Joseph W. Stucki

REDUCTION OF STRUCTURAL Fe(III) IN SMECTITE BY A PURE CULTURE OF SHEWANELLA PUTREFACIENS STRAIN MR-1

Shewanella putrefaciens is a species of metal-reducing bacteria with a versatile respiratory metabolism. This study reports that S. putrefaciens strain MR-1 rapidly reduces Fe(III) within smectite clay minerals. Up to 15% of the structural Fe within ferruginous smectite (sample SWa-1, Source Clays Repository of the Clay Minerals Society) was reduced by MR-1 in 4 h, and a range of 25% to 41% of structural Fe was reduced after 6 to 12 d during culture. Conditions for which smectite reduction was optimal, that is, pH 5 to 6, at 25 to 37 °C, are consistent with an enzymatic process and not with simple chemical reduction. Smectite reduction required viable cells, and was coupled to energy generation and carbon metabolism for MR-1 cultures with smectite added as the sole electron acceptor. Iron(III) reduction catalyzed by MR-1 was inhibited under aerobic conditions, and under anaerobic conditions it was inhibited by the addition of nitrate as an alternate electron acceptor or by the metabolic inhibitors tetrachlorosali-cylanilide (TCS) or quinacrine hydrochloride. Genetic mutants of MR-1 deficient in anaerobic respiration reduced significantly less structural Fe than wild-type cells. In a minimal medium with formate or lactate as the electron donor, more than three times the amount of smectite was reduced over no-carbon controls. These data point to at least one mechanism that may be responsible for the microbial reduction of clay minerals within soils, namely, anaerobic respiration, and indicate that pure cultures of MR-1 provide an effective model system for soil scientists and mineralogists interested in clay reduction. Given the ubiquitous distribution and versatile metabolism of MR-1, these studies may have further implications for bioremediation and water quality in soils and sediments.

Growth of Iron(III)-Reducing Bacteria on Clay Minerals as the Sole Electron Acceptor and Comparison of Growth Yields on a Variety of Oxidized Iron Forms

ABSTRACT Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe. While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor. Here we have demonstrated that a pure culture of Shewanella oneidensis strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor. Pure cultures of S. oneidensis were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors [smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen] in the same minimal medium. Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon). Cell counts showed that similar growth of S. oneidensis (108 cells ml−1) occurred with smectitic Fe(III) and on other Fe forms [amorphous Fe(III) oxyhydroxide, and Fe citrate] or oxygen as the electron acceptor. In contrast, cell yields of S. oneidensis measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions. Over a range of particle loadings (0.5 to 4 g liter−1), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced. From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the Bacteria (Clostridium and Desulfitobacterium) and the δ-Proteobacteria (members of the Geobacteraceae). Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available. Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments.

The impact of structural Fe(III) reduction by bacteria on the surface chemistry of smectite clay minerals

Abstract Although clay mineral reduction is thought to occur primarily as a result of the activity of indigenous microorganisms in soil, most research has focused on chemical mechanisms of Fe reduction within clay minerals. Here we show that bacteria isolated from soils and sediments catalyze the rapid reduction of structural Fe(III) in the smectite clay minerals. The extent of Fe(III) reduction is large, from 46% to >90%. Furthermore, the effects of structural Fe(III) reduction by bacteria on the surface chemistry of smectites are dramatic. Swelling pressure, as measured by water content, was shown to decrease by 40% to 44% in smectites reduced by bacteria as compared to unaltered or reoxidized smectites. Particle surface area decreased by 26% to 46% in response to bacterial reduction, and the surface charge density as measured by the ratio of cation exchange capacity to specific surface area increased over the same scale. Measurements of swelling pressure in smectite saturated with the organic cation trimethylphenylammonium (TMPA) indicated that the hydrophilic character of the clay mineral surface was enhanced upon reduction. The valence state of Fe in the octahedral layer of smectite, as revealed through reflectance spectra, correlated to the amount of Fe(III) reduced in bacterial cultures, providing information on the mechanism of intervalence electron transfer in bacterially reduced clay minerals. The extent of reduction and surface chemical effects catalyzed by bacteria in this study are similar in magnitude to those observed previously for potent inorganic reductants. Given that clay minerals dominate the solid phase of porous media and that Fe(III)-reducing bacteria are abundant in soils and aquatic sediments, these data suggest that bacterial clay mineral reduction may play an important role in soil biogeochemistry, affecting processes such as nutrient cycles and the fate of organic contaminants.

