Iron Complex Facilitated Copper Redox Cycling for Nitric Oxide

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Iron Complex Facilitated Copper Redox Cycling for Nitric Oxide Generation as Nontoxic Nitrifying Biofilm Inhibitor Vita Wonoputri,† Cindy Gunawan,†,‡ Sanly Liu,† Nicolas Barraud,§ Lachlan H. Yee,*,∥ May Lim,† and Rose Amal*,† †

Particles and Catalysis Research Group, School of Chemical Engineering, The University of New South Wales, Sydney, New South Wales 2052, Australia ‡ ithree Institute, University of Technology Sydney, Sydney, New South Wales 2007, Australia § Genetics of Biofilms Unit, Department of Microbiology, Institut Pasteur, 75015 Paris, France ∥ Marine Ecology Research Centre in the School of Environment, Science and Engineering, Southern Cross University, Lismore, New South Wales 2480, Australia S Supporting Information *

ABSTRACT: In this study, we developed poly(vinyl chloride) (PVC)-solvent casted mixed metal copper and iron complexes capable of catalytic generation of the antibiofilm nitric oxide (NO) from endogenous nitrite. In the absence of additional reducing agent, we demonstrated that the presence of iron complex facilitates a redox cycling, converting the copper(II) complex to active copper(I) species, which catalyzes the generation of NO from nitrite. Assessed by protein assay and surface coverage analyses, the presence of the mixed metal complexes in systems containing water industry-relevant nitriteproducing nitrifying biofilms was shown to result in a “nontoxic mode” of biofilm suppression, while confining the bacterial growth to the free-floating planktonic phase. Addition of an NO scavenger into the mixed metal system eliminated the antibiofilm effects, therefore validating first, the capability of the mixed metal complexes to catalytically generate NO from the endogenously produced nitrite and second, the antibiofilm effects of the generated NO. The work highlights the development of self-sustained antibiofilm materials that features potential for industrial applications. The novel NO-generating antibiofilm technology diverts from the unfavorable requirement of adding a reducing agent and importantly, the less tendency for development of bacterial resistance. KEYWORDS: biofilm, nitrifying bacteria, nitric oxide, copper, iron

1. INTRODUCTION The presence of surface-attached bacterial communities, which are commonly known as biofilms, are disadvantageous for the water industry. Growth of biofilms can decrease heat exchanger efficiency or foul water filtration membranes.1,2 Biofilms in water distribution pipelines can cause microbial-induced corrosion and often act as reservoirs for pathogens, compromising the microbiological quality of drinking water.1 Microbial growth as biofilms in water systems is usually controlled by addition of disinfectants, most commonly with chlorine and chloramine. In many cases, however, administration of these disinfectants have been shown to be ineffective at eradicating the biofilm growth due to their increased resistance compared to their free-floating (planktonic) counterparts.3 Chlorination, while effective in killing fecal pathogens, has been known to select for resistant pathogens in water distribution system, including Mycobacterium avium (cause of pulmonary disease).4 Biofilms with nitrifying bacteria as members have been known to flourish in chloraminated system (due to the release of ammonia from © XXXX American Chemical Society

chloramine decay) and in turn, further accelerating the inactivation and decay of chloramine.4,5 Therefore, there is a clear need for the development of novel antibiofilm technology capable of suppression of biofilm formation, with minimized tendency for inactivation and the occurrence of microbial resistance. Nitric oxide (NO) is a soluble free radical gas that has been shown to effectively control the formation of biofilms. NO affects biofilms either by inducing a toxic pathway (cell death) at high concentrations or via a nontoxic pathway at low concentrations that cause microbial members to disperse from biofilms and revert to a free-floating phase.6−8 These properties of NO have led to the development of novel strategies to control biofilms and biofilm-related infections, which could find broad applications in industrial water settings as well as in clinical settings, e.g., biofilms on prosthetic implants, catheters, Received: August 18, 2016 Accepted: October 19, 2016 Published: October 19, 2016 A

