Cadmium Ion Biosorption by the Thermophilic Bacteria Geobacillus stearothermophilus and G. thermocatenulatus

Abstract

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl₂ (as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd²⁺ adsorption experiments to characterize Cd²⁺ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd²⁺ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.

Various metal compounds can be either essential or toxic for living organisms, depending on the form and concentration. In important physiological catalytic reactions, many enzymes require metal ions as central atoms. In order to generate energy, several microorganisms depend on metal compounds that act as final electron acceptors during anaerobic respiration and as electron donors in chemolithotrophic metabolism. In contrast, nonessential metal ions, in particular, and metal ions (both nonessential and essential) at high concentrations, in general, harm the living cell by potentially displacing essential metal ions (in enzymes), competing with structurally related nonmetals in cell reactions and blocking functional groups in biomolecules (29). Therefore, organisms have developed several metal homeostasis and detoxification strategies, including an active efflux system, changes in ion permeability, adsorption, and intra- and extracellular complexation, biotransformation, and compartmentation (10, 21, 27, 31, 36).

Adsorption of metals by cell wall components is one of the more important interaction mechanisms, and it has been the subject of many studies (for review, see references 3, 15, 20, 34, 35, and 40). Several recent investigations have used surface complexation models (SCMs) to describe the extent of metal adsorption by bacteria as important physical, chemical, and biological parameters are independently varied (5, 8, 14, 16, 19, 23, 43). These SCMs are based upon a set of molecular-scale thermodynamic reactions, each describing adsorption of a particular dissolved chemical species to a particular type of cell wall functional group using a single stability constant, K. Some previous investigations indicate that different bacterial species display similar types of reactive surface functional groups (e.g., carboxyl, phosphoryl, hydroxyl, or amine) and exhibit broadly similar reactivities toward certain metals (5, 43).

The implication is that cell wall structure, while generally similar for the bacteria that have been studied to date, can still influence the nature and extent of metal interaction with the cell wall. Note that all previous SCMs have been developed for mesophilic bacteria; no single investigation has been ever published addressing metal adsorption onto thermophilic microorganisms. Metal adsorption reactions onto thermophilic microorganisms may therefore differ quantitatively and qualitatively from the mesophilic species that have been studied to date. A wide range of geological and anthropogenic thermal environments exhibit high concentrations of dissolved metals. In response to these conditions, microorganisms isolated from these habitats may have unique cell wall structures. Thus, studies of thermophilic microorganisms can supplement our present knowledge of metal biosorption, which is completely based on mesophilic organisms.

The aim of this investigation was to develop an SCM for cadmium adsorption by two thermophilic bacteria. Cadmium is considered to be a toxic heavy metal, despite the lack of a chemically sound definition for the term heavy metal (18) and an observed biological benefit of cadmium for zinc-limited marine diatoms (25). However, cadmium compounds are important environmental pollutants affecting most eukaryotic and bacterial cells. Cadmium was also selected in the present investigation because SCMs describing the adsorption of cadmium by various mesophilic bacteria are available for comparison (5, 8, 14, 16, 19, 43). In this study, we initially tested 26 thermophilic bacteria of the genera Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, and Geobacillus in cadmium ion toxicity experiments. Interestingly, the highest tolerance to cadmium (CdCl₂), 400 to 3,200 μM, was observed for species belonging to the genus Geobacillus. Geobacillus thermocatenulatus (22) and G. stearothermophilus (17) were selected for further investigation based on their tolerance to cadmium.

MATERIALS AND METHODS

Bacteria and culture conditions.

The following thermophilic bacteria were tested in toxicity experiments for cadmium tolerance (see also Table 1, below): Aneurinibacillus thermoaerophilus (DSM 10154); Anoxybacillus flavithermus (milk powder isolate); Bacillus sp. (DSM 2349), Bacillus caldolyticus (DSM 405), Bacillus caldotenax (DSM 406), Bacillus caldovelox (DSM 411), Bacillus coagulans (DSM 1, as mesophilic out-group for comparison), Bacillus licheniformis (milk powder isolate), Bacillus mycoides (DSM strain 299, as mesophilic out-group), Bacillus smithii (DSM 460 and 4216), Bacillus sphaericus (DSM 462 and 463); Brevibacillus brevis (DSM 5507), Brevibacillus borstelensis (DSM 6453, as mesophilic out-group); G. thermocatenulatus (DSM 730), G. thermodenitrificans (DSM 13147, 13148, and 13149), G. thermoglucosidasius (DSM 2543), G. thermoleovorans (DSM 5366), G. stearothermophilus (DSM 458, 6790, and 13240), and five environmental samples isolated from various hot springs in New Zealand (KP.A3 and Rt26.A1 [both from Rotorua], Wai21.A2 [from Waimangu], and Tok10.A3 and Tok13.A4 [both from Tokaanu]). Based on the cadmium toxicity results, G. stearothermophilus (DSM 6790) and G. thermocatenulatus (DSM 730) were selected for further acid-base titration and cadmium adsorption experiments.

