iE IDENTIFICATION OF ACID SULPHATE SOILS I N NORTH-WEST MALAYA

Robert F . A llbrook Universi ty o f Malaya Malaysia

The broad conditions under which acid sulphate soils are most frequently formed

are: marine deposit parent material (Watts 1960), (Wilshaw 1940), (Moormann 1963),

a tropical climate, flat topography, and a mangrove vegetation (Thornton and

Giglioli 1965), (Pons 1964), (Marius and Turenne 1968).

The Kedah/Perlis Plain in north-west Malaya, is derived from recent marine sedi-

ments, its topography is flat, the sediment is a heavy clay, and mangroves (Rhi-

zophora spp., Avicennia SPP. and Bruguiera spp.) are found along its shore.

During the field investigations reported by Allbrook (1972a) areas of acid sul-

phate soils were encountered. The soil profiles were described, sampled, and pH

readings taken.

The acid sulphate soil Profile description did not differ significantly from

that of the non acid sulphate soils from the same general area.

The basic soil colour of the A horizon is dark grey to brown depending on the

organic status. With increase in depth the colour changes to a greenish or bluish

grey to light grey. The mottles are variable, the surface horizon has usually

reddish brown mottles due to oxidation conditions set up by the rice roots; the

mottles can be seen to be on root channels. Lower down the mottles are redder,

particularly if a B horizon is Present. At depth bluish mottles may occur.Al1 sub-

soil mottles are confined to Structure faces. Yellow mottles characteristic of acid

sulphate soils were sometimes seen. These yellow mottles, reported as jarosite

(Slager et al. 1970), were found in Malaya to be natrojarosite (Allbrook

(1970 b). In view of the closeness to the sea of many acid sulphate soils (Profi-

le l , Tables l , 2, 3 ) , the presence of sodium rather than potassium in the basic

ferric sulphate is not surprising.

Laboratory studies

A comprehensive laboratory programme was undertaken and is fully reported else-

where (Allbrook 1972 a). Selected analyses are shown in Tables I , 2, and 3 .

Profiles 1 and 2 are acid sulphate soils. Profile 3 is a typical non acid sul-

phate soil from the same area. Profiles 4 and 5 are young soils under mangrove,

Rhizophora and Avicennia respectively.

131

Laborntoiy methods are as follows: ph using glass electrode; carbon by the Walk-

ley Black method; clay by using a Bouyoucos hydrometer after dispersion with

sodium metaphosphate plus NaOH; electrical conductivity on the saturated soil

paste. Exchangeable bases, calcium, magnesium, sodium, and potassium were esti-

mated by percolating with N NH4Ac at pH 7.0, aluminium by percolating with

N NHkC1 at pH 4 . 5 ; and iron by percolating with N NH4Ac at pH 4 . 5 . The concentra-

tions were then determined by atomic absorption techniques. Soluble aluminium

was extracted using a 1 : l soil water ratio. Total iron, manganese and sulphur

were estimated by X-ray fluorescence and clay minerals were estimated from a dif-

fractogram using cobalt radiation on orientated clays.

Discussion

In situ pH shows the soils are not extremely acid when in a waterlogged conditi-

on. pH in water and 1/100 M CaC12 are closely related, but due to the saline na- ture of some of the soils the pH in CaCl* is preferred.

Fresh pH, which is measured in the laboratory before drying, shows that extreme

acidity may be produced which is later neutralised. This fresh pH is, therefore,

rather dependent on time and moisture conditions between sampling and making the

pH determination. The figures therefore lack precision and may not be reproduce-

able. Soil pH under Rhizophora and Avicennia show a lower reading in the former.

Total elemental analysis on the clay fraction was carried out; no correlation

was found between acid sulphate condition and the content of silicon, aluminium,

iron, potassium, magnesium, calcium, or titanium. Manganese, however, was signi-

ficantly lower in acid sulphate soils due to loss brought about by high solubi-

lity at low Eh. Acid sulphate soils always had less than 100 p.p.m. Mn in the

upper horizons but Profiles 2 and 4 (Tab.3) showed accumulation in the lower ho-

rizons.

The carbon content shows the higher organic matter and buried A horizon in Pro-

files l and 2. A high C/N ratio is found in Profiles I , 2 and 4 , particularly

at depth in the buried A horizon. The significance of this high figure is that

it reflects the anaerobic conditions for organic matter breakdown. This break-

down to butyric acid is a fermentation process and is due to low Eh (Hesse 1961) ,

which in turn leads to reduction of sulphate to sulphide, resulting in a fixation

of sulphur in an insoluble form mainly as pyrite (Pons 1 9 6 4 ) .

The very high electrical conductivity figures for Profiles 4 and 5 are due to the fact that 4 is still covered by the sea twice daily and 5 is within a few me-

132

tres of the sea. The rise in electrical conductivity of the soil paste with depth

in Profiles 1 and 2 indicated the stagnant drainage due to the soils being in a

basin topography. The higher overall conductivity of Profile 1 compared with 2

is due to its being closer to the sea.

The erratic clay analyses in Profiles 1 and 3 illustrate the fact that the area

is made up of several deposits superimposed on each other (Allbrook, 1968) .

The low exchangeable bases in Profile 2 compared with the other profiles is be- cause it is more mature soil being further inland. Correlation of exchangeable

bases content with distance from the sea has been shown by Allbrook (1972 a).

