Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon [PDF]

Annarita Poli,1 Ida Romano,1 Gaetano Caliendo,4 Giancarlo Nicolaus,3 Pierangelo Orlando,2 Antonio de Falco,4. Licia Lama

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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 Amplified 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 (t d) 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 detectable 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 Chimica Biomolecolare (ICB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy. Tel: 39–081–8675245 Fax: 39–081–8041770 E-mail: [email protected]

Industrial composting is a microbial, aerobic, selfheating and solid-phase biodegradation process of organic-waste materials (Miller, 1996). During the thermogenic phase of the composting process, a large

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central zone remains at temperatures higher than 70°C for many weeks (Fugio and Kume, 1991; Strom, 1985a, b). In this “hot-zone” a high number of thermophilic bacteria, forming a different microbial community, belonging to Thermus, Bacillus and Hydrogenobacter species were identified (Beffa et al., 1996; Blanc et al., 1999; Lyon et al., 2000; Strom, 1985a). Thermophiles isolated from compost in recent years have become extremely interesting from a technological point of view (Lyon et al., 2000; Strom 1985b). Many are the studies undertaken for defining the principal metabolic pathways and the peculiar properties of their molecules. Hot compost is considered to offer a favorable habitat for thermophilic bacilli. Strom (1985b) isolated more than 750 heterotrophic sporeforming strains from compost. Most of these microorganisms grew under 60°C, and only Bacillus coagulans, Geobacillus stearothermophilus and Geobacillus toebii were isolated at 65°C (Strom, 1985a; Sung et al., 2002). Thermophilic bacteria related to the genus Geobacillus has been widely isolated from geothermal and man-made environments throughout the world (Maugeri et al., 2002; Nazina et al., 2001, 2005; Rhee et al., 2002; Sung et al., 2002). Thermophilic Bacillus species of the group 5 rRNA are a phenotypically and phylogenetic coherent group displaying very high similarity among their 16S rRNA sequences (98.5–99.2%) (Ash et al., 1991; Rainey et al., 1994; Sunna et al., 1997); this group was transferred to a new genus Geobacillus that comprised at that time 12 validated species G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius, G. thermodenitrificans, G. subterraneus, G. uzenensis, G. caldoxylosilyticus and G. toebii (Ahmad et al., 2000; Claus and Berkeley, 1986; Fortina et al., 2001a, b; Golovacheva et al., 1975; Logan and Berkeley, 1984; Manichini et al., 2000; Nazina et al., 2001; Priest et al., 1988; Sung et al., 2002; Suzuki et al., 1983; Tomita et al., 2003; White et al., 1993; Zarrilla and Perry, 1987). Other thermophilic established species belonged to Geobacillus group such as Bacillus thermantarcticus (Nicolaus et al., 1996) validated in Int. J. Syst. Evol. Microbiol. 2002 and G. vulcani (Caccamo et al., 2000; Gugliandolo et al., 2003; Maugeri et al., 2001; Nazina et al., 2004). The asporogenous Saccharococcus thermophilus representing a separate line of descent (Nazina et al., 2001; Nystrand, 1984) and more recently G.

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caldoproteolyticus (Chen et al., 2004), G. gargensis (Nazina et al., 2004), and G. jurassicus (Nazina et al., 2005) were assigned to the Geobacillus group. This paper describes the isolation of a new thermophilic Geobacillus strain, designated L1T, from thermogenic compost made from 12-week-old organic waste samples, able to grow at temperatures up to 75°C. The characteristics of this isolate were compared with its nearest neighbor. Data on L1T strain ability to grow using hydrocarbons as unique carbon sources and its resistance to heavy metals, were also reported. Materials and Methods