TABLE 1.

MICs of Cd²⁺ for the microorganisms studied and temperatures at which the toxicity experiments were conducted

Microorganism	Straina or source	Temp (°C)	MIC of Cd2+ (μM)
Bacillus mycoides	299	25	>3,200
B. coagulans	1	40	80
Brevibacillus borstelensis	6453	40	600
Aneurinibacillus thermoaerophilus	10154	55	80
Bacillus sp.	2349	55	80
B. licheniformis	Milk powder	55	80
B. smithii	460	55	80
B. smithii	4216	55	80
B. sphaericus	462	55	200
Brevibacillus brevis	5507	55	80
Geobacillus stearothermophilus	458	55	400
G. stearothermophilus	6790	55	600
G. thermocatenulatus	730	55	600
G. thermodenitrificans	13147	55	400
G. thermodenitrificans	13148	55	80
G. thermodenitrificans	13149	55	80
G. thermoglucosidasius	2543	55	600
G. thermoleovorans	5366	55	>3,200
KP.A3	Rotorua	55	80
Anoxybacillus flavithermus	Milk powder	60	200
Bacillus caldolyticus	405	60	400
B. caldotenax	406	60	400
B. caldovelox	411	60	200
B. sphaericus	463	60	200
Geobacillus stearothermophilus	13240	60	200
Tok10.A3	Tokaanu	60	80
Tok13.A4	Tokaanu	60	600
Rt26.A1	Rotorua	65	80
Wai21.A2	Waimangu	65	80

^aNumbers refer to the corresponding DSM strain (Deutsche Sammlung von Mikroorganismen, the German Collection of Microorganisms).

Prior to the titration and cadmium adsorption experiments, all bacteria strains were successively subcultured at least twice in new medium. The basal medium to grow Rt26.A1 and Wai21.A2 contained the following components (per liter): 0.25 g of CaCl₂ · 2H₂O, 0.50 g of MgSO₄ · 7H₂O, 0.20 g of (NH₄)₂SO₄, 2.00 g of yeast extract, 5.00 g of glucose, 3.00 g of KH₂PO₄, and trace element solution SL-6 (containing, per liter, 0.10 mg of ZnSO₄ · 7H₂O, 0.03 mg of MnCl₂ · 4H₂O, 0.30 mg of H₃BO₃, 0.20 mg of CoCl₂ · 6H₂O, 0.01 mg of CuCl₂ · 2H₂O, 0.02 mg of NiCl₂ · 6H₂O, and 0.03 mg of Na₂MoO₄ · 2H₂O). The final pH value of the medium was adjusted to 4. The culture medium for the other bacterial strains was 30 g/liter tryptic soy broth (TSB; Difco) adjusted to a pH value of 7.

For acid-base titration and cadmium adsorption experiments, G. stearothermophilus (DSM 6790) and G. thermocatenulatus (DSM 730) were grown in 1 liter of TSB at 60°C in an orbital mixer incubator at 100 rpm. Bacteria were harvested after 24 h in the exponential growth phase by centrifugation at 8,230 × g (Beckmann Coulter Avanti J-E centrifuge with fixed-angle rotor JLA-9.100) and washed five times in 250 ml 10 mM NaNO₃ to ensure complete removal of the growth medium and adsorbed cations from the bacterial surface. Between each wash step the bacterial suspension was centrifuged for 10 min at 8,230 × g to form a pellet and the supernatant was discarded. After the final wash the bacteria pellet was resuspended in a known weight of 10 mM NaNO₃ solution, and the optical density at 600 nm (Hach DR2010) relative to the electrolyte was determined.

Prior to the experiments to determine electrophoretic mobility, G. stearothermophilus (DSM 6790) and G. thermocatenulatus (DSM 730) were incubated in 200 ml TSB at 57°C at 100 rpm for 24 h. The optical density at 600 nm was measured, and cells were harvested by centrifugation (8 min; 9,820 × g; 4°C). The pellet was resuspended in 1 mM NaNO₃ solution, divided into three aliquots, and washed three times in 150 ml of 1 mM, 10 mM, or 100 mM NaNO₃, respectively. The final pellets were resuspended in 1 ml of the respective electrolyte concentration and kept on ice.

The ratio of bacterial biomass to optical density at 600 nm was determined using two different methods. First, a bacterial suspension of known optical density was filtered through a preweighed filter membrane (pore size, 0.45 μm; Millipore) and flushed with distilled water several times. The filter was dried at 80°C to constant weight. Second, aliquots of a bacterial suspension with known optical density and weight were centrifuged at 1,850 × g (Jouan CR 4.11) for 1 h, stopping three times to decant the supernatant. After 1 h, the wet weight of the bacterial pellet was determined. The dry mass of the pellet was recorded after drying at 80°C to constant weight.

