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Platinum recovery from synthetic extreme environments by halophilic bacteria Synthia Maes, Ruben Props, Jeffrey P. Fitts, Rebecca De Smet, ramiro VilchezVargas, Marius Vital, Dietmar Pieper, Frank Vanhaecke, Nico Boon, and Tom Hennebel Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05355 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Environmental Science & Technology

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Figure 1 Heatmap representing all OTUs present at a relative abundance ≥ 0.1% in at least one of the samples. The relative abundance is indicated by a color scale ranging from 0 to 85%. The clustering on top of the heatmap shows the difference in composition between the investigated halophilic cultures. Halophilic cells were cultivated at increasing salt concentrations (blue symbol: sea salts (S), red symbol: NH4Cl (A); salt concentration is indicated in g L-1) and different pH levels (low □, medium △, neutral ○). The Artemia cysts used as inoculum were included as well. The taxonomy of each species is shown at the phylum level (left column) and at the lowest determined level (right column). 333x254mm (205 x 205 DPI)


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Figure 2 Transmission electron microscopy (TEM) images of thin sections of halophilic cells loaded with platinum particles: precipitation of 100 mg L-1 Pt(II) and Pt(IV) by cultures S105L and S70N, respectively, in the presence of H2. 163x151mm (300 x 300 DPI)



Figure 3 X-ray absorption near edge spectroscopy (XANES) spectra of biomass pellet samples after Pt(II) recovery (100 mg L-1 Pt) by four halophilic mixed cultures: S105L, S105M, S70N and A70N.


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Figure 4 X-ray absorption near edge spectroscopy (XANES) spectra of biomass pellet samples after Pt(IV) recovery (100 mg L-1 Pt) by four halophilic mixed cultures: S105L, S105M, S70N and A70N.



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Platinum recovery from synthetic extreme environments by halophilic

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bacteria

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Synthia Maes1, Ruben Props1, Jeffrey P. Fitts2, Rebecca De Smet3, Ramiro Vilchez-

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Vargas1, Marius Vital4, Dietmar H. Pieper4, Frank Vanhaecke5, Nico Boon1, Tom

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Hennebel1 *

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1

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Links 653, B-9000 Gent, Belgium

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure

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NY 08544, USA

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9000 Gent, Belgium

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Microbiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany

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Gent, Belgium

Department of Civil and Environmental Engineering, Princeton University, Princeton,

Department of Medical and Forensic Pathology, Ghent University, De Pintelaan 185, B-

Microbial Interactions and Processes Research Group, Department of Medical

Department of Analytical Chemistry, Ghent University, Krijgslaan 281 (S12), B-9000

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* Corresponding author: Tom Hennebel, Laboratory of Microbial Ecology and

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Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium. Tel.

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+32(0)9 2645985 – Fax. +32(0)9 2646248. Email address: [email protected]

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For submission in: Environmental Science & Technology

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ABSTRACT

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Metal recycling based on urban mining needs to be established to tackle the increasing

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supply risk of critical metals such as platinum. Presently, efficient strategies are missing

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for the recovery of platinum from diluted industrial process streams, often

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characterized by extremely low pHs and high salt concentrations. In this research,

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halophilic mixed cultures were employed for the biological recovery of platinum (Pt).

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Halophilic bacteria were enriched from Artemia cysts, living in salt lakes, in different salt

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matrices (sea salt mixture and NH4Cl; 20 – 210 g L-1 salts) and at low to neutral pH (pH 3

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– 7). The main taxonomic families present in the halophilic cultures were

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Halomonadaceae, Bacillaceae and Idiomarinaceae. The halophilic cultures were able to

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recover > 98% Pt(II) and > 97% Pt(IV) at pH 2 within 3 – 21 hours (4 – 453 mg Ptrecovered

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h-1 g-1 biomass). X-ray absorption spectroscopy confirmed the reduction to Pt(0) and

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transmission electron microscopy revealed both intra- and extracellular Pt precipitates,

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with median diameters of 9 - 30 nm and 11 – 13 nm, for Pt(II) and Pt(IV), respectively.

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Flow cytometric membrane integrity staining demonstrated the preservation of cell

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viability during platinum recovery. This study demonstrates the Pt recovery potential of

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halophilic mixed cultures in acidic saline conditions.

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Key words: resource recovery, bioreduction, platinum precipitation, bacterial viability,

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XANES

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2



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INTRODUCTION

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Platinum is an increasingly important element in modern day technologies, but its

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critical supply leads to concerns. Although it is a crucial building block of catalysts in

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converters, in chemical industry, cancer therapy, electronics, etc.1, its primary mining is

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environmentally taxing and its deposits are geopolitically highly concentrated, mainly in

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South Africa and Russia2, endangering the supply to other regions3. Hence, platinum is

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defined as a critical raw material by both the European Union and the US Department of

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Energy4, 5. A more efficient use and recycling of platinum (e.g. urban mining2) will be

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essential to reduce primary mining and supply risks2, 6. Platinum’s high intrinsic value

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(av. 46297 $ kg-1 in 20147) stimulates the interest in and development of recycling

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strategies8, as could be seen by an estimated recycling rate of 60 – 70% in 20119.

