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Iron oxides in acid mine drainage environments and their association with bacteria
Chemical Geology, 74 (1989) 321-330
3') 1
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
[2]
IRON OXIDES IN ACID MINE DRAINAGE ENVIRONMENTS AND THEIR ASSOCIATION WITH BACTERIA F.G. FERRIS .1'.2, K. TAZAKI and W.S. FYFE Department of Geology, Faculty of Science, University of Western Ontario, London, Ont. N6A 5B7 (Canada) (Received December 15, 1987; revised and accepted September 27, 1988)
Abstract Ferris, F.G., Tazaki, K. and Fyfe, W.S., 1989. Iron oxides in acid mine drainage environments and their association with bacteria. Chem. Geol., 74: 321-330. A variety of iron oxides were identified by X-ray diffraction in sediments receiving acid drainage from mine tailing and coal refuse impoundments. Small amounts of goethite and hematite were found in the sediment samples. However, the major iron oxide species was ferrihydrite which gave diffuse diffraction bands at angles corresponding to d = 2.5, 2.2 and 1.5A. Main core line binding energies in Fe (2p) and 0 ( ls ) X-ray photoelectron spectra were consistent with the hydrous nature and predominance of ferrihydrite. Electron microscopy and energy-dispersive X-ray spectroscopy also showed that individual bacterial cells promoted the development of iron oxide mineralization. The bacterial associated iron oxides were similar to those in the bulk sediment samples, and exhibited structures conforming to the presence of chemisorbed sulfate or silicate anions.
1. I n t r o d u c t i o n
The formation of a variety of iron oxides is possible in natural sedimentary systems. In environments where high levels of Fe(III) are made available by the rapid oxidation ofFe (II), ferrihydrite precipitation is favored (Carlson and Schwertmann, 1981). This poorly ordered hydrous compound is thermodynamically unstable and usually converts with time to more stable crystalline forms, such as goethite or hematite (Fischer and Schwertmann, 1975; Schwertmann and Murad, 1983). The type of iron oxide mineralization that developes from *lAuthor to whom correspondence should be addressed. "2Present address: NOVA HUSKY Research Corporation, Biosciences Group, 292816th Street NE, Calgary, Alta. T2E 7K7, Canada.
0009-2541/89/$03.50
ferrihydrite is, however, strongly influenced by the physical and chemical nature of the depositional environment. For example, the conversion time for ferrihydrite increases dramatically with decreasing pH (Schwertmann and Murad, 1983). Similarly, dissolved inorganic anions which have a high affinity for the ferrihydrite surface not only inhibit goethite and hematite formation, but also suppress the crystallinity of the transformation products (Carlson and Schwertmann, 1981; Anderson and Benjamin, 1985; Brady et al., 1986; Cornell et al., 1987). Naturally occurring iron oxides therefore exhibit a broad range of morphological and crystalline states. Significant amount of organic matter are commonly found in natural goethites (Yapp and Poths, 1986) and fresh ferrihydrite precipitates
© 1989 Elsevier Science Publishers B.V.
322 (Carlson and Schwertmann, 1981), but information about the mechanism (s) reponsible for the inclusion of this carbonaceous material is rather limited. Several studies have, however, established that bacterial cells are capable of serving as nucleation sites for authigenic mineral formation during early sedimentary diagenesis (Degens and Ittekkot, 1982; Beveridge et al., 1983; Ferris et al., 1986,1987). In this paper we report on iron oxide mineralization associated with bacteria in sediments from acid mine drainage environments. Because of the complex nature in iron oxides, samples examined during this investigation were characterized not only with respect to bulk mineralogy, but also in terms of elemental composition, surface chemistry, and crystal morphology. The mineral content of the sediment samples was evaluated by X-ray diffraction (XRD) and secondary ion mass spectroscopy (SIMS) was used to determine elemental distributions. Electron spectrochemical surface analyses (ESCA) were done by X-ray photoelectron spectroscopy. Mineralization associated with individual bacterial cells was detected by electron microscopy, energy-dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (SAED). 2. Materials and m e t h o d s
2.I. Sample collection Sediment samples were collected in seepage areas at inactive mine tailing ponds at Rossport (on the north shore of Lake Superior), Burchell Lake (west of Thunder Bay) and Cranberry Lake (northwest of Sudbury), Ontario, Canada. Samples were also obtained near a coal refuse impoundment in Belmont County, Ohio, U.S.A. In these areas, the oxidation of pyrite and other metal sulfides typically generates acidic drainage (pH < 3.5 ) which supports high levels of dissolved Fe (5.0-500.0 ppm). After the samples were removed from the water, separate specimens were immediately placed into either empty 5.0-ml metal-free polypropylene
tubes, or tubes containing 1.0% (v/v) aqueous glutaraldehyde, a biological fixative for electron microscopy (Hayat, 1981). The surficial pH-values of the sediment samples were also checked in the field with pH paper, and again in the laboratory using a pH meter equipped with a standard combination electrode.
2.2. Secondary ion mass spectrometry (SIMS) and X-ray diffraction (XRD) Specimens for XRD and SIMS were ground extensively under deionized distilled water in a porcelain mortar. The resulting fine suspensions of sediment were then recovered with a pipette and dispersed evenly on glass slides. Powder XRD patterns were obtained from these thin sediment films using Cu-K~ radiation. A Cameca v IMS-3F spectrometer was used for SIMS; the samples were analyzed under a massfiltered 100-zm diameter ~ O - ion beam with primary and secondary accelerating voltages of 12.5 and 4.5 keV, respectively.
2.3. X-ray photoelectron spectroscopy Electron spectrochemical analyses (ESCA) were conducted using a SSX-IO0 ® X-ray photoelectron spectrometer equipped with a custom-designed vacuum system and sample treatment chamber. Sediment samples were mounted on copper grids and desiccated before being inserted into the high-vacuum chamber of the spectrometer. A monochromatized A1-K~ X-ray exciting beam was used, and specimen charging was controlled with a low-energy electron flood gun. All binding energies were referenced to C (ls) at 285.0 eV.
