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A dissertation Presented to The Graduate Faculty of The University of Louisiana at Lafayette In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy


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EFFECTS OF HERBIVORY AND COMPETITION ON GROWTH OF LEMNACEAE IN SYSTEMS FOR WASTEWATER TREATMENT AND LIVESTOCK FEED PRODUCTION



A Dissertation

Presented to

The Graduate Faculty of

The University of Louisiana at Lafayette

In Partial Fulfillment of the

Requirements for the

Degree

Doctor of Philosophy

Louis Landesman



Fall 2000

EFFECTS OF HERBIVORY AND COMPETITION ON GROWTH OF LEMNACEAE IN SYSTEMS FOR WASTEWATER TREATMENT AND LIVESTOCK FEED PRODUCTION



Louis Landesman



Approved:



________________________ ______________________

Mark Konikoff, Chair Robert G. Jaeger,

Associate Professor of Biology Professor of Biology

________________________ _______________________

Paul L. Klerks, Paul Richards,

Associate Professor of Biology Professor of Civil Engineering

________________________ _______________________

Nick Parker, Professor Lewis Pyenson

Texas Cooperative Fish and Dean, Graduate School

Wildlife Research Unit


Texas Tech University
EFFECTS OF HERBIVORY AND COMPETITION ON GROWTH OF LEMNACEAE IN SYSTEMS FOR WASTEWATER TREATMENT AND LIVESTOCK FEED PRODUCTION
Goals and Objectives
The main purpose of my research was to determine how species of Lemnaceae (duckweed) interact when growing in wastewater treatment systems and to see how grazing by fish (herbivory) effected the outcome of this interaction. Results of my research will help determine which species, or combination of species, of duckweed is best suited for use in wastewater treatment systems and which species will produce the most nutritious forage when grown on this wastewater. I conducted a series of experiments to answer the following questions:

1). For each of the duckweed species tested, Spirodela punctata, Lemna obscura, and Wolffia globosa, what concentration of treated wastewater produces the maximum quantity of leaf tissue (biomass)?

2). For each of the duckweed species examined, what concentration of treated wastewater produces the leaf tissue with the highest protein content?

3). How do these duckweed species respond to different factors such as aeration or light intensity?

4). What happens when these duckweed species grow together in media with varying concentrations of wastewater? How will the presence of competing duckweed species affect each species’ growth when compared to its growth in pure culture?

5). What happens when competing duckweed species growing in mixed cultures are exposed to herbivory? What effect will the presence of a grazer have on each species’ growth when compared to its growth when the grazer is absent?

6). Which equations can be derived from data on duckweed growth? What equations can I derive from the relationship between the nitrogen concentration of the media and the growth and protein content of duckweed leaves growing on that media?

Can these equations be used to predict the potential harvest and nutritional value of duckweed growing under field conditions?

7). Using my results along with those of other duckweed researchers, what management recommendations can I give to potential producers of duckweed for livestock feed or wastewater treatment?


ACKNOWLEDGMENTS

I begin by thanking my advisor, Mark Konikoff, whose patience and perseverance with my meanderings and uncertainties in my choice of a dissertation topic, allowed me to select a topic that I was able to complete in the time I had available. His constructive criticism and advice kept my doctoral program alive in spite of many obstacles and difficulties. I especially thank him for recommending me to Nick Parker who gave me the opportunity to do my research on duckweed at Texas Tech University. In Lafayette I need to thank my committee members, beginning with Bob Jaeger, whose advice and encouragement were so helpful over the years. Mike Walden, presently working at Virginia Polytechnic, gave me the opportunity to learn how to gather data for ecological model making and use it to construct a functional model with practical applications. I especially appreciated his sympathetic ear during a time of great financial and emotional stress. Paul Richards was kind enough to allow me to use the facilities of the Civil Engineering Department at The University of Louisiana at Lafayette while giving me valuable suggestions regarding the design and execution of my research. Paul Klerks also gave me useful feedback as to the design and implementation of my doctoral research. Staff members Penny Antley and Garrie Landry provided technical assistance in growing bacteria and identifying duckweed species.

