International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1384–1388

DOI 10.1099/ijs.0.058826-0

Raoultella electrica sp. nov., isolated from anodic biofilms of a glucose-fed microbial fuel cell Zen-ichiro Kimura,1 Kyung Mi Chung,1 Hiroaki Itoh,1 Akira Hiraishi2 and Satoshi Okabe1 Correspondence Satoshi Okabe [email protected]

1

Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North-13, West-8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan

2

Department of Environmental and Life Sciences, Toyohashi University of Technology, Toyohashi 441-8580, Japan

A Gram-stain-negative, non-spore-forming, rod-shaped bacterium, designated strain 1GBT, was isolated from anodic biofilms of a glucose-fed microbial fuel cell. Strain 1GBT was facultatively anaerobic and chemo-organotrophic, having both a respiratory and a fermentative type of metabolism, and utilized a wide variety of sugars as carbon and energy sources. Cells grown aerobically contained Q-8 as the major quinone, but excreted Q-9 and a small amount of Q-10 when cultured with an electrode serving as the sole electron acceptor. The G+C content of the genomic DNA of 1GBT was 54.5 mol%. Multilocus sequence typing (MLST) analysis showed that strain 1GBT represented a distinct lineage within the genus Raoultella (98.5–99.4 % 16S rRNA gene sequence similarity and 94.0–96.5 % sequence similarity based on the three concatenated housekeeping genes gyrA, rpoB and parC. Strain 1GBT exhibited DNA–DNA hybridization relatedness of 7–43 % with type strains of all established species of the genus Raoultella. On the basis of these phenotypic, phylogenetic and genotypic data, the name Raoultella electrica sp. nov. is proposed for strain 1GBT. The type strain is 1GBT (5NBRC 109676T5KCTC 32430T).

Microbial fuel cells (MFCs) are devices that are able directly to convert the chemical energy of organic compounds into electric energy by using micro-organisms (Bullen et al., 2006; Davis & Higson, 2007; Kim et al., 1999). Electricity generation in a mediator-less MFC is linked to the ability of certain bacteria, called exoelectrogens, to transfer electrons outside the cell to the anode electrode in the MFC (Logan & Regan, 2006). Previously, we constructed an MFC system by seeding activated sludge as the source of micro-organisms and feeding with glucose as the sole substrate, and analysed the microbial community of the anodic biofilm in the MFC (Chung & Okabe, 2009b). The results of our culture-independent approach revealed that species of the Klebsiella/Raoultella complex were present in abundance in the anodic biofilm community (Chung & Okabe, 2009b). From this MFC system, therefore, we isolated a bacterium (designated strain 1GBT), which was closely related to species of the genus Raoultella, created in 2001 to accommodate three Abbreviations: MFC, microbial fuel cell; MLST, multilocus sequence typing. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, gyrA, rpoB and parC gene sequences of strain 1GBT are AB762091, AB828204, AB828205 and AB828206, respectively. Four supplementary figures are available with the online version of this paper.

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species of the genus Klebsiella (Drancourt et al., 2001). In this paper, multilocus sequence typing (MLST) of housekeeping genes as well as phenotypic and chemotaxonomic studies were performed to characterize strain 1GBT more thoroughly taxonomically and phylogenetically. Based on the results of these studies, we propose the isolate should be assigned to a novel species of the genus Raoultella with the name Raoultella electrica sp. nov. Strain 1GBT was isolated by the standard dilution plating technique on LB agar (Difco, Becton Dickinson) at 25 uC. For comparison, Raoultella ornithinolytica strain NBRC 105727T, Raoultella planticola NBRC 14939T and Raoultella terrigena NBRC 14941T were used. Morphological, physiological and biochemical characteristics were investigated using standard cultivation techniques. All biochemical assays were performed at 25 uC because the optimum temperatures of all tested strains were slightly different. All strains grew sufficiently at this temperature for all biochemical assays. Growth at temperatures between 5 and 50 uC was measured by inoculating a loop full of young culture onto LB agar and incubating the inoculated plates at the required temperatures for 3 days. Electrochemical activity was evaluated using H-type MFCs that consisted of 500 ml anode and cathode glass chambers separated by proton exchange membranes (PEM; Nafion 117, Dupont) (Chung et al. 2011). The anode chamber was filled with 500 ml 058826 G 2014 IUMS Printed in Great Britain

