1
Table 5. Nitrogen content (mg N kg-1 soil) by
amino compound in Wisconsin Rosholt soil 28 d
after manure application. Autoclaving was for
either 16 h at 121oC or 90 min at 136oC.
Table 8. Nitrogen content (mg N kg-1 soil) by
amino compound in Illinois Catlin soil 0 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
Table 2. Nitrogen content (mg N kg-1 soil) by
amino compound in Oregon Adkins soil 0 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
INTRODUCTION A nationally coordinated team of
USDA-ARS scientists is developing predictions of
animal manure N availability in order to protect
water quality, make more efficient use of N
inputs, and enhance farm solvency. In an initial
set of studies in six states, animal manure was
applied to local soils and incubated in the
laboratory, following a coordinated protocol
(Honeycutt et al., 2005). Manure-N mineralization
was found to be affected more by soil type than
by temperature or moisture (Honeycutt et al.,
unpublished data). This study strives to
understand reasons for soil-specific effects on
manure-N mineralization. Chemical forms of amino
acids and amino sugars were analyzed at the
beginning and end of a 28-d laboratory incubation
for soil with and without manure amendment. An
analysis developed by D.A. Martens was used to
measure the amino compounds. Soil was extracted
by methanesulfonic acid, a non-oxidizing acid
that is thermally stable at elevated
temperatures. Detection was by anion
chromatography and pulsed amperometry, which
enable high sensitivity to amino compounds and
the simultaneous detection of 18 amino acids with
two amino sugars.
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 166.1 124.1 166.1 176.6 133.5 176.6
Lysine 56.0 66.8 66.8 61.2 73.1 73.1
Galactosamine 21.9 27.3 27.3 22.7 28.3 28.2
Glucosamine 50.3 47.5 50.3 50.9 48.7 50.9
Alanine 28.8 37.5 37.5 32.8 40.8 40.8
Threonine 17.5 22.2 22.3 19.6 26.0 26.0
Glycine 44.0 42.3 44.0 47.0 45.0 47.0
Valine 15.4 19.4 19.4 17.9 19.2 19.2
Hydroxyproline 1.6 1.0 1.6 1.7 1.0 1.7
Serine/Proline 19.3 17.1 19.3 21.4 18.5 21.4
Isoleucine 7.5 6.1 7.5 8.6 6.8 8.6
Leucine 11.9 10.5 11.9 14.4 12.0 14.4
Methionine 2.8 4.6 4.6 3.5 5.4 5.4
Histidine 16.7 4.9 16.7 22.4 2.9 22.4
Phenylalanine 3.8 0 15.3 8.7 0.3 8.7
Glutamate 24.5 15.3 24.5 19.1 18.1 19.1
Aspartate 35.1 21.5 35.1 31.7 23.2 31.7
Cystine 2.6 1.7 2.6 2.7 1.9 2.7
Tyrosine 1.9 1.9 1.9 1.6 2.7 2.7

Total 528 472 563 562 507 600
of total soil N 42 37 45 40 36 43
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 170.7 191.9 191.9 179.6 188.6 188.6
Lysine 75.0 98.5 98.5 78.4 97.2 97.2
.Galactosamine 34.1 54.7 54.7 34.8 48.6 48.6
Glucosamine 67.8 77.5 77.5 67.1 75.9 75.9
Alanine 45.4 77.4 77.4 48.5 79.0 79.0
Threonine 24.4 38.7 38.7 28.1 42.0 42.0
Glycine 58.2 74.3 74.3 65.2 77.8 77.8
Valine 20.1 23.5 23.5 21.7 26.6 26.6
Hydroxyproline 3.1 2.9 3.1 3.2 3.0 3.2
Serine/Proline 29.0 31.0 31.0 31.2 31.2 31.2
Isoleucine 13.8 11.9 13.8 15.8 13.1 15.8
Leucine 20.9 22.0 22.0 24.9 27.2 27.2
Methionine 7.5 7.4 7.5 7.9 7.0 7.9
Histidine 23.4 24.3 24.3 23.8 28.2 28.2
Phenylalanine 15.2 0 15.2 18.3 0 18.3
Glutamate 22.5 2.0 22.5 31.0 6.3 31.0
Aspartate 25.0 3.6 25.0 25.7 6.5 25.7
Cystine 2.0 2.1 2.1 4.5 2.4 4.5
Tyrosine 17.9 3.3 17.9 12.5 3.4 12.5

