AN ECOLOGICAL STUDY OF PHOTOAUTOTROPHS IN LAKE WORTH
by
Keren Bolter
A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment of the Requirements for the Degree of Master of Science
Florida Atlantic University
Boca Raton, Florida
August 2010
ABSTRACT
Author: Keren Bolter
Title: An Ecological Study of Photoautotrophs in Little Lake Worth
Institution: Florida Atlantic University
Thesis Advisor: Dr. J. William Louda
Degree: Master of Science
Year: 2010
Little Lake Worth (LLW) (800 m x 200 m x 8± m) is an artificially deep, monomictic marine basin. Pigments and other water quality parameters (O 2, pH, T, S,
- NO 3 . etc.) were utilized to characterize phototrophic communities and water quality.
The water column is dominated by diatoms except in the hypolimnon during stratification events (late Summer) when strong anoxia and H 2S favors abundant
Chlorobium sulfur bacteria. Results indicate nitrate-enriched freshwater baseflow indicative of septic tank seepage during the wet season. This also appears to lead to the accumulation of concentrated organic matter in the sediment. LLW is a potential threat to the health of the ecosystem and the humans using it recreationally. More research is required to verify the effectiveness of restoration options. The spatial and temporal distribution of Chlorobium (phaeovibiroides tent.) and their Bacteriochlorophyll-e homologues is described and compared to similar studies.
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DEDICATION
This manuscript is dedicated to my husband, Joe, to our sons, Roy and George, and to
my mom, Irit. I also dedicate this work to my wonderful advisor, Dr. J. William Louda,
and the rest of my committee, Drs. Tara Root and David Warburton. A special thanks to
the esteemed colleagues in the lab, Cidya Grant and Pam Mongkhonsri.
.
.
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Table of Contents
List of Tables ...... vii List of Figures ...... vii 1. Introduction...... 1 Objectives ...... 2 Human-Environment Interaction ...... 2 Bacterial and physiochemical considerations ...... 3 2. Study Area ...... 6 General History and Information: South Florida and the Everglades ...... 6 Palm Beach County ...... 9 Palm Beach County Climatology ...... 9 Population Trends and Statistics ...... 11 Land Use in PBC ...... 12 Lake Worth Lagoon ...... 13 History of Lake Worth Lagoon (LWL) ...... 14 Altered Hydroperiod ...... 15 Threat from Septic Systems ...... 16 Site Description: LLW ...... 19 3. Background ...... 21 Lake Classification ...... 21 Trophic Level ...... 21 Lake Stratification Classes ...... 22 Stratification in Saline Lakes ...... 24 Phototrophic Bacteria ...... 24 Photosynthesis: Oxygenic vs. anoxygenic ...... 24 Light and Depth ...... 26 Purple and Green Sulfur Bacteria ...... 26 The Chlorosome ...... 28
iv
Changes in GSB Composition as Photoadaptation to Changes in Light Intensity ...... 29 Carotenoids, Chlorophyll, and Bacteriochlorophyll Pigments ...... 30 Carotenoids...... 31 Chlorophylls ...... 35 Bacteriochlorophylls ...... 36 Breakdown - Senescence ...... 39 Chromatography ...... 41 4. Materials And Methods...... 42 Sampling Procedures ...... 42 Field Measurements and Water Sample Collection ...... 42 Water Sample Treatment ...... 43 Sediment Sampling, Storage, and Retrieval ...... 44 Pigment analysis ...... 45 Extraction Methods ...... 45 Ultraviolet-visible (UV/VIS) spectroscopy ...... 46 HPLC - High Performance Liquid Chromatography ...... 47 Analysis of HPLC Data ...... 48 HPLC Data Calculations ...... 49 Verification of Results ...... 50 5. Results And Discussion ...... 52 Water Column Studies ...... 52 Light Intensity ...... 58 Temperature and Oxygen ...... 59 Specific Conductance ...... 62 Rainfall and Density ...... 64 Nutrients, Chlorophyll, and PHEOs ...... 68 pH ...... 70 Chlorobium ...... 71
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Chlorobium Carotenoids ...... 74 BCHL-e Homologues ...... 76 Internal Seiches ...... 80 Sediment Studies ...... 80 Organic Matter (OM) ...... 81 Carotenoids...... 82 BCHL-e Homologues ...... 84 Accumulation ...... 85 6. Conclusions...... 86 Human-Environment Interaction ...... 87 Restoration Possibilities ...... 88 Bacterial and physiochemical considerations ...... 90 Chlorobium Homologues ...... 90 Chlorobium Carotenoids ...... 90 Applications and Further Research ...... 91 Human Environment ...... 91 Chlorobium Homologues ...... 92 7. References ...... 93 8. Appendix ...... 103 A1. Mechanism for Summer stratification in LLW ...... 103 A2. Retention times and UV/VIS (PDA) spectral data for chlorophylls, chlorophyll derivatives, carotenoids and scytonemin. For long (C18) column...... 103 A3. Normalized absorption spectra of chlorophylls -a and -b and bacteriochlorophylls -a, -b, -c, -d and -e in polar organic solution...... 108
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List of Tables
Table 1. Phosphorus and Chlorophyll Concentrations and Secchi Disk Depths Characteristic of the Trophic Classification of Lakes ...... 22 Table 2. Carotenoids in Chlorobium phaeobacteriodes with end group structures ...... 33 Table 3. Changes in the ratios of solvents throughout the 50 minute HPLC process...... 48 Table 4. Sediment core analysis results per section from LLW in September 2009...... 82
List of Figures
Figure 1. General locations of the major landscape types in the Everglades prior to human intervention ...... 6 Figure 2. The current main features of the South Florida environment ...... 8 Figure 3. Location of Palm Beach County ...... 9 Figure 4. Average climate in Palm Beach, Florida ...... 10 Figure 5. PBC population growth ...... 11 Figure 6. Land Uses in Palm Beach County ...... 12 Figure 7. Lake Worth Lagoon ...... 13 Figure 8. Onsite Sewage Treatment Systems in the northern section of LWL ...... 17 Figure 9. Bathymetric Plot of LLW ...... 20 Figure 10. Thermal classification scheme ...... 23 Figure 11. Absorption spectra of whole cells and acetone pigment ...... 30 Figure 12. β-carotene and Fucoxanthin/Fucoxanthinol structures...... 32 Figure 13. Hypothetical coupling between xanthophyll-cycle pools ...... 33 Figure 14. Structure of carotenoids and postulated carotenogenesis pathway in Chlorobium phaeobacteriodes ...... 34 Figure 15. structures of CHLs –a, -b, -c1, and -c2...... 36
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Figure 16. Chlorobium cell cross section ...... 37 Figure 17. Structural differences in BCHL-e homologues ...... 39 Figure 18. Structures of CHLs -a, -b, and -c, and their breakdown ...... 40 Figure 19. Image of LLW revised from Google maps ...... 43 Figure 20. Time average (Nov. 2008 – May 2009) chemotaxonomic estimate of phytoplankton community structure in LLW water column ...... 53 Figure 21. Profile of spatial and temporal chlorophyll-a concentrations ...... 54 Figure 22. Spatial and temporal chlorophyll-a breakdown (PHEOS) concentrations...... 55 Figure 24. Spatial bacteriochlorophyll-e profile for June and July 2009...... 56 Figure 23. July Dissolved Oxygen and Sulfide profiles...... 56 Figure 25. UV-Vis spectrum of sample taken from 7 meters on June 23, 2009...... 57 Figure 26. UV-Vis spectrum of a typical epilimnion sample extract...... 57 Figure 27. Spatial and temporal log of light intensity profile...... 58 Figure 28. Temperature profile throughout depth for 2009 ...... 60 Figure 29. Spatial profile of temperatures in June and July, during LLW stratification. .. 60 Figure 30. Spatial profile of changes in temperatures in June and July...... 60 Figure 31. Spatial profile of dissolved oxygen and changes in temperatures...... 61 Figure 32. Spatial profile of dissolved oxygen percent saturation, calculated from temperature and salinity at that depth...... 61 Figure 33. Spatial profile of SpC in LLW 2009...... 63 Figure 34. Water quality measurements over time (at station LWL-1, PGA Blvd.) ...... 64 Figure 35. Rainfall data over time at station S-44 (northern LWL) ...... 65 Figure 36. Spatial and temporal density profiles of LLW ...... 66 Figure 37. Nitrate Profiles for selected months in LLW ...... 67 Figure 38. Rainfall over time at station S-44 on Canal C-17 (northern LWL) ...... 68 Figure 39. Dissolved oxygen (DO), chlorophyll-a, and its breakdown measure “PHEO-a” over time (at station LWL-1, PGA Blvd.)...... 69
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Figure 40. Nutrients and chlorophyll over time at station LWL-1, PGA Blvd ...... 69 Figure 41. CHL-a and PHEO-a over time at station LWL-1, PGA Blvd ...... 70 Figure 42. The spatial and temporal pH variations in LLW...... 71 Figure 43. The BCHL-e homologues ...... 72 Figure 44. Chromatograms from Borrego et al. 1997 and from LLW June at 6 m ...... 74 Figure 45. Spectrum of trans -isorenieratene from a sample taken July at 7 m ...... 75 Figure 46. carotenoid/BCHL-e ratios throughout the metalimnion and hypolimnion in June and July ...... 76 Figure 47. Progression of secondary homologues, BCHL e2-e4 in basin III at a deeper part of the Lake Banyoles (max depth: 20 m), and BCHL e2-e4 in basin IV, a more shallow region (max depth: 15 m) ...... 78 Figure 48. BCHL-e homologue distribution in water column in June and July...... 79 Figure 49. Carotenoid composition in sediment core sections ...... 83 Figure 50. Algal taxonomy in sediment core sections ...... 83 Figure 51. BCHL-e Homologues in LLW Sediment Core Sample ...... 85
ix
1. Introduction
Human alteration of Earth is substantial and growing.
Our use of land alters the structure and functioning of
ecosystems, and it alters how ecosystems interact with the
atmosphere, with aquatic systems, and with surrounding
land.
- Vitousek, 1997
Florida's ecosystems have been severely altered by human development and
urbanization in the past century. Impacts include habitat loss, pollution, and changes to
hydrological flow. Historically, South Florida was dominated by wetlands, and the
Everglades were aptly nicknamed the “River of Grass”, describing the natural
southbound flow of water from Central Florida. In the early to mid 1900’s, extensive
dredging and draining was done to allow agricultural and urban development (Meindl
2003). This “reclamation” process was a complicated issue, as there were many failed
attempts to cut off and control the water flow in a way allowed the land to be
developed, and therefore valuable.
The focus of this study is a marine basin that was dredged to provide fill for
surrounding development in the 1940’s. The rectangular (800 m × 200 m) Little Lake
Worth (LLW), located in densely populated eastern Palm Beach County, was deepened
1
from about 2 meters to about 8 meters and connected to the saline Lake Worth Lagoon
(LWL). Historically, intense agricultural practices have been sending runoff with high
amounts of nutrients from fertilizers and animal waste to LWL. In recent decades,
upscale residential areas surrounding LWL have contributed to the nutrient overload
through sewage seepage from septic tank overflow and faulty septic systems,
particularly during the wet season (D. Meerof, unpublished data - personal
communication). These excess nutrients led to severe eutrophication events (FDEP
1997).
In 2000, samples taken from LLW and their subsequent analysis revealed the
existence of sulfur bacteria in the lower portion of the lake, but no futher study, until
now, was performed. (J.W. Louda, FAU Department of Chemistry and the Environmental
Sciences Program, unpublished data - personal communication). Given the depth of the
basin and the restricted circulation, one might expect a stratification to occur.
Objectives
The two main purposes of this study are 1) to explore the implications of human impacts on LLW and the surrounding environment, and 2) to correlate the physiological characteristics of the primary producers to the physiochemical conditions of the lake.
More specifically, the key objectives are divided into those two subgroups to answer the following:
Human-Environment Interaction
• What are the main anthropogenic inputs to Little Lake Worth?
2
• How does weather, particularly hurricanes, tropical storms, and extreme
wet seasons, affect physiological and physiochemical characteristics in
Little Lake Worth?
• What are potential benefits of restoring Little Lake Worth?
• Is reduction of incoming pollutant loads, active management to bury
existing polluted sediments (adding fill to the lake), or both more
important for restoration?
Bacterial and physiochemical considerations
• Is there a relationship between light availability and bacterial productivity
in LLW?
• What physiochemical changes, (other than temperature) occur if/when
stratification occurs?
• Which physiochemical parameters have the most impact on the
stratification of primary producers?
Stratification and anoxia events are natural in many cases. However, their occurrences around the world are increasing parallel to urban and agricultural development (Sklar 1998). The output of excessive nutrients from fertilizers and waste cause algal blooms, and initially there is a spike in dissolved oxygen due to increased photosynthesis. However, the bloom is short lived, and the bacterial decomposition of the pollutants and dead algae can deplete oxygen from the water body. This process, described as the oxygen sag curve, is characteristic of eutrophication. With sufficient 3 mixing, dissolved oxygen can be quickly replenished throughout the water column, but this is not the case when stratification exists. When sufficient depth and a corresponding temperature gradient have caused the water to separate into layers, an oxycline develops below which the water remains anoxic for the duration of the stratification. The top layer, termed the epilimnion, mixes within its boundaries, but not with the isolated, but mixed hypolimnion below. The area in between these strata is the metalimnion, as illustrated in Appendix 1.
As anthropogenically induced stratifications and anoxia are becoming more prevalent, the results of this study should be referenced for determining the most efficient way to avoid, decrease, or reverse persistent stratification. The results of this study should also assist government agencies’ management efforts to improve water quality, sustain flora and fauna, and improve the recreational value of for Little Lake
Worth. The physiochemical and bacterial data collected from Little Lake Worth should contribute to the limited information about the in situ restructuring of complex bacterial composition of as a response to environmental conditions.
Bacteria and algae are important indicators of ecosystem health for many reasons. The presence of bacteria that require anaerobic conditions indicate anoxia, and consequent threats to species with a high oxygen demand. They respond rapidly and predictably to a wide range of pollutants, providing useful early warning signals of deteriorating conditions and the possible causes (McCormick 1994). Since primary producers start the food webs, effects of eutrophication travel up to affect all aquatic life. Economic losses due to long term algal blooms include harm to fisheries, impeded
4 recreation, and potential contamination to drinking water (Havens et al. 1996). Since recreational activities such as windsurfing and fishing are a highlighted asset for the area's residents and tourists, it is important to ensure the water quality is not impaired.
