Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor

Accepted Manuscript Title: Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor Authors: Magnus Lindroos, David H¨ornstr¨om, Gen Larsson, Martin Gustavsson, Antonius J.A. van Maris PII: DOI: Reference:

S0304-3894(18)31000-8 https://doi.org/10.1016/j.jhazmat.2018.10.081 HAZMAT 19902

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

3-7-2018 17-9-2018 26-10-2018

Please cite this article as: Lindroos M, H¨ornstr¨om D, Larsson G, Gustavsson M, van Maris AJA, Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor, Journal of Hazardous Materials (2018), https://doi.org/10.1016/j.jhazmat.2018.10.081 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Continuous removal of the model pharmaceutical chloroquine from water

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using melanin-covered Escherichia coli in

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a membrane bioreactor

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Magnus Lindroos, David Hörnström, Gen Larsson, Martin Gustavsson, and Antonius J. A. van Maris*

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KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and

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Health, Department of Industrial Biotechnology, AlbaNova University Center, 114 21 Stockholm,

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Sweden

Magnus Lindroos - [email protected]

David Hörnström – [email protected]

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Gen Larsson – [email protected]

Martin Gustavsson – [email protected]

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*Corresponding author:

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Antonius J. A. van Maris – +4687909892, [email protected]

GRAPHICAL ABSTRACT

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HIGHLIGHTS

Melanized E. coli was implemented in a continuous membrane-bioreactor setup.

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Continuous removal of chloroquine was demonstrated for 26±2 reactor volumes (39±3 L).

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98.2 % removal with low effluent concentration (0.0018 mM) during first 20 hours.

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Successful in-situ regeneration was achieved using simple acidic conditions.

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Binding capacity of 140±6 mg/g did not decrease during three consecutive cycles.

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ABSTRACT

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Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical

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chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26±2 treated reactor volumes (39±3

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L). The average effluent concentration during the first 20 hours was 0.0018 mM, corresponding to 98.2 % removal. Up to 140±6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low-pH regeneration released all chloroquine without

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apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or

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manufacturing sites.

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Keywords: wastewater treatment, pharmaceuticals, membrane bioreactor, adsorption, surface

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expression

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1. INTRODUCTION The increasing global use of pharmaceutical- and personal-care products (PPCPs) is leading to the increasing contamination of surface- and groundwater [1]. Conventional wastewater treatment plants (WWTPs) are designed to remove solids, suspended solids, and easily (bio-)degradable organic material. Consequently, many of these PPCPs are only partially removed in current WWTPs [2–8],

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and their effluent concentrations remain within the range in which they raise a significant

ecotoxicological concern [2,3]. This problem is especially significant in developing countries, where

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untreated effluents from pharmaceutical industries result in the release of high quantities into the environment [9–11].

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In order to avoid future increasingly negative environmental effects of especially the persistent PPCPs

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in surface waters [12], different improvements of existing and developing treatment methods for PPCP removal are being investigated. Activated carbon is a known adsorbent for micropollutants in water

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and is considered a promising alternative. Pilot-scale investigations show that both powdered and

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granulated activated carbon can achieve a removal degree of up to 99 % [13,14] for a range of

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different pharmaceutically active compounds. Ozonation is another technique that has been investigated with promising results [15,16]. However, both of these techniques have their drawbacks. For example, the cost-effectiveness of activated carbon relies on regeneration, which is energy-

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demanding and problematic to implement in situ [17–19]. Ozonation, although effective for degradation of primary pharmaceutically active compounds, gives rise to degradation products that by

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themselves may prove (more) harmful [20,21]. Currently, no technique exists that is able to efficiently and completely remove all micropollutants from wastewater [22], thus creating a niche for the

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development of new, complementary, techniques for removal of PPCPs from diverse wastewater streams. The use of melanin as an adsorbent for PPCPs is one possible alternative to the aforementioned techniques. Melanin is a diverse and heterogeneous biopolymer, and the main pigment molecule in vertebrates [23]. Since the discovery of its affinity for phenothiazine [24], several studies have

