Lipids of soil humic acids. I. The hymatomelanic acid fraction

The Science of the Total Environment, 81/82 (1989) 409-420 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

409

LIPIDS OF SOIL HUMICACIDS. I. THE HYMATOMELANIC ACID FRACTION J. O. GRIMALT

1

and C. SAIZ-JIMENEZ

2

IDepartment of Environmental Chemistry (CID-CSIC), Jordi Girona, 18, 08034-Barcelona, Spain. 21nstitute of Natural Resources and Agrobiology (CSIC), P.O. Box I052, 41080-Sevilla, Spain.

SUMMARY Gas chromatography and gas chromatography-mass spectrometry have been used for the analysis of the hymatomelanic acids of four soil samples (a Typic Xerorthent, a Typic Rendoll and two Typic Xerochrepts, on shale and on granite) encompassing a wide diversity of properties in terms of pH, organic carbon, nitrogen content and associated vegetation. These humic fractions are largely composed of distributions of C12-C34 fatty acids where microbial and higher plant contributions may be observed. Mixtures of C27-C29 stera-3,5-dien-7-ones are also present, representing microbial oxidation products of aZ-sterols. Despite the wide diversity of soils considered no major qualitative differences have been observed. In all cases the corresponding hymatomelanic acids are composed of a rather uniform pattern of aerobic microbial components together with higher plant lipids.

INTRODUCTION As early as 1889 Hoppe-Sey]er (ref. I) introduced the term "hymatomelanic acid" for the definition of the ethanol-soluble fraction of humic acids. Since then few studies have been focussed on the analysis of this humic fraction and many uncertainties

concerning i t s

structure

and chemical composition s t i l l

remain to be clarified. Clark and Tan (ref. 2), based on infrared spectroscopy analysis (IR), stated that hymatomelanic acids are esters composed of humic substances linked to polysaccharides. Subsequent results from pyrolysis-mass spectrometry (PY-MS) showed that polysaccharides and lignin were most l i k e l y the major components (ref. 3). However, further pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS) studies have revealed that contributions from fatty acids and other aliphatic materials are also important and perhaps predominant (ref. 4). These latter results have evidenced the need for a detailed characterization of the individual molecular components of these humic fractions.

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1989 Elsevier Science Publishers B.V.

410

Gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS) may be particularly useful

in this

respect,

especially in view of the predominant

components identified by PY-GC-MS (ref.

4),

Surprisingly,

no reports

are

available on the use of these techniques for the study of hymatomelanic acid fractions. The use of GC and GC-MS represent an obvious l i m i t a t i o n to the analysis of the v o l a t i l e or v o l a t i l i z a b l e components, those generally involving MW < 600-700. However, this approach has been found particularly useful for the study of the

solvent soluble organic matter

of

sedimentary materials,

affording

significant information on the predominant inputs, depositional conditions and degree of transformation of the deposited organic matter. The use of these techniques for the analysis of hymatomelanic acids can also provide data on t h e i r " l i p i d composition" to be comparedwith that obtained from these previous studies. METHODS The soil samples were air-dried and powdered to pass a 2 mm sieve. Before extraction, plant debris was removed by f l o t a t i o n . Batches of 400 g of the sieved sample were extracted with a cold l : l NaOH under N2 .

solution of O.l M Na4P207 and O.l

The extraction was repeated with fresh

solvent

until

no

appreciable amounts of humic acid could be removed (about 9 treatments). The extracts were centrifuged at lO,O00 g, acidified to pH 2 with HCI, and the precipitated humic acid was removed by centrifugation. I t was dissolved again in O.l N NaOH, centrifuged at 15,000 g to remove mineral residues, then acidified and washed with O.l N HCI until colorless. After a de-ashing treatment with a d i l u t e HF/HCI solution (5 ml 48% HF + 5 ml 36% HCI + 990 ml H20) for 24 hours and subsequent centrifugation, i t was repeatedly washed with O.l N HCI, dialyzed against d i s t i l l e d water for one week and freeze-dried. The hymatomelanic acid fractions of these humic acids were obtained by Soxhlet extraction with ethanol. The process was carried out until no coloured material was observed when siphoning (about 72 hours). The ethanol solution was then