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Important secondary sources of platinum could be recovery from various liquid process

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and acidic waste streams from mine drainage or from extraction and separation

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processes in platinum group metal (PGM) refineries, waste streams from pharmaceutics,

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fine chemicals and jewelry, hospital wastewater from cancer therapy and sewage

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sludges10, 11.

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Conventional hydro-, pyro- and electrometallurgical processes are applied today

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for recovering metals from the concentrated process streams, but at the moment are

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insufficient or too expensive for the treatment of more dilute streams (< 100 mg Pt L-1)

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which contain lower economic value10. Biometallurgy, i.e. biotechnological processes

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based on microbe-metal interactions, could complement the conventional metallurgy

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and could be included as a sustainable and environmentally friendly process in a metal

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recovery technology flow sheet10. During the last decade, the bioreductive removal of

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platinum has been investigated both by using whole cells, e.g. Shewanella algae12, 3


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Acinetobacter sp.13 etc., as well as by using microbial products such as hydrogenases14.

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So far, these studies have focused on the removal of Pt under neutral pH and in a clean

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matrix13, 14, while in practice relevant industrial process streams offer more challenging

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conditions15, 16: high salt concentrations (40 – 300 g L-1), low pH (pH < 3) and elevated

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ammonium levels up to 50 g L-1 NH4+. Ammonium-based chemicals such as secondary or

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tertiary amines and ammonium chloride are used in PGM metal refinery processes17.

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The metal removal potential of selected bacteria from saline environments (e.g.

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Halomonas sp., Thalassospira sp. and Shewanella algae) was already demonstrated for

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Pd(II) and Pt(IV), but at circumneutral pH conditions12,

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characteristics such as solution pH and matrix solutes have proven to significantly

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influence the reduction potential and recovery efficiency of e.g. Pd species10,

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Nevertheless, Gauthier et al.15 as well demonstrated the removal of Pd and Pt by

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Cupriavidus sp. from a strongly acidic and saline stream (pH 1.4; 200 g L-1 salts).

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However, specific matrix

19, 20.

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For now, researchers mainly relied on well-known freshwater bacteria and

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adapted the harsh waste stream conditions to a point suitable for these species.

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Alternatively, specialized extremophilic bacteria such as halophiles originating from

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hypersaline environments could possibly cope with these conditions21, 22. Their unique

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environments (high salinity, often extreme pH) select for competitive stress tolerant

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organisms and result in high microbial diversity22.

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The aim of this research was to investigate the platinum recovery potential of

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halophilic mixed cultures under industrially relevant conditions (i.e. presence of low pH

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and salts). Halophilic cultures were enriched from Artemia cysts, originating from

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hypersaline environments. These were cultivated at increasing salt concentrations and

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from low to neutral pH and subsequently characterized by high throughput sequencing 4



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analysis. The bioreduction of platinum(II) and platinum(IV) was evaluated for four

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selected halophilic mixed cultures, with pure H2-gas as electron donor. The morphology,

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size distributions and metal speciation of the Pt particles were determined, as well as

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the viability of the halophilic cells during platinum recovery. This study aims at the

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recovery of platinum from a liquid stream and the simultaneous production of Pt loaded

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biomass, which can be further processed as one of the feeds of a precious metals

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refinery.

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MATERIALS AND METHODS

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Microbial cultures and growth conditions.

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Artemia cysts, obtained from a salt lake located in Vinh Chau, Vietnam, were used in this

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study as a source for the enrichment of halophilic bacteria. These cysts were obtained

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from the Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent

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University, Belgium. To isolate and actively grow the associated bacteria, the cysts were

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initially incubated aerobically in Lysogeny broth (LB-Lennox; 10 g L-1 tryptone, 5 g L-1

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yeast extract and 5 g L-1 NaCl) medium with 35 – 105 g L-1 additional sea salts (Instant

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Ocean®, a synthetic seawater mixture, referred to as “sea salts”) or 5 - 20 g L-1 NH4Cl at

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28°C and neutral pH. Instant Ocean® was chosen as salt mixture in order to represent as

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closely as possible the natural environment and consists of Na+ (462 mmol kg-1), K+ (9.4

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mmol kg-1), Mg2+ (52 mmol kg-1) and Ca2+ (9.4 mmol kg-1) as major cations and Cl- (521

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mmol kg-1) and SO42- (23 mmol kg-1) as major anions23. During a second and third

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enrichment, the resulting halophilic mixed cultures were transferred and grown in LB

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broth containing 35 - 210 g L-1 sea salts and 20 - 70 g L-1 NH4Cl, all at neutral pH. The

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halophilic mixed culture obtained from the 105 g L-1 growth condition was further

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enriched in LB broth containing 105 g L-1 sea salts at low (pH 3) and medium pH (pH 5),

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as well.

