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Mercury reduction encoded by the microbial mer operon is an efficient resistance mechanism that is widespread among gram-positive and gram-negative microorganisms (8, 11). Highly toxic water-soluble ionic mercury is taken up by the microorganisms and reduced to insoluble metallic mercury by the intracellular enzyme mercuric reductase, encoded by the merA gene. Metallic mercury subsequently diffuses out of the cells. The reduction process can be continuously performed within a submersed microbial biofilm on porous support material, resulting in accumulation of metallic mercury within the bioreactor (1, 12). Here the structure of mercury-reducing biofilms on the carrier material from packed-bed bioreactors was investigated by scanning electron microscopy and the elemental composition of droplets accumulating within the bioreactor was determined by electron-dispersive X-ray (EDX) analysis.

Industrial bioreactor operation is performed under nonsterile conditions. A monospecies biofilm initially established through inoculation may therefore be subject to colonization by ubiquitous mercury-resistant bacteria (8). The resulting multispecies biofilms may also contain organisms which are hard to cultivate separately. In this study, the invasion of monospecies mercury-reducing biofilms in model laboratory bioreactors operated with industrial waste water was analyzed (12). Genetic fingerprints of isolated effluent bacteria were determined using thermogradient gel electrophoresis (TGGE) of 16S rDNA fragments. This is a fast and sensitive screening technique which has a resolution limit intermediate between the species and genus levels for coryneform bacteria (4). It allows rapid differentiation between samples containing multiple isolates, which can subsequently be identified by 16S rDNA sequencing. However, even if applied with great care, isolation is always a subjective procedure since it relies on colony morphology to differentiate between strains. Moreover, uncultivable bacteria might be present in the biofilms. To analyze the complete community structure of the biofilms within the bioreactors independent of cultivation, 16S rDNA fragments were amplified directly from community DNA and separated by TGGE. In such a way, community fingerprints were generated, which could directly be compared to pure-culture fingerprints. This method also recognizes uncultured microorganisms within the biofilms as additional bands which do not match bands of cultivated bacteria.

Bioreactors and experimental design. Laboratory columns were filled with glass beads (20 ml, 1 to 5 mm in diameter; Schott Glaswerke, Mainz, Germany), and the beads were covered with carrier material (20 ml) and autoclaved. The carrier material in reactor 1 was Siran beads (SiO₂, 0.4 to 1.0 mm in diameter; Schott Glaswerke), the carrier material in reactors 2 and 3 was Lignocell (wood chips, 2.0 to 2.5 mm in diameter; Rettenmaier & Söhne GmbH & Co, Holzmühle, Germany), and the carrier material in reactors 4 to 6 was Arbocell (cellulose, fiber length of 700 µm, fiber diameter of 20 µm; Rettenmaier & Söhne GmbH & Co.). For inoculation, 500 ml of a pure culture of Pseudomonas putida Spi 3, a mercury-resistant isolate, was pumped through the column in upflow mode at 20 ml/h. Subsequently, sterile synthetic wastewater was pumped through the columns, followed by nonsterile chloralkali electrolysis wastewater, which had been aerated and neutralized prior to treatment. The wastewater was supplemented with nutrients (final concentration, 0.1 g of yeast extract per liter). Details of the experimental setup have been described (12).

Scanning electron microscopy and EDX. Samples of different column bed levels were conventionally fixed with 2.5% glutardialdehyde growth medium for 2 h or several days at 4°C, dehydrated with an acetone series, and critical-point dried with liquid CO₂ at 41°C and 85 atm. Samples were carbon coated to a thickness of 30 nm (sputter coater SCD 040; Balzers Union, Walluf, Germany) and analyzed with a field emission scanning microscope (DSM 982 Gemini; Zeiss, Oberkochen, Germany) at a working distance of 8 mm, an acceleration voltage of 20 kV, and a sampling area of 8 by 8 µm. Spectra were registered with the Link-ISIS system (Oxford Instruments, Munich, Germany), and intensities were equally scaled for direct comparison of spectral peaks.

