Hematite crystallization in the presence of organic matter: impact on

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Hematite crystallization in the presence of organic matter: impact on crystal properties and bacterial dissolution Svend Weihe, Marco Mangayayam, Karina Krarup Sand, and Dominique J. Tobler ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.8b00166 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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ACS Earth and Space Chemistry

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Hematite crystallization in the presence of organic matter: impact on crystal properties and bacterial

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dissolution

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Svend H.C Weihe1, Marco Mangayayam1, Karina K. Sand1,2 and Dominique J. Tobler1

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1Nano-Science

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(*Corresponding author: [email protected])

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2Aberystwyth

Center, Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark

University, Geography & Earth Sciences, SY23 3DB Aberystwyth, United Kingdom

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Abstract

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Microbial dissimilatory iron reduction (DIR) is a widespread process in oxygen poor sediments and waters,

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where it regulates Fe and C cycling and contributes to nutrient and trace metal distribution. This process is

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fairly well studied in terms of iron substrate, microbial DIR strain, and water chemistry. However, far less is

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known about DIR rates and yields of iron substrates that are tightly associated with organic matter, even though

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most iron substrates that form in nature are partially or completely covered by organic matter. Here, we

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assessed the impact of alginate on hematite crystallization and subsequently assessed the stability of so formed

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alginate-hematite precipitates in DIR experiments with Shewanella oneidensis MR-1. We found that during

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hematite synthesis via forced hydrolysis, the presence of alginate reduces hematite crystals and particle size

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and induces the formation of closely associated alginate-hematite composites. This is explained by alginate

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molecules acting as nucleation sites. Upon exposure of these composites to S. oneidensis MR-1, a ~30-50%

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decrease (depending on composites’ alginate content) in initial hematite reduction rate is observed compared

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to the alginate-free, pure hematite. However, while DIR rates ceased after 48h in the pure hematite system,

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reduction steadily progressed in the presence of the alginate-hematite composites, overall leading to a slightly

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higher DIR yield after 8 days. These trends are explained by alginate physically hindering direct contact

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between bacterial cells and hematite surfaces, thus lowering initial DIR rates. In turn, this lower rate potentially

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reduces quick passivation of cell and/or mineral surfaces by Fe(II) adsorption and/or surface precipitates as

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observed in the pure hematite system, thus enabling prolonged DIR reaction in the presence of alginate.

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Overall, this study highlights that a common natural organic molecule such as alginate can largely impact on

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hematite formation in natural settings leading to composites that show very different stabilities towards DIR

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compared to pure hematite. These are important considerations for predicting DIR processes and any

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associated element cycles in natural settings, as well as for potential use of DIR for biotechnical applications.

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Keywords: iron oxide, alginate, Shewanella oneidensis MR-1, biotic redox process, interface processes

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1. Introduction

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Organic matter (OM) is omnipresent in soils, sediments and waters, where it interacts with (in)organic ions,

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molecules and solid surfaces, highly impacting element and nutrient cycling, the reactivity of suspended

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particulate matter, as well as reactions on fixed surfaces (minerals and/or biota). Equally important in these

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settings are iron oxides (abbreviation used here for iron oxides, hydroxides and oxyhydroxides), because they

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constitute one of the most abundant and also most reactive mineral substrate in these environments. They

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commonly form as a result of silicate weathering or through oxidation/reduction reactions with other minerals

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and/or biota.1-3 Iron oxides have a high affinity towards OM due to their high reactive surface area and their

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positively charged surfaces at near neutral pHs.4-5 Similarly, Fe ions readily complex with OM functional

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groups (e.g., carboxyl, hydroxyl, carbonyl).6-8 Thus, when iron oxides form in natural environments, they are

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often either partially or completely covered by OM,9 which in turn changes their surface properties and hence

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reactivity in comparison to inorganic, pure iron oxides.10-12 While the potential impacts of associated OM on

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mineral properties have been postulated for several decades, it is only in recent years that studies have started

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to focus more on the role of OM on the stability and reactivity of iron oxides.1,13-15

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What is even less known is how microbial Fe redox processes are influenced by iron oxides that have formed

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in the presence of OM (i.e., OM- Fe oxide co-precipitates). This is important because microbial Fe redox

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processes take a central role in the cycling of Fe and C, and indeed any other compounds associated with iron

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oxides in near subsurface environments (e.g., phosphate, trace metals).2-3,16 Amongst these processes,

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dissimilatory iron reduction (DIR) is a widespread process in anaerobic settings,17 majorly contributing

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towards organic matter degradation and aqueous Fe(II) production, which in turn catalyzes redox reactions

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with (in)organic contaminants. DIR is also believed to be one of the oldest forms of respiration,18 thus

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understanding potential impacts of iron oxide-associated OM on these processes has also relevance for

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biogeochemical processes in ancient Earth settings.

