Download Sulfur compounds, potential turnover of sulfate and thiosulfate, and

FEMS Microbiology Ecology I8 ( 1995) 257-266
Sulfur compounds, potential turnover of sulfate and thiosulfate,
and numbers of sulfate-reducing bacteria in planted and unplanted
paddy soil
Thorsten Wind, Ralf Conrad
Received 7 April 1995: revised
*
IO August 1995: accepted 20 August 1995
Abstract
Sulfate reduction potentials (SRP), thiosulfate consumption potentials (TCP). numbers of sulfate-reducing bacteria (SRB)
and the vertical profiles of sulfate, thiosulfate, acid volatile sulfides (AVS) and chromium reducible sulfides (CRS) were
measured within IO-cm-deep l3-week-old planted and unplanted paddy soil microcosms. The soil pore water of unplanted
microcosms
showed sulfate concentrations
< I IO FM and no detectable thiosulfate. The upper layers of planted
microcosms. in contrast, showed concentrations
of sulfate and thiosulfate that reached > 300 PM and > IS0 PM,
respectively. indicating that oxidation of reduced sulfur was stimulated in this zone (the root zone of the rice plants). On the
other hand, concentrations of AVS were also much higher in the upper layers of planted versus unplanted microcosms
indicating that reduction of oxidized sulfur compounds was also stimulated in this zone. The highest AVS and CRS
concentrations were I.6 and I.3 pmol crne7 soil, respectively. Indeed, planted soils showed a two- to five-fold higher SRP
and TCP ( < 2.8 and < I .9 Fmol cmpi d- ‘, respectively) compared to unplanted microcosms ( < 0.56 pmol cm-” d- ’ 1.
Concentrations of acetate and lactate were also higher, especially in the uppermost soil layers. However, SRP and TCP were
only stimulated by the addition of hydrogen. SRB were enumerated by the MPN technique using hydrogen. acetate,
propionate, lactate, butyrate. succinate and benzoate as electron donors. Vertical profiles indicated that the SRB were
relatively homogenously distributed in the paddy soil microcosms. The SRB populations growing on HZ. propionate and
succinate appeared to be higher in planted than in unplanted paddy soil microcosms although at a relatively low statistic
significance ((u < 0.1). Enrichment cultures showed a relatively high diversity of sulfate-reducing bacteria with respect to
utilization of at least eight different substrates out of 21 substrates tested. The genera observed included Lksdfru~ihrio,
~e.sulforortzrcculcrrll,
Desu~fihdhus,
and D~~44~f0h0f44l44.s.
Our results indicate a very dynamic cycling of both reduced and
oxidized sulfur species in the rhizosphere of planted paddy soil.
K~\~~wY/.sc
Acid
volatile
sulfide: Chromium reducible sulfide: Paddy soil: Microcosm: Sulfate-reducing
bacteria; Thiosult’ate
1. Introduction
[email protected]
Detailed
studies of the sulfur
role of sulfate-reducing
bacteria
done for marine sediments
[l-.5].
Ol68-6496/95/$09.50
Societies. All rights reserved
~ Corresponding
.S.SDI 0 168.6396(
author.
0
Fax:
+49
(6471)
161470;
E-mail:
1995 Federation of European Microbiological
95 )00066-6
chemistry
and the
(SRB) have been
However,
compar-
258
T. Wind. R. Comd/
FEMS Microhiolog?_ Ecology 18 (1995) 257-266
atively little is known about the sulfur biogeochemistry and the effects of SRB in freshwater environments [6-l 11. In the littoral sediments of Lake Constance, for example, it has been shown, that despite
low sulfate concentrations,
sulfate reduction rates
can be relatively high [l I]. Ion chromatographic
measurements
on pore water in this lake sediment
revealed steep sulfate gradients directly below the
sediment surface [ 121. An intensive sulfate turnover
takes place in the narrow zone of the top few centimeters [l 11. Sulfate reduction has also been reported for several rice paddies [ 13- 171. but only a
few quantitative studies exist.