DOI: 10.1021/acsami.6b10357 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces or living tissues that can cause chronic infections. However, application of NO as antibiofilm agent is still limited due to its high reactivity and short half-life. Consequently, several research efforts have focused on the synthesis of materials that are capable of delivering NO to the target site.9 In general, materials that are capable of delivering NO can be divided into two types: NO-releasing materials and NOgenerating materials.10−12 NO-releasing materials utilize NO donors, such as N-diazeniumdiolates (NONOates) or Snitrosothiols (RSNO), incorporated into a nanomaterial delivery vehicle or coating. Such materials have limited NO release due to the finite reservoir of NO that can be loaded during synthesis. On the contrary, NO-generating materials appear more advantageous due to their ability to generate NO from endogenous sources, such as S-nitrosoglutathione (GSNO), S-nitrosoalbumin (AlbNO), and S-nitrosocysteine (CysNO), which are present in plasma and blood, or from nitrite ions which are present in water.8,13−15 NO-generating materials are usually copper or selenium based, which can catalyze the decomposition of endogenous NO donor to form NO.8,16 Copper(II) complexes that were embedded in a poly(vinyl chloride) (PVC), polyurethane (PU), or polycarbonate-urethane (PCU) matrix have been shown to be capable of generating NO at the polymer-solution interface.10,15,17,18 Other studies have reported the use of metallic copper nanoparticles or copper particles incorporated into polymeric films,13,19,20 zeolites or metal organic frameworks containing copper, to generate NO catalytically from endogenous sources.21,22 Organoselenium-based materials, such as selenocystine and 3,3-diselenodipropionic acid, have also been used to generate NO from GSNO.11,23 Importantly, the activity of the copper(II) and selenium as NO-generating catalysts is dependent on the presence of a reducing agent that can reduce copper(II) to copper(I) or selenium(II) to its reduced form, as the reduced form is the most active species in catalytic generation of NO.11,17 The presence of reducing agents in the bloodstream, including ascorbate and free thiol/thiolate may facilitate strategies based on the catalytic generation of NO to control biofilm formation or thrombosis on blood-contacting devices.17,24,25 Note that NO is known to play various physiological roles including in the vascular system regulating vasodilation and preventing blood platelet activation and adhesion, 10,26 which can be used in blood-contacting biomedical devices to prevent thrombosis.16 For generation of NO in water distribution system, however, the need for continuous addition of an external reducing agent is not desirable as it is costly and might affect the water composition and quality. Therefore, a reducing agent that can be immobilized together with the catalyst inside an appropriate polymeric matrix will be highly beneficial. One such reducing agent that has the potential to be embedded in a polymeric matrix is iron. Iron(II) has been reported to be able to reduce copper(II) to copper(I) in solution, which subsequently can react with nitrite to form NO.24,27 In this study, we synthesized mixed iron and copper complexes (FeDTTCT and CuDTTCT, respectively) immobilized onto a PVC matrix. (DTTCT: dibenzo[e,k]-2,3,8,9tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene, a nitrogen-based ligand; Scheme 1). We investigated the ability of the immobilized FeDTTCT in facilitating the reduction of copper(II) in CuDTTCT to the active copper(I) species and its subsequent generation of NO via nitrite reduction. The effectiveness of the mixed metal catalyst system in inhibiting

Scheme 1. DTTCT Structure

biofilm formation without any additional reducing agent and nitrite was also examined. Nitrifying bacteria, commonly found in water distribution systems, were chosen as a biofilm-forming model organism.