Compost characteristics. Compost samples were from “Pomigliano Environment” s.p.a., Pomigliano d’Arco (Campania Region, southern Italy). The first step, in the bio-fermentation process, is essentially based on a static heap covered by a sandwich constituted of two supports of polyester with interposed a foil of Gore-Tex® and on a forced airing cycle-treatment of 30 days. The process is monitored by a computerized system (BIOE® s.r.l, Milan, Italy) that checks the oxygen, the temperature and the humidity levels. The temperature of the inner core of the heap is above 60°C. At 30 days from pretreatment, the pile is moved to the threshing floor of maturation where, for a further 60 days, it is turned weekly to favor the oxygenation and consequently, the degradation of the more slowly biodegradable matrixes (lignin). The material is refined through a phase of drum sifting by obtaining the “compost.” The presence of heavy metals was determined by atomic spectrometry (Shimadzu AA6200) (Poli et al., 2005). Bacterial tests were performed by the methods of Koneman (Koneman, 1984). Sampling and isolation. Thirty grams of compost (fresh wt) were placed in 200 ml of sterile water, homogenized at room temperature on a shaker (150 rpm) for 20 min, and serially diluted (102 to 1010) in the TH medium (8 g/L peptone (Oxoid), 4 g/L yeast extract (Oxoid), 2 g/L NaCl (Applichem) at pH 7.0). For optimum temperature determination the cultures were incubated without agitation from 50°C to 80°C for 1 to 6 days. The pH dependence of growth was tested in the pH range 4.0 to 10.0. Pure strains were isolated at 68°C at pH of 7.2 on the TH medium solidified with agar (2%, wt/v). The first pure culture obtained was called L1T and studied in detail.

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Reference strains and media. The reference strains used were as follows: Geobacillus toebii DSM 14590T (Sung et al., 2002), Geobacillus thermoleovorans ATCC 43513T (Zarrilla and Perry, 1987) and Geobacillus caldoxylosilyticus ATCC 42125T. The media utilized were: TH medium; TH agar medium, containing (g/L) 20.0 agar (Oxoid); YN medium, containing (g/L) 6.0 yeast extract, 6.0 NaCl at pH 6.5. Other media were prepared as follows: M162 mineral medium containing: (g/L) 4.0 NaCl (Applichem), 0.53 NH4Cl (J.T. Baker), Solution A 60 ml/L, Solution B 20 ml/L, Solution C 100 ml/L; Solution A: (g/L) 35.58 Na2HPO4 · 2H2O (Applichem); Solution B: (g/L) 27.19 KH2PO4 (Carlo Erba); Solution C: (g/L) 1.0 Nitrilotriacetic acid (Applichem), 0.4 CaSO4 · 6H2O (Carlo Erba), 2.0 MgCl2 · 6H2O (Applichem), 2.5 Ferric citrate (Carlo Erba), Nitsch’s trace elements 5.0 ml/L. Minimal media were prepared using M162 medium plus either 1% glycerol, glucose, lactose, Na-acetate, mannose, xylose, galactose, sucrose, cellobiose, ribose, maltose, fructose, ethanol, EDTA, sorbose, raffinose, malic acid, citric acid or trehalose as sole carbon sources, at pH 7.2 (Maugeri et al., 2002). To evaluate the capacity of strain L1T to use different substrates as sole carbon sources, the medium M162 was supplemented with 1–2% of (w/v) n-decane (BDH—Poole England); pentadecane (Aldrich Chemie); n-esadecane (Schuchardt); toluene (Carlo Erba); SDS (Applichem); EDTA (Carlo Erba); tridecane (EGACHEMIE); squalane (FLUKA). All growth tests were done at 68°C and pH 7.2. The growth was scored as positive if the 540 nm absorbance was greater than 0.3 optical density, after 3 days of incubation. Morphological and physiological studies. Cellular morphology was determined by phase-contrast microscopy (Zeiss) and by scanning electron microscopy (SEM). For SEM analysis the samples were fixed for 24 h in 2.5% glutaraldehyde. Samples were dehydrated in ethyl alcohol, dried to critical point, gold coated by sputtering (SEM BALTECMED 020) and observed by a Philips XL 20 ESEM. Phenotypic characterization was performed after L1T incubation at 68°C for 3 days in TH medium or TH agar (Hudson et al., 1986; Maugeri et al., 2002; Santos et al., 1989). Sensitivity of the strain to antibiotics was tested by using the enrichment-solid medium TH and sensi-discs (6 mm, Oxoid). The following antibiotics were tested (m g): neomycin (5, 30), erythromycin (30), penicillin G

