Prepared in cooperation with the U.S. Bureau of Reclamation

Characterization of Cyanophyte Biomass in a

Bureau of Reclamation Reservoir

By Nancy S. Simon, Ahmad Abdul Ali, Kyle Michael Samperton, Charles S. Korson, Kris Fischer, and 

Michael L. Hughes

Open-File Report 2013–1156

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2013

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Suggested citation:

Simon, N.S., Ali, A.A., Samperton, K.M., Korson, C.S., Fischer, Kris, and Hughes, M.L., 2013, Characterization of 

cyanophyte biomass in a Bureau of Reclamation reservoir, U.S. Geological Survey Open-File Report 2013–1156, 

59 p., http://dx.doi.org/10.3133/ofr20131156. 

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ISSN 2331-1258 (online)

Cover: Anchor line through Aphanizomenon flos-aquae in water column of Upper 

Klamath Lake, Oregon, July 14, 2010. Photograph by Kyle Samperton, 

U.S. Geological Survey intern (2012) for the National Association of Geoscience 

Teachers.
iii

Contents

Abstract................................................................................................................................................................ 1

Introduction........................................................................................................................................................... 2

Objectives............................................................................................................................................................. 3

Study Description and Methods ............................................................................................................................. 3

Study Design..................................................................................................................................................... 3

Sampling Locations ........................................................................................................................................... 3

Sample Collection.............................................................................................................................................. 3

Sample Preparation........................................................................................................................................... 4

Sample Analysis................................................................................................................................................ 4

31P Nuclear Magnetic Resonance (NMR) Analysis .......................................................................................... 4

Carbon and Nitrogen Analysis ........................................................................................................................ 5

Total Elemental Analysis ................................................................................................................................ 5

Conversion of AFA Protein to Amino acids and Inactivation of Microcystin....................................................... 6

Analysis for Microcystin in Extracts of AFA...................................................................................................... 6

Coomassie Analysis for Protein Concentration in Digests of AFA..................................................................... 7

Results and Discussion......................................................................................................................................... 7

31P NMR Analysis.............................................................................................................................................. 7

Carbon and Nitrogen Analyses........................................................................................................................... 9

Total Elemental Analysis.................................................................................................................................... 9

Conversion of AFA Protein to Amino Acids and Inactivation of Microcystin .......................................................... 9

Coomassie Analysis for Protein Concentration in Digests of AFA......................................................................11

Summary ............................................................................................................................................................ 11

References Cited ................................................................................................................................................ 11

Figures

1. Map of Upper Klamath Lake, OR, showing sampling sites for Aphanizomenon flos-aquae (AFA) 

collected in July and August, 2010 .......................................................................................................... 15

2. Photograph of Aphanizomenon flos-aquae (AFA) samples were collected using a plastic sieve with 

a 0.5 millimeter screen............................................................................................................................ 16

3. Graph of an ideal spectrum of phosphorus-containing compounds. (Figure from Cade-Menum, 2005) ......17

4. Graph of 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae samples collected in 

July and August 2010 from Upper Klamath Lake. ....................................................................................18

Sample 1- Pelican Marina (PM) collected 7/27/2010

Spectrum 1a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 18

Spectrum 1b - 31P NMR of extraction with water only.........................................................................19

Spectrum 1c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 20
iv

Sample 2 - Pelican Marina North (PMN) collected 7/27/2010

Spectrum 2a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 21

Spectrum 2b - 31P NMR of extraction with water only.........................................................................22

Spectrum 2c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 23

Sample 3 - Pelican Marina (PM) collected 8/9/2010

Spectrum 3a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 24

Spectrum 3b - 31P NMR of extraction with water only.........................................................................25

Spectrum 3c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 26

Sample 4 - North Buck Island (NBI) collected 7/27/2010

Spectrum 4a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 27

Spectrum 4b - 31P NMR of extraction with water only.........................................................................28

Spectrum 4c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 29

Sample 5 - Howard Bay (HB) collected 8/9/2010

Spectrum 5a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 30

Spectrum 5b - 31P NMR of extraction with water only.........................................................................31

Spectrum 5c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 32

Sample 6 - Eagle Ridge (ER) collected 8/9/2010 

Spectrum 6a - 31P NMR of NaOH-EDTA extraction solution plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 33

Spectrum 6b - 31P NMR of extraction with water only.........................................................................34

Spectrum 6c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an internal 

standard ................................................................................................................... 35

Sample 7 - Eagle Ridge South (ERS) collected 8/12/2010 

Spectrum 7a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 36

Spectrum 7b - 31P NMR of extraction with water only.........................................................................37

Spectrum 7c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 38

Sample 8 - Shoalwater Bay (SB) collected 8/9/2010

Spectrum 8a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 39

 Spectrum 8b - 31P NMR of extraction with water only.........................................................................40

Spectrum 8c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 41
v 

Sample 9 - Agency Lake South (AS) collected 8/9/2010 (blue color).

Spectrum 9a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 42

Spectrum 9b - 31P NMR of extraction with water only.........................................................................43

Spectrum 9c - 31P NMR of extraction with water only – plus methylene diphosphonic acid, an 

internal standard....................................................................................................... 44

Sample 10 - Agency Lake South (AS) collected 8/9/2010 (green color).

Spectrum 10a - 31P NMR of NaOH-EDTA extraction solution – plus methylene diphosphonic acid, 

an internal standard .................................................................................................. 45

Spectrum 10b - 31P NMR of extraction with water only.......................................................................46

5. Graph of spectrum 5a - 31P Nuclear Magnetic Resonance spectrum of sample collected from 

Howard Bay (HB) in Upper Klamath Lake (UKL) on August 9, 2010 .........................................................47

6. Graph of ratio of concentration of organic phosphorus to the concentration of total phosphorus (TP) 

plotted against the percent carbon in each Aphanizomenon flos-aquae sample, R2 = 0.93....................48

7. Graph of the seven peptide areas of the microcystin molecule include (1) D-Alanine, (2) L-Leucine, 

(3) D-erythro-p-methyl Asparine, (4) L-Arginine, (5) Adda, 3-amino-9-methoxy2,6,8-trimethyl-10- 

phenyl-4,6-decadienoic acid, (6) D-Glutamic Acid, and (7) Mdha, N-methyldehydro Alanine .....................49

Tables

1. Sampling sites for Aphanizomenon flos-aquae collected in July and August 2012 from Upper Klamath 

Lake, Oregon ............................................................................................................................................ 50

2. Chemical Shift values for compounds that appear in 31P NMR spectra....................................................... 51

3. 31P NMR data for Aphanizomenon flos-aquDH samples collected July-August 2010 from Upper Klamath 

Lake, Oregon ............................................................................................................................................ 52

4. Chemical data for Aphanizomenon flos-aquae collected in July and August 2012 from Upper Klamath 

Lake, Oregon ............................................................................................................................................ 58

5. Degradation of microcystin in digest of Aphanizomenon flos-aquae......................................................... 59
vi

Acknowledgments

Samples were collected from Upper Klamath Lake, Oregon, for this study by Kyle Samperton, 

U.S. Geological Survey intern (2012) for the National Association of Geoscience Teachers. Kyle also 

took the photographs of Aphanizomenon flos-aquae in Upper Klamath Lake, Oregon.

Boat time and sampling site identification were provided by scientists from the Klamath Tribes 

Research Station in Chiloquin, Oregon. s.d.g.
vii

Conversion Factors

SI to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

nanometer (nm) 3.3937 x 10-8 inch (in) 

Volume

liter (L) 1.057 quart (qt)

cubic centimeters 0.033815 ounce, fluid (fl. oz)

milliliter 0.033814 ounce, fluid (fl. oz)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound avoirdupois (lb)

milligram (mg) 3.527 x 10-5 ounce

metric ton per year 3.527 x 10-8 ton per year (ton/yr) 

Density

3 gram per cubic centimeter (g/cm ) 62.4220 pound per cubic foot (lb/ft3

) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F-32)/1.8

Vertical coordinate information is referenced to the North American Vertical Datum of 1988 (NAVD 88).

Horizontal coordinate information is referenced to the North American Datum of 1983 (NAD 83).

Altitude, as used in this report, refers to distance above the vertical datum.

*Transmissivity: The standard unit for transmissivity is cubic foot per day per square foot times foot of aquifer thickness 

[(ft3

/d)/ft2]ft. In this report, the mathematically reduced form, foot squared per day (ft2/d), is used for convenience.

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 25°C).

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L) or micrograms per liter (μg/L).