If waste and sewage is getting into Little Lake Worth, it can bring pathogens, posing health threats to humans from bathing in the lake or from eating seafood from the area.
A comprehensive synopsis of Little Lake Worth’s recent history and current conditions will illustrate the intricacy of the unique environmental impacts from anthropogenic manipulations. We will write a manuscript for a journal to ensure the availability of the findings.
5
2. Study Area
General History and Information: South Florida and the Everglades
Historically, the Everglades marsh covered 10,000 km 2 in a 100-km-long basin
with an extremely low gradient (slope of only 3 cm · km -1). The region was a mosaic of
wetlands, uplands, estuarine, and marine ecosystems interconnected through the
regional hydrology (Obeysekera et al. 1999). The historic drainage basin’s hydrologic
features allowed surface and subsurface freshwater to flow freely from the meandering
Kissimmee River and its floodplain through Lake Okeechobee, and down to the
Everglades. The Everglades consisted of sawgrass plains and ridge and slough landscape,
Figure 1. Predrainage Everglades: General locations of the major landscape types in the Everglades prior to human intervention (USGS 50 km 2004).
6 with a peat-based system of dense sawgrass ridges with soil surfaces roughly 2 to 3 feet higher than adjacent and relatively open sloughs (Enright, 2004). The outflow went through Florida Bay to the Florida Keys and out to the reefs. The Atlantic Ocean was separated from the hydrologic flow on the east coast by the Atlantic Coastal Ridge.
Figure 2 shows how drastically the landscape has been changed by humans in the past century. The natural sheetflow and hydroperiod are no longer possible due to the series of canals and impoundments.
In the early 1900s, the land in South Florida went through a long and arduous process of digging and dredging for the purpose of making it functional for real-estate and farmland. The United States went through a long period in which wetland removal was not questioned. Indeed, it was considered the proper thing to do. While activists like Muir and Thoreau were standing for “wilderness,” they were working to conserve far different environments. A Miami Herald article in 1911 illustrated the mood of many people during this era, "Wetlands…as they are, are without value. In fact, they are a menace to health, being breeding places for malaria-carrying mosquitoes..." There were no legal environmental restrictions, and the ecological impacts were not taken into account (Meindl 2003).
7
LLW
Figure 2. The current main features of the South Florida environment (Ollis and Redfield 2010).
8
Palm Beach County
Palm Beach County (PBC) was created in 1911 and named after its palm trees and beaches (Figure 3). These palm trees sprouted from 20,000 coconuts that spilled onto the barrier island in the wreck of the Spanish ship, Providencia, in 1878 (Oyer
2001). Palm Beach County (PBC) is the state’s largest and wealthiest county with a land area of 5,113 km² (1,974 mi 2) and a median household income of $52,807, and a population estimated at 1.28 million (U.S. Census Bureau 2008). PBC joins Miami-Dade and Broward County to form the South Florida Metropolitan Area. The two most densely populated cities in PBC are West Palm Beach and Boca Raton.
Palm Beach County Climatology
Palm Beach County exceeds the national averages in terms of temperature, precipitation, and humidity (Figure 4).
N
200km
Figure 3. Location of Palm Beach County, from www.sunshinereview.org/index.php/Palm_Beach_County,_Florida.
9
Figure 4. Average climate in Palm Beach, Florida (City-Data).
The popular Köppen's scheme of climate classification defines a tropical moist climate as a non-arid climate in which all twelve months have mean temperatures above
18 °C (64 °F) and annual rainfall is large and exceeds annual evaporation (Bailey 1989).
Over a 30 year span (1971-2000), the lowest normal daily mean temperature in PBC was
19 °C (66.2 °F), occurring in January. For this period, the mean annual precipitation was
60.7 in (156 cm) (NOAA 2002).
10
Population Trends and Statistics
South Florida experienced an exponential increase in population in the 20 th century; the concurrent population increase in PBC is shown in Figure 5. However, there has been a 13% decline since 2000, possibly due to increased unemployment and the unstable housing market. The coastal areas of PBC are heavily developed and populated, and population density decreases inland. While many multi-million dollar single family homes along the intracoastal take up several thousand square meters, there are numerous high-rise condominiums that contribute to the elevated density in these areas. According to the 2000 census, PBC’s population density is 3856/km²
(1489/mi²). Within the 18 km² (7 mi²) that encompass the zip code for the coastal area surrounding Little Lake Worth, 33408, there were 17086 people in 2000 resulting in a population density of 7049/km² (2722/mi²) (U.S. Census Bureau 2009). This area is almost twice as populated per unit area as the county's average.
PBC Population Growth
14 12 10 Figure 5. PBC 8 population growth 6
(data from Forstall x people100000 4 2005 and U.S. Census 2 Bureau 2009). 0 1900 1920 1940 1960 1980 2000
11
Land Use in PBC
As population density increases towards the east, the land use becomes mainly
residential and commercial. Meanwhile, further west, there is a dominance of wetlands,
forest, and agriculture (Figure 6).
LLW
N
10 km
Figure 6. Land Uses in Palm Beach County from Florida Department of Environmental Protection (DEP) (2005).
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Lake Worth Lagoon
Lake Worth Lagoon (LWL) is the principal estuarine water body in Palm Beach
County (Figure 7). It is about 32 km (20 miles) long, 0.8 km (0.5 miles) wide, and
averages 2.4m (8 feet) deep.
N
10 km
Figure 7. Lake Worth Lagoon (FDEP 1997).
13
The main environmental issue is the freshwater runoff from the canals that drain into the lagoon. This runoff causes salinity fluctuations, excessive suspended matter, reduced water clarity, a high rate of sedimentation and nutrient loading (Crigger et al.
2005).
History of Lake Worth Lagoon (LWL)
LWL was historically a freshwater lake with inflow from wetlands along its western edge. A barrier island separated it from the Atlantic Ocean (Crigger et al. 2005).
In the late 1800s, when the first pioneers settled on the banks of LWL, they tried to connect the estuary to the ocean. After many failed attempts, a stable inlet to the
Atlantic Ocean was finally constructed in 1877, and later a southern inlet was established. In the 1890s, a navigation canal from the north end of LWL to Jupiter Inlet was dug, increasing freshwater discharges from the Loxahatchee River (FDEP 1999). In
1925, the West Palm Beach canal (C-51 Canal), was completed and began discharging
freshwater from the drainage and development of wetlands to the west (FDEP 1999).
The series of canals brought runoff from sewage and fertilizer that degraded the
water quality. In an attempt to improve water quality, 500,000 m 3 were dredged in
1940. This did little to help, because by 1950, about 10 million gallons of raw sewage were being discharged into the lagoon annually (FDEP 1997).
In Palm Beach County, several lakes around the Lake Worth Lagoon (LWL) were deepened from 1-2 meters to 7-9 meters, to provide fill for surrounding low lying areas.
More than 20 dredge holes still exist in the northern and central portions of the lagoon
(PBCERM 2003). The increased depth prohibits submerged aquatic vegetation (SAV),
14 such as seagrass and mangroves, which are important as nursery areas for the juvenile aquatic fauna (PBCERM 2010). This impact combined with increased pollution inputs drastically reduced fish populations during LWL development (Woodburn 1961). In
1940, the earliest seagrass survey found 17.3 km² cover in LWL. While the reliability of the survey is not solid, a survey in 1975 found only 0.65 km² of seagrass in the lagoon
(PBCERM 1998). This is a devastating decrease even if the 1940 figures were inflated.
Dames and Moore (1990) performed a survey which indicated that there were 8.5 km² of seagrass, indicating there had been much improvement. Mangroves serve to buffer the land from erosion, particularly during violent storms. LWL has experienced an 87% decrease of its mangrove acreage over the past 40 years, with 1.1 km² mangrove cover remaining (FDEP 2010). To facilitate and protect construction, the natural shoreline of
LWL was replaced by artificial seawalls. Currently, over 81% of the shoreline in LWL is bulkheaded (PBCERM 2007).
LWL ranges in depth from 0.3 m to 3.0 m. Most of the shallow areas are in the southern half, but the northeast has the bulk of the seagrass in some key shallow areas.
The reduced shallow areas along the North Palm Beach and West Palm Beach shorelines lead to less biological productivity, because it is more difficult for SAV to establish with
reduced light (PBCERM 2008).
Altered Hydroperiod
Many problems arose from the transition of LWL from a freshwater lagoon to a saltwater estuary. The variations in runoff that occur in the transition from wet seasons to dry seasons cause extreme fluctuations in salinity (CERP 2004). Current data shows
15 that from 1998 to 2003, around 1.36 billion liters per day were discharged into LWL from the C-51 canal during the summer months (CERP 2004). This variability of freshwater influx has implications for aquatic life that is sensitive to salinity levels
(Doering et al. 2002). Discharges during the winter are 43% lower but likely with higher per liter concentrations of pollutants (PBCERM 2006).
Runoff becomes more polluted as continued development of the highly urbanized coastal area increases the watershed’s impervious surface area (ISA). Most sediment has the ability to filter pollutants through percolation and aerobic metabolism, but this process is prevented due to flow increases if there are too many ISAs (Brabec et al. 2002). Untreated storm water (non-point source pollution) runs directly into the
lagoon or its tributaries, adversely impacting water quality. These unfavorable
conditions exist because most development of the LWL watershed occurred prior to the
creation of any storm water regulations.
Threat from Septic Systems
Continued dependence on aging and poorly sited septic tanks and drainfields (or other on-site sewage disposal systems, OSDS) in coastal counties may cause excess loading of nutrients and microbes to estuarine waters (Paul et al. 2000).
Dr. Daniel Meerof of FAU’s Engineering Dept. has been working with the Health
Department to isolate and classify sites that are still using septic tanks. The resulting
map (Figure 8) indicates that nearly every home around the lake is still on septic
systems, and most onsite sewage treatment and disposal systems are clustered around
the north end of LWL.
16
Little Lake Worth
N
5 km
Figure 8. Onsite Sewage Treatment and Disposal Systems in the northern section of LWL (Florida Dept. of Health 2009) .
17
The non-point pollution from the overflow of septic tank drainage fields is a serious problem. In Lake Worth Lagoon, during the wet season the water table can rise above the drainage fields, thus untreated sewage and potentially contaminated waste is leeched into the sediment (Dr. Daniel Meerof, unpublished data - personal communication). Tide induced water table oscillations contribute additional water during the flood, making coastal areas even more vulnerable to these threats (Li 2000).
There are human health hazards as well as threats to the ecosystem from the pathogens and organic matter (OM) in the leachate (Boesch 2002). Pathogens such as
Cryptosporidium and Giardia may cause infection through incidental ingestion of environmental waters during recreational swimming (Eisenberg et al. 2002). Humans
can also contract waterborne diseases by ingesting contaminated shellfish, in which
viruses and other pathogens may have bioaccumulated (Lipp et al. 2001). Symptoms of these pathogens can be infectious diarrhea, vomiting, or more severe gastrointestinal illnesses (Eisenberg et al. 2002).
Excess nutrients (nitrogen and phosphorous from OM) can significantly impact water quality causing anoxia, hypoxia, eutrophication, nuisance algal blooms, dieback of seagrasses and corals, and reduced populations of fish and shellfish (Howarth 1993).
The OM in sewage has extremely high biological and chemical oxygen demand (BOD and
COD) that can get up to 118,000 mg/L (Flanagan and Charles 1979). This is because microbes decompose the OM by oxidizing it, thus severely reducing dissolved oxygen. If the water body is shallow and well-mixed, the consequences are less severe as oxygen
18 can be replenished to the lower water column. Little Lake Worth has poor conditions for mixing, as will be described in the next section.
Site Description: LLW
Little Lake Worth was named when it was enclosed and had freshwater. Since it
is now connected to the Atlantic Ocean, it would more precise to call it a marine lagoon.
However, the name will be kept consistent and “lake” will be used from herein out in
text. LLW is located north of PGA Blvd. in Juno Ridge, Palm Beach, FL 33408 at 80°03.50
N and 26°51.00 W. LLW is unique because it is unnaturally steep and deep (Figure 9),
which is the first trigger that makes it susceptible to stratification and, as previously
discussed, hypoxia. The narrow and shallow inlet from LLW to Lake Worth Lagoon runs
under PGA Blvd. The bathymetry of LLW does not allow for a littoral zone, so there is no
submerged aquatic vegetation (SAV). Instead, a thick black anaerobic substrate called
muck accumulates, and this will be described in Core Analysis section of the Results and
Discussion.
Dr. J.W. Louda (FAU Department of Chemistry and the Environmental Sciences
Program) sampled LLW in 2008 while studying chlorophyll-a degradation (see Szymzak-
Zyla et al. 2008). Pigment analysis of these samples with High Performance Liquid
Chromatography indicated the likely presence of green sulfur bacteria. The presence of
reducing conditions and anaerobic bacteria in a lake, which in its natural condition
should be shallow, warm and mesotrophic, served as the impetus for this thesis
research.
19
Figure 9. Bathymetric Plot of LLW modified from Morgan and Eklund Inc. professional survey consultants. Contours represent depth below median stage in feet.
20
3. Background
Lake Classification
To understand and assess any type of stratification, a lake’s classification must first be considered. Lakes are characterized and categorized by trophic level, morphometric characteristics, and by occurrences of thermal stratification. Lake structure and ecology are generally described for naturally occurring temperate lakes
(Wehr and Sheath 2003). In South Florida, many canals and lakes were artificially dredged and thus do not fit in the traditional classification system for natural lakes.
Trophic Level
Classification of lakes in term of nutrients, such as nitrogen (N) and phosphorus
(P), ranges from oligotrophic lakes that have very low nutrients to nutrient rich eutrophic lakes that tend to have short-lived algal blooms. Mesotrophic lakes have moderate nutrients resulting in intermediate productivity. The concentrations of nutrients defining these categories vary and overlap (Table 1). During stratification, the concentrations of N and P tend to vary with depth, as the upwelling in the hypolimnion does not nourish the epilimnion when there is no mixing. This will cause decreases in chlorophyll a (CHL-a) in the epilimnion as nutrient limitation prevents algal growth.