Isolation of effluent bacteria. Reactor effluent samples were serially diluted in phosphate-buffered saline (PBS) (2.2 g of NaH₂PO₄ per liter, 6.0 g of Na₂HPO₄ per liter, 5.8 g of NaCl per liter [pH 7.2]). Aliquots (50 µl) of the appropriate dilution were spread on agar plates containing NaCl (10 g/liter) and yeast extract (1.5 g/liter). The plates were incubated at room temperature for 2 days and then inspected carefully, and colonies showing new morphologies were picked and analyzed further.

Determination of mercury resistance level. Isolates were grown in 5 ml of liquid growth medium [10 g of NaCl per liter, 2 g of yeast extract per liter, 4 g of sucrose per liter, 1 mg of Hg(II) per liter] for 1 day at 30°C on a rotary shaker at 180 rpm. Aliquots (100 µl) were serially diluted in PBS, and 50 µl of an appropriate dilution was spread on agar plates containing solid growth medium and various concentrations of Hg(II) [0, 1, 5, and 10 mg of Hg(II) per liter). For each resistance level, two dilutions were plated out in triplicate. The plates were incubated at room temperature for 4 days.

Extraction of DNA from pure cultures and biofilms. Total DNA was isolated by the modified method of Wilson (13, 14) from 2 ml of an overnight culture grown in Luria-Bertani medium supplemented with 1 mg of Hg(II) per liter at 30°C on a rotary shaker. For extraction of DNA from biofilms, 2 ml of the glass beads was transferred to 6 ml of PBS and vortexed (5 min). Then 2 ml of the suspended biofilm was used for extraction of total cellular DNA as described above.

Primers and PCR amplification. Primers for PCR were specific for conserved bacterial 16S rDNA sequences. PCR with primers R1401 and F968GC (3) amplified a bacterial 16S rDNA fragment from positions 968 to 1401 (Escherichia coli numbering). A GC-rich sequence was attached to the 5' end of primer F968GC. PCR amplification was performed as described previously (3).

TGGE. The TGGE system (Qiagen, Hilden, Germany) was used as described previously (3). A thermal gradient of 39 to 52°C was applied. A mixture of amplified 16S rDNA fragments of phylogenetically diverse bacteria was used as a reference pattern. The gels were silver stained (11), dried, and scanned (Umax Astra 1220U).

Determination of 16S rDNA sequences and species identification. DNA from pure cultures of effluent bacteria was extracted as described above. Nearly complete 16S rRNA genes were amplified by PCR using a forward primer hybridizing at positions 8 to 27 and a reverse primer hybridizing at the complement of positions 1525 to 1541 (E. coli 16S rRNA gene sequence numbering). PCR was performed as described previously (13). The sequence of the amplified 16S rDNA was determined directly using an Applied Biosystems 373A DNA sequencer (Perkin-Elmer, Applied Biosystems GmbH, Weiterstadt, Germany) and the protocols recommended by the manufacturer for Taq polymerase-initiated cycle sequencing with fluorescent-dye-labeled dideoxynucleotides and standard 16S rRNA sequencing primers (6). The resulting sequences were aligned with reference 16S rRNA and 16S rRNA gene sequences from the EMBL database by using FASTA (9).

Biofilm structure. Figure 1 shows a biofilm that had developed on the carrier material after 14 days of sterile reactor operation with model wastewater. A single microbial cell layer can be seen attached to the surface of the carrier material. The bacteria stuck firmly to the substratum and were not lost during bioreactor operation. This thin microbial biofilm acted as the catalyst for mercury removal, illustrating that it is not adsorption to biomass which is the main mechanism operating here. Figure 2a shows a larger field from a lower level of the reactor. Here, biomass had massively grown and filled the substratum cavity with an amorphous extracellular polysaccharide cell matrix, dotted with irregularly dispersed mercury droplets. EDX analysis of boxed areas, 64 µm² in size, revealed prominent Hg peaks (M¹ = 2,195 keV; L¹ = 9,987 keV), which correlated well with the droplet size and peak height. The number and size of mercury droplets increased during continuous operation of the reactor. Similar intensity ratios for O to Si to Ca to Na were found on the pure Siran surface (EDX data not shown) and around mercury droplets on the Siran surface (1:2.83:0.35:0.5 and 1:2.95:0.33:0.57, respectively [Fig. 2b]), contrasting with the element intensity ratios of the biofilm area (1:6.40:1.60:0.24 [Fig. 2c]). Siran not only is composed of SiO₂ but also contains small amounts of Na, Ca, Mg, and Al, and thus the corresponding EDX intensities are derived from the carrier material. Additionally, the biofilm matrix contained Fe (K¹ = 6,403 keV) from the yeast extract medium and chlorine (K¹ = 2,622 keV) from the NaCl present in the wastewater. Thus, mercury droplets of different sizes were identified throughout the carrier material, some of them in close contact with clumps of biomass glued together by extracellular polysaccharide material.

