1393 tósT*'; JfArch Microbiol (1996) 165:34-40 ORIGINAL PAPER © Springer-Verlag 1996 Trello Beffa • Michel Blanc • Michel Aragno Obligately and facultatively autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts Received: 20 July 1995 / Accepted: 25 September 1995 Abstract A variety of autotrophic, sulfur- and hydrogen- oxidizing thermophilic bacteria were isolated from ther- mogenic composts at temperatures of 60-80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non- spore-forming strains were gram-negative rods growing at 60-80°C, with an optimum at 70-75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7-37.6 mol%) and showed a high DNA:DNA homology (70-87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55-75° C, with an optimum at 65-70° C. These bacteria were able to grow heterotrophically, or au- totrophically with hydrogen; however, they oxidized thio- sulfate under mixotrophic growth conditions (e.g. pyru- vate or hydrogen plus thiosulfate). These strains had sim- ilar DNA G+C content (60-64 mol%) to and high DNArDNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermo- genic composts as habitats of thermophilic sulfur- and hy- drogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This con- trasts with the generally held belief that thermogenic com- posts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Key words Compost • Thermophilic bacteria • Hydrogenobacter ¦ Bacillus schlegelii ¦ Sulfur- and hydrogen-oxidizing bacteria T. Beffa (BI) • M. Blanc • M. Aragno Laboratoire de Microbiologie, Université de Neuchâtel, Rue Emile-Argand 11, CH-2007 Neuchâtel, Switzerland Tel. +41-38-232-261; Fax +41-38-232-231 Introduction Composting is a self-heating, aerobic, solid-phase biode- gradative process of organic waste materials (Waksmann et al. 1939; Finstein and Morris 1975; De Bertoldi et al. 1983). The composting process at the microbial level in- volves several interrelated factors, i.e., metabolic heat generation, temperature, ventilation (oxygen input), mois- ture content, and available nutrients. Temperature reflects both the prior and the current rate of microbial activity. The temperature increase involves a rapid transition from a mesophilic to a thermophilic mi- croflora. The compost ecosystem is limited by excessive heat accumulation. The terminal phase of composting is a cooling and maturation stage. The amount of readily available nutrients becomes a limiting factor that causes a decline in microbial activity and heat output. A high di- versity of bacteria, fungi, and Actinomycetes has been re- ported during the short initial mesophilic phase and the cooling or maturation phase (Finstein and Morris 1975; De Bertoldi et al. 1983). However, the present knowledge of microbial diversity during the thermogenic (> 60° C) phase is surprisingly poor. The high temperatures reached (60-80° C) are often considered to reduce the microbial diversity dramatically (Strom 1985a, b; Nakasaki et al. 1985a, b; Fujio and Kume 1991). At the highest tempera- tures considered (65-69° C), previous studies focused only on obligately heterotrophic bacteria, and only strains related to Bacillus stearothermophilus were identified (Strom 1985a, b). Microbial diversity could be expected during the ther- mogenic phase, where degradation and mineralization of complex organic matter also takes place (Schulze 1962; Nakasaki et al. 1985b). For example, autotrophic sulfur oxidizers oxidize (and, thus, detoxify) the hydrogen sul- fide generated through the mineralization of organic sul- fur compounds, whereas hydrogen-oxidizing bacteria use the molecular hydrogen produced by fermentative reac- tions during transitions from aerobic to anaerobic decom- position (Dugnani et al. 1986; Beffa et al. 1995). Such 35 transitions occur readily in the heterogeneous, biologi- cally active, and oxygen-poor matrix in composts (Miller 1989). The purpose of this study was to provide a better un- derstanding of the taxonomic and functional diversity of highly thermophilic, facultatively or obligately autro- trophic bacteria during the thermogenic stage of the com- posting process (> 600C). Materials and methods Compost facilities and sampling The industrial composting facilities studied (10 sites) represent the main types of composting systems used in Switzerland, such as classical open-air windrows, boxes in a semi-closed hall with au- tomated turning and aeration, or closed bioreactors with automated aeration. Organic materials subjected to composting varied consid- erably and consisted mainly of green waste, wood chips, and kitchen waste, or sewage sludge. The compost facilities were lo- cated 30-250 km from the authors' laboratory. None of the com- post facilities studied used seeding with a commercial compost- starter containing thermophilic bacteria. Enrichment and culture procedures Enrichment, parallel serial dilutions, and cultures were performed in basal mineral medium (Aragno 1991). One gram fresh-weight organic material from hot (60-800C) compost was placed in 10 ml of sterile basal mineral medium and shaken at 150 rpm for 30 min at room temperature. The parallel serial dilutions (lO-'-lO"10) and cultures were incubated at 700C for 1-14 days under micro- aerophilic conditions (5 kPa oxygen). Autotrophic hydrogen-oxi- dizing bacteria were grown under an atmosphere OfH2ZCO2ZO2 (35 kPa: 10 kPa:5 kPa, measured at room temperature) according to Aragno (1991). Autotrophic sulfur-oxidizing bacteria were grown with 20 mM thiosulfate or 5 g I"1 crystalline elemental sulfur (S0) under an atmosphere of N2ZCO2ZO2 (35 kPa: 10 kPa:5 kPa, mea- sured at room temperature). NaHCO3 (60 mM) or a few grains of CaCO3 were added to prevent excessive acidification under sulfur- oxidizing conditions. Pure colonies were isolated by successive plating on the same media solidified with 13 g I-1 agar-agar (Oxoid, Fakola AG, Basel, Switzerland) with or without 20 mM thiosulfate. Gas mixtures were the same as for liquid cultures except that O2 was at 2.5 kPa. Distinct colonies appeared after 1-7 days. Obligately autotrophic strains that oxidize both hydrogen and sulfur were able to form colonies only when the solid medium was supplemented with 20 mM thiosulfate, as previously reported for hydrogen-oxidizing, thermophilic bacteria (Alfredsson et al. 1986). The purity of each culture was routinely checked microscopically and by plating on solid mineral and organic media. We report the minimal and max- imal values of the parallel serial dilutions observed in hot compost samples from 10 different compost facilities. Heterotrophic growth of pure strains of spore-forming bacteria was assessed in liquid basal mineral medium supplemented with 0.2% wZv organic compounds (acetate, pyruvate, D-glucose) or with 0.8% wZv nutrient broth (Merck, Darmstadt, Germany) under an atmosphere of N2ZO2 (45 kPa:5 kPa, measured at room temper- ature) and under normal air conditions. Heterotrophic growth in liquid cultures of pure strains of non-spore-forming, obligately au- totrophic bacteria was determined under an atmosphere of N2ZO2 (45 kPa:5 kPa, measured at room temperature) in basal mineral medium at 65° C supplemented with 0.1% (wZv) organic com- pounds. The organic compounds tested were as follows: D-arabi- nose, D-fructose, D-galactose, D-glucose, D-maltose, D-mannose, D- xylose, soluble starch, acetate, citrate, formate, fumarate, ß-hy- droxybutyrate, gluconate, DL-lactate, malate, pyruvate, succinate, L-alanine, L-arginine, L-aspartate, L-glutamate, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-proline, L-serine, !.-trypto- phan, L-valine, ethanol, isopropanol, methanol, nutrient broth (Merck, Darmstadt, Germany), and yeast extract (Merck, Darm- stadt, Germany). The growth of obligately autotrophic, hydrogen-oxidizing ther- mophilic bacteria (e.g., Hydrogenobacter thermophilic) was se- verely inhibited by the presence of 20 mM pyruvate (Shiba et al. 1984). Accordingly, the effect of 2.5 and 25 mM pyruvate on the autotrophic growth of pure strains of non-spore-forming au- totrophic bacteria was determined in the basal mineral medium un- der an atmosphere of H2ZCO2ZO2 (35 kPa: 10 kPa:5 kPa, measured at room temperature). Cultures were incubated at 75° C during 10 days. Reference or type strains Thermophilic bacteria related to the genus Hydrogenobacter were isolated from the following geothermal sources: Hydrogenobacter thermophilic strain TK-H (Kyshu, Japan; Kawasumi et al. 1984); Hydrogenobacter strain MF-3 (Etna, Italy; Aragno 1992), Hy- drogenobacter strain T-3 (Tuscany, Italy; Bonjour and Aragno 1986), and Hydrogenobacter strain H-I (Borgarfjördur, Iceland; Kristjansson et al. 1985); Calderobacterium hydrogenophilum strain Z-829, (Kamtchatka, USSR; Kryukov et al. 1983). Bacillus schlegelii type strain (DSM 2000, Deutsche Sammlung von Mikroorganismen und Zellkulturen) was isolated from a cold en- vironment (lake sediment, Le Loclat, Switzerland; Schenk and Aragno 1979). Characterization of isolates Cell numbers and types were estimated by phase-contrast micro- scopical examination of enrichment cultures performed from serial dilutions and by the ability to form colonies on solid media, with either thiosulfate or hydrogen as electron source. Morphology and cell size were estimated on actively H2-growing cultures at 70° C using phase-contrast microscopy. Sensitivity to penicillin was tested during 5 days of incubation at 650C in liquid media con- taining 10 mg I"1 benzyl-penicillin (Fluka Chemie, Buchs, Switzer- land) according to the procedure described by Schenk and Aragno (1979). Temperature dependent growth was tested between 5O0C and 85° C at 5° C intervals. DNA base composition and hybridization DNA was isolated by the procedure (slightly modified) described by Jenni et al. (1987). Cells (1-2 g fresh weight) were collected by centrifugation at 6,000 x g for 20 min at 4° C and washed with 15 ml of 10 mM Tris-HCl buffer (pH 7.2) supplemented with NaCl (0.9% wZv). Washed cells were resuspended in 16 ml MUP buffer (7.5 M urea, 120 mM NaH2PO4, 120 mM Na2HP1O4, final pH 7.5) ,and broken by ultrasonic treatment using a Branson model 450 son- icator for 10 min at 35-40 W on ice. The broken cells were mixed with 5 g hydroxylapatite (Fluka AG, Buchs, Switzerland) equili- brated with MUP buffer and left for 1 h with occasional agitation at room temperature. The hydroxylapatite was decanted, washed with additional MUP buffer, and decanted again. The hydroxylapatite- DNA complex was then poured onto a GFZA filter (Whatman, Springfield Mill, UK) in a vacuum-filtration system. The hydroxyl- apatite-DNA was washed twice with 10 ml of MUP buffer and then 4 times with 10 ml of 14 mM sodium phosphate buffer (pH 6.8). The DNA was eluted with 400 mM sodium phosphate buffer (pH 6.8). The DNA fractions were pooled and concentrated with 2- butanol to about 4 ml. The purified DNA was then dialyzed against saline citrate buffer (15 mM NaCl - 1.5 mM Na-citrate). The ratio of absorbance of the DNA preparations at 260 and 280 nm was around 1.9, which indicates that they were essentially free of pro- teins. The concentrated DNA solutions were stored at -200C. DNA 36 mol% G+C content was determined by the melting point (Tm) method (Marmur and Doty 1962) and calculated according to the formula of Owen and Hill (1979). DNA-DNA homologies were measured spectrophotometrically by following the renaturation rates at Tm -200C according to De Ley et al. (1970). Protein gel electrophoresis Cells (about 1 g fresh weight) growing actively under autotrophic conditions (hydrogen) were collected and washed as described above. Washed cells were resuspended in 1-2 ml of 100 mM Tris- HCl buffer (pH 8.0). Cells were broken by ultrasonic treatment us- ing a Branson model 450 sonicator for 0.5-1.5 min at 10-20 W on ice. The broken-cell suspension was centrifuged at 12,000 x g for 10 min at 40C and the supernatant (cell-free extract) was stored at -20° C. Proteins (25-30 Hg per lane) were resolved by denaturing SDS-PAGE (5% stacking gel and 12.5% resolving gel) at 22° C ac- cording to Hames and Rickwood (1990). Gels were stained with Coomassie Brilliant blue R-250 after electrophoresis. Molecular mass standards (14.4-200 kDa) were purchased from Bio-Rad Laboratories (Hercules, Calif., USA). Respiratory activity measurements Cells for respiratory activity measurements were taken from ac- tively growing cultures, washed, and resuspended gently in basal mineral medium according to Beffa et al. (1991a). The respiratory activities were measured polarographically with an oxygen elec- trode (Hansatech, model CBH2, adjustable volume) at 600C as de- scribed previously (Beffa et al. 1991b, 1992). Oxygen consump- tion was calculated on the basis of 134 nmol O2 ml"1 in air-satu- rated medium at 600C according to Beffa et al. (1991b). The final cell concentration in the respiratory cuvette was 0.02-0.2 mg pro- tein ml"'. The respiratory substrates were supplied as follows (final concentration): 0.35 ug H2 ml-1, 10 mM thiosulfate; 10 mM hy- drophilic S0 (free of thiosulfate, obtained as described previously; Beffa et al. 1991a), 15 mM pyruvate. KCN (0.1 M) was freshly dissolved in 0.2 M sodium