The occurrence of SRB in paddy soils has been
described
elsewhere
[ 13- 171. Although
little is
known about the diversity of sulfate reducers in
paddy soils, DesulfoL~ibrio and Desu!fotornaculurn
seem to be the predominant species. Non-sporulating
forms account for the highest numbers [ 13-151. Lactate. pyruvate, acetate and propionate have been
reported as the predominant
substrates for SRB in
tropical paddy soil [14].
Several authors have shown that rice roots can
exudate various organic substrates which may serve
as electron donors for SRB [ 18.191. In flooded soils,
rice plants are able to transport oxygen via the
aerenchym system into their roots [20,21]. As rice
roots can occupy a large volume of the planted soil,
a significant fraction of it can become oxic and/or
exhibit increased redox potentials [22-241. It may be
assumed that these conditions allow the oxidation of
reduced sulfur compounds
and thus result in an
intensive sulfur cycling between oxidation and reduction processes at the oxic-anoxic interfaces. However, comparative studies about the sulfur chemistry
in planted and unplanted paddy soils are lacking.
Jorgensen [25,26] showed that thiosulfate is an
important intermediate in the sulfur cycle of sediments. Disproportionation
of thiosulfate can result in
sulfate production under anoxic conditions [25,26].
The disproportionation
of thiosulfate or sulfite to
sulfate and sulfide
was discovered
in several
sulfate-reducing
bacteria [27.28]. and is now known
to occur in sediments [29]. However, nothing is
known about the potential for thiosulfate consumption in paddy soil.
The aim of the present work was to measure
vertical profiles of different inorganic sulfur com-
pounds, to determine the consumption potentials of
sulfate and thiosulfate, and to assess the diversity
and density of sulfate-reducing
bacterial populations
in planted and unplanted paddy soil microcosms.
2. Materials
and methods
2. I. Soil microcosms
Soil samples were taken in the wetland rice fields
of the Italian Rice Research Institute in Vercelli,
located in the valley of the river PO. The soil was
sampled in April 1992 from as yet unflooded fields
and stored in a dry state at room temperature. The
dry soil was sieved (< 2 mm) and fertilized with
urea (I3 mg kg-’ 1, phosphate (20 mg P,O, kg- ’ >,
and KC1 (73 mg kg-‘), according to the local management practice [30]. Soil microcosms were prepared in 24 parallels using polystyrol plastic columns
(20 cm X 3.6 cm), which were sealed with rubber
stoppers at the bottom, and filled each with about
400 g dry weight (dw) paddy soil. The soil microcosms were then flooded with tap water until they
were waterlogged.
Rice seeds (0~~a satirw, var. Roma, type japonica) were germinated on water-soaked cotton wool at
25°C. When the young plants had reached a length of
30-50 mm, two shoots were planted in each of 12
microcosms;
I2 microcosms
were left unplanted.
The rice plants were grown for 13 weeks at 25°C
with an average light intensity of 40 W per rn-’
using a I?/ 12 h light/dark
cycle. At this time the
rice plants just started flowering. Weeds were regularly pulled from the soil. Soil parameters and sediment chemistry were determined in triplicate. while
SRP, TCP and population densities of SRB were
determined from both the planted and the unplanted
treatment using a pooled sample of three microcosms. SRB were counted in IO- and l3-week-old
microcosms.
2.2. Sediment chemistn
Soil pore water samples were obtained by slicing
the soil cores into segments of 1 cm thickness. The
pore water was collected by centrifugation of the soil
segments for 2 min at 10 000 ‘pm followed by
T. Wind, R. Conrad/
FEMS Micro&log!
membrane filtration (0.2 pm, regenerated cellulose)
of the supernatant.
The pore water samples were
stored at - 20°C until analysis. Dissolved anions
(sulfate and thiosulfate; given in molar concentrations) were measured by suppressed ion chromatography with conductivity detection (Sykam, Gilching,
Germany) [ 121.
