Lactivibrio Alcoholicus Gen. Nov., Sp. Nov., an Anaerobic, Mesophilic, Lactate-, Alcohol-, Carbohydrate- and Amino-Acid-Degradin

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International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2137–2145 DOI 10.1099/ijs.0.060681-0

Lactivibrio alcoholicus gen. nov., sp. nov., an anaerobic, mesophilic, lactate-, alcohol-, carbohydrate- and amino-acid-degrading bacterium in the phylum Synergistetes

Yan-Ling Qiu,1,2 Satoshi Hanada,3 Yoichi Kamagata,4 Rong-Bo Guo1 and Yuji Sekiguchi2

Correspondence 1Shandong Industrial Engineering Laboratory of Biogas Production & Utilization, Yan-Ling Qiu Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, [email protected] Chinese Academy of Sciences, Qingdao, Shandong Province 266101, PR China 2Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan 3Bioprocess Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan 4Bioprocess Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido 062-8517, Japan

A mesophilic, obligately anaerobic, lactate-, alcohol-, carbohydrate- and amino-acid- degrading bacterium, designated strain 7WAY-8-7T, was isolated from an upflow anaerobic sludge blanket reactor treating high-strength organic wastewater from isomerized sugar production processes. Cells of strain 7WAY-8-7T were motile, curved rods (0.7–1.0¾5.0–8.0 mm). Spore formation was not observed. The strain grew optimally at 37 6C (range for growth was 25–40 6C) and pH 7.0 (pH 6.0–7.5), and could grow fermentatively on yeast extract, glucose, ribose, xylose, malate, tryptone, pyruvate, fumarate, Casamino acids, serine and cysteine. The main end-products of glucose fermentation were acetate and hydrogen. In co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T, strain 7WAY-8-7T could utilize lactate, glycerol, ethanol, 1-propanol, 1-butanol, L-glutamate, alanine, leucine, isoleucine, valine, histidine, asparagine, glutamine, arginine, lysine, threonine, 2-oxoglutarate, aspartate and methionine. A Stickland reaction was not observed with some pairs of amino acids. Yeast extract was required for growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite and Fe (III) were not used as terminal electron acceptors. The G+C content of the genomic DNA was 61.4 mol%. 16S rRNA gene sequence analysis revealed that the isolate belongs to the uncultured environmental clone clade (called ‘PD-UASB-13’ in the Greengenes database) in the bacterial phylum Synergistetes, showing less than 90 % sequence similarity with closely related described species such as Aminivibrio pyruvatiphilus and Aminobacterium colombiense (89.7 % and 88.7 %, respectively).

The major cellular fatty acids were iso-C13 : 0, iso-C15 : 0, anteiso-C15 : 0,C18 : 1,C19 : 1,C20 : 1 and

C21 : 1. A novel genus and species, Lactivibrio alcoholicus gen. nov., sp. nov. is proposed to accommodate strain 7WAY-8-7T (5JCM 17151T5DSM 24196T5CGMCC 1.5159T).

The bacterial phylum Synergistetes was proposed recently, ‘subdivision A’ has been formally described as the class being subdivided into five major lines of descent as order- Synergistia (Jumas-Bilak et al., 2009). Based on culture- level lineages (i.e. ‘subdivisions A–E’), of which only dependent and culture-independent surveys, members of the phylum Synergistetes have been found in a wide range Abbreviations: FAME, fatty acid methyl ester; UASB, upflow anaerobic of anoxic ecosystems, such as anaerobic wastewater treat- sludge blanket. ment systems, soil, and gastrointestinal tracts (Vartoukian The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene et al., 2007). Members of this phylum have also been found sequence of strain 7WAY-8-7T is AB558582. in specimens from periodontal disease, gastrointestinal One supplementary figure is available with the online version of this paper. infections, genital tract and soft tissue infections, suggesting

