DRAFT
BIOTIC HYDROLYSIS OF ORGANIC PHOSPHORUS IN
EFFLUENTS OF STORMWATER TREATMENT AREAS
Final Report
October 2004
C-13986
Submitted to:
South Florida Water Management District
3301 Gun Club Road
West Palm Beach, Fl 33406
Submitted by
K. Sharma, P. Inglett, K. R. Reddy and A. Ogram
Soil and Water Science Department
University of Florida,
Gainesville, Fl. 32611-0510
R. G. Wetzel
Department of Environmental Sciences and Engineering
School of Public Health
The University of North Carolina
Chapel Hill, NC 27599-7431
W. T. Cooper, III
Department of Chemistry and Terrestrial Waters Institute
Florida State University
Tallahassee, Fl 32306-3006
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TABLE OF CONTENTS
EXECUTIVE SUMMARY
i
v
CHAPTER
1
INTRODUCTION
2
ENZYMATIC HYDROLYSIS OF DISSOLVED AND PARTICULATE ORGANIC
PHOSPHORUS (P) IN THE EFFLUENTS OF STORMWATER TREATMENT
AREAS (STAs) AND PERIPHYTON MATS.
2.1
2.2
2.3
2.4
2.5
3
INTRODUCTION
MATERIAL AND METHODS
2.2.1
Experimental site, sample collection and processing
2.2.2
Effect of C and N on APA
▪ Batch studies………………………………………………
▪ Flow through studies
2.2.3
Effect of light on APA
2.2.4
Alkaline phosphatase assay
RESULTS AND DISCUSSION
2.3.1
Effect of N-additions on APA in periphyton
2.3.2
Effect of C-additions on APA in periphyton
2.3.3
Effect of light on APA in periphyton
CONCLUSIONS
REFERENCE
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15
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16
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17
21
24
26
28
CHARACTERIZATION OF PHOSPHATASE-PRODUCING BACTERIAL
ASSEMBLAGES
3.1
3.2
3.3
INTRODUCTION
MATERIAL AND METHODS
3.2.1
Study site and sampling
3.2.2
Morphology of the periphyton mat
3.3.3
Microscopy
▪
Cryoembedding and cyosectioning
▪
Fluorescent staining
▪
Microscopy and image analysis
3.3.4
Phospholipid Fatty Acid analysis
▪
Sample handling
▪
Extraction
▪
Column chromatographic separation
▪
Transesterification
▪
Nomenclature of PLFA
RESULTS
3.3.1
Microbial characterization of Periphyton mat
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36
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3.4
3.5
3.6
3.7
4
INTRODUCTION
MATERIAL AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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60
ANALYTICAL SPECIATION OF ORGANIC P BY CAPILLARY
ELECTROPHORESIS WITH INDUCTIVELY COUPLED PLASMA MASS
SPECTROMETRY
5.1
5.2
5.3
5.4
5.5
6
38
38
42
44
44
46
DETERMINE THE ROLES OF PHOTOLYSIS IN HYDROLYZING WATER
COLUMN DISSOLVED ORGANIC P (DOP).
4.1
4.2
4.3
4.4
5
3.3.2
Screening of phoA
3.3.3
In situ PPO analysis
DISCUSSION
PROPOSED ASSOCIATION OF PPO AND CYANOBACTERIA
CONCLUSIONS
REFERENCES
INTRODUCTION
MATERIAL AND METHODS
5.2.1
Capillary Electrophoresis Separations
5.2.2
High Performance Liquid Chromatography
5.2.3
Inductively Coupled Plasma Mass Spectrometry
5.2.4
Time of Flight Mass Spectrometry
RESULTS AND DISCUSSION
5.3.1
Quantitatively reliability of Inductively Coupled Plasma Mass
Spectrometry
5.3.2
Capillary Electrophoresis Separation with ICP-MS Detection
5.3.3
High Performance Size Exclusion Liquid chromatography
5.3.4
Electrospray ionization time of mass Spectrometry
DISCUSSION
REFERENCES
63
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65
67
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68
68
68
69
71
73
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75
EXPERIMENTAL MESOCOSM SET-UP AND WATER QUALITY
MONITORING.
6.1
6.2
6.3
6.4
INTRODUCTION
MATERIAL AND METHODS
6.2.1
Study site
6.2.2
Effect of high glucose dosing (500 mg C l-1) on APA
▪ Mesocosm dosing
▪ Sample collection and processing
▪ Alkaline phosphatase assay
6.2.3
Effect of low glucose dosing (200 mg C l-1) and arginine on APA
RESULTS AND DISCUSSION
CONCLUSIONS
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6.5
REFERENCES
99
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EXECUTIVE SUMMARY
Stormwater treatment areas (STAs) are being used as buffers to reduce P in surface
runoff discharged from Everglades Agricultural Area (EAA) before they enter the Water
conservation areas(WCAs)1 and 2A. The watercolumn TP concentrations have to be in
compliance with those set by the Environmental Protection Agency (EPA) (TP ≤ 10 µg P l-1).
Technologies currently being used to treat the STA outflow waters are capable of decreasing TP
concentrations to approximately 20 µg P l-1 The dominant residual P in the effluents is in
organic form (DOP) that can be easily removed from the watercolumn by the microbial and algal
P assimilation once it is hydrolyzed to orthophosphate form by phosphatase. High production of
phosphatase in periphyton therefore enables us to employ these natural assemblages of bacteria
and algae as a tool to reduce residual P. In order to investigate means to augment the removal
rate of residual P from the STA effluents researchers from the University of Florida, (Soil and
Water Science Department), in cooperation with and the South Florida Water Management
District (SFWMD), planned to conduct several controlled field and laboratory experiments on
enzymatic hydrolysis of dissolved and particulate organic phosphorus (P) in the effluents of
stormwater treatment areas (STAs), and (2) characterize the microbial assemblages responsible
for production of enzymes capable of hydrolyzing organic P, and (3) evaluate new approaches to
induce phosphatase enzyme production in microbial mats and its effect on hydrolysis of organic
P in the effluent.
Additionally the role of photolysis in hydrolyzing water column DOP was investigated
by a group of researchers at the University of North Carolina (department of ES and
Engineering, School of Public Health). The analytical speciation of OP using Capillary
Electrophoresis with ICPMS was conducted by a research group at the Florida State University
(Department of Chemistry) The following report evaluates the potential of the biotic hydrolysis
of the DOP in effluents of the STA. The objectives of the study were to:
▪
▪
▪
▪
▪
▪
Analyze the current
Determine the potential of biotic hydrolysis of DOP using periphyton by labscale
experiments
Characterize the PPB in periphyton mats
determine the roles of photolysis in hydrolyzing water column dissolved organic P
(DOP).
analyze the speciation of Organic P by Capillary Electrophoresis with Inductively
Coupled Plasma Mass Spectrometry
monitor water quality by scaling up the dosing study to experimental mesocosm level.
The first objective was to analyze the current data for the
TP levels in water column were 15 ppb-20 ppb. The water from STA outflow was being
treated by the green technology however the residual P was in organic form therefore it
had to be removed.
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The second objective was to determine ways to increase the enzymatic hydrolysis of the
dissolved and particulate organic phosphorus (DOP and POP).
Periphyton mats were dosed with various sources of carbon and nitrogen with the
purpose of increasing P-stress in the system and inducing higher phosphatase activity.
Additions of glucose and alanine were more effective in increasing the alkaline
phosphatase activity (APA) in the periphyton mats when compared with nitrate and
ammonia. Increased APA was observed likely as a result of increased P demand that was
induced by the change in the C: N: P ratios. Carbon limitation in the system was
apparent when higher APA was recorded in the C dosed mats. However, whether this
observed increase in APA within the periphyton mat was due to the increased biomass or
increased expression of the single cells is not known at present. This increase may also be
a result of a shift in microbial population/ community toward a phosphatase producing
population Utilization of external carbon source for stimulation of APA also suggests that
heterotrophs may be the key players in AP production Role of light in the stimulation of
APA was only evident in the presence of C substrate (glucose). Results from batch and
flow-through incubation studies suggest that accumulated metabolic and photosynthetic
products may be a important carbon source for the phosphatase producing orgasnims
(PPO).
The third objective was to characterize the phosphatase-producing bacterial assemblages (Task 3
as per contract)
The overall significance of this study for the area of enzymatically removing DOP
from the water column is that this information will help identify the dominant groups of
phosphatase producing bacteria (heterotrophic and autotrophic) within periphyton mats.
The results of this study supported the following conclusions: (1) The site of Pase activity
is not evenly distributed within the Everglades periphyton mat matrix, but instead is
localized in certain regions of the mat interior. (2) Pase activity occurred on the surface
of, or closely associated with photosynthetic cyanobacterial cells/filaments or algae. (3)
Patterns of Pase activity associated with non-chlorophyll-containing areas indicate that
heterotrophic organisms are the dominant mat PPO. More information is required to
definitively document the role of the heterotrophs in the Pase production and further
research is required to identify the bacterial groups that form the dominant PPO and to
quantify the contribution of Pase by the autotrophic and the heterotrophic organisms. .
Based on these results, we propose a model describing the association of
cyanobacteria and heterotrophic organisms and based on the premise that mat
heterotrophs are largely C-limited. Additional support for this hypothesis is provided by
results from other experiments (not presented) where C additions to periphyton have
stimulated Pase activity.
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The fourth objective was to determine the roles of photolysis in hydrolyzing water column
dissolved organic P (DOP). (Robert G. Wetzel, William R. Kenan Distinguished Professor,
Department of Environmental Sciences and Engineering, School of Public Health, The
University of North Carolina, Chapel Hill, North Carolina 27599-7431)
Light plays an overriding role as an ecosystem modulator. Light is obviously the primary
energy source for synthesis of organic matter in photosynthesis but is simultaneously a
major agent of direct decomposition of organic matter without ever entering a
biochemical metabolic process or by rendering chemically recalcitrant organic
compounds more bioavailable by partial degradation of macromolecules (Wetzel 2002,
2003). Anthropogenic activities can alter climatic and other environmental properties,
such as elevating CO2 concentrations or increasing UV radiation by decreasing
absorptive gases such as ozone, that in turn change both the chemical composition of the
organic matter and the rates of decomposition. In the Everglades, the phosphorus
eutrophication is clearly enhancing the development of invasive species of macrophytes.
These plants function to shield the system from the natural photolytic processes. These
alterations can impact the thermodynamic stability of ecosystems via changes to the
organic carbon pools, their bioavailability, rates of nutrient recycling, and energy fluxes
throughout the ecosystem.
The fifth objective was to analyze the speciation of Organic P by Capillary Electrophoresis with
Inductively Coupled Plasma Mass Spectrometry (Florida State University)
We have concluded that high-resolution capillary electrophoresis is probably not a
technique suitably robust and sensitive for routine quantitation of organic phosphorus
compounds from an oligotrophic wetland system such as the Florida Everglades. We
believe that size exclusion liquid chromatography is much more amenable for such
analyses. When combined with ICP-MS detection, this separation approach can provide
reasonable molecular weight information on dissolved organic phosphorus, as well as
organic P extracts from soil and tissue samples. In addition, because of the size of
eluents provided by SEC-HPLC, we propose that direct coupling of the SEC column to
the ICP-MS instrument be combined with fraction collection and subsequent highresolution time-of-flight molecular mass spectrometry. This combined approach will
allow estimates of the molecular weights of organic P (SEC-ICP-MS) as well as chemical
formula identification (ESI-TOF-MS).
The following flow chart summarizes our suggestion for a comprehensive
analysis of organic phosphorus speciation in the Everglades. We are currently analyzing
samples from a P-dosing experiment being carried out by the Reddy group at UF to
demonstrate the utility of this approach.
The sixth objective was to monitor water quality by scaling up the dosing study (task 2) to
experimental mesocosm level.
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Addition of carbon stimulated the alkaline phosphatase activity in the periphyton mats. A
positive correlation between APA and periphyton TOC suggests that increase in APA is
likely to be a result of increase in bacterial biomass however, the mechanism for this
stimulation is unknown as yet. This observation is also partially supported by the
microscopic results in task 3, which demonstrated a predominance of APA in association
with bacteria living in the periphyton matrix.
If this is true, two hypotheses could be asserted regarding the observed APA increases
following C dosing. First, labile C additions could stimulate bacterial growth and
increase the standing stock of microbial biomass. These bacteria are the dominant
producers of alkaline phosphatase, therefore, higher APA is due to the presence of more
alkaline phosphatase producers. Alternatively, the environment created as a result of C
dosing (low O2, lowered pH, etc.) may have led to an altered species composition of the
microbial community, favoring species who maximize C consumption. These species,
which are likely facultative or anaerobic organisms, may exhibit higher rates of APase
production. Unfortunately, neither of these hypotheses was conclusively demonstrated in
the experiments of this task.
The observed increases in water column P levels in the dosed tanks implies that C
additions at this high concentrations are not a likely candidate as a potential organic P
removal strategy. Lower C doses may better preserve the natural periphyton function, and
thus, have a more positive effect on water column P levels. This conclusion may be
premature, however, especially considering the limitations of the current study. Among
these limitations is the short-term nature of these experiments which monitored only the
period of days to weeks following the onset of C dosing. During this time numerous
changes in water column chemistry and periphyton species composition were occurring,
and had likely not reached a steady state condition by the conclusion of the experiments.
If allowed sufficient time, the characteristics of the high C system at equilibrium (e.g.,
higher overall APA) may offset the short-term negative impacts (increased P export)
observed during the shift in community.
Based on the results of this experiment, we conclude that additions of labile C compounds such
as glucose and amino acids have the potential to increase the APA of periphyton of low-P
systems such as the Everglades. Many questions still remain about the potential for this strategy
to successfully lower water column P levels, however, future research is certainly warranted to
develop this concept. Such research could include (1) more sophisticated experimental
approaches (e.g., physiological or isotopic tracer studies) to better determine the relationship
between C and P cycling in these mats communities, (2) more systematic and long term
experiments (e.g., seasonal studies) to explore the types and concentrations of C which maximize
the process, and (3) more applied experiments to test the efficacy of this process in alone or in
conjunction with various other treatment designs (e.g., dosed/non-dosed cell combinations).
Also, because this process as tested is largely a function of microbial activity, it seems logical
that the C dosing idea should be investigated in other high microbial biomass systems, for
example litter decomposition, or even in batch or flow-through bioreactor systems.
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I. INTRODUCTION
University of Florida, (Soil and Water Science Department) researchers, in cooperation
with the researchers at the South Florida Water Management District (SFWMD), planned to
conduct controlled field and laboratory experiments: (1) on enzymatic hydrolysis of dissolved
and particulate organic phosphorus (P) in the effluents of stormwater treatment areas (STAs),
and (2) characterize the microbial assemblages responsible for production of enzymes capable of
hydrolyzing organic P, and (3) evaluate new approaches to induce phosphatase enzyme
production in microbial mats and its effect on hydrolysis of organic P in the effluent.
The biogeochemical processes occurring in surface waters of wetlands are dependent on
its chemical composition, microbial communities, mineral components and physicochemical and
biological factors. Stormwater treatment areas are now used as buffers to reduce P in surface
runoff discharged from the Everglades Agricultural Area (EAA). These are passive systems and
usually not managed. At present, all technologies used are capable of decreasing total P
concentrations to approximately 20 µg P/L. The composition of the water leaving these systems
is primarily in organic form. However, current requirements are to reduce TP concentrations
Fig 1. Schematic showing biotic and abiotic processes regulating dissolved organic P (DOP)
10 (Phosphatases); DIP, dissolved inorganic P;
in wetlands. EA, Extracellular enzyme activity
POP, particulate organic phosphorus.
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below 10 µg P/L. Several abiotic and biotic processes in the soil and water column are involved
in regulating the TP concentration of the STA effluents. Since the dominant form of P in the
effluent is in organic form, we hypothesize that stimulating the growth of microbial communities
involved in transforming organic P may aid in reducing DOP to levels below 20 µg P/L.
Specific hypothesis of this study is to create P stress in microbial communities by supplementing
with nitrogen, which will induce high phosphatase enzyme production in periphyton mats.
Increase in phosphatase activity will result in hydrolyzing DOP in soluble reactive P, which can
be readily removed either through coprecipitation or by uptake by periphyton communities.
Conceptualized algal and bacterial interactions related to hydrolysis of dissolved and particulate
organic P in the water column are shown in Fig 1and 2.
Fig 2. Schematic showing algal and bacterial interactions in hydrolyzing dissolved
and particulate organic phosphorus in surface waters
Many bacterial species export extracellular phosphatases that are likely to be induced
upon phosphorus limitation. Because of the broad phylogenetic groupings responsible for production
of phosphatases and the little available knowledge of the genetics of phosphatases, standard non11
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culturable approaches to studying microbial communities are not available. Instead, we will rely on
direct observation of phosphatase-producing bacteria and cultivable methods.
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2.0 ENZYMATIC HYDROLYSIS OF DISSOLVED AND PARTICULATE
ORGANIC PHOSPHORUS (P) IN THE EFFLUENTS OF STORMWATER
TREATMENT AREAS (STAs) AND PERIPHYTON MATS.
KANIKA SHARMA, P. W. INGLETT, and K.R. REDDY
Soil and Water Science Dept, University of Florida-IFAS, Gainesville, FL 32608
2.1
INTRODUCTION
Phosphorus cycling in low-P systems is extremely efficient and therefore, the biotic
productivity is often limited. P-compounds that are relatively resistant to degradation are
sequestered and labile forms are rapidly recycled. Since inorganic P (Pi) is rapidly assimilated
by the organisms, the majority of the labile P is comprised of organic phosphorus (Po) in these
systems. A common strategy adopted by bacterial and planktonic organisms to survive in
systems with limited Pi is to synthesize extracellular phosphatase enzyme to hydrolyze Po
compounds. Pi released following hydrolysis is bioavailable and quickly assimilated by
organisms. Phosphatase is an inducible enzyme that is regulated by the external P-concentration
in nature where under conditions of elevated phosphate, phosphatase synthesis and subsequent
rates of Po hydrolysis are repressed. For this reason, high phosphatase activity has been shown
to be an indicator of Pi deficiency (Fitzgerald & Nelson, 1966; Healy, 1973).
The Florida Everglades is an ecosystem based on extremely low total P concentrations.
Periphyton mats are a characteristic feature of this nutrient-limited environment that survive due
to their ability to efficiently recycle and retain the nutrients (Goldsborough and Robinson, 1996;
McCormick and Stevenson, 1998). The capacity to efficiently recycle nutrients is primarily due
to the close association between the cyanobacteria and the bacterial groups inhabiting the
periphyton mat. The uptake of P by cyanobacterial mats is dependent on P concentration in the
water column, antecedent P content in mat biomass, the forms of available P, and the growth
stage and thickness of the mat (Horner et al., 1983; Sand-Jensen, 1983; Cotner and Wetzel,
1992). The ability to synthesize phosphatase allows periphyton mat organisms to acquire P
needed for their growth and thrive in these low P Everglades systems (TP <10µg P L-1 in water
column; ~200 µg P Kgdw-1 biomass). As a result, high values of APA have been reported in
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these periphyton mats (Newman et al., 2003).