The cell number in relation to optical density was determined with a Thoma counting chamber (depth, 0.02 mm). Cell dimensions were recorded by evaluating images obtained from a scanning electron microscope.

Small-scale plasmid isolation according to the method of Ronimus (30), a derivation of the alkaline lysis method of Birnboim and Doly (4), was performed to detect the presence of extrachromosomal DNA. Plasmid-encoded genes for cadmium resistance are commonly found in bacteria. The species were grown in the presence of 200 μM CdCl₂ and without cadmium prior to the plasmid isolation.

Toxicity experiments.

All bacterial strains mentioned above were tested for tolerance to elevated cadmium ion levels. A 160-μl volume of medium per well in microtiter plates and 5 ml of medium in test tubes supplemented with different defined concentrations of CdCl₂ were inoculated using 5% inocula of freshly grown cultures. The growth was monitored in microtiter plates at a wavelength of 405 nm (FLUOstar Optima; BMG Labtechnologies) and in test tubes at 600 nm (Ultrospec 3000; Pharmacia Biotech). The MIC was defined as the absence of growth of the species after 16 h (microtiter plates) and 2 days (test tubes).

Acid-base titrations.

Acid-base titrations were conducted using an automated potentiometric titrator (Metrohm titrator 736 GP Titrino) with a glass electrode (Metrohm 6.0130.100) and a separate Ag/AgCl reference electrode (Metrohm 6.07.26.110). The CO₂-free NaOH and the HNO₃ titrants were calibrated against potassium hydrogen phthalate and standardized NaOH, respectively. A known weight (ca. 50 g) of homogenized bacterial suspension was placed in an air-tight polystyrene vessel, and the pH was adjusted to approximately 4 by addition of an aliquot of standardized HNO₃. The suspension was purged with humid N₂ gas for 30 min in order to remove dissolved CO₂. Then, the suspension of bacterial cells was titrated in an up-pH direction while continuously stirred and under a positive-pressure N₂ atmosphere. Following the first titration, the suspension was again adjusted to pH 4, and a second up-pH titration was performed to evaluate reproducibility and the reversibility of the bacterial surface protonation reactions. For each titration a stability of 5 mV/min was obtained prior to the addition of the next aliquot of titrant. All titrations were performed in a temperature-controlled room at 20 ± 2°C. At the end of each titration, the dry mass of the titrated bacteria was determined by drying the cells at 80°C, after accounting for the dry weight of the electrolyte solution (NaNO₃) and the added acid and base.

Metal adsorption.

Batch cadmium adsorption experiments were conducted for each bacterial species as a function of pH and of the bacteria/metal concentration ratio. A weighed aliquot from a bacterial parent solution was dried to constant weight at 80°C, as described above, to determine the dry weight of the bacteria. The remainder was used to prepare three suspensions (ca. 150 g each) that had 100%, 50%, and 25% of the bacterial concentration as the parent suspension. These three suspensions were supplemented with 1,000 ppm cadmium atomic absorption standard solution [Cd(NO₃)₂ in 0.5% HNO₃; Merck] to a final concentration of 5 ppm cadmium. After the addition of the metal, the optical density at 600 nm for each sample was recorded and each suspension was divided into 12 individual reaction vessels. The pH value in each reaction vessel was adjusted by adding small volumes of 100 mM NaOH solution to cover a pH range of 3.5 to 10.0 for each suspension. After a sufficient equilibration time of 2 h (6) in an orbital mixer incubator (Ratek) set at 100 rpm and 20°C, the samples were centrifuged at 6,870 × g (Beckmann Coulter Avanti J-E Centrifuge with swinging-bucket rotor JLA-5.3) for 15 min. A portion of the supernatant was decanted and acidified with concentrated HNO₃ solution (ARISTAR grade) for subsequent metal analysis by flame atomic adsorption spectroscopy (Perkin-Elmer Analyst 800 atomic adsorption spectrometer). The remaining supernatant was used for measurement of final equilibrium pH. The concentration of metal adsorbed to bacteria in each sample was calculated by subtracting the cadmium ion concentration remaining in the supernatant from the original concentration of 5 ppm.

Electrophoretic mobility measurements.

Electrophoretic mobility measurements were carried out using a Malvern Zetasizer Nano ZS at 25°C. Ten-milliliter volumes of 1 mM, 10 mM, and 100 mM NaNO₃ were prepared and adjusted to pH values between 1.8 and 9.5 by addition of HNO₃ and NaOH, respectively. Additionally, 10-ml volumes of 30 μM, 300 μM, and 3 mM Cd(NO₃)₂ solution with final pH values of 4.5 to 5.5 were prepared. The ionic strength of all cadmium solutions was maintained at 10 mM NaNO₃. Three to 100 μl of the bacterial suspension was added to the 10-ml electrolyte solutions, and the pH was measured. The cadmium samples were incubated for 2.5 to 5.0 h prior to measurement. The “auto” measurement duration was selected, which reports the electrophoretic mobility as an average of 10 to 30 runs. The isoelectric points were located by determining the pH value where the electrophoretic mobility of the bacteria was zero.