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DIR has received a lot of attention in recent years, with a vast literature describing reductions rates as a function

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of iron oxide type, bacterial strain and geochemical conditions,3,17,19-20 with several also investigated the effect

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of adsorbed OM on reduction.21-22 However, very few studies have investigated DIR of OM-Fe oxide

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composites.23-27 Those studies focused on the reduction of ferrihydrite (FHY) co-precipitated with humic acid

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or soil extract solutions (OM-FHY composite). They showed that OM-FHY reduction rates vary with the

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bacterial strain used. For example, Shewanella strains were able to reduce OM-FHY faster compared to organic

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free FHY, while for Geobacter strains, reduction rates were lower, particularly at larger OM loading. This is

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mainly explained by these two organisms having different means for electron transfer. Geobacter have been

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shown to use direct contact and conductive nanowire structures for electron transfer,28-29 thus in the presence

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of substantial organic coating, the surface is passivated. Shewanella instead, is able to use chelating agents and

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electron shuttles,30 thus can access electrons even if there is a thick organic coating. Other factors such as the

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type and number of redox-active groups in the mineral-associated OM further contributed to the observed

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differences in reduction rate.

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While OM effects have been studied for ferrihydrite, information on how other Fe substrates are affected is

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sparse. Hematite is one of the thermodynamically most stable iron oxides under typical near-surface

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temperature and pH conditions. Thus, it is often more abundant in soils and sediments than poorly ordered

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phases (e.g., FHY).31 Specifically, in warmer and drier pedoclimates (i.e., subtropical and tropical soils), FHY

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will readily transform to nanometer sized hematite.32-35 To the best of our knowledge, there is no study that

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has investigated how hematite-associated OM affects DIR processes or on how OM impacts hematite crystal

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formation. Such knowledge is not only important in terms of DIR processes but also for other (a)biotic Fe

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redox processes, as well as the mobility and reactivity of OM-hematite composites with other dissolved and

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solid phases in near surface environments.

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Here, we assessed the effect of OM on hematite formation and then tested the stability of the resulting OM-

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hematite composites in bacterial reduction experiments with Shewanella oneidensis MR-1, a widely used

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model strain in DIR studies.16 Alginate was chosen as a model OM because it is a polysaccharide that occurs

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widely in the environment, and it is often used to mimic extracellular polymeric substances (EPS) in biofilms

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and natural settings, where microbes are present.36-37 Important to note here that alginate (i.e., EPS) and most

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other OM are very large molecules (i.e., polymers), thus they cannot substitute into the crystal lattice during

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iron oxide formation. However, the presence of OM can greatly affect the crystallization process. For example,

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OM can act as nucleation site, thereby increasing the number of formed nuclei and decreasing crystallite

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size.6,38 OM can sorb to specific surface/nuclei sites, thereby inhibiting/directing crystal growth in specific

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directions.10,38 This in turn can increase the number of crystal defects and enhance incorporation of foreign

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ions and water. Studying the impact of alginate on hematite crystallization and DIR processes as performed

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here will give a first glimpse into how hematite formation in mature soils and sediments could be affected by

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the surrounding OM and how this then impacts on DIR processes and Fe and C biogeochemical cycling.

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2. Methods

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2.1 Synthesis of hematite ± alginate co-precipitates

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Hematite (-Fe2O3) nanoparticles were synthesized according to Bose et al.39 using forced hydrolysis of a

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Fe(III) salt solution.40 For this, solutions of 1.0 M Fe(NO3)39H2O and 10 gL-1 Na-alginate ([(C6H7O6Na)n])

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were prepared with reagent grade chemicals and deionized (DI) water (resistivity of 18 Mcm). 60 mL of 1.0

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M Fe(NO3)39H2O was placed in a dropping funnel and slowly dripped into 750 mL of boiling DI water under

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constant stirring (over a time span of ~30 min). For hematite-alginate co-precipitates, 0.1 and 1.0 gL-1 Na-

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alginate were added to the boiling DI water. Once Fe(III) addition was completed, the red-brown suspensions

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were removed from the heat and cooled to room temperature. The suspensions were dialyzed in standard-grade

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cellulose membrane tubes (Spectra/Por 6, 1000 MWCO) against DI until the resistivity was 18 Mcm.

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Successful hematite synthesis was confirmed by X-ray diffraction (Fig. S1). An aliquot of the synthesized

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hematite ± alginate suspension was frozen and then freeze-dried for solid characterization.

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2.2 Characterization of hematite ± alginate co-precipitates

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The Fe content ([Fe]tot) in pure hematite and hematite-alginate co-precipitates were determined by atomic

3

absorption spectroscopy (AAS, Perkin Elmer AAnalyst 800) of acid digested (1 M HCl) suspension samples.

