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［Japanese Journal of Water Treatment Biology Vol.46 No.4 191－199 2010］

Detection of Dissimilatory Iron-Reducing in Freshwater Sediments Using Ferrihydrite-Enriched Cultures and PCR-DGGE Analysis

TAKAHIRO SEKIKAWA1*, HIROKI HAYASHI2, and KEISUKE IWAHORI1

1Institute for Environmental Sciences, University of Shizuoka 2Graduate School of Nutritional and Environmental Sciences, University of Shizuoka /52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan

Abstract Dissimilatory Iron-Reducing Bacteria (DIRB) are known to produce magnetite in medium with amorphous iron oxide such as ferrihydrite that have been isolated from various sites including freshwater and marine sediments in the world. However, the isolation of DIRB from freshwater in Japan has not been reported. We attempted to detect DIRB from freshwater sediments collected at four sites in Shizuoka Prefecture using ferrihydrite-enriched cultures and PCR-denaturing gradient gel electrophoresis (DGGE) analysis. After 14 days of incubation, the ferrihydrite medium turned black and showed a magnetic response in vials containing the all samples. In the DGGE profiles, characteristic bands could be determined in all samples before and after incubation, and significant changes occurred in the microbial community after incubation in the ferrihydrite-enriched cultures. The main DGGE bands in the four samples after incubation showed 98% similarity with Bacterium ROME215Asa, thiogenes K1 (98%), Geobacter sp. T32 (100%), and PCA (100%), respectively. These results indicate that ferrihydrite-enriched cultures coupled with PCR-DGGE analysis are an effective means of detecting DIRB in the environment. Furthermore, this study revealed that several of DIRB exist with various kinds of bacteria in freshwater sediments in Shizuoka Prefecture, Japan.

Key words: dissimilatory iron-reducing bacteria, magnetite, ferrihydrite, freshwater sediments, PCR-DGGE

bacterium from sediments of the Arctic INTRODUCTION 6) Svalbard is able to grow at –2℃ . The Dissimilatory iron-reducing bacteria (DIRB) alkali-resistant bacterium 2+ 3+ are able to produce magnetite (Fe Fe 2O4) alkaliphilus isolated from salt lakes in Russia from the raw material of amorphous iron is able to grow at NaCl concentrations of oxide1–4). In addition to dissimilatory iron 0–50 g/l and its optimum pH is 8.67). The reduction, DIRB have various other thermophilic bacterium capabilities. Geopsychrobacter electrodiphilus ehrlichii was isolated from seafloor isolated from marine sediment have been hydrothermal vents, and its optimum growth used for the generation of electricity in temperature is 55℃8). Geobacter thiogenes a microbial fuel cell5). Desulfuromonas isolated from a river adjacent to a chemical svalbardensis isolated as a cryophilic plant is able to grow via reductive

*Corresponding author 192 Japanese J. Wat. Treat. Biol. Vol.46 No.4 dechlorination of trichloroacetate9). Geobacter (35°14’61”N, 138°70’01”E); industrial waste­ lovleyi has the capacity of reductive tetra- water is drained near the mouth of the River chloroethene dechlorination10). Thus DIRB Numa. The Sanaru sample was collected that are a variety of abilities, have been from a stream flowing into Lake Sanaru in exploring in the world. Hamamatsu City (37°72’03”N, 137°69’72”E); The methods for cultivation and isolation according to a nationwide survey released by of bacteria are dependent on the type of the Ministry of the Environment in 2001, the DIRB. For example, many species of chemical oxygen demand of Lake Sanaru was Shewanella have been isolated by using the the worst in Japan. The Tomoe sample was agar plate method11), whereas Geobacter collected from the upstream reach of the species have been generally isolated by River Tomoe in Shizuoka City (35°04’72”N, limiting dilution of the selective medium 138°41’00”E); the River Tomoe flows through containing amorphous iron oxide as an the urban area of Shimizu-ku, Shizuoka, into electron acceptor8). It was reported that the port of Shimizu and is a typical urban Geobacter, Bacteroides and Clostridium river. species were the dominant bacteria in the Enrichment conditions The ferrihydrite 3+ selective medium with ferrihydrite (Fe 5HO8 medium consisted of CH3COONa·3H2O (2.2