Exchangeable aluminium and iron do not, however, follow this pattern. Acid sulpha-

te soils show a rise in these exchangeable bases with depth, correlated with a

rise in acidity. Free (water soluble) aluminium was also found in these soils

(Tab.2). The high figure for iron is significant; this iron, to be sorbed on to

the exchange complex, must be in the soluble ferrous state. Thus a high exchan-

geable iron figure is an indication of the intense state of reduction. The cor-

relation between extractable iron, Z organic matter, and Eh and manganese was

shown by Tokkar (1969) . Soils having an exchangeable iron content of more than

1 m.e./100 g. soil were acid sulphate soils or potential acid sulphate soils.

The total sulphur content of the soils (Tab.3) has a tendency to increase with

depth (Profiles 1 and 4 ) . This feature was also found by Chow (1968) . Accumulati-

on of sulphur as insoluble Pyrite formed unter conditions of low Eh would result

in this pattern, if Eh falls with depth. The rise in organic carbon in Profiles

1 and 2 would lead to this effect (Tokkar 1969) . Comparison of soils under

Rhizophora and Avicennia spp., Profiles 4 and 5 respectively, show a higher sul-

phur content under Rhizophora confirming findings by Hesse (1961) in Sierra Leone

and the association of pyrite formation with Rhizophora in Malaya (Eswaren 1967) .

Most soils derived from marine sediments had more than 0.1% total sulphur. Some

soils, having a high pH due to the presence of sea shells, have a very high

sulphur content, present, it is believed, in the form of a gypsum/calcite or ara-

gonite complex (Williams 1960).

Examination of the clay minerals of acid sulphate soils showed the following:

kaolinite is the dominant mineral throughout the profile and quartz is rather

higher than average. However, appreciable quantities of 2:1 minerals are still present, montmorillonite and interstratified montmorillonite/vermiculite. The

presence of higher quartz content would suggest that it is a residual from the

breakdown of 2 :1 minerals.

133

c

Montmorillonite + montmorillonite/vermiculite + kaolinite + quartz.

The kaolinite dominance is in agreement with reports from Vietnam (Herbillon et

al. 1966), Guinea (Horn and Chapman 1968) and California (Lyn and Whittig 1966).

In the bottom horizon of the soils considerable loss of crystallinity is detecta-

ble, particularly in Profile 2 where the total crystalline clay minerals detected only accounts for 50% of the clay (Table 3 ) .

Conclusions

Acid sulphate soil may now be defined. As in all soil classifications we have

the fact that soil is a continuum. Thus in term of pH and sulphur content one can

find a complete range of soils from very acid to neutral, from high sulphur con-

tent to none. Is there, then, a key pH or sulphur content to divide acid sulpha-

te soils from other soils?

If one plots pH CaCl* against exchangeable aluminium, one finds, Graph 1, that one

obtains a sigmoid curve having an inflexion at pH 4 . Below this pH exchangeable aluminium rises steeply, and above it the exchangeable aluminium falls t3 zero at

about pH 5. Thus pH seems to be a pH buffered by the clay minerals when a range of 7-14 me/100 g soil, exchangeable aluminium may be found. Once the buffering capacity of the soil is exceeded, acid produced in the soil attacks the clay

minerals, liberating aluminium and silica with a marked fall in pH.

A soil therefore is an acid sulphate soil only if its pH is less than four when

determined in ]/I00 M CaClz. This determination is done on, the soil after drying

and crushing when oxidizing conditions have been set up, thus allowing the acid-

producing capacity of the soil to exert itself.

To distinguish acid sulphate soils by their sulphur content, discounting any

soils with high pH, one may recognize an accumulation of sulphur in the lower ho-

rizons. Soils having a total sulphur of 0.4% or more would be likely to be acid sulphate. However, the degree of acidity is not related to the sulphur content

(Graph 2 , Chow 1968).

It has been suggested that acid sulphate soils will not be found if 2 : 1 clay mine-

rals are present. Although these minerals are attacked and neutralize the acid

produced in the soil with the formation of kaolinite and exchangeable aluminium,

nevertheless they can continue to exist in conditions of low pH so that their pre-

sence is not a diagnostic feature.

From experience in Malaya it is known that an acid sulphate soil has a high orga-

134

nic matter content throughout the profile. Often the organic carbon content rises

in the bottom horizon samples indicating a buried A horizon, a feature also re-

cognized in Guyana (Marius and Turenne 1967), (Levèque 1962).

The occurrence of this distribution of organic matter seems to be related to

small topographic features. During the emergence of the coastline, lagoons were

formed that later formed backswamps. In the Kedah/Perlis Plain these swamps were

shallow and quickly dried up, forming the basin deposits of to-day (McGJalter

1956). The deposits were laid down in two stages, the earlier almost certainly

supported Rhizophora mangrove, the later may or may not have had mangrove,depend- ing on the salinity of the water. In some parts of Malaya the lagoons were deeper

and deep peat has accumulated but often with acid sulphate soils underneath

(Coulter 1952).

These back swamps are slightly lower than the surrounding areas and are very flat.

From aerial photographs they can sometimes be recognized by the meander courses

of streams crossing the area. Thus acid sulphate areas can be predicted to lie

on the inland side of a slight rise with poorer drainage than the rest of the

area.

In the field, an area of acid sulphate may sometimes be identified by a sulphu-

rous smell of free H z S when the soil is disturbed by digging or augering. Simi-

larly, the presence of yellow mottles of basic ferric sulphate is a sure sign of

acid sulphate, but not a l l s o i l s have them. A third feature is the change in co-

lour from light grey to black (Chenery 1 9 5 4 ) , (Coulter 1952), but this, too, was

not always recognized. Vegetation growing on acid sulphate soils may a l s o reflect

adverse conditions. Sedges and rushes were noted at the site of Profile 2 and

gelam (Melaleuca leucodendron) (Coulter 1952) has also been associated with acid

sulphate soils.