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These measurements were complemented by thermogravimetric analyses (TGA, Netzsch TG 209 Libra), in

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which the dry solids (~7 mg) were heated from 30 to 1000 °C at 10 °C/min, under N2 atmosphere. The size

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and shape of hematite particles was estimated by transmission electron microscopy, TEM (Philips CM 20, at

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200 kV, detection limit ~ 0.4 nm). For this, a droplet of hematite suspension was placed on a formvar/carbon

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coated copper TEM grid and left to dried. To resolve the short to medium range structure of the nanocrystalline

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hematite (±alginate) samples synchrotron based X-ray diffraction combined with pair distribution function

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(PDF) analysis was performed at Advanced Photon Source, Argonne National Laboratory, USA. High-energy

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X-ray scattering measurements were collected at the 11-1D-B beamline (58.6 keV, λ = 0.2114 Å) using a 40

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× 40 cm amorphous Si 2D detector (Perkin-Elmer) at a sample to detector distance of 16 cm. A CeO2 standard

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was used to calibrate the sample-to-detector distance and the tilt angle with respect to the beam path. The

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samples were packed into Kapton tubes and then measured alongside an empty Kapton tube for background

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correction. 2-D data integration and correction to 1D scattering data and further data processing to obtain PDFs

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are detailed in Tobler et al.41 The composition of hematite was assumed to be Fe2O3 • 1H2O based on total Fe

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of acid digested samples. Because the reduced scattering structure function, F(Q), i.e., Q[S(Q) − 1], for some

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samples contained spikes at Q > 22 Å−1, the Fourier transform included data only to Qmax = 21 Å−1. The

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software PDFgui was used to calculate PDFs based on the structure for hematite42 and the structural parameters

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and the size of the coherent scattering domains were fitted to minimize difference between calculated and

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measured patterns. All parameters were fitted in the order: 1) scale, unit cell dimension, 2) correlated atomic

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movement (2), isotropic atomic displacement parameters constrained within the space group of the hematite

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model (R-3c), and 3) spherical size (np) of coherent scattering domains. The first two steps were fitted using

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the PDF at distances (R) from 0 to 20 Å. Thereafter, the calculated parameters were fitted up to 40 Å by fixing

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the parameters from step one and two while fitting np (step 3).

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2.3 Bioreduction experiments with hematite ± alginate co-precipitates

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Bacterial hematite reduction experiments were set up with Shewanella oneidensis MR-1, using L-lactate as an

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electron donor and the synthesized hematite samples as an electron acceptor (in 0.05 M HEPES buffer at pH

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7):

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2Fe2O3 (s) + CH3CHOHCOO- (aq) + 7H+ (aq) → 4Fe2+ (aq) + CH3COO- (aq) + HCO3- (aq) + 4H2O

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For this, S. oneidensis was grown in sterile LB broth (20 g L-1) for 16 h (i.e., towards the end of the exponential

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growth phase), at 30 °C, under shaking. On the day of the experiments, cultures were harvested by

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centrifugation (4500 g, 4 ˚C) for 15 min. A threefold wash cycle was then performed, where the supernatant

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was discarded, the bacterial pellet re-suspended in sterile 0.05 M HEPES buffer, and then centrifuged again.

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After the last cycle the bacterial pellet was re-suspended in sterile HEPES and moved into the anaerobic

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chamber for the reduction experiments.

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Reduction experiments were performed inside a hard-walled anaerobic chamber under pure N2 conditions (O2

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levels < 0.1 ppm). A lactate stock solution (0.4 M) was prepared in 0.05 M HEPES buffer (pH 7) and then

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deoxygenated by bubbling with N2 for 2 h, prior transfer into the anaerobic chamber to equilibrate with the

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N2-atmoshpere overnight. The same procedure was applied to the hematite ± alginate suspensions, while the

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washed and OD600 adjusted bacterial suspension was only allowed to equilibrate for an hour inside the

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chamber. To start the reduction experiment, the hematite suspensions were mixed with the bacterial suspension

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and lactate to yield 1.27 mM hematite, 10 mM lactate and either 2x108 or 4x108 cfu/ml (cell number confirmed

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by plate count). All assays were run in triplicates. Tubes were sealed and incubated in the dark, at 24 ˚C, with

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slow gyratory shaking (25 rpm). At regular time-points over a time period of 192 h, 1 mL samples were taken

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to determine the dissolved Fe(II) ([Fe(II)]aq) and the total produced Fe(II) ([Fe(II)]tot). For [Fe(II)]aq, 0.5 mL

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of this solution sample was filtered (0.22 μm cellulose) and the filtrate then acidified (1.0 M HCl) for later

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analysis. For [Fe(II)]tot, the solution sample was mixed 1:1 with 1 M HCl and left to equilibrate for 24 h, to

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solubilize Fe(II)-organic complexes and Fe(II) sorbed to the hematite and bacteria,43 and then filtered (0.22

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m). [Fe(II)]aq and [Fe(II)]tot were determined using the ferrozine assay.44 To determine the amount of Fe(II)

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adsorbed to cell and hematite surfaces, [Fe(II)]aq was subtracted from [Fe(II)]tot.

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Abiotic controls were run for all tested conditions, and they confirmed that no Fe(II) was produced in the

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absence of S. oneidensis (Fig. S2). Noteworthy that the small amount of dissolved Fe(III) released during acid

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extraction for [Fe(II)]tot (as verified by AAS, section 2.2) interfered with the ferrozine method, similarly to a

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previous study.45 The [Fe(II)]tot data of the bacterial reduction experiments were therefore corrected for this

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interference.