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Platinum precipitation by halophilic cultures.

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The halophilic mixed cultures were harvested by centrifugation at 8200 g for 7 min, and

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were washed four times with phosphate buffered saline (PBS, 24.1 g L-1 total salts; 6.8 g

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L-1 KH2PO4, 8.8 g L-1 K2HPO4 and 8.5 g L-1 NaCl). The cells were suspended in PBS to a

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final optical density of 1.0 (at 610 nm) and added to 120 mL glass serum flasks (50 mL 6



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cell suspensions). After 20 repeated cycles of N2 overpressure and vacuum

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underpressure to remove all traces of oxygen, the headspace was replaced with 100%

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H2-gas, used as electron donor. Subsequently, K2Pt(II)Cl4 or K2Pt(IV)Cl6 was spiked to a

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final concentration of 100 mg L-1 Pt. During the entire experiment, the serum flasks were

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incubated, and continuously mixed at 100 rpm and 28°C. The pH was always set at 2.0.

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The pH was measured at the beginning and at the end of the experiments and remained

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within the same range (± 0.1 pH unit).

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Inductively coupled plasma optical emission spectrometry (ICP-OES).

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Platinum concentrations of aqueous samples were determined using ICP-OES.

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Therefore, the Pt containing biomass was separated from the liquid medium by

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centrifugation at 9000 g for 7 minutes. The supernatant samples were diluted 10 times

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with a 1% HNO3/1% HCl solution, and spiked with yttrium as internal standard (1 mg L-

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1).

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OES (Spectro Analytical Instruments GmbH, Kleve, Germany). Prior to analysis, the ICP-

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OES was optimized with a solution containing 2 mg L-1 Pb, As and Mn (in 2% HNO3). The

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ICP-OES parameters were: a plasma power of 1375 W (1100 – 1450 W), a pump flow

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rate of 30 rpm, a coolant flow of 12 L min-1, an auxiliary flow of 0.9 L min-1, and a

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nebulizer flow of 1.2 L min-1. Platinum peaks were measured at 177.708, 191.170,

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203.711 and 214.423 nm and yttrium at 371.030 and 377.433 nm. Argon peaks

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(404.442 and 430.010 nm) were used to indicate the potential influence of organic

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matrices. The detection limit was 0.5 mg L-1 Pt.

The resulting platinum concentrations were determined with a Spectro Arcos ICP-

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Illumina sequencing and data processing 7


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Samples (1 mL stationary culture) were collected of the different halophilic mixed

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cultures and total DNA was extracted as previously described24. Prokaryotic diversity

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was analysed using pair-end high throughput sequencing (MiSeq Illumina platform).

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Regions V5 – V6 of the 16S rRNA gene were amplified and targeted with suitable

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adapters and barcodes25, 26. A total of 764042 reads of 280 nucleotides length (forward

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and reverse reads) were obtained. After a quality filter25 and manual assembling,

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allowing zero mismatch, of the forward and reverse strands a total of 625853 reads

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were obtained and clustered into 291 unique phylotypes. These unique phylotypes were

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annotated using the ribosomal database with a 80% confidence threshold27. For further

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analysis sequencing depth was normalized to the minimum sequencing depth of 22274

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reads per sample. The vegan package of R (version 3.0.2) was used for principal

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component analysis, clustering the samples (using Bray Curtis dissimilarity), and

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building the rarefaction curves. The phyloseq package of R was used for normalizing to

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the minimum sequencing depth. The most relevant phylotypes were blasted in the NCBI

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database and the closest relative sequences were retrieved. Alignment of the V5 - V6

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region of the 16S rRNA gene was performed with muscle28 and the tree was built with

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MEGA5, using the Neighbor-joining method, Jukes-Cantor model and pairwise deletion

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with 1000 bootstrap replications.

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RESULTS AND DISCUSSION

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Cultivation and identification of halophilic bacteria from brine shrimp cysts.

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To tackle the challenging conditions of industrial process streams, specialized microbial

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cultures were enriched from hypersaline environments. Cysts of the brine shrimp

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Artemia, known to harbour a diverse community of halophilic microbial species22, were

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chosen in this study as the source of salt tolerant microbial communities. A salt lake

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located in Vinh Chau, Vietnam, characterized by salt concentrations of 80 – 90 g L-1 (pH

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7.5), was sampled for dormant encysted Artemia embryos. High throughput sequencing

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was utilized to reveal the microbial composition associated with the cyst inoculum

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sample and its subsequent enrichments (Figure 1). The bacteria most commonly

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identified in the cyst inoculum belonged to the genus Salinibacter and the order

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Oceanospirillales. The cysts contained archaeal species as well, belonging to the family

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Halobacteriaceae. Different species from the phyla Bacteroidetes, Firmicutes and

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Proteobacteria were present at a lower relative abundance.