2.4. Electron microscopy Whole mounts of unfixed and fixed material were prepared for electron microscopy by floating Formvar ® carbon-coated copper grids on small droplets of sediment suspended in deion-
323
ized distilled water. After several seconds, excess material was carefully removed from the grids with filter paper. For biological thin-sections, specimens were enrobed in 2.0% noble agar before dehydration through an ethanolpropylene oxide series and embedding in an epoxy resin. A Reichert-Jung ® Ultracut E ultramicrotome was used to cut thin-sect i o n s ~ 150 nm in thickness. Duplicate thinsections were prepared and mounted on Formvar ® carbon-coated grids; one specimen was routinely stained with uranyl acetate and lead citrate to enhance the electron contrast of biological material (Hayat, 1981 ). Specimens were examined using either a J E O L ® E M I00C or a Philips ® E M 400T equipped with an E D A X ® energy-dispersive Xray spectrometer. Both instruments were operated at 100 keV with liquid nitrogen cooled anticontamination devices in place at all times. EDS was conducted using electron beam spot sizes of~< 200 nm, and counts were collected for 100 s (live time). SAED patterns were calibrated using evaporated gold as a comparative standard. 3. R e s u l t s
3.1. Mineralogical and elemental composition The X R D patterns obtained from each of the sediment samples exhibited broad weak reflections suggestive of small crystal size, crystal disorder, a n d / o r amorphous structure. A large amount of hornblende and chlorite was detected in the Rossport samples with smaller amounts of quartz, feldspar, talc, gypsum and various iron oxides (Table I). In contrast, all other samples contained an abundance of ferrihydrite which gave two broad diffraction bands with maxima at angles corresponding to spacings of d = 2 . 5 , 2.2 and 1.5 ,~ (Chukhrov et al., 1973). The Burchell Lake sediment samples also contained high levels of quartz and a
TABLE
I
Mineral composition of acid mine drainage sediments as determined by X-ray diffraction Mineral
Sediment sample Rossport
Ohio
Hornblende Chlorite Feldspar Talc Gypsum Quartz
+ + + + + + + + tr. +
-
+ + +
+ tr.
Ferrihydrite Goethite Hematite
tr. + +
+ + + +
+ + + tr. tr.
+ + + tr. tr.
tr.=trace;
+
=
~<25 w t . % ; + +
Burchell Lake
CranberD'Lake
tr.
=
~<50 w t . % : + + +
=
~<75
wt.%.
trace of chlorite, whereas gypsum was present in material from Cranberry Lake. The surficial p H of the Burchell Lake and Cranberry Lake sediment samples ranged from 3.0 to 3.4. Higher pH-values of 4.3-4.8 were recorded for the Rossport and Ohio samples, and this probably reflects an increased dilution of the acid mine drainage by near-neutral p H groundwaters. A significant enrichment of C, Mg, AI, Si and Ca was revealed by SIMS in the Rossport samples as expected from both the bulk mineralogy of the sediment (i.e. predominantly hornblende and chlorite) and the presence of algae (i.e. organic carbon; revealed by light microscopy). In contrast, Fe was the major metallic element detected by SIMS in material from Burchell Lake, Cranberry Lake and Ohio. This paralleled the predominance of Fe mineralization in these samples, as revealed by XRD. Similarly, Si was found as a major nonmetallic element in the Burchell Lake samples as expected from the increased abundance of quartz in the sediment.
3.2. Surface chemistry The Fe (2p) ESCA spectra of material from Burchell Lake, Cranberry Lake and Ohio are
324 711.9
eV (Fe2Q), 711.9 eV (FeOOH) and minor components at 719.9 eV (FeOOH) (McIntyre and Zetaruk, 1977). A mean core line binding energy of 532.0 eV was observed in the O ( ls ) spectra (Fig. 2 ). This is shifted ~ 0.6 eV above the expected binding energy of 531.4 eV for hydroxyl oxygen in FeOOH, and probably reflects the presence of chemisorbed hydroxyl species in the Fe20:3 lattice of ferrihydrite (McIntyre and Zetaruk, 1977; Russel, 1979; Wada, 1981). The spectra are also relatively broad ( ~ 3 eV) and asymmetric to the low-energy side of the main core line. The broadening above 532.0 eV can be attributed to chemisorbed carbonyl species (i.e. organic matter), whereas the asymmetry arises from oxide oxygen in F%O:~ and FeOOH (McIntyre and Zetaruk, 1977). The O (ls) spectrum of the Ohio sample, in particular, exhibited a pronounced asymmetric shoulder around 530.0 eV that corresponds well with XRD data showing increased levels of goethite and hematite.
'I
711.0
%" ,
.L,
t
7224
1
7i64 71C,.Z, P!NEiNC ENERGY (eV)
Fig. 1. Fe (2p) X-ray photoelectron spectra of sediment samples from Ohio ( ), Cranberry Lake (- - - ) and Burchell Lake ( ..... ).
shown in Fig. 1. Analysis of the Rossport samples was not possible because of low Fe content. Nevertheless, evidence of several components is seen in the Fe (2p) spectra of the other samples. The main core lines are broad ( ~ 3 eV) and asymmetric to the high-energy side. In addition, weak satellites are observed ~ 8 eV above the mean binding energy of the core lines. This type of energy intensity distribution is characteristic of a mixture of iron oxide compounds with major core line binding energies at 711.0
3.3. Mineralization associated with bacteria
In thin-sectioned plastic embedded samples of sediment from Burchell Lake and Cranberry
-5320 T
..~530.0
I
5.374
I
I
533.4 5~.4 BIND[N@ ENERGY (eV)
Fig. 2. O (ls) X-ray photoelectron spectra of sediment samples from Ohio ( Lake ( ...... ).
), Cranberry Lake (- - - ) and Burchell
PLATE I A-D. Thin-section electron micrographs showing the progressive mineralization of bacteria by ferrihydrite in material from Cranberry Lake. E. Fibrous ferrihydrite and the corresponding SAED pattern in a whole mount of material from Cranberry Lake. (Scale bars represent 500 nm.)
326
PLATE II
C
D
m
I~ i:,:,,,.........
~, !:~::~iiiii~i:i:!:i:::: ¸¸¸~ ilh~U!!!~!iiii!'!!il '? Y ~iii~ :~ ~
IIII
IIII
•
I I
I
A. Granular ferrihydrite containing hematitic microcrystals surrounding a bacterium in a Rossport sediment sample. B. Elongated hematite crystals on the surface of a bacterium in material from Burchell Lake. C and D. Thin-section profile (C) and whole mount (D) showing the granular nature of bacterial-associated Fe mineralization in the Ohio sediment samples. E, Whole mount and corresponding goethite SAED pattern of the Ohio precipitates. (Scale bars represent 500 nm. )
327
~Fe
(A) PS
(~) S
A.