Carroll Cordes was gracious enough to allow me to use the facilities at the National Wetland Research Center while John McCoy, Bill Rizzo and Ron Boustany helped me start my duckweed experiments there. I also need to thank the staff at the St. Martinville and Sunset Wastewater Treatment Plants, who allowed me to collect duckweed and water samples at my convenience. Fred Johnson of Lemna USA was kind enough to introduce me to his company’s duckweed based wastewater treatment facilities in Broussard and St. Martinville, Louisiana, while John Barber of Tulane University gave me some useful advise on growing and working with duckweed species.

Special thanks are due to Nick Parker, Unit Leader of the Texas Cooperative Fish and Wildlife Research Unit, who gave me the opportunity to conduct my duckweed research at the Unit’s facilities in Lubbock and New Deal, Texas. His patience, practical advice and unflagging encouragement always inspired me to do my best, regardless of the obstacles in my way. Equally helpful to me was Cliff Fedler of the Civil Engineering Department of Texas Tech University. He helped me to design my experiments and to devise models to explain my results. He also provided encouragement and assistance to seeing my work done in a timely manner. Ron Bremer of the Department of Information Systems and Quantitative Sciences was more than helpful in helping me to design valid experiments and to understand the intricacies of statistics and experimental design. Richard Strauss and Mike Willig advised me on the subtleties of ecological modeling, and I need to give special thanks to Scott Holaday and David Tissue for letting me use the facilities of the Biology Department at Texas Tech. Thayne Montague of the Dept. of Plant and Soil Science helped and continues to help me to measure light and temperature in the greenhouses where I work, and I also thank Ambika Sridhara, of the Dept. of Animal Science and Food Technology, who helped me a lot in running Kjeldahl analyses on my duckweed samples, and Brad Thornhill, who helped me in analyzing my water samples.

Several students assisted me during my experiments and they include Dave Carlson, Tommy Tarango, and Chris Busch. Yohannes Kamm was particularly helpful in assisting me with my experiments while Patrick Benedict got me started in devising a functional model of duckweed growth and nitrogen uptake. The following students also assisted me in numerous ways with the construction and maintenance of test facilities: Troy White, Scott Modderman, Wesley Bowen, John Griffith, Greg Crabtree, as well as others to numerous to list.

Elias Landolt of the Zurich Polytechnic in Switzerland was kind enough to identify the duckweed species I sent to him while Jan Vermaat of the International Hydrological Institute and Gideon Oron of the Ben-Gurion University of the Negev helped and advised me from afar. I would also like to thank Mohamed Abdul Bashar for sending me articles on duckweed research in Bangladesh and Gerald B. Carr of the University of Hawaii at Manoa for allowing me to use his slide of three duckweed genera.

Throughout the long period of my doctoral program my parents have assisted me both financially and emotionally, encouraging me to finish no matter what obstacles need to be overcome. My strongest and most important supporter and champion was and will always be my wife Marilyn, who stuck by me through thick and thin and whose unfailing support made this dissertation possible. This dissertation is as much her achievement as mine. God bless her for her many sacrifices in helping me to finish this work. Last but not least I need to thank my two sons Gustav and Benjamin who do not complain about my absences and love me for who I am.

Partial funding for this project was provided by the Higher Education coordinating Board of Texas through the Advanced Technology Program No. 164 to Nick C. Parker and Clifford B. Fedler. Additional funds were provided by the Texas Cooperative Fish and Wildlife Research Unit, the Department of Civil Engineering, and the Biology Department of Texas Tech University.




TABLE OF CONTENTS

Goals and objectives……………………………………………………….….…….…iii


Acknowledgments………………………………………………………….….…….….v
Table of contents………………………………………………………… .…..….…...ix

.

List of tables……………….………………………………………………….….….….xii


List of figures…………………………………………………………………..….….…xv
List of abbreviations……………………………………………………….………..….xix

.

Chapter I. Introduction..…………………….………………………… ……..…...1





  1. Duckweed botany…………………………………………….…….…..1




  1. Ecological importance of duckweed..………………………….….....4

Chapter II. Practical applications of duckweed…..………………..….…….…...6


a). As a new source of livestock feed……………....………………......6
b). As an alternative means of wastewater treatment….………….…..8
c). As a means of recovering heavy metals from wastewater.……....10
. d). As an inexpensive and accurate way of toxicity testing…………..10
e). Miscellaneous uses……………………………………………….…..11

Chapter III. Ecology and composition of duckweed from Lemna Int.

wastewater treatment ponds……..……………….……………..…..13
a). Ecology of the duckweed layer……..…………………………….....13

