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Multispecies biofilms transform selenium oxyanions into elemental selenium particles: studies using combined synchrotron X-ray fluorescence imaging and scanning transmission X-ray microscopy Soo In Yang, Graham N. George, John R. Lawrence, Susan G. W. Kaminskyj, James J. Dynes, Barry Lai, and Ingrid J. Pickering Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04529 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Environmental Science & Technology

Multispecies biofilms transform selenium oxyanions into elemental selenium particles: studies using combined synchrotron X-ray fluorescence imaging and scanning transmission X-ray microscopy Soo In Yang,† Graham N. George,† John R. Lawrence,‡ Susan G. W. Kaminskyj,§ James J. Dynes,ǁ Barry Lai,⊥ Ingrid J. Pickering*† †

Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada



National Hydrology Research Centre, Environment Canada, Saskatoon, Saskatchewan S7N 3H5, Canada §

Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada

ǁ

Canadian Light Source, 101 Perimeter Road, Saskatoon, Saskatchewan S7N 0X4, Canada



Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, United States

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ABSTRACT

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Selenium (Se) is an element of growing environmental concern, as low aqueous concentrations

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can lead to biomagnification through the aquatic food web. Biofilms – naturally occurring

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microbial consortia – play numerous important roles in the environment, especially in

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biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally

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forming multispecies biofilms presents challenges for characterization since conventional

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microscopic techniques require chemical and physical modification of the sample. Here,

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multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray

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fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These

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complementary synchrotron techniques required minimal sample preparation and were applied

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correlatively to the same biofilm areas. Submicron XFI showed distributions of Se and

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endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of

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elemental Se (Se(0)). Nano-scale carbon K-edge STXM revealed the distributions of microbial

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cells, extracellular polymeric substances (EPS) and lipids using the protein, saccharide and lipid

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signatures, respectively, together with highly localized Se(0) using the Se LIII-edge.

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Transmission electron microscopy showed the electron-dense particle diameter to be 50-700 nm,

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suggesting Se(0) nanoparticles. The intimate association of Se(0) particles with protein and

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polysaccharide biofilm components has implications for selenium’s bioavailability in the

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environment.

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INTRODUCTION

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Selenium (Se, Z=34), an essential micronutrient in animals and certain microorganisms,

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serves as a catalytic element within the active site of several types of antioxidant enzymes such

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as thioredoxin reductase.1,2 In addition, although plants lack a requirement for this element, a

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positive role for selenium such as an increase in yield has been suggested for certain species.3,4

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Despite its beneficial role, selenium can be toxic at elevated levels and in most organisms tends

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to have a narrow margin between required and toxic levels.5 Selenium can cause toxicity by a

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number of mechanisms, including mimicking or disturbing sulfur metabolism6 and damaging

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cells by oxidative stress7 in most organisms, including humans. The environmental risks from

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selenium contamination are of increasing concern and can arise from a variety of sources,

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including mining, agricultural water use and power plant emissions.8-10 Relatively low levels of

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selenium entering the aqueous environment due to anthropogenic activities can be biomagnified

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through aquatic food webs11,12 with accumulated levels of selenium causing adverse effects

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especially in larval stages of oviparous vertebrates including birds and fish.13-15 The initial

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biotransformation of aqueous oxyanions selenate and selenite by microorganism communities is

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a key component of this biomagnification.11,12

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In the aqueous environment, microorganisms favor growing as communities known as

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biofilms by forming and incorporating a slimy matrix of extracellular polymeric substances

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(EPS). Microorganisms in biofilm communities have shown a high resistance to toxic elements

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compared with their free-living planktonic counterparts.16-18 One advantage for microorganisms

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living in community as a biofilm is the ease of metabolic control using cell-to-cell

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communication in response to environmental changes.19 Hence, the prevalent yet complex

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structure of biofilms typically confers increased tolerance to physical, chemical, and biological 3 ACS Paragon Plus Environment

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stress compared to free-living microorganisms.16-18 Therefore, biofilm formation is evolutionally

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significant for microorganisms, increasing their survival.

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Although advances in microscopy and molecular biological techniques have expanded our

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understanding of metabolic processes in biofilms, the investigation of biofilm interaction with,

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and resistance to toxic substances presents challenges. Characterization of both the structure and

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chemistry of the biofilm is especially challenging since these may be modified in conventional

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measurements. To address this, we employed a suite of complementary synchrotron-based

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transmission and fluorescence imaging techniques using Se LIII and K near-edge X-ray

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absorption spectroscopy to examine selenium biotransformation in multispecies biofilms. Our

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novel approach using synchrotron-based hard and soft X-ray imaging techniques on the same

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areas of the samples facilitated characterization of the chemistry of the accumulation and

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biotransformation of the selenium in relation to the biological structures.