2. EXPERIMENTAL SECTION 2.1. Synthesis of Dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10tetraaza-cyclododeca-1,3,7,9-tetraene Complex (DTTCT). The synthesis of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene (DTTCT) was as earlier described by Wonoputri et al.15 Briefly, benzil (0.05 mol; Aldrich 98%) and ophenylenediamine (0.05 mol; Aldrich, 99.5%) were refluxed in ethanol with few drops of hydrochloric acid (Ajax Finechem, 32%) for 6 h at 80 °C, and cooled overnight. The white precipitated crystals were filtered, washed with ethanol and dried in a vacuum desiccator. 2.2. Synthesis of Copper-DTTCT (CuDTTCT) and Iron-DTTCT (FeDTTCT) Complex. Copper complex was prepared following the method described by Wonoputri et al.15 In brief, dried DTTCT (0.01 mol) was mixed with copper acetate monohydrate (0.04 mol; Ajax APS) in ethanol and refluxed at 85 °C for 6 h. The resulting light blue precipitate was washed with ethanol and dried in vacuum desiccator. Synthesis of iron complex was carried out by mixing iron(III) chloride hexahydrate (0.05 mol; Ajax APS) with DTTCT (0.01 mol) in ethanol and refluxed at 85 °C for 6 h. After overnight cooling, the resultant precipitate was isolated by centrifugation, washed with cold ethanol and dried in vacuum desiccator yielding a dark brown powder. The coupons were synthesized by mixing the metal complex with predissolved PVC (provided by Chemson Pacific Pty Ltd.) in tetrahydrofuran (THF, Chem-Supply) solution (0.3 mL, 66 mg/L) in an ultrasonic bath. Round glass coverslips (18 mm diameter, ProSciTech) were washed with dilute nitric acid, acetone, and ethanol followed by overnight drying at 110 °C before being used as the cast template. Bare coupon (PVC without any metal complexes, typical weight ∼20 mg) was used as the control. To prepare the coupons that contain a mixture of the CuDTTCT and FeDTTCT metal complexes, CuDTTCT (0.25 mg, 0.5 mg, 0.75 mg, and 1 mg; 1.25, 2.5, 3.5, and 5 wt %) and FeDTTCT (equal loading) were mixed with the predissolved PVC in THF solution in an ultrasonic bath, and then casted on the round glass coverslips. All coupons were dried at 50 °C for 12 h and peeled off from the glass coverslips. 2.3. Characterization of Metal Complexes. Analysis of the structure was performed in deuterated dimethyl sulfoxide solvent by 1 H NMR spectroscopy using a Bruker Avance III HD 400 MHz. Thermal analysis of the ligands and metal complexes were carried out by Differential Scanning Calorimetry (DSC; PerkinElmer) to confirm the metal complex formation. Around 3 to 6 mg of the sample were placed in a closed aluminum pans, with empty closed aluminum pan as the reference. The temperature was increased at a rate of 10 °C/min under nitrogen environment (50 mL/min). Results shown are a representative thermogram obtained from duplicates. A field emission scanning electron microscopy (SEM; FEI Nova NanoSEM 230) equipped with an energy dispersive X-ray detector (EDS; Bruker Silicon Drift Energy Dispersive X-ray detector) was used to show the immobilization of copper and iron complex in PVC matrix. Prior to imaging, the samples were attached to 10 mm metal B


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Figure 1. DSC thermograms of DTTCT, CuDTTCT, and FeDTTCT with a heating rate of 10 °C/min.

Figure 2. (a) SEM images of mixed metal coupon (CuDTTCT+FeDTTCT) and its corresponding elemental mapping of (b) copper and (c) iron, (d) EDS spectra of the mixed metal coupon. Other peak labels are removed for clarity. mounts by carbon tape and sputter-coated with chromium. Data analysis was performed with Esprit EDS software. XPS measurements were performed using an ESCALAB 220iXL spectrometer with monochromated Al Kα (energy 1486.6 eV). Coupons samples were cleaned by brief immersion in toluene prior to analysis. Reaction was performed by immersing the precleaned sample in a 10 mM phosphate buffer solution pH 6 (Sigma, pH adjusted by concentrated hydrochloric acid) containing 1 mM sodium nitrite (Ajax Finechem) for 10 min. All coupons samples were dried in a vacuum desiccator for a minimum of 2 days before analysis. A random spot on the coupon with diameter around 0.5 mm was chosen for the analysis. All spectra were calibrated to the C 1s peak signal at 284.8 eV. 2.4. NO Generation Measurement. NO generation from the system was measured amperometrically using an Apollo TBR4100 Free Radical Analyzer (World Precision Instrument) equipped with an ISO-NOP 2 mm probe. Calibration was performed using S-nitroso-Nacetylpenicillamine (SNAP; Sigma) and copper sulfate solution according to the manufacturer’s protocol. The test coupon was placed at the bottom of a 20 mL glass vial equipped with stir bars and filled with 10 mL of 10 mM phosphate buffer solution pH 6. After a stable baseline was observed, 1 mM sodium nitrite was added to the glass vial. At the end of the measurement, an NO scavenger namely 2phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; Alexis Biochemicals) was added to re-establish the baseline. All experiments were performed in the presence of ambient oxygen. Results shown are a representative graph obtained from a minimum of three replicates. 2.5. Biofilm Assay. The effect of the mixed metal complex coupons on nitrifying biofilms was assessed as previously described.15