View larger version (164K): [in this window] [in a new window]   FIG. 1.   Scanning electron micrograph survey of a P. putida Spi3 biofilm on ceramic carrier material (Siran beads) from a packed-bed mercury-reducing bioreactor after 14 days of operation with sterile synthetic wastewater. A morphologically homogenous bacterial population is growing on the carrier as a monolayer. Little EPS can be observed (arrowheads).

View larger version (73K): [in this window] [in a new window]   FIG. 2.   EDX analysis of a P. putida Spi3 biofilm from a packed-bed mercury-reducing bioreactor after 14 days of operation with sterile synthetic wastewater. (a) Scanning electron micrograph of a biofilm aggregate, which fills a cavity of the carrier material (Siran beads) and contains several brightly shining droplets of mercury (black arrowheads). These droplets are larger when in direct contact with the carrier surface (white arrowhead, white-framed box). Boxed areas, i.e., Gp06 and Gp02, represent the detection area of the EDX analysis. (b) EDX spectrum of area Gp02 on Hg droplets in direct contact with the Siran surface. (c) EDX spectrum of area Gp06 as a characteristic in situ biomass sample.

TGGE fingerprints and identification of effluent bacteria. From the effluents of six laboratory bioreactors operated continuously with chloralkali electrolysis wastewater, 13 bacterial strains were isolated on the basis of differences in colony morphology. These strains could be grouped into four clearly distinguishable TGGE fingerprint types. The TGGE fingerprints of the inoculant strain, P. putida Spi3, and representatives of the four effluent TGGE fingerprint types are shown in Fig. 3. Most isolates generated single bands, indicating that their 16S rDNA amplicons showed no sequence heterogeneity, similar to results of Felske et al. (4) for coryneform bacteria. They could be distinguished from each other on the basis of their position relative to each other and to the standard. Two rDNA bands were observed for TGGE fingerprint type C. 

View larger version (81K): [in this window] [in a new window]   FIG. 3.   TGGE fingerprints of biofilm samples from six mercury-reducing bioreactors treating nonsterile chemical wastewater (lanes 1 to 6), effluent bacterial cultures (lanes A to D), and the inoculant strain, P. putida Spi3 (lane E). Ref, reference standard.

Biofilm community composition. Community fingerprints from the bioreactors are shown in Fig. 3, lane 1 to 6. The inoculant strain, P. putida Spi3, could clearly be detected in all bioreactors, and up to four additional bands were present. Community fingerprints from bioreactors 1, 2, 3, 4, and 6 showed a double band which was identical to fingerprint type C, identified as Paenibacillus amylolyticus. This organism was absent from the TGGE fingerprint of bioreactor 5, although it was isolated from its effluent. Bioreactors 1, 4, 5, and 6 showed a TGGE band corresponding to fingerprint type B, identified as B. cereus. TGGE fingerprint type A, identified as S. maltophilia, was detected only once, in bioreactor 6. TGGE bands of fingerprint type D, identified as C. freundii, were not seen in any of the community fingerprints.

Mercury resistance of invading strains. The mercury resistance levels of bacteria strongly depend on the number of cells used in the assay (2), with much higher resistance levels found at high cell densities, and on the buffering and complexing capacity of the test medium. Moreover, much higher resistance is generally observed on plates than in liquid cultures (5), because on plates the growth of the test strains can locally reduce mercury concentrations. The ability of a single cell to form a colony on an agar plate of a given mercury concentration was used here to determine in a rapid and reproducible way the resistance levels of our strains. The medium was unbuffered to obtain data which would be relevant to chemical wastewater conditions.