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investigated the binding affinity of melanin for a range of micropollutants, including inorganic ions [24,25], aminoquinolines [26,27], fluoroquinolones [28], and others [29–34]. In nature, synthesis of melanin from tyrosine is catalyzed by the enzyme tyrosinase, which previously has been expressed cytosolically in Escherichia coli, resulting in intracellular production of melanin [35]. However, producing melanin inside the cell inherently results in a barrier (i.e. the cell membrane) between the

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melanin and the adsorbate, in this case the PPCP. To circumvent this, we previously engineered a recombinant E. coli-strain as a self-replication production system which expresses tyrosinase, and subsequently melanin, on the cellular surface [36]. In this design the cell envelope functions as a

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carrier for the melanin-adsorbent, resulting in the simultaneous production and immobilization of the melanin polymer. In batch experiments, these pigmented non-growing E. coli cells showed >90 %

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removal of chloroquine [36], which is a model pharmaceutical with previously described

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ecotoxicological implications [37] and known adsorbate to melanin [26,27,38].

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Current conventional WWTPs are operated with a continuous in- and outflow of water streams. These

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WWTPs maintain high sludge concentrations and a clear effluent through either gravitational settling of the biomass or membrane-based separation. An example of the latter is the use of membrane

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bioreactors (MBR) using hollow fiber filtration for the continuous treatment of municipal wastewater [39–41]. In many cases, removal of PPCPs closer to the source (start-of-pipe solutions), such as hospitals or manufacturing facilities, might be a more economical way to minimize the environmental

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release of these compounds than conventional end-of-pipe solutions. Like current WWTPs, these

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potential future applications will likely also benefit greatly from continuous operation. This study aims, for the first time, to investigate the use of melanin-enveloped E. coli for continuous

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removal of the model pharmaceutical chloroquine in a membrane bioreactor-based process. To also study the kinetics of adsorption, which is especially relevant for continuous operation, these experiments were performed at moderate cell concentrations and followed until saturation. Furthermore, the efficacy of regeneration of the saturated E. coli cells was investigated through release of the bound chloroquine at low pH.

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2. EXPERIMENTAL 2.1. STRAIN, BIOMASS PRODUCTION AND HARVEST The bacterial surface-expression system used in this study was the autotransporter AIDA-I,

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which is a widely used system for these purposes, and has previously been used to express tyrosinase on the surface of E. coli [36], as well as other proteins [42–46]. The binding

experiments used E. coli K12 strain 017 (∆ompT, sup+, F-) [47], containing the tyrosinase

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surface-expression plasmid pAIDA1-Tyr1 [44]. As a negative control, E. coli K12 strain 017 containing the plasmid pAIDA1 was used; this strain expresses the AIDA-I C-terminal

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domain without any passenger protein.

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Cultivations were performed with a minimal salts medium as described by Gustavsson et. al

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[36]. Working cultures were taken from a -80 °C freezer and put into 250 ml shake-flasks

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with a working volume of 25 ml minimal medium. Cells were then incubated in an orbital shaker at 30 °C and 180 rpm until an optical density (OD600) of 1. An aliquot was then

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transferred into a 12 L steel bioreactor (Belach Bioteknik AB; Stockholm, Sweden) with a working volume of 10 L, using the abovementioned minimal salts medium with 15 g/L glucose. Further, cultivations were performed at 30 °C with a minimum dissolved oxygen

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tension (DOT) of 30 %. pH was kept at 7.5 by automatic titration using 25 % w/v NH4OH (Merck, 25 %) and 10 % w/v H3PO4 (Scharlau, 85 %).