· 4H2O), while Bradyrhizobium, Bacteroides, g/l), NaHCO3 (2.5 g/l), NH4Cl (1.5 g/l), KCl

Clostridium and Ralstonia species were the (0.1 g/l), NaCl (0.1 g/l), NaH2PO4·2H2O (0.8 dominant bacteria in the ferric citrate g/l), yeast extract (0.05 g/l, Nacalai Tesque), 12) medium . Furthermore, Geobacter and chemically synthesized ferrihydrite (Fe5HO8·

Desulfuromonas species such as Geobacter 4H2O [11.2 g/l]), and a stock solution (CaCl2· sulfurreducens, Geobacter metallireducens, 2H2O [0.1 g/l], MgCl2·6H2O [0.1 g/l], MnCl2· 13) Desulfuromonas alkaliphilus have been 4H2O [0.005 g/l], Na2MoO4·2H2O [0.001 g/l]) . known to produce magnetite in the medium The ferrihydrite was synthesized by neu­ with ferrihydrite as an electron acceptor and tralizing a 0.4 M solution of FeCl3 to a pH of 2–4) 13) acetic acid as an electron donor . 7 with NaOH . The reagents CH3COONa·

However, the isolation of DIRB from 3H2O, NaHCO3, NH4Cl, NaH2PO4·2H2O, KCl, freshwater in Japan has not been reported. NaCl, and yeast extract were dissolved in The purpose of this study is to clarify the distilled water and passed through a 0.2-μm diversity of DIRB in freshwater sediments in pore size membrane filter (Advantec) for Japan. We attempted to detect DIRB from sterilization. The ferrihydrite and the stock sediments collected at four sites (three rivers solution were autoclaved at 121℃ for 20 min. and a stream) in Shizuoka Prefecture using Under sterile conditions on a clean bench, ferrihydrite-enriched cultures and PCR- 27 ml of ferrihydrite medium and 3 ml of denaturing gradient gel electrophoresis each sediment sample were added to a 50-ml (DGGE) analysis. vial sterilized in advance by dry heating (180℃, 2 h). The vial was sealed with a butyl MATERIALS AND METHODS rubber stopper and an aluminum seal, and Sample collection and preparation the inside air was replaced with nitrogen gas Sediment samples were collected from three to produce anaerobic conditions. The vials rivers and a stream in Shizuoka Prefecture were incubated at 30℃ for 14 days under in June 2008. The samples were collected dark and static conditions. The formation of into 50-ml sterile tubes and were stored in a magnetite was then judged visually using a refrigerator until analysis. The Nakagochi neodymium magnet. sample was collected from the downstream DNA extraction and PCR A proper reach of the River Abenakagochi in Shizuoka quantity of oxalic acid solution (oxalic acid 15 City (35°11’60”N, 138°35’09”E); the River g/l, ammonium oxalate 28 g/l, pH 3.2) was Abenakagochi flows from the mountains and added to 400 μl of each sample in order to connects with the River Abe in Shizuoka completely dissolve insoluble iron oxides in City. The Numa sample was collected from the sample14). The treated sample was then the mouth of the River Numa in Fuji City centrifuged at 4,000 × g for 10 min, and the Detection of Dissimilatory Iron-Reducing Bacteria Using Ferrihydrite-Enriched Cultures 193 supernatant was discarded. The sample was from the gel using a sterile pipette tip. The then resuspended in sterile water (Invitrogen) DNA was extracted from the DGGE band and centrifuged at 4,000 × g for 10 min. using the High Pure PCR Product Purification Bacterial cells were collected from the sample Kit (Roche Diagnostics). The purified DNA by repeating the above procedures three times. was amplified with 341f and 907r primers for DNA was extracted and purified from the 30 PCR cycles, and amplified DNA was bacterial cells using a DNA extraction kit purified using the Purification Kit. The (DNeasy Tissue Kit, Qiagen). sequencing reactions were carried out by Extracted DNA was amplified with primers Macrogen Japan Corporation. 