U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2004-5217

Fractionation and Characterization of Organic Matter in 

Wastewater from a Swine Waste-Retention Basin
Cover. Photograph of swine waste-retention basin (photograph by Jason Smith, U.S. Geological Survey, Iowa District).
Fractionation and Characterization of 

Organic Matter in Wastewater from a 

Swine Waste-Retention Basin

By Jerry A. Leenheer and Colleen E. Rostad

Scientific Investigations Report 2004-5217

U.S. Department of the Interior

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

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia: 2004

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iii

Contents

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

Introduction ................................................................................................................................................... 1

Methods ......................................................................................................................................................... 2

Source ................................................................................................................................................... 2

Sample Collection ................................................................................................................................ 2

Organic Matter Fractionation ............................................................................................................ 2

Laboratory Equipment ......................................................................................................................... 2

Fourier Transform-Infrared (FT-IR) Spectrometry .......................................................................... 2

Solid State Cross Polarization Magic Angle Spinning (CPMAS) 13C-Nuclear Magnetic 

Resonance (NMR) Spectrometry ........................................................................................ 5

Fractionation of Organic Matter ................................................................................................................. 6

Compound-Class Spectral Analyses of Fractions .......................................................................... 9

Particulate Fractions .................................................................................................................. 9

Colloid Fractions ......................................................................................................................... 9

Dissolved Fractions .................................................................................................................. 10

Solvent-Extractable Fractions ............................................................................................... 12

Characterization of Organic Matter ......................................................................................................... 18

Summary and Conclusions ........................................................................................................................ 20

Acknowledgments ...................................................................................................................................... 20

References Cited ........................................................................................................................................ 20

Figures

1. Flow chart of total organic matter fractionation for north-side sample ............................. 3

2. Flow chart of solvent-extractable organic matter fractionation for south-side 

sample ........................................................................................................................................... 4

3. Fractionation of total organic matter of north-side sample .................................................. 6

4. Fractionation of particulate organic matter of north-side sample ...................................... 7

5. Fractionation of colloidal organic matter of north-side sample .......................................... 7

7. Fractionation of non-volatile dissolved organic matter in north-side sample ................... 8

6. Bar chart showing volatile and non-volatile dissolved organic matter of the 

north side sample ........................................................................................................................ 8

8. Organic solvent fractionation of south-side sample .............................................................. 9

9. Infrared spectra of particulate fractions from north-side sample .................................... 10

10. 13C-NMR spectra of particulate fractions from north-side sample. L= lipid, 

P = protein, C = carbohydrate .................................................................................................. 11

11. Comparison of infrared spectrum floc particulates extracted with methylene 

chloride with the infrared spectrum of peptidoglycan from Bacillus subtilis ................. 13

12. Infrared spectra of colloid fractions from north-side sample. ........................................... 13

13. 13C-NMR spectra of colloid fractions from north-side sample. .......................................... 14

14. Infrared spectra of dissolved fractions from north-side sample. CA = carboxylic 

acid, AI = amide 1 band, AII = amide 2 band. ........................................................................ 15
iv

15. 13C-NMR spectra of dissolved fractions from north-side sample. ..................................... 16

16. Infrared spectra of solvent-extractable fractions from south-side sample. ................... 17

17. 13C-NMR spectra of solvent-extractable fractions from south-side sample. .................. 17

Tables

1. Infrared frequency bands for various structures in DOM isolates ..................................... 5

2. Structural assignments for 13C-NMR spectra. ........................................................................ 5
Fractionation and Characterization of Organic Matter in 

Wastewater from a Swine Waste-Retention Basin

By Jerry A. Leenheer and Colleen E. Rostad

Abstract

Organic matter in wastewater sampled from a swine 

waste-retention basin in Iowa was fractionated into 14 fractions on the basis of size (particulate, colloid, and dissolved); 

volatility; polarity (hydrophobic, transphilic, hydrophilic); 

acid, base, neutral characteristics; and precipitate or flocculates (floc) formation upon acidification. The compound-class 

composition of each of these fractions was determined by 

infrared and 13C-NMR spectral analyses. Volatile acids were 

the largest fraction with acetic acid being the major component of this fraction. The second most abundant fraction was 

