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Effects of lead upon the actions of sulfate-reducing bacteria in the rice rhizosphere
Soil Biology & Biochemistry 42 (2010) 1038e1044
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Effects of lead upon the actions of sulfate-reducing bacteria in the rice rhizosphere Huirong Lin a, Jiyan Shi a, *, Xinai Chen a, Jianjun Yang a, Yingxu Chen a, Yidong Zhao b, Tiandou Hu b a
Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029, PR China b Institute of High Energy Physics, Chinese Academy of Science, Beijing Synchrotron Radiation Facility, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2009 Received in revised form 25 February 2010 Accepted 27 February 2010 Available online 16 March 2010
Microbeemineral interactions play an important role in affecting geochemical transformations of heavy metals in the soil environment. The formation of metal sulfide, which is mediated by sulfate-reducing bacteria (SRB) through contributing to sulfate reduction is an important pathway for heavy metal stabilization in anoxic soil. In oxic rice rhizospheres, there are abundant sulfur oxidizing bacteria (SOB) which can enhance sulfur oxidation and hence the availability of heavy metals, resulting in the uptake of such metals by the plant and a potential risk to human health. In this study, the potential existence of SRB in oxic rice rhizospheres, their contribution to sulfate reduction, and potential to reduce the availability of heavy metal was investigated. PCR-DGGE fingerprinting and real-time PCR results showed increasing numbers of SRB with Pb addition, which corresponded with increases in soil pH and reduction in Eh, suggesting the enhancement of sulfur reduction and SRB activity. Sulfur K-edge XANES, which characterized sulfur speciation in situ, revealed reduced states of sulfur. The SRB mediated the sulfate reduction and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide and reduction of Pb availability. In return, acclimated SRB populations developed in Pb-polluted conditions. Hence stabilization of reduced sulfur by Pb enhanced the activity of SRB and sulfate reduction in rice rhizosphere. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Sulfate-reducing bacteria Rice rhizosphere Sulfate reduction Pb PCR-DGGE XANES
1. Introduction Contamination of heavy metals in soil and its subsequent accumulation along the food chain is a potential risk to human health. Great concern has been paid to the behavior of metals in the soileplant system, particularly on the availability of such metals to plants and their consumers. Rice is an important food crop in China and paddy soils play a significant role in sulfur cycling. It is confirmed that cycling of sulfur takes play at the interface between the oxygenated rhizosphere and the anoxic bulk soil. Sulfur oxidizing bacteria (SOB) which enhance sulfur oxidation and the availability of heavy metals are abundant and active in rice rhizosphere soil. They could boost the uptake of heavy metal in rice, resulting in serious threats to human health through the food chain (Kayser et al., 2000; Wang et al., 2008; Wind and Conrad, 1995). By contrast, sulfate-reducing bacteria (SRB) are likely to exist in bulk soil, which in paddies is typically anoxic, as they are considered to be strict anaerobes. Bioprecipitation of heavy metals as metal sulfides mediated by SRB is a promising strategy for heavy metal * Corresponding author at: Department of Environmental Engineering, Huajiachi Campus, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, PR China. Tel.: þ86 571 86971424; fax: þ86 571 86971898. E-mail address: [email protected] (J. Shi). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.02.023
pollution control as it is a ubiquitous process in anaerobic environment (Hoa et al., 2007; Liamleam and Annachhatre, 2007). The oxygenated rhizosphere effect of rice can potentially prevent activity of SRB and hence the precipitation of heavy metals. However, some subgroups of SRB could defense oxygen. This feature may favor bioprecipitation of heavy metals in situ in oxic conditions (Dolla et al., 2006). On the other hand, heavy metals also can have great impact on SRB activity in polluted conditions. The objective of this study was to investigate the existence of SRB and the possibility of reduction of sulfate in the oxic rice rhizosphere, as well as their interaction with Pb. The investigation of SRB was conducted by molecular tools (nested PCR-DGGE and phylogenetic analysis of 16S rRNA gene) that specifically monitor the diversity of SRB. Sulfur K-edge XANES was conducted to detect sulfur species in situ effectively, attached to classical analytical methods (Eh, pH measurements). 2. Materials and methods 2.1. Experimental design and treatments Waterloggogenic paddy soil was collected from the top layer (0e15 cm) of a rice field in Shaoxing, Zhejiang Province, China.
H. Lin et al. / Soil Biology & Biochemistry 42 (2010) 1038e1044
The soil had an organic matter content ¼ 2.26%; pH 5.63 (water); total P ¼ 0.54 g kg1; total K ¼ 13.0 g kg1; total S ¼ 0.25 g kg1. Aliquots (2.5 kg) of air dried soil were put into 30 plastic pots and each treated with 0.4 g kg1 urea and 0.4 g kg1 K2HPO4. The experimental design was employed with Na2S2O3 and Pb(NO3)2. The chemicals were dissolved in distilled water, sprayed on the soil samples, and mixed evenly. Pb treatments were 0, 500 and 1200 mg kg1 dry soil separately. Sulfur treatments were 1 g kg1 dry soils on each Pb concentration, to provide substrate for SOB and SRB. Deionized water was added daily to maintain flooded conditions. After 3 months of soil aging, 3 rice seedlings (Oryza sativa L. Hybrid Zhenong 7) were transplanted to each nylon mesh bag per pot to keep roots in a small space as described by Lu et al. (2000). All treatments were carried out in triplicate in a greenhouse with a randomised block design. 2.2. Eh measurement, sampling and detection of physicalechemical characterization Eh measurements were carried out in situ before sampling by inserting the electrodes into the rhizosphere and bulk soil at the same depth using Orion Epoxy Sure-Flow Combination Rdeox/ORP (9678BN) following the manufacturer’s instructions. The rhizosphere soil was collected after 45 days growth. Part was stored at 20 C for PCR-DGGE and the rest was freeze-dried for detection of physical-chemical characterization and sulfur XANES analysis. Soil pH value was measured (H2O: soil ¼ 5). The speciation of Pb in the soil was determined by a sequential extraction procedure, which was based on the method reported by Chen et al. (2004). This sequential extraction procedure divided heavy metals in sediments into five binding forms, namely, exchangeable, carbonate-bound, Fe/Mn oxide-bound, organic matter-bound and residual. The Pb contents were determined by flame atomic absorption spectrophotometry (PerkinElmer AA100) after extraction with appropriate chemical agents associated with the forms above. 