Nitrogen-Containing Macromolecules in the Bio - American Chemical

Chapter 19

Organic Geochemical Studies of Soils from Rothamsted Experimental Station: III Nitrogen-Containing Organic Matter in Soil from Geescroft Wilderness 1,3

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Pim F . van Bergen , Matthew B. Flannery , Paul R. Poulton , and Richard P. Evershed

Downloaded by INDIANA UNIV BLOOMINGTON on May 9, 2015 | http://pubs.acs.org Publication Date: June 29, 1998 | doi: 10.1021/bk-1998-0707.ch019

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1

Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom Soil Science Deparment, IACR-Rothamsted, Harpenden, Herts A L 5 2JQ, United Kingdom 2

Three distinct soil horizons from a mature oak dominated woodland were studied in order to determine the changes in the molecular composition of nitrogen-containing organic matter down a soil profile. The total amount of nitrogen relative to soil organic carbon increased down the profile with most of the recognizable nitrogen-containing compounds in the leaf litter and humic horizon being either amino acid or amino sugar derived. In contrast, a significant proportion of the organic nitrogen moieties in the mineral horizon appeared to contain macromolecular-bound nitrogen which is believed to represent the so -called 'unknown' soil organic nitrogen and is not obviously related to known biomolecules. The increase in total amino acids in the humic and mineral horizons indicated contributions from sources other than the leaf litter. The increase in organic nitrogen-containing moieties, most probably amino acid derived, accounted for the less depleted δ C values observed in the mineral soil horizon. 13

Different forms of organic nitrogen have been recognized in soils including, proteins, amino acids, amino groups, ammonium ions, hexoamines and nucleic acids (1-4). However, it has long been known that substantial amounts, sometimes up to 50%, of the organic nitrogen occurring in the organic matter of soils are present as so-called 'unidentified' (3) or 'unknown' forms (2-5). The significance of these forms is that: (i) nitrogen potentially available for biological processes may become immobilized (3, 6), and (ii) that this organic nitrogen appears more difficult to assimilate (7). The nitrogen immobilization has been considered to affect soil fertility (7, 8) and to cause changes in the extent of litter decomposition, which in turn influences carbon dioxide 3

Present address: Organic Geochemistry Group, Faculty of Earth Sciences, Utrecht University, P.O. Box 80021, 3508 T A Utrecht, The Netherlands.

© 1 9 9 8 American Chemical Society

In Nitrogen-Containing Macromolecules in the Bio- and Geosphere; Stankiewicz, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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emission to the atmosphere (6). Despite the importance o f organic nitrogen, the origin and exact molecular composition o f 'unidentified' or 'unknown' structural moieties as well as the detailed molecular composition o f soil organic nitrogen i n general, are still poorly understood (5). The main problem with characterizing the 'unknown' or 'unidentified' organic nitrogen constituents o f soils is that they derive from complex biological macromolecules the structures o f which have been altered through a wide variety o f poorly understood complex chemical and microbiological soil processes. Treatment of whole soils or soil fractions with 6 M HC1 w i l l yield amino acids which can be analyzed by H P L C . Whilst the profiles produced by such analyses are o f value in assessing overall trends i n the degradation o f proteins and individual amino acids this approach is less useful i n determining the nature o f highly altered non-proteinaceous components (3, 4). Schnitzer was one o f the first to attempt to identify components present i n this nitrogen pool with 'unknown' Ν fractions being isolated using Sephadex gels o f various sizes (9). The results obtained indicated that heterocyclic N-components are major contributors to the 'unknown' N-pool. More recently, two other techniques have been applied to study the 'unidentified' N , namely pyrolysisgas chromatography-mass spectrometry (4, 5) and solid state N N M R (10, 11). Interestingly, the pyrolysis data largely corroborated Schnitzer's interpretations (4, 5) whereas the N M R results revealed mainly amides and terminal amino groups with only minor signals resulting from heteroaromatic structures. The few studies that have been performed have highlighted the fact that the chemical nature o f the 'unknown' N-containing components o f soil is a potentially highly complex field worthy o f further investigation. With this i n mind we decided to initiate a systematic study o f the nitrogenous components o f soil using a hierarchical analytical approach which sought to relate the bulk chemical properties, e.g. elemental composition, o f the soil to molecular and isotopic data obtained following various chemical treatments. 1 5