060681 G 2014 IUMS Printed in Great Britain 2137 Y.-L. Qiu and others that members of the phylum play a possible pathogenic role Greengenes database; McDonald et al., 2012) in the phylum (Horz et al., 2006; Vartoukian et al., 2007). Synergistetes ‘subdivision E’. Despite its habitat diversity, cultured strains representing Strain 7WAY-8-7T was originally obtained from the the phylum Synergistetes characterized to date largely share granular sludge of a mesophilic (35 uC) full-scale upflow similar phenotypic traits with one another. To date, thirteen anaerobic sludge blanket reactor (UASB) treating a high- genera with twenty-two species with validly published strength organic wastewater from isomerized sugar pro- names have been cultivated and characterized in the phylum duction processes (Narihiro et al., 2009). The medium used Synergistetes: all of the described species within this phylum for isolation and cultivation was prepared as described are strictly anaerobic, neutrophilic, Gram-negative rods that previously (Sekiguchi et al., 2000). Gently washed and (mostly) specifically ferment amino acids. The characteristic homogenized sludge was serially diluted 10-fold in anaer- of amino acid fermentation is also supported by represent- obic liquid medium supplemented with low concentrations ative genomes of members of the phylum Synergistetes, for of acetate (1 mM) and yeast extract (0.01 %, w/v) and which a greater average proportion of amino acid transport pH 7.0. Growth of cells was observed in the 1028 dilution and metabolism genes (COG functional category E) has tube after 1 month of incubation at 37 uC. Cells in the been predicted than for any bacterial phylum to date highest dilution were further purified by repeated serial (Hugenholtz et al., 2009). Carbohydrate fermentation dilution in glucose (1 mM) and yeast extract (0.01 %, w/v) is exceptionally exhibited by a few cultured species in medium, and then in the same agar roll tubes (2 %, w/v; the phylum Synergistetes, such as Thermanaerovibrio velox Difco Noble agar). Small, light brown, lens-shaped colonies, (Zavarzina et al., 2000) and Anaerobaculum spp. (Rees et al., were formed after 2–3 weeks of incubation at 37 uC. This 1997; Menes & Muxı´, 2002). These observations based on roll tube isolation step (transferring single colonies from cultured members of the phylum Synergistetes suggested solid medium to liquid medium) was repeated several times that members of the phylum are specialists with relatively and a purified strain, designated strain 7WAY-8-7T was shallow ecophysiological niches (Godon et al., 2005). obtained. However, recent cultivation and culture-independent studies Cell morphology was examined under a fluorescent micro- are highlighting the presence of more versatile metabolic scope (Olympus BX50F). Transmission electron micro- capabilities of members of the phylum Synergistetes.Recently, scopy was performed with a Hitachi H-7000 transmission a novel rumen bacterium within the phylum Synergistetes was electron microscope as described previously (Sekiguchi described that metabolizes fluoroacetate under anaerobic et al., 2003). The Gram staining reaction was performed by conditions, indicating the presence of reductive dehalogena- the method of Hucker (Doetsch, 1981). Cells were motile, tion capability for growth in the phylum Synergistetes (Davis curved rods, 0.7–1.0 mm wide and 5.0–8.0 mm in length et al., 2012). In addition, Delbe`s et al. (2001) reported a (Fig. 1). Cells became spirilloid in ageing cultures. Cells as marked increase in 16S rRNA of uncultured members of long as 15.0 mm were observed. Spore formation was not the phylum Synergistetes (AA56) following the addition of observed and Gram-staining was negative. lactate under anoxic conditions, indicating the involvement The physiological traits of strain 7WAY-8-7T were examined of members of the phylum Synergistetes in anaerobic lactate as described previously (Sekiguchi et al., 2000). Growth was degradation. Ito et al. (2011) suggested that an uncultured determined at 20–55 uC (at intervals of 5 uC), at pH 5.0–8.5 group of the phylum Synergistetes (named ‘Synergistes group (at intervals of 0.5 pH unit; 37 uC) and with 0–3.0 % (w/v) 4 anaerobic digester’) was one of the major acetate-utilizing NaCl (at intervals of 0.5 %). The growth of cells was taxa in an anaerobic digester, presumably indicating its evaluated on the basis of the increase in optical density at syntrophic acetate-oxidation capability with hydrogeno- 400 nm and the production of hydrogen. Unless otherwise trophic methanogens. However, no cultured strains repre- indicated, the organism was cultured anaerobically (N / senting the phylum Synergistetes capable of performing such 2 CO , 80 : 20, v/v) at 37 uC without shaking. Aerobic growth metabolism have been obtained so far, hampering further 2 was tested in a medium containing 2 mM glucose and confirmation of these additional metabolic traits of the 0.01 % (w/v) yeast extract under aerobic conditions without phylum Synergistetes. the addition of reducing agents. Strain 7WAY-8-7T grew To further explore and confirm the ecophysiological roles of anaerobically on glucose/yeast extract medium after 8 weeks members of the phylum Synergistetes besides amino acid of incubation at 25–40 uC (optimum, 37 uC), at pH 6.0–7.5 fermentation, we aimed at isolating members of the phylum (optimum, approximately pH 7.0) and with 0–2.0 % (w/v) Synergistetes that exhibit broader metabolic capabilities. NaCl, but did not grow at temperatures below 25 uCor Through these attempts, an anaerobic bacterium (strain above 40 uC. The isolate was a strictly anaerobic organism: it 7WAY-8-7T) belonging to the phylum Synergistetes was could not grow in the presence of oxygen (20 %, v/v, in the obtained that can anaerobically degrade lactate and alcohols gas phase) nor after N2/CO2 purging only (without addition in syntrophic association with hydrogenotrophs. Based of reducing agents). Yeast extract was strictly required for on the 16S rRNA gene phylogeny, strain 7WAY-8-7T growth. Strain 7WAY-8-7T could utilize sugars, lactate, was found to represent the uncultured clade ‘Synergistes amino acids and primary alcohols in pure culture or in group 4 anaerobic digester’ (named ‘PD-UASB-13’ in the syntrophic association with a methanogen. In the presence