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3. Results and Discussion

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3.1 Influence of alginate on hematite nanoparticle formation

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TEM analyses confirmed the nanoparticulate nature of the synthesized hematite samples and further showed

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that the average particle size decreased with increasing alginate content (Fig. 1): from 8.3 ± 2.2 nm in the

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alginate free system (pure hematite) to 6.5 ± 1.5 nm and 4.1 ± 1.1 nm in the 0.1 gL-1 and 1.0 gL-1 alginate

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system, respectively (Table 1). TEM also showed that the alginate was closely associated with hematite

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particles, covering particles with a thin film (Figure 1D).

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Figure 1: TEM images of synthesized (A) pure hematite and hematite co-precipitated with (B) 0.1 g L-1 alginate

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and (C) 1.0 g L-1 alginate. (D) Aggregate of alginate with hematite particles. Hematite aggregates were freed

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when alginate was burnt away under the exposure to the electron beam. The stated error represents one standard

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deviation.

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Table 1: Properties of pure hematite and alginate-hematite composites. Alginate content in hematite synthesis (gL-1)

TEM estimated particle diameter (nm)1

PDF estimated crystal size (nm)

TGA total weight loss (%)2

Acid digestion of TSS3

EA analyses4 (wt %)

H2O/OH ± alginate (%)

Fe2O3 (%)

C

H

H2O5

0

8.3 ± 2.2

6.9

7

6.8

93.2

0

0.81

7.5

0.1

6.5 ± 1.5

6.2

10

10.4

89.6

0.27

0.94

8.3

1.0

4.1 ± 1.1

5.2

31

24.6

75.4

5.58

1.67

7.6

1Average

value and standard deviation of 100 measured particles. weight loss recorded during heating from 30 to 1000 °C at 10 °C/min in N2 atmosphere 3Calculated from the amount of TSS and [Fe] determined by AAS analyses of acid digested hematite samples. tot 4Elemental (C, H) analyses were performed by combustion of solid hematite samples at 900 °C. 5Calculated under the assumption that all C stems from alginate and the C/H ratio in a pure alginate sample is 6.54. The remaining H was then assigned to water. 2Total

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The presence of alginate in the washed and dried hematite samples was also corroborated by thermogravimetric

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analyses (TGA). During heating of the solids from 30 to 1000 °C, the pure hematite showed a weight loss of

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7%, while the 0.1 and 1.0 g/L alginate hematite sample showed substantially larger weight losses (i.e., 10 and

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31%), particularly at the high alginate concentration (Table 1). Looking in more detail at the TGA profiles

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(Fig. S3), all samples lost around 4-6% of their weight when heated from 30 to ~100 °C. This can be explained

6


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by loss of loosely bound water. At T > 100 °C, the pure hematite lost a further 3% of its weight, which must

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be due to more strongly bound water and/or hydroxyl because no other additives were added to this sample.

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In the case of the alginate amended samples, the weight losses at T > 100 °C were significantly larger and

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proceeded to higher temperatures (>250 °C), similar to the TGA of a pure alginate sample (Fig. S3). This

5

indicated that while more water/hydroxyl may have been associated with the alginate amended samples

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(because alginates have a large capacity to absorb water),46 they likely also exhibited sorbed alginate,

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particularly the 1.0 g/L alginate sample (Fig. S3). The presence of phases other than hematite was also verified

8

by total Fe analyses of the dried solids by AAS analysis of acid digested samples, which matched well with

9

the recorded TGA weight losses (Table 1). Finally, to get some quantitative measure on the relative amounts

10

of alginate in these samples, elemental (C,H) analyses were performed and these suggested that the

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water/hydroxyl contents were fairly similar in all samples, with clear signals of C in the alginate amended

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hematite samples. A rough calculation of the amount of associated alginate was made, by subtracting the

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amount of water found in the pure hematite from the TGA total weight loss measured in alginate containing

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samples (assuming that the water content did not differ much between these samples, i.e., within 5% error of

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measurement). This showed that ~3 and 24 wt. % of alginate were associated with the hematite when

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synthesized in the presence of 0.1 and 1.0 g/L alginate, respectively.

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In terms of the hematite crystal structure, synchrotron-based X-ray scattering and pair distribution function

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(PDF) analyses showed that alginate was not only tightly associated with the hematite but also affected the

19

crystallinity of the formed hematite particles. The I(Q), which gives the total scattering as a function of the

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scattering angle Q, is shown for the three hematite samples in Figure 2A. All three samples exhibited the

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characteristic peaks expected for hematite,42 however, a clear decrease in peak intensity, i.e., peak broadening,

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was observed with increasing alginate content, particularly in the 1.0 g/L alginate hematite sample (Fig. 2A).