fine particulate organic matter that consisted of bacterial cells 

that were subfractionated into extractable lipids consisting 

of straight chain fatty acids, peptidoglycans components of 

bacterial cell walls, and protein globulin components of cellular plasma. The large lipid content of the particulate fraction 

indicates that non-polar contaminants, such as certain pharmaceuticals added to swine feed, likely associate with the particulate fraction through partitioning interactions. Hydrocinnamic 

acid is a major component of the hydrophobic acid fraction, 

and its presence is an indication of anaerobic degradation of 

lignin originally present in swine feed. This is the first study 

to combine particulate organic matter with dissolved organic 

matter fractionation into a total organic matter fractionation 

and characterization.

Introduction

In recent decades, animal production in the United States 

has shifted toward large confinement operations (United States 

General Accounting Office, 2003). This increase in large-scale 

operations has raised concerns of their potential environmental 

effects (Jongbloed and Lenis, 1998; Hanselman and others, 

2003). Many large-scale hog farms dispose of their waste in 

on-site retention basins. The waste-retention basins from these 

operations have become a topic of public concern due to their 

odors (Schiffman, 1998) and surface- and ground-water contamination (Campagnolo and others, 2002). Waste-retention 

basins are open-air pits dug into the earth to temporarily store 

and process the waste produced by the swine. These pits were 

designed to break down complex compounds so the waste 

could subsequently be applied to agricultural fields as a source 

of fertilizer to crops. Seepage from the waste-retention basins 

has become a major topic of study in itself (Raman and 

others, 2004, Chee-Sanford and others, 2001). These anaerobic lagoons contain chemical contaminants, pathogens, 

hormones, metals, bacteria, nitrates and parasites (Rubin, 

1998; Compagnolo and others, 2002; Raman and others, 2004; 

Kumar and others, 2004).

Although specific compounds have been studied in and 

nearby waste-retention basins, very few studies have characterized the overall organic material from these basins in detail. 

A comprehensive approach for dissolved organic matter fractionation and isolation was recently developed (Leenheer and 

others, 2000) that combines vacuum evaporation with dialysis 

to isolate colloidal organic matter, resin sorption to isolate dissolved hydrophobic and basic organic matter, and selective salt 

precipitations combined with vacuum evaporations to isolate 

dissolved hydrophilic organic matter. This dissolved organic 

matter isolation method has been applied to fresh surface 

water (Leenheer and others, 2000), and wastewater effluents 

and ground waters (Leenheer and others, 2001). However, it 

had not been applied to waste with substantial colloidal or 

solid material, such as that from a swine waste-retention basin. 

Because seepage, overflows, and land application of 

waste from waste-retention basins may transport contaminants 

to nearby surface and ground waters, information about the 

organic matter from a typical swine waste-retention basin 

that has been isolated and fractionated is important in order 

to determine its major chemical characteristics. This report 

describes a study that expands the dissolved organic matter 

fractionation analytical scheme of Leenheer and others (2000) 

to a total organic matter fractionation analytical scheme. The 

particulate, colloid and dissolved fractions were separated and 

compared in order to predict modes of contaminant transport 

and contaminant partitioning on the basis of their chemical 

composition. The waste-retention basin sampled in July 2003 

for this report was considered a typical storage structure for a 

swine operation (that is, for its size, waste input, and usage).
Methods

Source

The waste-retention basin sampled for this study (photograph on cover) was constructed in 1993 and is located in 

north central Iowa. The basin is permitted for 1.8 million 

gallons, with about 1.3 million gallons being pumped out 

in the spring and fall of each year. No fresh water is added 

to this basin as it fills with swine waste. This basin received 

waste from five buildings that housed about 5,700 swine at the 

time of sampling. The average weight per animal was about 

135 pounds at this time. In an effort to reduce the odors from 

this basin, the operator has added paper cuttings in previous 

years and soybean stover in the current year (2003).

Sample Collection

Two unfiltered 1-liter samples were collected in amber 

glass bottles in July, 2003. One sample was collected close to 

the waste in-flow pipe (north-side sample), and one was collected on the other side of the basin opposite the waste in-flow 

pipe (south-side sample). The samples were chilled on ice 

and shipped overnight to the U.S. Geological Survey National 

Water Quality Laboratory, and immediately analyzed with 

minimal refrigerated storage to minimize sample degradation.