2.3. Direct PCR and nested PCR-DGGE Total soil DNA of the rhizosphere soils was extracted and purified using a bead beating method (FastDNAÔ SPIN Kit for Soil, Bio101 Inc., USA) following the manufacturer’s instructions. A direct amplification with primers specific for the six different groups of SRB was attempted on the extracted DNA. The six pairs of the SRB group-specific primers were DFM140 and DFM842 for Desulfotomaculum (Group 1), DBB121 and DBB1237 for Desulfobulbus (Group2), DBM169 and DBM1006 for Desulfobacterium (Group 3), DSB127 and DSB1273 for Desulfobacter (Group 4), DCC305 and DCC1165 for DesulfonemaeDesulfocarcinaeDesulfococcus (Group 5), DSV230 and DSV838 for DesulfovibrioeDeuslfomicrobium (Group 6; Daly et al., 2000). A strategy of three-step nested PCR-DGGE was then used to analyze the SRB diversity in rice rhizosphere (Dar et al., 2005). Firstly, the nearly complete sequence of 16S rRNA was amplified using the primer GM3F/GM4R (Muyzer et al., 1995). Then a following amplification with each of the six pairs of the SRB group-specific primers was carried out using the product obtained as a template. Finally, the product obtained in Step 2 was used as a template for the amplification with primers F341GC and R518 (Muyzer et al., 1993) in order to generate products suitable for DGGE. Details of the different oligonucleotides and the reaction condition used in this study were as the description of Dar et al. (2005). The obtained PCR products were then subjected to DGGE analysis using a DcodeÔ Universal Detection System instrument according to the manufacturer’s instructions (Bio-Rad, USA). DGGE
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was performed using a denaturing gradient of 25%e60% denaturants (100% denaturant contained 7 M urea and 40% (v/v) formamide) in 8% polyacrylamide gel in 1TAE buffer (pH 8.0) at 60 C for 6.5 h at a constant voltage of 160 V (DcodeÔ Universal Detection System, Bio-Rad, USA). After electrophoresis, the gels were stained with SYBRÔ GREEN I (Sigma, USA) for 30 min following the manufacturer’s instructions. 2.4. Cloning, sequencing and phylogenetic analysis Individual bands were excised, reamplified. The product was then purified using Qiaquick PCR Clean-up columns (Qiagen, Valencia, CA). The purified products were ligated into the pMD19-T easy cloning vector (Takala, Japan) and transformed to E. coli DH5a competent cells. Clones grew for 12e16 h in LuriaeBertani agar adding 100 mg ml1 ampicillin, and were identified based on bluewhite screening. Plasmid DNA was purified with UNIQ-10 column Plasmid Mini-prep Kit (Sangon, Canada) and sequenced by the Invitrogen Corporation (USA). Sequences recovered from the excised bands were analyzed using Premier Version 5.0. Sequences were compared using the National Center for Biotechnology Information (NCBI) BLAST program and the Ribosomal Database Project II (RDPII) Chimera Check program. Sequences were aligned with the CLUSTAL X 1.83 program and the resulting alignments were optimized by using Paup v.4.0b.8.a (Sinauer Associates, Inc., Sunderland, Mass.) to construct phylogenetic trees. The neighbor-joining algorithms were used to generate optimal tree topologies, confirmed by 1000-fold bootstrapping. 2.5. Real-time PCR For the quantification SRB in the soil, two primers (B-F: 50 CCTACGGGAGGCAGCAGTG-30 ; B-R: 50 -TACCGCGGCTGCTGGCAC-30 ) were designed according to the results of cloning and sequencing with Primer Express 2.0 and Beacon designer. The sensitivities of PCR assays were determined with dilution series (105, 104, 103, 102 and 101) of template to make standard curve. The amplification was done in iQTM5 Multi-instrument real-time fluorescence quantitative PCR (Bio-Rad, the USA). The reaction was carried out as follows:10 s initial denaturation at 95 C, followed by 45 cycles of denaturation at 95 C for 5 s, annealing at 62 C for 20 s and DNA extension at 72 C for 1 min. The 16S rDNA target numbers were then calculated according to the standard curve. 2.6. Sulfur speciation using sulfur K-edge XANES Sulfur species in rhizosphere soil were analyzed by X-Ray absorption Spectroscopy at Beijing Synchrotron Radiation Facility, Institute of High Energy Physics of China. The freeze-dried soil samples were pressed into thin films before analysis. The storage ring was operated at the energy of 2.5 GeV with Si (111) double crystals. Spectra were recorded at 4B7A beamline (medium X-ray beamline, 2100e6000 eV) and scanned at step widths of 0.3 eV in the region between 2420 and 2520 eV, with fluorescence mode using a fluorescent ion chamber Si (Li) detector (PGT LS30135). Additional filters were placed between the sample and the detector to reduce the fluorescence signal derived from Si in the soil samples. Reference compounds for exploring different sulfur oxidation states and chemical structures are shown in Table 1. The X-ray energy was calibrated with reference to the spectrum of the highest resonance energy peak of Na2SO4 at 2.4804 KeV. The speciation of sulfur was identified by the energy position of Gaussian peak and the relative abundance of each sulfur species
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Table 1 Representative reference compounds used in this study.
160
Structure
Reference compounds
Oxidation state S atom
Sulfide Elemental S Organic polysulfide Organic disulfide
S2 S0 ReSeSeSeR0 ReSeSeR0
2 0 þ0.15 þ0.2
Thiol
ReSH
Thiosulfate Sulfite Sulfone Sulfonate
ReS(]O)eR0 SO23 ReS(]O)2R0 ReSO2OeX
Ester sulfate
ReOeSO3
Inorganic sulfate
SO2 4
Ferrous sulfide S Potassium persulfate Cystine; Oxidized glutathione Cysteine; Reduced glutathione Sodium thiosulfate Sodium sulfite Dimethyl sulfone Sodium diphenylamine sulfonate Sodium dodecyl sulfate (SDS) Sodium sulfate
þ0.5 2, þ6 þ3.68 þ4 þ5 þ6
100 80 60 40 20 0 500 -1 Pb added (mg kg )
1000
1500
Fig. 2. Distribution of Pb fractions in rhizosphere soil of rice under a gradient of Pb pollution defined by the sequential extraction procedure.
changes of the composition of Pb species in the rhizosphere with increasing Pb addition in the rhizosphere of rice. In all Pb gradients, residual Pb was in the majority (more than 50%).
