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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 References [I] Howarth. R.W. and Jorgensen. B.B. (1984) Formation of ‘.‘S-labelled elemental sulfur and pyrite in coastal marine sediments (Limfjorden and Kysing Fjord, Denmark) during short-term “SO:reduction measurements. Geochim. Cosmochim. Acta 48. 1807-1818. [2] Howarth. R.W. and Merkel, S. (1984) Pyrite formation and the measurement of sulfate reduction in salt marsh sediments. Limnol. Oceanogr. 29. 598-608. [3] Jorgensen, B.B., Bang, M. and Blackburn. T.H. (1990) Anaerobic mineralization in marine sediments from the Baltic Sea-North Sea transition. Mar. Ecol. Progr. Ser. 59. 39-54. [4] King, G.M., Howes. B.L. and Dacey. J.W.H. 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