Organic Matter Fractionation

The north-side sample was fractionated in a comprehensive scheme, presented in figure 1, designed to obtain 

compound class information for particulate, colloidal, and 

dissolved organic matter. This is the first comprehensive fractionation scheme designed to include volatile acid and particulate organic matter with the published scheme (Leenheer and 

others, 2000) for fractionation and analyses of colloidal and 

non-volatile dissolved organic matter. The south-side sample 

was used to evaluate a non-comprehensive solvent extraction 

scheme (fig. 2) for comparison of a typical solvent extraction 

analyses with the comprehensive analytical approach. This 

comparison assumes there were no significant differences 

between the north- and south-side samples as the pH and conductivity values (measured in the laboratory) were identical 

for these two samples.

Laboratory Equipment

Glass chromatography columns with Teflon end caps 

were obtained from Spectra/Chrom. Column dimensions for 

0.5-L resin-bed volume were 5 centimeter (cm) inside diameter (ID) X 30 cm; 80-milliliter (mL) resin-bed volumes were 

1.5 cm ID X 30 cm; and 20-mL resin-bed volumes were 1 cm 

ID X 28.5 cm. Excess volume in each column was left vacant 

to allow for resin-bed expansion during different chemical 

treatments. A reciprocating, ceramic piston pump (FMI Lab 

Pump Model QD) was used to pump water and reagents 

through the 1-L and 0.5-L resin-bed columns connected with 

3 millimeter (mm) ID, 6 mm outside diameter (OD) FEP Teflon tubing at a flow rate of 250 milliliters per minute 

(mL/min). A smaller pump (FMI Lab Pump Model RP-SY) 

was used to pump water and reagents through the 80-mL 

resin-bed column at a flow rate of 20 mL/min and through the 

20-mL resin-bed column at a flow rate of 10 mL/min. These 

smaller columns were connected to the pump with 1.5-mmOD, 3-mm-ID Teflon tubing. Selection, preparation, regeneration, capacities, and packing of resin adsorbents has been 

discussed previously (Leenheer, 1981).

Spectra/Por 3 regenerated cellulose dialysis membrane 

was used. It had a molecular weight cutoff of 3,500 Daltons. 

Prior to use, an appropriate length of membrane was cut to 

accommodate the volume to be dialyzed, and the membrane 

was washed by soaking it in 4 L of deionized water overnight. 

For dialysis, the bottom of the dialysis tubing was closed with 

a plastic snap closure, the solution was poured into the resulting bag using a funnel, and the top of the bag was closed with 

another closure. Dialysis was then conducted in 4-L Teflon 

beakers and the solution was stirred with a magnetic stirrer. 

For evaporations, a Buchi Rotavapor R had 1-L evaporation and condensation flasks, and its evaporation rate for water 

was about 0.5 L per hour for a water-bath temperature of 60°C 

at 750 mm mercury vacuum. The freeze dryer was a Labconco 

Lyph-Lock 6 Model.

For centrifuge separations, a Sorvall Superspeed RC-2B 

Refrigerated Centrifuge was used with an angle head. Centrifuge bottles were 250-mL Teflon bottles that were spun at 

5,000 rpms for 30 minutes.

Fourier Transform-Infrared (FT-IR) Spectrometry

Infrared spectra were collected using 2 to 5 milligrams 

(mg) of organic matter fraction isolates in potassium bromide 

pellets. The Perkin Elmer System 2000 FT-IR used an infrared 

source with a pulsed laser carrier and a deuterated triglycine 

sulfate detector. The instrument was setup to scan from 4,000 

to 400 cm-1 averaging 10 scans at 1.0 cm-1 intervals with 

a resolution of 4.0 cm-1. All spectra were normalized after 

acquisition to a maximum absorbance of 1.0 for comparative 

purposes. Peak frequency assignments for compound classes 

are given in table 1.

2 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 1. Flow chart of total organic matter fractionation for north-side sample.

Methods 3
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Figure 2. Flow chart of solvent-extractable organic matter fractionation for south-side sample.

