The Influence of Iron and Phosphate Mineral Inclusions

Micro-scale mineralogic controls on microbial attachment to silicate surfaces: the influence of iron and phosphate mineral inclusions

Jennifer A. Roberts & Brian T. Hughes Department of Geology, University of Kansas, Lawrence, Kansas, USA David A. Fowle Great Lakes Institute for Environmental Research/Department of Earth Sciences, University of Windsor, Windsor, Ontario, Canada

ABSTRACT: Microorganisms are ubiquitous in the shallow subsurface often attached to sediment surfaces where they alter the geochemical microenvironment and mediate weathering reactions. Microbial attachment on sediment grains, however, is heterogeneous and the variability of microbial occurrence cannot be easily explained. This study examined how phosphate mineral and iron oxide inclusions in feldspars influence mi- crobial attachment and distribution on the silicate surface using field colonization experiments in an anaerobic groundwater. Magnetite inclusions were preferentially colonized compared to the silicate groundmass while there was no discernable difference between colonization of phosphate mineral inclusions and the silicate sur- face. These differences are likely due to electrostatic interactions between the inclusion surfaces and cells as a function of the pH of the groundwater.

1 INTRODUCTION inclusions within silicate rocks in the spatial distri- bution of attached microorganisms. There is growing evidence that microbes are not merely passive occupants of many subsurface envi- 1.1 Microbial attachment ronments, but mediate biogeochemical reactions and perturb water-rock equilibria at the point of attach- Microbial attachment potentially impacts mineral ment (e.g. Liermann et al. 2000), potentially dissolv- weathering reactions, pore-scale heterogeneity, ing or precipitating mineral phases (e.g. Bennett et metal mobility through adsorption to the cell well al. 2001). The distribution of microorganisms on (e.g. Fowle & Fein 1999) and is a primary control in sediment grains and mineral surfaces, however, is microbial transport through porous media (Fletcher heterogeneous and the “patchy” nature of microbial & Murphy 2001). Efforts have been made to charac- occurrence, and associated surface reactions, are still terize microbial attachment to mineral surfaces ther- poorly understood. modynamically through surface and solution chem- Previous research on subsurface microbial colo- istry (e.g. Yee et al. 2000) or by using DLVO theory nization suggests that the distribution of microorgan- to model electrostatic interactions (Hermansson isms on different mineral surfaces in situ is related, 1999). These approaches provide guiding principles in part, to the nutrient content of the mineral. Micro- for cell-mineral interactions, but still do not effec- organisms preferentially colonize and destroy sili- tively describe the wide variability of microbial dis- cate minerals that contain limiting trace nutrients, tribution in subsurface environments. such as phosphorus and iron, occurring as trace apa- Feldspar and quartz surfaces, for example, are tite and iron oxyhydroxides (Rogers et al. 1998). uniformly negatively charged at environmental pHs, Colonizing cells release and utilize nutrients from as are bacteria, so attachment to those silicate sur- the silicate matrix, stimulating growth, biodegrada- faces must overcome some degree of coulombic re- tion rate, and silicate dissolution (Rogers & Bennett pulsion. In natural sediments, however, mineral 2004). It is still unclear, however, whether the min- grains are often coated with clay and Fe oxyhydrox- eralogy of the point of attachment is the nutrient-rich ides, which are positively charged at circum-neutral inclusion or silicate matrix. It is not known if these pH (pHzpc 8-9) compared to silicate surfaces (pHzpc inclusions impact only initial attachment, or if they 2-4 for quartz and feldspar; Stumm & Morgan influence irreversible attachment, subsequent growth 1996). Many studies have demonstrated that these and colonization, and surface etching. In this study oxide coatings account for the bulk of bacterial at- we investigated the role of nutrient-bearing mineral tachment to sediments that would otherwise have unfavorable attachment behavior (Ryan et al. 1999). In this study we used silicate surfaces that do not 2 EXPERIMENTAL APPROACH have surface coatings, but contain mineral inclusions 2.1 Mineral characterization and preparation of iron and phosphate minerals that possess different surface characteristics than the silicate groundmass. A suite of silicates rocks containing varying The surface expression of these inclusions is rela- amounts of P and