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Anal. Chem. 2006, 78, 6285-6290

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Epithermal Neutron Activation Analysis of Cr(VI)-Reducer Basalt-Inhabiting Bacteria Nelly Yasonovna Tsibakhashvili,† Marina Vladimirovna Frontasyeva,*,‡ Elena Ivanovna Kirkesali,‡ Nadezhda Gennadievna Aksenova,‡ Tamaz Levanovich Kalabegishvili,§ Ivana Georgievich Murusidze,† Ligury Mikhailovich Mosulishvili,§ and Hoi-Ying N. Holman|

Joint Institute for Nuclear Research, Dubna, Moscow Region, Russian Federation, Andronikashvili Institute of Physics and Chavchavadze State University, Tbilisi, Georgia, and Lawrence Berkeley National Laboratory, Berkeley, California 94720

Epithermal neutron activation analysis (ENAA) has been applied to study elemental composition of Cr(VI)-reducer bacteria isolated from polluted basalts from the Republic of Georgia. Cr(VI)-reducing ability of the bacteria was examined by electron spin resonance, demonstrating that the bacteria differ in their rates of Cr(VI) reduction. A wellpronounced correlation between the ability of the bacteria to accumulate Cr(V) and their ability to reduce Cr(V) to Cr(III) observed in our experiments is discussed. Elemental analysis of these bacteria also revealed that basalt-inhabiting bacteria are distinguished by relative contents of essential elements such as K, Na, Mg, Fe, Mn, Zn, and Co. A high rate of Cr(III) formation correlates with a high concentration of Co in the bacterium. ENAA detected some similarity in the elemental composition of the bacteria. The relatively high contents of Fe detected in the bacteria (140-340 µg/g of dry weight) indicate bacterial adaptation to the environmental conditions typical of the basalts. The concentrations of at least 12-19 different elements were determined in each type of bacteria simultaneously starting with the major to ultratrace elements. The range of concentrations spans over 8 orders of magnitude. Anthropogenic activity is a source of continual influx of heavymetal contaminants into the environment. A complex variety of abiotic and biotic processes affects their speciation and distribution. Some of these processes can be applied to removing * Corresponding author. E-mail: [email protected]. † Andronikashvili Institute of Physics and Chavchavadze State University. ‡ Joint Institute for Nuclear Research. § Andronikashvili Institute of Physics. | Lawrence Berkeley National Laboratory. 10.1021/ac051727e CCC: $33.50 Published on Web 08/23/2006

© 2006 American Chemical Society

environmental pollutants. Indigenous bacteria can be successfully used to either detoxify or immobilize toxic substances.1 Chromatereducing bacteria are under continuous investigation, and in-depth molecular understanding has been developed for some of them.1,2 Bacterial strains that were tolerant to Cr(VI) showed changes in the elemental composition of cells after exposure to Cr(VI).3,4 In the cells treated with other heavy metals, significant alterations in the cell composition were also observed.5 Recently, great attention has been paid to the elemental analysis of the major sites in cells where heavy metals are accumulated.3,6,7 Despite the intensive studies of the problem, the dependence between the ability of bacteria to reduce or immobilize metals and their elemental compositions still is not clear. The elemental composition of cells is studied for many bacteria that belong to various taxa.8-12 The bacteria have been found to differ significantly in their relative contents of different elements. (1) Bruins, M. R.; Kapil, S.; Oehme, F. W. Ecotoxicol. Environ. Saf. 2000, 45, 198-207. (2) Chen, J. M.; Hao, O. J. Crit. Rev. Environ. Sci. Technol. 1998, 28, 219251. (3) Vinze, G.; Vallner, J.; Balogh, A.; Kiss, F. Bull. Environ. Contam. Toxicol. 2000, 65, 772-779. (4) Keim, C. N.; Lins, U.; Farina, M. Can. J. Microbiol./Rev. Can. Microbiol. 2001, 47, 1132-1136. (5) Venkateswelu, G.; Stotzky, G. Can. J. Microbiol. 1986, 32, 654-662. (6) Vrede, K.; Heldal, M.; Norland, S.; Bratbar, G. Appl. Environ. Microbiol. 2002, 6, 2965-2971. (7) Dillon, C. T.; Lay, P. A.; Kennedy, B. J.; Stampfl, A. P.; Cai, Z.; Ilinski, P.; Rodrigues, W.; Legnini, D.; Lai, B.; Maser, J. J. Biol. Inorg. Chem. 2002, 7, 640-645. (8) Nikitin, D. I.; Sorokin, V. V.; Pitryuk, I. A.; Nikitina, E. S. Prikl. Biokhim. Mikrobiol. 1998, 34, 180-182. (9) Mulyukin, A. L.; Sorokin, V. V.; Loiko, N. G.; Suzina, N. E.; Duda, V. I.; Vorob′eva, E. A.; El-Registan, G. I. Microbiology 2002, 7, 31-40. (10) Goldberg, J.; Gonzalez, H.; Jensen, T. E.; Corpe, W. A. Microbios 2001, 106, 177-188. (11) Fagerbakke, K. M.; Norland, S.; Heldal, M. Can. J. Microbiol. 1999, 45, 304-311.