Alcohols (isopropanol
and ethanol) and organic
acids
(acetate,
propionate,
lactate.
succinate,
caproate, valerate, and butyrate; given in molar concentrations)
were analysed in pore water samples
(200 ~1) by high performance liquid chromatography (,HPLC) (Sykam, Gilching, Germany), using an
Aminex HPX (Bio-Rad; Miinchen, Germany) separatjon column (300 X 7.8 mm> at 65°C 1 mM H,SO,
at 0.8 ml mini’ as eluent, and a differential refractometer (Shimadzu, Kyoto, Japan) as detector.
Acid volatile sulfides (AVS: mainly FeS) and
chromium reducible sulfur (CRS; mainly FeS, based
on unpublished results; given in mol per volume of
soil) were determined in the soil segments as described by Howarth and Jorgensen [l]. The concentration of ZnS in subsamples of the Zn-acetate trap
was determined spectrophotometrically
[3 1,321. Separate experiments showed a total recovery of AVS of
> 93% (53.3%)
and of CRS of 100% (+7.0%).
Sediment bulk density was determined by measuring the weight of the core segments of known volumes. Organic matter was analysed as weight loss of
the dried sediment after ignition for 24 h at 450°C.
All determinations
were made in triplicate.
2.3. Sulfate
potentials
reduction
and thiosulfate
consumptioil
The potential consumption rates of sulfate (SRP)
and thiosulfate (TCP) were measured in homogenously mixed soil slurries prepared from soil segments at different depths. The slurries were prepared
by mixing the paddy soil segments with sulfate-free
mineral medium [33] at a ratio of 1:3 (w/w> using
120-ml serum bottles as incubation vessels. Sterile
sulfate or thiosulfate was added to a final concentration of 600-1000
PM, so that SRP and TCP were
not limited by electron acceptor. The bottles were
flushed with N1, sealed with black rubber stoppers
and incubated on a platform that was slowly rotating
in vertical direction (2 rpm> at 30°C. For determina-
Ecology IX f IYY51257-266
259
tion of sulfate and thiosulfate concentrations.
1.5 ml
samples were withdrawn with plastic pipettes, centrifuged for 2 min at 10 000 ‘pm, filtered through 0.2
pm cellulose acetate membrane filters, and immediately injected into the HPLC or stored at -20°C
until analysis. The consumption potentials were determined by linear regression from the depletion of
sulfate or thiosulfate in the water phase. The standard deviation of regression was calculated for each
potential rate by using MicroCal Origin (MicroCal
Software Inc., USA).
Experiments to stimulate SRP with various electron donors were performed as in the slurry experiments described above, except that the entire locm-deep paddy soil microcosm was used. The electron donors which were used in this experiment were
hydrogen (88% in CO,), or a mixture of the following substrates: acetate (2.3 mM), lactate (1.4 mM),
butyrate ( 1.2 mM), propionate (1.2 mM), and succinate (1.2 mM).
2.4. Counts of sulfate-reducing
bacteria
SRB were enumerated by the most probable number (MPN) technique using ten-fold dilutions in three
parallels of anoxic medium tubes [34]. Soil samples
were collected anoxically from different segments of
the paddy soil microcosms and diluted with freshwater medium [33] containing
sulfate (20 mM) and
either hydrogen (88% in CO,), acetate (10 mM),
propionate (7.7 mM) or succinate (5 mM) as electron
donor. Enumerations
were also carried out with IOweek-old microcosms, using a mixed sample of the
entire microcosm as inoculum and one of the following substrates: hydrogen (88% in CO?), acetate (10
mM). propionate (7.7 mM), buyrate (7 mM), succinate (5 mM), L-lactate (10 mM), or benzoate (2
mM). The tubes were incubated for three months at
30°C. Growth of SRB was assessed by detection of
the produced sulfide as CuS [35]. Statistical comparisons of the individual MPN were carried out by a
modified Student’s t-test [36]. Replicates of enumerations were evaluated with the Student’s t-test and
the nonparametric
Wilcoxon test using the logarithms of the bacterial numbers.