.

b). Species composition from St. Martinville and Sunset municipal



wastewater treatment ponds…..………………..………….….….....13
c). Protein content of duckweed from treatment ponds…………........15

Chapter IV. Duckweed and inorganic fertilizers………………………...……….17


a). Duckweed growth experiments on inorganic media …………..… 17
Methods……………………………………………………………..…17

Results……………………………………………………………..…..24


Discussion…………………………………………………………..…25

Chapter V. Duckweed and organic fertilizers …………………………….….....28


1). Duckweed growth on organic media in a growth chamber……...28

a). Duckweed growth in growth chambers…………………………..…33

Methods……………………………………………………………......33
Results………………………………………………………………....34

Discussion………………………………………………………..……40


b). Greenhouse experiments using organic media………………...…41
Methods………………………………………………………….….…41 Results…;;……...…………………………………………….…...…..49

Discussion………………………………………………………..……55


c). Effect of aeration on duckweed growth…………………..…….…..57
Methods………………………………………………………..………57

Results…………………………………………………………..……..58

Discussion……………………………………………………..………58
d). Effect of light intensity on duckweed growth………………......…..60
Methods…………………………………………………………..……60

Results………………………………………………………..………..61


Discussion……………………………………………………..….…...63

Chapter VI. Duckweed competition experiments.………………………...…......67

a). Concepts of competition theory………………………………..........67
b). Preliminary duckweed competition experiments in

Lafayette, Louisiana……………………………….……………..…...68


c). 1999 Greenhouse experiments………………………………..........69
Methods…………………………………………………....................71
Results…………………………………………………………….......72
Discussion…………………………………………………….….…...74

Chapter VII. Duckweed herbivory experiments……………………..……….…...79




  1. a). Concepts of herbivory theory……………………………...…….…...79



b). Duckweed grazing experiments…..……………...……….……....…80
Methods…..………………………………………………….…..….….80

Results………..…………………………………………………....…...84


Discussion……………………………………………………….….…..86

Chapter VIII. Modeling duckweed growth….……………..…………………...…….92


a). Benefits of modeling biological systems..….……....……………..…92
b). Model development experiments…..………………….…………..….93
Methods…….…………………………………………..………..…..….93



Results………..…………………………………………………..….….95
Discussion………………………………………………………...…….95
c). Model development………………………………………………...….95
d). Model verification……………………………………….……….….….98
e). Model application results.………..……………………….……..…….99
f). Growth chamber models………………………………….….....……106
g). Greenhouse models……..………………………………….….....….109

Chapter IX. Summary and recommendations to producers .………............…120

Summary......................................................................................120
Recommendations to producers ……………………...………...…130

Literature Cited…………………………… …………………………...….……...…...132

Abstract………………………………………………………….…...…..….……...….150

Biographical Sketch……………………………………………………..….….....…...152





LIST OF TABLES

Table 1. Fauna and flora found in the municipal wastewater

treatment plants at Broussard, St. Martinville and

Sunset, Louisiana employing duckweed in systems

designed by Lemna Technologies……………….…….…........….…16
Table 2 Individual leaf weight (frond or ramet) and occurrence of three