Currently, green technologies used in the Storm Water Treatment Areas (STAs) are
capable of reducing effluent P concentration to 15 to 20 ug l-1 with the majority of the P leaving
these systems being in the organic form. Compared with the natural Everglades, however, the
STA effluents still contain ~ 5-10 µg P l-1 of excess P. Due to the high uptake potential of
component organisms, periphyton mats can play an important role in removing dissolved
inorganic P (DIP) from the water column in wetlands and littoral habitats (Reddy et al., 1999;
Wetzel, 1999; McCormick et al., 2001). High levels of phosphatase also makes periphyton mats
a potential candidate for removing the residual Po from the water column. For this reason,
approaches which successfully stimulate phosphatase expression, may also provide an effective
means of removing the residual organic P from waters treated by the current STA methods.
The main objective of this study was therefore, to determine the external factors that
regulate the enzymatic hydrolysis of dissolved organic phosphorus (DOP) and particulate
organic phosphorus (POP) in periphyton mats. Among the various approaches adopted to induce
alkaline phosphatase in these mats was the stimulation of microbial biomass of PPO. Since the
microbial groups responsible for APA in these mats are not known therefore, common carbon
substrates (acetate, alanine, and glucose) were investigated. P demand in a system can also be
increased by addition of nitrogen (N), therefore the effect of nitrate and ammonium was also
investigated. We also investigated the effect of light on APA as some studies have found that
light availability also can affect alkaline phosphatase activity (APA) in some algal/bacterial
systems (Klotz 1985; Wynne and Rhee, 1988). In these cases, it is believed that light stimulates
phototrophic C exudation resulting in enhancement in microbial growth and/or enzyme activity.
2.2
MATERIALS AND METHODS
2.2.1
Experimental site, sample collection and processing.
At present we are maintaining periphyton mats in several experimental mesocosms
located at the south side of STA-1W. These mesocosms consist of fiberglass tanks (1 m wide x
3 m long x 1 m deep) with limestone substrate and are fed by the outflow water from the STA14
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1W. Periphyton mats typical of low-P Everglades systems are abundant in these tanks. Bulk
samples of this tank periphyton and STA-1W ouflow water were collected and transported to the
Wetland Biogeochemistry Laboratory, Soil and Water Science Department at University of
Florida, Gainesville, FL. All experiments were conducted in the greenhouse at the University of
Florida, Gainesville, Fl. Fresh mat samples were brought from the site for every experiment
conducted and experiments were started within 24 hours of sample collection. Periphyton mats
were stored at 4°C prior to use. To avoid sample heterogeneity and assist diffusion of substrates
in the samples, bulk periphyton were manually homogenized using a spatula to form floc.
2.2.2 Effect of C and N on APA.
Batch studies: To study the effect of acetate and nitrate on periphyton APA, two separate
batch studies were conducted during February and March, 2003, respectively. Both experiments
were conducted in acid washed 60 ml clear plastic syringe tubes that were closed with a stopcock
at the outflow end. Each study consisted of triplicate samples of two treatments (dosed or
control) for three sampling times (2 d, 4 d, and 7 d)(2 x 3 x 3, n=18). Preweighed (20 g wet wt.)
floc was placed in the syringe with 40 ml of site water (35 mg C l-1). Dosed treatments received
one initial addition of either acetate- (100 mg C l-1) or nitrate- (100 µg N l-1) amended site water,
while control samples received only unamended site water. Evaporative loss from the syringe
was replenished every alternate day with DI water. Incubations were conducted in a glasshouse
subjected to natural day/night cycles. Samples were collected periodically at the start of the
experiment (0d), the next day (1d) and after 1 week (7d) for laboratory analysis of APA.
Flow-through studies: Effect of continuous dosing of C and N substrates on APA in
periphyton was studied by establishing two experiments. The first study examined the effect of
acetate and nitrate as phosphatase-inducing substrates (June 2003) and the other study
investigated the effects of glucose, ammonia and alanine dosing on APA (August 2003). A flowthrough setup was constructed with slight modification of the batch setup including a continuous
supply of substrate to the floc containing syringes. This system functioned like a chemostat
wherein the substrate-amended water and non-amended water was added to the treatment- and
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control-floc, respectively, at a constant flow rate of 6 ml h (R.T. = 6.67 h). The water flow in
the syringe was directed upward to avoid entrapment of any air bubbles. Spent water was
flushed out of the system through an outlet tube located near the top of the syringe.
The experimental conditions were kept similar to those in the batch studies. Dosed
samples received one of the following substrates acetate (240 mg C l-1); nitrate (1mg N l-1);
glucose (100 mg C l-1); alanine (100 mg C l-1); or ammonia (5 mg N l-1). Periphyton samples
were collected for alkaline phosphatase analysis on 2nd, 4th, 7th and 14th day for nitrate and
acetate substrate experiments and on the 3rd and 6th day for the glucose, alanine and ammonia
dosing study. Periphyton samples were collected periodically at approximately the same time of
day (10:30 h) throughout the study to avoid any potential diel effect on APA.
2.2.3
Effect of light on APA:
The effect of light on APA in glucose-dosed (100 mg C l-1) and non-dosed periphyton
mats was assessed using floc samples exposed to conditions of either alternating light/dark
cycles or continuous darkness. Alternating light/dark cycles consisted of an initial 4 h period of
daylight (4 pm-8 pm), followed by a 10 h dark period (8 pm-8 am), and another exposure to
daylight for 7 h (8 am-2 pm). Samples for the dark treatment were kept in dark conditions for
the complete 21 h period. Each of the four sets included 3 replicate containers (n=12).
Preweighed floc (50 g wet wt.) was added to each container with 500 ml of substrate amended or
unamended filtered site water (0.45µm filter). The experiment was conducted in a glasshouse in
April, 2004. To prevent development of anoxic conditions in the containers, air was bubbled
through all the containers using a standard aquarium pump. Floc subsamples were collected
every 6 h using a spatula and stored under dry ice until analyzed for APA.
2.2.4
Alkaline phosphatase assay.
Triplicate periphyton subsamples were transferred to acid washed plastic containers and
thoroughly homogenized with a spatula. One gram (wet weight) of periphyton material was
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added to 9 ml of distilled-deionized water and homogenized with a hand-held biohomogenizer
(Tissue Tearor™, Racine, WI). The slurry was diluted accordingly to adjust APA values within
a previously determined range of detection. Methylumbelliferone phosphate (MUF-P) substrate
(100 mM) was added and allowed to react with APase present in the sample to form the
fluorogenic product. Rate of enzyme reaction in samples was then followed by determining the
fluorometric reaction over the period of 2 h. Rates are then expressed as mg MUF formed per
hour of reaction, and standardized per gram (dry weight) of periphyton assayed. Appropriate
dilutions of the standards were prepared with MUF. Subsamples of wet periphyton material were
analyzed for APA. Additional subsamples were dried at 70°C for 72 h to determine moisture
content.
2.3
RESULTS AND DISCUSSION
Although the tanks being maintained in STA-1W received the same inflow water, the
periphyton mats in these tanks did not exhibit the same relative APA (Fig 2.1). This variation in
APA can be attributed to the varied P demand on the periphyton in the different tanks and to the
different bacterial and algal communities that constitute the periphyton mat. Visually, there
were distinct differences in the periphyton mat morphology in these tanks which may be the
result of differences in microbial composition (not determined). Species composition of the mat
can be affected by the nutrient and the other biogeochemical conditions that exist around it.
Differences in tank conditions may be the result of (i) material that is applied to the tanks, such
as limerock versus shellrock, (ii) presence of macrophytes such as Eleocharis and Chara, and
(iii) differing levels of infestation and grazing by snails and crayfish etc.
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APA
TP
0.30
15
0.25
12
0.20
9
0.15
6
0.10
3
0.05
0
0.00
2
3
4
5
6
8
TP mg gdw-1
MUF mg gdw-1h-1
18
10 Raceway
Tank Samples
Fig 2.1. APA and total P (TP) in periphyton collected from the
experimental mesocosm tanks located at the STA-1W. Values of APA and
TP are indicated by bars and square symbols, respectively.
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The highest APA was exhibited by the periphyton samples collected from mesocosm
tank 2, which had a high density of periphyton mats. Density of periphyton mats in tanks 3, 4, 5
and nearby raceway was comparable, and they exhibited similar APA (Fig 2.1). The APA in
these mesocosms may be regulated by the density of mats in relation to volume of water and
availability of soluble reactive phosphorus (SRP). APA has also shown to be regulated by the
internal P level in organisms. This relationship was
`
also seen in the periphyton mat samples from tank, where samples with high levels of
total P exhibited low APA. Another factor that regulates the APA is the external Pi
concentration. The inverse relationship between SRP and APA has been well established in the
past. Likewise, when periphyton mats with high APA were dosed with high Pi concentrations
for a 24 h period, lowered APA was measured (Fig 2.2).
C, N, and P ratios are maintained in any given ecological system; therefore, the demand
of P can be increased by increasing any of the two other components.
y = 337.37x -0.2186
R2 = 0.1751
MUF mg gdw-1 h-1
4
3
2
1
0
0.001
0.01
0.1
1
10
SRP (mg l-1)
Fig 2.2 Increase in soluble reactive phosphorus (SRP) in water column
supresses the alkaline phosphatase activity in the periphyton mats.
19
DRAFT
20
DRAFT
2.3.2
Effect of N-addition on APA in periphyton.
Nitrate-dosed floc exhibited increased APA when compared with undosed control-floc
(Fig 2.3). In batch incubations, APA in amended samples increased by 15% within 1 day, and
by 30% after 7 days (Fig 2.3a). Possible reasons for this increase may be that nitrate additions
increased P demand within the organisms in the floc by increasing their N:P ratio. It is known
that many bacteria can increase their cellular
N-content by assimilatory processes where nitrate is utilized as an N-source. Increased N in
nitrate assimilating bacteria can result in P limitation which can induce phosphatase production.
If this is the case, then the observed stimulation of APA by nitrate may be the result of increased
N:P ratios via N assimilation.
It is also possible that nitrate addition indirectly stimulates the phosphatase producing
organisms (PPO) by dissimilatory processes. One such scenario, is that nitrate respiration by
denitrifying bacteria is coupled with oxidation of organic materials. Metabolic products of these
bacterial groups may then stimulate growth of other PPO which experience increased P stress as
their biomass increases. Alternatively, the denitrifying bacteria may, themselves, be directly
stimulated by nitrate which increases their activity/metabolism. In both of these situations, the
APA increase would be a result of an increase in cell number rather than increased enzyme
expression per cell.
Although the current experiments were not conducted under anaerobic conditions, there
is a possibility of that development of anaerobic microzones within the floc may have allowed
either one or both the above mentioned processes to occur. The significance of microzone
formation in these processes is further supported by the observation that nitrate dosing under the
flow through incubations was not as effective in stimulating APA as the batch experiments (Fig
2.3 b). Continuous flow of the water through the syringe may have prevented the formation of
the efficient anaerobic microzones, and thereby reduced the probability of the nitrate
assimilatory or dissimilatory processes from occurring. Continuous flow would also remove
metabolic products generated as a result of oxidation of complex organic material (e.g.
photosynthate), and thus, may have further prevented the stimulation of APA by C-starved PPO.
Addition of ammonium (5 mg N l-1) did not stimulate APA in floc (Fig. 2.4) moreover, a
21
DRAFT
continuous decrease in APA with time was observed. This result was surprising especially
considering that ammonia is a microbially-preferred N source that can be assimilated by the cells
to increase cellular N content and can increase the P demand in the cells.
2
Control
a
Nitrate
MUF mg gdw-1 h-1
1.6
1.2
0.8
0.4
0
day 0
18
day 1
day7
Control
b
Nitrate
MUF mg gdw-1 h-1
15
12
9
6
3
0
day 0
day 2
day 7
day 10
day 14
Fig 2.3. Effect of nitrate dosing (100 µg
22 N l-1 final concentration) on APA in floc
during (a) batch incubations and (b) flow- through incubations.
DRAFT
+
Since high concentrations of NH4 and aerobic conditions greatly favor growth of nitrifying
bacteria, increased P demand is likely to be observed. It has also been shown in the past that
cellular N fixation may be limited by the P deficiency. However, no increase in APA was
observed in NH4+-dosed floc indicating that either (1) the system was not P limiting, (2) NH4+
additions produced an inhibitory effect, or (3) nitrifying bacteria are not appreciably involved in
phosphatase production. A decrease in APA with time could be either due to the suppression of
enzyme or due to the dying microbial population. This may have been the result of the inability
of these groups of bacteria to acquire P may have eventually resulted in the decline of this
microbial population within the floc.
The fact that additions of two similar N sources, (NO3- and NH4+) resulted in such
different effects on APA could also provide evidence against the theory regarding APA and N
induction of P stress (i.e., increased TN:TP). Both NO3- and NH4+ are potentially available N
sources, therefore, if N additions act to raise periphyton TN:TP (resulting in P stress), there
should be approximately equal responses of periphyton APA to additions of either NO3- or NH4+.
Because we did not observe such similarity, we conclude that the response of NO3- is likely the
result of its use as a preferred electron acceptor, and that NO3-utilizing bacteria are potentially
the dominant population responsible for the observed APA increases.
2.3.3
Effect of C-additions on APA in periphyton.
The effect of C additions on APA in periphyton was investigated using acetate, glucose
and alanine as carbon substrates, and a mixed response was observed. While a stimulation of
APA in glucose- and alanine-dosed floc was observed after 6 days (Fig. 2.4), there was no
increase in APA in acetate-dosed floc (Fig. 2.5a). Besides, a decline in APA was observed in
samples that were incubated with acetate in a flow through setup (Fig 2.5 b). Carbon was added
to the mat with the intention of increasing APA due to the increased P demand caused by
increasing microbial biomass. In addition, studies have shown that C limitation can greatly
affect the utilization of P in bacteria (Cotner and Wetzel, 1992) thereby, affecting the synthesis
of AP. If this were the case, then an immediate response of APA would have been observed but
the stimulation of APA was observed only after a lag period of 6 days. This lag period in APA
23
DRAFT
response suggests that increased APA was a result of population shifts within the existing
microbial community or was due to an increase in biomass.
All samples (including the control) receiving substrate via a flow through setup showed a
steady decline in APA after 3 days. However, after 6 days of dosing, glucose-dosed floc showed
a 38% increase in APA followed by a 35.5% increase in alanine-dosed floc when compared with
the undosed control. This varied APA response may be due to the varied response of mat
organisms to different C substrates added. Glucose is a metabolic intermediate of cellulose, a
common C source in wetland systems. It can be utilized as a carbon source by several groups of
bacteria because the metabolic products (such as butyrate, propionate and acetate) generated
during glucose oxidation (by fermenting bacteria) can further be utilized as C and energy sources
by several other groups of bacteria. On the other hand, alanine is a source of both C and N,
where the amine group can also be assimilated.
Control
12
Glucose
MUF mg gdw-1h-1
Ammonia
Alanine
9
6
3
0
day 0
day 3
day 6
Figure 2.4. Alkaline phosphatase activity (APA) of floc periphyton in response
to dosing of C as Glucose (100 mg C l-1), N as ammonia (5 mg N l-1), and
combined C and N as alanine (100mg C l-1, 38 mg N l-1) during flow through
experiments.
24
DRAFT
In the flow-through studies, decreased APA in the control samples after 6 days could be
indicative of dying microbial populations. This observation contradicts that observed in the
experiments with nitrate- dosing (Fig. 2.3b) and acetate dosing (2.5b), where the APA level in
the control was maintained over 1 week. It is uncertain whether this is an artifact of the
experimental set up, or if the microbial species composition of the mat was different from those
used in the other experiments. The species composition may have differed significantly between
experiments as these experiments were conducted with periphyton mats collected at different
times of the year. Nonetheless, the higher APA in glucose treated sample is most likely a result
of increased biomass of PPO that was stimulated by additional carbon. Increased biomass of the
PPB would result in overall increased APA measured. The alternative explanation involves the
stimulation of a bacterial community that aids the PPO such as the denitrifiers or fermentating
bacteria.
Acetate as a choice for carbon source also stems from the fact that it is a commonly
produced bacterial metabolic product utilized by specific groups of heterotrophic organisms.
With the intention of identifying PPO, by observing increased APA as a result of stimulating the
growth of certain specific bacterial groups, effect of acetate was investigated. To our surprise,
there was no increase observed in APA relative to control with 100 µg C l-1 as acetate in the
batch experiment (Fig 2.5). Furthermore, there was a decline in APA when a higher dose of
acetate (240 µg C L-1) was administered continuously to periphyton with a flow through system
(Fig 2.5b). These results indicate that the acetate utilizing bacteria may not be the key players in
phosphatase production in periphyton. No change in APA as a result of one time substratedosing indicates that either the selected concentration (100 mg l-1) of acetate was not enough to
stimulate the PPO or that acetate utilizers are not the major phosphatase producers in a mat. The
decline in APA with continuous dosing of acetate at a higher concentration further substantiates
the fact that majority of organisms may not be capable of utilizing acetate as a carbon source to
directly or indirectly effect phosphatase activity. Decline in APA with time could also be a
25
DRAFT
result of dying microbial population that produces AP or simply a repression of the enzyme by
acetate. The direct or indirect mechanism of enzyme repression is unclear at this time.
26
DRAFT
2
Control
a
Acetate
MUF mg gdw-1 h-1
1.6
1.2
0.8
0.4
0
day 0
18
day 1
day7
Control
b
Acetate
MUF mg gdw-1h-1
15
12
9
6
3
0
day 0
day 1
day 2
day 4
day 7
Figure 2.5. Effect of acetate on alkaline phosphatase activity in periphyton.
Concentrations of acetate used for dosing were (a) 100 mg C l-1 in batch
experiment and, (b) 240 mg C l-1 in flow through experiment.
2.3.4
Effect of light on APA in periphyton
Although stimulation of APA was also observed under continuous dark period, response
27
DRAFT
time was twice that observed in samples that were exposed to light. This more rapid response to
C additions under light suggests there may be a synergism between photosynthesis, C, and APA
(Fig 2.6). It is well-documented that algae and cyanobacteria can also exhibit APA in addition to
bacteria (Strojsova et al., 2003), therefore, the positive effect of light could be indicative of a
direct stimulation of phosphatase derived from cyanobacteria (or other phototrophs). Similarly,
it is equally plausible that C additions are directly stimulating the heterotrophic organisms within
the mat. For these reasons, it is largely uncertain what groups of microorganisms within the mat
7.5
Light/Dark
-C
*
6.5
+C
6.0
5.5
(mg MUF gdw-1 h-1)
Alkaline phosphatase activity
7.0
5.0
4.5
4.0
8.0
Dark
7.5
-C
*
7.0
+C
6.5
6.0
5.5
5.0
4.5
4.0
0
3
6
9
12
Time (h)
respond to light and/or C.