NOTE TO USGS USERS: Use of hectare (ha) as an alternative name for square hectometer (hm2

) is restricted to the 

measurement of small land or water areas. Use of liter (L) as a special name for cubic decimeter (dm3

) is restricted to the 

measurement of liquids and gases. No prefix other than milli should be used with liter. Metric ton (t) as a name for 

megagram (Mg) should be restricted to commercial usage, and no prefixes should be used with it.
viii

Abbreviations

~ approximately

Δ interchangeable with ppm as chemical shift unit nuclear magnetic resonance 

spectroscopy spectra

Δ heat applied

μg microgram, 1 x 10-6 gram

μL microliter, 1 x 10-6 liter

μs microsecond, 1 x 10-6 second

31P NMR Nuclear Magnetic Resonance spectroscopy using the isotope 31P 

ACS American Chemical Society

AFA cyanophyte, Aphanizomenon flos-aquae

Al aluminum

AS Agency Lake South, sampling location

C carbon o

C degrees Celsius

Ca calcium

cc cubic centimeter

CS chemical shift; location of peak in NMR spectrum

CWA Clean Water Act 

D2O deuterium oxide

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid; metal complexing agent

ER Eagle Ridge, sampling location

ERS Eagle Ridge South, sampling location

Fe iron

g gram

H3PO4 phosphoric acid

HB Howard Bay, sampling location

Hz hertz, the measure of the frequency of an electromagnetic wave

ICP–AES Inductively Coupled Plasma detector for Atomic Emission Spectrometer 

Inorg. inorganic

Inter std internal standard

IP inorganic phosphorus 

K kilo bytes of data

M molarity, moles per liter of solution

MDPA methylene diphosphonic acid; internal standard for 31P analysis

MDT Mid-trench, sampling site

megohm 106 x SI unit for electrical resistance

mg milligram, 10-3 gram

mg g-1 milligrams per gram dry weight of sample

MHz megahertz, 106 Hz 

min minute

mL milliliter, 10-3 L 

mm millimeter, 10-3 m 
ix

mV millivolts, 10-3 volts

nm nanometer, 10-9 m 

N nitrogen

n.a. not available 

NaOH sodium hydroxide

NBI North Buck Island, sampling location

NMR Nuclear Magnetic Resonance spectroscopy o

C degrees Celsius o

K degrees Kelvin; degrees Kelvin = 273 + degrees Celsius

OP organic phosphorus

P phosphorus

PM Pelican Marina, sampling location

PMN Pelican Marina North, sampling location

ppm chemical shift unit in nuclear magnetic resonance spectroscopy spectra

RNA ribonucleic acid 

rpm revolutions per minute

s second 

SB Shoalwater Bay, sampling location

s.d.g. sola deo gloria

soln. solution

Ti titanium 

TP total phosphorus

UKL Upper Klamath Lake, Oregon

Zn zinc

+ internal standard added

x very small peak in spectrum 
1 

Characterization of Cyanophyte Biomass in a Bureau of 

Reclamation Reservoir

By Nancy S. Simon,1 Ahmad Abdul Ali, 2 Kyle M. Samperton,3 Charles S. Korson,4 Kris Fischer,5 and 

Michael L. Hughes6

Abstract

The purpose of this study was to characterize the cyanophyte Aphanizomenon flos-aquae (AFA) 

from Upper Klamath Lake, Oregon, (UKL) and, based on this description, explore uses for AFA, which 

would have commercial value. AFA collected from UKL in 2010 from eight sites during a period of 

approximately 2 weeks were similar in composition spatially and temporally. 31P nuclear magnetic 

resonance analysis of the samples indicated that the AFA samples contained a broad range of 

phosphorus-containing compounds. The largest variation in organic phosphorus compounds was found 

in a sample collected from Howard Bay compared with samples collected the sites at Pelican Marina, 

North Buck Island, Eagle Ridge, Eagle Ridge South, Shoalwater Bay, and Agency Lake South. 31P 

Nuclear Magnetic Resonance data indicated that the average ratio of inorganic phosphorus 

(orthophosphate) to organic phosphorus in the AFA samples was approximately 60:40 in extraction 

solutions of either water or a more rigorous solution of sodium hydroxide plus 

ethylenediaminetetraacetic acid. This indicates that when AFA cells senesce, die and lyse, cell contents 

added to the water column contain a broad spectrum of phosphorus-containing compounds 

approximately 50 percent of which are organic phosphorus compounds. The organic phosphorus content 

of AFA is directly and significantly related to the total carbon content of AFA. Total concentrations of 

the elements Al, Ca, Fe, Mg, Ti and Zn were similar in all samples with the exception of elevated iron in 

the July 27, 2010, sample from Pelican Marina. Iron concentration in the July 27, 2010, Pelican Marina 

sample was elevated; the concentration of iron in the August 9, 2010, sample from Pelican Marina was 

indistinguishable from iron in the other AFA samples that were collected. The carbon to nitrogen ratio 

in all AFA samples that were analyzed was 5.4 plus or minus 0.04 as compared with the Redfield ratio 

of carbon to nitrogen ratio of 6.6, which could be attributed to the large concentrations of nitrogen 

(protein) in AFA or to optimal growth rate.

In UKL there is a concern that microcystin, the toxin produced by microcystis, might be present 

in what appears to be predominantly AFA in the lake water. Experiments preformed as part of this study 

identified a process that reduces the toxicity of microcystin when it is present in water slurry containing 

 

1

 U.S. Geological Survey, nssimon@usgs.gov. 2 VirginiaTech Carilion School of Medicine 

 (formerly at James Madison University). 3

 Princeton University, Princeton, New Jersey, ksampert@princeton.edu. 4

 U.S. Bureau of Reclamation, ckorson@usbr.gov. 5 Klamath Tribe’s Research Station, Chiloquin, Oregon, fischerkris@msn.com. 6

 Oregon Institute of Technology, Klamath Falls, Oregon, Michael.Hughes@oit.edu.
2 

AFA. The process combines (1) the inhibition of the α, β-unsaturated carbonyl in microcystin with (2) 

the breakdown of proteins in AFA using the protease activity of plant enzymes. Protease enzymes can 

break peptide bonds in microcystin, which results in destruction of the cyclic structure of the 

microcystin polypeptide. Laboratory conditions used in this study resulted in the inactivation of 

approximately 60 percent of the activity of microcystin. 

Introduction

High density blooms of cyanobacterial algae adversely affect water quality, fish populations, and 

outdoor recreation at Bureau of Reclamation reservoirs in the western United States. Algal blooms 

degrade water quality when algal cells senesce and deplete lentic systems of oxygen necessary for biotic 

organisms to survive, release toxins that can threaten aquatic life and human health, and contribute large 

particulate organic loads to downstream waters (Sullivan and others, 2009). It may become 

economically viable to remove algal biomass from Bureau of Reclamation reservoirs or downstream 

receiving waters in quantities sufficient to remediate ecosystem effects if commercially valuable 

products can be derived from cyanobacterial algae. 

Bureau of Reclamation reservoirs in the western United States are often faced with water-quality 

impairment problems that result in Clean Water Act (CWA 303(d) listing status (California State Water 

Resources Control Board, 2010) for a variety of water-quality properties. Upper Klamath Lake (UKL), 

which is part of the Klamath Irrigation Project under the jurisdiction of the Mid-Pacific Region Klamath 

Basin Area Office, is a prime example of one reservoir with acute water-quality impairments and an 

Oregon CWA 303(d) (California State Water Resources Control Board, 2010) listing for dissolved 

oxygen, pH, and chlorophyll. Large populations of AFA in UKL are attributed the elevated 

concentrations of phosphorus (P) in the water column (Eldridge and others, 2012; Harke and others, 

2012). One approach to reducing the concentrations of P and AFA in the water column is removal of 

AFA biomass from the lake.

Additionally, the Klamath River downstream from UKL is 303 (d) listed for nutrients, organic 

enrichment/dissolved oxygen, and temperature in California. More recently (2010) reaches of the 

Klamath River in California also have been listed for microcystin. In the case of Bureau of 

Reclamation’s Klamath Irrigation Project, water-quality problems caused by nutrients coming from 

UKL are a major management concern because several fish species listed under the Federal Endangered 

Species Act rely on habitats in UKL (endangered Lost River and shortnose suckers) and downstream 

from UKL (threatened Coho salmon) in the Klamath River. Establishing commercial uses for AFA 

would improve the chances that a private company would be interested in removing AFA from UKL 

waters before they enter the Klamath River leading to an improvement of water quality in the Klamath 

River. This could provide direct benefits to the Klamath Irrigation Project by leading to the development 

of in-lake algae removal programs scaled to improve water-quality conditions in UKL.