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The elemental analysis of bacteria has been performed, in general, by inductively coupled plasma atomic emission spectrophotometry, atomic absorption spectrometry, mass spectrometry, and X-ray microanalysis (XRMA) with the transmission electron microscope. The epithermal neutron activation analysis (ENAA) has also been found to be suitable for multielement determination in biological samples.13 Recently, using ENAA, the multielement composition of Arthrobacter oxydans, which was isolated and cultivated from the Columbia (USA) basalt samples, has been characterized.14 In synchrotron radiation-based Fourier transform infrared spectromicroscopy experiments A. oxydans has been demonstrated to be a Cr(VI)-tolerant bacterium that can reduce Cr(VI) to Cr(III).15 A. oxydans was used as a model Cr(VI)-tolerant and reducing bacteria in our studies.16,17 Lately, several endolithic (rock/mineral inhabiting) bacterial strains were isolated from the most polluted regions in the Republic of Georgia.18 Here, we focus on the establishment of elemental composition of basalt-inhabiting bacteria with Cr(VI)-reducing ability for future elucidation of the Cr(VI) effect on this composition. This knowledge may turn out to be significant for understanding mechanisms of microbial heavymetal resistance. The aim of the present study was to characterize the baseline chemical composition of different Cr(VI)-reducer bacteria from Georgia basalts by applying ENAA. EXPERIMENTAL SECTION Chemicals. All chemicals were ACS-reagent grade and purchased from Sigma (St. Louis, MO). Bacteria and Treatment. The basalt samples were selected from the environmentally most deteriorated region of GeorgiaMarneuli. In this region, the samples from Kazreti mines of copper-disulfide and copper-zinc (Cu, Fe, Cr, C, Ni, Mo, Zn, Cd, etc.), the baryte-polymetallic (Pb, Zn, Cd, Ba, etc.), and gold ore (Au, Ag, Hg) types were taken along the Mashavera River gorge.18 All the rocks were basaltoids. From these samples, 121 endolithic bacteria were isolated by a previously described method.18 Among these isolates, 33 bacteria were found as Cr(VI) reducers. The reduction assay has been performed by electron spin resonance (ESR) for assessing chromium-reducing ability for fast identification of Cr(V). In the present work, the following Cr(VI)-reducer isolates were examined: Nos. 14, 61, 151, and 163. From their growth properties and morphology, they belong to the genus Arthrobacter (the exact identification of these bacteria by 16S rRNA is underway). Arthrobacter is of interest (12) Pitryuk, A. V.; Pusheva, M. A.; Sorokin, V. V. Microbiology 2002, 71, 2430. (13) Frontasyeva, M.; Steinnes, E. Proceedings of the International Symposium on Harmonization of Health Related Environmental Measurements Using Nuclear and Isotopic Techniques, IAEA, Hyderabad, India, 4-7 November, 1996, 1997; p 301. (14) Tsibakhashvili, N.; Mosulishvili, L.; Kalabegishvili, T.; Kirkesali, E.; Frontasyeva, M.; Pomyakushina, E.; Pavlov, S.; Holman, H.-Y. J. Radioanal. Nucl. Chem. 2004, 259, 527-531. (15) Holman, H.-Y.; Perry, D.; Martin, M.; Ganble, G.; McKinney, W.; HunterCevera, J. Geomicrobiol. J. 1999, 16, 307-324. (16) Kalabegishvili, T., Tsibakhashvili, N.; Holman, H.-Y. Environ. Sci. Technol. 2003, 37, 4678-4684. (17) Asatiani, N.; Abuladze, M.; Kartvelishvili, T.; Bakradze N.; Sapojnikova, N.; Tsibakhashvili, N.; Tabatadze, L.; Lejava, L.; Asanishvili, L.; Holman, H.-Y. Curr. Microbiol. 2004, 49, 321-326. (18) Tsibakhashvili, N.; Mosulishvili, L.; Kalabegishvili, T.; Pataraya, D.; Gurielidze, M.; Nadareishvili, G.; Holman, H.-Y. Fresenius Environ. Bull. 2002, 11, 352-361.