28
15
18
21
DRAFT
FIG. 2.6 Effect of light on APA in periphyton mats in presence and absence of glucose under (i)
light : dark : light (4h: 10h: 7h) period and, (ii) continuous dark period.
In the case of C additions, the effect on APA would likely only be observed in
populations of bacteria which utilize these labile C sources. If this were true, it would seem that
bacterial phosphatase production is largely C-limited. If bacteria are indeed C limited, it may
also be hypothesized that heterotrophs could be producing phosphatase in response to C derived
from mat photosynthesis.
In this case, short term exposure to light would supply photosynthetically-produced C
preferred by certain groups of PPO which may stimulate APA. Similarly, the absence of these
labile C products when photosynthesis is low may be offset by C additions in the dark period.
Based on this model, however, it is puzzling why the effect of light was not observed directly
between the unamended light and dark treatments (Fig 2.6).
Numerous studies suggest changes in pH influence phosphatase activity (Tabata et al.,
1988). Presence of light would result in photosynthesis and concurrent shifts in pH which may
then result in altered enzyme activities. Since small pH shifts do not inhibit the phosphatase
activity, it is unlikely that photosynthesis induced pH changes influenced the results of our
experiments. Increased dark periods can cause the pH to shift to acidic conditions, that is further
increased by the addition of glucose. This shift may dissolve the CaCO3 in the periphyton mats
thereby releasing Pi bound with CaCO3 which may further repress APA in periphyton. This did
not seem to be the case in these studies because the APA increased with time in samples
maintained under continuous dark period (Fig 2.6 ii).
2.4
CONCLUSIONS
Additions of glucose and alanine were more effective in increasing the APA in the
periphyton mats when compared with nitrate and ammonia. Increased APA was observed likely
as a result of increased P demand that was induced by the change in the C: N: P ratios by
increasing either the C or N concentration in the mat. Carbon limitation in the system was
apparent when higher APA was recorede in the C dosed mats. However, whether this observed
increase in APA within the periphyton mat was due to the increased biomass or increased
29
DRAFT
expression of the single cells is not known at present. A shift in microbial population/
community toward a phosphatase producing population may be responsible for the observed
change in APA as occurred gradually after a lag period. This increase in phosphatase synthesis is
suggestive of the carbon limitation in the system that prevents the complete APA activity.
Utilization of external carbon source for stimulation of APA suggests that heterotrophs may be
the key players in AP production
Our results with light and dark experiments suggest that in the absence of light,
stimulation of AP can be achieved by glucose additions. Bacteria may compensate for lower
labile carbon supplies by modifying phosphatase synthesis to maximize the probability of
acquiring phosphate through hydrolytic reactions.
Another noteworthy observation was the difference in APA response to the nitrate and
acetate dosing during batch and flow through experiments. Batch systems involve accumulation
of the biochemical products produced during the experiment. Unlike the batch experiments,
flow-through system set up involves continuous flushing with the fresh medium therefore
resulting in removal of any metabolic products produced as a result of various biochemical
reactions within a system. Lowered APA in these flow-through experiments may be a result of
dilution of the accumulated photosynthetic products that may be the important carbon source for
the PPO.
The methods of sample collection and preparation may have influenced the
measurements. Homogenization of periphyton mat samples may have allowed considerable cell
damage thereby resulting in liberating some surface bound cells. However this may or may not
have caused for lost activity. Based on these results C substrates were chosen for mesocosm
scale studies.
30
DRAFT
2.5
REFERENCES
Cotner, J. B. and Wetzel, R. G. (1992) Uptake of Dissolved Inorganic and Organic PhosphorusCompounds by Phytoplankton and Bacterioplankton. Limnology and Oceanography 37, 232243.
Fitzgerald, G. P., and Nelson, T.C. (1966) Extractive and enzymatic analyses for limiting or
surplus phosphorus in algae. Journal of Phycology 2, 32-37.
Goldsborough, L. G. and. Robinson, G. G. C. (1996) Pattern in wetlands. In Algal Ecology:
Freshwater Benthic Ecosystems., (eds.) R. J. Stevenson et al. pp 77-117.Academic Press. San
Diego, CA
Healey F.P. (1973). Characteristics of phosphorus deficiency in Anabaena. Journal of
Phycology. 9, 383-394
Horner, R. R.,Welch, E. B., and Veenstra, R. B. (1983) Development of nuisance periphytic
algae in laboratory streams in relation to enrichment and velocity. In Periphyton of
Freshwater Ecosystems (ed.) R. G Wetzel, pp 147-152, Dr W. Junk Publishers. The Hague,
The Netherlands.
Klotz, R. L. (1985) Influence of Light on the Alkaline-Phosphatase Activity of Selenastrum
capricornutum (Chlorophyceae) in Streams. Canadian Journal of Fisheries and Aquatic
Sciences 42, 384-388.
McCormick, P. V. and Stevenson, R. J. (1998) Periphyton as a tool for ecological assessment
and management in the Florida Everglades. Journal of Phycology 34, 726-733.
McCormick, P. V., O'Dell, M. B., Shuford, R. B. E., Backus, J. G. and Kennedy, W. C. (2001)
Periphyton responses to experimental phosphorus enrichment in a subtropical wetland.
Aquatic Botany 71, 119-139.
Newman S., McCormick, P. V. and Backus, J. G. (2003). Phosphatase activity as an early
warning indicator of wetland eutrophication: problems and prospects. Journal of Applied
Phycology, 15, 45-59
Reddy, K. R. White, J. R., Wright, A. and Chua, T. (1999) Influence of phosphorus loading on
microbial processes in the soil and water column of wetlands, p. 249- 273. in Phosphorus
biogeochemistry in subtropical ecosystems (eds.) Reddy et al., Lewis publishers, New York,
NY.
Sand-jensen, K. (1983) Physical and chemical parameters regulating growth of periphytic
communities. In Periphyton of Freshwater Ecosystems, ed R.G.Wetzel, pp. 63-71. Dr W.
Junk publishers, The Hague, The Netherlands.
31
DRAFT
Strojsova, A., Vrba, J., Nedoma, N., Komarkova, J. and Znachor, P. (2003) Seasonal study of
extracellular phosphatase expression in the phytoplankton of a eutrophic reservoir. European
Journal of Phycology 38, 295-306.
Tabata,M. Tachibana, W. Suzuki, S (1988) pH dependence of phosphatase activiy in freshwater
lakes. Jpn Journal of liminology 49, 93-98
Wetzel, R. G. (1999) Organic phosphorus mineralization in soils and sediments In Reddy et Al.
(eds.) Phosphorus Biogeochemistry of subtropical ecosystems., pp 225-245 CRC Press Inc.,
Boca Raton, FL..
Wynne, D. and Rhee, G. Y. (1988) Changes in Alkaline-Phosphatase Activity and PhosphateUptake in P-Limited Phytoplankton, Induced by Light-Intensity and Spectral Quality.
Hydrobiologia 160, 173-178.
32
DRAFT
3.0 CHARACTERIZATION OF PHOSPHATASE-PRODUCING
BACTERIAL ASSEMBLAGES
KANIKA SHARMA, P. W. INGLETT, A. V. OGRAM, AND K. R. REDDY
Soil and Water Science Department, University of Florida-IFAS, Gainesville, FL-32608
3.1
INTRODUCTION
Calcareous periphyton mats are a major component of the phosphorus (P) limited open
slough regions of the Florida Everglades ecosystem (Gleason and Spackman 1974). The term
periphyton generally refers to the microbial communities of attached microorganisms, both floral
and faunal, that grow on submerged surfaces. These communities are formed by complex
assemblages of cyanobacteria, eubacteria, diatoms and eukaryotic algae. In Everglades systems,
periphytic growths can occur in association with the benthos (benthic) or in association with
submersed and emergent macrophytes (epiphytic) such as Utricularia purpurea (Browder et al.
1994). Both epiphytic and benthic periphyton forms are also known to detach from their
substrata with the aid of trapped gases and float at the water surface. These periphytic forms can
be either thin films (ca 1-2 mm) or, more typically, well-developed, thick (ca 1-4 cm) growths
commonly referred to as floating and benthic ‘periphyton mats’ or epiphytic ‘sweaters’.
Structurally, mats are multilayered and characterized by vertical gradients in
physicochemical parameters such as light, oxygen, nutrients, and microbial metabolic products
(Fig. 3.1; Jorgensen et al., 1983). The presence of physical and chemical gradients directly
influences the abundance and distribution of different functional groups of microorganisms
within the mat structure (Stal et al., 1985, 1994). These organisms in turn regulate the nutrient
uptake, storage and release. They play an important role in biogeochemical cycles of carbon (C)(
Canfield and Marais, 1994), nitrogen (N) (Bebout et al. 1994) and P as they catalyze many
crucial steps using specific enzymes. Nutrient deprivation can induce several responses within
P-limited microbial mat communities such as those of the Everglades (McCormick et. al., 2001;
Neely and Wetzel, 1995). For example, temporary deprivation of a nutrient in the environment
can lead to elevated production of enzymes by bacteria. One such example is the extracellular
enzyme phosphatase (Pase) that is induced under P-limiting conditions. High Pase activity in
33
DRAFT
these calcareous mats are known to be early warning indicators of water quality in the
Everglades (Newman et. al., 2003), and increased Pase is an indicator of P-limited systems.
Phosphatases are mainly associated with the cell membranes where they hydrolyze the
organic phosphate and transport the phosphate ion inside the cell leaving the organic moiety
outside the cell (Chrost, 1991). Pase-producing organisms (PPO) in a periphyton mat are
therefore the key players for the phosphorus cycling in the system.
TOP
MIDDLE
BOTTOM
Figure 3.1 Cross section view of a floating periphyton mat from a nonimpacted site in Water
Conservation Area 2A of the Florida Everglades showing the vertical layering which occurs in
these mats.
Little is known about organisms within microbial mats that produce extracellular Pase, or
about the potential associations or dependency between PPO and other microbial groups in the
mat. Although cyanobacteria are dominant members of the mat and their phosphatase activities
have been reported in the past (Whitton et al., 1990,1991), it remains to be established if they are
the major producers of Pase in periphyton mats because currently, much of our knowledge of
Pase-producing organisms is based on pure cultures of organisms. It has been shown
that isolated strains are known to adapt genetically to environmental conditions which
they encounter, the physiological and biochemical properties of bacteria in isolation
(i.e., pure cultures) may not reflect those of organisms growing in natural consortia
(Caldwell et al., 1997; Deretic et al., 1994). Therefore, in such cases, it becomes
essential to study enzymatic processes in relation to the involved microbial groups as
34
DRAFT
they occur in the natural environment. Understanding the distribution of PPO in the mats
can reveal the location where transformations of organic P to inorganic P predominantly occurs.
Studying groups of PPO in their natural location without disrupting the natural associations will
facilitate the process of revealing microbial function within a mat system.
With this idea, we conducted the following study using calcareous floating periphyton
mats of the Florida Everglades. Our objectives were to (1) determine the spatial distribution of
the groups of organisms associated with Pase production within the mat structure, (2) screen the
phoA gene and attempt to phylogenetically characterize mat PPO, (4) microscopically examine
associations of PPO with autotrophs. In this study, we have attempted to demonstrate the site of
Pase production in the mat using the fluorescent substrate, ELF 97 (Molecular Probes) (Paragas
et al. 1997). Conclusions of this study have important implications for our understanding of the
microbial cycling of P within these and similar periphyton mat communities.
3.2
MATERIAL AND METHODS
3.2.1
Study site and sampling: Periphyton mat samples used in this study were
obtained from a site in the interior of Water Conservation Area 2A (WCA-2A) of the Florida
Everglades (Fig. 3.2). This area is typical of the P-limited areas of the Northern Everglades and
is characterized with ridges and open slough areas (Table 1). Vegetation of the ridges is
dominated by Cladium sp. while periphyton mats occur predominantly in open slough areas
dominated by Nymphaea, Eleocharis, and Utricularia spp. Floating periphyton mats of were
sampled in November 2002. Mat samples were collected and maintained at 4ºC while they were
transported to the Wetland Biogeochemistry Laboratory at the University of Florida, Gainesville,
FL. (ANOTHER PICTURE WOULD BE BETTER)
35
DRAFT
Fig. 3.2 Location of the sampling site in WCA 2A. The exterior eutrophic zone (impacted area)
is dominated by Cattail (Typha domingensis) and the interior pristine area (unimpacted site) is
dominated by periphyton mats and sawgrass (Cladium jamaisence).
Table 1. Description of major nutrient conditions at the WCA-2A area typical of the
sampling location used in this study.
*
Parameter
Soil*:
Soil Total N
Soil Total P
Porewater**:
NH4+
PO43Floodwater†:
Total K
Total Ca
Total Mg
Total Fe
Total SO4-2
Units
Concentration
Reference
g kg-1
g kg-1
27
0.6
Koch and Reddy (1992)
Koch and Reddy (1992)
mg N l-1
mg P l-1
2.3
0.1
DeBusk et al. (1994)
DeBusk et al. (1994)
mg l-1
mg l-1
mg l-1
mg l-1
mg l-1
6.9
70
25
0.02
49
McCormick et al. (1996)
McCormick et al. (1996)
McCormick et al. (1996)
McCormick et al. (1996)
McCormick et al. (1996)
Average of 0-5 and 5-10 cm values.
0-10 cm values.
† Measurements of samples filtered using 0.45µm membrane.
**
3.2.2
Morphology of the periphyton mat: Well-formed periphyton mats in P-limited
areas were 2-2.5 cm thick with three clearly-defined layers (Fig 3.1). - The top mat layers are
pale yellow to white in color, probably the result of photo bleaching at the water surface and/or
the presence of high concentrations of the pigment scytonemin (Dillon et al., 2003). The
underlying layers are removed from direct exposure to high intensities of solar radiation, and as a
result, these layers appear green from the dominance of photosynthetic cyanobacteria and green
36
DRAFT
algae. The bottom mat layer is grey black and likely contains remnants of the soils from the
benthic surface.
3.2.3
Microscopy
Cryoembedding and cryosectioning: Periphyton mat was sectioned within 24 hours of
collection. A periphyton mat exhibiting clear well developed layers in its vertical profile was
chosen for sectioning. The orientation of the mat on the cutting base was adjusted such that the
vertical profile of the mat was perpendicular to the plane of the blade. The sample was
cryoembedded in Tissue- Tek OCT compound (Miles Inc., Elkhart, IN) and frozen in liquid
nitrogen prior to sectioning. Embedded samples were sectioned with a model CM cryostat
(Lecia Inc., Deerfield, IL.). The 5µm thick sections were mounted on glass slides, treated with
formaldehyde (0.01%), and kept at 4ºC until further staining analysis.
Fluorescent staining: ELF-97 phosphatase substrate (ELF-P) is a water soluble, weakly
blue fluorescent stain that yields a water insoluble, photostable yellow-green alcohol (ELF-A)
upon enzymatic hydrolysis. The sites of Pase production can then be easily visualized by
epifluorescence microscopy. One of the major limitations faced while using this technique is
possibility of restricted diffusion of ELF-P through the mat structure. We have attempted to
address this issue by conducting several preliminary experiments to optimize both the
concentration of the added ELF-P substrate and the time required for the reaction to complete.
Mat sections were double stained with ELF-P ((5`-chloro-2`-phosphoryloxyphenyl)-6chloro-4-(3H)-quinazolinone) and DAPI (4`, 6-diamidino-2-phenyl-indole). Immediately prior
to use in this study, ELF-P was diluted 1:20 in ELF Detection Buffer (provided with the ELF
endogenous kit) and filtered through 0.2 µm spin filters to remove substrate precipitates. Each
pre-fixed mat section was incubated with 30µl of ELF-P for 30 min in the dark at room
temperature. Stained samples were then washed with 10 mM phosphate buffered saline (PBS) to
stop the reaction. Negative controls were prepared by treating the mat sections as described
above except that they were incubated with ELF detection buffer without ELF-P. Some mats
were doubly stained with DAPI for 5 min at room temp in dark.
37
DRAFT
Microscopy and image analysis: A fluorescent morphometric microscope was used to
examine prepared sections. The excitation spectrum of chlorophyll a & b and ELF-A are
different, therefore, the yellow-green signals of ELF-A and the red fluorescence of chlorophyll
can be visualized sequentially with appropriate filter sets. ELF-P-treated samples were
visualized with an Olympus type U filter. Filters used for ELF-A detection were 360±40 nm for
excitation and 530±25nm for emission. DAPI-stained samples were observed under a
fluorescent microscope equipped with long pass filter set (excitation 365±8 nm; emission >
420nm). The texas red filter was used for images of chlorophyll a autofluorescence (CHL
images). Images captured at the same spot by the different filters were digitized with a cooled
color charge-coupled device camera. ELF and CHL images of the same field were stored in a
single file and later merged to show the location of the chlorophyll containing organisms and the
Pase sites.
3.2.4
Phospholipid Fatty Acid Analysis (PLFA)
Knowledge of the microbial assemblages is essential in the development of hypotheses
concerning processes occurring in the complex structure of periphyton mats. It is well known
that the majority of bacteria are not cultivable, and for this reason, the molecular approach to target
the noncultivable bacteria in the environment are gaining in popularity (Sayler and Layton, 1990;
Vestal and White, 1989). In the case of Pase, however, standard molecular methods are not
available due to the high variability of the Pase gene (pho A) among organisms. Phospholipid fatty
acid analysis is another useful tool that can be used to broadly determine the diversity of the
organisms present in the periphyton mats. PLFA provides a quantitative measure of bacterial
and eukaryotic biomass. Cell death results in the hydrolysis of the phospholipids therefore the
presence of PLFA is a good indicator of the active, viable biomass in addition to providing the
community composition of the sample (Findlay and Dobbs, 1993).
Sample handling: Collected periphyton samples were frozen by storing with dry ice.
Samples were transported on dry ice to the Wetland Biogeochemistry Laboratory where they
were stored at -20ºC until analyzed.
38
DRAFT
Extraction: Lipids were extracted from periphyton material using the one-phase
extraction procedure of Bligh and Dyer (1959) modified by White et al. (1979). Wet sample (5
g) was extracted for 1 hr with extraction mixture chloroform, methanol; phosphate buffer
(1:2:0.8). The soil water content was subtracted from the amount of P-buffer that was added.
Tubes were centrifuged and the supernatant was collected. Lipids were reextracted by adding 5
ml of chloroform and 5 ml of phosphate buffer to the tubes and keeping them for 16 hrs in dark.
The organic phase containing lipid was then collected and the chloroform was evaporated under
nitrogen. Dried sample was stored under nitrogen at -20ºC.
Column –chromatographic separation: To isolate major classes of fatty acids, extracts
were separated using a silica gel column by the following. After conditioning the column with 1
volume of chloroform, separation of neutral, glyco- and phospholipids was carried out using 1
volume of chloroform, 2 volumes of acetone and 1 volume of methanol for eluting. The final
fraction was regarded as the phospholipid fraction.