SCMs.

SCMs were developed using the computer program FITMOD, a modified version of FITEQL (41). From the experimental acid-base titration data, FITMOD determines the concentrations and deprotonation constants of binding sites on the bacteria. The following deprotonation reaction of carboxylic groups is an example:

(1)

where R represents the cell to which the functional group is attached; the stability constant K can be determined using the corresponding mass action equation:

(2)

where a refers to the activity of the proton and square brackets represent surface site concentrations in moles per kg of solution. The adsorption of cadmium cations onto carboxylic surface binding sites can be expressed by the following equation:

(3)

FITMOD calculates the thermodynamic stability constants K for the metal complexes using the corresponding mass action equation:

(4)

All SCMs incorporated the Donnan electrostatic model (44).

SCM development required information about biophysical parameters, such as the cell wall thickness, the dry-to-wet mass ratio, and specific surface area (see Table 2, below). Methods to determine the first two parameters have been described above. The bacteria surface area, A, was calculated assuming that the cells were cylindrically shaped with a radius r, a length l, and a density p equal to 1 kg/liter by using the following equation:

(5)

where r and l are expressed in μm and A is in m²/g (wet weight) of bacteria (12).

TABLE 2.

Selected biophysical parameters for G. thermocatenulatus (DSM 730) and G. stearothermophilus (DSM 6790)

Parameter	G. thermocatenulatus	G. stearothermophilus
Dry-to-wet mass ratio	1:15	1:8
Isoelectric point (pH)	3.25	3.95
Cell wall thickness (nm)	49.5 ± 3.5	35.0 ± 1.6
Cell length (μm)	4.3 ± 1.9	3.1 ± 0.3
Cell width (μm)	0.4 ± 0.04	0.7 ± 0.02
Surface area (m2/g [wet wt] of cells)	8.96	6.38

RESULTS

Cadmium ion susceptibility tests.

The values of the MICs of Cd²⁺ for the bacterial species tested are displayed in Table 1. Note that all strains were assigned to the genus Bacillus until the last decade. As more and more information about DNA base compositions became available, a major reorganization for the genus Bacillus began, and changes in nomenclature occurred (45). It is noteworthy that the thermophilic species displaying the highest cadmium ion tolerance in our experiments belong to a phylogenetic group proposed in 1991 as “group 5” by Ash et al. (2) that defined later the genus Geobacillus (28).

In comparison to the mesophilic out-group B. mycoides, the thermophilic species were more sensitive to Cd²⁺, while G. thermoleovorans tolerated similar Cd²⁺ concentrations as B. mycoides, as high as 3.2 mM. However, no correlation between growth temperature and MIC was found. The reason for the high Cd²⁺ tolerance of G. thermoleovorans is unknown and needs further investigation. Although the diversity of growth media and conditions complicates a meaningful comparison with published studies, some of the results were higher than previously reported MICs of Cd²⁺ for thermophilic bacilli (26).

Subsequent studies of the species showing MICs of more than 500 μM Cd²⁺ revealed two different patterns of growth response (Fig. 1). An increasing concentration of Cd²⁺ resulted either in a decrease of the maximum optical density with no change in the onset of growth (B. mycoides, G. thermocatenulatus, and G. thermoleovorans) or, alternatively, a delay of the onset of growth but no significant effect on maximum optical density (G. stearothermophilus, G. thermoglucosidasius, and Tok13.A4). These results may indicate two different Cd²⁺ detoxification strategies. G. stearothermophilus and G. thermocatenulatus displayed the same MIC and had an identical optimal growth temperature, but different growth responses in the presence of Cd²⁺ were observed. These two species were therefore selected as representatives for each pattern.

FIG. 1.

Microbial growth monitored spectrophotometrically (optical density [OD]) at 405 nm as a function of concentration of Cd²⁺. Black circles, 0 μM; white triangles, 100 μM; black squares, 200 μM; white diamonds, 400 μM; black triangles, 600 μM; white hexagons, 800 μM. Growth curves are for G. thermoleovorans (DSM 730) (A) and G. stearothermophilus (DSM 6790) (B). For clarity, only every second data point is shown.

Although plasmid-encoded genes for cadmium resistance are widely distributed within the bacterial domain (36), no extrachromosomal DNA was obtained for G. stearothermophilus or G. thermocatenulatus cells by plasmid isolation. Regarding the known detoxification strategies summarized in the introduction, biotransformation and compartmentation can be excluded: cadmium only occurs in one oxidation state, and compartments could not be observed for G. stearothermophilus or G. thermocatenulatus cells. Due to the lack of plasmids, biosorption reactions might play an important role in microbial tolerance to cadmium ions either solely or in combination with the remaining possibilities, an efflux system and regulation of the ion permeability, which may be mediated by chromosomal genes.