Oxidation-reduction mechanism of iron in dioctahedral smectites: I. Crystal chemistry of oxidized reference nontronites

Abstract The crystal chemistry of Fe in four nontronites (Garfield, Panamint Valley, SWa-1, and NG-1) was investigated by chemical analysis, X-ray goniometry, X-ray absorption pre-edge spectroscopy, powder and polarized extended X-ray absorption fine structure (EXAFS, P-EXAFS) spectroscopy, and X-ray diffraction. The four reference nontronites have Fe/(Fe + Al + Mg) ratios ranging from 0.58 to 0.78, and are therefore representative of the different chemical compositions of dioctahedral ferruginous smectites. Pre-edge and powder EXAFS spectroscopy indicate that NG-1 contains 14 to 20% of tetrahedrally coordinated Fe3+, whereas the other three samples have no detectable IVFe3+. The partitioning of VIFe3+ between cis (M2) and trans (M1) sites within the octahedral sheet was determined from the simulation of X-ray diffraction patterns for turbostratic nontronite crystallites by varying the site occupancy of Fe. Based on this analysis, the four nontronite samples are shown to be trans-vacant within the detection limit of 5% of total iron. The in-plane and out-of-plane local structure around Fe atoms was probed by angular P-EXAFS measurements performed on highly oriented, self-supporting films of each nontronite. The degree of parallel orientation of the clay layers in these films was determined by texture goniometry, in which the half width at half maximum of the deviation of the c* axis of individual crystallites from the film plane normal, was found to be 9.9° for Garfield and 19° for SWa-1. These narrow distributions of orientation allowed us to treat the self-supporting films as single crystals during the quantitative analysis of polarized EXAFS spectra. The results from P-EXAFS, and from infrared spectroscopy (Madejova et al. 1994), were used to build a two-dimensional model for the distribution of Fe, and (Al,Mg) in sample SWa-l. In this nontronite, Fe, Al, and Mg atoms are statistically distributed within the octahedral sheet, but they exhibit some tendency toward local ordering. Fe-Fe and (Al, Mg)-(Al,Mg) pairs are preferentially aligned along the [010] direction and Fe-(Al,Mg) pairs along the [31̅0], and [3̅1̅0] directions. This distribution is compatible with the existence of small Fe domains separated by (Al,Mg), and empty octahedra, which segregation may account for the lack of magnetic ordering observed for this sample at low temperature (5K) (Lear and Stucki 1990).