Transesterification and Gas Chromatography: The dried phospholipid fraction was
resuspended in methanol: toluene (1:2). One milliliter of 0.2 N methanolic KOH was added and
the mixture was incubated for 15 minutes at 35ºC. The mixture was neutralized with 1 ml of
acetic acid. Water (1 ml) was added and the mixture vortexed for 30 seconds.
Hexane:chloroform (4:1) (2 ml) was added to the mixture. The suspension was centrifuged to
separate the FAME`s and non saponifiable phospholipids in the organic phase. The chloroform
fraction was evaporated under nitrogen. Dried FAME were then resuspended in hexane and
analyzed using a GC-FID (Shimadzu GC-14A). Peaks were identified using bacterial fatty acid
standards and MIDI peak identification software, (MIDI, Inc., Newark, DE).
Nomenclature of PLFA: Fatty acids are designated by total number of C atoms, and the
degree of unsaturation is indicated by a number separated by a colon from the chain length
number. The prefixes ‘cy’ and ‘d’ refer to cyclopropyl branching. Numbers preceded by “w”
indicate the position of hydroxyl groups from the aliphatic end of the FA. Cis and trans
geometry are indicated by the suffixes ‘c’ and‘t’. The prefixes ‘a’ and ‘i’ refer to the anteiso and
iso branching. ‘10 Methyl’ indicates a methyl group on the 10th carbon atom from the carboxyl
end of the chain.
39
DRAFT
3.3
RESULTS
3.3.1
Microbial characterization of periphyton mat
Comparison of PLFA of periphyton mats incubated in high- and low-Pase environments
can accurately mirror shifts in community composition. Samples from the surface, white layer
(UV-exposed) and middle, green layer revealed a difference in the dominant microbial
communities (Table 3.2). In a periphyton mat from a P-limited area, the green layer harbored
anaerobic bacteria including, a17:0, 17:1ω8c of sulfate reducing bacteria. In contrast, the surface
UV-impacted, white layer showed the presence of 16:0, 18:1ω9c, 18:1ω7c, 18:0 lipids indicative
of aerobic organisms. Fatty acid (i16:0) is an indicator of gram positive and anaerobic gram
negative bacteria; and 10 Me16:0 is an indicator of members of genus Desulfobacter sulfate
reducing bacteria and other anaerobic bacteria (Findlay and Dobbs, 1993). A marker for fungi
(18:3ω6c) was present only in the green layer of the periphyton. Presence of eukaryotic markers
18:1ω9c indicated the presence of microalgae.
Table 3.2. Structural composition of the top two layers of the periphyton mat as determined by
the Phospholipid fatty acid analysis (PLFA).
40
DRAFT
Indicator
Periphyton-White
layer
Anaerobic bacteria
Anaerobic bacteria
Anaerobic bacteria
Mycorrhizae
16:0
Sulfate reducing bacteria
Sulfate reducing bacteria
Sulfate reducing bacteria
Fungi
Microalgae
Aerobic bacteria
18:1 w9c
18:1 w7c
18:0
3.3.2
Periphyton-Green
layer
12:0
14:0 ISO
14:0
15:0 ISO
15:0 ANTEISO
15:0
16:0 ISO
16:1 w9c
16:1 w5c
16:0
16:0 10 methyl
15:0 3OH
17:0 ISO
17:0 ANTEISO
17:1 w8c
17:0
18:3 w6c (6,9,12)
18:1 w9c
18:1 w7c
18:1 w5c
18:0
11 methyl 18:1 w7c
19:0 CYCLO w8c
18:1 2OH
20:4 w6,9,12,15c
20:0 ISO
20:1 w7c
Screening of phoA.
The gene pho A encodes the periplasmic alkaline Pase (Kikuchi et al., 1981). Production of
alkaline Pase is transcriptionally controlled and mRNA encoding PhoA
accumulates only in P-deficient cells. Extensive screening of the sequences deposited in GenBank
database revealed that pho A is not conserved and therefore the probes targeting this gene could not
be constructed. Thus, this molecular approach for studying the PPO within the periphyton mat was
not feasible.
41
DRAFT
3.3.3
In Situ PPO Analysis
The fluorogenic substrate ELF (Enzyme-Labeled Fluorescence) reagent has demonstrated
great potential as a tool for detecting Pase activity in a variety of organisms in areas such as
histochemical studies and studies of biofilms and phytoplankton. To our knowledge, however,
ELF has never been employed in demonstrating localization of Pase in periphyton mat tissue
preparations.
Distribution of autotrophic organisms and phosphatase activity: The presence of
autotrophic organisms in the mat sections was indicated by high autofluorescence when
examined under the texas red filter (Fig. 3.2). Chlorophyll-containing cells dominated the mat in
all vertical sections, and were identified as filamentous cyanobacteria. When the same field
images taken with long pass and texas red filters were overlapped, dense aggregations of ELF-A
precipitates were observed to be in close association with the chlorophyll-containing cells (Fig1).
Most ELFA precipitation was seen attached to the empty sheaths of cyanobacteria cells
suggesting that other heterotrophic cells attached to cyanobacterial cells were the active sites of
Pase production. The matrix of the mat stained with periodic acid schiff stain (PAS), suggesting
that much of the mat was composed of polysaccharides.
To assess the distribution of Pase activity vertically within a periphyton mat, sites of Pase
production were determined by ELFA deposition. In this analysis, the majority of ELF-A
precipitation did not occur in the top, white layer of the mat (exposed at the water surface), but
instead occurred towards the middle and the lower sections of the mat. The ELF-A precipitation
appeared along with filamentous structures that may have been empty sheaths of the
cyanobacteria. There was no red fluorescence associated with the filamentous structures
indicative of empty cyanobacterial sheath.
Further screening of the ELF-stained slides revealed that the Pase activity was not closely
associated with the cyanobacterial or the algal cells (Fig. 3.2). The yellow-green fluorescence
was found associated with filamentous structures that did not contain chlorophyll. The
possibility of production of Pase by the cyanobacterial cell in order to scavenge the phosphorus
release from the dead cells cannot be ruled out. However, one plausible explanation for
observed ELF-A patterns, is that the Pase signatures are derived from bacteria degrading the
42
DRAFT
remnant, C-rich sheath material. Close proximity of these Pase-producing bacteria to
cyanobacterial cells suggest a potential interdependence between these different groups, perhaps
for N, C and P.
43
DRAFT
i
ii
F
Mat Top
AP
CF
AP
C
Mat Bottom
iii
iv
C
AP
10µm
v
Empty CS
vi
50µm
AP
AP
CF
C
C
Figure 3.2. Photomicrographs of vertical cryosections (5 µm thickness) of the mat stained
with fluorescent phosphate substrate (ELF® 97; Molecular Probes Inc, OR). Sites of
alkaline phosphatase (yellow-green fluorescence) and chlorophyll (red fluorescence) activity
are evident in: vertical mat cross section (i); localized aggregate of filamentous
cyanobacteria (ii); isolated film of polysaccharhide matrix (stained blue with DAPI) (iii);
isolated colony of coccoid dyanobacteria (iv); and along remnant cyanobacterial
sheaths/slime trails (v, vi) AP, Alkaline phosphatase; C, cyanobacterial cells; F,CF,
cyanobacterial filaments; CS, cyanobacterial sheath.
44
DRAFT
3.4
DISCUSSION
Through the production of Pase, periphyton are an important component in the P
chemistry of oligotrophic freshwater ecosystems such as the Everglades. Studies conducted in
the Florida Everglades have shown that the periphyton assemblage rapidly removes P from water
thereby maintaining the P-limiting conditions in the water column (Davis, 1982; McCormick et
al., 1998). Within periphyton mats, PPO are important for P cycling as they transform the nonutilizable, organic P into bioavailable orthophosphate (PO43-).
In agreement with previous studies of algal composition, our results with microscopy
show that cyanobacteria are the most dominant group that constitutes the periphyton of WCA-2A
(Gleason and Spackman, 1974). Examination of the ELF-stained sections revealed the localized
nature of extracellular Pase production in mats. The Pase activity was not evenly distributed
within the mat, and appeared to be concentrated within the lower mat sections. Most Pase
activity was associated with cell-like clusters on the surface of chlorophyll-containing cells
identified as cyanobacteria. This finding agrees with previous studies which have demonstrated
Pase production by axenic cyanobacteria cultures under P-limiting conditions (Whitton et al,
1991). Despite this finding, however, Pase activity was most frequently associated with nonchlorophyll-containing filament-like structures that appeared to be empty sheaths of
cyanobacteria. For this reason, we feel that heterotrophic organisms are responsible for the bulk
of Pase production within the mats of WCA-2A.
Due to the imprecise nature of the staining and microscopic techniques involved in this
work, we can only speculate that the phototrophic organisms (mainly the cyanobacteria and
diatoms) are not the dominant Pase producers in the mat. The association of Pase activity with
empty sheath-like structures and the relegation of Pase activity to surface edges of
photosynthetic regions support this as a plausible hypothesis. As a result, we propose a model
which links the C, N, and P cycling within the mat. In this model, bacterial production of Pase
results in the production of bioavailable P within the mat structure. Bacteria, which are known
to have a higher uptake affinity for P relative to larger algae, consume this available P until their
stoichiometric needs are satisfied (Wynne and Rhee, 1988). Addition P then becomes available
for algal uptake. These chlorophyll-containing algae and cyanobacteria have the ability to
45
DRAFT
photosynthesize and fix atmospheric N2. Algae are also known to maintain their colonial
structure by exudation of exopolysaccharides (EPS) such as mucilage and/or firm sheaths
(Browder et al., 1994). Production of EPS is representative of the bulk of the cyanobacterial
colony and can be a considerable diversion of C and N.
These active secretions, combined with products produced during cell death and
senescence become an important source of C and N for the heterotrophic bacteria. In this
manner, the algae and bacteria can cooperate to facilitate the existence of a periphyton mat
community under conditions of extreme P limitation.
CO2
N2
UV-IMPACTED MAT
Lysis
R-C
ALGAE
R-C
uptake
APA
Lysis
BACTERIA
Figure 3.5. Schematic diagram of proposed association between phosphatase-producing bacteria
and cyanobacteria in periphyton mats. Phosphatase producing bacteria live in close association
with the eukaryotic algae filaments and the cyanobacterial cells and filaments perhaps providing
them with inorganic P through activity of cell-bound phosphatase (APA).
46
DRAFT
Interactions between cyanobacteria and heterotrophic bacteria have been discussed in the past by
Marshall (1989)
Cooperative interactions are important when the population utilizes an insoluble substrate
for which the extracellular enzymes have to be produced. At higher population densities the
soluble product formed by enzyme hydrolysis can be used more efficiently in a communal
manner. Another benefit for heterotrophic organisms present in close proximity to cyanobacterial
cells is protection from proteases by the thick sheaths. This model may also explain that
association between the heterotrophic bacteria and the cyanobacteria is controlled by the C, N
and P cycling with in the mat.
3.5
PROPOSED ASSOCIATION OF PPO AND CYANOBACTERIA.
Metabolic pathways and the other physiological characteristics of the organisms are
studied in pure cultures. However, in nature the organisms do not live in isolation and they live
in mixed communities of various complexities where their activities are governed and regulated
by the presence of other neighboring organisms and the metabolic pathways of the carbon
source. Cyanobacterial periphyton mats are known to develop in environments with P limitation.
Cyanobacteria are a group of prokaryotic, oxygen evolving, photosynthetic, Gram-negative
bacteria that survive in wide ranges of environments. Cyanobacteria though have specialized
biochemical and ecological mechanisms to access essential nutrients that may limit growth.
Extracellular Pase is one of the enzymes that is produced by these organisms in the limitation of
P. So far, pure culture studies have demonstrated Pase production by several algae and
cyanobacteria that are present in the Everglades mats, however, little is known about the major
producers of the Pase in these systems. Our microscopic results suggest that the major producers
for Pase may be groups of heterotrophic organisms that form a symbiotic relationship with the
blue green algae. These Pase producers may be providing bioavailable P to the cyanobacteria
which in turn are providers of N and C. These concepts are illustrated in Fig. 3.5.
3.6
CONCLUSIONS
47
DRAFT
In conclusion, the results of this study support the following conclusions: (1) The site of
Pase activity is not evenly distributed within the Everglades periphyton mat matrix, but instead is
localized in certain regions of the mat interior. (2) Pase activity occurred on the surface of, or
closely associated with photosynthetic cyanobacterial cells/filaments or algae. (3) Patterns of
Pase activity associated with non-chlorophyll-containing areas indicate that heterotrophic
organisms are the dominant mat PPO. Based on these results, we propose a model describing the
association of cyanobacteria and heterotrophic organisms and based on the premise that mat
heterotrophs are largely C-limited. Additional support for this hypothesis is provided by results
from other experiments (not presented) where C additions to periphyton have stimulated Pase
activity.
More information is required to definitively document the role of the heterotrophs in the
Pase production and further research is required to identify the bacterial groups that form the
dominant PPO and to quantify the contribution of Pase by the autotrophic and the heterotrophic
organisms. The overall significance of this study for the area of enzymatically removing DOP
from the water column is that this information will help identify the dominant groups of
phosphatase producing bacteria (heterotrophic and autotrophic) within periphyton mats. These
conclusions have important implications for our understanding of the microbial diversity
involved in the phosphorus cycling in the mats. The eventual fate and ecological importance of
Pase produced by heterotrophic bacteria within such mat communities represents an exciting and
potentially important area of new research.
48
DRAFT
3.7
REFERENCES
Bebout, B. M. and H. W. Paerl, J. E. Bauer, D. E. Canfield and D. J. DesMarais (1994) Nitrogen
cycling in microbial mat communities: the quantitative importance of N-fixation and other
sources of N for primary productivity. in Microbial mats: Structure, Development, and And
Ecological Significance. (eds.) L. J. Stal and P. Caumette, pp 149-166 Springer-Verlag,
Berlin.
Bligh, E. G. and Dyer, W. J. (1959) Arapid method of total lipid extraction and purification
Canadian Journal of Biochemistry and Physiology 37, 911-917
Browder, J. A., Gleason, P. J. and Swift, D. R. (1994) Periphyton in the Everglades: Spatial
variation, environmental correlates, and ecological implications In Everglades: the ecosystem
and its restoration (eds.) S. M. Davis and J. C. Ogden, pp. 379-418, St Lucie Press, Delray
Beach FL.
Caldwell, D. E., Wolfaardt, G. M., Korber, D. R. and Lawrence, J. R. (1997) Do bacterial
communities transcend Darwinism? Advances in Microbial Ecology, 15, 105-191.
Canfield, D.E. and D.J. Des Marais (1994) Cycling of carbon, sulfur, oxygen and nutrients in a
microbial mat in Microbial mats: Structure, Development, and And Ecological Significance.
(eds.) L. J. Stal and P. Caumette, pp 149-166 Springer-Verlag, Berlin.
Chrost, R. J. (1991) Environmental Control of the synthesis and activity of aquatic microbial
ectoenzymes In Microbial Enzymes In Aquatic Environments (eds.) R. J. Chrost,. pp. 29-59,
Springer-Verlag, New York.
DeBusk, W.F., K.R. Reddy, M.S. Koch, and Y. Wang. 1994. Spatial distribution of soil nutrients
in a northern Everglades marsh: Water Conservation Area 2A. Soil Sci. Soc. Am. J. 58:543552.
Deretic, V., Schurr, M. J., Boucher, J. C., and Martin, D. W. (1994) Conversion of Pseudomonas
aeruginosa to Mucoidy in Cystic-Fibrosis - Environmental-Stress and Regulation of
Bacterial Virulence by Alternative Sigma-Factors. Journal of Bacteriology 176, 2773-2780.
DillonJ. G., Miller, S. R. and Castenholz, R. W., (2003) UV-acclimation responses in natural
populations of cyanobacteria (Calothrix sp.) Environmental Microbiology
5, 473Espeland, E. M. and Wetzel, R. G. (2001) Effects of photosynthesis on bacterial phosphatase
production in biofilms. Microbial Ecology 42, 328-337.
49
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Espeland, E. M., Francoeur, S. N. and Wetzel, R. G. (2002) Microbial phosphatase in biofilms:
A comparison of whole community enzyme activity and individual bacterial cell-surface
phosphatase expression. Archiv fur Hydrobiologie 153, 581-593.
Findlay, R. H. and Dobbs, F.C. (1993) Quantitative description of microbial communities using
lipid analysis,. In P.F. Kemp, B.F. Sherr, E.B. Sherr, and J. J. Cole (ed.) Handbook of
methods in Aquatic Microbial Ecology. p 271-284. Lewis publishers, Boca Raton, Fl.
Gleason, P. J. and Spackman, W. Jr. (1974) Calcareous periphyton and water chemistry in the
everglades. In Environments of south Florida: present and Past, Memoir No. 2, (ed.) P. J.
Gleason, Miami Geological Society, pp 146-181. Coral Gables, Fla.,
Jorgensen, B. B., Revsbech, N. P. and Cohen, Y. (1983) Photosynthesis and Structure of Benthic
Microbial Mats - Microelectrode and SEM Studies of 4 Cyanobacterial Communities.
Limnology and Oceanography 28, 1075-1093.
Kikuchi, Y., yoda, K, Yamasaki, M. and Tamura, G. (1981) The nucleotide sequence of the
promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of
Escherichia coli. Nucleic Acids Research. 9: 5671-5678.
Koch, M.S., and K.R. Reddy. 1992. Distribution of soil and plant nutrients along a trophic
gradient in the Florida Everglades. Soil Sci. Soc. Am. J. 56:1492-1499.
Marshall, K. C. (1989) Cyanobacterial-heterotrophic bacterial interaction. In Microbial mats:
Physiological ecology of benthic microbial communities, (eds.)Y. Cohen and E. Rosenberg,
pp. 239-245. American Society for Microbiology, Washington D. C.
McCormick, P. V. and Odell, M. B. (1996) Quantifying periphyton responses to phosphorus in
the Florida everglades: A synoptic-experimental approach Journal of the North American
Benthological Society 15, 450-468.
McCormick, P. V. and Stevenson, R. J. (1998) Periphyton as a tool for ecological assessment
and management in the Florida Everglades. Journal of Phycology 34, 726-733.
McCormick, P. V., O'Dell, M. B., Shuford, R. B. E., Backus, J. G. and Kennedy, W. C. (2001)
Periphyton responses to experimental phosphorus enrichment in a subtropical wetland.
Aquatic Botany 71, 119-139.
Neely, R. K. and Wetzel, R. G. (1995) Simultaneous Use of C-14 and H-3 to Determine
Autotrophic Production and Bacterial Protein-Production in Periphyton. Microbial Ecology
30, 227-237.
Newman S., P. V. McCormick and J. G. Backus. (2003). Phosphatase activity as an early
warning indicator of wetland eutrophication: problems and prospects. Journal of Applied
Phycology, 15, 45-59
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Paragas, V. B., Zhang, Y. Z., Haugland, R. P. and Singer, V. L. (1997) The ELF-97 alkaline
phosphatase substrate provides a bright, photostable, fluorescent signal amplification method
for FISH. Journal of Histochemistry & Cytochemistry 45, 345-357.
