the fall monitoring event,

irrigation/fertigation was applied at a rate of 3.875 L/h (1 gal/h), but for only one (1) 0.5 h cycle.

The total water volume applied during the fall 2004 event was 27,735 L (7,328 gal) (appendix

B).









Staged surface runoff events: bedding plant production nursery

Irrigation/fertigation was applied through overhead micro-sprinklers. Irrigation cycles were

generally 20 min per zone, while fertigation cycles were 10 min per zone. The irrigation well

pump was programmed to supply water at a rate of approximately 190 L/min (50 gal /min) to the

production area evaluated. During the spring 2005 event, an irrigation cycle was monitored at a

0. 112 ha (12,000 ft2) prOduction area with a total of six irrigation zones. Each zone was irrigated

for about 20 min, and the well pump supplied irrigation water at an average rate of 51.6 gal/min

for a total of 120 min. The total water volume applied during the irrigation event was 23,435 L

(6,192 gal.) (Appendix E). During the summer 2005 irrigation event, each zone was irrigated for

15 to 20 min, and the well pump supplied water at an average rate of 49.3 gal/min for a total of

90 min. During this event, zone 5 was not irrigated. The total water volume applied during the

irrigation event was 15,894 L (4, 199 gal) (Appendix E).

Results & Discussion

Foliage Plant Production Nursery

A total of 20,93 5 L (5,403 gal) of water drained from the monitored area during the

summer study. This runoff accounted for 36% of the total volume of water applied during the

irrigation/fertigation event. The runoff volume was highest at pipe 1 followed by pipes 2 and 3,

which were nearly equal (Figure 2-1). During the fall event, 13,668 L (3,611 gal) of water

drained from the application site. This total volume accounted for 49% of the water applied

during the event. In this event the runoff volume was also higher at pipe 1 followed by pipes 2

and 3 (Figure 2-1).

The total NO3-N load applied during the summer 2004 event was 4.862 Kg, while the total

NO3-N load applied during the fall 2004 irrigation/fertigation event was 2.976 Kg (appendix 3).

The NO3-N load during the fall was approximately 40% lower than the load observed during the









summer event since only one fertigation cycle was applied. NO3-N was inj ected into the

irrigation water as NH4NO3 and CaNO3 Soluble fertilizers. NO3-N concentrations in the

irrigation water ranged from 23 mg/L to 182.4 mg/L during the summer event and 23 mg/L to

160.5 mg/L during the fall event (appendix C). As expected NO3-N concentrations in the runoff

water varied depending on which zones were being fertigated. During the summer event, higher

NO3-N concentrations in the runoff water, ranging from 45.3 to 274.0 mg/L, were observed at

pipes 1 and 2; compared to 21.0 to 65.8 mg/L at pipe 3 (appendix 4). Likewise, during the fall

event NO3-N concentrations ranging from 74.2 to 252.7 mg/L were again highest in pipes 1 and

2 followed by pipe 3 (70.5 to 121.4 mg/L) (appendix 4). Pipes 1 and 2 collected leachate from

an area containing heavily fertigated Rhaphis exelsa palm trees, thus higher NO3-N

concentrations were expected at these pipes.

The total NO3-N load discharged from the production area during the summer event was

3.02 Kg (Figure 2-2 and appendix G). This load comprised 62% of the NO3-N that was applied

during the irrigation/fertigation event, indicating that more then half of the NO3-N applied to the

plants through micro-irrigation may have leached through the containers and left the production

area (appendix G). A portion of this load may have also been comprised of residual NO3-N from

previous applications. Only 3 8% of the NO3-N applied likely remained in the container, and was

possibly available for plant uptake. The total NO3-N load leaving the production area during the

fall event was 1.99 kg. This load comprised 67% of the NO3-N that was applied during the event

(appendix G).

Bedding Plant Production Nursery

The cumulative runoff estimates at this site are only representative of loses during the

monitored portion of discharge events since some water was flowing before the start of

monitoring. This particular site had drainage problems that resulted in ponding of water









throughout the production area. A total of 7,523 L (1,988 gal) of irrigation water was discharged

during the spring 2005 study (appendix G). Cumulative runoff volume was higher at pipe 1 than

pipe 2 (Figure 2-3). Flow rates for pipes 1 and 2 ranged from 6.7 to 59.2 L/min and 0.05 to 19

L/min, respectively, during this event. The total runoff water volume was 4,994 L for pipe 1 and

2,529 L for pipe 2.

During the summer 2005 irrigation event, a total of 8,190 L (2,164 gal) of irrigation water

was discharged during the 4.2 h period monitored. Likewise, discharge through pipe I was

higher than pipe 2 (Figure 2-3). Discharge from pipes 1 and 2 occurred for >24 h and 4.2 h,

respectively, with discharge rates ranging from 2.6 13.7 L/min at pipe 1 and 0.004 14.7

L/min at pipe 2.

The total NO3-N load applied to the crops during the spring irrigation and summer

irrigation events were estimated to be 17,053 and 37, 131 mg, respectively (appendix E). Ground

water pumped from a well was used for irrigation. NO3-N was present in the well water at

concentrations ranging from 0.47 to 1.24 mg/L (spring irrigation) and 1.45 3.69 mg/L (summer

irrigation). NO3-N concentrations in discharge water for the spring and summer irrigation events

ranged from 1.6 26.3 and 0.7 10 mg/L, respectively (appendix F). A total of 111,417 mg

NO3-N left the production site in runoff water during the monitored period of the spring

irrigation event, and 51,200 mg during the summer irrigation event (appendix G). Cumulative

NO3-N runoff loads for each discharge pipe for both spring and summer events are shown in

Figure 2-4. Total NO3-N losses from the Bedding Nursery production area were likely much

higher than reported in this study, because the area continued to drain from pipe 1 after the 6.6 h

monitored. Continuous monitoring beyond 6.6 hr was not possible, due to nursery operating

hours. Pipe 1 continued to drain into the next day at a flow rate of 4.3 L/min.










Conclusions

These results indicate that significant amounts ofNO3-N can leave the production sites in

normal irrigation runoff drainage water associated with both micro and overhead irrigation /

fertigation practices. Likewise, it can be assumed that rain fall events causing drainage through

containers and surface runoff from the production areas may result in similar losses. NO3-N

concentrations in the maj ority of the samples collected during the runoff events exceeded the 10-

mg/L drinking water limit set by the U.S. EPA. These high levels indicate a need for remedial

action if the drainage water interacts with drinking water sources. In addition, remedial action is

also needed to prevent adverse effects to natural water bodies. With regard to the overall proj ect

obj ective of developing a biofiltration system for removing NO3-N from plant nurseries surface

drainage water, this proj ect provided valuable information regarding expected, realistic loadings

and flow rates that must be considered.










Table 2-1. Exponential equations describing the discharge at the foliage nursery.
Pipe Equation R2
1 Y=0.1261X2.7123 0.999
2 Y=0.463 6X2.0732 0.997
3 Y=0.1681X2.4596 0.990
Note: Flow depth relationships. Y = Flow (L/min); X = Height (cm).

Table 2-2. Exponential equations describing the discharge at the bedding nursery.
Pipe Equation R2
1 Y=0.1854X2.8615 0.997
2 Y=0.6924X2.2017 0.992
Note: Flow depth relationships. Y = Flow (L/min); X = Height (cm).




















- -


15000

10000

5000

0


V


Pipe 3


Time (minutes)


15000

10000

5000

0


Pipe 3


--


Time (minutes)


Figure 2-1.Fertigation event cumulative water volume runoff for the flow studies at the foliage
nursery. A) Summer. B) Fall.















2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000


A


----Pipel1
--- --Pipe 2
Pipe 3


00000000000000000000000000000000o

Time (minutes)


2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0


El



----Pipel1
-- -- Pipe 2
Pipe 3


0 0 0 0 0 0 00 00 00 00 0 0 0 0

Time (minutes)




Figure 2-2.Cumulative NO3-N load for each discharge pipe during the flow studies at the foliage
nursery. A) Summer. B) Fall.



































7000
-6000 -
S5000
S4000
1- 3000
S2000
C2 1000 -



Time (minutes)


7000-
6000
S5000
S4000
S3000

S21000
100



Time (minutes)


- iPie 1
- Pipe 2
















- Pipe 1
- Pipe 2


Figure 2-3. Cumulative runoff volume for each discharge pipe during the flow studies at the
bedding nursery. A) Spring. B) Summer.
















100000-
900000-
80000
70000
60000
50000
40000
30000
20000 --
100 0 ...-
000000000000000000000000000000000

Time (minutes)






100000-
900000
80000
70000
60000
50000
40000
30000 -
20000
10000


A


- Pipe 1
- Pipe 2















B


- Pipe 1
- Pipe 2


000000000000000000000O

Time (minutes)



Figure 2-4. Cumulative NO3-N loads for each discharge pipe during the flow studies at the
bedding nursery. A) Spring. B) Summer.









CHAPTER 3
CHARACTERIZATION OF THE POTENTIAL BIOFILTRATION SYSTEM

Introduction

These studies were conducted in an effort to develop a bioremediation system for

removing NO3 fr0m surface runoff water associated with ornamental plant production. Most

research on NO3 TemOVal by biological and/or chemical processes has largely concentrated on

city and industrial wastewater treatment technology, which requires high infrastructure costs and

energy inputs. These models are not feasible for use in most nursery operations. Very little

research has addressed the use of biological and chemical processes to remove NO3 fr0m plant

nursery drainage water. Much of the research reported focuses on the use of constructed

wetlands and reed beds. However, constructed wetlands require extensive land areas, a relatively

long time period for establishment, and relatively long hydraulic retention times for nutrient

removal (Hume et al. 2000). These restrictions reduce the feasibility of constructed wetlands for

many small to medium-sized nurseries in regions such as South Florida were land value is high

and its availability is limited. Reed beds require less land area than constructed wetlands, around

200 m2 for each hectare of nursery area, and require a 2-day hydraulic retention time for efficient

nutrient removal (Headly et al. 2001). Besides relatively long hydraulic retention times (Stephens

2003), reed beds may discharge water during heavy rainstorm events and require excessive

maintenance.

Other research studies have focused on biological removal of NO3 by microbial mediated

denitrification. In order for denitrification to occur, a source of available carbon must be present.

Many studies have investigated different carbon sources such as methanol, corn silage, yeast,

whey, and spent sulfite liquor (Skrinde 1982). Aesoy (1998) investigated sludge and solid

organic waste as possible carbon sources, reporting that they were comparable to ethanol and









acetic acid. Constantin (1997) determined that acetic acid was more efficient for denitrifieation

than ethanol, because of its more directly assimilable structure. Hamersley (2002) reported that

increasing particulate organic carbon reduced the lag-phase for induction of denitrifieation and

increased denitrifieation rates. Menasveta et al. (2000) conducted a study on a closed

recirculating system using both nitrifieation and denitrification processes. NO3-N concentrations

were reduced with a hydraulic retention time (HRT) of 1-2 h from 165 mg/L to 25 mg/L using

methanol as the carbon source and crushed oyster shells as the substrate. Initially, aquaculture

designed plastic balls were used as substrate, but nitrate reduction did not occur at appreciable

levels. Other studies have focused on specific organisms that mediate denitrification such as

Pseudomona~s denitrificans (NBIMCC 1625) (Beschkov 2002), and Pseudomona~s sp. ASM-2-3

isolated from the Ariake Sea tideland (Kariminiaae-Hamedaani et al. 2003). Hamid et al. (2003)

compared the denitrification efficiency of the bacterium Pseudomona~s sp. ASM-2-3 relative to

simple structured, low molecular weight carbon sources such as succinate, acetate, citrate,

ethanol, and glucose. They reported that succinate, acetate, and citrate stimulated the removal of

nearly 25 mg/L NO3-N reduction in 20 to 24 h by denitrification.

Several factors directly related to the denitrification environment must be addressed when

considering bioremediation. These include: available carbon sources, appropriate physical

conditions (pH, redox, temperature etc.), and appropriate substrates. One objective of this study

was to develop a biofiltration model that can efficiently reduce the NO3-N present in plant

nursery runoff water. Within the context of ornamental plant production nurseries, a desirable

bioremediation system must be inexpensive, easy to maintain, simple to use, require very little

technical knowledge, and most importantly, it must function under the environmental conditions

present in a typical ornamental plant nursery. From an operational perspective, the system must









have capacity for expected NO3 l0ads, water flow volume and rates, and it must meet the needs

of the denitrifying microflora. The primary purpose of this research was not to perform an in-

depth study of denitrifieation processes, nor to perform a microbiological study and

characterization of the denitrifieation process. Instead, it was to use the well-understood

denitrification process to develop a simple and low cost tool that plant nursery managers can use

to reduce NO3 pOllution in their runoff water.

Obj ectives

* Capture native denitrifying microflora.

* Evaluate the effect of NO3-N availability (pulsing) on NO3-N removal potential using
captured native denitrifying microflora.

* Evaluate the effect of carbon availability (pulsing) on NO3-N removal potential using
captured native denitrifying microflora.

* Evaluate the effect of total water exchanges on NO3-N removal potential using captured
native denitrifying microflora.

Materials and Methods

Capture of Native Denitrifying Microflora

Native denitrifying microflora were captured from an irrigation/drainage ditch located in

the UF/IFAS-IRREC research farm, in Fort Pierce, Florida. Water within the ditch originated

from the King' s Highway canal. Kaldness media served as the substrate for the attachment of the

microflora. This bioailtration media, known as "bioailm carrier elements", is designed and

commonly used for the purpose of removing toxic ammonia waste in aquaculture recirculating

bioailtration systems. This media is constructed of polyethylene, has a large surface area (259

ft2 ft3), iS light, slightly positively buoyant, and self-cleaning. The self-cleaning action allows for

exfoliation of the older, less active bacterial layers, and eliminates the need for backwashing.

These characteristics should reduce clogging of the media and facilitate maintenance. The









elements are 7-mm long and 10-mm in diameter. The size and porosity of the media make it an

ideal candidate for developing a flow-through bioailtration system for treating discharge waters

from ornamental nursery production areas.

For initial inoculation, 189 L (50 gal) of media were placed in each of the 18-230 L (60

gal) polyethylene containers connected as shown in Figure 3-8. These containers were located

next to the IRREC irrigation/drainage ditch. Three (3) large submersible pumps (Big Versa

Pump, model VP3900) capable of pumping 13,815 L/h at a head of 1.5 meters were placed

inside a screened floating cage, and submerged in the ditch. The water was pumped into a large

circular 1,665 L holding tank, which was used to settle any suspended solids before feeding

water to the bioniltration media. Inside the holding tank, six submersible pumps (Big Versa

Pump, model VPl1225) capable of pumping 3,974 L/h at a head of 1.5 m supplied each of the 6

sets of containers with water at a rate of 37.9 L/min. Prior to the NO3 TemOVal studies, surface

water in the ditch was pumped through the media for several weeks. Once conditioned and

inoculated with native microflora, the media were mixed, and aliquots were used for the assays.

Experimental Units

Lab-scale bioHilters were created using 18.9 L (5 gal) polyethylene containers with lids.

Ten liters of biomedia and 10 liters of water were taken from the microflora harvesting

apparatus, and placed inside each container. To provide water circulation and improve water

contact with the biomedia, a submersible water pump (Resun, model SP-800) capable of

pumping 250 L/h was placed on the bottom of each container. Ten percent of the H20 volume in

each container was exchanged every other day with water from the microflora harvesting

apparatus in order to replenish necessary elements possibly needed by the microorganisms.

All NO3 TemOVal studies were conducted inside of a glass greenhouse. The greenhouse

allowed better control of climatic conditions such as temperature, rain, sunlight, and provided









electric power for the recirculation pumps. A shade cloth (60% shade) was placed over the study

area to reduce sunlight penetration and heat that could create excessive water loss due to

evaporation inside the experimental units.

Nitrate and Carbon Analysis

NO3-N samples were analyzed colorimetricaly using a Cary 300 Bio UV-visible

spectrophotometer (Walnut Creek, CA) and the NO3-N ultraviolet spectrophotometric screening

method described in Standard Methods for the Examination of Water and Wastewater (Clesceri

et al., 1998). Before each determination, the samples were centrifuged for 5 min at 3000 rpm to

remove any suspended solids. One hundred CIL of 1 N HCI was added to 5 ml of sample. The

light absorbance was read against nanopure water at 220 nm. In order to correct for interference

caused by organic matter, the sample absorbance (Abs) was measured at 275 nm. The corrected

UV-light abs of NO3-N in the sample was calculated using the equation: Abs (corr) = Abs (220)

- 2 x Abs (275). NO3-N concentrations were confirmed using a Dionex ion chromatograph

model: ICS-1000 (Dionex Corporation, Sunnyvale, CA) and USEPA method 300.6 protocols.