23

This is consistent with a decrease in the size of the coherent scattering domains (CSDs) with increasing

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alginate. The PDF can be extracted from I(Q), to give the distribution of characteristic distances, r, between

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atomic pairs (Fig. 2B). Note that the PDFs are scaled to have identical intensities at ~2 Å, which is the

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characteristic distance for first neighbor Fe-O pairs. The distance at 2.95 Å stems from first neighbor edge-

27

sharing Fe-Fe pairs, while 3.4 and 3.7 are from first neighbor corner-sharing Fe-Fe pairs (Fig. 2B). The peaks

28

at higher r have contributions from several atomic pairs. There is a clear trend with increasing alginate content,

29

in that the peak intensities decrease, thereby increasing peak widths and this is observed along the whole

30

spectra. Note that the peak positions are identical for the three samples, thus while the relative abundance of

31

atomic pairs is lower in alginate bearing hematite samples, alginate did not affect the distance between the

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atomic pairs. These features indicate that the hematite CSDs are becoming smaller when synthesized in the

33

presence of alginate. The changes in CSD size were quantified by fitting the measured PDF patterns to a

34

published structure of well crystallized hematite using the software PDFgui (Figure S4). The calculated

35

hematite pattern in all three samples showed similar lattice constants to the reference crystallized hematite

36

(Table S1) and the fitting showed a decrease in CSD from 6.9 nm to 6.2 and 5.2 with increasing alginate from

37

0 to 0.1 and 1.0 gL-1. This matched well with the TEM results (Table 1).

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Noteworthy that the PDF fitting showed a slight mismatch between the hematite samples and the well

2

crystallized hematite reference structure, particularly for the high alginate sample (Rw values in Fig. S4). This

3

is best explained by the nanocrystalline and hydrous nature of the hematite samples produced here. Moreover,

4

the number of crystal defects was likely higher in alginate-hematite composites because of the close association

5

of alginate with Fe(III) ions during synthesis.6 This is also supported by Eusterhues et al.39 who observed a

6

similar increase in iron oxide lattice distortion (i.e., increase in crystal effects) during the co-precipitation of

7

ferrihydrite with soil organic matter. Noteworthy that we checked for the possibility that some ferrihydrite

8

could have formed during hematite synthesis. However, no significant improvements in the PDF fits (i.e., Rw

9

value) were observed with the addition of ferrihydrite. Moreover, we did not spot any additional Fe phases

10

with TEM (Fig. 1) and XRD (Fig. S1) and previous studies that employed the exact same hematite synthesis

11

protocol did not observe additional phases.40,47-48

12 13

Figure 2: A) I(Q) for pure hematite and alginate-hematite co-precipitates. The peaks are annotated with

14

Miller indices of the hematite crystal lattice planes. B) G(r) for pure hematite and alginate-hematite co-

15

precipitates. The PDFs are scaled to have identical intensities at ~2 Å (first neighbour Fe-O pairs). The fit

16

with PDFgui and the residual are shown in Figure S4.

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Overall, the high-resolution characterizations performed on the pure hematite and alginate-hematite

2

composites clearly showed that the presence of alginate during the nucleation and growth of hematite can have

3

manifold effects on the properties of the final hematite crystals. Specifically, it was observed that alginate

4

reduced the size of hematite particle as well as crystal size. At the same time, a substantial amount of alginate

5

became associated with the hematite particles, particularly in the 1.0 gL-1 alginate system. Noteworthy that

6

alginate polymers are too large to become incorporated into the hematite crystal lattice. However, the strong

7

sorption affinity between the alginate’s functional groups and Fe3+ ions likely acted as nucleation seeds.

8

Specifically, it has been proposed that alginate and Fe3+ ions form Fe-OH bonds, which then upon further iron

9

loading (i.e., Fe3+ titration), can lead to a nucleation site for nanoparticulate iron oxide (Figure 6 in Horniblow

10

et al.6). This is supported by our recent findings (unpublished data) that demonstrated that alginate polymers

11

lower the nucleation barrier for ferrihydrite formation. Thus, we argue here that the presence of alginate

12

increases the number of nucleation sites, which in turn promotes smaller crystals. In contrast, if less or no

13

alginate is present, the nucleation barrier is higher, thus fewer nucleation events will occur and crystals will

14

grow larger. The direct nucleation of hematite on alginate likely also enhances the formation of defects during

15

crystallization,39 but overall leads to a close association of alginate molecules and hematite particles as

16

observed in TEM.

17 18

3.2 Influence of tightly associated alginate on hematite bioreduction

19

The effect of different cell densities (2 x 108 and 4 x 108 cfu mL-1) on the reduction of pure hematite (1.27 mM)

20

was examined first to get insights into the role of cell density on reduction kinetics. Doubling the amount of

21

bacteria had a clear impact on the initial reduction rate (Table 2), leading to about twice the amount of hematite

22

reduced after 24-48 h (Fig. 3A). At t ≥ 48 h, the reduction rate in the high cell density experiment quickly

23

approached zero, yielding a total reduction of 24% after 192 h (relative to the available hematite). In the low

24

cell density experiment, the reduction rate also dropped after 48 h, albeit less rapidly, yielding a total reduction

25

of 21%. These drops in reduction rate coincided with approaching the maximum adsorbed Fe(II)

26

concentrations, i.e., ~0.09 mM and ~0.15 mM in the low and high cell density experiment (after 192 h),