EFFECTS OF REDUCTION AND REOXIDATION OF STRUCTURAL IRON ON THE SURFACE CHARGE AND DISSOLUTION OF DIOCTAHEDRAL SMECTITES

The effect of Fe oxidation state on the surface charge (CEC) and solubility of smectites were studied using the <2-μ, Na+-saturated fraction of an Upton, Wyoming; a Czechoslovakian; and a New Zealand montmorillonite; and a Garfield, Washington, nontronite. The reduction of structural Fe3+ in the octahedral sheet of each clay produced a net increase in the negative surface charge of the clay. The observed cation-exchange capacities deviated from the linear relationship predicted by charge-deficit calculations, assuming changes only in the Fe2+/Fe3+ ratio, and reversibly followed Fe reduction according to a 2nd-degree polynomial function. The deviations suggest reversible changes in mineral structure and composition during Fe reduction.These clays were susceptible to partial dissolution in citrate bicarbonate (CB) and citrate-bicarbonate-dithionite (CBD) solutions. Small amounts of Fe and Si dissolved as a result of Fe reduction in CBD, but affected <1% of the total clay mass except for the Czechoslovakian clay in which 2% of the clay dissolved. Although slightly more Fe dissolved than Si, no change in surface charge was noted. Almost no dissolution of these elements was detected in CB solution. In contrast, significant Al was detected in the CB solution, suggesting a heterogeneous dissolution mechanism. The CEC, however, was unchanged by the CB treatment. These results may be explained by the adsorption of hydrogen ions into the vacated Al3+ sites in the mineral structure. Dissolution seems to have been independent of the effects of Fe oxidation state on surface charge.РезюмеИссделовался эффект состояния окисления Ре на повехностный заряд и растворяемость смектитов при использовании фракции размером <2 μм Na-насыщенных монтмориллонитов из Уптон, Вайоминг, из Чехословакии и из Новой Зеландии и нонтронита из Гарфельд, Вашингтон. Восстановление структурного Fe3+ в октаэдрическом пласте каждой глины вызывало результирующее увеличение отрицательного поверхностного заряда глины. Наблюдаемые значения катионообменной способности отклонялись от линейной зависимости, предсказанной по расчетам на основе недостатка заряда, предполагая изменения только в величине отношения Fe2+/Fe3+ и были обратно пропорцио¬нальны восстановлению Ре в соответствии с полиномом второй степени. Эти отклонения указывают на обратимые изменения в структуре и составе минерала во время восстановления Ре.Эти глины легко подвергались частичному растворению в растворах бикарбоната цитрата (БКЦ) и бикарбоната цитрата-дитионита (БКЦД). Небольшие количества Ре и 81 растворялись в результате восстановления Ре в БКЦД, но влияли только на < 1% всей массы глины, исключая глину из Чехо¬словакии, для которой 2% массы растворялось. Хотя немного больше Ре, чем §1 растворялось, то никаких изменений поверхностного заряда не наблюдалось. Почти никакого растворения этих эле¬ментов не обнаруживалось в растворе БКЦ. В противоположность, значительное количество Аl об¬наруживалось в растворе БКЦ, указывая на неоднородный механизм растворения. Катионообменная способность, однако, не изменялась в результате обработки в БКЦ. Эти результаты могут быть объяснены учитывая адсорбцию ионов водорода на пустых местах после Аl3+ в структуре минерала. Растворение кажется быть независимым от эффектов состояния окисления Ре на поверхностный заряд. [E.G.]ResümeeDie Auswirkung der Oxidationsstufe des Eisens auf die Oberflächenladung (CEC) und die Löslichkeit von Smektiten wurde untersucht, wozu die Na-gesättigte Fraktion eines Montmorillonits von Upton, Wyoming; eines Montmorillonits aus der Tschechoslowakei bzw. aus Neuseeland; und ein Non-tronit von Garfield, Washington, verwendet wurden. Die Reduktion des strukturellen Fe3+ in der Oktaederschicht verursachte bei allen untersuchten Tonen eine Gesamtzunahme der negativen Oberflächenladung. Die beobachteten Kationenaustauschkapazitäten wichen von der linearen Beziehund ab, die durch Ladungsdefizitberechnungen vorhergesagt wurden unter der Annahme, daß sich nur das Fe2+/Fe3+-Verhältnis ändert und folgten reversibel der Fe-Reduktion, die der 2. Ableitung einer polynomialen Funktion folgt. Die Abweichungen deuten auf reversibel Änderungen in der Mineralstruktur und in der Zusammensetzung während der Fe-Reduktion hin. Diese Tone waren teilweise in Citratbikarbonat (CB)- und Citrat-bikardonatdithionit (CBD)-Lösungen löslich. Kleine Mengen an Fe und Si wurden infolge der Fe-Reduktion in CBD gelöst, was jedoch nur <1% der gesamten Tonmenge betraf mit Ausnahme des tschechoslowakischen Tons, bei dem 2% der Gesamtmenge gelöst wurde. Obwohl etwas mehr Fe als Si gelöst wurde, wurde jedoch keine Veränderung der Oberflächenladung beobachtet. In CB-Lösungen fehlte eine Lösung dieser Elemente nahezu vollständig. Im Gegensatz dazu wurden beachtliche Mengen Al in der CB-Lösung gefunden, was auf einen heterogenen Lösungsmechanismus hindeutet. Die CEC wurde jedoch durch die Behandlung mit CB nicht beeinflußt. Diese Ergebnisse kann man durch die Adsorption von Wasserstoffionen an die leergewordenen Al-Plätze in der Mineralstruktur erklären. Die Auflösung scheint unabhängig von den Auswirkungen der Oxidationsstufe des Eisens auf die Oberflächenladung zu sein. [U.W.]RésuméOn a étudié l’effet de l’état d’oxidation Fe sur la charge de surface (CEC) et la solubilité de smectites utilisant la fraction <2 n»m, saturée de Na+, d’une montmorillonite d’Upton, Wyoming, de Tchécoslovaquie, et de Nouvelle Zelande, et d’une nontronite de Garfield, Washington. La réduction de Fe3+ de structure dans la feuille octaèdrale de chaque argile a produit une augmentation nette de la charge de surface négative de l’argile. Les capacités d’échange de cations observées ont dévié de la relation linéaire prédite par les calculs de déficit de charge, assumant des changements dans la proportion Fe2+/Fe3+ seulement, et elles ont réversiblement suivi la réduction Fe selon une fonction polynomiale du deuxième degré. Les déviations suggèrent des changements réversibles de la structure minérale et de la composition pendant la réduction Fe.Ces argiles étaient susceptibles à la dissolution partielle dans des solutions de citrate bicarbonate (CB) et de citrate-bicarbonate-dithionite (CBD). De petites quantités de Fe et Si se sont dissoutes résultant de la réduction de Fe dans CBD, mais elle n’ont affecté que <1% de la masse totale de l’argile, sauf dans le cas de l’argile tchécoslovaque dans laquelle 2% de l’argile s’est dissoute. Malgré qu’un peu plus de Fe s’est dissout que de Si, aucun changement n’a été remarqué dans la charge de surface. Pratiquement aucune dissolution de ces éléments n’a été détectée dans la solution CB. En contraste, des quantités significatives d’Al ont été détectées dans la solution CB, suggérant un méchanisme de dissolution hétérogène. Ces résultats peuvent être expliqués par Padsorption d’ions hydrogène dans les sites Al3+ évacués dans la structure minérale. La dissolution semble avoir été indépendante des effets de l’état d’oxidation Fe sur la charge de surface. [D. J.]