This report presents (1) a characterization of AFA removed from the lake in the summer of 

2010, and (2) a concept for breaking up protein in AFA to amino acids and reducing the toxicity of 

microcystin that can be found in some populations of cyanophytes growing in the water column with 

AFA. Any planned use of AFA for biological purposes would have to take into account the possibility 

that microcystin is present in the algal biomass removed from the lake.

The interest in reducing the toxicity of microcystin described in this report is focused solely on 

microcystin in the biomass of cyanobacteria collected from the water column. It is not focused on 

microcystin dissolved in the water column.
3 

Objectives

This research is needed to explore the potential for removing cyanobacterial algae on a 

commercial scale with the aim of improving water-quality conditions in Bureau of Reclamation 

reservoirs throughout the western United States. Cyanobacterial algae removal from Bureau of 

Reclamation reservoirs will yield multiple benefits. Water-quality conditions, if dramatically improved, 

would improve habitat conditions for aquatic organisms, slow the eutrophication process, reduce 

nutrient loading downstream, and help the Bureau of Reclamation ensure compliance with Clean Water 

Act requirements and Total Maximum Daily Limits (TMDL). (California State Water Resources 

Control Board, 2010).

This study explores the use of cyanobacterial biomass in UKL for commercially marketable 

products. Removal of some fraction of this algal biomass could reduce the negative effect of 

algal/cyanobacterial productivity on UKL dissolved oxygen and pH and improve water-quality 

conditions for the federally listed Lost River and shortnose suckers in UKL and Coho salmon 

downstream in the Klamath River. Moreover, biomass removal would eliminate a substantial pool of 

nutrients in the entire Klamath River system. Large populations of AFA in UKL are attributed to the 

elevated concentrations of P in the water column of UKL (Eldridge and others, 2012; Harke and others, 

2012). One approach to reducing the concentrations of P and AFA in the water column is removal of 

AFA biomass from the lake.

An objective of the study was to characterize AFA mats (dense blooms) because removal of 

AFA mats from the water is a more efficient method of algae removal than screening small colonies of 

AFA from the water column. Cyanobacteria blooms are present in other Bureau of Reclamation 

reservoirs and these methods are expected to have transferability to other settings.

Study Description and Methods

Study Design

To characterize AFA, this report provides the chemical composition of AFA and a description of 

the forms, inorganic and organic, of P in the AFA samples collected in the summer of 2010. Based on 

its large protein content, a promising use for AFA, was determined to be development as an agricultural 

feed stock. This study addressed detoxification of microcystin that might be included with AFA during 

AFA harvest from UKL. 

Sampling Locations 

AFA samples were collected from UKL, in July and August, 2010. A list of the sampling sites 

with collection dates and latitude/longitude values for each site appears in table 1. A map showing 

sampling site locations is shown in figure 1. The sampling locations for this study were selected from a 

list of established sampling sites where water quality samples are routinely collected by Klamath Tribes 

Research Station field personnel (Walker and others, 2012).

Sample Collection 

AFA sample collection was opportunistic. AFA material was collected when and where a 

surface bloom was present. A plastic sieve with a 0.5 millimeter (mm mesh screen was used to lift the 

floating algal mat from the water to the shipboard (fig. 2). The material in the sieve was transferred to a 

gallon-size plastic zip-lock bag. The plastic bags containing AFA samples were placed in a cooler with 
4 

ice and shipped the day of collection using overnight shipping service to the laboratory in Reston, 

Virginia, where they were frozen. 

A separate sample of AFA was collected on August 30, 2011, to be used for laboratory studies. 

This sample of AFA was collected at the water surface near the mid-trench site located near Eagle 

Ridge (fig. 1). The algae was freeze-dried and stored at -10 degrees Celsius (o

C). 

Sample Preparation

Frozen AFA was thawed and refrozen 3 times. This was done to maximize the lysing of the 

AFA cells, which breaks cell walls to expose cell contents for chemical analysis. After the third freezethaw cycle the AFA was transferred to glass jar(s) and refrozen. The frozen AFA was lyophilized. The 

dry AFA in screw cap glass jars was stored at -10 o

C. 

Sample Analysis 

31P Nuclear Magnetic Resonance (NMR) Analysis

Aliquots of the AFA were extracted with three different extraction solutions for 31P NMR 

analysis. One-tenth gram (g) of freeze-dried AFA was extracted with the following solutions:

1. 20 milliliters (mL) of 0.25 molar (M) NaOH plus 0.05 M EDTA. An internal standard is added 

before lyophilization Internal standards in NMR are used to quantify peaks in spectra of 

unknowns. An internal standard and the P-containing compounds in the sample will respond 

similarly to variations in the working conditions of the instrument. The concentration of the 

standard is known and this concentration is divided by the area of the peak produced by the 

internal standard to give a concentration per unit peak area. This value is used to quantify the 

peaks of the unknown. The peak of the internal standard should not have the same chemical shift 

as any peaks that appear in the spectrum of the unknown. 

2. 20 mL of 18 megohm water, and 

3. 20 mL of 18 megohm water (Internal standard added before lyophilization). 

Extraction with 0.25 M NaOH plus 0.05 M EDTA is the typical extraction solution for the 

preparation of 31P NMR samples because this solution extracts a maximum of P-containing compounds 

in biological samples (Cade-Menun and Preston, 1996). 

A comparison with water was chosen to demonstrate the compounds of P that are released to the 

water column when AFA cells die and lyse adding cell contents to surrounding water. Because 

phosphonate compounds of P have been found in cyanobacteria samples, extractions were prepared 

without and with the internal standard, which is a phosphonate compound. Absence of internal standard 

avoided overlap of the peak from the internal standard with a peak from phosphonate in the sample. The 

spectrum of the sample to which internal standard was added could be quantified by using the ratio of 

known concentration of the internal standard to its peak area. 

At the end of a 16 hour extraction period, the samples were centrifuged at 2,500 revolutions per 

minute (rpm) for 20 minutes (min). The supernatant was pipetted to a glass jar. An internal standard of 

methylene diphosphonic acid (50 micrograms (μg) P) was added to the supernatant from extraction 

steps (1) and (3). The contents of the jar were frozen, and lyophilized.

The dried extracts from treated samples were transferred to 2-cubic centimeters (cc) centrifuge 

tubes in the Reston laboratory. At the James Madison University Shenandoah Regional NMR Facility 

located at James Madison University, the material in the centrifuge tube was dissolved with a 

combination of 0.675 mL of 0.25 M NaOH–0.05 M EDTA and 0.2-mL of 10 M NaOH in D2O (99.9
5 

percent deuterium oxide, Sigma-Aldrich) immediately before analysis. The solution was mixed for ∼2 

min by using a vortex shaker, centrifuged and pipetted into 5 mm NMR tubes. The 31P NMR 

measurements were carried out on a Bruker Avance, 600 MHz (megahertz) spectrometer, equipped with 

a 5-mm broad band tunable probe operating at 242.9 MHz frequency for 31P. The proton decoupled 

spectra were acquired with a 30° pulse width (3.25 microseconds (μs)), acquisition time 1.35 second (s), 

relaxation delay 1 s, and sweep width 24,350 Hz (100 ppm). The sample temperature was maintained at 

293 °K (Kelvin) throughout the acquisition of 8,000 to 25,600 pulses, as required to achieve acceptable

signal to noise ratio. The 64-K (64 kilobytes) data point file was processed with 3 Hz line broadening 

and transformed to produce a 32-K data point spectrum. The chemical shifts were indirectly referenced 

to external 85% phosphoric acid (H3PO4) (at δ=0.; at chemical shift=0) by way of the lock signal. 

Spectral processing was performed with Bruker-BioSpin’s TopSpin 1.3 software.

Peak assignments in the 31P spectra that were obtained were made by using chemical shift data 

reported by Koopmans and others (2003); Turner et al. (2003); Cade-Menun (2005); and from 31P NMR 

experiments in the James Madison University laboratory. Chemical shift data are sensitive to solution 

pH. The relative separation of chemical shifts for P-containing compounds is more reproducible than the 

absolute values for chemical shifts for P-containing compounds. To compare NMR data from multiple 

studies, orthophosphate values are normalized to a chosen value and the chemical shifts for compounds 

flanking orthophosphate are calculated based on the arithmetic difference between the chemical shift 

value obtained for orthophosphate in the reported study and the chemical shift reported for 

orthophosphate in studies described in the literature. 

Carbon and Nitrogen Analysis

Algal samples were analyzed for total carbon (C) and total nitrogen (N) by using 20 milligrams 

(mg) freeze-dried AFA. Flash oxidation of each sample was followed by separation of the gaseous 

products using a Carlo Erba EA 1108 Elemental Analyzer. 