2.7. Statistical analysis All data were analyzed using Microsoft Excel 2003, Origin 7.5 and SPSS 11.5. The treatment effects were carried out with one-way ANOVA, the LSD multiple range test and Paired-Samples T Test by using SPSS 11.5 (SPSS for windows, Version 11.5, USA). Differences with values of P < 0.05 were considered statistically significant. 3. Results 3.1. Physicalechemical characterization of the rhizosphere soil The variations of Eh in rhizosphere and bulk soil of rice plants are shown in Fig. 1. Pb addition resulted in changes of Eh values in the soil. Eh values decreased dramatically with increasing Pb addition (P < 0.05). Consistently, Eh values in rhizosphere soil were higher than those in bulk soil (P < 0.05). Soil pH increased when Pb addition increased which exhibited dramatic changes between the samples under different Pb gradients (P < 0.05). Fig. 2 shows the
600 RS BS
500 400 300 200 100 0 500
3.2. PCR-DGGE analysis of 16S rRNA genes Direct PCR with six pairs of SRB specific primers did not succeed, suggesting that in oxic rice rhizosphere, the SRB groups accounted for only a small percentage of the total soil bacteria because target bacteria could not be detected by direct PCR when they were too few (Muyzer et al., 1993). Then a nested PCR strategy was attempted. Primers for Desulfotomaculum (Group 1), and Desulfobulbus (Group2) were successful, so the three-step nested PCRDGGE was conducted on them. The nested PCR-DGGE analysis indicated that there were some SRB in rice rhizosphere. There was an increase in the number of bands visualized on the DGGE gel with increasing Pb addition (Fig. 3). 3.3. Phylogenetic analysis of SRB and real-time PCR Closest matches of cloned DGGE bands to known species are shown in Table 2 (Fig. 4). A phylogenetic analysis was carried out using 16S rRNA sequences obtained with the primers used as shown in Fig. 5. Among these clones, B11, B12, B13 and B17 all had high similarities to gamma proteobacteria which had been proved to include bacteria associated with sulfur metabolism (Engel et al., 2007; Stubner, 2004). Some clones in our study (A3, A8, B15) were similar to Firmicutes. Some (e.g. A5, B2, B6, B8, B20) were closet relative to SRB or SRB enrichment clones. Besides, B7 was found to be similar to delta proteobacteria which mainly consist of sulfateand iron-reducing bacteria. Some of these subgroups of SRB corresponded with some of the previous studies conducted on paddy soil with other methods (Lu et al., 2006; Stubner, 2002; Stubner, 2004; Stubner and Meuser, 2000). There were significant differences among different treatments (P < 0.05). The target numbers increased with increasing Pb addition with target numbers of 3.31 102,1.90 103 and 2.97 103.
700
Eh (mV)
120
0 þ6
was determined from the peak area. Analysis of XANES was carried out using IFEFFIT package.
0
140
Percentage (%)
Sulfur species
residual organic matter-bound Fe/Mn oxide-bound carbonate-bound exchangeable
1000 -1
3.4. Sulfur speciation using sulfur K-edge XANES
Pb added (mg kg ) Fig. 1. Eh in rhizosphere and bulk soil of rice under a gradient of Pb pollution (RS, rhizosphere soil; BS, bulk soil). Bars indicate the standard deviation of the means (P < 0.05).
XANES spectra of some representative organic and inorganic sulfur reference compounds with different oxidation states (reduced and oxidized) as well as the rice rhizosphere soil samples
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Fig. 3. DGGE profiles of 16S rRNA fragments obtained after enzymatic amplification using different primer pairs and DNA from rhizosphere soil under a gradient of Pb pollution (a, Desulfotomaculum;b, Desulfobulbus).
Table 2 Closest match of cloned DGGE bands to known species. Bands Closest relative A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20
Alicyclobacillaceae bacterium JAM-FM0301 gene for 16S rRNA Bacillales bacterium BPC-C1/31-1 16S ribosomal RNA gene Sulfobacillus disulfidooxidans partial 16S rRNA gene Alicyclobacillaceae bacterium JAM-FM0301 gene for 16S rRNA Desulfotomaculum sp. 175 16S ribosomal RNA gene Uncultured soil bacterium clone JAB ST 77 16S ribosomal RNA gene Thermophilic bacterium sp. MP3 16S rRNA gene. Uncultured Firmicutes bacterium gene for 16S rRNA Sulfobacillus sp. C38-3 gene for 16S rRNA Desulfotomaculum sp. 175 16S ribosomal RNA gene Uncultured Chitinophaga sp. Uncultured Desulfobacteraceae bacterium Clostridiales Uncultured Chitinophaga sp. Bacillus drentensis strain Sulfate-reducing bacterium enrichment clone Uncultured delta proteobacterium clone Sulfate-reducing bacterium enrichment clone Uncultured Clostridiaceae bacterium clone Bacillus drentensis strain Gamma proteobacterium A40-1 16S ribosomal RNA gene Gamma proteobacterium TH-N59 partial 16S rRNA gene. Gamma proteobacterium TH-N59 partial 16S rRNA gene. Uncultured Acidobacterium group bacterium Uncultured Firmicutes bacterium clone Uncultured Acidobacterium sp. Uncultured gamma proteobacterium clone Bacillus sp. G2DM-33 Uncultured Desulfobulbaceae bacterium Uncultured Desulfobacca sp.
Similarity Acc nr 94%
AB362268
93%
DQ999995
96%
AJ871255
95%
AB362268
97%
AF295656
91%
EF495047
90% 91%
AJ607431 AB433145
90% 99%
AB059475 AF295656
96% 91% 98% 96% 100% 88% 93% 88% 98% 100% 99%
EU300389 EF187876 AB308100 EU300389 FJ174597 DQ903932 DQ676369 DQ903932 EU300070 FJ174597 AY049941
99%
AJ786005
100%
AJ786005
96% 97% 96% 100% 98% 93% 95%
AJ534690 EF123530 EU156148 DQ211474 DQ416793 AY711479 EF613479
revealed different sulfur species produced multiple peaks indicative of different oxidation states of the component S atoms (Fig. 5). Clear differences could be observed in different reference compounds. By comparing the X-ray absorption edge energies of samples with the reference compounds, sulfur XANES revealed the presence of multiple both oxidized and reduced states of sulfur in studied soil samples. The high absorption intensities of oxidized sulfur species were much greater than those of reduced forms in all the soil samples, indicating that oxidized sulfur accounted for a majority in the total sulfur though oxidized sulfur itself can cause higher peak than reduced states due to consequently increased orbital overlap the stronger transition dipole arising from polar bonding (Jalilehvand, 2006). The intense absorption positions of the studied soil samples were all around the peak of ferrous sulfide (FeS) (2.4705 KeV), indicating the presence of sulfide in rice rhizosphere. The percentage of sulfide increased concomitant with the Pb addition, indicating the enhancement of sulfate reduction (Table 3). 4. Discussion 4.1. Survival of SRB in rice rhizosphere and their role in Pb transformation The Eh results of the studied soil showed that rice rhizosphere was oxic, which is considered to be unsuitable to engender SRB activity. However, nested PCR-DGGE and phylogenetic analysis results indicated that there were some SRB in oxic conditions. With the success of nested PCR, different members of SRB were visualized by DGGE. Phylogenetic analysis results showed the main subgroups SRB in rice rhizosphere. The small number of SRB detected in the studied soil confirmed the importance of oxygenlimited environments within the soil. In rice rhizosphere, there could be anoxic microsites that housed SRB. The observation of SRB presence in these soils confirmed the presence of such microsite. In addition, several studies confirmed the presence of SRB in periodically oxic environments. In some
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A10 AF295656 Desulfotomaculum sp. A5 AB433145 Uncultured Firmicutes bacterium A3 99 AJ871255 Sulfobacillus disulfidooxidans A2 65 A7 51 A8 79 53 A1 A9 82 A4 A6 85 AJ607431 Thermophilic bacterium sp. 55 AB362268 Alicyclobacillaceae bacterium B3 B13 DQ676369 Uncultured delta proteobacterium 99 B7 74 B14 91 B15 96 54 B16 B9 72 99 EU300070 Uncultured Clostridiaceae bacterium B2 EF187876 Uncultured Desulfobacteraceae bacterium B6 77 100 B8 87 DQ903932 Sulfate-reducing bacterium enrichment clone 50 B19 88 AY711479 Uncultured Desulfobulbaceae bacterium B20 83 EF613479 Uncultured Desulfobacca sp. EU300389 Uncultured Chitinophaga sp. 100 B1 93 B4 B17 100 B11 100 AJ786005 Gamma proteobacterium B12 B10 AJ174597 Bacillus drentensis strain 100 B5 B18 10 Fig. 4. Phylogenetic analysis showing relationships of clones sequenced from 16S rRNA gene amplified from the rhizosphere soil. Bootstrap support values with 1000 replicates are given along the branches.