4 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
Solid State Cross Polarization Magic Angle 

Spinning (CPMAS) 13C-Nuclear Magnetic 

Resonance (NMR) Spectrometry

CPMAS 13C-NMR spectra were obtained on 5 to 200 mg 

of organic matter fractions. Freeze-dried samples were packed 

in ceramic rotors. CPMAS 13C-NMR spectra were obtained 

on a 200-megahertz (MHz) Chemagnetics CMX spectrometer with a 7.5-mm-diameter probe. The spinning rate was 

Table 1. Infrared frequency bands for various structures in DOM isolates

Compound class Frequencies (cm-1), structure (ø = aromatic carbon ring), and elements (C=carbon, H=hydrogen, 

N=nitrogen, O=oxygen)

Carbohydrates 3,400-3,300 (O-H), 1,100-1,000 (C-O)

Fulvic acid 3,400-3,300 (O-H), 2,700-2,500 (COOH), 1,760 (COOR), 1,720 (COOH), 1,660-1,630 (ø-C=O), 

1,280-1,150 (ø-O, COOH)

Aliphatic hydrocarbons 2,960 (CH3

), 2,940 (CH2), 1,460 (CH2), 1,380 (CH3)

Aromatic hydrocarbons ~1,600 (ø), ~1,500 (ø), 700-900, ø-H

Proteins 1,660 (amide 1 band, N-C=O), 1,540 (amide 2 band, N=C-O)

N-acetyl amino sugars 1,660 (amide 1 band, N-C=O), 1,550 (amide 2 band, N=C-O) 1,380 (CH3

)

Lipids 1,760 (COOR), 1,720 (COOH), 2960 (CH3

), 2,940 (CH2), 1,460 (CH2), 1,380(CH3)

Table 2. Structural assignments for 13C-NMR spectra.

[C, carbon; H, hydrogen; N, sulfur; O, oxygen; S, sulfur; ø, aromatic carbon ring]

Chemical linkage 

between elements Compound type

Chemical shift range 

(parts per million)

C-H Aliphatic hydrocarbon 0-55

C-N Amines, amides, proteins 40-55

O-CH3 Methoxy groups in tannins and lignins 55-60

C-O Aliphatic alcohols, ethers, and esters 60-90

O-C-O Anomeric carbon in carbohydrates, lactols 90-110

ø Aromatic carbon 95-165

ø-O Aromatic esters, ethers, and phenols 135-165

ø-SO3H Aromatic sulfonic acids 140-145 

O=C-O, O=C-N Carboxylic acids, esters, amides 160-190

O=C-C=C Flavones, quinones 170-200

O=C-C Aliphatic and aromatic ketones 190-220

5,000 Hz. The acquisition parameters included a contact 

time of 5 milliseconds, pulse delay of 1 second, and a pulse 

width of 4.5 microseconds for the 90° pulse. Variable contact 

time studies and comparison of liquid-state with solid state 

13C-NMR spectra by Wershaw and others (2000) indicate these 

are the optimum parameters for quantitatively determining 

different organic carbon structural group contributions to the 

NMR spectra. Organic structural assignments for peaks in the 

CPMAS 13C-NMR spectra are given in table 2.

Methods 5
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Figure 3. Fractionation of total organic matter of north-side sample.

Fractionation of Organic Matter

The total organic matter fractionation of the north-side 

sample is presented in the bar diagram of figure 3. Concentrations of particulate, colloidal, and dissolved organic matter are 

very large with the total summed organic matter concentration being 32,400 milligrams per liter (mg/L) (3.24 weight 

percent). In contrast, particulate organic carbon concentrations 

in major world rivers range from 0.6 to 14.2 mg/L (Ittekkot 

and Laane, 1991) and dissolved organic carbon concentrations range from 2 to 8 mg/L (Spitzy and Leenheer, 1991). 

