V. Nitrogen-, Phosphorus-, and Sulfur-Containing Components of SOM

A LIFETIME PERSPECTIVE



31



Nitrogen is essential for crop production as it is an important constituent of proteins, nucleic acids, porphyrins, and alkaloids. Nitrogen is the only essential plant

nutrient that is not released by the weathering of minerals in the soil. The main

source of soil N is the atmosphere, where dinitrogen (N2) is the predominant gas.

Only a few microorganisms have the ability to use molecular N2; all remaining living organisms require combined N for carrying out their life activities. Increases

in the level of soil N occur through the fixation of N2 by some microorganisms and

from the return of ammonia and nitrate in rain water; losses are due to the harvesting of crops, leaching, and volatilization. Atmospheric ammonia originates

from the volatilization from soil surfaces, lightening, fossil fuel combustion, and

natural fires.

While a considerable amount of research has been done over the years on soil

N, most of this work has been limited to the quantitative and qualitative determinations of proteinaceous materials, amino acids, amino sugars, ammonia, and

nitrates. Reviews on soil N summarize the known organic N forms in soils

(Stevenson, 1994) as well as their mineralization and importance in plant nutrition (Mengel, 1996). Because about one-half of total soil N remains unidentified

and poorly understood, there is a need for more research and information in this

area.



B. NITROGEN DISTRIBUTION IN SOILS

AND HUMIC SUBSTANCES

Sowden et al. (1977) determined the distribution of major N compounds in samples taken from soils formed under widely different climatic and geologic conditions on the earth’s surface. While the total N contents of the samples analyzed

ranged from 0.01 to 1.61%, the proportions of total N that could be hydrolyzed by

hot 6 M HCl were quite similar, ranging from 84.2 to 88.9%. Amino acid N varied from 33.1 to 41.7%, amino sugar N from 4.5 to 7.4%, and ammonia N from

18.0 to 32.0%. Proportions of unidentified hydrolyzable N ranged from 16.5 to

17.8%, whereas those of nonhydrolyzable N ranged from 11.1 to 15.8%. Estimates

of non-protein N ranged from 55% in tropical soils to 64% in arctic soils, averaging 61% of the total N in all soils (Sowden et al., 1977). From these data it appears

that about 60% of the total soil N is non-protein or, conversely, that 40% of the total soil N is protein N. To establish whether hydrolysis with hot 6 M HCl hydrolyzed all proteinaceous materials in soils and humic substances, Griffith et

al.(1976) hydrolyzed a number of soils and humic materials first with hot 6 M HCl

and then hydrolyzed separate samples of the acid-treated residues with either 0.2

M Ba(OH)2 or 2.5 M NaOH under reflux. The results obtained showed that hot 6

M HCl released almost all of the amino acids in the soil and humic substances in

24 hr.



32



M. SCHNITZER



C. AMINO ACIDS IN SOILS AND HUMIC SUBSTANCES

The amino acid composition of soils and humic substances is remarkably similar. Sowden et al. (1977) compared the amino acid composition of soils with those

of algae, bacteria, fungi, and yeasts and found that the amino acid composition of

soils was most similar to that of bacteria. This indicates that microbes play a major role in the synthesis of proteins, peptides, and amino acids in soils.

Stevenson (1994) lists the occurrence of the following ␣-amino acids in soils—

neutral amino acids: glycine, alanine, leucine, isoleucine, valine, serine, and threonine; secondary amino acids: proline and hydroxyproline; aromatic amino acids:

phenylalanine, tyrosine, and tryptophane; acidic amino acids: aspartic and glutamic acid; basic amino acids: arginine, lysine, and histidine. Other amino acids

first detected by Bremner (1967) are ␣-amino-n-butyric acid, ␣-⑀-diaminopimelic acid, ␤-alanine, and ␥-aminobutyric acid. Stevenson (1994) identified the amino

acids ornithine, 3,4-dihydroxyphenylalanine, and taurine in soils. These amino

acids are normally not protein constituents. According to Christensen (1996), diaminopimelic acid, which originates from cell wall peptidoglycans of prokaryotes,

may account for 0.5% of the total amino acid N.



D. AMINO SUGARS IN SOILS AND HUMIC SUBSTANCES

The most prominent amino sugars detected in soils and humic substances are dglucosamine and d-galactosamine, with the former usually occurring in greater

amounts (Stevenson, 1994). Other amino sugars found in small amounts are muramic acid, d-mannosamine, N-acetylglucosamine, and d-fucosamine.



