ARTICLE IN PRESS Systematic and Applied Microbiology 29 (2006) 404–413 www.elsevier.de/syapm Propionicicella superfundia gen. nov., sp. nov., a chlorosolvent-tolerant propionate-forming, facultative anaerobic bacterium isolated from contaminated groundwater Hee-Sung Baea, William M. Moeb,Ã, Jun Yanb, Igor Tiagoc, Milton S. da Costad, Fred A. Raineya aDepartment of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA bDepartment of Civil and Environmental Engineering, Louisiana State University, Baton Rouge, LA 70803, USA cDepartamento de Zoologia and Centro de Neurocieˆncias, Universidade de Coimbra, 3004-517 Coimbra, Portugal dDepartamento de Bioquı´mica and Centro de Neurocieˆncias, Universidade de Coimbra, 3001-401 Coimbra, Portugal Received 5 October 2005 Abstract A novel strain, designated as BL-10T, was characterized using a polyphasic approach after isolation from groundwater contaminated by a mixture of chlorosolvents that included 1,1,2-trichloroethane, 1,2-dichloroethane, and vinyl chloride. Stain BL-10T is a facultatively anaerobic bacterium able to ferment glucose to form propionate, acetate, formate, lactate, and succinate. Fermentation occurred in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations to at least 9.8 and 5.9 mM, respectively. Cells are Gram-positive, rod-shaped, non-motile, and do not form spores. Oxidase and catalase are not produced and nitrate reduction did not occur in PYG medium. Menaquinone MK-9 is the predominant respiratory quinone and meso-diaminopimelic acid is present in the cell wall peptidoglycan layer. Major cellular fatty acids are C15:0, iso C16:0, and anteiso C15:0. Genomic DNA G+C content is 69.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed strain BL-10T to fall within the radiation of genera Propionicimonas and Micropruina. On the basis of the results obtained in this study, it is proposed that strain BL-10T should be classified as a novel taxon, for which the name Propionicicella superfundia gen. nov., sp. nov. is proposed. The type strain of Propionicicella superfundia is BL-10T ( ¼ ATCC BAA-1218T, ¼ LMG 23096T).1 r 2005 Elsevier GmbH. All rights reserved. Keywords: Propionicicella superfundia; Facultative anaerobe; Propionate; Chlorosolvent tolerant; DNAPL; Dechlorination Introduction From 1969 to 1980, petrochemical wastes including à Corresponding author. Tel.: +1 225 578 9174 free-phase chlorinated solvents were disposed of by E-mail address: [email protected] (W.M. Moe). 1Nucleotide sequence accession number. Nucleotide sequence data direct discharge to earthen ponds at the Brooklawn site, reported are available in the DDBJ/EMBL/GenBank databases under one of two areas that comprise what is now known as the accession number DQ176646. the Petro-Processors of Louisiana, Inc. (PPI) EPA 0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.11.004 ARTICLE IN PRESS H.-S. Bae et al. / Systematic and Applied Microbiology 29 (2006) 404–413 405 Superfund Site, located approximately 10 miles north of differential interference contrast (DIC) microscopy Baton Rouge, LA (USA). Portions of the Brooklawn (Nikon microphot-Fxa) or transmission electron micro- area were capped in the early 1990s, and an array of scopy (JEOL 1000CX TEM). For TEM, cells were wells was installed to recover organic contaminants negatively stained using uranyl acetate (2%, v/v). Spore present in the subsurface as dense non-aqueous-phase formation and motility were observed using phase liquid (DNAPL) [9]. Residual DNAPL remains in the contrast microscopy (Nikon Optiphot). subsurface, and groundwater within the DNAPL source Gram staining was performed using a Gram Stain Kit zone contains high concentrations of chlorinated orga- (Difco) according to the manufacturer’s recommended nics including 1,1,2,2-tetrachloroethane, 1,1,2-trichloro- protocol. Catalase activity was assessed as described by ethane, 1,2-dichloroethane, 1,2-dichloropropane, hexa- Smibert and Krieg [36] using 3% hydrogen peroxide chloro-1,3-butadiene, and vinyl chloride. added to cells grown anaerobically on PYG agar plates. During a study aimed at characterizing the microbial Oxidase tests were performed using the tetramethyl- population present within the DNAPL source zone at p-phenylenediamine dihydrochloride filter paper techni- the PPI site in order to assess the potential for in situ que [36]. Aerobic growth was examined on several bioremediation, we isolated a novel bacterium desig- complex media including nutrient agar (Difco), PYG nated as strain BL-10T. To determine its taxonomic agar [4], plate count agar (Difco) and R2A (Difco) to position, a polyphasic approach, including phylogenetic, which no reducing agent was added. Nitrate reducing chemotaxonomic, and phenotypic assessment was con- ability in PYG cultures containing 10 mM sodium