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(10 U), chloramphenicol (10, 50), kanamycin (5, 30), tylosin (10, 30), ampicillin (25), gentamicin (10, 30), novobiocin (30), nystatin (100), cycloheximide (30), bacitracin (10 U), lincomycin (15), fusidic acid (10), vancomycin (30), streptomycin (25) and tetracycline (30, 50) (Nicolaus et al., 2000). Biochemical analysis. For the enzymatic assays, cells grown in YN medium were collected during stationary growth phase by centrifugation at 9,000g for 30 min. Wet cells (about 2.0 g) were suspended in 20 mM Tris-HCl at pH 8.0, lysed by ultrasonic treatment (Heat System Instrument) for 4 min, and centrifuged at 15,000g for 20 min. The supernatant (crude extract) was assayed for a -galactosidase and a -glucosidase enzymatic activities, by incubating for 10 min at 68°C a reaction mixture containing in 1 ml final volume: 0.1 ml of the crude extract, 40 mM Tris-HCl pH 7.0 and 1 mM of the following substrates: p-nitrophenyl-a -D-(glucopyranoside, galactopyranoside, maltoside, arabinoside); p-nitrophenyl- b -(glucopyranoside, xylopyranoside, galactopyranoside, lactopyranoside, maltoside); p-nitrophenyl N-acetyl-b -D-glucosamide and 2-nitrophenyl2-acetamido-2-deoxy a -glucopyranoside. The reaction was stopped by adding 1 ml of 1 M Na2CO3 followed by 3 ml of H2O. The released p-nitrophenol was measured as optical density at 420 nm (Nicolaus et al., 1998). Protein content was determined by the Bradford method using the BioRad protein assay with bovine serum albumin as standard (Bradford, 1976). Aminopeptidase activity was assayed with “Bactident Aminopeptidase Kit” from Merck (Germany) according to the manufacturer’s specifications. Hydrolysis of Nbenzoyl-arginine-p-nitroanilide (BAPA) stereoisomers was tested according to Oren and Galinski (1994). Lipid and fatty acid compositions. Cells, grown both in TH and hydrocarbon media, were harvested in the late exponential growth phase by centrifugation at 9,000g. Freshly harvested cells (5–10 g) were lyophilized and extracted by Soxhlet with CHCl3/MeOH (1 : 1 by vol.) for 5 h at 70°C (Nicolaus et al., 2001). Lipid spots were analyzed by thin layer chromatography (TLC) on silica gel (0.25 mm, F254, Merck) eluted with CHCl3/MeOH/H2O (65 : 25 : 4 by vol.). Lipids were detected by spraying the plates with 0.1% Ce(SO4)2 followed by heating at 100°C for 5 min. Staining tests for complex lipids were performed using specific reagents for phospho-, amino- and glycolipids. The total lipid extract was treated with two volumes of nhexane at 30°C for 12 h. The quinone content was an-