Potentiometric titration experiments.

Acid-base titration curves for G. stearothermophilus and G. thermocatenulatus cells in 10 mM NaNO₃ are displayed in Fig. 2. These figures depict the concentration of protons adsorbed onto the bacterial surface as a function of pH. Titration experiments were conducted in triplicate for G. thermocatenulatus and in duplicate for G. stearothermophilus. Individual titration curves consisting of two titrations from low to high pH values were in excellent agreement with each other after taking dilution into account, indicating reproducibility of the data and complete reversibility of the adsorption reactions during the experiments. The curves are normalized to the dry (Fig. 2A) and wet (Fig. 2B) cell mass of bacteria to allow direct visual comparison between the two tested species. In comparison to titrated electrolyte solution without bacteria (data not shown), G. stearothermophilus and G. thermocatenulatus cells significantly buffered pH over the entire range studied in this investigation, which is in agreement with published data for mesophilic microorganisms (13, 14, 19) and one thermophilic bacterium (42).

FIG. 2.

Acid-base titration data normalized to dry mass (A) and wet mass (B) obtained for G. stearothermophilus DSM 6790 (white circles) and G. thermocatenulatus DSM 730 (black circles). Solid lines represent the best-fitting discrete multisite model calculated by FITMOD. For clarity, only every third data point is displayed.

G. thermocatenulatus, when compared to G. stearothermophilus, shows a slightly higher buffering capacity against addition of the base when titration data are normalized to dry cell mass (Fig. 2A). In contrast, when data are normalized to wet cell mass, G. stearothermophilus displays a stronger buffering capacity than G. thermocatenulatus (Fig. 2B). The difference is due to the different bacterial dry-to-wet mass ratios, which were 1:15 and 1:8 for G. thermocatenulatus and G. stearothermophilus, respectively (Table 2). This result was unexpected, as the data show a smaller surface area and thinner cell wall for G. stearothermophilus, hence indicating a smaller volume that may adsorb cations. Bacterial concentrations during titration were approximately (3.8 ± 0.06) × 10⁸ cells/ml for G. thermocatenulatus and ranged from (4.8 ± 0.06) × 10⁸ to (5.3 ± 0.07) × 10⁸ cells/ml for G. stearothermophilus, representing a total bacterial dry mass of 1.59 ± 0.07 g. The bacterial surface areas calculated from biophysical parameters were 8.96 and 6.38 m²/g (wet weight) of cells for G. thermocatenulatus and G. stearothermophilus cells, respectively. The values are much lower than values reported in other SCM studies (44), where surface areas as high as 290 m²/g (wet weight) for B. subtilis have been reported (42).

To develop SCMs for the titration data, we took the electrophoretic mobility of the bacterial cells into consideration. In theory, the net concentration of supplied protons can be quantified by equation 6:

(6)

where [H⁺]_added and [OH⁻]_added refer to the known concentrations of the added HNO₃ and the NaOH titrants and where [H⁺]_adsorbed and [H⁺]_desorbed are the concentrations of protons consumed and released by the bacteria, respectively. The isoelectric point (IEP), when positive and negative charges of the chemical functional groups onto and within the microbial surface are in balance, can be described with the following relationship:

(7)

The net concentration of added protons at the IEP results from equations 6 and 7 and can be determined by equation 8:

(8)

which also represents the titration of a pure electrolyte solution. We assume titration data obtained without and with cells at the corresponding IEP to be identical. Therefore, titration curves for both studied strains were normalized to intersect the titration curve of a pure electrolyte solution at the measured IEP at pH values of 3.25 (G. thermocatenulatus) and 3.95 (G. stearothermophilus). IEP values commonly found for bacterial cells range from 2 to 4, depending on the species (24, 39). Although titrations at pH values below 4 or above 10 were avoided to minimize cell damage (13, 23), the titration data were constrained by the electrophoretic mobility of the cells by iteration (11).

A discrete multiple-site SCM can account for the buffering capacity of the bacterial species observed over the entire studied range between pH 4.0 and 10.0 (Fig. 2). Best-fit approximations of the titration data resulted in a three-site model for G. thermocatenulatus, whereas only two individual binding sites were necessary to fully interpret the titration data of G. stearothermophilus. Besides the excellent visual agreement of measured experimental data and calculated SCM, the quality of fit was quantified using the overall variance V(Y), which is the mean weighted sum of squares of errors Y in the stoichiometric equation. The program FITMOD computes the variance as an output parameter. Low V(Y) numbers indicate a better model fit to the data. For adsorption data, values of V(Y) between 1 and 20 indicate a reasonable fit (41). The V(Y) values for each SCM and corresponding pK values (negative log equilibrium constants for deprotonation reactions) and concentration c of proton-reactive surface sites are compiled in Table 3. The low variance values indicate a good fit to the experimental data. Values of pK are similar for both bacterial strains. The most abundant proton binding site has a pK value of approximately 3.8 and accounts for 66% and 80% of the total concentration of binding sites for G. thermocatenulatus and G. stearothermophilus, respectively.