Enrichment cultures were prepared with sulfatecontaining (20 mM) freshwater medium [33] and one
of 21 different substrates: Hz, acetate, propionate,
butyrate, iso-butyrate, valerate. iso-valerate. caproate.
caprinate. palmitate, benzoate, phenole, succinate.
pimelate, acetone, isopropanol, L-alanin. L-serin. Ltryptophane.
D-glucose. D-cellobiose. pyruvate. or
L-lactate.
S042-
[cIMI
3. Results
The unplanted
paddy soil microcosms
were
macroscopically
differentiated into three zones: ( I )
an oxidized brownish-colored
surface layer at the
uppermost 5 mm depth; (2) a black zone that was
due to high concentrations of FeS at 5-10 mm depth
(see below); and (3) a bluish-grey zone in deeper
sections ( > IO mm) of the sediment.
The surface layer (O-2 cm) of planted soil showed
a higher content of organic carbon and a lower bulk
density than that of unplanted soil (Fig. I ). This was
probably due to the presence of rice roots in the
planted microcosms,
especially in the upper soil
layers [23]. The pH in both planted and unplanted
microcosms increased with depth from pH 6.8 at I
cm depth to pH 7. I at IO cm depth. The pH of the
floodwater was pH 7.9.
Sulfate concentrations
were highest in the soil
surface layer and decreased gradually with depth.
They were significantly
higher in planted than in
unplanted paddy soil microcosms (Fig. 3a). In un-
.
.
‘;,I
,
.,2
3E4v5.
:
l
l
cn6
: 7
a9
1
IO-
’
l
3.0 3.5 4!0 4!5 do $5 610
org. C[ %]
Fig. I. Organic carbon content and bulk-density of 13.week-old
planted and unplanted rice microcosms. The average coet’ficicnt of
variation was approx. 10%. 0 unplanted.
*
planted.
a
1
,
.,
0
.,
50
,
loo
,
.,
150 MO250
b
1.
300
:
%42-~~Ml
Fig. 2. Pore
water concentrations of sulfate (a) and thioaulfate (b)
in 13.week-old
planted and unplanted paddy soil microcosms
(horizontal bars = standard error. II = 3: vertical bar5 = thickness
of soil segment). Thioalt’atc
detectable
( <
I
FM).
in
unplanted
0 unplanted,
*
microcosms
was
no1
planted.
planted microcosms, sulfate was no longer detectable
at depths > 5 cm, whereas in planted microcosms,
sulfate concentrations were generally > 30 PM. The
initial sulfate concentration
of the floodwater (tap
water) was 155 PM, and decreased during the period
of 13 weeks to about 90 PM, indicating a net uptake
of sulfate from the water into the soil. Since both
planted and unplanted microcosms were incubated in
the same water bath, it is not possible to distinguish
between the sulfate uptake of the two treatments.
Thiosulfate concentrations in unplanted cores were
in general below the detection limit (0.5 PM). In
planted microcosms, however. thiosulfate was found
in concentrations
of > 150 PM in the uppermost
centimeter. It decreased with depth to concentrations
of about IO PM at > 7 cm depth (Fig. 3b). In the
floodwater, thiosulfate was not detectable.
The vertical distribution of the insoluble sulfur
fractions. AVS and CRS. is shown in Fig. 3. AVS
T. Wind, R. Conrad/ FEMS Microbiology Ecology 15:(19951 257-266
AVS
0
500
lau
[nmol
1m
planted and unplanted microcosms. Low concentrations of acetate were measured in the upper Iayers of
unplanted,
in comparison
to planted microcosms.
The top layers of unplanted microcosms were also
low in propionate and lactate. Propionate concentrations were generally low ( < 18 PM), but lactate
concentrations
in planted paddy soil reached about
30 and 100 FM in the deepest and the uppermost
layers, respectively.
Caproate, butyrate,
valerate,
succinate, ethanol and isopropanol
were not detectable (< 10 PM). Hydrogen was not measured in
this series of microcosms, but showed no significant
difference between planted and unplanted soil in
another series of slightly different paddy soil microcosms (Dannenberg
and Conrad, manuscript
in
preparation).