24

20

16 d O P > 2 12 -J a r o X W

8

4

5

4

Graph I.

PH CaC12

Graph2

O 0.5 1.0 1.5 2.0 2.5 3.0 S 70

136

TABLE I

DH

PROFILE HORIZON AND in x NO. DEPTH (cm) situ fresh H 2 0 xc ‘IN clay

1 I . 0-10 2 . 10-30 3 . 30-60 4 . 6 0 +

2 I . 0-20 2 . 20-50 3 . 5 0 +

3 I . 0-20 2 . 20-50 3 . 50-100 4 . 100 +

4 . 2 4 . 4 4 . 2 3 . 9 2 .7 9 4 . 3 3.9 3 .7 3 . 5 2 . 2 16 4 . 4 3 . 6 3 . 4 3 . 2 3.1 2 0 4 .5 3 . 5 3.4 3.4 4 . 8 28

5 . 2 3 . 4 4.0 3.9 3 . 9 19 4 .6 2 . 0 4 .0 3 .8 1 . 3 14 4 . 5 2 . 0 3 . 2 3.1 3 .0 25

6.7 4.6 5 . 0 4 . 4 3 . 0 14 7 . 1 , 5 . 5 5.1 4 . 5 1.3 12 6 . 8 4 .8 4.7 4 . 2 0 . 4 15 6 . 0 4 . 8 4.7 4 . 5 0 . 5 8

6 0 60 5 8 5 0

5 0 6 2 66

57 54 67 81

4 I . 0-15 - 5 . 8 5 . 8 5 . 5 5 . 2 22 41 2 . 15-30 - 6 . 3 5 .7 5 .4 5 . 0 25 41

5 1 . 0 - 4 5 6 . 4 7 . 1 7 . 0 6 . 9 1.7 IO 5 4 2 . 4 5 + 7 . 7 7 . 4 7 . 1 6 .9 1.3 8 56

TABLE 2

PROFILE/ EL.COND. EXCHANGEABLE BASES m.e./100 g Soluble Al HORIZON “ h o s Ca Mg Na K Al Fe m.e.1100 g

Ill 2.0 4 . 8 10 .3 9 .0 2.1 3 . 5 1.0 / 2 3.2 5 . 4 13 .8 1 7 . 8 1.7 7 . 0 1.0 I 3 4 .5 6 . 0 14 .0 1 9 . 2 1 .8 10.0 1.5 I 4 7 . 0 9 . 9 17 .8 2 7 . 0 0 . 7 8 . 0 1.0

211 0 . 2 2 . 5 2 . 5 0 . 2 0 . 4 6 . 1 1.0 I 2 0 . 3 3 . 5 3 . 5 0 . 3 0 . 4 9 .8 0 . 4 I 3 1.5 3 . 0 5 .3 0 .4 0 . 6 23 .7 4 . 6

311 0 . 2 6 . 3 5 . 6 0 .9 1 . 1 2.4 1.0 I 2 0.1 7 . 9 10 .3 0 . 7 1.0 1 . 2 0 . 3 I 3 0 . 2 5 . 6 7 . 8 0 .7 0 . 5 1 . 5 tr I 4 0.1 6 . 3 6 .7 0 . 9 0 . 9 1.2 0 . 6

411 10.5 7 . 3 17 .0 4 9 . 0 4 . 6 4 . 5 2.7 I 2 12 .3 12 .0 3 1 . 8 7 5 . 0 4 . 2 4 . 5 2 .3

I

511 10.0 8 . 2 26.7 7 4 . 0 5 .5 O 0.1 I 2 9 . 0 8.4 25 .7 6 9 . 0 4 .5 O tr

W .I

O O

1 . 1 8.5

O O

3.9

O O O O

1.8 4 . 2

0 . 3 O

TABLE 3

PROFILE/ T O T A L CLAY MINEMLS %

Fe % Mn ppm. S% K. M. S. I. Q.

I l l 2 . 0 55 0.6 45 tr 20 5 10

I 2 2.7 6 0 0 . 6 4 5 5 35 IO 5

I 3 1.4 90 1 . 1 6 0 5 15 15 5

I 4 3 .4 27 3.0 35 5 5 10 5

211 1 . 1 6 0 0.4 7 0 5 IO 10 5

I 2 2.1 45 0 . 2 6 5 er 10 5 5

I 3 2.0 107 0 . 4 20 tr IO IO 10

311 1.6

I 2 1.0

I 3 2.1

I 4 2 . 0

125 0 . 2 25 tr 20 5 0 5

95 0.1 IO tr IO IO 5

77 0 . 2 15 tr 25 4 0 tr

137 0.1 IO tr 20 25 tr

411 2 . 0

I 2 2 .2

8 5 2 . 9 25 5 15 20 5

110 3.5 30 5 10 50 tr

511 3.8

I 2 3.8

120 0 . 5 35 5 20 25 5 170 0 . 3 25 5 25 20 5

K = kaolinite M = mica

S = smectite I = interstratified Q = quartz

REFERENCES

ALLBROOK, R.F. 1968. The Kedah/Perlis Plain, how many deposits? An.Gnrl.Yeeting

Geol.Soc.Malays. (unpublished)

ALLBROOK, R.F. 1972 a. Investigation into the genesis and characteristics of

soils derived from recent marine sediments in North-west Malaysia. Ph.D. Univ. Malaya.

ALLBROOK, R.F. 1972 b. Natrojarosite in acid sulphate soils of Malaya. Geol.Soc.

Malays. Newsletter (in press).