The particulate organic matter fractionation is presented in the 

bar diagram of figure 4. The majority of particulate organic 

matter (86 percent) is composed of <61 μm particulates that 

formed larger floc when acidified to pH 2 with HCl. When this 

floc particulate fraction was extracted with methylene chloride, 40.3 percent of the mass was extractable as lipids. The 

three colloid fractions generated by the fractionation scheme 

of figure 1 are presented in the bar diagram of figure 5. The 

floc-derived colloid fraction was initially soluble in water, 

but it precipitated inside the dialysis bag when salts and HCl 

were removed from the sample during dialysis. The precipitate 

colloid fraction was initially soluble in the wastewater sample, 

but it precipitated when the sample was acidified and concentrated during vacuum evaporation. It remained as a precipitate 

during dialysis to remove salt and acid. The soluble colloid 

fraction remained soluble during the dialysis step. Most (90 

percent) of the dissolved organic matter consists of sodium 

salts of volatile organic acids as shown in figure 6. Thus, nonvolatile dissolved organic matter concentration is lower than 

particulate and colloidal organic matter concentrations when 

figure 6 is compared to figure 3. The non-volatile dissolved 

organic matter fractionation is presented in figure 7 with the 

hydrophobic neutrals, hydrophobic acids, and base fractions 

being the major fractions.

Results from the solvent-extractable fractionation of the 

south-side sample are presented in the bar diagram of figure 8. 

Solvent-extractable acids constitute the large majority of this 

fraction, with solvent extractable bases being nearly absent. 

As the base fraction was a major fraction in the comprehensive 

fractionation of the north-side sample, this difference indicates 

that most bases in this wastewater are not solvent extractable. 

Solvent-extractable organic matter constitutes only 12.1 percent of total organic matter. It is unlikely that the north-side 

and south-side samples had significantly different concentrations of total organic matter as both samples had identical 

specific conductance (24.7 millisiemens per centimeter at 

25°C) and pH (7.1) values.

6 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 4. Fractionation of particulate organic matter of north-side sample.

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Figure 5. Fractionation of colloidal organic matter of north-side sample.

Fractionation of Organic Matter 7
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Figure 7. Fractionation of non-volatile dissolved organic matter in north-side sample.

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Figure 6. Bar chart showing volatile and non-volatile dissolved organic matter of the north 

side sample.

8 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 8. Organic solvent fractionation of south-side sample.

Compound-Class Spectral Analyses of Fractions

Particulate Fractions

Infrared spectra of the particulate fractions are presented 

in figure 9 and 13C-NMR spectra are presented in figure 10. 

Compound-class assignments were made by comparing the 

spectra of figures 9 and 10 with spectral peak assignments 

presented in tables 1 and 2. Spectral analyses of the coarse 

particulate fraction indicate a mixture of lipid, protein, and 

carbohydrate that is characteristic of plant and animal biomolecules. Visual examination of this fraction showed short 

white hairs from swine, and plant and grain fragments from 

undigested feed. The medium particulate fraction was greatly 

depleted in protein (peak at 55 parts per million (ppm) in the 

13C-NMR spectra) as compared to the coarse particulate fraction. This loss in protein is likely caused by the removal of 

swine hair (mostly protein) by the sieving process. The fine 

particulate fraction was depleted of both protein and carbohydrate constituents compared to the larger-size fractions, with 

lipid components being dominant. Carbohydrates are nearly 

absent in the floc particulate fraction, with aliphatic lipids 

constituting the majority of the carbon. The lipids extracted 

from the floc particulates are dominated by straight chain fatty 

acids as evidenced by the sharp methylene peaks at 2,920 cm-1

and 1,460 cm-1 in the infrared spectrum and by the 34 ppm 

peak in the 13C-NMR spectrum. After extraction by methylene chloride, the infrared spectrum of the floc particulates 

closely matched the spectrum of peptidoglycans derived from 

a pure culture of the bacterium, bacillus subtilis, as shown in 

figure 11. Peptidoglycans are the major component of bacterial cell walls, and finding these peptidoglycan components in 

the floc particulate fraction indicates a bacterial origin for this 

fraction. The infrared spectrum of the humic acid fraction in 

figure 9 are similar to the infrared spectrum of the extracted 

lipid fraction, indicating these humic acids are also lipidderived.

