ῐ
J. Jpn. Soc. Soil Phys.
ῐ῎῍ῑῒ῏
No. +*,, p. 1+ῌ12 ῍,**0῎

Population Dynamics of Thiobacillus ferrooxidans inDi#erent Soil Moisture Conditions of a Pyritic Soil
Kaoru UENO*, Tadashi ADACHI** and Hajime NARIOKA**** Research Institute for Biological Functions, Chubu University,+,** Matsumoto-cho Kasugai, Aichi .21ῌ2/*+, Japan** Faculty of Environmental Science and Technology, Okayama University,-ῌ+ῌ+ Tsushima-naka, Okayama 1**ῌ2/-*, Japan*** Faculty of Bioresources, Mie University, +/11 Kurimamachiya-cho,Tsu-shi, Mie /+.ῌ2/*1, Japan
Abstract
Iron-oxidizing bacteria (e.g. Thiobacillus ferrooxidans) are known as an inﬂuential catalyst of soil
acidiﬁcation in primary oxidization of pyritic soils. We have reported that multiplication rate of
the bacteria is inﬂuenced by soil moisture. In this paper, we tried to estimate the conﬁguration of
the microhabitat of T. ferrooxidans under three di#erent soil moisture conditions (ῌ-./ kPa, ῌ-/kPa, and ῌ-*** kPa) to look into the mechanism of bacterial oxidation in detail measuring thenumber of adsorbed and free form of the bacteria (AFT and FFT) using incubation and centrifugation method. At the beginning of the soil incubation, most T. ferrooxidans were counted as
AFT in every soil moisture condition. Multiplication rate of both forms of bacteria under thecondition of ῌ-/ kPa was highest of all conditions. Under the condition of ῌ-./ kPa, the survival
rates of both forms of bacteria were higher especially in AFT. And under the condition of ῌ-***kPa, FFT could not be counted and a small number of AFT remained at the ﬁrst stage of incubation, which did not live long. These data indicate that population dynamics of T. ferrooxidans
in soil matrix is inﬂuenced by soil moisture conditions.
Key words : pyrite, soil moisture, bacterial oxidation, Thiobacillus ferrooxidans, population dynamics+.

Introduction

Bacterial activity is an important factor tocontrol natural soil conditions, and this is thesame in pyritic soils. Pyritic soils are one of theworldwide problem soils because of its sulfur
production in the process of water drainage. It
is di$cult to control acidiﬁcation without
using a large amount of water, and the soil
improvement method washing out the acidfrom the soil causes water pollution. Thiobacillus ferrooxidans which is an iron-oxidizing
bacterium is known as an inﬂuential catalystof pyrite oxidation (Silverman and Ehrlich,+30. ; Singer and Stumm, +31*). Pyrite in sedi-ments is produced and piled on the bottom ofsea coast or lakes with brackish water. The
sediments contain a large amount of , : + typeclay minerals (Yoneda, +3/2 ; Fujii and Yasuda,+31+ ; Attanandana et al., +32+ ; Dixson et al.,+32,), and during the drainage, they generatemany cracks and aggregates. These soil structures improve gas exchanges and promote
pyrite oxidation, because the bacteria catalyzing the oxidation are aerobic and chemoautotroph.
Degree of bacterial oxidation in pyritic soils
is inﬂuenced by soil moisture condition(Adachi et al., +33, ; Ueno et al., ,**, a b). A soil
moisture condition of ῌ-/ kPa is suitable for῏ΐῐ῎῔῕ῑ ῒ +*, ῏ ῌ,**0῍

72

Fig. +

Outline of sampling site.

multiplication of T. ferrooxidans (Ueno et al.,,**-). Bacterial activity in soil is also inﬂuenced by soil moisture conditions (e.g.
Tanaka and Sakamoto, +31, ; Mielnich andDugas, ,***), but details about the movementof bacteria or their microhabitats in soil matrix
have not been elucidated. Hattori (+310) suggested that bacterial activity results in a big
di#erence in the adsorption of soil particles.
Soil moisture condition is assumed to havee#ective inﬂuences on the adsorptions of bacteria to the soil. Understanding on the mechanism of bacterial multiplication and their
movement in soil matrix under drying condition contribute in controlling pyritic soils. We
examined changes in the adsorption and multiplication rate of T. ferrooxidans under di#erent
soil moisture conditions, and also the e#ects ofsoil moisture conditions on bacterial microhabitat.
,.

