Laura Vaugon

Annie Finiels

Thomas Cacciaguerra

Vasile Null Hulea

Anne Galarneau

Cindy Aquino

Jean-Pierre Dath

Delphine Minoux

Corine Gerardin

Francois Fajula

Impact of Pore Architecture on the Hydroconversion of Long Chain Alkanes over Micro and Mesoporous Catalysts

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Professor Patrick Rossignol
email: p.rossignol@chru-nancy.fr

Mitja Lainscak

Maria G Crespo-Leiro

Cécile Laroche

Massimo F Piepoli

Gerasimos Filippatos

Giuseppe Mc

Gianluigi Savarese

Stefan D Anker

Frank Ruschitzka

Giuseppe Mc Rosano

Andrew Js Coats

Alexandre Mebazaa

Theresa Mcdonagh

Ana Sahuquillo

Maria Penco

Aldo P Maggioni

Lars H Lund

Data from 9222 chronic heart failure patients of the ESC-HFA-EORP Heart Failure Long-Term Registry

Keywords: Hyperkalaemia, Hypokalaemia, Heart Failure, Renin Angiotensin Aldosterone System inhibitors, Prognosis-mineralocorticoid receptor antagonists

come

Unravelling the interplay between hyperkalaemia, renin-angiotensin-aldosterone inhibitor use and clinical outcomes.

Introduction

Inhibitors of the renin-angiotensin-aldosterone system (RAASi) (i.e. angiotensin-converting enzyme inhibitors [ACEs], angiotensin receptor blockers (ARBs), mineralocorticoid-receptor antagonists [MRAs], and angiotensin receptor neprilysin inhibitors [ARNis]), and β blockers are the cornerstones of therapy in patients with chronic heart failure (CHF) with reduced ejection fraction (HFrEF). Based on the highest level of evidence, they are strongly recommended in international guidelines (class IA(1)) for improving survival, decreasing sudden death, and preventing heart failure hospitalisations in patients with HFrEF [START_REF] Rossignol | Heart failure drug treatment[END_REF].

International registry data have shown that adherence to guideline-recommended medications in HFrEF is associated with improved outcomes [START_REF] Komajda | Physicians' guideline adherence is associated with better prognosis in outpatients with heart failure with reduced ejection fraction: the QUALIFY international registry[END_REF]. However, guidelines are not being implemented [START_REF] Greene | Medical Therapy for Heart Failure With Reduced Ejection Fraction: The CHAMP-HF Registry[END_REF], both in terms of overall use and target doses, often because of actual, or concerns for potential adverse events (2) [START_REF] Savarese | Factors associated with underuse of mineralocorticoid receptor antagonists in heart failure with reduced ejection fraction: an analysis of 11 215 patients from the Swedish Heart Failure Registry[END_REF][START_REF] Lund | Association between enrolment in a heart failure quality registry and subsequent mortality-a nationwide cohort study[END_REF]. Hyperkalaemia, hypotension, deterioration of renal function, higher age and poor access to cardiologist care are the most frequently cited reasons for underdosing, underuse and discontinuation of RAAS inhibitors in patients with HFrEF [START_REF] Rossignol | Heart failure drug treatment[END_REF][START_REF] Komajda | Physicians' guideline adherence is associated with better prognosis in outpatients with heart failure with reduced ejection fraction: the QUALIFY international registry[END_REF][START_REF] Savarese | Factors associated with underuse of mineralocorticoid receptor antagonists in heart failure with reduced ejection fraction: an analysis of 11 215 patients from the Swedish Heart Failure Registry[END_REF][START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF][START_REF] Trevisan | Incidence, predictors and clinical management of hyperkalaemia in new users of mineralocorticoid receptor antagonists[END_REF] and at the same time, K is poorly monitored [START_REF] Nilsson | A real-world cohort study on the quality of potassium and creatinine monitoring during initiation of mineralocorticoid receptor antagonists in patients with heart failure[END_REF], even though benefits of RAASi agents appear as important in patients with severe CKD [START_REF] Edner | Association between renin-angiotensin system antagonist use and mortality in heart failure with severe renal insufficiency: a prospective propensity score-matched cohort study[END_REF] and older age [START_REF] Savarese | Association between reninangiotensin system inhibitor use and mortality/morbidity in elderly patients with heart failure with reduced ejection fraction: a prospective propensity score-matched cohort study[END_REF]. The ESC HFA EORP Heart Failure Long-Term Registry (HF-LT) identified hyperkalaemia, impaired kidney function and hypotension as the main causes for the non-prescription or underdosing of RAASi [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF].