phosphate buffer at pH 7.5 and used at 1 mM final concentration. Cells were pre-incubated for 3 min at 60° C prior to addtion of the substrate, and the respiratory activities were measured from constant respiratory slopes. Results are ex- pressed as nmol O2 (mg protein)'1 min-1; corrected for the low en- dogenous oxygen uptake. Analytical methods Growth was followed turbidimetrically at 436 nm using 1-cm cu- vettes in a Perkin-Elmer Lambda 6 spectrophotometer. Protein concentration was determined by the method of Bradford (1976) with bovine serum albumin as the standard. Results and discussion We report here the first evidence for the occurrence of suIt fur- and hydrogen-oxidizing, facultatively or obligately chemoautotrophic bacteria growing between 700C and 75° C in thermogenic (60-800C) composts. Thermophilic bacteria numbers in compost samples were estimated by enrichment of serial dilutions: 10^-106 cells (g compost dry weight)-1 for non-spore-forming, obligately au- totrophic sulfur- and hydrogen-oxidizing bacteria, and 10^108 cells (g compost dry weight)-1 for facultatively autotrophic hydrogen-oxidizing bacteria forming spheri- cal spores. Obligately autotrophic bacteria could grow only under microaerophilic conditions (<10 kPa oxygen). They could not grow with the organic compounds and media tested as the sole energy and carbon sources. All isolates except strain THS-13 grew autotrophically on hydrogen in the presence of 25 mM pyruvate. In addition, organic com- pounds did not seem to significantly inhibit autotrophic growth with hydrogen and thiosulfate because the enrich- ments of these bacteria from compost samples that proba- bly contained high concentrations of organic matter scored positive even at the first serial dilution. In contrast, the growth of obligately autotrophic, thermophilic hydro- gen-oxidizing bacteria isolated from geothermal environ- ments (e.g., Hydrogenobacter thermophilus TK-6) was strongly inhibited, particularly by 20 mM pyruvate (Shiba et al. 1984). Facultatively autotrophic bacteria were able to grow on organic compounds or nutrient broth under mi- croaerophilic and normal air pressure conditions. Bacteria related to the latter were unable to grow with inorganic re- duced sulfur compounds or sugars as the sole electron donors. All strains presented in this study were penicillin G sensitive, which proves that they belong to the Bacteria Table 1 Main characteristics of hydrogen- and sulfur-oxidizing autotrophic, thermophilic Bacteria strains isolated from hot compost piles (+ growth, - no growth) Strains Shape and size Spores Sensitivity Gram stain Motility Growth temperature (0C) (min) (max) moI% G+C Growth substrates to penicillin H2 Thiosulfate S0 Acetate Pyruvate Glucose Obligate autotrophic sulfur- and hydrogen-oxidizers THS-Il Rod 0.5 x 2-3.5 urn - + + 60 80 35 + + + THS-13 Rod 0.5 x 2-3.5 um - + - + 60 80 34.7 + + + - - - THS-25 Rod 0.5 x 2-3.5 u.m - + - + 60 80 35.6 + + + - - - TS-63 Rod 0.5 x 3-6 urn - + - - 60 80 37.6 + + + - - - TS-17 Rod 0.5 x 1.5-3 urn - + - + 60 80 36 + + + - - - Facultative autotrophic hydrogen-oxidizers THS-44 Rod 0.6 X 3-6 urn + + ± 55 75 60 + + + TH-102 Rod 0.6 X 2.5-7 um - + ± + 55 75 60.4 + - - + + - 37 100 90 80 Homology (%] 70 60 50 40 30 20 THS 25 THS 11 THS 13 TS 17 T-3 TSG3 MF-3 H-1 TK-H 2-829 Fig.] Percent homology based on DNA:DNA hybridization be- tween five strains isolated from compost (THS-25, THS-I I. THS- 13, TS-17, TS-63) and reference strains related to Hydrogenobac- ler isolated from gcothermal areas in the world. Values are means of 3-5 runs. Hydrogenobacler tberniophllits strain TK-H (Kyshu. Japan: Kawasumi et al. 1984); Hydrogenobacler strain T-3 (Tus- cany, Italy; Bonjour et Aragno 1986, Aragno 1992); Hydro- genobactcr strain MF-3 (Etna, Italy; Aragno 1992); Hydro- genobacler strain H-I (Borgarfjördur, Iceland; Kristjansson et al. 1985); Calderobacterium hydrogenophihiin strain Z-829 (Kamtchatka, Rusia; Kryukov el al. 1983) domain. The main features of these strains are presented in Table 1. All sulfur- and hydrogen-oxidizing, obligately autotro- phic strains had a 35-37.6 mol% G+C content (Table 1), which is similar to those published for the strains related to Hydrogenobacler that have been isolated from gco- thermal areas (see Aragno 1991 and 1992 for a review). Strains THS-II, THS-Ï3. THS-25, and TS-17 shared a high DNA:DNA homology (71-92%) with each other and with Hydrogenobacler reference strain T-3 (Fig. 1). This strain belongs to a DNA:DNA homology group found in geothermal springs in Italy and in the USA (Aragno 1991. 1992; M. Marchiani, Laboratory of Microbiology, Univer- sity of Neuchâtel, Switzerland, personal communication). Strain TS-63 showed no significant homology with strain T3. but a high homology (86¾) with Hydrogenobacler reference strain MF-3, which belongs to another DNA: DNA homology group found in geothermal springs in Italy and in the Azores (Aragno 1991. 1992; M. Marchi- ani, personal communication). Protein profiles showed that Hydrogenobacter-rehucd strains divide into two groups (Fig.2). Strains THS-II. THS-13, THS-25, and TS-17 showed patterns similar to that of strain T-3. However, slight differences among these strains appeared among proteins of 40-45 kDa. Strain TS-63 showed a profile similar to that of strain MF- 3 if one takes into account that the differences observed probably depend on band intensities. Strains Z-829, TK- H, and H-I showed significantly different profiles (data not shown). These results confirmed the DNA:DNA hy- bridization results. Studies recently have been carried out on the phyloge- netic position of the genus Hydrogenobacler, based on the 16S rRNA complete sequence similarities between three strains of Hydrogenobacler (including strain T-3) and Aquifex pyrofilus (strain Kol5a) (Piuille et al. 1994). The Fig. 2 Coomassic brilliant blue-stained SDS-polyacry- lamide gel showing proteins of total cell extracts of ther- mophilic bacteria related to the genus Hydrogenobacler iso- lated from geothermal areas (strains T-3 and MF-3) and from hot composts (strains THS-I I, THS-13, THS-25, TS- 17, and TS-63) Protein molecu- lar si7.c markers are indicated on the right THS-11 THS-13 THS-25 TS-17 T-3 Protein TS-63 MF-3 standards kDa 200 21.5 14.4 ft 18 Table 2 Respirator) activities of five strains isolated from compost |nmol Oj consumed (mg protein) ' min~'|. Cells were grown for 2-3 days al 700C in basal mineral medium supplied with thiosulfate, Na- pyruvate, or H2 as the energy and electron source Strains Growth Respi iratory substrates and activities substrates [nmo I O2 (mg protein) ~' min"1] Hydrogen Hi !('sulfate Elemental Pyruvate sulfur THS-25 Hydrogen 132 97 12 < 1 Thiosulfatc < I 488 358 < I TS-17 Hydrogen I IS < I < 1 < I Thiosulfate < I 514 436 < 1 TS-63 Hydrogen 120 < 1 < 1 < 1 Thiosulfatc < I 694 135 < I THS-44 Hydrogen 154 266 < 1 < I Pyruvate 67 330 < 1 82 TH-102 Hydrogen I 13 22 < 1 < 1 Pyruvate 148 75 < 1 91 present authors have confirmed that the genera Aquife.\ and Hydrogenobacter arc closely related and support the proposal that the Aquifex-Hydrogenòbacter complex be placed in the new order "Aquificales" and that the genera Aquifex and Hydrogenobacter belong to the same family. the "Aquificaceae" (Burggraf el al. 1992). Our results present the first evidence that highly ther- mophilic, chemolithoautotrophic Bacteria related to the genus Hydrogenobacter occur in hot composts, which are short-term, high-temperature habitats and are not confined to the highly specialized, sparse geothermal habitats. The eight facultatively autotrophic strains isolated from hot composts all showed similar G+C (60-63 mol%) and high DNA homology (75-90'¾-) with each other and with the reference strain of Bacillus schlegelii DSM 2000. I'his continus thai all the strains isolated belong to the same species regardless of their geographical origin and environment (Aragno 1992). Table 1 shows the main tax- onomic and metabolic features of one of the spore-form- ing strains, THS-44, and those of the non-spore-forming strain, TH-102. Bacillits-schlegelii-relaled isolates also proved to grow well in nutrient broth under air. When grown with thiosulfate as the sole energy source, strains isolated from compost and related to Hydro- genobacter possessed high thiosulfate- and elemental sul- fur-oxidizing activities (Table 2). In contrast to strains TS- 17 and TS-63, strain THS-25 possessed constitutive thio- sulfate-oxidizing activity when grown on hydrogen. All respirator)' activities were almost totally inhibited (> 85%) by 1 mM KCN, an inhibitor of the terminal cytochrome oxidase of the respiratory chain. The number of types of thermophilic (optimum tem- perature > 65°C), aerobic, chemolithoautotrophic sulfur- oxidizing bacteria is very limited. Hydrogenobacter and related isolates, Aquifex pyrofilm (Huber et al. 1992) and Thermothrix thiopara (Caldwell et al. 1976; Mason et al. 1987) are the only well-characterized Bacteria to date. T. thiopara is however, no longer available from strain col- Fig.3 Phase-contrast micro graphs of three strains isolated from compost and related to Hydrogenobacter (A TS-17; It THS-25; C TS-63) Cultures were incubated at 65°C on solid basal mineral medium supplemented with thiosulfatc and Hi + CO2 + O2 as gas phase. When grown with H2 .nul thiosulfate, cells released sulfur which appeared as white, réfringent pellets {large arrows) and/or sheaths (small arrows) Bar: 10 um I lections, and seems to have been lost (Krisljansson et al. 1994). Thermophilic, aerobic, autotrophic sulfur-oxidiz- ing Bacteria are now most probably represented only by the strains related to Hydrogenobactcr spp. and Aquifex pyrofdus. The strains related to Bacillus schlegelii (THS-44 and TH-102) did not grow autotrophically with inorganic sul- fur compounds as the sole energy and electron source. They possessed a constitutive thiosulfate-oxidizing activ- ity but lacked elemental sulfur-oxidizing activity (Table 2). Some mesophilic heterotrophs have been reported to be able to oxidize thiosulfate (Mason and Kelly 1987). These bacteria may play a dominant role in the oxidation of sulfur compounds in soils and marine environments (Vishniac and Santer 1957; Tuttle and Jannasch 1972). To date, strains related to B. schlegelii constitute the first ev- idence of thermophilic, heterotrophic spore-forming bac- teria able to oxidize sulfur compounds. AU autotrophic hydrogen oxidizers that grow on solid medium with thiosulfate alone or with thiosulfate plus hy- drogen released sulfur, which appeared as white, réfrin- gent sheaths and/or pellets (Fig. 3). Reference strains of Hydrogenobaaer and B. schlegelii grown under the same conditions also released S0 in the growth medium (Beffa et al. 1993). The results presented in this study contrast with the generally held belief that thermogenic composts at tem- peratures above 600C support only a very low diversity of obligately heterotrophic thermophiles related to Bacillus stearothermophilus (Strom 1985a, b). The presence of high numbers of thermophilic, obligately and facultatively sulfur- and hydrogen-oxidizing bacteria in hot composts suggest that they may play a part in mineralization, and particularly in inorganic sulfur compound oxidation dur- ing the thermogenic phase (> 60°) of the composting pro- cess. Acknowledgements We are indebted to Drs. T. Kodama, J. Kristjansson, K.A. Malik, G.A. Zavarzin, and F. Bonjour for send- ing us strains, and to J. Lott Fischer for correcting the manuscript. This research was supported by grants 31-28597-90 and 5002- 038921 of the Swiss National Science Foundation. 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Syst Appi Microbiol 9:347-253 Kawasumi T, Igarashi Y, Kodama T, Minoda Y (1984) hy- drogenobacter tlicrmophilus n. gen. n. spec, an extremely ther- mophilic aerobic hydrogen-oxidizing bacterium. Int J Syst Bacteriol 34:5-10 Kristjansson JK, Ingason A, Alfredsson GA (1985) Isolation of thermophilic autotrophic hydrogen-oxidizing bacteria similar to Hydrogenobacter thermophilus from Icelandic hot springs. Arch Microbiol 140:321-325 Kristjansson JK, Hjörlcifsdottir S, Martcinsson VT. Alfredsson GA (1994) Thermus scotoductus sp. nov.. a pigmentcd-produe- ing thermophilic bacterium from hot lap water in Iceland and including Thermus sp. X-I. Syst Appi Microbiol 17:44-50 Kryukov VR, Savclcva ND, Pusheva MA (1983) Calderobac- lerium hydrogenophilum n. gen. n. spec, an extremely ther- mophilic hydrogen bacterium and its hydrogenase activity. Mi- crobiology 52:611—618 Marmur J. Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temper- ature. J MoI Biol 5:109-118 Mason J, Kelly DP (1987) Thiosulfate oxidation by obligately het- erotrophic bacteria. Microb Ecol 15:123-134 Schulze KL (1962) Continuous thermophilic composting. Appi Microbiol 10:108-122 Shiba H, Kawasumi T, Igarashi Y, Kodama T, Minoda Y (1984) Effect of organic compounds on the growth of an obligately au - totrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. Agric Biol Chem 48:2809-2813 Strom PF (1985a) Effect of temperature on bacterial species diver- sity in thermophilic solid waste composting. Appi Environ Mi- crobiol 50:899-905 Strom PF (1985b) Identification of thermophilic bacteria in solid waste composting. Appi Environ Microbiol 50:906-913 Tuttle JM, Jannasch HW (1972) Occurrence and types of thio- bacillus-like bacteria in the sea. Limnol Oceanog 17:532- 543 Vishniac W, Santer M (1957) The thiobacilli. Bacteriol Rev 21: 195-213 Waksmann SA, Cordon TC, Hulpoi N (1939) Influence of temper- ature upon the microbiological population and decomposition processes in composts of stable manure. Soil Sci 47:83-114 International Journal of Systematic Bacteriology, Oct. 1997, p. 1246-1248 Vol. 47. No. 4 0020-7713/97/S04.00+0 Copyright © 1997, International Union of Microbiological Societies NOTES Rapid Identification of Heterotrophic, Thermophilic, Spore-Forming Bacteria Isolated from Hot Composts MICHEL BLANC,* LAURENT MARILLEY, TRELLO BEFFA, and MICHEL ARAGNO Laboratoire de Microbiologie, Université de Neuchâtel, CH-2007 Neuchâtel, Switzerland The restriction enzyme profiles of 16S ribosomal DNAs (rDNAs) amplified by PCR from thermophilic heterotrophic bacterial strains isolated from composts were compared with those of reference strains. This allowed us to assign all but 1 of 16 strains to four different Bacillus species (namely, Bacillus stearothermophilus, Bacillus pallidus, Bacillus thermoglucosidasius, and "Bacillus lhermodenitrificans"). This study showed that PCR restriction analysis of 16S rDNA contributes to rapid and reliable identification of newly isolated strains belonging to recognized species. A few studies have reported the presence of thermophilic bacteria in hot compost (3,4, 7,19, 20). Strom (19, 20) isolated more than 750 heterotrophic spore-forming strains from com- post; very few of these strains grew at temperatures above 60°C, and growth at 65°C was restricted to Bacillus coagulons (type A) and Bacillus stearothermophilus. Until recently, only strains related to B. stearothermophilus were identified from the hottest compost samples screened (65 to 69°C) (7, 19, 20). The great diversity of thermophilic bacteria related to the genus Bacillus has frequently been emphasized (16, 22), but it appears that only a few of the isolates have properly been identified to date. The morphology of sporulating cells, the shape of colonies, and growth abilities have proved to be in- sufficient for unequivocal identification of Bacillus strains (10, 11). The purpose of the present study was to identify hetero- trophic, thermophilic, spore-forming strains isolated from hot composts by using a rapid molecular method based on the restriction profiles of 16S ribosomal DNA (rDNA) amplified by PCR. Serial dilutions of compost sample suspensions were carried out in five different media. B and DN media consisted simply of nutrient broth (Merck, Darmstadt, Germany); DN medium was supplemented with 2 g of KNO3 per liter. GA, P, and PN media were synthetic media composed of a basal mineral me- dium (1) supplemented with various growth substrates at a concentration of 2 g liter" ' [GA medium contained D-glucose and sodium acetate; P medium contained sodium pyruvate; PN medium contained sodium pyruvate and (NH4)2S04)]. The cultures were incubated under air at 65°C for 1 to 6 days, and pure colonies were isolated by repeated streaking on the same media solidified with agar. Colonies varying in appearance were picked deliberately to try to increase the number of dif- ferent species isolated (the second and third letters of the compost strain designations in Fig. 1 refer to the isolation medium). Pure strains were then routinely cultivated at 600C on B medium supplemented with 2 g yeast extract per liter and solidified with agar (NAY medium). The type and reference strains are listed in Table 1. * Corresponding author. Mailing address: Laboratoire de Microbi- ologie, Université de Neuchâtel, Rue Emile-Argand 11, CH-2007 Neu- châtel, Switzerland. Phone: 41-32-718.22.60. Fax: 41-32-718.22.31. E- mail: Michel.Blanc@bota.unine.ch. Metabolic tests were carried out at 55°C with API 20 NE strips (BioMérieux, Marcy-l'Etoile, France) by using a few fresh colonies suspended in the basal mineral medium supple- mented with 0.1 g of yeast extract per liter and 0.1 g of peptone per liter, unless indicated otherwise. Starch hydrolysis was tested in the basal mineral medium as described by Smibert and Krieg (17). Anaerobic growth (denitrification) was tested on NAY medium plates supplemented with 10 g of KNO1 per liter. Cultures were incubated for 4 days at 60°C in desiccator jars; oxygen was eliminated by using an Anaerocult IS bag (Merck). DNA extraction and purification were carried out as previ- ously described (4), except that the cells were treated with lysozyme prior to guanidium thiocyanate DNA extraction (13). The 16S rDNA was selectively amplified by using oligonucle- otide primers designed to anneal to bacterial 16S rRNA genes as previously described (4). The reaction conditions were as follows: 1 to 5 ng of template DNA, 0.8 U of Goldstar Taq DNA polymerase (Eurogentec, Seraing, Belgium), 5 u.1 of 1Ox Goldstar PCR buffer, 1.5 mM (final concentration) MgCl2, 0.25 U.M forward primer, 0.25 u.M reverse primer, and each deoxynucleoside triphosphate at a concentration of 170 u.M were combined in a total volume of 50 u.1. Amplification was carried out in a model PTC-100 thermal cycler (MJ Research, Inc., Watertown, Mass.) with the following program: a prelim- inary denaturation step was carried out at 95°C for 1 min and was followed by 35 cycles consisting of 30 s at 940C (denatur- ation), 30 s at 62°C (except for the three first touchdown cycles, which were successively at 68, 66, and 64°C), and 1 min at 72°C (extension). For restriction enzyme digestion, 50 ng of the PCR product was mixed with 2 U of Hae\\\ or 1 U of Hinf\ (New England Biolabs, Inc., Beverly, Mass.), Taq\, or Rsa\ (Gibco BRL) and incubated for 4 h according to the manufac- turer's instructions. PCR products and restriction digests were separated by electrophoresis as previously described (4). Phenotypic characterization. The strains studied were iso- lated from 2- to 80-day-old composts as previously described (4); the sample temperatures ranged from 57 to 78°C. All of the strains were rods and formed oval endospores. Spore po- sition varied from central to terminal. These strains were ini- tially supposed to belong to the genus Bacillus, according to the description of Gordon et al. (8). Their maximum growth tem- peratures were between 65 and 72°C. Except for the Bacillus pallidus group, as pointed out previously (22), all of the strains 1246 Voi.. 47. 1997 NOTES 1247 TABLE 1. Reference strains used in this study EMBL I6S Species Strain" rDNA sequence accession no. IS. stcurothermopliilus DSM 22'1'( = ATCt 1298(1'') X60640 (2) B. sieumllicnnopliilus DSM 494 (12) NA' IS. lhcnitoitlucosiiltisiiis DSM 2542'1'( = ATCC 43742"') X60641 (2) (21) Bacillus sp. DSM 6499 (IS) NA "IS. ihcmiodcniiiijicans" DSM 465 (= ATCC 29492) (9) Z26928(14) IS. /xillkliix DSM 3670''' ( = ATCC 51176T) Z26930(14) (15) Bacillus sp. DSM 2349 (5) Z26929(14) " DSM. Deutsche Sammlung von Mikroorganismen. Braunschweig. Germany: ATCC. American Type Culture Collection. Rockville. Md. ''The numbers in parentheses are reference numbers. '' NA, not available. could grow anaerobically with nitrate as a respiratory sub- strate. Glucose, mannosc. and maltose were utilized as growth substrates by all but two strains, as reported previously for most thermophilic bacilli (22), and starch was hydrolyzed by most strains (data not shown). PCR restriction analysis (PRA) of 16S rDNA. The restric- tion fragment length polymorphism profiles of 16S rDNAs (digested with Hae\\\) of the strains isolated from composts and of the reference strains listed in Table 1 formed five groups with distinctive patterns (Fig. 1). To avoid confusion with primer dimer bands, and because of the detection threshold, restriction fragments shorter than 90 bp were disregarded. FIG. I. I'RA profiles of I6S rDNAs, selectively amplified and digested with lineili, of Bacillus reference strains and of 16 strains isolated from hot composts. 0. undigested amplification product: simularli. 'I>X174RF digested with //«<•] 11. See Table I for reference strains. These profiles permitted us to group all but 1 of the 16 strains with the seven reference strains belonging to four species (namely, B. stearothennophilus, B. pallidus, Bacillus themioglu- cosidasius and "Bacillus thennodenitrificans"). The profiles ob- tained with restriction enzymes Hind and Rsal showed no distinctive patterns for the strains tested (data not shown). Theoretical restriction profiles calculated from 16S rDNA sequences available in the EMBL nucleotide sequence data- base were compared with the results obtained for our strains and for the reference strains. Within the first group, the pro- files of reference strains DSM 22 and DSM 494 matched the theoretical profile calculated from the corresponding se- quence of B. stearothennophilus (EMBL accession no. X60640). In the second group, the profile of reference strain DSM 2542 matched the theoretical profile of the sequence EMBL X60641. No 16S rDNA sequence could be found for strain DSM 6499. However, the restriction profiles did show that this strain was related to B. thennoglucosidasius DSM 2542, as described pre- viously (18). In the third group, the profile of the reference strain "B. thennodenitrificans" DSM 465 matched the theoretical profile of the corresponding sequence EMBL Z26928, except for the calculated 206-bp fragment that appeared to be 240 ± 5 bp long on the gel, and this was also true for the four related compost strains. This may have been due to the lack of recog- nition of a restriction site that caused a 25-bp fragment to remain attached to the 206-bp fragment. Taqi restriction pro- files confirmed that this group was distinct from the three other groups (data not shown). In the fourth group, the profiles of reference strains DSM 3670 and DSM 2349 matched the theoretical profiles of the corresponding sequences EMBL Z26930 and EMBL Z26929. respectively. Strain DSM 2349 could be linked to B. pallidus DSM 3670, as has been proposed in previous studies (14, 22). The profile of strain TP-84 could not be related to any 16S rDNA sequence available for thermophilic bacilli. PRA profiles are a powerful tool for identifying new strains related to heterotrophic thermophilic bacilli. The results ob- tained showed, however, that in some cases (e.g., the "B. ther- modenitiificans" group [see above)) the digestion profiles of new strains should be compared with profiles obtained exper- imentally from reference strains, and the restriction profiles calculated from published sequences should not be relied on. By using five different isolation media, we showed that there is a taxonomically and metabolically diverse population of het- erotrophic thermophilic spore-forming bacteria in thermo- genic composts. Few growth tests had diagnostic value for any single group of strains; only the strains related to B. pallidus had common features (particularly the absence of growth un- der denitrificating conditions) that clearly distinguished them from the other groups. Strain TP-84, despite its lack of reac- tivity with most of the substrates tested, like Bacillus thenno- cloacae strains (22), could not be related to this species by its PRA profile. Phenotypic tests can therefore not be relied on for taxonomic grouping of new Bacillus strains, while PRA of 16S rDNA appears to be a promising tool for rapid and reliable identification of newly isolated strains of recognized species. This work was supported by grant 5002-038921 from the Swiss Na- tional Science Foundation. Wc are grateful to Nicole Jeanncret, Johanna Lott Fischer, Pierre- François Lyon, and Valeric Mauron for collaboration and technical assistance. Wc thank Catherine Fischer, Claudio Valsangiacomo, Jean Mariaux. and Jean-Marc Neuhaus for their help. 1248 NOTES Int. J. Syst. Bacteriol. REFERENCES 1. Aragno, M. 1992. Thermophilic, aerobic, hydrogen-oxidizing (Knallgas) bac- teria, p. 3917-3933. In A. Balows, H. G. Triipcr, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotcs, 2nd ed. Springer-Verlag, New York, NY. 2. Ash, C, J. A. E. Farrow, S. Wallbanks, and M. D. Collins. 1991. Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Leu. Appi. Microbiol. 13:202-206. 3. Beffa, T., M. Blanc, and M. Aragno. 1996. Obligately and facultatively au- totrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts. Arch. Microbiol. 165:34-40. 4. Beffa, T., M. Blanc, P.-F. Lyon, G. Vogt, M. Marchiani, J. LoIt Fischer, and M. Aragno. 1996. Isolation of Thermus strains from hot composts (60 to 80°C). Appi. Environ. Microbiol. 62:1723-1727. 5. Daron, H. H. 1973. Nutritional alteration of the fatty acid composition of a thermophilic Bacillus species. J. Bacteriol. 116:1096^1099. 6. Donk, P. J. 1920. A highly resistant thermophilic organism. J. Bacteriol. 5:373-374. 7. Fujio, Y., and S. J. Kume. 1991. Isolation and identification of thermophilic bacteria from sewage sludge compost. J. Ferment. Bioeng. 72:334-337. 