CHENERY, E.M. 1954. Acid sulphate soils in Central Africa. Trans.5th Int.congr.

Soil Sci. Vo1.4, 195-198.

CHOW, W.T. 1968. A preliminary study on acid sulphate soils in W.Malaysia.

Proc.3rd Malays. Soil Conf. Kuching.

COULTER, J.K. 1952. Gelam soils. Malayan Agric.J. Vol.35, 22-35.

ESWAREN, H. 1967. The micromorphology of a "cat-clay'' soil. Pédologie, 17, 259-265.

HERBILLON, A.J., PECRC)T, A., VIELVOYE, L. 1966. Aperçu sur la minéralogie des fractions fines de quelques grands groupes de sols du Vietnam. Pédologie,

XVI, 1, 5-16.

HESSE, P.R. 1961. Some differences between soils of Rhizophora and Avicennia

mangrove swamps in Sierra Leone. Pl.and Soil, 14, 335-346.

HORN, M.E., CHAPMAN, S.L. 1968. Clay mineralogy of some acid sulphate soils

of the Guinea Coast. Trans. 9th Int. Congr. Soil Sci., 3, 31-40.

LEVEQUE, A . 1962. Mémoire explicatif de la carte des sols des des Terres Bas-

ses de Guyane Française. Mem. ORSTOM 3, Paris.

LYNN, W.C., WITTIG, L.D. 1966. Alteration and formation of clay minerals during

cat clay development. Clays and Clay Minerals: 14th Nat.Conf.241-248.

MARIUS, C., TURENNE, J.F. 1968. Problèmes de classification et de caractgrisation

des sols formés sur alluvions récentes marines dans les Guyanes. Cah.ORSTOM, sér. Pédol., Vol.VI., No.2.

Mc WALTER, A.R. 1956. The development of soil profiles in the rice growing areas

of the Kedah/Perlis Coastal Plain of North-west Malaya. Proc. 6th Int. Congr.

Soil. Sci.. Paris.

I38

MOORMANN, F . R . 1963. Acid sulphate soils. Soil Sci. Vo1.95: 271-275.

PONS, L.J. 1964. A quantitative microscopical method of pyrite determination

in soils. In: Soil Micromorphology, ed.A.Jongerius, Elsevier Publ.

SLAGER, S . , JONGMANS, A.G., PONS, L.J. 1970. Micromorphology of some tropical

alluvial clay soils. J.Soil Sci. 21, 231-241.

THORNTON, I., GIGLIOLI, M.E.C. 1965. The mangrove swamps of Keneba, Lower Gam-

bia River basin. 11. Sulphur and pH in the profile of swamp soils. J.App1.

Ecol. 2: 257-269.

TOKKAR, P.N. 1969. Effect of organic matter on soil iron and manganese. Soil

Sci. 108, 108-112.

WATTS, J.C.D. 1960. Sea water as the primary source of sulphate in tidal swamp

soils of Sierra Leone. Nature, 186, 308-309.

WILSHAW, R.G.H. 1940. Note on the development of high acidity in certain coastal

clays of Malaya. Malayan Agric. J. Vol . 28: 352-357.

WILLIAMS, C.H., WILLIAMS, E.G., SCOTT, N.M. 1960. Carbon, nitrogen, sulphur and

phosphorus in some Scottish soils. J.Soil Sci. 1 1 : 334-347.

139

Summary

Acid sulphate soils in north-west Malaya are i d e n t i f i e d b y having a pH below 4

in 1/100 M.CaCl2 and more than 0 .4% total sulphur.

Résume'

Les sols sulfate's acides du nord-ouest de Malaisie sont i d e n t i f i é s p a r un pH i n f é r i e u r à 4 dans l / l O O CaCl2 combine' avec un t a m de soufre t o t a l supérieur à

o. 4%.

Resmen

Los suelos de s u l f a t o s ác idos del noroeste de l a penis la Malaya se i d e n t i f i c a

por un valor pH i n f e r i o r de 4 en 1/100 M.CaCl2 en unidn con un contenido en azufre

t o t a l superior de 0.4%.

Zusamenfassung

Die sul fatsauren Böden Nordwest-Malakkas werden i d e n t i f i z i e r t durch d i e pH-Zahl

( l / l O O M.CaCl21 4 s m t 0.4% des gesamten S.

140

THE USE OF A RESPIROMETRIC METHOD FOR THE EVALUATION OF SULFUR OXIDATION I N SOILS

J . Baldensperger Loboratoru of S o i l Wkroh?:ologu, ORSTOM Dakar, Senegal

Introduction

The dynamics of sulfur in soils through gaseous, liquid, or solid phase, organic

or inorganic compounds, oxidized or reduced states, is generally represented by

a cycle (BUTLIN 1953, LA RITTIERE 1966, KAISER 1966, NICHOLAS 1 9 6 7 ) . Although

there are a variety of reactions involved in the "sulfur cycle", it may be divi-

ded into two principal pathways: 1 ) the reduction of sulfate to sulfide, and

2) the oxidation of reduced compounds to sulfate.

Biological oxidation and reduction of inorganic sulfur compounds in soils are

brought about by a relatively select group of microorganisms (ROY and TRUDIXER

1970). However, chemical reactions may also occur, due to the relative jnstahj-

lity of certain sulfur compounds even under the physical and chemical conditions

of natural environments (POSTGATE 1 9 6 3 ) .