The objective o f this paper is to study the organic nitrogen fraction present i n solvent-insoluble organic matter from three soil horizons at Geescroft Wilderness, Rothamsted Experimental Station. The history o f this site and the annual input of organic matter to it are well-documented (11-14). The solvent-insoluble material was treated with base and, subsequently, acid to determine the mode o f occurrence o f the organic nitrogen. Elemental analyses, bulk stable isotopes, amino acid analyses and flash pyrolysis-gas chromatography-mass spectrometry were used to investigate the molecular composition o f nitrogen-containing soil organic matter down this soil profile. Sample Description Samples were taken from three soil horizons o f Geescroft Wilderness at Rothamsted Experimental Station, Harpenden, Herts., U K , i n M a y 1995. This site is located i n a small area o f once-arable land (1.3 ha). The land had long been cultivated as shown on a map dated 1623. The present site was part o f an experimental field growing beans from 1847 to 1878, with frequent breaks towards the end o f that period.


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Subsequently it was fenced off and allowed to revert to natural woodland i n 1883 (72, 13). The site has never been chalked, except for receipt o f a little lime on occasions prior to reversion. This is reflected i n the decreasing soil p H (in H 0 ) 7.1, 6.1, 4.5 and 4.2 i n 1883,1904,1965 and 1991 respectively. The soil organic carbon input has been estimated at 2.5 t h a yr" , with leaf-fall being the main contributor (estimated 1.57 t ha" yr" ; 13). Dead roots, dead mycorrhizae, root exudates etc. have been suggested as minor contributors to the soil organic matter (13). For additional information about this site the reader is referred to references 12-14. The top horizon sampled was a leaf litter predominantly composed o f Quercus robur leaves. The underlying soil was sampled using a 2 c m diameter auger to a depth o f 23 cm. The soil sample was subsequently subdivided into a humic rich brown top horizon (ca. 5 cm) and a light brown mineral soil horizon (ca 18 cm). Replicates o f the underlying soil were taken at approximately 5 m from the first sampling location. The soil at the site is a silty clay loam also classified as Chromic Luvisol (75) or A q u i c Paleudalf (16). 2

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1

1

1

Experimental Soil and litter samples were initially oven dried at 60°C. Samples were crushed with a pestle and mortar and sieved over 2 m m and 75 μ ι η sieves; the litter sample was only sieved over 2 mm. The leaf litter was solvent extracted (dichloromethane/acetone, 9:1 v/v) by ultrasonication whereas the soils were Soxhlet extracted for 24 h using the same solvent mixture. The insoluble residues obtained were vacuum dried. Extracted residues were base treated using a 1 M potassium hydroxide solution i n 96% methanol. The suspensions, under N , were heated to reflux for one hour at 70°C. After cooling, the reaction mixtures were acidified to p H 3 using 2 M hydrochloric acid i n methanol/water (1:1 v/v). After centrifugation (1 min, 3000 rpm), the residues were extracted with water (3x), methanol/water (1:1 v/v; l x ) , methanol (2x) and dichloromethane (3x). After each extraction step the suspension was centrifuged and the supernatant was removed. The residues after base treatment were vacuum dried. 2

A c i d treatment was performed by suspending the residue after base extraction in a 4 M hydrochloric acid solution i n water. The samples were heated for six hours at 105°C. After cooling the samples were neutralized using a 16 M potassium hydroxide solution i n water. Mixtures were centrifuged (1 m i n at 3000 rpm) and the residues obtained were again saponified and extracted as described above. These final residues were vacuum dried. A l l residues (insoluble, base and acid treated) were studied using elemental analysis ( E A ) , bulk stable carbon isotope measurements ( I R M S ) and flash pyrolysis-gas chromatography/mass spectrometry ( P y - G C / M S ) . The insoluble residues and residues after base treatment were also studied for their amino acid contents. Individual free amino acids were obtained using an acid treatment in an evacuated reaction tube sealed with a P T F E lined cap (6 M H C I 1:500 w/v, 100°C, 24 hr) (7 7). Excess acid was evaporated at 40°C using a stream o f dry N and 30% o f the 2