E. NUCLEIC ACID BASES IN SOILS AND

HUMIC SUBSTANCES

Anderson (1957, 1958, 1961) identified guanine, adenine, cytosine, thymine,

and traces of uracil in acid hydrolysates of HAs extracted from Scottish soils. At

a later date, Cortez and Schnitzer (1979) determined the distribution of purines

(guanine and adenine) and pyrimidines (uracil, thymine, and cytosine) in soils and

humic materials. Quantitatively, the distribution in soils was guanine Ͼ cytosine

Ͼ adenine Ͼ thymine Ͼ uracil. HAs were richer in guanine and adenine but poorer in cytosine, thymine, and uracil than FAs. The ratio of guanine ϩ cytosine to

adenine ϩ thymine was >2 for soils and humic substances. The absence of methylcytosine suggested that the nucleic acid bases extracted from soils and humic substances were of microbial DNA origin. An average of 3.1% of total N in agricultural soils, but only 0.3% of total N in organic soils, occurs in nucleic acid bases.



A LIFETIME PERSPECTIVE



F.



15N



33



NMR ANALYSES OF SOILS AND

HUMIC SUBSTANCES



Knicker and Luedemann (1995) reported that the major peak in the 15N NMR

spectrum of a wheat compost prior and after hydrolysis is due to amide/peptide

structures with minor resonances due to amino acid-and amino sugar N and

minute signals arising from indoles, pyrroles, and imidazoles. Similar 15N NMR

spectra of N compounds in peats, plant composts, whole soils, and humic materials have been published by Preston et al. (1982), Benzing-Purdie et al.

(1983, 1986), Almendros et al. (1991), Zhuo et al. (1992, 1995), Zhuo and Wen

(1992), Knicker et al. (1993, 1995, 1996), Knicker and Luedemann (1995), and

Steelink (1994). Preston (1996) noted that in all studies done so far on soils,

humic substances, and composts, 15NMR spectra recorded are very similar and

remarkably simple, consisting of one major peak due to amide/peptide and a

few minor signals arising from indoles, pyrroles, and amino acid N. Along the

same lines, Zhuo and Wen (1992) reported that in the 15N NMR spectrum of

15N-labeled HA, 86.4% of the total area is due to amide/peptide, 4.3% to

aliphatic and/or aromatic amines, and only 5.4% to pyrrole N. Similarly, Knicker et al. (1993) reported that 85% of the signal intensity in 15N NMR spectra of

15N-enriched composts and recently formed humic materials is due to amide/

peptide and that no signals in the range typical of heteroatomic N compounds

are detected. From 15N spectra recorded periodically on 15N-enriched rye

grass–wheat composted for 600 days, Knicker and Luedemann (1995) concluded that most of the detectable N is present in amide/peptide structures and

that spectra do not reveal any 15N signals that could be ascribed unequivocally

to N heterocyclics.

In contrast to the 85% of the total N in soils and humic substances occurring as

protein N as revealed by 15N NMR, chemical methods show that only 40% of the

total N occurs as protein N in these materials (Sowden et al., 1977). What are the

reasons for these wide divergencies? To provide answers to this question, it may

be useful to consider the following: because natural 15 N abundance levels in soils

and humic materials are low (0.4%), direct analysis by 15N NMR is very difficult.

Another problem is the small gyromagnetic ratio of the 15N nucleus. To overcome

these difficulties, 15N concentrations in soils and humic substances are increased

by adding 15N-labeled salts such as (15NH4)2SO4 and incubating. However, as the

work of Knicker and Luedemann (1995) shows, even incubation for 600 days does

not produce the same array of 15N compounds as those synthesized in the soil in

the presence of reactive (catalytic) surfaces over a period of hundreds or thousands

of years. It is likely that during the early stages of incubation, the microbial synthesis of proteins is the predominant reaction but that of heterocyclic N compounds

may take a much longer time.



34



M. SCHNITZER



G. DETECTION OF NITROGEN COMPOUNDS IN SOILS

AND HUMIC SUBSTANCES BY PYROLYSIS GC/MS

Using Py-FIMS and Curie-point GC/MS, Schulten and Schnitzer (1998) identified over 100 N compounds in soils and humic substances. The N compounds

identified included nonsubstituted and substituted pyrroles, pyrrolidines, imidazoles, pyrazoles, pyridines, pyrazines, nitriles, indoles, quinolines, benzothiazole,

and pyrimidines. Low-mass N compounds identified were hydrocyanic acid, dinitrogen, dinitrogen monoxide, isocyanomethane, acetamide, and hydrazoic acid. A

number of soil-specific N derivatives of benzene were also identified, including

benzeneamine, benzonitrile, and isocyanomethylbenzene. None of the latter three

compounds has so far been reported to occur in plants and microbial substances.

