J. Gen. Appl. Microbiol., 52, 223–234 (2006)

Full Paper

Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon-degrading, heavy metal resistant bacterium from hot compost

Annarita Poli,1 Ida Romano,1 Gaetano Caliendo,4 Giancarlo Nicolaus,3 Pierangelo Orlando,2 Antonio de Falco,4 Licia Lama,1 Agata Gambacorta,1 and Barbara Nicolaus1,*

1 Istituto di Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy 2 Istituto di Biochimica delle Proteine (IBP), CNR, Napoli, Italy 3 Istituto di Ricerche di Biologia Molecolare “P. Angeletti” IRBM, Pomezia, Rome, Italy 4 Pomigliano Ambiente s.p.a., Napoli, Italy

(Received December 14, 2005; Accepted August 4, 2006)

A thermophilic, spore-forming bacterial strain L1T was isolated from hot compost “Pomigliano Environment” s.p.a., Pomigliano, Naples, Italy. The strain was identiﬁed by using a polyphasic taxonomic approach. L1T resulted in an aerobic, gram-positive, rod-shaped, thermophilic with an optimum growth temperature of 68°C chemorganotrophic bacterium which grew on hydrocarbons as unique carbon and energy sources and was resistant to heavy metals. The GC DNA content was 43.5 mol%. Phylogenetic analysis of 16S rRNA gene sequence and Random Ampli- ﬁed Polymorphic DNA-PCR (RAPD-PCR) analysis of L1T and related strains showed that it forms within Geobacillus toebii, a separate cluster in the Geobacillus genus. The composition of cellular fatty acids analyses by Gas-Mass Spectroscopy differed from that typical for the genus Geobacillus in that it is lacking in iso-C15 fatty acid, while iso-C16 and iso-C17 were predominant. Isolates grew on a rich complex medium at temperatures between 55–75°C and presented a

doubling time (td) of 2 h and 6 h using complex media and hydrocarbon media, respectively. Among hydrocarbons tested, n-decane (2%) was the more effective to support the growth (1 g/L of wet cells). The microorganism showed resistance to heavy metal tested during the growth. Furthermore, intracellular a-galactosidase and a-glucosidase enzymatic activities were de- tectable in the L1T strain. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA-DNA hybridization, we propose assigning a novel subspecies of Geobacillus toebii, to be named Geobacillus toebii subsp. decanicus subsp. nov., with the type strain L1T (DSM 17041ATCC BAA 1004).

Key Words——alkanes; DNA-DNA hybridization; fatty acid; Geobacillus; heavy metals; hot compost; lipid; PCR ﬁnger print; thermozymes