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The sampled Artemia cysts were initially incubated in salt containing LB-broth

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medium, thus selecting for fast growing bacteria. Subsequently, the microorganisms

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associated with the Artemia cysts were cultivated at increasing salt concentrations and

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different pH levels by means of sequential enrichments. The process streams of interest

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are always characterized by high salt levels but can vary in pH (pH 2 - 7). Therefore, we

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opted to select for salt tolerant cultures, which were further enriched at different pH

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levels. To mimic the saline and acidic conditions of PGM refinery streams, cultures were

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grown in the presence of 35 – 210 g L-1 sea salts (S), or 20 – 70 g L-1 NH4Cl (A) and at low

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(pH 3, L), medium (pH 5, M) or neutral pH (pH 7, N). Cultures in this study are presented

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by a code indicating the salt matrix (S or A), the salt concentration in g L-1 (20 – 210) and

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the pH (L, M or N).

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The microbial composition present in the samples, grown under 13 different

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combinations of previously mentioned conditions, was determined as well (Figure 1). In

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contrast to the initial Artemia sample, members of the genus Halomonas were dominant

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in most of these cultures, except under the conditions of cultures A70M, A70L and

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S105L. The importance of the genus Halomonas in Artemia associated microbiomes was

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demonstrated by several studies29, 30. The influence of the salt concentration, NH4Cl and

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pH on the microbial composition of the cultures was examined by principal component

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analysis (PCA, Figure SI1). Increased salt levels mainly induced the presence of genera

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Halomonas and Idiomarina, while the genera Bacillus and Marinobacter were present as

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well, but to a lower degree. Acidic environments favored the presence of Idiomarinaceae

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species, demonstrated by the increased relative abundance in samples S105L (67%) and

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A70L (27%). Additionally, an acidic pH induced the presence of species from the families

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Flavobacteriaceae and Sphingobacteriaceae, as well as from the genus Bacillus. Strongly

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acidic conditions (pH 3) decreased the total relative abundance of identified Halomonas

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species. Finally, ammonium mainly induced the presence of the genus Psychrobacter

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which preferred the lower NH4Cl concentration range (20 – 35 g L-1, samples A20N and

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A35N). Overall, the salt matrix and low pH were the primary drivers determining the

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microbial community composition (Figure 1 and Figure SI1). The Bray Curtis

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dissimilarity among the different cultures was quantified based on the relative

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abundances between samples, represented by the clustering in Figure 1. The strongly

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acidic pH (pH 3) differentiated the microbial composition the most, as cultures S105L

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and A70L cluster together, regardless of the salt matrix. The salt matrix was 10



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determinative at medium to neutral pH since all NH4Cl grown cultures clustered

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separately from the sea salt samples. The clustering also clearly demonstrates the

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microbial diversity between the Artemia inoculum sample and the enriched cultures.

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After the broad microbial screening, a small selection of diverse and industrially

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relevant cultures was subjected to platinum recovery experiments. Cultures were

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chosen so that an experimental design consisting of both salt matrices and three pH

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levels (pH 3, 5 and 7) across a range of moderately high salt levels (70 – 105 g L-1) was

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possible. Based on the microbial diversity and the differentiated growth conditions,

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cultures S105L, S105M, S70N and A70N were selected as models for halophilic

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enrichment cultures. The heatmap and cluster analysis (Figure 1) highlight the

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differences in microbial diversity between these four cultures. The differences among

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the community assemblies were driven by the abiotic growth conditions, as shown in

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the PCA plot (Figure SI1).

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To ensure sustained treatment of process streams, growth parameters are vital

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criteria for selecting the right microbial biomass. The bacterial surface area and cell

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density will impact the metal recovery capacity of any biological technology.

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Furthermore, platinum recovery reactors will have to be fed by new active biomass on a

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regular basis. Therefore, fast growing bacteria would offer a big advantage for a

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biologically driven reactor technology. The morphology and growth kinetics of the

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selected cultures differed considerably (Figure SI4); mean cell sizes ranged from 0.79

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µm (S105M) up to 2.57 µm (S105L). Culture S105L, cultivated at a strongly acidic pH,

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was characterized by a very broad cell size distribution, covering a 1.15 - 6.13 µm range

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(5% and 95% percentile). Culture S105M was characterized by the smallest cells on

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average and the highest specific growth rate by far (0.47 d-1). In comparison, the 11


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halophilic cells adapted to NH4Cl (A70N) showed the lowest growth rate, about one

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order of magnitude lower (0.04 d-1) than culture S105M, and a lower cell density;

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4.3*108 cells mL-1 compared to about 1.1*109 cells mL-1 for cultures S105M and S70N

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(OD610 nm = 1). Their possible advantage of being adapted for application in ammonium-

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loaded streams diminishes based on the growth kinetics. Overall, the low pH strongly

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affected the composition and morphology of the microbial community, and lowered the

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growth rate (0.06 d-1) and cell density (7.2*107 cells mL-1) of the adapted culture S105L

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considerably. Additionally, the added stress of ammonium salts induced an even more

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specialized, narrow sized culture, consisting of very slow growing cells.