The vertical profiles of the microbial sulfate reduction potentials (SRP) and thiosulfate consumption
cme3]
m
2500
0
2
2
x
+
:
n
2
3
4
5
6
7
8
a
9
10
0
&
0
3
z4
x5
a”
6
7
8
9
10
J
Acetate [ PM]
0
CRS
[nmol
261
200
400
600
600
cmm3]
Fig. 3. Depth distribution of acid volatile sulfides (AVS, (a)) and
chromium reducible sulfides (CRS, (b))of 13.week-old planted
and unplanted rice paddy microcosms (horizontal bars = standard
error. n = 3; vertical bars = thickness of soil segment). q unplanted. * planted.
lg
‘:
_c 3E'
if! 4:
567-
exhibited maximum values in the upper centimeters
of the microcosms. The AVS concentration
at l-4
cm depth was significantly higher in planted than in
unplanted microcosms. In unplanted microcosms, the
AVS concentration
averaged for 10 cm depth was
only 59.8% of the planted microcosms. However, the
AVS concentrations in the uppermost 5- 10 mm zone
of unplanted microcosms
were exceptionally
high
exceeding 1600 nmol cmm3 soil. At this depth there
was a visible layer of precipitated black FeS. The
concentrations
of CRS (e.g. pyrite) were slightly
higher than those of AVS. However, except for the
top centimeter, CRS showed almost similar values in
planted and unplanted microcosms (Fig. 3). On average. the total amount of CRS in unplanted compared
to planted microcosms was 13% higher.
Vertical profiles of fatty acids are shown in Fig.
4. Acetate was the most abundant fatty acid. It
reached concentrations
of about 800 PM in both
E9x),
Fig. 4. Concentration profiles of acetate. lactate and propionate in
13-week-old unplanted and planted paddy soil microcosms. Note
the different scales of fatty acid concentrations.
The average
coefficient of variation was approx. 30%. A acetate. W: lactate,
0 : propionate.
T. Wind. R. Conrad / FEMS Microbiology
TCP[pnml
0.0
1,
0.0
0.5
1.0
1.5
1.0
1.5
d-l]
2.0
2.5
I.
,’
2.0
2.5
3.0
I.,
I,,
0.5
cm3
Ecology 18
f 199.5)
257-266
cosms, SRP and TCP decreased with depth. In unplanted microcosms TCP showed a similar pattern,
while SRP had a local maximum in the S-10 mm
zone. The dependence of SRP on different electron
donors was studied by time course experiments in
presence of additional substrates. A significant stimulation was recorded only in slurries to which hydrogen was added. The stimulation was 40% in unplanted and 28% in planted paddy soil microcosms.
TCP was also stimulated with hydrogen by 62% and
67% in planted and unplanted microcosms, respectively.
Since SRP in the upper layers of planted and
unplanted paddy soil differed by almost one order of
magnitude, it was interesting to determine the numbers of SRB. Consistent with earlier work done on
tropical paddy soil [ 15,371, a comparison of numbers
of SRB in dry soil. and in IO- and 13-week-old
paddy soil indicates a high dynamic of the populations of SRB during the development
of the rice
plants (Table 1). Vertical profiles of the numbers of
SRB in planted and unplanted paddy soil microcosms using hydrogen, acetate, propionate and succinate as electron donors indicated a relatively homogenous distribution with depth (Fig. 6). Due to
the relatively large confidence limits (i.e. about one
order of magnitude) of the MPN technique, pairwise
t-test [36] showed no significant differences between
different depths. There were also no significant differences between planted and unplanted microcosms,
except for the OS-2 cm depth segment and hydro-
3.0
SRP[ pm1 crrf3 d-l]
Fig. 5. Vertical distribution of (a) sulfate reduction potentials
(SRP) and (b) thiosulfate consumption potentials (TCP) of 13week-old planted and unplanted paddy soil microcosms (horizontal bars = standard error of the regression; vertical bars = thickness
of soil segment, analysis of one time course experiment).
q
unplanted, . planted
potentials (TCP) in 13-week-old planted and unplanted paddy soil microcosms are shown in Fig. 5.