Introduction * Address reprint requests to: Dr. Barbara Nicolaus, Istituto di Industrial composting is a microbial, aerobic, self- Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy. heating and solid-phase biodegradation process of Tel: 39–081–8675245 Fax: 39–081–8041770 organic-waste materials (Miller, 1996). During the ther- E-mail: [email protected] mogenic phase of the composting process, a large 224 POLI et al. Vol. 52 central zone remains at temperatures higher than caldoproteolyticus (Chen et al., 2004), G. gargensis 70°C for many weeks (Fugio and Kume, 1991; Strom, (Nazina et al., 2004), and G. jurassicus (Nazina et al., 1985a, b). In this “hot-zone” a high number of ther- 2005) were assigned to the Geobacillus group. mophilic bacteria, forming a different microbial commu- This paper describes the isolation of a new ther- nity, belonging to Thermus, Bacillus and Hy- mophilic Geobacillus strain, designated L1T, from ther- drogenobacter species were identified (Beffa et al., mogenic compost made from 12-week-old organic 1996; Blanc et al., 1999; Lyon et al., 2000; Strom, waste samples, able to grow at temperatures up to 1985a). 75°C. The characteristics of this isolate were com- Thermophiles isolated from compost in recent years pared with its nearest neighbor. Data on L1T strain have become extremely interesting from a technologi- ability to grow using hydrocarbons as unique carbon cal point of view (Lyon et al., 2000; Strom 1985b). sources and its resistance to heavy metals, were also Many are the studies undertaken for defining the prin- reported. cipal metabolic pathways and the peculiar properties of their molecules. Hot compost is considered to offer Materials and Methods a favorable habitat for thermophilic bacilli. Strom (1985b) isolated more than 750 heterotrophic spore- Compost characteristics. Compost samples were forming strains from compost. Most of these microor- from “Pomigliano Environment” s.p.a., Pomigliano ganisms grew under 60°C, and only Bacillus coagu- d’Arco (Campania Region, southern Italy). The first lans, Geobacillus stearothermophilus and Geobacillus step, in the bio-fermentation process, is essentially toebii were isolated at 65°C (Strom, 1985a; Sung et based on a static heap covered by a sandwich consti- al., 2002). Thermophilic bacteria related to the genus tuted of two supports of polyester with interposed a foil Geobacillus has been widely isolated from geothermal of Gore-Tex® and on a forced airing cycle-treatment of and man-made environments throughout the world 30 days. The process is monitored by a computerized (Maugeri et al., 2002; Nazina et al., 2001, 2005; Rhee system (BIOE® s.r.l, Milan, Italy) that checks the oxy- et al., 2002; Sung et al., 2002). gen, the temperature and the humidity levels. The tem- Thermophilic Bacillus species of the group 5 rRNA perature of the inner core of the heap is above 60°C. are a phenotypically and phylogenetic coherent group At 30 days from pretreatment, the pile is moved to the displaying very high similarity among their 16S rRNA threshing floor of maturation where, for a further 60 sequences (98.5–99.2%) (Ash et al., 1991; Rainey et days, it is turned weekly to favor the oxygenation and al., 1994; Sunna et al., 1997); this group was trans- consequently, the degradation of the more slowly ferred to a new genus Geobacillus that comprised biodegradable matrixes (lignin). The material is refined at that time 12 validated species G. stearother- through a phase of drum sifting by obtaining the “com- mophilus, G. thermocatenulatus, G. thermoleovorans, post.” The presence of heavy metals was determined G. kaustophilus, G. thermoglucosidasius, G. thermo- by atomic spectrometry (Shimadzu AA6200) (Poli et denitrificans, G. subterraneus, G. uzenensis, G. cal- al., 2005). Bacterial tests were performed by the meth- doxylosilyticus and G. toebii (Ahmad et al., 2000; ods of Koneman (Koneman, 1984). Claus and Berkeley, 1986; Fortina et al., 2001a, b; Sampling and isolation. Thirty grams of compost Golovacheva et al., 1975; Logan and Berkeley, 1984; (fresh wt) were placed in 200 ml of sterile water, ho- Manichini et al., 2000; Nazina et al., 2001; Priest et al., mogenized at room temperature on a shaker (150 rpm) 1988; Sung et al., 2002; Suzuki et al., 1983; Tomita et for 20 min, and serially diluted (102 to 1010) in the TH al., 2003; White et al., 1993; Zarrilla and Perry, 1987). medium (8 g/L peptone (Oxoid), 4 g/L yeast extract Other thermophilic established species belonged to (Oxoid), 2 g/L NaCl (Applichem) at pH 7.0). For opti- Geobacillus group such as Bacillus thermantarcticus mum temperature determination the cultures were in- (Nicolaus et al., 1996) validated in Int. J. Syst. Evol. cubated without agitation from 50°C to 80°C for 1 to 6 Microbiol. 2002 and G. vulcani (Caccamo et al., 2000; days. The pH dependence of growth was tested in the Gugliandolo et al., 2003; Maugeri et al., 2001; Nazina pH range 4.0 to 10.0. Pure strains were isolated at et al., 2004). The asporogenous Saccharococcus ther- 68°C at pH of 7.2 on the TH medium solidified with mophilus representing a separate line of descent (Naz- agar (2%, wt/v). The first pure culture obtained was ina et al., 2001; Nystrand, 1984) and more recently G. called L1T and studied in detail. 2006 Geobacillus toebii subsp. decanicus subsp. nov. 225

Reference strains and media. The reference (10 U), chloramphenicol (10, 50), kanamycin (5, 30), strains used were as follows: Geobacillus toebii DSM tylosin (10, 30), ampicillin (25), gentamicin (10, 30), 14590T (Sung et al., 2002), Geobacillus thermoleovo- novobiocin (30), nystatin (100), cycloheximide (30), rans ATCC 43513T (Zarrilla and Perry, 1987) and bacitracin (10 U), lincomycin (15), fusidic acid (10), Geobacillus caldoxylosilyticus ATCC 42125T. The vancomycin (30), streptomycin (25) and tetracycline media utilized were: TH medium; TH agar medium, (30, 50) (Nicolaus et al., 2000). containing (g/L) 20.0 agar (Oxoid); YN medium, con- Biochemical analysis. For the enzymatic assays, taining (g/L) 6.0 yeast extract, 6.0 NaCl at pH 6.5. cells grown in YN medium were collected during sta- Other media were prepared as follows: M162 min- tionary growth phase by centrifugation at 9,000g for eral medium containing: (g/L) 4.0 NaCl (Applichem), 30 min. Wet cells (about 2.0 g) were suspended in

0.53 NH4Cl (J.T. Baker), Solution A 60 ml/L, Solution B 20 mM Tris-HCl at pH 8.0, lysed by ultrasonic treatment 20 ml/L, Solution C 100 ml/L; Solution A: (g/L) 35.58 (Heat System Instrument) for 4 min, and centrifuged at Na2HPO4 ·2H2O (Applichem); Solution B: (g/L) 27.19 15,000 g for 20 min. The supernatant (crude extract)