Total Elemental Analysis 

Samples weighing 0.07 to 0.1 g were digested in concentrated nitric and hydrofluoric acids by 

using microwave digestion equipment (Simon and others, 2009). Hydrofluoric acid was used in addition 

to nitric acid because it is possible that diatoms were included in the whole mat sample. An acid blank 

and reference standard Community Bureau of Reference BCR® - 60 certified Reference Material 

(aquatic plant, Lagarosiphon major) (European Commission, 2007) were included with each set of 

sediment digests. Ten milliliters (mL) of concentrated nitric acid were added approximately 16 hours 

before 2 mL of hydrofluoric acid were added to the Teflon vessels containing the weighed AFA 

samples. Soaking the samples in nitric acid helped wet the samples. The penetration of sample particles 

with acid contributes to efficient dissolution of sample particles. Immediately after addition of 

hydrofluoric acid, the vessels were closed, placed in the instrument round table, and placed in a CEM 

Microwave Accelerated Reaction System (MARS). After the digestion sequence in the microwave, the 

samples were brought up to a volume of 100 mL using double deionized water. 

Diluted aliquots of microwave digests of AFA samples were analyzed for P concentrations by 

using the method of Murphy and Riley (1962). Microwave digests were diluted 1:20 with 0.5 percent 

nitric acid and analyzed for Al, Ca, Fe, Mg, Ti, and Zn by using a Perkin-Elmer Model 5100 Inductively 

Coupled Plasma-Atomic Emission spectrometer ICP–AES. 

Data from a set of microwave digestion samples were considered acceptable if the data for a 

sample of the aquatic plant, Lagarosiphon major BCR® 60, certified Reference Material (European 
6 

Commission, 2007) analyzed with the set of sediment samples were within two standard deviations of 

the certified values reported for the standard. 

Conversion of AFA Protein to Amino acids and Inactivation of Microcystin 

Aliquots from one large sample of freeze-dried AFA were used for all experiments. The AFA 

was collected from Upper Klamath Lake, Oregon, on August 30, 2011, and shipped to the laboratory in 

Reston, Va. The bulk AFA was frozen and lyophilized upon arrival at the laboratory. This material was 

stored in a glass jar at -10 o

C. All experiments combined 0.1 g of freeze dried AFA with 20 mL of 

water. When testing the effect of experimental conditions on concentrations of microcystin, 1 

microgram (μg) of microcystin in 1 mL methanol was added to AFA slurries. 

Bromelain is a protease enzyme that will break down large protein molecules to short chain 

polypeptides and amino acids. Microcystin is a polypeptide and AFA contains approximately 60 percent 

protein. This study examined the efficiency of bromelain in degrading the proteins in AFA and breaking 

down the microcystin molecule. Cysteine was added to increase the activity of bromelain. In these 

experiments 0.1 g of bromelain and 0.1 g of cysteine were added to slurries of AFA when studying the 

effects of enzyme digestion on the concentration of microcystin in the mixture. Digestions were run in a 

rotating flask in a water bath at 60o

C for a period of 1 hour. At the end of the digestions, the sample in 

the flask was transferred to test tubes and centrifuged for 20 minutes at 2,500 rpm. The supernatant was 

freeze dried and extracted with 5 mL of methanol. 

Analysis for Microcystin in Extracts of AFA 

The method used for the detection of microcystin is based on the method published by Lo and 

others (2002). This method is an electrochemical technique that can be used for detecting and 

quantifying analytes in complex environmental samples because the detector that applies 300 millivolts 

(mV) in this analysis is ‘blind’ to most compounds in environmental samples which require larger 

potentials for oxidation. 

This method uses the reaction between α, β−unsaturated carbonyl functional groups and 

6-ferrocentylhexanthiol. All homologs of microcystin contain a α, β−unsaturated carbonyl functional 

group so this method is sensitive to all homologs of microcystin present in a sample. The published 

method (Lo and others, 2002) was developed for use with solutions of microcystin. In this study the 

method was expanded for use with methanol extracts of protease digests of AFA.

Chemicals used in the study experiments were American Chemical Society (ACS) grade with 

the following exceptions, bromelain, microcystin-LR and 6-ferrocentylhexanthiol (Sigma Chemicals). 

High purity methanol was required. Other sources of methanol contained electroactive species that 

produced a high background in the solvent and interfering peaks in the chromatograms when samples 

dissolved in methanol were injected.

Instrumentation: A Bioanalytical Systems liquid chromatograph with an electrochemical 

detector was operated at 300 mV. Initially, chromatographs were obtained by using a trichloroacetic 

acid eluent at a pH of 2. Because the electrochemical reaction in this method is oxidation, the buffer was 

changed to ammonium acetate at a pH of 5. Concentrations of buffer ranging from 0.05 to 0.2 were 

tested and ratios of methanol concentrations of 35 to 50 percent were compared. The most effective 

buffer for separating the components of the samples was a 1:1 ratio of 0.05 M ammonium acetate buffer 

at a pH of 5.0 and methanol.

Several chromatographic columns were tested. A low-flow rate is required for a good separation 

of the peaks produced when AFA extract samples are injected onto the column. An Agilent Zorbax 

Poroshell 120 SB-C18 column (2.1x30 mm, 2.7-micron particles) gave the best separation of 
7 

microcystin in AFA extracts within a one-half hour time period when the eluent flow is 0.2 cc per min 

and the column was heated to 70 o

C. 

Instrument working conditions

Electrode: 300 mV

Eluent: 50% 0.05 M ammonium acetate pH 5.0: 50% methanol

Flow: 0.2 mL per minute

Column heater: 70o C 

Injection loop: 20 microliters (μL) 

Coomassie Analysis for Protein Concentration in Digests of AFA

The method published by Bradford (1976) was used to determine the percent change of proteins 

in AFA to polypeptides and amino acids during protease digestion. The Coomassie Brilliant Blue dye 

has the formula C47H48N3NaO7S2 and formula weight 854.02. When mixed with a protein solution, an 

acidic Coomassie-dye reagent changes color from brown to blue in proportion to the amount of protein 

present in the sample. Protein determinations are made by comparison of the color response of the 

sample at wavelengths 610 (nanometer) nm and 595 nm. The smaller the difference of the absorbance of 

the sample at the two wavelengths, the smaller the concentration of protein in the sample. The 

Coomassie blue dye reagent is made by diluting 10 mg Coomassie Brilliant Blue dye in 5 mL of ethanol 

plus10 mL of phosphoric acid to 100 mL with water. To 5 mL of this solution, 100 μL of AFA digest 

was added. The AFA digests were 0.1 g samples of AFA in 20 mL of water that were (a) not heated, (b) 

heated to 60o C for 1 hour, or (c) heated to 60 o

C for 1 hour with 0.1g cysteine and 0.1g bromelain

Results and Discussion

31P NMR Analysis

Nuclear magnetic resonance (NMR) is a form of absorption spectrometry similar to infrared or 

ultraviolet spectrometry ( Silverstein and others, 1974). Like other methods of spectrometry, NMR 

measures the interaction between molecules with energies from the electromagnetic spectrum. Under 

appropriate conditions in NMR, a sample can absorb electromagnetic radiation in the radio-frequency 

region at frequencies governed by the characteristics of the sample. Chemical shift is the difference in 

the frequency of absorption of a reference proton and the frequency of absorption of a proton in the 

compound being analyzed. The applied frequency used in the analysis must be given as hertz, if the 

chemical shift is not calculated by dividing the frequency at which the peak from the compound 

appeared, by the applied frequency, and multiplying by 106

. The units of chemical shift from this 

calculation are δ (dimensionless) or parts per million (ppm). Energy absorption is a function of certain 

nuclei in the molecule. A plot of the frequencies of the absorption peaks as compared to peak intensities 

constitutes an NMR spectrum. (Silverstein and others, 1974). Chemical shift data for multiple 31Pcontaining functional groups are listed in table 2. An ideal spectrum of P-containing compounds is 

shown in figure 3. 