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because of its high potential for the reduction and immobilization of chromium in aerobic environments.17 The bacteria were grown in the following nutrient medium: 10 g of glucose, 10 g of peptone, 1 g of yeast extract, 2 g of caseic acid hydrolysate, 6 g of NaCl, and 1 L of distilled water. Bacterial cells were grown in 250-mL Erlenmeyer flasks as a suspension. The medium was inoculated with 0.1 mL of overnight broth and incubated at 21 °C during continuous shaking. After being cultivated for 5 days, the cells were harvested by centrifugation (10 000 rpm, 15 min, 4 °C) and rinsed twice in a 20 mM phosphate buffer. This wet biomass was then placed in an adsorption-condensation lyophilizer and dried following a procedure reported elsewhere.19 The dry native biomass was finally pelletized into 5-mm pills using a special titanium press form. The elemental composition of the bacterial biomass was determined by ENAA. To determine Cr(V) and Cr(III) content in cells by ESR, bacteria were cultivated in the same nutrient medium. At the early stationary phase of growth, 35 mg/L Cr(VI) (as K2CrO4) was added to the nutrient medium. Bacterial cells were harvested at 1 and 49 h by centrifugation prior to analysis. Epithermal Neutron Activation Analysis. ENAA was conducted at the IBR-2 pulsed fast reactor in FLNP, JINR, Dubna, which is characterized by a very high ratio (∼100) of epithermal neutrons to thermal ones. The IBR-2 reactor provides activation with the whole fission spectrum: thermal, epithermal, and fast neutrons. Thermal NAA takes advantage of the high intensity of neutrons available from the thermalization of fission neutrons and the large thermal neutron cross sections for most isotopes. Epithermal is taken to be neutrons with energies from the Cd “cutoff” of 0.55 eV up to ∼1 MeV. ENAA is a useful extension of thermal (conventional) NAA in that it enhances the activation of a number of trace elements relative to the major matrix elements. ENAA is particularly advantageous for radionuclides produced from a stable isotope with a high resonance activation integral relative to its thermal neutron activation cross section. In general, the activation cross sections of the matrix elements of environmental samples are inversely proportional to the neutron energy (1/v law). The trace elements also follow this general trend, but many of them have large activation cross sections at specific energies in the epithermal energy region. In our case, the following advantages are evident: (i) improved detection limits for As, Br, Rb, Sr, and Sb; (ii) reduction of high matrix activity, e.g., from 28Al, 56Mn, 24Na, 46Sc, smf 60Co (see relevant nuclear data in Table 1 20). Bacterial samples of ∼0.5 g were packed in aluminum cups for long-term irradiation and were heat-sealed in polyethylene foil bags for short-term irradiation. The neutron flux density characteristics and temperature in the irradiation channels are given in Table 2.21 Long-lived isotopes were determined using the cadmiumscreened irradiation channel. The samples were irradiated for 5 days, repacked, and then counted twice after decays of 4 and 20 days. The counting time varied from 1.5 to 10 h. (19) Mosulishvili, L.; Kirkesali, E.; Belokobilsky, A.; Khizanishvili, A.; Frontasyeva, M.; Gundorina, S.; Opera, C. J. Radioanal. Nucl. Chem. 2002, 252, 15-20. (20) De Corte, F.; Simonits, A.; De Wispelaere, A. J. Radioanal. Nucl. Chem. 1989, 133, 131-151. (21) Frontasyeva, M.; Pavlov, S. Analytical Investigations at the IBR-2 Reactor in Dubna, Dubna, 2000; JINR preprint E14-2000-177.

