APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2008, p. 1019–1029 Vol. 74, No. 40099-2240/08/$08.00�0 doi:10.1128/AEM.01194-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

pH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganismsin Coal Mining-Associated Lake Sediments�†

Marco Blothe,1‡ Denise M. Akob,2 Joel E. Kostka,2 Kathrin Goschel,1Harold L. Drake,1 and Kirsten Kusel1,3*

Department of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany1; Department of Oceanography,Florida State University, Tallahassee, Florida 323062; and Limnology Research Group,

Friedrich Schiller University Jena, 07743 Jena, Germany3

Received 29 May 2007/Accepted 29 November 2007

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) andsulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amountsof reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study,the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, andmolecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zoneI and III sediment microcosms at rates that were approximately 710 and 895 nmol cm�3 day�1, respectively.A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditionsinhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones(approximately 44%) belonged to the Acidobacteria phylum. Since moderately acidophilic Acidobacteria specieshave the ability to oxidize Fe(II) and since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from2 to 5, this group appeared to be involved in the cycling of iron. PCR products specific for species related toAcidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium orAcidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associatedbacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained.Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zoneIII than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichmentcultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betapro-teobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment wasinhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic condi-tions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to beactive under acidic conditions.

Very phylogenetically diverse prokaryotes are capable ofdissimilatory Fe(III) reduction or respiratory growth withFe(III) as the sole electron acceptor (49, 50). The thermody-namic energy available from Fe(III) reduction varies based ona variety of parameters, including the Fe(III) form, crystallin-ity, and pH. The energetics of Fe(III) reduction under neu-tral-pH conditions differ substantially from those under acidicconditions (72). While Fe(III) exists predominantly in the solidphase as oxyhydroxide minerals at circumneutral pH, Fe(III) ismore soluble and should be an electron acceptor for microbialgrowth under low-pH conditions. Reduction of both solid andsoluble forms of Fe(III) becomes more thermodynamicallyfavorable with decreasing pH. Since proton concentrations se-lect for acidophilic microorganisms at low pH, it is not surpris-ing that Fe(III) reduction in neutral-pH and acidic environ-ments is carried out by different microbial populations (50, 72).

During the last two decades, Fe(III) reduction in neutral-pHenvironments and neutrophilic Fe(III)-reducing microorgan-isms have been studied intensively (13, 14, 47, 49, 51). How-ever, the process in acidic environments has received littleattention (26, 34, 38, 41, 60).

Some acidophilic bacteria have the capacity to reduceFe(III) (39, 63). The chemolithoautotrophs Acidithiobacillusthiooxidans and Acidithiobacillus ferrooxidans can couple theanaerobic oxidation of elemental sulfur to the reduction ofFe(III) (11, 20, 62). Heterotrophic acidophiles belonging to thegenus Acidiphilium can also catalyze the reduction of Fe(III)and appear to be widely distributed in metal-rich acidic envi-ronments (30, 31, 32, 39, 41, 42). In anoxic environments theseorganisms may contribute to iron cycling by redissolvingFe(III) minerals which precipitate when the pH is increased bymixing with groundwater or surface water. However, the rateand extent of Fe(III) reduction vary significantly in the isolatesthat have been examined (30). Acidiphilium cryptum JF-5 wasthe first acidophile isolated under Fe(III)-reducing conditionsat pH 3 from an acidic coal mining-associated lake (41). Coalmining-associated lakes receive high concentrations of Fe(II),protons, and sulfate due to the oxidation of pyrite in the minetailings (27, 59). Subsequently, Fe(II) is oxidized biologically(e.g., by A. ferrooxidans) and precipitates as poorly crystallineFe(III) oxyhydroxysulfate to the sediment (59). Whereas most

* Corresponding author. Mailing address: Limnology ResearchGroup, Institute of Ecology, Friedrich Schiller University Jena, Dorn-burgerstrasse 159, 07743 Jena, Germany. Phone: (49) (0)3641-949461.Fax: (49) (0)3641-949462. E-mail: Kirsten.Kuesel@uni-jena.de.

‡ Present address: Department of Geology and Geophysics, Univer-sity of Wisconsin—Madison, Madison, WI 53706.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 14 December 2007.

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studies have shown that the optimum pH for growth of Acidi-philium is 3 to 4, A. cryptum ATCC 33463 was recently shownto reduce small amounts of solid-phase Fe(III) at pH 5 (8).Since most cultivated Fe(III)-reducing prokaryotes are neutro-philic and have a negligible capacity to reduce Fe(III) at acidicpHs (pH � 5.5), we have only a marginal understanding of themicroorganisms that drive the reduction of Fe(III) under mod-erately acidic conditions. Thus, the objectives of this studywere to (i) explore the microbial diversity of coal mining-associated lake sediments, (ii) investigate the impact of pH onFe(III)-reducing potentials, and (iii) elucidate the Fe(III)-re-ducing microbial community in acidic (pH 3) and slightly acidic(pH 5) sediment zones.

MATERIALS AND METHODS

Field site description and sampling. The acidic coal mining-associated lakethat we studied (Lake 77) is located in the Lusatian mining area in east centralGermany. The pH of the lake water and the maximum summer temperature ofthe upper sediment were approximately 3 and 12°C, respectively (59). Sedimentcores were obtained using a boat and a gravity corer in Plexiglas tubes (insidediameter, 5.9 cm), always at the deepest location (7 m). On each sampling date(November 2001, July 2003, November 2003, and February 2004) 5 to 12 coreswere obtained, transported to the laboratory, and sectioned based on sedimentstratification under an N2 atmosphere within 24 h. Sediments were fairly uniformhorizontally in the area of the lake examined.