Dissolved Organic Carbon (DOC) was analyzed colorimetricaly using a Hach DR/4000

UV-visible spectrophotometer (Hach Co., Loveland, CO) and the Hach Direct Method 10173,

(mid range; 15 150 mg/L C). Every sample was first centrifuged for 5 min at 3000 rpm to

remove suspended solids. Ten ml of sample was placed in a 50 ml Erlenmeyer flask containing a

stir bar, followed by the addition of 0.4 ml of buffer solution (pH 2.0) and stirring for 10 min.

While the samples were stirring, a persulfate powder pillow was added to each acid digestion

vial and properly labeled. One ml of each sample was then added to each digestion vial and

swirled gently. Blue ampules were rinsed with deionized water, wiped with a soft, lint-free wipe,

and opened before lowering into the vial contents. The digestion vials were capped, and allowed

to digest in a heating block for 2 h at a temperature ranging from 103-105 oC. Following









digestion, the vials were allowed to cool for 1 h, and then read colorimetricaly at multiple

wavelengths of 598 and 430 nm using the spectrophotometer.

Dissolved organic carbon (DOC) consumption was determined by performing a linear

regression from the day carbon was dosed to the day before carbon was re-dosed. The DOC

consumption per day was calculated by dividing the difference between day-today DOC loads by

the number of days included in the regression. Finally, the carbon to nitrogen ratio (C:N) was

calculated by the DOC consumption per day over the amount of NO3-N reduced daily.

Physical/Chemical Measurements

The pH and redox potential (Eh) were monitored using a portable Accumet model AP 63

meter (Fisher Scientific, Singapore). Dissolved oxygen (DO) was monitored using a portable

YSI model 95 meter (Yellow Springs Instruments, Yellow Springs, Ohio). Greenhouse air

temperature and the water temperatures inside the experimental units were monitored at 15-min

intervals during NO3 TemOVal studies using two Hobo model H18 data loggers.

Experimental Design

Effects of NO3-N availability (pulsing) on NO3-N removal potential

The effect of NO3-N availability on the achievement of optimal NO3-N removal rates was

investigated in the summer of 2004 from July 14 to July 30. A total of 36 small-scale bioreactors

were assembled to evaluate three NO3-dosing scenarios: 1) daily dosing, 2) dosing every 3rd day,

and 3) dosing every 5 days (Fig. 3-1). A total of 12 replicate experimental units were used for

each treatment. All experimental units were assembled as previously described. On the day of

initial treatment, each unit was dosed with 1,515 mg of calcium nitrate (CaNO3) to achieve a

NO3-N concentration of 25 mg/L in 10 L of water. The carbon source used for these studies was

laboratory-grade sucrose. Initially, 10 g of sucrose was added to each bioreactor to achieve a









concentration of 420 mg/L of dissolved organic carbon (DOC). DOC was maintained at a

minimum of 2:1 ratio (C:N) during the entire study.

NO3 dosings were performed at 8 AM for the duration of the experiment. Following

dosing, media and H20 within each experimental unit was mixed using a clean plastic pipe. After

mixing, a sample was collected for the analysis ofNO3-N to confirm the initial concentration.

DOC was analyzed daily to provide a baseline concentration.

Following the initial dose and sampling (referred to as time "O"), a 10 ml sample was

collected from three randomly selected units/treatments, and then after 1, 2, 3, 4, 8, and 24 h to

determine the rate of NO3-N removal. It was neither practical nor feasible to collect samples

from all units at each interval. However, an equal number of samples were collected from each

unit by the end of the study. After the 24 h sampling, each replicate was dosed again with CaNO3

on its corresponding day (i.e. daily or every 3 or 5 days). Measurements for redox potential

(Eh), dissolved oxygen (D.O.), pH, and temperature were taken during the experiment on a daily

basis using the previously described equipment.

A ten percent water exchange was performed every-other-day on all replicates to replenish

nutrients and minerals possibly needed by the microorganisms. The fresh water added to each

bioreactor came from the same surface water source where the microbes were harvested. This

investigation continued for a total of 16 days; after which the same replicates were used to

conduct an investigation into the effects of pulsing carbon on optimal NO3-N removal rates.

Effects of carbon availability (pulsing) on NO3-N removal potential

Once optimal denitrification rates were established under saturated carbon conditions, this

investigation assessed the impact of carbon depletion on NO3-N removal potential. During this

investigation DOC was allowed to fall below a 2: 1 carbon:nitrogen ratio for a period of 5 days

before adding more carbon. This investigation was conducted in the summer of 2004 from









August 1st through the 18th. The same replicates used in the previously described study were

used. In this study, half of the replicates (6 experimental units) within each of the NO3-dosing

treatments continued to receive a carbon source above the 2:1 carbon:nitrogen ratio, while the

other half (6 experimental units) were allowed to fall below the 2: 1 carbon:nitrogen ratio for a

total of 4 consecutive days before re-dosing with 420 mg/L of DOC as sucrose on the 5th Day.

A 10% water exchange was performed every other day on all replicates as previously described.

In this study, samples for NO3-N analysis were collected at the same intervals previously

described. In this case, three samples were randomly collected for each DOC treatment

(saturated vs. pulsed) within each of the three NO3-N pulsing treatments.

Confirmation of NO3-N removal rates under optimal NO3-N and DOC dosing scenarios

This investigation was conducted in the spring of 2005 (March 15 through April 1st). The

experimental bioreactor setup was similar to that previously described except that an aquarium

heater was added to each bioreactor to stabilize temperatures. These heaters were necessary

because of hurricane (Francis and Jeanne) damage that prevented proper functioning of the

glasshouse temperature controls. The aquarium heaters were set to maintain optimal water

temperatures ranging between 29- 30 oC inside the experimental units.

During this investigation NO3 WAS added on a daily basis at 25 mg/L NO3-N and DOC was

maintained at saturated (2: 1 C:N ratio) concentrations by addition of sucrose at 1000 mg/L (420

mg/L DOC). Samples were collected at the time of dosing, and then at 4, 8, and 24 h after daily

dosing in order to determine the rate of NO3-N removal. Fifteen replicate bioreactors were used

for this study. Samples for carbon analysis were collected each day before dosing.

Effect of total water exchange on NO3-N removal potential

The purpose of this study was to evaluate the effect a total water exchange would have on

optimal NO3-N removal potential. Results would provide information on the biofilter' s capability









in a plug-flow system; the most ideal system to treat NO3 pOlluted discharge waters from plant

nursery operations that irrigate or fertigate on a daily basis.

After the maximum NO3-N removal rate was achieved, all of the replicates received a

100% water exchange with fresh surface water, followed by addition of 1000 mg/L of sucrose,

and the initial 25 mg/L of NO3-N. Samples were collected at the previously described time

intervals 0, 4, 8 and 24 h to determine NO3-N concentrations and removal rates. This water

exchange evaluation was conducted for 2 days (Days 16 and 17).

Data Analysis

Least squares linear regression analysis was used to estimate NO3-N removal rates based

on the log-transformed NO3-N concentration data. This analysis was performed for each day of

sampling, and was based on the respective sampling intervals. Once the linear regression model

was determined, the time required to remove 25, 50, 75, and 90% of the NO3-N load in the

experimental bioreactors was estimated.

Results and Discussion

Effects of NO3-N Availability (Pulsing) on NO3-N Removal Potential

Results indicate that optimal NO3-N removal rates were achieved only with treatment one

(1) where NO3-N was dosed daily into the bioreactors. With this treatment, maximum NO3

removal rates were achieved by Day 16; where 98% of the NO3-N dosed was removed within 8

h. In comparison, only 54% of the nitrate load was removed for the 3rd day pulse treatment at

Day 15, and 10.9% for the 5th day pulsed treatment at Day 15 (Figure 3-3).

Comparable NO3-N removal rates were not achieved for the treatments where NO3-N was

dosed every 3 or 5 days. This is likely a response to the lower total loads of NO3-N in each

respective treatment limiting denitrifying microflora establishment. Interestingly, after 9 days of

pulsing nitrate every 3rd day, over 90% of the NO3-N was removed within 24 h of dosing,









indicating that denitrifying microflora populations were increasing relative to the initial day of

study. Likewise, over 90% of the NO3-N added every 5th day was removed within 24 h after 15

days (Figure 3-3).

A summary of the estimated NO3-N removal rates (linear regression slope) for the daily

NO3-dose treatment is shown in Table 3-1. Linear regression slopes were only possible for the

daily NO3-dosed treatment. For this treatment, removal rates were similar from Day 1 though

Day 1 1; generally more than 15 h was needed to remove 90% of the nitrate load added. After

Day 11 a significant decrease was apparent. On Days 12-16 only 4.7 6.7 h was needed to

remove 90% of the NO3 l0ad. These results indicate a long lag phase of 17 days after day of

treatment is needed to achieve the maximum NO3-N reduction rate. During the lag phase, NO3-N

reduction improves on a 24 h basis as the denitrifying microbial populations increase.

Physical Conditions

A summary of the dissolved oxygen (D.O.) concentrations, redox potentials (Eh), and pH

is shown in Figure 3-4. D.O. concentrations dropped from 1.4 mg/L to < 0.4 mg/L anoxicc)

within the first 24 h for all treatments. Eh dropped from +1 dV to < -4 dV within the first 24 h

for all treatments, and remained negative during the duration of the study with the exception of

the 5th day treatment which rose to positive levels near the end of the study. Negative Eh values

indicated constant activity by the microbial populations as electron acceptors were reduced.

The average pH for each treatment appeared to vary. The NO3 daily dosed treatment pH

was consistently higher than the 3rd and 5th day dosing treatments. Mean pH for each treatment

over 17 days was 7.0, 5.9, and 5.7 for the daily, 3rd, and 5th day NO3-dosing treatments,

respectively. Because the initial pH of all treatments was approximately 7.5, some type of

buffering capacity reducing activities were apparent in the 3rd and 5th day dosing treatments.

The optimal pH for denitrification is approximately 7.0 (Aesoy 1998). The higher pH observed









in the daily-dosed treatment is likely because denitrification results in the recovery of alkalinity

(calcium carbonate), and denitrification rates were much higher in the daily-dosed treatment

followed by 3rd and 5th day treatments (Jeyanayagam 2000). The water temperatures within

each treatment were similar, averaging 290C.

Effect of Carbon Availability (Pulsing) on NO3-N Removal Potential

Because optimal NO3-N removal rates were not achieved with the treatments where NO3-N

was dosed every 3rd or 5th day, those treatments were not evaluated in this study. For the

replicates maintained with saturated carbon, NO3-N removal continued on Days 17-34. However,

NO3-N removal rates began to significantly decrease after Day 21 of the study (Day 5 of the

DOC evaluation). This decrease in NO3-N removal rates may be due to a variety of factors,

including limited nutrients and co-factors not replenished by the partial water changes. The

estimated time required to remove 25, 50, 75, and 90% of the NO3-N load for the replicates

maintained with saturated carbon during the pulsed carbon study is shown in Table 3-2. The

reduction in nitrate removal rate is reflected in the increases in the time needed to remove each

respective portion of the NO3-N load.

NO3-N removal was significantly reduced when carbon became limited. Because of this

reduction, a slope analysis was not possible. This is expected since carbon is a necessary

ingredient for the denitrification process. Carbon is the energy source for the denitrifying

bacteria (Hamersley 2002). In addition, NO3-N removal rates remained low even after

restoration of saturated carbon conditions. This is important because in the field, steps will have

to be taken to ensure that carbon levels do not become limited. Otherwise, the efficiency of the

biofilter may be compromised.

Figures 3-5 and 3-6 compare the amount of NO3-N reduced during the pulsed carbon

study. Results depict how the saturated carbon treatment reduced significantly higher amounts









of NO3-N (Figure 3-5) than the pulsed carbon treatment (Figure 3-6). These results confirm that

carbon amounts of at least a 2: 1 ratio with nitrogen must be maintained for optimal NO3-N

removal to occur.

Confirmation of NO3 Removal Rates under Optimal NO3 and DOC Dosing Scenarios

A summary of the time required to remove 90% of NO3-N (25 mg/L) on a daily basis is

shown in Figure 3-7. A lag phase of 6 days was observed during this study before achieving

maximal NO3-N removal rates after 7 days. These maximal NO3-N removal rates persisted

throughout the study (from 7-17 Days). These rates were also nearly twice as fast as those

observed during the summer 2004 studies. The optimal removal rate from 7-17 Days ranged

from 5.7 to 6.2 mg NO3-N/h. At these rates, the estimated time required to remove 90% of the

nitrate load ranged from 3.6-3.8 h (Table 3-3).

Linear regressions for carbon consumption were also calculated during this study. On

average a C:N ratio of 1.84: 1 was needed during the spring 2005 confirmation study (Table 3-4).

A C:N ratio of 7.8 was needed during the total water exchange treatment from Day 16 to Day 17.

The high C: N ratio seen during this last treatment may have been caused by the possible

reduction of other electron acceptors such as iron, manganese, and sulfate, which may have been

present in high quantities when the fresh ditch water was added to the system. A strong hydrogen

sulfide smell was apparent from Day 16 to 17, indicating high reduction activity of sulfate (SO4)

into hydrogen sulfide gas (H2S).

Effect of Total Water Exchange on NO3-N Removal Potential

Results demonstrated that performing 100% water exchanges did not adversely affect NO3-

N removal potential during the 2-day evaluation. Total water exchanges appeared to slightly

improve NO3-N removal rates.









Conclusions

Pulsing NO3-N negatively affected the lag phase for induction of denitrifieation and the

NO3-N removal rates in the bioreactors. Denitrifying microorganisms are facultative anaerobes

meaning that they can respire in both aerobic and anaerobic conditions by using both 02 and

NO3-N as electron acceptors (Jones et al. 2000). When both NO3-N and 02 are absent,

denitrifying microorganisms cannot continue their metabolic activities, because of the lack of a

terminal electron acceptor needed by their electron transport chain during energy generation.

Pulsing DOC in the bioreactors appeared to halt denitrification processes, due to the

absence of carbon. Carbon is used by denitrifying bacteria for both an energy and carbon source.

Without carbon, denitrifieation metabolic activities cannot function. Pulsing DOC prolonged the

lag phase. In this case, the denitrifyers are either killed or could not continue to proliferate. The

results in this study suggest that a carbon source cannot be absent from the biofilter system for

NO3-N removal to occur. Maintaining constant optimal water temperatures of 290C to 30oC

inside the bioreactors may have significantly increased the rate of NO3-N removal, and reduced

the lag phase for denitrifieation induction. This indicates that regions with lower temperatures,

and a high degree of temperature fluctuations may have prolonged lag phases, and reduced NO3-

N removal rates. Performing water exchanges of the total water volume of the bioreactors does

not appear to adversely affect the denitrifying microbial populations. This opens the possibility

of using the bioreactors as a plug flow system, in which they may be used to treat nursery

runoff water at nurseries that irrigate or fertigate on a daily basis.

The DOC consumption after maximum NO3-N removal rates have been achieved

indicated that sucrose was a highly efficient carbon source. Sucrose may be a very practical

carbon source for NO3-N removal at plant nursery operations due to its relatively low cost as

compared to more costly sources such as ethanol, acetic acid, glucose, and others (Aesoy 1998).










Table 3-1. Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load
for the daily NO3-dosing.
Day T25 T50 T75 T90 Slope Intercept
DOT 5.2 5.6 14.4 25.9 -0.03 1.90
1 3.8 6.6 11.3 17.6 -0.06 2.51
2 5.2 10.4 19.2 30.9 -0.03 2.41
3 4.1 6.5 10.7 16.2 -0.07 2.57
4 1.8 5.0 10.6 17.8 -0.05 2.37
5 3.8 7.1 12.7 20.1 -0.05 2.43
6 2.2 4.8 9.3 15.2 -0.07 2.44
7 2.9 5.5 9.9 15.7 -0.07 2.45
8 2.1 4.7 9.0 14.8 -0.07 2.40
9 3.4 7.2 13.6 22.0 -0.05 2.43
10 2.4 5.7 11.5 19.1 -0.05 2.44
11 2.6 6.3 12.7 21.1 -0.05 2.42
12 1.2 2.3 4.2 6.7 -0.16 2.57
15 1.5 2.7 4.8 7.6 -0.14 2.57
16 0.6 1.4 2.8 4.7 -0.21 2.37
Note: Slope and intercept apply to the linear regression models using the log-transformed NO3-
N concentration data.

Table 3-2. Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load
for the daily NO3-dosing during the pulsed carbon study.
Day T25 T50 T75 T90 Slope Intercept
17 0.3 0.9 2.0 3.4 -0.28 2.31
18 1.1 2.0 3.6 5.6 -0.19 2.40
19 1.2 2.1 3.6 5.7 -0.19 2.57
20 0.8 1.6 3.1 5.0 -0.21 2.45
21 0.8 2.1 4.4 7.5 -0.13 2.40
22 0.5 1.8 4.2 7.3 -0.13 2.35
25 1.0 2.2 4.2 6.9 -0.15 2.19
33 1.0 2.8 5.8 9.8 -0.10 2.44
34 1.2 3.0 6.2 10.4 -0.10 2.42
Note: Slope and intercept apply to the linear regression models using the log-transformed NO3-
N concentration data.











































Days r2 Slope (mg/L) day C:N ratio
7 -9 0.87 -77 77 38.5 1.54
10 -14 0.85 -80 80 20.0 0.80
15 -16 1.00 -79 79 79.0 3.20
Averages 0.91 -79 79 46.0 1.84
Notes: Carbon consumption for the total water exchange treatment (Day 16 to 17) was 196 mg/L,
C:N ratio of 7.8.