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At OD600=1, isopropyl ß-D-1-thiogalactopyranoside (IPTG, VWR International, ≥98 %) was added to a concentration of 200 µM to induce the respective plasmids. Simultaneously, a

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sterile CuSO4-solution (Merck, ≥99.0 %) was added to a concentration of 10 µM, as the expressed tyrosinase requires Cu2+ for activity. When the batch phase ended, indicated by a sharp increase of DOT, production of melanin was initiated by addition of L-tyrosine (SigmaAldrich, ≥ 97%) to a concentration of 1 g/L, as previously described [36]. Melanin production continued for 60 hours, at the abovementioned settings. Lastly, the cells were harvested by

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centrifugation (3000 g, 15 min, 4 °C) using an Avantitm J-20 XP centrifuge (Beckman Coulter; Stockholm, Sweden), and the cell-pellet was washed once and re-suspended in phosphate buffered saline (PBS) [43]. Cultivations were monitored by collecting samples for cell dry weight (CDW) and OD600. CDW-samples were collected in triplicate by withdrawing 5 ml of cell suspension into pre-

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weighed, and dried, conical glass tubes that were centrifuged at 4500 rpm for 5 minutes using

a Z 206 A centrifuge (Hermle Labortechnik GmbH; Wehingen, Germany). The cell pellet was

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dried in an oven at 105 °C overnight, and finally weighed.

2.2. MEMBRANE BIOREACTOR SET-UP

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Binding experiments were performed in a glass bioreactor (Ant, Belach Bioteknik AB;

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Stockholm, Sweden) stirred at 600 rpm, with an operating volume of 1.5 L. Cell-suspensions were diluted with PBS to a concentration of 10 g/L CDW and added to the bioreactor.

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Throughout the binding experiments, the concentration of non-growing cells remained stable.

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A 0.1 mM solution of chloroquine diphosphate (Sigma-Aldrich; Stockholm, Sweden; solid,

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≥98 %) in water was prepared in 20 L batches and continuously pumped into the system using a peristaltic pump (120 U, Watson-Marlow; Stockholm, Sweden). The treated water was continuously removed from the reactor as permeate from a polysulfone hollow fiber

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membrane (HFM, inside-out mode) with a pore size of 0.1 µm, a fiber inner diameter of 1 mm, a cartridge outer diameter of 1.9 cm, and a membrane area of 420 cm2 (Xampler CFP-1-

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E-4MA, GE Healthcare Life Sciences; Stockholm, Sweden). The nominal flow path was 30 cm and the connections were 0.5” tri-clamp connections for feed- and retentate ports, as well

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as 0.375” tubing nipples for the permeate ports. The permeate flow was set to 0.02 L/min using a valve on the permeate tubing. Recirculation (2 L/min) of the cell suspension/chloroquine mixture through the HFM was done using a gear-pump (MV-Z, Ismatec; Wertheim, Germany). A constant volume of 1.5 L was kept within the bioreactor by weight-based feedback-control of the feed-pump. Pressure gauges were connected to feed-,

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retentate- and permeate-ports for off-line monitoring of pressure build-up due to membrane fouling.

2.3. ADSORPTION, REGENERATION AND ANALYSIS Before the start of the first binding cycle for each of the duplicate experiments, cells obtained from independent cultivations were washed by flowing PBS buffer through the system for a

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total of 5 reactor volumes (7.5 L). Binding studies were then started by continuously feeding the chloroquine solution through the system. Samples of the permeate, approximately 10 ml,

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were collected every 30 to 60 minutes for up to 48 hours, at which point saturation of the cells had occurred in all experiments. An in-house built autosampler meant that each sample sat in room temperature, in an open plastic tube, for a maximum of eight hours before handling. A

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stability test was performed, confirming that the chloroquine concentration was stable during

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this time span (data not shown). Each sample was measured by spectrophotometer (Genesys

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20, Thermo Fisher Scientific; Uppsala, Sweden) at 343 nm [48] for chloroquine and 400 nm

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for melanin [49]. The pH within the reactor was kept at 7.5 by titration with 0.1 M citric acid (Merck, ≥99.5 %) and 0.1 M NaOH (Merck, ≥99.0 %).

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Cell regeneration was initiated by switching off the pumps supplying the chloroquine solution and the subsequent lowering of the pH in the system to 3 using 0.1 M citric acid as the titrant. Continuous-flow washing was then performed by pumping in water at a rate of 0.02 L/min,

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whilst maintaining a pH of 3 through titration with 0.1 M citric acid. Although not

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representative of final process configuration, this mode of regeneration enables accurate determination of regeneration potential and kinetics. During regeneration, samples were

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collected each 30th minute and each regeneration ran for 24 hours before moving on to the next binding cycle. In total, three consecutive adsorption/regeneration-cycles were performed per experiment.