cases, sulfate reduction has actually been detected in supposedly oxic zones of microbial mats and marine sediments. It was found that some SRB possess a relatively high degree of oxygen tolerance (Baumgartner et al., 2006; Dolla et al., 2006). SRB hold various enzymatic protection mechanisms against oxidative stress (Dolla et al., 2006).The sulfur cycle in these soils was probably sustained by re-oxidation of reduced S-compounds and the stabilization of reduced S-compounds by heavy metal between rhizosphere soil and bulk soil (Haitzer et al., 2003; Hu et al., 2007; Karlsson and Skyllberg, 2003; Morse and Luther, 1999). Most sulfur transformations are fundamentally controlled by specialized metabolism of microorganisms (Engel et al., 2007). Rice has an ability to grow in flooded, anaerobic soils because it can transport oxygen to respiring root tissues via internal gas channels. O2 is present via the aerenchymal system of the rice plants in the rhizosphere and a small layer (about 2e3 mm) at the soil surface (Frenzel et al., 1992). It is generally considered that reduced sulfur is oxidized by SOB in rhizosphere of rice while sulfate is reduced by SRB in bulk soil. The oxygen depletion in flooded paddy soil leads to changes in the bacterial community. The active respiration processes of the microbial community at the rhizosphere and bulk soil interface in
conjunction with the reduced diffusivity of oxygen leads to a rapid depletion of oxygen. With high Eh (Fig. 1), rice rhizospheres represent an oxic zone. As a result, oxidation states of sulfur were in the majority accordingly as they were the main end product of sulfur oxidation controlled by SOB. Nevertheless, the sulfur speciation showed both oxidized and reduced states in the studied soil samples (Fig. 5). The presence of oxidized and reduced states of sulfur might correlate to the presence of SOB abundant in rice rhizosphere that oxidized reduced sulfur and SRB contributing to sulfate reduction respectively. The reduced sulfur observed in the XANES spectra of rhizosphere soil samples further confirmed that the action of SRB promoted the reduction of oxidized sulfur which led to the formation of sulfide. Sulfide generated by sulfate reduction then reacted with Pb in the flooded soil forming stable metal sulfide.
4.2. The role of Pb in the activity of SRB in rice rhizosphere Pb in the studied soil influenced the number of SRB accordingly. With increasing Pb addition there was a dramatic rise of soil pH and
H. Lin et al. / Soil Biology & Biochemistry 42 (2010) 1038e1044
2.4804
2.4705
bacterial metallothioneins and heavy metal-transporting ATPases to exist in toxic heavy metal ions and contribute to sulfate reduction (Naz et al., 2005). These Pb tolerant indicated promising prospect for controlling of availability in high Pb pollution environment.
potassium persulfate sodium thiosulfate reduced glutathione cytine oxidized glutathione S cyteine
5. Conclusions
dimethyl sulfone ferrous sulfide sodium sulfite sodium diphenylamine sulfonate sodium sulfate
Absorption (a.u.)
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sodium dodecyl sulfate
Our results indicated the existence of SRB in oxic rice rhizosphere. The SRB mediated the reduction of sulfate and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide. In return, stabilization of reduced sulfur by Pb led to increasing numbers of SRB in rice rhizosphere. The presence of lead-tolerant SRB represented promising prospects of reducing the availability of Pb in environments polluted with this element. Acknowledgments This work was supported by the National Natural Science Foundation of China (40601086, 20777066) and the Natural Science Foundation of Zhejiang Province (Y506063). We gratefully thank Lei Zheng, Chenyan Ma, Yong Han at the Beijing Synchrotron Radiation Facility (BSRF) medium-energy beamline station for their generous help. We thank Dr. Adam Gillespie in Department of Soil Science, University of Saskatchewan for the improvement in the language.
-1
0 mg kg
-1
500 mg kg
-1
1200 mg kg
References
2.460 2.465 2.470 2.475 2.480 2.485 2.490
Energy (KeV) Fig. 5. Comparison of sulfur K-edge XANES spectra of reference compounds and rice rhizosphere soil samples. a.u., arbitrary units.
a significant drop of Eh. These results indicated the promotion of sulfate reduction and were consistent with previous report on the relationship of pH and sulfate reduction (Stein et al., 2007). As shown in Fig. 3, there was a concomitant rise of the number of bands in DGGE fingerprint with increasing Pb addition. Real-time PCR results further confirmed this. It may be due to the fact that increasing addition of Pb resulted in the formation of metal sulfide. The peak area of reduced sulfur increased along the Pb addition. This result indicated the enhancement of sulfate reduction corresponding to DGGE results. The precipitation of metal sulfide stabilized sulfide. As a result, the number of SRB increased which led to the enhancement of sulfate reduction. Microbial sulfate reduction is of great importance in influencing the transformation of heavy metals. As a result, great attention has been paid to SRB due to their important role in the mobility of heavy metal. The application of SRB in controlling the mobility of heavy metals requires heavy-metals-tolerant SRB which can grow in heavy-metals polluted environment. Our results indicated that there were some efficient and heavy metal tolerant SRB existed in comparatively high concentration of Pb. They may possess genetic determinants for metal resistance encoding
Table 3 Percentage of different S species as assessed by S K-edge XANES in rice shizosphere soil (atom% S). Pb added (mg kg1)
Sulfide
Organic monosulfide/ Organic disulfide
Sulfonate
Sulfate
0 500 1200
1.40 6.96 19.11
0 1.42 9.78
4.6 4.14 0
93.99 87.47 7.11
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Stubner, S., Meuser, K., 2000. Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses. FEMS Microbiology Ecology 34, 73e80. Stubner, S., 2002. Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection. Journal of Microbiological Methods 50, 155e164. Stubner, S., 2004. Quantification of Gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR. Journal of Microbiological Methods 57, 219e230. Wang, Y.P., Li, Q.B., Wang, H., Shi, J.Y., Lin, Q., Chen, X.C., Chen, Y.X., 2008. Effect of sulphur on soil Cu/Zn availability and microbial community composition. Journal of Hazardous Materials 159, 385e389. Wind, T., Conrad, R., 1995. Sulfur compounds, potential turnover of sulfate and thiosulfate, and numbers of sulfate-reducing bacteria in planted and unplanted paddy soil. FEMS Microbiology Ecology 18, 257e266.