KH2PO4 (Carlo Erba); Solution C: (g/L) 1.0 Nitrilotri- was assayed for a-galactosidase and a-glucosidase acetic acid (Applichem), 0.4 CaSO4 ·6H2O (Carlo enzymatic activities, by incubating for 10 min at 68°C a

Erba), 2.0 MgCl2 ·6H2O (Applichem), 2.5 Ferric citrate reaction mixture containing in 1 ml final volume: 0.1 ml (Carlo Erba), Nitsch’s trace elements 5.0 ml/L. of the crude extract, 40 mM Tris-HCl pH 7.0 and 1 mM Minimal media were prepared using M162 medium of the following substrates: p-nitrophenyl-a-D-(glucopy- plus either 1% glycerol, glucose, lactose, Na-acetate, ranoside, galactopyranoside, maltoside, arabinoside); mannose, xylose, galactose, sucrose, cellobiose, ri- p-nitrophenyl-b -(glucopyranoside, xylopyranoside, bose, maltose, fructose, ethanol, EDTA, sorbose, raffi- galactopyranoside, lactopyranoside, maltoside); p-ni- nose, malic acid, citric acid or trehalose as sole carbon trophenyl N-acetyl-b-D-glucosamide and 2-nitrophenyl- sources, at pH 7.2 (Maugeri et al., 2002). To evaluate 2-acetamido-2-deoxy a-glucopyranoside. The reaction T the capacity of strain L1 to use different substrates as was stopped by adding 1 ml of 1 M Na2CO3 followed by sole carbon sources, the medium M162 was supple- 3 ml of H2O. The released p-nitrophenol was mea- mented with 1–2% of (w/v) n-decane (BDH—Poole sured as optical density at 420 nm (Nicolaus et al., England); pentadecane (Aldrich Chemie); n-esade- 1998). Protein content was determined by the Bradford cane (Schuchardt); toluene (Carlo Erba); SDS (Ap- method using the BioRad protein assay with bovine plichem); EDTA (Carlo Erba); tridecane (EGA- serum albumin as standard (Bradford, 1976). CHEMIE); squalane (FLUKA). All growth tests were Aminopeptidase activity was assayed with “Bactident done at 68°C and pH 7.2. The growth was scored as Aminopeptidase Kit” from Merck (Germany) according positive if the 540 nm absorbance was greater than 0.3 to the manufacturer’s specifications. Hydrolysis of N- optical density, after 3 days of incubation. benzoyl-arginine-p-nitroanilide (BAPA) stereoisomers Morphological and physiological studies. Cellular was tested according to Oren and Galinski (1994). morphology was determined by phase-contrast mi- Lipid and fatty acid compositions. Cells, grown croscopy (Zeiss) and by scanning electron microscopy both in TH and hydrocarbon media, were harvested in (SEM). For SEM analysis the samples were fixed for the late exponential growth phase by centrifugation at 24 h in 2.5% glutaraldehyde. Samples were dehy- 9,000g. Freshly harvested cells (5–10 g) were drated in ethyl alcohol, dried to critical point, gold lyophilized and extracted by Soxhlet with CHCl3/MeOH coated by sputtering (SEM BALTECMED 020) and ob- (1 : 1 by vol.) for 5 h at 70°C (Nicolaus et al., 2001). served by a Philips XL 20 ESEM. Lipid spots were analyzed by thin layer chromatogra- T Phenotypic characterization was performed after L1 phy (TLC) on silica gel (0.25 mm, F254, Merck) eluted incubation at 68°C for 3 days in TH medium or TH with CHCl3/MeOH/H2O (65 : 25 : 4 by vol.). Lipids were agar (Hudson et al., 1986; Maugeri et al., 2002; San- detected by spraying the plates with 0.1% Ce(SO4)2 tos et al., 1989). followed by heating at 100°C for 5 min. Staining tests Sensitivity of the strain to antibiotics was tested by for complex lipids were performed using specific using the enrichment-solid medium TH and sensi-discs reagents for phospho-, amino- and glycolipids. The (6 mm, Oxoid). The following antibiotics were tested total lipid extract was treated with two volumes of n- (mg): neomycin (5, 30), erythromycin (30), penicillin G hexane at 30°C for 12 h. The quinone content was an- 226 POLI et al. Vol. 52 alyzed by high-performance liquid chromatography containing (1%) glucose as sole carbon source. The (HPLC) using an RP-18 