Oxidation-reduction mechanism of iron in dioctahedral smectites: II. Crystal chemistry of reduced Garfield nontronite

Abstract The crystallochemical structure of reduced Garfield nontronite was studied by X-ray absorption pre-edge and infrared (IR) spectroscopy, powder X-ray diffraction, polarized extended X-ray absorption fine structure (P-EXAFS) spectroscopy, and texture goniometry. Untreated and highly reduced (>99% of total Fe as Fe2+) nontronite samples were analyzed to determine the coordination number and the crystallographic site occupation of Fe2+, changes in in-plane and out-of-plane layer structure and mid-range order between Fe centers, and to monitor the changes in structural and adsorbed OH/H2O groups in the structure of reduced nontronite. Contrary to earlier models predicting the formation of fivefold coordinated Fe in the structure of nontronites upon reduction, these new results revealed that Fe maintains sixfold coordination after complete reduction. In-plane PEXAFS evidence indicates that some of the Fe atoms occupy trans-sites in the reduced state, forming small trioctahedral domains within the structure of reduced nontronite. Migration of Fe from cisto trans sites during the reduction process was corroborated by simulations of X-ray diffraction patterns which revealed that about 28% of Fe2+ cations exist in trans sites of the reduced nontronite, rather than fully cis occupied, as in oxidized nontronite. Out-of-plane P-EXAFS results indicated that the reduction of Fe suppressed basal oxygen corrugation typical of dioctahedral smectites, and resulted in a flat basal surface which is characteristic of trioctahedral layer silicates. IR spectra of reduced nontronite revealed that the dioctahedral nature of the nontronite was lost and a band near 3623 cm-1 formed, which is thought to be associated with trioctahedral [Fe2+]3OH stretching vibrations. On the basis of these results, a structural model for the reduction mechanism of Fe3+ to Fe2+ in Garfield nontronite is proposed that satisfies all structural data currently available. The migration of reduced Fe ions from cis-octahedra to adjacent trans-octahedra is accompanied by a dehydroxylation reaction due to the protonation of OH groups initially coordinated to Fe. This structural modification results in the formation of trioctahedral Fe2+ clusters separated by clusters of vacancies in which the oxygen ligands residing at the boundary between trioctahedral and vacancy domains are greatly coordination undersaturated. The charge of these O atoms is compensated by the incorporation of protons, and by the displacement of Fe2+ atoms from their ideal octahedral position toward the edges of trioctahedral clusters, thus accounting for the incoherency of the Fe-Fe1 and Fe-Fe2 distances. From these results, the ideal structural formula of reduced Garfield nontronite is Na1.30[Si7.22Al0.78] [Fe2+3.65Al0.32Mg0.04]O17.93(OH)5 in which the increased layer charge due to reduction of Fe3+ to Fe2+ is satisfied by the incorporation of protons and interlayer Na.