8. Gordon, R. E., W. C. Haynes, and C. Hor-Nay Pang. 1973. The genus Bacillus. Agricultural handbook no. 427. Agricultural Research Service, U.S. Department of Agriculture, Washington, D.C. 9. Klaushofer, H., and F. Hollaus. 1970. Zur Taxonomie der Hochthermo- philen, in Zuckerfabrikssaften vorkommenden aeroben Sporenbildner. Z. Zuckerind. 20:465-^170. 10. Logan, N. A., and R. C. W. Berkeley. 1981. Classification and identification of members of the genus Bacillus using API tests, p. 105-140. In R. C. W. Berkeley and M. Goodfellow (ed.). The aerobic endospore-forming bacteria. Academic Press, Ltd., London. United Kingdom. 11. Logan, N. A., and R. C. W. Berkeley. 1984. Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130:1871-1882. 12. Manachini, P. L., A. Craveri, and A. Guicciardi. 1968. Composizione in basi dell'acido desossiribonucleico di forme mesofile tcrmofacoltativc e termofile dell'genere Bacillus. Ann. Microbiol. Enzimol. 18:1. 13. Pitcher, D. G., N. A. Saunders, and R. J. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appi. Microbiol. 8:151-156. 14. Rainey, F. A., D. Fritze, and E. Stackebrandt. 1994. The phylogenetic diver- sity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis. FEMS Microbiol. Lett. 115:205-212. 15. Scholz, T., W. Demharter, R. Henzel, and O. Kandier. 1987. Bacillus pallidus sp. nov., a new thermophilic species from sewage sludge. Syst. Appi. Micro- biol. 9:91-96. 16. Sharp, R. J., P. W. Riley, and D. White. 1991. Heterotrophic thermophilic bacteria, p. 19-50. In J. K. Kristjansson (ed.), Thermophilic bacteria. CRC Press, Inc., Boca Raton, FIa. 17. Smibert, R. M., and N. R. Krieg. 1994. Phenotypic characterization, p. 607-654. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C. 18. Stalder, V., M. Marchiani, M. Aragno, and R. Bachofen. 1994. Character- ization and identification of a new strain of esterase-producing Bacillus thermoglucosidasius "EAEC" from an aerated thermophilic sewage sludge. Microbiol. Res. 149:1-7. 19. Strom, P. F. 1985. Effect of temperature on bacterial species diversity in thermophilic solid-waste composting. Appi. Environ. Microbiol. 50:899-905. 20. Strom, P. F. 1985. Identification of thermophilic bacteria in solid-waste composting. Appi. Environ. Microbiol. 50:906-913. 21. Suzuki, Y., T. Kishigami, K. Inoue, Y. Mizoguchi, N. Eto, M. Takagi, and S. Abe. 1983. Bacillus thermoglucosidasius sp. nov., a new species of obligately thermophilic bacilli. Syst. Appi. Microbiol. 4:487-495. 22. White, D., R. J. Sharp, and F. G. Priest. 1993. A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie van Leeu- wenhoek 64:357-386. International Journal of Systematic Bacteriology, Oct. 1997, p. 1246-1248 Vol. 47. No. 4 0020-7713/97/$04.00+0 Copyright © 1997, International Union of Microbiological Societies NOTES Rapid Identification of Heterotrophic, Thermophilic, Spore-Forming Bacteria Isolated from Hot Composts MICHEL BLANC,* LAURENT MARILLEY, TRELLO BEFFA, and MICHEL ARAGNO Laboratoire de Microbiologie, Université de Neuchâtel, CH-2007 Neuchâtel, Switzerland The restriction enzyme profiles of 16S ribosomal DNAs (rDNAs) amplified by PCR from thermophilic heterotrophic bacterial strains isolated from composts were compared with those of reference strains. This allowed us to assign all but 1 of 16 strains to four different Bacillus species (namely, Bacillus stearothermophilus, Bacillus pallidus, Bacillus thermoglucosidasius, and "Bacillus thermodenitrificans"). This study showed that PCR restriction analysis of 16S rDNA contributes to rapid and reliable identification of newly isolated strains belonging to recognized species. A few studies have reported the presence of thermophilic bacteria in hot compost (3, 4, 7,19, 20). Strom (19, 20) isolated more than 750 heterotrophic spore-forming strains from com- post; very few of these strains grew at temperatures above 600C, and growth at 65°C was restricted to Bacillus coagulons (type A) and Bacillus stearothermophilus. Until recently, only strains related to B. stearothermophilus were identified from the hottest compost samples screened (65 to 690C) (7, 19, 20). The great diversity of thermophilic bacteria related to the genus Bacillus has frequently been emphasized (16, 22), but it appears that only a few of the isolates have properly been identified to date. The morphology of sporulating cells, the shape of colonies, and growth abilities have proved to be in- sufficient for unequivocal identification of Bacillus strains (10, 11). The purpose of the present study was to identify hetero- trophic, thermophilic, spore-forming strains isolated from hot composts by using a rapid molecular method based on the restriction profiles of 16S ribosomal DNA (rDNA) amplified by PCR. Serial dilutions of compost sample suspensions were carried out in five different media. B and DN media consisted simply of nutrient broth (Merck, Darmstadt, Germany); DN medium was supplemented with 2 g of KNO3 per liter. GA, P, and PN media were synthetic media composed of a basal mineral me- dium (1) supplemented with various growth substrates at a concentration of 2 g liter"" ' [GA medium contained D-glucose and sodium acetate; P medium contained sodium pyruvate; PN medium contained sodium pyruvate and (NHj2SO4)]. The cultures were incubated under air at 650C for 1 to 6 days, and pure colonies were isolated by repeated streaking on the same media solidified with agar. Colonies varying in appearance were picked deliberately to try to increase the number of dif- ferent species isolated (the second and third letters of the compost strain designations in Fig. 1 refer to the isolation medium). Pure strains were then routinely cultivated at 6O0C on B medium supplemented with 2 g yeast extract per liter and solidified with agar (NAY medium). The type and reference strains are listed in Table 1. * Corresponding author. Mailing address: Laboratoire de Microbi- ologie, Université de Neuchâtel, Rue Emile-Argand 11, CH-2007 Neu- châtel, Switzerland. Phone: 41-32-718.22.60. Fax: 41-32-718.22.31. E- mail: Michel.Blanc@bota.unine.ch. Metabolic tests were carried out at 55°C with API 20 NE strips (BioMérieux, Marcy-l'Etoile, France) by using a few fresh colonies suspended in the basal mineral medium supple- mented with 0.1 g of yeast extract per liter and 0.1 g of peptone per liter, unless indicated otherwise. Starch hydrolysis was tested in the basal mineral medium as described by Smibert and Krieg (17). Anaerobic growth (denitrification) was tested on NAY medium plates supplemented with 10 g of KNO1 per liter. Cultures were incubated for 4 days at 60°C in desiccator jars; oxygen was eliminated by using an Anaerocult IS bag (Merck). DNA extraction and purification were carried out as previ- ously described (4), except that the cells were treated with lysozyme prior to guanidium thiocyanate DNA extraction (13). The 16S rDNA was selectively amplified by using oligonucle- otide primers designed to anneal to bacterial 16S rRNA genes as previously described (4). The reaction conditions were as follows: 1 to 5 ng of template DNA, 0.8 U of Goldstar Taq DNA polymerase (Eurogentec, Seraing, Belgium), 5 |jl! of 1Ox Goldstar PCR buffer, 1.5 mM (final concentration) MgCI2, 0.25 u.M forward primer, 0.25 yM reverse primer, and each deoxynucleoside triphosphate at a concentration of 170 p.M were combined in a total volume of 50 u,l. Amplification was carried out in a model PTC-100 thermal cycler (MJ Research, Inc., Watertown, Mass.) with the following program: a prelim- inary denaturation step was carried out at 95°C for 1 min and was followed by 35 cycles consisting of 30 s at 94°C (denatur- ation), 30 s at 620C (except for the three first touchdown cycles, which were successively at 68, 66, and 64°C), and 1 min at 720C (extension). For restriction enzyme digestion, 50 ng of the PCR product was mixed with 2 U of HaelU or 1 U of Hinfl (New England Biolabs, Inc., Beverly, Mass.), Taql, or Rsa\ (Gibco BRL) and incubated for 4 h according to the manufac- turer's instructions. PCR products and restriction digests were separated by electrophoresis as previously described (4). Phenotypic characterization. The strains studied were iso- lated from 2- to 80-day-old composts as previously described (4); the sample temperatures ranged from 57 to 780C. All of the strains were rods and formed oval endospores. Spore po- sition varied from central to terminal. These strains were ini- tially supposed to belong to the genus Bacillus, according to the description of Gordon et al. (8). Their maximum growth tem- peratures were between 65 and 72°C. Except for the Bacillus pallidus group, as pointed out previously (22), all of the strains 1246 Voi.. 47. 1997 NOTES 1247 TABLE I. Reference strains used in this study Species Strain" EMBL IdS rDNA sequence accession no. Ii. mnirothmiiopliiliis DSM 22T( = ATCC 1298(1''') X60640 (2) (ft)" Ii. xinirolhcimophilttx DSM 494 (12) NA' Ii. llwmwxluamdasiwi DSM 2542'1' (= ATCC 437421) X60641 (2) (2D Bacillus sp. DSM 649«) ( US) NA "B. tlicnnodeiiilrìjicans" DSM 465 (= ATCC 29492) (9) Z26928(14) lì. pullulili DSM 3670'1'(= ATCC 51176T) Z26930(14) (15) Bacillus sp. DSM 2349 (5) Z26929(14) " DSM. Deutsche Sammlung von Mikroorganismen. Braunschweig. Germany: ATCC. American Type Culture Collection. Rockvillc. McI. ''The numbers in parentheses are reference numbers. '' NA. not available. could grow anaerobically with nitrate as a respiratory sub- strate. Glucose, mannosc, and maltose were utilized as growth substrates by all but two strains, as reported previously for most thermophilic bacilli (22), and starch was hydrolyzed by most strains (data not shown). PCR restriction analysis (PRA) of 16S rDNA. The restric- tion fragment length polymorphism profiles of 16S rDNAs (digested with Hae\\\) of the strains isolated from composts and of the reference strains listed in Table 1 formed five groups with distinctive patterns (Fig. 1). To avoid confusion with primer dimer bands, and because of the detection threshold, restriction fragments shorter than 90 bp were disregarded. FlG. I. I*RA profiles of 16S rDNAs, selectively amplified and digested with //«clll. of Bacillus reference strains and of 16 strains isolated from hot composts. G. undigested amplification product; mandarti. 'I>X174RF digested wilh //urlìi. See Table I for reference strains. These profiles permitted us to group all but 1 of the 16 strains with the seven reference strains belonging to four species (namely, B. steamthennopliilus, B. pallidas, Bacillus thermoglu- cosidasius and "Bacillus thennodenitrificans"). The profiles ob- tained with restriction enzymes Hinü and Rsal showed no distinctive patterns for the strains tested (data not shown). Theoretical restriction profiles calculated from 16S rDNA sequences available in the EMBL nucleotide sequence data- base were compared with the results obtained for our strains and for the reference strains. Within the first group, the pro- files of reference strains DSM 22 and DSM 494 matched the theoretical profile calculated from the corresponding se- quence of B. stearothennophilus (EMBL accession no. X60640). In the second group, the profile of reference strain DSM 2542 matched the theoretical profile of the sequence EMBL X60641. No 16S rDNA sequence could be found for strain DSM 6499. However, the restriction profiles did show that this strain was related to B. thermoglucosidasius DSM 2542, as described pre- viously (18). In the third group, the profile of the reference strain "B. thennodenitrificans" DSM 465 matched the theoretical profile of the corresponding sequence EMBL Z26928, except for the calculated 206-bp fragment that appeared to be 240 ± 5 bp long on the gel, and this was also true for the four related compost strains. This may have been due to the lack of recog- nition of a restriction site that caused a 25-bp fragment to remain attached to the 206-bp fragment. Taql restriction pro- files confirmed that this group was distinct from the three other groups (data not shown). In the fourth group, the profiles of reference strains DSM 3670 and DSM 2349 matched the theoretical profiles of the corresponding sequences EMBL Z26930 and EMBL Z26929. respectively. Strain DSM 2349 could be linked to B. pallidas DSM 3670, as has been proposed in previous studies (14, 22). The profile of strain TP-84 could not be related to any 16S rDNA sequence available for thermophilic bacilli. PRA profiles are a powerful tool for identifying new strains related to heterotrophic thermophilic bacilli. The results ob- tained showed, however, that in some cases (e.g., the "B. ther- modenitrificans" group [see above]) the digestion profiles of new strains should be compared with profiles obtained exper- imentally from reference strains, and the restriction profiles calculated from published sequences should not be relied on. By using five different isolation media, we showed that there is a taxonomically and metabolically diverse population of het- erotrophic thermophilic spore-forming bacteria in thermo- genic composts. Few growth tests had diagnostic value for any single group of strains; only the strains related to B. pallidas had common features (particularly the absence of growth un- der denitrificating conditions) that clearly distinguished them from the other groups. Strain TP-84, despite its lack of reac- tivity with most of the substrates tested, like Bacillus therrno- cloacae strains (22). could not be related to this species by its PRA profile. Phenotypic tests can therefore not be relied on for taxonomic grouping of new Bacillus strains, while PRA of 16S rDNA appears to be a promising tool for rapid and reliable identification of newly isolated strains of recognized species. This work was supported by grant 5002-038921 from the Swiss Na- tional Science Foundation. Wc are grateful to Nicole Jeanneret, Johanna Lott Fischer, Pierre- François Lyon, and Valeric Mauron for collaboration and technical assistance. Wc thank Catherine Fischer, Claudio Valsangiacomo, Jean Mariaux, and Jean-Marc Neuhaus for their help. Ì 1248 NOTES Int. J. Syst. Bacteriol. REFERENCES 1. Aragno, M. 1992. Thermophilic, aerobic, hydrogen-oxidizing (Knallgas) bac- teria, p. 3917-3933. In A. Balows, H. G. Triipcr, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer-Verlag, New York, NY. 2. Ash, C, J. A. E. Farrow, S. Wallbanks, and M. D. Collins. 