As to the origins of the N compounds identified, it is possible that some of these

compounds are pyrolysis products of amino acids, peptides, or polypeptides (Martin et al., 1979) or originate from the microbial decomposition of plant lignins and

other phenolics in the presence of ammonia (Bremner, 1967) or the pyrolysis of

porphyrin, a component of chlorophyll (Bracewell et al., 1987). However, there is

considerable evidence that N heterocyclics are significant components of soil N

compounds rather than degradation products of other molecules produced by pyrolysis. Arguments in favor of N heterocyclics as genuine SOM components are:

(a) some heterocyclics are formed by microbial synthesis in the soil from plant

residues or remains of animals that contain carbohydrates, proteinaceous materials, aromatic compounds, and lipids. (b) In aquatic humic substances and dissolved

organic matter (DOM) at pyrolysis temperatures of only 200 –300ЊC, Schulten et

al. (1999) have identified unsubstituted and substituted N heterocyclics such as

pyrroles, pyrrolidines, pyridines, pyrans, and pyrazoles. (c) The identification of

N heterocyclics such as those referred to earlier in soils and humic substances has

also been made without pyrolysis by gel chromatography–GC/MS after reductive

acetylation (Schnitzer and Spiteller, 1986), by X-ray photoelectron spectroscopy

(Patience et al., 1992), and by spectroscopic, chromatographic, chemical, and isotopic methods (Ikan et al., 1992).

Further research is needed to identify additional N heterocyclics in soils and humic substances and to determine whether the heterocyclis are present in the soil and

humic substances in the forms in which they were identified or whether they originate from more complex structures. If the latter is correct, we need to isolate these

complex N molecules and identify them. It is likely that many of many of the N heterocyclics identified by Schulten and Schnitzer (1998) occur in soils and humic substances in low concentrations only so that 15N NMR in its current state of development is unable to detect them. It is hoped that with substantial improvements in

instrumental design and procedures, the gulf between results obtained by 15N NMR

and chemical and mass spectrometric methods will eventually narrow.

On the basis of their data, Schulten and Schnitzer (1998) proposed the follow-



A LIFETIME PERSPECTIVE



35



ing distribution of total N in soils and humic substances: proteinaceous materials

(proteins, peptides, amino acids), 40%; amino sugars, 5 –6%; heterocyclic N (including purines and pyrimidines), 35%; and NH3-N, 19%. Thus, proteinaceous

materials and N heterocyclics are the major N components.



H. PHOSPHORUS IN SOILS AND SOM

According to Stevenson (1994), up to 75% of the total P in soils occurs in organically bound forms but less than one-half of this P has been identified so far.

Principal organic P forms include inositol phosphates (the major components),

phospholipids, nucleic acids, and traces of phosphoproteins and metabolic phosphates (Stevenson, 1994).

Inositol phosphates are esters of hexahydrocyclohexane (inositol). These esters

can occur as mono-, di-, tri-, tetra-, penta-, and hexaphosphates. Inositol phosphates form insoluble complexes with metal ions, which stabilize them so that they

tend to accumulate in the soil.

Phospholipids identified in soils include glycerophosphatides, phosphatidyl inositol, phosphatidyl choline or lecithin, phosphatidyl serine, and phosphatidyl

ethanolamine. Other organic P compounds detected in soils are glucose-1-phosphate and phosphorylated carboxylic acids (Stevenson, 1994).

The following organic P compounds have been identified in soils and soil extracts by 31P NMR: alkylphosphonic ester (RCH2PO3R1R11), alkylphosphonic

Ϫ2

acid (RCH2POϪ2

3 ), choline phosphate [(CH3 )3 N(CH2 )2PO4 ], orthophosϪ2

phate monoester (ROPO3 ), inositol phosphates, and orthophosphate diester

31P NMR include hydroxy(RO)(RO1)POϪ1

2 . Inorganic phosphates identified by

apatite [Ca5(PO4)3OH], crandallite [CaAl3(PO4 )2(OH)2(H2O)], orthophosphate

Ϫ4

Ϫ3

(POϪ3

4 ), pyrophosphate (P2O7 ), polyphosphate and trimetaphosphate (P3O9 )

(Wilson, 1990).