Colloid Fractions

Infrared spectra of the colloid fractions are presented in 

figure 12 and 13C-NMR spectra are presented in figure 13. The 

spectra of the floc-derived colloid fraction are indicative of a 

glycoprotein. The solubility behavior of this fraction (soluble 

with salt water, insoluble in deionized water) indicates this 

material is a globulin, a major constituent of cellular plasma 

(White and others, 1964). The spectra of the precipitate colloid 

fraction is nearly identical to the spectra of the peptidoglycans 

(fig. 11) discussed previously. Therefore, precipitate colloids 

are free peptidoglycans in which cellular lipid constituents 

have been removed by biodegradation or extraction by the 

Fractionation of Organic Matter 9
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Figure 9. Infrared spectra of particulate fractions from north-side sample.

isolation procedure. Peaks of the infrared spectrum of the 

soluble colloid fraction match the peaks for N-acetyl amino 

sugars listed in table 1, and the 13C-NMR spectrum of this 

fraction also matches the 13C-NMR spectra of N-acetyl amino 

sugars colloids isolated from natural surface waters (Leenheer 

and others, 2000). N-acetyl amino sugars are derived from 

degradative removal of the peptide from peptidoglycans.

Dissolved Fractions

Infrared spectra of the dissolved fractions are presented 

in figure 14 and 13C-NMR spectra are presented in figure 15. 

The major acid in the volatile acid fraction is acetic acid in 

the form of its sodium salt as indicated by comparison of 

the major spectral peaks with the published infrared spectra 

(Pouchart, 1985) ( peaks at 1,576, 1,011, and 619 cm-1) and 13C-NMR spectra (Simons, 1983) (peak at 22 ppm). In the 

13C-NMR spectrum, the broad envelope peak from 0-60 ppm 

that includes the methyl peak at 13 ppm is indicative of a 

mixture of longer chain and branched volatile fatty acids. 

Branched fatty acids, such as isovaleric acid, are partially 

responsible for the unpleasant odor of swine manure (Gralapp 

and others, 2001).

For the non-volatile dissolved organic matter fractions, 

the hydrophobic neutral and transphilic acid fractions were 

misplaced, and the 13C-NMR spectra were not obtained. The 

infrared spectrum of the hydrophobic neutral fraction (fig. 14) 

indicates the major components are ortho-substituted (peaks 

at 750 and 673 cm-1) aromatic hydrocarbons and aromatic carboxylic acids. Aliphatic hydrocarbons are minor components 

as indicated by the relatively minor peak at 2,920 cm-1. The 

infrared spectrum of the hydrophobic acid fraction indicates 

the major component is hydrocinnamic acid by the exact 

matches of several spectral peaks with the published spectrum 

(Pouchart, 1985). The 13C-NMR spectrum of the hydrophobic acid fraction (fig. 15), confirms the hydrocinnamic acid 

10 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 10. 13C-NMR spectra of particulate fractions from north-side sample. L= lipid, P = protein, 

C = carbohydrate.

Fractionation of Organic Matter 11
assignment. The transphilic acid plus neutral fractions have 

infrared peaks ( fig. 14) assigned to carboxylic acid, amide I 

and amide II bands which indicates this fraction is derived 

from degradation of proteins and/or N-acetyl amino sugars. 

The hydrophilic acid plus neutral fractions have the same 

infrared peaks as the transphilic acid plus neutral fractions, 

but the carbohydrate component (1,100–1,000 cm-1) is much 

greater. The 13C-NMR spectrum of the hydrophilic acid plus 

neutral fraction has a sharp methyl peak at 22 ppm ( fig. 15) 

that is indicative of N-acetyl hydroxyl acids derived from 

degradation of N-acetyl amino sugars (Leenheer and others, 

2004). The base fraction contains hydrocinnamic acid as a 

major component. The hydrophobic acid component partially 

fractionated into the base fraction because it adsorbed on 

the hydrophobic styrene-divinylbenzene matrix of the cation 

exchange resin during the fractionation procedure. The small 

broad peak near 55 ppm in the 13C-NMR spectrum of the base 

fraction (fig. 15) is characteristic of basic peptides with the 

amide carbonyl peak of these peptides being at 177 ppm.

Solvent-Extractable Fractions 

Infrared spectra of the dissolved fractions are presented 

in figure 16 and 13C-NMR spectra are presented in figure 17. 