Biofilm was formed from a mixed inoculum of nitrifying bacteria that is available commercially for the purification of aquarium water and is composed of ammonia-oxidizing bacteria Nitrosospira multiformis, nitrite-oxidizing bacteria Nitrospira marina, and heterotrophic bacteria Bacillus sp. (Aquasonic Bio-Culture). One mililiter of the mixed inoculum was added into 100 mL of the nitrifying medium (ATCC medium 2265 consisting of three different stock solutions−stock 1 (final composition in the medium mixture): 25 mM (NH4)2SO4, 3 mM KH2PO4, 0.7 mM MgSO4, 0.2 mM CaCl2, 0.01 mM FeSO4, 0.02 mM EDTA, 0.5 μM CuSO4; stock 2:40 mM KH2PO4, and 4 mM NaH2PO4, adjusted to pH 8 by 10 M NaOH; stock 3:4 mM Na2CO3) and incubated for 3 days in the dark (30 °C, 100 rpm). The three-dayold cultures were inoculated into fresh medium and 2 mL aliquots (OD600 = 0.008) were added into 12-well plates with the presence of a bare coupon (control) or a mixed metal coupon. The plates were then incubated in the dark for 3 days at 30 °C, 100 rpm. At the end of incubation, the bacteria supernatants were removed and centrifuged at 12 000 rpm for 15 min and washed in phosphate buffer saline (PBS; Sigma) once to separate planktonic bacteria. The supernatants were filtered through a 0.2 μm membrane and used to measure the extent of copper and iron leaching from the coupons by ICP-OES (PerkinElmer OPTIMA 7300). The biofilms were washed twice to remove the loosely attached cells. The biomass was measured by quantifying total protein content using the bicinchoninic acid method (BCA assay; Sigma). BCA working reagent (2 mL) was added into each of the biofilm and planktonic cells, followed by incubation for 30 min at 37 °C, 100 rpm. A preliminary experiment has shown that the presence of a mixed metal coupon did not interfere with the assay, even after the C


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ACS Applied Materials & Interfaces incubation step. Standard solutions as per manufacturer’s instructions were used for each experiment. Surface coverage analysis and cell viability assessment were performed using confocal laser scanning microscopy (Olympus FluoView FV1000) of LIVE/DEAD-stained biofilm cells. For microscopic observation, an aliquot of the nitrifying culture was added into a 35 mm culture dishes with coverglass bottom (internal glass diameter 22 mm, ProSciTech) with the presence of bare coupon (control) or mixed metal coupon, followed by incubation in the dark for 3 days at 30 °C, 100 rpm. At the end of incubation, the supernatants were removed and biofilms attached on the glass substratum were washed twice with PBS. Adhered cells were stained with SYTO-9 and propidium iodide according to manufacturer’s instruction (LIVE/DEAD BacLight bacterial viability kits L-7007, Molecular Probes Inc.) and incubated at room temperature for a minimum of 15 min. Twelve pictures across the glass bottom were obtained and surface coverage analysis was performed on live cells (green channel) using image analysis software (Fiji/ImageJ). All statistical analyses were performed on GraphPad Prism 6 (GraphPad Software) using one-way ANOVA method followed by Dunnett’s posthoc analysis.

Figure 3. Amperometric measurements of NO generation from mixed metal coupon containing a mixture of metal complexes (CuDTTCT +FeDTTCT; 1 + 1 and 0.5 + 0.5) with the addition of 1 mM nitrite. Three different controls were used (bare coupon, 1 mg CuDTTCT coupon, 1 mg FeDTTCT coupon) in the presence of 1 mM nitrite solution. PTIO (100 μM; black arrow) was added at the end of measurement to re-establish the baseline.