Materials and Methods,. + Soil
Nakaumi-Yasugi soil, a redoxed and a poten-

tial acid sulfate soil in Shimane Prefecture, wasused (Fig. +). The area of the collection was areclamation area, but the sampling point had
never been used as an agricultural ﬁeld but asa material yard. The soil was collected from adepth of +2/ to ,+* cm. Sampling and treatment methods were the same as in the previous
study (Ueno et al., ,**-). Physical and chemicalsoil properties are shown in Table +. As mentioned above, most pyritic soils in Japan contain , : + type clay minerals. We did not analyze the type of clay minerals in the samplingsoil, but have presumed to contain them because .*ῌ of the soil was clay and showed acharacter of swelling and shrinkage. In addition, Fujii and Yasuda (+31+) analyzed sediments of Lake Nakaumi and indicated that thesoil include high rates of montmolilonite andkaorinite. Our sampling point was in a reclamation area of Lake Nakaumi, and the sampled
soil showed similar characteristics as the sediments.
,. , Experiments,. ,. + Soil Moisture Conditions

ῒῑ : Population Dynamics of Thiobacillus ferrooxidans in Di#erent Soil Moisture Conditions of a Pyritic Soil 73

Table +

Fundamental physical and chemical propertiesof sample soil.

soil texture+῍
density of soil particle (rS, Mg m῎-),῍natural soil moisture content (kg kg῎+)
liguid limit (wL, kg kg῎+)plastic limit (wP, kg kg῎+)
shrinkage limit (wS, kg kg῎+)plasticity index, Ip
pH (H,O ; + : /)pH (H,O,)-῍
EC+ : / (mS cm῎+)
pyrite (mg kg῎+).῍
Feo (mg kg῎+)/῍Fed (mg kg῎+)0῍
sulfate ion (mg kg῎+)SiC (Clay .*ῌ)
,4/2
+4-,
+4/3
*4/0
*4-,
+*,4..
04,/
,4*/
.4.+
.343
*43
,-43
14,

Hydrometer-method (international system), ,῍ Picnometermethod, -, .῍ Murakami (+30+), /῍ Tamm (+3,,), 0῍ Mehra andJacson (+30*).
+῍

Three moisture conditions were prepared bydrying the soil at -*ῐ in an incubator.
+) Matric potential ῎-./ kPa, water content+.-2 kg kg῎+, wet condition over liquid limit,
pasted.
,) Matric potential ῎-/ kPa, water content*.1* kg kg῎+, near the soil moisture condition ofplastic limit, the best condition for the multiplication of T. ferrooxidans (Ueno et al., ,**-),
aggregated.
-) Matric potential ῎-*** kPa, water content *.-* kg kg῎+ῌ near the shrinkage limit,
allows low multiplication of the bacteria (Uenoet al., ,**-), aggregated.
Methods for drying, treatments of the soil
after adjusting moisture condition, and theway of converting water content to matric potential were the same as in Ueno et al., (,**-).
,. ,. , Incubating Conditions
Twenty grams of moisture conditioned soil
sample was put in a sterilized petri dish, 3* mmin diameter and ,* mm in height, and the dishwas wrapped with Paraﬁlm. Samples wereincubated at -*ῐ for six weeks, and bacterial
count, soil pH (H,O), and electric conductivity(EC+ : /) were checked every week from the startof the incubation. We opened the dishes every
three days to exchange air.
,. ,. - Bacterial CountWe took four grams of wet soils in a centrifuge tube (/* mL), added .* mL of sterilized
distilled water, shook one minute by hand, left
it still for ﬁve minutes, and then centrifuged (῏
1/* gῌ Nioh and Furusaka, +31,) it for +/ minutes. The supernatant were collected to countthe “free form of T. ferrooxidans (FFT)”. Weagain added .*mL of sterilized distilled water
to the residue, shook by hand for one minute,and treated with supersonic vibration for threeminutes. All the soil solution was collected tocount the “adsorbed form of T. ferrooxidans(AFT)”. FFT and AFT counts were obtainedby Most-Probable-Number method using
Silverman 3k liquid culture and +./mL tubes(Ueno et al., ,**-).
,. ,. . Soil pH (H,O) and EC+ : /Dishes for measuring soil pH (H,O) and EC+ : /were prepared and incubated as in the bacterial
counting. Ten grams of wet soil were suspended in distilled water and the pH (H,O, + : /)of the suspension was checked with electroprobe (HORIBA F-,-). The soil solution was  +*,74