1991. Phylogenese hclcrogcncity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett. Appi. Microbiol. 13:202-206. 3. Beffa, T., M. Blanc, and M. Aragno. 1996. Obligately and facultatively au- totrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts. Arch. Microbiol. 165:34-40. 4. Beffa, T., M. Blanc, P.-F. Lyon, G. Vogt, M. Marchiani, J. Loti Fischer, and M. Aragno. 1996. Isolation of Thermus strains from hot composts (60 to 80°C). Appi. Environ. 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Abe. 1983. Bacillus thermoglucosidasius sp. nov., a new species of obligately thermophilic bacilli. Syst. Appi. Microbiol. 4:487-495. 22. White, D., R. J. Sharp, and F. G. Priest. 1993. A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie van Leeu- wenhoek 64:357-386. ELSEVIER MICROBIOLOGY ECOLOGY FEMS Microbiology Ecology 28 (1999) 141-149 Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods Michel Blanc *, Laurent Marilley, Trello Beffa, Michel Aragno Laboratoire de Microbiologie, Université de Neuchâtel, Rue Emile-Ârgand 11, CH-2007 Neuchâtel, Switzerland Received 3 February 1998; received in revised form 29 September 1998; accepted 27 October 1998 Abstract Thermogenic composts are known to host a variety of thermophilic micro-organisms that were recently investigated by cultural means and identified as Thermits thermophilus, Bacillus spp., and Hydrogenobacter spp. In this paper, we present a classical, cultural enumeration of thermophilic populations on the one hand, and a molecular investigation of the bacterial community by restriction enzyme analyses of a clone library of bacterial 16S rRNA genes on the other hand. Bacterial diversity, revealed by the clone analyses of four samples, was shown to undergo a dramatic change between the young (13-18- day) and the old (39-41-day) samples, possibly linked to the general decrease in temperature and the physicochemical evolution of organic matter during the composting process. Among the 200 clones investigated, 69 clones could be identified as Thermits thermophilus and thermophilic Bacillus spp. These results proved both taxa to be among the dominant bacterial populations at the highest temperatures reached by thermogenic composts. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Compost; Thermophilic Bacteria; 16S rDNA clone library; Thermits; Thermophilic Bacillus 1. Introduction Composting is a self-heating, aerobic, solid-phase process, during which organic waste materials are biologically degraded [1-3]. Among the factors which condition the development of microbial pop- ulations in compost, such as oxygen and nutrient * Corresponding author. Present address: Research & Development, Philip Morris Europe, CH-2003 Neuchâtel, Switzerland. Tel: +41 (32) 888 7616; Fax: +41 (32) 8? e-mail : blanc.michel@pmintl.ch Abbreviations: MPN, most probable number; OTU, opera- tional taxonomic unit availability, the temperature increase up to 65-800C results in a rapid transition from a mesophilic to a thermophilic community [4-6]. This thermogenic phase is followed by a slow temperature decrease where the diversity of micro-organisms increases, fungi and mesophilic bacteria re-establish them- selves, and further biotransformations of the organic matter occur [1,2]. Recently, we developed a set of media adapted to the enumeration of thermophilic bacterial popula- tions. Relatively high numbers of autotrophic bacte- ria, growing at temperatures above 7O0C, were iso- lated from hot composts and characterized [7], as well as large numbers of thermophilic heterotrophic 0168-6496/99/$ 19.00 © 1999 Published by Elsevier Science B.V. All rights reserved. PIl: S0168-6496(98)00099-3 142 M. Blanc el al. IFEMS Microbiology Ecology 28 (1999) 141-149 bacteria related to Thermus thermophilus [8] and Ba- cillus spp. [9-11]. Methods that rely on bacterial cultivation are cur- rently thought to identify only a small fraction (0.01- 10%) of the micro-organisms in natural environ- ments [12-14]. A molecular alternative, that involves DNA extraction followed by PCR amplification and subsequent cloning of 16S rRNA genes, was devel- oped to alleviate the limitation associated with cul- tural approaches, although it is anticipated that this approach may also introduce bias. This technique has been used successfully for marine bacterioplank- ton [15,16], soil environments [17,18], hydrothermal vent systems [19], and for a peat bog sample [20]. In this study, we investigated four hot compost samples, two of them being taken from the mid and late thermogenic phases, respectively. We carried out a most probable number (MPN) determination of thermophilic populations based on the methods previously published [7,8]. In parallel, we estimated the diversity of the community in each sample by a molecular cloning approach. The bacterial 16S rRNA genes were amplified by PCR of DNA ex- tracted by directly lysing the micro-organisms in the compost matrix, and these amplicons were then used to construct a clone library that was subse- quently analyzed by restriction enzyme profiles and partial sequencing of dominant clones. This clone library was compared to the restriction profiles of strains previously isolated from hot composts [8- 10] and of sequences available in the EMBL gene database. 2. Materials and methods 2.1. Composting facility and sampling The industrial composting facility consisted of classic open air windrows (1.5-m high) made up of 45% (v/v) grass, 25-35% kitchen and garden waste, and 10-25% shredded wood. The large pieces of wood retained by the sieving of the mature compost piles were recycled by mixing with fresh organic ma- terial and accounted for 5% of the mixture. The windrows were turned daily. Oxygen and carbon di- oxide were measured using electrochemical cells (M ulti warn P detectors, Drägerwerk, Lübeck, Ger- many). Measurements were made immediately prior to sampling. Compost samples were taken from the core (40 cm from the top) of four different windrows, before turning (August 1996). Each sample (approximately 1 kg) was homogenized by sterile hand-mixing and divided into two subsamples: one was used for MPN determination (inoculation within 3 h), and one was deep frozen in liquid nitrogen and stored at —800C for subsequent DNA extraction. For the dry weight determination, about 100 g of fresh compost was dried for 24 h at 7O0C under vacuum. 2.2. MPN determination To determine the MPN of culturable thermophilic bacteria, 30 g samples of compost (fresh weight) was added to 270 ml of a sterile 0.9% NaCl solution and shaken at 150 rpm for 30 min at room temperature. The samples were then serially diluted (I0-2-10-13) in a basal mineral medium [21] supplemented with nutrient broth and yeast extract, as previously de- scribed (MNY medium, [8]), and in the basal mineral medium supplemented with 20 mM Na2S2O3 (BTM medium). Eight parallel 200-u.l microplate wells were filled with each dilution. MNY plates were incubated under air without shaking, either for 2 days at 6O0C, or for 6 days at 750C, to favor either heterotrophic spore-forming bacteria, or highly thermophilic, heterotrophic non- spore-formers [8]. Wells were scored positive when a distinct cell pellet was visually detected. BTM plates for autotrophic, hydrogen- and sulfur- oxidizing bacteria were incubated without shaking for 8 days at 7O0C under an atmosphere of H2:C02:02 (25:10:3 kPa, measured at room tem- perature) according to Aragno [21]. To avoid confu- sion with oligocarbophilic growth, and taking into account that known hydrogen-oxidizing thermo- philic populations in compost also oxidized thiosul- fate [7], wells were checked for thiosulfate oxidation by adding 25 ul of 0.1 g I-1 bromocresol purple. Wells were considered positive when they turned yel- low as a result of acidification. Cell types were ex- amined with a phase-contrast microscope. MPN values were calculated using the program of Schneider [22] and expressed per gram (dry weight) of compost. M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 143 2.3. Genomic DNA extraction and purification All reagents and glassware were autoclaved or sterile filtered before use. Samples (including an empty tube as negative control) were extracted in duplicates by a modification described below of the procedure published by Lee et al. [23]. Frozen compost material (4.0 g (fresh weight)) was thawed and suspended in 10 ml of a 0.12 M sodium phosphate buffer (pH 8.0), left to stand at room temperature for 10 min with occasional mixing, then centrifuged at 7500Xg for 10 min. The super- natant was discarded and the pellet was suspended for another washing cycle. A volume of 8 ml of lysis solution I (0.15 M NaCl, 0.1 M EDTA (pH 8.0), 10 mg lysozyme ml-1) were added to the washed pellet and incubated at 37°C with occasional mixing for 90 min, and then 8 ml of lysis solution Il (0.1 M NaCl, 0.5 M Tris-HCl (pH 8.0), 10% sodium dodecyl sul- fate) were added. The sample was frozen at -8O0C for 20 min and thawed in a 65°C water bath for 20 min, and this freezing and thawing cycle was re- peated three times [24]. The lysate was centrifuged at 7500 X g at room temperature for 10 min. The supernatant was transferred to fresh tubes and brought to a final concentration of 0.7 M NaCl and 1% CTAB (cetyltrimethyl-ammonium bromide; Serva, Heidelberg, Germany). The lysate was mixed and incubated at 65°C for 10 min, followed by ex- traction with an equal volume of CHQa-isopentylal- cohol (24:1) and centrifugation at 3000 Xg for 5 min. The supernatant was transferred to a fresh tube and 15 ml of 13% polyethylene glycol (MW = 6000, pre- pared in 1.6 M NaCl) were added. This solution was held on ice for 10 min, before centrifugation at 25 000 X g for 25 min. The pellet was then washed once with 70% ethanol, briefly dried at room temper- ature and dissolved in 750 u.1 of TE (10 mM Tris- HCl, 1 mM EDTA (pH 8.0)). A 190-ul volume of 10 M ammonium acetate was added to a final con- centration of 2.5 M ammonium acetate, and the sample was incubated on ice for 10 min. The mixture was centrifuged in a microcentrifuge at 13 000 X g for 10 min. To obtain amplifiable DNA, it proved necessary to repeat the CTAB step to remove contaminating substances, such as humic acids (Andrew Ogram, personal communication; [24]). Consequently, 750 u.1 of the supernatant were brought to a final concen- tration of 1% CTAB and 0.7% NaCl, incubated and extracted with CHCl3-isopentylalcohol as described above. Seven hundred and fifty microliters of the upper aqueous phase was recovered and one volume of ice-cold isopropanol was added. The precipitated DNA was removed from the liquid phase after 10 min on a Pasteur pipette, gently washed in 70% etha- nol, and allowed to dry in open air. The dried pellet was then suspended in 400 u.1 TE and allowed to dissolve overnight at 4°C. Spectrophotometric measurements showed that the DNA extraction yielded 10-35 ug of nucleic acids per g (fresh weight) of compost. The integrity of the DNA was checked by horizontal gel electro- phoresis in 0.8% agarose gels in 0.5 X TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA (pH 8.3)), containing ethidium bromide (0.5 mg I-1). 