2138 International Journal of Systematic and Evolutionary Microbiology 64 Lactivibrio alcoholicus gen. nov., sp. nov.

(a) 0.9 mol hydrogen; electron recovery: 98 %). In the presence of a hydrogenotrophic methanogen, Methanospirillum hungatei DSM 864T, growth of co-culture and methane production were observed with the following substrates (5 mM each): lactate, glycerol, ethanol, 1-propanol, 1-butanol, and amino acids such as glutamate, alanine, leucine, isoleucine, valine, histidine, asparagine, glutamine, arginine, lysine, threonine, 2-oxoglutarate, aspartate and methionine. None of the following substrates supported the growth of strain 7WAY-8-7T in pure culture or co-culture with M. hungatei (5 mM unless specified): crotonate, H2/ (b) CO2 (1 atm, 80 : 20, v/v) plus acetate, acetate, propionate, butyrate, iso-butyrate, succinate, methanol, fructose, maltose, arabinose, galactose, mannose, raffinose, sucrose, starch (1 g l21), pectin, citrate, glycine, proline, phenylalanine, phenol (1 mM), hydroquinone (2 mM) or benzoate (2 mM). Table 1 shows several typical substrates, i.e. ethanol, lactate, alanine and glutamate used and the end products formed by strain 7WAY-8-7T in the presence and absence of a hydrogen-consuming methanogen. In pure culture, ethanol and lactate were partially fermented, hydrogen accumulated at approximately 0.02 atm (2 kPa) and 0.05 atm (5 kPa) during lactate and ethanol degrada- tions, respectively, and the accumulated hydrogen may Fig. 1. Phase-contrast micrograph (a) and thin-section electron have inhibited further fermentation. Similarly, only a minor micrograph (b) of strain 7WAY-8-7T in pure culture grown on fraction of alanine and glutamate were fermented in pure glucose/yeast extract. Bars, 10 mm (a) and 1.0 mm (b). culture of strain 7WAY-8-7T, where the hydrogen partial pressure rapidly reached at 1.7 kPa and 1.8 kPa, respectively, and then further fermentation was stopped. In the of yeast extract (0.01 %, w/v), growth of pure culture and presence of the hydrogenotrophic methanogen M. hungatei, hydrogen production were observed with the following 10 mM ethanol was degraded completely and transformed substrates (at 5 mM unless indicated) under optimum into acetate and methane within 10 days of incubation. conditions (37 uC, pH 7.0): yeast extract (0.05 %, w/v), Lactate and alanine were degraded relatively slowly to form glucose, ribose, xylose, malate, tryptone (0.05 %, w/v), acetate and methane; glutamate was degraded to acetate, pyruvate, fumarate, Casamino acids (0.05 %, w/v), serine propionate and methane by the syntrophic co-culture in and cysteine. In the medium supplemented with glucose, 1–2 months of incubation (Table 1). The oxidation and the major end-products were acetate and hydrogen (1 mol reduction of amino acids via the Stickland reaction was glucose was converted to approximately 2.7 mol acetate and not observed in a basal medium containing alanine as an

Table 1. Ethanol, lactate, glutamate and alanine degradation by strain 7WAY-8-7T

Culture Substrate (mM) Products formed (mM) Degradability (%) Incubation condition time (days) Initial concn Degraded Acetate Propionate Hydrogen*