Visually and geochemically, the sediment could be sectioned into four differ-ent zones. The upper orange sediment zone (zone I, 0 to 7 cm) had a pH of2.8 to 3.1 and was enriched with reactive iron, mainly schwertmannite[Fe8O8(OH)x(SO4)y] (40, 59). In the next zone, a yellowish-brownish sedimentzone (zone II, 7 to 10 cm), the pH varied from 3.4 to 4.7. The third zone (zoneIII, 10 to 16 cm) had a pH of 5.2 to 5.7 and was enriched in goethite (�-FeOOH),which is consistent with the fact that schwertmannite is solved or transformed inmore stable minerals, such as goethite, under changing physicochemical condi-tions (7). Below 16 cm the sediment pH ranged from 5.3 to 6.1. Only smallamounts of reactive iron were present in this zone, zone IV; thus, we focused onthe upper three zones. The sediment dry weights were approximately 8.2, 14.6,22.4, and 36.3% of the wet weights in zones I, II, III, and IV, respectively. Moredetailed geochemical descriptions of the sediment have been given elsewhere(40, 59).

Preparation of sediment microcosms. For zones I and III, sediment fromreplicate cores was pooled under anoxic conditions, and 40 g (wet weight) ofsediment was transferred to sterile 150-ml infusion bottles (Merck ABS,Dietikon, Switzerland) inside a Mecaplex O2-free chamber (100% N2 gas phase).The bottles were closed with rubber stoppers and screw-cap seals, flushed withsterile argon for 15 min, and incubated in the dark at 15°C with an initialoverpressure of 20 to 25 kPa argon at room temperature. No electron donorswere added. The pH was adjusted with sterile solutions of 10 N HCl and 10 NNaOH. Three replicates of all microcosms were prepared. Samples were re-moved with sterile, argon-flushed syringes. Rates of Fe(II) formation were cal-culated only for the time period when a linear increase in the Fe(II) concentra-tion was observed.

Media and enumeration studies. One representative sediment core obtainedin November 2001 was sectioned to obtain 1.5-cm sediment depth zones under anN2 atmosphere. Subsamples were taken from every 1.5-cm sediment depth zonewith sterile syringes to determine the pH and sulfate and Fe(II) contents of thepore water. One milliliter of wet sediment was transferred into a sterile 50-mlpolycarbonate tube, diluted fourfold with sterile filtered 4% paraformaldehyde,and incubated for 2 h at 4°C. Fixed samples were diluted 500-fold in sterilefiltered 1� phosphate-buffered saline (130 mM NaCl, 10 mM NaPi; pH 7.4),homogenized, and incubated for 15 min in an ultrasonic water bath. Five micro-liters of 4�,6-diamidino-2-phenylindole (DAPI) (100 �g ml�1) and 0.5 ml of 1�phosphate-buffered saline were added to the fixed samples, which were thenincubated for 15 min on ice. Stained samples were filtered on black polycarbon-ate filters (Millipore GTBP 047000) and washed with 10 to 20 ml of filter-sterilized distilled H2O. For counting, each filter was covered with Citifluor, andcells were examined using an epifluorescence microscope (Zeiss Axioskop) witha high-pressure mercury bulb (50 W) and 02 filter set (G365, FT395, LP420;Zeiss).

For pure-culture studies, A. ferrooxidans DSM 583 and Acidobacterium capsu-latum DSM 11244 were used. Cells of A. ferrooxidans were cultivated in Thio

medium (62) under a CO2 gas phase. Autoclaved, washed flowable sulfur (StollerChemical Company, Inc., Houston, TX) was added to a final 10 mM concentra-tion. Cells of A. capsulatum were cultivated in Acido medium that contained (perliter) 2.0 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of MgSO4 � 7H2O, and 0.1 g ofKCl. The pH was adjusted with NaOH to obtain a final pH between 2.2 and 5.The incubation temperature was 30°C. A. ferrooxidans and A. capsulatum cultureswere grown first under oxic conditions with FeSO4 (10 mM) and glucose (2 mM)as the electron donors, respectively. After 2 weeks, cells were centrifuged,washed three times, and transferred to anoxic media to obtain a final opticaldensity at 660 nm of 0.3 to 0.4. The optical density at 660 nm was determinedwith a Spectronic 501 photometer (Bausch & Lomb Inc., Rochester, NY). Sol-uble Fe(III) was added as ferric sulfate [Fe(III)2(SO4)3] from a sterile, anoxicstock solution. Goethite, schwertmannnite, and FeOOH were prepared as pre-viously described (43) and added at a final concentration of 40 mM.

Numbers of cultured Fe(III) reducers were determined by the most-probable-number (MPN) technique using a 10-fold dilution series with three replicatetubes per dilution and incubation at 15°C in selective media. Zone III sediment(pH 5.6) from two cores obtained in November 2003 was sectioned and pooledunder an N2 atmosphere. Five grams (wet weight) was transferred to a bottlecontaining 95 ml of dilution buffer (43) and some 0.2-mm-diameter glass beads.The bottle was sealed and mixed using an end-over-end shaker for 1 h. Dilutionseries were prepared, and media were inoculated using the dilutions. The me-dium contained (per liter) 40 mM amorphous ferric hydroxide [Fe(OH)3] (51),2.0 g of (NH4)2SO4, 0.5 g of K2HPO4, 0.5 g of MgSO4, 0.1 g of KCl, 5 ml of atrace metal solution (23), and 5 ml of a vitamin solution (23). The pH of eachmedium was adjusted to pH 5.5 with 0.5 N HCl or 0.1 M NaOH. The gas phasewas N2-CO2 (80:20, vol/vol). Either ethanol (10 mM), lactate (5 mM), or H2 (10ml) in combination with succinate as a carbon source (1 mM) was added as anelectron donor. Tubes containing H2 were incubated horizontally. Tubes wereconsidered positive based on the formation of Fe(II) and the consumption ofelectron donors compared to uninoculated controls. MPN values were calculatedfrom standard MPN tables and were within 95% certainty (3).