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Our multispecies biofilms were cultured as previously20 using water sampled from a lentic

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environment receiving effluent from a coal mine with elevated selenium levels.21 The biofilms

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thus represent previously-characterized environmentally-relevant communities that have adapted

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to cope with elevated levels of selenium. Our results show that specific regional communities

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within the biofilms carry out selenium detoxification, as indicated by the localized distribution of

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reduced selenium species. In particular, elemental selenium was found to be deposited as sub-

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micron-sized particles closely associated with the microorganisms.

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EXPERIMENTAL SECTION

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Biofilm growth. Water for biofilm cultivation was collected as previously described by Yang

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et al.20 from Goddard Marsh, British Columbia, Canada, a lentic environment receiving effluent 4 ACS Paragon Plus Environment

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from a coal mine with high levels of selenium.21 The community of microorganisms was

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previously reported20 to include the following selenium-resistant microorganisms: Rhodococcus

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sp. (Gene Bank ID, AP0008957); Pseudomonas sp. (FJ652605.1); Bacillus sp. (GQ495047); and

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Arthrobacter sp. (FJ517623.1). Biofilms were cultivated as described by Yang et al.20 with the

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following modifications. The inoculum was 1 % of a microbial pre-culture incubated overnight

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in 10 % tryptic soy broth (TSB) at room temperature (RT, 22 ± 3 °C). Biofilm growth occurred

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on the surface of polycarbonate strips (10 cm × 1 cm) for 10 days or 1 month. Biofilms were

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subsequently transferred to test tubes containing 16 mL of fresh 10 % TSB, which also contained

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selenate or selenite (0.63 – 63 mM). Tubes were further incubated aerobically on a gyratory

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incubator at 100 rpm for 20 days or 2 months. Thus, total incubation periods were 1 or 3 months.

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The viability of the biofilms during this time period was verified by morphological examination

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using confocal laser scanning microscopy, as previously described.20 All experimental

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procedures were carried out in sterile conditions.

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X-ray fluorescence imaging (XFI). Biofilm samples were rinsed at least 3 times using

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autoclaved 10 % TSB, followed by removal of excess liquid. Biofilms were then harvested using

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a sterile scraper made from silicon tubing. For each sample a 1 µL aliquot of the collected

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biofilm was applied onto a silicon nitride window (0.5 mm × 0.5 mm, NX5050B, Norcada Inc,

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AB, Canada) and allowed to air-dry. The prepared biofilm samples were brought to beamline 2-

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ID-D of the Advanced Photon Source, USA. Regions of interest were recorded using an optical

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light microscope (Leica DMR, Leica, IL, USA). The silicon nitride window was mounted onto

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an aluminum frame positioned at 15° to the incident beam with a Vortex® silicon drift detector

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(SII Nano Technology USA Inc., CA, USA) located at 90° to the incident beam to measure

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fluorescence emission spectrum. The beam size was focused to 200 nm × 200 nm using a Fresnel 5 ACS Paragon Plus Environment

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zone plate. The sample was maintained in a helium gas filled environment and raster scanned at

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0.5 s per pixel. Data were processed using MAPS software.22 Further image processing was

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conducted using the aXis2000 software.23 Co-localization maps of elements were processed

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using the MAPS software.22 Mander’s overlap coefficients24 were calculated using macros

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created in the Image J software.25

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Determination of selenium concentration. To calibrate Se fluorescence emission from the

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biofilm samples, X-ray fluorescence from a multi-element reference provided by National

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Bureau of Standards26 was collected under the same geometrical configuration. Due to the thin

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nature of the prepared biofilm samples (1~3 µm), thickness effects were disregarded. Among the

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recorded Se concentrations, pixels having intensities less than 10 % of the maximum

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concentration were disregarded in determining selenium concentration in both XFI and STXM

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data. Following the usual XFI convention, Se concentrations are expressed in terms of the areal

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density (µg/cm2) instead of a volume density (µg/cm3) since there is some uncertainty in the

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thickness of the sample.