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Effects of lead upon the actions of sulfate-reducing bacteria in the rice rhizosphere Huirong Lin a, Jiyan Shi a, *, Xinai Chen a, Jianjun Yang a, Yingxu Chen a, Yidong Zhao b, Tiandou Hu b a
Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029, PR China b Institute of High Energy Physics, Chinese Academy of Science, Beijing Synchrotron Radiation Facility, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2009 Received in revised form 25 February 2010 Accepted 27 February 2010 Available online 16 March 2010
Microbeemineral interactions play an important role in affecting geochemical transformations of heavy metals in the soil environment. The formation of metal sulfide, which is mediated by sulfate-reducing bacteria (SRB) through contributing to sulfate reduction is an important pathway for heavy metal stabilization in anoxic soil. In oxic rice rhizospheres, there are abundant sulfur oxidizing bacteria (SOB) which can enhance sulfur oxidation and hence the availability of heavy metals, resulting in the uptake of such metals by the plant and a potential risk to human health. In this study, the potential existence of SRB in oxic rice rhizospheres, their contribution to sulfate reduction, and potential to reduce the availability of heavy metal was investigated. PCR-DGGE fingerprinting and real-time PCR results showed increasing numbers of SRB with Pb addition, which corresponded with increases in soil pH and reduction in Eh, suggesting the enhancement of sulfur reduction and SRB activity. Sulfur K-edge XANES, which characterized sulfur speciation in situ, revealed reduced states of sulfur. The SRB mediated the sulfate reduction and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide and reduction of Pb availability. In return, acclimated SRB populations developed in Pb-polluted conditions. Hence stabilization of reduced sulfur by Pb enhanced the activity of SRB and sulfate reduction in rice rhizosphere. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Sulfate-reducing bacteria Rice rhizosphere Sulfate reduction Pb PCR-DGGE XANES
1. Introduction Contamination of heavy metals in soil and its subsequent accumulation along the food chain is a potential risk to human health. Great concern has been paid to the behavior of metals in the soileplant system, particularly on the availability of such metals to plants and their consumers. Rice is an important food crop in China and paddy soils play a significant role in sulfur cycling. It is confirmed that cycling of sulfur takes play at the interface between the oxygenated rhizosphere and the anoxic bulk soil. Sulfur oxidizing bacteria (SOB) which enhance sulfur oxidation and the availability of heavy metals are abundant and active in rice rhizosphere soil. They could boost the uptake of heavy metal in rice, resulting in serious threats to human health through the food chain (Kayser et al., 2000; Wang et al., 2008; Wind and Conrad, 1995). By contrast, sulfate-reducing bacteria (SRB) are likely to exist in bulk soil, which in paddies is typically anoxic, as they are considered to be strict anaerobes. Bioprecipitation of heavy metals as metal sulfides mediated by SRB is a promising strategy for heavy metal * Corresponding author at: Department of Environmental Engineering, Huajiachi Campus, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, PR China. Tel.: þ86 571 86971424; fax: þ86 571 86971898. E-mail address: [email protected] (J. Shi). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.02.023
pollution control as it is a ubiquitous process in anaerobic environment (Hoa et al., 2007; Liamleam and Annachhatre, 2007). The oxygenated rhizosphere effect of rice can potentially prevent activity of SRB and hence the precipitation of heavy metals. However, some subgroups of SRB could defense oxygen. This feature may favor bioprecipitation of heavy metals in situ in oxic conditions (Dolla et al., 2006). On the other hand, heavy metals also can have great impact on SRB activity in polluted conditions. The objective of this study was to investigate the existence of SRB and the possibility of reduction of sulfate in the oxic rice rhizosphere, as well as their interaction with Pb. The investigation of SRB was conducted by molecular tools (nested PCR-DGGE and phylogenetic analysis of 16S rRNA gene) that specifically monitor the diversity of SRB. Sulfur K-edge XANES was conducted to detect sulfur species in situ effectively, attached to classical analytical methods (Eh, pH measurements). 2. Materials and methods 2.1. Experimental design and treatments Waterloggogenic paddy soil was collected from the top layer (0e15 cm) of a rice field in Shaoxing, Zhejiang Province, China.
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The soil had an organic matter content ¼ 2.26%; pH 5.63 (water); total P ¼ 0.54 g kg1; total K ¼ 13.0 g kg1; total S ¼ 0.25 g kg1. Aliquots (2.5 kg) of air dried soil were put into 30 plastic pots and each treated with 0.4 g kg1 urea and 0.4 g kg1 K2HPO4. The experimental design was employed with Na2S2O3 and Pb(NO3)2. The chemicals were dissolved in distilled water, sprayed on the soil samples, and mixed evenly. Pb treatments were 0, 500 and 1200 mg kg1 dry soil separately. Sulfur treatments were 1 g kg1 dry soils on each Pb concentration, to provide substrate for SOB and SRB. Deionized water was added daily to maintain flooded conditions. After 3 months of soil aging, 3 rice seedlings (Oryza sativa L. Hybrid Zhenong 7) were transplanted to each nylon mesh bag per pot to keep roots in a small space as described by Lu et al. (2000). All treatments were carried out in triplicate in a greenhouse with a randomised block design. 2.2. Eh measurement, sampling and detection of physicalechemical characterization Eh measurements were carried out in situ before sampling by inserting the electrodes into the rhizosphere and bulk soil at the same depth using Orion Epoxy Sure-Flow Combination Rdeox/ORP (9678BN) following the manufacturer’s instructions. The rhizosphere soil was collected after 45 days growth. Part was stored at 20 C for PCR-DGGE and the rest was freeze-dried for detection of physical-chemical characterization and sulfur XANES analysis. Soil pH value was measured (H2O: soil ¼ 5). The speciation of Pb in the soil was determined by a sequential extraction procedure, which was based on the method reported by Chen et al. (2004). This sequential extraction procedure divided heavy metals in sediments into five binding forms, namely, exchangeable, carbonate-bound, Fe/Mn oxide-bound, organic matter-bound and residual. The Pb contents were determined by flame atomic absorption spectrophotometry (PerkinElmer AA100) after extraction with appropriate chemical agents associated with the forms above. 