ducted. Results indicate that strain BL-10T should be nitrate was determined according to Smibert and Krieg classified as a novel genus and novel species, for which [36]. Additional biochemical and physiological charac- the name Propionicicella superfundia, gen. nov, sp. nov. terization was conducted using Rapid ID 32A kits is proposed. (bioMe´rieux) with preparation and incubation per- formed in the anaerobic chamber. The cell suspension used to inoculate Rapid ID 32A kit was produced by Materials and methods flooding a CASB agar plate grown anaerobically at 30 1C for 14 days with API Suspension Medium provided in the kit. Isolation and bacterial strains Carbon utilization tests were performed in 16-mL Hungate tubes containing 10 mL of PY medium [4] and Groundwater samples were collected from well a headspace of N and CO (95:5, v/v). Each substrate W-1024-1 located in the DNAPL source zone at the 2 2 was added at a concentration of 5 or 10 g LÀ1 for mono-, Brooklawn portion of the PPI site. Sterile, 1.0 L glass di-, and polysaccharides and sugar alcohols. Organic sample collection bottles were filled with groundwater acids were added at a concentration of 15 or 30 mM. leaving little or no headspace, and placed on ice during Cultures were inoculated using a 1% (v/v) of an transport to the laboratory (approximately 1 h). After exponentially growing seed culture. Tubes showing an serial dilution with a potassium phosphate buffer increase in OD of more than 0.1 OD unit above the (100 mM, pH 7.0) in an anaerobic chamber (Coy 660 controls without added carbon were recorded as Laboratory Products Inc., MI), 0.1 mL aliquots were positive. The pH range for growth was evaluated over spread on Columbia Anaerobic Sheep Blood agar plates the range pH 3.0–10.0 using buffers comprised of (CASB, BBL). Plates were incubated in the anaerobic 100 mM acetate buffer for pH 3.0–6.0, 100 mM potas- chamber (headspace 90% N ,5%CO, and 5% H )at 2 2 2 sium phosphate buffer for pH 6.0–8.0, and Tris buffer 30 1C for up to 5 weeks. Isolated colonies were for pH 8.0–10.0. Growth in the NaCl concentration subcultured on CASB agar and purity verified by range of 0–5% (w/v) and the temperature range microscopy. Propionicimonas paludicola strains WdT 10–40 1C was determined in Hungate tubes containing and Wf were kindly provided by Professor Atsuko Ueki 10 mL of PYG. of the Yamagata University, Japan. These reference The ability to use chlorosolvents as electron acceptors strains were maintained on CASB agar or PYG agar [4] was examined in 25-mL serum bottle containing 10 mL in the anaerobic chamber. The isolates were stored at of anaerobic basal medium [39] or in 1/10 strength PY À80 1C in PYG with 15% (v/v) glycerol and 5% (v/v) broth. The anaerobic medium was amended with dimethylsulfoxide. acetate, lactate, and pyruvate (each 2 mM) and one of the following chlorosolvents: 1,1,2-trichloroethane, Growth, morphological, biochemical, and tolerance 1,2-dichloroethane, tetrachloroethene, trichloroethene, characteristics cis-1,2-dichloroethene, or 1,2-dichloropropane (each 2.0 mmol). Serum bottle headspace gas consisted of N2, Growth of the organisms was examined in CASB and CO2, and H2 (80:10:10, v/v/v). The ability to degrade PYG media. Cell morphology was observed using aromatic compounds was also tested in serum bottles. ARTICLE IN PRESS 406 H.-S. Bae et al. / Systematic and Applied Microbiology 29 (2006) 404–413 Each bottle was amended with 0.5 mmol of benzene, cellulose plates using the solvent system of Rhuland toluene, ethylbenzene, or p-xylene. Serum bottle head- et al. [31], at DSMZ. space gas consisted of N2,CO2, and H2 (80:10:10, v/v/v) for anaerobic basal medium and N2 and CO2 (95:5, v/v) Analytical methods for PY/10. Serum bottles were sealed with butyl rubber stoppers and aluminum seals. Uninoculated bottles were Fermentation products from PYG grown cultures 1 used as negative controls. Incubation was at 30 C. were analyzed by ion chromatography (IC) using a The ability of strain BL-10 to grow in the presence of Metrohm peak 761 Compact IC equipped with a 1,2-dichloroethane (1,2-DCA) or 1,1,2-trichloroethane Metrosep Organic Acid Column (25 cm  7.8 mm) and (1,1,2-TCA) was examined in 120-mL serum bottles a conductivity detector. Isocratic elution was performed containing 80 mL of PYG supplemented with various À1 with 0.5 mM H2SO4 at a flow rate of 0.5 mL min and concentrations (ranging to 9.8 mM in the aqueous constant temperature of 25 1C. Retention times and phase) of 1,2-DCA or 1,1,2-TCA. The headspace was peak areas of products were compared to those of filled with a mixture of N2 and CO2 (95:5%, v/v). authentic standards that included acetic, butyric, formic, Growth based on OD was measured using a Smart TM lactic, isovaleric, propionic, pyruvic, and succinic acids.