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Applied and Environmental Microbiology, October 2006, p. 6819-6821, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00694-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Effect of Octamethylcyclotetrasiloxane on Methylation of Bismuth by Methanosarcina barkeri

Klaus Michalke,^* Jörg Meyer, and Reinhard Hensel

Department of Microbiology, University of Duisburg-Essen, Universitaetsstr. 4, D-45177 Essen, Germany

Received 26 March 2006/ Accepted 21 July 2006

Top Abstract Introduction References

 
Octamethylcyclotetrasiloxane (OMCTS), a common constituent of household products, triggers the transformation of bismuth to the volatile toxic derivative trimethylbismuth by Methanosarcina barkeri, which is a representative member of the sewage sludge microflora. Comparative studies with the ionophores monensin and lasalocid, which induce effects similar to those observed for OMCTS, indicated that the stimulation of bismuth methylation is not specific for the siloxane and suggested that the stimulation observed is mainly due to facilitated membrane permeation of the metal ion.

Top Abstract Introduction References

 
Methylation of metals and metalloids by living cells, mainly prokaryotic microorganisms, is a well-known phenomenon with notable ecotoxicological impact and health concerns, since a great many of the methylated derivatives released into the environment are volatile and more toxic than their inorganic educts (1, 7, 14, 17). Despite the relevance of these processes, the underlying biochemical reactions and their dependence on general or specific environmental conditions are far from being understood, and thus a reliable assessment of the risks of these processes cannot be given yet.

One example of the susceptibility of these processes to single chemical compounds was the surprising observation that cyclic polydimethylsiloxanes trigger the methylation of bismuth to the volatile toxic derivative trimethylbismuth (TMBi) by Methanosarcina barkeri (18), a representative of the methanogenic microflora of anaerobically operated sewage sludge treatment facilities (10, 12). In contrast to other prokaryotes, such as the methanoarchaeon Methanobacterium formicium or the peptolytic bacterium Clostridium collagenovorans, which methylate bismuth without special agents (6), M. barkeri was found to produce the permethylated derivative TMBi only in the presence of cyclic polydimethylsiloxanes, such as octamethylcyclotetrasiloxane (OMCTS) (18).

Cyclic polydimethylsiloxanes are high-volume chemicals used in a wide range of industrial and personal care products (9a) and are believed to be nontoxic (9). Disposal of these compounds results in an increasing load in wastewater treatment facilities and finally in the environment (3). For OMCTS an average concentration of 4 µg liter^–1 (13 nM) in wastewater streams has been reported (8). Although the bismuth concentration in sewage sludges is low (1 to 5 mg kg [dry weight]^–1) (6) compared to the concentrations of other metal(loid)s which might undergo biomethylation, TMBi is one of the most abundant volatile metal(loid)s in sewage gasses (6), which might be explained by the stimulating effect of OMCTS on the biomethylation of bismuth. Furthermore, since increases in the loads of bismuth in wastewater and in sewage sludge can be expected due to the increased use of this metal in technological processes, increased emission of volatile toxic TMBi (13) is expected.

Since previous investigations using deuterium-labeled OMCTS indicated that OMCTS does not have a role as a methyl donor in bismuth methylation by M. barkeri (18), we aimed at characterizing its adjuvant function in the biomethylation of bismuth. Here we compared the efficiency of OMCTS in stimulating bismuth methylation by M. barkeri with the efficiencies of other compounds that resemble OMCTS chemically in order to examine the mechanistic background for this stimulation.

Liquid cultures of M. barkeri DSMZ 800^T (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were grown under strictly anaerobic conditions in 120-ml serum bottles with butyl rubber stoppers containing 50 ml of medium DSMZ120. The culture media were reduced by addition of L-cysteine (0.3 to 0.5 g liter^–1), pressurized with CO₂-H₂ (20:80, vol/vol; 200 kPa), and incubated in the dark in a rotary shaker (150 rpm) at 37°C. In the early exponential growth phase the cultures were supplemented with different concentrations of inorganic bismuth [1 µM to 20 µM Bi(NO₃)₃] and/or with different concentrations of OMCTS (1.7 pM to 34 nM; 98%; ABCR, Karlsruhe, Germany), monensin (1.5 pM to 150 nM; Sigma, Deisenhofen, Germany), or lasalocid (1.7 pM to 170 nM; Sigma, Deisenhofen, Germany). A 100 mM Bi(NO₃)₃ stock solution was prepared in 1,2-propanediol and diluted in double-distilled water to obtain a concentration of 1 mM Bi(NO₃)₃ in 5 mM EDTA and 5 mM Tris, pH 7.0. Dilutions of OMCTS were prepared in dimethyl sulfoxide (Merck, Darmstadt, Germany). Data for OMCTS concentrations less than 1.7 pM showed that the standard deviations for the production of TMBi were high, probably due to undefined adsorption on the inner glass walls of the cultivation vessels, and therefore were not evaluated. Dimethyl sulfoxide had no effect on the methane production by M. barkeri at concentrations of 1 to 5 mg/ml (data not shown). The physiological activities of the cultures were determined before and after addition of the compounds mentioned above by measuring the production of methane by gas chromatography with flame ionization detection (7). TMBi was analyzed by a modified purge-and-trap gas chromatographic system coupled to an inductively coupled plasma mass spectrometer (Fisons VG PlasmaQuad II) (6). Methane production and TMBi production were monitored for approximately 60 days at intervals of 12 to 48 h. After each analysis of volatile bismuth compounds, the gas phase was exchanged with CO₂-H₂ (20:80, vol/vol; 200 kPa), and the cultures were incubated further. To avoid contamination with bacteria, ampicillin (100 µg/ml) was added to M. barkeri cultures. All experiments were performed at least in triplicate.