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alyzed by high-performance liquid chromatography (HPLC) using an RP-18 Lichrospher (2504 mm) column eluted with n-hexane/ethylacetate (99 : 1 v/v) with a flow rate of 1.0 ml/min. Compounds were identified by 1H Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) as previously described. Lipid hydrolysis was performed by acid methanolysis. GasChromatography and Mass Spectroscopy (GC-MS) analyses were performed with an HP5890 series II plus-5989B equipped with an HP-V column with a flux of 45 ml/min. Fatty acid methyl esters were detected using the temperature program of 120°C (1 min), from 120 to 250°C at 2°C/min. The identification of the compounds was performed by parallel runs of pure standards (Sigma), and by interpretation of mass spectra. Degradation of hydrocarbons. The evaluation of growth rate and hydrocarbon hydrolysis for the L1T strain was carried out at 68°C and pH 7.2 in the M162 medium supplemented by 1–2% (w/v) n-decane or tridecane by a 3 liter fermenter (Chemap), with low mechanical agitation (100 rpm) and an aeration flux of 20 ml min1 for liter of broth. Cell density was estimated by measuring absorbance at 540 nm by direct insertion of culture tubes into DMS 90 VARIAN UV/VIS spectrophotometer. Hydrocarbon depletion was evaluated using GC-MS and NMR analyses on cell free supernatants at interval times of growth. The supernatants were extracted with dichloromethane (v/v) and dried under vacuum. GC-MS was performed on a Hewlett-Packard 5890-5970 instrument, equipped with an HP-V column. The analysis was performed at flow rate of 0.7 ml min1; the temperature program used was: 60°C for 2 min, ramping from 60 to 280°C at 3°C min1 and final step of 10 min at 280°C. NMR spectra were recorded on a Bruker AMX-500 instrument (500.13 MHz for 1H NMR) using CDCl3 as solvent for hydrocarbon samples. Heavy metal resistance. The chemicals assessed for toxicity were NiSO4 · 6H2O (Carlo Erba), ZnSO4 · 7H2O (Aldrich), Co(NO3)2 · 6H2O (J.T. Baker), HgCl2 (J.T. Baker), MnCl2 · 4H2O (J.T. Baker), Cr(NO3)3 · 9H2O (J.T. Baker), K2Cr2O7 (J.T. Baker), CuSO4 · 5H2O (J.T. Baker), FeCl3 (Carlo Erba), CdSO4 (Aldrich). Solutions were prepared by dissolving in distilled water and subsequently filtered. The chosen metal concentrations were calculated as mg/L (ppm part per million) of each metal (Poli et al., 2005). Cells were grown by inoculating 90 ml media in a 250-ml Erlenmeyer flask using the minimal medium

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containing (1%) glucose as sole carbon source. The culture was incubated for 12 h, after which the cells were transferred to fresh media to get an optical density of 0.1 (A540 nm). Each heavy metal was added to fresh media at appropriate concentrations at To (time). Bacterial growth controls and other samples were checked by measuring spectrophotometrically at 540 nm, using UV Spectrophotometer (Varian DMS 90). Cells were harvested in late exponential growth phase by centrifugation at 9,000g for 30 min. Genetic studies. The almost complete 16S rRNA gene sequence was determined by direct sequencing of PCR-amplified 16S rDNA. Genomic DNA extraction, PCR mediated amplification of the 16S rDNA and purification of PCR products were carried out as described previously (Rainey et al., 1996). Purified PCR products were sequenced using the ABI PRISMtm Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Germany) as specified in the manufacturer’s protocol. Sequence reactions were electrophoresed using the Applied Biosysthems 373A Sequencer (Rainey et al., 1994). The resulting sequence data were put into the alignment editor ae2 software and compared with representative 16S rDNA sequences of organisms belonging to the Geobacillus group (Maidak et al., 1999). For comparison 16S rDNA sequences were obtained from the EMBL data base (Maidak et al., 1999). The 16S rDNA sequence of strain L1T has been deposited in EMBL database under the accession number AJ966346. The results of alignments are presented, in RESULTS AND DISCUSSION, as similarity matrix and phylogenetic tree (Saitou and Nei, 1987). The 16S rDNA similarity values were calculated by pair-wise comparison of the sequences within the alignment. For construction of the phylogenetic dendrogram, the PHYLIP package was used (Felsenstein, 1993); pair-wise evolutionary distances were computed from percent similarities by the correction of Jukes and Cantor (1969) and the phylogenetic tree was constructed by the neighbour-joining method (Higgins et al., 1992). The GC content was determined by HPLC method (Mesbah et al., 1989; Tamaoka and Komagata, 1984). The calibration was performed with non-methylated Lambda-DNA (Sigma) (GC content 49.85 mol%) and with Halomonas pantelleriensis DNA (GC content 65.02 mol%). Random Amplified Polymorphic DNA-PCR (RAPDPCR) assay was used to produce fingerprint patterns

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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 m l 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 m M dNTPs, 0.5 m M of OPR-2 primer (5-CACAGCTGCC-3) or OPR-13 primer (5GGACGACAAG-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 m l of PCR products were electrophoresed on 2% agarose gel (Agarose-1000, Invitrogen) in 1 TAE buffer at 5 V/cm for 4 h. Ethidium bromide (0.1 m g/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 French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 66 multicell changer and a temperature controller with in situ temperature probe (Varian). Results and Discussion

Compost parameters The compost was obtained by using 50% of organic urban waste and 50% green brush waste. Figure 1 summarizes the flow-chart of the process. During the fermentation, the temperature ranged from 60 to 70°C, the oxygen concentration was 10–20% (v/v), the pH ranged from 6.0 to 7.5 and the humidity decreased from 60 to 20%. Chemical analysis of compost material was reported in Table 1. Zinc, Copper, and Lead were the main heavy metal detected. Plastic material was found in a much lower amount. Among bacterial tests performed, only Enterobacteriaceae and Strepto-

Fig. 1.