Analytical techniques. The reduction of Fe(III) was determined by determin-ing the amount of Fe(II) formed after acid extraction. Aliquots (0.2 ml) of themedium or the sediment suspension were removed with sterile syringes, trans-ferred to 9.8 ml of 0.5 N HCl, and then incubated for 1 h at room temperature(41). The Fe(II) formed was measured after addition of acetate by the phenan-throline method (73). The pH was determined with an Ingold U457-S7/110combination pH electrode (Ingold-Me�technik, Switzerland). Short-chain ali-phatic acids and alcohols were measured with Hewlett-Packard 1090 series IIhigh-performance liquid chromatographs (44). Headspace gases (H2 and CO2)were measured with 5890 series II gas chromatographs (Hewlett-Packard Co.,Palo Alto, CA) (43).

DNA extraction. DNA was extracted from 10 g of sediment from zones I, II,and III by first adjusting the pH to 9 with sterile 5 N NaOH and incubating thepreparations overnight at 4°C in 100 ml of sterile distilled H2O to dissolve humicsubstances. After incubation, the sediment was centrifuged at 8,000 � g for 20min, the supernatant was discarded, and the pellet was resuspended in 4 ml ofsterile extraction buffer (100 mM EDTA, 10 mM Tris [pH 8.0], 1% sodiumdodecyl sulfate). Samples were incubated at 70°C for 30 min with occasionalmixing and then centrifuged at 10,000 � g for 15 min. Each supernatant wastransferred to a clean centrifuge tube, and each pellet was washed once with 10ml of extraction buffer. The supernatant was retained and pooled with thesupernatant resulting from the previous centrifugation. Nucleic acids were pre-cipitated overnight at �20°C with isopropanol and centrifuged to pellet nucleicacids. The solution was centrifuged again. Potassium acetate was added to thepellet to a final concentration of 0.5 M, and the solution was incubated on ice for2 h and centrifuged at 5,000 � g for 5 min. The DNA yield was determined bygel electrophoresis on 1.5% agarose gels. All gels were stained for 20 min withSYBR gold (Molecular Probes, Hamburg, Germany) and documented using ascanner (Storm 860 molecular imager; Molecular Dynamics, Sunnyvale, CA) andthe ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA). DNA wasextracted from MPN culture cell pellets using an Ultra Clean soil DNA kitaccording to the manufacturer’s instructions (Mo Bio Laboratories, SolanaBeach, CA).

PCR amplification of 16S rRNA genes. Aliquots of DNA from zone I, II, andIII sediment samples and MPN cultures were PCR amplified using Bacteriadomain-specific (57) and group-specific (14, 21, 22, 25, 28, 70) 16S rRNA geneprimers (see Table S1 in the supplemental material). DNA extracts from zone Iand III sediment samples were PCR amplified with primers GM3 and GM4 in areaction mixture consisting of 20 pmol of each primer, 200 �M deoxynucleosidetriphosphates, 300 �g of bovine serum albumin, 1� PCR buffer, and 1 U of TaqDNA polymerase (Eppendorf, Hamburg, Germany). Template DNA (4 �l) was

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preheated to 70°C prior to addition to the PCR mixture. Thermocycling wasperformed with a T-Gradient cycler (Biometra, Gottingen, Germany), and theprogram consisted of 35 cycles of 95°C for 60 s, 42°C for 60 s, and 72°C for 90 s,followed by a final extension step of 72°C for 7 min. PCR amplification of DNAextracts from zone I, II, and III sediments using group-specific primers forAcidiphilium, bioleaching-associated bacteria, Geobacter, Geothrix, and Sh-ewanella was performed as described above using an annealing temperature of55°C. The primer set for acidophilic bioleaching-associated bacteria is specific forsix bacterial phylotypes (A. ferrooxidans, A. thiooxidans, Acidithiobacillus caldus,Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans, A. cryptum, andAcidiphilium organovorum) which are involved in the bioleaching of mineral ores(21). DNA extracts from MPN cultures were PCR amplified using the Bacteriadomain-specific primers 8F and 1392R in a mixture containing 10 to 50 ng ofDNA, 1� LA PCR buffer II (TaKaRa Mirus Bio, Madison, WI), 2.5 mMdeoxynucleoside triphosphates, 0.5 �M forward primer, 0.5 �M reverse primer,2.0 �g of bovine serum albumin, and 0.03 U of LA Taq polymerase (TaKaRaMirus Bio, Madison, WI). Thermocycling was performed as follows: incubationat 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°Cfor 30 s and then a final extension step of 72°C for 10 min.

Clone library construction. PCR amplicons produced with the Bacteria do-main-specific primer pair GM3/GM4 and group-specific 16S rRNA gene primersfrom zone I and III sediments were cloned using the pGEM-T vector andEscherichia coli JM109 competent cells according to the manufacturer’s instruc-tions (Promega, Madison, WI). Zone I (pH 3) and zone III (pH 5.5) wereselected due to the difference in pH. Clones in libraries constructed with primersGM3 and GM4 were screened by denaturing gradient gel electrophoresis(DGGE) (48) and grouped into phylotypes based on migration behavior. Clonelibraries constructed with group-specific amplicons were screened by restrictionfragment length (RFLP) analysis. Cloned inserts were PCR amplified with thegroup-specific primer pair and verified by gel electrophoresis on a 1.5% agarosegel (Roth, Darmstadt, Germany). Five microliters of PCR product was digestedin a single reaction with 1 U of restriction enzymes HaeIII and CfoI (Promega,Madison, WI) at 35°C for 2 h. The digested PCR products were separated by gelelectrophoresis in 7% polyacrylamide (Bio-Rad Laboratories, Hercules, CA) in1� TAE buffer (40 mM Tris-acetate [pH 7.4], 20 mM sodium acetate, 1 mMEDTA) at 23°C at 100 V for 3.5 h with the D-Code system (Bio-Rad Labora-tories, Hercules, CA).