Table 3-3. Predicted time intervals (h) for removing 25%, 50%, 75%, and 90% of the NO3-N
load for the daily NO3-dosing treatment for the spring 2005 study.
Day T25 T50 T75 T90 Slope R2 (transf)
1 1.479 3.750 7.414 12.769 -0.078 0.997
2 1.063 3.003 6.135 10.710 -0.091 0.982
3 1.063 3.003 6.135 10.710 -0.091 0.982
4 1.201 3.116 6.207 10.723 -0.092 0.995
5 0.950 2.432 4.822 8.316 -0.119 0.998
6 0.969 1.944 3.517 5.817 -0.181 0.986
7 1.047 2.094 3.141 3.769 -5.970 1.000
8 1.036 2.073 3.109 3.731 -6.030 1.000
9 1.036 2.073 3.109 3.731 -6.030 1.000
10 1.028 2.056 3.084 3.701 -6.080 1.000
11 1.054 2.108 3.162 3.794 -5.930 1.000
12 1.096 2.193 3.289 3.947 -5.700 1.000
13 1.025 2.049 3.074 3.689 -6.100 1.000
14 1.054 2.108 3.162 3.794 -5.930 1.000
15 1.054 2.108 3.162 3.794 -5.930 1.000
16 1.008 2.016 3.024 3.629 -6.200 1.000
17 1.003 2.006 3.010 3.612 -6.230 1.000
Note: Slope and intercept apply to the linear regression models using the log-transformed NO3-
N concentration data.

Table 3-4. Linear regression coefficients, DOC consumption, and C:N ratios during optimal
NO3-N removal period (Day 7 to 17).
TOC TOC


consumption


consumption /









Concentrations: Pulsing: NO3-N Replicates:

(5m/ aNO3-


day
DOC saturated
(2:1 C: N ratio) day~ ,l r r r r v~



Figure 3-1. NO3-pulSing experimental design.


Concentrations: Pulsed NO3-N / Saturated DOC / Pulsed DOC 5 days after DOC depleted


1
day
3


~r~rrrr`m


~~hh~hh,


>0000000


25 mg/L
N03-N


DOC a
sturateN a

ratio)

Figure 3-2. DOC-pulsing experimental design.













A



0 Daily
-O 3rd day
- 5th day


-70


1


1 23 45 6 7


8 9 10 11 12 13 14 15 16


100


-70
60
go 5
o 40
S30
20
10
0
-10
-20


U
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16


Days after treatment



Figure 3-3. Percentage of NO3 TemOVal during the summer 2004 studies. A) 8 h and B) 24 h after
dosing with 25 mg/L of NO3-N and DOC constantly saturated.


Days after treatment


O Daily
O 3rd day
H5th day















-*-daily
-m-3rd day
5th day


1.6 -
1.4 -n
1.2 -

S0.8 -
8 0.6-
0.4-
0.2 -


Time (hours)


3
2
1
g0o

-c -1,
-0 -2

-4
-5


5th day


4 -( -daily

2 -m-3rd day
5th day




Time (min)




Figure 3-4. Summary of A) dissolved oxygen concentrations. B) Redox potential. C) pH
measurements during the summer 2004 studies.


Time (min)
















gl-Aug
g2-Aug
03-Aug
04-Aug
g5-Aug
g6-Aug
g7~-Aug
g9-Aug
gl0-Aug
011i-Aug
012-Aug
gl3-Aug
gl'5-Aug
gl'7-Aug
gl8-Aug


4 8 24


Time (hours)


Figure 3-5. NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N
during the summer 2004 studies with DOC saturated.




gl-Aug

g2-Aug

03-Aug


120

100

80

60

40

20

0


04-Aug

g5-Aug

g6-Aug

g7~-Aug

g9-Aug

gl0-Aug

O 11-Aug
012-Aug

gl3-Aug

gl5-Aug

gl7~-Aug


0 4 8


gl8-Aug


Figure 3-6. NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N
during the summer 2004 studies with DOC depleted.


Time (hours)














NO03-N removal (25 mg/L)


15
14
13
12
011
10
8
6
59


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Days after treatment


Figure 3-7. Time required for removing 90% of 25 mg/L NO3-N during the spring 2005
confirmation study under optimal NO3 and DOC scenarios.












Return


Ditch



Hoses




Holding tank
Pumns


Hoses

































100% water exchange




Figure 3-8. Native microflora harvesting apparatus.










APPENDIX A
FERTIGATION EVENT WATER VOLU1VE INPUT AT THE FOLIAGE NURSERY FLOW
STUDIES. 1) SU1V1VER. 2) FALL


Table A-1
Zone Emitter volume/zone (Liters)
1 5.56
2 4.35
3 5.31
4 4.33
5 5.60
6 4.15
7 3.90
8 3.20
9 4.38
10 4.35
11 3.75
12 4.43
Total


Emitters/zone
576
2,208
960
264
560
960
2,340
2,688
320
960
1,400
708


Total volume/zone (Liters)
3,203
9,605
5,098
1,142
3,136
3,984
9,126
8,602
1,400
4,176
5,250
3,133
57,853


Table A-2
Zone
1
2
3


Emitter volume/zone


(Liters)
2.54
2.05
1.88
2.17
2.53
2.17
1.75
2.29
2.59
2.05
1.92
1.98


Emitters/zone
576
1,152
960
264
660
1,060
2,340
2,688
420
1,060
1,400
708


Total volume/zone (Liters)
1,463
2,356
1,800
573
1,667
2,300
4,095
6,142
1,086
2,168
2,688
1,398
27,735


Total









APPENDIX B
NO3-N LOADINGS APPLIED TO CROPS DURING THE FLOW STUDIES AT THE
FOLIAGE NURSERY. 1) SUMMER. 2) FALL


Table B-1
Zone
1
2
3
4
5
6
7
8
9
10
11
12
Total


Total water vol


(Liters)
3,203
9,605
5,098
1,142
3,136
3,984
9,126
8,602
1,400
4,176
5,250
3,133
57,853


NO3-N conc. (mg/L)
41.0
56.0
46.0
23.0
41.3
29.5
110.5
182.4
42.0
28.7
111.3
110.3
Average: 69 +/- 49


NO3-N conc. (mg/L)
30.0
23.0
27.1
31.1
82.1
104.4
138.0
160.5
92.8
86.5
144.2
148.5
Average: 89 +/- 52


Total NO3-N load (mg)
131,305.0
537,868.8
234,489.6
26,261.4
129,516.8
117,528.0
1,008,423.0
1,568,931.8
58,800.0
119,851.2
584,325.0
345,558.9
4,862,860 =4.86 Kg


Total NO3-N load (mg)
43,891
54,184
48,780
17,817
136,820
240,141
565,110
985,804
100,753
187,506
387,610
207,648
2,976,064 =2.98 Kg


Table B-2
Zone Total water vol
1
2
3
4
5
6
7
8
9
10
11
12
Total


(Liters)
1,463
2,356
1,800
573
1,667
2,300
4,095
6,142
1,086
2,168
2,688
1,398
27,736










APPENDIX C
NO3-N RUNOFF CONCENTRATIONS AT THE FOLIAGE NURSERY. 1) SUMMER. 2)
FALL

Table C-1


Time (min)
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
310
320
Average
Standard deviation
Minimum
Maximum


Pipe 1 (m/L)
46.6
47.7
50.2
51.3
49.3
48.1
52.8
51.0
56.6
54.0
134.7
159.6
196.5
243.2
236.7
211.5
189.6
192.2
141.3
121.0
129.1
133.7
139.4
201.5
153.7
167.6
230.2
240.3
274.0
250.7
256.0
251.3
253.7
152.0
79.3
46.6
274.0


Pipe 2 (mg/L)
52.8
49.3
47.7
48.0
45.3
48.9
53.6
56.3
58.1
56.8
52.8
149.9
154.7
161.0
151.9
85.3
76.9
76.2
76.6
80.7
83.1
96.8
99.7
115.6
153.6
153.4
144.1
159.6
156.3
160.2
162.0
158.0
159.6
102.6
46.4
45.3
162.0


Pipe 3 (mg/L)
27.6
30.8
37.5
38.8
39.2
32.3
39.7
38.9
38.9
40.6
39.6
50.1
50.1
50.0
65.8
52.6
54.9
55.4
55.5
47.3
50.0
44.5
50.1
21.0
45.1
40.0
42.7
43.1
42.6
42.1




43.6
9.2
21.0
65.8










Table C-2
Time (min)

10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
310
320
Average
Standard deviation
Minimum
Maximum


Pipe 1 (mg/L)
113.8
188.5
210.7
252.7
244.8
241.2
252.5
141.4
115.7
111.6
123.8
136.9
146.6
144.5
136.4
139.7
143.1
150.9
150.2
152.2
155.2
155.5
161.8
163.9
171.8
160.7
165.6


Pipe 2 (mg/L)
81.6
141.5
163.8
155.7
168.2
160.5
167.0
159.0
90.3
74.2
89.0
97.6
99.2
99.1
95.3
89.7
99.9
101.9
106.3
105.2
101.7
110.9
105.4
110.0
108.6
108.6


Pipe 3 (mg/L)
100.4
82.9
70.5
81.8
89.1
97.3
100.3
100.6
102.7
104.6
102.6
109.1
109.2
116.5
111.9
118.4
117.8
121.4
121.4


















103.1
14.2
70.5
121.4


164.1
41.4
111.6
252.7


115.0
29.1
74.2
168.2


























Table D-2
Zone Total water volume (Liters) NO3-N conc. (mg/L) Total NO3-N load (mg)
1 3,524 2.13 7,505
2 2,874 1.98 5,691
3 3,493 1.65 5,763
4 3,193 2.91 9,291
6 2,810 3.16 8,881
Total 15,894 Average = 2.37 37,131


APPENDIX D
NO3-N LOADINGS APPLIED TO CROPS DURING THE FLOW STUDIES AT THE
BEDDING NURSERY. 1) SPRING. 2) SUMMER


Table D-1
Zone
1
2
3
4
5
6
Total


Total water volume (Liters)
3,962
3,879
3,856
3,914
3,961
3,863
23,435


NO3-N conc.(mg/L)
0.51
0.52
0.55
0.47
1.24
1.05
Average = 0.73


Total NO3-N load (mg)
2,022.6
2,026.5
2,118.1
1,915.1
4,897.2
4,073.7
17,053.2











APPENDIX E
NO3-N RUNOFF CONCENTRATIONS AT THE BEDDINGNURSERY. 1) SPRING. 2)
SUMMER


Table E-1
Time (min)

10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
310
320
330
340
350
360
370
380
390
400
Average
Standard deviation
Minimum
Maximum


Pipe 1 (mg/L)
20.6
20.3
19.1
16.0
18.7
21.3
20.4
23.8
26.3
21.4
24.1
12.3
15.6
15.0
14.3
15.6
16.3
13.9
14.3
12.6
14.8
17.3
17.9
19.1
14.7
19.6
16.4
19.2
19.5
19.5
19.8
19.6
13.2


Pipe 2(mg/L)
3.2
1.7
1.6
1.7
1.6
2.0
2.1
4.2
7.6
10.1
11.6
15.1
15.7
16.0
15.3
17.9
19.7
18.7
16.7
15.3
16.3
16.7
11.3
13.1
13.8
14.8


18.0
3.4
12.3
26.3












Table E-2
Time (min)

10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
310
320
330
340
350
360
370
380
390
400
Average
Standard deviation
Minimum
Maximum


Pipe 1 (mg/L)
0.9
0.8
0.8
0.7
5.3
8.5
7.6
7.6
7.8
7.9
7.7
7.5
7.1
6.9
6.6
6.4
6.1
5.9
5.6
5.3
5.1
4.8
4.6
4.4
4.2
4.1
4.0
3.8
3.7
3.5
3.3
3.2
3.0
2.9
2.8
2.7
2.6
2.6
2.6
2.5
2.4
5.3
2.3
0.7
8.5


Pipe 2 (mg/L)
1.2
5.1
7.7
7.3
6.9
5.9
5.7
6.2
7.5
7.9
8.5
8.3
9.0
9.9
10.0
9.9
9.5
8.3
7.5
7.4
7.5
8.0
8.4
8.6
8.8
8.9




















7.7
1.8
1.2
10.0









APPENDIX F
AND NO3-N LOADS FOR ALL FLOW STUDIES

FN- Fall BN Spring (Irr) BN-Summer (Irr)


SUMMARY TABLE FOR WATER

FN- Summer


NO3-N input
NO3-N runoff
NO3-N runoff/acre
NO3-N runoff %
Water input
Water runoff
Water runoff %


4.80 Kg
3.00 Kg
1.92 Kg
62%
57,861 L
20,935 L
36%


2.98 Kg
1.99 Kg
0.80 Kg
67%
27,736 L
13,668 L
49%


17.05 g
111.42 g
405.00 g
665%
23,435 L
7,523 L
32%


37.1 g
51.2 g
186.0 g
138%
15,894 L
8,190 L
52%









APPENDIX G
DISSOLVED ORGANIC CARBON (DOC) RESULTS DURING THE SPRING 2005 NO3-N
REMOVAL RATES STUDIES UNDER OPTIMAL DOSING SCENARIOS


Days 1-DOC (mg/L) 1- STDEV
Day oftreatment 302 29
Day 1 (3/16) 204 70
Day 2 (3/17) 239 22
Day 3 (3/18) 555 36
Day 4 (3/19) 379 46
Day 5 (3/20) 403 12
Day 6 (3/21) 342 30
Day 7 (3/22) 300 27
Day 8 (3/23) 275 26
Day 9 (3/24) 146 26
Day 10 (3/25) 467 49
Day 11 (3/26) 334 63
Day 12 (3/27) 291 92
Day 13 (3/28) 300 20
Day 14 (3/29) 84 28
Day 15 (3/30) 565 43
Day 16 (3/31) 486 44
Day 17 (4/1) 290 87


2-DOC (mg/L) 2-STDEV 3-DOC
481 80
396 49
327 31
929 57
611 32
626 34
510 31
457 50
396 24
254 25
194 18
178 9
172 26
791 121
323 24
480 45
429 34
223 49


(mg/L) 3-STDEV
679 79
1671 312
1501 154
1371 104
1224 61
1331 102
1193 88
1208 73
1128 100
995 105
816 59
1090 38
1071 86
923 43
990 37
1145 58
242 21
75 37











APPENDIX H
REGRESSIONS OBTAINED DURING THE CALIBRATIONS OF THE V-NOTCH WEIRS
AT THE FOLIAGE NURSERY. A) PIPE 1. B) PIPE 2. C) PIPE 3


160-
/*A


150
140-
130-
120-
110-
100-
90-
80 -
70-
60 -
50
40-
30-
20-
10-
On
0


y = 0. 1261K''
S= 0.9986


1 2 3 4 5 6 7 8 9 10 11 12 13 14

Height (cm)


y = O 4636~'';
R = O995


2 3 4 5 6 7 8 9 10
Height (cm)


APPENDIX H. Continued











25
24
23
22
21
20
19





[]12
11
10
9
8
7
6
5
4
3
2*
1
0
0 1 2 3 4 5

He 9 (a n)


y = 0. 16810
R:= 0.9902


6 7 8 9 10











APPENDIX I
REGRESSIONS OBTAINED DURING THE CALIBRATIONS OF THE V-NOTCH WEIRS
AT THE BEDDING NURSERY


40




20


y = 0. 1854~c "
F'= 0.9967


0 05 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9
He~ight (cm)


.E~0


-20


y = 0.6924p o':
FT = 0.992


0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Heght (cm)










LIST OF REFERENCES


Aesoy, A., Odegaard, H., Bach, K., Pujol, R. and Hamon, M. (1998). Denitrification in a packed
bed biofilm reactor (biofor)-experiments with different carbon sources. Water Research,
32(5), 1463-1470.

Alexander, S. (1993). Pollution control and prevention at containerized nursery operations.
Water Science & Technology, 28, 509-517.

Amos, R. (1993). Reduction of nitrates in nursery surface and ground waters. Combined
Proceedings hIternational Plant Propagators Society, 43, 244-248.

Beschov, V., Velizarov, S.N., Agathos. and Lukova, V. (2002). Bacterial denitrification of
wastewater stimulated by constant electrical field. Biochentical Engineering Journal,
17(2), 141-145.

Bigelow, C., Bowman, D. and Cassel, D. (2001). Nitrogen leaching in sand-based root zones
amended with inorganic soil amendments and sphagnum peat. Journal of the American
Society ofI~orticultural Sciences, 126, 151-156.