Raoultella electrica sp. nov.

sterilized mineral medium (Chung & Okabe, 2009a) supplemented with glucose as the sole electron donor. The cathode chamber was filled with 500 ml 80 mM phosphate buffer (pH 7.1) containing 50 mM ferricyanide and with the carbon electrode as the sole electron acceptor. The H-type MFCs were operated at 25±2 uC. Utilization of various substrates, enzymic activities and other physiological and biochemical properties were tested using API 20E and API 50CHE (bioMe´rieux) according to the manufacturer’s instructions.

reconstruction are listed in Table S1 (available in the online Supplementary Material).

Whole-cell fatty acid analysis was performed at the stationary phase of growing cells [on YPG agar (Difco, Becton Dickinson) for 48 h at 30 uC] by GLC according to the instructions of the Microbial Identification System (MIDI) Sherlock version 6.0 (Sasser, 1990) with the RTSBA6 MIDI database. Quinones were extracted with a chloroform/methanol (2:1, v/v) mixture, purified by TLC and analysed by reversed-phase HPLC, as described previously (Hiraishi, 1988).

Cells of strain 1GBT were Gram-stain-negative, non-sporeforming, non-motile, rod-shaped, and 0.3–0.4 mm63.0– 6.0 mm. Colonies grown on LB agar were circular, smooth, glistening, light yellow and 5.0 mm in diameter after 2 days of incubation at 37 uC. Growth occurred at temperatures from 10 to 41 uC, but not at 5 or 50 uC (Table 1). Images of Gram-staining and colony morphology are shown in Fig. S4 (A, B). Strain 1GBT was facultatively anaerobic and chemo-organotrophic, having both a respiratory type and a fermentative type of metabolism with sugars and other simple organic compounds acting as sole carbon and energy sources (Table 1 and species description). Also, strain 1GBT could generate electrical current via oxidation of glucose in a pure culture MFC (Fig. S1).

Three housekeeping genes gyrA (383 bp), rpoB (512 bp) and parC (319 bp)) were used for species-level identification of members of the Klebsiella/Raoultella complex (Brisse & Verhoef, 2001; Drancourt et al., 2001). The 16S rRNA, gyrA, rpoB and parC genes of strain 1GBT were amplified by PCR with a thermal cycler (GeneAmp PCR System 9700, Life technologies). The PCR amplification conditions were as follows: 3 min at 95 uC (initial denaturation), followed by 30 cycles of 94 uC for 1 min, 53 uC for 1 min, and 72 uC for 1 min, finishing with an extension step of 72 uC for 5 min. The following primers were used for sequence analysis: 27-F (59-AGAGTTTGATCCTGGCTCAG-39) and 1492-R (59-GGTTACCTTGTTACGACTT-39) for 16S rRNA gene; gyrA-F (59-CGCGTACTATACGCCATGAACGTA-39) and gyrA-R (59ACCGTTGATCACTTCGGTCAGG-39) for gyrA; rpoB-F (59-AACCAGTTCCGCGTTGGCCTGG-39) and rpoB-R (59-CCTGAACAACACGCTCGGA-39) for rpoB; parC-F (59-ATGGACCGTGCGTTGCCGTTTAT-39) and parC-R (59-CGGCAATACCGGTGGTGCCGTT-39) for parC (Brisse & Verhoef, 2001; Drancourt et al., 2001; Lane, 1991). Purified PCR products were sequenced directly with an ABI model PRISM 3100-Avant Genetic Analyzer (Life technologies). All sequences obtained were compared with reference 16S rRNA gene sequences available in the GenBank/EMBL/DDBJ databases using the BLAST search. Sequence data were compiled with the BioEdit program (Hall, 1999). Multiple alignment of the sequence data, calculation of the corrected evolutionary distances (Kimura, 1980), and reconstruction of neighbour-joining phylogenetic trees (Saitou & Nei. 1987) were performed using CLUSTAL W program version 1.83 (Thompson et al., 1994). An MLST analysis was performed as described by Urwin & Maiden. 2003). A phylogenetic tree of concatenated sequences (gyrA+rpoB, 895 bp; gyrA+rpoB+ parC, 1214 bp) including fragments of three genes [gyrA (383 bp), rpoB (512 bp), parC (319 bp)] of the isolate was reconstructed based on the neighbour-joining method. All the gene sequences used for phylogenetic tree http://ijs.sgmjournals.org