16S rRNA gene amplicons from MPN cultures were purified using a QIAquickPCR purification kit (Qiagen, Valencia, CA) and were cloned into the TOPO TAcloning vector pCR 2.1 according to the manufacturer’s instructions (Invitrogen,Carlsbad, CA). Cloned inserts were PCR amplified using the vector-specificprimers M13F and M13R and were digested with restriction enzyme HaeIII (0.25U �l�1; New England Biolabs, Beverly, MA) for 2 h at 37°C. The digested PCRproducts were separated by gel electrophoresis on a 3.5% MetaPhor agarose gel(Cambrex, Rockland, ME) in 1� Tris-borate-EDTA buffer at 4°C for 3 h. Allclones screened using RFLP analysis were grouped into phylotypes on the basisof the RFLP banding patterns.

DGGE fingerprinting. Community DNA extracted from zone I, II, and IIIsediments was fingerprinted using DGGE. DNA extracts were PCR amplifiedwith the universal primers GM5-clamp and 907RM (56) as described above forprimer pair GM3/GM4 with an annealing temperature of 55°C. PCR productswere separated on a 7% polyacrylamide gel with a 40 to 60% denaturant gradient(100% denaturant was 7 M urea and 40% formamide) in 1� TAE buffer usingthe D-Code system (Bio-Rad Laboratories, Hercules, CA). DGGE gels were runfor 18 h at 60°C and 100 V.

Dilution PCR analysis of sediment DNA extracts. The relative levels of mem-bers of the genus Acidiphilium, the bioleaching-associated bacteria, the familyGeobacteraceae, the genus Geothrix, and the genus Shewanella in sediment sam-ples were determined using a modified MPN-PCR technique (61). In contrast toquantitative PCR, the dilution PCR method provided only rough comparativeestimates for PCR products from different sediment zones amplified with thesame primer sets. DNA extracts from zones I, II, and III were adjusted to aconcentration of 10 �g ml�1 and serially diluted 10-fold (100 to 10�4) in sterilewater. PCRs were performed as a single replicate for each dilution as describedabove using an annealing temperature of 55°C and 4 �l of each dilution as thetemplate. PCR amplicons were detected by 1.5% (wt/vol) agarose gel electro-phoresis using TAE buffer at 80 V for 45 min. Gels were stained with SYBR goldfor 20 min and documented with the Storm 860 molecular imager. The corre-sponding dilution level was considered positive if a PCR product was present.

Phylogenetic and statistical analyses. Representative clones for each RFLPphylotype were sequenced bidirectionally using a Big-Dye Terminator v3.1 cyclesequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosys-tems 3100 genetic analyzer with capillary electrophoresis. Sequences were as-

sembled using Sequencher v4.5 (Gene Codes Corp., Ann Arbor, MI), and priorto phylogenetic analysis, vector sequences flanking the 16S rRNA gene insertswere removed. Previously identified sequences with high sequence similarity tothe clones obtained in this study were determined using the BLAST algorithmwith the GenBank database available from the National Center for Biotechnol-ogy Information (4). Clone sequences were checked for chimeras using theprogram CHIMERA_CHECK from Ribosomal Database Project II. All clonesequences and reference sequences were aligned with the ARB software packageusing the Fast Aligner algorithm, incorporating ribosomal secondary structuredata. Dendrograms were constructed with the ARB software package by adding16S rRNA sequences to the distance matrix tree using PARSIMONY_INTERAKTIVwithout changing the overall tree topology (52). The coverage of the clonelibraries was calculated by using the equation described by Singleton et al. (69),and the sampling efficiency in clone libraries was assessed using the AnalyticaRarefaction 1.3 software (http://www.uga.edu/strata/software/) originally de-scribed by Heck et al. (33).

Nucleotide sequence accession numbers. The 16S rRNA gene sequences de-termined in this study have been deposited in the EMBL database under acces-sion numbers AM712138 to AM712178 and AM713378 to AM713401.

RESULTS

Vertical geochemical profiles. The pore water pH increasedfrom 3 in the upper 7.5 cm of the sediment to 5.5 at a depth of8 to 10 cm (Fig. 1). The concentrations of Fe(II) and sulfateincreased with increasing depth from approximately 20 to 50mM and from 8 to 15 mM, respectively. Similar profiles wereobtained with other sediment cores (40, 59). Due to the distinctvertical geochemical gradients, the sediment was separatedinto an upper acidic zone (pH 3; zone I), a transition zone(zone II), and a slightly acidic zone (pH 5.5; zone III). Thehighest density of DAPI-stained cells (1.5 � 109 cells ml�1)was detected in zone I, whereas the cell density decreased to0.75 � 108 cells ml�1 in zone III (Fig. 1).

16S rRNA gene-based community analysis. PCR productsobtained from zones I, II, and III produced different DGGEpatterns (see Fig. S1 in the supplemental material). Fourteen, 11,and 13 bands were differentiated for zones I, II, and III, respec-tively. Only five bands appeared to have similar migration behav-iors in all zones, suggesting that there were differences in themicrobial community structures of the three zones.

Clone libraries of Bacteria domain-specific amplicons (ob-tained with GM3/GM4) contained 185 and 95 clones fromzone I and III sediments, respectively. DGGE screening of theclone libraries (48) revealed the presence of 40 different phy-lotypes in zone I, whereas zone III contained 42 differentphylotypes (data not shown). Bacterial 16S rRNA gene clonelibraries derived from zone I and III sediments showed 85 and65% coverage, respectively. No PCR product was obtainedwith Archaea domain-specific primers. Many of the clones(45% of the clones from zone I and 43% of the clones fromzone III) belonged to the phylum Acidobacteria (Fig. 2). Othersequences detected in both zones were related to gene se-quences of Nitrospira, Cytophagales, and Alpha-, Gamma-, andDeltaproteobacteria groups along with some 18S rRNA genesequences. Sequences related to Firmicutes, Actinomyces, andBetaproteobacteria were obtained only from zone I. Sequencesrelated to Verrumicrobia subdivision 5, Thermus/Deinococcus,Bacillus/Clostridium, and an uncultured group were detectedonly in zone III.