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Sub-micron Se K-edge X-ray absorption spectroscopy. Se K near-edge X-ray absorption

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spectra from the intense selenium spots were recorded using a 200 nm × 200 nm beam by

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scanning energies from 12,620 to 12,740 eV in duplicate. Each spectrum was examined for any

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photo-damage (oxidation or reduction) by checking for any spectral changes between duplicate

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data collection scans. Spectra were processed using EXAFSPAK,27 and were compared with

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spectra previously reported by Yang et al.20

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Scanning transmission X-ray microscopy (STXM). STXM images and spectroscopy were

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carried out at the Soft X-ray Spectromicroscopy beamline (SM) at the Canadian Light Source

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(CLS). Data were collected using point, line scan, spectral, and whole scanning modes at a 6 ACS Paragon Plus Environment

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sequence of energies and processed using the aXis2000 software.23,28 For the quantitative

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analyses of selenium and biofilm macromolecular structures, singular value decomposition

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(SVD)29 was used. Spectra of selenium references including selenate, selenite, selenomethionine,

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selenocysteine, selenocystine, and elemental selenium were collected under the same conditions

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and were used for quantitative analysis of selenium in biofilms. Carbon K-edges were collected

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from 280 to 320 eV.28 Selenium LIII-edge spectra were collected from 1420 to 1470 eV. Spectra

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from the carbon K and selenium L-edge were extracted, background subtracted, and normalized

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to the highest absorbance points.

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Transmission electron microscopy (TEM). TEM studies were carried out using Phillips CM 10 transmission electron microscope according to the method previously reported by Shi et al.30

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RESULTS AND DISCUSSION

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Elemental distribution in selenate-amended multispecies biofilms. We used synchrotron-

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based X-ray fluorescence imaging (XFI) to examine the distribution of selenium and other

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elements within the biofilms. Figure 1 shows examples of the images for different concentrations

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of amended selenate or control, with fields of view corresponding to one or a few

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microorganisms. We observed highly localized selenium in selenate-amended multispecies

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biofilms incubated for three months with the highest concentration (63 mM) of selenate (Figure

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1). These intense selenium spots, appearing as just one to two pixels across, are consistent with

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highly concentrated selenium in objects of 400 nm diameter or less which are substantially

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smaller than the cells; the lower limit on diameter is less than the 200 nm beamsize. The 6.3 mM

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selenate treatment resulted in a peak selenium areal concentration which was three orders of

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magnitude smaller than that of the 63 mM treatment (Table S1) and did not show evidence for

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the highly localized selenium objects.

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XFI allows the simultaneous measurement of distributions of other elements in the biofilm. Cu

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and Zn were highly co-localized in certain regions of the biofilms. Mander's overlap coefficients

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of Cu and Zn distribution images calculated for 0.63 and 63 mM selenate amended biofilms

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(Figure 1) were 0.87 and 0.95, respectively (1 indicating the highest and 0 absence of overlap).

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The morphologies of the Cu and Zn rich areas suggest that they are not distributed in the EPS,

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but instead are restricted to cells. In contrast, P is evenly distributed throughout the biofilms. The

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P distribution also differs from that of microbiologically important nonmetal elements S and Ca

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which appear highly co-localized with each other (Mander’s overlap coefficients of 0.99 and

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0.97 for 0.63 and 63 mM selenate-amended biofilms, respectively) and elevated in concentration

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relative to the control (Table S1, Figure 1).

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Elemental distribution in selenite amended multispecies biofilms. When multispecies

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biofilms were incubated under selenite conditions (0.63 – 6.3 mM) for either 1 or 3 months

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(Figure 2), we observed an elemental distribution pattern similar to that shown in selenate-

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amended biofilms. However, in the case of selenite, the selenium concentrations were

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considerably greater and more intensely localized within the biofilm (Figure 2, Table S1),

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suggesting a highly concentrated phase. They also occurred at a much lower substrate

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concentration (0.63 mM) compared with that for selenate (63 mM). Se K X-ray absorption near-

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edge structure spectra measured with a sub-micron beam from these intense selenium spots

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indicated that their chemical form is elemental selenium (Figure 3). This is consistent with our

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observation of a red precipitate at the bottom of the bioreactors, suggesting that biofilms also

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release elemental selenium particles into the environment. A consequence of the highly 8 ACS Paragon Plus Environment

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concentrated selenium was that self-absorption distortions of the fluorescence signal caused a

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reduction in intensity of the prominent 1s→4p transition (Figure 3). This intensity reduction was

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more obvious in pt 2 and 3 than pt 1, which had much lower concentration. The relative

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comparison, using spectral edge jump of the near-edge spectrum at each point, suggested pt 2

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and 3 are 3.5 and 10.4 times more concentrated compared to pt 1. The relation between the

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distortion in fluorescence signal and selenium particle size was previously simulated by

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Wiramanaden et al.,31 with larger particle sizes of elemental selenium causing greatly decreased

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fluorescence intensities of spectra at the peak compared with the edge jump.