Lichrospher (2504 mm) col- culture was incubated for 12 h, after which the cells umn eluted with n-hexane/ethylacetate (99 : 1 v/v) with were transferred to fresh media to get an optical den- a flow rate of 1.0 ml/min. Compounds were identified sity of 0.1 (A540 nm). Each heavy metal was added to 1 by H Nuclear Magnetic Resonance (NMR) and Mass fresh media at appropriate concentrations at To (time). Spectroscopy (MS) as previously described. Lipid hy- Bacterial growth controls and other samples were drolysis was performed by acid methanolysis. Gas- checked by measuring spectrophotometrically at Chromatography and Mass Spectroscopy (GC-MS) 540 nm, using UV Spectrophotometer (Varian DMS analyses were performed with an HP5890 series II 90). Cells were harvested in late exponential growth plus-5989B equipped with an HP-V column with a flux phase by centrifugation at 9,000g for 30 min. of 45 ml/min. Fatty acid methyl esters were detected Genetic studies. The almost complete 16S rRNA using the temperature program of 120°C (1 min), from gene sequence was determined by direct sequencing 120 to 250°C at 2°C/min. The identification of the com- of PCR-amplified 16S rDNA. Genomic DNA extraction, pounds was performed by parallel runs of pure stan- PCR mediated amplification of the 16S rDNA and pu- dards (Sigma), and by interpretation of mass spectra. rification of PCR products were carried out as de- Degradation of hydrocarbons. The evaluation of scribed previously (Rainey et al., 1996). Purified PCR growth rate and hydrocarbon hydrolysis for the L1T products were sequenced using the ABI PRISMtm Dye strain was carried out at 68°C and pH 7.2 in the M162 Terminator Cycle Sequencing Ready Reaction Kit (Ap- medium supplemented by 1–2% (w/v) n-decane or tri- plied Biosystems, Germany) as specified in the manu- decane by a 3 liter fermenter (Chemap), with low me- facturer’s protocol. Sequence reactions were elec- chanical agitation (100 rpm) and an aeration flux of trophoresed using the Applied Biosysthems 373A Se- 20 ml min1 for liter of broth. Cell density was esti- quencer (Rainey et al., 1994). The resulting sequence mated by measuring absorbance at 540 nm by direct data were put into the alignment editor ae2 software insertion of culture tubes into DMS 90 VARIAN UV/VIS and compared with representative 16S rDNA se- spectrophotometer. Hydrocarbon depletion was evalu- quences of organisms belonging to the Geobacillus ated using GC-MS and NMR analyses on cell free su- group (Maidak et al., 1999). For comparison 16S rDNA pernatants at interval times of growth. The super- sequences were obtained from the EMBL data base natants were extracted with dichloromethane (v/v) and (Maidak et al., 1999). The 16S rDNA sequence of dried under vacuum. GC-MS was performed on a strain L1T has been deposited in EMBL database Hewlett-Packard 5890-5970 instrument, equipped with under the accession number AJ966346. The results of an HP-V column. The analysis was performed at flow alignments are presented, in RESULTS AND DISCUSSION, rate of 0.7 ml min1; the temperature program used as similarity matrix and phylogenetic tree (Saitou and was: 60°C for 2 min, ramping from 60 to 280°C at Nei, 1987). The 16S rDNA similarity values were cal- 3°C min1 and final step of 10 min at 280°C. NMR culated by pair-wise comparison of the sequences spectra were recorded on a Bruker AMX-500 instru- within the alignment. For construction of the phyloge- 1 ment (500.13 MHz for H NMR) using CDCl3 as sol- netic dendrogram, the PHYLIP package was used vent for hydrocarbon samples. (Felsenstein, 1993); pair-wise evolutionary distances Heavy metal resistance. The chemicals assessed were computed from percent similarities by the correc- for toxicity were NiSO4 ·6H2O (Carlo Erba), tion of Jukes and Cantor (1969) and the phylogenetic