Rejmankova, E. and Komarkova, J. (2000) A function of cyanobacterial mats in phosphoruslimited tropical wetlands. Hydrobiologia 431, 135-153.
Sayler, G.S., and Layton, A. C. (1990) Environmental application of nucleic acid hybridization.
Annual Reviews in Microbiology. 44:625-648.
Stal, L. J. (1994) Microbial Mats in coastal environments. In Microbial Mats: Structure,
Development and Environmental Significance, ed. L. J. Stal and P. Caumette, pp. 21-32.,
Spinger-Verlag Berlin Heidelberg, Germany
Stal, L. J., Van Gemerden, H. and Krumbien, W. E. (1985) Structure and development of a
benthic marine microbial mat. FEMS Microbiology Ecology 31:111-125
Vestal, J. R. and White, D. C. (1989) Lipid analysis in microbial ecology. Bioscience 39, 535541.
White, D. C., Davis, W. M., Nickles, J. S., King, J. D., and Bobbie, R. J. (1979) Determination
of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40, 51-62.
Whitton, B. A., Grainger, S. L. J., Hawley, G. R. W. and Simon, J. W. (1991) Cell bound and
extracellular phosphatase activities of cyanobacterial isolates. Microbial Ecology 21, 85-98
Whitton, B. A., Potts, M., Simon, J. W. and Grainger, S. L. J. (1990) Phosphatase activity of the
blue green alga (cyanobacteria) Nostoc commune UTEX 584. Phycologia 29, 139-145.
Wynne, D. and Rhee, G. Y. (1988) Changes in Alkaline-Phosphatase Activity and PhosphateUptake in P-Limited Phytoplankton, Induced by Light-Intensity and Spectral Quality.
Hydrobiologia 160, 173-178.
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4.0 DETERMINE THE ROLES OF PHOTOLYSIS IN HYDROLYZING
WATER COLUMN DISSOLVED ORGANIC P (DOP)
ROBERT G. WETZEL, William R. Kenan Distinguished Professor
Department of Environmental Sciences And Engineering, School of Public Health,
The University of North Carolina,
Chapel Hill, North Carolina 27599-7431
4.1
INTRODUCTION
As aquatic macrophytes grow and particularly during plant senescence, appreciable nutrient
content is translocated to rooting tissues, particularly among herbaceous perennials that
dominate among aquatic plants. Some senescence and degradation of leaf tissues, however,
results in the loss of cellular integrity and leaching of nutrients and dissolved organic compounds
from the leaves. Labile components of these leachates, particularly amino acids, for example,
are readily utilized and sequestered by the periphytic microflora (e.g., Cunningham and Wetzel,
1989; Bicudo et al., 1998). Similarly, as leaching of dissolved organic matter from senescent
macrophyte vegetation under various stages of degradation occurs, a selective utilization of more
labile constituents by attached microflora occurs as the dissolved organic matter moves through
the wetland communities. The result is an increasing recalcitrance of the dissolved organic
compounds, and particularly the humic substances. These phenolic substances have a high
aromaticity, particularly from lignin of structural tissues, and are difficult for microbes to
hydrolyze.
Leaching of dissolved organic carbon (DOC) is greater from leaves of some species than
from others. Bacterial production rates are significantly different utilizing leachates from tissues
of these different species. Some of the DOC leaching from the tissues and undergoing partial
degradation is assimilated and immediately converted to CO2 under aerobic conditions and to
CH4 under anoxic reducing conditions within compacted macrophyte and sediment detritus. The
more recalcitrant organic compounds, however, can be transported down gradient with the
wetlands, continually being exposed to different environmental conditions.
A major habitat change among different plant communities in the Everglades wetlands is
52
DRAFT
light availability. This investigation evaluated such differences and what effects light has on
alteration of the chemical bioavailability of dissolved organic compounds, including: (a) organic
carbon compounds, (b) dissolved organic nitrogen, and (c) dissolved organic phosphorus. As
such bioavailability is changed, alterations in bacterial growth should occur, and increased
bacterial metabolism should enhance rates of nutrient regeneration and availability for utilization
by other components of the ecosystem.
In ancillary studies by the PI, when the DOC was exposed under sterile conditions to
sunlight, under both natural conditions or with a solar simulator at 80% of full summer sunlight,
two major transformations of the DOC occurred. First, a large number of volatile fatty acids
(acetic, levulinic, succinic, formic, pyruvic, tartaric, others) were cleaved from the humic
macromolecules. The photolytic responses were much larger (+200%) from leachates emanating
from plant tissues grown on elevated CO2 concentrations than those (+100%) from leachates
from tissues grown on ambient CO2 concentrations (Wetzel and Tuchman 2004). In addition,
several examples were shown that quantified significant photolysis of the DOC by UV-A (320400 nm) and by photosynthetically active radiation (PAR; 400-720 nm). Depending upon the
optical characteristics of the water, on the average ca. 25% of the total photolysis was
accomplished by UV-B (285-320 nm), 40-60% by UV-A, and the remainder by PAR. In many
but not all cases, PAR was nearly as effective as UV-A.
A second transformation was to photolytically degrade some DOC completely to CO2.
Again, it was demonstrated that a significant portion of the DOC was fully oxidized
photolytically under sterile conditions directly to CO2 (reviewed in Wetzel 2001; Wetzel and
Tuchman 2004). Similarly to the generation of fatty acids, PAR and UV-A were found to be
responsible for a majority of the photodegradation, in part because of the rapid attenuation of
UV-B in water.
4.2
MATERIALS AND METHODS:
Water samples utilized in these experimental analyses were collected along the well-
studied 10-km eutrophicational gradient along the northern portion of Water Conservation Area
2A in collaboration with the program operations of Prof. K.R. Reddy at the University of
53
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Florida. Samples were obtained at stations 0, 4, 7, and 10 km from the loading canal at 0 km
during two times of the year (high water vs. low water seasons). Water was immediately sterile
filtered in the field (Nalgene 0.22 µm pore size) and stored at 4°C in the dark.
In the laboratory, water was again sterile filtered under subdued light and aseptically
transferred to sterile quartz incubation chambers (30 cm in length, 5 cm in diameter) that
contained sterile sampling ports with septa. The samples were incubated in a solar simulator
(Atlas Suntest SXL, Chicago) at 14040 KJ m2 6 hr-1, equivalent to approximately 80% of full
sunlight in summer at that latitude. Along with a control incubated under identical conditions
but opaqued from any light, samples were exposed to full sunlight (Quartz tubes, UV-B + UV-A
+ PAR, 285 to 720 nm), UV-A and PAR only (Mylar-D filter, 320 to 720 nm), and to
photosynthetically active radiation (PAR) only (acrylic OP-2, 400 to 720 nm) (Fig. 1). Samples
were incubated for six hours, and subusamples were removed aseptically at hour intervals for
analyses of changes in DOC, DON, DOP, and other organic analyses.
Figure 4.1. Selective filters utilized experimentally for evaluating the individual photolytic
effects of UV-B, UV-A, and photosynthetically active radiation (PAR).
Analyses were performed as follows:
54
DRAFT
1. DOC and DON: High temperature combustion Shimadzu TOC-V-6500 analyzers that
were passed through precombusted (500°C) Whatman glass-fiber filters (GF/F, nominal
pore size, 0.7 µm; effective pore size <0.5 µm) within an all-glass filtration system.
2. Organic acids: Samples were filtered (0.22 µm pore size (Millex-GV) and analyzed with
a Waters HP liquid chromatography (Waters 1525 and 2847 detectors).
3. Fluorescent properties: Automated excitation and emission fluorometry of the organic
compounds were analyzed with a Hiatchi Scanning Fluorometer (4500).
4. NO3, NH4 and PO4: These ions were analyzed using standard methods (Standard
Methods for the Examination of Water and Wastewater, 2001) with a Technicon auto
analyzer system.
The responses of bacteria to photolytic changes in substrate and nutrient availability were
analyzed by evaluating relative responses of natural bacterial consortia (from the Everglades) to
media after exposure to light under different conditions. Incubations were sampled after 24, 48,
and 72 hours of growth at constant 25°C in the dark. Changes in bacterial biomass were
evaluated by flow cytometry (special flow cytometer developed specifically for enumerating and
sizing bacteria of natural samples; Dr. J. M. Burkholder, North Carolina State University).
4.3
RESULTS
The nutrient pollution of P and N from the agricultural areas south of Lake Okeechobee
has clearly induced the eutrophication and invasion of the cattail (Typha domingensis Pers.).
The growths of these essentially monocultures are so dense that light is severely attenuated and
less that 15% of surface insolation reaches diffusely to the water (e.g., Grimshaw et al. 1997).
Water was sampled along the transect at 0 km (within the Typha), at 4 km (a mixture of Cladium
and Typha but with markedly increased insolation reaching the water) and at 10 km in a largely
Cladium-dominated habitat. The responses of the dissolved organic matter (DOM) to light can
be summarized as follows:
1) Photolytic generation of simple organic substrates from DOM, largely humic
substances from higher plant origins, was greatest with the DOM from the more shaded Typha
55
DRAFT
portions of the transect (Fig. 4.2). The rate of photolytic generation of volatile fatty acids was
much faster with DOM from the shaded region than from further down the gradient. Note the
very high levels of simple organic acids, excellent bacterial substrates, that were produced
photolytically under these sterile conditions.
Fig 4.2. Phytolytic degradation of humic substances of sterile DOM from the Everglades
transect.
2) The photodegradation can be seen in the rapidity with which primary fluorophore
regions of the excitation-emission matrix (SSM) spectroscopy of dissolved organic compounds
changed (Figures 4.3 and 4.4). It should be noted in Figure 4.4 the marked differences in the
scales, with appreciably less photodegradation in the water from the Cladium site (10 km).
56
DRAFT
Fig 4.3
57
DRAFT
Fig 4.4
Figs 4.3 & 4.4. Photodegradation of the primary fluorophore regions of the excitation-emission
matrix (EEM) of DOM from the three transect stations after being exposed to sunlight (80% of
full sunlight; 14,040 KJ m2 6 hr-1).
3) The same trend could be seen in the progressive decay of the ultraviolet absorption
characteristics of DOM from the stations of the transect (Figure 4.5). Although this technique of
relative optical absorbance at 260 nm gives only a rough approximation of changes in total
chromophoric dissolved organic matter, the progressive photolytic degradation was consistent
with the other more detailed analyses (discussed above) both among the transect stations and the
effectiveness of the different spectral components of light..
58
DRAFT
Fig 4.5. Changes in absorbance (OD260) of DOM of the three stations upon photolysis by natural
light (14040 KJ m2 6 hr-1).
4) In addition to partial photolytic degradation of natural DOM to simple organic acids,
a portion of the DOM is phytodegraded completely to CO2. Here again, as with the generation
of organic acids, photolytic degradation to CO2 was much less at the 10-km site than among
samples from the more shaded habitats (Fig 4.6). The effectiveness of photolysis by PAR was
highly significant, and was of much greater impact on the DOM of the shaded site (0 km) than in
the others.
59
DRAFT
Fig 4.6. Photodegradation of dissolved organic matter (normalized to 30 mg C L-1 at each
station) from different stations of the Everglades transect to CO2.
5) The photolytic degradation of dissolved organic nitrogen (DON) compounds was
conspicuous (Figures 7 and 8). The generation of significant amounts of nitrate and, to a lesser
extent, ammonium was consistently more evident when water from the enriched but shaded
station (0 km) was exposed to light under sterile conditions.
6) The photolytic effects on generation of phosphate from dissolved organic compounds
were inconclusive (Figure 8). Changes were not significant at this time of the year (November).
60
DRAFT
Figs 4.7.& 4.8 Phytolytic generation of nitrate and ammonium from dissolved organic matter
along the transect from heavily shaded habitat (0 km) to less shaded habitats.
Fig 4.8. Photolytic induction of phosphate from dissolved organic matter along the transect in
the Everglades.
61
DRAFT
7) The work on evaluating bacterial responses to water exposed to various photolytic light
treatments is continuing but preliminary results indicated that bacterial growth was greatly
enhanced as the dissolved organic matter moved into the more open portions of the wetland
habitat (e.g., 4 km and 10 km stations) (Figure 9). There was some indication (trend but not
significantly different) that the more mildly energetic portion of the spectrum (PAR only) was
more effective in altering the substrate and nutrient bioavailability that the more energetic UV
portions of the spectrum. These aspects are being evaluated with additional flow cytometric
techniques for biomass differences of the bacteria as well as live vs dead cells (membrane
compromised cells) by specific fluorometric staining techniques.
Fig 4.9. Bacterial densities grown on water from the different transect stations of the Everglades
that was exposed for 6 hours to full sunlight (quartz; 14040 KJ m2 6 hr-1), UV-A+ PAR (Mylar),
and PAR only (acrylic) over a 72-hour growth period (dark at 25°C).
62
DRAFT
Light plays an overriding role as an ecosystem modulator. Light is obviously the
primary energy source for synthesis of organic matter in photosynthesis but is simultaneously a
major agent of direct decomposition of organic matter without ever entering a biochemical
metabolic process or by rendering chemically recalcitrant organic compounds more bioavailable
by partial degradation of macromolecules (Wetzel 2002, 2003). Anthropogenic activities can
alter climatic and other environmental properties, such as elevating CO2 concentrations or
increasing UV radiation by decreasing absorptive gases such as ozone, that in turn change both
the chemical composition of the organic matter and the rates of decomposition. In the
Everglades, the phosphorus eutrophication is clearly enhancing the development of invasive
species of macrophytes. These plants function to shield the system from the natural photolytic
processes. These alterations can impact the thermodynamic stability of ecosystems via changes
to the organic carbon pools, their bioavailability, rates of nutrient recycling, and energy fluxes
throughout the ecosystem.
4.4
REFERENCES
Bicudo, D. C., Ward, A. K. and Wetzel, R. G. (1998) Fluxes of dissolved organic carbon within
attached aquatic microbiota. Verhandlungen Internationale Vereinigung der Limnologie
26, 1608-1613.
Cunningham, H. W. and Wetzel, R. G. (1989). Kinetic analysis of protein degradation by a
freshwater wetland sediment community. Applied and Environmental Microbiology 55,
1963-1967.
Grimshaw, H. J., R. G. Wetzel, M. Brandenburg, K. Segerblom, L. J. Wenkert, G. A. Marsh, C.
Charnetzky, J. E. Haky, and C. Carraher. (1997). Shading of periphyton communities by
wetland emergent macrophytes: Decoupling of algal photosynthesis from microbial
nutrient retention. Archiv für Hydrobiologie 139, 17-27.
Wetzel, R. G. (2001). Limnology: Lake and River Ecosystems. Academic Press, San Diego. pp
1006.
63
DRAFT
Wetzel, R. G. (2002). Dissolved organic carbon: Detrital energetics, metabolic regulators, and
drivers of ecosystem stability of aquatic ecosystems. In: S. Findlay and R. Sinsabaugh,
Editors. Aquatic Ecosystems: Interactivity of Dissolved Organic Matter. Academic Press,
San Diego. pp. 455-477.
Wetzel, R. G. (2003). Solar radiation as an ecosystem modulator. In: E. W. Helbling and H.
Zagarese, Editors. UV Effects in Aquatic Organisms and Ecosystems. Comprehensive
Series in Photochemical and Photobiological Sciences.
European Society of
Photobiology, Cambridge. Pp. 3-18.
Wetzel, R. G. (2004). Periphyton in the aquatic ecosystem and food webs. In: Azim, E.,
Verdegem, M., van Dam, A., and Beveridge, M., Editors. Periphyton: Ecology,
Exploitation, and Management. CABI Publishing, London, UK. (In press)
Wetzel, R. G. and Tuchman, N. C. (2004). Effects of atmospheric CO2 enrichment on the
production of plant degradation products and their natural photodegradation and biological
utilization. Archiv für Hydrobiologie (In press)
64
DRAFT
5.0 ANALYTICAL SPECIATION OF ORGANIC P BY CAPILLARY
ELECTROPHORESIS WITH INDUCTIVELY COUPLED PLASMA MASS
SPECTROMETRY
WILLIAM COOPER, Department of Chemistry and Terrestrial Waters Institute, Florida State
University, Tallahassee, Fl 32306-3006
5.1
INTRODUCTION
The coupling of CE separations with mass spectrometry (MS), and particularly element-
specific inductively coupled plasma mass spectrometry (ICP-MS) is a relatively new hyphenated
technique, but its utility for determining metal and non-metal speciation information has already
been demonstrated (Olesik et al., 1996; Michalke and Schramel, 1996; Liu et al., 1995). Olesik
and co-workers (1996) were particularly successful at separating different polyatomic anions of
the same element, as well as different charge states of ions. These investigators also pointed out
the advantage of using element-specific detection such as ICP-MS, since the CE separation
resolution of P-containing compounds can be compromised to some extent, since the ICP-MS
detector "sees" only P. This allows a trade-off in resolution for speed, producing rapid elemental
speciation separations.
Although no CE separations of environmental organic phosphorus have appeared in the
literature, previous separations of inorganic forms of P by conventional gel electrophoresis
indicate that orthophosphate, the "assumed" principal form of dissolved reactive phosphorus
(DRP), and condensed phosphates (DAHP) have significantly different electrophoretic
mobilities. While no organic P compounds were included in that study, the additional mass of
these compounds suggest a large difference in electrophoretic mobilities between inorganic and
organic phosphorous, and a rapid and efficient P-speciation separation should be possible. This
idea has been confirmed by applications of CE for separating organic P compounds from various
matrices, such as inositol phosphates in fermentation broths (Buscher et al., 1994) and
65
DRAFT
physiological fluids (Henshall et al., 1992). Integration of the CE with the ICP-MS has been
done previously for element speciation, although none has been specifically designed for
phosphorus.
Because of the very low levels of DOP found in the Everglades, a sensitive and
selective detection system is required. For this project the Finnigan MAT Element ICP-MS
located in the Geochemistry Laboratory at the National High Magnetic Field Laboratory was
used. Elemental mass spectrometry normally requires only low-resolution analyzers because
unit mass resolution should be adequate. However, there are several molecular species that
specifically interfere with phosphorus at 30.974 Da, including NO (30.0064) and O2 (31.998).
Bandura et al. (2002) have shown how these interferences could be removed by converting P+ to
PO+ in a reaction cell. However, that approach is specific to P. The high resolution MAT
Element is an instrumental approach to overcome these interferences that is applicable to most
elements.
The ICP-MS detection system is designed for quantitative phosphorous analysis and thus
requires a well-defined separation step (e.g. CE) beforehand in order to obtain speciation
information. A more direct approach is mass spectrometry combined with a “soft” ionization
process. If the mass spectrometer is sufficiently powerful, molecular formula information can be
obtained. Because we have access to two of the most powerful types of molecular mass
spectrometers, we also carried out studies of the possibility of determining individual organic P
compounds by direct mass spectrometry, employing the most gentle ionization technique now
available, electrospray ionization (ESI).