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Platinum precipitation by halophilic mixed cultures.

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The four selected halophilic cultures were subsequently used to recover platinum from

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the aqueous phase and concentrate it on the bacterial biomass. The platinum recovery

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potential was investigated for both aqueous platinum(II) and platinum(IV) species at pH

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2. All tested halophilic communities were able to recover Pt(II) from the aqueous phase

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with > 98% efficiency in the presence of hydrogen gas as electron donor (Figure SI5).

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The sorption control (absence of electron donor) and heat-killed control (with electron

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donor) removed only 5.0 and 7.9% Pt(II), respectively. Abiotic reduction by H2-gas was

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negligible for both Pt(II) and Pt(IV) (1.3%).

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The recovery rate of Pt was very different across the studied cultures; whereas

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the precipitation of platinum(II) was induced within the first hour using the S105L

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culture (452.5 mg Ptrecovered h-1 g-1 biomass), other halophilic mixtures required 3 to 6

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hours (49.6 and 13.0 mg Ptrecovered h-1 g-1 biomass for cultures A70N and S105M resp.),

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and even longer for culture S70N (7 to 24 h; 4.1 mg Ptrecovered h-1 g-1 biomass). The 12



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biological recovery of platinum(IV) appeared to be slower (8.5 – 21 hours), but was as

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effective as Pt(II) recovery (97.1 – 99.5%; 4.1 – 10.7 mg Ptrecovered h-1 g-1 biomass, 64.4

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mg Ptrecovered h-1 g-1 biomass for S105L) (Figure SI5).

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Previous studies using enzymatically active microorganisms have only reported

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Pt removal efficiencies under neutral to alkalic pH14,

31.

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marine bacterium Shewanella algae reduced 90% Pt(IV) within the first hour, using

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lactate as electron donor (185.3 mg Ptrecovered h-1 g-1 biomass, pH 7)12. Sulphate-reducing

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bacteria were reported to remove 44% Pt(II) (0.6 mg Ptrecovered h-1 g-1 biomass, pH 7.6)

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and 95% Pt(IV) (1.2 mg Ptrecovered h-1 g-1 biomass, pH 7.6), within 8 hours and in the

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presence of H2-gas31. However, most freshwater and known metal reducing microbes

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would not cope easily with strongly acidic and saline conditions, making them

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inapplicable for metal refinery process streams. To our knowledge, no halophilic mixed

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microbial cultures have been developed for the biological reduction of PGMs. Previous

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researchers already applied single strain halophilic isolates, e.g. Halomonas sp., Bacillus

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sp. and Thalassospira sp. for a 100% removal of Pd(II) within 24 h. However, they aimed

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at producing living palladized cells for the dehalogenation of polluted marine sediments

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and did not investigate Pd reduction in acidic or saline conditions. Few studies have

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investigated biological PGM recovery from real waste streams. Mabbett et al.16 reported

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the recovery of palladium, platinum and rhodium by Desulfovibrio desulfuricans and

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Escherichia coli species from acidic leachates, derived from PGM metal scrap. However,

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palladized cells, i.e. cells containing Pd precipitates produced by the use of a commercial

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Pd(II) solution and H2, were required because the harsh conditions of the PGM metal

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stream (acidic Cu-containing aqueous wastes) inhibited metal recovery by non-

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palladized cells. Additionally, the acidic leachates had to be diluted and the pH adjusted

The metal-ion reducing and

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prior to treatment. In contrast, Cupriavidus necator and Cupriavidus metallidurans

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species were able to recover Pd with high efficiency from a mixed metal acidic leachate,

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without any pre-dilution or pH adjustment (pH 1.4)15. Moreover, unpalladized cells

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achieved a similar Pd recovery efficiency as palladized cells; 96 – 100%. The complete

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Pd removal in the abiotic control revealed the possibility of an abiotic, non-enzymatic

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process. Nevertheless, a simple desalting protocol was still applied, based on freeze-

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thaw cycles, to lower the salt level (decrease from 300 to 200 g L-1). Platinum and

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rhodium exhibited recoveries of 74 and 57%, respectively. The removal of other

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elements present in the leachate (Ag, Au, Ir, Ru, Pb and Cu) was not reported. In

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summary, previous studies were required to reduce process stream toxicity by means of

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dilution, pH adjustment, desalination or the application of palladized biomass using a

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commercial metal source.