Therefore, trophic classifications are often inadequate to characterize a stratified lake.
21
Table 1. Phosphorus and Chlorophyll Concentrations and Secchi Disk Depths Characteristic of the Trophic Classification of Lakes (data from Wetzel, 1983).
Measured Parameter Oligotrophic Mesotrophic Eutrophic
Total Phosphorus Ave rage 8 26.7 84.4
(mg/m 3) Range 3.0 -17.7 10.9 -95.6 16 -386
Average 1.7 4.7 14.3 Chlorophyll-a (mg/m 3) Range 0.3 -4.5 3-11 3-78
Average 9.9 4.2 2.45 Secchi Disk Depth (m) Range 5.4 -28.3 1.5 -8.1 0.8 -7.0
Lake Stratification Classes
The morphometric characteristics for stratification are a steep basin wall, sulfide rich hypolimnion, temperature gradient, and increased depth (Rodrigo et al. 2001). The oxic/anoxic boundary of many lakes tends to be around 7 m below the surface, so it is rare for any stratified lake to be less than 9 m deep.
Classification of lake stratification was originally a thermally based classification when Forel (1901) described the tendencies of polar, temperate, and tropical holomictic
(entire mixing) lakes. This method was extremely insufficient and inefficient, as it did not consider altitude or depth. In 1956, Hutchinson and Löffler published a paper that emphasized the importance of mixing patterns as they revised the system. Mixis, or
“turn-over”, is mostly vertical circulation and it is a function of temperature, wind, area, and depth. There are many kinds of mixis; from meromixis, partial or incomplete
22 mixing, to holomixis, entire mixing. Holomixis has subcategories based on how many times per year the mixing occurs (Figure 10). Inputting the depth and latitude of LLW into Figure 10 indicates that the lake should be continuous warm polymictic, mixing continuously throughout the year except for brief (hours) periods of stratification.
However, as these classes are generalizations based on naturally occurring lakes, LLW could possibly stay stratified for longer lengths of time.
100
Cold Warm Dimictic Monomictic Monomictic
Amictic 50 Lake DepthLake (m)
Discontinuous Discontinuous Warm Cold Polymictic Polymictic
Continuous Continuous Cold Warm Polymictic Polymictic LLW (-26.5, 9) 0 90 80 70 60 50 40 30 20 10 0
Adjusted Latitude
Figure 10. Thermal classification scheme modified from Hutchinson and Löffler (1956) and Hutchinson (1957) as modified by Lewis (1983) showing the parameters of LLW.
23
Stratification in Saline Lakes
Some stratification occurs primarily due to a strong halocline, as seen in the
Eastern Antarctic marine basins (Gibson 2004). Most often there are other factors at
play. Water becomes denser with increases in salinity and specific conductivity and with
decreases in temperature (until ~4°C). Specific conductivity is the measure of how well
water can conduct an electrical current; so many ions other than sodium and chloride
contribute to its magnitude. These include nitrate, phosphate, and calcium present in
runoff. Therefore, conductivity can be influenced by the balance between seawater
flushing, runoff, and leaching. The specific conductivity of standard mean ocean water
(SMOW), 50,000 μS/cm (microSiemens per cm), is higher than the specific conductivity
of typical freshwater. Storm water runoff can lower the specific conductivity by diluting
the seawater brought in by the tide. When considering these factors, it is important to
look at weather conditions that correlate to the sampling date, and to consider
precipitation when interpreting the data.
Phototrophic Bacteria
Photosynthesis: Oxygenic vs. anoxygenic
Photosynthesis means “light putting together.” Photosynthesis, along with
respiration and decomposition, are the biogeochemical processes affecting the
chemistry of lakes. They are all connected in the carbon cycle. Photosynthesis is the
ultimate source of carbon in all the organic compounds within organisms' bodies, except
in the cases of chemosynthesis (such as around hydrothermal vents). Photoautotrophs
24
12 convert around 1x10 tons of carbon (from CO 2) into biomass per year (Field et al.
1998). While oxygenic photosynthesis consumes carbon and produces oxygen as a waste product (CO 2 + 6H 2O → CH 2O + ½ O2), anoxygenic photosynthesis uses different reduced molecules as an electron source, such as H 2, H 2S, S, and organic molecules
(e.g. CO 2 + 2H 2S → CH 2O + H 2O + 2S). Plants, algae, and cyanobacteria build organic compounds by transporting their electrons through the Calvin cycle, while anaerobic phototrophic bacteria, such as Chlorobium , use the reverse Krebs cycle. The former group keeps its chlorophyll in organelles called chloroplasts in the photosynthetic reaction center (RC), while the latter have bacteriochlorophyll (BCHL) in ellipsoidal vesicles called chlorosomes (Grimm et al. 2006).
All photosynthesis has 3 main functions: light absorption, energy transfer, and
ATP generation. Chlorophylls are able to carry out these duties because they have intense light absorptions and long lived excited states. Photons are transferred over tens of nm from the site of excitation to the RC, where the energy is used to yield ATP, which can be stored for later use (Ort and Yocum 1996). The assemblage of diverse antennae is able to increase the absorption cross section of the reaction center by orders of magnitude at most wavelengths of the visible spectrum. The only limitation that algae have is around 500 nm, in the “green gap”. Bacteria are able to exist at the lowest depths because they have adapted to efficiently absorb these “leftover” wavelengths with specialized bacteriochlorophylls (BCHLs) and carotenoids (Grimm et al. 2006).
25
Light and Depth
When sunlight hits the surface of a water body, it is attenuated by absorption and by scattering. The rate at which the light is attenuated determines the depth to which the light can reach. The degree of attenuation increases with the wavelength of the visible spectrum. If the absorption coefficient is constant in water, the light intensity decreases exponentially with distance (Stewart 2005). Photosynthetic organisms can often regulate light intensity by adjusting their buoyancy to increase or decrease their depth.
There are two mechanisms by which many unicellular photosynthetic organisms are known to utilize buoyancy to increase efficiency. Some organisms can increase buoyancy of cells by forming gas vesicles (Walsby 1994). The other option is the alteration of cellular production to decrease cell density. This is achieved by synthesis of cells with low carbon numbers such as alkanes and fatty acids. Models can calculate density changes in cyanobacteria and determine constant rates of vertical migration that are dependent on light intensity as well as time (Walsby and Deacon 1990).
Purple and Green Sulfur Bacteria
Stratified saline lakes show poor diversity of phototrophic bacteria species and tend to have one phototrophic bacteria dominating completely (Miracle et al. 1992).
While there are a variety of bacteria that use photosynthesis as their energy source, only the sulfur bacteria will be focused on here. Sulfur bacteria can be purple (PSB) or green (GSB). There is also a brown-color species of the GSB, called brown sulfur bacteria
(BSB). GSB’s photosynthetic system is located in chlorosomes that are independent of
26 the cytoplasmic membrane (Imhoff 1995). PSB’s photosynthetic system is located in spherical or lamellar membrane systems that are continuous with the cytoplasmic membrane (Grimm et al. 2006). PSB can tolerate some oxygen and require more light,
so they tend to be found in metalimnetic mats growing just above blooms of GSB. The
PSB layer acts as a biological filter for light, and as such is a factor that contributes to
self-shading (Borrego et al. 1999a). The PSB absorbs wavelengths of light similar to GSB,
resulting in a relative decrease in GSB (Itoh et al. 2003). Both Lake Banyoles and Lake
Kinneret have microbial plates of Chromatium , a PSB species that develops above the
Chlorobium blooms (Borrego et al. 1997; Yacobi et al. 2000). GSB requires strongly reducing conditions and will not be found outside of hypoxic conditions. The Chlorobium genus of GSB is the most modelled one and will be focused on.
The presence of sulfate is obviously vital for sulfate reduction by bacteria
(S 6+ + 8e - → S2-), which results in the production of sulfide in the lower layers of lakes.
This sulfide provides reducing power (S 2- → S0+ 2e -) for the phototrophic sulfur bacteria and allows their development if enough light reaches the anaerobic waters (Miracle et al. 1992). This sulfate uptake will increase sulfur bacteria biomass and lead to more reducing conditions throughout stratification. Concentrations of sulfur bacteria blooms have been found to follow the logistic growth curve (Rimmer et al. 2008), but with
enough light, they will grow at rates that far exceed algae. This is because phototrophic
sulfide oxidation is approximately 150 times faster than auto-oxidation (Eckert et al.
1990).
27
The Chlorosome
The photosynthetic complex in most plants and algae is the chloroplast, but some green filamentous anoxygenic phototrophs and all GSB have chlorosomes instead.
Chlorosomes are the light-harvesting antenna organelles consisting of aggregated BCHLs
-c, -d, or -e, carotenoids, isoprenoid quinones (lipid molecules), and minute amounts of
BCHL-a (Friggard and Bryant 2006). The chlorosomes of GSB are exceedingly proficient
at growing at the lowest of light intensities (Blakenship and Matsuura 2003) and are
known to live at 100 m in the Black Sea with light intensities of 0.0022 (2.2 nmol) -
0.00075 (750 pmol) μmol quanta/m·sec (Manske et al. 2005). The peripheral light
harvesting antennae is packed with extreme BCHL concentrations yet devoid of any
proteins. In the chloroplast of oxygenic phototrophs, there is an abundance of proteins,
and most interactions are CHL - protein ones. Meanwhile, the chlorosome is
characterized by its BCHL - BCHL interactions; energy is saved by avoiding protein
synthesis, and light absorption per unit volume is increased. In some cases, the dense
packing of BCHL can lead to “concentration quenching”; where a defective pigment
molecule is a productivity sink. One BCHL’s short-lived excited state drags down the
others if they are tightly coupled. The GSB avoid this and manage to achieve high
fluorescence by “nonphotochemical quenching”, in which carotenoids act as safety
valves (Grimm et al. 2006).
The chlorosome is the largest antenna structure known, yet very little is known
about its synthesis. Large efforts have been devoted to understanding the structural
28 organization and energy transfer characteristics of the BCHL aggregates because they serve as models of self-assembling systems.
Changes in GSB Composition as Photoadaptation to Changes in Light Intensity
The earliest GSB studies noted a light-dependent increase in the size of the
chlorosomes (Broch-Due et al . 1978; Fuhrmann et al. 1993). Many species of GSB were grown at a range of low light (0.1 to 0.5 μmol quanta/m·sec) and only BSB were able to grow at critical intensities (Blankenship et al. 1995). Many studies have provided clues on how BSB may adapt to low light with a drastic increase in the content of highly alkylated pigment molecules, thereby improving efficiency of energy transfer towards the RC (Borrego and Garcia-Gil 1994; Borrego et al. 1997; Airs et al. 2000). The lamellarly
organized BCHL in GSB are highly aggregated and have a variety of homologues, varying
in their degree of alkylation. The highly alkylated homologues may enhance productivity
by increasing wavelength absorption, energy transfer, and cell density (Borrego 1997).
More recent studies observed minimal to no changes in homologues but, rather,
increases in particular carotenoids at low light intensities (Yacobi 2000; Glaeser 2002;
Hirabayashi 2004). The in vivo light absorption of GSB is shown here (Figure 11) as
recorded by Manske and others (2005).
29
Figure 11. Absorption spectra of whole cells (—) and acetone pigment.
Carotenoids, Chlorophyll, and Bacteriochlorophyll Pigments
Light harvesting antennae are grouped into four kinds of pigments:
Bacteriochlorophyll (BCHL), Chlorophyll (CHL), carotenoids, and phycobiliproteins.
Phycobiliproteins are water soluble and therefore not extracted by the organic solvents used in the analysis of chlorophylls and carotenoids, and they are not encountered during typical HPLC pigment analysis (Wright 2006). For this reason, biliproteins will not be discussed further.
The chlorophylls are light harvesting pigments that have chemical structures composed of conjugated rings and double bonds. This arrangement allows transfer of electrons to higher energy states as each pigment absorbs its specific wavelengths
(Grimm et al. 2006). CHL and BCHL are both tetrapyrrole porphyrin macrocyclic ring structures surrounding an atom of magnesium. Small variations in the structure, such as
30 at the alkyl side chains or added carbonyl moieties, lead the absorption of different wavelengths and of different efficiencies (Glaeser et al. 2002).
Carotenoids
Carotenoids are tetraterpenoid molecules, long carbon chains with conjugated double bonds. Their chemical structure is divided into two types. Xanthophylls, such as lutein and zeaxanthin, contain oxygen. Carotenes, such as β-carotene (Figure 12), are aliphatic hydrocarbon chains. Carotenoids are also grouped based on function, involving the absorption and transfer of photons or photoprotection. For example, zeaxanthin is a pigment that found in cyanobacteria and serves to protect the reaction center from auto-oxidation. Photoprotective carotenoids become more prevalent with increased light availability and water clarity, as energy quenching becomes more necessary. For instance, the surface layer of the lake is more likely to contain photoprotective pigments due to photoinhibition (Mackey et al. 1996). Meanwhile, photosynthetic carotenoids, such as fucoxanthin in diatoms, have relatively stable ratios to CHL-a, irrespective of depth. Fucoxanthin (Figure 12), a main pigment in the Chromophyta is the most abundant carotenoid on earth and diatoms are the dominant plankton group in the ocean (Singhe-Damsté and Koopmans 1997).
31
Figure 12. β-carotene and Fucoxanthin/Fucoxanthinol structures.
Photoprotection is typically regulated by xanthophyll cycles, in which stimulated energy is dissipated by increasing and altering carotenoids through the enzymatic removal of epoxy groups (Falkowski and Raven 1997). In diatoms and dinoflagellates, the xanthophyll cycle consists of the pigment diadinoxanthin, which is transformed into diatoxanthin (in diatoms) or dinoxanthin (in dinoflagellates) at high light in the diadinoxanthin (Ddx) cycle (Jeffrey et al. 2005). In the other xanthophyll cycle, the violaxanthin (Vx) cycle, violaxanthin changes to antheraxanthin (Ax) and then into zeaxanthin at high light. The hypothesized biosynthetic pathways for the formation of fucoxanthin depend on high or low light conditions (Figure 13). The absorption maximum of zeaxanthin is longer than that of violaxanthin. In 2004, Hirabayashi and others found that photoadaptation of Chlorobium phaeobacteroides differed from these cycles in that there was a shift to shorter wavelengths at high light. The study also found
32 that with high light only the carotenoids with cyclohexenyl (β) end groups increased, while carotenoids with aryl (Φ) end groups decreased (Table 2).