27

respectively. These trends indicated that once hematite and/or cell surfaces became saturated with adsorbed

28

Fe(II), the reduction rate drastically decreased. Similar reduction trends have been observed in previous

29

studies31,49-50 and these are often discussed as a two stage process, where the first, initial fast reduction is

30

kinetically controlled, and the second, slower reduction stage is controlled by one or multiple of the following

31

processes: i) mass transfer limitations, ii) loss of thermodynamic driving force due to produced Fe(II), iii)

32

physical blocking of electron transfer between the cell and the hematite by adsorbed Fe(II) and/or surface

33

Fe(II) precipitate (i.e., secondary biominerals), and iii) decrease in cell viability due to adsorbed Fe(II). In the

34

experiments discussed here, the bacterial density was the only parameter that differed, and it clearly controlled

35

the initial kinetic reaction, with a 2-fold increase in initial rate with the addition of twice the amount of bacteria

36

(Table 2). Note that lactate was not a limiting factor in these experiments as shown by identical Fe(II)

37

production rates and yields observed in experiments that only differed in the amount of lactate (10 vs. 20 mM

9



Page 10 of 21

1

lactate, Fig. S5). Also hematite was still highly in excess after 24-48 hours, thus likely did not inhibit this

2

initial stage.

3

In terms of the second, slower reduction stage, it is more difficult to pinpoint the exact process(es) that led to

4

this trend (Fig. 3A). Looking at the final Fe(II) adsorption yields in these experiments, it seems however, that

5

the cells adsorbed a considerable amount of the produced Fe(II), which could have affected the electron

6

transfer chain and potentially also cell viability under the performed non-growth conditions. Specifically, the

7

Fe(II) adsorption yield was almost twice as high in the high cell density experiment compared to the low cell

8

density experiment. Since the same amount of hematite was added to these experiments, this almost 2-fold

9

increase in Fe(II) adsorption is best explained by the doubling in cell density, i.e., bacterial surface site. Thus,

10

the bacterial cells were likely smothered by Fe(II) which physically blocked the reduction reaction. As pointed

11

out by previous studies,21,49-50 other mass transfer limitations and loss of thermodynamic driving force could

12

have also contributed to the observed drop in reduction but the individual contributions cannot be determined

13

from the data at hand.

14

15 16

Figure 3: Total acid extractable Fe(II) and adsorbed Fe(II) in reduction experiments with hematite and S.

17

oneidensis: A) the effect of different cell densities: high (4x108 cells mL-1) vs low (2x108 cells mL-1) and B)

18

the effect of co-precipitated alginate (0, 0.1 and 1.0 g/L alginate; cell density = 4x108 cells mL-1). All

10


Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

experiments contained 1.27 mM hematite (e-acceptor) and 10 mM lactate (e-donor). Each experiment was

2

performed in triplicate; error bars show one standard deviation.

3 4

Table 2: Experimental conditions of hematite reduction experiments by Shewanella oneidensis MR-1 and the

5

total extent of bio-reduced FeIII. Run

Hematite type

Cell density

Initial reduction rate

Total Fe(III)

(1.27 mM)

(cfu mL-1)

(x10-2 mM FeIII h-1)1

reduction yield (%)2

R1

Pure

2 x 108

0.80

21

R2

Pure

4 x 108

1.62

24

R3

0.1 gL-1 Alg

4 x 108

1.18

26

R4

1.0 gL-1 Alg

4 x 108

0.81

26

6 7 8 9

fit (forced through zero) to Fe(II) data (Fig. 3) for initial 24 hours (R2 values >98.5). to the amount of initially added hematite, i.e., 1.27 mM. Statistical tests showed that the variations in observed yields are not significant (p-values > 0.5).

10

The initial reduction rates for alginate-hematite composites were distinct from the rate measured for pure

11

hematite. The initial rate was highest for the pure hematite system and decreased the more alginate was

12

associated with the hematite (Table 2, Fig. 3B). Interestingly, the reduction rate in the alginate-hematite

13

systems did not cease after 48 h as it did for the pure hematite system. Instead reduction progressed steadily

14

for a few days more, particularly in the 1.0 gL-1 alginate system. This eventually led to a slightly higher

15

reduction yield in the alginate bearing experiment (26%) after 192 h compared to the pure hematite (24%).

16

Interestingly, the amount of adsorbed Fe(II) did not differ as much between different alginate treatments: all

17

reaching similar values after 192 h. The main difference was that it took a longer time in the high alginate

18

experiment to reach the same adsorption values as in the other two experiments, mirroring the trends observed

19

for the total produced Fe(II).

20

Because the cell density, the lactate and the hematite concentrations were identical in all experiments, the

21

differences in reduction rate and yield were due to the presence of increasing amounts of alginate associated

22

with the hematite and maybe also due to the hematite particles getting smaller. Seeing however, lower initial

23

reduction rates for alginate-hematite composites, it seems most plausible that alginate hinders direct contact

24

between the bacteria and the hematite. This is also supported by TEM images, that show the close association

25

of alginate with hematite, particularly at the highest alginate concentration (Fig. 1D). The bacteria would have

26

had to rely on alternate mechanisms (e.g. electron shuttles, nanowires) to transfer the electrons to the hematite.