Total 676 747 821 720 764 835
of total soil N 28 31 34 27 29 31
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Higher value 16 hour at 121oC 90 min at 136oC Higher value
Arginine 54.4 61.9 61.9 62.0 61.3 62.0
Lysine 24.2 24.2 24.2 27.8 25.9 27.8
Galactosamine 5.5 6.6 6.6 5.8 6.8 6.8
Glucosamine 10.1 12.5 12.5 10.9 12.5 12.5
Alanine 15.8 16.8 16.8 19.5 18.8 19.5
Threonine 7.6 9.3 9.3 11.0 12.2 12.2
Glycine 29.8 25.8 29.8 34.2 27.0 34.2
Valine 10.2 13.4 13.4 13.3 13.2 13.3
Hydroxyproline 0.7 0.8 0.8 0.8 0.9 0.9
Serine/Proline 6.9 8.9 8.9 8.8 9.3 9.3
Isoleucine 4.0 3.6 4.0 5.5 4.6 5.5
Leucine 5.3 6.0 6.0 7.6 7.1 7.6
Methionine 4.0 3.9 4.0 5.2 5.0 5.2
Histidine 7.0 7.4 7.4 8.3 8.1 8.3
Phenylalanine 1.4 9.0 1.4 1.6 1.3 1.6
Glutamate 14.2 9.7 14.2 19.0 11.7 19.0
Aspartate 25.9 21.7 25.9 28.2 28.1 28.2
Cystine 0.1 0.4 0.4 0.2 0.5 0.5
Tyrosine 3.4 1.9 3.4 3.7 2.6 3.7

Total 230 236 251 274 256 278
of total soil 33 34 36 46 43 46

• RESULTS
•  
• A sample chromatogram is shown in Figure 2.
  Some amino compounds had more distinct and
  reproducible peaks than did others.
• Extraction by MSA allowed identification of
  26-47 of total N for these four soils and 26-60
  for all nine soils, with a mean of 41 (Tables
  2-9).
• Among selected soil properties, those that were
  best correlated with the percent of soil N that
  was identified were percent sand (r 0.88), total
  N content (r -0.87), and percent silt (r
  -0.84).
• Variability among the four lab replicates was
  often high, especially for compounds with small
  peaks (data not shown).
• By both autoclaving techniques, manure
  application was found to result in more amino-N
  for all four soils. This increase resulted
  largely from Arg, Lys, Ala, Thr, Gly, Glu, and
  Asp, which were abundant in the manure (data not
  shown). The magnitude of this increase did not
  change consistently during the 28-d incubation.
• The magnitude of the increase was similar in
  three of the four soils (not Maine Caribou),
  despite differences in soil properties.
• Several amino compounds contributed to this
  increase in amino levels. Glu and Asp contributed
  disproportionately to the increase for all soils
  except Maine Caribou.
• The amino sugars galactosamine and glucosamine
  accounted for 6-8 of the identified amino-N in
  the Adkins soil, 13-15 in the Rosholt and
  Caribou soils, and 15-17 in the Catlin soil.
  They are normally associated with fungal mass,
  hence suggesting different proportions of fungal
  and bacterial populations in these soils.
• Although the two amino sugars should not be
  abundant in manure, their soil levels were
  somewhat higher in the manured soils than in the
  unmanured soils, and more so at 28 d than 0 d.
  These trends suggest that the manure supported
  fungal growth during the incubation.
• The two autoclaving techniques provided
  supplementary results. The longer,
  lower-temperature autoclaving generally enabled
  better extraction of Ser/Pro, Iso, Phe, Glu, Asp,
  and hydroxyproline. The shorter,
  higher-temperature enabled better extraction of
  the two amino sugars, Lys, Ala, and Val.
  Extraction efficiency for the other amino
  compounds varied between the two autoclaves in
  patterns that depended on the soil. For each
  comparison of the two autoclaves, the greater
  value of extracted amino-N is shown in color in
  Tables 2-9.
• The increase in amino levels with manuring was
  greater with long autoclaving at 121oC than with
  short autoclaving at 136oC at both sampling times
  for three of the soils (not Maine Caribou).