Table 1.

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Flow-chart of composting process.

Characteristics of hot compost from “Pomigliano Environment.”

Parameters

Cadmium Chromium VI Mercury Nickel Lead Copper Zinc Plastics3.33 mm Plastics3.33 mm Salmonella Enterobacteriaceae Streptococcus faecalis Nematode Trematode Cestode Infestant seeds

Unity

Standard values

mg/kg d.c. 1.5 mg/kg d.c. 0.5 mg/kg d.c. 1.5 mg/kg d.c. 50 mg/kg d.c. 140 mg/kg d.c. 150 mg/kg d.c. 500 % d.c. 0.45 % d.c. 0.05 — Absent /25 g CFU/g 1100 NPM/g 11,000 NPM/50 g Absent NPM/50 g Absent NPM/50 g Absent N Absent

Measured values 1.34 0.5 1.0 11.6 78.5 129.2 256.9 0.01 0.01 Absent 1,800 200 Absent Absent Absent Absent

d.c., dry compost; CFU, colony forming units; NPM, numeric ponderable media; N, number.

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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 m m long and 0.5 m m wide (Fig. 2). Spores oval, located terminally, first appeared on medium TH with 2% MnCl2. Growth of strain L1T occurred at 55–75°C with an optimum of 68°C (no growth was observed at 50°C and 80°C). Until now no strains of thermophilic bacilli isolated from hot compost were able to grow at temperatures above 70°C. At optimum temperature, growth occurred between pH 5.0–9.0 with an optimum at pH 7.2. The isolate was able to utilize a large variety of sugars and hydrocarbons. The bacterium utilized synthetic media and did not require any growth factors and vitamins. Considerable growth was observed on TH agar and YN agar. The isolate was catalase, tyrosine decomposition, hippurate and gelatine hydrolysis, posi tive. Isolate L1T was able to reduce NO 3 but not NO2 and was sensible to lysozyme while it was oxidase, casein and starch hydrolysis, indole production, phenylalanine deamination and urease negative. Isolate L1T was negative for xylanase, b -glucosidase, a -mannosidase, a -amylase, esterase, b -galactosidase activities while it possessed a -galactosidase and a glucosidase activities. The isolate L1T utilized a wide range of carbon sources including ribose, glucose, glycerol, trehalose, maltose, cellobiose, ethanol and raffinose. The isolate was sensitive to kanamycin (5 m g), bacitracin (10 U), novobiocin (30 m g), streptomycin (25 m g), tetracycline (30 m g) and penicillin G (10 U). Lipid and fatty acid compositions Strain L1T possessed complex lipids based on fatty acid. The total lipid contents accounted for 10% and 9% of dry weight for culture grown in TH medium and hydrocarbon media, respectively. Under these conditions three major phospholipids and one phosphoglycolipid were present. In particular, using n-decane

Fig. 2. Scanning electron microscopy of strain L1T grown on n-decane medium, in fermenter culture at 68°C. The sample morphology is observed by using a SEM Philips XL 20 series microscope.