27

Similar observations were made in a recent study where Fe(III) oxides were entrapped within alginate beads

28

to prevent contact between the bacteria and the Fe(III) oxide. No reduction was seen unless AQDS was

29

provided.51

30

Interestingly, while alginate hinders direct contact between the cell and hematite, it did not affect the extent

31

and longevity of the reduction process. This is seen by the slightly higher overall reduction yields and the

1Linear

2Relative

11



Page 12 of 21

1

apparent ongoing reduction reactions in the alginate-hematite samples beyond 8 days (Fig. 3B), particularly in

2

the high alginate sample. In contrast, reduction seemed to have ceased in the pure hematite sample after 4 days.

3

This is best explained by alginate preventing quick passivation of the electron transfer chain by the produced

4

Fe(II) as observed in the pure hematite system. Potentially, this could be due to alginate acting as a a sorption

5

site (i.e., complexing agent) for the produced Fe(II) and/or alginate itself gets released during the reduction

6

process, thus ensuring some cell and hematite surface sites remain free for electron transfer. Another potential

7

controlling factor could be the speed of the initial reduction phase. If a high amount of Fe(II) accumulates

8

within a short time (like for the pure hematite system), conditions for the formation of Fe(II) precipitates

9

become more favorable and thus also the likelihood for hematite surfaces to get passivated increases.52 This in

10

turn will impact the second, slower reduction stage negatively and ultimately also decrease the overall

11

reduction yield. Another factor that may have also influenced the reduction trends is the lower hematite

12

crystallinity (i.e., higher number of crystal defects, smaller crystal sizes) observed with increasing alginate.

13

This is argued because the solubility of nanoparticles increases with decreasing crystal size (i.e., increasing

14

surface area), which in turn may enhance bacterial reduction. However, while size could be a controlling factor,

15

hematite aggregation mechanism can have an even higher impact on reduction.40 This is also indicated by the

16

data presented here, where alginate seems to enhance particle aggregation, lowering hematite surface area, and

17

thereby also initial reduction rate. Finally, it may be possible that cell viability under the tested non-growth

18

conditions is stimulated by the presence of alginate, allowing for a higher portion of the hematite to be reduced.

19

Indeed alginate hydrogels have been shown to create conditions similar to a biofilm,53 and these are known to

20

enhance viability of bacterial cells.54

21

As mentioned in the introduction, a few other studies have evaluated the influence of OM-Fe oxide composites

22

on bacterial DIR rates.23-27 However, they all focused on a different mineral substrate (e.g., mostly ferrihydrite),

23

and they also differed in terms of the used associated organic material (e.g., humic acid, soil extract solution,

24

extracted extracellular polymeric substances) and/or the bacterial strain (e.g., Shewanella putrefaciens and

25

Geobacter strain, soil community), making bacterial DIR rate and yield comparisons between studies difficult.

26

For example, ferrihydrite (FHY) is a poorly ordered Fe(III) oxyhydroxide, with a higher surface area and

27

smaller particles sizes compared to the hematite tested here. As such the relative solubility of FHY is larger,55

28

making it more susceptible towards bacterial DIR.26 Also, some of the previously tested organic substances

29

have functional groups that facilitate electron transfer processes (e.g., humic acid, soil extracts), while the here

30

tested alginate molecule does not. Lastly, no nutrient media was added to the experiments here, which is

31

contrary to the OM-FHY studies discussed above, thus, further complicating comparison between these

32

previous studies and the work presented here. Interestingly however, despite all these experimental differences,

33

there is a clear agreement in that the presence of closely associated organic molecules seems to enhance the

34

overall reduction of nanoparticulate iron oxides by a Shewanella strain, whether it is highly or poorly soluble

35

iron oxide (i.e., FHY vs. hematite). Moreover, while humic substances act as potential electron shuttles,

36

exopolymeric substances such as alginate are less likely to transfer electrons under the here tested conditions.56

37

Instead, they seem to sustain long-term reduction through a combination of processes including Fe(II)

38

complexation and by protecting the bacteria from Fe adsorption, i.e., cell death.

12


Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Conclusions

3

To better constrain carbon and iron biogeochemical processes in soils and sediments, this study aimed first to

4

assess the effect of organic matter on iron oxide formation and second, how the formed OM-iron oxide

5

composite then interacts with the biota. This was done here, with a case study on the effects of alginate on

6

hematite particle formation, followed by stability assessment of these alginate-hematite composites in

7

reduction experiments with S. oneidensis.