Fig. ,The number of adsorbed and free form of T. ferrooxidans under three soil moisture conditions.

ﬁltrated using /B ﬁlter paper and EC+ : / wasmeasured using electro probe (HORIBA DS-+.).
-.

Results

Number of adsorbed and free form of
T. ferrooxidans
Changes in the number of T. ferrooxidans areshown in Fig. ,. In every soil moisture condition, a larger number of bacteria were counted
in AFT than FFT at * week of the incubation.
The largest number was counted under the soil
moisture condition of -./ kPa in both forms.
Under the condition of -./ kPa, the di#erencein the population size between the forms was
small at * week. After a logarithmic phase(from the second to ﬁfth week), the population
size decreased in FFT, but remained constantin AFT. Under the condition of -/ kPa, from* week to the second week the counts were lessthan those of -./ kPa, and the di#erence between the two forms of bacteria was larger.
The multiplication rates at the logarithmic
phase were higher than those of -./ kPa in
both forms, especially in FFT. After the logarithmic phase, the number declined in bothforms, and the declining rates were higher thanthose of -./ kPa, especially in FFT. Under the
-. +

,**0

condition of -*** kPa, T. ferrooxidans was notdetectable in FFT, and the number did notincrease throughout the incubation period in
AFT.
-. , Soil pH (H,O) and EC+ : /Changes in the soil pH (H,O) are shown in
Fig. -. Under the condition of -./ kPa, thevalue dropped to ,.12 after a week, gradually
declined to ,.++ from the second to fourth week,
and then slightly increased in the ﬁfth week.
Under the condition of -/ kPa, the value
dropped linearly from the start to the second
week, gradually declined until the third week
and marked a minimum of ,.+3, and then slightly increased. Under the condition of -***kPa, the value declined in the ﬁrst week,reached a minimum of -.**, and slightly increased thereafter.
Changes in the soil EC+ : / are shown in Fig. ..
The EC tended to increase while pH value
decreased. The result was the same as previous
reports and this phenomenon is assumed tocause production of sulfate ion and soluble iron
(Ueno et al., ,**, a b, ,**-).

῎῍ : Population Dynamics of Thiobacillus ferrooxidans in Di#erent Soil Moisture Conditions of a Pyritic Soil 75

Fig. -

Changes in soil pH in the incubating
period.

..

Discussion.. + Microhabitat of T. ferrooxidans
In order to analyze the relation between
physical condition of soil matrix and microhabitat of T. ferrooxidans in soil matrix, themicrohabitats were modeled considering thenumber of FFT and AFT as shown in Fig. /.
The model is simpliﬁed to indicate only , : +clay mineral, H,O, and bacteria. Although thecomponents are drawn nearly to scale, except
for the distance between clay minerals andmacropore size, the model may not reﬂect theexact microhabitat because it has not been conﬁrmed by a direct way such as by electric
microscope. A more precise state should beresearched by some direct method. In themodel, however, at least the number of FFT
represents that of weakly adsorbed bacteria toclay minerals, and the number of AFT represents that of bacteria adsorbed more tightly
than FFT. From the di#erence in the adsorption rate and their number, we can easily imagine where in the soil matrix the bacteria conceivably stay and multiply in each soil moisture condition.
Under the soil moisture condition of ῌ-./kPa, a large number of T. ferrooxidans wasFig. .