3. RESULTS AND DISCUSSION 3.1. Confirmation of Metal Complex Formation. Copper complex (CuDTTCT) and iron complex (FeDTTCT) were synthesized and characterized. 1H NMR analysis of the metal complexes shows similar spectra as that observed for DTTCT, thus confirming the presence of DTTCT in the metal complex (Figure S1 of the Supporting Information). Thermal analysis by DSC shows that ligand DTTCT only exhibited one strong endothermic peak at 124 °C, while further complexation with copper generated four endothermic peaks at 121 °C, 156 °C, 204 °C, and the sharpest at 269 °C (Figure 1), which could be ascribed to the melting point-decomposition of CuDTTCT sample. This shift in melting point from 124 °C (DTTCT) to 269 °C confirmed the successful formation of the complex. For FeDTTCT sample, one exothermic peak at 153 °C was observed, followed by 3 endothermic peaks at 165 °C, 193 °C, and 248 °C. The exothermic peak at 153 °C is possibly due to decomposition of the sample, as has been previously observed by Breviglieri et al.28 The absence of sharp endothermic peak at 124 °C again confirmed the formation of metal complex.28−30 Finally, SEM-EDS mapping was performed on the PVC coupon that contains both CuDTTCT and FeDTTCT (mixed metal coupon). Copper was uniformly detected throughout the samples, while iron mapping showed aggregated distribution (Figure 2). The observations confirmed the presence of copper and iron in the complexes. 3.2. NO Generation from CuDTTCT+FeDTTCT Coupon in the Presence of Nitrite. Evaluation of NO release in systems containing mixed CuDTTCT+FeDTTCT metal complexes coupon (along with their associated set of controls) was performed amperometrically, where NO peak can be observed upon addition of 1 mM nitrite into the systems (Figure 3). Low and stable levels of NO were generated in the various control systems. In the case of the bare PVC coupon (without metal complex) and coupon with 1 mg FeDTTCT loading, up to 25 nM NO was generated, which is thought to result from the formation of nitrous acid from nitrite and its subsequent decomposition in acidic pH.31 A higher level of ∼50 nM NO was generated in the presence of coupon with 1 mg CuDTTCT loading. The interactions of copper(II) with various NO donors, such as nitrite and S-nitrosothiols, have been reported to generate low levels of NO.24,32,33 Interestingly, when the combination of CuDTTCT+FeDTTCT mixed

metal coupon at (0.5 + 0.5) mg loading was used, a surge of up to 125 nM NO was generated (in the first ∼750 s), followed by a slow decrease in NO concentration. Addition of the NO scavenger PTIO at the end of the measurement caused a sudden decrease in the signal, confirming the prior generation of NO. The significant generation of NO from nitrite in the presence of the mixed metal complexes is most likely facilitated by copper(II)/copper(I) redox cycling with iron complex acting as reducing agent, as suggested by the following equations: Cu 2 +(DTTCT) + Fe2 +(DTTCT) ⇌ Cu+(DTTCT) + Fe3 +(DTTCT)

(i)

Cu+(DTTCT) + NO2− + H 2O ⇌ NO + 2OH− + Cu 2 +(DTTCT)

(ii)

Earlier studies, including those of our group, have reported NO generation from nitrite via copper(II)/copper(I) redox cycling in the presence of a reducing agent (including ascorbic acid), whereby copper(I) species, that formed from reduction of copper(II) species by the reducing agent, is the most active species for NO production from nitrite.15,17 Copper(II) reduction by iron species and importantly, the generation of NO from nitrite via reaction with the formed copper(I) species, have been observed by several studies.24,27,34−37 Up to this stage, copper(II) reduction by iron(II) has been reported in solution form, either homogeneously (both metals as dissolved species) or heterogeneously (with one metal in solution form).36 Eventually, when the formed copper(I) is oxidized to copper(II) and iron-facilitated reduction of copper(II) does not happen anymore, NO generation will stop. Doubling the mixed metal complexes loading to (1 + 1) mg caused an up to 2-fold increase in NO generation in the presence of nitrite, also detected within similar time frame as that of the lower loading of metal complexes (in the first ∼750 s). This was again followed by a similar slow decrease in NO concentration prior to PTIO addition. The enhanced NO generation was due to the presence of more catalytic active sites with the increasing amount of metal complexes. D


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Figure 4. (a) XPS spectra of Fe 2p region of CuDTTCT+FeDTTCT (mixed) coupon before and after reaction. (b) XPS spectra of Cu 2p region of CuDTTCT+FeDTTCT (mixed) coupon before and after reaction. Reaction was performed in the presence of 1 mM of nitrite for 10 min.

