Shewanella oneidens microcosms laboratory data 
Creators: name: Leiser, Rico
Publisher: name: Helmholtz-Zentrum für Umweltforschung
Year: 2022
Subjects: Laboratory Data

description: Iron reduction by Shewanella oneidensis does not release microplastics from organo-metallic aggregates

Abstract
The presented study is a laboratory study elucidating the potential of the freshwater bacteria Shewanella oneidensis to reduce microplastic containing iron-organo aggegrates, and to release microplastics from the flocs through this reduction. The data were obtained by incubating 5 individual iron-organo flocs inside of microcosm (20 individual) with added S. oneidensis for 120 days and samplings conducted after 0, 20, 40, 60 and 120 days. 

General study design
Laboratory study with controlled conditions (constant 28° C, dark, no agitation) in microcosms (small serum bottles, Volume: 25 ml filled with 20 ml of media) 
Method description
1.	Iron-organo floc formation (see manuscript)
2.	Cultivation of Shewanella oneidensis (after BCCM, Xiao et al., 2018 doi.org:10.1021/acs.est.7b05098
3.	Microcosm incubation and sampling (see manuscript and below for detailed instrumentation and steps)
4.	Confocal laser scanning microscopy (Imaging was conducted with a TCS SP5X upright microscope equipped with white laser and water-immersible lens (25x/0.95), controlled by LAS AF version 2.4.1 (Leica, Germany). Aleuria aurantia lectin (Vector Laboratories, USA) labeled with Alexa Fluor 633 (Thermo Fisher Scientific, USA) was used to visualize the extracellular polymeric substances (EPS) of the flocs. SYBR Green was used to visualize cells. )
Laboratory methods
Field
Surface water was collected from Bautzen reservoir on the 4th of September 2020. The water was sampled from the surface by a bucket.
Laboratory
The water was filtered over 10 µm stainless steel filters on the 4th of September 2020 and stored in the dark. The water was amended to pH 9.5 with addition of some drops of NaOH (1M) using a pH meter (PP-50, Sartorius). 10 mg of polyethylene spheres (RHBPMS-0.98 106-125 µm, Cospheric USA), followed by 300 µl FeSO4 stock-solution (500 mM, pH 1.8) were added to 500 ml of filtered surface water. The bottle was quickly closed and transferred to a tumbling roller incubator (3 – 4 rpm, 24 h, RM5, M. Zipperer GmbH, Germany) at 20° C in a temperature controlled room until floc formation.
20 x 25 ml Serum-bottles (autoclaved, sealed) were filled with 20 ml sterile mineral media for Shewanella spp. (Burlage et al., 1998) under a flow bench. For each of these bottles 5 flocs were visually and randomly selected from bottle which was incubated at the roller incubator. The flocs were transferred by an inverted pipette into tap water and washed 3x by renewal of the water. Afterwards the flocs were added to the Serum-bottles inside of the flow bench, by using sterilized inverted pipettes. This was followed by the addition of Na-lactate (concentration inside the Serum-bottle 10 mM) and S. oneidensis cells (please the manuscript for detailed cultivation and clean-up procedure). The bottles were closed with caps and butyl rubber stoppers and bubbled with N2  (unkown flow rate, 1 hour) to remove oxygen.
The Serum-bottles (microcosms) were sampled on the 15.09.2020, 5.10.2020, 13.11.2020 and the 13.1.2020. On each sampling date 5 microcosms were randomly selected and opened for the sampling. Hence, different microcosms were sampled on each sampling date
Water samples (100 µL) were retrieved from the microcosms punching a syringe through the butyl rubber stopper. The samples were directly injected into acid (1 M HCl, 200 µL) and measured with ferrozine or further transferred to hydroxylammonium chloride – HCl (0.5 / 1 M) and measured with ferrozine. For 3 of the 5 sampled microcosms, the flocs were gently removed from the microcosms with an inverted pipette and added to 1 M HCl (10 ml). They were variously shaken (orbital shaker, ~50 rpm) for 1 hour to disintegrate the flocs. Afterwards 100 µL of this solution was treated with hydroxylammonium chloride – HCl (0.5 / 1 M) followed by measurement of Fe(II) and Fe(III) via ferrozine assay. Polyethylene spheres inside of the 1 M HCl solution were counted by 10 times sieving 100 µL of the solution onto stainless steel sieves (10 µm). The concentrations were then calculated from the mean of the counting. The concentrations of liberated polyethylene sphere inside of the remaining liquids of the microcosms were counted by sieved the whole liquid of the sieves, followed by counting. All counting was conducted with a light microscope (Zeiss Axioplan). 
The remaining two microcosms were treated in a different way, by gently transferring the flocs into formaline (2 % v/v) and storing in the fridge (4° C). The flocs were than stained with fluorescent dyes and visualized using a laser scanning microscope (see  Leiser et al., 2021, 2020; Neu et al., 2001 for detailed description)