(¢)
4Fe
si (D)
L
{ CO
,-,Fe
I
J
5 12 X-RAY ENERSY (KEY)
10 2L
Fig. 3. Energy-dispersive X-ray spectra from bacterial-associated iron oxide precipitates in thin-sectioned material from: (A) Burchell Lake, P, S and Fe (Ks and Kz) peaks; (B) Cranberry Lake, S and Fe (K~ and Kz) peaks; (C) Ohio, Si and Fe (K~ and K/~) peaks; and (D) Rossport, Si and Fe (K,~ and Kz) peaks.
Lake, individual bacterial cells and their remains were found in successive stages of mineral encrustation by ferrihydrite (Plate I). This mineralization had clearly developed in association with the bacteria, and typically appeared as elongated fibers extending away from the cell surface. Similar fibrous networks of ferrihydrite giving diffuse prismatic reflections at d=2.5 and 1.5 A were evident in SAED patterns from whole mounts of these specimens [Plate I, (E) ]. The Fe mineralization associated with bacteria in thin-sections of the Rossport samples was sparse and restricted to a poorly developed granular material containing small hematitic microcrystals [Plate II, (A) ]; the algae in these samples were not mineralized by Fe. In contrast, elongated hematite crystals giving strong SAED reflections at d--2.69 and 1.45 A were observed in some specimens from Burchell Lake [Plate II, (B) ]. Bacterial cells in the Ohio samples were also encrusted by granular iron oxides
which generated goethitic SAED patterns with d-spacings of 4.2, 2.5, 2.4 and 1.8 A [Plate II, (C)-(E)]. Several minor-element impurities in the bacterial-associated iron oxides were detected by EDS (Fig. 3). The Burchell Lake samples contained low levels of sulfur and phosphorus. Similar amounts of sulfur were detected in material from Cranberry Lake, but this element was not found in iron oxide precipitates associated with bacteria in the Rossport or Ohio samples. Instead, Si was present as an indigenous minor-element impurity. Binding energies for S (2p) and Si (2p) in the corresponding ESCA spectra suggested that these elements were chemisorbed by the iron oxides as sulfate or silicate anions, repsectively. 4. D i s c u s s i o n
The results show that an abundance of poorly crystallized iron oxides are commonly produced in acid mine drainage sediments. Similar Fe-rich precipitates have been observed in other areas where ochreous deposits arise from acidic waters draining mine tailing and coal refuse impoundments (Chukhrov et al., 1973; Crosby et al., 1983; Brady et al., 1986). Under these conditions, where the supply and oxidation rate of Fe (II) is high, ferrihydrite developes preferentially. In most natural systems, this hydrous compound serves as a precursor for the formation of more stable anhydrous iron oxides, such as goethite and hematite (Carlson and Schwertmann, 1981 ). However, low pH-values tend to increase the conversion time of ferrihydrite and decrease the crystallinity of the transformation products (Schwertmann and Murad, 1983). This accounts for both the predominance of ferrihydrite and formation of other poorly ordered iron oxides in surficial sediments from acid mine drainage environments. Ferrihydrite appears to have a hematite-like structure with a hexagonal unit cell and bulk composition of approximately 5F%O3"9H20
328 (Wada, 1981). However, data from infrared spectroscopic studies of synthetic ferrihydrites indicate that structural hydroxyl groups may be present, and suggest a formula of Fe203" 2FeOOH-2.6H20 (Russel, 1979). Additional evidence in favor of O H - constituents in ferrihydrite was provided by the ESCA spectra from our samples. The major core line binding energies observed in the Fe (2p) spectra are consistent with the presence of both Fe203 and FeOOH species (McIntyre and Zetaruk, 1977). However, relatively small amounts of goethite and hematite were detected by XRD and electron microscopy. Also, mean core line binding energies of 532.0 eV in the O (ls) ESCA spectra are suggestive of a partial replacement of lattice oxygen in Fe20:~ by O H - , and supportive of high levels of chemisorbed water (McIntyre and Zetaruk, 1977). Thus, it seems reasonable to view ferrihydrite as a complex hydrous oxide-oxyhydroxide phase (Russel, 1979). In each of the samples examined during this investigation, bacterial cells were found encrusted in successive stages by ferrihydrite and other iron oxides. These bacteria were not only serving as templates for the initial deposition of Fe, but their organic remains were clearly being trapped and incorporated into the mineral precipitates during crystal growth. There are numerous acidophilic bacterial genera and species present in acid mine drainage environments (Harrison, 1984). Some acquire their energy for growth through the direct oxidation of Fe (II), such as Thiobacillus ferrooxidans, and actively promote iron oxide precipitation (Lazaroff et al., 1982). Conversely, many are conventional heterotrophs which utilize organic compounds to grow (Harrison, 1984). These bacteria are not usually viewed as being capable of directing the precipitation of Fe. However, the Fe mineralization associated with bacteria in our samples was not restricted to any specific morphological group, and commonly developed on lysed (i.e. dead) cells. This indicates that bacteria, regardless of physiological state, are capable of serving as passive nucleation elements for iron oxides in acidic sediments.
The ability of bacteria to act as nucleation sites for mineral formation depends primarily on the inherent capacity of their anionic surface polymers to bind metallic ions (Ferris and Beveridge, 1985; Beveridge and Fyfe, 1985). Once immobilized, cellularly complexed metals can be precipitated by hydrolysis, a change in oxidation state, or through reactions with other counter ions in solution (Degens and Ittekkot, 1982; Beveridge et al., 1983; Ferris et al., 1986,1987 ). The subsequent growth of the mineral precipitates formed initially on the surfaces of bacterial cells may then proceed via homogeneous crystal nucleation reactions (Berner, 1980). Eventually, this later process would contribute to the complete encrustation of a bacterial cell within a mineral matrix. A similar series of events starting with the oxidation and hydrolysis of cell-bound ferrous or ferric iron accounts for the pattern of mineralization associated with the bacteria in our sediment samples. Recent carbon isotope studies suggest that the organic matter trapped in natural iron oxides may be a useful indicator of paleoenvironmental and paleobiological conditions (Yapp and Poths, 1986). Most biogenic substances in sediments are enriched in 12C, but sizable differences in the extent of isotopic fractionation can arise through modifications induced biologically by degradative heterotrophic microorganisms, or abiotically during diagenetic maturation (Schidlowski, 1987). Interpretations of carbon isotope fractions are thereibre strengthened by some knowledge of the geochemical history of sedimentary organic matter. In the case of iron oxides in acidic environments, heterotrophic bacteria and their remains appear to be a major source of mineralized carbon. These bacteria usually cause a preferential loss of isotopically distinct groups, and bring about a slight enrichment of 1~C relative to their primary organic substrates (Schidlowski, 1987). The recognition of this phenomenon in iron oxides could prove to be useful in depositional environment analyses. Variations in the morphological characteris-
329
tics of the bacterial-associated mineralization correlate well with the trace-element content of the precipitates. Synthesis experiments have shown that the specific adsorption of small quantities of sulfate or silicate not only suppress the ordering of ferrihydrite, but also hinder the formation of goethite and hematite (Wada, 1981; Anderson and Benjamin, 1985; Brady et al., 1986; Cornell et al., 1987). The exact mechanisms by which chemically adsorbed inorganic anions influence the development of iron oxides have not yet been elucidated. However, the available evidence seems to suggest that negatively charged ions essentially crosslink colloidal particles of ferrihydrite, thus fbrming a relatively unreactive immobile phase (Cornell et al., 1987). In the case of sulfate, this results in the formation of fibrous networks of ferrihydrate similar to those observed in association with bacteria in material from Burchell Lake and Cranberry Lake {Brady et al., 1986). On the other hand, siliceous ferrihydrites generally exhibit a more granular morphology reminiscent of the bacterial-induced Fe precipitates in the Rossport and Ohio samples (Carlson and Schwertmann, 1981). These observations are interesting as the sulfate-bearing ferrihydrites developed under rather low pH conditions (3.0-3.4), whereas the siliceous varieties formed at higher pH-values (4.3-4.8). At. present, it is not known whether such compositional differences persist during later stages of diagenesis, but they could be used as indicators of environmental pH. Further studies are needed, however, to sustain this possibility.