Before any molecular identifications of individual DOP compounds in an oligotrophic
system such as the Everglades can be accomplished, concentration of DOP from sub-ppb levels
must be achieved. Tangential Cross Flow Filtration (CFF) has become a popular method for
fractionating natural dissolved organic matter into molecular weight fractions (Hilger et al.,
1999; Buesseler et al., 1996). CFF can also concentrate organic species retained by the
membrane (i.e. the “retentate”) by up to 20-fold. Unfortunately, this improvement is not
sufficient to characterize organic phosphorus compounds in oligotrophic systems at sub-ppb
levels. Furthermore, we have found that the background dissolved organic carbon (DOC)
66
DRAFT
component isolated by CFF is typically 1000-fold higher in concentration than DOP. This
background organic matter is a serious interferent for direct mass spectral analysis by
electrospray ionization. We have thus modified and added to a method originally developed by
McKercher and Anderson (1968a; 1968b) for the extraction of inositol penta- and
hexaphosphates from soil samples. Our method both concentrates DOP from water and isolates it
from most of the background organic matter (Llewlyn et al., 2002). This combined approach
provides concentration factors of about 200 and will be used for all DOP speciation studies.
Previous attempts to develop robust separation techniques for determining organic P
speciation in natural, oligotrophic waters were unsuccessful because the concentrations of all
forms of P, including organic P, are too low in such systems. When total P concentrations are
below 100 µg/L, detection of individual compounds is very difficult, even following selective
concentration steps (Llewlyn et al. 2002). We thus carried out experiments to develop a high
performance liquid chromatography (HPLC) separation in parallel with the CE studies. HPLC
does not offer the very high resolving power of CE, but sample capacity is ~ 1000X higher and
thus HPLC provides detection of individual solutes at much lower concentrations.
5.2
EXPERIMENTAL
5.2.1
Capillary Electrophoresis Separations. Experiments were performed on a Beckman
P/ACE 5510 equipped with a UV Detector and 254 nm filter. The fused silica capillary was 37
cm total length (30cm length to detector (LD)), 75 µm i.d. and maintained at 25 oC by the
instrument. Injections were made using the low pressure injection function of the instrument (5
seconds at 0.5psi). The capillary was initially conditioned by rinsing with 0.1 N NaOH for two
hours followed by a water rinse. The daily conditioning routine consisted of a 2 minute 0.1 N
NaOH rinse, a 2 minute water rinse, and a 2 minute buffer rinse. Between runs, when the
analysis buffer was changed, the capillary was rinsed and allowed to stand in the new buffer for
2 minutes. Prior to sample injection, the capillary was rinsed again with the analysis buffer for
one minute. Between runs of the same analysis buffer, the capillary was rinsed with separation
buffer for 1 minute. Electropherograms were obtained at 10kV applied voltage. Electroosmotic
flow (EOF) was measured by injecting a solution of acetone (100µL/4mL).
67
DRAFT
Two types of run buffers, Borate and Tris, that spanned a pH range of ~4.7 to 9.35 were
prepared. The Tris buffers were prepared in two stock solutions of 15 mM Tris-HCl
(Trizma Hydrochloride), pH 4.69 and 15 mM Tris-base (Trizma Base), pH 9.35. A pH
scale was prepared by combining various ratios of the two stock solutions as shown in
Table 5.1. The borate buffers were prepared in the same manner from stock solutions of 5
mM Tetraborate and 20 mM Boric Acid.
Table 5.1 CE buffers used in the study of Humic Acids and buffer pH. T-HCl is Tris-HCl, TBase is Tris Base; BA is Boric Acid, TB is Tetraborate. These ratios represent solutions of 15
mM Tris-HCl and15mM Tris Base or 20 mM Boric Acid and 5mM Sodium Tetraborate.
T-HCl:T-Base
pH
BA:TB
pH
100:0
4.69
100:0
5.49
99:1
5.73
99:1
6.00
95:5
6.41
98:2
6.34
90:10
7.09
97:3
6.66
75:25
7.22
96:4
6.76
50:50
7.73
95:5
6.91
25:75
8.50
90:10
7.36
0:100
9.35
75:25
7.87
50:50
8.30
0:100
8.86
The CE column was connected to the ICP source via a specially designed home-built
interface. Cross micro channels (0.067”) were drilled in a block of Kel-F
(Chlorotrifluoroethylene, an inert plastic with high tolerances for temperature, electricity,
68
DRAFT
compression and machining) and screw holes of 10-32 were tapped for each. A CE capillary
(37cm x 360µm x 50µm) was threaded through one channel of the interface and held in place at
on end by a conical neoprene compression ferrule and PEEK nut. A short length of 1/16” Teflon
sheath tubing was threaded over the other end of the capillary and attached to the interface with a
PEEK ferrule and nut. A Pt wire was inserted into one of the crossing channels and held with a
neoprene ferrule and PEEK nut. The other opening of the cross channel was fitted with 1/16”
Teflon tubing attached to a peri-pump for a flow of makeup solution. The makeup solution
flowed through the interface, where it contacted the Pt electrode, and out the sheath tubing where
it met the outlet of the CE column, thus closing the CE electrical circuit. The inlet end of the
capillary is inserted into a buffer vial with a Pt electrode and high voltage applied. This interface
is depicted in Figure 5.1 below.
Pt Electrode
Neoprene Ferrules
Kel-F Cross Connector
Compression Nut
and Ferrule
CE Capillary
Meinhard
Nebulizer
1/16” Tubing
To Peri Pump
Fig 5.1 Special interface for connecting the capillary electrophoresis column to the Meinhard
nebulizer for on-line CE-ICP-MS analysis.
5.2.2
High Performance Liquid Chromatography. The size exclusion high performance
liquid chromatography system utilized for this project was modeled after that described by
69
DRAFT
Reemtsma and These (2003). A Beckman System Gold liquid chromatograph with Beckman
diode array multiwavelength UV-Vis detector was used for all experiments. This chromatograph
can be interfaced directly to a JEOL AccuTOF time-of-flight mass spectrometer through an
atmospheric pressure electrospray ionization source. A PL Aquagel-OH 30 SEC column, 250 X
4.6 mm i.d. 8 µm particle diameter, was the separation medium. This column has a nominal
molecular weight range of 100 – 30,000 Dalton. The flow rate was maintained at 0.3 mL/min.
The mobile phase was 80/20 (v/v) water/methanol with ammonium bicarbonate buffer (pH =
8.2). This buffer concentration was varied from 0.01 to 0.1M.
5.2.3
Inductively Coupled Plasma Mass Spectrometry. The high-resolution inductively
coupled plasma mass spectrometer (ICP MS) was an Element (Finnigan MAT, Bremen,
Germany). The instrument is located in the Geochemistry Laboratory at the National High
Magnetic Field Laboratory (NHMFL). Briefly, instrument design is a reversed Nier – Johnson
geometry that utilizes magnetic and electrostatic sectors to achieve mass resolving power up to
8,000. For phosphorus measurements the instrument is operated in the medium resolution mode
(m/∆m = 3,000), which is sufficient to resolve elemental P ions from interfering oxides [NO, O2]
that are present at high concentrations.
5.2.4
Time-of-Flight Mass Spectrometry. Time-of-flight mass spectrometry experiments
were carried out on a JEOL AccuTOF high resolution spectrometer located in the Department of
Chemistry’s mass spectrometry laboratory. This high resolution spectrometer uses the reflectron
geometry and has a nominal mass resolution of 6,000. It is equipped with several ionization
sources; for this work, the standard atmospheric pressure electrospray ionization source was
used.
5.3
RESULTS
5.3.1
Quantitative Reliability of Inductively Coupled Plasma Mass Spectrometry. The
quantitative reliability of ICP-HRMS for phosphorus determinations was judged by comparing it
to the standard total dissolved phosphorus (TDP) (Cooper et al. 2004). That method includes a
persulfate oxidation step to convert all forms of phosphorus to the ortho-phosphate form that is
then suitable for the molybdenum blue reaction. Using the same set of standards, calibration
70
DRAFT
curves for both methods were developed over a range of 0 – 200 µg-P/L. Figures of merit for the
two methods are included in Table 5.2. The ICP-MS method is clearly superior to the classical
method over this concentration range.
The two methods were also compared using actual data on field samples generated by
both methods. Figure 5.2 is a plot in which results of the two methods are regressed against each
other, with a regression line of unit slope included. From this plot it appears that the colorimetric
method overestimates P concentrations at low levels due to interferences and background
absorption, and underestimates higher concentrations due to incomplete reactions or color
development.
Table 5.2. Analytical Figures of Merit for the ICP-MS and Colorimetric Methods of Phosphorus
Determinations.
Figure of Merit
ICP-MS Method
Colorimetric
Method
0.004 ± 0.0004
1.70
~ 3.5 ppb P
~ 12 ppb P
~ 12 ppb P
Calibration Sensitivity (m)a
38.1 ± 0.152
b
316
Analytical Sensitivity (γ)
Limit of Detection (LOD)c
~ 0.4 ppb P
Limit of Quantitation (MQL)d
~ 1.7 ppb P
d
Limit of Quantitation (MQL)
~ 1.7 ppb P
a
slope of the calibration curve
b
slope of the calibration curve by the standard deviation of the measurement
c
the concentration that produces a signal equal to the average plus 3 standard deviations of the
blank signal
d
the concentration that produces a signal equal to the average plus 10 standard deviations of the
blank signal
71
DRAFT
100
90
ppb P Colorimetric
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
90
100
ppb P ICP-MS
Fig 5.2. Comparison of total phosphate data determined by from ICP-MS and colorimetric
method; solid line is linear regression, indicating a 1:1 relationship.
5.3.2
Capillary Electrophoresis Separation with ICP-MS Detection. The combined CE-
ICP-MS separation system was optimized using a mixture of standard organic phosphates: ophosphate, creatine phosphate (CP), tyrosine phosphate (TP), phosphor(enol)pyruvate (PEP),
and andenosine triphosphate (ATP). Individual organic phosphates standards were prepared in
distilled water. Their mobilities were measured by CE-ICP-MS by injecting them individually
and monitoring the P signal at 30.974 Da. The samples were then combined in proportions to
produce a solution that was ~150 ppb P in each standard. The standard separation was good but
difficult to interpret due to limit of detection. There were four peaks detected at 6.8, 8.1, 9.0 and
10.2 minutes, indicating TP, CP, PEP and o-phosphate respectively.
This system was tested for its applicability for analyzing filed samples using a
DOP sample that had been isolated and concentrated by the combined CFF and phosphorus
isolation system described previously (Lleweyln et al., 2002). The water sample was obtained
from Water Conservation Area 2A. The resulting electropherogram (Figure 5.3) shows distinct
peaks sitting on a background between 4 and 6 minutes, indicating a broad distribution of diffuse
72
DRAFT
charged dissolved organic material containing some P. Other retentate samples were analyzed
with similar or no P detection and the permeate and filtered samples yielded no P detection due
to the very low P concentration.
4000
Phosphates
3500
3000
CPS P
2500
2.32, ESF
2000
1500
1000
500
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Figure 5.3. Capillary Electropherogram with ICP-MS detection of organic phosphorus isolated
from Water Conservation Area 2A. Separation done on 50 M i.d. x 50 cm capillary, 15mM tris
buffer, pH 7.5, at 10kV with 15 second siphon injection. Detection by single ion monitoring at
30.974 Da.
Although a working CE-ICP-MS system was designed and standard organic phosphates
successfully separated, detection limits for this system are much too high for routine analyses of
organic phosphorous in oligotrophic systems like the Everglades. Another separation technique
which delivers more sample to the ICP-MS is needed to achieve the necessary detection and
quantitation limits.
5.3.3
High Performance Size Exclusion Liquid Chromatography. Previous experiments by
Reemtsma and These (2003) demonstrated that SEC columns separate natural organic matter
better as the ionic strength of the buffer increases. However, this presents a dilemma when ICP
or electrospray ionization mass spectrometry will be used for detetction, since high ionic
strengths decrease signal intensities in these ionization techniques. Thus, the primary
optimization experiments involved trying to find a buffer strength that was high enough for good
size exclusion separations but not too high so as to decrease ionization efficiencies in the mass
spectrometry detection. We chose a relatively new buffer system for this work, water/methanol
73
DRAFT
with ammonium bicarbonate, because of its unique volatility. Poly(styrene)sulfonate standards
(PSS) with nominal molecular weights of 1,640, 7,900, 16,600 and 70,000 Da. were used in
these experiments.
Figure 5.4 are the calibration curves for size exclusion chromatograms of mixtures of
PSS at ammonium bicarbonate concentrations of 0.01 M (top) and 0.1 M (bottom). Clearly the
0.1 M mobile phase produces a better separation, as indicated by the much broader distribution
of retention times. These experiments confirmed the conclusions of Reemtsma and These (2003)
and indicate that effective molecular weight separations of dissolved organic matter, including
DOP, can be accomplished with this SEC-HPLC system.
Calibration curve for PSS standards, ionic strength = 0.01 M
5
4.8
4.6
4.4
log Mw
4.2
4
3.8
3.6
3.4
3.2
3
15.5
15.6
15.7
15.8
15.9
16
Tr (min)
74
16.1
16.2
16.3
16.4
DRAFT
Calibration curve for PSS standards, ionic strength = 0.1 M
5
4.8
4.6
4.4
log Mw
4.2
4
3.8
3.6
3.4
3.2
3
16
17
18
19
20
21
22
23
24
Tr (min)
Fig 5.4. Calibration curves (log MW vs. retention time Tr) for SEC separation of PSS standards
using 0.01 M (top) and 0.1 M (bottom) ammonium bicarbonate buffer mobile phases.
Next, we looked at the electrospray ionization efficiency of solutes in ammonium
bicarbonate buffers of various concentrations. A standard mixture of surfactants was used in
these experiments. As expected, we noted that signal intensity decreased by about 50% as the
ammonium bicarbonate concentration increased from 0.01 to 0.1 M. However, we were able to
recapture much of the signal loss by increasing solute concentration. Because the SEC column
has such a large sample capacity, we have concluded that the best combination is a high-ionic
strength ammonium bicarbonate buffer and relatively concentrated samples in which ICP and
ESI ionization efficiencies are reasonable.
5.3.4
Electrospray Ionization Time-of-Flight Mass Spectrometry. In previous studies of
DOP speciation in the Everglades we noted that electrospray ionization was not inherently very
efficient for the organic P. We therefore carried out a series of experiments in which the cone
voltage within the ESI source was varied. Sufficiently high cone voltages are necessary to ionize
target analytes, but too high a voltage can result in fragmentation. For these experiments we used
a mixture of inositol monophosphate (IP1; [C6H6(HnPO4)1]) and inositol hexaphosphate (IP6, or
phytic acid; [C6H6(HnPO4)6] dissolved in an appropriate ESI solvent. Surprisingly, we found
75
DRAFT
that alone, the phytic acid was efficiently ionized, with an optimum cone voltage of about 60 V.
Unfortunately, at all voltages we sampled, we noted significant fragmentation of the IP6 phytic
acid, with the 1-5 congeners also appearing in the mass spectrum (Figure 5.5). We believe that
the phosphate groups in organic P may be relatively labile, and intact molecular ions may thus be
difficult to detect.
Fig 5.5. ESI-TOF mass spectrum of a mixture of inositol monophosphate (IP1) and inositol
hexaphosphate (IP6)
5.4
DISCUSSION
We have concluded that high-resolution capillary electrophoresis is probably not a
technique suitably robust and sensitive for routine quantitation of organic phosphorus
compounds from an oligotrophic wetland system such as the Florida Everglades. We believe that
size exclusion liquid chromatography is much more amenable for such analyses. When
combined with ICP-MS detection, this separation approach can provide reasonable molecular
weight information on dissolved organic phosphorus, as well as organic P extracts from soil and
tissue samples. In addition, because of the size of eluents provided by SEC-HPLC, we propose
that direct coupling of the SEC column to the ICP-MS instrument be combined with fraction
collection and subsequent high-resolution time-of-flight molecular mass spectrometry. This
76
DRAFT
combined approach will allow estimates of the molecular weights of organic P (SEC-ICP-MS) as
well as chemical formula identification (ESI-TOF-MS).
The following flow chart summarizes our suggestion for a comprehensive analysis of
organic phosphorus speciation in the Everglades. We are currently analyzing samples from a Pdosing experiment being carried out by the Reddy group at UF to demonstrate the utility of this
approach.
Fig 5.6. Proposed sample concentration, separation and mass spectral identification scheme for
defining organic phosphorus speciation in the Everglades.
5.5
REFERENCES
Bandura, D.R., Baranov, V.I. and Tanner, S.D. 2002. Detection of ultratrace phosphorus and
77
DRAFT
sulfur by quadrupole ICPMS with dynamic reaction cell. Anal. Chem. 74, 1497-1502.
Buesseler, K. O., Bauer, J. E., Chen, R. F., Eglinton, T. I., Gustafsson, O., Landing, W., Mopper,
K., Moran, S. B., Santschi, P. H., Vernon, C. R. and Wells, M. L. (1996). An
intercomparison of cross-flow filtration techniques used for sampling marine colloids:
Overview and organic carbon results. Marine Chem. 55, 1-31.
Buscher, B. A. P., Irth, H., Anderson, E., Tjaden, U. R. and van der Greef, J. (1994).
Determination of Inositol Phosphates in Fermentation Broth using Capillary Zone
Electrophoresis with Indirect UV Detection. J. Chromatogr A, 678, 145-150.
Cooper, W. T., Lleweyln, J. M., Bennett, G. L., Stenson, A. C., and Salters, V. J. M. "Organic
Phosphorus Speciation in Natural Waters by Mass Spectrometry", in Organic
Phosphorus in the Environment, CAB International, Wallingford, UK, in press.
Henshall, A., Harrold, M. P. and Tso, J. M. (1992). Separation of Inositol Phosphates by
Capillary Electrophoresis, J. Chromatogr. 608, 413-419.
Hilger, S., Sigg, L. and Barbieri, A. (1999). Size fractionation of phosphorus (dissolved,
colloidal and particulate) in two tributaries to Lake Lugano. Aquatic Science. 61, 337353.
Liu, Y., Lopez-Avilla, V., Zhu, J. J., Wiederin, D. R. and Beckert, W. (1995). Capillary
Electrophoresis Coupled On-Line with Inductively Coupled Plasma Mass Spectrometry
for Elemental Speciation, Analytical. Chemistry 67, 2020-2025.
Llewyln, J. M., Landing, W. M., Marshall, A. G. and Cooper, W. T. (2002). Electropsray
Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Dissolved
Organic Phosphorus Species in a Treatment Wetland after Selective Isolation and
Concentration. Anal. Chem. 74, 600-606.
McKercher, R.B. and Anderson, G. (1968a). Characterization of the inositol penta- and
hexaphosphate fractions of a number of Canadian and Scottish soils. Journal of. Soil
Science. 19, 302-309.
McKercher, R.B. and Anderson, G. (1968b). Content of inositol penta- and hexaphosphates in
some Canadian soils. Journal of. Soil Science 19, 47-54.
Michalke, B. and Schramel, P. J. (1996). Hyphenation of capillary electrophoresis to inductively
coupled plasma mass spectrometry as an element-specific detection method for metal
specification. J. Chrom., A. 750: 51-62.
