191 ［Japanese Journal of Water Treatment Biology Vol.46 No.4 191－199 2010］ Detection of Dissimilatory Iron-Reducing Bacteria 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, Geobacter thiogenes K1 (98%), Geobacter sp. T32 (100%), and Geobacter sulfurreducens 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 species 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 Desulfuromonas 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 Geothermobacter 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.