species of duckweed along with Azolla caroliniana collected in

South Louisiana. All weights are of wet, fresh plant material………......18

Table 3 Composition of Hoagland’s Medium…………………….……...........23

Table 4. Summary of growth rates of Lemna obscura and Spirodela

punctata in Lafayette, Louisiana………………………………..…...........27

.
Table 5. Growth chamber results for Lemna obscura, Spirodela



punctata and Wolffia globosa at high light intensities…......…..……......37

Table 6. Growth chamber results of Lemna obscura, Spirodela



punctata and Wolffia globosa at low light intensities……..........……….38

Table 7. Greenhouse growth responses for Lemna obscura and



Wolffia globosa in February 1999…………………….….............…….…39

Table 8. Greenhouse growth responses for Wolffia globosa

in April 1999……………………………………..…...……............….…….50

Table 9. Greenhouse growth responses for Lemna obscura

and Wolffia globosa in May 1999. ………………………..........………..50

Table 10 Greenhouse growth responses for Lemna obscura and



Wolffia globosa in June 1999……….…………………….............… …..51

Table 11. Percent protein in duckweed grown during February 1999............……53


Table 12. Percent protein in duckweed grown during April 1999……............……53

Table 13. Percent protein in duckweed grown during May 1999.…............…….54

Table 14. Percent protein in duckweed grown during June 1999….…............…..54

Table 15. Aeration experiment results for Lemna obscura and

Wolffia globosa during March 1999……………………..…..........…….59

Table 16. Greenhouse light level experimental results for Lemna



obscura and Wolffia globosa in June 1999…………………..…..….....64

Table 17. Outdoor light level results for Lemna obscura and



Wolffia globosa during September 1999………………..….....….….....65

Table 18. Percent protein in duckweed grown during June

light experiment………………………………………...………….......….65

Table 19. Pooled greenhouse competition results for Lemna



obscura and Wolffia globosa during the spring of 1999……...........…73

Table 20. Monthly greenhouse competition results for Lemna



obscura and Wolffia globosa during the spring of 1999…..…….…….75

Table 21. Growth in greenhouse of Lemna obscura in competition

with Wolffia globosa (present) or without competition

(W. globosa absent) at nine levels of TN..................................…....…76

Table 22. Growth in greenhouse of Wolffia globosa in competition

with Lemna obscura (present) or without competition

(L. obscura absent) at nine levels of TN.....................................…..….78

Table 23. Results from fish palatability experiment……………....…...............…...82


Table 24. Herbivory experimental results for Lemna obscura

and Wolffia globosa in outdoor tanks, summer 1999……......….....…..85

Table 25. Pooled herbivory experimental results for Lemna



obscura and Wolffia globosa in greenhouse tanks

September and October 1999…………..........…………………………85

Table 26. Herbivory experimental results for Lemna obscura

and Wolffia globosa in greenhouse tanks partitioned

by grazing, September and October 1999………………..........………87

Table 27. Average daily production (g/m2) of Lemna obscura

in flow-through cells receiving anaerobically digested cattle

manure at four concentrations of total nitrogen (TN)…………...........103

Table 28. RGR, PWG and mean daily gain (g/m2) Lemna obscura

in flow-through cells receiving anaerobically digested cattle

manure at four concentrations of total nitrogen (TN)……………...…103

Table 29. Regression equations and R2 values of three duckweed

species grown in environmental growth chambers…….……………..107

Table 30. Multiple regression analysis of 1999 greenhouse

experimental results…………………………………………...........…...107

Table 31. List of findings……………………………………………...…….....….…127



LIST OF FIGURES
Figure 1. Three genera of duckweed: Spirodela, Wolffia and Lemna……………………………………………………….........….2

Figure 2. Proportion of Lemna, and Wolffia biomass in the

Sunset Wastewater Treatment Plant from February

1998 to December 1999.......................………..………........…..19

Figure 3. Proportion of Lemna, and Wolffia biomass in the

St. Martinville Wastewater Treatment Plant from February

1998 to December 1999..................….………......……….……..20

Figure 4. Protein content of duckweed mixtures from Louisiana

Lemna Technology treatment ponds……………..………..........21

Figure 5. Duckweed biomass in a Lafayette, Louisiana,

greenhouse from October to December, 1997.

Duckweed growing on an inorganic media (Peter’s

Water Soluble Fertilizer) with a TN concentration of

15mg/L………………………………………………………........…29

Figure 6. Duckweed biomass in the NWRC growth chamber.

Duckweed growing on an inorganic media (Hoagland’s

medium) with a TN concentration of 350 mg/L………...…..........30

Figure 7. Lemna and Spirodela biomass in the NWRC growth

Chamber. Duckweed growing on dilutions of an

inorganic (Hoagland’s) medium. TN concentrations

were 0, 35, and 175 mg/L….………………………….......….…...31

Figure 8. Linear regression between TN content and percent ADCM in

tap water – ADCM mixtures…..…… …………………........…….36

Figure 9. Growth of Spirodela punctata on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to high light intensities at the top of the

growth chamber (34.8 W/m2)………………………............….….43

.
Figure 10. Growth of Spirodela punctata on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to low light intensities at the bottom of the

growth chamber (4 W/m2)………………..……………….........…44

Figure 11. Growth of Lemna obscura on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to high light intensities at the top of the

growth chamber (34.8 W/m2)……………….……............….…...45
..