Changes in soil EC in the incubating
period.

observed at the ﬁrst stage of incubation. They
increased during the logarithmic phase regardless of the adsorbed amount. These multiplication rates were lower throughout the soil
matrix than the condition of ῌ-/ kPa, but thesurvival rate was higher in both forms of bacteria. Under the condition of ῌ-/ kPa, themultiplication rates of both bacterial forms
were the highest of all conditions and thenumber of FFT declined at highest ratio.
Under the condition of ῌ-*** kPa, bacterial
activity was very low.
Although factors causing these statuses
should be studied by other experiments, we
may assume that ; under the condition of ῌ-./kPa, there is low stress for bacterial survival
because of the wide space to move aroundbetween the clay minerals ; and under the condition of ῌ-/ kPa, soil forms aggregates with a
lot more macro pores, generating larger speciﬁc surface area than the soil which had nosoil structure as in the condition of ῌ-./ kPa(Fig. /). These physical conditions are supposedly favorable for T. ferrooxidans to acquire
CO,, O,, and nutrients, and to move and spreadin the soil matrix. The signiﬁcant decrease in
FFT under the condition of ῌ-/ kPa may be
explained by the lack of nutrient and space toΐῐ῎῔῕ῑ ῒ +*, ῏ ῌ,**0῍

76Fig. /

Schematic model of microhabitat of T. ferrooxidans in di#erent soil moisture conditions.

῎῍ : Population Dynamics of Thiobacillus ferrooxidans in Di#erent Soil Moisture Conditions of a Pyritic Soil 77

live after rapid increase in bacterial population.
Under the condition of ῌ-*** kPa, the soil
moisture condition was nearly at the shrinkage
limit, and distances between clay minerals
should be extremely small (Fig. /). In this condition, it is assumed that T. ferrooxidans iscompletely adsorbed to clay minerals and is
not able to move against the molecular force
and the van der Waals force. In addition, molecular di#usion of nutrients, water, and gasses
may be extremely low, and material circulationin this dry and high-pressured condition isalmost impossible that bacteria cannot survive.
.. , Soil Acidiﬁcation and Microhabitat ofT. ferrooxidans
Soil acidiﬁcation nearly stopped with the termination of the logarithmic phase under thecondition of ῌ-./ kPa and ῌ-/ kPa. The minimum pH value was ,.++ and ,.+3, respectively.
These pH values were nearly the level of H,O,
(Table +). On the other hand, the minimum pH
value in ῌ-*** kPa was -.**. This di#erence inthe minimum pH value was presumed to becaused by the di#erence in the bacterial activity. Adachi et al. (+33,) indicate that over the
drying conditin of pF -.* (ῌ32.+ kPa) soil acidiﬁcation is caused mainly by chemical reaction.
Ueno et al. (,**, a) shows that under the condition of ῌ-+.* kPa bacterial oxidation is superior to the chemical oxidation. The present
study revealed that, under the condition of
ῌ-*** kPa, chemical oxidation seems to be themain factor for the soil acidiﬁcation, and thatT. ferrooxidans largely contributes to soil acidiﬁcation under the condition of ῌ-./ kPa andῌ-/ kPa.
/.

Conclusion

In this study, we obtained following ﬁndings.
+) At the beginning of soil incubation, mostT. ferrooxidans lived in the adsorbed form.
,) Under the condition of ῌ-./ kPa and ῌ-/kPa, the bacteria multiplied both in the adsorbed and free form, and the survival rate washigher in the adsorbed form.
-) Multiplication rate of both forms werehigh under the condition of ῌ-/ kPa, andtherefore soil acidiﬁcation seemed to take placein the whole soil matrix under this condition.
.) Under the condition of ῌ-*** kPa, bacteria were adsorbed tightly to the soil and could
not live long, and the entire bacterial activity
was very low.
These data indicate that population dynamics of T. ferrooxidans in soil matrix is seriously
inﬂuenced by soil moisture conditions.
Acknowledgments
Authors sincerely thank Shuji Hamazaki in
Shimane Prefecture for collecting soil samples,and students in the Lithosphere Management
Lab. in Okayama University for lending their
help in the experiments.
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