Acknowledgments This work was supported by funds from the Ontario Geological Survey and a Natural Science and Engineering Research Council (NSERC) of Canada Postdoctoral Fellowship awarded to F.G.F. Technical assistance was provided by R. Humphrey, NSERC Regional STEM Facility, Department of Microbiology, University of Guelph, and M. Hyland, Surface
Science Western, Ontario.
University
of Western
References
Anderson, P.R. and Benjamin, M.M., 1985. Effect of silicon on the cr~/stallization and adsorption properties of ferric oxides. Environ. Sci. Technol., 19: 1048-1053. Berner, R.A., 1980. Early Diagenesis: A Theoretical Approach. Princeton University Press, Lawrenceville, N.J., 241 pp. Beveridge, T.J. and Fyfe, W.S., 1985. Metal fixation hy bacterial cell walls. Can. J. Earth Sci., 22: 1892-1898. Beveridge, T.J., Meloche, J.D., Fyf'e, W.S. and Murray, R.G.E., 1983. Diagenesis of metals chemically complexed to bacteria: laboratory formation of metal phosphates, sulfides, and organic condensates in artificial sediments. Appl. Environ. Microbiol., 45:1094 1108. Brady, K.S., Bigham, J.M., Jaynes, W.F. and Logan, T.,I., 1986. Influence of sulfate on Fe-oxide formation: comparisons with a stream receiving acid mine drainage. Clays Clay Miner., 34: 266-274. Carlson, L. and Schwertmann, U., 1981. Natural ferrihydrites in surface deposits from Finland and their ass()ciation with silica. Geochim. Cosmochim. Acta, 45:42 ! 429. Chukhrov, F.V., Zvyagin, B.B., Ermilova, L.P. and Gorshov, A.I., 1973. New data on iron oxides in the weathering zone. In: J.M. Serratosa (Editor), Proceedings of the 1972 International Clay Conference, Div. Cienc. C.S.I.C. (Cons. Super. Invest. Cient. ), Madrid, pp. 333 341. Cornell, R.M., Giovanoli, R. and Schindler, P.W., 1987. Effect of silicate species on the transformation of ferrihydrite into goethite and hematite in alkaline media. Clays Clay Miner., 35: 21-28. Crosby, S.A., Glasson, D.R., Cuttler, A.H., Butler, I., Turner, D.R., Whitfield, M. and Millward, G.E., 1983. Surface areas and porosities of Fe (III) - and Fe (II)-derived oxyhydroxides. Environ. Sci. Technol., 17: 709-713. Degens, E.T. and Ittekkot, V.I.~ 1982. In situ metal staining of biological membranes in sediments. Nature (L(mdon), 298: 262-264. Ferris, F.G. and Beveridge, T.J., 1985. Functions of bacterial cell surface structures. BioScience, 35: 172-177. Ferris, F.G., Beveridge, T.J. and Fyfe, W.S., 1986. Ironsilica crystallite nucleation by bacteria in a geothernml sediment. Nature (London), 320: 609-611. Ferris, F.G., Fyfe, W.S. and Beveridge, T.J., 1987. Bacteria as nucleation sites for authigenic minerals in a metalcontaminated lake sediment. Chem. Geol., 63: 225-232. Fischer, W.R. and Schwertmann, U., 1975. The formation
330 of hematite from amorphous iron (III) hydroxide. Clays Clay Miner., 23: 33-37. Harrison, A.P., 1984. The acidophilic Thiobacilli and other acidophilic bacteria that share their habitat. Annu. Rev. Microbiol., 38: 265-292. Hayat, M.A., 1981. Principles and Techniques of Electron Microscopy: Biological Applications. University Park Press, Baltimore, Md., 522 pp. Lazaroff, N., Sigal, W. and Wasserman, A., 1982. Iron oxidation and precipitation of ferric hydrosulfates by resting Thiobacillus ferrooxidans cells. Appl. Environ. Microbiol., 43: 924-938. McIntyre, N.S. and Zetaruk, D.G., 1977. X-ray photoelectron spectroscopic studies of iron oxides. Anal. Chem., 49: 1521-1529. Russel, J.D., 1979. Infrared spectroscopy studies of ferri-
hydrite: evidence for the presence of structural hydroxyl groups. Clay Miner., 14: 190-214. Schidlowski, M., 1987. Application of stable carbon isotopes to early biochemical evolution on earth. Annu. Rev. Earth Planet. Sci., 15: 47-72. Schwertmann, U. and Murad, E., 1983. Effect ofpH on the formation of goethite and hematite from ferrihydrite. Clays Clay Miner., 31: 277-284. Wada, K., 1981. Amorphous clay minerals - - Chemical composition, crystalline state, synthesis, and surface properties. In: H. van Olphen and P. Veniale (Editors), Developments in Sedimentology, Vol. 35. Proceedings of the VII International Clay Conference. Elsevier, Amsterdam, pp. 385-398. ¥app, C.J. and Poths, H., 1986. Carbon in natural goe~ thites. Geochim. Cosmochim. Acta, 50: 1213-1220.