Microbial oxidation of reduced inorganic sulfur compounds in s o i l s can be medi-

ated by: 1 ) dissimilatory oxidation by the colorless sulfur bacteria, 2) oxida- tion by the photosynthetic sulfur bacteria and 3 ) incidental oxidation by vari-

ous heterotrouhic microorganisms. The relative importance of the chemical ox;-

dation and of the three different microbial processes is discussed by various

workers (STARKEY 1950, KUZNETSOV et al. 1963, LA RITTIERE 1 9 6 6 ) .

Although there are no means of measuring in situ the actual contribution of

each process in sulfur oxidation in soils (POSTGATE 1966),it is generally agreed

that the chemoautotrophic sulfur oxidizers of the ThiobacLllus genus are of

greater importance (VISHNIAC and SANTER 1957, STARKEY 1966, FRENEY 1967) .

The evaluation of the importance of the incidental oxidation by non snecific mi-

croorganisms is not possible, due to the absence of any selective medium. For

the contingent photoautotrophs or chemoautotrophs, several media are available,

but in previous experiments on acid sulfate soils in Senegal we have shown that

conventional dilution procedures, based on the highest active dilution, were not

convenient to estimate the concentration of these germs, due to the fact that a

unique cell was not able to grow in the highest dilution tube.

The existence of a minimum mass of inoculum is well known in bacteriology (WNnD

141

1 9 4 2 ) hut this phenomenon is more important in autotronhic bacteria. The minimum

mass of inoculum differs for each species of Thjohacillus with the physico-

chemical conditions of the medium.

On the other hand, no data have heen aubljshed on the quantitative corresnonden-

ce between total counts of Thiohacilli and plate counts on thiosulfate apar

plates.

In conclusion, the evaluation of the sulfur oxidizing activity i n soils cannot

he monitored solely by the count of the responsihle microorganisms. One could

think that the concentration of the sulfur compounds in the soil, and snecially

the concentration of sulfate, would reflect the activity of the different path-

ways of the sulfur cycle. Previous experiments on acid sulfate Soil5 from Senegal

have shown that this assumntion was erroneous. In fact, a s in an ideal cycle an

intermediate compound is utilized as soon as it is formed and does not accumula-

te, no correlation can he expected between the sulfate concentration i n the s o i l

and the activity of the sulfur oxidizers.

Therefore, an attempt was made to use a respirometric method for estimating the

total sulfur oxidizing actitivity, the results obtained with acid sulfate S o i l s

from Senegal are presented i n this uaper.

MATEWALS AND WTHODS

S amp 1 es

Vangrove soils were collected from the estuary of the river Casamance, in Sene-

gal, in a sequence of soils descrihed and studied by VIEILLEF0N (196s ) . Three

representative s o i l s of this sequence, numbered 57, 55 and 53, were used in our

experiments:

57 is a sediment of a mud-bank in the middle of the river, and represents the parent material of the s o i l s of the whole sequence. This sediment has a low den-

sity due to a high water content (250%) and a neutral pH.

55 is a real mangrove s o i l , covered by Phizophora, containine about 15032 of wa-

ter, of neutral or slightly acid reaction. vangrove soils in Senegal are charac-

terized by their daily submersion hy the tide, and two phenomena predominate i n

their pedogenesis: the physical ripening of the material and the reduced condi-

tions caused by the suhmersion.

53 is a highly saline soil devoid of vegetation, or wjth a sparse vegetation o €

Heliocharis. This s o i l i s not submerged by the tide, but is annually flooded in

the rainy season. In the dry season the surface layer i s rapidly oxidized, as can

1 4 2

be noticed by the rapid decrease of the pH.

Another series of soil samples was collected from the estuary of the Senegal ri

ver from three different basins where trials are performed for the development

of rice output.') These acid sulfate soils are former mangroves soils, but of flu-

vial origin, and are not subjected to the steady alternation of submersion and

drainage as are the mangroves soils of Casamance.

All samples were collected in the dry season at 0-20 cm depth. Principal physi-

co-chemical characteristics and determinations of sulfur fractions of these

soils are summarized in Table 1 .

Plate counts of Thiobacilli

The enumeration of Thiobacilli in the samples of soils were made by spreading

various dilutions of the samples on thiosulfate agar plates.

Respirometric methods

Measurements were carried Out with a conventional WARBURG apparatus, model V 166

(B. BRAUN Melsungen, W. Germany).

Except when otherwise specified, 10 g of dry soil was introduced into a 130 ml

Warburg vessel, and moistened to the appropriate humidity either with water or

with a 1 % ( V / V ) solution Of Tween 80, which is a wetting agent.

Immediately after the solutions were added, the vessels containing the samples were put into a dessiccator and vacuum created three times. Details about the

method of damp-drying the sample in the vessel will be discussed together with

the results.

When elemental sulfur was used as a substrate for sulfur oxidation, the appro-

priate amount of sublimed sulfur (MERCK) was mixed with the dry soil prior to

the introduction in the vessel.

When thiosulfate was the substrate used, a thiosulfate solution of known concen-

tration was placed in the side arms of the Warburg vessels and tipped into the

soil at zero time after thermic equilibration.

Oxygen consumption was measured at regular intervals for a period of 50 hours. Counting frames, described previously (BALDENSPERGER 1969), were used for the di-

rect determination of the vessels' coefficients from the weight, the density, and

the final humidity of the sample. The results given are the means of three repe-

tit ions.

3 + ) Samples marked: RT, BN, KS, G o , GI and G

1 4 3

To remove the CO2 released, 1 ml of a 40% (V/V) solution of KOH was put into the central well of the vessels. This quantity of KOH can theorically occlude

400 ml of CO2, and one can consider that the occluding is complete for the first

40 O00 U1 of C O Z .

As the quantity of CO2 released is about equivalent to the quantity of 0 2 , it is

possible to measure the consumption of 40 000 ~1 of O 2 before changing the KOH

of the central well, which is enough for 10 or 20 g of dry soil.