Pure culture Ethanol 9.5 2.8 4.8 0 6.4 29 30 Lactate 10.9 2.1 1.6 0 2.0 19 30 Alanine 9.7 1.1 2.1 0 1.9 11 60 Glutamate 5.7 0.6 1.6 0.6 2.0 10 60 Acetate Propionate Methane* Co-culture with Ethanol 8.9 8.9 9.6 0 8.3 100 30 M. hungatei Lactate 7.7 4.3 4.1 0 7.9 56 30 Alanine 9.2 3.4 4.5 0 4.3 37 60 Glutamate 5.6 3.0 1.4 4.5 6.8 53 60

The medium contained 0.01 % (w/v) yeast extract. *Values are expressed as mmol of methane or hydrogen formed in 1 l of culture. http://ijs.sgmjournals.org 2139 Y.-L. Qiu and others electron donor and glycine, arginine, serine or proline analysis was run with 100 bootstrap replicates. A total of (10 mM) as an electron acceptor, respectively. Strain 1443 base pairs of the 16S rRNA gene from strain 7WAY-8- 7WAY-8-7T did not use any of the following electron 7T were obtained. Phylogenetic analysis revealed that strain acceptors within 6 weeks of incubation on glucose/yeast 7WAY-8-7T was affiliated with the phylum Synergistetes extract medium (mM): nitrate (5), sulfate (5), thiosulfate ‘subdivision E’, in which the three genera Aminivibrio, (2), elemental sulfur (5), Fe (III) nitrilotriacetate (2) or Aminobacterium and Fretibacterium are classified (Jumas- sulfite (1). Bilak et al., 2009; Vartoukian et al., 2013; Honda et al., 2013). Strain 7WAY-8-7T fell into the uncultured group For DNA G+C content determination, DNA was extracted Synergistetes ‘PD-UASB-13’ (Hugenholtz et al., 2009), with and purified according to Kamagata & Mikami (1991). The DNA G+C content was determined by HPLC (Shimadzu 97 % sequence similarity with environmental 16S rRNA LC-6A) with a UV detector (Shintani et al., 2000). The DNA gene sequence PD-UASB-13 (accession no. AY261810), which was retrieved from an ambient UASB reactor G+C content was 61.4 mol% (SD: ±0.15 mol%). For fatty acid analysis and polar lipids analysis, cells were harvested degrading phenol wastewater (Zhang et al., 2005). The most at exponential phase from cultures grown anaerobically closely related micro-organisms cultured and characterized on 2 mM glucose and 0.01 % (w/v) yeast extract at 37 uC. so far were Aminivibrio pyruvatiphilus (89.7 % 16S rRNA Fatty acids of cells were converted to methyl esters (FAMEs) gene sequence similarity), an amino acid- and organic acid- using HCl/methanol and identified by GC-MS (Hitachi degrading bacterium isolated from rice field soil (Honda M7200AFC/3DQMS system) (Hanada et al., 2002). Strain et al., 2013), and Aminobacterium colombiense (88.7 %), an 7WAY-8-7T contained a complex FAME profile, the major anaerobic amino acid-degrading bacterium isolated from an anaerobic wastewater treatment plant (Baena et al., 1998; cellular fatty acids were iso-C13 : 0 (13.9 %), iso-C15 : 0 2000) (Fig. 2). Because members of the ‘PD-UASB-13’ lineage (10.0 %), anteiso-C15 : 0 (9.8 %), C18 : 1 (7.1 %), C19 : 1 formed a robustly monophyletic group in all the phylogeny (8.5 %), C20 : 1 (7.8 %) and C21 : 1 (9.3 %). Polar lipids were extracted and analysed by the methods of Tindall (1990) inference analyses exhibiting an independent genus level clade from other known taxa, we concluded that the strain by using two-dimensional TLC (Merck no. 5554 silica gel T 60 F254 plates, layer thickness 0.2 mm). The polar lipids 7WAY-8-7 represents a new taxon at least at the genus level profile of strain 7WAY-8-7T consisted of six unknown in the ‘subdivision E’ of the phylum Synergistetes (Fig. 2). phospholipids, four unknown aminophospholipids, three Comparative phenotypic analyses of strain 7WAY-8-7T unknown polar lipids, and one unknown aminolipid (Fig. and closely related genera are shown in Table 2. Among the S1, available in the online Supplementary Material). known members of the phylum Synergistetes, strain 7WAY- T For 16S rRNA gene sequencing, the genomic DNA of 8-7 exhibited the most versatile metabolic capability, strain 7WAY-8-7T was extracted according to the method of such as degradation of carbohydrates, amino acids, lactate Hiraishi (1992). 16S rRNA genes were amplified by PCR and alcohols (Table 2). Similar to the other described T with Taq polymerase (Promega). The PCR primers used members of the phylum Synergistetes, strain 7WAY-8-7 in the amplification were the bacterial domain universal was a Gram-stain-negative, strictly anaerobic, rod-shaped, primer 8F and the prokaryote universal primer 1490R amino acids-degrading bacterium. Phenotypically, strain T (Weisburg et al., 1991). The PCR product was sequenced 7WAY-8-7 was most similar to Aminivibrio pyruvatiphilus directly on a Beckman CEQ-8000 DNA sequencer using a and Aminobacterium spp. in the same ‘subdivision E’ CEQ DTC quick start kit (Beckman Coulter). 16S rRNA (Baena et al., 1998, 2000; Honda et al., 2013). They shared gene sequences of interest were aligned with the Greengenes several common traits as follows: they are mesophilic alignment data (McDonald et al., 2012) with PyNAST anaerobes and can degrade amino acids by fermentation or (Caporaso et al., 2010), imported in ARB, and the alignments syntrophic association with hydrogen-consuming metha- T were manually corrected in the ARB EDIT tool. For recon- nogens. Furthermore, strain 7WAY-8-7 showed a similar structing the alignment data, representative taxa (with DNA G+C content and FAME profile to Aminivibrio sequences .1300 nt) were selected and their alignment data pyruvatiphilus: both have relatively high DNA G+C T were exported from ARB with Lane mask filtering. A base content (61.4 mol% for strain 7WAY-8-7 ; 61.9 mol% tree was made based on the masked alignments with the for Aminivibrio pyruvatiphilus) and contain a complex neighbour-joining method (Saitou & Nei 1987) implement- FAME profile, with straight- and branched-chain, saturated ed in the ARB program (Ludwig et al., 2004). Bootstrap and unsaturated FAMEs (Honda et al., 2013). Our study neighbour-joining trees were calculated from the masked corroborates findings in other studies of Cloacibacillus alignments with LogDet distance estimation using PAUP*4.0 evryensis, Synergistes jonesii and Pyramidobacter piscolens (Swofford, 2003) with 100 bootstrap resampling. Maximum- that the FAME profiles of members of the phylum likelihood trees were calculated using RAxML v7.7.8 Synergistetes can exhibit significant variation and comple- (Stamatakis, 2006) (GTR and Gamma models+I) with xity (Ganesan et al., 2008; Downes et al., 2009) (Table 2). rapid 100-time bootstrapping. Maximum-parsimony trees However, strain 7WAY-8-7T is clearly distinct from Amini- were calculated using PAUP*4.0. An heuristic search was used vibrio pyruvatiphilus and species of the genus Aminobac- with a random stepwise addition sequence of 10 replicates terium in terms of cell size, and lactate, carbohydrates and nearest-neighbour-interchange swapping. A further and alcohol degradation. Among members of the phylum