Table 1. Nuclear Data for the Determined Elements by (n, γ) Reactions element

T1/2 of product nuclide

Io/σo20

(A) Favorable by ENAA 26.3 h 74Se 119.8 d 81Br 35.3 h 85Rb 18.7 d 84Sr 64.8 d 109Ag 249.7 d 121Sb 2.70 d 123Sb 60.2 d 133Cs 2.06 y 130Ba 11.8 d 152Sm 46.7 h 197Au 2.7 d 75As

As Se Br Rb Sr Ag Sb Sb Cs Ba Sm Au Na Mg Al K Ca Sc Cr Mn Fe Co Zn

target nuclide

13.6 10.0 19.3 14.8 13.2 17.5 33.0 28.8 12.7 24.8 14.4 15.7

(B) Favorable by Thermal (Conventional) NAA 23Na 15.0 h 26Mg 9.46 m 27Al 2.24 m 42K 12.4 h 48Ca 8.72 m 45Sc 83.8 d 50Cr 27.7 d 55Mn 2.58 h 58Fe 44.5 d 59Co 5.27 y 64Zn 243.9 d

0.59 0.64 0.71 0.97 0.45 0.43 0.53 1.05 0.97 1.99 1.91

Table 2. Flux Parameters of Irradiation Positions21 irradiation position

Φth ‚1012,a n cm-2 s-1

Φth ‚1012,b n cm-2 s-1

Φth ‚1012,c n cm-2 s-1

T, °C

Ch1 (Cd-screened) Ch2

0.023 1.23

3.3 2.9

4.2 4.1

70 60

a

E ) 0 ÷ 0.55 eV. b E ) 0.55 ÷ 105 eV. c E ) 105 ÷ 25 × 106 eV.

To determine the short-lived isotopes of Mg, Al, Ca, and Mn, the conventional irradiation channel was used. The samples were irradiated for 3 min and measured twice for 5-8 and 20 min after 3-5- and 20-min decay, respectively. γ-Ray spectra were measured using a large-volume Ge(Li) detector with a resolution of 1.96 keV at the 1332.4-keV line of 60Co with an efficiency of 30% relative to a 3 in. × 3 in. NaI detector for the same line. The data processing and element concentration determination were performed on the basis of certified reference materials and comparators using software developed in FLNP JINR.22 Three certified reference materials (CRMs), namely, IAEA Lichen-336, IAEA Bottom Sediments SDM-2T, and Nordic Moss DK-1, were used for quality assurance purposes. Electron Spin Resonance Spectrometry. The ESR investigations were carried out on a RE 1306 radiospectrometer with 100kHz modulation at 9.3 GHz.16 Detection of Cr(V) was carried out at liquid nitrogen temperature (77 K) to avoid a decrease in sensitivity of the ESR spectrometer caused by the water content in bacterial samples. The detection of the broad line for Cr(III) was complicated at low temperatures due to the presence of (22) Ostrovnaya, T. M.; Nefedyeva, L. S.; Nazarov, V. M.; Borzakov, S. B.; Strelkova, L. P. Proceedings, Activation Analysis in Environment Protection, Dubna, 1993; p 319, preprint D-14-93-325.

Figure 1. Formation of Cr(V) and Cr(III) in different isolates.

oxygen impurity in liquid nitrogen, which shifts the zero line. To avoid this problem, we measured Cr(III) at room temperature after drying the samples at 100 °C. RESULTS AND DISCUSSION After mixing the bacterial cells with the chromate solution, the ESR line with a g-factor of 1.980 and a width of 12 G appeared in a few minutes. This line is similar to that detected in A. oxydans and is characteristic of the Cr(V) complexes with diol-containing molecules.16 Thus, Cr(VI) reduction begins at the surface of endolithic bacteria. The macromolecules at the cell wall of bacteria could act as an electron donor to Cr(VI) to form a stable Cr(V) complex. Control experiments also revealed that some of these bacteria produce a stronger Cr(V) line than A. oxydans. These cultures were chosen for further investigations and tested for the time course of both Cr(V) decomposition and Cr(III) formation in them. We observed that the Cr(III) ESR line had the following parameters in all tested bacteria: a g-value of 2.02 and a line width of 650 G. The relative intensities of Cr(V) and Cr(III) ESR signals, which are in direct proportion to the concentration of Cr(V) and Cr(III) complexes, respectively, are given in Figure 1. It can be seen that the level of Cr(V) was significantly different in different bacteria after 1 h of Cr(VI) action, suggesting that there is a difference in the lifetimes of Cr(V) complexes (in the figure, bacteria are Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