2.4. ADSORPTION AND REGENERATION CALCULATIONS

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Breakthrough curves were constructed by plotting the amount of bound chloroquine against time. Bound amount of chloroquine at time t (Nbound,t in mmol) was calculated according to mass-balance-derived equation 1. 𝑁𝑏𝑜𝑢𝑛𝑑,𝑡 = 𝑓 · 𝑐𝑖𝑛 · (𝑡 − 𝑡0 ) − ∑𝑡𝑖=1 𝐶𝑜𝑢𝑡 · 𝑓𝑜𝑢𝑡 · (𝑡𝑖 − 𝑡𝑖−1 ) − (𝐶𝑜𝑢𝑡 ∙ 𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟 ) (1)

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Due to feedback regulation of pumps, fin = fout =f (in L/min). Cin was the concentration (mM) of chloroquine in the influent and was constant at 0.1 mM. The start time was defined as t0. Cout was the measured chloroquine concentration (mM) at the indicated sample times (tn).

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Vreactor was the volume of the reactor, 1.5 L. The dissolved chloroquine concentration in the bioreactor was assumed to be equal to that in the permeate. The binding capacity of

(𝑁𝑏𝑜𝑢𝑛𝑑,𝑡 ∙ 𝑀𝑐𝑞 ) 𝐶𝑐𝑒𝑙𝑙 ∙ 𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟

(2)

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𝐵𝐶𝑡 =

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chloroquine to the E. coli cells (BCt in mg/g) was calculated according to equation 2.

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Where Mcq is the molecular mass of chloroquine phosphate, Mcq=515.86 mg/mmol. Ccell was

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the initial cellular concentration in the reactor which was 10 g/L.

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The time for breakthrough was defined as the time (tb in hours) when the chloroquine concentration Cout was equal to 10 % of Cin. For the subsequent regeneration of the E. coli cells, the regeneration capacity (RC), i.e. the fraction of previously bound chloroquine, Nbound,

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that was released from the cells during a regeneration cycle, was calculated according to

𝑅𝐶 = 𝑁

𝑁𝑟𝑒𝑔𝑒𝑛. 𝑏𝑜𝑢𝑛𝑑,𝑡𝑒𝑛𝑑

∙ 100

(3)

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equation 3.

Where Nregen. was the amount of chloroquine that was released during the present regeneration cycle and was calculated analogously to Nbound. Nbound, tend was the accumulated amount of chloroquine (mmol) at the end time (tend) of the preceding binding cycle.

2.5. DETERMINATION OF CHLOROQUINE ADSORPTION ISOTHERMS

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Adsorption data was fitted with Langmuir- and Freundlich isotherms [50–53], according to equation 4 and 5. 𝑞

𝐾 𝐶

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= 1+𝐾𝐿

𝐿𝐶

𝑞 = 𝐾𝐹 𝐶 1/𝑛

(4)

(5)

In both equations, q is the amount adsorbed (mg/g) and C is the equilibrium concentration of

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adsorbate in solution (mg/L). In equation 4, KL (L/mg) is the model specific constant for binding affinity and free energy of adsorption, and q0 is the theoretical maximum binding

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capacity (mg/g) [49]. In equation 5, 1/n is the heterogeneity index and KF (L/g) is the Freundlich constant.

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3. RESULTS

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3.1. CONTINUOUS CHLOROQUINE BINDING

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To investigate the potential of melanin-enveloped E. coli for continuous removal of PPCPs, a bioreactor with external hollow fiber membrane (HFM) was chosen as the experimental set-

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up (Fig. 1). This enabled the continuous feeding of a chloroquine solution and the removal of the treated water as the membrane permeate, while at the same time retaining the recombinant E. coli within the bioreactor. The biomass concentration (10 g/L) and ingoing chloroquine

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concentration (0.1 mM) were chosen to facilitate the determination of the kinetics of chloroquine adsorption as well as regeneration of the E. coli cells. Prior to evaluating the

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adsorptive properties of the melanized E. coli, a control experiment was performed without cells in the bioreactor, which confirmed that there was no chloroquine binding in the absence

