acetylacetone extraction of soil organic sulphur and ... - Science Direct

0038-07 f 7190 $3.00 + 0.00 Copyright C 1990 Pergamon Press plc

Soil Bid. Biochem. Vol. 22. No. 1, pp. 97-104, 1990

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ACETYLACETONE EXTRACTION OF SOIL ORGANIC SULPHUR AND FRACTIONATION USING GEL CHROMATOGRAPHY J. I. KEER,* R. G. McLmENt

and

R. S. SwIFrt

Edinburgh School of Agriculture, West Mains Road, Edinburgh EHS 33G, Scotland (Accepted

16 June 1989)

Summary-An aqueous solution of acetylacetone (2 M, pH 8.0) in combination with ultrasonic treatment, was used for the extraction of organic sulphur (S) from soils. Between 80-100% of the total soil organic sulphur was extracted and the proportions of the extracted S occurring in hydriodic acid (HI)-reducible forms were similar to those determined for the whole soiis. Schemes were developed for the fractionation of acetylacetone extracted S using gel permeation chromatog~phy, either on its own or in combination with conventional separation of fractions into humic and fulvic acids. The procedures developed were used to examine the organic S present in (a) paired samples of arable and pasture soils, in (b) a mineral compared to a peat soil and in (c) a soil with recently incorporated organic S labelled with lsS. There appeared to be no consistent major differences between S concentrations in the various organic matter fractions from the arable compared to the pasture soils. In all these soils, 75% or more of the S present in the fraction with a molecular weight (MW) nominally >ZOO,OOODa, was present in HI-reducible forms. For fractions with MW nominally lXlO’

Fraction

E

J. I. KEERet al.

100

Table I. Effect of soil:extractant ratio and ultrasonic treatment on the proportion of sulphur extracted from soils % Of total S extracted

Organic Soil

(2,

Kilmarnock pasture Kilmarnock arable Stirling pasture Stirling arable

4.65 2.60 5.56 2.15

Total s &gSg-‘) 705 352 632 530

Without ultrasonification

With ultrasonification

I :45*

1:9*

1:45*

1:9*

60 31 41 49

24 36 38 54

78 80 101 loo

56 71 67 89

lSoil:extractant ratio.

were quite similar and hence the C: S ratios of the extracted materials were similar to those of the whole soils. This was not found to be the case in the present study, C : S ratios of the extracted materials were all lower than for the intact soils (Table 2). The decrease in C: S ratio was particularly marked for the Stirling soil. It has been shown (McLaren and Swift, 1977) that a large proportion of the organic S extractable from this soil (the arable soil in particular) is present in the fulvic acid fraction, an observation confirmed later in this study. Fulvic acid has a much lower C content than other humic materials (Hayes and Swift, 1978) and, together with the low C:S ratio of the whole soil, this could account for the extremely low C:S ratio of the material extracted from the Stirling arable soil. The difference between our results and those obtained by Scott and Anderson (1976) could be due to differences in the nature of the organic matter in the soils used. The soils examined by Scott and Anderson (1976) were all freely drained and tended to be considerably more acidic than the soils used in this present study. In spite of the difference in the proportions of C and S extracted by acetylacetone, the ratios of HIreducible to C-bonded S extracted, were similar to those in the whole soils (Table 2). However, there was a tendency for the proportion of HI-reducible S to be higher in extracts prepared at a soil:extractant ratio of 1: 9. The similarity of the ratios of HI-reducible to C-bonded S between whole soils and extracts suggests that both forms of organic S are equally easily extracted by acetylacetone and that the extraction process does not cause significant conversion between the two forms. or breakdown to sulphate, as has been noted with other extractants (Freney et al., 1969). Comparison of arable and pasture soils