As shown in Table 1, the amount of TMBi produced in the culture assays depended on the concentration of both the metal and the siloxane. At an OMCTS concentration of 3.4 nM, the TMBi production was maximal with low concentrations of Bi(NO₃)₃ [1 and 5 µM Bi(NO₃)₃], but it was significantly reduced with 20 µM Bi(NO₃)₃ (Table 1), probably due to inhibiting or damaging effects of Bi³⁺ on essential cell components, such as membrane-associated or integrated proteins of M. barkeri (11).

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TABLE 1. Effects of OMCTS, monensin, and lasalocid on TMBi production by M. barkeri in the presence of different Bi(NO3)3 concentrations over 60 days of incubation

This general inhibiting or damaging effect of the metal ion on the cell physiology, especially at higher concentrations, could also be demonstrated by the inhibitory action of Bi(NO₃)₃ on methanogenesis. Increasing the concentration of Bi(NO₃)₃ from 1 µM to 20 µM resulted in reducing the methane production by growing cultures to 20% (1 µM), 77% (5 µM), and 87% (20 µM) of the production by control cultures (28 ± 6 mmol CH₄ produced over 30 days). No effect on methane production was observed with NaNO₃ at concentrations up to 60 µM, indicating that the inhibition was due to the metal ion.

When the concentration of OMCTS in the presence of 5 µM Bi(NO₃)₃ was varied (Table 1), the largest amounts of TMBi were produced with 0.017 to 0.17 nM OMCTS, and there was significantly reduced TMBi production at lower (0.0017 nM) and higher (0.34 to 34 nM) concentrations of OMCTS. As assumed for reduced TMBi production at higher Bi(NO₃)₃ concentrations, the lower TMBi yields at higher concentrations of OMCTS were probably caused by inhibiting or damaging effects on cell constituents. Because of lipophilic properties of the siloxane, cytotoxic interactions with the cell membrane probably cause disturbances of the membrane potential and permeation processes (16), thus inhibiting cell physiology in general, like higher concentrations of metal ions.

The crown ether-like structure of OMCTS led us to suppose that the influence of this compound on bismuth methylation is, for the major part, due to complexation of the metal ion. Since no data are available yet about the ability of this compound to bind Bi³⁺, we included structurally related compounds with known cation complexation capabilities, such as monensin and lasalocid, in our studies and investigated their influence on the stimulation of bismuth methylation. Both ionophores build cage-like structured metal complexes and are structurally similar to OMCTS with regard to not only the complexation of cations but also their lipophilic moieties favoring membrane interactions. Both compounds preferentially bind monovalent cations such as Na⁺ or K⁺ but are also able to complex di- and trivalent cations (4). Mainly due to their disturbing effect on transmembrane cation gradients, both compounds act as strong antibiotics, although only at concentrations higher than 3 to 4 nM (2).

As shown in Table 1, both ionophores stimulated the bismuth methylation by M. barkeri, and in both cases a very similar dependence on the concentrations of the agent and the metal ion was observed for the interaction studies with OMCTS. With 5 µM Bi(NO₃)₃, the largest amounts of TMBi were produced with rather low concentrations of the ionophores similar to the concentrations identified for OMCTS (0.075 nM monensin, 0.17 nM lasalocid). As also observed for OMCTS, the production of TMBi deceased significantly with 20 µM Bi(NO₃)₃ in the presence of the additives.

The striking similarity between the effects of OMCTS and the effects of the ionophores monensin and lasalocid in terms of their stimulating effects on TMBi production suggests that the main reason for the observed stimulation by OMCTS is complexation of the metal ion allowing facilitated membrane permeation and thus providing the prerequisite for intracellular methylation.

The dependence of M. barkeri on adjuvants during the methylation of bismuth could be explained by the assumption that the membrane of this organism is itself impermeable to bismuth ions, possibly due to the absence of nonspecific cation uptake systems, but allows permeation of bismuth only in the presence of lipophilic, ion-complexing compounds as a prerequisite for methylation in the cytoplasm. This explanation, based on facilitated membrane permeation, does not rely on a specific interaction between the adjuvant and the microorganism, which would restrict the effect to a special group of organisms. As confirmation for this assumption, a stimulating effect of OMCTS similar to that found for the methanoarchaeon M. barkeri was also observed for Desulfovibrio vulgaris (5) using a comparable culture design [50-ml cultures in serum bottles; TMBi produced only in the presence 300 µM OMCTS; 30 fmol TMBi with 20 µM Bi(NO₃)₃ in 1 week]; the latter organism belongs to the domain Bacteria and differs from Archaea by having a different membrane composition.

Furthermore, provided that the assumption that there is a rather nonspecific reaction of the stimulating agents is correct, we could not exclude the possibility that organisms which methylate without adjuvants (e.g., M. formicicum or C. collagenovorans [7]) also are affected by these agents and react (at least at low agent concentrations) with increased methylation activity.

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG).

We thank L. Hartmann for critically reading the manuscript and the working group of A. V. Hirner, Department of Environmental and Analytical Chemistry, University of Duisburg-Essen, for analytical assistance.

 
* Corresponding author. Mailing address: Department of Microbiology, University of Duisburg-Essen, Universitaetsstr. 4, 45177 Essen, Germany. Phone: 49 201 183-4707. Fax: 49 201 183-3990. E-mail: klaus.michalke{at}uni-essen.de.

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Applied and Environmental Microbiology, October 2006, p. 6819-6821, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00694-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Michalke, K. Articles by Hensel, R. Search for Related Content PubMed PubMed Citation Articles by Michalke, K. Articles by Hensel, R. Agricola Articles by Michalke, K. Articles by Hensel, R.