 
MATERIALS AND METHODS Soil sampling and
incubation Soil was collected from
unfertilized sites in grasslands or margins of
agricultural fields at nine locations in seven
states (AL, IL, ME, MS, NE, OR, WI). Results
from four representative locations (Table 1) are
presented here. Soils were sampled in late
spring from the 0-15 cm depth. Samples were
maintained at field moisture and transported to a
central laboratory. Then 50 g of each soil was
packed at field bulk densities into vials (four
replicates) and amended with dairy manure at a
rate of 300 kg N ha-1, including 130 kg organic N
ha-1. Samples were incubated for 28 d, and
aerobic moisture conditions were maintained.
Further details are provided by Fortuna et al.
(Presentation 282-2, this conference). To
analyze the soils for their amino compounds, soil
was sub-sampled at 0 d and 28 d and then
air-dried and finely ground.
Table 9. Nitrogen content (mg N kg-1 soil) by
amino compound in Illinois Catlin soil 28 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
Table 3. Nitrogen content (mg N kg-1 soil) by
amino compound in Oregon Adkins soil 28 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 172.5 174.2 174.2 174.7 187.3 187.3
Lysine 78.9 90.7 90.7 77.7 102.0 102.0
Galactosamine 34.9 46.1 46.1 35.9 45.9 45.9
Glucosamine 70.5 72.8 72.8 68.5 76.6 76.6
Alanine 49.0 75.6 75.6 52.6 74.2 74.2
Threonine 27.9 37.1 37.1 29.2 42.5 42.5
Glycine 68.9 75.5 75.5 71.9 80.0 80.0
Valine 21.7 25.0 25.0 23.1 26.5 26.5
Hydroxyproline 3.2 2.9 3.2 3.0 2.4 3.0
Serine/Proline 31.5 8.4 31.5 31.8 31.7 31.8
Isoleucine 14.7 11.5 14.7 15.7 12.6 15.7
Leucine 23.3 20.5 23.3 24.2 34.5 34.5
Methionine 7.3 6.7 7.3 8.3 4.9 8.3
Histidine 25.7 22.6 25.7 25.4 8.5 25.4
Phenylalanine 0 0 0 52.4 0 52.4
Glutamate 22.5 5.4 22.5 31.0 6.0 31.0
Aspartate 26.2 4.2 26.2 37.8 7.0 37.8
Cystine 2.9 2.6 2.9 3.7 3.0 3.7
Tyrosine 10.5 2.9 10.5 5.0 4.8 5.0

Total 692 705 765 772 750 884
of total soil N 29 29 32 31 30 36
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Higher value 16 hour at 121oC 90 min at 136oC Higher value
Arginine 50.8 60.3 60.3 60.0 63.7 63.7
Lysine 25.5 23.2 25.5 26.5 25.3 26.5
Galactosamine 5.7 6.7 6.7 5.9 7.0 7.0
Glucosamine 10.1 12.6 12.6 11.4 14.1 14.1
Alanine 14.5 15.9 15.9 17.9 18.8 18.8
Threonine 7.3 8.8 8.8 9.2 10.4 10.4
Glycine 28.0 25.4 28.0 32.2 28.2 32.2
Valine 11.1 13.0 13.0 12.8 13.0 13.0
Hydroyxproline 0.6 0.9 0.9 0.8 1.0 1.0
Serine/Proline 6.6 7.8 7.8 8.2 9.3 9.3
Isoleucine 3.6 3.4 3.6 4.7 4.1 4.7
Leucine 5.1 5.3 5.3 6.4 6.7 6.7
Methionine 3.6 4.0 4.0 4.6 4.6 4.6
Histidine 6.7 6.9 6.9 7.6 8.6 8.6
Phenylalanine 1.0 0.4 1.0 1.3 0.9 1.3
Glutamate 9.8 9.1 9.8 18.1 11.8 18.1
Aspartate 21.0 22.0 22.0 27.2 24.2 27.2
Cystine 0.1 0.4 0.4 0.3 0.5 0.5
Tyrosine 1.8 3.6 3.6 2.5 2.3 2.5