TABLE 3.

Average model parameters for G. thermocatenulatus and G. stearothermophilus in comparison to other bacterial species

Species (reference)	Model	Parameter	Site 1	Site 2	Site 3	Site 4	V(Y)d
G. thermocatenulatus	Donnan	pKa	3.84 ± 0.03	5.97 ± 0.08	8.70 ± 0.30		3.4
G. thermocatenulatus	Donnan	cb	7.03 ± 0.14	1.20 ± 0.10	2.50 ± 0.52
G. thermocatenulatus	Donnan	log Kc	1.30 ± 0.22				1.8
G. stearothermophilus	Donnan	pKa	3.73 ± 0.04	6.46 ± 0.07			4.9
G. stearothermophilus	Donnan	cb	9.74 ± 0.03	2.40 ± 0.04
G. stearothermophilus	Donnan	log Kc	2.20 ± 0.16				5.9
TOR-39 (42)	Constant capacitance	pKa,f	4.5 ± 0.2	5.8 ± 0.1	8.2 ± 0.1		0.9
TOR-39 (42)	Constant capacitance	cb	6 ± 2	5 ± 1	3 ± 1
Anoxybacillus flavithermus (11)	Donnan	pKa	4.94 ± 0.35	6.85 ± 0.57	7.85 ± 0.27		15.5
Anoxybacillus flavithermus (11)	Donnan	cb,e	3.57 ± 0.58	1.20 ± 0.56	0.95 ± 0.20
Bacillus subtilis (16)	Constant capacitance	pKa	4.80 ± 0.1	6.49 ± 0.3	8.52 ± 0.6		8.5-13.5
Bacillus subtilis (16)	Constant capacitance	cb	6.78 ± 3.9	4.35 ± 2.6	6.17 ± 2.2
Bacillus subtilis (16)	Constant capacitance	log Kc	3.62 ± 0.5	4.11			11
Avg model on nine individual bacterial species (43)	Constant capacitance	pKa	5.0 ± 0.2	7.2	9.7		NAg
Avg model on nine individual bacterial species (43)	Constant capacitance	cb,e	20 ± 8	8	17
Avg model on nine individual bacterial species (43)	Constant capacitance	log Kc	4.0 ± 0.3	NA	NA		3.3-34.6
Avg model on five natural bacterial consortia (5)	Nonelectrostatic	pKa	3.12 ± 0.13	4.70 ± 0.11	6.57 ± 0.17	8.99 ± 0.21	NA
Avg model on five natural bacterial consortia (5)	Nonelectrostatic	cb	6.65 ± 1.96	6.78 ± 2.65	3.68 ± 1.71	4.47 ± 2.19
Avg model on five natural bacterial consortia (5)	Nonelectrostatic	log Kc	2.83 ± 0.30	2.70 ± 0.47	3.95 ± 0.22	5.22 ± 0.40	NA

^aNegative logarithm of the deprotonation constant for subscripted surface functional groups with 1σ errors.

^bConcentration of subscripted surface sites with 1σ errors in 10⁻⁵ mol/g of wet cells.

^cLogarithm of the stability constant for Cd²⁺ complexation reaction with 1σ errors.

^dVariance computed by FITMOD. A lower value indicates a better model fit.

^eBurnett et al. (11) and Yee and Fein (43) published concentrations of surface sites in mol/g of dry bacteria. The dry-to-wet mass ratio for A. flavithermus is 1:6.7, and for the nine bacterial species it is 1:9.9.

^fThe Wightman et al. (42) determinate deprotonation constant at 50°C.

^gNA, not applicable.

Despite the present knowledge of the cell wall composition and structure of gram-positive bacteria (32, 33) such as Geobacillus species, the clear identification of a proton-reactive binding site solely based on its pK value remains complicated. Stability constants of individual functional groups can vary significantly, depending on various parameters, such as temperature, ionic strength (8), and relative position of the functional group in the biomolecule. Due to this interpretation problem, we will not assign the pK values to possible functional groups and refer to the detected reactive binding sites as sites 1, 2, and 3. The best-fitting SCMs included deprotonation reactions onto the bacterial surface that can be characterized for site 1 and 2 by equation 9:

(9)

and for site 3 by equation 10:

(10)

where L represents the functional group acting as a ligand. These formulations are consistent with the electrophoretic mobility data, which indicate that both bacteria maintain a positive cell surface charge at pH values below their IEP and negative surface charge at pHs above their IEP. Although site 3 is required for both Geobacillus strains to explain the positive charge under acidic conditions, it does not play an active role in proton adsorption for G. stearothermophilus under the conditions covered in our experiments.