Effect of pH on microbial formation of Fe(II) in sedimentsand by pure cultures. Fe(II) was formed in zone I sedimentmicrocosms prepared from sediment cores obtained in July

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2003 at a rate of linear production that was approximately 710nmol cm�3 day�1 (Fig. 3A). When the pH was changed fromthe in situ pH (pH 2.9) to 5.3, Fe(II) was formed after 5 daysof incubation at a rate of 1,550 nmol cm�3 day�1. In zone IIIsediment microcosms at the in situ pH, Fe(II) was formed at arate of approximately 895 nmol cm�3 day�1 (Fig. 3B). Whenthe pH was decreased to 2.9, Fe(II) was formed after 5 days ofincubation at a rate of 557 nmol cm�3 day�1. Thus, a shift toslightly acidic conditions increased Fe(II) formation in zone Isediment by a factor of 2, but a shift to acidic conditions inzone III sediment inhibited Fe(II) formation, although thesoluble pool of Fe(III) that was assumed to be easily bioavail-able should have been larger at pH 2.9 than at pH 5.3. Similarresults were obtained with sediment cores obtained in October2000 (data not shown).

The rates of Fe(II) formation under in situ pH conditionscalculated on a dry weight basis were approximately 8.7 and 1.9nmol mg�1 day�1 in zones I and III, respectively, due to thesmaller amount of sediment (on a dry weight basis) in zone I thanin zone III. The rates of Fe(II) formation under in situ pH con-ditions for sediment cores obtained in November 2001 were ap-proximately 595 and 1,405 nmol cm�3 day�1 for zones I and III,respectively, which equaled 7.3 and 3.0 nmol mg�1 day�1 on a dryweight basis (data not shown). These rates were determined inlong-term incubation experiments. Fe(II) formation was linearfor 45 and 58 days of incubation for zones I and III, respectively,and resulted in maximum Fe(II) concentrations of 38 and 108mM in zones I and III, respectively.

The total amounts and rates of Fe(II) formation from schw-ertmannite by cultures of A. ferroxidans decreased with increas-ing pH (Fig. 4A). At pH 3, 4, and 5, the rates were approxi-mately 84, 21, and 3 �mol liter�1 day�1, respectively. Similar

results were obtained with FeOOH (data not shown). Smallamounts of goethite were reduced at pH 3 but not at pH 5 byA. ferrooxidans. The rates of Fe(II) formation from FeOOH bycultures of A. capsulatum supplemented with 2 mM glucoseunder anoxic conditions were similar (approximately 57 �molliter�1 day�1) at initial pH values of 3 and 5 (Fig. 4B). Fe(II)formation from goethite was delayed at pH 5. However, similartotal amounts of Fe(II) were present at the end of incubationat pH 3 and 5. Fe(II) was also formed from solubleFe(III)2(SO4)3 at pH 2.2. In general, the number of DAPI-stained cells did not increase during Fe(II) formation (data notshown). Glucose was fermented to acetate, ethanol, and traceamounts of succinate and lactate. The recovery of reducingequivalents theoretically obtained from the oxidation of glu-cose varied between 6 and 14%.

Dilution PCR analysis. Dilution PCR performed with sedi-ment samples from zones I, II, and III yielded PCR productswith 16S rRNA primers specific for acidophiles belonging tothe genus Acidiphilium or the bioleaching-associated bacteriaand for neutrophiles belonging to the genus Geobacter (Table1). PCR products of acidophiles were obtained at the samedilution for all three sediment zones. For bioleaching-associ-ated bacteria, a PCR product was obtained only with undilutedDNA. PCR products of neutrophiles with specific primer setsGM3/Geo825R and Gb564/Gb1290 could be obtained athigher dilutions (10�2) with zone III sediments. Use of Gb564/Gb1290 also yielded a PCR product at the 10�2 dilution forzone II sediments.

When a primer set specific for Geothrix was used, a PCRproduct was obtained only with zone II sediment. No PCRproducts were obtained with a primer set specific for Sh-ewanella. A PCR product was obtained with the undiluted

FIG. 1. Vertical biogeochemical characterization of the sediment of acidic coal mining-associated Lake 77. One representative core obtainedin November 2001 was used. (A) Symbols: E, Fe(II); f, sulfate. (B) Symbols: Œ, pH; ‚, cell counts. The cell count data are the means standarddeviations for three replicate sediment samples.

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DNA only when nested PCR was performed after a first cyclewith universal bacterial primer set GM3/GM4.

RFLP and comparative sequence analysis. A total of 50Acidiphilium 16S rRNA gene clones (22 clones from zone I and28 clones from zone III) were screened by RFLP analysis, andeight different phylotypes were differentiated. Four of theseeight phylotypes were present in zones I and III, while fourphylotypes were present only in zone III. Comparative se-quence analyses indicated that the four phylotypes present inboth zones had 99% similarity at the nucleotide level to cul-tured Acidiphilium species. The remaining four sequencesfrom zone III were 95% similar to cultured Acidiphilium or

Acidisphaera species sequences. Comparative phylogeneticanalysis of the sequences revealed two distinct groups (Fig. 5).For the bioleaching-associated bacteria, 20 clones from zone Iand 28 clones from zone III were screened. Although theprimer set used for bioleaching-associated bacteria obtains se-quences specific for A. thiooxidans, A. caldus, S. thermosulfido-oxidaans, L. ferrooxidans, and other groups (21), only onephylotype with 99% similarity to A. ferrooxidans was obtained.Nonetheless, other mesophilic bacteria potentially involved inbioleaching might be not amplified with this specific primer set.

When primer pair Gb564/Gb1290 specific for Geobacter wasused, screening yielded 38 and 52 phylotypes from zones I and

FIG. 2. Frequencies of bacterial phylogenetic lineages detected in 16S rRNA gene-based clone libraries derived from zones I and III.Calculations were based on the total number of clones associated with phylotypes of sequenced representatives.