Casella, S. and Payne, W.J. (1997). Potential denitrifiers for soil environmental protection.
FEM\~S M~icrobiology Letters, 140, 1-8.

Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (1998). NO3- N ultraviolet spectrophotometric
screening method. In: Standar~d Methodd~~~dd~~~dds o the Examination of Water and Wastewater,
20th edn, Washington DC, USA, pp. 249-250.

Constantin, H. and Fick, M. (1997). Influence of C-sources on the denitrification rate of a high-
nitrate concentrated industrial wastewater. Water Research, 31(3), 583-589.

Cresswell, G.C. (1995). Improving nutrient and water management in nurseries. Combined
Proceedings hIternational Plant Propagators Society, 45, 1 12-1 16.

Dumroese, R., Wenny, D. and Page-Dumroese, D. (1995). Nursery waste water: The problem
and possible remedies. National proceedings, Forest and Conservation Nursery
Association, Colorado, USA, p. 89-97.

Escobar, F.R., Benlloch, M.,Herrera, E. and Garcia-Novelo, J.M. (2003). Effect of traditional
and slow release N fertilizers on growth of olive nursery plants and N losses by leaching.
Scientia Horticulturae, 101, 39-49.

Hamersley, R.M. and Howes, B.L. (2002). Control of denitrification in a septage-treating
artificial wetland: the dual role of particulate organic carbon. Water Research, 36(17),
4415-4427.

Headley, T.R. (2001). The removal of nutrients from plant nursery irrigation runoff in subsurface
horizontal-flow wetlands. Water science and technology, 44, 77-84.











Hume, N.P. (2000). Plant carbohydrate limitation on nitrate reduction in wetland microcosm.
Water Research, 36(3), 577-584.

James, E.A. (1995). Water quality of stored and runoff water in plant nurseries and implications
for recycling. Combined Proceedings International Planzt Propagators Society, 45, 1 17-
120.

Jeyanayagam, S, Rodieck, A. and Husband, J. (2000). BNR 101. Water Environment &
Technology, 12(8), 85-88.

Jones, L.M., Liehr, S.K.,Classen, J.J. and Robarge, W. (2000). Mechanisms of dinitrogen gas
formation in anaerobic lagoons. Advances in Environmental Research, 4, 13-139.

Kariminiaae-Hamedaani, H., Kanda, K. and Kato, F. (2003). Denitrification activity of the
bacterium Pseudomonas sp. ASM-2-3 isolated from the Ariake Sea tideland. Journal of
Bioscience and Bioengineering, 97(1), 39-44.

Mancino, C. and Troll, J. (1990). Nitrate and ammonium leaching from N fertilizers applied to
penncross creeping bentgrass. Hortscience, 25(2), 194-195.

Menasveta, P., Panritdam, T., Sihanonth, P., Powtongsook, S., Chuntapa, B. and Lee, P. (2000).
Design and function of a closed, recirculating seawater system with denitrification for the
culture of black tiger shrimp broodstock. Aquacultural Engineering, 25(1), 3 5-49.

Skrinde, J.R. and Bhagat, S.K. (1982). Industrial wastes as carbon sources in biological
denitrification. J. Wat. Pollut. ControlFed., 54(4), 370-377.

Stephen, R. (2003). Reed beds clean up nursery run-off water. The Nursery Papers. Issue n:
2003/05.

Yeager, T.H. and Knox, G.W. (1991). Alternative irrigation strategies. The Woody
Ornamentalist, 16(2), 1-3.

Yeager, T.H, Fare, D., Gilliam, C., Niemiera, A., Bilderback, T. and Tilt, K. (1997). Best
management practices guide for producing container-grown plants. Sonlrlhein Nursery
Association, 1000 Johnson Ferry Rd. Suite E-130, Marietta, Georgia, 30068.

Ziebarth, A. (1991), NF91-49, Well Water, Nitrates and the "Blue Baby" Syndrome
Methemoglobinemia; Lincoln, NE; University of Nebraska Cooperative Extension.









BIOGRAPHICAL SKETCH

Miguel A. Mozdzen was born in a small village called el Pao, located within the Amazon

basin, in Venezuela. Miguel lived and enjoyed his childhood within the wilderness while his

father helped construct the third largest hydroelectric plant in the Americas. After his father

returned to Chicago, USA (and later Florida), Miguel continued his fascination for the

environment and natural sciences and after completing his active military service in the U.S.

Army he attended the University of Florida and graduated in May 2003 with a Bachelors degree

in Horticultural Sciences and in August 2007 earned a Masters degree in Environmental Sciences

from the Soil and Water Science Department, while he continued to serve in the U.S. Army

Reserves. Miguel currently serves as a biologist proj ect manager for the U. S Army Corps of

Engineers, Jacksonville District's Regulatory Division helping protect and conserve the nation' s

aquatic environment. Miguel's passion is his lovely wife and daughter.





PAGE 1

1 POTENTIAL BIOREMEDIATION SYSTEM FOR NITRATE REMOVAL FROM PLANT NURSERY RUNOFF WATER By MIGUEL ANGEL MOZDZEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Miguel Angel Mozdzen

PAGE 3

3 To my deceased father Walter Joseph Mozdzen, da d, mentor and friend; and to my daughter and wife who were my principa l driving force throughout.

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4 ACKNOWLEDGMENTS This research was supported by US DA/ARS projects 6618-13000-002-00D and 661813000-002-04S. Project S is supported by th e USDA-ARS Floriculture and Nursery Research Initiative. I would like to thank the Floriculture and Nursery Research Initiative (FNRI), USDA-ARS, and the Horticulture Research Institute (HRI) for funding this research. I would also like to thank the members of my graduate committee: Patrick Wilson, Thomas Yeager, Joseph Albano, and Andrew Ogram for allo wing me to work on this fascinating project and for their support, patience, and guidance du ring my graduate program. I would like to specially thank my chair: Patrick Wilson, for alwa ys keeping his door open for me to discuss and correct any aspects of my thesis project, and I truly appreciate all of the time he dedicated to me. He always pushed me to excel, and encouraged me during the rough times. I would also like to thank Patrick Wilsons and Joseph Albanos labora tory staff for their as sistance and support. I would like to give sp ecial thanks to my wife Sonia who supported me, especially when I was up late at night, and gone during weekends. Her love, patience, and support allowed me to succeed in my masters program. Als o, I would like to dedicate this project to my parents who always encouraged, and supported me during my scholarly pursuits.

PAGE 5

5 TABLE OF CONTENTS Page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................10 Background..................................................................................................................... ........10 Objectives..................................................................................................................... ..........12 2 CHARACTERIZING NITRATE CONCEN TRATIONS AND LOADINGS IN SURFACE RUNOFF WATERS FROM TW O ORNAMENTAL PLANT NURSERIES....13 Introduction................................................................................................................... ..........13 Materials and Methods.......................................................................................................... .14 Site Description...............................................................................................................14 Discharge Measurements.................................................................................................15 Sampling....................................................................................................................... ...16 Analysis....................................................................................................................... ....17 Staged surface runoff events: foliage plant production nursery...............................18 Staged surface runoff events: bedding plant production nursery.............................19 Results & Discussion........................................................................................................... ...19 Foliage Plant Production Nursery...................................................................................19 Bedding Plant Production Nursery..................................................................................20 Conclusions.................................................................................................................... .........22 3 CHARACTERIZATION OF THE POTEN TIAL BIOFILTRATION SYSTEM..................28 Introduction................................................................................................................... ..........28 Objectives..................................................................................................................... ..........30 Materials and Methods.......................................................................................................... .30 Capture of Native Denitrifying Microflora.....................................................................30 Experimental Units..........................................................................................................31 Nitrate and Carbon Analysis...........................................................................................32 Physical/Chemical Measurements...................................................................................33 Experimental Design.......................................................................................................33 Effects of NO3-N availability (pulsing) on NO3-N removal potential.....................33 Effects of carbon availability (pulsing) on NO3-N removal potential.....................34

PAGE 6

6 Confirmation of NO3-N removal rates under optimal NO3-N and DOC dosing scenarios...............................................................................................................35 Effect of total water exchange on NO3-N removal potential...................................35 Data Analysis.................................................................................................................. .36 Results and Discussion......................................................................................................... ..36 Effects of NO3-N Availability (Pulsing) on NO3-N Removal Potential.........................36 Physical Conditions.........................................................................................................37 Effect of Carbon Availability (Pulsing) on NO3-N Removal Potential..........................38 Confirmation of NO3 Removal Rates under Optimal NO3 and DOC Dosing Scenarios......................................................................................................................39 Effect of Total Water Exchange on NO3-N Removal Potential......................................39 Conclusions.................................................................................................................... .........40 APPENDIX A FERTIGATION EVENT WATER VOLUME INPUT AT THE FOLIAGE NURSERY FLOW STUDIES. 1) SUMMER. 2) FALL............................................................................49 B NO3-N LOADINGS APPLIED TO CROPS DUR ING THE FLOW STUDIES AT THE FOLIAGE NURSERY. 1) SUMMER. 2) FALL....................................................................50 C NO3-N RUNOFF CONCENTRATIONS AT THE FOLIAGE NURSERY. 1) SUMMER. 2) FALL...............................................................................................................51 D NO3-N LOADINGS APPLIED TO CROPS DUR ING THE FLOW STUDIES AT THE BEDDING NURSERY. 1) SPRING. 2) SUMMER............................................................53 E NO3-N RUNOFF CONCENTRATIONS AT THE BEDDING NURSERY. 1) SPRING. 2) SUMMER...................................................................................................................... .....54 F SUMMARY TABLE FOR WATER AND NO3-N LOADS FOR ALL FLOW STUDIES........................................................................................................................ ........56 G DISSOLVED ORGANIC CARBON (DOC) RESULTS DURING THE SPRING 2005 NO3-N REMOVAL RATES STUDIES UNDE R OPTIMAL DOSING SCENARIOS........57 H REGRESSIONS OBTAINED DURING TH E CALIBRATIONS OF THE V-NOTCH WEIRS AT THE FOLIAGE NURSERY. A) PIPE 1. B) PIPE 2. C) PIPE 3........................58 I REGRESSIONS OBTAINED DURING TH E CALIBRATIONS OF THE V-NOTCH WEIRS AT THE BE DDING NURSERY..............................................................................60 LIST OF REFERENCES............................................................................................................. ..61 BIOGRAPHICAL SKETCH.........................................................................................................63

PAGE 7

7 LIST OF TABLES Table Page 2-1 Exponential equations describing the discharge at the foliage nursery.............................23 2-2 Exponential equations describing th e discharge at the bedding nursery...........................23 3-1 Predicted time intervals (h) to re move 25%, 50%, 75% and 90% of the NO3-N load for the daily NO3-dosing...................................................................................................41 3-2 Predicted time intervals (h) to re move 25%, 50%, 75% and 90% of the NO3-N load for the daily NO3-dosing during the pulsed carbon study..................................................41 3-3 Predicted time intervals (h) for re moving 25%, 50%, 75%, and 90% of the NO3-N load for the daily NO3-dosing treatment for the spring 2005 study...................................42 3-4 Linear regression coeffi cients, DOC consumption, and C:N ratios during optimal NO3-N removal period (Day 7 to 17)................................................................................42

PAGE 8

8 LIST OF FIGURES Figure Page 2-1. Fertigation event cumulative water volume r unoff for the flow studies at the foliage nursery. A) Summer. B) Fall..............................................................................................24 2-2. Cumulative NO3-N load for each discharge pipe during the flow studies at the foliage nursery. A) Summer. B) Fall..............................................................................................25 2-3 Cumulative runoff volume for each discharg e pipe during the flow studies at the bedding nursery. A) Spring. B) Summer...........................................................................26 2-4 Cumulative NO3-N loads for each discharge pipe during the flow studies at the bedding nursery. A) Spring. B) Summer...........................................................................27 3-1 NO3-pulsing experimental design......................................................................................43 3-2 DOC-pulsing experimental design.....................................................................................43 3-3 Percentage of NO3 removal during the summer 2004 studies. A) 8 h and B) 24 h after dosing with 25 mg/L of NO3-N and DOC constantly saturated........................................44 3-4 Summary of A) dissolved oxygen con centrations. B) Redox potential. C) pH measurements during th e summer 2004 studies................................................................45 3-5 NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N during the summer 2004 studies with DOC saturated.......................................................46 3-6 NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N during the summer 2004 studi es with DOC depleted........................................................46 3-7 Time required for removing 90% of 25 mg/L NO3-N during the spring 2005 confirmation study under optimal NO3 and DOC scenarios..............................................47 3-8. Native microflora harvesting apparatus.............................................................................48

PAGE 9

9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science POTENTIAL BIOREMEDIATION SYSTEM FOR NITRATE REMOVAL FROM PLANT NURSERY RUNOFF WATER By Miguel Angel Mozdzen August 2007 Chair: Chris Wilson Major: Soil and Water Science High levels of nitrate (NO3) in ornamental plant nursery runoff water can contribute to euthrophication of water bodies, a nd to human health issues such as baby blue syndrome. The objectives of these studies were to characterize th e range of nitrate concen trations and loadings in surface runoff water from two typical ornamental plant nurseries, and to evaluate the potential use of a common aquaculture biofiltration media for removing nitrate from nursery generated runoff water. A foliage plant nursery and a beddi ng plant nursery were used to evaluate NO3-N losses. Losses of NO3-N at the foliage nursery during a t ypical fertigation cycle ranged from 0.8 to 1.2 kg per acre, with concentrations ranging fr om 70 to 274 mg/L. Losses at the bedding plant nursery ranged from 186 to 405 g per acre with concentrations ranging from 0.7 to 26.3 mg/L. The bioremediation experiment s indicated that pulsing NO3-N considerably increased the lag phase for induction of optimal NO3-N removal when dissolved organic carbon (DOC) was not limited. Results also indicated that pulsing DOC once optimal nitrate removal was achieved reduced NO3-N removal rates signifi cantly. Using sucrose as th e source of carbon, optimal nitrate removal rates of 5.97 mg and 3.06 mg of NO3-N/h were achieved after 7 and 16 days in separate studies. Performing total water exchan ges did not appear to affect the rate of NO3-N removal once the optimal rate was achieved.

PAGE 10

10 CHAPTER 1 INTRODUCTION Background The ornamental plant production industry is among the fastest growing segments of American agriculture. Much of th e plant material marketed in th e United States is produced in containers in nurseries located in the Southern and Pacific coas t states (Alexander 1993). These nursery operations may use large amounts of nutri ents and water, which can lead to nutrient leaching and losses in runoff water. High levels of NO3 in nursery leachate and runoff waters can contribute to environmental problems rela ted to euthrophication of water bodies. NO3 is also known to cause methemoglobinemia (blue baby syndrome) in infants (Ziebarth 1991). Plant nurseries use diverse irrigation a nd fertilization systems. Overhead and microirrigation are the most common irriga tion systems used today. The most common fertilization strategies include incorporation in the substrate, top-dr essing, and application through the irrigation system. Fertilizer formula tions range from controlled-release to watersoluble. Regardless of the fertilizer type, nitr ate leaching and losses in surface water can occur. Even controlled-release fertilizers (CRF) have a high potential for nitrate leaching under conditions of high temperatures and excessive moisture. Also, mishandling CRF may cause cracking of the granule coatings and result in a quick releas e of nitrate (Dumroese 1995). According to Mancino ( 1990), nitrate-nitrogen (NO3-N) is the predominant form of nitrogen leached from plant containers containing at least 20% of an organic amendment such as sphagnum peat. The high leaching potential of NO3 is due to its negative charge. Most substrates used for ornamental plant production have a very high cation exchange capacity and a very low anion exchange capacity, resulting in little abso rptive capacity of the negatively charged NO3 ion, and a very high absorptive capacity for the positively charged ammonium ion (NH4). Due to

PAGE 11

11 the substrates low ani on holding capacity, the NO3 molecules can move freely through the substrates, into surface waters, and/or ground waters. Alexander (1993) reported that NO3-N concentrations in r unoff waters from plant nurseries in the Southern United States range d from 1.6 55.0 mg/L. Studi es have also shown NO3-N levels in the upper 3 feet of soil under some benches and near retention ponds to exceed 2,245 kg/ha (2000 Lbs/acre), in nur series that have been ope rating for over 10 years (Amos 1993). Best management practices (BMPs) can reduce NO3 leaching and losses in runoff, and may include the use of retention structures and wa ter recycling. Retention st ructures are designed to retain a fraction of runoff wa ter on site, allowing opportunity fo r natural processes to remove nutrients and sediments. Alexander (1993) reported that a lined retention structures used alone was only 20% effective in removing nutrients, while a system that included a lined retention structures, water treatment, and water recy cling was 80% effectiv e in removing nitrogen. However, once filled to capacity, retention struct ures may discharge surface runoff water before natural processes are able to remove NO3. In addition, NO3 leaching can also occur in areas with high water tables where liners for retention structures cannot be used, such as in many areas of South and Central Florida. Further more, as wa ter is recycled, salts and some nutrients may increase with each cycle causing a significant reduction in production, and other problems associated with over fertiliz ation, including weed and nuisa nce algae growth on production surfaces and in water collection structures (A lexander 1993). Treatment of recirculated water may alleviate some of these problems. Due to the recent increase in nursery/greenhous e operations in the Southern U.S., and the high potential for NO3 leaching and runoff, federal and state governments are evaluating NO3

PAGE 12

12 sources in nursery operations (Amos 1993). The ma nagement of nitrogen to prevent surface and ground water contamination may affect many nurse ry operations in the future. Many BMPs are now available that can help reduce NO3 leaching and runoff (Yeage r et al. 1997). However, NO3 leaching and runoff losses can occur even when us ing appropriate BMPs. In this event, other strategies must be considered to help reduce NO3 loads in runoff waters. One possible strategy is the use of biofiltra tion systems originally developed for the aquaculture industry. These biofiltr ation systems have a relatively small footprint, use relatively low cost infrastructure and operating costs, and are designed to remove large quantities of NH4N through nitrification processes. Because these systems are designed to treat relatively large volumes of water within a short period of time, a similar syst em might me useful for in-situ NO3N removal from runoff water near production areas. However, NO3-N and NH4-N removal by biofiltration occurs under very diffe rent conditions. In order for NO3-N removal to occur by biofiltration, appropriate redox c onditions must be met, and a s ource of carbon must be provided at a minimum of a 2:1 ratio C: N (Aesoy 1998). Th e following studies were designed to address the following objectives: Objectives 1. Characterize the range of NO3-N concentrations and loadings likely in surface runoff water from ornamental plant nurseries. 2. Capture native denitrifying microflora fr om surface water drainage systems using biofiltration substrate. 3. Evaluate the effect of NO3-N availability (pulsing) on NO3-N removal potential using captured native denitrifying microflora. 4. Evaluate the effect of car bon availability (pulsing) on NO3-N removal potential using captured native denitrifying microflora. 5. Evaluate the effect of total water exchange on NO3-N removal potentia l using captured native denitrifying microflora.