Acetylacetone extracts obtained from the Kilmarnock and Stirling soils were fractionated using a column of Sephadex G-200 gel, and the included

fraction separated into humic and fulvic acid fractions as described in fractionation procedure 1. excluded The fractions (MW nominally >200,000 Da) contained a high proportion of mineral matter, probably very fine colloidal clay, or sesquioxide material intimately combined with organic matter. The existence and possible bonding mechanisms of such complexes have been discussed by Greenland (1971). The humic acid fractions were relatively low in ash but the fulvic acids had large ash contents (28-55% ash). Scott and Anderson (1976) observed that acetylacetone extracted substantial amounts of iron and aluminium from soils, and suggested that much was bound to organic matter in the extracts rather than complexed by acetylacetone. They also reported high ash contents, and correspondingly high iron and aluminium contents, in some of the organic matter fractions they obtained by gel chromatography (Scott and Anderson, 1976). In our study, with the procedure for fractionating organic matter extracts into humic and fulvic acids it is likely that iron or aluminium accumulated in the fulvic acid fraction. Larger amounts of organic matter were extracted from the Kilmarnock pasture soil compared with its arable counterpart (Fig. 2a), which reflected the difference in total organic matter content between the two soils. However, although there was a similar difference in organic matter content between the pair of samples from the Stirling soil (McLaren and Swift, 1977), the arable soil contained the slightly greater amounts of extracted organic matter in both the excluded and humic acid fractions. Figure 2b shows the amount of S present in the individual organic matter fractions of each soil. Comparison of the two Kilmamock soils shows that cultivation has reduced the amount of S in all three fractions whereas with the Stirling soils only the fulvic acid fraction showed any significant decrease in S content. The concentrations of S in the individual organic matter fractions are shown in Table 3. S

Table 2. Properties of whole soils and acelylacetone extracts

Soil Kilmarnock pasture Kilmamock arable Stirling pasture Stirlinn arable lSoil:extractant ratio.

HI-reducible S in extracts (“h)

C:S ratio of organic matter in whole soil

C : S ratio of extracted organic matter 1:45*

HI-reducible S in whole soils (%)

1:45’

1:9*

66 68 88 40

40 45 49 16

41 53 50 52

40 48 57 46

50 60 54 58

Extraction and fractionation

Kilmamock

:ilmarnwk

Stirling

Stirling

pasture

arable

pasture

arable

exdudad fraction

F 5 -

q

0.4

included humic acid included fulvic acid

fJ.3 I

0 Kilmarnock

Kilmamock

Stirling

Stirling

pasture

arable

pasture

arable

Fig. 2. Yields of organic matter and S in fractions from Kilmarnock and Stirling soils. in the excluded organic matter fractions (MW nominally > 200,000 Da) were similar for both soils and appeared unaffected by cultivation. The greatest S concentrations were found in the fulvic acid fractions, especially those from the Stirling soils, and the lowest in the humic acid fractions. There appeared to be no consistent major differences between S concentrations in the fractions from the arable compared with the pasture soils. The proportion of S in each fraction occurring as HI-reducible S is also shown in Table 3. In all soils 75% or more of the S present in the excluded fraction (MW nominally >200,000 Da) was present in HIreducible forms. Bettany ef al. (1979. 1980), in S fractionation studies, noted that clay-associated humic acid contained a higher percentage of HIreducible S than humic acid completely solubilized by alkaline extractants such as sodium hydroxide and sodium pyrophosphate. The clay-associated fraction of Bettany et al. (1979, 1980) would be very similar

concentrations

Table

3. S concentrations

in organic

S as % of organic

matter

matter

MW

pasture

Kilmarnock

arable

Stirling

pasture

Stirling

arable

*Nominal

MW

values

(Da).

The Stirling pasture soil and the fen peat were extracted with acetylacetone as described above, and fractionated using the step-wise fractionation procedure shown in Fig. 1. The relative yields of organic matter and S in the various fractions are shown in Fig. 3. The proportions of the S in each fraction occurring as HI-reducible or C-bonded forms are shown in Fig. 4. No data is available for the > 10,000 Da nominal MW fraction of the peat soil because insufficient material for analysis was found in the included fraction when using the Sephadex G-50 gel. For both the mineral and peat soils, there were considerable differences in the amounts and the proportions of S and organic matter present in individual MW fractions. In neither soil did the distribution of S between fractions completely parallel the distribution of organic matter, indicating that there are variation in S concentration with the MW of organic matter. However, on examination, there appeared to fractions

from

Kilmarnock HI-reducible

and Stirling

soils

S as % of total

fraction

S in fraction

Included

< 200.000*

MW

fraction

< 200,000*

fraction

Humic

Excluded fraction MW > 200.000’