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Figure 2. Elemental distribution in lyophilized samples of different isolates.

arranged in ascending order of their Cr(V) ESR line intensities). Though the amount of Cr(V) registered is a combined effect of the formation Cr(VI) f Cr(V) and decay Cr(V) f Cr(III) processes, one should take into account that the rate of formation of Cr(V) is much higher than that of decay. Consequently, the effect of decomposition, i.e., the decay of Cr(V) complexes and generation of Cr(III), will appear later. Indeed, Figure 1 demonstrates that the amount of Cr(III) accumulated by bacteria during 2 days correlated positively with the level of Cr(V) after 1 h of Cr(VI) action. This correlation suggests that bacteria characterized by a higher rate of the Cr(V) f Cr(III) process had lower level of Cr(V) at 1 h of Cr(VI) action. On the other hand, the great amount of Cr(III) generated in the bacteria may slow the Cr(V) f Cr(III) process. Really, the alteration in the Cr(V) level observed after 2 days of Cr(VI) action gives sound evidence in favor of this mechanism. The level of Cr(V) decreased less in the bacteria that accumulated more Cr(III) than in those that accumulated less Cr(III). Thus, the comparative study of paramagnetic chromium formation and decomposition processes in different bacteria was found to be very useful in order to characterize the mechanisms of Cr(VI) detoxification in bacteria. The obtained results show that at the beginning of chromate action isolate 61 has a higher rate of Cr(VI) reduction than other bacteria. Besides, it was observed that isolate 163 exhibits dynamics of Cr(V) decomposition and Cr(III) formation different from other isolates. Thus, we can say that the reduction of Cr(VI) proceeds in different ways in different bacteria. It is well known that bacterial resistance to metals is heterogeneous in both their genetic and biochemical bases.3 At the biochemical level, at least six different mechanisms are responsible for the resistance. A detailed investigation of mechanisms of Cr(VI) reduction by bacteria from Georgia basalts is underway. 6288

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In the next set of experiments, we investigated the elemental composition of selected bacteria to gain insight into the dependence between the Cr(VI)-reducing ability of bacteria and their elemental composition. Several aspects of cellular metabolic activities of bacteria can be evaluated knowing the elemental composition of cells. For example, C/N/P ratios provide information on environmental nutrient conditions. Osmotic and energy conversion can be studied by investigating elements such as sodium, potassium, magnesium, and calcium.6,10 Recently, using XRMA analysis, the haloalkaliphilic acetogenic bacteria and the alkaliphilic sulfate-reducing bacteria were found to differ significantly in their relative contents of S, K, P, and Cl.12 Elemental analysis of the cells also revealed that the distinctive feature of viable resting forms of bacteria was their low P/S ratio and high Ca/K ratio.6 In the present work, we are focused on the determination of metal contents in bacteria, although the concentrations of some other elements were also established. Metals play an integral role in the life processes of microorganisms. Some metals, such as Ca, Co, Cr, Cu, Fe, Na, Mg, Mn, K, Ni, and Zn, are required nutrients and are essential. Others have no definite biological function (Ag, Al, Cd, Au, Hg) and are nonessential.23 In Figure 2, results of ENAA determinations of elemental contents of bacteria isolated from the Georgian basalts are presented. The concentrations from 12 to 19 elements of the following set, Na, Mg, Al, K, Ca, Sc, Cr, Mn, Fe, Co, Zn, As, Br, Rb, Sr, Ag, Sb, Cs, Ba, Sm, and Au, were determined in the bacterial cells. The maximum number of elements was detected in isolate 163; and the minimum one, in isolate 151. The (23) Hoghes, M. N., Peele, R. K., Eds. Metals and Microorganisms; Chapman and Hall: London, 1989.