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hydrolysate was transferred to a reaction vial. The solvents were removed by evaporation. Free amino acids contained i n the hydrolysate were derivatized to the phenylisothiocyanate (PITC) derivatives by means o f the P i c o - T A G method (18). The reagent mixture added to each sample consisted o f 2.0 μΐ triethylamine, 2.0 μΐ double distilled H 0 , 2.0 μΐ phenylisothiocyanate, and 15 μΐ HPLC-grade methanol. The vial was sealed with a P T F E lined cap, the reagents agitated to m i x and left to react for 10 minutes at room temperature. Excess reagent mixture was removed by evaporation. Separation o f the amino acids was accomplished using a 30 c m PicoTag column with an elution profile as described by Suleiman (19). A m i n o acids were detected at 254 nm using a Waters 486 U V detector. Quantification o f the amino acids was based on the peak areas o f standard amino acids o f known concentration and normalized to the dry weight o f the samples.


2

Elemental analyses were performed using a Perkin Elmer 240C elemental analyzer. Values stated are based on duplicate analyses (Table I). I R M S analyses o f the residues were performed i n duplicate using a Carlo Erba N C 2 5 0 0 elemental analyzer coupled to a Finnigan M A T Delta S instrument. A l l ô C values have been corrected against a N I S T sucrose standard and are relative to the P D B standard. P y - G C / M S analyses were performed using a Carlo Erba 4130 gas chromatograph, equipped with a C D S 1000 Pyroprobe device (Chemical Data System, Oxford, Pennsylvania) connected to a Finnigan M A T 4500 mass spectrometer. Samples were loaded into quartz tubes into the C D S 1000 Pyroprobe. The temperature o f the pyrolysis interface temperature and G C injector was set at 250°C. Pyrolysis time was 10 seconds at 610°C. The G C oven was programmed from 35°C (5 min) to 310°C (10 min) at a rate o f 4°C min" . Separation was achieved using a fusedsilica capillary column (50 m χ 0.32 mm) coated with CPSil-5 C B (film thickness 0.4 μιη). Helium was used as the carrier gas. The M S was operated at 70 e V scanning the range m/z 35-550 at a cycle time o f 1 s. Compound identifications were based on mass spectral data and retention time comparisons with reference samples and data reported in the literature (20-24). 1 3

1

Results Elemental and Isotope Measurements. The replicate soil samples o f the mineral layer were similar i n overall elemental composition whereas those o f the humic layer showed a certain disparity i n the amounts o f C and N (Table I) which is most likely due to differences i n the contributions o f minerals (58.8% vs. 75.8% ash). The elemental compositions o f all three soil horizons clearly revealed the reduction o f total Ν and organic C content o f the residues following the base and acid treatments. This is, however, not revealed i n the C / N o f the residues after base treatment due the greater loss o f carbon relative to total N . o r g

t o t a l

13

ô C values o f insoluble residues of the humic rich top layer are very similar those o f the leaf litter. In contrast, the organic matter present i n the mineral soil relatively enriched i n C when compared with the overlying horizons. The ô values o f the various samples show no specific trend as a result o f base treatment. 1 3


13

to is C In


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Table I. Bulk elemental and stable carbon isotope data from the three soil horizons. N.D. = not determined. N/A = not applicable as N , = 0. Values in parenthesis are corrected for the weight loss during the base and acid treatments. toU

Sample

c .(%) org

Leaf litter

insoluble 46.08 base treated 43.40 (20.83) base and acid treated 48.55 (9.08)

N

total

1

19.29/9.59 1.47/0.91 14.77/6.33 1.32/0.82 (12.26)/(5.63) (1.10)/(0.73) base and acid treated 9.80/4.08 0.23/0.14 (6.27)/(2.49) (0.15)/(0.09) 1

1

C/N

5 C (%>)

0.63 N.D. N.D.

28.7 34.0 103.9

-27.9 -27.3 -29.4

58.,78/75.84 N..D./N.D.

15.3 /12.3 13.1/ 9.1

-27.8/-28.0 -27.8/-27.9

N..D./N.D.

49.7/35.3

-29.4/-29.7

93.,28/92.31 N,.D./N.D.

