 Springer-Verlag 1996

Appl Microbiol Biotechnol (1996) 46: 593–596

ORIGINAL PAPER

A. Mutzel · U. M. Reinscheid · G. Antranikian R. Mu¨ller

Isolation and characterization of a thermophilic bacillus strain, that degrades phenol and cresols as sole carbon source at 70 ºC

Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996

Abstract A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed.

Introduction Aromatic compounds are common pollutants in many industrial waste waters produced from oil refineries, petrochemical plants, coal-conversion plants and phenolic-resin industries. Aromatic substances are hazardous pollutants. Their toxicity to microorganisms means that aromatic compounds often result in the breakdown of waste-water treatment plants by inhibi-

tion of microbial growth even at relatively low concentrations (Hinteregger et al. 1992). Concentrations as low as 0.25 mM have been reported to inhibit growth of mesophilic microorganisms in waste-water treatment plants (Gurujeyalakshmi and Oriel 1989). The degradation of aromatic compounds has been investigated in detail under mesophilic conditions, especially in gram-negative bacteria, but little information is available on thermophilic microorganisms degrading environmental contaminants. This is surprising, given the biotechnological importance of thermophilic bacteria as sources of thermostable enzymes and other products of industrial interest. Higher temperatures in the treatment of contaminated wastes have the advantages of increasing the solubility of the substrates and lowering the risk of contamination by pathogenic microorganisms. The aim of this study was to isolate bacteria that grow at high temperatures on phenols without the addition of complex supplements to the medium, that show tolerance towards higher concentrations of phenols and, at the same time, use a wider range of phenols as substrates.

Materials and methods Media and culture conditions

A. Mutzel · U. M. Reinscheid · R. Mu¨ller (&) Department of Biotechnology II, Technical Biochemistry, Technical University of Hamburg-Harburg, Denickestraße 15, 21073 Hamburg, Germany. Fax: 0049 40 7718-2127 email: [email protected] G. Antranikian Department of Biotechnology I, Technical Microbiology, Technical University of Hamburg-Harburg, Hamburg, Germany

Bacillus sp. A2 was enriched on medium A containing, per litre 1.0 g NH4Cl, 0.42 g Na2HPO4, 0.18 g NaH2PO4·H2O, 0.1 g MgCl2·7H2O, 0.1 g CaCl2 · 2H2O, 0.4 g KCl, 1 mg FeSO4·7H2O 10 ml trace element solution according to Balch et al. (1979), 10 ml solution of vitamins according to Wolin et al. (1964), 0.1 g tryptone and 0.1 g yeast extract. To test for growth on aromatic compounds as sole carbon and energy source, medium A was used without tryptone and yeast extract. Liquid cultures were incubated at 70 °C in a shaking incubator rotating at 150 rpm in tightly closed vials. Solid media, containing 1.5% agar, were also incubated at 70 °C; the petri dishes were sealed in plastic bags to reduce evaporation. All chemicals used were of analytical grade from commercial sources.

594 Isolation and maintenance of Bacillus sp. A2 Strain A2 was isolated from a water and mud sample from a hot spring of the Hveragerdi area in the south of Iceland. An aliquot of the sample was diluted tenfold with medium A then phenol was added to a final concentration of 1 mM; 5 ml vials were incubated at 70 °C for 1 week. The phenol disappeared during this treatment. A 100-ll sample of the culture was then transferred into 2 ml medium A containing 1 mM phenol and similar culture samples were subsequently transferred every day. Pure bacterial cultures were obtained by dilution of the final cultures and inoculation on agar plates containing medium A with 1 mM phenol. Single colonies were isolated and transferred to nutrient broth to test purity. The strain obtained was either stored on agar plates containing medium A at room temperature and transferred every 4 weeks or frozen at )80 °C in Microbank vials. (Mast Diagnostica, Reinfeld, Germany). The strain has been deposited at DSM, Braunschweig under the accession number DSM 11100.

Results Isolation of Bacillus sp. A2 Samples from various sources (geyser area, Iceland; Hveragerdi hot spring, Iceland; hot spring, Bulgaria; hot spring, California; compost, Hamburg) where temperatures above 60 °C prevailed were incubated in a medium containing traces (0.01%) of yeast extract and peptone and 1 mM phenol as major carbon source. After the disappearance of the phenol, an aliquot was transfered to fresh medium. After several transfers an aliquot was spread onto agar plates containing phenol as carbon source. By transfer of single colonies, a pure culture was finally obtained from one sample from an Icelandic hot spring. The strain was named Bacillus sp. A2.