8

The presence of increasing amounts of alginate impacted hematite formation in several ways; the particle and

9

crystal sizes decreased and the amount of sorbed alginate increased. Exposure of these alginate-hematite

10

composites to the dissimilatory iron reducing bacteria S. oneidensis showed that while the bacteria may be

11

physically hindered to get direct access to hematite surfaces, due to the tightly associated alginate, reduction

12

readily occurred, and over a far longer time span (≥ 8 days) compared to the system with no alginate present

13

(≤ 2 days). This highlights that a common natural organic molecule such as alginate takes a key role in

14

sustaining reduction over longer time periods. Thus, in the presence of alginate, a higher portion of hematite

15

will likely be reduced. Such considerations are critical when estimating rates and yields of dissimilatory iron

16

reduction in soils and sediments, i.e., their contribution towards Fe and C cycling. Moreover, seeing that overall

17

more Fe(II) is produced in the presence of alginate, this then also suggests that a greater amount of pollutants

18

(e.g., Cr(VI)) could potentially be reduced and immobilized through this process. Such considerations could

19

be useful for biotechnological applications of DIR for contaminant remediation.

20 21

Supporting Information

22

Additional details on methods that includes conventional XRD of hematite samples (Fig. S1), details on

23

ferrozine method and on abiotic control experiments (Fig, S2); additional details on TGA results (Fig. S3),

24

PDF fitting with PDFgui (Fig. S4, Table S1) and reduction experiments with varying lactate concentrations

25

(Fig. S5).

26 27

Acknowledgements

28

The authors thank Olaf Borkiewicz and Kevin A. Beyer for support with X-ray total scattering measurements

29

at APS beamline 11 ID-B, Argonne, USA. We also thank Stanislav Jelavić for advice with the synthesis of

30

nanoscale hematite. The work was supported by the Metal-Aid Innovative Training Network (ITN), supported

31

by a grant from the European Commission (EC) Marie Skɫowdowska Curie Actions (MSCA) program under

32

project number 675219. DJT also acknowledges financial support from the Marie Curie Intra-European

33

Fellowship (IEF) grants MIRO (PIEF-GA-2013-624619). KKS is grateful for funding from the Danish Council

34

for Independent Research on their Sapere Aude Program (0602-02654B), the EC MSCA H2020 research and

35

innovation program (grant agreement No. 663830), and the Welsh Government and Higher Education Funding

36

Council for Wales through the Sêr Cymru National Research Network for Low Carbon, Energy and

13



Page 14 of 21

1

Environment. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office

2

of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Support for travel to

3

the synchrotron facilities came from the Danish Council for Independent Research (via DANSCATT).

4 5

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6 7

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26 27

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28 29

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54 Prakash, B.; Veeregowda, B. M.; Krishnappa, G. Biofilms - A survival strategy of bacteria. Curr. Sci. 2003, 85, 1299–1307.

32 33

55 Kraemer, S. M.; Butler, A.; Borer, P.; Cervini-Silva, J. Siderophores and the dissolution of iron-bearing minerals in marine systems. Rev. Mineral. Geochem. 2005, 59, 53-84.

34 35

56 Hassan (El-Moushy), R. M.; Khairou, K. S.; Awad, A. M. In Polymer gels. Gels horizons: from science to smart materials, V. T. , M. T, Eds.; Springer: Singapore, 2018; pp. 275-35.

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1 2 3 4

For TOC only

5

6 7 8 9

17


A 1 2 3 4 5 6C 7 8 9 10 11 12 13

ACS Earth and Space B ChemistryPage 18 of 21

100 nm

100 nm

D alginate clump ACS Paragon Plus Environment hematite 100 nm

5 um

Page 19 of 21


A

pure hematite

(104)

0.1 g/L alginate hematite

(110)


Relative Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

(116)

(300) (214)

(024) (012)

1

2

(113)

3

4

5

6

7

8

Q(Å-1)

B

pure hematite 0.1 g/L alginate hematite

G(r)


Fe-Fe Fe-O

1

0

10

2

3

4

5

20 ACS Paragon 30 Plus Environment 40 50

r(Å)

6

7

8

60

9

10

70

A

Produced Fe(II) (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

Produced Fe(II) (mM)

B


0,7

Page 20 of 21

0,6 0,5 0,4

high cell density - total Fe(II) high cell density - adsorbed Fe(II)

0,3

low cell density - total Fe(II) low cell density - adsorbed Fe(II)

0,2 0,1 0 0

25

50

75

100

125

150

175

200

Time (h) 0,7 0,6 0,5 pure hematite - total Fe(II) pure hematite - adsorbed Fe(II)

0,4

0.1 g/L Alg hematite - total Fe(II) 0.1 g/L Alg hematite - adsorbed Fe(II)

0,3

1.0 g/L Alg hematite - total Fe(II) 1.0 g/L Alg hematite - adsorbed Fe(II)

0,2 0,1 0 0

25

50

ACS Paragon Environment125 75 Plus 100

Time (h)

150

175

200

Fe(III) reduced by Shewanella oneidensis MR-1 (mM)

Page 21 0,7 of 21


0,6

1 0,5 2 0,4 3 4 0,3 5 6 0,2 7 0,1 8 9 0 10 0 11 100 nm 12

pure hematite 20 nm alginate – hematite 20 nm composite ACS Paragon Plus Environment 25 50 75 100 125 150

Time (hours)

175

200

Hematite crystallization in the presence of organic matter: impact on

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