4.7/ 5.5 2.8/ 5.4

-26.4/-26.6 -27.1/-26.2

N,.D./N.D.

13.6/N/A

-27.9/-27.3

1.88 1.15(0.55) 0.55 (0.10)

Humic layer insoluble base treated

Mineral soil insoluble base treated

Ash (%)

(%)

1.77/1.78 1.20/1.28 (1.08)/(1.14) base and acid treated 0.70/0.52

0.44/0.38 0.49 / 0.26 (0.44)/(0.23) 0.06/0.00

(0.57)/(0.33)

(0.05)/(0.00)

,3

Values for the replicate soil samples are reported separately


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contrast, the residues after both base and acid treatment were depleted i n

1 3

C


compared with the untreated soil (Table I). Pyrolysis. The pyrolysis products o f insoluble residue o f the leaf litter (Fig. la) are primarily dominated by methoxyphenols, furan and pyran derivatives indicative of dicotyledonous angiosperm ligno-cellulose (25). Only relatively few nitrogencontaining products have been detected including, acetamide (18, note that this product is present as a hump rather than a single peak), pyrrole (1), benzeneacetonitrile (16), indole (8) and diketodipyrrole (23; Table II). Other relatively abundant pyrolysis products, without nitrogen, which may have been derived from nitrogen-containing moieties include toluene and phenol (26) although these may originate from other sources, including ligno-cellulose. The pyrolysate of insoluble residue o f the humic layer (Fig. l b ) resembles that o f the leaf litter revealing evidence o f a significant ligno-cellulose component. A substantial number o f nitrogen-containing products are also identified including, pyrrole (1), (^-pyridine (7), indole (8) and diketodipyrrole (23; Table II). Toluene, styrene and phenol are also present. The most abundant N-containing products were detected i n the pyrolysate o f insoluble residue o f the mineral soil (Fig. l c ; Table II) including pyrrole (1), pyridine (6), benzonitrile (14) and diketodipyrrole (23). The most abundant pyrolysis products are, toluene and phenols, as well as styrene and Cj phenols. Notably, this sample also revealed the presence o f a number o f alkyl nitriles (C , C , C and C ; 27-30) which were not detected i n any of the overlying layers. 14

1 8

1 6

20

The pyrolysates o f the residue after base treatment (Fig. 2) were similar i n composition to those o f the extracted insoluble residues (Fig. 1). The most significant differences observed are the increase i n the abundance o f cyclopentenones (Fig. 2) and a significant reduction i n the abundance o f levoglucosan and 4-ethenylphenol in the leaf litter. The distribution o f nitrogen-containing pyrolysis products changed little except for a decrease i n relative abundance o f the alkyl nitriles i n the mineral soils (cf. F i g . 2c vs. Fig. l c ) . The principal changes i n the macromolecular composition o f the organic matter i n the samples occur after the acid treatment. The pyrolysates o f the leaf litter residue after acid treatment (Fig. 3a) still shows evidence o f lignin based on the methoxyphenols but the characteristic polysaccharide component is now undetectable. However, it should be noted that the distribution o f the specific lignin markers is drastically altered when compared with the original litter (cf. F i g . la). N containing compounds are virtually absent with the exception o f pyrrole (1), N methylpyrrole (2), pyridine (6) and diketodipyrrole (23; not shown). The pyrolysis products o f residue after acid treatment o f the humic layer (Fig. 3b) showed the same chemical alteration o f the lignin-derived markers (drastic change o f distribution pattern) as observed i n that o f the leaf litter (Fig. 3a). Nitrogen compounds similar to those i n the base treated and extracted residues were still detectable, although i n much lower relative amounts. Important non-nitrogenous pyrolysis products, phenol, toluene and styrene, are still relatively abundant (22, 26). The dominant pyrolysis products o f the mineral soil after base and acid treatment (Fig. 3c) are N -


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Quercus

leaf litter a) x


A?-alk-1-enes • n-alkanes

Humic layer b)

Mineral soil C )

p=9 OH

G= 9+10

T^OCHj OH

g

s=

JUL HjCO

T^OCH

3

Γ 20

40

Retention time (min)