Fulvic

acid

Fulvic acid

Humic

MW > 200.000*

acid

acid

I .08

0.32

1.61

15.0

30.0

Excluded

Kilmarnock

Fractionation of organic S extracted from a mineral soil and from a peat soil

in fraction

Included

Soil

101

in nature to the excluded organic matter fraction obtained in our study. Although it has been proposed that HI-reducible forms of S are more likely to be readily mineralized than C-bonded forms (McLaren and Swift, 1977; McLaren et al., 1985), Bettany et al. (1979) have suggested that the intimate association of HI-reducible S with fine clay can protect it from microbial transformation. Within the included organic matter fraction (MW nominally < 200,000 Da) for all soils, the proportion of S present as HI-reducible forms was much greater in the fulvic acid compared with the humic acid fraction. This result again agrees with the work of Bettany et al. (1979) who, using a different extractant, also found that humic acid S was largely C-bonded. These workers proposed that HI-reducible S is not readily incorporated into the aromatic units which are major constituents of humic acid molecules. There appeared to be no difference between the Kilmarnock pasture and arable soils with respect to the proportion of S occurring as HI-reducible forms in any of the three organic matter fractions examined. However for the Stirling soil the proportions of HI-reducible S in the arable soil were higher in both fulvic and humic acid fractions and lower in the excluded fraction than for the pasture counterpart (Table 3). McLaren and Swift (1977) have observed that a greater proportion of the total S is present in HI-reducible forms in arable compared to pasture soils.

(b) sulphur

E v)

of soil organic S

I

66.9

1.16

0.5

1.68

75.6

30.0

66.9

I.38

0.40

5.93

84.3

21.2

45.6

I .22

0.40

4.17

16.2

25.0

85 0

102

J. I. KEER et 01. (a) Stirling eoil

(a) Stirling soil

aor

z f g .Y ‘53 5g P

20 Fraction A

B

D

c

E

i$ z! I 1 H z

Fraction A J P .L 2 c ‘-

(b) Fen peat

C

D

E

B

C

D

E

(b) Fen oeat

00

Fraction

B

!%I

B

c

organic matter

D

mcreasingmolecularweight

E

b

Fig. 3. Distribution of organic matter and S in MW fractions from Stirling pasture and peat soils. (See Fig. 1 for nominal MW range for each fraction.) be no clear relationship between S concentration and MW, although for both soils, S concentrations were lowest in the 100,00~200,000 Da nominal MW range. Swift and Posner (1972) fractionated alkaline pyrophosphate soil extracts, using gel chromatography and found that the S concentrations in humic acids did not vary significantly with MW. For both soils in our study a large proportion of the extracted S was present in the fractions within the lOO,OOO-200,000Da nominal MW range (57 and 76% of the total extracted S for the mineral and peat soils respectively). All the prepared fractions had relatively low ash content except for the highest MW fraction from the Stirling soil. The high ash content of this fraction was almost certainly due to the presence of very fine mineral particles intimately associated with the organic matter. In both soils there was a clear trend in the proportion of S in the fractions occurring in HI-reducible forms, which increased with increasing molecular weight (Fig. 4). This presents something of an anomaly since it has been observed above (Table 3) and in previous studies (e.g. Bettany et al., 1979, 1980) that fulvic acids, considered to be relatively low MW humic substances, often have a higher proportion of their S in HI-reducible forms than higher MW humic materials. Indeed, as noted above, Bettany et al. (1979) have suggested that it is unlikely that a large proportion of HI-reducible S could be incorporated into the predominantly aromatic units of humic acids. The data shown in Fig. 4 would clearly appear to contradict this hypothesis. Similarly, the suggestion made above, that the high proportion of HIreducible S in the highest “MW” fractions was due to the presence of low MW materials in intimate association with fine colloidal clay, is certainly not true in the case of the peat. Even the highest MW fraction (nominal MW > 1 x lo6 Da) from the peat soil had an extremely low ash content indicating the