Interpretation of the spectra of the solvent-extractable acid 

fraction indicates a mixture of aliphatic straight and branchedchain fatty acids, and hydrocinnamic acid as the aromatic 

acid. Acetic acid, the dominant volatile acid, was not detected 

because it was lost during evaporation of the solvent. The solvent extractable neutral fraction is mostly long-chain aliphatic 

fatty acids that did not back-extract into water as sodium salts. 

The ortho-disubstituted aromatic hydrocarbons found in the 

hydrophobic neutral fraction also were found in this solventextractable neutral fraction. The infrared spectrum of the 

small solvent extractable base fraction indicated straight chain 

aliphatic amines.

12 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 11. Comparison of infrared spectrum floc particulates extracted with methylene chloride with the 

infrared spectrum of peptidoglycan from bacillus subtilis.

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Figure 12. Infrared spectra of colloid fractions from north-side sample.

Fractionation of Organic Matter 13
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Figure 13. 13C-NMR spectra of colloid fractions from north-side sample.

14 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 14. Infrared spectra of dissolved fractions from north-side sample. CA = carboxylic acid, 

AI = amide 1 band, AII = amide 2 band.

Fractionation of Organic Matter 15
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16 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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Figure 16. Infrared spectra of solvent-extractable fractions from south-side sample.

Figure 17. 13C-NMR spectra of solvent-extractable fractions from south-side sample.

Fractionation of Organic Matter 17
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Characterization of Organic Matter

Organic matter in wastewater from a swine waste-retention basin mainly consists of volatile fatty acids and bacterial cellular constituents (aliphatic straight-chain lipids and 

peptidoglycans). Volatile fatty acids, especially acetic acid, 

are produced as metabolites in both aerobic and anaerobic 

(fermentation) biodegradation processes (White and others, 

1964; Gibson, 1984). Fermentation of organic wastes also produces large concentrations of volatile alcohols, aldehydes, and 

hydrocarbons, but these compound classes were not recovered 

or analyzed by this study.

Natural hormones (Raman and others, 2004; Hanselman and others, 2003) as well as veterinary pharmaceuticals 

(Pierini and others, 2004; Kumar and others, 2004; Cromwell, 

2002; Campagnolo and others, 2002; Boxall and others, 2003) 

are associated with swine wastes. Significant concentrations of 

non-polar hormones and pharmaceuticals, such as tetracycline, 

likely partition into non-polar aliphatic straight chain lipids 

found in the particulate fractions. Thus, measuring hormone 

and pharmaceutical concentrations of filtered liquid swine 

waste from a waste-storage basin could greatly underestimate 

total residues actually present in such samples.

Peptidoglycans are unique constituents of microbial cells, 

especially the N-acetyl muramic acid monomeric constituent. Peptidoglycan constituents found in the wastewaters may 

simply be the fragments of bacterial cell walls after the cells 

are attacked by bacteriophage viruses as shown in the following reaction sequence:

18 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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The detection of globulins in the silt-derived colloid fraction indicates that plasma proteins in living bacterial cells were 

released by the analytical procedure. The multiple extraction 

and freeze-drying steps of figure 1 rupture cellular membranes 

and release cellular plasma constituents such as globulins into 

solution. Thus, much of the particulate and colloidal material 

in the wastewater likely consists of living microbial biomass.

Animal feed residues in these wastewaters should be 

detectable as polysaccharide and lignin components in the 

particulate fractions; however, the particulate fractions contain 

only small amounts of polysaccharide and no detectable 

amounts of lignin. The fate of lignin components is indicated 

by the detection of hydrocinnamic acid in the hydrophobic 

acid fraction.

Lignin is known to bacterially degrade under anaerobic 

conditions to produce methane and other by-products (Young, 

1984). A proposed pathway (Healy and others, 1980) for the 

degradation of ferulic acid, a lignin monomer, is presented in 

the following reaction.

Characterization of Organic Matter 19
report. Robert Wershaw assisted in the acquisition of the 

13C-NMR spectral and also reviewed the report. The authors 

also acknowledge the U.S. Geological Survey Toxic Substances Hydrology Program for its support of this research.