The cell wall thickness, the bacterial surface area, and the concentration of the dominant site 1 indicate densities of 232.6 and 322.0 sites/nm³ for G. thermocatenulatus and G. stearothermophilus, respectively. Theoretically, an ionic radius for cadmium of 0.1 nm results in a maximum density of 238.7 Cd²⁺/nm³ that may be complexed by functional groups in or on the bacterial cell wall. Therefore, we assume that all reactive groups of site 1 that are accessible by protons are also accessible by the larger cadmium ion.

Cadmium ion biosorption.

Cadmium ion adsorption experiments performed as a function of pH and biomass revealed that both parameters strongly influenced the adsorption onto the microbial surfaces (Fig. 3). Experiments were performed in 10 mM NaNO₃ in the presence of 5 ppm CdCl₂ (44.5 mM) with bacteria-to-metal concentration ratios of 1:1, 1:2, and 1:4, resulting in total bacterial dry mass of 1.15 ± 0.20 g, 0.57 ± 0.09 g, and 0.30 ± 0.05 g, respectively. At a given pH, an increasing concentration of organic mass resulted in an increase in Cd²⁺ adsorption. G. thermocatenulatus cells (1.31 g [dry mass]) were able to adsorb up to 95% Cd²⁺ (Fig. 3A), whereas G. stearothermophilus cells (1.32 g [dry mass]) absorbed up to 85% (Fig. 3B). At a given biomass concentration, increases in the pH result in increased Cd²⁺ adsorption. Depending on the biomass concentration, most adsorption occurred in the pH range of 3.5 to 5.5, demonstrating the importance of site 1 (pK = 3.8). This pH dependence is due to the deprotonation of cell wall functional groups that occurs with increasing pH, progressively resulting in increasing Cd²⁺ biosorption until saturation of binding sites occurs. Negative control experiments (no bacterial cells) indicated that there was a significant loss of Cd²⁺ at pH values above 8 within 2 h (data not shown). Thermodynamic calculations (1) suggest that for a solution containing 5 ppm Cd²⁺ in equilibrium with the atmosphere, otavite (CdCO₃) should precipitate at pHs above 7.

FIG. 3.

Percentage of Cd²⁺ ions adsorbed onto G. thermocatenulatus (A) and G. stearothermophilus (B) cells as a function of pH and dry biomass. Black circles, 1.3 g; white triangles, 0.6 g; black squares, 0.3 g. Solid lines represent the best-fitting discrete one-site model developed by FITMOD. For clarity, for each species only data from one of three individual cultures are shown.

The computer code FITMOD, incorporating the Donnan electrostatic model, was used to develop SCMs to describe the Cd²⁺ adsorption data. The SCMs used the concentrations and deprotonation constants of cell wall surface functional groups obtained from the potentiometric titration experiments (Table 3). SCMs did not consider data obtained at pH values above 8, due to the possibility of bias caused by loss of Cd²⁺ by precipitation. For both bacterial species, three individual cultures each with three different biomass/metal ion ratios were investigated, resulting in a total of nine data subsets per bacterium.

Initially, SCMs with unique thermodynamic stability constants (K) for the metal complexes were calculated for each individual subset. A discrete one-site SCM fit all data subsets. Additionally, for the two data sets displaying the lowest biomass/metal ion ratio for G. stearothermophilus (0.26 and 0.34 g [dry mass]), a two-site SCM could be applied. The multisite model for both data sets just slightly improves the fit in terms of lower V(Y) values. However, it indicates that at lower cell concentrations site 1 may be saturated; hence, the Cd²⁺ complexation reaction significantly occurs at site 2 (14).

Subsequently, a single one-site SCM was developed to fit all of the data for each bacterium. These one-site SCMs account for variation in pH and the metal-to-bacteria concentration ratio. Good agreement with the analytical data was obtained, yielding overall V(Y) values of 1.8 and 5.9 for G. thermocatenulatus and G. stearothermophilus, respectively (Table 3). The higher log K value of 2.20 ± 0.16 obtained for G. stearothermophilus indicates a stronger interaction between Cd²⁺ and site 1, compared to G. thermocatenulatus (log K = 1.30 ± 0.22). The fit of the single best-fitting SCM to the experimental data is displayed in Fig. 3.

DISCUSSION

This study uses a combined approach of metal ion susceptibility tests and complexation modeling analysis. Initial cadmium ion toxicity experiments revealed high MICs of Cd²⁺ for G. stearothermophilus (DSM 6790) and G. thermocatenulatus (DSM 730) in comparison to 24 other studied thermophilic bacteria.