3') 1
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
[2]
IRON OXIDES IN ACID MINE DRAINAGE ENVIRONMENTS AND THEIR ASSOCIATION WITH BACTERIA F.G. FERRIS .1'.2, K. TAZAKI and W.S. FYFE Department of Geology, Faculty of Science, University of Western Ontario, London, Ont. N6A 5B7 (Canada) (Received December 15, 1987; revised and accepted September 27, 1988)
Abstract Ferris, F.G., Tazaki, K. and Fyfe, W.S., 1989. Iron oxides in acid mine drainage environments and their association with bacteria. Chem. Geol., 74: 321-330. A variety of iron oxides were identified by X-ray diffraction in sediments receiving acid drainage from mine tailing and coal refuse impoundments. Small amounts of goethite and hematite were found in the sediment samples. However, the major iron oxide species was ferrihydrite which gave diffuse diffraction bands at angles corresponding to d = 2.5, 2.2 and 1.5A. Main core line binding energies in Fe (2p) and 0 ( ls ) X-ray photoelectron spectra were consistent with the hydrous nature and predominance of ferrihydrite. Electron microscopy and energy-dispersive X-ray spectroscopy also showed that individual bacterial cells promoted the development of iron oxide mineralization. The bacterial associated iron oxides were similar to those in the bulk sediment samples, and exhibited structures conforming to the presence of chemisorbed sulfate or silicate anions.
1. I n t r o d u c t i o n
The formation of a variety of iron oxides is possible in natural sedimentary systems. In environments where high levels of Fe(III) are made available by the rapid oxidation ofFe (II), ferrihydrite precipitation is favored (Carlson and Schwertmann, 1981). This poorly ordered hydrous compound is thermodynamically unstable and usually converts with time to more stable crystalline forms, such as goethite or hematite (Fischer and Schwertmann, 1975; Schwertmann and Murad, 1983). The type of iron oxide mineralization that developes from *lAuthor to whom correspondence should be addressed. "2Present address: NOVA HUSKY Research Corporation, Biosciences Group, 292816th Street NE, Calgary, Alta. T2E 7K7, Canada.
0009-2541/89/$03.50
ferrihydrite is, however, strongly influenced by the physical and chemical nature of the depositional environment. For example, the conversion time for ferrihydrite increases dramatically with decreasing pH (Schwertmann and Murad, 1983). Similarly, dissolved inorganic anions which have a high affinity for the ferrihydrite surface not only inhibit goethite and hematite formation, but also suppress the crystallinity of the transformation products (Carlson and Schwertmann, 1981; Anderson and Benjamin, 1985; Brady et al., 1986; Cornell et al., 1987). Naturally occurring iron oxides therefore exhibit a broad range of morphological and crystalline states. Significant amount of organic matter are commonly found in natural goethites (Yapp and Poths, 1986) and fresh ferrihydrite precipitates
© 1989 Elsevier Science Publishers B.V.
322 (Carlson and Schwertmann, 1981), but information about the mechanism (s) reponsible for the inclusion of this carbonaceous material is rather limited. Several studies have, however, established that bacterial cells are capable of serving as nucleation sites for authigenic mineral formation during early sedimentary diagenesis (Degens and Ittekkot, 1982; Beveridge et al., 1983; Ferris et al., 1986,1987). In this paper we report on iron oxide mineralization associated with bacteria in sediments from acid mine drainage environments. Because of the complex nature in iron oxides, samples examined during this investigation were characterized not only with respect to bulk mineralogy, but also in terms of elemental composition, surface chemistry, and crystal morphology. The mineral content of the sediment samples was evaluated by X-ray diffraction (XRD) and secondary ion mass spectroscopy (SIMS) was used to determine elemental distributions. Electron spectrochemical surface analyses (ESCA) were done by X-ray photoelectron spectroscopy. Mineralization associated with individual bacterial cells was detected by electron microscopy, energy-dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (SAED). 2. Materials and m e t h o d s
2.I. Sample collection Sediment samples were collected in seepage areas at inactive mine tailing ponds at Rossport (on the north shore of Lake Superior), Burchell Lake (west of Thunder Bay) and Cranberry Lake (northwest of Sudbury), Ontario, Canada. Samples were also obtained near a coal refuse impoundment in Belmont County, Ohio, U.S.A. In these areas, the oxidation of pyrite and other metal sulfides typically generates acidic drainage (pH < 3.5 ) which supports high levels of dissolved Fe (5.0-500.0 ppm). After the samples were removed from the water, separate specimens were immediately placed into either empty 5.0-ml metal-free polypropylene
tubes, or tubes containing 1.0% (v/v) aqueous glutaraldehyde, a biological fixative for electron microscopy (Hayat, 1981). The surficial pH-values of the sediment samples were also checked in the field with pH paper, and again in the laboratory using a pH meter equipped with a standard combination electrode.
2.2. Secondary ion mass spectrometry (SIMS) and X-ray diffraction (XRD) Specimens for XRD and SIMS were ground extensively under deionized distilled water in a porcelain mortar. The resulting fine suspensions of sediment were then recovered with a pipette and dispersed evenly on glass slides. Powder XRD patterns were obtained from these thin sediment films using Cu-K~ radiation. A Cameca v IMS-3F spectrometer was used for SIMS; the samples were analyzed under a massfiltered 100-zm diameter ~ O - ion beam with primary and secondary accelerating voltages of 12.5 and 4.5 keV, respectively.
2.3. X-ray photoelectron spectroscopy Electron spectrochemical analyses (ESCA) were conducted using a SSX-IO0 ® X-ray photoelectron spectrometer equipped with a custom-designed vacuum system and sample treatment chamber. Sediment samples were mounted on copper grids and desiccated before being inserted into the high-vacuum chamber of the spectrometer. A monochromatized A1-K~ X-ray exciting beam was used, and specimen charging was controlled with a low-energy electron flood gun. All binding energies were referenced to C (ls) at 285.0 eV.