SRP and TCP were significantly
higher in planted
than in unplanted microcosms.
In planted micro-
Table 1
Most probable numbers (MPN) of sulfate-reducing
bacteria (10’ cell (g dry weight)-’ 1 in dry paddy soil. and in planted and unplanted
flooded paddy soil microcosms of different age. Counts were performed with different electron donors. The numbers of the 13.week-old
microcosms give the range (median) of the vertical profile shown in Fig. 6
Substrate
(concentration)
Dry soil
H,/C02
@S/12%)
Acetate (10 mM)
Propionate (7.5 mM)
Succinate (5 mM)
Lactate (I0 mM)
Butyrate (7 mM)
Benzoate (2 mM)
9.2
0.5
0.9
4.8
0.2
0.9
9.2
Paddy soil unplanted
Paddy soil planted
10 weeks
13 weeks
IO weeks
13 weeks
I5
0.9
4.8
24
9.2
22
0.9
I-50 d (7)
2- 150 (53)
2-5 .’ (5)
2-l 1 ‘I (7)
n.d.
n.d.
n.d.
22
2.2
9.2
4.8
0.9
4.8
0.9
46-460 a (460)
3-llOO(15)
I O-70 a (43)
24-l 10 d (78)
n.d.
n.d.
n.d.
a Values with the same electron donor are statistically
the logarithm of the MPN.
n.d. = not determined.
different (a. < 0.
I) in the Student’s r-test or the nonparametric
Wilcoxon
test using
T. Wind, R. Conrad/
FEMS Microbiology
Ecology 18 (1995) 257-266
263
lus, Desulfobulbus, and Desulfotomaculum.
Further
characterization
of these strains is in progress.
0
1
-2
E
0
3
-4
4. Discussion
'5
8:
-1 a
”
--~
1
-
2-
1
E3
04
1
AZ5
X6
w7
0
a
9
!
10 I--_
I
l.OXld
I
l.OXld
.......1
1.0~18
.,!...-
l.oXld
.’
acetate
“I
1.0x1$
“”
I
“’
l.Oxlo6 6
106
Cell Number [cell counts g-’ dw]
Fig. 6. Profiles of MPN of sulfate-reducing
bacteria ORB) of
13-week-old planted and unplanted paddy soil microcosms, enumerated on hydrogen, propionate, acetate and succinate. Numbers
are given in cell counts per g dry weight. Confidence limits for the
MPN were approximately
one order of magnitude. 0 unplanted,
* planted (vertical bars = thickness of soil segment).
gen as electron donor ((Y < 0.05). When the logarithms of the numbers were averaged over depth, a
significant difference between planted and unplanted
microcosms could only be found for hydrogen, propionate and succinate as electron donors, though at a
low (a < 0.1) significance (Table 1). However, the
numbers of acetate-utilizing
SRB were not significantly different in planted and unplanted paddy soil
microcosms (Table 1).
Enrichment
cultures on 21 different substrates
resulted in the isolation of 8 strains of SRB on the
following substrates: hydrogen, acetate, propionate,
iso-butyrate,
succinate,
pimelate,
and
butyrate,
caproate. Spore-forming bacteria of the genus Desulfotomaculum were obtained on acetate and pimelate.
Physiological
tests were performed
according
to
Widdel and Bak [33]. They revealed at least 4 different genera of SRB, i.e. DesulfoLGbrio, Desulfobotu-
Our study showed that the average concentration
of dissolved sulfur compounds (sulfate and thiosulfate) was 20- to 40-fold higher in planted than in
unplanted microcosms. Since the sulfate concentration in the floodwater decreased by about 70 PM
over the time of 13 weeks, it is possible that sulfatecontaining floodwater was taken up into the soil by a
convective transport that was driven by the evapotranspiration of the rice plants. In unplanted microcosms, where evapotranspiration
was absent, a convective transport of sulfate into the soil was not
possible. However, convective transport is not an
explanation
for the occurrence
of thiosulfate
in
planted microcosms as thiosulfate was absent in the
floodwater.