16S rRNA 341f-GC (5’-GC clamp [5’-CGC CCG CCG sequences were determined based on the CGC GCG GCG GGC GGG GCG GGG GCA nucleotide sequence databank of Japan CGG GGG-3’]-CCT ACG GGA GGC AGC AG- (DDBJ: http://www.ddbj.nig.ac.jp). High se­ 3’) and primer 907r (5’-CCG TCA ATT CCT quence similarity with known bacteria was TTG AGT TT-3’) for the 16S rRNA V3–V5 investigated using a gene sequence database region (586 bp) using the Gene Amp PCR BLAST (http://blast.ddbj.nig.ac.jp/top-j.html) System 9700 (Applied Biosystems)15). The search. Sequences from bacteria closely PCR was carried out with PCR reagent related to the cultured types were estimated FastStart Taq DNA Polymerase, dNTPack from the results of the similarity search; (Roche Diagnostics) consisted of FastStart alignment was performed by MEGA 5.0 Taq DNA polymerase (5 U/μl), PCR-Grade (http://www.megasoftware.net/beta/index.php) Nucleotide Mix, 10× PCR buffer including 20 was used to create a phylogenetic tree. The mM-MgCl and distilled water. Each 25-μl estimation of the phylogenetic tree was reaction contained the following: 0.2 μl carried out by the neighbor-joining method. FastStart Taq DNA polymerase, 0.5 μl PCR- Analytical procedures As pretreatment Grade Nucleotide Mix, 2.5 μl 10× PCR buffer, for the measure of metals, each sample was 20.3 μl distilled water, 0.5 μl each forward passed through a 0.45-μm pore size membrane and reverse primer (10 μM), and 0.5 μl DNA filter (DISMIC-13HP, Advantec). Standard template. The PCR conditions were as curves were created using 1,000 ppm standard follows: preheating at 95℃ for 4 min for hot- solutions (Wako) of Ca, Fe, K, Mg, Mn, and start PCR; 35 cycles of denaturing at 95℃ Na. The sample was diluted with 2% nitric for 30 sec, annealing at 56℃ for 30 sec, and acid, and concentrations of metals were extension at 72℃ for 1 min; and final measured by an inductively coupled plasma extension at 72℃ for 7 min. optical emission spectrometer (730-ES, Varian). DGGE analysis Amplified 16S rRNA Total organic carbon (TOC) was measured gene fragments were analyzed by DGGE using a total carbon analyzer (TOC-5000A, with the Dcode Universal Mutation Detection Shimadzu). The analytical methods for total System (Bio-Rad) and Power Station 1000XP nitrogen (TN) and total phosphate (TP) were (Atto) as the power supply. PCR products those of Japanese Industrial Standard (JIS) were separated on an 8% polyacrylamide gel, K010016). and a linear gradient of the denaturants Nucleotide sequence accession numbers urea and formamide increasing from 30% at The sequences generated in this study have the top of the gel to 60% at the bottom for been deposited in the GenBank database the separation of the 16S rRNA fragments. under accession numbers AB568586 to For each sample, 40 μl of PCR product was AB568596. loaded, and the gel was run for 12 h at 100 RESULTS AND DISCUSSION V and 60℃. After electrophoresis, the gel was incubated for 30 min in Milli-Q Physico-chemical properties of samples (Millipore) water containing ethidium The physico-chemical properties of the water bromide (1.0 mg/l) and removed from the in the sediment samples are shown in Table plastic plate to a UV-transparent gel scoop. 1. For the Nakagochi, Sanaru, and Tomoe Sequencing and phylogenetic analysis samples, TOC was less than 1 mg/l. However, DGGE bands containing DNA were punched the TOC of the Numa sample containing 194 Japanese J. Wat. Treat. Biol. Vol.46 No.4 industrial wastewater was 82 mg/l. at the bottom of the vial, and the ferrihydrite Furthermore, the concentrations of Mg and showed no magnetic response. After 14 days of Na in the Numa sample were signifi cantly incubation, however, the ferrihydrite medium higher than those of the other samples, because turned black and showed a magnetic response seawater containing high concentrations of in vials containing the Nakagochi, Numa, these elements was mixed with water at the Sanaru, or Tomoe sample (Fig. 2), indicating Numa site. The Ca concentrations in the Numa that the black substance was magnetite. and Tomoe samples were high: 150 and 130 Because DIRB can produce magnetite 2+ 3+ 3+ mg/l, respectively. Only the Numa sample (Fe Fe 2O4) from ferrihydrite (Fe 5HO8·4H2O), contained Mn (0.59 mg/l), whereas the Mn these results indicate that DIRB survived in concentration of the other samples was less all samples. than the detection limit of 0.01 mg/l. Closest matches to excised and sequenced Magnetite production by ferrihydrite- 16S rRNA-derived DGGE bands In the enriched cultures Figure 1 illustrates the DGGE profi les, characteristic bands could be ferrihydrite medium before incubation. The determined in all samples before (bands A1– brown amorphous ferrihydrite was precipitated A6) and after (bands B1–B5) incubation (Fig. 3). Signifi cant changes occurred in the