2.4. Electron microscopy Whole mounts of unfixed and fixed material were prepared for electron microscopy by floating Formvar ® carbon-coated copper grids on small droplets of sediment suspended in deion-
323
ized distilled water. After several seconds, excess material was carefully removed from the grids with filter paper. For biological thin-sections, specimens were enrobed in 2.0% noble agar before dehydration through an ethanolpropylene oxide series and embedding in an epoxy resin. A Reichert-Jung ® Ultracut E ultramicrotome was used to cut thin-sect i o n s ~ 150 nm in thickness. Duplicate thinsections were prepared and mounted on Formvar ® carbon-coated grids; one specimen was routinely stained with uranyl acetate and lead citrate to enhance the electron contrast of biological material (Hayat, 1981 ). Specimens were examined using either a J E O L ® E M I00C or a Philips ® E M 400T equipped with an E D A X ® energy-dispersive Xray spectrometer. Both instruments were operated at 100 keV with liquid nitrogen cooled anticontamination devices in place at all times. EDS was conducted using electron beam spot sizes of~< 200 nm, and counts were collected for 100 s (live time). SAED patterns were calibrated using evaporated gold as a comparative standard. 3. R e s u l t s
3.1. Mineralogical and elemental composition The X R D patterns obtained from each of the sediment samples exhibited broad weak reflections suggestive of small crystal size, crystal disorder, a n d / o r amorphous structure. A large amount of hornblende and chlorite was detected in the Rossport samples with smaller amounts of quartz, feldspar, talc, gypsum and various iron oxides (Table I). In contrast, all other samples contained an abundance of ferrihydrite which gave two broad diffraction bands with maxima at angles corresponding to spacings of d = 2 . 5 , 2.2 and 1.5 ,~ (Chukhrov et al., 1973). The Burchell Lake sediment samples also contained high levels of quartz and a
TABLE
I
Mineral composition of acid mine drainage sediments as determined by X-ray diffraction Mineral
Sediment sample Rossport
Ohio
Hornblende Chlorite Feldspar Talc Gypsum Quartz
+ + + + + + + + tr. +
-
+ + +
+ tr.
Ferrihydrite Goethite Hematite
tr. + +
+ + + +
+ + + tr. tr.
+ + + tr. tr.
tr.=trace;
+
=
~<25 w t . % ; + +
Burchell Lake
CranberD'Lake
tr.
=
~<50 w t . % : + + +
=
~<75
wt.%.
trace of chlorite, whereas gypsum was present in material from Cranberry Lake. The surficial p H of the Burchell Lake and Cranberry Lake sediment samples ranged from 3.0 to 3.4. Higher pH-values of 4.3-4.8 were recorded for the Rossport and Ohio samples, and this probably reflects an increased dilution of the acid mine drainage by near-neutral p H groundwaters. A significant enrichment of C, Mg, AI, Si and Ca was revealed by SIMS in the Rossport samples as expected from both the bulk mineralogy of the sediment (i.e. predominantly hornblende and chlorite) and the presence of algae (i.e. organic carbon; revealed by light microscopy). In contrast, Fe was the major metallic element detected by SIMS in material from Burchell Lake, Cranberry Lake and Ohio. This paralleled the predominance of Fe mineralization in these samples, as revealed by XRD. Similarly, Si was found as a major nonmetallic element in the Burchell Lake samples as expected from the increased abundance of quartz in the sediment.
3.2. Surface chemistry The Fe (2p) ESCA spectra of material from Burchell Lake, Cranberry Lake and Ohio are
324 711.9
eV (Fe2Q), 711.9 eV (FeOOH) and minor components at 719.9 eV (FeOOH) (McIntyre and Zetaruk, 1977). A mean core line binding energy of 532.0 eV was observed in the O ( ls ) spectra (Fig. 2 ). This is shifted ~ 0.6 eV above the expected binding energy of 531.4 eV for hydroxyl oxygen in FeOOH, and probably reflects the presence of chemisorbed hydroxyl species in the Fe20:3 lattice of ferrihydrite (McIntyre and Zetaruk, 1977; Russel, 1979; Wada, 1981). The spectra are also relatively broad ( ~ 3 eV) and asymmetric to the low-energy side of the main core line. The broadening above 532.0 eV can be attributed to chemisorbed carbonyl species (i.e. organic matter), whereas the asymmetry arises from oxide oxygen in F%O:~ and FeOOH (McIntyre and Zetaruk, 1977). The O (ls) spectrum of the Ohio sample, in particular, exhibited a pronounced asymmetric shoulder around 530.0 eV that corresponds well with XRD data showing increased levels of goethite and hematite.
'I
711.0
%" ,
.L,
t
7224
1
7i64 71C,.Z, P!NEiNC ENERGY (eV)
Fig. 1. Fe (2p) X-ray photoelectron spectra of sediment samples from Ohio ( ), Cranberry Lake (- - - ) and Burchell Lake ( ..... ).
shown in Fig. 1. Analysis of the Rossport samples was not possible because of low Fe content. Nevertheless, evidence of several components is seen in the Fe (2p) spectra of the other samples. The main core lines are broad ( ~ 3 eV) and asymmetric to the high-energy side. In addition, weak satellites are observed ~ 8 eV above the mean binding energy of the core lines. This type of energy intensity distribution is characteristic of a mixture of iron oxide compounds with major core line binding energies at 711.0
3.3. Mineralization associated with bacteria
In thin-sectioned plastic embedded samples of sediment from Burchell Lake and Cranberry
-5320 T
..~530.0
I
5.374
I
I
533.4 5~.4 BIND[N@ ENERGY (eV)
Fig. 2. O (ls) X-ray photoelectron spectra of sediment samples from Ohio ( Lake ( ...... ).
), Cranberry Lake (- - - ) and Burchell
PLATE I A-D. Thin-section electron micrographs showing the progressive mineralization of bacteria by ferrihydrite in material from Cranberry Lake. E. Fibrous ferrihydrite and the corresponding SAED pattern in a whole mount of material from Cranberry Lake. (Scale bars represent 500 nm.)
326
PLATE II
C
D
m
I~ i:,:,,,.........
~, !:~::~iiiii~i:i:!:i:::: ¸¸¸~ ilh~U!!!~!iiii!'!!il '? Y ~iii~ :~ ~
IIII
IIII
•
I I
I
A. Granular ferrihydrite containing hematitic microcrystals surrounding a bacterium in a Rossport sediment sample. B. Elongated hematite crystals on the surface of a bacterium in material from Burchell Lake. C and D. Thin-section profile (C) and whole mount (D) showing the granular nature of bacterial-associated Fe mineralization in the Ohio sediment samples. E, Whole mount and corresponding goethite SAED pattern of the Ohio precipitates. (Scale bars represent 500 nm. )
327
~Fe
(A) PS
(~) S
A.