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The maximum concentration of selenium was substantially higher than those of other

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biologically significant elements present in both selenite and selenate-amended biofilms. The

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maximum concentration of the elemental selenium particles ranged from 14.7 to 301 µg/cm2 in

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the examined biofilm areas (Figure 2, Table S1).

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bioaccumulation as a consequence of bio-detoxification or utilization of selenite as an electron

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acceptor by members of the multispecies biofilms.

This may suggest either selenium

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Biotransformation of selenite to elemental selenium has been observed using conventional

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analytical methods in many free-living microorganisms, such as Rhodospirillum rubrum,

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Rhodobacter sphaeroides, Bacillus subtilis, Exiguobacterium sp, Rhizobium sp strain B1,

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Pseudomonas sp. strain CA5, and Escherichia coli.32-37 However, there is limited information on

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production of elemental selenium from selenite in complex multispecies biofilms. Indeed,

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complex multispecies biofilms, which are inherently more challenging to characterize, likely

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more closely represent how microbial consortia may cope with these toxic selenium oxyanions.

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In general, selenite is more biochemically reactive than selenate in biofilms34-36 and thus

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microorganisms may convert it to less reactive elemental selenium as a result of detoxification

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and utilization.

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Biotransformations of selenium oxyanions and biofilm macromolecular structure. We

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used synchrotron-based scanning transmission X-ray microscopy (STXM) to investigate the

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biotransformation products of selenium oxyanions and their possible associations with the

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biofilm structures. STXM can specifically image for proteins, polysaccharides, and lipids that are

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used respectively as markers for microbial cells, EPS and lipid in biofilms28,38 by using the

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carbon K edge.39 In addition, imaging using the Se LIII-edges can give information about the

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selenium distribution and speciation. Thus, information from STXM can be used to examine how

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selenium oxyanions and their biotransformed species are associated with microbial cells and EPS

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without the need for any physical or chemical modifications of biofilms.

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The biofilm areas (Figure 4) that were initially investigated using XFI were subsequently

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examined with STXM. Comparisons (Figure 4) between the selenium map (XFI) and the

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elemental selenium/bio-macromolecule map (STXM-B) indicated that elemental selenium

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particles are closely localized with microorganisms and tend to be deposited in biofilms. The

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elemental selenium particles are seen to be surrounded by both microbial cells (protein) and EPS

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(saccharide) (STXM-B), suggesting that the elemental selenium is embedded in the biofilm

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structure. In an additional STXM map (Figure 5), the elemental selenium particles are again seen

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to be associated with both EPS (green) and microbial cells (blue). Further investigation with least

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square curve fitting of the observed Se LIII–edge spectrum in Figure 5 demonstrated that it is

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indeed elemental selenium with less than 2 % error. Thus, our results clearly indicated that the

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amended selenium oxyanions were biotransformed by the biofilms and that the majority of

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selenium species present under the exposed incubation is the reduced product, elemental

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selenium.

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As shown in Figure 4, lipid (red, STXM-A) appeared to be closely associated with elemental

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selenium (red, STXM-B), suggesting the coexistence and interaction of Se0 and lipid in highly

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localized regions, although this observation may be complicated by the highly absorbing nature

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of the dense elemental selenium particle. There have been a large number of reports on the role

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of sulfur containing peptides or proteins in elemental selenium synthesis in vitro,37,40 but not on

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the role of lipids. Although the reduction of selenium oxyanions can induce lipid peroxidation

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through the generation of reactive oxygen species, it is not known whether the associated lipid

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was oxidized during the reduction process of selenium oxyanions. However, the possible strong

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association of lipid with elemental selenium may suggest that lipid is involved in the formation

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and stabilization of elemental selenium in a similar fashion as thiol groups.37,41

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Investigation of selenium chemical species using X-ray absorption spectroscopy.