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The pH and salt level strongly determine bacteria-metal interactions. Increased

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ionic strength and proton concentrations affect the membrane charge of a bacterial

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surface, and the protonation and activity of its functional groups, affecting metal

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sorption20, 32. Furthermore, metal cation speciation, activity and affinity for binding sites

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at the cell surface change as a function of pH and ionic strength19, 20. For example, at

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increased chloride levels and acidic pH, Pt(II) will mainly be present as negatively

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charged platinum-chloride complexes33. The optimal pH-range for Pt(II) and Pt(IV)

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sorption was observed to be 2.0 – 3.034. However, the effect of the different governing

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parameters (i.e. pH and ionic strength) on the metal recovery will ultimately depend on

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the metal and microbial consortium of interest10. For example, the adsorption of metals

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Cd, Pb and Sr onto G- bacteria decreased as a function of increased ionic strength (0.01 –

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0.5 M), mainly due to changes in the activity of the aqueous metal cations20. Although the 14



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acidic conditions (pH 2) in the current study promoted sorption, the high saline

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conditions resulting in an ionic strength of 0.52 M likely decreased platinum sorption on

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the G- Halomonas sp. (as an important constituent of the used cell culture). Since near

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complete recoveries were obtained and sorption could not explain for the entire Pt

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recovery percentages, the removal mechanism was studied with transmission electron

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microscopy (TEM).

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TEM provides insight into the presence, spatial distribution, and morphology of

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platinum precipitates recovered by the biomass. The TEM micrographs of halophilic

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cells exposed to either 100 mg L-1 Pt(II) or Pt(IV) in the presence of H2 are shown in

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Figure 2 (cultures S105L and S70N) and Figure SI6 (cultures S105M and A70N). The

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location, size and morphology of the platinum particles were observed to be very

324

diverse. All samples from these four cultures showed Pt particles mainly ranging from 2

325

to 224 nm, but the size distribution and number of particles per cell differed. Particle

326

size distributions for all conditions are reported in Figure SI7, Figure SI8 and Figure

327

SI9.

328

TEM micrographs show that after culture S105L was exposed to Pt(II), platinum

329

precipitated exclusively on the cell wall and formed bigger clusters. The Pt suspension

330

was characterized by an exceptionally broad particle size distribution, covering a 2.1 –

331

224.2 nm range (5% and 95% percentile), with a median diameter of 27.7 nm. In

332

contrast, TEM micrographs of cultures S105M and A70N incubated with Pt(II) revealed

333

much smaller nanoparticles, ranging mainly from 2 to 30 nm, and characterized by a

334

median diameter of 10.6 and 11.5 nm, respectively. Most of the particles were

335

precipitated on the cell wall and in the periplasmic space. While the S70N culture

15


Page 20 of 32

Page 21 of 32


336

accumulated platinum mainly intracellularly, forming larger Pt clusters inside the

337

halophilic cells (median diameter of 9.4 nm).

338

After exposure to Pt(IV), all cultures showed both intra- and extracellular

339

precipitation. Particles were more randomly spread over the cells, and characterized by

340

a median size of 10.6 – 13.4 nm. In general, similar (S105M and A70N) or greater

341

numbers (S105L) of smaller particles were observed after platinum(IV) recovery

342

compared to Pt(II) recovery. From the TEM pictures, it is clear that only subpopulations

343

within the enrichments interact with platinum. For recovery applications, the use of

344

balanced mixed cultures is still preferred because of the ease of use and cultivation and

345

their flexibility towards varying applications.

346

The main genera detected in the halophilic mixed cultures of this study, i.e.

347

Halomonas and Bacillus, were investigated before by Hosseinkhani et al. for the

348

bioreduction of palladium18. Halomonas sp. (G- species) formed very small Pd

349

nanoparticles (< 10 nm) both intra– and extracellularly, while Bacillus sp. (G+ species)

350

deposited particles up to 50 nm, mainly extracellularly on the cell wall18. Hosseinkhani

351

et al.18 indicated the importance of a thinner peptidoglycan layer of G- species, possibly

352

promoting metal transport to the cytoplasm and thus enhancing intracellular

353

precipitation. Differences in Pt immobilization in the studied mixed cultures might be

354

due to the involvement of several species, possibly characterized by a different cell

355

envelope structure (e.g. involvement of G+ Bacillus sp. in culture S105L).

356 357

Platinum speciation analysis

358

X-ray absorption spectroscopy was used to determine the predominant oxidation state

359

of the Pt recovered by the halophilic biomass and the nature of Pt bonding within the 16



360

nanoparticles observed in the TEM images. The X-ray Absorption Near Edge Structure

361

(XANES) region of the spectra shown in Figure 3 compare water saturated halophilic

362

biomass samples produced by Pt recovery with electron donor (H2) from aqueous

363

K2Pt(II)Cl4 solutions (0.1 g L-1 Pt, pH 2, 24.1 g L-1 PBS salts). The white line position,

364

which is defined as peak intensity, of all Pt(II)-biomass spectra (11,565 – 11,566 eV) are

365

shifted relative to peak intensity of the Pt(II)-aquo cation in solution (11,568 eV). These

366

whiteline shifts are all consistent with reduction to zerovalent platinum. The zoomed

367

inset in Figure 3 shows variable shifts to lower energy of all Pt(II)-biomass samples

368

relative to Pt(0) reference that could indicate incomplete reduction and/or disordered

369

bonding with the Pt(0) nanoparticles35. In contrast, XANES spectra of biomass samples