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of cells (supplementary Fig. S1). To study the kinetics of chloroquine binding, the tyrosinase-containing E. coli strain (K12 0:17 ΔompT [47] pAIDA1-Tyr1 [44]) and a non-tyrosinase-containing control strain (K12 0:17 ΔompT [47] pAIDA1) were grown in stirred-tank bioreactors prior to induction of, respectively, the pAIDA1-Tyr1 and pAIDA1 vectors with IPTG. The resulting cells

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containing the pAIDA1-Tyr1 were enveloped by melanin, while the reference cells, lacking the Tyr1 passenger protein, were not, in accordance with previous results [36]. Both strains were harvested, washed, and transferred to the MBR, before starting duplicate continuous chloroquine-removal experiments and determination of adsorption kinetics (Fig. 2). Upon starting of the pumps, the initial rates of removal were sufficient as the chloroquine was

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removed to below the detection limit of 0.001 mM, which corresponds to >98% removal. The effluent concentration remained low throughout approximately the first 20 hours of the

experiment, with an average concentration of 0.0018 mM (Fig. 2A). Comparing the amount

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of bound chloroquine to the cumulatively added amount of chloroquine (Fig. 2C) shows that, up until approximately 25 treated reactor volumes, >98 % of the added chloroquine (top x-

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axis) is bound to the cells. This equals a total of approximately 4 mmol of chloroquine. From

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this point onwards, the residual chloroquine concentration slowly increased until, after 31±3 hours, the effluent concentration reached 10 % of the inlet concentration. At this point a sharp

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increase of chloroquine was observed in the permeate concentration. In a real-life application, when the effluent concentration reaches undesirable values the

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effluent would either be led through a 2nd fresh tank of cells in series to achieve complete removal or the influent would be switched to fresh cells altogether. However, to determine the saturation kinetics and binding capacity of chloroquine, the adsorption experiment was

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monitored until the effluent concentration approached the inlet concentration, indicating complete saturation of the cells (Fig. 2A). This concentration was reached after 47±2 hours at

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which point 140±7 mg chloroquine per gram of cells was absorbed to the melanized cells (Fig. 2B). After saturation, the binding experiment was terminated and the cells were

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regenerated (see section 3.2) and used for two more cycles of identical adsorption experiments for each replicate in order to evaluate the re-usability of the cells. In previous work with batch adsorption of chloroquine it was shown that the empty-vector control-strain adsorbed about 15-20 % of the amount of chloroquine that was adsorbed by the melanized cells. To assess if this is influenced by the operational mode, i.e. continuous versus

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batch, identical adsorption experiments were also performed with the empty vector control strain. The adsorption in the reference experiments (Fig. 2D) follows the same pattern, where all added chloroquine was bound to the cells until approximately 13 treated reactor volumes. This means that in this continuously operated MBR set-up the reference cells demonstrated approximately 50 % of the chloroquine binding capacity of the melanin-enveloped cells.

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3.2. REGENERATION OF CELLS

To assess the reusability of the cells, in situ regeneration was performed by lowering the pH

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to 3 and switching the influent to water, at a flowrate of 0.02 L/min. Consequently, the bound chloroquine was released and washed from the system (Fig. 3). The majority of chloroquine was released in the first couple of wash volumes and almost complete regeneration was

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achieved after washing with 16±6 % of the previously treated total water volume. The ‘nicks’

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in the graphs of cycle 2 and 3 were caused by wear on the stirrer motor and ensuing required

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adjustments. That this did not influence the regeneration outcome was shown by the

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maximum amount of chloroquine bound in each of the binding cycles at 133 mg/g for the first

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cycle, 142 mg/g for the second cycle and 146 mg/g for the third cycle.