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Investigation of selenium chemical species using two synchrotron-based techniques on the same

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areas of the biofilm demonstrated complementary and consistent results. In Figure 6, X-ray

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absorption near-edge structure collected following XFI (Se K) and using STXM (Se LIII) from

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selenium intense spots in biofilms both showed elemental selenium to be the intense selenium

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species. Using STXM, selenium concentrations in the examined area were determined to be 152

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and 62.9 µg/cm2 at maximum and average intensity, respectively. XFI on the same area, scanned

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using the energy of the maximum intensity of the elemental selenium K absorption edge,

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indicated 135 and 52.1 µg/cm2 at the most intense spot and at the average concentration,

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respectively. Thus, the two methods provided results that were consistent and reproducible over

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Biogenic selenium nanoparticles. Further investigation using transmission electron

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microscopy (TEM) with selenite-treated biofilm sections of 70 nm thickness suggested the

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presence of biogenic electron dense particles, consistent with the substantial presence of

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elemental selenium. The electron dense particle sizes observed using TEM ranged from 50 to

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700 nm in diameter, a size-range that including nanoparticles (Figure 7). Some electron-dense

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particles were found inside of the cells although they were mostly within the EPS. This

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distribution agrees with our observation of the locations of elemental selenium particles from

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STXM (Figure 4 and Figure 6). Combining the results of XFI, STXM and TEM we conclude the

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presence of elemental selenium particles, in the size range 50 to 700 nm, both inside cells and

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also associated with the EPS. The association of selenium particles with EPS and the range of

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particle sizes is consistent with the observations of Jain et al.42

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In contrast, selenate-amended biofilms, similar to the control, lacked the high electron density

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regions suggesting the absence of elemental selenium particles (Figure S2). This result agrees

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with our XFI results (Figures 1 and 2) suggesting that the biotransformation of selenite is more

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facile that that of selenate, since in the XFI the small particles of elemental selenium were

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observed at high concentrations in treatment concentrations down to 0.63 mM selenite whereas

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for selenate they were only apparent at 63 mM.

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Complementarity of XFI and STXM. Here we have used two complementary synchrotron

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X-ray methods – XFI and STXM – to provide information about the multispecies biofilms.

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STXM typically measures the absorption of the sample in transmission, although fluorescence

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detection increasingly is available.43 Transmission is ideal for locally concentrated species, such

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as the elemental selenium particles observed in this study. By measuring stacks of images using a

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series of energies across the Se LIII-edge, chemical information on Se for the entire image can be 12 ACS Paragon Plus Environment

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obtained. In addition, measuring the C K-edge energy range of the same area allows the

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identification of biological moieties (bacterial cells, EPS and lipid), enabling the Se particles to

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be localized with respect to the biofilm structures. XFI measures the fluorescent photons44 and

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typically works at higher X-ray energies than STXM. Instrumentally, good spatial resolution is

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easier to obtain at low X-ray energies and thus hard X-ray XFI generally has poorer spatial

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resolution than STXM. Conversely, hard X-ray XFI typically can detect much more dilute

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species per pixel than can STXM, since fluorescence yield is higher for K-edges than for L-edges

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as well as increasing with atomic number.45 In addition, Se XFI allows the simultaneous

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measurement of the distributions of metals and other elements with absorption edges below that

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of the Se K-edge (Figures 1 and 2), and is useful for scanning a wider area of the sample than is

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feasible using STXM. Following collection of XFI maps, X-ray absorption spectroscopy at select

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pixels can provide information on the Se chemical form. Alternatively, by tuning the incident

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energy to characteristic features in the near-edge, maps of individual Se chemical forms can be

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generated.46 Together, STXM and XFI provide complementary probes of complex multispecies

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biofilms.

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Any method that uses ionizing radiation runs the risk of sample damage, and both XFI and

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STXM have potential to damage samples. In our case XFI measurements were conducted first

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and STXM second. One reason for this order was that XFI is better suited for range-finding than

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XFI, in that it maps a larger spatial area much more quickly than STXM. Moreover, the higher

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energy X-rays of the XFI measurement have more than four orders of magnitude lower X-ray

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mass absorption coefficient since they are more penetrating and result in a lower radiation dose

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to the sample than lower energy X-rays. Hence, at the lower energies of the C K-edge used on

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STXM, the beam is strongly absorbed making radiation damage more likely. Thus, we employed 13 ACS Paragon Plus Environment

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high energy XFI first followed by low energy STXM to minimized both damage to biofilms and

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photoreduction of selenium oxyanions. Since photoreduction of selenium oxyanions can be quite

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facile,47 the effects of beam damage would appear earliest in the Se K near-edge spectra of these

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species. Thus, we investigated possible photo-damage of selenium species by collecting multiple

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Se K-edge spectra from spots of selenate and selenite powder samples, and observed no

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significant changes (Figure S1). This demonstrates that our methodology, of XFI followed by

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STXM, minimizes photo-damage and allows correlative measurements to be made on the same

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sample. If samples had sustained substantial damage from exposure to X-rays during the XFI

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then, as previously reported,48 STXM should show the formation of peaks at 285 and 286.6 eV

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(ketones and phenols) and substantially reduced lipid, polysaccharide and protein features. This

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was not observed in our data, and we therefore feel reasonably confident that the samples were

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relatively damage free at the start of the STXM experiment, but such damage cannot be

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rigorously excluded at the time of writing. In any case, this novel combination of methods may

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be useful for other research in microbiology and cell biology for example, in which tools with

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high spatial resolution and chemical sensitivity with minimal sample destruction would be an

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asset.