(¢)
4Fe
si (D)
L
{ CO
,-,Fe
I
J
5 12 X-RAY ENERSY (KEY)
10 2L
Fig. 3. Energy-dispersive X-ray spectra from bacterial-associated iron oxide precipitates in thin-sectioned material from: (A) Burchell Lake, P, S and Fe (Ks and Kz) peaks; (B) Cranberry Lake, S and Fe (K~ and Kz) peaks; (C) Ohio, Si and Fe (K~ and K/~) peaks; and (D) Rossport, Si and Fe (K,~ and Kz) peaks.
Lake, individual bacterial cells and their remains were found in successive stages of mineral encrustation by ferrihydrite (Plate I). This mineralization had clearly developed in association with the bacteria, and typically appeared as elongated fibers extending away from the cell surface. Similar fibrous networks of ferrihydrite giving diffuse prismatic reflections at d=2.5 and 1.5 A were evident in SAED patterns from whole mounts of these specimens [Plate I, (E) ]. The Fe mineralization associated with bacteria in thin-sections of the Rossport samples was sparse and restricted to a poorly developed granular material containing small hematitic microcrystals [Plate II, (A) ]; the algae in these samples were not mineralized by Fe. In contrast, elongated hematite crystals giving strong SAED reflections at d--2.69 and 1.45 A were observed in some specimens from Burchell Lake [Plate II, (B) ]. Bacterial cells in the Ohio samples were also encrusted by granular iron oxides
which generated goethitic SAED patterns with d-spacings of 4.2, 2.5, 2.4 and 1.8 A [Plate II, (C)-(E)]. Several minor-element impurities in the bacterial-associated iron oxides were detected by EDS (Fig. 3). The Burchell Lake samples contained low levels of sulfur and phosphorus. Similar amounts of sulfur were detected in material from Cranberry Lake, but this element was not found in iron oxide precipitates associated with bacteria in the Rossport or Ohio samples. Instead, Si was present as an indigenous minor-element impurity. Binding energies for S (2p) and Si (2p) in the corresponding ESCA spectra suggested that these elements were chemisorbed by the iron oxides as sulfate or silicate anions, repsectively. 4. D i s c u s s i o n
The results show that an abundance of poorly crystallized iron oxides are commonly produced in acid mine drainage sediments. Similar Fe-rich precipitates have been observed in other areas where ochreous deposits arise from acidic waters draining mine tailing and coal refuse impoundments (Chukhrov et al., 1973; Crosby et al., 1983; Brady et al., 1986). Under these conditions, where the supply and oxidation rate of Fe (II) is high, ferrihydrite developes preferentially. In most natural systems, this hydrous compound serves as a precursor for the formation of more stable anhydrous iron oxides, such as goethite and hematite (Carlson and Schwertmann, 1981 ). However, low pH-values tend to increase the conversion time of ferrihydrite and decrease the crystallinity of the transformation products (Schwertmann and Murad, 1983). This accounts for both the predominance of ferrihydrite and formation of other poorly ordered iron oxides in surficial sediments from acid mine drainage environments. Ferrihydrite appears to have a hematite-like structure with a hexagonal unit cell and bulk composition of approximately 5F%O3"9H20
328 (Wada, 1981). However, data from infrared spectroscopic studies of synthetic ferrihydrites indicate that structural hydroxyl groups may be present, and suggest a formula of Fe203" 2FeOOH-2.6H20 (Russel, 1979). Additional evidence in favor of O H - constituents in ferrihydrite was provided by the ESCA spectra from our samples. The major core line binding energies observed in the Fe (2p) spectra are consistent with the presence of both Fe203 and FeOOH species (McIntyre and Zetaruk, 1977). However, relatively small amounts of goethite and hematite were detected by XRD and electron microscopy. Also, mean core line binding energies of 532.0 eV in the O (ls) ESCA spectra are suggestive of a partial replacement of lattice oxygen in Fe20:~ by O H - , and supportive of high levels of chemisorbed water (McIntyre and Zetaruk, 1977). Thus, it seems reasonable to view ferrihydrite as a complex hydrous oxide-oxyhydroxide phase (Russel, 1979). In each of the samples examined during this investigation, bacterial cells were found encrusted in successive stages by ferrihydrite and other iron oxides. These bacteria were not only serving as templates for the initial deposition of Fe, but their organic remains were clearly being trapped and incorporated into the mineral precipitates during crystal growth. There are numerous acidophilic bacterial genera and species present in acid mine drainage environments (Harrison, 1984). Some acquire their energy for growth through the direct oxidation of Fe (II), such as Thiobacillus ferrooxidans, and actively promote iron oxide precipitation (Lazaroff et al., 1982). Conversely, many are conventional heterotrophs which utilize organic compounds to grow (Harrison, 1984). These bacteria are not usually viewed as being capable of directing the precipitation of Fe. However, the Fe mineralization associated with bacteria in our samples was not restricted to any specific morphological group, and commonly developed on lysed (i.e. dead) cells. This indicates that bacteria, regardless of physiological state, are capable of serving as passive nucleation elements for iron oxides in acidic sediments.
The ability of bacteria to act as nucleation sites for mineral formation depends primarily on the inherent capacity of their anionic surface polymers to bind metallic ions (Ferris and Beveridge, 1985; Beveridge and Fyfe, 1985). Once immobilized, cellularly complexed metals can be precipitated by hydrolysis, a change in oxidation state, or through reactions with other counter ions in solution (Degens and Ittekkot, 1982; Beveridge et al., 1983; Ferris et al., 1986,1987 ). The subsequent growth of the mineral precipitates formed initially on the surfaces of bacterial cells may then proceed via homogeneous crystal nucleation reactions (Berner, 1980). Eventually, this later process would contribute to the complete encrustation of a bacterial cell within a mineral matrix. A similar series of events starting with the oxidation and hydrolysis of cell-bound ferrous or ferric iron accounts for the pattern of mineralization associated with the bacteria in our sediment samples. Recent carbon isotope studies suggest that the organic matter trapped in natural iron oxides may be a useful indicator of paleoenvironmental and paleobiological conditions (Yapp and Poths, 1986). Most biogenic substances in sediments are enriched in 12C, but sizable differences in the extent of isotopic fractionation can arise through modifications induced biologically by degradative heterotrophic microorganisms, or abiotically during diagenetic maturation (Schidlowski, 1987). Interpretations of carbon isotope fractions are thereibre strengthened by some knowledge of the geochemical history of sedimentary organic matter. In the case of iron oxides in acidic environments, heterotrophic bacteria and their remains appear to be a major source of mineralized carbon. These bacteria usually cause a preferential loss of isotopically distinct groups, and bring about a slight enrichment of 1~C relative to their primary organic substrates (Schidlowski, 1987). The recognition of this phenomenon in iron oxides could prove to be useful in depositional environment analyses. Variations in the morphological characteris-
329
tics of the bacterial-associated mineralization correlate well with the trace-element content of the precipitates. Synthesis experiments have shown that the specific adsorption of small quantities of sulfate or silicate not only suppress the ordering of ferrihydrite, but also hinder the formation of goethite and hematite (Wada, 1981; Anderson and Benjamin, 1985; Brady et al., 1986; Cornell et al., 1987). The exact mechanisms by which chemically adsorbed inorganic anions influence the development of iron oxides have not yet been elucidated. However, the available evidence seems to suggest that negatively charged ions essentially crosslink colloidal particles of ferrihydrite, thus fbrming a relatively unreactive immobile phase (Cornell et al., 1987). In the case of sulfate, this results in the formation of fibrous networks of ferrihydrate similar to those observed in association with bacteria in material from Burchell Lake and Cranberry Lake {Brady et al., 1986). On the other hand, siliceous ferrihydrites generally exhibit a more granular morphology reminiscent of the bacterial-induced Fe precipitates in the Rossport and Ohio samples (Carlson and Schwertmann, 1981). These observations are interesting as the sulfate-bearing ferrihydrites developed under rather low pH conditions (3.0-3.4), whereas the siliceous varieties formed at higher pH-values (4.3-4.8). At. present, it is not known whether such compositional differences persist during later stages of diagenesis, but they could be used as indicators of environmental pH. Further studies are needed, however, to sustain this possibility.