Fe, including anorthoclase (Wards tively small, ~10-50 um in diameter, but collectively # 46E0575), two different microclines (Wards # they may function like macroscopic charge hetero- 46E5125, Keystone, South Dakota and Wards geneity; therefore, enabling models of the inclusions #46E5120, Ontario, Canada), and quartz (Wards # with a bulk uniform electrostatic potential (Song et 46E6605) were used to investigate the role of spatial al. 1994). These inclusions are also potential sources compositional heterogeneity as it relates to microbial of essential nutrients or terminal electron acceptors attachment. Silicate rock specimens have been char- that may be otherwise scarce in solution and limiting acterized previously using light microscopy, trace to the indigenous microbial population. metal and whole rock analysis. Rocks were prepared as thin sections with four rock types on each slide. Anorthoclase, S.D. micro- 1.2 Silicates as nutrient sources cline, quartz, and O. microcline were mounted in ~1 Because of their positive charge at neutral pH and cm2 chunks, sectioned to ~35 um and probe- their ubiquitous nature as coatings in natural sedi- polished. Thin sections were utilized in all field and ments, the role of Fe oxides in colloid deposition has laboratory experiments to minimize surface rough- been studied extensively. Fe can be scarce in many ness or microtopography as attachment variables, groundwaters because of the low solubility of Fe and to provide an ideal surface for spatial analysis of oxyhydroxides at neutral pH but it is still necessary the trace and major element geochemistry (LA-ICP- for microorganisms in cellular electron transport. MS), and microbial attachment (SEM). Additionally, Fe-oxidizing bacteria derive energy Thin sections were analyzed for the initial spatial from the oxidation of Fe2+ to Fe3+, while facultative distribution of trace and major elements in the min- and obligate anaerobes derive energy from the eral phases via LA-ICP-MS. A ThermoElemental Fe3+/Fe2+ redox couple (+0.77 V) using iron as a X7 ICP-MS was utilized coupled with Nd:YAG terminal electron acceptor (TEA). Many dissimila- (266 nm) laser ablation setup which has been de- tory iron reducing bacteria (DIRB) require contact scribed in detail elsewhere (e.g. Crowe et al. 2003). with Fe to achieve reduction (e.g. Lovley & Phillips Laser transects (7 micron spot size) were conducted 1988) and may use flagella to detect and attach to across apatite, biotite, and magnetite phases in the Fe-rich surfaces (e.g. Caccavo & Das 2002). host rock. Trace element concentrations in the apa- Few if any studies, however, have investigated tite phases were calculated based on the stoichiome- the role of phosphate minerals in colloid deposition. try of CaO in fluroapatite as an internal standard and Like the Fe oxides, phosphate minerals are sparingly a NIST 610 glass as an external standard. soluble at neutral pH and exhibit pHzpc ranging from 6.4 to 8.5 (Stumm & Morgan 1996). Bioavailable P is commonly lacking in many subsurface environ- 2.2 Field colonization studies ments and can diminish cell growth and metabolic Thin sections were incubated in situ in a carbon- efficiency. P is a fundamental macronutrient needed rich, anaerobic groundwater using a stainless-steel by microorganisms for synthesis of nucleic acids, holder that was placed in the water well and left un- nucleotides, phosphoproteins, and phospholipids disturbed for twelve months. The sectioned rocks (e.g. Ehrlich 2002), compounds used as the energy were exposed to the native microbial consortium, source for driving biosynthetic reactions in the cell. which is dominated by DIRB, methanogens, and Because P plays a fundamental role in microbial life fermenting bacteria (Bekins et al. 1999). The sam- functions, microorganisms significantly impact the ples were retrieved and tissues were fixed in the distribution and cycling of P in subsurface environ- field using a chemical critical point drying method ments. The major reservoir for inorganic P in these (Vandevivere & Bevaye 1993; Nation 1983). The geologic settings are minerals such as apatite, and slides were stub-mounted, gold sputter coated and microorganisms may access this mineral-bound P imaged on a LEO 1550 field emission scanning elec- from both detrital (e.g. Goldstein 1986) and igneous tron microscope at 20 kV. Samples were imaged us- sources (Taunton et al. 2000). We hypothesize that ing secondary electron (SED) and backscatter elec- microorganisms will preferentially attach to surface tron detectors (BSD) to optimize for imaging of exposures of Fe- or P-bearing mineral inclusions attached microorganisms and mineralogy, respec- rather