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Environmental implications. While the accumulation of elemental selenium particles has

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been observed in planktonic forms of pure bacterial cultures,32-37 our work demonstrates this

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process for complex multispecies biofilms. We have achieved this using a suite of

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complementary synchrotron-based tools, which are widely applicable to other biofilm and

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cellular-level structures of environmental relevance. The reduction of selenium to low solubility

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elemental selenium may suggest that such multispecies biofilms represent a pathway for

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sequestration of selenium as a low bioavailability form and even for bioremediation of selenium 14 ACS Paragon Plus Environment

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contaminated areas, such as mine impacted sites. In support of this, elemental selenium, likely

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produced by multispecies biofilms, was observed as a significant component of selenium present

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in sediments near uranium mining and milling operations.12,31 However, caution is warranted

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since nanoparticles have different properties compared with the bulk form48 and synthetic

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elemental selenium nanoparticles have been observed to be highly bioavailable to benthic

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invertebrates.50 Moreover, biogenic selenium nanoparticles have been reported to show

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antimicrobial activity in effectively inhibiting biofilm formation in other microbes,51

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demonstrating that elemental selenium nanoparticles are not inert. Here we observe that the

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elemental selenium nanoparticles are intimately associated with the microbial cells and EPS

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component of the multispecies biofilm. This association may modify both the stability of the

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nanoparticles and their bioavailability in the environment.

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ASSOCIATED CONTENT

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Supporting Information.

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Maximum and average areal elemental concentrations in multispecies biofilms (Table S1),

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selenium K near-edge spectra of powdered selenate and selenite as a function of time of

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irradiation (Figure S1), and TEMs of control and selenate-amended biofilms (Figure S2). The

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Supporting Information is available free of charge on the ACS Publications website at DOI:

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10.1021/acs.est.xxxxxxx.

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AUTHOR INFORMATION

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Corresponding Author

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*Phone: 306-966-5706; fax: 306-966-5683; e-mail: [email protected]

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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The authors thank members of the Pickering/George group at the University of Saskatchewan

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and George Swerhone at Environment Canada. This research is supported by a Natural Sciences

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and Engineering Research Council of Canada (NSERC) Discovery Grant (to Pickering), by

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Saskatchewan Innovation and Science Fund (to Pickering) and by Environment Canada.

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Pickering and George are Canada Research Chairs and Yang is a CIHR-THRUST Fellow. The

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Canadian Light Source is supported by the Canada Foundation for Innovation, NSERC, the

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University of Saskatchewan, the Government of Saskatchewan, Western Economic

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Diversification Canada, the National Research Council Canada, and the Canadian Institutes of

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Health Research. Use of the Advanced Photon Source, an Office of Science User Facility

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operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National

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Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.

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23. Hitchcock, A. P. (2010) aXis2000 is written in interactive data language (IDL) and available at http://unicorn.mcmaster.ca/aXis2000.html 24. Manders, E. M. M.; Verbeek, F. J.; Aten, J. A. Measurement of co-localization of objects in dual-colour confocal images. J. Microscopy 1993, 169, 375–382. 25. Abramoff, M. D.; Magelhaes, P. J.; Ram, S. J. Image processing with Image J. Biophoton. Int. 2004, 11, 36–42. 26. Pella, P. A.; Newbury, D. E.; Steel, E. B.; Blackburn, D. H. Development of National Bureau of Standards thin glass films for x-ray fluorescence spectrometry. Anal. Chem. 1986, 58, 1133–1137. 27. George, G. N.; Pickering, I. J. (2001) EXAFSPAK: A suite of computer programs for analysis