2.4. Amplification and cloning of 16S rDNA The 16S rDNA was selectively amplified from the purified genomic DNA by PCR using universal oli- gonucleotide primers designed to anneal to con- served positions in the 3'- and 5'-regions of bacterial 16S rDNA, as previously published [8], with minor modifications: 2.5 U Taq DNA polymerase pur- chased from Appligene-Oncor (Gaithersburg, MD) was added in a total volume of 100 u.1, in 1 X reaction buffer according to the manufacturer's instructions and 1-10 ng of compost DNA was used as template. Also, a final extension step at 72°C for 10 min was added to the protocol. PCR products were checked by electrophoresis in 1.3% agarose gels as described above and they appeared as single bands, about 1.4 kbp in size. To test for possible contamination of glassware and solutions by foreign DNA, the above-mentioned negative controls were used as tem- plate DNA and gave no detectable amplification sig- nal. PCR products were excised from 2% low melting agarose (Sigma, St. Louis, MO), the DNA was pu- rified using a Geneclean II kit (Bio 101, La Jolla, CA) or a Qiaex II kit (Qiagen, Hilden, Germany) and amplicons were then ligated into a pGEM-T vector (Promega, Madison, WI). The molar ratio of the ligation reaction mixture was 3:1, in a total volume of 35 l.1. This ligation mixture was used to 144 M. Blanc el al. IFEMS Microbiology Ecology 28 (1999) 141-149 perform 1-7 parallel transformations in Escherichia coli competent cells according to the manufacturer's instructions. Plasmid preparation of 70 randomly picked colo- nies of E. coli per sample was performed, using the alkaline lysis method followed by chloroform extrac- tion [25,26]. Plasmid preparations were checked by electrophoresis in 0.8% agarose gels as described above. Plasmid vectors that did not contain the in- sert migrated faster and were discarded. 2.5. 16S rDNA restriction profile analysis The 16S rDNA genes ligated in the pGEM-T vec- tor were amplified as described previously [9]. Three tetrameric enzymes were used separately for the 16S rDNA restrictions. Aliquots (5 ul) of the PCR prod- ucts were digested in 20 ul reaction volumes, either with 2 U HaeiU restriction endonuclease, 2 U of Hha\ or 2 U of Rsal, according to the manufactur- er's instructions (Gibco BRL, Life Sciences, Bethes- da, MD). The restriction fragments were then sepa- rated by gel electrophoresis in 2.5% agarose gels as described above. Fragments shorter than 80 bp were not taken into consideration, because they were too near the detection threshold. The Shannon diversity index H was calculated from the number of clones in each OTU with the following formula [27]: JV-' -Z-JVj (logJVi-logJV) where N1 is the number of clones per OTU, and JV is the total number of clones per sample. 2.6. Determination of nucleotide sequences Partial sequences of some 16S rDNA genes ligated Table 1 Main features of the four windrow spots sampled" Sample Age (days) Temperature (°C) I 13 68 II 18 82 III 39 64 IV 41 67 in the pGEM-T vector were made by Microsynth (Balgach, Switzerland) using an analytical sequencer. Two hundred and fifty to 690 nucleotides were se- quenced from one side with a T7 primer (single run) and compared to 16S rDNA sequences available in the nucleotide databases by using the BLAST soft- ware [28]. These sequences were deposited at the EMBL database under accession numbers AJOl 1355-AJ011368. 3. Results 3.1. Compost samples Four compost samples were taken from the hottest part of four different compost windrows. Samples I and II were taken from young windrows, whereas samples III and IV were taken from older windrows. The main features of the samples are listed in Table 1. 3.2. MPN of thermophilic bacteria The values calculated are reported in Fig. 1. Phase-contrast microscopic observations of wells showed that the heterotrophic populations growing for 2 days at 60°C were almost exclusively rods, 2—12 urn in length, some of them forming oval endo- spores, as previously reported [8,9,11]. MNY plates incubated at 750C yielded only non- spore-forming rods, filaments and rotund bodies re- lated to Thermus spp. strains as previously described [8,29]. BTM plates wells scored positive for autotrophic bacteria yielded only non-spore-forming, short rods, suggesting them to be Hydrogenobacter spp. strains (data not shown). O2 content (v/v) CO2 content (v/v) 17.8% 3.9% 4.1% 23% 2.8% 21% 13.7% 9.1%, "Measurements were made just prior to sampling. M. Rione et ai IFEMS Microbiology Ecology OH (19W) 141-149 145 i WWiWopMc acoretomwn ¦ I Itmoaopufc non iponjloinm» O Autotrophic baenria Compost samples I ig. I. Most probable numbers (MPN, single values) of three thermophilic bacterial populations in four hoi compost samples. I Ik- sample feattOM are listed in Table 1. Error bars are confi- dence intervals (0.05) with U)O(K) bootstraps [22]. 3.3. Analysis of the clone library A total of 50 clones from each sample were chosen after 16S rDNA recombinant plasmids tested posi- tive for PCR amplification. The clones with identical patterns for the three restriction profiles were grouped in discrete operational taxonomic units (OTUs) as shown in Fig. 2. No restriction profile 35 30 25 Sample Il 20 1 6 11 16 21 26 Operational Taxonomic Units among the clones resembled the E. coli 16S rDNA profile. The Shannon diversity indexes were 0.54 and 1.30 for compost samples I and II, and 1.56 and 1.60 for samples III and IV. Dominant OTUs appeared in the four compost samples shown in Fig. 2 and some of them were identified (Table 2). The identification of OTUs A, E. G and L was based on the comparison with pre- viously published restriction profiles [8.9] and con- firmed by partial sequencing. The restriction patterns of OTU C matched those of the thermophilic Bacil- lus sp. strain TP-84 that we had previously isolated from hot compost [9]. The sequence of TP-84 was deposited at the EMBL database under accession number AJ002I54 and showed 96.2% homology with Bacillus thermosphaericus1* (sequence X90640). The identification of OTU F and OTU J was based on the theoretical profiles calculated from I6S rDNA sequences available in the EMBL data- base for the thermophilic aerobic species Sacchaio- coccus thermophllus (sequence X70430) and Rhodo- thermus mariniti (sequence X77140). respectively. These matches were confirmed by partial sequencing. No other profile could be identified on the basis of Sample III 6 11 16 21 26 31 36 Operational Taxonomic Units 41 » 15 o 10 o L™n B Sample UU I I I I I 1 4 7 10 13 Operational Taxonomic Units Sample IV I I I Il I I I I M I I I I I I I I I I 1 I I I I I I..... II 16 21 26 31 36 41 Operational Taxonomic Units Fig. 2. Distribution among operational taxonomic units (OTUs) of bacterial I6S rDNA clones from four hot compost samples. Letters identify OTUs referred to in the text. 146 M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 Table 2 Identification of OTUs as defined in Fig. 2 OTU Identification based on restriction profiles Closest relatives based on partial sequence homology (%) A B C D E F J K L Thermus lliermoj>hilusTS [8] n.m. Bacillus sp. TP-84 [9] n.m. 'Bacillus thermodenitrificans^ [9] Saccharococcus thermophilusTS Bacillus pallidas''^ [9] n.m. Rhodothermus marinus1 s n.m. Bacillus lliermoglucosidasiusTS [9] Thermus thermophilus'rs (99%) Bacillus sp. TP-84 (98%) Bacillus sp. TP-84 (98%) Thermus thermophilusTS (99-100%) 'Bacillus thermodenitrificans' (99%) Members of the genus Bacillus (92-95%) Saccharococcus lhermophilus1 s (92%) Ammoniphilus oxalalicus (98%) Members of the genus Bacillus (93-96%) Thermophilic members of the genera Thermoanaerohacler, Clostridium, Destilfolomaculum, Bacillus (85-90%) Rhodothermus marinus15 (98%) Members of Micrococcaceae and nocardioforms (89-95%) Bacillus firmusTS (98%) The numbers in square brackets are reference numbers for restriction profiles. TS, type strain, n.m., no match with available sequence profiles of thermophiles. the available thermophilic aerobic Bacteria profiles (genus Bacillus [9], Thermus [8], and unpublished profiles of Hydrogenobacter, Calderobacterium or Aquifex strains). 4. Discussion The MPN values for heterotrophic and autotro- phic thermophiles were shown to be within the range previously reported in similar compost samples [7,8]. However, significant differences appeared between young and old compost samples. The MPN of ther- mophilic aerobic micro-organisms (Fig. 1) showed a 100-fold decrease in Thermus spp. populations be- tween young and old samples. This is probably due to an increased competition with less thermophilic, fast-growing strains when the temperature drops be- low the optimal temperature range for T. thermophi- Ius (70-750C [8,30]). Thermophilic spore-forming bacterial numbers remained almost within the same order of magnitude in both samples. Nevertheless, since thermophilic Bacillus species cannot grow at the temperature measured in sample II (820C) [10,31,32], the high numbers of colony forming units (approximately 7.5 X 109 per g (dry wt.)) detected in this sample were probably endospores rather than actively growing cells, as previously suggested [8]. Autotrophic hydrogen- and sulfur-oxidizer numbers were three to five orders of magnitude fewer than heterotrophs, as previously reported for hot compost [7]. Even if their numerical importance could be con- sidered as minor among the thermophilic popula- tions studied, their specific role might actually prove to be significant for the detoxification of sulfur com- pounds, as previously suggested [7]. Among the 200 restriction profiles investigated during the study, 38 clones of compost I, 22 clones of compost II, 6 clones of compost III and 1 clone of compost IV could be identified to the species level. The identification of OTUs F and G was not posi- tively confirmed by the partial sequence data, but this could conceivably be due to the fact that only part of the 16S rRNA gene was sequenced. The par- tial sequence of the OTU L proved to be closer to the sequence reported for the mesophile Bacillus firmusTS (sequence X60616) than to any other Bacil- lus sp., whereas the theoretical restriction profiles calculated from the sequence of B. firmusTS were identical to those reported for Bacillus lhermo- glucosidasiusTS [9]. In this particular case, the sequencing step showed that the identification of this OTU L based on the restriction profiles was not reliable. To our knowledge, this paper is the first report of clones related to 5. thermophilus [33] and R. marinus [34] to be found in environments other than sugar beet factories or marine hydrothermal vents, respectively. The number of clones investigated determines the threshold of detection. In this case, it is limited to the M. Blanc el al. IFEMS Microbiology Ecology 28 (1999) 141-149 147 most abundant bacterial OTUs. This explains that profiles for hydrogen-oxidizers were not detected, although the strains were shown to be present by the MPN determination. More specific primers could be designed, though, to enhance the detectability of particular strains [14,35]. Moreover, it should be em- phasized that biases in DNA extraction and amplifi- cation [19,35-37] can be reduced using a procedure designed to maximize the extraction of bacterial DNA, along with a subsequent PCR amplification program that proved adapted to poorly amplifiable, highly thermophilic DNA [8], A previous work used random amplified polymor- phic DNA (RAPiD) fingerprinting for characteriza- tion of compost microbial communities [36]. This study yielded characteristic fingerprints of the com- munity at different times and suggested fast changes in population composition in the first days of the composting process in a pilot-scale reactor. Never- theless, the RAPiD technique could not be used to identify discrete, previously known populations among the community. Recently, Hellmann and coworkers [38] and Herrmann and Shann [39] studied the microbial community changes during composting based on the phospholipid fatty acid (PLFA) analysis. These authors showed the PLFA profiles to evolve in a consistent and predictable manner, proving the profiles to be characteristic of specific stages of composting. OTUs distinguished by a combination of three re- striction profiles using different tetrameric enzymes were shown in a model data set to correspond to discrete species, with P > 0.99 [40]. In our study, the bacterial diversity revealed by the clone analysis was therefore shown to undergo a dramatic change between the young and the old samples. As a matter of fact, the indisputable dominance of T. thermophi- lus in the young composts (50% of the clones, OTUs A and D) was no longer observed in the older com- posts (Fig. 2). Also, only four OTUs (A, B, C and G) could be found in more than one sample. It has to be stressed, though, that the samples were taken from four different windrows. Consequently, the differen- ces observed between samples I and II, for instance, account for the variability and the heterogeneity of the compost matter at the same step of the process (about 2 weeks). The physicochemical evolution of organic matter during the composting process (such as depletion of solubles within the first weeks [I]) possibly plays a major role in this population shift. It may be that the higher diversity in the old compost (1.56/1.6 vs. 0.54/ 1.30) indicates that the decreasing temperatures fa- vor the recolonization of the maturing compost by a broader range of micro-organisms. Those micro- organisms were reported to degrade more diverse and less easily degradable materials [1-4,39]. Although composting is essentially an aerobic process, anaerobic decomposition is known to occur in micro-environments of the heterogenous, oxygen- poor matrix of the compost itself [41,42]. Moreover, a decreasing gradient of temperature establishes from the core to the outer part of the compost wind- row within a few hours after turning [2,43]. Given that the windrows are turned each day, the bacterial populations colonizing a particular spot in the pile daily undergo a complete redistribution, a factor which must be taken into account when discussing the biodiversity assessed in a single spot. The media and incubation conditions chosen for the MPN de- termination did not allow potential mesophilic or anaerobic micro-organisms to be revealed. It should be emphasized that numbers of clones found in a clone library are a rough estimate for abundance. Indeed, the study of the true microbial community structure would require hybridization techniques applying specific oligonucleotide probes for taxa of interest. Therefore, further work is needed to better understand how bacterial popula- tions, not only aerobic thermophiles, but also ther- morésistant mesophiles and anaerobes, vary as a function of time and space in a compost pile. Our molecular approach confirms, though, that T. ther- mophilus strains and, to a lesser extent, thermophilic Bacillus spp. are the dominant bacterial populations at the higher temperatures reached by thermogenic composts. Interestingly, the most abundant Bacillus sp. strain, TP-84 (OTU C), is not related to any validly described species and could thus form a new species. Acknowledgments We are indebted to Gudrun Vogt, Noémie Matile, Muriel Anderegg, Antoinette Blanc and to the per- 148 M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 sonnel of Vollenweider AG (Grenchen) for their col- laboration and technical assistance. Many thanks to Andrew Ogram for communicating to us his DNA extraction method before publishing it, to Pierre- François Lyon and Johanna Lott Fischer for provid- ing us with unpublished data and to Catherine Fisch- er for correcting the manuscript. References [I] de Bertoldi, M., Valdini, G. and Pera, A. (1983) The biology of composting: a review. Waste Manag. Res. 1, 157-176. [2] Finstein, M.S. and Morris, M.L. (1975) Microbiology of mu- nicipal solid waste composting. Adv. Appi. Microbiol. 