Therefore, it is more likely that the
increased concentrations of thiosulfate and sulfate in
the upper soil layers of the planted microcosms were
due to oxidation of reduced sulfur compounds driven
by the release of oxygen from rice roots.
Thiosulfate was reported to be the main product
of chemical oxidation of sulfide both under anoxic
and oxic conditions [26]. Thiosulfate was proposed
to function as an important intermediate in the sulfur
cycle [26]. The chemically unstable thiosulfate can
readily be oxidized to sulfate, or can be reduced to
sulfide [26]. There are only a few measurements of
thiosulfate in freshwater sediments which all exhibited concentrations
< 1 PM [38]. Here we report for
the first time vertical profiles of thiosulfate in paddy
soil with concentrations
reaching > 150 PM. Since
thiosulfate was only detected in the planted microcosms, we assume that it was an oxidation product of
sulfide mediated by the oxidation potential of the
plant roots. The release of oxygen from the rice roots
due to diffusive transport through the aerenchyma of
the plant [23,24,39] may have resulted in the chemical or biological oxidation of sulfide. Sulfur-oxidizing bacteria such as Beggiatoa and Thiobacillus are
known to occur at the rhizoplane
of rice roots
[ 13,16,40]. Alternatively,
sulfate and thiosulfate may
have been produced by chemical oxidation of sulfide
with oxygen, or by oxidation of sulfide with Fe(III),
MnW)
or oxidized N-compounds,
which presumably also exist adjacent to the root surface. Indeed,
increased redox potentials [22,24] and detectable
concentrations
of oxygen [23] were reported for the
rhizosphere of planted microcosms.
However, the presence of plants did not only
result in increased concentrations
of oxidized but
also of reduced sulfur compounds, i.e. AVS. AVS
(mainly FeS) and CRS (mainly FeS,) concentrations
in the paddy soil microcosms were similar to those
in other freshwater sediments [I I], but IO-100 times
smaller than in marine sediments [1.5]. AVS was
relatively high in the upper part of the planted
microcosms,
where the SRP and TCP were also
relatively high. Preliminary
experiments
with soil
slurries indicated that AVS was the major fraction of
the reduced sulfur compounds that were formed as
the short-term end-products of sulfate reduction (data
not shown). CRS concentrations differed not much in
unplanted
and planted microcosms.
On average,
however, unplanted microcosms showed a slightly
higher CRS concentration
than planted ones. This
could be due to the oxidation potential of the rice
roots or to root exudation resulting in CRS dissolution. For example. it has been shown that Fe0111 can
be solubilized by organic ligands (e.g. root exudates)
and then act as an oxidant for reduced iron sulfides
[41,42]. However, our analyses do not provide a
complete budget of sulfur in paddy soil, since S” and
organic sulfur were not determined.
SRP and TCP that were determined by slurry
experiments do not represent the real in situ rates of
sulfate reduction and thiosulfate consumption. However, our experiments show the relative distribution
of the potential activities in planted and unplanted
paddy soil. The SRP that were measured in the
unplanted microcosms were comparable to those in
freshwater
sediments
[6,9,11]. In planted microcosms, however, SRP were especially high indicating that the conditions for SRB were more suitable
in planted than in unplanted paddy soil.
The paddy soil microcosms also showed a relatively high TCP. However. the pathway of thiosulfate consumption remains unknown. Anaerobic pathways of thiosulfate consumption
include the reduction of thiosulfate to sulfide or the disproportionation
to sulfate and sulfide [26-291. Depth profiles of TCP
showed a similar pattern to those of SRP. As with
sulfate reduction, the TCP was stimulated by hydrogen in planted and unplanted systems. Preliminary
experiments with paddy soil slurries showed only a
small increase of sulfate during thiosulfate consumption, indicating that reduction rather than disproportionation was the main consumption pathway. However, further work on the pathways of thiosulfate
consumption in rice paddy soil has to be done.