Figure 12. Growth of Lemna obscura on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to low light intensities at the bottom of

the growth chamber (4 W/m2)………………...………….........…46

Figure 13. Growth of Wolffia globosa on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to high light intensities at the top of the

growth chamber (34.8 W/m2).……………….…….............……..47

Figure 14. Growth of Wolffia globosa on ACDM mixtures

with concentrations of 0, 4.4, 10.1, 18.7 and 35.8 mg/L

when exposed to low light intensities at the bottom of

the growth chamber (4 W/m2).…………...........……………...….48

Figure 15. Photograph of experimental greenhouse tank

arrangement for competition, aeration, light level,

and optimum wastewater TN concentration level

experiments……………………………………………..................56

Figure 16. Light effects on the growth of Lemna obscura and



Wolffia globosa in greenhouse tanks, June 1999…....…...........66

Figure 17. Duckweed competition trial on Lake Martin water in

Lafayette, Louisiana, TN 4 mg/L………………………...........….70

Figure 18. Biomass change of Lemna obscura when exposed to

fish grazing in outdoor tanks……..……………..........……….….88

Figure 19. Biomass change of Wolffia globosa when exposed to

fish grazing in outdoor tanks …………………………...........…..89
Figure 20. Biomass change of Lemna obscura when exposed to

fish grazing in greenhouse tanks……….….............…………..90

Figure 21. Biomass change of Wolffia. globosa when exposed to

fish grazing in greenhouse tanks………………………...........…91




Figure 22. Arrangement of the 16 linear troughs in groups of

four with one headtank (circular tank) per series

of four troughs…………………………………………............…100


Figure 23 Daily biomass gain of Lemna obscura cultured on

different ADCM mixtures with TN concentrations

varying from 0 to 54.6 mg/L between August and

November 1998……………………………..…….…............…..101

Figure 24. Graph of the model relationship to predict daily

biomass gain of Lemna obscura as a function of

total nitrogen (TN) concentration of the medium……………...102

Figure 25. Relationship between Lemna obscura leaf protein

content and the total nitrogen content (TN) of the

medium in mg/L………………………………………................105

Figure 26. Relationship between Wolffia globosa leaf protein

content and the total nitrogen content (TN) of the

medium in mg/L……………………………….…….............…..108

Figure 27. Non-linear regression lines relating the relative growth

rates (RGR) of three duckweed species to the media

total nitrogen content in the growth chamber, 1999.

Light intensity = 34.8 W/m2…..…………………...............…….110

Figure 28. Non-linear regression lines relating the relative growth

rates (RGR) of three duckweed species to the media

total nitrogen content in the growth chamber, 1999.

Light intensity = 4 W/m2….…………………………..................113

Figure 29. Non-linear regression lines relating the daily wet

biomass gain of two duckweed species to the media

total nitrogen content in the greenhouse, 1999…...................114

Figure 30. Non-linear regression lines relating the relative

growth rates (RGR) of two duckweed species to the

media total nitrogen content in the greenhouse, 1999…….….115


Figure 31. Non-linear regression lines relating the daily dry

biomass gain of two duckweed species to the

media total nitrogen content in the greenhouse, 1999…...…..116


Figure 32. Non-linear regression lines relating the daily

protein production of two duckweed species to the

media total nitrogen content in the greenhouse, 1999....….....117

Figure 33. Relationship between Lemna obscura relative growth

rate (RGR) and the total media nitrogen concentration,

and Julian date for the year 1999.……………..............……....118

Figure 34. Relationship between W. globosa relative growth rate

(RGR) and the total media nitrogen concentration, and

Julian date for the year 1999…...........………………………....119


LIST OF ABBREVIATIONS


ADCM…………..Anaerobically digested cattle manure
GLM…………… General linear model
MERV…………...Model evaluation and research verification
PAR……………..Photosynthetically active radiation, i.e. solar radiation between

400 and 700 nanometers wavelength. Usually measured in micro Einsteins per meter2 per second (mE/m2/second).


PVC....................Polyvinyl chloride
PWG… Percent weight gain, defined as the ratio of the fresh weight change divided by the initial weight divided by the length of the experiment in days.
RGR… Relative growth rate, defined as the natural logarithm of the final weight minus the natural logarithm of the initial weight divided by the length of the experiment in days.
SAS…………… Statistical Analysis System, developed by the SAS Institute,

Cary, North Carolina.


TN………………Total nitrogen in a solution expressed in milligrams per liter (mg/L).



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