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Olesik, J. W., Kinzer, J. A. and Olesik, S. V. (1996). Capillary Electrophoresis Inductively
Coupled Plasma Spectrometry for Rapid Elemental Speciation, Analytical. Chemistry.
67:1-12.
Reemtsma, T. and These, A. (2003). On-line Coupling of Size Exclusion Chromatography with
Electropsray Ionization-Tandem Mass Spectrometry for the Analysis of Aquatic Fulvic
and Humic Acids. Analytical. Chemistry. 75, 1500-1507.
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6.0 EXPERIMENTAL MESOCOSM SET-UP AND WATER QUALITY
MONITORING
P. W INGLETT, KANIKA SHARMA, K. R. REDDY
Soil and Water Science Department, University of Florida-IFAS, Gainesville, FL-32608
6.1
INTRODUCTION
In P-limited freshwater systems, P is rapidly recycled and any form of readily labile P is
immediately taken up by the organisms. While inorganic P is immediately assimilated by the
organisms, organic P requires enzymatic hydrolysis to liberate inorganic phosphate for plant and
microbial uptake. One of the major extracellular enzymes responsible for this hydrolysis is
phosphatase. High values of phosphatase activity have been reported (Newman et al.
2003) in P-limited systems. Production of phosphatase (by microorganisms and algae) is
stimulated in presence of low inorganic P conditions. Under conditions of elevated inorganic P
(orthophosphate), however, phosphatase synthesis and consequent rates of organic P hydrolysis
are repressed. This almost certainly leads to accumulation of organic P in forms that are
potentially readily degradable if P limitation was increased. This induction can be stimulated by
increasing the C: P and C: N: P ratio and creating additional P-stress on the microorganisms
present in the system.
Our preliminary greenhouse experiments investigated the effect of two N-sources (NH4+
and NO3-) and three C-sources including a simple sugar (glucose), an organic acid (acetate), and
an amino acid (alanine) on alkaline phosphatase (APA) (refer to Task 2). Results demonstrated
that nitrogen amendments did not appear to stimulate the enzyme activity in the absence of
additional C. However, elevated APA was observed in the periphyton material in response to C
additions. Among the three C sources, glucose and the alanine appeared to stimulate the APA.
Based on these results, these two carbon sources were selected for further study in the field scale experiments. The amino acid alanine was substituted with arginine because it has a higher
(3:2) C: N ratio as compared to alanine (3:1). Concentrations of carbon substrates used for
80
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dosing were also determined by preliminary experiments. The field study was designed to
accommodate dosing concentrations of 500 mg C l-1 and 200 mg C l-1 for glucose and 200 mg C
l-1 for arginine. This objective was accomplished by establishing controlled mesocosms at the
outflow of the Effluent Nutrient Removal (ENR) Project site. These systems consisting of
rectangular, aboveground of tanks fed with ENR outflow water mimic the STAs, as they consist
of macrophytes and are operated as flow-through systems.
Results from two experiments allowed us to compare (i) the effects of C at two different
concentrations, (ii) the effects of two different C sources and, (iii) the effect of C with and
without N, on APA and ultimately on the concentrations of P in the water column.
6.2
MATERIAL AND METHODS
This task was accomplished through two studies (Phase I and Phase II). The Phase I
study involved assessing the effect of glucose amendment on APA (500 mg C l-1). The Phase II
study examined and contrasted the effect of C by itself (as glucose, 200 mg C l-1) with that of C
added with and organic N source (as arginine, 200 mg C l-1; 155 mg N l-1).
6.2.1
Study site. At present we are maintaining periphyton mats in several experimental
mesocosms located at the south side of STA-1W. These mesocosms consist of fiberglass tanks
(1 m wide x 3 m long x 1 m deep) with limestone substrate and are fed by the outflow water
from the STA-1W. Water depth is maintained at 30cm above the bottom of the limestone
surface. Establishment of periphyton in these systems has been difficult occurring over a period
of two years. Initially, macrophytes were removed by hand, and approximately 2-5 cm of
additional limestone substrate (coarse gravel) was applied in each tank to redevelop the benthic
surface. The tanks were then seeded from periphyton collected from other tanks as well as a
nearby periphyton raceway experiment and allowed to colonize.
Growth rates of periphyton mats within these mesocosms have been shown to vary
significantly even though the tanks were stocked with approximately equal amounts of mats.
Regrowth of macrophytes (primarily Eleocharis sp.) was curtailed by hand removal of stems,
however, efforts to control the spread of Chara sp. met with minimal success. In addition, some
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mesocosms were infested with snails and crayfish which grazed large proportions of periphyton.
In particular, two tanks were completely grazed (leaving only minimal floc on the limestone
surface) by the inhabiting crayfish. For these reasons, only 4 of the initial 12 mesocosm tanks
showed sufficient periphyton cover to be used in the studies described here.
6.2.2
Effect of high glucose dosing (500 mg C l-1) on alkaline phosphatase activity.
Four tanks were selected for this study based on similar morphological periphyton types. Two of
these tanks served as unamended controls, while two served as amended treatment tanks.
Experiments were conducted from January 2004-April 2004. Periphyton and water samples were
collected from these tanks at appropriate sampling times as indicated in Table 1.
Mesocosm dosing. Stock solution of glucose was prepared to dose the mesocosm to reach a final
concentration of 500 mg C l-1. Mesocosms are designed to hold 1200L of water. Based on initial
data collected the DOC level in STA outflow water (water received by mesocosms) was 30 mg C
l-1. Stock solution of glucose was introduced in the mesocosms at several points along its length.
The tank was then lightly mixed to ensure homogeneous addition of glucose to the water column
and to minimize the disruption of the biota and periphyton mats.
Table 1a. Phase I mesocosm tank dosing experiment sampling schedule.
Activity
Date (2003-2004)
12/3
Periphyton, APA
Periphyton, Nutrients
Water, Nutrients
Hydrolab Data
Tank Dosing
12/10
12/18
1/14
1/22
1/29
2/12
2/27
3/10
3/24
4/8
4/21
5/6
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
XXXXXXXXXXXXXXXXXXXXXXX
Table 1b. Phase II mesocosm tank dosing experiment sampling schedule.
Activity
Periphyton, APA
Periphyton, Nutrients
Date (2004)
7/15
7/22
7/23
7/29
5-Aug
12-Aug
18-Aug
26-Aug
X
X
X
X
X
X
X
X
X
X
X
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Water, Nutrients
Hydrolab Data
Tank Dosing
X
X
X
X
X
X
X
X
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
X
X
Sample collection and processing. Water pH, temperature, dissolved oxygen (DO),
specific conductance were continuously monitored using Hydrolabs deployed at mid-water column
depth in each tank. Sampling of periphyton and tank inflow and outflow water occurred
approximately once per week during the study. Samples were collected at approximately noon on
different sampling dates throughout the study to avoid any potential diel effect on the samples.
Water and periphyton samples were collected into polyethylene bottles or bags, respectively, and
stored on ice for transport to the Wetland Biogeochemistry Laboratory, Soil and Water Science
Department at University of Florida, Gainesville, FL.
Triplicate samples of all three periphyton forms (epiphytic, benthic and floating) were
collected separately from all 4 tanks. Subsamples of wet periphyton material were analyzed for
alkaline phosphatase activity (APA). Additional subsamples were dried at 70°C for 72 h to
determine moisture content and ground for nutrient analysis. Total N (TN) and total carbon (TC)
were measured simultaneously using a Carlo-Erba NA-1500 C/N elemental analyzer (HaakBuchler Instruments, Saddlebrook, NJ). Loss in weight after combustion at 550°C for 5 h (LOI)
was used to calculate for ash content (American Public Health Association, 1992). Total P was
determined on the residual ash by ashing method. Total inorganic carbon (TIC) was measured on
periphyton samples using an acid dissolution/pressure calcimeter method for total carbonate
(Loeppert and Suarez, 1996).
Unfiltered water samples were analyzed for TP, DRP, TDP, and DOC while samples for
DRP, TDP, TDKN, NH4+, and NO3- were filtered with 0.45µm syringe filters. Samples collected for
TP, TDP, DOC, TKN, TDKN, NH4+, and NO3- nutrients were acidified to pH~2 using concentrated
H2SO4. DRP was determined according to a colorimetric, ascorbic acid method (USEPA, 1993;
Method 365.1) using a Shimadzu UV-160 spectrophotometer equipped with a 10-cm long path
cuvette. TP and TDP were measured as ortho-phosphate following potassium persulfate
digestion using a Technicon Autoanalyzer (Terrytown, NY) according to USEPA Method 365.1.
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+
Dissolved NH4 was analyzed by the salicylate-nitroprusside technique (USEPA, 1993; Method
350.1) using a Technicon Autoanalyzer. Dissolved NO3- + NO2- was analyzed colorimetrically
using an Alpkem RFA according to EPA Method 353.2. TKN sample digests were analyzed for
NH4+ using a Technicon Autoanalyzer within 30 days of the digestion (USEPA, 1993; Method
351.2). DOC was determined using a Dohrman DC 190 carbon analyzer (Santa Clara, CA).
Alkaline phosphatase assay (APA). Triplicate periphyton subsamples were transferred to
acid washed plastic containers and thoroughly homogenized with a spatula. Dry weight of the
samples was determined by subjecting a weighed amount of wet material to 70ºC for 72 hrs. One
gram (wet weight) of periphyton material was added to 9 ml of distilled-deionized water and
homogenized with a hand-held biohomogenizer (Tissue Tearor™, Biospec Products Inc, Racine,
WI). The slurry was diluted accordingly to adjust APA values within a previously determined
range of detection. Methylumbelliferone phosphate (MUF- P) substrate (100 mM) was added
and allowed to react with APase present in the sample to form the fluorogenic product. Rate of
enzyme reaction in samples was then followed by determining the fluorometric reaction over the
period of 2 h. Rates are then expressed as mg MUF formed per hour of reaction, and
standardized per gram (dry weight) of periphyton assayed. Appropriate dilutions of the
standards were prepared with MUF.
6.2.3 Effect of low glucose dose (200 mg C l-1) and arginine on alkaline phosphatase
activity
Three tanks were selected for this study based on similar morphological periphyton
types. One tank served as unamended control and two served as amended treatment tanks.
Treatment tanks were amended with either glucose (200 mg C /L) or arginine (200 mg C/L;155
mg N l-1). Experiments were conducted from July through August, 2004 (Table 1). Tanks were
dosed in the 2nd week to initiate the experiment, and again in the 5th week of sampling when the
DOC dropped to the background levels. Periphyton and water samples were collected from these
tanks 2 weeks prior to dosing to provide a baseline for all parameters being analyzed during the
experiment. Water samples for DOC analysis were collected before and after dosing the same
day in order to know the amount of additional C being added to the tank. Water samples were
84
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analyzed for NH4-N, NO3 -N, TKN, TDKN, TP, DRP, TDP, DOC. Water pH, temperature,
dissolved oxygen (DO), and specific conductance were also continuously monitored in situ.
Periphyton samples were collected and analyzed for alkaline phosphatase activity (APA), ash
content, TP, TN, TC and TIC as described above.
6.3
RESULTS AND DISCUSSION
The effects of high C additions to the mesocosm tanks were severe. Visually, these
effects could be seen in the changing appearance of the tank components following dosing (Fig.
1a, b). These visual changes included an increased formation of mucilaginous layer on both the
surface of the mat and at the water surface, cloudiness of the water column and, the
disappearance of the floating mats either by settling to the bottom of the mesocosm or by
85
DRAFT
Tank 3
Tank 4
Tank 6
Tank
10
Fig 1a. Pictures showing conditions inside the STA-1W outflow site tanks before (left column)
and after Phase I glucose dosing (right column). Tanks 4 & 6 represent control tanks (un-dosed)
86
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while Tanks 3 & 10 are dosed treatments. Pre-dosing pictures were taken 12/18/04 while after
dosing pictures were recorded on 4/27/04.
Tank 3
Tank 4
Tank 6
Figure 1b. Pictures showing conditions inside the STA-1W outflow site tanks before (left
column) and after Phase II glucose/arginine dosing (right column). Tank 3, 4 and 6 represent
87
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arginine-dosed, glucose-dosed, and control tanks, respectively. Pre-dosing pictures were taken
5/06/04 while after dosing pictures were recorded on 8/12/04.
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Tank 4 - Temp (C)
35
50
Tank 4 - Control
Tank 3 - Glucose
45
30
40
25
35
20
30
15
25
10
20
5
0
15
Tank 3 - Temp (C)
40
10
10
9
pH
8
7
6
4
2000
1600
1200
800
400
0
120
Tank 4 – Dissolved
Oxygen (%Sat)
100
80
60
40
20
0
Dec-03
Jan-04
Feb-04
Glucose
Dosing
Glucose
Dosing
Mar-04
Apr-04
180
160
140
120
100
80
60
40
20
0
May-04
Tank 3 – Dissolved
Oxygen (%Sat)
Specific Conductance (mS)
5
Date
Figure 2a. Water column chemical parameters (specific conductance, temperature, pH, and
89
DRAFT
dissolved oxygen) observed for one control (Tank 4) and one dosed (Tank 3) treatment during
the Phase I dosing experiment. Dates of carbon dosing are denoted by arrows.
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Water Temp (C)
Tank 3 - Arginine
Tank 4 - Glucose
Tank 6 - Control
40
38
36
34
32
30
28
26
24
22
20
12
pH
11
10
9
8
Specific Conductance (mS)
7
6
1200
1000
800
600
400
200
0
250
DO (%Sat)
200
150
100
50
0
8-Jul
15-Jul
22-Jul
29-Jul
5-Aug
12-Aug
19-Aug
26-Aug
Date (2004)
Dosing
Dosing
Figure 2b. Water column chemical parameters (specific conductance, temperature, pH, and
dissolved oxygen) observed for one control (Tank 4) and one dosed (Tank 3) treatment during
91
DRAFT
the Phase I dosing experiment. Dates of carbon dosing are denoted by arrows.
92
DRAFT
disintegration of the mat structure into floc. With these visual changes in tank appearance,
distinct differences were also noted in the water chemistry parameters determined during the
experiments (Fig 2a,b). In the case of glucose, dosing events resulted in the lowering of tank pH
by approximately one unit (to pH 7). Lowered pH remained while DOC levels were high, then as
DOC levels declined; pH of the dosed tank increased and eventually maintained a higher pH
than the control tank (8.4). These pH trends were similar in both Phase I and II experiments and
this likely occurred as a result of increased CO2 production during bacterial respiration of the
added C substrates. In the Phase II study, the addition of arginine resulted in a similar pH shift
to that observed for glucose, however, in the case of arginine addition, tank pH increased slightly
(to pH of 10) during the overnight period immediately following dosing. This increase is likely
the result of microbial hydrolysis of the amine functional group of arginine resulting in the
release of carbonate anions which act to raise pH. This high pH was gradually reduced to
normal pH level (8.2 to 9.2) as DOC was reduced to background levels (Fig. 2b).
In addition to reducing pH, increased respiration in the C-dosed tanks also had the effect
of reducing tank dissolved oxygen. In both phase I and II experiments, glucose additions
resulted in a reduction of the diel fluctuation in DO eventually leading to anaerobic conditions
within the tank. DO levels began to increase following the reduction of watercolumn DOC.
Similar patterns were observed for the arginine dosing of the Phase II experiment. With the
reduction of DO and lowering of tank pH, little change was noted in specific conductance after
dosing with glucose. This result was consistent for both Phase I and II glucose dosings (Fig.
2a,b), however, a drastic lowering of conductance was observed for the tank dosed with arginine
in Phase II (Fig. 2b). It is uncertain if this result is a function of the arginine addition or simply a
result of probe fouling/failure.
Tank water column nutrient levels also showed the effects of C additions. In the case of
P, TP concentrations in the tank inflow water (derived from STA-1W) ranged between 20-40 µg
P l-1 (Dec 2003-May 2004) and 30-70 µg P l-1 (July -August 2004), while water TP in control
tanks for both studies ranged between 10-13 µg P l-1. TDP levels closely approximated those of
TP, reflecting the mostly organic nature of P in these systems. With C dosing, however, all P
93
DRAFT
species (DRP, TDP and TP) were observed to increase relative to controls (Fig. 3a,b).
94
DRAFT
Tank 3 (Dosed)
Tank 10 (Dosed)
Tank 4 (Control)
Inflow1
Tank 6 (Control)
Inflow2
18
DRP (µg P l-1)
15
12
9
6
3
0
60
TDP (µg P l-1)
50
40
30
20
10
TP (µg P l-1)
0
120
100
80
60
40
20
0
300
DOC (ppm)
250
200
150
100
50
0
Dec-03
Jan-04
Feb-04
Mar-04
Apr-04
May-04
Glucose
Date
Dosing
Glucose
Dosing
Figure 3a. Water column values of dissolved reactive P (DRP), total dissolved P (TDP), total P
(TP), and dissolved organic C (DOC) observed during the Phase I dosing experiment in the
undosed control (Tanks 4 and 6) and glucose-dosed (Tanks 3, and 10) STA-1W outflow
95
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mesocosm tanks. Points represent the mean of three replicate samples. Dates of carbon dosing
are denoted by arrows.
96
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Tank 6-Control
Inflow
4.0
60
3.2
50
40
2.4
30
1.6
20
0.8
10
0.0
20
0
70
Inflow DRP (µg L-1)
Tank 4-Glucose
60
16
50
12
40
8
30
20
4
10
Inflow TDP (µg L-1)
TDP (µg L-1)
DRP (µg L-1)
Tank 3-Arginine
0
0
100
TP (µg L-1)
80
6
0
40
20
DOC (mg L-1)
0
250
200
150
100
50
0
7/15
7/22
Dosing
7/29
8/5
Dosing
8/12
8/19
8/26
Date (2004)
Figure 3b. Water column values of dissolved reactive P (DRP), total dissolved P (TDP), total P
(TP), and dissolved organic C (DOC) observed during the Phase II dosing experiment in the
undosed control (Tanks 6), arginine-dosed (Tank 3) and glucose-dosed (Tanks 4) mesocosm
97
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tanks. Points represent the mean of three replicate samples. Dates of carbon dosing are denoted
by arrows.
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Tank 3-Arginine
Tank 4-Glucose
Tank 6-Control
40
20
0.080
15
10
0.040
5
0
0.200
0.040
0.160
0.030
0.120
0.020
0.080
0.010
0.040
0.000
10
0.000
160
TDKN (mg L-1)
0.000
0.050
8
120
6
80
4
40
2
TKN (mg L-1)
0
10
Arginine
Treatment NO3-N
(mg L-1)
25
0.120
Arginine
Treatment TDKN
(mg L-1)
30
Arginine
Treatment NH4-N
(mg L-1)
35
0.160
0
160
8
120
6
80
4
40
2
0
Arginine
Treatment TKN
(mg L-1)
NH4-N (mg L-1)
0.200
NO3-N (mg L-1)
Inflow
0
7/15
7/22
Dosing
7/29
8/5
Dosing
8/12
8/19
8/26
Date
(2004)
Figure 3c. As for Figure 3b, water column values of dissolved ammonium (NH4+), dissolved
nitrate (NO3-), total dissolved Kjeldhal N (TDKN), and total Kjeldhal N (TKN) observed during
the Phase II dosing experiment in the undosed control (Tanks 6), arginine-dosed (Tank 3) and
glucose-dosed (Tanks 4) mesocosm tanks. Points represent the mean of three replicate samples.
