In this study, we isolated five strains capable of degrading 14 C-labeled sulfamethoxazole to 14 CO 2 from a membrane bioreactor acclimatized to sulfamethoxazole, carbamazepine, and diclofenac. Of these strains, two belonged to the phylum Actinobacteria, while three were members of the Proteobacteria.T he antibiotic sulfamethoxazole (SMX) undergoes only partial removal in municipal wastewater treatment plants (WWTP) and is often detected in treated effluents and receiving water bodies at concentrations up to 1.9 g/liter (6, 8). It is suspected to promote the development of antibacterial resistance in water bodies (3). Little information on the bacterial and fungal transformation/degradation of SMX is available. The versatile peroxidase of Bjerkandera adusta was demonstrated to transform SMX to 3-amino-5-methylisoxazole and a range of oxidation products (4). Previous studies reported the transformation of SMX by bacteria from microorganism collections. Rhodococcus rhodochrous and Pseudomonas aeruginosa were shown to cometabolize SMX (5, 7). Only Rhodococcus equi led to the transformation of SMX without glucose as a cosubstrate (7). Among the tentatively identified biotransformation products were a deamination product (5) and acetyl and hydroxyacetyl conjugates of SMX. Nevertheless, until now, no evidence for mineralization of SMX by axenic strains has been reported. This is especially important because removal of SMX can be biased by choosing unfavorable sampling conditions.In the present study, five strains capable of SMX mineralization were isolated from a lab-scale membrane bioreactor (MBR) acclimatized with a synthetic effluent loaded with pharmaceuticals. The 1.5-liter MBR was operated continuously as described in reference 1. The reactor was fed with a complex medium adapted from DIN ISO 11733 (2) and spiked with SMX, carbamazepine, and diclofenac to a final concentration of 100 g/liter (each). It was operated under steady conditions for 10 months before sampling of the biomass to inoculate enrichment cultures. The hydraulic retention time was 12 h, the sludge retention time was infinite, and the total concentration of solids was 6 g/liter, on average. At the time of biomass sampling, the average SMX removal rate in the MBR was 52% (data not shown).Enrichment cultures were prepared in Erlenmeyer flasks containing 100 ml of mineral salts medium (10) containing 0.5 mM SMX as the sole carbon source (MSM-S) and were inoculated with 2-ml samples of biomass from the MBR before incubation at 28°C on a rotary shaker at 130 rpm. Twice, after a 1-month incubation each time, half of the culture volume was replaced with fresh enriched MSM-S. After another month, 3 ml of enrichment culture was used to inoculate 100 ml of fresh MSM-S. A month later, the SMX-degrading enrichment culture was diluted in 0.85% (wt/vol) NaCl and plated on plate count agar (PCA) (medium 464; DSMZ, Germany) and, to possibly select for isolated fungi, Sabouraud agar (Sigma-Aldrich, Switzerland). Plates were incubated overnight at 28°C, and six...

High numbers (10 7 to 10 10 cells per g [dry weight]) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82؇C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windrows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80؇C, with optimum growth between 65 and 75؇C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70؇C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80؇C) of the composting process.

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 thermodenitrijicans") . 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 compost; very few of these strains grew at temperatures above 60"C, and growth at 65°C was restricted to Bacillus coagulans (type A) and Bacillus stearothennophilus. Until recently, only strains related to B. stearothennophilus 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 insufficient for unequivocal identification of Bacillus strains (10, 11). The purpose of the present study was to identify heterotrophic, 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 KNO, per liter. GA, P, and PN media were synthetic media composed of a basal mineral medium (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 different 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 60°C 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 Metabolic tests were carried out at 55°C with API 20 NE strips (BioMkrieux, Marcy-l'Etoile, France) by using a few fresh colonies suspended in the basal mineral medium supplemented with 0

SummaryA great variety and high numbers of aerobic thermophilic heterotrophic and/or autotrophic bacteria growing at temperatures between 60-80°C have been isolated from thermogenic (temperature 60-80°C) composts in several composting facilities in Switzerland. They include strains related to the genus Thermus (T. thermophilus, T. aquaticus. and several other new strains). Bacillus schlegelii, Hydrogenohacter spp., and of course heterotrophic sporeforming Bacilli. This contrasts with the generally held beliefthat thermogenic composts (> 60°C) support only a very low diversity of heterotrophic thermophiles. This biodiversity suggests efficient decomposition of organic matter at temperatures above 60°C, and a good thermo-hygienization.During the terminal cooling or maturation phase of composts high numbers and a great metabolic diversity of mesophilic bacteria was observed, including nitrogen-fixers, sulfur-oxidizers, hydrogen-oxidizers, nitrifyiers, and producers of extracellular polysaccharides or bacterial humin. This microbial diversity plays an essential roJe for compost stabilization. It is suggested that mature compost application improves soil chemistry and microbiology, and can thus be regarded beneficial for agriculture. lntroductionAmong the various processes used to manage organic wastes (landfiiL incineration), only the biological process of composting can bring about a stabilization of * To whom correspondence should he addressed. M. de Bertoldi et al. (eds.), The Science of Composting

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