medium an additional phospholipid was detected. The fatty acid compositions, determined from cells grown in TH medium or n-decane medium, were characterized by the abundance of branched acyl chains; in particular strain L1T contained major amounts of iso-C16:0 (46%) and iso-C17 : 0 (28%) when cultured in TH medium, and iso-C16 : 0 (55%), iso-C17 : 0 (11%), nC17 (16%) using the n-decane medium. The membrane lipids are characterized by an elevated number of phospholipids composed of branched fatty acids and by the presence of a quinone MK7 type. IsoC16 : 0 and iso-C17 : 0 fatty acids were predominant while iso-C15 : 0 was absent, in contrast with the typical fatty acid composition of bacteria from the genus Geobacillus. The pattern of the complex lipids has allowed to establish the taxonomic position of L1T by assigning it to the Bacteria Domain. The phenotypic characteristics are coherent for strain L1T belonging to the genus Geobacillus. Growth on hydrocarbons Growth studies of strain L1T on hydrocarbon media revealed its ability to utilize different hydrocarbon substrates such as pentadecane, tridecane, n-decane, squalane, and toluene, as unique carbon and energy sources (Fig. 3). In particular n-decane supported efficiently the growth reaching 1 g/L of wet cells. Growth curves of L1T strain in the TH and n-decane media are compared (Fig. 4). The doubling time (t d) of strain L1T was 2 h in TH medium and 6 h using hydrocarbon sub-

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

Fig. 4. Growth curves of strain L1T on TH medium () and on M162 medium containing n-decane ().

strate. Figure 5 showed 1H NMR spectra of the cell free supernatant dichloromethane extract at time 0 (Fig. 5a) and the supernatant dichloromethane extract at time 24 h (Fig. 5b), respectively. The characteristic chemical shifts of n-decane (Fig. 5a) disappeared completely after 24 h of incubation (Fig. 5b). These data were also confirmed by GC-MS analyses (data not reported). Heavy metal resistance The isolate L1T has been grown on media containing various concentrations of heavy metals. The microorganism showed resistance to all heavy metal tested during the growth (Fig. 6). Only Cd2 and Zn2 caused a decrease of the growth when added at low concentrations. In particular Cd2 affected the growth

Fig. 5. The 1H NMR spectra of the cell free supernatants extracted with dichloromethane (v/v), obtained by a) the extraction was performed at time 0 of incubation; b) the extraction was performed at time 24 h of growth. The 1H NMR spectra were recorded of a solution of 3 mg of extract in 0.6 ml of CDCl3 at 25°C. The resonances for the n-decane compound appeared at d 0.88, d 0.90 and 1.26 ppm (Fig. 5a).

(up to 80%) of strain L1T at 40 ppm and Zn2 caused a decrease of growth (up to 70%) at 60 ppm (Fig. 6). No significant differences were noted for the strain grown in the presence of Mn2, Fe2, and Cu2 up to 300 ppm of concentration (Fig. 6). Strain L1T was not susceptible to the presence of Cr3 at 300 ppm while the presence of Cr6 at 60 ppm caused 50% of growth inhibition. This study confirmed that thermophilic Geobacillus strain plays an important role in organic matter degradation during the compost process. Microorganisms inhabiting extreme environments are often producers of

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unusual enzymes and biomolecules in order to survive high temperatures and high concentrations of heavy metals. Several thermotolerant and thermophilic Bacillus spp. show a different pattern of heavy metal resistance and a different ability to grow on hydrocarbon compounds. Indigenous hydrocarbon degraders are of

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special significance for the bioremediation of oil-polluted water and soil. Strain L1T displaying the ability to grow on media contained hydrocarbons as n-decane, squalane, and tri-decane, as sole carbon sources, meaning resistance 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.

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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 Geobacillus 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 MATERIALS AND METHODS. Both OPR-2 (Fig. 8, lines 1, 2, 3) and OPR-13 (Fig. 8, lines 4, 5, 6) primers produced fingerprints patterns characterized by relevant bands within 2,500–400 bp. L1T and G. toebii differed at level of bands 500 and 700 bp utilizing OPR-2 primer and at level of band 2,500 bp using OPR-13 primer. L1T and Geobacillus caldoxylosilyticus showed a different fingerprints using both primers. The phylogenetic analysis (16S rDNA % of similarity and PCR-fingerprint) confirms that strain L1T belongs to the genus Geobacillus (Stackebrandt and Goebel, 1994). DNA-DNA hybridization experiments between strain L1T and G. toebii SK-1T, showing low 16S rDNA similarity level (97%), revealed a border line re-association value between the new isolate with G. toebii SK-1T (73.7%) and a lower value with Geobacillus caldoxylosilyticus (55.9%) (Wayne et al., 1987). Table 2 indicates the difference in the phenotypic and biochemical characteristics between strain L1T and G. toebii. Strain L1T differed from G. toebii in optimal growth temperature, pH range, reduction of NO 2, utilization of substrates, casein and gelatin hydrolysis and fatty acid compositions (Table 2). On the basis of these results, we propose that thermophilic Geobacillus strain L1T is a novel subspecies of Geobacillus toebii, for which the name Geobacillus toebii subsp. decanicus is proposed.

