GEOLOGICA CARPATHICA, 50, 5, BRATISLAVA, OCTOBER 1999
MICROBIAL ACTIVITY IN SOILS UNDER THE INFLUENCE
OF PYRITE WEATHERING
MARIANA VÝBOHOVÁ, ALEXANDRA IMONOVIÈOVÁ, PAVEL DLAPA and
Department of Soil Science, Faculty of Science, Comenius University, Mlynská dolina, 842 15 Bratislava, Slovak Republic
(Manuscript received February 12, 1998; accepted in revised form December 9, 1998)
Abstract: The chemical and microbial characteristics of soils at seven research sites four affected by an acid
sulphate weathering and three intact, undisturbed have been investigated. The extremely low soil pH, the increased
electrical conductivity and high exchangeable aluminium contents are characteristic features of the affected soils.
These chemical changes were accompanied by the inhibition of the soil respiration and enzyme activities denoted the
microbial activity in soils. The small microbial biomass and the abundance of bacteria and microscopic fungi indi-
cated the worsening of living conditions for the soil microorganisms. In spite of this some representatives of micro-
scopic fungi (Deuteromycotina) isolated from the damaged soils gave evidence of their adaptability to the extreme
Key words: acid sulphate weathering, soil acidification, microscopic fungi, respiration and enzyme activity changes.
The quartzite quarring has been practiced on the obov hill
near Banská tiavnica since the year 1956. The waste materi-
al containing pyrite has been accumulated on a dump pile
next to the quarry. The weathering of pyrite leads to the pro-
duction of extremely acid and mineralized water solutions
within the dump pile. The acid solution with high concentra-
tions of iron and sulphatic ions has damaged soils and the
vegetation downslope. The soil acidification, the precipita-
tion of the characteristic secondary minerals, the destruction
of the soil structure, and consequently the absence of the
plant cover and the strong erosion of topsoils are the degra-
dational features on the slope of obov hill.
The first symptoms of the damage appeared in 1978. The
area of about 15 ha is influenced at present. The geological,
chemical and biological aspects of the affected territory has
been studied by a number of authors (Holub et al. 1991; Holub
et al. 1993; Kriáni et al. 1994a; Kriáni et al. 1994b; Forgáè
et al. 1995; imonovièová et al. 1996; imonovièová et al.
1997; Dlapa et al. 1997; ucha et al. 1997). The processes of
the acid sulphate weathering in the volcanic complex in
Vtáènik Mts. was described by Èurlík & Forgáè (1983).
The aim of this paper is to compare the microbiological
characteristics of the original topsoils with those affected by
the acid and mineralized water originating from the pyrite
oxidation. The soil microbial characteristics, like the soil res-
piration or enzyme activities, inform about the microbial ac-
tivity in the soils. In addition, the microbial biomass, the
abundance of bacteria and microscopic fungi, the occurrence
of microscopic fungi species and genera testify to the quality
of the soil environment.
Material and methods
The obov hill is situated NE of Banská tiavnica town in
the tiavnické vrchy region. The soils are Eutric Cambisol
(FAO) with transitions to Planosol, in depressions. Detailed
mapping of soils was done in the area affected by the water
leaking out from the dump pile. Seven sampling sites were
studied four influenced (P1, P2, P3, P4) and three without
degradational features (N5, N6, N7). The samples were taken
from the topsoils (020 cm) for the chemical and the micro-
Soil samples were air-dried and fraction < 2 mm was taken
for chemical analyses. Soil pH values (H
O, 1 M KCl) and
exchangeable Al were determined using a soil : solution ratio
of 1 : 2.5. Total organic carbon was measured by oxidation
and titration of non-reduced chromate.
Electrical conductivity was determined in the saturation soil
extracts, after allowing the pastes to stand for 24 h.
The microbiological investigation was realized with the
fresh soil samples. The soil fraction < 2 mm was used for
The microbiological analyses include:
-evolution (the soil respiration) basal and po-
tential after adding 1 % of glucose (Kopèanová et al. 1990);
the enzyme activities: carboxymethylcellulase (CMC),
xylanase and saccharase (Schinner et al. 1993);
the amount of carbon in microbial biomass (C
(Schinner et al. 1993);
the abundance of bacteria and microscopic fungi (Ko-
pèanová et al. 1990);
the composition of microscopic fungi species and gen-
era (Fassatiová 1979).