Genomic DNA was extracted and purified according to the method of Marmur (1961). The guanine plus cytosine (G+ C) ratio of genomic DNA was determined by HPLC with external nucleotide standards, as described by Mesbah et al. (1989). DNA–DNA hybridization studies were performed using quantitative dot-blot hybridization with the Alkphos Direct Labelling and Detection System with CDP-star (GE healthcare) as described previously (Kubota et al., 2005)

The major isoprenoid quinone detected in aerobically grown cells of strain 1GBT was ubiquinone-8 (Q-8) (8.98 nmol, 64.3 mol%), in accordance with previously reported quinone

Table 1. Differential characteristics of strain 1GBT and type strains of established species of the genus Raoultella Strains: 1, 1GBT; 2 R. ornithinolytica NBRC 105727T; 3, R. planticola NBRC 14939T; 4, R. terrigena NBRC 14941T. +, Positive; –, negative. Characteristic Growth at: 5 uC 10 uC 41 uC 50 uC Urea hydrolysed Indole production L-Ornithine decarboxylase Utilization of: L-Arabinose L-Rhamnose D-Fructose Melezitose Gentiobiose Glycogen Starch Glucose Gluconate Citrate DNA G+C content (mol%)

1

2

3

4

2 + + 2 + 2 2

+ + + 2 + + +

+ + + 2 + 2 2

+ + 2 2 2 2 2

+ 2 2 + + 2 2 + 2 + 54.5

+ + + 2 2 + + + + + 57–58

2 + + 2 2 2 2 2 + + 55.4

+ + + + 2 2 2 2 2 2 56.7

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systems of the genera Klebsiella and Raoultella (OyaizuMasuchi & Komagata, 1988; Whistance et al., 1969). Menaquinone-8 (MK-8) (4.50 nmol, 32.2 mol%) and demethylmenaquinone (DMK-8) (0.48 nmol, 3.4 mol%) were also detected when cultured anaerobically with a fermentative substrate (e.g. glucose) (Fig. S2a). Interestingly, in addition to intracellular quinones, extracellular quinones with Q-9 (0.30 nmol, 2.6 mol% intracellularly; 0.28 nmol, 53.2 mol% extracellularly) as the major homologue and a small amount of Q-10 (0.27 nmol, 2.5 mol% intracellularly; 0.24 nmol, 46.7 mol% extracellularly) were detected when cultured with the anode electrode as the sole electron acceptor (i.e. MFC conditions) (Fig. S2b, c). These ubiquinones may play a role in being an extracellular electron shuttle (Kimura and others unpublished observations). Production of 2,6-di-tert-butyl-p-benzoquinone by Klebsiella pneumoniae strain L17 as the electron shuttle has been previously reported (Deng et al., 2010). However, our attempts to detect extracellular quinones in previously described species of the genera Raoultella and Klebsiella were negative in this study. The major fatty acids of strain 1GBT were C16 : 0, summed feature 3 (C16 : 1v7c and/or iso-C15 : 02-OH) and C18 : 1v7c (Table 2). These fatty acid profiles are similar to those of recognized species of the genera Klebsiella and Raoultella. The chemotaxonomic properties of strain 1GBT warrant classifying it into the Klebsiella/Raoultella complex. A nearly complete sequence of the 16S rRNA gene (1452 bp) and partial sequences of the gyrA (383 bp), rpoB (512 bp) and parC (319 bp) genes of strain 1GBT were determined (Table S1). A neighbour-joining phylognetic tree based on 16S rRNA gene sequences showed that strain 1GBT is closely related to members of the genus Raoultella with the type strain of R. ornithinolytica its nearest phylogenetic neighbour (Fig. 1). The similarity between strain 1GBT and the type strain of R. ornithinolytica is 99.4 %, which is above the threshold value (98.7–99.0 %) for grouping bacterial strains as a genetically single species (Stackebrandt & Ebers, 2006). To obtain a more comprehensive understanding of the taxonomic position of strain 1GBT, therefore, we performed the MLST analysis according to the MLST scheme reported for differentiation of species of the genus Klebsiella species (Diancourt et al., 2005) (Fig. S3). The sequence similarity levels of gyrA, rpoB, parC and the concatenated sequences of these 3 housekeeping genes between strain 1GBT and the most closely related strain (R. ornithinolytica NBRC 105727T) were 97.1 %, 95.5 %, 97.8 % and 96.5 %, respectively (Fig. S3). All sequence similarities of related strains of species of the Klebsiella/Raoultella complex with strain 1GBT are shown in Table S2. A phylogenetic analysis of the concatenated sequences of the three housekeeping genes showed that strain 1GBT forms a distinct lineage within the genus Raoultella with R. ornithinolytica and R. planticola its nearest phylogenetic neighbours. The similarity levels between strain 1GBT and 1386