Dissolution of hectorite in inorganic acids

The effect of acid type and concentration on the reaction rate and products of dissolution of hectorite in inorganic acids was investigated. The dissolution of hectorite in hydrochloric (HCl), nitric (HNO3) and sulphuric (H2SO4) acids was characterized using quantitative chemical analysis, infrared (IR) and multinuclear MAS NMR spectroscopies. The rate of dissolution increased with acid concentration and decreased in the order HCl ≥ HNO3 = H2SO4 at the same molar concentration. No differences were found in the reaction products of hectorite treated with the three acids. The rate of Li dissolution was slightly greater than that of Mg at lesser acid concentrations (0.25 M), indicating that protons preferentially attack Li octahedra. The gradual changes in the Si-O IR bands reflects the extent of hectorite dissolution. The analysis of 29Si MAS NMR spectra relative peak intensities with dissolution time and acid concentration provided direct dissolution rates for tetrahedral (Q3) Si. After acid dissolution, most Si was bound in a three dimensional framework site (Q4), but a substantial part also occurred in the Si(OSi)3OH (Q31OH) and Si(OSi)2(OH)2 (Q220H) environments. These three sites probably occur in a hydrous amorphous silica phase. Both AlJV and AlVt rapidly disappeared from 27Al MAS NMR spectra of the dissolution products with acid treatment. The changes in IR and MAS NMR spectra of hectorite due to acid dissolution are similar to those of montmorillonite.

REDUCTION AND REOXIDATION OF NONTRONITE: EXTENT OF REDUCTION AND REACTION RATES

The reduction and reoxidation of three nontronite samples, GAN (API H-33a, Garfield, Washington), SWa-1 (ferruginous Washington smectite), and NG-1 (Hohen Hagen, Federal Republic of Germany) were studied with visible absorption and Mössbauer spectroscopy. The intensity of the intervalence electron transfer (IT) band at 730 nm in these nontronites was monitored during reduction and reoxidation at 277, 294, and 348 K. The results showed that the intensity of the band followed the number of Fe(II)-O-Fe(III) groups in the clay crystal, increasing to a maximum at about Fe(II): total Fe = 0.4; upon complete reduction, the band decreased to about the intensity of the unaltered, oxidized sample. With reoxidation of the sample with O2, the intensity of the band increased sharply, followed by a gradual decay back to the original, oxidized intensity. The ultimate level of Fe reduction achieved was at least 92%. Concomitantly, the color changed from yellow through green, blue-green, dark blue, light blue, and light gray as the Fe(II) content increased. The GAN nontronite was more difficult to reduce than the SWa-1 or NG-1 samples. The rate and level of reduction increased with the amount of reducing agent added.

Functional Diversity and Electron Donor Dependence of Microbial Populations Capable of U(VI) Reduction in Radionuclide-Contaminated Subsurface Sediments

ABSTRACT In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.

Effects of iron oxidation state on the surface and structural properties of smectites

The oxidation state of iron (Fe) in the crystal structure of smectite clay minerals profoundly alters their physical-chemical properties. Among the properties affected are layer charge, cation exchange and fixation capacity, swelling in water, particle size, specific surface area, layer stacking order, magnetic exchange interactions, octahedral site occupancy, surface acidity, and reduction potential. Also affected is the surface chemistry of the clay, which alters clay–water and clay–organic interaction mechanisms. Rates and extents of degradation of pesticides are increased in the presence of reduced smectites compared to oxidized and reduced-reoxidized counterparts. A hypothesis regarding the mechanism for Fe reduction in clay minerals was first developed in 1963, and subsequent modifications have been proposed periodically through the present time. Recent studies clearly reveal that the process of Fe reduction involves more than the mere transfer of an electron to octahedral Fe(III) in the clay crystal. Ancillary reactions occur that produce significant structural modifications, some of which are reversible and others of which are not. Such changes in the crystal-chemical environment of structural Fe are thought to play a dominant role in altering the clay surface chemistry.