390 VÝBOHOVÁ, IMONOVIÈOVÁ, DLAPA and MADARAS
The number of repeated samplings (310) was chosen in
accordance with the methods used. The arithmetical means
and the mean deviations have been calculated and illustrated
in the Figs. 14.
Figures on Pls. III were taken on Scanning Electron Mi-
croscopy (SEM) model Tesla BS 301 by J. Blahutiaková (In-
stitute of Experimental Phytopathology and Entomology,
Slovak Academy of Sciences, Ivanka pri Dunaji). Before
that, the samples were air-dried and coated with gold.
Results and discussion
The chemical properties of investigated topsoils reveal
degradation processes as follows from Table 1. A drop in the
soil pH to about 3 is typical. The soil buffering mechanisms
are insufficient and almost ineffective because acid water en-
tering the soil profiles contains high concentrations of iron and
sulphatic ions (in contrast to acid rains with only very low
concentrations of salts). The salinity of saturated soil extracts
increases due to the dissolution of secondary minerals which
are formed during the interaction of acid water with the origi-
nal soil. Exchangeable base cations (Ca
, and K
displaced by Al
(Dlapa et al. 1997). The low nutrient status
and aluminium toxicity will cause the inhibition of all bio-
way from the plant or litter bioorganic compounds to the col-
loidal humic substances is very complex involving a number
of degradative and condensation reactions. These reactions
involve numerous enzymes, since non-enzymatic reactions in
soils are very slow (Lähdesmäki & Piispanen 1988). The first
components to be removed, by the combined action of mi-
crobes and leaching, are water-soluble compounds, such as
sodium and potassium salts and sugars. After these, more
complex substances such as starch and proteins are attacked.
Whereas many of these components can be decomposed dur-
ing a few months, more complex structural ones, such as lig-
nin or keratin, may persist in soils for several years (Gray &
Williams 1971). The enzyme activities saccharase, xyla-
nase and carboxymethylcellulase (CMC) from the carbon
cycle of organic matter circulation in the natural environ-
ment, were clearly detected. It has been observed that the
highest saccharase and xylanase activities (5 170.48 694.6
µg GLC· g
dry soil · (3 h)
and 648.03 522.5 µg GLC · g
dry soil · (24 h)
) are at the places N5N7, that is at the plac-
es without degradational attributes (Fig. 2). The saccharase
Fig. 2. Saccharase, xylanase and carboxymethylcellulase activities.
Fig. 1. Basal and potential respiration of microorganisms.
Table 1: Basic analytical data of the studied soils (P1P4 influ-
enced, N5N7 not influenced by acid sulphate weathering).
µS . cm
mg . kg
EC electrical conductivity of saturated soil extract
Al exchangeable aluminium (1M KCl).
The study of carbon cycling is necessary to understand the
functioning of the ecosystems. The carbon fluxes from the
soil and litter compartments to the atmosphere (collectively
referred to as soil respiration) are major pathways of the car-
bon cycle (Redmann 1978). The evolution of CO
is an indi-
cator of intensified processes of organic matter decomposi-
tion in the environments (Kubicka 1973). The carbon dioxide
output of soil samples reflects the overall metabolic activity
of the microflora in soil (Gray & Williams 1971). The high-
est values of CO
-evolution (112.0 and 73.8 mg CO
soil · (24 h)
) were measured at the chemically undisturbed
places N6 and N7 (Fig. 1). The potential soil respiration (the
respiration after adding of glucose as the source of energy)
was more intensive than the basal soil respiration in all sam-
ples (Fig. 1). Such results testify to the fact, that the quanti-
ties of available nutrients had a direct and the greatest influ-
ence on the activity of microorganisms.