Partial 16S rDNA sequence determination The partial sequences of the 16 S rRNA gene were determined by direct sequencing of the polymerase-chain-reaction (PCR)-amplifed 16S rDNA by Fred Rainey at DSM, Braunschweig. Genomic DNA extraction, PCR-mediated amplification of the 16S rDNA and purification of PCR products were carried out as described previously (Rainey et al. 1992; Rainey and Stackebrandt 1993). Purified PCR products were sequenced using the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Germany) as directed in the manufacturer’s protocol. The products were electrophoresed using the Applied Biosystems 373 A DNA sequencer. The partial 16S rDNA sequences were aligned against representative sequences of members of the genus Bacillus.

Analysis of phenol and cresols Phenol and related compounds were measured by HPLC with a Merck 50943 LiChroCART 125–4, LiChrospher 100 RP 18, 5-mm column with detection at 275 nm.The mobile phase was methanol water acetic acid (60: 40: 1) at a flow rate of 1 ml/min. Elucidation of the mode of ringcleavage of catechol Bacillus sp. A2 was incubated overnight on Luria broth agar plates. A 0.5 M catechol solution was sprayed onto the colonies. The formation of an intense yellow colour indicated the presence of catechol 2,3-dioxygenase, as described by Buswell (1974). For detection of an ortho-cleavage mechanism the Rothera reaction was carried out according to Reichardt (1978). Growth on aromatic carbon sources The following substrates were tested for their ability to support growth: phenol, benzoate, p-hydroxybenzoate, o-cresol, m-cresol, p-cresol, toluene, o-xylene, m-xylene or p-xylene. Catechol is unstable at 70 °C and was not used for growth experiments. To ensure that the chosen concentrations were not toxic for the bacteria, growth of the cultures in Luria broth in the presence of the substrates was tested. Since no toxic effects were observed at concentrations of 1 mM, all aromatic carbon sources were tested at this concentration.

Characteristics of Bacillus sp. A2 Bacillus sp. A2 formed circular, smooth colonies on Luria broth and phenol medium. The cells of the isolate were gram-positive, endospore-forming rods of 1 lm in the log phase and up to 4 lm in the stationary phase of the culture. Chains of two or more cells were observed. The spores were ellipsoidal and located terminally. The sporangium was slightly swollen. The catalase test was positive. Bacillus sp. A2 grew between pH 5 and pH 8 with optimal growth at pH 6. 16S rDNA sequence data were compared with currently available sequences of organisms belonging to the domain Bacteria. The results showed, that Bacillus sp. A2 belongs to the bacillus rRNA group 5. The partial sequence of 16S rDNA was identical to the closest relatives of Bacillus sp. A2: Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermolevorans. By partial sequencing it was not possible to distinguish between these three species.

Growth characteristics of Bacillus sp. A2 on phenol as sole carbon source Strain A 2 was able to use phenol as the sole carbon and energy source. The degradation of phenol is shown in Fig. 1. Phenol in a 1 mM solution was degraded completely within 12 hours. Bacillus sp. A2 degraded phenol completely up to concentrations of 5 mM. Growth inhibition of Bacillus sp. A2 in mineral medium occurred at phenol concentrations higher than 10 mM. Degradation of phenol by Bacillus sp. A2 occurred at temperatures between 55 °C and 70 °C. Optimal growth of strain A 2 was observed at 65 °C. At 70 °C the growth rate was reduced from 0.2/h to 0.17/h. No growth was detected at 75 °C. At this temperature spores were formed. Cultures incubated at 40 °C for 1 week showed no growth, but, after transfer to 70 °C, the immediate growth of the cultures demonstrated survival of cells.