concentrated

by

vacuum rotary

evaporation

to

almost dryness

and

re-dissolved with a small volume of ethyl ether. Solutions of diazomethane in ethyl ether were used for e s t e r i f i c a t i o n . GC-MS analyses were performed before and after methylation of the extracts. The GC analyses were performed using a Carlo Erba 4160 GC instrument equipped with a 20 m x 0.5 mm i . d . fused s i l i c a column coated with SE-54. Hydrogen was the carrier gas. The temperature was programmed from 60°C to 310°C at 6°C/min (detector temperature: 340°C). Injection was on column. The GC-MS analyses were carried

out

with

a

Hewlett-Packard Model 5995 instrument

coupled to

a

Hewlett-Packard Model 300 data system. The chromatographic conditions were similar to those described above except for the carrier gas which was helium.

411

The mass spectrometer temperatures were: transfer oven 300°C, ion source 200°C and m u l t i p l i e r 230°C. The quadrupole was scanned from m/z 40 to 540 at l s per decade. The elemental composition of the humic and hymatomelanic acids was determined using two Carlo

Erba Elemental Analyzers,

models ll06

and 1500. Several

replicates per sample were analyzed u n t i l the observed dispersion of the results had comparable values to the instrumental precision (around 0.I-0.3%). RESULTS AND DISCUSSION The soil samples used in this study were taken from the A horizons of a Typic Rendoll, a Typic Xerorthent and two Typic Xerochrepts on different parent rocks (granite and shale). These selected examples encompass a wide d i v e r s i t y of soil types in terms of pH, organic carbon, nitrogen content and associated vegetation (see Table l ) . All the soils were located in the northern part of the province of Huelva, southwestern Spain.

TABLE 1 Some properties and characteristics of the soils included in the present study.

Soil

Location

pH

6~

N~

(H20)

Altitude

Typic Xerorthent Caflaveral Leon 5~6

6.9

1.2

720

Typic Rendoll

3.3

0.3

512

HiEuera Sierra 7.7

VeEetation

(m)

Typic Xerochrept

Santa Olalla

5.6

3.5

0.4

480

(Eranite) Typic Xerochrept (shale)

Puerto Moral

6.8

1.2

0.1

575

Quercus coccifera, Clstu8 albidus, C. ealvifolius Cistue albidus, C. ladaniferus Prairie of gramineous, Medica||o and Trifolium Quercus ilex, Lavandula sp. Cistus salvlfolius,

C. crispus

The elemental composition of the humic acids isolated from these soils is presented in Table 2. The percentage values are similar to those reported for other humic substances (refs. 3,5) and f a l l within the ranges and means reported for humic acids of soils from widely d i f f e r i n g climatic zones (ref.

6).

The

composition of the corresponding hymatomelanic acids is also shown in Table 2. No major differences are observed between these hymatomelanic fractions and the bulk humic acids. However, the N/C ratio of the ethanol-soluble material

is

s i g n i f i c a n t l y lower than t h a t of the whole humic extract, suggesting that peptides and other nitrogen-containing molecules tend to remain in association with the unextractable polymeric structure. A higher H/C ratio is observed in the hymatomelanic acids but the difference with respect to the bulk material is only s i g n i f i c a n t in one case (Typic Xerorthent).

humic

412

TABLE 2 Elemental composition of the hymatomelanic acid fraction and total humic acids of the soil samples studied.

Typic Typic Typic Typic

C Xerorthent 50.55 Rendoll 56.56 Xerochrept (granite) 57.27 Xerochrept (shale) 61.88

HYMATOMELANIC H N 7.05 1.28 6.05 2.24 6.84 1.34 7.97 1.28

S 0.34 0.43 0.43 0.55

ACIDS H/C 1.7 1.3 1.4 1.5

N/C 0.020 0.032 0.019 0.017

Typic Typic Typic Typic

C Xerorthent 54.55 Rendoll 55.50 Xerochrept (granite) 52.76 Xerochrept (shale) 49.95

HUMIC H N 5.58 3.55 5.55 4.23 5.97 4.37 5.87 4.21

ACIDS 0 36.22 34.72 36.90 39.97

H/C 1.2 1.2 1.4 1.4

N/C 0.057 0.065 0.071 0.072

The gas chromatograms

of the unmethylated

hymatomelanic

acid

fractions

isolated from the soils considered in this study are shown in Figures ] and 2. Fatty acid ethyl ester components represent about 90% of the resolved peaks. These are dominated by a group of CI4 -C20

n-alkanoic

and 2-alkenoic ethyl

esters with even to odd carbon number preference. The occurrence of fatty acids in this carbon number range is usua]ly attributed to autochthonous sources (ref. 7). Thus,

the fatty acid composition of many algae (refs. 8,9) and bacteria

(refs. 10,]]) is dominated by n-hexadecanoic acid and contains large amounts of monounsaturated

hexadecenoic

branched fatty acids,

straight

chain components,

namely iso- and anteiso-pentadecanoic

acids.