In a recent observational study including all Stockholm citizens initiating MRA therapy, the development of hyperkalaemia within a year was associated with a four-fold significantly higher risk in overall mortality, with the results being consistent in the subpopulation of patients with HF. Following the occurrence of hyperkalaemia, 47% discontinued MRA whereas only 10% reduced the prescribed dose. Strikingly, when MRA was discontinued, most patients (76%) were not reintroduced to MRA therapy during the subsequent year [START_REF] Trevisan | Incidence, predictors and clinical management of hyperkalaemia in new users of mineralocorticoid receptor antagonists[END_REF].

Reintroduction of RAASi is less likely the longer the break in therapy [START_REF] Gjesing | Patient adherence to evidence-based pharmacotherapy in systolic heart failure and the transition of follow-up from specialized heart failure outpatient clinics to primary care[END_REF]. Therefore, hyperkalaemia, beyond its potentially lethal proarrhythmogenic properties, may be both a biomarker of RAASi under-use and under-dosing, a barrier to RAASi use, and a trigger for subsequent RAASi discontinuation, with all potentially being associated with poor outcomes in various populations [START_REF] Rossignol | A new area for the management of hyperkalaemia with potassium binders: clinical use in nephrology[END_REF], including HF [START_REF] Epstein | Evaluation of the treatment gap between clinical guidelines and the utilization of renin-angiotensinaldosterone system inhibitors[END_REF].

We hypothesize that hyperkalaemia-associated increased death may be related to RAASi withdrawal. Therefore, using the ESC HFA EORP Heart Failure Long-Term Registry (HF-LT), we aimed to assess the interplay between hyperkalaemia (and its determinants), RAASi prescription and dosing at baseline, RAASi discontinuation, and associations with outcomes in out-patients with CHF enrolled in the HF-LT registry.

Methods

Data Source and Study Population

The primary objective of the ESC-HFA EORP HF-LT registry is the description of the clinical epidemiology of HF out-patients and in-patients in ESC and affiliated countries, as well as the diagnostic and therapeutic processes used in the care of these patients. All national cardiology societies affiliated to the ESC were invited to participate [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF][START_REF] Maggioni | EURObservational Research Programme: regional differences and 1-year follow-up results of the Heart Failure Pilot Survey (ESC-HF Pilot)[END_REF]. The ESC HFA EORP HF-LT registry (HF-LT) is a prospective, multicentre, observational registry of patients referred to a broad range of cardiology centres in 31 countries. The 31 countries are Belarus, Bulgaria, Croatia, Czech Republic, Georgia, Hungary, Moldova, Poland, Romania, Serbia, Slovakia, Slovenia (Eastern countries), Israel (Middle East), Egypt (North Africa), Denmark, Estonia, Latvia, Lithuania, Sweden (Northern), Bosnia Herzegovina, Cyprus, Greece, Italy, Kosovo, Macedonia, Portugal, Spain, Turkey (Southern), Austria, France, Switzerland (Western). The study design has previously been described [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF][START_REF] Crespo-Leiro | European Society of Cardiology Heart Failure Long-Term Registry (ESC-HF-LT): 1-year followup outcomes and differences across regions[END_REF]. Briefly, there were no specific exclusion criteria, with the exception of age, which had to be greater than 18 years. Patients were followed up in accordance with the usual practice of the centres, with the exception of a mandatory follow-up visit at 12 months to collect information on morbidity and mortality. In cases where the patient was unable to reach the clinical centre, a phone survey replaced this follow-up clinical visit. The HF-LT registry was approved by each local ethics board and written informed consent obtained in accordance with each country's legislation. Random audits were conducted in each participating country. ESC-HF-LT registry enrolled all the out-patients with CHF seen at the clinics and those admitted to hospital for AHF (either pre-existing or new-onset HF) for whom intravenous HF therapy (diuretics, inotropes or vasodilators) was used [START_REF] Crespo-Leiro | European Society of Cardiology Heart Failure Long-Term Registry (ESC-HF-LT): 1-year followup outcomes and differences across regions[END_REF]. Patients were enrolled 1 day per week for 12 consecutive months [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF].