The high rates of SRP and TCP may be due to the
relatively high numbers of SRB in planted soil (see
below). The presumably oxidized conditions in the
soil surface layer and in the root zone of the rice
plants ([23.24], and unpublished
results on other
microcosms from Italian paddy soil) obviously did
not inhibit the development of populations of SRB,
since a relatively high rate of SRP and TCP was
found in these zones. SRB are able to survive in oxic
environments
[43.44]. Moreover. it was found that
SRB are able to respire oxygen under microaerophilic conditions [45]. Strategies of active growth
in oxic environments are seen in the establishment of
anoxic microniches such as the formation of iron
sulfide aggregates
[46,47]. These aggregates are
likely to form around particles of organic compounds, where oxygen could locally be removed
rapidly due to high respiration
rates. In planted
paddy soil, decaying roots could provide such environments that make high sulfate reduction potentials
reasonable.
Jaqc et al. [14] determined
higher
amounts of ferrous iron and sulfides in the pore
water solution of planted than in unplanted flooded
soils. The authors concluded that, despite the aeration processes of the roots. iron reduction and sulfate
reduction are stimulated by the roots.
Populations
of SRB seem to be higher at the
flowering stage of the vegetation cycle [ 15,371. This
may be explained by increasing amounts of senescing or decaying roots that supply organic substrates
as electron donors to the SRB [ 141, by root exudation
[ 18,191, or by a decrease of the redox potential due
to insufficient
oxidative power of senescing roots
[37]. Although depth profiles of the MPN of SRB
with hydrogen.
propionate
and succinate always
showed higher numbers in planted than in unplanted
microcosms (Fig. 6), the difference was at a statistically low significance (a < 0. I ). Only the OS-l.5
cm depth segment of planted microcosms exhibited
significantly ( CY< 0.05) higher numbers of H 2-utiliz-
T. Wind. R. Conrad/
FEMS Microbiology
ing SRB than the unplanted microcosms. Despite the
statistic uncertainties, our results are consistent with
field observations where the numbers of SRB were
almost always higher in planted than in unplanted
paddy soil [ 13- 15,371.
In agreement with earlier studies 148,491, acetate
was observed to be the most abundant fatty acid in
the Italian paddy soil microcosms. However, this is
not necessarily an indication for acetate being an
important substrate of SRB. Quite in contrast, attempts to stimulate sulfate reduction with acetate
(included in a palette of different electron donors)
failed. This result is consistent with earlier observations in paddy soil slurries [50] and may be explained by the failure of Desulfotomaculum
spores
to germinate and then utilize acetate. It is possible
that the germination
process takes longer than the
incubation time used. In fact, acetate-utilizing
SRB
seemed to be more numerous after 13 than after 10
weeks of incubation (Table I ), but the difference was
statistically not significant. In contrast to acetate, the
SRP was stimulated by HZ, which is consistent with
the relatively high numbers of SRB enumerated with
hydrogen even in dry soil samples (Table 1). However. more research is necessary to find out which
are the main substrates of SRB in paddy soil.
Enrichment
cultures on several electron donors
revealed a relatively high metabolic capability of the
sulfate reducers present in paddy soil. Eight strains
of SRB could be isolated on different substrates.
Further work has to be done to characterize the
isolates. but preliminary
experiments
indicate that
the SRB belong to the genera DesdfoLibrio,
Desulfobotulus, Desuljhomaculum,
and Desuvobulbus.
In conclusion, our results indicate that the turnover
of sulfur is more intensive in planted than in unplanted paddy soil. The intensification
of sulfur
turnover is probably the result of increased oxidation
of reduced sulfur to sulfate and of increased reduction of sulfate to sulfide. Both reactions are likely to
be stimulated by the plant roots which provide an
interface for oxidation processes and supply electron
donors for reduction processes.
Acknowledgements
We thank the late Dr. Friedhelm Bak who initiated this work. We also thank Dr. P. Janssen for
Ecology
18 (19YS) 257-266
helpful comments. Financial support
the Fonds der Chemischen Industrie.
265
was given by
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