TAXONOMIC DESCRIPTION Zhou et al., Int J Syst Evol Microbiol 2018;68:1914–1918 DOI 10.1099/ijsem.0.002766 Propionicimonas ferrireducens sp. nov., isolated from dissimilatory iron(III)-reducing microbial enrichment obtained from paddy soil Guo-Wei Zhou,1,2 Xiao-Ru Yang,1,* Mohammed A. M. Wadaan,3 Wael N. Hozzein,3 Bang-Xiao Zheng,1,4 Jian-Qiang Su1 and Yong-Guan Zhu1,2 Abstract A novel strain, designated Y1A-10 4-9-1T, with Gram-stain-positive and rod-shaped cells, was isolated from paddy soil in Yingtan, Jiangxi, China. Cells were 0.15–0.2 µm wide and 1.5–3.3 µm long. The optimal growth temperature was 30 C and the optimal pH was 7.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain is closely related to Propionicimonas paludicola JCM 11933T (98.57 %). The genomic DNA G+C content was 63.9 mol%. The predominant menaquinone was MK-9(H4) and meso-diaminopimelic acid was present in the cell-wall peptidoglycan layer. The major polar lipids were diphosphatidylglycerol, one unidentified phospholipid and two unidentified lipids. The dominant cellular fatty acids T detected were anteiso-C15 : 0 and iso-C16 : 0. The phylogenetic and phenotypic results supported that strain Y1A-10 4-9-1 is a novel species of the genus Propionicimonas, for which the name Propionicimonas ferrireducens sp. nov. is proposed. The type strain is Y1A-10 4-9-1T (=CCTCC AB 2016249T=KCTC 15566T=LMG 29810T). The anaerobic microbial communities found in paddy soil propionate-producing group in the family Propionibacteria- have attracted wide interest from researchers, and ceae was fully characterized and is proposed as a new spe- several novel anaerobic micro-organisms have been isolated cies of the genus Propionicimonas in this study. and identified, including iron(III)-reducing bacteria, iron The paddy soil for the ferrihydrite incubation was collected (II)-oxidizing bacteria, methanogens, organic matter-hydro- from Yingtan (116 82¢ E, 28 2¢ N), Jiangxi Province, China. lysing micro-organisms and fermentative anaerobic micro- It is a typical soil found in Southern China, in which the organisms [1–9]. Iron(III) oxides, especially amorphous ferrihydrite, are abundant and exist ubiquitously in paddy acid and red soil is rich in Fe(III) (oxyhydr)oxide. Three grams of the paddy soil was suspended in 50 ml anoxic dis- soil environments [10]. Dissimilatory iron(III)-reducing bacteria (DIRB) and related organic matter-decomposing tilled water and shaken at 120 r.p.m. for 2 h at 25 C. Two bacteria use these Fe(III) oxides and organic matter [10, 11]. millilitres of the well-mixed slurry was inoculated into 50 ml serum vials containing 20 ml DIRB medium (sterilized and In order to analyse the composition of the culturable anaer- anoxic) and incubated at 25 C in the dark without shaking. – Á obic microbial community in the Fe(III) oxides-abundant The DIRB medium (pH 6.8 7.2) contained MgCl2 6H2O À1 Á À1 À1 paddy soil, anaerobic bacteria were previously isolated from (0.4 g l ), CaCl2 H2O (0.1 g l ), NH4Cl (0.027g l ), KH2 À1 À1 À1 a paddy soil enrichment incubated with ferrihydrite. Anaer- PO4 (0.6 g l ), 1 ml l vitamin solution, 1 ml l trace ele- À obic bacterial isolates from these incubations were phyloge- ment solution, 30 mmol l 1 bicarbonate, acetate (2 mmol À1 À1 netically affiliated on the basis of 16S rRNA gene sequences. l ) and ferrihydrite (10 mmol l ) and purged with N2/ The results indicated that Fe(III)-reducing bacteria predo- CO2 (80/20 %). The vitamin solution [12], trace element minated the microbial community and a minor proportion solution [12] and sodium acetate were added from stock of methanogens were detected from the ferrihydrite enrich- solutions sterilized with a 0.22 µm filter, respectively. Other ment [10]. Out of these bacterial isolates, one strain of the solutions were autoclaved for 20 min at 120 C. The Author affiliations: 1Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, PR China; 2State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China; 3Bioproducts Research Chair, Zoology Department, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia; 4University of Chinese Academy of Sciences, Beijing 100049, PR China. *Correspondence: Xiao-Ru Yang, [email protected] Keywords: Propionicimonas ferrireducens; taxonomy; dissimilatory iron(III)-reducing microbial enrichment; paddy soil. Abbreviations: DIRB, dissimilatory iron(III)-reducing bacteria; JCM, Japan Collection of Microorganisms; meso-DAP, meso-diaminopimelic acid; RP- HPLC, reversed-phase high-performance liquid chromatography; SEM, scanning electron microscopy; TLC, thin-layer chromatography. Five supplementary figures are available with the online version of this article. 