than to the nutrient-poor silicate groundmass tively. Fe and phosphate mineral inclusions were lo- in which they are included. Attachment is likely cated using BSD and inclusion composition was promoted by the positive surface charge of the in- confirmed using EDAX and LA-ICP-MS. The inclu- cluded minerals interacting with negatively charged sions and the surrounding silicate groundmass were cell membranes. then imaged at a range of magnifications to discern Figure 1. Reflected light photomicrograph of mineral assem- Figure 2. SEM photomicrograph of anorthoclase surface after blage in anorthoclase, including apatite (near center, hexago- reaction in anaerobic groundwater for twelve months. An apa- nal), magnetite, biotite, and amphibole. Scale bar is 250 µm. tite inclusion intergrown with magnetite is shown. Scale bar is 10 µm. changes in attachment density as a function of dis- are heavily colonized with an average of 2x103 cells tance away from the inclusion. These images were mm-2 compared to the silicate groundmass which then analyzed using Opti Analysis imaging software supports ~2x102 cells mm-2, likely due to favorable for quantification of attached cells as a function of electrostatic interactions between negatively charged surface mineralogy. cells and slightly positively charged oxide surface (pHzpc ~7; magnetite). The phosphate mineral and apatite inclusions, 3 RESULTS AND DISCUSSION however, did not exhibit colonization densities that 3.1 Mineralogy and inclusion chemistry were significantly different from the silicate groundmass. Figure 2 shows an SEM photomicro- Quartz and the O. microcline were free of inclusions graph of an apatite inclusion adjacent to magnetite. and therefore served as colonization controls for Few cells are visible on the apatite surface, while S.D. microcline and anorthoclase, both of which ex- several cells can be seen on the magnetite. hibited abundant mineral inclusions. S.D. microcline Preferential attachment to both inclusion types contained phosphate mineral inclusions, which occur was expected because the groundwater pH was be- as elongate terminating prisms. Anorthoclase con- low that of the pHzpc of the minerals. The pHzpc val- tained assemblages of magnetite, biotite, amphibole, ues used for apatite were reported for hydroxyapatite and apatites. but LA-ICP-MS analyses revealed that the inclu- Laser ablation of phosphate inclusions in the S.D. sions are not hydroxyapatite, but rather REE- microcline indicate a shift away from stoichiometric enriched apatites (anorthoclase) and Pb phosphates apatite, with enrichment of Pb ~1,000 times chron- (S.D. microcline). These compositional differences drite. Apatite inclusions in anorthoclase are enriched likely impact the surface charge characteristics of in REE, U, and Th (light REE ~1,000 times chon- the minerals and it is possible that the inclusions are drite; heavy REE ~10,000 times chondrite). Prelimi- negatively charged at the groundwater pH. These nary analyses of the magnetite inclusions indicate compositional differences may also be a deterrent to they have incorporated some Zn and alkali metals. microbial attachment by serving as a source of toxic REE element enrichment is limited to the apatite. elements (Pb) or radionuclides (U, Th) which inhibit Figure 1 shows an inclusion-rich zone within anor- microbial activity. thoclase. Smaller apatites are commonly included The close association between apatite and mag- within amphibole while larger apatite inclusions oc- netite on the anorthoclase surface (Figs. 1 & 2) pre- cur in close association with magnetite inclusions. sents another possible explanation for the lack of colonization on apatite. If the apatites are dissolving, 3.2 Microbial colonization of mineral inclusions which is likely with less than 1 µm dissolved PO4 in solution, then PO4 may adsorb to the proximal oxide Groundwater in the study zone is anaerobic with a surface. Figure 3 is a speciation diagram of phospho- pH of 6.74, 3.4 mM DOC, 0.66 mM Fe2+, 0.74 mM rus sorption on hydrous ferric oxide (HFO) as a CH4, and 1.2 mM Si. The water also contains 4.58 function of pH, showing substantial sorption of PO4 2+ 2+ - mM Ca , 1.35 mM Mg , 12.4 mM HCO3 and in the pH range for the groundwater. The diagram <0.01 mM of Al, K, Na, SO4, NO3 and PO4. After was generated using the geochemical speciation pro- twelve months in the groundwater, only magnetite gram J-Chess 2.0 (van der Lee 1998) using input pa- inclusions showed substantial colonization by the rameters of 10 g/L hydrous ferric oxide, 1 umol kg-1 native microbial consortium. Magnetite inclusions Caccavo, J.R. & Das, A. 2002. 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