Soft X-Ray Spectromicroscopy Studies of Microbial Processes at Environmental Interfaces
Gordon E. Brown Jr.1, Karim Benzerara2, Tae Hyun Yoon3, Juyoung Ha4, Carmen D. Cordova5, Alfred M. Spormann5, Guillaume Morin3, Georges Calas3, and Tolek Tyliszczak6
1Department of
Geological and Environmental Sciences and Stanford Synchrotron Radiation
Laboratory, Stanford University
2Institut de Minéralogie et
Physique des Milieux Condensés, University of Paris
3Department
of Chemistry, Hanyang University
4Department of Geological and
Environmental Sciences, Stanford University
5Department of
Civil and Environmental Engineering, Stanford University
6Chemical
Sciences Division, Lawrence Berkeley National Laboratory
Environmental interfaces are the locations of most chemical reactions occurring in the environment and commonly juxtapose minerals, aqueous solutions, atmospheric and soil gases, microbial organisms, and/or organic matter (including black carbon, plant litter, and xenobiotic organics). Fundamental understanding of the chemical and biological processes occurring at such interfaces is limited because of their complexity and the need to study them at appropriate spatial scales under realistic environmental conditions. We have used scanning transmission x-ray microscopy (STXM) to study various environmental interfaces with the aim of defining reaction products and conditions in several natural and model systems. The Molecular Environmental Science STXM beam station 11.0.2.2 at the Advanced Light Source, which is capable of 25-30 nm spatial resolution over the energy range 75-2150 eV, was used for imaging and NEXAFS spectroscopy in the following interfacial systems: (1) an orthopyronene-filamentous microbe interface from the Tathouine meteorite, (2) aragonite-bacteria-EPS interfaces in a modern microbialite from Lake Van, Turkey, (3) Shewanella oneidensis MR-1-hematite interfaces in pH 7.4 solutions; (4) Gallionella-Fe(II) solution interfaces in an acid mine drainage (AMD) containing 350 mg/l of As(III) in Gard (Carnoulès), France, (5) black carbon surfaces before and after interaction with polychlorinated biphenyls (PCBs), and (6) hydroxyapatite-protein interfaces in human calcifications. These studies have shown, respectively, that (1) microbial organisms create distinct microenvironments in their immediate vicinity at mineral surfaces that result in rapid bioweathering of these surfaces; (2) the unusual morphology of microbialite aragonite crystals are likely due to crystal growth in a polysaccharide matrix; (3) differences in hematite particle size influence S. oneidensis cell activity and iron reduction rates; (4) Gallionella are capable of Fe(II) oxidation but not As(III) oxidation, resulting in the precipitation of tooeleite [Fe(II)6(AsO3)4(SO4)(OH)4•4H2O] in the Carnoulès AMD system; (5) chemical heterogeneities in black carbon materials influence the sorption of hydrophobic organics like PCBs, which are found to adsorb preferentially to surface regions of black carbon with the highest content of aromatic functionalities; and (6) “nanobacteria” don’t appear to be involved in the precipitation of hydroxyapatite in human calcifications such as those found in heart valves.
The proposed ERL at CHESS will result in significantly smaller beam sizes and significantly higher brightnesses than are currently possible with Fresnel zone plates on STXM beamlines (15 nm is current minimum at the ALS) or with K-B mirrors on hard x-ray mXAFS or mXRF beamlines at 3rd-generation light sources (≈ 1mm is typical limit at the APS). Thses attributes should make it possible to conduct spectromicroscopy studies of even smaller regions within microbial cells and at environmental interfaces where bacteria have a major impact on the breakdown of solids and cause the precipitation of biominerals. The benefit of such studies includes the ability to more quantitatively define nano-environments at microbe-solid interfaces, including compositional gradients, redox gradients, and the identity, morphology, spatial location, and phase association of biomineralization products such as nanocrystalline iron oxides, calcium carbonates, or calcium phosphates that form at microbial cell walls or in protein matrices. An example of variations in redox microenvironments at a filamentous microbe-orthopyroxene interface observed using the ALS 11.0.2.2 STXM endstation is shown in the attached figure (from Benzerara et al. (2005) Proc. Nat. Acad. Sci. USA 204, 979), which illustrates changes in Fe(II)/Fe(III) ratios with location at this complex interface. Another advantage of the ERL over current soft x-ray STXM beamlines at 3rd-generation light sources is the far broader energy range that will be accessible with the ERL. This attribute will permit spectroscopic and imaging studies of a greater range of elements, including the K-edges of the biologically important elements phosphorous and sulfur, which are not currently accessible on the STXM beamlines at the ALS. Compared with the SLAC LCLS, which has a limited bandwidth that precludes normal NEXAFS (or EXAFS) spectroscopy, the proposed ERL should permit normal NEXAFS spectroscopy studies at both soft and hard x-ray energies, which typically require tunability over a 100-200 eV region. In addition, the higher energy resolution of the ERL will result in higher energy resolution NEXAFS spectra than are currently attainable using electron energy loss (EEL) spectroscopy on transmission electron microscopes equipped with EEL spectrometers. Another advantage of an ERL-STXM beamline relative to a TEM equipped with EELS in studies of microbial-solid interfaces is that the former would permit spectroscopy and imaging to be done on under ambient or in situ rather than ultra-high vacuum conditions, as is required by conventional TEM, and would require minimal sample preparation. The short pulse length of the CHESS ERL, although not as short as that proposed for the LCLS (1 fs) would also permit time-resolved NEXAFS spectroscopic studies of chemical reactions at microbe-solid interfaces, such as changes in iron oxidation state as an iron-reducing bacteria (FERB) such as Shewanella oneidensis interacts with hemtaite (a-Fe2O3) surfaces. Such time-resolved measurements would help resolve the current controversy surrounding different proposed electron transfer mechanisms between FERB and mineral surfaces. Electron transfer reactions at redox-sensitive mineral surfaces such as hematite involving bacteria and organic matter result in their dissolution, which can release toxic metalloids such as As(V).
Additional potential applications of the ERL include (1) higher spatial resolution x-ray standing wave fluorescent yield spectroscopy studies of biofilm-coated solids following reactions with heavy metals such as lead and metalloids such as arsenic, which provide information on the partitioning of these ions between the biofilm and the solid surface under in situ conditions, (2) nano-XAFS characterization studies of biominerals in such biofilms, (3) nano-XRD characterization studies of biominerals, and (4) nano-XRF imaging studies of metal distributions within bacteria, plants, and cells from higher organisms.
Figure Caption: Left: TEM image (top) of the cross section showing the microorganism (arrow), the CaCO3 cluster, and the orthopyroxene (Opx) and equivalent STXM image (bottom) at 707.8 eV. Right: Iron L3-edge NEXAFS spectra from the orthopyroxene (area 1 in STXM image), representing the Fe2+ endmember, the CaCO3 cluster (area 2 in STXM image), the microorganism (area 3 in STXM image), and reference hematite, representing the Fe3+ end member. Dashed lines represent the positions of iron L3 maxima for Fe2+ and Fe3+ at 707.8 and 709.5 eV, respectively. The black scale bars in the TEM and STXM images are 1mm in length.