Table 2. Cellular fatty acid composition (%) of strain 1GBT and type strains of established species of the genus Raoultella Strains: 1, 1GBT; 2, R. ornithinolytica NBRC 105727T; 3, R. planticola NBRC 14939T; 4, R. terrigena NBRC 14941T. Fatty acid components accounting for ,0.1 % of the total are omitted. 2, Not detected. Fatty acid Straight-chain C10 : 0 C11 : 0 C12 : 0 C13 : 0 C14 : 0 C15 : 0 C16 : 0 C17 : 0 C18 : 0 C19 : 0 Unsaturated C15 : 1v8c C16 : 1v5c C17 : 1v6c C17 : 1v8c C18 : 1v5c C18 : 1v7c Hydroxy C12 : 0 3-OH C15 : 0 3-OH Cyclopropane fatty acids Cyclo-C17 : 0 Cyclo-C19 : 0v8c Summed features* 1 2 3

1

2

3

4

2 2 3.2 2 6.9 2 28.4 0.1 2 2

0.1 2 3.1 0.4 5.7 2.4 21.3 2.0 0.2 2

0.1 0.1 3.0 0.5 5.5 3.0 21.5 2.4 0.2 2

2 2 2.5 2 5.2

2 0.2 2 2 2 20.8

2 0.2 2 0.4 2 26.2

0.1 0.3 2 0.6 2 24.8

2 0.2 2 2 2 24.6

27.0 0.2 2

0.03 2

0.04 0.12

2 2

2 2

4.2 0.1

6.5 0.2

6.7 0.1

12.9 0.5

2 7.1 27.7

0.4 7.8 21.4

0.5 7.1 21.7

2 8.2 16.5

*Summed feature 1 comprises C13:0 3-OH, iso-C15:1 H and/or isoC15:1 I. Summed feature 2 comprises iso-C16:1 I and/or C14:0 3-OH. Summed feature 3 comprises C16:1 v7c and/or iso-C15:0 2-OH.

R. ornithinolytica or R. planticola based on concatenated sequences of the 3 housekeeping genes were 94.0–96.6 %; low enough to justify the classification of strain 1GBT as a representative of a novel species of the genus Raoultella. To confirm further the taxonomic position of strain 1GBT as a distinct genospecies of the genus Raoultella, genomic DNA–DNA hybridization studies were performed. Strain 1GBT exhibited relatively low levels of DNA–DNA relatedness to R. ornithinolytica NBRC 105727T (43±9 %), R. planticola NBRC 14939T (33±6 %) and R. terrigena NBRC 14941T (7±3 %). The DNA G+C content of strain 1GBT was 54.5 mol%. As shown in Table 1, strain 1GBT differed clearly from previously established species of the genus Raoultella in several of its phenotypic characteristics. In view of this, together with the phylogenetic distinctiveness, it could be International Journal of Systematic and Evolutionary Microbiology 64