3.3. ADSORPTION MODELLING

To characterize the chloroquine/melanin-binding and help classify the nature of the

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adsorption [54], experimental data from each binding cycle with the melanin-covered cells were fitted to two common adsorption isotherms: Langmuir (homogeneous adsorption) and

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Freundlich (heterogeneous adsorption). Plotting and fitting the bound amount of chloroquine (mg/g cells) to the dissolved concentration (mg/L), showed a better fit for the melanin-

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covered cells with the Freundlich isotherm (R2>0.90) than with the Langmuir isotherm (R2<0.83) (Fig. 4, Table 1). Since the heterogeneity index (1/n) in the Freundlich isotherm is a measure of surface heterogeneity [31,51], where a lower value implies a more heterogeneous surface, this might suggest that the melanized cells display a (mostly) heterogeneous type of adsorption, which is in line with the irregular conformation and configuration of the melanin

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polymer [55] as well as the different types of binding-sites and -mechanisms therein [29]. The Freundlich constant (KF), although not a direct measurement of maximum binding capacity, gives an indication of the adsorption capacity of a heterogeneous system [51], and was 2-fold higher for the melanized cells (106.4±4.9 L/g) than for the reference cells (46.6±14.9 L/g). Adsorption by the reference cells displayed the best fit with the Langmuir isotherm, although

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also showing a reasonably good fit with the Freundlich isotherm. The theoretical monolayer saturation capacity, q0 (in the Langmuir isotherm), for the melanin-enveloped cells was

(Fig. 2B), was just under 2 times that of the reference cells.

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14312 mg/g, which, consistent with the results from the continuous binding experiments

TABLE 1: CONSTANTS OBTAINED FROM FITTING EXPERIMENTAL DATA FROM BINDING EXPERIMENTS, TO

0.98 103.0 0.10

0.93 104.2 0.11

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Freundlich R2 KF (L/g) 1/n

0.61 6.4 141.1

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0.83 5.9 132.8

Reference binding, cycle # 1 2

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0.72 6.1 156.1

0.99 0.8 69.9

0.99 1.8 89.7

0.99 0.3 86.6

0.90 112.1 0.12

0.93 39.9 0.15

0.97 63.7 0.10

0.94 36.2 0.22

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Langmuir R2 KL (L/g) q0 (mg/g)

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Melanin binding, cycle # 1 2

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THEORETICAL ADSORPTION ISOTHERMS.

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4. DISCUSSION This study shows that using melanin-covered E. coli in an MBR-setting has potential for the

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continuous removal of pharmaceuticals from wastewater. The ability for melanin to bind to chemicals are widely reported in literature [24,28,30,31,55] but the possibility to utilize melanin as an adsorbent in wastewater-treatment has not been investigated aside from smallscale batch-experiments [36]. The MBR-based system presented here is an interesting option for continuous water-treatment with melanin-enveloped cells.

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Surprisingly, the maximum observed capacity for chloroquine binding in this study (140±7 mg/g) was about 3-fold higher than the previously observed 45 mg/g observed for the same strain in batch conditions [36]. It is possible that the slow feeding and low residual concentrations of chloroquine in the continuous experiments facilitate a more optimal use of the (different types of) binding sites present in the heterogeneous melanin structure [29,55].

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This is supported by the modelling of the adsorption kinetics, where the melanin-covered cells showed a better fit to the Freundlich isotherm, which describes a heterogeneous layer of adsorption not confined to a monolayer [50]. The Freundlich isotherm is indicative of a

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competitive binding mechanism in which each site binds unequally strong to the adsorbate. In contrast, the reference cells without melanin, showed excellent fit to the Langmuir isotherm.

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This model describes a homogeneous monolayer adsorption mechanism [52], where each site

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represents an equal, individual, point of adsorption where no interaction with adjacent binding sites occurs [50]. Although, the reference strain showed increased (reversible)

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uptake/adsorption, this quantitatively cannot explain the full increase for the melanized cells.