2140 International Journal of Systematic and Evolutionary Microbiology 64 Lactivibrio alcoholicus gen. nov., sp. nov.

UASB sludge clone PD−UASB−13, AY261810 Anaerobic digester sludge clone QEDN10DG10, CU925137 Lactivibrio T Lactivibrio alcoholicus 7WAY−8−7 , AB558582 (PD-UASB-13 group) Anaerobic digester sludge clone 11SN, EU887774 Aminobacterium colombiense DSM 12261T, AF069287 Subdivision E 0.02 Aminobacterium mobile DSM 12262T, AF073521 Aminivibrio pyruvatiphilus DSM 25964T, AB623229 Fretibacterium fastidiosum DSM 25557T, GQ149247 Dethiosulfovibrio acidaminovorans DSM 12590T, AY005466 Dethiosulfovibrio marinus DSM 12537T, AF234544 Dethiosulfovibrio russensis DSM 12538T, AF234542 Dethiosulfovibrio peptidovorans DSM 11002T, U52817 Subdivision B Dethiosulfovibrio salsuginis DSM 21565T, EU719657 Jonquetella anthropi ADV 126T, EF436500 Pyramidobacter piscolens DSM 21147T, EU309492 Thermovirga lienii DSM 17291T, DQ071273 Subdivision D1 Anaerobic fluoroacetate-degrading bacterium strain MFA1, GQ291322 Cloacibacillus porcorum DSM 25858T, JQ809697 Cloacibacillus evryensis DSM 19522T, CU463952 Synergistes jonesii ATCC 49833T, L08066 Synergistia Thermanaerovibrio acidaminovorans DSM 6589T, AF071414 (Subdivision A) Thermanaerovibrio velox DSM 12556T, AF161069 Aminomonas paucivorans DSM 12260T, AF072581 Aminiphilus circumscriptus DSM 16581T, AY642589 Subdivision D2 Anaerobaculum mobile DSM 13181T, AJ243189 Acetomicrobium flavidum DSM 20664T, FR733692 Subdivision C Anaerobaculum thermoterrenum DSM 13490T, U50711 Anaerobaculum hydrogeniformans DSM 22491T, FJ862996