Generated NO. The potential of CuDTTCT+FeDTTCT mixed metal complexes as NO-generating material for antibiofilm application was investigated on the ubiquitously occurring nitrifying bacteria biofilms. Nitrifying bacteria of the Nitrosomonas and Nitrosospira genera are commonly known as ammonia oxidizing bacteria or AOB, capable of oxidizing ammonia to nitrite. Nitrite production from ammonia occurs via a two-step process involving first, the conversion of ammonia to hydroxylamine catalyzed by the enzyme ammonia monooxygenase (Amo) and second, the conversion of hydroxylamine to nitrite by the enzyme hydroxylamine oxidoreductase (Hao). Here, we investigated for the first time the ability of the mixed metal complexes to catalytically utilize the nitrite produced by bacterial biofilms for NO release through redox cycling. Biofilms containing the AOB Nitrosospira spp. was grown in an ammonia-loaded culture medium in the presence of bare (PVC-only) coupon and after 3 days, ∼16 mM nitrite was detected in the system (by ion chromatography). A low level of ∼0.1 mM nitrate was also detected in the system, potentially due to the presence of nitrite-oxidizing bacteria (NOB) Nitrospira spp. in the inoculum, capable of oxidizing nitrite to nitrate. The biofilm growth was characterized by detection of ∼300 μg/mL protein on the well and bare coupon surfaces, while ∼100 μg/mL protein was detected in the suspended planktonic phase. In such self-produced nitrite environment, the mixed metal systems exhibited a unique antibiofilm effect, as described in the following. The extent of biofilm and planktonic growth in the presence of increasing mixed metal loading of (0.25 mg Cu + 0.25 mg Fe), (0.5 mg Cu + 0.5 mg Fe) and (0.75 mg Cu + 0.75 mg Fe) were investigated. The presence of (0.25 + 0.25) mg mixed metal complexes was benign to the bacterial growth, with comparable biofilm and planktonic biomass detected as those in the presence of bare coupon as control (Figure 5a). Increasing the metal loading to (0.5 + 0.5) mg caused a 20% reduction in biofilm biomass relative to the control growth and in parallel, a 20% increase in planktonic biomass. Ultimately, at (0.75 +

The iron complex-facilitated copper(II)/copper(I) redox cycling is supported by the observed changes in the binding energy of iron and copper species, as revealed by XPS analysis of the mixed metal complexes before and after addition of nitrite and the subsequent NO generation (Figure 4). The XPS spectra of FeDTTCT in the mixed metal coupon can be deconvoluted into two Fe 2p3/2 peaks at 711.3 and 713.9 eV, with a satellite peak at 719.4 eV (Figure 4a top/before reaction), which is comparable to those of the as-synthesized FeDTTCT (Figure S2). Note that to the best of our knowledge, there is currently no published data on the XPS spectra of FeDTTCT, and the position of iron(II) and iron(III) peaks in Fe 2p region could differ depending on the ligand species.38 The CuDTTCT in the mixed metal coupon mainly exists as copper(II) species, with detection of Cu 2p3/2 peak at 934.7 eV along with two satellite peaks at 940.1 and 943.7 eV (Figure 4b top/before reaction). Following NO generation in the presence of nitrite, a significant Fe 2p3/2 peak shift from 711.3 to 712.1 eV was observed, along with the disappearance of the second Fe 2p3/2 peak (the satellite peak also shifts to 718.6 eV) (Figure 4a bottom/after reaction). This shift of Fe 2p3/2 peak to higher binding energy indicates oxidation of the iron complex.38,39 Post reaction XPS analysis also revealed the emergence of copper(I) peak at 933.9 eV (Figure 4b bottom/ after reaction), which as stated previously, is the active species in NO generation reaction. Therefore, this observation implies the role of the iron complex as a reducing agent, facilitating the catalytic activity of copper through copper(II)/copper(I) redox cycling for generation NO from nitrite. Finally, comparative studies on catalytic NO generation in solutions containing nitrite and ascorbic acid (as reducing agent) detected a significantly higher NO concentration in the presence of CuDTTCT than that of FeDTTCT (Figure S3). This further validates the role of FeDTTCT in facilitating the reduction of copper(II) to copper(I) in the mixed metal systems, rather than catalyzing the reduction of nitrite to NO. 3.3. Antibiofilm Activity of CuDTTCT+FeDTTCT Mixed Metal Coupon by the Involvement of Catalytically E


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observed with the presence of the corresponding leached soluble species from the mixed metal systems (Table 1note the comparable total leaching of ∼0.5 mg/L Cu and Fe with the increasing loading of the mixed metal systems) and with the presence of the Cu or Fe metal complex alone. As shown in Figures S4 and S5, the presence of up to 2 mg FeDTTCT (with 0.6 mg/L leached Fe, Table S1) alone, or up to 2 mg CuDTTCT (with 1.2 mg/L leached Cu, Table S2) alone, did not alter the extent of biofilm growth and the presence of planktonic biomass relative to the control. Therefore, this apparent “dispersal-type” antibiofilm activity is most likely to result from the presence of NO, which in this case was catalytically generated by the mixed metal complexes from endogenous nitrite, whereby the latter was produced by the AOB-containing biofilms. To confirm the involvement of the catalytically generated NO in the observed antibiofilm activity, the well-characterized NO scavenger PTIO40,41 was added into the mixed metal systems. Commonly used to provide insight into the physiological function of NO,41 we validated the scavenging activity of 100 μM PTIO toward catalytically generated NO (from added nitrite) in the mixed metal CuDTTCT +FeDTTCT systems (Figure 3). The PTIO concentration, as also reported in earlier studies,40 only had minimal effect on the biofilm growth (Figure S6). Here, the presence of 100 μM PTIO in the mixed metal systems were found to eliminate the antibiofilm effects previously seen with the (0.5 + 0.5) mg or (0.75 + 0.75) mg mixed metal complexes, as comparable amount of biofilm and planktonic biomass as those of the control growth was observed (p value =0.99 vs the control or control+PTIO; Figure 6).