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Fig. 1 Growth and degradation of phenol by Bacillus sp. A2 in mineral salts medium A without yeast extract or peptone at 65 °C. Cells were incubated in closed 100-ml vials containing 20 ml medium and were shaken at 150 rpm on a rotary shaker. Phenol concentration (r) was measured by HPLC and the cell count (❍) was determined under the microscope

Fig. 3A, B Degradation of cresols (A) and growth (B) of Bacillus sp. A2. Growth conditions and measurements were identical to those described in Figure 1. ❏ Mixture of all isomers, ❍ m-cresol, e ocresol, 3 p-cresol

Degradation pathway and induction of the phenol-degrading enzymes Fig. 2 Temperature dependence of growth of Bacillus sp. A2 on phenol. Growth conditions were the same as described in Fig. 1, only the temperatures were varied. The division rates were calculated from the logarithmic growth phase

Figure 2 shows the effect of temperature on growth of Bacillus sp. A2. Degradation of other aromatic compounds by Bacillus sp. A2 Bacillus sp. A2 was able to use all isomers of cresol (Fig. 3), the growth and degradation rates being practically identical for all three isomers. In a mixture containing all three isomers, simultaneous degradation occurred. In all cases complete degradation occurred within 10 h and no yeast extract or peptone was required. No growth was observed with benzoate, p-hydroxybenzoate, toluene, xylene or o-phenylphenol as the sole source of energy and carbon.

When cells of Bacillus sp. A2 grown on phenol were incubated with catechol a transient yellow colour appeared with a maximum at 375 nm, indicating that phenol is degraded via the meta-cleavage pathway. When colonies of cells grown on nutrient broth agar were sprayed with catechol, an intense yellow colour also appeared, indicating that the enzymes for phenol degradation were constitutively expressed. Even in the presence of 5 g/l glucose, the strain degraded the phenol present in the medium without a lag phase. In the Rothera reaction no purple colour was detected, indicating that Bacillus sp. A2 does not use the ortho-pathway for cleaving catechol.

Discussion In 1974 Buswell described the isolation of a thermophilic bacillus degrading phenol. In later years several thermophilic bacilli with similar properties were found (Adams and Ribbons 1988; Buswell and Twomey 1975; Gurujeyalakshmi and Oriel 1989). According to the partial 16S rRNA sequence, Bacillus sp. A2 isolated in this study belongs to the rRNA group 5 of the bacillus. All of the thermophilic bacilli that degrade aromatic compounds isolated so far have been classified as Ba-

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cillus stearothermophilus, but no 16S rDNA sequences are available. The taxonomy of all the thermophilic bacilli is still a field of intense research. Even White et al. (1993), who did a polyphasic taxonomic study of thermophilic bacilli, stressed the need for more research. Rainey et al. (1994) demonstrated the phylogenetic diversity of the thermophilic members of the genus Bacillus by 16S rDNA analysis. Clearly more work on the taxonomy of thermophilic degraders is necessary to close the gaps in the phylogeny of thermophilic bacilli. The bacilli isolated in previous studies showed optimum growth at temperatures around 55 °C (Adams and Ribbons 1988; Buswell and Twomey 1975; Gurujeyalakshmi and Oriel 1989). Most of these bacteria required the addition of complex substrates, such as yeast extract or peptone, to the medium for the degradation of phenol (Adams and Ribbons 1988). Which compounds in these supplements were really necessary for growth remains unresolved. In contrast, Bacillus sp. A2 isolated in this study grew at significantly higher temperatures, up to 70 °C, on phenols as sole carbon source without the addition of yeast extract or peptone. Furthermore Bacillus sp. A2 tolerates a wide pH range from pH 5 to pH 8, a versatility that is important for treating waste waters with pH fluctuations. There have been two reports (Dong et al. 1992; Natarajan et al. 1994) of thermophilic strains degrading phenol without yeast extract, but, in contrast to Bacillus sp. A2, these strains failed to degrade other aromatic compounds such as cresols without complex supplements. In our Bacillus sp. A2 the enzymes for the degradation of phenols seem to be constitutively expressed, a feature that has not been found in the other strains. Recent petroleum processes, improved in terms of energy reduction, are associated with ever higher effluent temperatures. Therefore, the treatment of effluents with thermophilic organisms will become more and more attractive (Yanase et al. 1992). The unique properties of Bacillus sp. A2 mean that it could be applied to the treatment of such hot industrial effluents contaminated with phenols and cresols. Acknowledgement This work was supported by the European Community (Programme Environment Contract EV5V-CT940540) and by a fellowship of the Graduiertenkolleg Biotechnology

to U. M. Reinscheid and by the City of Hamburg (HSP II) to A. Mutzel.

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