Besides

these

and heptadecanoic

acids and iso- and 10-methyl- hexadecanoic and octadecanoic acids, are present in the form of ethyl esters.

They may occur in algae (ref. ]2) but they are

common in cultures of bacteria] species (refs. 13-15) and sedimentary microbial populations

(refs.

]0,ll)

so that their

occurrence

in important

amounts

is

associated with microbial inputs (refs. 10,11,]6,]7). Another group of fatty acids found in the samples analyzed is formed by a modal distribution of even carbon numbered C22-C30 saturated homologs. These are constituents occurrence

of is

cuticular indicative

waxes of

of

higher

organic

plants

materials

(refs. derived

18,]9) from

and

their

terrestrial

vegetation. In addition to these unsubstituted monocarboxylic acids, a series of C22-C24 a-hydroxyacids

maximized

at C24, sometimes

with

slight

even

carbon

number

predominance, has been found in all samples. ~-Hydroxyacids are present in a wide diversity of organisms such as bacteria (ref. 20), yeasts (refs. 21,22), algae (ref. 23) and higher plants (ref. 23). Their biochemical formation process usually requires molecular oxygen so that they are mostly produced by aerobic biota.

413

G

215

" t "1

T

I ~ Je~d )

'

32

~ 2"II:

Figure i. Gam chromatograms of the hymatomelanic acids of a Typic Xerorthent and a Typic Rendoll (B) soilm: Peak identifications in Table 3.

(A)

414

55

38 8

7

52 47 6O

31

3

5

+

I

G

1 I

25

2"~

i

28

21 1

~

Z

j

Figure 2. Gas chromatograms of the hymatomelanic acids of Typic Xerochrept (A, on shale; B, on granite). Peak identification in Table 3.

soils

415

TABLE 3 List of identified components in the hymatomelanic acids. COMPONENTS i.2.3.4.5.6.7.8.9.i0.ii.12.13.14.15.16.17.18.19.20.21.22.23.24.25.26.27.28.29.30.31.32.33.34.35.36.37.38.39.40.41.42.43.44.45.46.47.48.49.50.51.52.53.54.55.56.-

n-dodecanoate ethyl ester iso-tridecanoate ethyl ester anteiso-tridecanoate ethyl ester n-tridecanoate ethyl ester iso-tetradecanoate ethyl ester n-tetradecenoate ethyl ester n-tetradecanoate ethyl ester iso-pentadecanoate ethyl ester anteiso-pentadecanoate ethyl ester ~-pentadecanoate ethyl ester n-hexadecanoate methyl ester iso-hexadecanoate ethyl ester n-hexadecenoate ethyl ester n-hexadecenoate ethyl ester n-hexadecanoate ethyl ester heptadecenoate ethyl ester lO-methylhexadecanoate ethyl ester is___~o-heptadecanoate ethyl ester anteiso-heptadecanoate ethyl ester ~-heptadecenoate ethyl ester ~-heptadecenoate ethyl ester ~-heptadecanoate ethyl ester iso-octadecanoate ethyl ester n-octadecadienoate ethyl ester n-octadecenoate ethyl ester n-octadecenoate ethyl ester n-octadecanoate ethyl ester ~O-methyloctadecanoate ethyl ester n-nonadecenoate ethyl ester n-nonadecanoate ethyl ester n-hexadeca-l,16-dioate ethyl ester n-eicosanoate ethyl ester iso-heneicosanoate ethyl ester ~-pentacosane n-heneicosanoate ethyl ester n-docosanoate methyl ester ~-octadecen-l,18-dioate ethyl ester n-docosanoate ethyl ester ~-heptacosane n-tricosanoate ethyl ester ~-hydroxydocosanoate ethyl ester n-nonacosane n-tetracosanoate ethyl ester ~-hydroxytricosanoate ethyl ester ~-pentacosanoate ethyl ester a-hydroxytetracosanoate ethyl ester n-hexacosanoate ethyl ester ~P9 steradiene C-- steradiene 29 C^_ steradiene n-~entriacontane ~-heptacosanoate ethyl ester n-octacosanoate ethyl ester arborane cholesta-3,S-dien-7-one n-tritriacontane