Table 1 Physico-chemical properties of the water in the sediment samples

TOC TN TP Metals (mg/l) Sample name pH (mg/l) (mg/l) (mg/l) Ca Fe K Mg Na Mn Nakagochi 7.34 0.55 2.5 0.18 56 <0.01 1.2 14 3.4 <0.01 Numa 6.78 82 48 2.2 150 0.11 48 310 1400 0.59 Sanaru 7.18 0.64 11 2.9 16 0.05 3.4 5.7 12 <0.01 Tomoe 7.14 <0.05 5.6 0.11 130 <0.01 3.5 5.5 5.6 <0.01

1 2

3 4 A B

Fig. 1 The magnetic response of the ferrihydrite Fig. 2 The magnetic response of the ferrihydrite medium (A) before incubation, as detected medium after the 14-day incubation of sediment using a neodymium magnet (B) samples from: (1) Nakagochi, (2) Numa, (3) Sanaru, and (4) Tomoe Detection of Dissimilatory Iron-Reducing Bacteria Using Ferrihydrite-Enriched Cultures 195

microbial community after incubation in the which was isolated from Antarctic sediment17), ferrihydrite-enriched cultures. Bands A1 and and band A2 showed 97% similarity with A5 showed strong signals in the Nakagochi bacterium CNRF05. and Sanaru samples before incubation, Burkholderiales are an order of respectively. Bands B2 (Nakagochi), B3 that contains several pathogenic bacteria, (Numa), B4 (Sanaru), and B5 (Tomoe) were including species of Burkholderia and found in each sample after incubation. The Bordetella. The phylogenetic tree of 16S bacterial strains showing the greatest similarity rRNA gene sequences before incubation (Fig. with bands A1 to A6 and B1 to B5 are listed 4) shows that bands A1 and A2 are closely in Table 2. Three species of Geobacter were related species. The bacterium in the found in the samples after incubation. Nakagochi sample after incubation (band B2) In the Nakagochi sample, band A1 showed showed 98% similarity with Bacterium 99% similarity with Antarctic bacterium CA1, ROME215Asa (Table 2), and 95% similarity with D. alkaliphilus Z-0531. Bacterium ROME215Asa has not been reported to produce magnetite, although both D. Nakagochi Numa Sanaru Tomoe alkaliphilus can produce magnetite from D1 D2 D3 D4 D5 D6 D7 D8 ferrihydrite7). Based on the phylogenetic tree B1 of 16S rRNA gene sequences after incubation (Fig. 5), band B2 was in the same cluster as Desulfuromonas and species. Band B1 of the Nakagochi sample after incubation A3 showed 98% similarity with Mollicutes A4 A5 B5 bacterium pACH93. As shown in Figure 5, B4 M. bacterium pACH93 is distantly related to A1 A6 B3 Geobacter, Desulfuromonas and Pelobacter A2 species18). Although M. bacterium pACH93 has not been reported to have the ability to

B2 reduce ferrihydrite, our results indicate that this strain was able to grow in the ferrihydrite Fig. 3 DGGE profi les of 16S rRNA fragment amplifi ed medium. from DNA extracts from the sediment samples In the Numa sample before incubation, (lanes D1, D3, D5, D7) and after samples were incubated for 14 days on ferrihydrite-enriched band A3 showed 98% similarity with medium (lanes D2, D4, D6, D8) tsuruhatensis BM90, which is a denitrifi er