More recently, Bedrock et al. (1994) determined organic and inorganic P in a

HA separated from a Scottish blanket peat. The following organic P compounds

were identified: (1) phosphonate, (2) inositol hexaphosphate, (3) phosphate monoester (major component), (4) aromatic phosphate diester and nucleic acid P, and

(5) phosphate diester. The only inorganic P compound detected was orthophosphate. There is considerable potential for the use of 31P NMR for structural studies of P in humic substances.



I. SULFUR COMPOUNDS IN SOILS AND HUMIC SUBSTANCES

Plants require S for the production of proteins, vitamins, chlorophyll, glycosides, and structurally and physiologically important sulfide linkages in cell walls



36



M. SCHNITZER



and sulfhydryl groups. Most plant-available S in soils comes from the weathering

of minerals (Biederbeck, 1978). Over 90% of the total S in most noncalcareous

soils is in organic forms. The latter can be differentiated into (a) organic S that is

reduced to H2S on treatment with HI; these S forms include phenolic sulfates, sulfated polysaccharides, choline sulfate, and sulfated lipids, all of which are considered to be the most labile S forms; (b) organic S that is reduced to inorganic sulfide by Raney Nickel and which consists mainly of S-containing amino acids

(cystine and methionine); and (c) organic S that is not reduced by either HI or

Raney Nickel and which is considered to occur in the form of highly stable C–S

linkages in organic compounds.

The following organic S- containing compounds are known to occur in soils

(Stevenson, 1994): cystathionine, choline sulfate, djenkolic acid, taurine, biotine,

and thiamine. In poorly drained soils, the decomposition of organic S compounds

produces volatile S compounds such as carbon disulfide, carbonyl sulfide, methyl

mercaptan, diethyl sulfide, dimethyl sulfide, and dimethyl disulfide. So far, 34S

NMR has been of little help in identifying organic S compounds in SOM. Significant advances in 34S NMR are needed before this method can assist SOM

chemists in this respect.



VI. COLLOID CHEMICAL CHARACTERISTICS

OF HAS AND FAS

A. SURFACE TENSION, SURFACE PRESSURE, AND VISCOSITY

MEASUREMENTS ON HAS AND FAS

To obtain information on molecular sizes, shapes, and weights of HAs and FAs,

Chen and Schnitzer (1978) and Ghosh and Schnitzer (1980) did surface tension,

viscosity, and surface pressure measurements at different pHs and at varying concentrations of humic materials and neutral salts. Some of the data obtained by

Ghosh and Schnitzer (1980) are shown in Table IV, which demonstrate the effect

of pH on the molecular characteristics of FA. Note that at pH 2.0, the molecular

¯ n and M

¯ v) is four times as high as that at pH 6.5 and 9.5. At

weight of FA (both M

pH 2.0, four molecules of FA appear to combine to form an aggregate. The molec¯ 2)1/2 are also significantly greater

ular area (Aô) and the end-to-end separation (R

at pH 2.0 than at the higher pH values. Ghosh and Schnitzer (1980) concluded that

the three parameters that control the molecular characteristics of HAs and FAs are

the concentration of the humic material, the pH of the system, and the ionic

strength of the medium. From viscosity and surface pressure measurements,

Ghosh and Schnitzer (1980) infer that HAs and FAs are rigid uncharged spheroids

at (1) high sample concentration; (>3.5 to 5.0 g literϪ1), (2) low pH (6.5 for HA

and Ͻ3.5 for FA), and (3) electrolyte concentrations of 0.05 M and higher. How-



37



A LIFETIME PERSPECTIVE

Table IV

Effects of pH on Molecular Characteristics of a Spodosol FAa

pH



– nb

M



A0c (m2mgϪ1)



– 2 )1/2d (nm)

(R



– ve

M



2.0

3.5

6.5

9.5



4270

1180

1020

1080



0.044

0.030

0.024

0.026



3.03

2.34

2.10

2.27



9720

2580

2290

2450



a From



Ghosh and Schnitzer (1980).

molecular weight.

cMolecular area.

dEnd-to-end separation.

eViscosity-average molecular weight.

bNumber-average



ever, both HAs and FAs are flexible, linear polyelectrolytes at (1) low sample concentrations (Ͻ3.5 g literϪ1), (2) pH Ͼ 6.5 for HA and Ͼ3.5 for FA, and (3) electrolyte concentrations Ͻ0.05 M. The different molecular configurations are summarized in Fig. 18. In soil solutions and fresh waters, where normally both humic

and salt concentrations would be expected to be low, HA (at pH Ͼ 6.5) and FA (at



Figure 18 Macromolecular HA and FA configurations at different pH values and electrolyte concentrations. From Ghosh and Schnitzer (1980), with permission of the publisher.