Fig. 8. RAPD-PCR fingerprint patterns of L1T and reference strains. PCR analysis was performed as described in MATERIALS AND METHODS by OPR-2 primer (5-CACAGCTGCC-3) (lanes 1–3) or OPR-13 primer (5-GGACGACAAG-3)(lanes 4–6). MW, 100 bp DNA ladder (Invitrogen); lanes 1 and 4, Geobacillus toebii DSM 14590T; lanes 2 and 5, strain L1T; lanes 3 and 6, Geobacillus caldoxylosilyticus ATCC 42125T.

Table 2.

Comparison of the phenotypic characteristics of strain L1T with related species.

Characteristic

Temperature range (°C) Optimum temperature (°C) pH range Gelatin hydrolysis Casein hydrolysis Fermentation of glucose Denitrification G+C content (mol%) FAME composition (%): nC15 : 0 iC15 : 0 iC16 : 0 nC16 : 0 iC17 : 0 nC17 : 0 nC18 : 0

Strain L1

Geobacillus toebii strain SK-1a

60–70 68 5.0–9.0     43.5

45–70 60 6.0–9.0     44.0

6.2  46.1 8.6 28.1 6.1 4.8

 34.03 17.46  34.86  

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

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Description of Geobacillus toebii subsp. decanicus subsp. nov. G. toebii subsp. decanicus (de. ca. ni. cus. N.L. n. decane common name for hydrocarbon; able to grow on n-decane; N.L. masc. adj. decanicus pertaining to n-decane). Gram positive, aerobic, motile, spore forming bacterium, 2.0–3.0 m m long and 0.5 m m wide, growth occurs at 55–75°C with optimum growth occurring at 68°C. It grows at pH 5.0–9.0 (optimal pH 7.2). Grows on M162 mineral medium up to and in 1–2% n-decane, tridecane, pentadecane, or squalane as sole carbon sources. It is able to grow on glucose, ribose, trehalose, glycerol, maltose, cellobiose, ethanol, raffinose. It is resistant to different heavy metals added during the growth: Cu2, Mn2, Fe2, Co2, Hg2 and Ni2. It is catalase, tyrosine decomposition, hippurate  and gelatine hydrolysis positive, reduces NO 3 to NO2 ; it is sensitive to lysozyme, negative for oxidase, starch and casein hydrolysis, urease, indole production, phenylalanine deamination. Positive results are obtained for a -galactosidase and a -glucosidase activities. MK7 is the predominant quinone. i C16 : 0 and i C17 : 0 are the major fatty acids (74% of total fatty acids). The following antibiotics inhibit the growth: kanamycin (5 m g), bacitracin (10 U), novobiocin (30 m g), streptomycin (25 m g), tetracycline (30 m g) and penicillin G (10 U). The mol% GC content of DNA is 43.5 mol%. The EMBL accession number for the 16S rDNA sequence of strain L1T is AJ 966346. Type strain is L1T (DSM 17041TATCC BAA 1004T). Isolated from hot compost “Pomigliano Environment” s.p.a., Pomigliano, Naples, Italy. Acknowledgments This work was partially supported by Regione Campania. We thank Valeria Calandrelli and Eduardo Pagnotta for technical assistance, Ottavio De Luca for LC-MS analyses, Vincenzo Mirra and Salvatore Zambardino for NMR service. References Ahmad, S., Scopes, R. K., Rees, G. N., and Patel, B. K. C. (2000) Saccharococcus caldoxylosilyticus sp. nov., an obligately thermophilic, xylose-utilizing, endospore-forming bacterium. Int. J. Syst. Evol. Microbiol., 50, 517–523. Ash, C., Farrow, J. A. E., Wallbanks, S., and Collins, M. D. (1991) Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA. Lett. Appl. Microbiol., 13, 202–206.

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