ZnSO4 ·7H2O (Aldrich), Co(NO3)2 ·6H2O (J.T. Baker), tree was constructed by the neighbour-joining method

HgCl2 (J.T. Baker), MnCl2 ·4H2O (J.T. Baker), (Higgins et al., 1992). Cr(NO3)3 ·9H2O (J.T. Baker), K2Cr2O7 (J.T. Baker), The G C content was determined by HPLC method

CuSO4 ·5H2O (J.T. Baker), FeCl3 (Carlo Erba), CdSO4 (Mesbah et al., 1989; Tamaoka and Komagata, 1984). (Aldrich). Solutions were prepared by dissolving in dis- The calibration was performed with non-methylated tilled water and subsequently filtered. The chosen Lambda-DNA (Sigma) (GC content 49.85 mol%) and metal concentrations were calculated as mg/L (ppm with Halomonas pantelleriensis DNA (GC content part per million) of each metal (Poli et al., 2005). 65.02 mol%). Cells were grown by inoculating 90 ml media in a Random Amplified Polymorphic DNA-PCR (RAPD- 250-ml Erlenmeyer flask using the minimal medium PCR) assay was used to produce fingerprint patterns 2006 Geobacillus toebii subsp. decanicus subsp. nov. 227 of L1T and of reference strains Geobacillus toebii DSM 14590T and Geobacillus caldoxylosilyticus ATCC 42125T, according to Ronimus et al. (1997). DNA amplification was performed in a 50 ml PCR reaction mixture containing: 50–200 ng of genomic DNA, 1 PCR buffer (supplied as component of the DNA polymerase kit), 3 mM MgCl2, 250 mM dNTPs, 0.5 mM of OPR-2 primer (5-CACAGCTGCC-3) or OPR-13 primer (5- GGACGACAAG-3) and 2.5 units of Platinum® Taq DNA polymerase (Invitrogen). The mixtures were amplified in a thermocycler iCycler® (BIO RAD). The amplification profile consisted of an initial denaturation of 2 min at 92°C and 35 cycles of 15 s at 94°C, annealing for 15 s at 36°C (previously optimized by temperature gradient amplification) and elongation for 2 min at 72°C. A final extension of 7 min was carried out at 72°C. Ten to 20 ml of PCR products were elec- trophoresed on 2% agarose gel (Agarose-1000, Invit- rogen) in 1 TAE buffer at 5 V/cm for 4 h. Ethidium bromide (0.1 mg/ml) was included both in the gel and electrophoresis buffer and PCR products were detected by UV visualization and recorded on Polapan 55 films (Polaroid). DNA-DNA hybridization. DNA was isolated using a Fig. 1. Flow-chart of composting process. French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA hybridiza- Table 1. Characteristics of hot compost from tion was carried out as described by De Ley et al. “Pomigliano Environment.” (1970) under consideration of the modifications described by Huss et al. (1983) using a model Cary Standard Measured Parameters Unity 100 Bio UV/VIS-spectrophotometer equipped with a values values Peltier-thermostatted 66 multicell changer and a temperature controller with in situ temperature probe Cadmium mg/kg d.c. 1.5 1.34 (Varian). Chromium VI mg/kg d.c. 0.5 0.5 Mercury mg/kg d.c. 1.5 1.0 Nickel mg/kg d.c. 50 11.6 Results and Discussion Lead mg/kg d.c. 140 78.5 Copper mg/kg d.c. 150 129.2 Compost parameters Zinc mg/kg d.c. 500 256.9 The compost was obtained by using 50% of organic Plastics3.33 mm % d.c. 0.45 0.01 urban waste and 50% green brush waste. Figure 1 Plastics3.33 mm % d.c. 0.05 0.01 summarizes the flow-chart of the process. During the Salmonella — Absent /25 g Absent fermentation, the temperature ranged from 60 to 70°C, Enterobacteriaceae CFU/g 1100 1,800 the oxygen concentration was 10–20% (v/v), the pH Streptococcus faecalis NPM/g 1 1,000 200 ranged from 6.0 to 7.5 and the humidity decreased Nematode NPM/50 g Absent Absent Trematode NPM/50 g Absent Absent from 60 to 20%. Chemical analysis of compost mate- Cestode NPM/50 g Absent Absent rial was reported in Table 1. Zinc, Copper, and Lead Infestant seeds N Absent Absent were the main heavy metal detected. Plastic material was found in a much lower amount. Among bacterial d.c., dry compost; CFU, colony forming units; NPM, numeric tests performed, only Enterobacteriaceae and Strepto- ponderable media; N, number. 228 POLI et al. Vol. 52 coccus faecalis tests resulted positive (Table 1).