PAGE 13

13 CHAPTER 2 CHARACTERIZING NITRATE CONCENTRAT IONS AND LOADINGS IN SURFACE RUNOFF WATERS FROM TWO ORNAMENTAL PLANT NURSERIES Introduction One overall objective of this program was to design a biofiltration system for use in ornamental plant production areas. In order to accomplish this, some knowledge regarding NO3N concentrations and loadings is needed. NO3 is an important source of nitrogen, because it is the main form that most plants assimilate directly. NO3 is commonly used by the ornamental plant industry, and is applied through either soluble (such as a mmonium nitrate (NH4NO3) and calcium nitrate (CaNO3)), or controlled-release fertiliz ers (CRF) (such as Osmocote and Nutricote). The most common soluble fertilizer-containing NO3-N is NH4NO3. It is widely used by the industry because of its high solubility and ease of application throu gh irrigation systems. NO3 molecules have a high potential for leach ing in nursery substrates due to their negative charge, and the absence of anion holdi ng capacity in most substrates used by the industry. High amounts of NO3 leaching from plant nurseries can result in serious environmental problems when it enters natural aquatic ecosy stems in high concentrations, resulting in euthrophication. Euthrophication often results in hypoxia, which can lead to fish kills and a rapid degradation of more nutrient-poor aquatic ecosystems. Health c oncerns can also arise when infants drink ground water with high levels of NO3 causing Blue Baby Syndrome (Ziebarth 1991). Fertilizer Formulations vary in their suscepti bility to leaching. The use of CRF can reduce NO3 leaching by regulating its release (Dumroes e 1995). Escobar et al. (2004) investigated nitrogen leaching from olive trees grown in plas tic pots in which total nitrogen losses were higher when NH4NO3 and CaNO3 were applied, and lower with controlled-release fertilizers. However, high temperatures and moisture can in crease the release rate of nitrate from CRFs,

PAGE 14

14 making them more susceptible to leaching. Misha ndling CRFs can also cau se the fertilizers granule coating to crack, resul ting in a quick release of NO3 (Mancino 1990). The amount of NO3 that leaches and is lost in runoff water from plant nursery operations may be influenced by several factors includi ng irrigation and nutrition management practices. Many nursery operations use inefficient overhead irrigation systems, which can result in the delivery of up to 80% of the irrigation wate r to non-target area s (Alexander 1993). To compensate for the inefficiencies in water pl acement, growers may incr ease the volume of irrigation water applied, increasing the risk of NO3 losses by leaching and runoff. Other operations use microirrigation systems with soluble fertilizers for fertigation. Improperly managed, these systems can result in leaching losses of more than 72% of the water applied per container (Dumroese 1995), and up to 95% of the NO3 applied (Bigelow 2001). According to Mancino (1990), NO3-N is the predominant form of nitrogen leached from plant containers containing at least 20% of an organic amendmen t such as sphagnum peat. Other leaching studies found similar results where substrat es incorporated with inorganic and organic amendments such as sphagnum peat leached more than 95% of the NO3 applied, while NH4 leaching was negligible (Bigelow 2001). Hypothesis: Nitrate loss occurs in surface runoff water from container ornamental plant nurseries. Objective: To characterize nitrate losses in su rface runoff water from two container nurseries. Materials and Methods Site Description Two nurseries were selected for investigation of NO3 losses in surface runoff/drainage water. Both nurseries were located in Sout h Florida (St. Lucie a nd Martin Counties). The nursery located in St. Lucie County produces high quality foliage plants using fertigation through

PAGE 15

15 a drip irrigation system. This nursery will be refe rred to in this study as the Foliage Nursery (FN). This nursery employs a water recycling system and the water used to irrigate/fertigate is pumped from a non-lined water reservoir. The tota l area of the drained production area studied at this nursery was 1 hectare (ha) (2.5 acres or 108,900 ft). This area was drained through three discrete discharge pipes. The nursery located in Martin County produ ces high quality bedding plants using an overhead irrigation/fertigation micro-sprinkler sy stem, coupled with the addition of controlledrelease fertilizers in the container substrate. This nursery will be referred to in this study as the Bedding Nursery (BN). This nurse ry does not recycle water. Water used to irrigate/fertigate is pumped from an onsite well. The nursery co llects its runoff water in a small non-lined pond. During storm events runoff water leaves the nu rsery site once the pond reaches capacity. The total nursery area studied at th is location was 0.112 ha (0.28 acres or 12,000 ft), and was drained through two discrete drainage pipes. Discharge Measurements V-notch weirs were constructed and used at both nursery sites to estimate the instantaneous and cumulative flow volumes of runoff water during monitored events. The respective weirs were constructed of PVC sewer pipe by cutting a vnotch into the pipe at angles varying from 30 to 60. Plexiglass panels with a thickness of 5 mm were marked at 1-cm in tervals, and glued with clear silicon to each side of th e v-notch. The constructed v-notch weirs were attached to 90 elbows, which were attached to the downstr eam end of each discharge pipe evaluated. Three 25.4 cm (10-in) discharge pipes drained the FN study area, while a 10 and 15 cm (4 and 6-in) discharge pipe draine d the BN study area. In order to estimate discharge volumes during monitored events, a flow rate versus de pth of flow (through each weir) relationship was derived by regression analysis. Each weir was manually calibrate d at 1 cm depth increments

PAGE 16

16 throughout the useable range. Regression equations used for each nursery are shown in Table 2.1. Water depth readings were taken every 5 mi n, and the flow (L/min) was calculated using the regression equation determined during each weirs calibration. The weir calibrations were checked during each irrigation/fertigation event to confirm accuracy. The total amount of water discharged through each pipe during the irrigation/ fertigation event was calculated by averaging the flows every 5 min. The average flow was then multiplied by the time (5 min) to determine the water volume discharged during each 5-min interval. Water discharge volumes for each 5min interval during the entire duration of the runoff event were summed to estimate total discharge per drainage pipe. Total discharge from the production area was estimated by summing the total volume for each pipe. Sampling To determine the amount of water and NO3 loads applied during an irrigation/fertigation event, sampling containers were randomly placed throughout the different irrigation zones within the production area monitored. For the FN, five gall on buckets were used as sampling containers to collect irrigation/fertigati on water. Two buckets were randomly placed inside each zone within the area being studied, and one microirri gation emitter was placed inside each bucket. There were a total of twelve z ones in the production area with two buckets per zone for a total of 24 buckets. At the end of the irrigation/fertigatio n event the total volume of water per bucket was measured, the average water volume applied per emitter within each zone was determined, and then multiplied by the total number of functional emitters present in each respective zone during the irrigation/fertigation event. The total water volume applied per zone was summed to determine the total volume of water applied during the event at th e production area being evaluated.

PAGE 17

17 For the BN, plastic cups were used as sampli ng containers to collect irrigation/fertigation water. Two cups were randomly placed inside each zone within the area be ing studied to directly collect the water applied by the ove rhead micro sprinkler irrigation system. There were a total of six (6) zones in the area with two (2) cups per zo ne for a total of 12 cups. The total volume of water applied for all zones during the irrigation/ fertigation event was determined using a flow meter at the well. Flow measurements were taken every 5 min, and the flow was multiplied by the time or duration the pumping occurred in or der to determine the total water volume applied. To determine the NO3-N loads applied during the irrigation/fertigation event, a water sample was collected from each bucket or cup. Samples were preserved by adding 2 drops of 11 N sulfuric acid to lower the pH below 2, and we re immediately cooled (4 C) with ice. Samples were refrigerated until analysis. The average of the NO3-N concentrations per zone was determined, and then multiplied by the total volume of water applied per zone to determine the total load applied per zone. The total NO3-N load applied during an irrigation/fertigation event was determined by adding the loads of all zones within the production area. The total NO3-N load discharged during the irriga tion/fertigation event was estimated by sampling for NO3-N at 10-min intervals from the water di scharged at the v-notch weirs. The total water volume in liters discharged at the weir during each 10-min interval was multiplied by the NO3-N concentration (mg/L) sampled at each particular interval to estimate the total load in milligrams of NO3-N. Finally, the total NO3-N load lost during the irrigation/fertigation event was calculated by adding all of the 10-min interval loads for the entire runoff event. Analysis NO3-N samples collected from the nurseries ru noff events were centrifuged for 5 min at 3000 revolutions per minute (rpm) to remove any suspended solids. Then, 100 L of 1 N hydrochloric acid (HCl) was added to 5 ml of each centrifuged sample prior to

PAGE 18

18 spectrophotometric analysis us ing a Cary model 300 Bio UV-vis ible spectrophotometer at 220 nanometers (nm) (Walnut Creek, CA). All NO3-N samples were analyzed using the NO3-N ultraviolet spectrophotometric screening met hod described under Standard Methods for the Examination of Water and Wastew ater (Clesceri et al 1998). In order to account for possible interference from dissolved orga nic materials, light absorbance was also measured at 275 nm. The corrected UV-light absorbance (Abs) of NO3-N in the sample was calculated using the following equation: Abs (corr) = Abs (220) 2 x Abs (275) Results from NO3-N screening method were confirme d using a Westco auto-analyzer (model: Smartchem) and USEPA method 353.1 fo r colorimetric determination of NO3-N. Results were also confirmed using a Dione x ion chromatograph model: ICS-1000 (Dionex Corporation Sunnyvale, CA) and operating under conditions outlined in USEPA method 300.6. Staged surface runoff events: folia ge plant production nursery Runoff sampling events were staged on two separate occasions at the FN. Irrigation/fertigation was applied thr ough emitters at a rate of 3.875 L/h Each irrigation/fertigation cycle was 0.5 h in durati on. During the summer event, two cycles were performed for each of the 12 zones of the producti on area, resulting in a total application of 3.8 L (1gal) per emitter. The total water volume app lied during the summer 2004 irrigation/fertigation event was 57,853 L (15,285 gal) (appendix A). During the fall monitoring event, irrigation/fertigation was applied at a rate of 3.875 L/h (1 gal/h), but for only one (1) 0.5 h cycle. The total water volume applied during th e fall 2004 event was 27,735 L (7,328 gal) (appendix B).

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19 Staged surface runoff events: bedd ing plant production nursery Irrigation/fertigation was appl ied through overhead micro-sprinkl ers. Irrigation cycles were generally 20 min per zone, while fertigation cy cles were 10 min per zo ne. The irrigation well pump was programmed to supply water at a rate of approximately 190 L/min (50 gal /min) to the production area evaluated. During the spring 2005 even t, an irrigation cycle was monitored at a 0.112 ha (12,000 ft) production area with a total of six irrigation z ones. Each zone was irrigated for about 20 min, and the well pump supplied irriga tion water at an average rate of 51.6 gal/min for a total of 120 min. The tota l water volume applied during the irrigation event was 23,435 L (6,192 gal.) (Appendix E). During the summer 2005 ir rigation event, each zone was irrigated for 15 to 20 min, and the well pump supplied water at an average rate of 49.3 gal/min for a total of 90 min. During this event, zone 5 was not irri gated. The total water vol ume applied during the irrigation event was 15,894 L (4,199 gal) (Appendix E). Results & Discussion Foliage Plant Production Nursery A total of 20,935 L (5,403 gal) of water dr ained from the monitored area during the summer study. This runoff accounted for 36% of the total volume of water applied during the irrigation/fertigation event. The runoff volume wa s highest at pipe 1 followed by pipes 2 and 3, which were nearly equal (Fi gure 2-1). During the fall event, 13,668 L (3,611 gal) of water drained from the application site. This total volume accounted for 49% of the water applied during the event. In this event the runoff volum e was also higher at pipe 1 followed by pipes 2 and 3 (Figure 2-1). The total NO3-N load applied during the summer 2004 event was 4.862 Kg, while the total NO3-N load applied during the fall 2004 irrigati on/fertigation event was 2.976 Kg (appendix 3). The NO3-N load during the fall was approximately 40% lower than the load observed during the

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20 summer event since only one fertigation cycle was applied. NO3-N was injected into the irrigation water as NH4NO3 and CaNO3 soluble fertilizers. NO3-N concentrations in the irrigation water ranged from 23 mg/L to 182.4 mg/L during the summer event and 23 mg/L to 160.5 mg/L during the fall event (a ppendix C). As expected NO3-N concentrations in the runoff water varied depending on which zones were bei ng fertigated. During the summer event, higher NO3-N concentrations in the runoff water, rangi ng from 45.3 to 274.0 mg/L, were observed at pipes 1 and 2; compared to 21.0 to 65.8 mg/L at pipe 3 (appendix 4). Likewise, during the fall event NO3-N concentrations ranging from 74.2 to 252.7 mg/L were again highest in pipes 1 and 2 followed by pipe 3 (70.5 to 121.4 mg/L) (appendix 4). Pipes 1 and 2 collected leachate from an area containing heavily fertigated Rhaphis exelsa palm trees, thus higher NO3-N concentrations were expected at these pipes. The total NO3-N load discharged from the produ ction area during the summer event was 3.02 Kg (Figure 2-2 and appendix G). Th is load comprised 62% of the NO3-N that was applied during the irrigation/fertigation event, i ndicating that more then half of the NO3-N applied to the plants through micro-irrigation may have leached through the cont ainers and left the production area (appendix G). A portion of this load ma y have also been comprised of residual NO3-N from previous applications. Only 38% of the NO3-N applied likely remained in the container, and was possibly available for plant uptake. The total NO3-N load leaving the pr oduction area during the fall event was 1.99 kg. This load comprised 67% of the NO3-N that was applied during the event (appendix G). Bedding Plant Production Nursery The cumulative runoff estimates at this site are only representative of loses during the monitored portion of discharge events since some water was flowing before the start of monitoring. This particular site had drainage problems that resulted in ponding of water

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21 throughout the production area. A total of 7,523 L ( 1,988 gal) of irrigation water was discharged during the spring 2005 study (appendix G). Cumulati ve runoff volume was higher at pipe 1 than pipe 2 (Figure 2-3). Flow rates for pipes 1 and 2 ranged from 6.7 to 59.2 L/min and 0.05 to 19 L/min, respectively, during this event. The tota l runoff water volume was 4,994 L for pipe 1 and 2,529 L for pipe 2. During the summer 2005 irrigation event, a to tal of 8,190 L (2,164 gal) of irrigation water was discharged during the 4.2 h period monito red. Likewise, discharg e through pipe 1 was higher than pipe 2 (Figure 2-3). Discharge fr om pipes 1 and 2 occurred for >24 h and 4.2 h, respectively, with discharge rates ranging from 2.6 13.7 L/min at pipe 1 and 0.004 14.7 L/min at pipe 2. The total NO3-N load applied to the crops dur ing the spring irrigation and summer irrigation events were estimated to be 17,053 and 37,131 mg, respectively (appendix E). Ground water pumped from a well was used for irrigation. NO3-N was present in the well water at concentrations ranging from 0.47 to 1.24 mg/L (spr ing irrigation) and 1.45 3.69 mg/L (summer irrigation). NO3-N concentrations in disc harge water for the spring and summer irrigation events ranged from 1.6 26.3 and 0.7 10 mg/L, respec tively (appendix F). A total of 111,417 mg NO3-N left the production site in runoff wate r during the monitored period of the spring irrigation event, and 51,200 mg during the summer irrigation event (appendix G). Cumulative NO3-N runoff loads for each discharge pipe for both spring and summer events are shown in Figure 2-4. Total NO3-N losses from the Bedding Nurser y production area were likely much higher than reported in this study, because the area continued to dr ain from pipe 1 after the 6.6 h monitored. Continuous monitoring beyond 6.6 hr wa s not possible, due to nursery operating hours. Pipe 1 continued to drain into th e next day at a flow rate of 4.3 L/min.