References Cited

Boxall, A.B.A., Kolpin, D.W., Halling-Sorensen, Brent, and 

Tolls, Johannes, 2003, Are veterinary medicines causing 

environmental risks?: Environmental Science and Technology, v. 37, p. 286A-294A.

Chee-Sanford, J.C., Aminov, R.I., Krapac, I.J., GarriguesJeanjean, Natalie, and Mackie, R.I., 2001, Occurrence and 

diversity of tetracycline resistance genes in lagoons and 

groundwater underlying two swine production facilities: 

Applied and Environmental Microbiology, v. 67, 

p. 1494-1502.

Cromwell, G.L, 2002, Why and how antibiotics are used in 

swine production?: Animal Biotechnology, v. 13, p. 7-27.

Compagnolo, E.R., Johnson, K.R., Karpati, Adam, Rubin, 

C.S., Kolpin, D.W., Meyer, M.T., Esteban, J.E., Currier, R.W., Smith, Kathleen, Thu, K.M., and McGeehin, 

Michael., 2002, Antimicrobial residues in animal waste and 

water resources proximal to large-scale swine and poultry 

feeding operations: The Science of the Total Environment, 

No. 299, p. 89-95

Gralapp, A.K., Powers, W.J., and Bundy, D.S., 2001, Comparison of olfactometry, gas chromatography, and electronic 

noise technology for measurement of indoor air from swine 

facilities: Transactions of American Society of Agricultural 

Engineers, v. 16, p. 621-627.

Gibson, D.T., ed., 1984, Microbial Degradation of Organic 

Compounds: New York, Marcel Dekker, Inc., 

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v. 24, p. 5471-5478

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Other lignin monomers, such as caffeic acid and homoprotocatechuic acids, also lost ring substituents during 

anaerobic biodegradation (Young, 1984). These losses of ring 

substituents and propane side chain result in the loss of lignin 

identity with regards to spectra characterizations, but hydrocinnamic acid is an intermediate metabolite that indicates 

lignin as a precursor.

The solvent extraction scheme that was applied to the 

south-side sample only recovered 3,927 mg of organic matter 

as compared to 32,413 mg of organic matter recovered for the 

comprehensive fractionation scheme for the north-side sample. 

If the organic matter concentrations of the two samples are 

equivalent, the solvent extraction scheme of figure 2 recovered 

only 12 percent the organic matter as the comprehensive fractionation scheme of figure 1. Particulate, colloidal, and nonextractable polar dissolved organic matter was not recovered 

by the solvent extraction scheme.

Summary and Conclusions

Fractionation and spectral analyses of particulate, colloid, 

and dissolved organic fractions isolated from wastewater from 

a swine waste-retention basin indicate the bulk of the organic 

matter consists of microbial cellular constituents and their 

degradation products. This total organic matter fractionation 

analysis was the first comprehensive analysis that included 

particulate organic matter. The compound-class composition 

of each of these fractions was determined by infrared and 

13C-NMR spectral analyses. Volatile acids were the largest 

fraction with acetic acid being the major component of this 

fraction. The second most abundant fraction was fine particulate organic matter that consisted of bacterial cells that were 

subfractionated into extractable lipids consisting of straight 

chain fatty acids, peptidoglycans components of bacterial cell 

walls, and protein globulin components of cellular plasma. 

This inclusion of particulate organic matter in the analytical approach provided valuable insights about the origin and 

processes that produce particulate, colloid, and dissolved 

organic matter in this wastewater. The large lipid content of 

the particulate fractions indicates that non-polar contaminants, 

such as certain pharmaceuticals added to swine feed, likely 

associate with the particulate organic matter through partitioning interactions. The finding of hydrocinnamic acid indicates 

an anaerobic process of lignin degradation in this wastewater.

Acknowledgments

Use of trade names is for identification purposes only and 

does not constitute endorsement by the U.S. Geological Survey (USGS). The authors would like to thank Kent Becher and 

Jason Smith, USGS, Iowa District, for collecting the swine 

waste samples used for this study. Dana Kolpin, USGS, Iowa 

District, provided background references, and review of the 

20 Fractionation and Characterization of Organic Matter in Wastewater from a Swine Waste-Retention Basin
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J.R., 2000, Structural-group quantitation by CP/MAS 

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