SAMPLES i 2 3 4 1400 b 720 540 440 270 360 160 180 52 72 52 40 1200 150 ii0 180 480 680 370 360 -400 320 520 5200 3300 2200 2100 2800 2700 2700 2600 1900 2600 2000 2000 960 640 540 840 I00 160 120 180 2100 2000 1700 2400 2400 2200 1700 1600 1300 2800 3100 8000 24000 14000 ii000 17000 440 480 290 320 ii00 1900 i000 ii00 640 680 720 800 720 llO0 360 600 640 760 280 520 640 400 800 600 640 680 480 680 380 280 440 440 4 360 80 48 5600 4800 2700 4400 2900 4000 2100 3600 3400 2300 2300 3800 680 640 720 640 2100 1400 ii00 4000 140 96 52 160 3700 1700 920 1500 5200 1700 920 960 640 720 200 350 260 140 64 130 880 480 290 440 20 40 i0 26 3900 2400 640 680 5600 1300 1900 6000 ii0 600 80 300 2500 3800 540 1200 1800 800 440 1400 620 500 300 1200 7200 2900 1400 2300 1500 800 480 i000 2400 2500 ii00 2400 3300 2000 720 2200 2000 880 680 ii00 200 140 60 220 I0 ii0 80 200 i0 40 40 180 400 800 180 340 380 1700 250 i000 1500 1200 290 600 6 30 60 300 700 1200 1900 1500 160 200 i00 i00

416 TABLE 3 (continued) 57.58.5g.60.61.62.63.64.65.-

n-nonacosanoate ethyl ester ~4-methylcholesta-3,5-dien-7-one n-triacontanoate ethyl ester 24-ethylcholesta-3,5-dien-7-one n-hentriacontanoate ethyl ester friedelan-3-one n-dotriacontanoate ethyl ester n-tritriacontanoate ethyl ester n-tetratriacontanoate ethyl ester

TOTAL CONCENTRATION

(mE/g)

a) i, Typic Xerorthent; 2, Typic Rendoll; 4, Typic Xerochrept (shale).

380 600 1400 400 160 360 160 160 20

800 880 1900 540 140 220 180 160 40

250 340 320 860 120 40 40 40 20

400 840 460 340 120 i00 120 120 20

Ii0

88

60

92

3, Typic Xerochrept

(granite);

b) The Concentrations are expressed in ~g/g, for the fatty acids they refer to the sum of the corresponding methyl and ethyl esters.

~-Hydroxyacids in algae and bacteria are currently predominated by short chain homologs (Clo -C20). I t is therefore unlikely that the components found in the present study originate from these organisms. The :-hydroxyacids of yeasts encompass homologs in the C18-C26 range with maxima at C26 (refs. 21,22), which represents a distribution closer to that of Figs. l and 2. However, the most remarkable parallelism is observed with the

~-hydroxyacidcomposition reported

for some higher plant detritus, with predominance of C22-C 24 components and maxima at C 24 (refs. II,25). In this latter case, contributions of parasitic fungi living on these remains cannot be excluded (ref. 25). In consequence, vascular vegetation debris and/or the corresponding microbial transformation products appear as l i k e l y sources for

the hydroxyacids observed in these

hymatomelanic acid fractions, although direct microbial inputs such as yeast cell components cannot be ruled out. Dicarboxylic acids have a l s o been found in these hymatomelanic acid fractions, namely n-hexadeca-l,16-dioate and n-octadecen-l,18-dioate ethyl esters. In principle, ~m-dicarboxylic acids may originate from higher plants (refs. 11,26-27) or from the microbial oxidation of sedimented fatty acids (refs. 28,29). In the samples of Table l , the dicarboxylic acid composition parallels the predominant monocarboxylic fatty acids, n-hexadecanoic and n-octadecenoic acids, pointing to microbial oxidation of these major lipids as the most l i k e l y process for their occurrence. All these carboxylic acids are found as ethyl esters when analyzed in the untreated ethanol extracts. Only a small amount of hexadecanoic acid has been found in the form of methyl ester (peak I I in Figs. l and 2), However, after derivatization with diazomethane, a distribution of fatty acid methyl esters is observed in the GC trace of each sample. T h i s parallels that of the corresponding ethyl ester components and, from a quantitative point of view,