Fig. 2. Neighbour-joining phylogenetic inference of strain 7WAY-8-7T based on 16S rRNA gene sequence. The tree was inferred on the basis of 16S rRNA gene sequences from the Greengenes database and a sequence obtained in this study. 16S rRNA gene sequences of Geovibrio ferrireducens ATCC 51996T (accession no. X95744), Deferribacter abyssi DSM 14873T (AJ515881) and Chrysiogenes arsenatis DSM 11915T (X81319) were used to root the tree. The phylogenetic robustness of each node is indicated by a symbol on the node: filled circle (node was resolved in .90 % of all the tree calculations including neighbour-joining, maximum-parsimony and maximum-likelihood inferences); open circle (resolved in .80 % of all the calculations); open square (resolved in .70 % of all the calculations). Bar, 2 % estimated sequence divergence. Subphylum (class)-level clades are bracketed on the right.

Synergistetes, those of some genera such as Anaerobaculum lactate and primary alcohols in syntrophic association with and Thermanaerovibrio can utilize carbohydrates fermenta- hydrogenotrophic methanogens is a notable feature of strain tively, but they cannot degrade alcohols and lactate under 7WAY-8-7T, because these compounds are often important anoxic conditions. Strain 7WAY-8-7T also differs from the intermediates in anaerobic decomposition of organic matter genera Anaerobaculum and Thermanaerovibrio in temperature in the system (Salminen & Rintala, 2002; Schink & Stams, range for growth; members of the genera Anaerobaculum and 2006). However, strain 7WAY-8-7T does not exhibit syntro- Thermanaerovibrio are thermophiles (Rees et al., 1997; Baena phic acetate oxidation with hydrogenotrophic methanogens T et al., 1999), whereas strain 7WAY-8-7 is a mesophile. These as suggested for some ‘PD-UASB-13’ members by Ito observations suggested significant differences not only in et al. (2011). This may be because strain 7WAY-8-7T is phylogeny but also in phenotypic features for strain 7WAY-8- phylogenetically distant from a previously suggested acetate- T 7 from others in the phylum Synergistetes, further justifying utilizing phylotype (88 % 16S rRNA gene sequence similar- creation of a novel species of a new genus. ity with clone RSg13-6, AB603841), suggesting differences in Strain 7WAY-8-7T is the first cultured member of the phenotypic features between them. phylum Synergistetes to exhibit anaerobic lactate and In conclusion, this study further expands the metabolic primary alcohol degradation, further supporting the asso- capability of members of the phylum Synergistetes.Onthe ciation of the phylum Synergistetes with lactate degradation basis of the phylogenetic, genetic and physiological pro- in anoxic ecosystems as suggested previously (Delbe`s et al., perties, it is evident that strain 7WAY-8-7T should be 2001). Members of the phylum Synergistetes have been classified as a representative of a novel species of a novel detected frequently in anaerobic digesters treating municipal genus in the phylum Synergistetes ‘subdivision E’. Therefore, wastewater sludge (Chouari et al., 2005; Godon et al., 1997, we propose the name Lactivibrio alcoholicus gen. nov., sp. nov. 2005), and anaerobic sludge treating wastewater (Narihiro et al., 2009). However, the ecophysiological function of these Description of Lactivibrio gen. nov. members of the phylum Synergistetes in the anaerobic ecosystems is poorly understood. In regard to ecophysiolo- Lactivibrio (Lac.ti.vi9bri.o. L. neut. n. lact, lactis milk; N.L. gical roles in these treatment systems, the ability to degrade masc. n. vibrio vibrio, that which vibrates, the vibrating, http://ijs.sgmjournals.org 2141 2142 others and Qiu Y.-L.