RESULTS

I ) Influence of the treatment of the sample

In previous experiments (BALDENSPERGER 1969) we have shown that drying of the soil was necessary to get a representative sample of a sulfate soil for respiro-

metric studies. However, the respiration rate of a check sample, without additi-

on of any sulfur compound, appears to be strongly influenced by the drying

treatment.

The differences are more important when the total sulfur content of the soil is

high. For exemple, the respiration rate of soil 55, containing 3 , 4 % of total

sulfur, is respectively:

- 20 pl/02/g/h. for the air-dried sample

- 25 lil/Oe/g/h. if the soil is dried rapidly under a draught and Infra Red 1 amps

- 48 l/O?/g/h. if the soil is dried under vacuum.

This important difference can be explained for soil 55 by the difference in pH

just after drying:

- air-dried sample, pH with 1/2.5 water: 3.8 - soil dried under a draft and I.R. lamps, pH 4 . 4

- soil dried under vacuum, pH 4 . 8

The oxidation of an important fraction of the reduced sulfur compounds in soil

55 occurs during the dessiccation. But it can be seen on Figure 1 that if the

indigenous respiration differs for each treatment, the respiration due to the

sulfur substrate added (here 0.5 m Yole of thiosulfate) is not significatively modified by the drying treatment.

T h u s the most simple drying technique was preferred, and all samples were obtai-

ned by drying a freshly collected soil under Infra Red lamps, with a draught to

144

maintain the temperature below 40 O C .

Immediately after the wetting solutions were added, the vessels containing the

samples were put into a dessiccator and vacuum was created three times. This

operation allowed the water or the wetting solution to soak the sample correct-

ly, and the oxygen consumption was enhanced by this procedure. The results were

also more reproducible.

For example, the respiration rate of soil 55 was:

- 25 'role 02fgfh. if the sample is moistened under vacuum to a final humidi- ty of 40%.

- 1 7 "ble Ol/g/h. if the sample is moistened at the atmospheric pressure.

The possible explanation may be that the mangrove soils acquire hydrophohic

characteristics during the drying out, due to their high content in organic mat-

ter. For this reason, all samples were placed under vacuum prior to the resniro-

metric experiment.

2) Influence of the final humidity of the soil in the vessel

Experiments were carried out with soil G3 in order to determine the optimum

humidity for the respirometric measurement of sulfur oxidation. The sample was

enriched either with 5m 'role of elemental sulfur, or with 0.5 m "ole of thiosul-

fate, and moistened to a final humidity of 3 0 % , 4 n Z , and 60% (on dry weight

basis).

Figure 2A and 2B show the cumulative differences between the oxygen consumption

of the enriched sample and the check. It can be seen that the ontimum humidity

for a high respiration rate and reproducible results is about 40% for this acid

sulfate soil. At 30% the respiration rate is higher but the measurements are

poorly reproducible.

The final humidity of 40% is close to the "moisture equivalent" for the acid

sulfate soils from Casamance or Senegal, and was used for the further experiments.

3 ) Oxidation of thiosulfate

In this experiment the dry sample is moistened by water and the solution of thio-

sulfate to a final humidity of 40%. Previous experiments had shown that a final

concentration of 0.5 m Itole of thiosulfate for 10 g of dry soil in the vessel

was the optimum concentration to get a maximum oxygen consumption.

Summation curves of the differences between the enriched s o i l and the check are

145

L

seen in Figure 3, for the different samples. It can be seen that oxygen consump-

tion due to the substrate added is higher for soil 55 (real mangrove s o i l ) than

for soil 57 (primary material) and soil 53 (developed acid sulphate soil). One

can notice that the oxygen consumption due to the substrate stops after 24 hours

for acid sulfate soils from Casamance, and is very important during the first

6 hours.

Reciprocally, the oxygen consumption due to the thiosulfate stops only after 48 hours of experiment for samples from Senegal but the f i n a l efficiency is much

lower.

In Table 2 are summarized the rates of oxygen consumption during the linear pha-

se, the final efficiency calculated in % of thiosulfate oxidized in sulfate, and

the counts of Thiobacilli for each sample.

4 ) Oxidation of elemental sulfur

a) Use of Tween 80 as wetting agent

It has been reported by several workers (COOK 1964, KODA' and MOR1 1968, TAYLOR

1968) that the oxidation rate of elemental sulfur was improved by the use of

Tween 80 as a wetting agent. This result was confirmed by our experiments. This

can be seen from Figure 4A, which shows the oxygen consumption rates of soil BN.

Respiration rates were as follows:

- Check sample moistened by water 0.14 UiMole 02/g/h. - Enriched sample (0.5 d o l e So/g dry soil)

0.24 VMole Oz/g/h.

- Check sample moistened by 1 % (v/v) Tween 0.31 )Plole 02/g/h.

- Enriched sample moistened by Tween 0.56 UMole 02/g/h.

moistened by water

Summation curves of the difference between enriched and check sample with water

and Tween are shown in Figure 4 B .

It can be seen that the oxygen consumption due to the elemental sulfur added is

significantly increased by the Tween. In addition, the results are more reprodu- cible when Tween is employed as wetting solution.

b ) E f f e c t of the quantity of etementat s u l f u r added

Soil G3 from Senegal was enriched with different amounts of elemental sulfur:

0.1, 0.5, 2.5, and 5.0 d o l e So/g.d.w. of soil, and moistened by a 1 % (v/v) solu-

tion of Tween under vacuum to a final humidity of 4 0 % . Figure 5 shows the respi-

146

ration rates corresponding to each quantity of sulfur added.