002766 ã 2018 IUMS Downloaded from www.microbiologyresearch.org by IP: 210.72.152.171914 On: Tue, 21 May 2019 02:26:06 Zhou et al., Int J Syst Evol Microbiol 2018;68:1914–1918 ferrihydrite was synthesized as before [13]. The ferrihydrite Spin Kit (MP Biomedical) according to the manufacturer’s was almost completely reduced and the enrichments were instructions. The 16S rRNA genes of the strains were PCR- transferred (10 %) to fresh media monthly for four genera- amplified using universal primers (27F and 1492R) [14] and tions. To isolate the iron reduction-related bacteria, the then sequenced. The 16S rRNA gene sequences were aligned DIRB enrichment was serially diluted to 10000-fold and against sequences of related taxa by using the EzBioCloud 50 µl was spread onto DIRB agar plates (agar, 2 %; w/v) (www.ezbiocloud.net; [15]) Phylogenetic analyses with the neighbour-joining, minimum-evolution and maximum- under anaerobic conditions (Bactron IV, Shel Lab; N2 : CO2 T likelihood methods (bootstrap values based on 1000 replica- :H2,90 : 5 : 5). Strain Y1A-10 4-9-1 was one of the growing colonies on the DIRB plates. A single colony was selected tions) were calculated with MEGA version 6.0 based on the aligned 16S rRNA gene sequences using CLUSTAL_X [16, 17]. and inoculated onto a fresh DIRB agar plate to purify a col- T ony-forming strain. This procedure was repeated three The results showed that strain Y1A-10 4-9-1 shared over times to ensure reliable colony purification for further work. 95 % 16S rRNA gene sequence similarity to various genera T of the family Propionibacteriaceae (Friedmanniella, Nau- Strain Y1A-10 4-9-1 was also observed to grow in the mannella, Microlunatus, Micropruina, Propionicicella and DIRB agar medium without ferrihydrite addition after Propionicimonas) (Fig. 1). The closest phylogenetic relatives 3 days at 25 C. were P. paludicola DSM 15597T (98.57 %) and M. glycogen- Propionicimonas paludicola JCM 11933T and Micropruina ica Lg2T (96.65 %). The phylogenetic tree based on the glycogenica JCM 10248T were purchased from the Japan neighbour-joining method suggested that strain Y1A-10 4- Collection of Microorganisms (JCM) and used as reference 9-1T was affiliated to the genus Propionicimonas (Fig. 1). strains. Total genomic DNA of strain Y1A-10 4-9-1T and The results from the maximum-likelihood and minimum- the two reference strains was extracted using a FastDNA evolution methods also supported the affiliation of strain 81 Friedmanniella spumicola ACM 5121T (AF062535) 86 Friedmanniella antarctica DSM 11053T (Z78206) 97 Friedmanniella sagamiharensis FB2T (AB445456) Friedmanniella okinawensis FB1T (AB445455) 64 T 50 Friedmanniella flava CGMCC 4.6856 (jgi.1085040) Friedmanniella lucida FA2T (AB445454) 82 Friedmanniella luteola DSM 21741T (LT629749) 97 0.02 Friedmanniella lacustris EL-17AT (AJ132943) 81 94 T 57 Friedmanniella capsulata Ben 108 (AF084529) Friedmanniella aerolata 7515T-26T (KR869781) 63 Friedmanniella endophytica 4Q3S-3T (KU168417) Microlunatus panaciterrae Gsoil 954T (AB271051) Microlunatus soli DSM 21800T (LT629772) 70 Microlunatus ginsengisoli Gsoil 633T (AB245389) 77 Microlunatus phosphovorus NM-1T (AP012204) 79 T 100 Microlunatus aurantiacus YIM 45721 (EF601828) Auraticoccus monumenti MON 2.2T (LT629688) Aestuariimicrobium kwangyangense DSM 21549T (ATXE01000005) Luteococcus peritonei CCUG 38120T (AJ132334) 84 Brooklawnia cerclae BL-34T (DQ196625) 52 81 Tessaracoccus oleiagri CGMCC 1.9159T (jgi.1058080) 88 Tessaracoccus flavus RP1T (FNPU01000023) 93 Tessaracoccus rhinocerotis YIM 101269T (KT215777) 63 T 95 Tessaracoccus flavescens SST-39 (AM393882) Naumannella halotolerans WS4616T (FR832425) Naumannella huperziae CPCC 204135T (KR184192) 99 T 100 Naumannella cuiyingiana AFT2 (KF041479) Propionicicella superfundia DSM 22317T (KE384022) Micropruina glycogenica Lg2T (AB012607) 98 Propionicimonas ferrireducens LMG 29810T (MG775313) 67 T 84 Propionicimonas paludicola DSM 15597 (FR733712) Escherichia coli ATCC 11775T (NZKK583188) Fig. 1. Neighbour-joining tree showing the phylogenetic position of strain Y1A-10 4-9-1T within related genera of the family Propioni- bacteriaceae based on 16S rRNA sequences. Filled circles indicate that the corresponding nodes are recovered in the trees generated with the maximum-likelihood and minimum-evolution methods. Bootstrap values (%) are shown on nodes in bootstrap analysis of 1000 replicates. Bar, 2 nucleotide substitutions per 100 nucleotide positions. Bootstrap values above 50 % given at the node. The sequence of Escherichia coli was used as an outgroup. Downloaded from www.microbiologyresearch.org by IP: 210.72.152.171915 On: Tue, 21 May 2019 02:26:06 Zhou et al., Int J Syst Evol Microbiol 2018;68:1914–1918 Y1A-10 4-9-1T (Fig. S1, available in the online version of The Gram reaction to the strains was tested by using 3 % this article). KOH [20]. Gram staining showed that strain Y1A-10 4-9-1T T was Gram-stain-positive. Cell morphology was examined by The DNA G+C content of strain Y1A-10 4-9-1 determined by reversed-phase high-performance liquid chromatography using the LabRAM Aramis confocal Raman microscope (RP-HPLC) [18] was 63.9 mol%. This value is lower than (HORIBA) with an integrated Olympus BXFM microscope the G+C value for other related species (Table 1).