Figure 13 . Hypothetical coupling between xanthophyll -cycle pools; HL=high light; LL=low light (Lohr and Wilhelm 1999).
Table 2. Carotenoids in Chlorobium phaeobacteriodes (Hirabayashi 2004) with th e structures of their end groups.
Identification End Group Absorption Maximum (nm) trans -Isorenieratene Φ,Φ 451 cis -Isorenieratene Φ,Φ 451 trans -Chlorobactene Φ,Ψ 451 cis -Chlorobactene Φ,Ψ 451 trans -β-Isorenieratene β,Φ 451 cis -β-Isorenieratene β,Φ 451 trans -β-Zeacarotene β,Ψ 426 trans -β-Carotene Β,β 451 cis -β-Carotene β,β 451 trans -7,8 -Dihydro -β-carotene β,β 426 cis -7,8 -Dihydro -β-carotene β,β 426
33
It was not until 1956 that Goodwin and Lang first confirmed the existence of carotenoids in GSB when they were isolated and identified in three Chlorobium species.
That study found that all three contained ɣ-carotene as the main carotenoid pigment
(85% of the total).
Anoxygenic phototrophic bacteria come in a wide spectrum of colors, ranging from shades of pink, red, yellow, green, brown, and purple. These hues depend on the type of BCHL and carotenoid pigments they contain (Imhoffe 1995). Most GSB contain the carotenoid chlorobactene. However, the brown colored species of the green sulfur bacteria (GSB) is distinguished by its brown color, which is actually caused by extremely high content of the carotenoids, β-carotene and isorenieratene (Isr) (Olsen 1998) (Figure
14). While most of the carotenoid accessory pigments do function as Light Harvesting
Complexes (LHC), it has been debated whether this is true in the phototrophic bacteria.
Figure 14. Structure of carotenoids and postulated carotenogenesis pathway in Chlorobium phaeobacteriodes (Hirabayashi 2004).
34
Chlorophylls
Chlorophylls’ structure is known as a porphyrin, a chlorin subgroup of four pyrrole molecules surrounding centrally complexed magnesium (Figure 15). “CHL-a is
the common phototrophic currency of the photoautotrophic world” (Loitz 1999). That
quote applies only to oxygenic species. Since it is omnipresent in both prokaryotes and
eukaryotes, CHL-a is used more for quantifying primary productivity rather than for
taxonomic purposes. CHL and BCHL can also be very useful during core analysis to
determine past oxygenic/anoxygenic ratios, respectively (Baker and Louda 1986; Louda
et al. 2000). CHL-a has intense but restricted absorption maxima at 440 nm and 675 nm
in vivo . It needs other chlorophylls and carotenoids to fill the wavelength gaps in order
to maximize efficiency. CHL-b is only produced by three taxa of photoautotrophs;
prochlorophytes, chlorophytes, and higher plants. CHLs –c1 and –c2 are
phytoporphyrins found in chromophyte algae, specifically diatoms (Bacilliariophyceae)
and dinoflagellates (Pyrrhophyta).
35
Chlorophyll A
C1 C2
Chlorophyll B
Figure 15. structures of CHLs –a, -b, -c1, and -c2.
Bacteriochlorophylls
BCHL-a is the most abundant BC HL in PSB, while BCHLs -c, -d, and -e are more abundant in gree n, brown, and non -sulfur bacteria (Schlegel 1993; Neto et al. 1996). At least a small amount of BCHL -a is found in all anoxygenic bacterial photoautotr ophs except the h eliobacteria.
36
BCHLs -c, -d, and -e are structurally similar but differ in terms of stereochemistry,
methylation, and esterifying alcohols. Despite their limited contribution to worldwide
photosynthesis, their structures actually comprise more than half of the 100 CHL and
BCHL structures that have been discovered. The chlorosome of the GSB contains a core
with 1000s of densely packed BCHL that are organized into 10-30 rods that run along the
cigar-shaped structure (Figure 16).
Figure 16. Chlorobium cell cross section. The white ovoid shapes around the edge are chlorosomes.
BCHL –d was originally named Chlorobium 650 (Holt and Hughes 1961, Purdie and Holt 1965) referring to its low energy absorption band at 650 nm in acetone (Holt et al. 1962, 1966). Consequently, BCHL –c was called Chlorobium 660, after band I absorption at 660 nm, when it was isolated as a homologous mixture (Hughes and Holt
1962) in some strains (such as Chlorobium limnicola) and as single homologue in other strains (such as Chloroflexus aurantiacus ) (Smith 1994). BHL-d tends to be found as a homologous mixture, for example in Chlorobium vibrioforme (Hughes and Holt 1962).
37
Bacteriochlorophyll-e (BCHL-e) is the most recently discovered BCHL, and it was isolated
by thin layer chromatography in 1974 from representatives of the brown-colored GSB
species (Gloe et al 1974). This was a result of Chlorobium phaeobacteriodes first being analyzed in a sample from meromictic Lake Blankvann in Norway (Pfennig 1968). Gloe and others (1974) described at least 3 different homologues which differ from each other different substituents on the pyrrol rings II and III, similar to BCHLs -c and -d. The light-harvesting BCHLs -c, -d and -e of green sulfur bacteria are chlorins that contain only one reduced pyrrole ring (dihydroporphyrin) and therefore exhibit spectral properties similar to CHLs -a and -b but different from BCHLs -a, -b and -g ,which are
tetrahydroporphyrins (Smith 1994). BCHLs -c, -d and -e have mixtures of homologues
which differ with regard to their alkyl substituents at positions C-8 and C-12 of the
porphyrin ring system. Now that the structures have all been depicted, the different
challenge is to determine the role of variations in assembly and functions of the
chlorosome core. Glaeser and others (2002) described a total of 23 different BCHL-e
structures. The four homologous porphyrin ring systems differ at C-8 and C-12 and with
eight different esterifying alcohols tails (Figure 17).
38
Figure 17. Structural differences in BCHL-e homologues (Glaeser 1998).
Breakdown - Senescence
Pigments degrade both in the water column and following deposition, but the process is much faster (Damste and Koopmans 1997) if bottom waters and sediments are oxic (Baker and Louda 1986, 2002). Anoxia greatly slows pigment destruction (Louda et al. 1998, 2002). CHL and BCHL breakdown occurs during natural processes including chemically or microbially-mediated oxidation, herbivory, bacterial degradation, cell lysis, and enzymatic metabolism during senescence (Louda et al. 1998; Szymczak-Zyla et al.
2008). Decomposition can also occur during handing and processing of samples.
Degradation of a CHL molecule can differ quite variably, depending on the process by
39 which it is being broken down (Figure 18). As such, there are a series of single transformations or a combination of them. While healthy cells contain chloro-pigments
(which are green and have magnesium 2+ ), senescent cells contain chloro-pigments plus pheo-pigments (brown) indicating that they no longer have chelated magnesium. If magnesium is the only portion missing, it is pheophytin, but if the phytol tail is lost as well, it becomes pheophorbide. Further loss of a carbomethoxy group yields pyropheophorbide (Baker and Louda 1986, 2002; Louda et al. 1998, 2002). Another possibility is for the magnesium to remain while only the phytol tail is cleaved, which occurs during chlorophyllase, and leads to chlorophyllide (Leavett and Hodgson 2006).
The presence of water may result in breakdown of CHL via chlorophyllase (Jeffrey et al.
2005).
Figure 18. Structures of CHLs -a, -b, and -c, and their breakdown (Louda et al. 2002).
40
The aging of a cell is also indicated by carotenoids converting from their trans to cis forms and then to colorless compounds (Leavitt 1993; Singhe-Damsté and Koopmans
1997). After sedimentation and burial through geological forces, further modifications to the pigments create a wide variety of geoporphyrins (Baker and Louda 1986, 2002;
Callot 1991). Pheophytins and pheophorbides are common in most sedimentary systems (Baker and Louda 1986, 2002; Louda and Baker 1981).
Chromatography
Chromatography embodies a variety of laboratory techniques that separate mixtures by utilizing a stationary and mobile phase. A compound will transition from the stationary phase to the mobile phase based on its particular partition coefficient. The time it takes the compound to travel through the system is the retention time. In liquid chromatography, the mobile phase is a solvent that carries the compounds through the column, and it comes out as eluate. The diversity in size, charge, polarity and structure of the pigments described allows them to be “fingerprinted” by their retention time and absorbance spectrum (Mantoura and Llewellyn 1983, references in Jeffrey et al. 2005).
The Beer-Lambert law draws a linear relationship between absorbance and
concentration so that the amount of each pigment can be calculated from the time
(equal volume relative to flow rate) integrated absorption at a specific wavelength. It is
also possible to separate and quantify the homologues of BCHL (Yacobi et al. 1990). High
Performance Liquid Chromatography (HPLC) was used in this study and will be described
in further detail in the next chapter.
41
4. Materials And Methods
Sampling Procedures
Routine sampling occurred at the surface ( 10-20 cm) and then at successive 1.0 meter intervals at a consistent sampling site in which maximum depth ranged from 8.5 to 9 meters, depending on tidal stage. In July 2009, samples were taken at 0.5 m increments. Coordinates of the main sampling site were 80°03.50’ N and
26°51.00’ W (Figure 19). This area was toward the south end of the lake and closer to the eastern shore. This site was chosen due to its location in the deepest basin of the lake (see Figure 11).
Field Measurements and Water Sample Collection
A Hach Hydrolab multiparameter sonde was used to measure temperature,
dissolved oxygen (DO), pH, specific conductance, and nitrate. Light was measured with a
LiCor 4π Spherical Quantum Radiometer Light Meter, Model LI-250. Measurements
were recorded and samples were collected monthly from September 2008 to November
2009. Water column samples were taken with a LaMotte water sampler, model JT-1,
code 1077. The water was transferred into 2 L opaque dark brown Nalgene bottles.
These bottles were kept shaded and at ambient temperatures during transport directly
42 to the lab at Florida Atlantic University in Boca Raton, FL where they were immediately filtered and treated, as described in the next section.
N
400m
Figure 19. Image of LLW revised from Google maps. Red star indicates sampling site.
Water Sample Treatment
Once in the lab, the volume of water from each Nalgene bottle was measured with a polyethylene graduated cylinder. The samples were vacuum filtered onto
Whatman GF/F filters (0.7 micron pore size borosilicate glass fiber). These filters were folded in half, blotted between paper towels, folded again, and wrapped in aluminum foil before placement into the freezer (-20°C) for at least 24 hours before extraction for
High Performance Liquid Chromatography (HPLC) analysis.
43
Any time after collection that the sample was exposed to light, it was a subdued yellow light in order to avoid photo-oxidative changes and isomerization of the pigments. These controls were applied to both water and sediment samples.
Sediment Sampling, Storage, and Retrieval
In September 2009, a core sample was taken from the sediment at the central
sampling site with a modified Wildco hand core sediment sampler (Forestry Suppliers
Cat #77408). The modifications included the removal of the handles and adding two 6 x
8 inch stabilizing fins. This allowed attachment of a line to the device so that it could be
used as a free-falling gravity corer. Once the sediment core was retrieved, it was
immediately capped and transferred to a specially made wooden case containing dry ice
for initial cool down and preservation. This arrangement also kept the core vertical for
less mixing of the semi-consolidated sediments. After transport to the lab, the core was
frozen for over 48 hours. While still frozen, the core was extruded using an acrylic rod
(mm less in diameter than the inner dimensions of the core liner) with a Buna-N “O” ring
near the tip for a tight seal during the process. The still frozen core was next cut into
four 10 cm sections.
The four segments were thawed in 0.5 L glass jars that were labeled respectively to represent 0-10 cm, 10-20 cm, 20-30 cm, and 30-40 cm. They were shaken thoroughly to ensure the sample was completely mixed before individual 2-5 mL amounts were withdrawn to be analyzed for ash free dry weight (AFDW) and chemical oxygen demand
(COD). The sample was dried at 115°C for 18-24 hours to determine water content. The dried sediment was then combusted at 450°C for 24-48 hours to obtain loss on ignition
44
(LOI) (Schumacher 2002). The difference between AFDW and LOI was taken as organic matter. The process of extracting the sample from the sediment for analysis by HPLC required more than filtering, as it was more solid. This will be covered in the following section.
Pigment analysis
This study utilized High Performance Liquid Chromatography (HPLC) with photodiode array spectrometric detection (PDA) as the primary method for analysis of the pigments in the water samples taken throughout the water column and sediments of Little Lake Worth. By this method, a lake sample containing a variety of organisms can be analyzed to determine its chemotaxonomy (Mille et al. 1993).
Extraction Methods
Samples were removed from freezer, unwrapped, and transferred to pre-chilled glass homogenizing test tubes (Kontes ‘‘Duall’’ TM, 15 mL) that will herein be referred to as tubes. The tubes remained in an ice bath during grinding to minimize frictional heat and to enhance preservation. The extractant solvent used was acetone/methanol/dimethylformamide/water (30:30:30:10 v,ν,ν,ν; aka “MADW”:
Hagerthey et al. 2006) as well as a trace of internal standard (IS) (0.1-0.2 µg/mL) for
quality control purposes that are explained in the final section of this chapter. MADW
solvent (3.00 μL) was added to each tube and filter. A stainless steel rod with Teflon tip
tissue grinder was inserted into the test tube and rotated at about 350-750 rpm for 60
seconds, until the filter and MADW had formed a green/brown slush. The tube was then
45 sonicated in an ice bath several times in 10 seconds “spurts”. The tube/extract mix was placed in the refrigerator (2–3° C) to steep for 1 hour. Then, the tube was centrifuged for 3 minutes, and the supernatant was decanted into a glass vial, the crude extract taken into a 3.0 mL syringe and filtered through a 0.45 µm PFTE filter to remove any remaining filter or sediment particles (see Jeffrey and Vesk 1997; Jeffrey et al. 2005).