Acknowledgments This work was supported by funds from the Ontario Geological Survey and a Natural Science and Engineering Research Council (NSERC) of Canada Postdoctoral Fellowship awarded to F.G.F. Technical assistance was provided by R. Humphrey, NSERC Regional STEM Facility, Department of Microbiology, University of Guelph, and M. Hyland, Surface
Science Western, Ontario.
University
of Western
References
Anderson, P.R. and Benjamin, M.M., 1985. Effect of silicon on the cr~/stallization and adsorption properties of ferric oxides. Environ. Sci. Technol., 19: 1048-1053. Berner, R.A., 1980. Early Diagenesis: A Theoretical Approach. Princeton University Press, Lawrenceville, N.J., 241 pp. Beveridge, T.J. and Fyfe, W.S., 1985. Metal fixation hy bacterial cell walls. Can. J. Earth Sci., 22: 1892-1898. Beveridge, T.J., Meloche, J.D., Fyf'e, W.S. and Murray, R.G.E., 1983. Diagenesis of metals chemically complexed to bacteria: laboratory formation of metal phosphates, sulfides, and organic condensates in artificial sediments. Appl. Environ. Microbiol., 45:1094 1108. Brady, K.S., Bigham, J.M., Jaynes, W.F. and Logan, T.,I., 1986. Influence of sulfate on Fe-oxide formation: comparisons with a stream receiving acid mine drainage. Clays Clay Miner., 34: 266-274. Carlson, L. and Schwertmann, U., 1981. Natural ferrihydrites in surface deposits from Finland and their ass()ciation with silica. Geochim. Cosmochim. Acta, 45:42 ! 429. Chukhrov, F.V., Zvyagin, B.B., Ermilova, L.P. and Gorshov, A.I., 1973. New data on iron oxides in the weathering zone. In: J.M. Serratosa (Editor), Proceedings of the 1972 International Clay Conference, Div. Cienc. C.S.I.C. (Cons. Super. Invest. Cient. ), Madrid, pp. 333 341. Cornell, R.M., Giovanoli, R. and Schindler, P.W., 1987. Effect of silicate species on the transformation of ferrihydrite into goethite and hematite in alkaline media. Clays Clay Miner., 35: 21-28. Crosby, S.A., Glasson, D.R., Cuttler, A.H., Butler, I., Turner, D.R., Whitfield, M. and Millward, G.E., 1983. Surface areas and porosities of Fe (III) - and Fe (II)-derived oxyhydroxides. Environ. Sci. Technol., 17: 709-713. Degens, E.T. and Ittekkot, V.I.~ 1982. In situ metal staining of biological membranes in sediments. Nature (L(mdon), 298: 262-264. Ferris, F.G. and Beveridge, T.J., 1985. Functions of bacterial cell surface structures. BioScience, 35: 172-177. Ferris, F.G., Beveridge, T.J. and Fyfe, W.S., 1986. Ironsilica crystallite nucleation by bacteria in a geothernml sediment. Nature (London), 320: 609-611. Ferris, F.G., Fyfe, W.S. and Beveridge, T.J., 1987. Bacteria as nucleation sites for authigenic minerals in a metalcontaminated lake sediment. Chem. Geol., 63: 225-232. Fischer, W.R. and Schwertmann, U., 1975. The formation
330 of hematite from amorphous iron (III) hydroxide. Clays Clay Miner., 23: 33-37. Harrison, A.P., 1984. The acidophilic Thiobacilli and other acidophilic bacteria that share their habitat. Annu. Rev. Microbiol., 38: 265-292. Hayat, M.A., 1981. Principles and Techniques of Electron Microscopy: Biological Applications. University Park Press, Baltimore, Md., 522 pp. Lazaroff, N., Sigal, W. and Wasserman, A., 1982. Iron oxidation and precipitation of ferric hydrosulfates by resting Thiobacillus ferrooxidans cells. Appl. Environ. Microbiol., 43: 924-938. McIntyre, N.S. and Zetaruk, D.G., 1977. X-ray photoelectron spectroscopic studies of iron oxides. Anal. Chem., 49: 1521-1529. Russel, J.D., 1979. Infrared spectroscopy studies of ferri-
hydrite: evidence for the presence of structural hydroxyl groups. Clay Miner., 14: 190-214. Schidlowski, M., 1987. Application of stable carbon isotopes to early biochemical evolution on earth. Annu. Rev. Earth Planet. Sci., 15: 47-72. Schwertmann, U. and Murad, E., 1983. Effect ofpH on the formation of goethite and hematite from ferrihydrite. Clays Clay Miner., 31: 277-284. Wada, K., 1981. Amorphous clay minerals - - Chemical composition, crystalline state, synthesis, and surface properties. In: H. van Olphen and P. Veniale (Editors), Developments in Sedimentology, Vol. 35. Proceedings of the VII International Clay Conference. Elsevier, Amsterdam, pp. 385-398. ¥app, C.J. and Poths, H., 1986. Carbon in natural goe~ thites. Geochim. Cosmochim. Acta, 50: 1213-1220.