2.3. Direct PCR and nested PCR-DGGE Total soil DNA of the rhizosphere soils was extracted and purified using a bead beating method (FastDNAÔ SPIN Kit for Soil, Bio101 Inc., USA) following the manufacturer’s instructions. A direct amplification with primers specific for the six different groups of SRB was attempted on the extracted DNA. The six pairs of the SRB group-specific primers were DFM140 and DFM842 for Desulfotomaculum (Group 1), DBB121 and DBB1237 for Desulfobulbus (Group2), DBM169 and DBM1006 for Desulfobacterium (Group 3), DSB127 and DSB1273 for Desulfobacter (Group 4), DCC305 and DCC1165 for DesulfonemaeDesulfocarcinaeDesulfococcus (Group 5), DSV230 and DSV838 for DesulfovibrioeDeuslfomicrobium (Group 6; Daly et al., 2000). A strategy of three-step nested PCR-DGGE was then used to analyze the SRB diversity in rice rhizosphere (Dar et al., 2005). Firstly, the nearly complete sequence of 16S rRNA was amplified using the primer GM3F/GM4R (Muyzer et al., 1995). Then a following amplification with each of the six pairs of the SRB group-specific primers was carried out using the product obtained as a template. Finally, the product obtained in Step 2 was used as a template for the amplification with primers F341GC and R518 (Muyzer et al., 1993) in order to generate products suitable for DGGE. Details of the different oligonucleotides and the reaction condition used in this study were as the description of Dar et al. (2005). The obtained PCR products were then subjected to DGGE analysis using a DcodeÔ Universal Detection System instrument according to the manufacturer’s instructions (Bio-Rad, USA). DGGE
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was performed using a denaturing gradient of 25%e60% denaturants (100% denaturant contained 7 M urea and 40% (v/v) formamide) in 8% polyacrylamide gel in 1TAE buffer (pH 8.0) at 60 C for 6.5 h at a constant voltage of 160 V (DcodeÔ Universal Detection System, Bio-Rad, USA). After electrophoresis, the gels were stained with SYBRÔ GREEN I (Sigma, USA) for 30 min following the manufacturer’s instructions. 2.4. Cloning, sequencing and phylogenetic analysis Individual bands were excised, reamplified. The product was then purified using Qiaquick PCR Clean-up columns (Qiagen, Valencia, CA). The purified products were ligated into the pMD19-T easy cloning vector (Takala, Japan) and transformed to E. coli DH5a competent cells. Clones grew for 12e16 h in LuriaeBertani agar adding 100 mg ml1 ampicillin, and were identified based on bluewhite screening. Plasmid DNA was purified with UNIQ-10 column Plasmid Mini-prep Kit (Sangon, Canada) and sequenced by the Invitrogen Corporation (USA). Sequences recovered from the excised bands were analyzed using Premier Version 5.0. Sequences were compared using the National Center for Biotechnology Information (NCBI) BLAST program and the Ribosomal Database Project II (RDPII) Chimera Check program. Sequences were aligned with the CLUSTAL X 1.83 program and the resulting alignments were optimized by using Paup v.4.0b.8.a (Sinauer Associates, Inc., Sunderland, Mass.) to construct phylogenetic trees. The neighbor-joining algorithms were used to generate optimal tree topologies, confirmed by 1000-fold bootstrapping. 2.5. Real-time PCR For the quantification SRB in the soil, two primers (B-F: 50 CCTACGGGAGGCAGCAGTG-30 ; B-R: 50 -TACCGCGGCTGCTGGCAC-30 ) were designed according to the results of cloning and sequencing with Primer Express 2.0 and Beacon designer. The sensitivities of PCR assays were determined with dilution series (105, 104, 103, 102 and 101) of template to make standard curve. The amplification was done in iQTM5 Multi-instrument real-time fluorescence quantitative PCR (Bio-Rad, the USA). The reaction was carried out as follows:10 s initial denaturation at 95 C, followed by 45 cycles of denaturation at 95 C for 5 s, annealing at 62 C for 20 s and DNA extension at 72 C for 1 min. The 16S rDNA target numbers were then calculated according to the standard curve. 2.6. Sulfur speciation using sulfur K-edge XANES Sulfur species in rhizosphere soil were analyzed by X-Ray absorption Spectroscopy at Beijing Synchrotron Radiation Facility, Institute of High Energy Physics of China. The freeze-dried soil samples were pressed into thin films before analysis. The storage ring was operated at the energy of 2.5 GeV with Si (111) double crystals. Spectra were recorded at 4B7A beamline (medium X-ray beamline, 2100e6000 eV) and scanned at step widths of 0.3 eV in the region between 2420 and 2520 eV, with fluorescence mode using a fluorescent ion chamber Si (Li) detector (PGT LS30135). Additional filters were placed between the sample and the detector to reduce the fluorescence signal derived from Si in the soil samples. Reference compounds for exploring different sulfur oxidation states and chemical structures are shown in Table 1. The X-ray energy was calibrated with reference to the spectrum of the highest resonance energy peak of Na2SO4 at 2.4804 KeV. The speciation of sulfur was identified by the energy position of Gaussian peak and the relative abundance of each sulfur species
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Table 1 Representative reference compounds used in this study.
160
Structure
Reference compounds
Oxidation state S atom
Sulfide Elemental S Organic polysulfide Organic disulfide
S2 S0 ReSeSeSeR0 ReSeSeR0
2 0 þ0.15 þ0.2
Thiol
ReSH
Thiosulfate Sulfite Sulfone Sulfonate
ReS(]O)eR0 SO23 ReS(]O)2R0 ReSO2OeX
Ester sulfate
ReOeSO3
Inorganic sulfate
SO2 4
Ferrous sulfide S Potassium persulfate Cystine; Oxidized glutathione Cysteine; Reduced glutathione Sodium thiosulfate Sodium sulfite Dimethyl sulfone Sodium diphenylamine sulfonate Sodium dodecyl sulfate (SDS) Sodium sulfate
þ0.5 2, þ6 þ3.68 þ4 þ5 þ6
100 80 60 40 20 0 500 -1 Pb added (mg kg )
1000
1500
Fig. 2. Distribution of Pb fractions in rhizosphere soil of rice under a gradient of Pb pollution defined by the sequential extraction procedure.
changes of the composition of Pb species in the rhizosphere with increasing Pb addition in the rhizosphere of rice. In all Pb gradients, residual Pb was in the majority (more than 50%).