It can be seen that the respiration rate during the linear phase is not increa-

sed if the quantity of sulfur added exceeds 0.5 d o l e of elemental sulfur per g.

of soil, and reaches 0.34 uMole/g./h. for sample G 3 .

After 30 hours the curves differentiate and a second phase begins, corresponding

probably to a multiplication of the sulfur oxidizing bacteria in the vessel. We

may assume that the rate of oxygen consumption of the first linear phase reflects

the initial population of sulfur oxidizers as no augmentation of this rate can

be obtained if the amount of sulfur added to the sample is increased.

A s the natural concentration of elemental sulfur in G3 was 0.015% an enrichment

of 0.5 mMole So/g. corresponds to a final concentration of 1.6% thus to one hun-

dred times the initial concentration.

Even in the mangrove samples from Casamance, addition of this quantity of elemen-

tal sulfur was found to be adequate for the measurement of the sulfur oxidizing

activity . c) Ozygen consumption due t o t h e a d d i t i o n of suZfur f o r d i f f e r e n t ac id

s u l f a t e s o i l s from Senegal

Summation curves of the oxygen consumption due t o the addition of 0.5 d o l e of

elemental sulfur per dry gram of soil are shown in Figures 6A and 6B.

Mangrove soil 55 differs from all other samples because of its high sulfur oxi-

dation rate. This result is in agreement with the high population of aerobic sul-

fur oxidizers found in this sample. The oxygen consumption rates due to the sulfur

added for the different samples are summarized in Table 2 .

DISCUSSION

We have noticed that the respirometric response of the acid sulfate soils to an

enrichment in elemental sulfur was on the whole more quick-acting than the response

to thiosulfate. This may be due to the fact that elemental sulfur is a substrate

present under natural condition, whereas thiosulfate could not be detected in

the samples. Thus one can imagine that the oxidation of the elemental sulfur

added for the measurement required little or no adaptation for the responsible

organisms, while an adaptation was necessary for thiosulfate.

The oxygen consumption observed after addition of thiosulfate was not the result

of a chemical reaction. A s can be seen in Figure 1 no response to thiosulfare was

1 4 7

observed with soil sterilized for 1 hour of time during three consecutive days.

Oxidation of elemental sulfur cannot occur chemically under the conditions of

temperature and pression of our experiments, and also the oxygen consumption

observed as a response to addition of sulfur must be ascribed to the activity

of sulfur oxidizing microorganisms in the sample.

A discussion is necessary to compare the results with samples GO, G I , and G3.

These samples were taken from experimental plots undertaken by FAO searchers in

order to study the desalinization of the soils of the basin o f Boundoum in the

delta of Senegal (S.de RAAD and WTSAARS 1971) . GO soil was the check plot, GI

was enriched with ZO T/ha of gypsum, and G3 enriched with 4 T/ha of gypsum. In

all plots a system of drain-pipes was placed at 1.5 m depth. The enrichment in gypsum was made in order to increase the permeability of the soils to the water

provided for desalinization.

After two months of drainage, a little acidification (0.5 unit of pH) was noti-

ced in plots GO (check) and GI (ZO T of gypsum) while a notable acidification has occurred in plot G 3 ( 4 T of gynsum, fall of 1 . 5 unit of p H ) . But the SO,

concentration in G I and G 3 were not higher than in the check GO. Plot 6 3 was

even found to have a lower concentration of total sulfur and SO4 than the check

devoid of gypsum (see Table 1 ) . In the G3 plot, mortality of rice due to the sul-

fate- reduction was observed at the germination and the seedling phase o f the

crop.

Respirometric measurements as well as Thiobacilli counts show that also the sul-

fur oxidizing activity of soil G3 was higher than the sulfur oxidizing activity

of the check soil. It seems that the initial enrichment in gypsum has

activated the sulfur cycle especially i n this soil, causing the diseases on rice

plants by sulfate reduction and the final fall of pH. After 2 months of drainage

the gypsum added was completely leached out of the soils, and the residual effect

was an increased amount of sulfur oxidizers (noticed by the respirometric method

and plate counts) and a dron in pH.

-

CONCLUSION

A respirometric method was used to estimate the sulfur oxidizing activity of

soils. This method is lengthy and requires special apparatus and materials, hut

it shows significant differences in the sulfur oxidizing activity of acid sulfate

soils from Senegal that cannot be predicted by chemical analyqis. Plate counts

of aerobic Thjobacilli were in agreement with the respirometric results. The

method's sensitivity was sufficient to separate different treatments of the same

soil.

148

s -* v1 v a

i L. Vlo

m m m N m m C - h m c c C O C " . . . - . . . . . . .

149

c u r v e 1 : s t e r i l i z e d s o i l and w a t e r 2 : s t e r i l i z e d s o i l and 0 . 5 m blole S203 3 : a i r d r y e d s o i l and w a t e r 4 : a i r d r y e d s o i l and 0 . 5 m Mole S 2 0 3 5 : s o i l d r y e d u n d e r vacuum and w a t e r 6 : s o i l d r y e d u n d e r vacuum and 0 . 5 m Mole S 2 0 3 -

F i g u r e 1 : i n f l u e n c e o f t h e t r e a t m e n t o f t h e sample

I50

1

Figure ZA: influence of the humidity on sulfur oxidation

IJ mole O , / g

1 0 4

oxygen consumption due to 0.05 m Mole thiosulfate added I

. I Figure ZB: influence of the humidity on thiosulfate oxidation

151

i

VI b 3

r: 2 .3 u

O *

o M

o N

a, u n u, ri

3 VI

O .i

c v

W O 0

c .i u m a x O

-

c,

e, k

3 u) .i

L L

152

tween and So

F i g u r e 4 A : oxygen consumpt ion w i t h s u l f u r

153

M \

M O \ V I

i n m u o

O

d

Z E E m i n m

m i O z

i

O 5 10 1 5 2 0 25 30 35 time h o u r s

Figure 6 : oxydation o f elemental s u l f u r

155

REFERENCES

BALDENSPERGER, 3 . 1969. Etude de la sulfo-oxydation dans les s o l s formé$ sur alluvions fluvio-marines en milieu tropical. Doc. Multigraph. Centre

ORSTOFI, Dakar. 54 pp.