The soil microorganisms play a very important role in the
decomposition of dead organic matter. The chemical path-
/ kg soil / 24 h
mg GLC / g soil
CMC (after 24 h)
xylanase (24 h)
saccharase (3 h)
GLC - glucose equivalent
MICROBIAL ACTIVITY IN SOILS UNDER THE INFLUENCE OF PYRITE WEATHERING 391
Plate I: Fig. 1. Absidia sp., (Zygomycotina), 3900
. Fig. 2. Zygorhynchus sp., (Zygomycotina), 1300
. Fig. 3. Acremonium sp., (Deuter-
. Fig. 4. Aspergillus sp., (Deuteromycotina), 640
. Fig. 5. Cladosporium cladosporioides, (Deuteromycotina), 440
Fig. 6. Paecilomyces sp., (Deuteromycotina), 2200
392 VÝBOHOVÁ, IMONOVIÈOVÁ, DLAPA and MADARAS
and xylanase activities were explicitly different between the
groups of the unaffected and the affected topsoils. Their mea-
sured values were higher than those of CMC activity. Low
CMC activities were observed at all places (37.4303.9 µg
GLC · g
dry soil · (24 h)
). The differences between the
studied groups of undamaged and devastated soils were not
ascertained (Fig. 2). It is known that the decomposition of
cellulose is rather slow in the soil, whereas, sugars, carbohy-
drate polymers as well as starch, pectin, and hemicellulose
decompose more rapidly (Lähdesmäki & Piispanen 1988).
A large amount of energy within the soil is accumulated in
the form of humic substances, plant tissue residues, and as
microbial biomass, too. The microbial biomass is important
in the cycling of matter and energy and it determines the
character and the intensity of biological processes within the
soil (Bernát et al. 1984). The amount of carbon in microbial
7.214.3 mg · (100 g)
dry soil, Fig. 3) and the
abundance of bacteria (0.01.4 · 10
CFU · g
Plate II: Fig. 1. Penicillium sp., (Deuteromycotina), 1600
. Fig. 2.
Penicillium vulpinum, (Deuteromycotina), 40
. Fig. 3. Chaetomium
sp., (Ascomycotina), 90
Fig. 4. Bacteria and microfungi abundances.
Fig. 3. The amount of carbon in microbial biomass.
g / 100 g
P1 P2 P3 P4 N5 N6 N7
10000 CFU / g soil (bacteria)
10000 CFU / g soil (microfungi)
MICROBIAL ACTIVITY IN SOILS UNDER THE INFLUENCE OF PYRITE WEATHERING 393
Table 2: Species and genera of soil micromycetes observed in the
P1 P2 P3 P4 P5 P6 P7
Fig. 4) determined at the affected places (P1P4) were of
lower values in comparison with those at unaffected places
43.7101.3 mg· (100g)
dry soil, the bacteria abun-
dance 18.433.6 · 10
CFU · g
dry soil). The abundance of
microscopic fungi (Fig. 4) was comparable in all samples
(0.8 6.3 · 10
CFU · g
dry soil). The sensitivity of micro-
scopic fungi to decreased soil pH is lower than the sensitivity
of bacteria (Holub et al. 1993).
Fifteen genera and species of microfungi were isolated and
identified in the studied topsoils (Table 2, Pls. III). The high-
est number of genera and species was isolated at the places N5
(6), N6 (6), and N7 (8). The representatives of classes ZYGO-
MYCOTINA (Absidia sp. Tiegh., Mucor sp. P. Micheli ex St.-
Amans, Zygorhynchus sp. Vuill.), DEUTEROMYCOTINA
(Acremonium sp. Link ex Fresen., Aspergillus sp. P. Micheli
ex Fresen., Aureobasidium sp. Viala ex Boyer, Cladosporium
cladosporioides (Fresen.) N.F. de Vries, Cladosporium mac-
rocarpum Preuss, Geotrichum sp. Link ex Léman, Paecilo-
myces sp. Bainier, Paecilomyces lilacinus (Thom) Samson,
Penicillium sp. Link ex Fresen., Penicillium vulpinum
(Cooke et Massee) Seifert et Samson, Verticillium sp. Nees
ex Link) and ASCOMYCOTINA (Chaetomium sp. Kunze ex
Fresen.) were determined. The representatives of the class
Zygomycotina exist in the topsoils and they require sufficient
amounts of easily decomposable organic substances. Their
absence reflects the insufficiency of soil organic matter and
the deterioration of decomposing processes at the places P2
and P3. Penicillium sp. and Cladosporium cladosporioides
of the class Deuteromycotina were found at almost all af-
fected places. The survival of the representatives of DEU-
TEROMYCOTINA under the physiologically extreme con-
ditions confirms their high adaptability to the adverse soil
The observed degradation processes represent the extreme
case of soil acidification. It has been found that the chemical
deterioration within the investigated soils (extremely low
pH, high contents of exchangeable aluminium) was accom-
panied by significant changes in the soils microbiological
properties. The inhibition of the microbial activity, as the soil
respiration or the enzyme activities, was observed in the soils
influenced by the acid sulphate weathering. The microbial
biomass, the abundance of microorganisms, as well as the di-
versity of microscopic fungi genera were reduced in the af-
fected soils, too. Only tolerant groups of microorganisms
survive in the chemically degraded soils on the slope of
Bernát J., Braunová O. & Mirèink T.G., 1984: Determination of
the microfungal biomass. Acta FRNUC Microbiologia XI,
Èurlík J. & Forgáè J., 1983: The phenomena of the sulphur-acid
weathering and its depth changes in the volcanic rocks in the
Vtáènik Mts. Geol. Práce, Spr., 79, 197217 (in Slovak).