Dates of carbon dosing are denoted by arrows.
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100
DRAFT
With the decrease in water column DOC during Phase I, P levels returned to predosing levels,
while in Phase II, P levels remained high up to two weeks after the return of background DOC
levels (DRP), and TP and TDP were not observed to return to pre-dosing concentrations.
Similar to P, the dominant form of N in these systems is also organic in nature, however,
C dosing in the form of glucose did not appreciably affect N distributions or concentration
during this experiment. Not surprisingly, arginine dosing did result in substantial alteration of
water column concentrations of all N species. A significant portion (~20%) of the added organic
N (as arginine) was rapidly converted to NH4+ within the first week following dosing. Increased
NH4+ levels then led to accumulations of NO3- through the process of nitrification.
Despite the increases in water column P levels, relative increases in APA were observed
in both benthic and floating mats as a result of carbon dosing (Fig. 4a,b). In general, the effect
was an increase of approximately 3-5 mg MUF g DW-1 h-1 or an approximate doubling of the
existing rates. This observation is an apparent contradiction to general theory that increased P
availability will lead to a general suppression of phosphatase activity. The fact that the
enhancement observed during this study occurred following C dosing lends credibility to the
hypothesis in this study that C additions will increase P stress in periphyton (and subsequently
increase APA production/activity), however, the peak in APA and DOC levels were poorly
correlated, with APA lagging behind C dosing by up to two weeks in both experiments. During
this time, many other conditions within the tank environment were also affected by the C
additions (pH, DO, etc), therefore, the exact physiological or biogeochemical reason for this
observed enhancement is unknown.
In addition, the enhancement of APA by C seemed to be specific to periphyton type.
Initially, it was noted that visually-different periphyton types were present in the tanks. The
difference in periphyton types may be the result of differences in species composition between
different tanks (samples to confirm this are being processed), and this difference may explain the
drastic differences in APA between Tank 3 (where there was an increase) and Tank 10 (where
there was no change) following C dosing in Phase I.
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Tank 3-Glucose
Tank 4-Control
Tank 6-Control
Tank 10-Glucose
12
Benthic
10
8
6
4
2
APA, MUF (mg gdw-1 h-1)
0
12
Epiphytic
10
8
6
4
2
0
12
Floating
10
8
6
4
2
0
Dec-03
Jan-04
Glucose
Dosing
Feb-04
Mar-04
Apr-04
May-04
Date
Glucose
Dosing
Figure 4a. Alkaline phosphatase activity (APA) determined using the MUF-P hydrolysis method
for periphyton samples collected during the Phase I dosing experiment from the undosed control
(Tanks 4 and 6) and glucose-dosed (Tanks 3, and 10) mesocosm tanks. Points represent the
102
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mean of three replicate samples. Dates of carbon dosing are denoted by arrows.
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DRAFT
Tank 3-Arginine
Tank 4-Glucose
12
Tank 6-Control
Benthic
10
8
6
APA, MUF (mg gdw-1 h-1)
4
2
0
12
Floating
10
8
6
4
2
0
7/15
7/22
7/29
8/5
8/12
8/19
8/26
Date (2004)
Dosing
Dosing
Figure 4b. As for Figure 4a, alkaline phosphatase activity (APA) determined using MUF-P
hydrolysis for benthic and floating periphyton samples collected during the Phase II dosing
experiment from the undosed control (Tank 6), arginine-dosed (Tank 3), and glucose-dosed
(Tank 4) mesocosm tanks. Points represent the mean of three replicate samples. Dates of carbon
dosing are denoted by arrows.
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Table 2a. Results of Phase I dosing experiment of STA-1W outflow periphyton tanks. Measured parameters of benthic floating and
epiphytic tank periphyton including alkaline phosphatase (APA) measured as MUF-P hydrolyis, loss on ignition (LOI), total P (TP),
total N (TN), total C (TC), and total inorganic C (TIC). Values represent the means (± Standard Deviation) of three replicate
samples.
Benthic
APA
MUF ug g-1dw h-1
LOI
% dw
TP
mg kg-1
TN
% dw
TC
% dw
TIC
% dw
Floating
APA
MUF ug g-1dw h-1
LOI
% dw
TP
mg kg-1
TN
% dw
TC
% dw
TIC
% dw
Epiphytic
APA
MUF ug g-1dw h-1
LOI
% dw
TP
mg kg-1
TN
% dw
TC
% dw
Sampling Date (2004)
12-February
14-January
Parameter Units
8-April
Tank 3Glucose
Tank 4Control
Tank-6
Control
Tank 10Glucose
Tank 3Glucose
Tank 4Control
Tank-6
Control
Tank 10Glucose
Tank 3Glucose
Tank 4Control
Tank-6
Control
Tank 10Glucose
5266±1191
2919±498
3307±581
2759±625
9586±250
4282±464
5505±761
3450±258
6645±547
4152±304
8385±1460
4911±251
42.1±1.1
40.1±1.1
42.1±1.1
35.1±2.1
52.1±1.1
41.1±2.1
43.1±1.1
39.1±2.1
45.1±1.1
37.1±1.1
44.1±1.1
38.1±1.1
185±6
158±8
189±15
167±24
248±10
178±3
158±9
193±9
236±32
193±12
171±8
214±16
1.01±0.01
1.21±0.11
1.11±0.01
1.01±0.11
1.21±0.01
1.31±0.01
1.11±0.01
1.01±0.01
1.21±0.01
1.11±0.01
1.11±0.01
1.11±0.01
22.1±0.1
22.1±0.1
21.1±0.1
19.1±1.1
25.1±1.1
23.1±1.1
24.1±0.1
22.1±1.1
24.1±0.1
22.1±0.1
23.1±0.1
21.1±1.1
5.51±0.11
6.31±0.21
6.01±0.21
6.01±0.11
4.71±0.51
6.21±0.21
6.01±0.21
5.71±0.11
5.81±0.41
6.61±0.01
5.51±0.21
6.11±0.01
3283±67
2034±755
2960±207
3016±488
7288±243
4309±375
5501±913
3504±368
4647±329
3644±365
4233±759
3653±442
45.1±1.1
41.1±2.1
39.1±1.1
37.1±2.1
46.1±2.1
42.1±1.1
40.1±1.1
49.1±2.1
50.1±1.1
41.1±1.1
41.1±1.1
43.1±1.1
208±35
145±24
182±8
163±17
211±32
205±14
167±4
181±13
205±16
176±12
160±6
177±9
1.11±0.11
1.21±0.11
1.01±0.01
1.01±0.01
1.01±0.01
1.31±0.11
1.01±0.01
1.11±0.01
1.31±0.01
1.21±0.01
1.01±0.01
1.21±0.11
23.1±0.1
22.1±1.1
20.1±1.1
20.1±0.1
23.1±1.1
24.1±1.1
23.1±1.1
24.1±0.1
25.1±0.1
24.1±1.1
22.1±0.1
23.1±0.1
5.31±0.11
5.81±0.61
5.91±0.21
6.11±0.41
5.41±0.31
6.41±0.31
6.01±0.11
4.91±0.51
5.21±0.21
6.41±0.31
5.91±0.31
5.81±0.21
3891±329
2959±873
4934±786
2824±210
7054±533
5084±419
4645±637
4189±148
5838±575
4579±948
7517±648
4072±112
40.1±1.1
--
39.1±1.1
34.1±2.1
47.1±1.1
42.1±0.1
39.1±1.1
41.1±1.1
47.1±1.1
42.1±2.1
43.1±1.1
38.1±1.1
188±16
--
169±12
134±8
183±14
212±8
144±23
172±14
302±23
272±12
142±21
172±2
1.01±0.01
--
1.11±0.11
1.01±0.01
1.01±0.01
1.41±0.01
1.01±0.01
1.11±0.01
1.31±0.01
1.41±0.01
1.01±0.11
1.01±0.01
22.1±0.1
--
21.1±0.1
19.1±1.1
25.1±0.1
25.1±0.1
22.1±0.1
23.1±1.1
26.1±0.1
24.1±0.1
23.1±0.1
21.1±1.1
105
DRAFT
TIC
% dw
6.41±0.11
--
6.31±0.31
6.51±0.21
5.31±0.51
106
6.11±0.41
6.11±0.31
5.81±0.11
5.41±0.41
6.11±0.11
6.01±0.41
6.21±0.31
DRAFT
Table 2b. Results of Phase II dosing experiment of STA-1W outflow periphyton tanks. Measured parameters of benthic and floating
tank periphyton including alkaline phosphatase (APA) measured as MUF-P hydrolyis, loss on ignition (LOI), total P (TP), total N
(TN), total C (TC), and total inorganic C (TIC). Values represent the means (± Standard Deviation) of three replicate samples.
Parameter
Sampling Date (2004)
Units
15-July
5-August
Tank 3-Arginine Tank 4- Glucose Tank-6 Control
Benthic
APA
Tank 3-Arginine Tank 4- Glucose
15-August
Tank-6 Control
Tank 3-Arginine Tank 4- Glucose Tank-6 Control
MUF ug g-1dw h-1
4497±244
3330±284
4729±950
9261±1028
5961±449
3665±325
6537±474
5823±140
3749±283
LOI
% dw
38.1±1.1
40.1±2.1
37.1±0.1
39.1±1.1
37.1±1.1
43.1±2.1
42.1±1.1
43.1±1.1
33.1±1.1
TP
mg kg-1
154±16
174±7
172±27
168±2
153±3
167±10
178±0
236±9
128±5
TN
% dw
1.01±0.01
1.21±0.11
1.11±0.11
1.31±0.01
1.21±0.11
1.21±0.01
1.51±0.01
1.61±0.01
1.01±0.01
TC
% dw
24.1±0.1
24.1±0.1
23.1±1.1
24.1±0.1
24.1±1.1
24.1±0.1
25.1±0.1
25.1±0.1
22.1±0.1
TIC
% dw
6.51±0.21
6.41±0.11
6.91±0.41
6.61±0.11
7.01±0.41
5.81±0.51
6.11±0.21
5.81±0.11
7.31±0.11
MUF ug g-1dw h-1
4189±211
3755±191
4303±98
6923±269
7102±1665
3727±70
5340±91
5119±231
3791±221
LOI
% dw
42.1±1.1
37.1±1.1
38.1±0.1
40.1±4.1
36.1±1.1
39.1±2.1
42.1±2.1
38.1±0.1
36.1±1.1
TP
mg kg-1
155±7
206±7
121±5
136±12
145±9
195±10
163±7
193±15
100±20
TN
% dw
1.01±0.01
1.21±0.01
1.01±0.01
1.21±0.01
1.01±0.01
1.21±0.11
1.41±0.01
1.41±0.01
1.01±0.01
TC
% dw
24.1±0.1
23.1±0.1
23.1±0.1
24.1±0.1
22.1±0.1
24.1±1.1
24.1±1.1
24.1±0.1
22.1±0.1
TIC
% dw
6.11±0.31
7.01±0.21
6.81±0.11
6.31±0.21
7.01±0.11
6.61±0.21
5.91±0.21
6.41±0.31
6.71±0.21
Floating
APA
107
DRAFT
108
DRAFT
Alkaline Phosphatase Activity
Periphyton TP
Periphyton TN
Periphyton LOI
Periphyton TC
Periphyton TIC
Water NH4+
Log(Water DOC)
Log(Water DRP)
Figure 5. Linear regressions of measured periphyton and water chemistry variables with APA
measured during the combined Phase I and Phase II dosing experiments. See Table 3 for
summary of regression parameters.
109
DRAFT
110
DRAFT
Table 3. Linear regression model parameters for correlations of nutrient variables with
periphyton APA collected during Phase I and II experiments*.
Variable
LOI
TP
TN
TC
TIC
Water DRP **
Water NH4+ *
Water DOC **
r
0.344
0.164
0.176
0.491
-0.177
-0.195
-0.005
0.017
r²
Regression Parameter (Variable vs. APA)
p
N
Constant
0.119
0.027
0.031
0.241
0.031
0.038
0.000
0.000
9.35 x 10-6
.0392
.0266
5.18 x 10-11
.0270
5.64 x 10-5
0.956
0.753
158
158
159
159
157
423
126
333
-1207
3481
2554
-7865
7962
4638
4177
4188
Slope
147
7
1990
553
-516
-170
-1
35
* Water NH4+ regression is based on samples collected during Phase II experiment only.
** Regressions for Water DRP and Water DOC were performed on LOG transformed data.
Increased APA also seemed to be specific to periphyton growth form as evidenced by the
different response between benthic and floating versus epiphytic mats in the Phase I experiment.
In this case, the floating mats and benthic mats showed an equally stimulated response as APA to
the amendments while epiphytic forms were only minimally increased. Although the enzymatic
activity of the benthic mat remained stimulated longer than the floating mats, the enzymatic
activity of floating mats appeared to be stimulated earlier than the benthic mats. The results from
the Phase II study, did not show a lag in activity between the benthic and floating mats, however,
there was a difference in response time for both mat types as compared with Phase I (Fig. 4). In
Phase I, periphyton from tank 3 (floating and benthic) responded very quickly (within 1 week) to
glucose additions, while in the Phase II study, a lag period of up to two weeks was required for
the increased APA. These results strongly suggest that seasonal time of sampling (i.e., seasonal
community/species differences) also affects the response of tank periphyton APA to C. The
microbial composition of the periphyton mats have been shown to respond to seasonal changes
by changing the dominant populations.
Despite the inconsistencies between tanks and periphyton types, however, it is apparent
that C addition has some effect on the activity of APA within the tank periphyton. Though the
exact nature of this effect remains unexplained, the presence of the lag period (prior to increases
in APA) perhaps suggests that a shift in species composition is perhaps involved. During this
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DRAFT
lag period, bacterial populations undoubtedly begin to increase in response to the addition of
labile C source. As the newly adapted microbial population increases, the nonadjusting
population dies off. This die off and subsequent cell lysis may release P into the water column
and explain the increasing water column TDP and TP observed in this study. Increased
bacterial/heterotrophic populations are evidenced by the depletion of O2 in amended systems as
well as by the increase in C content in the mat material following dosing (Phase I, Table 2) and a
significant positive correlation between mat TC and APA (Fig. 5, Table 3).
The positive correlation of APA with TC and no correlation with TIC indicates that the
increase in APA activity was not a function of weight changes through dissolution of calcium
carbonate, but rather, is a function of increased organic carbon (determined as difference
between TC and TIC) in the periphyton mats. This relationship with TC was also reflected in the
significant positive association between APA and loss on ignition (LOI), and was by far the most
significant correlation with APA observed in this study explaining approximately 24% of the
variance in APA (Table 3). A significant negative correlation was also observed between APA
and the DRP concentration of the tank water column (Fig 5, Table 3), and this relationship
agrees with the general theory that increasing levels of available P act to suppress phosphatase
activity. APA was not shown to correlate with any of the other measured periphyton or water
column nutrient parameters in either the Phase I or II experiments.
6.5
CONCLUSIONS
Addition of carbon stimulated the alkaline phosphatase activity in the periphyton mats.
The mechanism for this stimulation is unknown as yet, however, a positive correlation between
APA and periphyton TOC suggests that increase in APA is likely to be a result of increase in
bacterial biomass. This observation is also partially supported by the microscopic results in task
3, which demonstrated a predominance of APA in association with bacteria living in the
periphyton matrix. If this is true, two hypotheses could be asserted regarding the observed APA
increases following C dosing. First, labile C additions could stimulate bacterial growth and
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DRAFT
increase the standing stock of microbial biomass. These bacteria are the dominant producers of
alkaline phosphatase, therefore, higher APA is due to the presence of more alkaline phosphatase
producers. Alternatively, the environment created as a result of C dosing (low O2, lowered pH,
etc.) may have led to an altered species composition of the microbial community, favoring
species who maximize C consumption. These species, which are likely facultative or anaerobic
organisms, may exhibit higher rates of APase production. Unfortunately, neither of these
hypotheses was conclusively demonstrated in the experiments of this task.
The observed increases in water column P levels in the dosed tanks implies that C
additions are not a likely candidate as a potential organic P removal strategy. This conclusion
may be premature, however, especially considering the limitations of the current study. Among
these limitations is the short-term nature of these experiments which monitored only the period
of days to weeks following the onset of C dosing. During this time numerous changes in water
column chemistry and periphyton species composition were occurring, and had likely not
reached a steady state condition by the conclusion of the experiments. If allowed sufficient time,
the characteristics of the high C system at equilibrium (e.g., higher overall APA) may offset the
short-term negative impacts (increased P export) observed during the shift in community.
Also, because the rates of C dosing used in this study were drastically in excess of what
could be normally tolerated by the algal mat system, conclusions that would assume ‘normal’
biological activity in the mat are prevented. The fact that the normal activity assumption is false
is evidenced not only by the visual changes observed as soon as one week after dosing, but also
in the rapid shifts in pH and DO within the tanks. Such drastic alteration of the periphyton mat
would likely result in significant changes to the existing biotic structure (affecting C cycling) as
well as storages and cycling of P. Therefore, lower C doses may better preserve the natural
periphyton function, and thus, have a more positive effect on water column P levels.
Based on the results of this experiment, we conclude that additions of labile C
compounds such as glucose and amino acids have the potential to increase the APA of
periphyton of low-P systems such as the Everglades. Many questions still remain about the
potential for this strategy to successfully lower water column P levels, however, future research
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DRAFT
is certainly warranted to develop this concept. Such research could include (1) more
sophisticated experimental approaches (e.g., physiological or isotopic tracer studies) to better
determine the relationship between C and P cycling in these mats communities, (2) more
systematic and long term experiments (e.g., seasonal studies) to explore the types and
concentrations of C which maximize the process, and (3) more applied experiments to test the
efficacy of this process in alone or in conjunction with various other treatment designs (e.g.,
dosed/non-dosed cell combinations). Also, because this process as tested is largely a function of
microbial activity, it seems logical that the C dosing idea should be investigated in other high
microbial biomass systems, for example litter decomposition, or even in batch or flow-through
bioreactor systems.
6.5
REFERENCES
Loeppert, R. H., and D.L. Suarez. 1995. Carbonate and gypsum. In: D.L. Sparks (ed.) Methods
of Soil Analysis, Part 3: Chemical Analysis. Soil Science Society of America Book Series
No. 5, Madison, WI.
Newman S., P. V. McCormick and J. G. Backus. 1993. Phosphatase activity as an early warning
indicator of wetland eutrophication: problems and prospects. Journal of Applied Phycology,
15, 45-59
United States Environmental Protection Agency (EPA). 1993. Methods for the Determination
of Inorganic Substances in Environmental Samples.
114