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30. Shi, C.; Kendall, S. C.; Grote, E.; Kaminskyj, S.; Loewen, M. C. N-terminal residues of the yeast pheromone receptor, Ste2p, mediate mating events independently of G1-arrest signaling. J. Cell Biochem. 2009, 107, 630–638. 31. Wiramanaden, C. I. E.; Liber, K.; Pickering, I. J. Selenium speciation in whole sediment using X-ray absorption spectroscopy and micro X-ray fluorescence imaging. Environ. Sci. Technol. 2010, 44, 5389–5394. 32. Kessi, J.; Ramuz, M.; Wehrli, E.; Spycher, M.; Bachofen, R. Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum. Appl. Environ. Microbiol. 1999, 65, 4734–4740. 33. Van Fleet-Stalder, V.; Chasteen, T. G.; Pickering, I. J.; George, G. N.; Prince, R. C. Fate of selenate and selenite metabolized by Rhodobacter sphaeroides. Appl. Environ. Microbiol. 2000, 66, 4849–4853. 34. Ikram, M.; Faisal, M. Comparative assessment of selenite (SeIV) detoxification to elemental selenium (Se0) by Bacillus sp. Biotechnol. Lett. 2010, 32, 1255–1259. 35. Hunter, W.; Kuykendall, L. Reduction of selenite to elemental red selenium by Rhizobium sp. strain B1. Curr. Microbiol. 2007, 55, 344–349. 36. Hunter, W.; Manter, D. Reduction of selenite to elemental red selenium by Pseudomonas sp. strain CA5. Curr. Microbiol. 2009, 58, 493–498. 37. Dobias, J.; Suvorova, E. I.; Bernier-Latmani, R. Role of proteins in controlling selenium nanoparticle size. Nanotechnology 2011, 22, 195605.

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38. Lawrence, J. R.; Swerhone, G. D. W.; Leppard, G. G.; Araki, T.; Zhang, X.; West, M. M.; Hitchcock, A. P. Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms. Appl. Environ. Microbiol. 2003, 69, 5543–5554. 39. Brandes, J. A.; Lee, C.; Wakeham, S.; Peterson, M.; Jacobsen, C.; Wirick, S.; Cody, G. Examining marine particulate organic matter at sub-micron scales using scanning transmission X-ray microscopy and carbon X-ray absorption near edge structure spectroscopy. Mar. Chem. 2004, 92, 107–121. 40. Zhang, J.; Gao, X. G.; Zhang, L.; Bao, Y. Biological effects of a nano red elemental selenium. Biofactors 2001, 15, 27–38. 41. Ogasawara, Y.; Lacourciere, G.; Stadtman, T. C. Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: Possible role of a protein perselenide in a selenium delivery system. Proc. Natl. Acad. Sci. USA 2001, 98, 9494– 9498. 42. Jain, R.; Jordan, N.; Weiss, S.; Foerstendorf, H.; Heim, K.; Kacker, R.; Hübner, R.; Kramer, H.; van Hullebusch, E. D.; Farges, F.; Lens, P. N. L. Extracellular polymeric substances govern the surface charge of biogenic elemental selenium nanoparticles. Environ. Sci. Technol. 2015, 49, 1713−1720. 43. Hitchcock, A. P.; Obst, M.; Wang, J.; Lu, Y. S.; Tyliszczak, T. Advances in the detection of As in environmental samples using low energy X-ray fluorescence in a

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scanning transmission X-ray microscope: Arsenic immobilization by an Fe(II)oxidizing freshwater bacteria. Environ. Sci. Technol. 2012, 46, 2821–2829. 44. Pushie, M. J.; Pickering, I. J.; Korbas, M.; Hackett, M. J.; George, G. N. Elemental and chemically specific X-ray fluorescence imaging of biological systems. Chem. Rev. 2014, 114, 8499–8541. 45. Hubbell, J. H.; Trehan, P. N.; Singh, N.; Chand, B.; Mehta, D.; Garg, M. L.; Garg, R. R.; Singh, S.; Purl, S.A Review, bibliography, and tabulation of K, L, and higher atomic shell X-ray fluorescence yields. J. Chem. Phys. Ref. Data 1994, 23, 339–364. 46. Pickering, I. J.; Prince, R. C.; Salt, D. E.; George, G. N. Quantitative, chemically specific imaging of selenium transformation in plants. Proc. Natl. Acad. Sci. 2000, 97, 10717–10722. 47. George, G. N.; Pickering, I. J.; Pushie, M. J.; Nienaber, K.; Hackett, M. J.; Ascone, I.; Hedman, B.; Hodgson, K. O.; Aitken, J. B.; Levina, A.; Glover, C.; Lay, P. A. X-rayinduced photo-chemistry and X-ray absorption spectroscopy of biological samples. J. Synch. Radiat. 2012, 19, 875–886. 48. Gillespie, A. W.; Phillips, C. L.; Dynes, J. J.; Chevrier, D.; Regier, T. Z.; Peak, D. Advanced in using soft X-ray spectroscopy for measurements of soil biogeochemical processes. Adv. Agron. 2015, 133, 1-32. 49. Buzea, C.; Pacheco, I. I.; Robbie, K. Nanomaterials and nanoparticles: Sources and toxicity. Biointerphases 2007, 2, MR17–MR71.