The 31P data in table 3 are from the samples collected for this study. The table includes chemical 

shift (CS, position of peaks in the NMR spectrum) data based on normalization of other peaks with the 

peak for orthophosphate in each spectrum, and peak area and concentration calculations based on an 

internal standard. The ratios of concentrations of inorganic and organic P in all NMR spectra averaged 

3:2. Slightly smaller concentrations of organic P were extracted by water than were extracted by 0.25 M 

NaOH plus 0.05 M EDTA. 
8 

All of the spectra for the AFA samples collected for this study are presented in figure 4. A 

comparison of these spectra with the descriptive 31P NMR spectrum presented in figure. 3 (CadeMenun, 2005) indicates that a suite of organic P compounds are present in AFA cells. The one organic P 

compound that was observed in only two (Howard Bay and Eagle Ridge) of the nine AFA samples was 

polyphosphate. Biological organisms use polyphosphates to store surplus phosphate. No phosphonate 

was observed in samples that did not contain the internal standard used in our studies. The largest 

inorganic peaks were orthophosphate monoesters, which include phosphate sugars (glucose-phosphate), 

phospholipids, and phytic acid. Orthophosphate diesters include deoxyribonucleic acid (DNA) and 

ribonucleic acid (RNA). 

Included in figure 4 are three 31P NMR spectra for each AFA sample. The first extraction was 

done by using 0.25 M NaOH plus 0.05 M EDTA, which extracts a maximum of the P-containing 

compounds in biological samples. This is the typical extraction solution for the preparation of 31P 

samples. The samples contain the internal standard methylene diphosphonic acid (MDPA) and the 

chemical shifts (peak position) for peaks in these spectra are directly comparable with spectra from 

other laboratories. 31P NMR data for algae in the literature are dominated by studies of marine 

organisms. A limited amount of data exists for freshwater algae. The few studies of freshwater 

cyanophytes that have been published focus on a limited number of P-containing compounds. Selig and 

others (2002), for example, used 31P NMR to study polyphosphates and phospholipids in the cyanophyte 

algae in Lake Bützow (Germany). Selig and others interpreted the presence of polyphosphate in the 

algae as an indicator that there was adequate phosphate for algal growth. In Upper Klamath Lake, 

polyphosphates were found only in samples from the Howard Bay and Eagle Ridge sampling sites. 

The second and third extractions were done by using deionized water. The same internal 

standard (MDPA) that was added to the 0.25 M NaOH plus 0.05 M EDTA extraction was added to one 

of the two water extractions; no internal standard was added to the second water extraction. Using water 

as an extractant indicated which P-containing compounds would be released to the water column when 

the AFA senesced and cells lysed. In general, the pattern in spectra from 0.25 M NaOH plus 0.05 M 

EDTA extractions matched the spectra from the water extractions for each sample. The single exception 

was for the P compound, pyrophosphate, which did not appear in spectra of water extracts. 

Pyrophosphates are generally considered stable in water solution. Polyphosphates are not stable in 

0.25 M NaOH plus 0.05 M EDTA extraction solutions of environmental samples (Hupfer, 1995) and the 

pyrophosphate in the 0.25 M NaOH plus 0.05 M EDTA extractions might have formed by the 

degradation of polyphosphates in the AFA samples. 

31P NMR analyses of the samples indicated that the largest variation in organic P compounds 

were determined in a sample collected from Howard Bay compared with samples collected the sites at 

Pelican Marina, Buck Island, Shoalwater Bay, and Agency Lake South. All P-containing compounds 

that appear in the ideal 31P NMR spectrum (fig. 3) also appear in spectrum 13, Howard Bay (fig. 5). The 

phosphonate shown in spectrum 13 is the internal standard. The AFA in UKL is a rich source of 

inorganic and organic P-containing compounds. 

When the AFA population senesces and the cells lyse, inorganic and organic P are released to 

the water column. The form of P preferentially used as a nutrient by AFA is inorganic P, that is, 

orthophosphate. Microcystis is able to use inorganic and organic P. The enrichment of the water column 

with inorganic P and organic P could favor the development of populations of microcystis (Vahtera and 

others, 2007; Shi and others, 2011; Eldridge and others, 2012). This might help to explain the pattern of 

algal species development in UKL. After the senescence of the first large AFA bloom of the summer, 

numbers of microcystis cells increase resulting in microcystis developing into a substantial percentage 

of water column phytoplankton (Eldridge and others, 2012). Possibly the enrichment of the water 
9 

column with inorganic and organic P when an AFA bloom dies and the availability of organic P for 

microcystis growth are reasons for the increased numbers of microcystis cells in the water column of 

UKL. 

Carbon and Nitrogen Analyses

Chemical data for AFA collected in July–August 2010 from Upper Klamath Lake, Oregon, are 

presented in table 4. The average concentration of carbon in the AFA samples analyzed in this study 

was 44 percent. The average concentration of nitrogen in the AFA samples analyzed in this study was 

9.5 percent. Multiplying by the factor 6.25 (protein is 16 percent nitrogen), gives an average 

concentration of protein in our AFA samples of 59 percent. The organic P content of AFA is directly 

and significantly related to the total carbon content of AFA (fig. 6). 

The carbon to nitrogen ratio in all AFA samples that were analyzed was 5.4 plus or minus 0.04 

compared with the Redfield ratio of carbon to nitrogen, which is 6.6. This difference could be related to 

the large concentration of protein in AFA or to an optimal growth rate of AFA. Benthic algae (including 

cyanophytes) have been reported to produce C:N ratios that decreased with increasing growth rate 

(Hillebrand and Sommer, 1999). 

Total Elemental Analysis

Chemical data for AFA collected in July–August 2010 from Upper Klamath Lake, Oregon, are 

presented in table 4. Total concentrations of P, Al, Ca, Fe, Mg, P, Ti and Zn were similar in all AFA 

samples analyzed in this study. The one exception was the Fe concentrations in the Pelican Marina 

samples collected on July 27, 2010. Iron concentrations were approximately 6 to10 times larger than in 

any other AFA samples including the sample from Pelican Marina collected on August 9, 2010. 

Titanium analyses were included in the suite of metals to identify samples that might have suspended 

sediment included in the algal mat. Inclusion of suspended sediment is not indicated for any of the 

samples. 

Conversion of AFA Protein to Amino Acids and Inactivation of Microcystin 

Two potential uses for AFA were considered and dismissed because results were not promising. 

The oil content of AFA is small, approximately 3 percent. This portends a low return for development 

of a biofuel. Attempts to ferment AFA in the laboratory were unsuccessful. The use of AFA for ethanol 

production was set aside. The use of AFA as a food stock was more promising and experiments to 

determine the usefulness of AFA as a protein or amino acid source were run. 

Cyanophytes contain L-amino acids and D-amino acids, which sets them apart from most 

biological organisms, which contain only L-amino acids (Lam and others, 2009). We did not investigate 

the possibility that AFA might be a commercial source of D-amino acids.

AFA cells contain more than 50 percent protein. Using study data (table 4) for the average 

percent nitrogen in samples, the average protein content in AFA samples is calculated to be 59 percent. 

Based on the large concentration of protein in AFA, we decided to examine the use of AFA for animal 

feed. 

Because microcystis, a source of the toxin microcystin, is often found growing with AFA in 

UKL, there is a persistent concern that microcystin could be present in AFA harvested from the lake. 

Our approach to determining a method to remove microcystis from AFA biomass was to consider only 

procedures that would make the AFA end-product suitable for use as a feed stock. The toxicity of 
10 

microcystin is attributed to several structural features of the microcystin molecule (fig. 7) as listed 

below:

1. Microcystin inactivates phosphatase enzymes. It does this when the α, β−unsaturated carbonyl 

functional group of microcystin binds with sulfur-containing amino acids in phosphatase 

molecules (Billam, 2006). 

2. The Adda group, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, in the 

microcystin molecule is considered toxic. 

3. The cyclic structure of the microcystin molecule is reported to contribute to the toxicity of the 

molecule.

Several treatments of microcystin in slurry of AFA were tested in an effort to minimize the 

toxicity of the microcystin molecule. These treatments include the following: 

1. Complexation of the active functional group, α, β−unsaturated carbonyl, by a compound 

containing a sulfydryl group was done to inactivate the α, β−unsaturated carbonyl in 

microcystin. Cysteine has been reported to detoxify microcystin (Zhang-Qiang, 2012).

2. The toxicity of the Adda group is linked with the loss of toxicity of the microcystin molecule 

when that Adda functional group is destroyed by using very strong oxidizing agents such as 

chlorine gas or ozone. When the Adda group is destroyed by using these strong oxidizers, the α,

β−unsaturated carbonyl functional group also will be destroyed. Publications have not reported 

using a procedure where the Adda group was destroyed while leaving the α, β−unsaturated 

carbonyl functional group unaffected. Also, one study indicated that toxicity was not connected 

with the Adda group (Harada and others, 2004).