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22 Conclusions These results indicate that significant amounts of NO3-N can leave the production sites in normal irrigation runoff dr ainage water associated with bot h micro and overhead irrigation / fertigation practices. Likewise, it can be assume d that rain fall events causing drainage through containers and surface runoff from the production areas may result in similar losses. NO3-N concentrations in the majority of the samples co llected during the runoff events exceeded the 10mg/L drinking water limit set by th e U.S. EPA. These high levels indicate a need for remedial action if the drainage water inter acts with drinking water sources. In addition, remedial action is also needed to prevent adverse e ffects to natural water bodies. With regard to the overall project objective of developing a biofiltration system for removing NO3-N from plant nurseries surface drainage water, this project prov ided valuable information regard ing expected, realistic loadings and flow rates that must be considered.

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23 Table 2-1. Exponential equations describi ng the discharge at the foliage nursery. Pipe Equation R 1 Y=0.1261X2.71230.999 2 Y=0.4636X2.07320.997 3 Y=0.1681X2.45960.990 Note: Flow depth relationships. Y = Flow (L/min); X = Height (cm). Table 2-2. Exponential equations describi ng the discharge at the bedding nursery. Pipe Equation R 1 Y=0.1854X2.8615 0.997 2 Y=0.6924X2.2017 0.992 Note: Flow depth relationships. Y = Flow (L/min); X = Height (cm).

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24 A0 5000 10000 150000 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360Time (minutes)Cumulative Runoff (L) Pipe 1 Pipe 2 Pipe 3 B0 5000 10000 150000 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250Time (minutes)Cumulative Runoff (L) Pipe 1 Pipe 2 Pipe 3 Figure 2-1. Fertigation event cumulative water volu me runoff for the flow studies at the foliage nursery. A) Summer. B) Fall.

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25 A0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 24000000 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320Time (minutes)NO3-N Runoff Load (mg) Pipe 1 Pipe 2 Pipe 3 B0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 24000000 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250Time (minutes)NO3-N Runoff Load (mg) Pipe 1 Pipe 2 Pipe 3 Figure 2-2. Cumulative NO3-N load for each discharge pipe during the flow studies at the foliage nursery. A) Summer. B) Fall.

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26 A 0 1000 2000 3000 4000 5000 6000 70000 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320Time (minutes)Runoff (Liters) Pipe 1 Pipe 2 B0 1000 2000 3000 4000 5000 6000 70000 20 40 60 80 100 120 140 160 190 210 230 250 270 290 310 330 350 370 390Time (minutes)Runoff (Liters) Pipe 1 Pipe 2 Figure 2-3. Cumulative runoff volum e for each discharge pipe duri ng the flow studies at the bedding nursery. A) Spring. B) Summer.

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27 A0 10000 20000 30000 40000 50000 60000 70000 80000 90000 1000000 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320Time (minutes)NO3-N Runoff (mg/L) Pipe 1 Pipe 2 B0 10000 20000 30000 40000 50000 60000 70000 80000 90000 1000000 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400Time (minutes)NO3-N Runoff (mg/L) Pipe 1 Pipe 2 Figure 2-4. Cumulative NO3-N loads for each discharge pipe during the flow studies at the bedding nursery. A) Spring. B) Summer.

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28 CHAPTER 3 CHARACTERIZATION OF THE POTEN TIAL BIOFILTRATION SYSTEM Introduction These studies were conducted in an effort to develop a bioremediation system for removing NO3 from surface runoff water associated with ornamental plant production. Most research on NO3 removal by biological and/or chemical processes has largely concentrated on city and industrial wastewater treatment technology, which require s high infrastructure costs and energy inputs. These models are not feasible for use in most nursery operations. Very little research has addressed the use of biologi cal and chemical processes to remove NO3 from plant nursery drainage water. Much of the research reported focuses on the use of constructed wetlands and reed beds. However, constructed we tlands require extensive land areas, a relatively long time period for establishment, and relativ ely long hydraulic reten tion times for nutrient removal (Hume et al. 2000). These restrictions re duce the feasibility of constructed wetlands for many small to medium-sized nurseries in regions such as South Florida were land value is high and its availability is limited. Reed beds require less land area than c onstructed wetlands, around 200 m for each hectare of nursery area, and requir e a 2-day hydraulic retention time for efficient nutrient removal (Headly et al. 2001 ). Besides relatively long hydr aulic retention times (Stephens 2003), reed beds may discharge water during he avy rainstorm events and require excessive maintenance. Other research studies have fo cused on biological removal of NO3 by microbial mediated denitrification. In order for denitr ification to occur, a source of available carbon must be present. Many studies have investigated di fferent carbon sources such as methanol, corn silage, yeast, whey, and spent sulfite liquor (Skrinde 1982). Aesoy (1998) investigated sludge and solid organic waste as possible carbon sources, reporting that they were comparable to ethanol and

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29 acetic acid. Constantin (1997) dete rmined that acetic acid was more efficient for denitrification than ethanol, because of its more directly assimilable structure. Hamersley (2002) reported that increasing particulate organic carbon reduced th e lag-phase for induction of denitrification and increased denitrification rates. Menasveta et al. (2000) conducted a study on a closed recirculating system using both nitrifi cation and denitrific ation processes. NO3-N concentrations were reduced with a hydraulic retention time (HRT) of 1-2 h from 165 mg/L to 25 mg/L using methanol as the carbon source and crushed oyster sh ells as the substrate. Initially, aquaculture designed plastic balls were used as substrate, but nitrate reducti on did not occur at appreciable levels. Other studies have focu sed on specific organisms that me diate denitrification such as Pseudomonas denitrificans (NBIMCC 1625) (Beschkov 2002), and Pseudomonas sp. ASM-2-3 isolated from the Ariake Sea tideland (Karimin iaae-Hamedaani et al. 20 03). Hamid et al. (2003) compared the denitrification efficiency of the bacterium Pseudomonas sp. ASM-2-3 relative to simple structured, low molecular weight carbon sources such as succinate, acetate, citrate, ethanol, and glucose. They reported that succinat e, acetate, and citrate st imulated the removal of nearly 25 mg/L NO3-N reduction in 20 to 24 h by denitrification. Several factors directly related to the denitr ification environment must be addressed when considering bioremediation. These include: av ailable carbon sources, appropriate physical conditions (pH, redox, temperature etc.), and appropriate substrat es. One objective of this study was to develop a biofiltration model that can efficiently reduce the NO3-N present in plant nursery runoff water. Within th e context of ornamental plant production nurseries, a desirable bioremediation system must be inexpensive, easy to maintain, simple to us e, require very little technical knowledge, and most im portantly, it must function unde r the environmental conditions present in a typical ornamental plant nursery. From an operational perspective, the system must

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30 have capacity for expected NO3 loads, water flow volume and rates, and it must meet the needs of the denitrifying microflora. The primary purpose of this research was not to perform an indepth study of denitrification processes, nor to perform a microbiological study and characterization of the denitr ification process. Instead, it was to use the well-understood denitrification process to develop a simple and low cost tool that plant nursery managers can use to reduce NO3 pollution in their runoff water. Objectives Capture native denitrifying microflora. Evaluate the effect of NO3-N availability (pulsing) on NO3-N removal potential using captured native denitrifying microflora. Evaluate the effect of car bon availability (pulsing) on NO3-N removal potential using captured native denitrifying microflora. Evaluate the effect of total water exchanges on NO3-N removal potentia l using captured native denitrifying microflora. Materials and Methods Capture of Native Denitrifying Microflora Native denitrifying microflora were captured fr om an irrigation/draina ge ditch located in the UF/IFAS-IRREC research farm, in Fort Pierce, Florida. Water within the ditch originated from the Kings Highway canal. Kaldness media served as the substrate for the attachment of the microflora. This biofiltration media, known as biofilm carrier elemen ts, is designed and commonly used for the purpose of removing toxi c ammonia waste in aquaculture recirculating biofiltration systems. This media is constructe d of polyethylene, has a large surface area (259 ft2/ft3), is light, slightly positively buoyant, and self-cleaning. The self-cleaning action allows for exfoliation of the older, less act ive bacterial layers, and elimin ates the need for backwashing. These characteristics should re duce clogging of the media and f acilitate maintenance. The

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31 elements are 7-mm long and 10-mm in diameter. The size and porosity of the media make it an ideal candidate for developing a flow-through biof iltration system for treating discharge waters from ornamental nursery production areas. For initial inoculation, 189 L (50 gal) of me dia were placed in each of the 18-230 L (60 gal) polyethylene containers connected as show n in Figure 3-8. These containers were located next to the IRREC irrigation/dr ainage ditch. Three (3) large submersible pumps (Big Versa Pump, model VP3900) capable of pumping 13,815 L/ h at a head of 1.5 meters were placed inside a screened floating cage, and submerged in the ditch. The water was pumped into a large circular 1,665 L holding tank, whic h was used to settle any suspended solids before feeding water to the biofiltration media. Inside the holding tank, six submersible pumps (Big Versa Pump, model VP1225) capable of pumping 3,974 L/h at a head of 1.5 m supplied each of the 6 sets of containers with water at a rate of 37.9 L/min. Prior to the NO3 removal studies, surface water in the ditch was pumped through the media for several weeks. Once conditioned and inoculated with native microflo ra, the media were mixed, and aliquots were used for the assays. Experimental Units Lab-scale biofilters were created using 18.9 L (5 gal) polyethylene containers with lids. Ten liters of biomedia and 10 liters of water were taken from the microflora harvesting apparatus, and placed inside ea ch container. To provide water circulation and improve water contact with the biomedia, a submersible wa ter pump (Resun, model SP-800) capable of pumping 250 L/h was placed on the bottom of each container. Ten percent of the H2O volume in each container was exchanged every other day with water from the microflora harvesting apparatus in order to replenis h necessary elements possibly ne eded by the microorganisms. All NO3 removal studies were conducted inside of a glass greenhouse. The greenhouse allowed better control of clima tic conditions such as temperatur e, rain, sunlight, and provided

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32 electric power for the recirculation pumps. A sh ade cloth (60% shade) was placed over the study area to reduce sunlight penetra tion and heat that could creat e excessive water loss due to evaporation inside the experimental units. Nitrate and Carbon Analysis NO3-N samples were analyzed colorimetr icaly using a Cary 300 Bio UV-visible spectrophotometer (Walnut Creek, CA) and the NO3-N ultraviolet spectr ophotometric screening method described in Standard Methods for the Ex amination of Water and Wastewater (Clesceri et al., 1998). Before each determination, the samp les were centrifuged for 5 min at 3000 rpm to remove any suspended solids. One hundred L of 1 N HCl was added to 5 ml of sample. The light absorbance was read against nanopure water at 220 nm. In orde r to correct for interference caused by organic matter, the sample absorbance (Abs) was measured at 275 nm. The corrected UV-light abs of NO3-N in the sample was calculated usi ng the equation: Abs (corr) = Abs (220) 2 x Abs (275). NO3-N concentrations were confirmed using a Dionex ion chromatograph model: ICS-1000 (Dionex Corpor ation, Sunnyvale, CA) and USEPA method 300.6 protocols. Dissolved Organic Carbon (DOC) was analy zed colorimetricaly using a Hach DR/4000 UV-visible spectrophotometer (Hach Co., Love land, CO) and the Hach Direct Method 10173, (mid range; 15 150 mg/L C). Every sample wa s first centrifuged for 5 min at 3000 rpm to remove suspended solids. Ten ml of sample was placed in a 50 ml Erlenmeyer flask containing a stir bar, followed by the addition of 0.4 ml of buffer solution (pH 2.0) and stirring for 10 min. While the samples were stirring, a persulfate powder pillow was added to each acid digestion vial and properly labeled. One ml of each samp le was then added to each digestion vial and swirled gently. Blue ampules were rinsed with deionized water, wi ped with a soft, lint-free wipe, and opened before lowering into th e vial contents. The digesti on vials were capped, and allowed to digest in a heating block for 2 h at a temperature ranging from 103-105 C. Following

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33 digestion, the vials were allowed to cool for 1 h, and then read colorimetricaly at multiple wavelengths of 598 and 430 nm using the spectrophotometer. Dissolved organic carbon (DOC) consumption was determined by performing a linear regression from the day carbon was dosed to th e day before carbon was re-dosed. The DOC consumption per day was calculated by dividing the difference between day-today DOC loads by the number of days included in the regression. Fi nally, the carbon to nitrogen ratio (C:N) was calculated by the DOC consumption per day over the amount of NO3-N reduced daily. Physical/Chemical Measurements The pH and redox potential (Eh) were mon itored using a portable Accumet model AP 63 meter (Fisher Scientific, Singa pore). Dissolved oxygen (DO) wa s monitored using a portable YSI model 95 meter (Yellow Springs Instrume nts, Yellow Springs, Ohio). Greenhouse air temperature and the water temperatures inside th e experimental units were monitored at 15-min intervals during NO3 removal studies using two H obo model H8 data loggers. Experimental Design Effects of NO3-N availability (pulsing) on NO3-N removal potential The effect of NO3-N availability on the achievement of optimal NO3-N removal rates was investigated in the summer of 2004 from July 14 to July 30. A total of 36 small-scale bioreactors were assembled to evaluate three NO3-dosing scenarios: 1) daily dos ing, 2) dosing every 3rd day, and 3) dosing every 5 days (Fig. 3-1). A total of 12 replicate experimental units were used for each treatment. All experimental units were asse mbled as previously described. On the day of initial treatment, each unit was dosed with 1,515 mg of calcium nitrate (CaNO3) to achieve a NO3-N concentration of 25 mg/L in 10 L of water. The carbon source used for these studies was laboratory-grade sucrose. Initially, 10 g of su crose was added to each bioreactor to achieve a

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34 concentration of 420 mg/L of dissolved organic carbon (DOC). DOC was maintained at a minimum of 2:1 ratio (C:N) during the entire study. NO3 dosings were performed at 8 AM for the duration of the experiment. Following dosing, media and H2O within each experimental unit was mi xed using a clean pl astic pipe. After mixing, a sample was collected for the analysis of NO3-N to confirm the initial concentration. DOC was analyzed daily to provi de a baseline concentration. Following the initial dose and sampling (refer red to as time ), a 10 ml sample was collected from three randomly selected units/tr eatments, and then after 1, 2, 3, 4, 8, and 24 h to determine the rate of NO3-N removal. It was neither practical nor feasible to collect samples from all units at each interval. However, an equal number of samples were collected from each unit by the end of the study. After the 24 h samp ling, each replicate was dosed again with CaNO3 on its corresponding day (i.e. daily or every 3 or 5 days). Measurements for redox potential (Eh), dissolved oxygen (D.O.), pH, and temperatur e were taken during the experiment on a daily basis using the previously described equipment. A ten percent water exchange wa s performed every-other-day on all replicates to replenish nutrients and minerals possibly needed by the microorganisms. The fresh water added to each bioreactor came from the same surface water so urce where the microbes were harvested. This investigation continued for a tota l of 16 days; after which the same replicates were used to conduct an investigation into the e ffects of pulsing carbon on optimal NO3-N removal rates. Effects of carbon availability (pulsing) on NO3-N removal potential Once optimal denitrification ra tes were established under satu rated carbon conditions, this investigation assessed the impact of carbon depletion on NO3-N removal potential. During this investigation DOC was allowed to fall below a 2: 1 carbon:nitrogen ratio for a period of 5 days before adding more carbon. This investiga tion was conducted in the summer of 2004 from