In the current analysis only HF out-patients (i.e. chronic HF) were considered.

Compared to the previous publications where 7401 [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF] (16) CHF patients were described, a total of 9222 patients could now be described at the time the present research question was first addressed (2017). RAASi target doses were defined as in the ESC HF guidelines related to HFrEF [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF]. Patients who were receiving a RAASi dose equal to or above 50% of the target dosage were compared to those who received less than 50% or no treatment. This threshold was based on the recent observation in the European setting that patients reaching <50% of the recommended ACE-inhibitor/ARB (and beta-blocker) dose displayed an increased risk of death and/or heart failure hospitalisation. Patients reaching 50-99% of the recommended ACE-inhibitor/ARB and/or beta-blocker dose had a comparable risk of death and/or heart failure hospitalisation to those reaching 100% [START_REF] Ouwerkerk | Determinants and clinical outcome of uptitration of ACE-inhibitors and beta-blockers in patients with heart failure: a prospective European study[END_REF].

Statistical Analyses

Descriptive statistics were used to summarise frequency tabulations (n, %) and distributions [median, interquartile range (IQR)]. Categorical variables were compared using the Chi-square test or Fisher exact test and continuous variables with a non-parametric test (Kruskal-Wallis test).

Predictors of MRA use

Because MRAs is the RAASi category most affecting K homeostasis [START_REF] Bandak | Hyperkalemia After Initiating Renin-Angiotensin System Blockade: The Stockholm Creatinine Measurements (SCREAM) Project[END_REF] and also the least likely to be started and the most likely to be stopped in HF [START_REF] Thorvaldsen | Use of evidence-based therapy and survival in heart failure in Sweden 2003-2012[END_REF] we assessed predictors of 1) non-use or at low dose at baseline 2) MRA discontinuation during the follow-up. In order to investigate why patients with low doses of MRAs or without MRAs at baseline or with

subsequent MRA discontinuation at one-year follow-up, logistic regression models were performed with a stepwise procedure, using a p-value < 0.05 to allow entry in the model and a p-value < 0.05 to remain in the updated model. Independent variables that were significant at univariable analysis (p<0.10) as well as variables considered of relevant clinical interest were included at the beginning in the multivariable model. Potential interactions between baseline characteristics and baseline kalaemia (as a categorical variable) were tested. For the logistic regression model, MRA discontinuation corresponds to patients with any MRA agent at baseline but not at one-year follow-up (i.e. stopped during the 1-year follow-up or at the 1year follow-up visit).

Outcomes

For all-cause and cardiovascular death, Cox regression models including baseline potassium level were performed using the same stepwise procedure as in the logistic regression models. Two different models were performed : 1) including ARBs, ACEi and MRA, separately, as use vs. non-use at the baseline; 2) comparing RAASi use at the baseline but not at the followup (i.e. discontinued) and, separately, no use of RAASi at the baseline and at the follow-up with RAASi use at baseline and at 1-year follow-up. In addition, natural cubic splines were presented for the unadjusted association between potassium levels and outcomes.

Finally, three mediations analyses were performed [START_REF] Lange | A simple unified approach for estimating natural direct and indirect effects[END_REF] for ACEi, ARB or MRA discontinuation, where the "direct effect" was the direct effect of the potassium level on overall/cardiovascular mortality, meaning if significant that the potassium level directly impacts the mortality. The indirect effect means that the potassium level has an impact on MRA/ACEi/ARB discontinuation, which has its own impact on mortality. P-value <0.05 was used as a cut-off for statistical significance. All analyses were performed in SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA). 1. In this mixed population of HFrEF (60.6%), HF with mid-range (HFmrEF) (22.9%) and preserved ejection fraction (HFpEF) (16.5%), with the latter two without any guideline recommendation for RAASi (2), most patients were treated with an ACEi or an ARB (88.3%), an MRA (58.7%) or a combination thereof (53.2%), mostly at doses above or equal to 50% (ACEi: 61.8% ≥ 50% of the target dose; ARB: 64.7% ; MRA: 90.3%). After one year, the frequency was 57.5%, 23.5%, 79.1% and 54.3% for ACEi, ARB, ACEi/ARB and MRA respectively.