Fraction

increasingmolecularweight

n

HI-reducible S

q

b

Carbon-bonded S

Fig. 4. Proportions of S in individual MW fractions present as HI-reducible (HI-S) or C-bonded S (C-S). (See Fig. 1 for nominal MW range for each fraction.)

absence of significant amounts of inorganic colloidal materials. The discrepancy in the distribution of HI-reducible forms of S between the conventional humic-fulvic acid separation, and the MW fractionation outlined above, requires some explanation. In highly alkaline solutions, as for example those used by Bettany et al. (1980) to extract organic matter from soils, Freney et al. (1969) showed evidence of a release of HIreducible S, formerly associated with humic acid, together with a corresponding increase in HIreducible S in fulvic acid fractions. However, given the relatively mild extraction conditions in our study, such as transformation would seem unlikely. A more likely explanation for the above discrepancy is that a proportion of the HI-reducible S in soils occurs as non-humic substances, such as high MW sulphated polysaccharides. Such compounds would remain in solution when acetylacetone extracts were acidified to precipitate humic acid, and thus would have appeared in the fulvic acid fraction. However, they would have been eluted with the relatively high MW fractions using gel chromatography. It is clear that great care should be taken in interpreting the results of organic matter fractionation studies. If the forms of organic S which appear in a particular fraction are artifacts of the particular fractionation scheme, the usefulness of that scheme in characterizing soil organic S could be extremely limited. Fractionation of recently incorporated organic S

Samples of Whitsome series soil which had been incubated with “S-1abelled sulphate in the presence

Extraction and fractionation

103

of soil organic S

addition, ~30% of the S present in the included fraction was present in C-bonded forms (i.e. not HI-reducible) and thus could not have been inorganic sulphate. Therefore, overall at least 65% and possibly all of the S present in the original low MW fraction must have been in organic forms. Acknowledgement-We acknowledge the support of an Agricultural Research Council grant for these studies.

REFERENCES (b) withaddedglucose

F : ii

3.0

I

A

-

-----

otpmicma”l a”lph”P35

SO

1

40

^

! L?

i 20

Archer E. E. (1956) The determination of small amounts of sulphate by reduction to hydrogen sulphide and titration with mercuric or cadmium salts with dithizone as indicator. Analyst, London 81, 181-182. Bettany J. R., Stewart J. W. B. and Saggar S. (1979) The nature and forms of sulfur in organic matter fractions of soils selected along an environmental gradient. Soil Science Society of America Journal 43, 981-985.

Bettany J. R., Saggar S. and Stewart J. W. B. (1980) Comparison of the amounts and forms of sulfur in soil organic matter fractions after 65 years of cultivation. Soil Science Society of America Journal 44, 7&75.

Fig. 5. The fractionation of organic matter and “S m extracts from Whitsome soil using Sepharose 6B. V,, = void volume, V, = total column volume.

Ensminger L. E. and Frtney J. R. (1966) Diagnostic relationships for determining sulphur deficiencies in crops and soils. Soil Science 101, 283-290. Fox R. L., Olsen R. A. and Rhoades H. F. (1964) Evaluating the sulphur status of soil by plant and soil tests. Soil Science Society of America Proceedings 28, 243-246.