Dlapa P., Madaras M., Výbohová M. & imonovièová A., 1997:
Soil degradation processes accelerated by products of pyrite
weathering. Proceedings of International Conference Analy-
ses for Geology and Environment 97. Slovak Geological
Survey, Spiská Nová Ves, O/16.
Fassatiová O., 1979: Moulds and fibrous fungi in technical micro-
biology. SNTL, Praha, 1211 (in Czech).
Forgáè J., Streko V. & kvarka L., 1995: Occurrence of toxic ele-
ments in the Banská tiavnica area (Central Slovakia). Miner.
slovaca, 27, 4556 (in Slovak).
Gray T.R.G. & Williams S.T., 1971: Soil micro-organisms. Edin-
Holub Z., Banásová V. & Kriáni I., 1991: The influence of soil
acidification on vegetation in surrounding of quarzite mine
(obov). Proceedings of workshop Geology and environ-
ment, Donovaly 126.96.36.1991, 114121 (in Slovak).
Holub Z., imonovièová A. & Banásová V., 1993: The influence
of acidification on some chemical and microbiological prop-
erties of soil, those determining plant viability. Biológia, 6,
Kriáni I., Jeleò S. & Háber M., 1994a: The resistible and second-
ary minerals of the historical depositions in the ore field of
tiavnické vrchy Mts. the environmental and technologi-
cal aspects. In: Mineralogie, geochemie a ivotní prostøedí,
Ostrava-Poruba 28.30. 6. 1994, 99100 (in Slovak).
Kriáni I., Kunierová M., Andráò P. & Jeleò S., 1994b: The dump
pile of obov the spontaneous geobioreactor. In: Biohy-
drometalurgia III., Koice 4.5.10. 1994, 5055 (in Slovak).
Kopèanová ¼., Øehoøková V. & Bumbala ¼., 1990: The instruc-
tions for the microbiological practice for the phytotechni-
cians. Príroda, Bratislava, 1128 (in Slovak).
Kubicka H., 1973: The evolution of CO
in two meadow commu-
nities. Ekol. Pol., 5, 116.
Lähdesmäki P. & Piispanen R., 1988: Degradation products and
the hydrolytic enzyme activities in the soil humification pro-
cesses. Soil Biol. Biochem., 3, 287292.
394 VÝBOHOVÁ, IMONOVIÈOVÁ, DLAPA and MADARAS
Redmann R.E., 1978: Soil respiration in a mixed grassland ecosys-
tem. Canad. J. Soil Sci., 58, 119124.
Schinner F., Öhlinger R., Kandeler E. & Margesin R., 1993: Boden-
biologische Arbeitsmethoden. Springer-Verlag, Berlin, 1389.
imonovièová A., Franková E. & Výbohová M., 1996: Microfungi
of acidificated region of Banská tiavnica-obov. Miner. slo-
vaca, 28, 355356.
imonovièová A., Forgáè J., Streko V. & Výbohová M., 1997:
The change of soil microbiota under the influence of mining
activities. Miner. slovaca, 29, 431438.
ucha V., Kraus I., Zlocha M., Streko V., Gaparovièová M.,
Lintnerová O. & Uhlík P., 1997: Acidification in the obov
region (tiavnické vrchy Mts.): Manifestation and causes.
Miner. slovaca, 29, 407416 (in Slovak).
Note: This paper was presented at the Conference of the IGCP Project #405 ENVIVEATH, held in Bratislava from 24th to 26th November, 1997