3. The methods that have success in degrading microcystin have generally been complex or 

expensive or both. Microcystin is a peptide. In the literature, reports of proteases being been 

used to decompose microcystin, animal proteases were used because the question being 

addressed was whether microcystin could be broken down in the digestive tract of an animal

(Rohrlack and others, 2004). In published reports (Bourne and others, 1996) the animal proteases 

used in the studies have had limited effectiveness in breaking down the microcystin molecule. In 

this study, plant proteases were tested. Plant proteases (including bromelain and papain, which 

were activated with cysteine) were tested and both were determined to be effective in breaking 

down proteins in AFA. Bromelain was selected for detailed work because it is particularly 

effective in breaking arginine peptide bonds (Chloe and others, 2006). Arginine is one of the Lamino acids in the cyclic ring structure of microcystin. 

In this study, the activity of microcystin is considered to be related to active α, β−unsaturated 

carbonyl functional groups in the microcystin molecule. Deactivation of the α, β−unsaturated carbonyl 

destroys the toxicity of the microcystin molecule. Using experimental conditions in this study 

(bromelain plus cysteine), the average loss of active microcystin at a concentration of 1 μg of 

microcystin in a water slurry of AFA was approximately 60 percent (table 5). Preliminary experimental 

results indicate that cysteine plays a role in the inactivation of microcystin. Whereas the average 

inactivation of microcystin in solution is approximately 60 percent using bromelain or cysteine (table 5), 

cysteine alone inactivated approximately 30 percent of 1 μg of microcystin in an AFA slurry (data not 

shown).

A common analytical procedure for the analysis of microcystin is liquid-chromatography massspectroscopy. Liquid-chromatography mass-spectroscopy instrumentation was not available in the 

Reston, Va. Biogeochemistry Lab. This complication, coupled with the need for instrumentation that
11 

could be used immediately after a digestion was completed, dictated that a sensitive analytical technique 

be available in our laboratory. The electrochemical technique was chosen because the derivatization step 

provides an electroactive form of the analyte detectable at small concentrations and employs a low 

voltage to the electrochemical cell making the detector ‘blind’ to compounds in complex extraction 

solutions that could complicate a chromatogram. 

Coomassie Analysis for Protein Concentration in Digests of AFA

Using the Coomassie protein analysis, protein determinations were made for water slurries of 

AFA, and for water slurries that had been heated, and with water slurries that were heated with 

bromelain and cysteine. Compared with the AFA sample that was not heated; the AFA sample that was 

heated lost 15 percent of the protein in the sample, and the AFA sample that was heated with cysteine 

and bromelain lost 99 percent of the protein in the sample.

Summary

The Aphanizomenon flos-aquae (AFA) bloom in 2010 started to develop in the middle of July. 

The chemical data from all AFA samples collected from Upper Klamath Lake, Oregon, in 2010 were 

similar in composition spatially and temporally. The lack in temporal variability might reflect the fact 

that all of the samples collected for this study were collected between the end of July and the middle of 

August. 

The carbon to nitrogen ratio in all AFA samples that were analyzed was 5.4 + 0.04 as compared 

with the Redfield ratio of 6.6 for carbon to nitrogen ratio, which could be related to the large 

concentrations of proteins in AFA or to optimal growth rate for AFA. 31P Nuclear Magnetic Resonance data indicated that both inorganic and organic phosphorus were 

present in AFA samples that were collected in August and September 2010, at an approximate average 

ratio of 3:2. This was true for all nine samples collected at different sites on dates in August and 

September 2010. The ratio of inorganic to organic phosphorus in AFA might be related to the pattern of 

algae growth in Upper Klamath Lake when a substantial growth of microcystis follows the senescence 

of a large population of AFA. AFA favors inorganic phosphorus as a nutrient, whereas microcystin can 

readily breakdown organic phosphorus as well as use inorganic phosphorus. The phosphorus contents of 

lysed AFA cells might favor the growth of microcystis. 

The experimental conditions used in this study indicated that the average deactivation of 1 

microgram of microcystin in water slurries of AFA was approximately 60 percent. The experimental 

conditions include a combination of the protease bromelain and the amino acid cysteine. 

Laboratory studies to examine the possibility of using a plant protease and sulfhydryl-containing 

compound to (a) detoxify microcystin that might be present in AFA collected from the water column, 

and (b) break down proteins in AFA to short chain peptides and amino acids, indicate that both goals are 

achievable. 

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15 

Figure 1. Map of Upper Klamath Lake, Oregon, showing sampling sites for Aphanizomenon flos-aquae (AFA) 

collected in July and August, 2010. Sites include: Pelican Marina (PM), Pelican Marina North (PMN), North Buck 

Island (NBI), Howard Bay (HB), Eagle Ridge (ER), Eagle Ridge South (ERS), Shoalwater Bay (SB) and Agency 

Lake South (AS). Also shown is Mid-Trench (MDT) where AFA was collected in September, 2011, for use in 

laboratory experiments.
16 

Figure 2. Aphanizomenon flos-aquae (AFA) samples were collected using a plastic sieve with a 0.5 millimeter 

screen. Image is of scant AFA in surface water at North Buck Island on June 29, 2010. There was insufficient AFA 

to collect a sample.
17 

Figure 3. An ideal spectrum of phosphorus-containing compounds. (Figure from Cade-Menum, 2005). The peak 

at a chemical shift of 7 is orthophosphate. Organic phosphorus compounds range from phosphonates at a 

chemical shift of 18 to polyphosphates at a chemical shift of -20. 
18 

1a 31P NMR 

PM - Pelican Marina, AFA collected 7/27/2010, extracted with 0.25M NaOH plus 0.05M EDTA.

MDPA added NMR sample in 10M NaOH in D2

0.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide] 
19 

1b. 31P NMR

PM - Pelican Marina, AFA collected 7/27/2010, extracted with water.

No MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
20 

1c. 31P NMR

PM - Pelican Marina, AFA collected 7/27/2010, extracted with water.

MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
21 

2a. 31P NMR

PMN - Pelican Marina North, AFA collected 7/27/2010, extracted with 0.25M NaOH.

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
22 

2b. 31P NMR.

PMN - Pelican Marina North, AFA collected 7/27/2010, extracted with water only.

No MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
23 

2c. 31P NMR.

PMN - Pelican Marina North, AFA collected 7/27/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
24 

3a. 31P NMR

PM - Pelican Marina, AFA collected 8/9/2010, extracted with 0.25M NaOH 

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
25 

3b. 31P NMR

PM - Pelican Marina, AFA collected 8/9/2010, extracted with water.

No MDPA added. NMR sample in 10M NaOH in D2

O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
26 

3c. 31P NMR

PM - Pelican Marina, AFA collected 8/9/2010, extracted with water.

MDPA added. NMR sample in 10M NaOH in D2O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
27 

4a. 31P NMR

NBI - North Buck Island, AFA collected 7/27/2010, extracted with 0.25M NaOH.

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
28 

4b. 31P NMR

NBI - North Buck Island, AFA collected 7/27/2010, extracted with water.

No MDPA added. NMR sample in 10M NaOH in D2O. On the X axis are chemical shift values.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
29 

4c. 31P NMR

NBI - North Buck Island, AFA collected 7/27/2010, extracted with water.

MDPA added. NMR sample in 10M NaOH in D2O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
30 

5a. 31P NMR

HB - Howard Bay AFA collected 8/9/2010, extracted with 0.25M NaOH.

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response. Bold values in black are peak areas calculated by

 the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
31 

5b. 31P NMR

HB - Howard Bay AFA collected 8/9/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2O.

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response.

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
32 

5c. 31P NMR

HB - Howard Bay AFA collected 8/9/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
33 

6a. 31P NMR

ER - Eagle Ridge, AFA collected 8/9/2010, extracted with 0.25M NaOH.

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green.

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
34 

6b. 31P NMR

ER - Eagle Ridge, AFA collected 8/9/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
35 

6c. 31P NMR

ER - Eagle Ridge, AFA collected 8/9/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
36 

7a. 31P NMR

ERS - Eagle Ridge South, AFA collected 8/12/2010, extracted with 0.25M NaOH. 

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
37 

7b. 31P NMR

ERS - Eagle Ridge South, AFA collected 8/12/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
38 

7c. 31P NMR

ERS - Eagle Ridge South, AFA collected 8/12/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
39 

8a. 31P NMR

SB - Shoalwater Bay, AFA collected 8/9/2010, extracted with 0.25M NaOH. 

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
40 

8b. 31P NMR

SB - Shoalwater Bay, AFA collected 8/9/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
41 

8c. 31P NMR

SB -Shoalwater Bay, AFA collected 8/9/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
42 

9a. 31P NMR

AS - Agency Lake South Blue, AFA collected 8/9/2010, extracted with 0.25M NaOH. 

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
43 

9b. 31P NMR

AS - Agency Lake South Blue, AFA collected 8/9/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
44 

9c. 31P NMR

AS - Agency Lake South Blue, AFA collected 8/9/2010, extracted with water. 