Calculations of SCMs for both microorganisms were performed using the Donnan model. In theory, the advantage of the Donnan model over other electrostatic models is that it considers the cell wall as a volume that is ion penetrable, whereas other electrostatic models (e.g., the constant capacitance and Stern models) only account for ion-impenetrable planar surfaces (44). Although the Donnan model assumes the surface binding sites to be uniformly distributed over the cell wall volume, some previous research on bacilli suggests that binding sites may be concentrated on the cell poles (37, 38). We acknowledge that additional research is warranted to better define the most appropriate format for SCMs for bacterial surfaces, in particular with regard to the modeling of the effects of cell wall electric charge. However, in the absence of contradictory data, we employ the Donnan model under the assumption of uniform site distribution to quantify electrostatic interactions between aqueous metal ions and chemically functional groups within and on the cell walls.

Some of the model parameters are similar to previously published results (Table 3), although the diversity of biosorbants and modeling approaches described in the literature somewhat limit the comparisons that can be made. The deprotonation constant pK values determined for site 2 and site 3 for both Geobacillus strains are in good agreement with recent results reported for the thermophilic species TOR-39 (42) and Anoxybacillus flavithermus (11), the mesophilic bacterium Bacillus subtilis (16), and five natural bacterial consortia (5). The pK values of site 1 for G. stearothermophilus and G. thermocatenulatus are considerably lower than those reported for the other bacteria and may indicate that a different type of functional group is involved in complexation reactions for species of the genus Geobacillus. Interestingly, the average of the pK values for site 1 and site 2 of the four-site model for the bacterial consortia (5) is almost equivalent to the deprotonation constant for site 1 for the Geobacillus strains. Yee and Fein (43) proposed that metal-bacteria adsorption is independent of the bacterial species involved and, therefore, a generalized adsorption model may be applied. We acknowledge that the differences between the SCMs developed in this investigation and the generalized model of Yee and Fein (43) may relate to the different experimental methods or modeling approaches employed, but it is also possible that the generalized model is simply inappropriate for the two studied Geobacillus strains and therefore not universally applicable.

Although in all of the above-mentioned previous investigations site 1 is proportionally the most abundant binding site, only for the thermophiles A. flavithermus, G. thermocatenulatus, and G. stearothermophilus does it account for more than 62% of the total concentration of binding sites. The significantly lower proportions of site 1 observed for the other bacteria (below 44%) may indicate that A. flavithermus, G. thermocatenulatus, and G. stearothermophilus as thermophiles differ in cell wall composition from the other mesophilic bacteria and therefore expose different chemically reactive functional groups on and within their cell walls.

As mentioned above, we did not assign site 1 (pK = 3.8) to a particular identity or functional group structure. Many studies assume binding sites with low pK values to be carboxylic, because short carboxylic acids have pK values below 4.5 and carboxylic acids are present in the cell wall. However, it must be borne in mind that functional groups can only be reliably identified by other methods, such as X-ray spectroscopy analysis. X-ray adsorption spectroscopy experiments conducted by Boyanov and colleagues (9) suggested that Cd²⁺ adsorption onto Bacillus subtilis at a pH of 3.4 is predominated by phosphoryl ligands, whereas carboxylic ligands are the dominant binding sites in the pH range of 5.0 to 7.8.

The thermodynamic stability constant K for cadmium ion binding are not as high for the Geobacillus strains as for the other bacteria described in the literature (5, 11, 16, 42, 43). We acknowledge that the differences may be due to different modeling approaches employed. Alternatively, this may indicate a weaker interaction between the cadmium ions and the reactive surface sites of the Geobacillus strains compared to other species investigated to date.

The variation between the SCM parameters of the two Geobacillus strains investigated here are too small to permit identification of any relationship between biosorption and growth responses to elevated concentrations of Cd²⁺. G. thermocatenulatus and G. stearothermophilus may have evolved several metal ion detoxification strategies that account for the observed types of growth curves. The genome of G. stearothermophilus (DSM 13240) is being sequenced at the present time. A search of the genome data currently available shows evidence that G. stearothermophilus (DSM 13240) contains sequence information required for cadmium ion efflux, such as cadmium-transporting ATPases (http://www.genome.ou.edu/bstearo.html). When completely sequenced, the genome may reveal even more cadmium ion detoxification systems.

This study demonstrates that the extent of cadmium ion adsorption by G. thermocatenulatus and G. stearothermophilus can be predicted for a range of pH values and metal-to-bacteria concentration ratios. SCMs can be applied to describe a cation-bacterium interaction under a given set of conditions and may be valuable for development and optimization of bioremediation schemes. Investigations of microbial biosorption can deepen the insight for metal mobilization in the environment (such as contamination of drinking water) and can be used to improve waste treatment of metal-polluted water and soil. Further studies should evaluate the adsorption of other metals by the species studied here and by other thermophiles, for example, to determine if systematic metal affinity series exist, as have been observed for mesophiles (7, 43).