Table 2 Closest matches to excised and sequenced 16S rRNA-derived DGGE bands Band Length of Closest match in Genbank database Similarity Accession sequence (bp) (Accession No.) (%) No. A1 492 Antarctic bacterium CA1 (EU636023) 99 AB568586 A2 581 Burkholderiales bacterium CNRF05 (GU300542) 97 AB568587 A3 529 Delftia tsuruhatensis BM90 (EU779949) 98 AB568588 A4 505 Desulfomicrobium sp. La1.1 97 AB568589 A5 423 Desulfofrigus oceanense ASv26 ( AF099064) 84 AB568590 A6 586 Delftia tsuruhatensis BM90 (EU779949) 100 AB568591 B1 557 Mollicutes bacterium pACH93 (AY297808) 98 AB568592 B2 576 Bacterium ROME215Asa (AY998187) 98 AB568593 B3 570 Geobacter thiogenes K1 (AF223382) 98 AB568594 B4 584 Geobacter sp. T32 (GQ463728) 100 AB568595 B5 578 Geobacter sulfurreducens PCA (AE017180) 100 AB568596 196 Japanese J. Wat. Treat. Biol. Vol.46 No.4

49 Delftia tsuruhatensis BM90 (EU779949) 98 A3 (Numa) 74 A6 (Sanaru) 97 ACM 498T (AF078774) 99 Comamonas sp. R7 (AJ002810) A2 (Nakagochi) 100 95 A1 (Nakagochi) 57 Antarctic bacterium CA1 (EU636023) Burkholderiales bacterium CNRF05 (GU300542) Holdemania filiformis ATCC 51649T (Y11466) 100 A4 (Numa) 94 Desulfomicrobium sp. La1.1 (AF228113) Desulfovibrio profundus DSM 11384T (AF418172) A5 (Sanaru) 90 73 Desulfuromonas alkaliphilus Z-0531T (DQ309326) 30 Geobacter sulfurreducens PCAT (AE017180) 52 Desulfobulbus propionicus DSM 2032T (AY548789) T 65 Desulfofrigus oceanense ASv26 (AF099064) 0.02 79 Desulfobacterium autotrophicum DSM 3382T (AF418177)

Fig. 4 Phylogenetic tree of 16S rRNA gene sequences of the DNA extracts from sediment samples. A phylogenetic tree was constructed for 446-bp aligned sequences from neighbor-jointing method positions using MEGA 5.0 software. Robustness of derived groupings was tested by bootstrap using 1,000 replications. The scale bar represents 0.02 substitutions per nucleotide site.

91 B2 (Nakagochi) 45 bacterium ROME215Asa (AY998187) T 17 Desulfuromonas alkaliphilus Z-0531 (DQ309326) 100 bacterium Tc37 (AB260047) 21 Geothermobacter ehrlichii SS015T (AY155599) Geopsychrobacter electrodiphilus A1T (AY187303) 39 32 DSM 2380T (CP000142) 50 Gra PEG1T (U41562) Desulfuromonas thiophila DSM 8987T (Y11560) T 96 Desulfuromonas svalbardensis 112 (AY835388) 98 Desulfuromonas acetoxidans DSM 684T (AY187305) 99 99 B4 (Sanaru) 99 Geobacter sp. T32 (GQ463728) 82 B5 (Tomoe) 99 Geobacter sulfurreducens PCAT (AE017180) Geobacter metallireducens GS-15T (CP000148) T 97 58 Geobacter chapelleii 172 (U41561) Rf4T (CP000698) 51 B3 (Numa) T 87 SZ (CP001089) 91 Geobacter thiogenes K1T (AF223382) Delftia acidovorans ACM 498T (AF078774) 42 Solobacterium moorei RCA59-74T (AB031056) Holdemania filiformis ATCC 51649T (Y11466) 100 Erysipelothrix rhusiopathiae ATCC 19414T (AB034200) 52 B1 (Nakagochi) 0.01 99 Mollicutes bacterium pACH93 (AY297808)