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35 August 1st through the 18th. The same replicates used in the previously described study were used. In this study, half of th e replicates (6 experimental units) within each of the NO3-dosing treatments continued to recei ve a carbon source above the 2:1 carbon:nitrogen ratio, while the other half (6 experimental units ) were allowed to fall below the 2:1 carbon:nitrogen ratio for a total of 4 consecutive days before re-dosing w ith 420 mg/L of DOC as sucrose on the 5th Day. A 10% water exchange was performe d every other day on all replicat es as previously described. In this study, samples for NO3-N analysis were collected at the same intervals previously described. In this case, three samples were randomly collected for each DOC treatment (saturated vs. pulsed) within each of the three NO3-N pulsing treatments. Confirmation of NO3-N removal rates under optimal NO3-N and DOC dosing scenarios This investigation was conducted in the spri ng of 2005 (March 15 through April 1st). The experimental bioreactor setup was similar to that previously described except that an aquarium heater was added to each bioreactor to stabi lize temperatures. These heaters were necessary because of hurricane (Francis and Jeanne) dama ge that prevented proper functioning of the glasshouse temperature controls. The aquarium heaters were set to maintain optimal water temperatures ranging between 2930 C inside the experimental units. During this investigation NO3 was added on a daily basis at 25 mg/L NO3-N and DOC was maintained at saturated (2:1 C: N ratio) concentrations by add ition of sucrose at 1000 mg/L (420 mg/L DOC). Samples were collected at the time of dosing, and then at 4, 8, and 24 h after daily dosing in order to determine the rate of NO3-N removal. Fifteen repli cate bioreactors were used for this study. Samples for carbon analysis were collected each day before dosing. Effect of total water exchange on NO3-N removal potential The purpose of this study was to evaluate the effect a total water exchange would have on optimal NO3-N removal potential. Results would provide information on the biofilters capability

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36 in a plug-flow system; the most ideal system to treat NO3 polluted discharge waters from plant nursery operations that irrigate or fertigate on a daily basis. After the maximum NO3-N removal rate was achieved, all of the replicates received a 100% water exchange with fresh surface water, followed by addition of 1000 mg/L of sucrose, and the initial 25 mg/L of NO3-N. Samples were collected at the previously described time intervals 0, 4, 8 and 24 h to determine NO3-N concentrations and removal rates. This water exchange evaluation was conducted for 2 days (Days 16 and 17). Data Analysis Least squares linear regression analysis was used to estimate NO3-N removal rates based on the log-transformed NO3-N concentration data. This analysis was performed for each day of sampling, and was based on the respective sampling intervals. Once the linear regression model was determined, the time required to remove 25, 50, 75, and 90% of the NO3-N load in the experimental bioreactors was estimated. Results and Discussion Effects of NO3-N Availability (Pulsing) on NO3-N Removal Potential Results indicate that optimal NO3-N removal rates were achie ved only with treatment one (1) where NO3-N was dosed daily into the bioreact ors. With this treatment, maximum NO3 removal rates were achieved by Day 16; where 98% of the NO3-N dosed was removed within 8 h. In comparison, only 54% of the nitrate load wa s removed for the 3rd day pulse treatment at Day 15, and 10.9% for the 5th day pulsed treatment at Day 15 (Figure 3-3). Comparable NO3-N removal rates were not achie ved for the treatments where NO3-N was dosed every 3 or 5 days. This is likely a response to the lower total loads of NO3-N in each respective treatment limiting denitrifying microflora establishment. Interestingly, after 9 days of pulsing nitrate every 3rd day, over 90% of the NO3-N was removed within 24 h of dosing,

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37 indicating that deni trifying microflora populations were incr easing relative to th e initial day of study. Likewise, over 90% of the NO3-N added every 5th day was removed within 24 h after 15 days (Figure 3-3). A summary of the estimated NO3-N removal rates (linear re gression slope) for the daily NO3-dose treatment is shown in Table 3-1. Linear regression slopes were only possible for the daily NO3-dosed treatment. For this treatment, re moval rates were similar from Day 1 though Day 11; generally more than 15 h was needed to remove 90% of the nitrate load added. After Day 11 a significant decrease was apparent. On Days 12-16 only 4.7 6.7 h was needed to remove 90% of the NO3 load. These results indicate a long lag phase of 17 days after day of treatment is needed to achieve the maximum NO3-N reduction rate. Duri ng the lag phase, NO3-N reduction improves on a 24 h basis as the deni trifying microbial populations increase. Physical Conditions A summary of the dissolved oxygen (D.O.) c oncentrations, redox potentials (Eh), and pH is shown in Figure 3-4. D.O. concentrations dropped from 1.4 mg/L to < 0.4 mg/L (anoxic) within the first 24 h for all treatments. Eh dropped fr om +1 dV to < -4 dV within the first 24 h for all treatments, and remained negative during the duration of the study with the exception of the 5th day treatment which rose to positive levels near the end of the study. Negative Eh values indicated constant activity by the microbial popu lations as electron acceptors were reduced. The average pH for each treatment appeared to vary. The NO3 daily dosed treatment pH was consistently higher than the 3rd and 5th da y dosing treatments. Mean pH for each treatment over 17 days was 7.0, 5.9, and 5.7 for the daily, 3rd, and 5th day NO3-dosing treatments, respectively. Because the initial pH of all treatments was approximately 7.5, some type of buffering capacity reducing activities were appare nt in the 3rd and 5th day dosing treatments. The optimal pH for denitrification is approxi mately 7.0 (Aesoy 1998). The higher pH observed

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38 in the daily-dosed treatment is likely because denitr ification results in the recovery of alkalinity (calcium carbonate), and denitrif ication rates were much higher in the daily-dosed treatment followed by 3rd and 5th day treatments (Jeyan ayagam 2000). The water temperatures within each treatment were similar, averaging 29C. Effect of Carbon Availability (Pulsing) on NO3-N Removal Potential Because optimal NO3-N removal rates were not achiev ed with the treatments where NO3-N was dosed every 3rd or 5th day, those treatmen ts were not evaluated in this study. For the replicates maintained with saturated carbon, NO3-N removal continued on Days 17-34. However, NO3-N removal rates began to si gnificantly decrease after Day 21 of the study (Day 5 of the DOC evaluation). This decrease in NO3-N removal rates may be due to a variety of factors, including limited nutrients and co-factors not re plenished by the partial water changes. The estimated time required to remove 25, 50, 75, and 90% of the NO3-N load for the replicates maintained with saturated carbon during the pulsed carbon study is shown in Table 3-2. The reduction in nitrate removal rate is reflected in th e increases in the time needed to remove each respective portion of the NO3-N load. NO3-N removal was significantly reduced wh en carbon became limited. Because of this reduction, a slope analysis was not possible. This is expected since carbon is a necessary ingredient for the denitrificat ion process. Carbon is the en ergy source for the denitrifying bacteria (Hamersley 2002). In addition, NO3-N removal rates remained low even after restoration of saturated carbon conditions. This is important because in the field, steps will have to be taken to ensure that carbon levels do not become limited. Otherwise, the efficiency of the biofilter may be compromised. Figures 3-5 and 3-6 compare the amount of NO3-N reduced during the pulsed carbon study. Results depict how the saturated carbon treatment reduced significantly higher amounts

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39 of NO3-N (Figure 3-5) than the pulsed carbon treatmen t (Figure 3-6). These results confirm that carbon amounts of at least a 2:1 ratio with ni trogen must be maintained for optimal NO3-N removal to occur. Confirmation of NO3 Removal Rates under Optimal NO3 and DOC Dosing Scenarios A summary of the time required to remove 90% of NO3-N (25 mg/L) on a daily basis is shown in Figure 3-7. A lag phase of 6 days wa s observed during this study before achieving maximal NO3-N removal rates after 7 days. These maximal NO3-N removal rates persisted throughout the study (from 7-17 Days). These rates were also nearly twic e as fast as those observed during the summer 2004 studies. The op timal removal rate from 7-17 Days ranged from 5.7 to 6.2 mg NO3-N/h. At these rates, the estimated time required to remove 90% of the nitrate load ranged from 3.6-3.8 h (Table 3-3). Linear regressions for carbon consumption were also calc ulated during this study. On average a C:N ratio of 1.84:1 was needed during the spring 2005 confirmation study (Table 3-4). A C:N ratio of 7.8 was needed dur ing the total water exchange treatment from Day 16 to Day 17. The high C: N ratio seen duri ng this last treatment may ha ve been caused by the possible reduction of other electron acceptors such as iron, manganese, and sulfate, which may have been present in high quantities when the fresh ditch water was added to the system. A strong hydrogen sulfide smell was apparent from Day 16 to 17, in dicating high reduction ac tivity of sulfate (SO4) into hydrogen sulfide gas (H2S). Effect of Total Water Exchange on NO3-N Removal Potential Results demonstrated that performing 100% wa ter exchanges did not adversely affect NO3N removal potential during the 2-day evaluation. Total water exchanges a ppeared to slightly improve NO3-N removal rates.

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40 Conclusions Pulsing NO3-N negatively affected the lag phase fo r induction of denitr ification and the NO3-N removal rates in the bior eactors. Denitrifying microorgani sms are facultative anaerobes meaning that they can respire in both aerobi c and anaerobic conditi ons by using both O2 and NO3-N as electron acceptors (Jones et al. 2000). When both NO3-N and O2 are absent, denitrifying microorganisms cannot c ontinue their metabolic activitie s, because of the lack of a terminal electron acceptor needed by their el ectron transport chain during energy generation. Pulsing DOC in the bioreactors appeared to halt denitrification processes, due to the absence of carbon. Carbon is used by denitrifying bacteria for both an energy and carbon source. Without carbon, denitrification metabolic activi ties cannot function. Pulsing DOC prolonged the lag phase. In this case, the denitrif yers are either killed or could not continue to proliferate. The results in this study suggest that a carbon source cannot be absent from the biofilter system for NO3-N removal to occur. Maintaining constant optimal wate r temperatures of 29C to 30C inside the bioreactors may have sign ificantly increased the rate of NO3-N removal, and reduced the lag phase for denitrification induction. This indicates that re gions with lower temperatures, and a high degree of temperature fluctuations may have prolonged lag phases, and reduced NO3N removal rates. Performing water exchanges of the total water volume of the bioreactors does not appear to adversely affect the denitrifying microbial populations This opens the possibility of using the bioreactors as a pl ug flow system, in which they may be used to treat nursery runoff water at nurseries that irrigate or fer tigate on a daily basis. The DOC consumption after maximum NO3-N removal rates have been achieved indicated that sucrose was a highly efficient carbon source. Sucrose may be a very practical carbon source for NO3-N removal at plant nursery ope rations due to its relatively low cost as compared to more costly sources such as etha nol, acetic acid, glucose, and others (Aesoy 1998).

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41 Table 3-1. Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load for the daily NO3-dosing. Day T25 T50 T75 T90 Slope Intercept DOT 5.2 5.6 14.4 25.9 -0.03 1.90 1 3.8 6.6 11.3 17.6 -0.06 2.51 2 5.2 10.4 19.2 30.9 -0.03 2.41 3 4.1 6.5 10.7 16.2 -0.07 2.57 4 1.8 5.0 10.6 17.8 -0.05 2.37 5 3.8 7.1 12.7 20.1 -0.05 2.43 6 2.2 4.8 9.3 15.2 -0.07 2.44 7 2.9 5.5 9.9 15.7 -0.07 2.45 8 2.1 4.7 9.0 14.8 -0.07 2.40 9 3.4 7.2 13.6 22.0 -0.05 2.43 10 2.4 5.7 11.5 19.1 -0.05 2.44 11 2.6 6.3 12.7 21.1 -0.05 2.42 12 1.2 2.3 4.2 6.7 -0.16 2.57 15 1.5 2.7 4.8 7.6 -0.14 2.57 16 0.6 1.4 2.8 4.7 -0.21 2.37 Note: Slope and intercept apply to the linear re gression models using the log-transformed NO3N concentration data. Table 3-2. Predicted time intervals (h) to remove 25%, 50%, 75% and 90% of the NO3-N load for the daily NO3-dosing during the pulsed carbon study. Day T25 T50 T75 T90 Slope Intercept 17 0.3 0.9 2.0 3.4 -0.28 2.31 18 1.1 2.0 3.6 5.6 -0.19 2.40 19 1.2 2.1 3.6 5.7 -0.19 2.57 20 0.8 1.6 3.1 5.0 -0.21 2.45 21 0.8 2.1 4.4 7.5 -0.13 2.40 22 0.5 1.8 4.2 7.3 -0.13 2.35 25 1.0 2.2 4.2 6.9 -0.15 2.19 33 1.0 2.8 5.8 9.8 -0.10 2.44 34 1.2 3.0 6.2 10.4 -0.10 2.42 Note: Slope and intercept apply to the linear re gression models using the log-transformed NO3N concentration data.

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42 Table 3-3. Predicted time intervals (h) fo r removing 25%, 50%, 75%, and 90% of the NO3-N load for the daily NO3-dosing treatment for the spring 2005 study. Day T25 T50 T75 T90 Slope R2 (transf) 1 1.479 3.750 7.414 12.769 -0.078 0.997 2 1.063 3.003 6.135 10.710 -0.091 0.982 3 1.063 3.003 6.135 10.710 -0.091 0.982 4 1.201 3.116 6.207 10.723 -0.092 0.995 5 0.950 2.432 4.822 8.316 -0.119 0.998 6 0.969 1.944 3.517 5.817 -0.181 0.986 7 1.047 2.094 3.141 3.769 -5.970 1.000 8 1.036 2.073 3.109 3.731 -6.030 1.000 9 1.036 2.073 3.109 3.731 -6.030 1.000 10 1.028 2.056 3.084 3.701 -6.080 1.000 11 1.054 2.108 3.162 3.794 -5.930 1.000 12 1.096 2.193 3.289 3.947 -5.700 1.000 13 1.025 2.049 3.074 3.689 -6.100 1.000 14 1.054 2.108 3.162 3.794 -5.930 1.000 15 1.054 2.108 3.162 3.794 -5.930 1.000 16 1.008 2.016 3.024 3.629 -6.200 1.000 17 1.003 2.006 3.010 3.612 -6.230 1.000 Note: Slope and intercept apply to the linear re gression models using the log-transformed NO3N concentration data. Table 3-4. Linear regression co efficients, DOC consumption, and C:N ratios during optimal NO3-N removal period (Day 7 to 17). Days r2 Slope TOC consumption (mg/L) TOC consumption / day C:N ratio 7 9 0.87 -7777 38.5 1.54 10 14 0.85 -8080 20.0 0.80 15 16 1.00 -797979.0 3.20 Averages 0.91 -797946.0 1.84 Notes: Carbon consumption for the total water ex change treatment (Day 16 to 17) was 196 mg/L, C:N ratio of 7.8.

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43 Concentrations: Pulsing: NO3-N Replicates: Figure 3-1. NO3-pulsing experimental design. Concentrations: Pulsed NO3-N / Saturated DOC / Pulsed DOC 5 days after DOC depleted Figure 3-2. DOC-pulsing experimental design. 25 mg/L NO3N (Media) 1 day 3 day 5 da y DOC saturated (2:1 C: N ratio) 25 mg/L NO3-N 1 day 3 day 5 da y DOC saturated (2:1 C: N ratio )

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44 A0 10 20 30 40 50 60 70 80 90 10012345678910111213141516Days after treatment% removal l Daily 3rd day 5th day B-20 -10 0 10 20 30 40 50 60 70 80 90 10012345678910111213141516Days after treatment% removal l Daily 3rd day 5th day Figure 3-3. Percentage of NO3 removal during the summer 2004 studies. A) 8 h and B) 24 h after dosing with 25 mg/L of NO3-N and DOC constantly saturated.

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45 A 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.61 8 3 2 56 80 10 4 1 28 152 176 200 22 4 248 272 296 32 0 3 44 368 392Time (hours)DO (mg/ L daily 3rd day 5th day B -6 -5 -4 -3 -2 -1 0 1 2 3-1 8 32 56 80 10 4 128 152 176 200 2 24 2 48 272 296 3 20 344 368 392Time (min)Redox (d V daily 3rd day 5th day C0 2 4 6 8 10-1 8 32 56 80 104 128 152 176 200 224 248 272 296 320 344 368 392Time (min)p H daily 3rd day 5th day Figure 3-4. Summary of A) di ssolved oxygen concentrations. B) Redox potential. C) pH measurements during the summer 2004 studies.

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46 Figure 3-5. NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N during the summer 2004 studies with DOC saturated. 0 20 40 60 80 100 120 04824 Time (hours)NO3-N (mg/L 1-Aug 2-Aug 3-Aug 4-Aug 5-Aug 6-Aug 7-Aug 9-Aug 10-Aug 11-Aug 12-Aug 13-Aug 15-Aug 17-Aug 18-Aug Figure 3-6. NO3-N (mg/L) removed within 4 h, 8 h, and 24 h after dosing with 25 mg/L NO3-N during the summer 2004 studi es with DOC depleted. -10 0 10 20 30 40 50 04824Time (hours)NO3-N (mg/L 1-Aug 2-Aug 3-Aug 4-Aug 5-Aug 6-Aug 7-Aug 9-Aug 10-Aug 11-Aug 12-Aug 13-Aug 15-Aug 17-Aug 18-Aug

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47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 151234567891011121314151617Days after treatmentTime (hrs) for 90% NO3-N removal NO3-N removal (25 mg/L) Figure 3-7. Time required for removing 90% of 25 mg/L NO3-N during the spring 2005 confirmation study under optimal NO3 and DOC scenarios.