19, 113-151. [3] Waksman, S.A., Cordon, T.C. and Hulpoi, N. (1939) Influ- ence of temperature upon the microbiological population and decomposition processes in composts of stable manure. Soil Sci. 47, 83-114. [4] Beffa, T., Blanc, M., Marilley, L., Lott Fischer, J., Lyon, P-F. and Aragno, M. (1996) Taxonomic and metabolic microbial diversity during composting. 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(1990) Fundamentals and application of windrow composting. J. Environ. Eng. 116, 746-763. BO m ELSEVIER MICROBIOLOGY ECOLOGY FEMS Microbiology Ecology 28 (1999) 141-149 Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods Michel Blanc *, Laurent Marilley, Trello Beffa, Michel Aragno Laboratoire de Microbiologie, Université de Neuchâtel. Rue Emile-Argund 11, CH-2007 Neiicbàtel. Switzerland Received 3 February 1998: received in revised form 29 September 1998: accepted 27 October 1998 Abstract Thermogenic composts are known to host a variety of thermophilic micro-organisms that were recently investigated by cultural means and identified as Thermits thermophilus, Bacillus spp., and Hydrogenobacter spp. In this paper, we present a classical, cultural enumeration of thermophilic populations on the one hand, and a molecular investigation of the bacterial community by restriction enzyme analyses of a clone library of bacterial 16S rRNA genes on the other hand. Bacterial diversity, revealed by the clone analyses of four samples, was shown to undergo a dramatic change between the young (13-18- day) and the old (39-41-day) samples, possibly linked to the general decrease in temperature and the physicochemical evolution of organic matter during the composting process. Among the 200 clones investigated. 69 clones could be identified as Thermits thermophilus and thermophilic Bacillus spp. These results proved both taxa to be among the dominant bacterial populations at the highest temperatures reached by thermogenic composts. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Compost; Thermophilic Bacteria; 16S rDNA clone library; Thermits: Thermophilic Bacillus 1. Introduction Composting is a self-heating, aerobic, solid-phase process, during which organic waste materials are biologically degraded [1-3]. Among the factors which condition the development of microbial pop- ulations in compost, such as oxygen and nutrient * Corresponding author. Present address: Research & Development, Philip Morris Europe, CH-2003 Neuchâtel, Switzerland. Tel: +41 (32) 888 7616; Fax: +41 (32) 888 6808; e-mail; blanc.michel@pmintl.ch Abbreviations: MPN, most probable number; OTU, opera- tional taxonomic unit availability, the temperature increase up to 65-80°C results in a rapid transition from a mesophilic to a thermophilic community [4-6]. This thermogenic phase is followed by a slow temperature decrease where the diversity of micro-organisms increases, fungi and mesophilic bacteria re-establish them- selves, and further biotransformations of the organic matter occur [1,2]. Recently, we developed a set of media adapted to the enumeration of thermophilic bacterial popula- tions. Relatively high numbers of autotrophic bacte- ria, growing at temperatures above 700C, were iso- lated from hot composts and characterized [7], as well as large numbers of thermophilic heterotrophic 0168-6496/99/S19.00 © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S0168-6496(98)00099-3 142 M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 bacteria related to Thermus thermophilus [8] and Ba- cillus spp. [9-11]. Methods that rely on bacterial cultivation are cur- rently thought to identify only a small fraction (0.01- 10%) of the micro-organisms in natural environ- ments [12-14]. A molecular alternative, that involves DNA extraction followed by PCR amplification and subsequent cloning of 16S rRNA genes, was devel- oped to alleviate the limitation associated with cul- tural approaches, although it is anticipated that this approach may also introduce bias. This technique has been used successfully for marine bacterioplank- ton [15,16], soil environments [17,18], hydrothermal vent systems [19], and for a peat bog sample [20]. In this study, we investigated four hot compost samples, two of them being taken from the mid and late thermogenic phases, respectively. We carried out a most probable number (MPN) determination of thermophilic populations based on the methods previously published [7,8]. In parallel, we estimated the diversity of the community in each sample by a molecular cloning approach. The bacterial 16S rRNA genes were amplified by PCR of DNA ex- tracted by directly lysing the micro-organisms in the compost matrix, and these amplicons were then used to construct a clone library that was subse- quently analyzed by restriction enzyme profiles and partial sequencing of dominant clones. This clone library was compared to the restriction profiles of strains previously isolated from hot composts [8- 10] and of sequences available in the EMBL gene database. 2. Materials and methods 2.1. Composting facility and sampling The industrial composting facility consisted of classic open air windrows (1.5-m high) made up of 45% (v/v) grass, 25-35% kitchen and garden waste, and 10-25% shredded wood. The large pieces of wood retained by the sieving of the mature compost piles were recycled by mixing with fresh organic ma- terial and accounted for 5% of the mixture. The windrows were turned daily. Oxygen and carbon di- oxide were measured using electrochemical cells (Multiwarn P detectors, Drägerwerk, Lübeck, Ger- many). Measurements were made immediately prior to sampling. Compost samples were taken from the core (40 cm from the top) of four different windrows, before turning (August 1996). Each sample (approximately 1 kg) was homogenized by sterile hand-mixing and divided into two subsamples: one was used for MPN determination (inoculation within 3 h), and one was deep frozen in liquid nitrogen and stored at —800C for subsequent DNA extraction. For the dry weight determination, about 100 g of fresh compost was dried for 24 h at 700C under vacuum. 2.2. MPN determination To determine the MPN of culturable thermophilic bacteria, 30 g samples of compost (fresh weight) was added to 270 ml of a sterile 0.9% NaCl solution and shaken at 150 rpm for 30 min at room temperature. The samples were then serially diluted (10~2-10-13) in a basal mineral medium [21] supplemented with nutrient broth and yeast extract, as previously de- scribed (MNY medium, [8]), and in the basal mineral medium supplemented with 20 mM Na2S2Os (BTM medium). Eight parallel 200-|il microplate wells were filled with each dilution. MNY plates were incubated under air without shaking, either for 2 days at 600C, or for 6 days at 75°C, to favor either heterotrophic spore-forming bacteria, or highly thermophilic, heterotrophic non- spore-formers [8]. Wells were scored positive when a distinct cell pellet was visually detected. BTM plates for autotrophic, hydrogen- and sulfur- oxidizing bacteria were incubated without shaking for 8 days at 7O0C under an atmosphere of H2:CO2:02 (25:10:3 kPa, measured at room tem- perature) according to Aragno [21]. To avoid confu- sion with oligocarbophilic growth, and taking into account that known hydrogen-oxidizing thermo- philic populations in compost also oxidized thiosul- fate [7], wells were checked for thiosulfate oxidation by adding 25 ul of 0.1 g I-1 bromocresol purple. Wells were considered positive when they turned yel- low as a result of acidification. Cell types were ex- amined with a phase-contrast microscope. MPN values were calculated using the program of Schneider [22] and expressed per gram (dry weight) of compost. M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 143 2.3. Genomic DNA extraction and purification All reagents and glassware were autoclaved or sterile filtered before use. Samples (including an empty tube as negative control) were extracted in duplicates by a modification described below of the procedure published by Lee et al. [23]. <. Frozen compost material (4.0 g (fresh weight)) was thawed and suspended in 10 ml of a 0.12 M sodium phosphate buffer (pH 8.0), left to stand at room temperature for 10 min with occasional mixing, then centrifuged at 7500 X g for 10 min. The super- natant was discarded and the pellet was suspended for another washing cycle. A volume of 8 ml of lysis solution I (0.15 M NaCl, 0.1 M EDTA (pH 8.0), 10 mg lysozyme ml-1) were added to the washed pellet and incubated at 37°C with occasional mixing for 90 min, and then 8 ml of lysis solution II (0.1 M NaCl, 0.5 M Tris-HCl (pH 8.0), 10% sodium dodecyl sul- fate) were added. The sample was frozen at —800C for 20 min and thawed in a 65°C water bath for 20 min, and this freezing and thawing cycle was re- peated three times [24]. The Iysate was centrifuged at 7500 X g at room temperature for 10 min. The supernatant was transferred to fresh tubes and brought to a final concentration of 0.7 M NaCl and 1% CTAB (cetyltrimethyl-ammonium bromide; Serva, Heidelberg, Germany). The lysate was mixed and incubated at 65°C for 10 min, followed by ex- traction with an equal volume of CHCl3-isopentylal- cohol (24:1) and centrifugation at 3000 X g for 5 min. The supernatant was transferred to a fresh tube and 15 ml of 13% polyethylene glycol (MW = 6000, pre- pared in 1.6 M NaCl) were added. This solution was held on ice for 10 min, before centrifugation at 25 000 X g for 25 min. The pellet was then washed once with 70% ethanol, briefly dried at room temper- ature and dissolved in 750 julI of TE (10 mM Tris- HCl, 1 mM EDTA (pH 8.0)). A 190-ul volume of 10 M ammonium acetate was added to a final con- centration of 2.5 M ammonium acetate, and the sample was incubated on ice for 10 min. The mixture was centrifuged in a microcentrifuge at 13 000 X g for 10 min. — ¦ - ~ TT, To obtain amplifiable DNA, it proved necessary to repeat the CTAB step to remove contaminating substances, such as humic acids (Andrew Ogram, personal communication; [24]). Consequently, 750 fxl of the supernatant were brought to a final concen- tration of 1% CTAB and 0.7% NaCl, incubated and extracted with CHCl3-isopentylalcohol as described above. Seven hundred and fifty microliters of the upper aqueous phase was recovered and one volume of ice-cold isopropanol was added. The precipitated DNA was removed from the liquid phase after 10 min on a Pasteur pipette, gently washed in 70% etha- nol, and allowed to dry in open air. The dried pellet was then suspended in 400 ul TE and allowed to dissolve overnight at 4°C. Spectrophotometric measurements showed that the DNA extraction yielded 10-35 Ug of nucleic acids per g (fresh weight) of compost. The integrity of the DNA was checked by horizontal gel electro- phoresis in 0.8% agarose gels in 0.5 X TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA (pH 8.3)), containing ethidium bromide (0.5 mg I-1). .»rs 2.4. Amplification and cloning of 16S rDNA *\ The 16S rDNA was selectively amplified from the purified genomic DNA by PCR using universal oli- gonucleotide primers designed to anneal to con- served positions in the 3'- and 5'-regions of bacterial 16S rDNA, as previously published [8], with minor modifications: 2.5 U Taq DNA polymerase pur- chased from Appligene-Oncor (Gaithersburg, MD) was added in a total volume of 100 ul, in 1 X reaction buffer according to the manufacturer's instructions and 1-10 ng of compost DNA was used as template. Also, a final extension step at 72°C for 10 min was added to the protocol. PCR products were checked by electrophoresis in 1.3% agarose gels as described above and they appeared as single bands, about 1.4 kbp in size. To test for possible contamination of glassware and solutions by foreign DNA, the above-mentioned negative controls were used as tem- plate DNA and gave no detectable amplification sig- nal. PCR products were excised from 2% low melting agarose (Sigma, St. Louis, MO), the DNA was pu- ~~rified using a Geneclean II kit (Bio 101, La Jolla, CA) or a Qiaex II kit (Qiagen, Hilden, Germany) and amplicons were then ligated into a pGEM-T vector (Promega, Madison, WI). The molar ratio of the ligation reaction mixture was 3:1, in a total volume of 35 ul This ligation mixture was used to 144 M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 perform 1-7 parallel transformations in Escherichia coli competent cells according to the manufacturer's instructions. Plasmid preparation of 70 randomly picked colo- nies of E. coli per sample was performed, using the alkaline lysis method followed by chloroform extrac- tion [25,26]. Plasmid preparations were checked by electrophoresis in 0.8% agarose gels as described above. Plasmid vectors that did not contain the in- sert migrated faster and were discarded. 