Ultraviolet-visible (UV/VIS) spectroscopy
The 1.00 mL filtered sample was placed in a cuvette for a UV/VIS reading of the
visible absorption spectrum from 330 nm to 800 nm. This allowed a spectrophotometric
estimate of total chlorophyll concentration as well as a glimpse of the peaks in the
spectrum. The peaks indicate the most prominent pigments. Ultraviolet-visible spectra
were measured with a Perkin-Elmer Model “Lambda-2” UV-Vis spectrophotometer. It is
calibrated for wavelength (vs. holmium oxide) and for absorbency (vs. potassium
chromate in aqueous KOH : Rao, 1967). The background correction was 90% MAD in
water with no internal standard (IS). The IS was quantified within the raw extraction and
recovery following extraction and HPLC allowed a procedural correction factor (CF) to be
determined. Typically, this CF ranged from 1.1 to 1.3 and was then applied to all
pigment calculations.
Ultraviolet/Visible spectrophotometry (UV/Vis) uses the Beer-Lambert
relationship that relates concentration to spectral absorption. Application to the crude
extract allowed a rough spectrophotometric estimation of the CHL and BCHL contents. If
the absorption level at any wavelength was above 0.100 AU (absorption units), the
extract was quantitatively diluted in order to not overwhelm the system. This was
46 particularly important when analyzing samples from the hypolimnion during the bloom of brown bacteria, because the samples were so concentrated that 0.5 L samples needed to be diluted up to three times. Typical 2.0 L water samples at other times of the year did not need to be diluted.
HPLC - High Performance Liquid Chromatography
The HPLC system is made up of a Consta Metric 4100 series Quaternary Solvent
Delivery Systems pump (Thermo Separation Products, Riviera Beach, FL), a Rheodyne model 7125 syringe loading sample injector fitted with a 100µL injection loop, a 300 mm long Waters Nova Pak ® C18 column with an internal diameter of 3.9mm (4 µm spherical particle size, 60°A pore size, 120m 2·g-1 surface area, 0.3 mL·g-1 pore volume)(part #
Wat011695) and a Waters 990 Photodiode Array Detector (PDA: 190-800nm). The
Waters 990 software was used on the connected computer.
Prior to injection into the loop, the 1.0 mL sample was mixed with 0.125 mL of
ion pairing (IP). A 1.00 L volume of IP is made from 15.0 g tetrabutyl ammonium acetate,
and 77.0 g ammonium acetate, in nanopure water (Mantoura and Llewellyn, 1983). IP is
an ion pairing (suppressing) reagent, which is known to improve separation of
compounds with polar units on reversed phase HPLC (Poole and Poole, 1991).
Once IP had been added, 200-220 µL of the sample was taken into a 250 µL
syringe. The HPLC had previously been prepped through a system function check,
calibration, and 10 minutes of the initial 60/40/0 mix (Table 3). Once the sample was
loaded from the syringe into the 100 μL injection loop, the valve was opened, allowing
the mobile phase to flow through the loop and “inject” the sample into the column
47
(stationary phase). The change in the ratio of solvents works to partition the sample between the mobile and stationary phase thus separating into the various pigments along the way was induced. Table 3 shows how the ratios of the solvents change throughout the 50 minute process.
Table 3. Changes in the ratios of solvents throughout the 50 minute HPLC process.
Time (min) A/B/C Pigments eluted 0 60/40/0 Chlide-a, Chl-c 5 60/40/0 fucoxanthinol, pyrochlide-a, peridinin 10 0/100/0 all other carotenoids, Chl-a, Chl-b, pheophytins 40 0/30/70 all other carotenoids, Chl-a, Chl-b, pheophytins 45 0/30/70 all other carotenoids, Chl-a, Chl-b, pheophytins 46 0/0/100 flush 47 0/100/0 polarity readjustment 48 60/40/0 re-establishment of initial conditions
Solvents: A = 0.5M Ammonium Acetate in Methanol/water, 85:15 B = Aceonitrate/water, 90:10 C = Ethyl acetate, 100%
Each change in the gradient program occurs gradually from one time step to the
next. The ratios change slowly so that each interval ratio is reached at a specific
retention time, at which a particular pigment is known to elute. 100% solvent D
(Methanol/water, 85:15) is passed through the column for 15 minutes for the column to
be stored in before the instrument is shut down and stored in solvent “D”.
Analysis of HPLC Data
HPLC analysis of each sample, filtered directly from the water or indirectly from the core as described above, provided data on the concentration of each pigment in
48 that sample (Millie 1993, Louda 2008). By knowing the type of algae that each pigment is a marker for and its general ratio to CHL-a, it was possible to estimate the
“chemotaxonomy” of each sample. In addition, quantifying the proportion of senescent cells (e.g. percent pheopigments, see Louda 2008) gives an idea of the state of “health” of the sampled community.
Comparing the algal and bacterial concentrations and divisions to the changes in temperature, oxygen, and other independent variables should allow an estimation of the mechanisms by which they all interact.
HPLC Data Calculations
As the ratios of the solvent gradually changed, a pigment’s solubility would increase at that certain ratio, mobilizing that individual pigment. The pigment then eluted to the end of the column, where the PDA constantly records the UV/Vis spectrum
300-800 nm of the eluate. The variety in structure and number of carbon double bonds
(C=C) is what gives each carotenoid and chlorophyll a signature chromophore by which it can be “fingerprinted”. Further aiding the process, the absorption spectrum can be paired with the retention time at which the pigment elutes. This allows for a two dimensional analysis to be used when identifying the pigments. A list of pigments with their retention times and UV/Vis can be found in Appendix 1. This list was compiled based on comparison to the FAU Organic Geochemistry Group’s library of standards as well as from a sample of standards mix that was purchased from VKI (Denmark). The standard compounds were run on the same HPLC system in order to ground truth the
49 analyses (viz. QA/QC). Absorbance spectrum of various chlorophylls and bacteriochlorophylls are illustrated in Appendix 2.
HPLC software automatically integrates each peak, allowing quantification of each of the pigments. The area under the peak is calculated as time based absorbance units ·mL (AU ·min) as the flow rate used was 1.00 mL/min. The chromatogram was integrated at 410, 440, and 395 nm for quantification of pheopigments, chlorophylls plus carotenoids, and the internal standard, respectively. There were many methods for troubleshooting. When there were small peaks that were not auto-integrated, or peaks were grouped together by the computer, manual integration was required.
Once each peak had been differentiated and assigned an AU ·min value, this
number was input to “PIGCALC”, short for pigment calculation. This is an Excel®
spreadsheet that was developed by the FAU Organic Geochemistry Group, using
standardized equations to calculate the concentration of each pigment based on its
absorption coefficient. Any minute change in concentration for the overall sample was
corrected for by inputting the AU ·min for IS which calculated and applied the procedural correction factor as discussed. The spreadsheet also determines ratios of pigments and converts results to taxon-specific chlorophyll-a and bacterial chlorophyll contributions.
A chemotaxonomic estimate (percent of each taxon) results.
Verification of Results
Copper mesoporphyrin-IX dimethyl ester was used as internal standard IS to mimic chlorophyll, and it acted as a correction factor throughout the extraction and chromatography. This was achieved by recording the amount of IS before and after
50 going through the HPLC column. The IS added was quantified by measuring the absorbance spectrum of the solution of MADW being used as the extractant with ultraviolet-visible
(UV/VIS) spectroscopy. The background correction was MADW with no IS. This allowed the IS peak to be quantified during each analysis for the correction factor. The absorbance of the IS at 394 nm was recorded into PIGCALC.
The IS eluted from the HPLC column around 24.5 minutes and had a sharp peak at 394nm. If it came earlier or later, the other retention times could be corrected for.
The IS pigment was identified on the chromatogram and the AU ·min of its integrated peak was recorded as IS found . This ensured accuracy throughout the process as the
UV/VIS peaks, retention time, and AU min of the IS were recorded and used to calibrate
the other pigments. PIGCALC calculated a correction factor with the equation:
Correction Factor = IS added /IS found
51
5. Results And Discussion
Water Column Studies
During most of the year, LLW was dominated by diatoms throughout the water
column, and taxonomic divisions remained constant throughout depth and throughout
the year, without any drastic changes spatially or temporally (Figure 20). Maximum CHL-
a concentrations were reached from September to January with closely correlated
peaks in CHL-a breakdown products (Figures 21 and 22). LLW stratified in the summer,
during which a chemocline occurred, as the dissolved oxygen level dropped below 2
mg/L and hydrogen sulfide increased (Figure 23).
The stratification was pronounced in June and July. In June, the metalimnion was
between 5 and 6 meters, and the maximum concentration of BCHL-e occurred at 7
meters. In July, the metalimnion was between 4.5 and 5.5 meters, and the maximum
concentration of BCHL-e occurred at 6 meters (Figure 24). Mixis occurred in the fall,
most likely due to the decrease in temperature causing changes in density, advanced
further by wind. UV-Vis spectra of extracted hypolimnion samples during stratification
events consistently showed peaks at 469 nm and 654 nm (Figure 25), in contrast to the
434 nm and 665 nm that were typical during mixis or in the epilimnion (Figure 26).
52
Taxonomic Avgs During Mixis
GREENS -0.1
-1 DIATS -2
-3 DINOS -4
depth(m) -5 CRYPTOS -6
-7 CYANO -8
0% 20% 40% 60% 80% 100%
Taxon Percent
Figure 20. Time average (Nov. 2008 – May 2009) chemotaxonomic estimate of phytoplankton community structure in LLW water column.
Key: Greens (green algae), diats (diatoms), dinos (dinoflagelattes). cry ptos (cryptophyte), and cyano (cyanobacteria) .
53
Figure 21. Profile of s patial and temporal chlorophyll
9 LLW CHL-a Breakdown
8
7
6
5 ug/g
4
3
2 -8 1 -6 -4 0
-2 Depth(m)
-0.1
0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9
Figure 22. Spatial and temporal chlorophyll -a breakdown (PHEOS) concentratio ns.
55
0.0 LLW July -1.0 Dissolved Oxygen -2.0
-3.0 Sulfide -4.0
Depth (m) Depth -5.0
-6.0
-7.0
-8.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Concentration (mg/L) Figure 23. July Dissolved Oxygen and Sulfide profiles.
-3.5 BCHL-e Profiles -4.0 -4.5 June -5.0 July -5.5
depth (m) depth -6.0
-6.5
-7.0
-7.5 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Concentration (mol/L e 10 )
Figure 24. Spatial bacteriochlorophyll-e profile for June and July 2009.
56
Figure 25. UV-Vis spectrum of sample taken from 7 meters on June 23, 2009.
Figure 26. UV-Vis spectrum of a typical epilimnion sample extract.
57
Light Intensity
Light was found to be an important factor affecting the bacterial composition and biomass in LLW. The trends of log of light intensity had a much higher slope for April and October than September and July (Figure 27). This indicates a much higher degree of light attenuation during stratification, as the largest decrease in light intensity occurred when it passed through the depth at which the GSB was at a maximum.
Increases in light intensity as the metalimnion became shallower caused increases in
GSB biomass.
0.5 Light Intensity -0.5 LLW -1.5
-2.5
-3.5
Sep -08 depth(m) -4.5
Apr-09 -5.5
Jul-09 -6.5 Oct -09 -7.5
-8.5 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 log of light (W·m −2 )
Figure 27. Spatial and temporal log of light intensity profile.
58
Temperature and Oxygen
The temperature was reasonably homogenous during the mixis period (Figure
28). The thermocline during stratification differed from June to July (Figure 29). In June, the change in temperature was deeper and more drastic; the greatest change was 2.2°C from 6 m to 7 m below the surface. Later, July had a more steady and gradual slope that began three meters down and did not exceed 1°C/m. The first derivative of the change in temperature throughout depth for June and July is shown in Figure 30. While July lacked a strong thermocline, it did have a distinct shallow oxycline (Figure 31). Oxygen saturation profiles year-round are shown in Figure 32.
2009 LLW Temperature Profiles
-0.5
-1.5 Jan -2.5 Apr
-3.5 May
-4.5 June depth(m) -5.5 July Aug -6.5 Oct -7.5
-8.5 18.0 20.0 22.0 24.0 26.0 28.0 30.0 Temp (°C)
59
Figure 28. Temperature profile throughout depth for 2009
-0.5 Thermocline -1.5 Progression 2009 -2.5 -3.5 -4.5
depth(m) -5.5 -6.5 June -7.5 July -8.5 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 temp (°C)
Figure 29. Spatial profile of temperatures in June and July, during LLW stratification.
1.0 July Thermocline 1.0 June Thermocline -1.0 -1.0
-3.0 -3.0
-5.0 -5.0 depth (m) depth depth (m) depth -7.0 -7.0
-9.0 -9.0 -2.5 -1.5 -0.5 0.5 -0.9 -0.7 -0.5 -0.3 -0.1 dT/dz dT/dz
Figure 30. Spatial profile of changes in temperatures in June and July.
60
0.5 Oxycline vs. Thermocline -0.5
-1.5
-2.5
-3.5
-4.5 depth(m) -5.5 JUNE DO (mg/L) JUNE dT/dz -6.5 JULY DO (mg/L) -7.5 JULY dT/dz -8.5
0 2DO mg/L and dT/dz 4 6
Figure 31. Spatial profile of dissolved oxygen and changes in temperatures.
0.0 MAY Dissolved Oxygen Profiles -1.0 JUNE JULY -2.0 AUG OCT -3.0
-4.0 depth(m) -5.0
-6.0
-7.0
-8.0 0.0 20.0 40.0 60.0 80.0 100.0 % Saturation
Figure 32. Spatial profile of dissolved oxygen percent saturation, calculated from temperature and salinity at that depth.
61
The summer of 2009 had ninety-one days in which temperatures above 90°F were recorded (NOAA 2009). The extreme solar radiation may have penetrated deeply enough to heat the hypolimnion. While the stratification remained intact, the thermocline was weakened. This is supported by the temperature increase at 7 m, which went up from 26.4°C in June to 27°C in July. The oxygen profile for July is the only one with pronounced photoinhibition. Less photosynthesis occurred and thus less oxygen was produced at the surface due to the intense radiation in that period.