Figure 5. (a) Protein measurements of nitrifying bacteria biomass grown in the presence of mixed metal coupons containing various loading of CuDTTCT and FeDTTCT. All values shown are normalized to the bacteria biomass grown in the presence of bare coupon (control, inset). Error bars indicate standard error between replicates (n = 4); *p ≤ 0.05 against the control. (b) Surface coverage analysis of LIVE/DEAD-stained nitrifying biofilm. All values shown are normalized to the surface coverage of control. Error bars indicate standard error between replicates (n = 3); *p ≤ 0.05 against the control. Confocal laser scanning microscopy images of nitrifying biofilm grown in the presence of (c) bare coupon, (d) mixed metal coupon with (0.25 + 0.25) mg loading, (e) (0.5 + 0.5) mg loading, and (f) (0.75 + 0.75) mg loading. Scale bar =100 μm.

0.75) mg metal loading, a 36% decrease in biofilm biomass was observed, which was followed by the detection of a 70% increase in planktonic biomass relative to the control. The increase in planktonic biomass detected in the (0.75 + 0.75) system indicates that growth inhibition is unlikely. Surface coverage analysis by confocal laser scanning microscopy (CLSM) (Figure 5b) further confirmed the above-mentioned trend from the protein assay. Although significant reduction in biofilm surface coverage was not found for the (0.25 + 0.25) mg and (0.5 + 0.5) mg mixed metal systems (p values of 0.40 and 0.59, respectively), a 56% overall reduction in biofilm surface coverage relative to the control was observed for the (0.75 + 0.75) mg system. Note that the CLSM analysis was carried out to account for potential interference of biofilm EPS in the protein assay. Further, only minimal toxicity effects were

Figure 6. Protein measurements of nitrifying bacteria biomass grown in the presence of mixed metal coupons containing various loading of CuDTTCT and FeDTTCT with the addition of PTIO (100 μM). All values shown are normalized to the bacteria biomass grown in the presence of bare coupon (control, inset). Error bars indicate standard error between replicates (n = 4); *p ≤ 0.05 against the control.

Table 1. ICP-OES Measurements of Leached Copper and Iron Ions from the Mixed Metal Coupons after 3 Days Incubation with Nitrifying Bacteria samples

leached copper (mg/L)

leached iron (mg/L)