370

collected from Pt(IV) reduction experiments shown in Figure 4 are more closely aligned

371

with Pt(0) reference spectrum. However, the shape of the XANES spectra and the

372

periodicities of the oscillations within the Extended X-ray Absorption Fine Structure

373

(EXAFS) region of the spectra of biomass samples following either Pt(II) or Pt(IV)

374

recovery are dominated by Pt-Pt bonds (2.74 - 2.76; see supporting information S4 and

375

Figure SI10 and Figure SI11), which is consistent with previously reported bonds

376

lengths in Pt-Pt metal catalysts36. Furthermore, the EXAFS spectrum of the Pt(II)

377

recovered by A70N was the only spectrum to show evidence of Pt-Cl or Pt-O bonding37.

378

While the XANES did not produce any truly novel information, we are able to provide

379

more confidence to the particle size distributions derived from the TEM images given

380

that the XANES spectra average over millions of Pt particles within each sample.

381 382

Membrane integrity of halophilic cultures during platinum recovery

17


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Page 23 of 32


383

Halophilic mixed cultures were cultivated at high salt concentrations and acidic pH to

384

mimic and adapt them to the challenging conditions of the process streams. However,

385

the exposure to platinum has the potential to induce an additional physiological stress

386

during metal recovery applications. As a consequence, the biologically driven recovery

387

processes and efficiency could be affected. It is thus important that the viability of the

388

microbial community is optimally preserved. Viable biomass also offers the potential

389

benefits of in situ growth and biomass renewal under challenging conditions. To

390

evaluate the viability of the halophilic cultures during platinum recovery we monitored

391

the bioreductive process through flow cytometry based membrane integrity staining,

392

selected as indicator for structural cell integrity38. This staining made it possible to

393

quantify the amount of cells with an intact cellular membrane during platinum recovery

394

experiments. Cells that maintained their membrane integrity will be further referred to

395

as intact cells. It needs to be noted that mixed cultures are inherently variable

396

populations, so a relative interpretation of the results is preferred. Initiation of the

397

recovery experiment by dosing of the platinum solution and lowering the pH to 2

398

induced a near instantaneous 1.6 and 1.9 log10 decrease in intact cells mL-1 in cultures

399

S70N and S105M respectively (Figure SI12). Culture A70N showed a higher survival

400

(0.6 log10 decrease in intact cells mL-1). The amount of intact cells further decreased

401

during platinum recovery, resulting in approximately 106 intact cells mL-1 for cultures

402

S70N and S105M after 6 h. The viability of culture A70N was only slightly affected

403

throughout the experiment, suggesting its higher resistance towards platinum and

404

acidic pH. Culture S105L, grown at pH 3, could withstand the acidic metal stream the

405

most; a small decrease of 0.5 log10 (intact cells mL-1) was measured during reduction.

406

Even though the cultures were clearly affected, these halophilic microbial communities 18



Page 24 of 32

407

could cope with the challenging conditions by maintaining a viable population (7.9*105 –

408

1.8*107 intact cells mL-1). The viability assessments directly correlate with the platinum

409

recovery performance (Figure SI5); culture S105L was the most resistant and fastest

410

platinum reducing culture, indicating the importance of intact cells for efficient

411

biologically mediated platinum reduction. The more severe a culture was affected by the

412

experimental conditions, the lower the Pt recovery rate. Still, we assume that both intact

413

and non-intact cells participate in the precipitation of platinum. Furthermore, a non-

414

quantified part of the remaining intact cells do not interact with platinum and are thus

415

not interesting for Pt recovery. This might be due to the differences in cell envelope

416

structure and intrinsic metal reduction capacities of the different species. However,

417

mixed cultures are preferred over specific pure cultures since they include generally

418

more balanced populations and are economically more feasible and practical to

419

cultivate.

420

The intracellular precipitation of platinum nanoparticles has been previously

421

shown to affect cell viability of Acinetobacter calcoaceticus, in which the cultivable

422

fraction decreased by approximately 0.4 log10 colony forming units (cfu) mL-1

423

Rashamuse et al.39 observed an increased permeability of the cell membrane of

424

sulphate-reducing bacteria, due to exposure to Pt(IV) ions. The acidic pH (pH 2) will also

425

affect the viability of the cultures. Control samples (e.g. halophilic cultures at pH 2) were

426

characterized by a similar decrease in intact cells as the Pt recovery samples; a 0.3 log10

427

up to 2.8 log10 decrease in intact cells mL-1 was observed for cultures S105L and S70N,

428

respectively. The strongly acidic pH had the greatest effect on the viability of halophilic

429

cultures in this study. Yet, halophilic species belonging to the genera Bacillus and

430

Halomonas have shown to be highly resistant to heavy metals, e.g. As5+, Pb2+ or Cu2+ 40, 41, 19


13.

Page 25 of 32


431

and are thus suitable for application under metal and salt stressed conditions, as long as

432

pH effects remain limited.