Total 213 230 236 258 254 270
of total soil N 42 46 47 44 43 46
Table 6. Nitrogen content (mg N kg-1 soil) by
amino compound in Maine Caribou soil 0 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 189.1 175.4 189.1 194.8 181.7 194.8
Lysine 54.5 69.6 69.6 56.8 76.3 76.3
Galactosamine 22.6 30.9 30.9 23.0 30.7 30.7
Glucosamine 49.3 47.3 49.3 50.8 49.3 50.8
Alanine 46.8 59.4 59.4 47.2 62.6 62.6
Threonine 29.8 35.0 35.0 32.3 41.8 41.8
Glycine 63.8 73.1 73.1 66.5 77.9 77.9
Valine 27.7 23.8 27.7 25.4 29.0 29.0
Hydroxyproline 6.6 4.5 6.6 7.0 5.4 7.0
Serine/Proline 31.7 29.5 31.7 34.3 31.4 34.3
Isoleucine 11.7 9.8 11.7 12.7 11.3 12.7
Leucine 17.8 16.9 17.8 18.0 21.5 21.5
Methionine 5.9 7.0 7.0 6.7 8.2 8.2
Histidine 0 8.8 8.8 0 15.0 15.0
Phenylalanine 0 8.5 8.5 0 17.5 17.5
Glutamate 4.0 13.2 13.2 5.7 9.8 9.8
Aspartate 5.6 10.4 10.4 7.0 7.9 7.9
Cystine 1.5 1.0 1.5 1.7 1.3 1.7
Tyrosine 2.5 6.4 6.4 3.2 4.1 4.1

Total 581 630 668 605 683 715
of total soil N 32 34 36 29 33 34
Table 1. Selected soil properties. All units
are g kg-1 except for pH.
State Soil series Sand content Clay content Soil organic C Total soil N pH
Oregon Adkins 85 4 6 0.6 6.7
Wisconsin Rosholt 53 12 13 1.2 7.3
Maine Caribou 52 9 20 1.7 5.6
Illinois Catlin 16 20 35 2.5 7.0
   
Table 4. Nitrogen content (mg N kg-1 soil) by
amino compound in Wisconsin Rosholt soil 0 d
after manure application. Autoclaving was for
either 16 h at 121oC or 90 min at 136oC.
Table 7. Nitrogen content (mg N kg-1 soil) by
amino compound in Maine Caribou soil 28 d after
manure application. Autoclaving was for either 16
h at 121oC or 90 min at 136oC.
DISCUSSION Useful results included (i) depiction
of an effect of a one-time manure application on
soil N forms, (ii) a proportion of total soil N
that was identified that was as high as is
achieved by the conventional HCl extraction
(30-45), and (iii) detection of two amino sugars
that enabled conjecture regarding fungal growth.
The two autoclaving techniques provided
supplementary information. The proportions of
soil N that we identified as amino compounds were
less than that (51) reported by Martens and
Loeffelmann (2003) and Martens et al. (2006).
This difference might be due to the smaller
amounts of plant biomass that had been
incorporated annually into our soils. These
results will be integrated with simultaneous
measurements by Fortuna et al. (Presentation
282-2) on microbial populations and N
transformations.  
Soil particle size was determined by the
method of Kettler et al. (2001). Carbon and N
contents were determined by dry combustion. Soil
pH was measured in a solution with 11
(massvolume) 0.01 M CaCl2.
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 166.7 121.7 166.7 184.2 125.5 184.2
Lysine 56.6 66.1 66.1 60.0 66.1 66.1
Galactosamine 21.6 25.1 25.1 23.3 25.8 25.8
Glucosamine 52.2 45.7 52.2 52.7 46.5 52.7
Alanine 31.8 35.7 35.7 35.0 34.6 35.0
Threonine 19.4 20.6 20.6 21.0 23.5 23.5
Glycine 52.8 41.5 52.8 50.9 41.5 50.9
Valine 20.8 19.3 20.8 19.3 19.4 19.4
Hydroxyproline 1.6 0.9 1.6 1.9 0.9 1.9
Serine/Proline 20.3 16.3 20.3 21.9 17.7 21.9
Isoleucine 8.0 5.9 8.0 9.5 6.6 9.5
Leucine 12.7 9.9 12.7 17.1 11.5 17.1
Methionine 31.8 4.4 4.4 3.6 5.2 5.2
Histidine 16.4 0 16.4 18.8 0.8 18.8
Phenylalanine 7.3 0.9 7.3 7.3 0 7.3
Glutamate 21.7 11.7 21.7 22.2 13.2 22.2
Aspartate 37.0 16.9 37.0 36.2 18.4 36.2
Cystine 2.7 1.4 2.7 3.9 2.3 3.9
Tyrosine 4.0 1.4 4.0 3.9 2.3 3.9