Results

Between

Factors associated with RAASi prescription and dosing at baseline, or RAASi discontinuation

Patients with hyperkalaemia at baseline were older, more frequently men, with diabetes and lower eGFR compared to normokalaemic patients (4.0-5.0 mmol/L). There were no significant differences in the proportion of patients with HFrEF, HFmrEF or HFpEF across the potassium strata (Table 1). ACEi use was more common in mildly hyperkalaemic (5-5.5 mmol/L) patients while MRA use was more common in normokalaemic patients. The coprescription of ACEI/ARB and MRA was less common in patients with serum potassium 5.5 mmol/L. While the percentage of ACEi or ARB target dose at baseline did not differ significantly across the potassium categories, hyperkalaemic patients were 2-3 times more frequently prescribed an MRA at < 50% of the target dose at baseline (Table 1).

Hyperkalaemic patients were also more prone to undergo ACEI/ARB or MRA discontinuation during follow-up (table1).

Multivariable analyses did not confirm an association between baseline potassium categories and no use or low dose use of ACEI or ARB at baseline, while lower systolic blood pressure (SBP), ejection fraction (EF) >50% (vs. <40%) (for ACEI drug class) and renal dysfunction (for both drug classes) were found significantly and independently associated with no use or low dose use at baseline (data not shown). In multivariable analysis, hyperkalaemic patients (5 -5.5 mmol/L vs. 4 -5 mmol/L): ≥5.5 mmol/L vs. 4 -5 mmol/L, and patients with renal dysfunction, along with those with higher SBP were more prone to receive lower doses or no MRA at baseline (Figure 1).

In multivariable analyses, lower BP and kidney dysfunction were significantly associated with subsequent discontinuation of ACEI/ARB while potassium levels were not (data not shown). In contrast, renal dysfunction and potassium levels (hyperK 5.5 mmol/L) were associated with MRA discontinuation, along with other factors such as older age, EF >50 % vs. <40 %, and comorbidities (Figure 2).

Factors associated with clinical outcomes

In univariable analysis, a U-shaped relationship was observed between baseline potassium and risk of all-cause death (Figure 3A), with a statistically significant association above approximately 5 mmol/ for HK and below 4 mmol/L for hypokalaemia. In multivariable analysis, HK 5.5 mmol/L remained associated with greater risk of death [HR 1.40 (1.02-1.92), p=0.038], as was hypokalaemia [(3 -4 mmol/L), HR 1.26 (1.02-1.55), p= 0.031], while ACEI and/or ARB prescriptions at baseline were associated with better outcomes (HR 0.70 (0.57-0.86), p<0.001; HR 0.70 (0.56-0.89), p=0.004). MRA prescription was not retained in the model (p= 0.476). (Figure 4) When considering RAASi (ACEi, ARB, MRA) baseline percent of target doses instead of only use vs. non-use, the non-prescription of ACEi performed significantly worse compared to the prescription of ACEi at lower than 50% of the target doses (HR 1.42 (1.13;1.79), p=0.003), while the comparison between <50% and ≥50% target doses did not reach statistical significance. In this model, only baseline hypokalaemia was associated with all-cause mortality (HR 1.25 (1.01-1.56), p=0.042).

When considering subsequent discontinuation of RAASi for adjustments (instead of baseline use or dose) and serum potassium at baseline, hyperkalaemia was no longer associated with risk of all-cause death while all RAASi (ACEI, ARB, or MRA) discontinuations, regardless of class, were strongly associated with the outcomes (Figure 5).

In a sensitivity univariable analysis, ACEI, ARB or MRA discontinuations were found associated with all-cause death across the ejection fraction strata (i.e. EF <40, 40-50, >50 %: data not shown)

In further sensitivity analyses, CV rather than all-cause deaths were considered. In univariable analysis, a U-shaped relationship was observed between baseline potassium and risk of CV death (Figure 3B), with a significant association for HK and for hypokalaemia.