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In these proof-of-principle experiments, a low loading of cells (10 g/L) was used. Any future

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application is likely to use much higher cell loading, resulting in both process intensification, energy savings and perhaps most importantly a (much) larger volume of water that can be treated before regeneration is required. Another parameter coupled to the cost-effectiveness is

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the number of cycles the cells are able to be regenerated. The melanized E. coli cells in the MBR-system, were able to remove almost all of the fed 0.1

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mM chloroquine (51.6 mg/L) down to the detection limit of around 0.001 mM. Because the ingoing chloroquine concentration used in this study is high compared to the concentration

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range that is often found in wastewaters [2,56,57], the capacity for the volume of treated water per bioreactor volume would even increase further. Since it is, in general, more difficult to remove the low concentrations observed in end-of-pipe solutions, it might be desirable to remove PPCPs further upstream where they occur in higher concentrations, such as in the wastewater from hospitals or factories. For operation at concentrations below the detection

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limit in this current study (0.001 mM), this would require further investigation on how well the melanized E. coli-based system performs on concentrations below this limit. The simple proof-of principle regeneration-experiments under mild acidic conditions in this study, supported by the constant binding capacity, showed little to no decrease of its regenerative ability over the course of the three experimental cycles. Combined with the

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foreseen operation at increased biomass concentrations, this provides a basis for design of a process with continuous ‘counter-current’ removal of PPCPs by freshly-regenerated cells,

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whilst the saturated cells are simultaneously regenerated to a percentage (<100 %) that

enables fast regeneration with low volumes of concentrated PPCPs for sustainable disposal. However, before moving on towards real-life trials, treating wastewater streams from

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WWTPs, further studies into competitive binding between different compounds as well as

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binding capacity towards other types of PPCPs needs to be investigated. Nevertheless, our

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results show that this compact MBR-system, with an easily performed in situ regeneration-

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ACKNOWLEDGMENTS

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step, has potential for the continuous binding and regeneration of PPCPs from wastewater.

The authors thank Anders Kihl and Gustav Sjöberg for scientific discussion and Gustav

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Sjöberg for advice on construction of the autosampler. This research is partially funded by

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grant VR-621-2014-5293 from the Swedish Research council.

CONFLICTS OF INTEREST

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Gen Larsson is inventor on a patent application describing the melanin-producing E. coli strain.

REFERENCES

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FIGURES

Fig. 1. Schematic representation of the membrane bioreactor setup used for continuous water-

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treatment. (1) 20 L feed-container with 0.1 mM chloroquine. (2) Molecular structure of the

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chloroquine pharmaceutical. (3) Feedback-regulated peristaltic pump, connected to the

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stirred-tank bioreactor system. (4) Gear pump used to circulate the cell suspension over the

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within the system. (6) Sampling port.

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hollow-fiber membrane. (5) Hollow-fiber membrane, 0.1 µm pore-size; used to retain cells

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Fig. 2. Adsorption of chloroquine onto melanin-enveloped E. coli. The solid lines in panels A, C and D represent the 1st adsorption cycle, the dashed lines the 2nd cycle and the dotted

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lines the 3rd. Panel A) Change in chloroquine concentration over time for three consecutive binding cycles with melanized cells. Panel B) The final amount of chloroquine bound per cell

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(mg/g) for each of the three binding cycles of melanized cells (black) compared to the reference cells (white). Panel C-D) The cumulative amount of chloroquine bound to cells and dissolved in the permeate for melanized cells (C) or reference cells (D). The bottom x-axis shows the treated reactor volumes (1 reactor volume = 1.5 L), whilst the top x-axis shows the cumulative amount of chloroquine added to the system. The lines titled ‘Bound’ shows the cumulative bound amount of chloroquine (mmol); whereas, the lines titled ‘Outflow’ shows

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the cumulative released amount of chloroquine from the system. The figure describes one

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representative set-of experiments (replicate shown in Fig. S2).

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Fig. 3. Chloroquine released from the melanized cells during three regeneration cycles.

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The released chloroquine is shown as a fraction of the total bound chloroquine in the preceding binding cycle, plotted against the volume of wash solution (dilute citric acid,

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pH=3) shown as a fraction of the treated water volume in the previous binding cycle. Solid line represents cycle 1 whereas dashed line represents cycle 2 and dotted line,

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cycle 3.

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Fig. 4. Adsorption isotherms, Langmuir (dotted lines) and Freundlich (dashed lines) fitted to data (solid line) for both melanized cells (top of graph) and control without melanin (bottom

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of graph). (A) represents cycle 1, (B) represents cycle 2 and finally, (C) represents cycle 3.

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