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III, respectively. Twenty clones from zone I and 40 clones fromzone III were sequenced. Only one short sequence which wasderived from zone III showed 97% similarity to Geobacter spp.The remaining sequences were similar to Acidobacterium sp.(89 to 97%), Streptococcus (90 to 99%), Propionibacterium(97%), Ferrimicrobium (92%), and Actinomyces (94%). Whenprimer pair GM3/Geo825R, also specific for Geobacter, wasused, 33 different phylotypes were obtained. Comparative se-quence analysis revealed that four of the sequences obtainedshowed high sequence identity to Geobacteraceae sequences;one showed 90% similarity to Geobacter grbicium (15), andthree were related to Geobacter sp. strain CdA-2 (93 to 97%)(Fig. 6). The remaining sequences were similar to Syntro-phobacteraceae (91 to 96%), Desulfobacteraceae (91 to 93%),Magnetobacterium sp. (85%), Acidobacterium sp. (92%), andActinomyces sp. (91%).

When the primer pair specific for Geothrix was used, 12clones were obtained from zone II, and 9 of them were se-quenced without previous RFLP screening. Only one sequencewas related to Geothrix fermentans (95%). When the primerpair specific for Shewanella was used, eight white clones weresequenced without previous RFLP screening. One sequencewas related to Shewanella algae (93%), and the remaining 7clones were related to Sterolibacterium denitrificans (95%)and Chromobacterium sp. (89 to 95%).

Enumeration and characterization of Fe(III) reducers.Pooled sediment from three cores obtained in March 2003 wasused to enumerate the Fe(III)-reducing microorganisms in zoneIII capable of growing at pH 5.5. The MPN of Fe(III)-reducingmicroorganisms were approximately 104 to 105 cells g (wet

weight)�1, i.e., approximately 0.5% of the total DAPI-stainedmicroorganisms present in zone III. The following MPN valueswere obtained for Fe(III) reducers from sediment zone III cul-tured at pH 5.5: for H2-utilizing Fe(III) reducers, 2.3 � 104 cellsg (wet weight) of sediment�1 (confidence limits, 4.9 � 103 to 1.1 �105 cells g�1); for ethanol-utilizing Fe(III) reducers, 4.0 � 105

cells g (wet weight) of sediment�1 (8.6 � 104 to 1.9 � 106 cellsg�1); and for lactate-utilizing Fe(III) reducers, 2.3 � 104 cells g(wet weight) of sediment�1 (4.9 � 104 to 1.1 � 105 cells g�1). InMPN tubes scored positive for Fe(II) formation, the consumptionof ethanol and lactate yielded acetate as the main organic endproduct. Four MPN enrichment cultures that exhibited highFe(III)-reducing activities were selected for further 16S rRNAgene-based community analysis [one MPN 10�4 dilution and oneMPN 10�3 dilution of ethanol-utilizing Fe(III) reducers, oneMPN 10�3 dilution of lactate-utilizing Fe(III) reducers, andone MPN 10�1 dilution of H2-utilizing Fe(III) reducers].Rarefaction curves indicated that there was saturation ofsampling (data not shown). The majority (63%) of sequenced 16SrRNA gene clones of the four clone libraries were most closelyrelated to members of the Betaproteobacteria (Fig. 7), primarily tospecies of Thiomonas (see Table S2 in the supplemental mate-rial), a genus which is closely related to the family that containsThiobacillus. Members of the Deltaproteobacteria and Firmicutescomprised 19 and 18% of all the clones, respectively. Phylotypesrelated to the class Deltaproteobacteria included some clones re-lated to Geobacter species, whereas most phylotypes related to theFirmicutes were related to Desulfosporosinus. Phylotypes relatedto Acidobacteria were detected only in H2-supplemented enrich-ment cultures and showed 96% similarity to G. fermentans.

FIG. 3. Effect of pH on microbe-catalyzed Fe(II) formation in zones I (A) and III (B). Sediment from replicate cores obtained in July 2003was used. Symbols: F, Fe(II) formation at the in situ pH (pH 2.9 in zone I and pH 5.3 in zone III); E, Fe(II) formation under altered pH conditions.The data are the means standard deviations of three replicates.

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DISCUSSION

Raising the water table after open-cast lignite mines wereclosed led to the formation of numerous acidic mining-associ-ated lakes in wide areas of Germany, Poland, and the CzechRepublic (67). Coal mining-associated lakes are characterizedby moderate temperatures, small amounts of toxic metals, anda pH not less than 2 (27). The more extreme acid mine drain-age (AMD)-impacted habitats contain fewer prokaryotic lin-eages (two archaeal and eight bacterial divisions) than manyother environments (5, 9, 74). This study demonstrated thatcoal mining-associated lake sediments were inhabited by adiverse bacterial community that differed from the communi-ties in extreme AMD environments. No PCR product wasobtained with Archaea domain-specific primers, while 10 and12 bacterial lineages were detected in sediment zones I and III,respectively. Phylogenetic analyses of 16S rRNA genes placedmany of the sequences obtained in the Proteobacteria, Nitro-

spira, Firmicutes, and Acidobacteria groups, similar to the re-sults for AMD and other bioleaching sites (5, 74) and uranium-contaminated sediments (2, 70). However, in contrast to theprevious studies, many of the clones (46 and 45% in zones Iand III, respectively) were related to sequences which clus-tered within the Acidobacteria. Many of the phylotypes wererelated to G. fermentans, an Fe(III)-reducing heterotroph, andto one of the three cultured and described representatives ofthe phylum Acidobacteria (12, 16). Members of the Acidobac-teria have been detected in a great variety of ecosystems, in-cluding soils, peatlands, geothermal vents, AMD sites, anduranium-contaminated environments (6, 12, 36, 54, 66). Al-though the physiological capabilities of the vast majority ofAcidobacteria remain unclear, some moderate acidophilic Ac-idobacterium spp. have been shown to oxidize Fe(II) (31). A.capsulatum formed Fe(II) under anoxic conditions at pHsranging from 2.2 to 5 during glucose fermentation. Since the

FIG. 4. Effect of pH on microbe-catalyzed Fe(II) formation from schwertmannite by A. ferrooxidans DSM 583 at pH 3 (F), pH 4 (E), and pH5 (f) with elemental sulfur (10 mM) as the electron donor (A) and by A. capsulatum DSM 11244 at pH 5 (filled symbols) and pH 3 (open symbols)from FeOOH (F and E) or goethite (Œ and ‚) or from soluble Fe(III)2(SO4)3 at pH 2.2 (�) with glucose (2 mM) as the electron donor (B). Thedata are the means standard deviations of three replicates.