2.7. Statistical analysis All data were analyzed using Microsoft Excel 2003, Origin 7.5 and SPSS 11.5. The treatment effects were carried out with one-way ANOVA, the LSD multiple range test and Paired-Samples T Test by using SPSS 11.5 (SPSS for windows, Version 11.5, USA). Differences with values of P < 0.05 were considered statistically significant. 3. Results 3.1. Physicalechemical characterization of the rhizosphere soil The variations of Eh in rhizosphere and bulk soil of rice plants are shown in Fig. 1. Pb addition resulted in changes of Eh values in the soil. Eh values decreased dramatically with increasing Pb addition (P < 0.05). Consistently, Eh values in rhizosphere soil were higher than those in bulk soil (P < 0.05). Soil pH increased when Pb addition increased which exhibited dramatic changes between the samples under different Pb gradients (P < 0.05). Fig. 2 shows the
600 RS BS
500 400 300 200 100 0 500
3.2. PCR-DGGE analysis of 16S rRNA genes Direct PCR with six pairs of SRB specific primers did not succeed, suggesting that in oxic rice rhizosphere, the SRB groups accounted for only a small percentage of the total soil bacteria because target bacteria could not be detected by direct PCR when they were too few (Muyzer et al., 1993). Then a nested PCR strategy was attempted. Primers for Desulfotomaculum (Group 1), and Desulfobulbus (Group2) were successful, so the three-step nested PCRDGGE was conducted on them. The nested PCR-DGGE analysis indicated that there were some SRB in rice rhizosphere. There was an increase in the number of bands visualized on the DGGE gel with increasing Pb addition (Fig. 3). 3.3. Phylogenetic analysis of SRB and real-time PCR Closest matches of cloned DGGE bands to known species are shown in Table 2 (Fig. 4). A phylogenetic analysis was carried out using 16S rRNA sequences obtained with the primers used as shown in Fig. 5. Among these clones, B11, B12, B13 and B17 all had high similarities to gamma proteobacteria which had been proved to include bacteria associated with sulfur metabolism (Engel et al., 2007; Stubner, 2004). Some clones in our study (A3, A8, B15) were similar to Firmicutes. Some (e.g. A5, B2, B6, B8, B20) were closet relative to SRB or SRB enrichment clones. Besides, B7 was found to be similar to delta proteobacteria which mainly consist of sulfateand iron-reducing bacteria. Some of these subgroups of SRB corresponded with some of the previous studies conducted on paddy soil with other methods (Lu et al., 2006; Stubner, 2002; Stubner, 2004; Stubner and Meuser, 2000). There were significant differences among different treatments (P < 0.05). The target numbers increased with increasing Pb addition with target numbers of 3.31 102,1.90 103 and 2.97 103.
700
Eh (mV)
120
0 þ6
was determined from the peak area. Analysis of XANES was carried out using IFEFFIT package.
0
140
Percentage (%)
Sulfur species
residual organic matter-bound Fe/Mn oxide-bound carbonate-bound exchangeable
1000 -1
3.4. Sulfur speciation using sulfur K-edge XANES
Pb added (mg kg ) Fig. 1. Eh in rhizosphere and bulk soil of rice under a gradient of Pb pollution (RS, rhizosphere soil; BS, bulk soil). Bars indicate the standard deviation of the means (P < 0.05).
XANES spectra of some representative organic and inorganic sulfur reference compounds with different oxidation states (reduced and oxidized) as well as the rice rhizosphere soil samples
H. Lin et al. / Soil Biology & Biochemistry 42 (2010) 1038e1044
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Fig. 3. DGGE profiles of 16S rRNA fragments obtained after enzymatic amplification using different primer pairs and DNA from rhizosphere soil under a gradient of Pb pollution (a, Desulfotomaculum;b, Desulfobulbus).
Table 2 Closest match of cloned DGGE bands to known species. Bands Closest relative A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20
Alicyclobacillaceae bacterium JAM-FM0301 gene for 16S rRNA Bacillales bacterium BPC-C1/31-1 16S ribosomal RNA gene Sulfobacillus disulfidooxidans partial 16S rRNA gene Alicyclobacillaceae bacterium JAM-FM0301 gene for 16S rRNA Desulfotomaculum sp. 175 16S ribosomal RNA gene Uncultured soil bacterium clone JAB ST 77 16S ribosomal RNA gene Thermophilic bacterium sp. MP3 16S rRNA gene. Uncultured Firmicutes bacterium gene for 16S rRNA Sulfobacillus sp. C38-3 gene for 16S rRNA Desulfotomaculum sp. 175 16S ribosomal RNA gene Uncultured Chitinophaga sp. Uncultured Desulfobacteraceae bacterium Clostridiales Uncultured Chitinophaga sp. Bacillus drentensis strain Sulfate-reducing bacterium enrichment clone Uncultured delta proteobacterium clone Sulfate-reducing bacterium enrichment clone Uncultured Clostridiaceae bacterium clone Bacillus drentensis strain Gamma proteobacterium A40-1 16S ribosomal RNA gene Gamma proteobacterium TH-N59 partial 16S rRNA gene. Gamma proteobacterium TH-N59 partial 16S rRNA gene. Uncultured Acidobacterium group bacterium Uncultured Firmicutes bacterium clone Uncultured Acidobacterium sp. Uncultured gamma proteobacterium clone Bacillus sp. G2DM-33 Uncultured Desulfobulbaceae bacterium Uncultured Desulfobacca sp.
Similarity Acc nr 94%
AB362268
93%
DQ999995
96%
AJ871255
95%
AB362268
97%
AF295656
91%
EF495047
90% 91%
AJ607431 AB433145
90% 99%
AB059475 AF295656
96% 91% 98% 96% 100% 88% 93% 88% 98% 100% 99%
EU300389 EF187876 AB308100 EU300389 FJ174597 DQ903932 DQ676369 DQ903932 EU300070 FJ174597 AY049941
99%
AJ786005
100%
AJ786005
96% 97% 96% 100% 98% 93% 95%
AJ534690 EF123530 EU156148 DQ211474 DQ416793 AY711479 EF613479
revealed different sulfur species produced multiple peaks indicative of different oxidation states of the component S atoms (Fig. 5). Clear differences could be observed in different reference compounds. By comparing the X-ray absorption edge energies of samples with the reference compounds, sulfur XANES revealed the presence of multiple both oxidized and reduced states of sulfur in studied soil samples. The high absorption intensities of oxidized sulfur species were much greater than those of reduced forms in all the soil samples, indicating that oxidized sulfur accounted for a majority in the total sulfur though oxidized sulfur itself can cause higher peak than reduced states due to consequently increased orbital overlap the stronger transition dipole arising from polar bonding (Jalilehvand, 2006). The intense absorption positions of the studied soil samples were all around the peak of ferrous sulfide (FeS) (2.4705 KeV), indicating the presence of sulfide in rice rhizosphere. The percentage of sulfide increased concomitant with the Pb addition, indicating the enhancement of sulfate reduction (Table 3). 4. Discussion 4.1. Survival of SRB in rice rhizosphere and their role in Pb transformation The Eh results of the studied soil showed that rice rhizosphere was oxic, which is considered to be unsuitable to engender SRB activity. However, nested PCR-DGGE and phylogenetic analysis results indicated that there were some SRB in oxic conditions. With the success of nested PCR, different members of SRB were visualized by DGGE. Phylogenetic analysis results showed the main subgroups SRB in rice rhizosphere. The small number of SRB detected in the studied soil confirmed the importance of oxygenlimited environments within the soil. In rice rhizosphere, there could be anoxic microsites that housed SRB. The observation of SRB presence in these soils confirmed the presence of such microsite. In addition, several studies confirmed the presence of SRB in periodically oxic environments. In some
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A10 AF295656 Desulfotomaculum sp. A5 AB433145 Uncultured Firmicutes bacterium A3 99 AJ871255 Sulfobacillus disulfidooxidans A2 65 A7 51 A8 79 53 A1 A9 82 A4 A6 85 AJ607431 Thermophilic bacterium sp. 55 AB362268 Alicyclobacillaceae bacterium B3 B13 DQ676369 Uncultured delta proteobacterium 99 B7 74 B14 91 B15 96 54 B16 B9 72 99 EU300070 Uncultured Clostridiaceae bacterium B2 EF187876 Uncultured Desulfobacteraceae bacterium B6 77 100 B8 87 DQ903932 Sulfate-reducing bacterium enrichment clone 50 B19 88 AY711479 Uncultured Desulfobulbaceae bacterium B20 83 EF613479 Uncultured Desulfobacca sp. EU300389 Uncultured Chitinophaga sp. 100 B1 93 B4 B17 100 B11 100 AJ786005 Gamma proteobacterium B12 B10 AJ174597 Bacillus drentensis strain 100 B5 B18 10 Fig. 4. Phylogenetic analysis showing relationships of clones sequenced from 16S rRNA gene amplified from the rhizosphere soil. Bootstrap support values with 1000 replicates are given along the branches.