Characteristic of the isolate Phase-contrast microscopy observations of compost samples show the presence of high numbers of oval bacterial spores. Bacterial growth occurred up to a 107 dilution of 15% (w/v) compost in TH medium, and the strain L1T was isolated from this dilution. Strain L1T exhibited morphological and chemical characteristics that are consistent with those found in the genus Geobacillus. Cells of strain L1T are aerobic and gram-positive motile rods, 2.0–3.0 mm long and 0.5 mm wide (Fig. 2). Spores oval, located terminally, T first appeared on medium TH with 2% MnCl2. Growth Fig. 2. Scanning electron microscopy of strain L1 grown of strain L1T occurred at 55–75°C with an optimum of on n-decane medium, in fermenter culture at 68°C. 68°C (no growth was observed at 50°C and 80°C). The sample morphology is observed by using a SEM Philips Until now no strains of thermophilic bacilli isolated from XL 20 series microscope. hot compost were able to grow at temperatures above 70°C. At optimum temperature, growth occurred be- medium an additional phospholipid was detected. The tween pH 5.0–9.0 with an optimum at pH 7.2. fatty acid compositions, determined from cells grown in The isolate was able to utilize a large variety of sug- TH medium or n-decane medium, were characterized ars and hydrocarbons. The bacterium utilized synthetic by the abundance of branched acyl chains; in particu- media and did not require any growth factors and vita- lar strain L1T contained major amounts of iso-C16:0 mins. Considerable growth was observed on TH agar (46%) and iso-C17 : 0 (28%) when cultured in TH and YN agar. The isolate was catalase, tyrosine de- medium, and iso-C16 : 0 (55%), iso-C17 : 0 (11%), n- composition, hippurate and gelatine hydrolysis, posi- C17 (16%) using the n-decane medium. The mem- T tive. Isolate L1 was able to reduce NO3 but not NO2 brane lipids are characterized by an elevated number and was sensible to lysozyme while it was oxidase, ca- of phospholipids composed of branched fatty acids sein and starch hydrolysis, indole production, phenyl- and by the presence of a quinone MK7 type. Iso- alanine deamination and urease negative. C16 : 0 and iso-C17 : 0 fatty acids were predominant Isolate L1T was negative for xylanase, b-glucosidase, while iso-C15 : 0 was absent, in contrast with the typi- a-mannosidase, a-amylase, esterase, b-galactosidase cal fatty acid composition of bacteria from the genus activities while it possessed a-galactosidase and a- Geobacillus. The pattern of the complex lipids has al- glucosidase activities. lowed to establish the taxonomic position of L1T by as- The isolate L1T utilized a wide range of carbon signing it to the Bacteria Domain. The phenotypic sources including ribose, glucose, glycerol, trehalose, characteristics are coherent for strain L1T belonging to maltose, cellobiose, ethanol and raffinose. The isolate the genus Geobacillus. was sensitive to kanamycin (5 mg), bacitracin (10 U), novobiocin (30 mg), streptomycin (25 mg), tetracycline Growth on hydrocarbons (30 mg) and penicillin G (10 U). Growth studies of strain L1T on hydrocarbon media revealed its ability to utilize different hydrocarbon sub- Lipid and fatty acid compositions strates such as pentadecane, tridecane, n-decane, Strain L1T possessed complex lipids based on fatty squalane, and toluene, as unique carbon and energy acid. The total lipid contents accounted for 10% and sources (Fig. 3). In particular n-decane supported effi- 9% of dry weight for culture grown in TH medium and ciently the growth reaching 1 g/L of wet cells. Growth hydrocarbon media, respectively. Under these condi- curves of L1T strain in the TH and n-decane media are T tions three major phospholipids and one phosphogly- compared (Fig. 4). The doubling time (td) of strain L1 colipid were present. In particular, using n-decane was 2 h in TH medium and 6 h using hydrocarbon sub- 2006 Geobacillus toebii subsp. decanicus subsp. nov. 229

Fig. 3. Biomass of strain L1T grown on M162 medium supplemented with different carbon sources, measured at A540 nm.

Fig. 5. The 1H NMR spectra of the cell free supernatants extracted with dichloromethane (v/v), obtained by a) the extrac- Fig. 4. Growth curves of strain L1T on TH medium () and tion was performed at time 0 of incubation; b) the extraction on M162 medium containing n-decane (). was performed at time 24 h of growth. The 1H NMR spectra were recorded of a solution of 3 mg of 1 strate. Figure 5 showed H NMR spectra of the cell extract in 0.6 ml of CDCl3 at 25°C. The resonances for the n-de- free supernatant dichloromethane extract at time 0 cane compound appeared at d0.88, d0.90 and 1.26 ppm (Fig. 5a) and the supernatant dichloromethane extract (Fig. 5a). at time 24 h (Fig. 5b), respectively. The characteristic chemical shifts of n-decane (Fig. 5a) disappeared (up to 80%) of strain L1T at 40 ppm and Zn2 caused a completely after 24 h of incubation (Fig. 5b). These decrease of growth (up to 70%) at 60 ppm (Fig. 6). No data were also confirmed by GC-MS analyses (data significant differences were noted for the strain grown not reported). in the presence of Mn2 , Fe2 , and Cu2 up to 300 ppm of concentration (Fig. 6). Strain L1T was not sus- Heavy metal resistance ceptible to the presence of Cr3 at 300 ppm while the The isolate L1T has been grown on media contain- presence of Cr6 at 60 ppm caused 50% of growth in- ing various concentrations of heavy metals. The mi- hibition. croorganism showed resistance to all heavy metal This study confirmed that thermophilic Geobacillus tested during the growth (Fig. 6). Only Cd2 and Zn2 strain plays an important role in organic matter degra- caused a decrease of the growth when added at low dation during the compost process. Microorganisms in- concentrations. In particular Cd2 affected the growth habiting extreme environments are often producers of 230 POLI et al. Vol. 52 unusual enzymes and biomolecules in order to survive special significance for the bioremediation of oil-pol- high temperatures and high concentrations of heavy luted water and soil. metals. Several thermotolerant and thermophilic Bacil- Strain L1T displaying the ability to grow on media lus spp. show a different pattern of heavy metal resis- contained hydrocarbons as n-decane, squalane, and tance and a different ability to grow on hydrocarbon tri-decane, as sole carbon sources, meaning resis- compounds. Indigenous hydrocarbon degraders are of tance to heavy metals could result in relevant biotech-

Fig. 6. Heavy metal effects on microorganism growth. g/L, grams of dry cells/liter; ppm, part per million.