Raoultella electrica sp. nov. Klebsiella pneumoniae subsp. ozaenae ATCC 11297

736

594

Klebsiella pneumoniae subsp. rhinoscleromatis ATCC 13884T

975

Klebsiella pneumoniae subsp. pneumoniae ATCC 1000

1000 1000

13883T

Klebsiella variicola At-22 Klebsiella oxytoca ATCC 13182T

991 361

Klebsiella oxytoca KCTC 1686 833

Klebsiella michiganensis W14T 989

Raoultella terrigena NBRC 14941T 522

393 560

0.002

Raoultella planticola NBRC 14939T

1000

627

Raoultella ornithinolytica NBRC 105727T

1000

Raoultella electrica 1GBT

0.01

Serratia liquefaciens ATCC 27592T

gyrA (383bp)+rpoB (512bp)+parC (319bp)

16S rRNA (1370bp)

Fig. 1. Phylogenetic trees reconstructed from comparative analysis of 16S rRNA genes and three housekeeping gene (gyrA, rpoB and parC) sequences showing the relationship of strain 1GBT with related species. Serratia liquefaciens was used as an outgroup. These trees were reconstructed using the neighbour-joining method (Saitou & Nei, 1987) with a Kimura (1980) two-parameter distance matrix and pairwise deletion. Closed circles show the nodes supported by .80 % bootstrap probabilities with 1000 replicates. Bar, 0.2 % and 1 % nucleotide substitution rate (Knuc).

concluded that strain 1GBT represents a novel species of the genus Raoultella. We propose the name Raoultella electrica sp. nov. Description of Raoultella electrica sp. nov. Raoultella electrica [e.lec9tri.ca. L. n. electrum amber; L. suff. -icus -a -um suffix used with the sense of pertaining to; N.L. fem. adj. electrica referring to generation of electricity (electron so called because it first was generated by rubbing amber)]. Cells are Gram-stain-negative, non-spore-forming rods measuring 0.3–0.4 mm63.0–6.0 mm. Colonies on LB agar are circular, smooth, glistening, light yellow and 5.0 mm in diameter after 2 days of incubation at 37 uC. The temperature range for growth is 10–41 uC (optimum 37 uC). Facultatively anaerobic and chemo-organotrophic, having both a respiratory type and a fermentative type of metabolism using sugars and other simple organic compounds as carbon and energy sources. Catalasepositive and oxidase-negative. Voges–Proskauer reaction is positive. H2S and indole are not produced. Arginine dihydrolase and lysine decarboxylase are present. Ornithine decarboxylase and tryptophan deaminase are absent. In the API 20E and API 50CH systems, the following compounds are utilized as carbon sources: D-glucose, D-mannitol, Dsorbitol, sucrose, melibiose, D-amygdalin, L-arabinose, glycerol, D-ribose, D-xylose, D-adonitol, D-galactose, D-fructose, D-mannose, L-sorbose, cellobiose, maltose, Melezitose, lactose, trehalose, raffinose, gentiobiose, D-arabitol, 6-ketogluconate, http://ijs.sgmjournals.org

inositol, methyl a-D-glucopyranoside, N-acetylglucosamine, arbutin, aesculin, citrate and salicin. Not utilized are L-rhamnose, D-arabinose, L-xylose, turanose, D-lyxose, Dtagatose, D-fucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate, erythritol, methyl b-D-xylopyranoside, dulcitol, methyl a-D-mannopyranoside, inulin, starch, glycogen and xylitol. The major quinones are Q-8 and MK-8. A small amount of DMK-8 is present. The type strain is 1GBT (5NBRC 109676T5KCTC 32430T), isolated from anodic biofilms of a microbial fuel cell fed with glucose as an electron donor. The DNA G+C content of the type strain is 54.5 mol% (determined by HPLC).

Acknowledgements We are grateful to Keiko Okamura for her guidance in genomic DNA–DNA hybridization. This study was supported by a Core Research for Evolutional Science and Technology (CREST) project from the Japanese Science and Technology Agency (JST) and a Grantin Aid for Scientific Research from the Japanese Society for the Promotion of Science (JSPS) (23656324). Z. K. was supported by a grant from the JSPS.

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