MDPA added. NMR sample in 10M NaOH in D2

O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
45 

10a. 31P NMR

AS - Agency Lake South Light green, AFA collected 8/9/2010, extracted with 0.25M NaOH. 

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2

O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software. 

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
46 

10b. 31P NMR

AS - Agency Lake South, Light green, AFA collected 8/9/2010, extracted with water. 

No MDPA added. NMR sample in 10M NaOH in D2

O. 

Chemical shift values are on the X axis. Chemical shifts of individual peaks are in green. 

The Y axis indicates the intensity of response. 

Bold values in black are peak areas calculated by the TopSpin software.

Figure 4. 31P Nuclear Magnetic Resonance data for Aphanizomenon flos-aquae (AFA) samples collected in July 

and August 2010 from Upper Klamath Lake. [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; 

AFA, cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; 

EDTA, ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide]—Continued
47 

 31P NMR

HB - Howard Bay AFA collected 8/9/2010, extracted with 0.25M NaOH.

plus 0.05M EDTA. MDPA added. NMR sample in 10M NaOH in D2

O. 

Chemical shift values are on the X axis. The Y axis indicates the intensity of response. 

Values in green are peak areas calculated by the TopSpin software.

Figure 5. Spectrum 5a - 31P Nuclear Magnetic Resonance spectrum of sample collected from Howard Bay (HB) 

in Upper Klamath Lake (UKL) on August 9, 2010. Peak at chemical shift (CS) 17.8 is the internal standard 

methylene diphosphonic acid (MDPA). [P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy; AFA, 

cyanophyte, Aphanizomenon flos-aquae; M, molarity, moles per liter of solution; NaOH, sodium hydroxide; EDTA, 

ethylenediaminetetraacetic acid; MDPA, methylene diphosphonic acid; D2

0, deuterium oxide] 
48 

♦

♦

♦

♦ ♦♦♦

4

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

43 43.2 43.4 43.6 43.8 44 44.2 44.4 44.6

Ratio of organic P/Total P X 10

Percent organic carbon

f(x) = 5.662E-1*x + -

2.044E+1

R^2 = 9.263E-1

AFA

Figure 6. Ratio of concentration of organic phosphorus to the concentration of 

total phosphorus (TP) plotted against the percent carbon in each Aphanizomenon 

flos-aquae (AFA) sample, R2 = 0.93. [P, phosphorus]
49 

Figure 7. The seven peptide areas of the microcystin molecule include (1) D-Alanine, (2) L-Leucine, 

(3) D-erythro-p-methyl Asparine, (4) L-Arginine, (5) Adda, 3-amino-9-methoxy2,6,8-trimethyl-10-phenyl-4,6-

decadienoic acid, (6) D-Glutamic Acid, and (7) Mdha, N-methyldehydro Alanine. Mdha contains an alpha-beta 

unsaturated carbonyl functional group. The X and Z amino acids vary in the homologs of microcystin but are 

L-leucine and L-arginine in microcystin-LR, the most common form of microcystin.

Illustration from Fewer (2007; available at http://www.biomedcentral.com/content/pdf/1471-2148-7-

183.pdf). 
50 

Table 1. Sampling sites for Aphanizomenon flos-aquae collected in July and August 2012 from Upper Klamath Lake, Oregon.

Sample

Date of 

collection Site Coordinates Decimal degrees

1 7/27/2010 PM- Pelican Marina N 42° 14' 16.892 W 121° 48' 37.341 N 42.24156° W121.82341°

2 7/27/2010 PMN - Pelican Marina North N 42° 14' 16.892 W 121° 48' 37.341 N 42.24156° W121.82341°

3 8/9/2010 PM - Pelican Marina N 42° 14' 16.892 W 121° 48' 37.341 N 42.24156° W121.82341°

4 7/27/2010 NBI - North Buck Island N 42° 18' 30.2 W 121° 51' 22.4 N 42.30823° W121.85733°

5 8/9/2010 HB - Howard Bay N 42° 19' 35.0 W 121° 55' 11.9 N 42.32623° W121.92108°

6 8/9/2010 ER - Eagle Ridge N 42° 25' 19.5 W 121° 56' 35.8 N 42.42192° W121.94439°

7 8/12/2010 ERS - Eagle Ridge South N 42° 25' 19.5 W 121° 56' 35.8 N 42.42192° W121.94439°

8 8/9/2010 Shoalwater Bay N 42° 24' 26.1 W 121° 57' 47.1 N 42.40709° W121.96420°

9a 8/9/2010 AS - Agency Lake South (Blue color) N 42° 31' 24.9 W121° 59' 03.4 N 42.52342° W121.98539°

9b 8/9/2010 AS - Agency Lake South ( Green color) 
51 

Table 2. Chemical shift values for compounds that appear in 31P NMR spectra.

[P, phosphorus; NMR, Nuclear Magnetic Resonance spectroscopy] 

Compound

Chemical shift:

Literature values

Adjustment of chemical shift using 

difference between chemical shift of 

orthophosphate observed in this 

experiment and the chemical shift of 

orthophosphate published by other 

authors

phosphonate 

methylenediphosphonic acid sodium salt 18.1*** 18.1

orthophosphate monoesters 3 to 6* 4.1 to 7.1

adenosine 5 monophosphate 4.65 5.65

orthophosphate

5.7-6.1* 6.1** 

7.0 to 7.1***

7.0 

7.0 to 7.1

glucose-1- phosphate 3.2* 4.1

myo inositol hexakisphosphate (5.85, 4.9, 4.6, 4.4)* 6.9, 5.9, 5.6, 5.4

orthophosphate diesters 0.25 to (-1)* 1.15 to (-.1)

DNA from salmon (-0.5) average of two values** 0.4

RNA

0.68 and 0.56 (30 minutes after 

preparation) ** 0.77 to 0.65

pyrophosphate (-3.6 to -3.7)* (-4.3 to -4.4)** (-2.5 to -2.6) (-3.4 to -3.5)

inorganic polyphosphate (-19.2 to -20.5)*** (-19.2 to -20.5)

Koopmans and others, 2003.*** 

Turner and others, 2003.** 

Cade-Menum, 2005.* 
52 

Table 3. 31P NMR data for Aphanizomenon flos-aquDH samples collected July–August 2010 from Upper Klamath Lake, Oregon.

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

 

area 

of 

peak

conc of 

P mg g-1 

area 

of 

peak

conc of 

P mg g-1 

area of 

peak

conc of 

P mg g-1 

area 

of 

peak

conc of 

P mg g-1 

area 

of 

peak

conc of 

P mg g-1 

area 

of 

peak

conc of 

P mg g-1 % IP % OP

PM - 

Pelican 

Marina

PM - Pelican 

Marina 

7/27/10 

NaOHEDTA with 

MDPA 1a

NaOHEDTA + 0.11 5.66 2.58 5.81 2.65 0.47 0.22 5.45 0.47 0.53

7/27/2010

PM - Pelican 

Marina 

7/27/10 

Water only 1b Water 0.112

PM - Pelican 

Marina 

7/27/10 

Water plus 

MDPA 1c Water + 0.114 7.40 3.26 4.14 1.82 5.09 0.64 0.36

PMN - 

Pelican 

Marina 

North

PMN - 

Pelican 

Marina 

North 

7/27/10 

NaOHEDTA with 

MDPA 2a

NaOHEDTA + 0.113 6.11 2.71 4.65 2.06 0.41 0.18 4.95 0.55 0.45
53 

Table 3. 31P NMR data for Aphanizomenon flos-aquDH samples collected July–August 2010 from Upper Klamath Lake, Oregon.—Continued

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P 

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1 % IP % OP

7/27/2010

PMN - 

Pelican 

Marina 

North 

7/27/10 

Water only 2b Water 0.105

PMNPelican 

Marina 

North 

7/27/10 

Water plus 

MDPA 2c Water + 0.108 6.75 3.12 3.61 1.67 4.79 0.65 0.35

PM - 

Pelican 

Marina

PM - Pelican 

Bay Marina 

8/9/10 

NaOHEDTA with 

MDPA 3a

NaOHEDTA + 0.098 7.56 3.88 4.48 2.30 0.67 0.34 6.51 0.59 0.41

8/9/2010

PM - Pelican 

Marina 

8/9/10 

Water only 3b Water 0.101

PM - Pelican 

Bay Marina 

8/9/10 

Water plus 

MDPA 3c Water + 0.109 #### 4.71 2.71 1.24 5.95 0.79 0.21
54 

Table 3. 31P NMR data for Aphanizomenon flos-aquDH samples collected July–August 2010 from Upper Klamath Lake, Oregon.—Continued