417

represents about the same order of concentration. According to these features, both the methyl and ethyl ester mixtures originate from a single distribution of free fatty acids, which is esterified in part during ethanol extraction. For simplicity, the unmethylated extracts are displayed in Figures l and 2. Besides these fatty acids, the other class of compounds present in important proportion in the chromatograms of Figs. l and 2 consists of a series of C27-C2g stera-5,7-dien-7-ones. They have been found in all the samples, being the predominant components among those of steroid structure. A uniform distribution of homologs is not observed but cholesta-3,5-dien-7-one predominates in most cases. These steroids are considered to originate from the microbial oxidation 5 of a -sterols via hydroperoxide intermediates and subsequent dehydratation at C-3 (refs. 30,31). Accordingly, they represent an additional

indication of

microbial a c t i v i t y in the soil samples of Table l , involving the transformation of organic materials originating from other sources. In addition to these l i p i d constituents other minor components have been identified, namely friedelan-3-one and distributions of C25-C33 ~-alkanes with odd to even carbon number predominance. These are known indicators of higher plant contributions (refs. 32,33) and their presence is consistent with the occurrence of other lipids originating from the same source, such as the above reported C22-C30 fatty acids. The uniform composition of these hymatomelanic acids deserves particular attention (see Table 3). Although changes in the relative proportion of some components may be observed, no significant qualitative differences are found. This result is in principle unexpected, especially in view of the diversity of samples selected for this study (Table l ) .

I t is however consistent with the

similarity of the elemental composition (in humic and hymatomelanic acids, see Table 2) as well as with the uniformity of the solid state nuclear magnetic resonance spectra of the corresponding humic acids (ref. 34). Obviously, more soil samples should be analyzed in order to obtain a more representative overview. These preliminary data suggest that the l i p i d signature present in the hymatomelanic acid fractions of soils essentially reflects a uniform pattern of aerobic microbial components occurring along with higher plant compounds. Finally, another aspect to be addressed concerns the representativeness, on a quantitative basis, of these l i p i d components. As indicated in Table 3, their total concentration represents between 6 and If% of the hymatomelanic acid extracts. This result is to some extent surprising in view of the components identified by PY-GC-MS (ref. 4). In any case i t indicates that only a small proportion of the ethanol-extracted material is sufficiently volatile for gas chromatographic analysis. In order to obtain some insight into the remaining fraction, the elemental composition may be considered (see Table 2). In the case that this would be constituted by very heavy aliphatic l i p i d components, H/C ratios in the order of 1.8-2 would be expected. However, the H/C values are much

418

lower, close to those of the corresponding humic acids. An important portion of the hymatomelanic acids is perhaps constituted by ethanol-extractable fragments of the same structure as the humic acids.

CONCLUSIONS Fatty acid constitute the predominant components of all hymatomelanic acid fractions analyzed. They encompass distributions in the C12-C34 range where microbial and higher plant contributions are recognized. The former are represented by mixtures of C14-C20 ~-alkanoic and n-alkenoic acids with a high proportion of branched components, namely iso and anteiso-pentadecanoic acids, and the l a t t e r by distributions of C22-C30~-alkanoic acids. The occurrence of am -dicarboxylic acids (compounds No. 31 and 37 in Table 3) and C27 "C29 stera-3,5-dien-7-ones is indicative of microbial oxidation of the predominant monocarboxylic acids and of A5-sterols. C22-C24 a-hydroxy acids constitute the only free hydroxy components identified, and they l i k e l y originate from vascular plant remains although direct contributions from microbial sources, namely yeast, cannot be excluded. Despite the wide diversity of soil samples analyzed no major qualitative differences have been found in their hymatomelanic acid extracts. In all cases a rather uniform pattern of aerobic microbial components together with higher plant lipids have been observed.

ACKNOWLEDGEMENTS We thank Prof. Jan W. de Leeuw (Delft University of Technology) for his useful comments, and Pilar Domenech (C.I.D., C.S.I.C.) for her dedication in the analysis of the elemental composition of the extracts.

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