Table 2. Characteristics of strain 7WAY-8-7T and members of the related genera in the phylum Synergistetes

Taxa: 1, Strain 7WAY-8-7T (this study); 2, Aminobacterium spp. (data from Baena et al., 1998, 2000); 3, Aminivibrio pyruvatiphilus DSM 25964T (Honda et al., 2013); 4, Fretibacterium fastidiosum DSM 25557T (Vartoukian et al., 2013); 5, Aminomonas paucivorans DSM 12260T (Baena et al., 1999); 6, Cloacibacillus evryensis DSM 19522T (Ganesan et al., 2008); 7, Synergistes jonesii ATCC 49833T (Allison et al., 1992; Ganesan et al., 2008); 8, Thermanaerovibrio velox DSM 12556T (Zavarzina et al., 2000); 9, Dethiosulfovibrio acidaminovorans DSM 12590T (Surkov et al., 2001; Jumas- Bilak et al., 2007); 10, Jonquetella anthropi CIP 109408T (Jumas-Bilak et al., 2007); 11, Pyramidobacter piscolens DSM 21147T (Downes et al., 2009); 12, Anaerobaculum mobile DSM 13181T (Menes & Muxı´, 2002); 13, Aminiphilus circumscriptus DSM 16581T (Dı´az et al., 2007); 14, Thermovirga lienii DSM 17291T (Dahle & Birkeland, 2006). None of the strains perform amino acid degradation via the Stickland reaction, although some strains were not determined. +, Compound metabolized; 2 not metabolized; ±, poor growth; ND, not determined.

Characteristic Subdivision E Class Synergistia (subdivision A) Subdivision B Subdivision Subdivision D C

12 3 4 5 67 8 91011121314

Morphology Curved Straight/ Curved Curved Curved Straight Rods Curved Curved Straight Bacilli Straight Curved Straight rods curved rods rods rods rods rods rods rods rods rods rods rods Motility ++/2 ++ 222++–– +++ Temp. range 25–40 20–42 20–42 25–42 20–40 20–50 39 45–70 15–40 37–42 25–42 35–65 25–55 37–68 (optimum, uC) (37) (37) (37-40) (37) (35) (37) (60–65) (28) (37) (30–37) (55–60) (42) (58) pH range 6.0–7.5 6.6–8.5 6.4–8.4 6.0–8.0 6.7–8.3 6.5–10.0 ND 4.5–8.0 5.5–8.0 6.8–7.0 ND 5.4–8.7 5.5–8.5 6.2–8.0 (optimum) (7.0) (7.3) (7.3) (7.0) (7.5) (7.0) (7.3) (6.5–7.0) (6.6–7.3) (7.1) (6.5–7.0) Requirement for + + + + + – ND +++ND + + + yeast extract DNA G+C 61.4 44–46 61.9 63 43 55.8 58 54.6 51 59.4 59 51.5 52.7 46.6 content

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Major cellular iso-C13 : 0 , iso-C15 : 0 iso-C13 : 0 , C14 : 0 , ND iso-C15 : 0 , C17 : 0 , ND iso-C15 : 0 , iso-C15 : 0 , C13 : 0 , ND ND ND fatty acids iso-C15 : 0 , iso-C15 : 0 , C18 : 0 , iso-C15 : 0 C17 : 1 v6c, C18 : 0 C16 : 0 C14 : 0 anteiso- C19 : 1 C16 : 0 3-OH, C20 cyc C15 : 0 , C17 : 1 v6c C18 : 1 , C19 : 1 , C20 : 1 , C21 : 1 Substrate utilization Carbohydrates +* 22222ND +* 222+* 22 Lactate + 22ND 2 ND ND 22ND ND 222 Ethanol + 22ND 22ND 22ND ND ND 22 Glycerol + 22ND 22ND 222ND +* +* 2 Yeast +* ND ND +* ++* ND +* +* +* +* +* +* +* Tryptone/ +* ±* ±* +* ±* +* ND +* +* ND +* +* +* +* peptone Pyruvate +* +* +* ND 2 ND ND 2 +* ND ND +* +* +* Fumarate +* 2 + ND 22ND ND 2 ND ND 2 +* 2 Malate +* 2 ±* ND 2 ND ND 2 +* ND ND +* +* 2 Casamino acids +* ±* ±* ND ±* +* +* +* +* ND ND +* +* +* Cysteine +* ±* +* ND ±* ±* 2 ND +* ND 2 ND 2 ND + + + + + + + 64 Serine * * * ND 2 * ND * * ND ND ND 2 * Lactivibrio alcoholicus gen. nov., sp. nov.