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48 Ditch 100% water exchange Figure 3-8. Native microflora harvesting apparatus. Return Holding tank Pum p s Biofilters Hoses Hoses Cage/pumps

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49 APPENDIX A FERTIGATION EVENT WATER VOLUME INPU T AT THE FOLIAGE NURSERY FLOW STUDIES. 1) SUMMER. 2) FALL Table A-1 Zone Emitter volume/zone (Liters) Emitters/zone Total volume/zone (Liters) 1 5.56576 3,203 2 4.352,208 9,605 3 5.31960 5,098 4 4.33264 1,142 5 5.60560 3,136 6 4.15960 3,984 7 3.902,340 9,126 8 3.202,688 8,602 9 4.38320 1,400 10 4.35960 4,176 11 3.751,400 5,250 12 4.43708 3,133 Total 57,853 Table A-2 Zone Emitter volume/zone (Liters) Emitters/zone Total volume/zone (Liters) 12.545761,463 22.051,1522,356 31.889601,800 42.17264573 52.536601,667 62.171,0602,300 71.752,3404,095 82.292,6886,142 92.594201,086 102.051,0602,168 111.921,4002,688 121.987081,398 Total 27,735

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50 APPENDIX B NO3-N LOADINGS APPLIED TO CROPS DUR ING THE FLOW STUDIES AT THE FOLIAGE NURSERY. 1) SUMMER. 2) FALL Table B-1 Table B-2 Zone Total water vol (Liters) NO3-N conc. (mg/L) Total NO3-N load (mg) 1 1,463 30.0 43,891 2 2,356 23.0 54,184 3 1,800 27.1 48,780 4 573 31.1 17,817 5 1,667 82.1 136,820 6 2,300 104.4 240,141 7 4,095 138.0 565,110 8 6,142 160.5 985,804 9 1,086 92.8 100,753 10 2,168 86.5 187,506 11 2,688 144.2 387,610 12 1,398 148.5 207,648 Total 27,736 Average: 89 +/52 2,976,064 =2.98 Kg Zone Total water vol (Liters) NO3-N conc. (mg/L) Total NO3-N load (mg) 1 3,203 41.0 131,305.0 2 9,605 56.0 537,868.8 3 5,098 46.0 234,489.6 4 1,142 23.0 26,261.4 5 3,136 41.3 129,516.8 6 3,984 29.5 117,528.0 7 9,126 110.5 1,008,423.0 8 8,602 182.4 1,568,931.8 9 1,400 42.0 58,800.0 10 4,176 28.7 119,851.2 11 5,250 111.3 584,325.0 12 3,133 110.3 345,558.9 Total 57,853 Average: 69 +/49 4,862,860 =4.86 Kg

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51 APPENDIX C NO3-N RUNOFF CONCENTRATIONS AT THE FOLIAGE NURSERY. 1) SUMMER. 2) FALL Table C-1 Time (min) Pipe 1 (m/L) Pipe 2 (mg/L) Pipe 3 (mg/L) 0 46.6 52.8 27.6 10 47.7 49.3 30.8 20 50.2 47.7 37.5 30 51.3 48.0 38.8 40 49.3 45.3 39.2 50 48.1 48.9 32.3 60 52.8 53.6 39.7 70 51.0 56.3 38.9 80 56.6 58.1 38.9 90 54.0 56.8 40.6 100 134.7 52.8 39.6 110 159.6 149.9 50.1 120 196.5 154.7 50.1 130 243.2 161.0 50.0 140 236.7 151.9 65.8 150 211.5 85.3 52.6 160 189.6 76.9 54.9 170 192.2 76.2 55.4 180 141.3 76.6 55.5 190 121.0 80.7 47.3 200 129.1 83.1 50.0 210 133.7 96.8 44.5 220 139.4 99.7 50.1 230 201.5 115.6 21.0 240 153.7 153.6 45.1 250 167.6 153.4 40.0 260 230.2 144.1 42.7 270 240.3 159.6 43.1 280 274.0 156.3 42.6 290 250.7 160.2 42.1 300 256.0 162.0 310 251.3 158.0 320 253.7 159.6 Average 152.0 102.6 43.6 Standard deviation 79.3 46.4 9.2 Minimum 46.6 45.3 21.0 Maximum 274.0 162.0 65.8

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52 Table C-2 Time (min) Pipe 1 (mg/L) Pipe 2 (mg/L) Pipe 3 (mg/L) 0 113.8 81.6 100.4 10 188.5 141.5 82.9 20 210.7 163.8 70.5 30 252.7 155.7 81.8 40 244.8 168.2 89.1 50 241.2 160.5 97.3 60 252.5 167.0 100.3 70 141.4 159.0 100.6 80 115.7 90.3 102.7 90 111.6 74.2 104.6 100 123.8 89.0 102.6 110 136.9 97.6 109.1 120 146.6 99.2 109.2 130 144.5 99.1 116.5 140 136.4 95.3 111.9 150 139.7 89.7 118.4 160 143.1 99.9 117.8 170 150.9 101.9 121.4 180 150.2 106.3 121.4 190 152.2 105.2 200 155.2 101.7 210 155.5 110.9 220 161.8 105.4 230 163.9 110.0 240 171.8 108.6 250 160.7 108.6 260 165.6 270 280 290 300 310 320 Average 164.1 115.0 103.1 Standard deviation 41.4 29.1 14.2 Minimum 111.6 74.2 70.5 Maximum 252.7 168.2 121.4

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53 APPENDIX D NO3-N LOADINGS APPLIED TO CROPS DUR ING THE FLOW STUDIES AT THE BEDDING NURSERY. 1) SPRING. 2) SUMMER Table D-1 Table D-2 Zone Total water volume (Liters) NO3-N conc. (mg/L) Total NO3-N load (mg) 1 3,524 2.13 7,505 2 2,874 1.98 5,691 3 3,493 1.65 5,763 4 3,193 2.91 9,291 6 2,810 3.16 8,881 Total 15,894 Average = 2.37 37,131 Zone Total water volume (Liters) NO3-N conc.(mg/L) Total NO3-N load (mg) 1 3,962 0.51 2,022.6 2 3,879 0.52 2,026.5 3 3,856 0.55 2,118.1 4 3,914 0.47 1,915.1 5 3,961 1.24 4,897.2 6 3,863 1.05 4,073.7 Total 23,435 Average = 0.73 17,053.2

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54 APPENDIX E NO3-N RUNOFF CONCENTRATIONS AT THE BEDDING NURSERY. 1) SPRING. 2) SUMMER Table E-1 Time (min) Pipe 1 (mg/L) Pipe 2 (mg/L) 0 20.6 3.2 10 20.3 1.7 20 19.1 1.6 30 16.0 1.7 40 18.7 1.6 50 21.3 2.0 60 20.4 2.1 70 23.8 4.2 80 26.3 7.6 90 21.4 10.1 100 24.1 11.6 110 12.3 15.1 120 15.6 15.7 130 15.0 16.0 140 14.3 15.3 150 15.6 17.9 160 16.3 19.7 170 13.9 18.7 180 14.3 16.7 190 12.6 15.3 200 14.8 16.3 210 17.3 16.7 220 17.9 11.3 230 19.1 13.1 240 14.7 13.8 250 19.6 14.8 260 16.4 270 19.2 280 19.5 290 19.5 300 19.8 310 19.6 320 13.2 330 340 350 360 370 380 390 400 Average 18.0 10.9 Standard deviation 3.4 6.4 Minimum 12.3 1.6 Maximum 26.3 19.7

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55 Table E-2 Time (min) Pipe 1 (mg/L) Pipe 2 (mg/L) 0 0.9 1.2 10 0.8 5.1 20 0.8 7.7 30 0.7 7.3 40 5.3 6.9 50 8.5 5.9 60 7.6 5.7 70 7.6 6.2 80 7.8 7.5 90 7.9 7.9 100 7.7 8.5 110 7.5 8.3 120 7.1 9.0 130 6.9 9.9 140 6.6 10.0 150 6.4 9.9 160 6.1 9.5 170 5.9 8.3 180 5.6 7.5 190 5.3 7.4 200 5.1 7.5 210 4.8 8.0 220 4.6 8.4 230 4.4 8.6 240 4.2 8.8 250 4.1 8.9 260 4.0 270 3.8 280 3.7 290 3.5 300 3.3 310 3.2 320 3.0 330 2.9 340 2.8 350 2.7 360 2.6 370 2.6 380 2.6 390 2.5 400 2.4 Average 5.3 7.7 Standard deviation 2.3 1.8 Minimum 0.7 1.2 Maximum 8.5 10.0

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56 APPENDIX F SUMMARY TABLE FOR WATER AND NO3-N LOADS FOR ALL FLOW STUDIES FNSummer FNFall BN Spring (Irr) BN-Summer (Irr) NO3-N input 4.80 Kg 2.98 Kg 17.05 g 37.1 g NO3-N runoff 3.00 Kg 1.99 Kg 111.42 g 51.2 g NO3-N runoff/acre 1.92 Kg 0.80 Kg 405.00 g 186.0 g NO3-N runoff % 62% 67% 665% 138% Water input 57,861 L 27,736 L 23,435 L 15,894 L Water runoff 20,935 L 13,668 L 7,523 L 8,190 L Water runoff % 36% 49% 32% 52%

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57 APPENDIX G DISSOLVED ORGANIC CARBON (DOC) RESULTS DURING THE SPRING 2005 NO3-N REMOVAL RATES STUDIES UNDER OPTIMAL DOSING SCENARIOS Days 1-DOC (mg/L) 1STDEV 2-DOC (m g/L) 2-STDEV3-DOC (mg/L) 3-STDEV Day of treatment 30229 48180679 79 Day 1 (3/16) 20470 396491671 312 Day 2 (3/17) 23922 327311501 154 Day 3 (3/18) 55536 929571371 104 Day 4 (3/19) 37946 611321224 61 Day 5 (3/20) 40312 626341331 102 Day 6 (3/21) 34230 510311193 88 Day 7 (3/22) 30027 457501208 73 Day 8 (3/23) 27526 396241128 100 Day 9 (3/24) 14626 25425995 105 Day 10 (3/25) 46749 19418816 59 Day 11 (3/26) 33463 17891090 38 Day 12 (3/27) 29192 172261071 86 Day 13 (3/28) 30020 791121923 43 Day 14 (3/29) 8428 32324990 37 Day 15 (3/30) 56543 480451145 58 Day 16 (3/31) 48644 42934242 21 Day 17 (4/1) 29087 2234975 37

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58 A y = 0.1261x2.7123R2 = 0.99860 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 01234567891011121314 Height (cm)Flow (L/min) B y = 0.4636x2.0732R2 = 0.99650 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30012345678910 Height (cm)Flow (L/min) APPENDIX H REGRESSIONS OBTAINED DURING THE CA LIBRATIONS OF THE V-NOTCH WEIRS AT THE FOLIAGE NURSERY. A) PI PE 1. B) PIPE 2. C) PIPE 3 APPENDIX H. Continued

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59 C y = 0.1681x2.4596R2 = 0.99020 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 012345678910Height (cm)Flow (L/min)

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60 APPENDIX I REGRESSIONS OBTAINED DURING THE CA LIBRATIONS OF THE V-NOTCH WEIRS AT THE BEDDING NURSERY Ay = 0.1854x2.8615R2 = 0.99670 10 20 30 40 50 60 70 00.511.522.533.544.555.566.577.588.59Height (cm)Flow (L/min) B y = 0.6924x2.2017R2 = 0.9920 1 0 20 30 40 50 60 70 00.511 .522.533.544.555.566.577.588.59Height (cm)Flow (L/min)

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61 LIST OF REFERENCES Aesoy, A., Odegaard, H., Bach, K., Pujol, R. and Hamon, M. (1998). Denitrification in a packed bed biofilm reactor (biofor)-experimen ts with different carbon sources. Water Research, 32 (5), 1463-1470. Alexander, S. (1993). Pollution control and pr evention at containeri zed nursery operations. Water Science & Technology, 28 509-517. Amos, R. (1993). Reduction of nitrates in nursery surface and ground waters. Combined Proceedings International Plant Propagators Society 43 244-248. Beschov, V., Velizarov, S.N., Agathos. and Lukova, V. (2002). Bacterial denitrification of wastewater stimulated by constant electrical field. Biochemical Engineering Journal, 17 (2), 141-145. Bigelow, C., Bowman, D. and Cassel, D. (2001) Nitrogen leaching in sand-based root zones amended with inorganic soil amendments and sphagnum peat. Journal of the American Society of Horticultural Sciences, 126 151-156. Casella, S. and Payne, W.J. (1997). Potential de nitrifiers for soil environmental protection. FEMS Microbiology Letters, 140 1-8. Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (1998). NO3N ultraviolet spectrophotometric screening method. In: Standard Methods for the Examina tion of Water and Wastewater, 20th edn, Washington DC, USA pp. 249-250. Constantin, H. and Fick, M. (1997) Influence of C-sources on the denitrification rate of a highnitrate concentrated industrial wastewater. Water Research 31 (3), 583-589. Cresswell, G.C. (1995). Improving nutrien t and water management in nurseries. Combined Proceedings International Plant Propagators Society 45 112-116. Dumroese, R., Wenny, D. and Page-Dumroese, D. (1995). Nursery waste water: The problem and possible remedies. National proceedi ngs, Forest and Conservation Nursery Association, Colorado, USA, p. 89-97. Escobar, F.R., Benlloch, M.,Herrera, E. and Ga rcia-Novelo, J.M. (2003). Effect of traditional and slow release N fertilizers on growth of o live nursery plants and N losses by leaching. Scientia Horticulturae 101 39-49. Hamersley, R.M. and Howes, B.L. (2002). Contro l of denitrification in a septage-treating artificial wetland: th e dual role of partic ulate organic carbon. Water Research, 36 (17), 4415-4427. Headley, T.R. (2001). The removal of nutrients from plant nursery irrigati on runoff in subsurface horizontal-flow wetlands. Water science and technology 44 77-84.

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62 Hume, N.P. (2000). Plant carbohyd rate limitation on nitrate re duction in wetland microcosm. Water Research 36 (3), 577-584. James, E.A. (1995). Water quality of stored and runoff water in plant nurse ries and implications for recycling. Combined Proceedings International Plant Propagators Society 45 117120. Jeyanayagam, S, Rodieck, A. and Husband, J. (2000). BNR 101. Water Environment & Technology, 12 (8), 85-88. Jones, L.M., Liehr, S.K.,Classen, J.J. and Roba rge, W. (2000). Mechanisms of dinitrogen gas formation in anaerobic lagoons. Advances in Environmental Research 4 13-139. Kariminiaae-Hamedaani, H., Kanda, K. and Kat o, F. (2003). Denitrifi cation activity of the bacterium Pseudomonas sp. ASM-2-3 isolated from the Ariake Sea tideland. Journal of Bioscience and Bioengineering 97 (1), 39-44. Mancino, C. and Troll, J. (1990). Nitrate and a mmonium leaching from N fertilizers applied to penncross creeping bentgrass. Hortscience 25 (2), 194-195. Menasveta, P., Panritdam, T., Sihanonth, P., Po wtongsook, S., Chuntapa, B. and Lee, P. (2000). Design and function of a closed, recirculating se awater system with denitrification for the culture of black tiger shrimp broodstock. Aquacultural Engineering, 25 (1), 35-49. Skrinde, J.R. and Bhagat, S.K. (1982). Industr ial wastes as carbon s ources in biological denitrification. J. Wat. Pollut. Control Fed., 54 (4), 370-377. Stephen, R. (2003). Reed beds cl ean up nursery run-off water. The Nursery Papers Issue n: 2003/05. Yeager, T.H. and Knox, G.W. (1991). Alternative irrigation strategies. The Woody Ornamentalist 16(2), 1-3. Yeager, T.H, Fare, D., Gilliam, C., Niemiera, A., Bilderback, T. and Tilt, K. (1997). Best management practices guide for pr oducing container-grown plants. Southern Nursery Association 1000 Johnson Ferry Rd. Suite E-130, Marietta, Georgia, 30068. Ziebarth, A. (1991), NF91-49, Well Water, Nitrates and the "Blue Baby" Syndrome Methemoglobinemia; Lincoln, NE; Universi ty of Nebraska Cooperative Extension.

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63 BIOGRAPHICAL SKETCH Miguel A. Mozdzen was born in a small village called el Pao, located within the Amazon basin, in Venezuela. Miguel lived and enjoyed his childhood within the wilderness while his father helped construct the thir d largest hydroelectric plant in the Americas. After his father returned to Chicago, USA (and later Florida) Miguel continued his fascination for the environment and natural sciences and after completing his active military service in the U.S. Army he attended the University of Florida a nd graduated in May 2003 with a Bachelors degree in Horticultural Sciences and in August 2007 earne d a Masters degree in Environmental Sciences from the Soil and Water Science Department, while he continued to serve in the U.S. Army Reserves. Miguel currently serves as a biologis t project manager for the U.S Army Corps of Engineers, Jacksonville District s Regulatory Division helping pr otect and conserve the nations aquatic environment. Miguels passion is his lovely wife and daughter.