433 434

Implications for biomass recovery of critical metals

435

This study represents an important step towards the use of mixed halophilic cultures for

436

the microbial recovery of platinum from process and waste streams. The cell viability,

437

platinum reduction kinetics and growth rate will determine the overall performance and

438

capability of a culture for platinum recovery applications. High growth rates and cell

439

densities (of cells reacting with platinum) will increase the metal recovery capacity in a

440

biologically driven reactor technology. In this study, culture S105M proved to be the

441

most promising culture based on the growth and platinum recovery kinetics. Cultures

442

can be further adapted to specific process and waste streams characterized by different

443

conditions to obtain an even more effective platinum recovery. In order to obtain similar

444

recovery rates as current alternative technologies such as Smopex®

445

recovery within 0.5 – 1 h) a biomass concentration of 7.7 g L-1 of culture S105M would

446

be required to treat a 0.1 g L-1 Pt solution in batch. These batches are supplied with a

447

100% hydrogen gas headspace, as is common practice in industrial reduction reactions

448

(to stay above the upper explosion limit), but at lower pressures typically encountered

449

in industry.

450

The current study focuses on the recovery of Pt from liquid streams and the

451

simultaneous precipitation on halophilic cells and does not aim at developing any

452

postprocessing technologies (e.g. Pt extraction from the biomass). The Pt loaded

453

biomass product is considered as our final product which can be separated from the

454

streams with typical biomass separation technologies such as settling, centrifugation or 20


42

(complete


455

filtration. This product can be further processed as one of the feeds of a precious metals

456

refinery, and needs to cope with certain requirements of which the Pt content will be the

457

most important one. Based on preliminary results (data not shown), the Pt loaded

458

biomass can be reused to treat multiple batches of a Pt stream and reach Pt levels that

459

are interesting for refineries to take in. However, the reuse of the Pt loaded biomass and

460

the maximal metal loading of the studied biomass should be further explored with more

461

detail.

462 463

Supporting information

464

The SI provides additional information on: high throughput sequencing (S1), cell size

465

distributions and growth kinetics (S2), transmission electron microscopy (S3), X-ray

466

absorption spectroscopy (µXAS, S4) and flow cytometry (S5). This material is available

467

free of charge via the Internet at http://pubs.acs.org.

468 469

21


Page 26 of 32

Page 27 of 32


470

ACKNOWLEDGEMENTS

471

Synthia Maes is supported by the Agency for Innovation by Science and Technology

472

(IWT Flanders). Tom Hennebel was supported by a postdoctoral fellowship from the

473

Research Foundation Flanders (FWO Flanders). Ramiro Vilchez-Vargas was supported

474

as a postdoctoral fellow by the Belgian Science Policy Office (BELSPO). BNL and

475

the NSLS operate under US DOE Contract No. DE-AC02-98CH10886. Additional

476

support for Beamline X27A comes from the US DOE under DE-FG02-92ER14244 to the

477

University of Chicago – CARS. The authors thank Asha Debrabandere for the ICP

478

measurements, Gilbert Van Stappen and the Laboratory of Aquaculture & Artemia

479

Reference Center, Ghent University, for providing Artemia cyst samples and Silke Kahl

480

and Iris Plumeier for their technical assistance (high throughput sequencing). The

481

authors also thank Sam Van Nevel, Jo De Vrieze and James Barazesh for critically reading

482

the manuscript and Tim Lacoere for his contribution to the graphical abstract.

483

22



484

FIGURE CAPTIONS

485

Figure 1: Heatmap representing all OTUs present at a relative abundance ≥ 0.1% in at

486

least one of the samples. The relative abundance is indicated by a color scale ranging

487

from 0 to 85%. The clustering on top of the heatmap shows the difference in

488

composition between the investigated halophilic cultures. Halophilic cells were

489

cultivated at increasing salt concentrations (blue symbol: sea salts (S), red symbol:

490

NH4Cl (A); salt concentration is indicated in g L-1) and different pH levels (low ,

491

medium , neutral ). The Artemia cysts used as inoculum were included as well. The

492

taxonomy of each species is shown at the phylum level (left column) and at the lowest

493

determined level (right column).

494 495

Figure 2: Transmission electron microscopy (TEM) images of thin sections of halophilic

496

cells loaded with platinum particles: precipitation of 100 mg L-1 Pt(II) and Pt(IV) by

497

cultures S105L and S70N, respectively, in the presence of H2.

498 499

Figure 3: X-ray absorption near edge spectroscopy (XANES) spectra of biomass pellet

500

samples after Pt(II) recovery (100 mg L-1 Pt) by four halophilic mixed cultures: S105L,

501

S105M, S70N and A70N.

502 503

Figure 4: X-ray absorption near edge spectroscopy (XANES) spectra of biomass pellet

504

samples after Pt(IV) recovery (100 mg L-1 Pt) by four halophilic mixed cultures: S105L,

505

S105M, S70N and A70N.

506

23


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Page 29 of 32

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552


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