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50. Gallego-Gallegos, M.; Doig, L. E.; Tse, J. J.; Pickering, I. J.; Liber, K. Bioavailability, toxicity and biotransformation of selenium in midge (Chironomus dilutus) larvae exposed via water or diet to elemental selenium particles, selenite, or selenized algae. Environ. Sci. Technol. 2013, 47, 584–592. 51. Zonaro, E.; Lampis, S.; Turner, R. J.; 2, Qazi, S. J. S.; Vallini, G. Biogenic selenium and tellurium nanoparticles synthesized by environmental microbial isolates efficaciously inhibit bacterial planktonic cultures and biofilms. Front. Microbiol. 2015, 6, 584.

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Figure 1. False color micro-XFI elemental distribution maps for biofilms grown for 3 months in the presence of 0 (Se-free control), 6.3 and 63 mM selenate. Incident energy was 12,680 eV. Left panels show optical microscopic images of the scanned areas. Scale bars show 2 μm. The false color scale represents concentrations, with the maximum for each panel shown in Table S1.

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Figure 2. False color micro-XFI elemental distribution maps for biofilms grown for 3 months in the presence of 0.63 mM selenite, and 1 month and 3 months in the presence of 6.3 mM selenite. Incident energy was 12,680 eV. Left panels show optical microscopic images of the scanned areas. Scale bars show 2 μm. The false color scale represents concentrations, with the maximum for each panel shown in Table S1.

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Figure 3. Selenium distribution in biofilm (left) incubated for 1 month under 6.3 mM of selenite. The scale bar shows 2 μm and the selenium intensity bar is shown at the bottom of the image. Normalized Se K near-edge spectra (right) from 200 nm spots as indicated by pt 1 to pt 3, in comparison with reference spectra of selenite and elemental selenium (Se0). The broken line indicates the peak position of elemental selenium.

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Figure 4. Optical, XFI and STXM images of the same areas of biofilms treated with selenite at 0.63 mM for 3 months (also shown in Figure 2), and at 6.3 mM for 1 and 3 months. XFI results show the upper 90 % and lower 10 % of the selenium intensity in red and green, respectively. STXM-A shows tricolor maps of the distribution of lipid (Lpd, red), polysaccharide indicating EPS (Psd, green) and protein showing bacterial cells (Prt, blue). STXM-B combines elemental selenium (Se0, red) with macromolecular distributions of EPS (Psd, green) and microbial cells (Prt, blue). The areas were investigated by XFI followed by STXM. Scale bars indicate 1 μm.

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Figure 5. STXM image (left) and observed selenium LIII-edge spectra (right) from biofilm amended with 6.3 mM selenite for 1 month. The STXM image shows elemental selenium (red), extracellular polymeric substances modelled as polysaccharide (green), and microbial cells modelled as protein (blue). Scale bar indicates 1 μm. The yellow broken line outlines the region from which the observed spectrum was extracted. The least square fitting result of the observed spectrum to an elemental selenium standard is shown as a dotted line.

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Figure 6. XFI and STXM images (left) of biofilm amended with 6.3 mM selenite for 1 month. The same area of biofilm was investigated by XFI followed by STXM. Scale bars indicate 1 μm. XFI image shows selenium distribution on a relative intensity scale (white = high). STXM tricolor image shows selenium (red), protein (blue) and polysaccharide (green). Se X-ray absorption spectra (right) of the biofilm are compared with spectra of selenite and elemental selenium (Se0) standards, showing good correspondence with the latter. Nano Se K X-ray absorption near-edge spectra (upper right), were recorded from pt 1 and pt 2 in the XFI image. The “Intense” Se LIII near-edge spectrum (lower right) was extracted from the areas of intense selenium shown in red in the STXM image. The dotted lines indicate the peak position of elemental selenium.

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Figure 7. Thin sectioned (70 nm) transmission electron micrographs of biofilms amended with 6.3 mM (a) and 63 mM selenite (b) for 3 months. Electron dense areas shown in black suggest elemental selenium nanoparticles. Scale bars indicate 0.5 μm.  

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Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy.

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food...
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