2.5. 16S rDNA restriction profile analysis The 16S rDNA genes ligated in the pGEM-T vec- tor were amplified as described previously [9]. Three tetrameric enzymes were used separately for the 16S rDNA restrictions. Aliquots (5 u.1) of the PCR prod- ucts were digested in 20 u.1 reaction volumes, either with 2 U Haelll restriction endonuclease, 2 U of Hhal or 2 U of Rsal, according to the manufactur- er's instructions (Gibco BRL, Life Sciences, Bethes- da, MD). The restriction fragments were then sepa- rated by gel electrophoresis in 2.5% agarose gels as described above. Fragments shorter than 80 bp were not taken into consideration, because they were too near the detection threshold. The Shannon diversity index H was calculated from the number of clones in each OTU with the following formula [27]: Af-1 -Z-TVi (logTVi-logAO where TV( is the number of clones per OTU, and TV is the total number of clones per sample. 2.6. Determination of nucleotide sequences Partial sequences of some 16S rDNA genes ligated Table 1 '; - Main features of the four windrow spots sampled" in the pGEM-T vector were made by Microsynth (Balgach, Switzerland) using an analytical sequencer. Two hundred and fifty to 690 nucleotides were se- quenced from one side with a T7 primer (single run) and compared to 16S rDNA sequences available in the nucleotide databases by using the BLAST soft- ware [28]. These sequences were deposited at the EMBL database under accession numbers AJOl 1355-AJ011368. • • 3. Results 3.1. Compost samples Four compost samples were taken from the hottest part of four different compost windrows. Samples I and II were taken from young windrows, whereas samples III and IV were taken from older windrows. The main features of the samples are listed in Table 1. 3.2. MPN of thermophilic bacteria The values calculated are reported in Fig. 1. Phase-contrast microscopic observations of wells showed that the heterotrophic populations growing for 2 days at 6O0C were almost exclusively rods, 2-12 urn in length, some of them forming oval endo- spores, as previously reported [8,9,11]. MNY plates incubated at 75°C yielded only non- spore-forming rods, filaments and rotund bodies re- lated to Thermus spp. strains as previously described [8,29]. ¦ ¦- .-. BTM plates wells scored positive for autotrophic bacteria yielded only non-spore-forming, short rods, suggesting them to be Hydrogenobacter spp. strains (data not shown). < " • ¦ -> ¦*'>- ''¦'- .>-.f. ..r-. -------r,:. _r"9.1% „jSMIj~I.M.|iMflH.|.hl>T7 6 11 16 21 26 Operational Taxonomic Units V Sample I £ B l-M-M-l I ITTI 1 4 7 10 13 Operational Taxonomic Units 3 I- Q U) (D C .2 O Sample I Ihl.l I I i l l.ll.l Il IH Il M.I.UI.IjI 6 11 16 21 26 31 36 Operational Taxonomic Units 41 J K Sample IV / IIM-III!.!!.!.!.!.!.!.!.!,!,!.!,!.!.!,!,!.!,!,!!!!:!.!.! 11 16 21 26 31 36 Operational Taxonomic Units 41 Fig. 2. Distribution among operational taxonomic units (OTUs) of bacterial I6S rDNA clones from four hot compost samples. Letters identify OTUs referred to in the text. 146 M. Blanc et al. IFEMS Microbiology Ecology 28 (1999) 141-149 Table 2 Identification of OTUs as defined in Fig. 2 OTU Identification based on restriction profiles Closest relatives based on partial sequence homology (%) A B Ç D E F J K L Thermits ihermophilusrs [8] n.m. Bacillus sp. TP-84 [9] n.m. 'Bacillus thermodenitrificems' [9] Saccharococcus thermopliilusrs Bacillus pallidusK [9] Rhodotliermus marinusTS n.m. Bacillus thermoglucosidasiusTS [9] Tliermus ihermopliilus15 (99'Mi) Bacillus sp. TP-84 (98%) Bacillus sp. TP-84 (98'½) Thermus ihermopliilusrs (99-100%) 'Bacillus lliermodenilrificans' (99%) Members of the genus Bacillus (92-95%) Saccharococcus thermophilusTS (92%) Ammoniphilus oxalalicus (98'½) Members of the genus Bacillus (93-96%) Thermophilic members of the genera Thermoanaerohacler. Clostridium. Desulfotomaculum. Bacillus (85-90%) Rhodotliermus marinus1^ (98%) Members of Micrococcaceae and nocardioforms (89-95%) Bacillus firmus™ (98%) The numbers in square brackets are reference numbers for restriction profiles. TS. type strain, n.m.. no match with available sequence profiles of thermophiles. the available thermophilic aerobic Bacteria profiles (genus Bacillus [9], Thermus [8], and unpublished profiles of Hydrogenohacter, Calderobacterium or Aquifex strains). 4. Discussion The MPN values for heterotrophic and autotro- phic thermophiles were shown to be within the range previously reported in similar compost samples [7,8]. However, significant differences appeared between young and old compost samples. The MPN of ther- mophilic aerobic micro-organisms (Fig. 1) showed a 100-fold decrease in Thermus spp. populations be- tween young and old samples. This is probably due to an increased competition with less thermophilic, fast-growing strains when the temperature drops be- low the optimal temperature range for T thermophi- lus (70-750C [8,30]). Thermophilic spore-forming bacterial numbers remained almost within the same order of magnitude in both samples. Nevertheless, since thermophilic Bacillus species cannot grow at the temperature measured in sample II (82°C) [10,31,32], the high numbers of colony forming units (approximately 7.5 X 109 per g (dry wt.)) detected in this sample were probably endospores rather than actively growing cells, as previously suggested [8]. Autotrophic hydrogen- and sulfur-oxidizer numbers were three to five orders of magnitude fewer than heterotrophs, as previously reported for hot compost [7]. Even if their numerical importance could be con- sidered as minor among the thermophilic popula- tions studied, their specific role might actually prove to be significant for the detoxification of sulfur com- pounds, as previously suggested [7]. Among the 200 restriction profiles investigated during the study, 38 clones of compost I, 22 clones of compost II, 6 clones of compost III and 1 clone of compost IV could be identified to the species level. The identification of OTUs F and G was not posi- tively confirmed by the partial sequence data, but this could conceivably be due to the fact that only part of the 16S rRNA gene was sequenced. The par- tial sequence of the OTU L proved to be closer to the sequence reported for the mesophile Bacillus firmusrs (sequence X60616) than to any other Bacil- lus sp., whereas the theoretical restriction profiles calculated from the sequence of B. firmusrs were identical to those reported for Bacillus thermo- glucosidasiusTS [9]. In this particular case, the sequencing step showed that the identification of this OTU L based on the restriction profiles was not reliable. To our knowledge, this paper is the first report of clones related to S. thermophilus [33] and R. marinus [34] to be found in environments other than sugar beet factories or marine hydrothermal vents, respectively. The number of clones investigated determines the threshold of detection. In this case, it is limited to the M. Blanc el ai IFEMS Microbiology Ecology 28 (1999) 141-149 147 most abundant bacterial OTUs. This explains that profiles for hydrogen-oxidizers were not detected, although the strains were shown to be present by the MPN determination. More specific primers could be designed, though, to enhance the detectability of particular strains [14,35]. Moreover, it should be em- phasized that biases in DNA extraction and amplifi- cation [19,35-37] can be reduced using a procedure designed to maximize the extraction of bacterial DNA, along with a subsequent PCR amplification program that proved adapted to poorly amplifiable, highly thermophilic DNA [8]. A previous work used random amplified polymor- phic DNA (RAPiD) fingerprinting for characteriza- tion of compost microbial communities [36]. This study yielded characteristic fingerprints of the com- munity at different times and suggested fast changes in population composition in the first days of the composting process in a pilot-scale reactor. Never- theless, the RAPiD technique could not be used to identify discrete, previously known populations among the community. Recently, Hellmann and coworkers [38] and Herrmann and Shann [39] studied the microbial community changes during composting based on the phospholipid fatty acid (PLFA) analysis. These authors showed the PLFA profiles to evolve in a consistent and predictable manner, proving the profiles to be characteristic of specific stages of composting. OTUs distinguished by a combination of three re- striction profiles using different tetrameric enzymes were shown in a model data set to correspond to discrete species, with P > 0.99 [40]. In our study, the bacterial diversity revealed by the clone analysis was therefore shown to undergo a dramatic change between the young and the old samples. As a matter of fact, the indisputable dominance of T. thermophi- lus in the young composts (50% of the clones, OTUs A and D) was no longer observed in the older com- posts (Fig. 2). Also, only four OTUs (A, B, C and G) could be found in more than one sample. It has to be stressed, though, that the samples were taken from four different windrows. Consequently, the differen- ces observed between samples I and II, for instance, account for the variability and the heterogeneity of the compost matter at the same step of the process (about 2 weeks). The physicochemical evolution of organic matter during the composting process (such as depletion of solubles within the first weeks [I]) possibly plays a major role in this population shift. It may be that the higher diversity in the old compost (1.56/1.6 vs. 0.54/ 1.30) indicates that the decreasing temperatures fa- vor the recolonization of the maturing compost by a broader range of micro-organisms. Those micro- organisms were reported to degrade more diverse and less easily degradable materials [1-4,39]. Although composting is essentially an aerobic process, anaerobic decomposition is known to occur in micro-environments of the heterogenous, oxygen- poor matrix of the compost itself [41,42]. Moreover, a decreasing gradient of temperature establishes from the core to the outer part of the compost wind- row within a few hours after turning [2,43]. Given that the windrows are turned each day, the bacterial populations colonizing a particular spot in the pile daily undergo a complete redistribution, a factor which must be taken into account when discussing the biodiversity assessed in a single spot. The media and incubation conditions chosen for the MPN de- termination did not allow potential mesophilic or anaerobic micro-organisms to be revealed. It should be emphasized that numbers of clones found in a clone library are a rough estimate for abundance. Indeed, the study of the true microbial community structure would require hybridization techniques applying specific oligonucleotide probes for taxa of interest. Therefore, further work is needed to better understand how bacterial popula- tions, not only aerobic thermophiles, but also ther- morésistant mesophiles and anaerobes, vary as a function of time and space in a compost pile. Our molecular approach confirms, though, that T. ther- mophilus strains and, to a lesser extent, thermophilic Bacillus spp. are the dominant bacterial populations at the higher temperatures reached by thermogenic composts. Interestingly, the most abundant Bacillus sp. strain, TP-84 (OTU C), is not related to any validly described species and could thus form a new species. Acknowledgments We are indebted to Gudrun Vogt, Noémie Matile, Muriel Anderegg, Antoinette Blanc and to the per- 148 M. Blanc el til. IFEMS Microbiology Ecology 28 (1999) 141-149 sonnel of Vollenweider AG (Grenchen) for their col- laboration and technical assistance. 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