concentration range was over 8 orders of magnitude, from major to ultratrace levels. The ENAA results revealed some similarity in the elemental composition of the isolates studied (Figure 2). In all bacteria, potassium and sodium were the dominant elements (together more than 90% of the total content of the elements determined). The concentrations of both Na and K were in the range of 104 µg/g. The content of Mg was ∼1 order of magnitude lower than those Na and K and ∼1 order higher than those of Fe and Al. In general, the concentration of Zn was a little less than these latter ones. The concentrations of all other elements were much lower. The high content of Na and K in the bacterial cells can be explained by the fact that bacteria were freeze-dried in a Na-K phosphate buffer at pH 7, which could increase concentrations of Na and K. As known, the membranes in lyophilized bacteria lose their barrier function.24 Therefore, during lyophilization Na and K ions must diffuse along their concentration gradients, and as a result, the lyophilization in media with a high concentration of either of these elements must lead to high content of this element in the cell. This result is in agreement with the data obtained recently by Mulyukin et al.9 It was observed that, indeed, the lyophilized Micrococcus luteus contained larger amounts of K than the vegetative cells and viable resting cells. Potassium is an essential metal for living organisms and is required for regulation of intracellular osmotic pressure. K is also involved in nonspecific activation of many enzymes, in bacterial energy metabolism (as a coupling ion), and in the regulation of intracellular pH.23 Iron is a most important metal biologically. It is constituent of complex molecules with a wide array of functions.23 In tested bacteria, the concentration of Fe was relatively higher (in the range of 140-340 µg/g) than that in other bacteria.3,11,25 For comparison, the concentration of Fe in the Pseudomonas strain was less than 30 µg/g.3 In autotrophic and heterotrophic bacteria, it constituted 40-50 µg/g.25 This result suggests that the chemical composition of basalts influenced the element composition of bacteria. Really, the Georgia basalt samples from the studied sites are rocks with high content, which is due to the abundance of ferromagnesium mineralsspyroxene [(Ca,Na,Mg,Fe)(Al,Si)O3], olivine (Mg1.8Fe0.2SiO4), and magnetite (Fe2+Fe3+2)O4.18 It seems that, in the tested bacteria, active transport systems exist for iron, and as a result, high concentrations of Fe were found in all the isolates. As is known, iron easily enters bacterial cells mainly by way of the magnesium transport system.23 The high content of Al (which is also present in large quantity in basalts) in isolates also confirms this consideration. However, in basalt-inhabiting bacteria, the content of magnesium, which is known to stabilize the cell wall in addition to being involved in the catalysis of various reactions in the cell, was not high. This indicates that the high content of magnesium in basalts does not influence the Mg content in bacteria. In the tested bacteria, the concentration of magnesium was in the range detected for other bacteria (it ranged from 1.4 × 103 µg/g in isolate 151 to 2.17 × 103 µg/g in isolate 14). In most bacteria, the content (24) Suzina, N. E.; Mulykhin, A. L.; Loiko, N. G.; Kozlova, A. N.; Dmitriev, V. V.; Shorokhova, A. P.; Gorlenko, V. M.; Duda, G. I. Mikrobiologiya 2001, 70, 776-787. (25) Kurbanov, I. S.; Zlatkin, I. V.; Nikitin, D. I.; Mordvintsev, P. I,; Aliev, D. I.; Vanin, A. F. Izv. Akad. Nauk. SSSR. Biol. 1990, 3, 443-447.