TABLE 1. Dilution PCR products obtained with specific primer sets for Acidiphilium, bioleaching bacteria, Geothrix, Shewanella, andGeobacter from DNA extracted from sediment zones I, II, and III

Sedimentzone

Dilution at which products were obtained

Acidiphilium (primersAcido594F andAcido1150R)

Bioleaching bacteria(primers Ferro458F

and Ferro1473R)

Geothrix (primersGx182F and

Gx472R)

Shewanella (primersShw783F andShw1245R)

Geobacter (primersGM3 andGeo825R)

Geobacter (primersGb564F andGb1290R)

I 10�3 10�1 NPa NP 10�1 10�1

II 10�3 10�1 10�2 NP 10�1 10�2

III 10�3 10�1 NP NP 10�2 10�2

a NP, no product.

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cell counts of A. capsulatum did not increase and only smallamounts of reducing equivalents were transferred to Fe(III),the dissimilatory reduction of Fe(III) appeared not to be cou-pled with growth. Nonetheless, Acidobacteria seemed to play arole in the cycling of iron in coal mining-associated lake sedi-ments. Clones related to Betaproteobacteria, Firmicutes, andActinomycetes were detected only in zone I, whereas clonesrelated to Verrucomicrobia, Thermus/Deinococcus, or the Ba-cillus-Clostridium group were detected only in zone III. Thediversity was greater in zone III than in zone I, which mighthave been due to the less acidic conditions. The low coverage(65%) of the rarefaction analysis indicated that the samplingefficiency was low; thus, the diversity in zone III might be evenhigher.

The dominant lithoautotrophic Fe(II) oxidizer in coal min-ing-associated lake sediments appeared to be A. ferrooxidans.Oligonucleotide probe-based studies have indicated thatLeptospirillum strains often dominate microbial communitiesin hot and extremely acidic AMD environments (9, 24, 68),although some species are mesophiles and have been isolatedfrom cold and cool environments. Nonetheless, coal mining-associated lake sediments appear to be a more suitable habitatfor A. ferrooxidans due to their moderately acidophilic nature(37).

Large amounts of amorphous Fe(OH)3, goethite, andFe(III) oxyhydroxysulfates, like schwertmannite or jarosite,precipitate to the sediments of coal mining-associated lakes(43, 59). Reduction of Fe(III) is often the dominant electron-accepting process for the oxidation of organic matter, whereassulfate reduction is restricted to sediments with a pH greaterthan 5 (40, 53, 59). Although the primary production rate inthese lakes is low (29, 58), Fe(III) reduction occurred at highrates and was linear for up to 60 days of incubation, indicatingthat both the pool of bioavailable Fe(III) and the pool of labilecarbon were sufficient.

Culture-dependent techniques used in previous studies dem-onstrated that heterotrophic and autotrophic acidophilic bac-teria capable of reduction of Fe(III) were present in the sed-iments (41, 53). Due to the absence of elemental sulfur in the

upper sediment zone (59), heterotrophic species appear to beresponsible for the reduction of Fe(III) under acidic condi-tions. Surprisingly, PCR products of Acidiphilium-relatedstrains were obtained to the same DNA dilution level in allthree sediment zones, and a higher number of phylotypes re-lated to cultured Acidiphilium or Acidisphaera species wereobtained from zone III than from zone I. Acidisphaera rubri-faciens is a moderately acidophilic aerobic heterotroph thatwas isolated from an AMD site (35). The optimum pH forgrowth of A. cryptum JF-5 is approximately pH 3.2 (41), but sixmembers of the genus Acidiphilium are adapted to pH valuesfrom 1.5 to 6.0 (37). A. cryptum ATCC 33463 has the capacityto reduce small amounts of Fe(III) at pH 5 (8), and A. capsu-latum formed Fe(II) at pHs ranging from 2.2 to 5. Althoughthe metabolic roles of the phylotypes detected only in zone IIIremain unknown, we can speculate that these organisms mightbe moderate acidophiles or well adapted to pH 5 conditions.

Acidophilic Fe(III) reducers likely do not have to attach tothe surface of Fe(III) minerals to transfer electrons to Fe(III);instead, they can utilize the small pool of soluble Fe(III) forreduction (10). Thus, the reduction of Fe(III) should be lessfavorable at higher pHs, because the solubility of the Fe(III)minerals decreases with increasing pH. The concentration ofsoluble Fe(III) can reach 10 mM under extremely acidic con-ditions, whereas only 10�15 mM Fe(III) is present under neu-tral-pH conditions (46). In addition, the amount of Fe(II)adsorbed to mineral surfaces (17, 47, 64, 65) should increase atelevated pHs. This advanced surface passivation would inhibitthe microbial reductive dissolution at higher pHs. Nonetheless,Fe(II) formation increased in zone I after a small lag phasewhen the pH was increased from 2.9 to 5.3. This lag phasecould have resulted from acclimation to different chemicalconditions caused by pH adjustment or could indicate a shift inthe microbial Fe(III)-reducing population. Thus, the capacityof some acidophiles to reduce Fe(III) at pH 5 combined withthe activity of moderate acidophilic Fe(III) reducers might beresponsible for the elevated rates of Fe(III) reduction in zoneI at pH 5.3. The increase in pH might also have resulted inbetter availability of carbon sources. At higher pHs, schwert-

FIG. 5. Phylogenetic tree of Acidiphilium-related 16S rRNA gene sequences (indicated by boldface type) determined by phylogenetic distanceanalysis with a maximum likelihood algorithm.