Fig. 7. Phylogenetic dendrogram indicating the position of the strain L1T in relation to phylogenetically related Geobacillus, S. thermophilus and Bacillus species. The root of the tree was determined by including the 16S rDNA sequence of Brevibacillus centrosporus into the analysis. The scale bar below the dendrogram indicates 0.1 nucleotide substitution per 100 nucleotides. Bootstrap values (%) are indicated at the branches from 1,000 replications. Only bootstrap values greater than 50% are shown. 2006 Geobacillus toebii subsp. decanicus subsp. nov. 231 nological importance in industrial uses. One example is the possibility to use such microorganisms to clean up oil spills in high temperature environments.

Genetic studies Comparison of 16S rDNA strain sequence in data bank (EMBL AJ966346), revealed that strain L1T falls within the radiation of the cluster comprising Geobacil- lus species and forms a coherent cluster with Geobacillus toebii DSM 14590T and Geobacillus caldoxylosilyticus ATCC 42125T (Fig. 7). The level of 16S rDNA sequence similarity between strain L1T and G. toebii SK-1T was 97.0%. The sequence similarity to other species within the genus Geobacillus (with validly published names) was less than 95.0%. The GC content of the DNA of strain L1T was 43.5 mol%. RAPD-PCR was used to produce fingerprint patterns of L1T isolate and related strains, as described in Fig. 8. RAPD-PCR fingerprint patterns of L1T and refer- MATERIALS AND METHODS. Both OPR-2 (Fig. 8, lines 1, ence strains. 2, 3) and OPR-13 (Fig. 8, lines 4, 5, 6) primers pro- PCR analysis was performed as described in MATERIALS AND duced fingerprints patterns characterized by relevant METHODS by OPR-2 primer (5 -CACAGCTGCC-3 ) (lanes 1–3) or OPR-13 primer (5-GGACGACAAG-3)(lanes 4–6). MW, 100 bands within 2,500–400 bp. L1T and G. toebii differed bp DNA ladder (Invitrogen); lanes 1 and 4, Geobacillus toebii at level of bands 500 and 700 bp utilizing OPR-2 DSM 14590T; lanes 2 and 5, strain L1T; lanes 3 and 6, primer and at level of band 2,500 bp using OPR-13 Geobacillus caldoxylosilyticus ATCC 42125T. primer. L1T and Geobacillus caldoxylosilyticus showed a different fingerprints using both primers. The phylogenetic analysis (16S rDNA % of similarity Table 2. Comparison of the phenotypic characteristics of and PCR-fingerprint) confirms that strain L1T belongs strain L1T with related species. to the genus Geobacillus (Stackebrandt and Goebel, Geobacillus toebii 1994). Characteristic Strain L1 strain SK-1a DNA-DNA hybridization experiments between strain T T L1 and G. toebii SK-1 , showing low 16S rDNA simi- Temperature range (°C) 60–70 45–70 larity level (97%), revealed a border line re-association Optimum temperature (°C) 68 60 T value between the new isolate with G. toebii SK-1 pH range 5.0–9.0 6.0–9.0 (73.7%) and a lower value with Geobacillus caldoxy- Gelatin hydrolysis losilyticus (55.9%) (Wayne et al., 1987). Casein hydrolysis Table 2 indicates the difference in the phenotypic Fermentation of glucose and biochemical characteristics between strain L1T Denitrification G+C content (mol%) 43.5 44.0 and G. toebii. Strain L1T differed from G. toebii in opti- FAME composition (%): mal growth temperature, pH range, reduction of NO, 2 nC15 : 0 6.2 utilization of substrates, casein and gelatin hydrolysis iC15 : 0 34.03 and fatty acid compositions (Table 2). iC16 : 0 46.1 17.46 On the basis of these results, we propose that ther- nC16 : 0 8.6 mophilic Geobacillus strain L1T is a novel subspecies iC17 : 0 28.1 34.86 of Geobacillus toebii, for which the name Geobacillus nC17 : 0 6.1 toebii subsp. decanicus is proposed. nC18 : 0 4.8