Additionally, warmer surface waters can be expected to more efficiently outgas in accord with Claussius-Clapyron constraints acting on Henry’s Law.
Specific Conductance
Measurements for specific conductance taken (SpC) at LLW are shown in Figure
31. The specific conductivity of standard mean ocean water (SMOW) is 50,000μS/cm
(Cox et al. 1970), and the lake is consistently higher than this value in May 2009. The
previous winter had set a record for the second driest 6-month period (November 2008-
April 2009) in West Palm Beach (NOAA 2009). This drought reduced the normal annual
pattern of incoming ocean water dilution, so the SpC increased in accord with salinity.
Salinity increases from evaporation could be the reason that SpC was above SMOW in
May. These data are further confirmed by South Florida Water Management District
SFWMD) surface water measurements taken at a sampling site at the south of the inlet
to LLW at PGA Blvd (Figure 34). The six month drought period shows elevated SpC
corresponding very closely to salinity. There are consistent turbidity peaks in August of
2008 and 2010, and another one from February to April 2009, towards the end of the
62 drought. These maxima are most likely due to resuspension of sediment caused by the turbulence of wind and heavy rains plus terrestrial runoff.
The lowest consistent SpC values were recorded in November 2008, after the halocline had been broken long enough to stabilize the water column. This was the culmination of a particularly wet season, so the heavy precipitation all summer and fall diluted the water to below 47,000 μS/cm.
Specific Conductance Profile
-0.5
-1.5
-2.5
-3.5
-4.5 NOV depth(m) -5.5 JUNE
-6.5 JULY AUG -7.5 MAY -8.5 41500 43500 45500 47500 49500 51500 (uS/cm)
Figure 33. Spatial profile of SpC in LLW 2009.
63
6 SFWMD PGA inlet
5 Sp Cond S/cm 4 TURB NTU
3 SAL %
2 TSS (various units(various in legend) (cg/L)
1
0 Apr-08 Jul-08 Oct-08 Feb-09 May-09 Aug-09 Dec-09 Mar-10
Figure 34. Water quality measurements over time (at station LWL-1, PGA Blvd.) Data from SFWMD’s DYBHYDRO database downloaded February 12, 2010 from http://www.sfwmd.gov/dbhydroplsql/show_dbkey_info.main_menu
Rainfall and Density
The last two weeks of May 2009 had the highest rainfall (Figure 35) during the sampling period. The 6-month dry period ended the day after the May sampling trip, and the rest of May had from 0.2 up to 2 inches of rain nearly every day. The inundation of freshwater was possibly a trigger for the subsequent stratification in density (Figure
36). Density of water increases with salinity and reaches its maximum at 4.0°C. The density values in Figure were calculated based on these variables (CGS 2010). The less dense freshwaters layered as they could not mix below the denser seawater in the
64 lower depths of the lake and a pycnocline was formed. The density profile in June shows the greatest variance.
Figure 35. Rainfall data over time at station S-44 (northern LWL). Red stars indicate sampling dates.
Data from SFWMD’s DYBHYDRO database downloaded February 12, 2010 from http://www.sfwmd.gov/dbhydroplsql/show_dbkey_info.main_menu
65
LLW Density Profiles 2009 0
-1
-2
-3
-4 May 12 -5 depth(m) June 22 -6 July 16 -7 Aug 31 -8 Oct 27 -9 1014 1016 1018 1020 1022 1024 1026 density (kg/m 3)
Figure 36. Spatial and temporal density profiles of LLW.
The density profiles for July and August show a dip in density at the lowest depth
of the lake. One possible explanation for this is that although the less dense water could
not penetrate the hypolimnion from above, there was a significant amount of
freshwater baseflow (groundwater seepage) coming in.
Substantial rainfall can cause the water table to rise above the drainage fields of
septic systems, releasing sewage and waste before it has been properly treated and
percolated through the soil. The leaching of nitrates from fertilizers and sewage could
have been accelerated by the heavy rains. There was a marked variance in nitrate levels
throughout depth in relation to time. As shown in Figure 37, the nitrates drop to a
minimum at 7-8 meters, only to increase sharply again at the lowest depth. The
66 heightened nitrates could be from the baseflow containing polluted runoff, or it could just be resuspension of nutrient-rich sediment. There was no direct evidence, due to
infrequency of sampling and lack of knowledge about travel times for groundwater.
LLW Nitrate Profiles 2009 0 -1 -2 -3 -4 May -5 June -6 July Depth (m) Depth -7 Aug -8 Oct -9 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 Nitrates (μg/L)
Figure 37. Nitrate Profiles for selected months in LLW.
The green sulfur bacteria (GSB) utilize nitrates and this appears to explain why
the greatest decline with depth occurs during the stratification, which allows the GSB to
thrive at depth. The one exception is October, when sampling occurred after a relatively
dry period, and nitrates were at a record high at the surface, only to decline sharply at
the bottom. The GSB that had been flourishing all summer in the hypolimnion were now
restricted to lay dormant in the anoxic soil until the next summer. It is possible that they
were still using nitrates from the water in order to maintain themselves.
67
Nutrients, Chlorophyll, and PHEO s
Tropical Storm Faye made la ndfall on the east coast of Florida on Aug 21 st , 2008, bringing severe winds and five inches of rain that flooded the area (Figure 38).
Figure 38. Rainfall over time at station S-44 on Canal C-17 (northern LWL ). Red stars indi cate LLW sampling dates.
Data from SFWMD’s DYBHYDRO database downloaded February 12, 2010 from http://www.sfwmd.gov/dbhydroplsql/show_dbkey_info.main_menu
The first sampling tri p was on September 16, 2008. It appears the storm did not have enough impact to break the stratification, as phototrophic bacteria were still in the bottom portion of LLW . However, there were significant effects on nutrient and chlorophyll levels. South Flori da Water Management District (SFWMD ) sampling results from the PGA station during this period show that there were substantial spikes in ammonia and phosphates correlating to eutrophication events(Figures 39 and 40 ).
68
14 SFWMD Oxygen Sag Curves PGA inlet 12 D.O. mg/L 10 PHEO -a mg/m3 8
6
4
(various units(various indicated in legend) 2
0 Jul-07 Oct-07 Feb -08 May-08 Aug-08 Dec-08 Mar-09 Jun-09 Sep -09 Jan-10
Figure 39. Dissolved oxygen (DO), chlorophyll -a, an d its breakdown measure “PHEO -a” over time (at station LWL -1, PGA Blvd.).
140 SFWMD Nutrients vs Chl at PGA inlet CHLOR cg/m3 120
NOX µg/L 100 NH4 µg/L 80 TPO4 µg/L 60
40
20
0 Jul-07 Oct-07 Feb-08 May-08 Aug-08 Dec-08 Mar-09 Jun-09 Sep -09 Jan-10 date
Figure 40. Nutrients and chlorophyll over time at station LWL-1, PGA Blvd. Nutrients: NOx (nitrates), NH4 (ammonia ) and TPO4 (total phosphates) correlated to chlorophyll - a (CHLORO). Data for Figures 39 and 40 from SFWMD’s DYBHYDRO database downloaded February 12, 2010 from www. sfwmd.gov/dbhydroplsql/show_dbkey_info.main_menu
69
Data were also obtained from Florida’s Department of Environmental Protection
(FDEP) for the same station. Although they were less detailed, they dates much further back to 2002, and allowed levels of chlorophyll-a (CHL-a) and its breakdown measure
“PHEO-a” to be correlated to many more storms, which are labeled in Figure 41. The
sampling period goes back to 2001, but it stopped in 2007 before taking a final 5
samples in 2008. The two final samples taken in August and October 2008 reflect the
peaks in CHL-a measured both by the author and the SFWMD.
Figure 41. CHL-a and PHEO-a over time at station LWL-1, PGA Blvd. Tropical storms and hurricanes are indicated. Data from the FDEP/EPA’s STORET database downloaded February 26, 2010 from www.epa.gov/storet
pH
70
The pH decreased with depth during the stratification mainly due to hydrogen
sulfide, but the trend is seen to a lesser extent even in the other months (Figure 42). A
possible explanation is increased respiration (organic matter (OM) + O 2 → CO 2) with depth generating carbonic acid. There is a pronounced slump in pH and nitrates for July, particularly at the surface. In addition, the photoinhibition causes a decrease in photosynthesis and thus less carbon dioxide utilization in the water. This will lower pH as well because carbonic acid equilibrium equates carbon dioxide with carbonic acid.
pH Profiles 2009 0.5
-0.5 JAN
-1.5 APR
-2.5 MAY
-3.5 JUNE
depth(m) -4.5 JULY
-5.5 AUG -6.5
-7.5
-8.5 7.0 7.2 7.4 7.6 7.8 8.0 8.2 pH
Figure 42. The spatial and temporal pH variations in LLW.
Chlorobium
The most obvious indicator that the bacteria were in fact brown was their color.
Samples from oxygenated water remained light green during the entire process of pigment extraction. When the samples from the hypolimnion were filtered in the lab,
71 the retentate was a dark brown. This color in BSB has been attributed to the carotenoids mitigating the green of the chlorophyll (BCHL-e) (Olsen 1998). However, when the GSB cells underwent lysis in the grinder, they became a bright green.
BCHL-e is the signature pigment of the three species of brown sulfur bacteria
(BSB), Chlorobium phaeobacteroides, Chlorobium phaeovibrioides, and Pelodeicttyon phaeum. These bacteria all contain a collection of the various homologues in a fairly similar arrangement. All of the homologues have identical UV-Vis spectra with peaks at
466 nm and 654 nm (see Figure 14), but they elute at different times based on their degree of alkylation and the esterified alcohol (i.e. geranyl, farnesyl, hexadecanol:
Manske et al. 2005) (Figure 43). Therefore, the only way to distinguish between them is by retention time or through the use of liquid chromatography-mass spectrometry (LC-
MS). The retention times were very consistent throughout the samples.
Figure 43. The BCHL-e homologues (Manske et al. 2005) (ethyl/methyl [E,M]-BCHL-eG, ethyl/ethyl [E,E]-BCHL-eG, propyl/ethyl [P,E]-BCHL-eG and isobutyl/ethyl [I,E]-BCHL eG eluted first. Homologues esterified with farnesol were ethyl/methyl [E,M]-BChl eF, ethyl/ethyl [E,E]-BChl eF, propyl/ethyl [P,E]-BChl eF, isobutyl/ethyl [I,E]-BCHL eF. Trace amounts of the secondary homologs were also detected. Carotenoids,Isr1 and Isr2 = isorenieratene.)
72
In the Banyoles region of Spain, the meromictic freshwater lakes’ dominant species is Chlorobium phaeobacteroides whereas in the meromictic, brackish coastal lagoons, the main species identified are Chlorobium phaeovibrioides and Pelodeicttyon phaeum (Borrego et al. 1997). The comparison in BCHL-e homologue peaks between
Chlorobium phaeobacteroides and Chlorobium phaeovibrioides is illustrated in Figure 44, showing that they are quite similar. Considering the salinity and conditions of LLW, the bacteria found is either Chlorobium phaeovibrioides or Pelodeicttyon phaeum. The latter species is distinguished by the fact that it is capable of forming gas vesicles. (Borrego,
1997) Overman and Pfennig published a study of these two strains in 1989 which described Pelodeicttyon phaeum to exhibit a rosy red shine in transparent light. This
characteristic was not observed in the GSB of LLW.
Comparing differences in chromatograms of BCHL-e homologues of the GSB in
LLW to those of Borrego (Figure 44) suggests that the GSB in LLW is Chlorobium phaeovibrioides. Unfortunately, without mass spectroscopy or a known sample, the identification is not conclusive.
73
C LLW
Figure 44. A and B chromatograms from Borrego et al. 1997; C from LLW June at 6 m.
Chlorobium Carotenoids
The dominant carotenoid in Chlorobium phaeobacteroides is isorenieratene
(Hirabayashi et al . 2004, Glaeser et al . 2002). This was the only carotenoid detected in
the hypolimnion throughout the study. There were only two isorenieratene peaks found
in the water column samples. The first peak had maxima in the UV-VIS spectrum at 458
nm and 482 nm (Figure 45). The following peak had a blue shift to maxima at 448 nm
74 and 472 nm, as well as a spike towards the UV end of the spectrum. This indicates that it is the cis form of isorenieratene .
Figure 45. Spectrum of trans -isorenieraten e from a sample taken July at 7 m.
75
Analysis of the ratios of each form of isorenieratene to BCHL-e throughout depth
showed that there was only a strong dominance of trans -isorenieratene in the
uppermost sections of the BSB bloom (Figure 46). The cis form of a carotenoid is more
prevalent in older cells, therefore indicating degradation. The younger Chlorobium are
advantage to these novel cells. The maximum concentration of the bloom rose from 7 m
June carotenoid/Bchl e ratios July carotenoid/Bchl e ratios -4.0 -4.0 (a) -4.5 -4.5 (b)
-5.0 -5.0
-5.5 -5.5 -6.0 -6.0 transIsr:Bchl e depth(m) transIsr:Bchl e -6.5 -6.5 cisIsr:Bchl e -7.0 -7.0 cisIsr:Bchl e -7.5 -7.5 0 0.01 0.02 0.03 0.04 0.05 0 0.01 0.02 0.03 0.04 0.05 ratio ratio to 6 m from June to July, increasing accessibility to light. In June, the ratios converge to
a 1:1 ratio at 7.5 m, where in July they diverge from 5.5 m and below. The apparent self-
shading mechanism of the bloom in July caused a drop at 7 m. This could explain the
increasing proportion of cis carotenoids, as there were potentially more unhealthy cells.
Figure 46. carotenoid/BCHL-e ratios throughout the metalimnion and hypolimnion in (a) June and (b) July
BCHL-e Homologues
Samples taken in June and July from the hypolimnion were found to contain a bloom of the brown phototrophic sulfur bacterium. This was indicated by 14
76 homologues of BCHL-e. To quantify the homologues in PIGCALC, the extinction coefficient used for BCHL-e was 155.6 mM -1 (Borrego et al. 1999b).
GSB possibly have a photo-adaptive mechanism based on the regulation of homologue synthesis as a response to self-shading effects (Borrego et al. 1995). The changes in BCHL-e’s degree of alkylation cause the shifts in retention time for each peak.