cases, sulfate reduction has actually been detected in supposedly oxic zones of microbial mats and marine sediments. It was found that some SRB possess a relatively high degree of oxygen tolerance (Baumgartner et al., 2006; Dolla et al., 2006). SRB hold various enzymatic protection mechanisms against oxidative stress (Dolla et al., 2006).The sulfur cycle in these soils was probably sustained by re-oxidation of reduced S-compounds and the stabilization of reduced S-compounds by heavy metal between rhizosphere soil and bulk soil (Haitzer et al., 2003; Hu et al., 2007; Karlsson and Skyllberg, 2003; Morse and Luther, 1999). Most sulfur transformations are fundamentally controlled by specialized metabolism of microorganisms (Engel et al., 2007). Rice has an ability to grow in flooded, anaerobic soils because it can transport oxygen to respiring root tissues via internal gas channels. O2 is present via the aerenchymal system of the rice plants in the rhizosphere and a small layer (about 2e3 mm) at the soil surface (Frenzel et al., 1992). It is generally considered that reduced sulfur is oxidized by SOB in rhizosphere of rice while sulfate is reduced by SRB in bulk soil. The oxygen depletion in flooded paddy soil leads to changes in the bacterial community. The active respiration processes of the microbial community at the rhizosphere and bulk soil interface in
conjunction with the reduced diffusivity of oxygen leads to a rapid depletion of oxygen. With high Eh (Fig. 1), rice rhizospheres represent an oxic zone. As a result, oxidation states of sulfur were in the majority accordingly as they were the main end product of sulfur oxidation controlled by SOB. Nevertheless, the sulfur speciation showed both oxidized and reduced states in the studied soil samples (Fig. 5). The presence of oxidized and reduced states of sulfur might correlate to the presence of SOB abundant in rice rhizosphere that oxidized reduced sulfur and SRB contributing to sulfate reduction respectively. The reduced sulfur observed in the XANES spectra of rhizosphere soil samples further confirmed that the action of SRB promoted the reduction of oxidized sulfur which led to the formation of sulfide. Sulfide generated by sulfate reduction then reacted with Pb in the flooded soil forming stable metal sulfide.
4.2. The role of Pb in the activity of SRB in rice rhizosphere Pb in the studied soil influenced the number of SRB accordingly. With increasing Pb addition there was a dramatic rise of soil pH and
H. Lin et al. / Soil Biology & Biochemistry 42 (2010) 1038e1044
2.4804
2.4705
bacterial metallothioneins and heavy metal-transporting ATPases to exist in toxic heavy metal ions and contribute to sulfate reduction (Naz et al., 2005). These Pb tolerant indicated promising prospect for controlling of availability in high Pb pollution environment.
potassium persulfate sodium thiosulfate reduced glutathione cytine oxidized glutathione S cyteine
5. Conclusions
dimethyl sulfone ferrous sulfide sodium sulfite sodium diphenylamine sulfonate sodium sulfate
Absorption (a.u.)
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sodium dodecyl sulfate
Our results indicated the existence of SRB in oxic rice rhizosphere. The SRB mediated the reduction of sulfate and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide. In return, stabilization of reduced sulfur by Pb led to increasing numbers of SRB in rice rhizosphere. The presence of lead-tolerant SRB represented promising prospects of reducing the availability of Pb in environments polluted with this element. Acknowledgments This work was supported by the National Natural Science Foundation of China (40601086, 20777066) and the Natural Science Foundation of Zhejiang Province (Y506063). We gratefully thank Lei Zheng, Chenyan Ma, Yong Han at the Beijing Synchrotron Radiation Facility (BSRF) medium-energy beamline station for their generous help. We thank Dr. Adam Gillespie in Department of Soil Science, University of Saskatchewan for the improvement in the language.
-1
0 mg kg
-1
500 mg kg
-1
1200 mg kg
References
2.460 2.465 2.470 2.475 2.480 2.485 2.490
Energy (KeV) Fig. 5. Comparison of sulfur K-edge XANES spectra of reference compounds and rice rhizosphere soil samples. a.u., arbitrary units.
a significant drop of Eh. These results indicated the promotion of sulfate reduction and were consistent with previous report on the relationship of pH and sulfate reduction (Stein et al., 2007). As shown in Fig. 3, there was a concomitant rise of the number of bands in DGGE fingerprint with increasing Pb addition. Real-time PCR results further confirmed this. It may be due to the fact that increasing addition of Pb resulted in the formation of metal sulfide. The peak area of reduced sulfur increased along the Pb addition. This result indicated the enhancement of sulfate reduction corresponding to DGGE results. The precipitation of metal sulfide stabilized sulfide. As a result, the number of SRB increased which led to the enhancement of sulfate reduction. Microbial sulfate reduction is of great importance in influencing the transformation of heavy metals. As a result, great attention has been paid to SRB due to their important role in the mobility of heavy metal. The application of SRB in controlling the mobility of heavy metals requires heavy-metals-tolerant SRB which can grow in heavy-metals polluted environment. Our results indicated that there were some efficient and heavy metal tolerant SRB existed in comparatively high concentration of Pb. They may possess genetic determinants for metal resistance encoding
Table 3 Percentage of different S species as assessed by S K-edge XANES in rice shizosphere soil (atom% S). Pb added (mg kg1)
Sulfide
Organic monosulfide/ Organic disulfide
Sulfonate
Sulfate
0 500 1200
1.40 6.96 19.11
0 1.42 9.78
4.6 4.14 0
93.99 87.47 7.11
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