%mass of copper leached out

%mass of iron leached out

0.25 + 0.25 0.5 + 0.5 0.75 + 0.75

0.11 ± 0.04 0.21 ± 0.06 0.40 ± 0.01

0.34 ± 0.04 0.28 ± 0.03 0.12 ± 0.02

0.27 0.25 0.29

0.82 0.40 0.09

F


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ACS Applied Materials & Interfaces Taken together, the findings infer to the capability of the CuDTTCT+FeDTTCT mixed metal complexes to catalytically convert nitrite that was endogenously produced by nitrifying bacteria biofilm, to NO. The NO in this case subsequently suppressed the biofilm formation, while confining bacterial growth to the planktonic phase. Such NO-mediated biofilm dispersal has been reported in a wide spectrum of biofilmforming microorganisms.2,6,42,43 A common mechanism underlying the biofilm to planktonic phase transition is via the NOregulated alteration of intracellular levels of the secondary messenger cyclic di-GMP (c-di-GMP), already observed in various bacteria, including Pseudomonas aeruginosa and Shewanella oneidensis.44−46 In general, low cellular c-di-GMP levels tend to induce biofilm dispersal, activate cell’s motility and promote planktonic growth, while an increase in cellular cdi-GMP level promote cell attachment to surfaces and biofilm formation.4 In Nitrosomonas europaea, which is one of the major members of AOB, the link between c-di-GMP signaling and NO-mediated dispersal has yet been fully established. Instead, NO-mediated dispersal in the bacteria has been shown to involve up-regulations of 11 proteins, such as motility (flagellar) and flagellar assembly proteins.47 Due to the involvement of signaling pathways in NO-mediated dispersal, cellular membrane damage is not expected to occur, as confirmed by the confocal images shown earlier (Figure 5c−f, no propidium iodide (PI)-positive red fluorescent cells were observed; note that PI only enters cells with compromised cytoplasmic membrane). Such “nontoxic” mode of biofilm dispersal, as described in the following, often associates with a unique characteristic of NO release. The antibiofilm effects were observed over relatively long period of 3 days and given the short half-life of NO in biological systems, this suggests continual slow release of low concentrations of NO by the mixed CuDTTCT+FeDTTCT metal systems. The slow release is a likely consequence of interactions between the redox cycling of the complexes’ metallic centers and the activity of the slow growing AOB as nitrite producer. Further, the iron center could act as an electron mediator that receive electrons from bacteria,48 such as Bacillus spp. (also present in the bacterial inoculum), which have been shown to utilize (“breathe”) insoluble substrates such as iron(III).49 Such cellular respiration can occur via secretion and subsequent transfer of extracellular redox metabolites from the bacteria known as electron shuttles, to the iron substrate, or alternatively can involve direct contact between cell appendages (e.g., pili proteins or EPS components) with the iron substrate.50 The effects could restart the redox cycling of iron and copper and subsequently, the generation of NO from nitrite produced by the AOB. As observed in the current study, the indicated slow release of NO at low concentrations tend to induce a dispersal effect converting bacteria to the free-floating planktonic phase, which will otherwise induce lethal effects on biofilms if rapidly generated at high levels.14,51,52 Due to its nongrowth inhibiting or noncell killing antibiofilm effect, the development of bacterial resistance to NO is unlikely.53,54 This importantly implies minimal emergence of highly tolerant biofilm with the use of CuDTTCT+FeDTTCT metal complexes as NO-based antibiofilm technology. It remains unclear, however, as with the exact mechanisms of the prolonged catalytic generation of NO in the mixed metal systems. Future research will be directed toward a more detailed understanding of this process.

4. CONCLUSIONS An iron complex (FeDTTCT) was successfully synthesized and incorporated into PVC matrix together with an active copper species (CuDTTCT) for catalytic generation of antibiofilm NO from nitrite. Amperometric measurement showed that the mixed metal complexes (CuDTTCT+FeDTTCT) in nitrite solution was able to generate NO without additional reducing agent. NO was generated from nitrite through copper(II)/ copper(I) redox cycling, whereby nitrite reduction to NO is via reaction with copper(I), the latter was formed from reduction of copper(II) facilitated by the iron complex, as shown by XPS analysis. Here, we found that the mixed metal complexes were capable of converting nitrite that was endogenously produced by nitrifying bacteria biofilms, into NO. The generated NO subsequently exhibited a “dispersal-type” antibiofilm activity or in other words, suppressing the biofilm formation without inhibiting bacterial growth. The involvement of NO in the antibiofilm activity was validated by the use of NO scavenger, whereby its presence in the mixed metal system canceled the antibiofilm effects. The current study highlights the discovery of metal complexes-based material capable of sustained catalytic generation of NO from endogenous nitrite for antibiofilm application.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10357. DTTCT NMR spectrum, XPS spectra of as-synthesized FeDTTCT, NO generation measurement from the metal complex in the presence of nitrite and ascorbic acid, the effect of FeDTTCT and CuDTTCT coupon and its corresponding iron and copper ions leached from the coupons, effect of PTIO on nitrifying bacteria growth (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.H.Y.). *E-mail: [email protected] (R.A.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported under Australian Research Council’s Linkage Projects funding scheme (LP110100459). We gratefully acknowledge the provision of polyvinyl chloride from Chemson Pacific Pty. Ltd., and the financial support from Australian Water Quality Centre (SA Water) and Western Australia Water Corporation. We would like to thank Mark Wainwright Analytical Centre for their assistance, especially for XPS analysis (Dr. Bill Gong) and ICP-OES analysis (Dr. Rabeya Akter and Dr. Dorothy Yu)

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REFERENCES

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Iron Complex Facilitated Copper Redox Cycling for Nitric Oxide

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