Fig. 5 Phylogenetic tree of 16S rRNA gene sequences of the DNA extracts from samples incubated for 14 days on ferrihydrite-enriched medium. A phylogenetic tree was constructed for 446-bp aligned sequences from neighbor-jointing method positions using MEGA 5.0 software. Robustness of derived groupings was tested by bootstrap using 1,000 replications. The scale bar represents 0.01 substitutions per nucleotide site. Detection of Dissimilatory Iron-Reducing Bacteria Using Ferrihydrite-Enriched Cultures 197 that can reduce nitrate to nitrogen gas19). phylotypes Bands of the Tomoe, Sanaru, Band A4 showed 97% similarity and Numa samples after incubation were with Desulfomicrobium sp. La1.1. closely related to Geobacter species, although Desulfomicrobium species belong to the Band B3 of the Numa sample belongs to a Desulfomicrobiaceae and has a sulfate- different phylogenetic cluster than Bands B4 reducing ability20). Band B3 after incubation of the Sanaru sample and B5 of the Tomoe showed 98% similarity with both G. thiogenes sample (Fig. 5). Band B2 of the Nakagochi K1 and G. lovleyi SZ. Geobacter thiogenes K1 sample was placed in a separate cluster with was isolated from a river close to chemical Desulfuromonas and Pelobacter species. Thus, plants and is able to reductively dechlorinate our phylogenetic analysis indicates that the trichloroacetate9). Geobacter lovleyi SZ, which species of DIRB were in all sediment samples. was isolated from river sediment in the The results in Table 1 show that there is no suburb of Seoul City, South Korea, is able to correlation between the species of DIRB and reductively dechlorinate tetrachloroethene the physico-chemical properties of the water and produce magnetite from ferrihydrite10). in the samples. A previous study reported Our findings suggest that bacterial growth the Geobacteraceae were diverse and condition such as water quality in a river abundant in lake sediments, regardless of may influence which species of DIRB are metals content23). DIRB were in the Nakagochi able to survive in an environment, because and Tomoe samples despite soluble Fe Band B3 most closely related to G. thiogenes concentrations being less than the detection K1 was detected only from the Numa limit of 0.01mg/l in the samples, because sediment sample in this study, which was they are able to use insoluble iron compounds mixed with industrial discharge. for growth. In the Sanaru sample before incubation, Bands B4 of the Sanaru sample and B5 of band A5 showed 84% similarity with the Tomoe sample belong to a phylogenetic Desulfofrigus oceanense ASv26, a filamentous cluster that contains two species isolated anaerobic sulfate-reducing bacterium 21). from a river and a stream: Gobacter sp. T32 Band A6 showed 100% similarity with Delftia and G. sulfurreducens PCA (Fig. 5)3). Band tsuruhatensis BM90, which was isolated from B3 of Numa sample belongs to a cluster that the Tyrrhenian Sea. Delftia tsuruhatensis contains two species isolated from rivers with has the ability to break down various phenolic industrial discharge: G. thiogenes K1 and G. compounds22). As shown in the phylogenetic lovleyi SZ9, 10). tree in Figure 4, band A6 of the Sanaru Band B2 of the Nakagochi sample was sample and band A3 of the Numa sample are closely related to Bacterium ROME215Asa, closely related. Band B4 of the Sanaru which was closely related to D. palmitatis sample after incubation showed 100% isolated from seawater. Many Desulfuromonas similarity with Geobacter sp. T32, which was species including D. palmitatis, D. closely related to G. sulfurreducens PCA. svalbardensis, D. ferrireducens, and D. There was no strong band in the DGGE acetoxidans have been isolated from seawater profile of the Tomoe sample before incubation sediments2, 6, 24), on the other hand D. (Fig. 3, D7). Our analyses indicate that the michiganensis and D. thiophila have been viable number of bacteria in the Tomoe isolated from freshwater sediments 25, 26). sample was very low compared with the Although seawater-isolated D. acetoxidans other samples, because the TOC concentration was able to reduce Fe(III) and produce in the sample was less than the detection magnetite in the seawater medium, it hardly limit of 0.05 mg/l. Band B5 of the Tomoe reduced Fe(III) in the freshwater medium24). sample after incubation showed 100% Geobacter metallireducens reduced Fe(III) similarity with G. sulfurreducens PCA, which only in the freshwater medium24). It is not was isolated from the sediment of a clear why there is a difference in the bacterial freshwater canal contaminated with iron-reducing ability between freshwater and hydrocarbons3). seawater. Distribution and diversity of DIRB 198 Japanese J. Wat. Treat. Biol. Vol.46 No.4

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