Total 549 445 574 592 461 604
of total soil N 39 32 41 39 31 40
Amino compound Unmanured soil Unmanured soil Unmanured soil Manured soil Manured soil Manured soil
Amino compound 16 hour at 121oC 90 min at 136oC Greater value 16 hour at 121oC 90 min at 136oC Greater value
Arginine 181.6 176.2 181.6 187.9 184.0 187.9
Lysine 53.0 74.9 74.9 56.4 77.6 77.6
Galactosamine 22.0 28.1 28.1 22.2 33.7 33.7
Glucosamine 50.7 49.0 50.7 52.2 50.9 52.2
Alanine 44.7 62.2 62.2 46.7 64.2 64.2
Threonine 29.0 34.8 34.8 31.2 38.0 38.0
Glycine 64.7 77.3 77.3 66.6 81.4 81.4
Valine 22.5 23.4 23.4 24.8 26.3 26.3
Hydroxyproline 6.7 4.5 6.7 6.2 4.7 6.2
Serine/Proline 31.7 28.7 31.7 32.1 30.4 32.1
Isoleucine 10.4 9.7 10.4 12.1 10.7 12.1
Leucine 15.8 17.2 17.2 15.6 19.3 19.3
Methionine 5.2 6.8 6.8 6.6 7.7 7.7
Histidine 0 12.3 12.3 0 9.9 9.9
Phenylalanine 0 8.3 8.3 0 8.7 8.7
Glutamate 4.6 8.9 8.9 4.2 4.7 4.7
Aspartate 6.2 8.5 8.5 5.8 7.2 7.2
Cystine 1.6 1.6 1.6 1.5 0.8 1.5
Tyrosine 2.6 3.6 3.6 2.7 3.3 3.3

Total 563 636 659 585 665 685
of total soil N 26 29 30 26 30 31
Analysis for amino compounds Following the
procedure of Martens and Loeffelmann (2003), soil
samples (250 mg) were extracted in 2 mL of 4 M
methanesulfonic acid during autoclaving. We
performed the conventional 16-hour extraction at
121oC and compared the results with autoclaving
for 90 min at 136oC as recommended by Martens and
Loeffelmann (2003). Extracts were neutralized,
diluted, and injected into a Dionex DX-500 ion
chromatograph equipped with an AminoPac PA-10
column for anion chromatographic separation.
Detection was by pulsed amperometry as each
amino compound passed by a gold working
electrode, it was oxidized in an electrical
field, generating current that was measured.
Concentrations of the amino compounds were
calculated through a 5-point standard calibration
curve. Amino compounds that were detected
included 18 amino acids and two amino sugars,
galactosamine and glucosamine (Fig. 1). Peaks
for serine and proline co-eluted, so the combined
concentrations of these compounds are reported.
REFERENCES Honeycutt CW, Griffin TS, Wienhold BJ,
Eghball B, Albrecht SL, Powell JM, Woodbury BL,
Sistani KR, Hubbard RK, Torbert HA, Eigenberg RA,
Wright RL, and Jawson MD. 2005. Protocols for
nationally coordinated laboratory and field
research on manure nitrogen mineralization.
Comm. Soil Sci. Plant Anal. 362807-2822. Kettler
TA, Doran JW, and Gilbert TL. 2001. Simplified
method for soil particle size determination to
accompany soil quality analyses. Soil Sci. Soc.
Am. J. 65849-852. Martens DA and Loeffelmann KL.
2003. Soil amino acid composition quantified by
acid hydrolysis and anion chromatography-pulsed
amperometry. J. Agric. Food Chem
516521-6529. Martens DA, Jaynes DB, Colvin TS,
Kaspar TC and Karlen DL. 2006. Soil organic
nitrogen enrichment following soybean in an Iowa
corn?soybean rotation. Soil Sci. Soc. Am. J.
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Figure 2. Representative chromatogram of a soil
sample.