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P 

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1 % IP % OP

NBI - 

North 

Buck 

Island 

NBI - North 

Buck Island 

7/27/10 

NaOHEDTA with 

MDPA 4a

NaOHEDTA + 0.103 5.93 2.87 5.30 2.56 0.25 0.12 5.55 0.52 0.48

7/27/2010

NBI - North 

Buck Island 

7/27/10 

Water only 4b Water 0.108

NBI - North 

Buck Island 

7/27/10 

Water plus 

MDPA 4c Water + 0.106 6.13 2.88 3.98 1.87 4.75 0.61 0.39

HB 

Howard

Bay 

HB - 

Howard Bay 

8/9/10 

NaOHEDTA with 

MDPA 5a

NaOHEDTA + 0.102 6.28 3.07 4.33 2.11 0.06 0.03 0.13 0.07 x 5.27 0.58 0.42

8/9/2010

HB - 

Howard Bay 

8/9/10 

Water only 5b Water 0.103

HB - 

Howard Bay 

8/9/10 

Water plus 

MDPA 5c Water + 0.104 7.58 3.65 3.66 1.76 0.06 0.03 0.10 0.05 x 5.49 0.67 0.33
55 

Table 3. 31P NMR data for Aphanizomenon flos-aquae samples collected July–August 2010 from Upper Klamath Lake, Oregon.—Continued

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P 

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1 % IP % OP

ER 

Eagle 

Ridge

ER - Eagle 

Ridge 

8/9/10 

NaOHEDTA with 

MDPA 6a

NaOHEDTA + 0.116 7.57 3.26 6.71 2.89 0.07 0.03 0.14 0.06 6.24 0.52 0.48

8/9/2010

ER - Eagle 

Ridge 

8/9/10 

Water only 6b Water 0.105

ER - Eagle 

Ridge 

8/9/10 

Water plus 

MDPA 6c Water + 0.105 8.63 4.12 4.56 2.18 x 6.30 0.65 0.35

ERS 

Eagle 

Ridge 

South

ERS- Eagle 

Ridge South 

8/12/10 

NaOHEDTA with 

MDPA 7a

NaOHEDTA + 0.107 8.53 3.98 6.28 2.93 6.91 0.58 0.42

8/12/2010

ERS - Eagle 

Ridge South 

8/12/10 

Water only 7b Water 0.108

ERS- Eagle 

Ridge South 

8/12/10 

Water plus 

MDPA 7c Water + 0.109 #### 4.62 4.51 2.08 6.70 0.69 0.31
56 

Table 3. 31P NMR data for Aphanizomenon flos-aquae samples collected July–August 2010 from Upper Klamath Lake, Oregon.—Continued

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P 

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1 % IP % OP

SB 

Shoalwate

r Bay 

SBShoalwater 

Bay 8/9/10 

NaOHEDTA with 

MDPA 8a

NaOHEDTA + 0.102 6.51 3.19 5.59 2.74 5.93 0.54 0.46

8/9/2010

SB - 

Shoalwater 

Bay 8/9/10 

Water only 8b Water 0.105

SB - 

Shoalwater 

Bay 8/9/10 

Water plus 

MDPA 8c Water + 0.102 7.97 3.89 4.93 2.40 6.29 0.62 0.38

AS 

Agency 

Lake 

South 

AS - Agency 

Lake South 

8/9/10 (blue 

color) 

NaOHEDTA with 

MDPA 9a

NaOHEDTA + 0.105 4.49 2.13 5.55 2.63 4.76 0.45 0.55

Blue 

AS - Agency 

Lake South 

8/9/10(blue 

color) 

Water only 9b Water 0.103 x
57 

Table 3. 31P NMR data for Aphanizomenon flos-aquae samples collected July–August 2010 from Upper Klamath Lake, Oregon.—Continued

[CS, chemical shift, location of peak in NMR spectrum; EDTA, ethylenediaminetetraacetic acid, metal complexing agent; Inorg., inorganic; IP, inorganic phosphorus; 

MDPA, methlyene diphosphonic acid, internal standard for 31P NMR analysis; Inter std, internal standard; mg g-1, milligrams per gram dry weight of sample; NaOH, 

sodium hydroxide; NMR, Nuclear Magnetic Resonance spectroscopy; OP, organic phosphorus; Org., organic; P, phosphorus; soln., solution; +, internal standard 

added; x, very small peak in spectrum] 

Site and 

date of 

collection

Label on 

spectrum

31P 

NMR 

spectrum

Extract 

soln.

Inter 

std

sample 

weight 

g

NMR 

CS 7

NMR CS 

7

NMR 

CS 

4-6

NMR CS 

4-6

NMRCS 

2

NMR CS 

2

NMR 

CS 0

NMR CS 

0

NMR 

CS

-4

NMR CS 

-4

NMR 

CS 

-18

Chemical 

analysis 

TP NMR NMR 

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1

area 

of 

peak

conc of 

P mg g-1 % IP % OP

8/9/2010

AS - Agency 

Lake South 

8/9/10(blue 

color) 

Water plus 

MDPA 9c Water + 0.101 5.45 2.70 4.48 2.22 x 4.92 0.55 0.45

AS 

Agency 

Lake 

South 

AS Agency 

Lake South 

8/9/10 (green 

color) 

NaOHEDTA with 

MDPA 10a

NaOHEDTA + 0.075 3.62 2.42 3.38 2.26 4.68 0.52 0.48

Green

AS Agency 

Lake South 

8/9/10 

(green color) 

Water only 10b Water 0.024

8/9/2010
58 

Table 4. Chemical data for Aphanizomenon flos-aquae collected in July and August 2012 from Upper Klamath 

Lake, Oregon.

[TP, total phosphorus; Ca, calcium; Mg, magnesium; Ti, titanium; Al, aluminum; Fe, iron; Zn, zinc; mg g-1, milligrams per 

gram; wt, weight; C, carbon; N, nitrogen; C/N, ratio of carbon to nitrogen; %, percent; PM, Pelican Marina; PMN, Pelican 

Marina North; NBI , North Buck IslandI; HB, Howard Bay; ER, Eagle Ridge; ERS, Eagle Ridge South; SB, Shoalwater Bay; 

AS, Agency Lake South; na, not available; nd, not detectable] 

Site and date of 

collection

Digest 

TP 

mg g-1 

dry wt

Digest 

Ca 

mg g-1

dry wt

Digest 

Mg

mg g-1 

dry wt

Digest 

Ti 

mg g-1

dry wt

Digest 

Al 

mg g-1 

dry wt

Digest 

Fe 

mg g-1 

dry wt

Digest 

Zn 

mg g-1 

dry wt %C %N C/N

PM - Pelican Marina 5.24 6.15 1.96 0.01 0.05 2.98 0.05 43.9 10.0 5.1

7/27/2010

PMN - Pelican 

Marina North 5.21 6.65 1.86 0.00 0.04 2.57 0.02 43.3 9.9 5.1

7/27/2010

PM - Pelican Marina 6.05 5.81 1.69 0.00 0.07 0.38 0.02 43.3 9.2 5.5

8/9/2010

NBI - North Buck 

Island 5.33 6.06 2.00 0.00 0.02 0.43 nd 44.6 10.4 5

7/27/2010

HB - Howard Bay 5.42 7.12 1.61 0.00 0.07 0.50 0.02 43.7 8.5 6

8/9/2010

ER - Eagle Ridge 5.88 5.67 1.80 0.00 0.02 0.38 0.02 43.9 10.0 5.1

8/9/2010

ERS - Eagle Ridge 

South 6.29 6.64 1.88 0.00 0.07 0.48 0.02 44.0 9.7 5.3

8/12/2010

SB - Shoalwater Bay 5.94 5.50 1.82 0.00 0.08 0.28 0.01 43.8 9.6 5.3

8/9/2010

AS - Agency Lake 

South 4.66 5.83 1.66 0.00 0.00 0.38 0.02 43.8 8.0 6.4

Blue color

8/9/2010

AS - Agency Lake 

South 4.53 6.28 1.87 0.00 0.02 0.43 0.02 na

Green color

8/9/2010
59 

Table 5. Degradation of microcystin in digest of Aphanizomenon flos-aquae. 

Date of Analysis

Calculated 

percent 

degradation

September 2, 2012 67

September 3, 2012 45

September 4, 2012 43

September 29, 2012 57

September 28, 2012 76

58 Average

14 Standard deviation 
ISSN 2331-1258 (online)

DOI URL http://dx.doi.org/10.3133/ofr20131156