60

•

Figure 1. TICs o f the pyrolysates o f the solvent insoluble residues o f a) Quercus leaf litter, b) humic rich top horizon and c) mineral soil horizon. K e y : Ρ = phenol; 3+4P = co-eluting 3- and 4-methylphenol; C F A = hexadecanoic acid; 1-Pr:l = Prist- 1-ene; H E = Hemicellulose marker (4hydroxy-5,6-dihydro-(2//)-pyran-2-one); L G = Levoglucosan; C N i = hexadecanenitrile; x= «-alk-1-enes; · = w-alkanes; * = contaminants. Numbers i n bold refer to compounds listed i n Table II. Side chains (attached at positions 4) o f phenol- (P), 2-methoxyphenol- (guaiacyl; G ) and 2,6-dimethoxyphenol- (syringyl-; S) components are indicated. For additional information and source of the material see text. 1 6

1 6


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Table Π. Nitrogen-containing pyrolysis products detected in the pyrolysates of the three different soil horizons.


Pyrolysis products

Origin

a

Mw

Leaflitter^

Humic layer^ Mineral soil^

1

2

3

1

2

3

1

2

3

+ +++ +++ ++ + + + +++ + + + + + + + + + + + +

Pro, Hyp, Glu AA / unknown Pro, Hyp Pro, Hyp Hyp

67 81 81 81 95

+ + -

+ + + + -

+ + -

++ + + + -

++ + + + +

Pyridine C\ -pyridines

AS/Ala AS /Ala

79 93

+ -

+ -

+ -

+ ++

+ +

+ +

++ +

++ +

+ +

8 9 10

Indole 3-Methylindole C\ -Indole

Trp Trp unkown

117 131 131

+ +

+ +

-

+ +

++ +

+ + +

++ + +

++ + +

+ -

11 12

Quinoline ? Isoquinoline ?

unknown unknown

129 129

13 14 15 16 17

Benzenamine Benzonitrile Ci-Benzonitriles Benzenacetonitrile Benzenepropanenitrile

unknown unknown unknown Phe Phe

18 19 20 21 22

Acetamide Acetylpyrrolidone 3-Acetamido-5-methylfuran 3-Acetamido-4-pyrone or 3 - Acetam ido-2-pyrone Oxazoline structures

23 24 25 26

Diketodipyrrole 2,5-Diketopiperazines der. 2,5-Diketopiperazines der. 2,5-Diketopiperazine

27 28 29 30

Tetradecanenitrile Hexadecanenitrile Octadecanenitrile Eicosanenitrile

unknown unknown unknown unknown

209 237 265 293

1 2 3 4 5

Pyrrole N-methylpyrrole 2-Methylpyrrole 3-Methylpyrrole C2-pyrroles

6 7

-

-

-

-

-

-

-

+ +

+ +

+ +

+ +

-

-

93 103 117 117 131

+

-

-

+

+

+ +

+

-

+ +

+ +

? ++ + + +

++ + + +

++ + +

-

+ + + +

AS AS AS AS

59 127 139 153

+ -

+ -

-

+ + -

+ + + +

_

+ -

+ -

AS

185

-

?

-

+

+

-

-

-

-

Hyp-Hyp Pro-Val, Pro-Arg Pro-Ala Pro-Pro

186 154 168 194

+ -

+ + -

+ -

++ + + +

++ + + +

+ + -

++ +

++ + +

? ? -

+ + -

_

-

+ + + +

-

-

-

-

-

-

-

-

-

A A = amino acid; AS = amino sugar; Pro = Proline; Hyp = Hydroxyproline; Glu = Glutamine; Ala = Alanine; Trp = Tryptophan; Phe = Phenylalanine; Val = Valine; Arg = Arginine - = not detected, + = present, ++ = abundant, +++ = very abundant.


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Quercus leaf litter

x

n-alk-1-enes


• n-alkanes

CO

c ω > '·*—·

ω α:

Retention time (min) Figure 2. T I C s o f the pyrolysates o f the residues after saponification o f a) Quercus leaf litter, b) humic rich top horizon and c) mineral soil horizon. For key to symbols see caption o f Figure 1.


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Quercus leaf litter a)

x

G

n-alk-1-enes

• n-alkanes

G


Nitrogen-Containing Macromolecules in the Bio - American Chemical

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