Geobacillus toebii strain SK-1T. a Data were obtained from Sung et al. (2002). 232 POLI et al. Vol. 52

Description of Geobacillus toebii subsp. decanicus Beffa, T., Blanc, M., Lyon, P. F., Vogt, G., Marchiani, M., Fisher, subsp. nov. J. L., and Aragno, M. (1996) Isolation of Thermus strain G. toebii subsp. decanicus (de. ca. ni. cus. N.L. n. from hot composts (60–80°C). Appl. Environ. Microbiol., decane common name for hydrocarbon; able to grow 62, 1723–1727. Blanc, M., Marilley, L., Beffa, T., and Aragno, M. (1999) Ther- on n-decane; N.L. masc. adj. decanicus pertaining to mophilic bacterial communities in hot composts as re- n-decane). vealed by most probable number counts and molecular Gram positive, aerobic, motile, spore forming bac- (16S rDNA) methods. FEMS Microbiol. Ecol., 28, 141–149. terium, 2.0–3.0 mm long and 0.5 mm wide, growth oc- Bradford, M. M. (1976) A rapid and sensitive method for quanti- curs at 55–75°C with optimum growth occurring at tation of microgram quantities of protein using the princi- 68°C. It grows at pH 5.0–9.0 (optimal pH 7.2). Grows ples of protein-dye binding. Anal. Biochem., 72, 248–254. on M162 mineral medium up to and in 1–2% n-dec- Caccamo, D., Gugliandolo, C., Stackebrandt, E., and Maugeri, ane, tridecane, pentadecane, or squalane as sole car- T. L. (2000) Bacillus vulcani sp. nov., a novel thermophilic species isolated from a shallow marine hydrothermal vent. bon sources. It is able to grow on glucose, ribose, tre- Int. J. Syst. Evol. Microbiol., 50, 2009–2012. halose, glycerol, maltose, cellobiose, ethanol, raffi- Cashion, P., Hodler-Franklin, M. A., McCully, J., and Franklin, nose. It is resistant to different heavy metals added M. (1977) A rapid method for base ratio determination of 2 2 2 2 2 during the growth: Cu , Mn , Fe , Co , Hg and bacterial DNA. Anal. Biochem., 81, 461–466. Ni2 . It is catalase, tyrosine decomposition, hippurate Chen, X. G., Stabnikova, O., Tay, J. H., Wang, J. Y., and Tay, S. and gelatine hydrolysis positive, reduces NO3 to NO2 ; T. L. (2004) Thermoactive extracellular proteases of it is sensitive to lysozyme, negative for oxidase, starch Geobacillus caldoproteolyticus, sp. nov., from sewage and casein hydrolysis, urease, indole production, sludge. Extremophiles, 8, 489–498. phenylalanine deamination. Positive results are ob- Claus, D. and Berkeley, R. C. W. (1986) Genus Bacillus Cohn 1872, 174AL. In Bergey’s Manual of Systematic Bacteriol- tained for a-galactosidase and a-glucosidase activi- ogy, ed. by Sneath, P. H. A., Mair, N. S., Sharpe, M. E., ties. MK7 is the predominant quinone. iC16 : 0 and Holt, J. G., Williams & Wilkins, Baltimore, Vol. 2, pp. iC17 : 0 are the major fatty acids (74% of total fatty 1105–1139. acids). The following antibiotics inhibit the growth: De Ley, J., Cattoir, H., and Reynaerts, A. (1970) The quantita- kanamycin (5 mg), bacitracin (10 U), novobiocin (30 mg), tive measurement of DNA hybridization from renaturation streptomycin (25 mg), tetracycline (30 mg) and penicillin rates. Eur. J. Biochem., 12, 133–142. G (10 U). The mol% GC content of DNA is 43.5 mol%. Felsenstein, J. (1993) PHYLIP Phylogeny Inference Package, The EMBL accession number for the 16S rDNA se- version 3.5.1. Department of Genetics, University of Wash- ington, Seattle, USA. quence of strain L1T is AJ 966346. Type strain is L1T Fortina, M. G., Mora, D., Schumann, P., Parini, C., Manichini, P. (DSM 17041TATCC BAA 1004T). Isolated from hot L., and Stackebrandt, E. (2001a) Reclassification of Sac- compost “Pomigliano Environment” s.p.a., Pomigliano, charococcus caldoxylosilyticus as Geobacillus caldoxylosi- Naples, Italy. lyticus (Ahmad et al. 2000) comb. nov. Int. J. Syst. Evol. Mi- crobiol., 51, 2063–2071. Acknowledgments Fortina, M. G., Pukall, R., Schumann, P., Mora, D., Parini, C., Manichini, P. L., and Stackebrandt, E. (2001b) Ureibacillus This work was partially supported by Regione Campania. 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