or absence of added glucose (McLaren et al., 1985) were extracted with acetylacetone and fractionated using Sepharose 6B gel as described above (procedure 3). Figures 5a and b show the organic matter and “S distribution patterns for the two samples. “S was incorporated into organic molecules of all MW within the range 10,000-l x lo6 with distinct peaks both in the excluded fraction (high MW) and just prior to the total column volume (low MW). The extract from the soil with added glucose showed a greater degree of “S incorporation than for the soil without glucose, especially in the excluded, high MW peak (Fig. 5b). As noted with the previous fractionation, this peak could be due to either high MW compounds or possibly lower MW compounds associated with fine clay particles. For both extracts there was a noticeable difference between the distributions of organic matter and 35Sin the included portions of the elution curves. In particular the included organic matter peak in both cases was eluted prior to the included peak for 3sS. This would suggest that a distinct low MW, relatively high S concentration fraction of recently formed organic S had been produced during incubation of the soils. However, since these peaks were eluted very close to the total column volume it was thought that they may have been due to “S-1abelled inorganic sulphate released by the extraction and ultrasonic dispersion procedures. In order to eliminate this possibility, the material from this low MW peak was refractionated using a column of Sephadex G-25 gel, which has a MW exclusion limit of only 5000 Da. Approximately 50% of the material fractionated appeared in the excluded fraction

and hence had a nominal

MW > 5000 Da. In

Freney J. R., Melville G. E. and Williams C. H. (1969) Extraction, chemical nature and properties of soil organic sulphur. Journal of the Science of Food and Agriculture 20, 44cL445.

Freney J. R., Melville G. E. and Williams C. H. (1970) The determination of carbon bonded sulphur in soil. Soil Science 189, 310-318. Greenland D. J. (1971) Interactions between humic and fulvic acids and clays. Soil Science 111, 34-41. Halstead R. L., Anderson G. and Scott N. M. (1966) Extraction of organic matter from soils by means of ultrasonic dispersion in aqueous acetylacetone. Nature, London 211, 1430-1431. Hayes M. H. B. and Swift R. S. (1978) The chemistry of Soil Organic colloids. In The Chemistry of Soil Consfituents (D. J. Greenland and M. H. B. Hayes, Eds), pp. 179-320. John Wiley, Chichester. Laing D. (1976) The soils of the country around Perth, Arbroarh and Dundee. Memoirs of the Soil Survey of Great Britain. H.M.S.O. McL.aren R. G. and Swift R. S. (1977) Changes in soil organic sulphur fractions due to the long term cultivation of soils. Journal of Soil Science 28, 445-453. McLaren R. G., Keer J. 1. and Swift R. S. (1985) Sulphur Transformations in soils using sulphur-35 labelling. Soil Biology & Biochemistry 17, 73-79.

Ragg J. M. (1960) The soils of [he country around Kelso and Lauder. Memoirs of the Soil Survey of Great Britain. H.M.S.O. Ragg J. M. and Futty D. W. (1967) The soils of the counfry round Haadingron and Eyemouth. Memoirs of the Soil Survey of Great Britain. H.M.S.O. Scott N. M. and Anderson G. (i976) Sulphur, carbon and nitrogen contents of organic fractions from acetylacetone extracts of soils. Journal of Soil Science 27, 324-330. Sinclair A. G. (1973) An autoanalyser method for determination of exchangeable sulphate in soil. New Zealand Journal of Agriculrural Research 16, 287-292. Spencer K. and Freney J. R. (1960) A comparison of several procedures for estimating the sulphur status of soils. Australian Journal of Agriculrural Research 11, 948-959.

J. I. KEERet al. Steinbergs A., iismaa O., Freney J. R. and Barrow N. J. (1962) Determination of total sutphur in soil and plant material. Rnaiytiea Chimicu Acta SO, 158-164. Stevenson F. J. (1982) Organic phosphorus and sulfx compounds. In Humus Chemistry, pp. 120-14.5. John Wiley, New York. Swift R. S. (1985) Fractionation of soil humic substances. In Humic Substances in Soil, Sediment and Water

(G. R. Aiken, D. M. McKnight, R. L. Wershaw and P. MacCarthy, Eds), pp. 387-408. John Wiley, New York. Swift R. S. and Posner A. M. (t971) Gel chromato~aphy of humic acid. Journal of Soil Science 22, 237-249. Swift R. S. and Posner A. M. (1972) Nitrogen, phosphorus and sulphur contents of humic acids fractionated with respect to molecular weight. Journnl cf Soil Science 23, 5&57.