It has been suggested that the saturation of the antenna with highly alkylated homologues could be useful during episodes of light limitation. Yacobi and others (1990) described the BCHL-e homologues of Chlorobium phaeobacteriodes in Lake Kinneret and identified two clusters. Three small initial peaks were termed 1-2-3, and the large triplet peaks that follow were named 4-5-6 and were described as a “trident”. The trident is followed by two other peaks from another GSB, Prosthecochloris , and then pigments from two species of purple sulfur bacteria (PSB); Thiocapsa and Rhodopseudomonas .
While the ratio of the trident peaks remained constant throughout depth in situ, this was not the case in lab experiments and in other lakes (Borrego et al. 1997).
Borrego and co-workers studied the BCHL-e in Chlorobium phaeobacteriodes in the Lake Banyoles (LB), Spain. The lake was sampled fortnightly, and in the deepest part.
The initial four BCHL-e peaks were named e 1-e4, respectively, and the subsequent homologues are clustered in the group named “secondary homologues” (SH) (Figure
44). The e 2-e4 peaks are the three towering ones that correlate to the “trident”. The
“SH” are thought to be end products of BCHL-e biosynthesis, with different esterifying alcohols than farnesyl (Borrego et al. 1997). These have been identified by Mankse et al.
(2005) as alkylated BCHL-e homologues esterified with hexadecanol, a straight chain
77 saturated C-C alcohol. These peak names were kept in the current study to compare the relationships among BCHL-e homologues in LLW with other studies. There was an extra
initial peak, thus it was named e 0.
In LLW, the percent content of secondary homologues increased with depth in
July but in June this was less obvious. There is a distinct increase in secondary
homologues at 7 meters in June, which corresponds to the BCHL-e maximum. This indicates that the bloom was in a cogent growth stage at that depth, while it had stabilized by July. These results are incongruous with those of Borrego and others
(1997), in which SH increased over time (Figure 47). The peaks e 3 and e 4 are the more
highly alkylated pigments, and their percent content also increased over time in Lake
Banyoles (Borrego and Garcia-Gil 1994) (Figure 47). This trend was also the opposite in
LLW, as BCHLs e1 and e2 became more predominant (Figure 48).
(a) (b)
Figure 47. Progression of (a) secondary (c) homologues, (b) BCHL e2-e4 in basin III at a deeper part of the Lake Banyoles (max depth: 20 m), and (c) BCHL e2-e4 in basin IV, a more shallow region (max depth: 15 m) (Borrego and Garcia-Gil 1994).
78
(a) June BCHL -e Homologues
-2.0
-3.0 e0 -4.0 e1 -5.0 e2 -6.0 e3 e4 -7.0 2ndary -7.5
-8.0
0% 20% 40% 60% 80% 100%
(b) July 2009 BCHL-e Homologues -4.0
-4.5
-5.0
-5.5 e0 e1 -6.0 e2 -6.5 e3 e4 -7.0 2ndary
-7.5
-8.0
0% 20% 40% 60% 80% 100%
Figure 48. BCHL-e homologue distribution in water column (a) June, and (b) July.
79
Internal Seiches
It was hypothesized that a PSB plate should occur above the Chlorobium bloom in LLW, and this is why samples were taken at half meter increments in July. PSB contain
BCHL-a or BCHL-b, and these pigments were not detected at any time during the study.
There is still a possibility that PSB has yet to be discovered in LLW. However, it is
possible that an internal seiche could exist in the lake and move the oxycline in waves,
thus disrupting PSB plate formation. Studying this is beyond the scope of this thesis
research.
Internal seiches also serve to cause resuspension of sediment, leading to an
increase in algal productivity as a result of the injection of nutrient laden hypolimnetic
waters into the epilimnion (Ostrovsky et al. 1996). This frequent migration and mixis of
the metalimnion might also explain the trace amounts of BCHL-e found in the upper layers in June. The hypothesis that an internal seiche could exist comes from the fact that LLW is basically a bowl with strong tidal flood and ebb at a single small inlet at the southwest corner.
Sediment Studies
Bacterial carotenoids are preserved in sediments to a higher degree than that of algal carotenoids (Villanueva et al. 1994). This was supported by data, in which the ratio of algal to bacterial carotenoids went from 8:1 in 0-10 cm to 4:1 in 30-40cm.
Interestingly, there was no trace of CHL-a or its breakdown product below 10 cm.
Comparing BCHL-e : -CHL-a molar ratios gives clues as to whether the pre-depositional
environment was oxic or anoxic. More oxic conditions in the water column give way to
80 more bioturbation and reworking of surface sediments by the burrowing and feeding of benthic organisms, decreasing the abundance of OM and intact pigments entering the deeper layers of the sediment. Anoxic sediments ensure conditions of higher preservation by avoiding many of these destructive forces. The absence of CHL-a in deeper layers indicates that the lake’s sediment was previously more oxic. The bottom water samples ( 8.0-8.5 m) containing sediment were taken throughout the year by closing the sampler as it hit bottom. All of these samples contained substantial amounts of BCHL-e.
Organic Matter (OM)
Organic Carbon (% Corg ), actually pyrolyzable matter, by depth is shown in Table
4 as measured from the core sample taken in September 2009. When organic matter
has been deposited at the water column-sediment interface, the environment is quite
unstable due to bioturbation and post-depositional diagenetic reactions. Once it has
been buried below subsequent surficial oxic sediments it is a more stable component of
the, often anoxic, sedimentary column.
The section of the core that was closest to the surface, as well as the deepest section, both had close to three quarters in their composition as OM (Table 4). This indicates high sediment content of OM likely as septic seepage, as these percentages are found in raw sewage. The top section does have around 5% more OM, but this does not imply that the conditions have gotten worse, because decomposition with increased depth must be taken into consideration. Additionally, chemical oxygen demand (COD) was higher than the highest limit of COD that the Hach water analysis could measure,
81 indicating it was at least 150,000 mg/L. This is also akin to sewage's COD levels (Lipp et
al. 2001)
Table 4. Sediment core analysis results per section from LLW in September 2009.
% OM by AFDW weight TOP 0.20 77.5 0-10 cm 0.16 78.9 0.21 78.6 BOTTOM 0.25 74.0 30-40cm 0.25 73.7 0.26 74.0
Carotenoids
There are a variety of pigments throughout the sections, as shown in Figure 49, representing the algal groups shown in Figure 50. Peridinin, indicative of dinoflagellates, is only present in the 10-30 cm sections. Alloxanthin, indicative of cryptophytes, was absent from 10-20 cm, but found in 0-10cm and at z > 20 cm.
There is an apparent absence of diatoms in the core despite their dominance in the water column and this is likely related to the fact that their main carotenoid, fucoxanthin, degrades quickly (Louda et al. 2002). Fucoxanthin is generally traced by its
anoxic sedimentary transformation remnant, loliolide, which is not detected in HPLC
(Repeta 1989). The molecular structure of fucoxanthin causes it to decompose more
than other marine biomarkers including alkenones and long-chain diol/keto-ol
structures (Singhe-Damsté and Koopmans 1997). In addition to structure, burial
82 efficiency is depen dent on the amount of time that accumulating particles are exposed to molecular ox ygen in sedimentary pore waters (Singhe-Damsté and Koopman s 1997).
Core Carotenoids
Peridinin 0-10cm Fucoxanth
Alloxanthin
10 -20cm Diato Lutein
Depth Zeaxanthin 20 -30cm Tns-B-Car
Tns Isr
30 -40cm Cis Isr
0% 20% 40% 60% 80% 100% Percent Compostion
Figure 49. Carotenoid composition in sediment core sections (LLW, September 200 9).
Core Algal Taxonomy
0-10cm
10 -20cm CYANOS DIATS 20-30cm DINOS CRYPTOS 30 -40cm
0% 20% 40% 60% 80% 100% Percent Compostion
Figure 50. Algal taxonomy in sediment core sections (LLW, September 2009) .
83
One would expect a much higher degree of preservation of the pigments in
LLW’s anoxic sediment. If oxygenated freshwater is seeping in from the baseflow, particularly in the wet season, significant degradation in the deeper sediment layers may result. However, there were no increased dissolved oxygen levels measured in the sediment throughout the year to support this theory.
BCHL-e Homologues
The abundance of the BCHL-e homologues in the core varied considerably more than those of the water column and this can be attributed to differences in degradation rates based on structural differences. The e 2 homologue was the dominant one in most
water samples, yet it consistently had the lowest concentration in the core. The
opposite is seen for e1, as it greatly surpasses BCHLs e 2 and e3, yet was not abundant in
the water column (Figure 51). BCHLs e2 and e3 are likely to be the least well preserved
homologues, while e 1 is the most resilient.
84
BCHL-e Homologues in LLW Core Sample 0
-5
-10
-15 Bchl e1 -20
Depth(cm) Bchl e2 -25 Bchl e3 -30 Bchl e4 -35 0.0 0.5 1.0 1.5 2.0 2.5 conc (µmol/L)
Figure 51. BCHL-e Homologues in LLW Sediment Core Sample from September 2009.
Accumulation
A Lake Worth Lagoon (LWL) Muck Sediment Monitoring Study determined accumulation rates through the analysis of concentrations of the isotopes Cs-137 and
Pb-210 (PBCERM 2003). The sedimentation rates calculated from core samples at four sites in LWL but outside LLW varied from 0.1 to 1.0 cm/yr. Amount of sedimentation depends on the amount of suspended solids in the water and the degree of stagnation that allows those solids to be preserved. There was/is obviously more stagnation in LLW than the rest of LWL due to the depth and lack of tidal mixing. With 1.00 cm/yr as a minimum, the core represents between 40 and 400 years of accumulation. However, as
LLW was excavated in the 1950s, this accumulation can only date back to that period
(~60 years).
85
6. Conclusions
The data supports that the stratification within Little Lake Worth (LLW) is induced by the formation of a pycnocline. It is evident that, while June exhibited a stronger difference in conductivity and temperature, July had a more pronounced stratification according to DO, pH, and nitrates. If the chronological order of intensification were interpreted to indicate causality, it would infer that the stratification was initiated due to changes in specific conductivity and temperature (viz. thermohaline stratification). Both of these are the factors that contribute to density.
Once the stratification had been established, it likely promoted by other physiochemical parameters such as the oxycline and chemocline formation.
During stratification, the hypolimnion of LLW was replete with hydrogen sulfide and BCHL-e. This indicates that the dominant species was the strictly anaerobic and obligately photoautotrophic Chlorobium . Although it cannot be concluded without a doubt, pigment results indicate that the species is most likely Chlorobium phaeovibrioides , a non-motile, brown-colored GSB (width: 0.6 to 0.8 µm; length: 1.3 to
2.7 µm) that forms rod-shaped cells.
86
Human-Environment Interaction
Nitrates were the only pollutants measured for that could be direct indicators of
anthropogenic inputs to Little Lake Worth. Data from the South Florida Water
management District, from a sampling station at PGA Blvd, indicated a range of
concentrations of nitrates, phosphates and ammonia. The peaks in these nutrients occur
during heavy rains, possibly from runoff contributing to significant baseflow
(groundwater seepage) that leaches these pollutants. High nutrient levels result in
eutrophication, which causes concurrent peaks in Chl-a and dissolved oxygen levels. The
swift reduction in salinity coupled with a spike in nitrates measured at the lake’s bottom
during June, July, and August could support the baseflow hypothesis. However, the
increased nitrates could be from the sediment, and the reduced salinity could be from a
leaking pipe close to the sampling site. Unfortunately, specific conductance and nitrates
were not measured in other areas to determine if the observed changes were consistent
throughout the lake.
Extreme weather such as hurricanes and tropical storms cause severe wind and
rain that exacerbate the situation further by decreasing salinity and increasing turbidity.
Both of these effects are harmful to the ecosystem because they threaten the organisms
and SAV. Many species of seagrasses have different salinity ranges that are threatened
by the drastic fluctuations (Doering et al. 2002). Reduced clarity attenuates the light and
inhibits photosynthesis.
87
Restoration Possibilities
There are several potential benefits of restoring Little Lake Worth, depending on which method of restoration was utilized. Reduction of incoming pollutant loads by imposing limits on fertilizers and backfilling septic tanks in conjuction with sanitary sewer hookups will aid in reducing nutrient loading and levels. This will decrease the eutrophication in the entire LWL, as nutrients are not fully flushed out during tidal cycles. The ecosystem improvements attained will provide increased water clarity and biodiversity, increasing the area’s recreational value. In a recent restoration project, the town of Hypoluxo at the south end of LWL had 99 single family homes’ septic systems removed. This and having the homes connected to the municipal sewer line cost
$900,000. Some of these systems had been over 50 years old and many were within 3 meters of the lagoon (PBCERM 2006). As shown in Figure 8, there is a high density of homes with septic systems around LLW, where similar conditions possibly exist.
Nutrient reduction alone will not stop the hydrogen sulfide and anoxia produced during stratification, and the health hazards present from the sewage. The existing bottom sediment has such a high Chemical Oxygen Demand (it measured over the limit of 150,000 mg/L on the Hach Water Analysis), that it alone is enough to reduce dissolved oxygen in nearby water for an indefinite period of time. The installation of a lake aeration system would increase circulation and dissolved oxygen, but it can also bring dangerous pathogens closer to the surface, increasing human exposure. Active management to bury existing polluted sediments by adding fill to the lake would likely
88 terminate the hypoxia, hydrogen sulfide, and sulfur bacteria blooms, but baseflow could still bring pollutants right up through the sand. It is not the sulfur bacteria that impair the waters, but their presence is significant because it indicates stratification with anoxia. BCHL-e is constant throughout the 50 cm sediment core, suggesting the presence of sulfur bacteria, and therefore anoxic conditions began shortly after Little
Lake Worth was deepened (considering the accumulation rate minimum was estimated at 1 cm/yr). Reducing the depth of the water is the only way to get SAV back to the ecosystem.
An example of successful restoration within close proximity is the Twelve Oaks
Dredge Hole, a 25-acre area in NW Lake Worth Lagoon. It was dredged for spoil the
1940’s, but then filled in 1997 with over 100,000 m3 of material. Since then, monitoring has shown that benthic biodiversity has increased dramatically (DEP 2007).