BUTLIN, K.R. 1953. The bacterial sulfur cycle. Research (London), fí:

184-1 91 . COOK, T.Y. 1964. Growth of Thiobacillus thiooxidans in shaken cultures.

J . Bacter. 88 (3): 620-623.

DE RAAD, S . , 'WTSAARS, Y. 1977. Compte rendu d'expérimentation sur le des-

salement de Boundoum Ouest. Doc. Polygraph. h4 PD.

FRENEY, J.R. 1967. Oxidation of sulfur in soils. Mineralium Denosita 2:

181-1 87.

KAISER, P. 1966. Ecologie des bactéries vhotosynthétiques. Rev.6col.biol.

301. III, 3: 409-472.

KODAMA, A. and "IORI, T. 1968. Studies on the metabolism of a sulfuroxidizing bacterium. V: Comparative studies on sulfur and sulfite oxidizing systems of

Thiobacillus thiooxidans. Plant cell physiol. 9: 725-734.

KUZNETSOV, S.I., IVANOV, M.B., LYALIKOVA, N.N. 1963. Introduction to geolo-

gical microbiology. WcGraw-Hill Book Comp. Inc. New York. 1 1 6 np .

LARIVIERE, J . W . M . 1966. The microbial sulfur cycle and some of its implica-

tions for the geochemistry of sulfur isotopes. Geol. Runds. 55: 568-582.

MOONOD, 3 . 1942. Recherche sur la croissance des cultures hactériennes.

Hermann. Paris. 2nd ed.

NICHOLAS, D.J.D. 1967. Biological sulphate reduction. vineralium Deposita.

2: 169-180.

POSTGATE, J.R. 1963. The examination of sulfur autotrophs: A warning. J.Gen.

Yicrobiol. 30: 481-484.

POSTGATE, J . R . 1966. "ledia for sulphur bacteria. Lab. Pract. 15 (11) :1239-

1244.

ROY, A.B., TRUDINGER, P.A. 1970. The biochemistry of inorganic compounds of

sulfur. Chap.9: 207. Cambridge Univ. Press.

15) STAFXEY, R.L. 1950. Relations of microorganisms to transformations of

sulfur in soils. Soil Sci. 70: 55-65.

16) STARKEY, R.L. 1966. Oxidation and reduction of s u l f u r compounds in soils.

Soil Sci. I01 ( 4 ) : 297-306.

17) TAYLOR, B.F. 1968. Oxidation of elemental sulfur by an enzyme system of

Thiohacillus neapolitanus. Biochem. Biophys. Acta 1 7 0 (1 ) : 112-122.

18) VIEILLEFON, J. 196s. La pédogenèse dans les mangroves tropicales. Un

exemple de chronoséquence. Science du s o l 2:115-148.

19) VISHNIAC, W.,SANTEP, v. 1957. The Thiobacilli. Bacteriol. Qev.21: 195-213.

157

Summary

The conventional Warburg re sp i ra t ion technique has been used t o characterize the

sulphur-oxidizing a c t i v i t y o f soils. Respiration ijas measured f o r so?: 1 s a m l e s

i A t h and without enrichment of elementar sulphur and .from the d i f f e r e n c e the ra t e

o f sulphur-oxidation was calculated. The optimal operational conditions iJlere de-

termined. The optimal operational condi t ions were determined. The results ?,)ere

checked w i th enumerations of relevant bacteria.

Résumé

i/n procédé e s t élaboré à déterminer l ' a c t i v i t é o z ida t r i ce du sol par ranpor t au

soufre, sur l a base de l a méthode re sp i ra to i re de Varhurg. Fn partant des mesura-

ges de l a re sp i ra t ion d 'échant i l lons respectivement avec e t sans enrichissement

avec soufre élémental, un taux d 'oxidat ion de soufre du s o l e s t calculé . C e taux

correspond avec numérations des bac te r i e s relevantes .

Resume n

Se elabora un procedimiento pma caracterizar l a act iv idad de oxidación de azufre

en e l suelo, partiendo d e l método resp i ra tor io de Warhurg. Se mide l a resniración

de muestras respectivamente sin y con e l anadir de l azufre elemental, 14 de l a d i - ferencia se calcula l a tasa de ozidacicSn de azufre del suelo. E l procedimiento a

s i d o emprobado con cuentas de la s bacterias per t inen te s .

Zusamenfassung

Zur Messung d e r A k t i v i t ä t der Schwe,fe Zoxidation i m Boden wurde e i n rlerfahren

m i t Verwendung der Wartburg-Respirationsmethodik ausgearhei te t . Bei den Boden-

proben ( m i t und ohne Schwefeli wird d ie Respiration gemessen und aus der Di f f e renz

wird dann der Schwefeloxidationsgrad abge l e i t e t . Die Prüfung des 1fer;fahrens

Sand s m t Ziihlungen einschlägiger Bakterien s t a t t .

158