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mannite transforms to goethite, which leads to a reduction inthe surface area. An increase in pH alters the charge of theiron oxide surfaces to more negative values so that negativelycharged organic matter can desorb (45) and more surfaceFe(III) might be accessible for Fe(III) reduction.

Zone I was highly enriched by freshly precipitated schwert-mannite resulting from high Fe(III) sedimentation rates (570 gm�2 day�1) (59). The Fe(III) content of the solid phase inzone I was approximately 200 to 350 g kg�1, and 60 to 100%was reactive iron. In zone III, less than 90 g kg�1 was present,

FIG. 6. Phylogenetic tree of Deltaproteobacteria-related 16S rRNA gene sequences determined by phylogenetic distance analysis with amaximum-likelihood algorithm. The designations for clone sequences derived from dilution PCR analysis with group-specific primers begin with“D-PCR,” and the designations for clone sequences from sediment community clone libraries begin with “Common.”

FIG. 7. Frequencies of bacterial phylogenetic lineages detected in 16S rRNA gene clone libraries from four MPN enrichment cultures ofFe(III)-reducing bacteria derived from zone III.

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and the main Fe(III) oxide was goethite. When the pH of zoneIII was decreased from 5.3 to 2.9, Fe(II) formation was re-duced by 30 to 40% despite the better solubility of Fe(III)under low-pH conditions. The lower rate of Fe(III) reductionsuggested that the majority of Fe(III) reducers inhabiting zoneIII had minor capacities to reduce Fe(III) under acidic condi-tions and/or acidophilic Fe(III) reducers did not dominate thisFe(III)-reducing community. The pH and geochemical gradi-ents in the sediment appeared to be responsible for the occur-rence of two distinct Fe(III)-reducing communities.

PCR products obtained using primer sets specific forGeobacteraceae were obtained with higher DNA dilutions fromzone III than from zone I. However, dilution PCR allowed onlyrough comparative estimates for zone III and zone I. Only afew sequences related to cultured Fe(III) reducers were de-tected. With primer pair Gb564/Gb1290, one sequence thatwas related to the family Geobacteraceae was obtained from 31clones, and four sequences were obtained from 45 clones withprimer pair GM3/Geo825R. The latter four sequences wererelated to an isolate obtained from a mining-impacted sedi-ment designated Geobacter sp. strain CdA-2 that can reduceFe(III) at a broad pH range (pH 5.5 to 8.1) (19). Both specificclone libraries revealed that these PCR primer sets are notspecific for the Geobacteraceae group, as reported previously(18). Similar observations for acidic (pH � 4) uranium-con-taminated sediments revealed that members of the Betapro-teobacteria accounted for a large portion of the microbial com-munity, whereas the levels of Geobacteraceae were below thedetection limit (2). Geobacteraceae were detected only in en-richment cultures of acidic uranium-contaminated sedimentsincubated under neutral-pH conditions (60). Firmicutes-re-lated clones had high sequence similarity to the spore-forminggenus Desulfosporosinus and the endospore-forming generaPaenibacillus and Sporomusa (1, 71, 75). Using both cultiva-tion-dependent and -independent techniques, these three gen-era have been shown to inhabit acidic coal mining-associatedlake sediments (43, 53) and acidic uranium-contaminated sed-iments (2, 60).

Shewanella or Geothrix species appeared to not contributesubstantially to the reduction of Fe(III) in coal mining-associ-ated lake sediments. Clonal analysis of the highest positivedilutions of Fe(III)-reducing communities enriched from zoneIII at pH 5.5 revealed that the majority of the clones wererelated to Thiomonas species (55), independent of the electrondonor used. Some clone sequences obtained from our cultureswere related to known Fe(III) reducers, like Geobacter sp.clone Fe_P3-19 or G. fermentans, but they were also related tocultured sulfate reducers, like Desulfosporosinus species, whichare known to inhabit acidic coal mining-associated lake sedi-ments (43, 53). Sulfate reducers can reduce Fe(III) eitherdirectly or indirectly through generation of sulfide (50). Toobtain a better understanding of the cycling of Fe in otherslightly acidic environments, more suitable cultivation tech-niques should be developed to study the ecophysiology of newacidophilic or acid-tolerant isolates.

ACKNOWLEDGMENTS

We thank Winfrid Gade for his help with collecting the sedimentcores and Stefan Peiffer for constructive discussions.

Financial support for this study was provided by the Deutsche For-schungsgemeinschaft (grant KU 1367/1-2) and the German Ministry ofEducation, Science, Research, and Technology (grant PT BEO 51-0339476C).

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AUTHOR’S CORRECTION

pH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganisms inCoal Mining-Associated Lake Sediments

Marco Blothe, Denise M. Akob, Joel E. Kostka, Kathrin Goschel, Harold L. Drake, and Kirsten KuselDepartment of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany; Department of Oceanography,Florida State University, Tallahassee, Florida 32306; and Limnology Research Group, Friedrich Schiller University Jena,

07743 Jena, Germany

Volume 74, no. 4, p. 1019–1029, 2008. Page 1019, Abstract, lines 9 to 11: “Since moderately acidophilic Acidobacteria specieshave the ability to oxidize Fe(II) and since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, this groupappeared to be involved in the cycling of iron.” should read “Since Acidobacterium capsulatum reduced Fe oxides at pHs rangingfrom 2 to 5, Acidobacteria might be involved in the cycling of iron.”

Page 1025, column 2, lines 14 and 15: “some moderate acidophilic Acidobacterium spp. have been shown to oxidize Fe(II)”should read “a novel Acidobacterium species was isolated from ferruginous drainage water samples, using a ferrous-iron-containingoverlay medium.”

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