of magnesium ranges from 145 to 12 × 103 µg/g.12,26 One can presume that the obtained result indicates that the demand for magnesium is low in basalt-inhabiting bacteria. There are other types of bacteria in which the demand for magnesium is insignificant.12,26,27 For example, the growth of alkaliphilic bacterium N. acetigena depends on the content of magnesium salts in the medium.26 A 5-fold increase of magnesium concentration in the medium stimulated N. acetigena growth and delayed cell lysis, although calculations showed that the amount of soluble Mg changed insignificantly when the content of magnesium salts in the medium was increased. five- or 10-fold. Similar data were obtained for Archaehalobacterium sp., the growth of which was observed in a magnesium-deficient medium (less than 50 µM), although the optimal magnesium concentration ranged from 0.1 to 2 mM at pH 9.5.27 Thus, we propose that the relatively high concentrations of Fe and Al in basalt-inhabiting bacteria may result from bacterial adaptation to the environmental conditions. Similar effects are well known in other bacteria.6,12 The study of cell physiology of alkaliphilic bacteria isolated from soda lakes revealed a demand for definite elements (Mn, Co, Ni). This demand results from the bacterial adaptation to the environmental conditions typical of soda lakes.12 The physiological adaptation of marine bacteria to high internal concentrations of chloride was also detected earlier.6 XRMA analysis showed that growing marine bacterioplankton have an internal environment in which chloride is the dominating anion. The concentrations of other essential metals such as Mn and Zn were also within the range detected for other bacteria.8,12 Zn stabilizes various enzymes and DNA through electrostatic forces. It is also a part of complex molecules with a wide array of functions. Mn is an important trace element with low toxicity.23 The elemental analysis of basalt-inhabiting bacteria revealed a series of other metals necessary for bacterial activity. Metals displaying changes in valence, especially Ni, Cu, Cr, and Co, participate in electron transport and redox reactions in bacteria.23 Cu was not detected in the tested bacteria due to poor detection limit. Trace amount of Ni (0.1 µg/g) was observed only in A. oxydans.14 At the same time, the basalt-inhabiting bacteria were found to have an obligate requirement for Co. Co was detected in all the isolates. Intracellular Co content in the tested bacteria differed significantly from each other. In two isolates, 61 and 163, we also detected trace amounts of Cr (∼2 µg/g). As, Sb, Br, and Rb were determined in all bacteria. The concentrations of Br and Rb were ∼1 µg/g. The concentrations of As and Sb were much less. In some isolates, we also detected Ba and Sr and ultratrace amounts of Au, Ag, Cs, and Sm. All of these elements have no beneficial function but have to be considered by cells as toxins. We did not reveal any significant difference between levels of Na, K, Mg, Fe, Zn, and Mn in A. oxydans and in isolates from the Georgian basalts. However, in A. oxydans, a large amount of Ca (210 µg/g) was detected.14 The same amount of this element was observed only in isolate 151. Comparison of elemental composition of different isolates revealed that, in isolate 61, which had the maximum rate of Cr(III) formation, the concentration of Co was much larger (0.33 (26) Murray, T.; Popham, D.; Setlow, P. J. Bacteriol. 1998, 180, 4555-4563. (27) Tindall, B.; Mills, A.; Grant, W. J. Gen. Microbiol. 1980, 116, 257-260.

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The concentrations of both Fe and Mn were the highest in isolate 14, and again, in other bacteria the contents of these elements were similar. In isolate 151, the maximum amount of Zn was observed (106 µg/g). At the same times, the concentrations of Na, K, Mg, Fe, Mn, and Co in this sample were the lowest detected. We did not detect any significant feature in the elemental composition of isolate 163. For more illustration, the distribution of some elements, such as Fe, Co, and Zn, in different isolates is presented in Figure 3. The distribution of elements is changed in bacteria after exposure to Cr(VI). These alterations give insight into the mechanism of bacterial resistance. These results will be presented in our next work. CONCLUSION Cr(VI) is a widespread environmental pollutant. It has been discharged into the environment primarily from industries such as metal plating, leather tanning, and wood preservation. Indigenous bacteria have high potential for the reduction and detoxification of chromium in the environment. However, the molecular mechanisms of Cr(VI) resistance in bacteria still are not clear. In this work, the multielement composition of several Cr(VI)-reducer basalt-inhabiting bacteria has been characterized for the first time for future elucidation of Cr(VI) effect on this composition. Using ENAA, the concentrations of 12-19 elements were determined simultaneously in each bacteria strain. Our experiments revealed that basalt-inhabiting bacteria can be distinguished by their relative distribution of essential elements. However, some similarity in their elemental compositions was also detected. ENAA measurements revealed that the tested bacteria have an obligated requirement for cobalt, which participates in electron transport and redox reaction in bacteria. Co was detected in all bacteria. Besides, in the isolate showing the maximum rate of Cr(III) formation, the concentration of Co was found to be larger than in other bacteria. The experimental data presented in this study may turn out to be significant for understanding the mechanisms that can potentially sustain the capability of Cr(VI) resistance and reduction in bacteria.

Figure 3. Concentrations of Fe, Co, and Zn in different isolates. 1 corresponds to isolate 61; 2 to isolate 14; 3 to isolate 151, and 4 to isolate 163.

µg/g) than in other bacteria. A similar amount of Co was detected in A. oxydans as well.14 It should be noted that the concentrations of Co were similar in all bacteria (Figure 3).

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ACKNOWLEDGMENT This work was funded through Project GE-B2-2597-TB-03 from the U.S. Civilian Research and Development Foundation (CRDF). We gratefully acknowledge Drs. D. Pataraya and M. Gurielidze for providing bacterial samples. Received for review September 27, 2005. Accepted February 13, 2006. AC051727E