In 2001, there was a surplus of spoil material available in the area, and it was considered for transport north to fill LLW. The plan was abandoned due to transportation issues. A barge will not fit under the PGA bridge to LLW, and some type of pumping system would have had to be arranged (Dr. Roberts of the FAU Geosciences
Dept, personal communication). In addition, the volume required to fill LLW is substantial, with 160,000 m 3 needed to fill just 1 meter. Recently, Palm Beach County has proposed to cap the muck by filling LLW with sand, and estimated the cost between
$5 million and $10 million. However, it was classified as a low priority action plan (ERM
2009).
89
Bacterial and physiochemical considerations
As changes in bacterial productivity and composition were measured throughout depth as well as time, there are different dimensions by which to analyze the results.
Light availability decreases with depth. To that effect, light increases as the stratification persists and the bloom rises closer to the surface. Although there were some indistinct trends relating light availability with bacterial productivity and composition, most would need much more frequent sampling to confirm.
Chlorobium Homologues
The mechanism by which the chlorosomes can organize their antennae to optimize light-harvesting at critical light conditions is generally accepted (Borrego et al.
1997). This is achieved by having high content of alkylated, long wavelength absorbing pigments that improve energy transfer and increase absorption. The results do support this hypothesis that GSB increase efficiency by adjusting the ratio of homologues and carotenoids in response to reductions in light intensity. However, the alkylated pigments increased with depth in Little Lake Worth, contrary to the results found by Borrego et al.
(1997) in the Banyoles lakes.
Chlorobium Carotenoids
Another interesting comparison is the spatial and temporal differences in carotenoid content in the chlorosomes. Chlorobium species are characterized by a lack
of proteins and high carotenoid content. The ratios differ considerably, as do the
opinions on the function of these carotenoids (Arellano et al. 2001). Some authors have
90 suggested that the carotenoids are photoprotective, while some argue their importance as light harvesting capabilities. None of the results were supportive of either argument, as the only bacterial carotenoid found was isorenieratene, coupled with its cis form. The prevalence of cis isorenieratene increased with depth, indicating that older cells were more predominant. This is attributed to newly formed fully active bacterial cells’ ability to decrease cell density in order to increase buoyancy and therefore light availability, maximizing photosynthetic efficiency.
Applications and Further Research
Human Environment
A useful follow up study would be one that targets finding specific data about
nutrient and fecal coliform levels and groundwater flow. This would confirm that
baseflow contributes an observable nitrate load to LLW. For example, a long term study
quantifying groundwater flow around and into the lake, and the corresponding nutrients
levels throughout the lake’s bottom would give sound evidence to support the pollution
seepage. It would be useful to collect more specific data on the septic systems, such as
age, condition, and distance from the lake. This is important because the results would
better correlate the baseflow to its source. If a direct connection between septic tanks
and pollution in the lake were found, it could help drive regulation and management to
take action for restoration.
Another useful study would be one that explored the other remediation options
that were discussed and performed a cost benefit analysis of some sort. If pollution
91 sources could be pinpointed and quantified, increases in ecosystem value could be determined by estimating the benefit of each restoration plan. This would help determine which projects were the most cost efficient ones to invest it.
Chlorobium Homologues
Although the homologue distribution of a given species is not fully considered a taxonomical feature (see Borrego et al. 1994), it appears to be a useful tool in eco- physiological studies, assuming that pigment composition of a population may reflect both the physiological status of cells and the light regime under which the population has been grown (see Borrego et al. 1995).
The real advantage of studies such as the present is that they allow the researcher to go beyond taxonomic classifications and into specific physiological aspects of a single population. The pigments eluted in HPLC during mixis, such as the carotenoid, fucoxanthin, occur in a variety of species, many times overlapping into many groups.
The input of these pigment concentrations into PIGCALC merely gives an idea of the abundance of each of four algal groups, and has limited use for studying a particular species. The dominance and diversity of the Chlorobium allows for a more precise and detailed analysis of their response mechanisms to various conditions, namely light availability. The trends that are found can be a useful tool in making eco-physiological connections between the population, the physiological status of cells, and the light regime under which the population has been grown (Borrego et al. 1995).
92
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8. Appendix
A1. Mechanism for Summer stratification in LLW
A2. Retention times and UV/VIS (PDA ) spectral data for chlorophylls, chlorophyll derivatives, carotenoids and scytonemin. For long (C18) column.
______PIGMENT ______TIME(min.) ____UV/VIS(nm) ______Solvent Front ~2.9 N/A Scytonemin-like 3.47 372, 440, 562 Bacteriochlorophyllides-d unkn 412, 428 , 616, 658 “P468” 3.66 472 “P457” 4.45 460
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Scytonemin (Reduced) unkn Chlorophyllinde-b unkn 464, 654 Chlorophyllide-a 4.75 426, 582, 616, 660 Chlorin-e6 free acid unkn 414 514, 554, 606, 660 Chlorophylls-c1/-c2 5.95 446, 582, 628 Scytonemin (oxidized form) unkn 388 Fucoxanthinol 11.21 452 Cu-chlorophyllin unkn 406, 508, (575), 628 Pyro-Chlorophyllide-a* 9.65 426, 582, 616, 660 Peridinin 10.85 474 Pyropheophorbide-b unkn 438, 530, 600, 656 Vaucheriaxanthin (19-hydroxy-neoxanthin) unkn (422), 440, 476 19’-butanoyloxyfucoxanthin unkn 446, 470 Siphonoxanthin* unkn 448, (468) Pheophorbide-a 13.21 408, 506, 534, 610, 668 Fucoxanthin 13.78 452 Neoxanthin 14.55 414, 438, 466 Bacteriopheophorbides-d unkn 408, 426 614, 656 “Polar” MYXO (= aphanizophyll ?) unkn (448), 476, 508 19’-hexanoyloxyfucoxanthin unkn 446, 468 Pyropheophorbide-a 16.95 412, 510, 540, 608, 666 Violaxanthin 17.50 418, 442, 470 Prasinoxanthin unkn 454 Pheophorbide-b ME unkn 436, 526, 598, 654 Pheophorbide-b’ ME unkn 436, 526, 598, 654 Myxoxanthophyll (MYXO) 20.21 (448), 476, 508 Astaxanthin unkn 480 Cu-Pheophorbide-a-ME unkn 408 500, 540, (590), 642 Dinoxanthin 18.25 418, 442, 470 cis-Fucoxanthin 17.11 320 , 440, (462) Diadinoxanthin 19.35 (426), 448, 476 Cu-Mesopyropheophorbide-a-ME unkn 418, 544, 592, 636 Bacteriochlorophyll-d(1) unkn 408, 428 614, 656 Antheraxanthin* 20.55 446, 473 Cu-Chlorine-e6-TME unkn 406, 500, 634 Pyropheophorbide-b ME unkn 436, 526, 598, 654 Bacteriochlorophyll-d(2) unkn 408, 428 614, 656 BCHL-c3 (7%:4n-Pr, 5Et, 2S)* unkn 434 630, 666 Pheophorbide-a ME unkn 410, 508, 538, 608, 666 Cu-Chlorin-p6-TME unkn 406, 500, 538, 640 Phoenicoxanthin unkn 480 Alloxanthin unkn (426), 454, 482 BCHL-c5 (71%: 4Et, 5Et, 2R)* unkn 434, 630, 666 Pheophorbide-a’ ME unkn 410, 508, 538, 608, 666
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Diatoxanthin unkn (426), 454, 484 BCHL-c4 (17%: 4n-Pr, 5Et, 2R)* unkn 434, 630, 666 BCHL-c1 (5%: 4iBu, 5Et, 2S)* unkn 434, 630, 666 Monadoxanthin unkn (422), 448, 476 Bacteriochlorophyll-d(3) unkn 408, 428 614, 656 Pyropheophorbide-a ME unkn 410, 508, 538, 608, 666 Cu-Purpurin-18-ME unkn 416, 504, 544, 622, 670 Phoenicoxanthin unkn 476 Lutein (β,ε-carotene-3,3’-diol) 22.55 (422), 446, 476 Isozeaxanthin (β,β-carotene-4,4’-diol) unkn (424), 454, 480 Zeaxanthin (β,β-carotene-3,3’-diol) 23.05 (424), 454, 480 Bacteriochlorophyll-d(4) unkn 408, 428 , 614, 656 4’-hydroxy-echinenone* 23.24 462 (7-?) cis-zeaxanthin* unkn 336, (426), 448, 474 Siphonein* unkn 334, 452, (478) Bacteriochlorophyll-d(5) unkn 408, 428 614, 656 Bacteriochlorophyll-agg unkn 360, 580, 770 Canthaxanthin (β,β-carotene-4,4’-dione) 23.40 472 Cu Mesoporphyrin-IX DME (Int.Std .) 23.80 394, 524, 558 Gyryoxanthin Diester (422) 448,470 Bacteriopheophytin-c3 * ( 7%) unkn 412, 518, 550, 614, 668 Monodemethylated spirilloxanthin * unkn 468, 494, 530 Rhodovibrin* unkn 458, 484, 518 Bacteriopheophytin-d(1) unkn 424, 520, 612, 652 Bacteriopheophytin-c5 * ( 62%) unkn 412, 518, 550, 614, 668 Bacteriopheophytin-c4* ( 27%) unkn 412, 516, 552, 614, 668 Bacteriopheophytin-d(2) unkn 424, 520, 612, 652 Bacteriochlorophyll-ap unkn 358, 580, 772 Chlorophyll-b 25.15 458, 596, 646 Cyclopyropheophorbide-a-enol unkn 360,426, 628, 686 3,4-Didehydrorhodopin* unkn (458), 486, 520 Crocoxanthin unkn (422), 448, 476 Bacteriopheophytin-c1 * ( 4%) unkn 412, 518, 550, 614, 668 Rhodopin* unkn 474, (505) Spirilloxanthin unkn 470, 496, 530 Chlorophyll-b’ (epimer) 26.95 458, 596, 646 1 - 13 -oxydeoxo-Chlorophyll-a (prep:BH 4 .) unkn 416, 514, 562, 606, 654 Chlorophyll-a-allomer (“13 2-OH-Chl-a” ) 27.20 430, 582, 616, 662 Cryptoxanthin unkn (428), 456, 480 Isocryptoxanthin unkn (428), 456, 480 Chlorophyll-a 27.90 430, 582, 616 662 Echinenone (β,β-caroten-4-one) 28.50 462 Chlorophyll-a’ (epimer) 28.55 430, 582, 616, 662 Anhydrorhodovibrin * unkn 460, 482, 518 105
Pheophytin-b-allomer (“13 2-OH-PP-b”) unkn 436, 528, 598, 656 Bacteriopheophytin-agg unkn 358, 526, 750 Bacteriopheophytin-ap unkn 358, 526, 750 Pheophytin-b unkn 436, 528, 598, 656 Bacteriopheophytin-ap'(epimer) unkn 358, 526, 750 Pheophytin-b’ (epimer) unkn 436, 528, 598, 656 Astaxanthin esters (Panulirus argus ) ~ unkn 478 Lycopene unkn 448, 474, 506 Pheophytin-a-allomer (“13 2-OH-PP-a”) 29.85 410, 502, 536, 610, 666 Pyrobacteriopheophytin-ap unkn 358, 526, 750 Pyropheophytin-b unkn 436, 528, 598, 656 Pheophytin-a 30.65 410, 502, 536, 610, 666 γγγ-Carotene unkn 440, 465, 495 Pheophytin-a’ (epimer) 30.95 410, 502, 536, 610, 666 ααα-Carotene 32.55 (422), 448, 476 βββ-Carotene (all-trans, all-E) 32.80 (428), 456, 482 cis-βββ-Carotene (15-Z, tent.) 32.91 338, (424), 448, 476 Purpurin-18-phytyl Ester * unkn 360, 408 546, 696 Pyropheophytin-a 33.06 410, 502, 536, 610, 666 Pheophorbide-a-steryl ester (s) ~ unkn 410, 502, 536, 610, 666 Pyropheophorbide-a-steryl esters * ~ unkn 410, 502, 536, 610, 666
FOOTNOTES: -- Retention times are from injection. Solvent peak is typically at 1.0-1.2 minutes. UV/VIS spectral maxima are given in the eluting solvent as recorded via the PDA. Compounds given in bold type are those for which authentic knowns and/or known culture distributions were utilized. The bacteriochlorophyll-c and bacteriopheophytin-c series is an authentic known assemblage with only the structural isomer positioning being estimated. Compounds marked with an asterisk were tentatively identified based upon UV/Vis spectra and chromatographic positioning in relation to literature reports and ‘expected’ physicochemical behavior. Compounds without an asterisk were for single known standards (Sigma-Aldrich Chemical Co., DHI) and/or in known mixtures of a pure algal culture (Carolina Biological Supply and CCMP). Derivatives of knowns ( e.g. pheophytin-a, pyropheophytin-a, etc. ) were formed in vitro by accepted methods and products verified by appropriate molecular spectrometric determination (UV/VIS, MS: see Louda, 1993). For runs which incorporated a guard column, add retention times change +0.27 minutes at CHL-c to +1.17 minutes at Diadinoxanthin and then decrease to + 0.42 minutes at pyroPheophytin-a. Degraded columns or other minor alterations can lead to shorter retention times before about 20 minutes, after which retention times are relatively unaffected by column / solvent subtleties.
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A3. Normalized absorption spectra of chlorophylls -a and -b and bacteriochlorophylls - a, -b, -c, -d and -e in polar organic solution.
The pigments were derived from various photosynthetic organisms and the spectra were recorded as the pigments eluted from an C 18 HPLC column with a solvent consisting of methanol, acetonitrile, ethyl acetate and water in varying proportions. The spectra in the graph below resemble the spectra in methanol or ethanol.
Pigment Chl a Chl b BChl a BChl b BChl c BChl d BChl e
Color in graph above black red magenta orange cyan blue green
Absorption maxima 430, 463, 373, 469, 364, 770 434,666 427,655 (nm) 663 648 795 654
408, 367, 412, 406, 435, Absorption maxima of ND 357, 746 corresponding 664 776 666 657 665 pheophytin (nm)
Available from http://www.bio.ku.dk/nuf/resources/scitab/chlabs/index.htm
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