darting organism; N.L. masc. n. Lactivibrio a vibrio that degrades lactic acid). + + + + + ND Oil reservoir

respiratory Strictly anaerobic, mesophilic, non-spore-forming, Gram- stain-negative, motile, curved rods. Yeast extract is required for growth. Able to utilize carbohydrates, alcohols, lactate, * * * * Subdivision D – Sulfur, cystine 2 22 2 pyruvate, fumarate and amino acids. Oxygen, nitrate, + + + + sludge

Anaerobic sulfate, sulfite, thiosulfate, elemental sulfur and Fe (III) Fermentative Fermentative, nitrilotriacetate do not serve as electron acceptors for growth. The type species is Lactivibrio alcoholicus. C 2 + ND ND ND sludge respiratory thiosulfate, Anaerobic

sulfur, cysteine Description of Lactivibrio alcoholicus sp. nov. Lactivibrio alcoholicus (al.co.ho9li.cus. N.L. n. alcohol * 2 2

+ alcohol; L. suff. -icus suffix used with the sense of pertaining Human oral cavity to; N.L. masc. adj. alcoholicus belonging to alcohol, referring to the substrate alcohols, which are metabolized by the species). – – Crotonate, 2 NDND ND ND ND ND ND sample clinical Human Exhibits the following properties in addition to those given Fermentative Fermentative Fermentative, in the genus description. Cells are motile, curved rods (0.7– 6 m

* 1.0 5.0–8.0 m). Gram-stain-negative. Colonies on agar 222 2 2 + + mats sulfur

Sulfur are light brown and oval shaped after cultivation at 37 uC respiratory

Fermentative, for 2–3 weeks. Grows at 25–40 uC (optimum 37 uC), at pH 6.5–7.5 (optimum pH 7.0) and with 0–2.0 % (w/v)

* NaCl. Utilizes some carbohydrates, primary alcohols, 2 mat ND ND +

Sulfur Thiosulfate, lactate and various amino acids for growth. Ferments yeast respiratory

Cyanobacterial extract, glucose, ribose, xylose, malate, tryptone, pyruvate, fumarate, Casamino acids, serine and cysteine. The main * * end-products of glucose fermentation are acetate and ND NDNDND ND ND ND ND ND ND ND ND ND ND + + Goat rumen hydrogen. In syntrophic co-culture with M. hungatei, (subdivision A) Subdivision B Subdivision oxidizes lactate, glycerol, ethanol, 1-propanol, 1-butanol, glutamate, alanine, leucine, isoleucine, valine, histidine, * * * * * ± ± ± + ± asparagine, glutamine, arginine, lysine, threonine, 2- digester Synergistia Anaerobic

Fermentative Fermentative Fermentative, oxoglutarate, aspartate and methionine. The following substrates are not used by the pure culture or co-culture * * * with M. hungatei: crotonate, H2/CO2 plus acetate, pro- 2 22 22 2 + + + sludge pionate, butyrate, iso-butyrate, succinate, methanol, fruc- syntrophy Anaerobic tose, maltose, arabinose, galactose, mannose, raffinose, sucrose, starch, pectin, citrate, glycine, proline, phenylalanine, phenol, hydroquinone, and benzoate. A Stickland 2 ND ND ND ND ND Human

oral cavity reaction is not observed. The main fatty acids of the type Fermentative Fermentative, strain of the type species are iso-C13 : 0, iso-C15 : 0, anteiso- C15 : 0,C18 : 1,C19 : 1,C20 : 1 and C21 : 1. The polar lipid profile *

+ of the type strain consists of six unknown phospholipids, Rice field soil syntrophy four unknown aminophospholipids, three unknown polar Fermentative, lipids, and one unknown aminolipid. T T * The type strain is 7WAY-8-7 (5JCM 17151 5DSM 222

+ T T sludge 24196 5CGMCC 1.5159 ), isolated from a mesophilic syntrophy Anaerobic

Fermentative, full-scale anaerobic reactor treating wastewater from isomerized sugar production processes. The DNA G+C 12 3 4 5 67 8 91011121314

–– –––– content of the type strain is 61.4 mol%. 2 ++ + ++ + ±+ + ++ + ++ + + sludge syntrophy Acknowledgements cont. We thank Mizuho Muramatsu in the National Institute of Advanced Industrial Science and Technology (AIST) for the determinations Glutamate Valine Aspartate Alanine Leucine Arginine Glycine

acceptors of FAMEs and DNA base composition. This work was supported Electron Characteristic Subdivision EMetabolism Fermentative, Class Habitat Anaerobic

Table 2. *By fermentation. by Taishan Scholar Program of Shandong Province, Knowledge http://ijs.sgmjournals.org 2143 Y.-L. Qiu and others

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