However, serum potassium concentrations were not identified as a predictor of CV death in the multivariable models, while use of ACEI or ARB at baseline were associated with better outcomes. (Online data supplement Table 1). When considering RAASi (ACEi, ARB, MRA) baseline target doses instead, only the non-prescription of ACEi or ARB performed significantly worse compared to the prescription of ACEi or ARB lower than 50% of the target doses (Online data supplement Table 2) Again, as for all-cause death, ACEI, ARB or MRA discontinuations were all associated with a higher risk also of CV death (online data supplement Table 3). In a sensitivity univariable analysis, ACEI, ARB or MRA discontinuations were found associated with CV death across the ejection fraction strata (i.e. EF <40, 40-50, >50 %: data not shown) Finally, three multivariable mediation analyses (for ACEi, or ARB, or MRA discontinuation, respectively) were performed. Hyperkalaemia (>5.5 mmol/L) was associated with mortality and CV mortality by mediating discontinuation of ACEi (marginally significant for all-cause death: P= 0.059), ARB (not statistically significant), and MRA (P= 0.007 for total mortality; P= 0.080 for CV death) (online data supplement Table 4).

Discussion

In this contemporary European out-patient HF cohort, with a high rate of patients treated with RAASi compared to other HF populations, the prevalence of hyperkalemia was high (16.6%), while the 1-year outcome was poor (overall and CV death: 8.6% and 4.4%, respectively).

Similar to a number of surveys in post-discharge post-acute HF [START_REF] Nunez | Long-Term Potassium Monitoring and Dynamics in Heart Failure and Risk of Mortality[END_REF][START_REF] Legrand | Association between hypo-and hyperkalemia and outcome in acute heart failure patients: the role of medications[END_REF], chronic HFrEF [START_REF] Vardeny | Incidence, Predictors and Outcomes Related to Hypo and Hyperkalemia in Severe Heart Failure Patients Treated with a Mineralocorticoid Receptor Antagonist[END_REF] and HFpEF [START_REF] Nishihara | Serum Potassium and Cardiovascular Events in Heart Failure With Preserved Left Ventricular Ejection Fraction Patients[END_REF] patients, a U-shape relationship between baseline serum potassium and allcause death and CV death was observed over one-year follow-up in univariate analysis.

In a multivariable model considering all deaths, both hypokalaemia and HK were associated with poor outcomes, while ACEi/ARB but not MRA prescription was found to be independently associated with better outcomes. The absence of association between MRA use and outcomes has been previously described in observational settings and is most likely due to residual unmeasured confounding, such as higher use of MRAs in patients with refractory oedema, ascites, liver disease, diuretic resistance, high dose diuretic use, and/or other factors associated with poor outcomes. However, in additional multivariable analyses, when considering RAASi doses at baseline or subsequent RAASi discontinuation -instead of considering RAASi prescription at baseline -only RAASi discontinuations (ACEI, ARB, or MRA) were found consistently associated with all-cause and CV death, independent from serum potassium at baseline and, importantly, hyperkalaemia was no longer associated with the clinical outcomes. Moreover, in multivariable analyses, hyperkalaemia at baseline was found to be associated with MRA non-prescription at baseline and with its subsequent discontinuation. Additional mediation analyses showed that hyperkalaemia above 5.5 mmol/L was associated with mortality and CV mortality by mediating discontinuation of ACEi, ARB, MRA, although it was not statistically significant for all. This was actually much more significant for MRA and suggests that the hyperkalaemia is more important through MRA discontinuation than through ACEi/ARB discontinuation.

Altogether, these data provide insights into the interplay between hyperkalaemia, RAASi use and clinical outcomes in real-life HF settings. Indeed, they clearly depict the current dilemma facing the implementation of these life-saving drugs, in particular MRAs, in routine practice. The present findings complement numerous previous reports from The HF-LT Registry and from the Swedish Heart Failure Registry studying the reasons for nonprescription of RAASi [START_REF] Savarese | Factors associated with underuse of mineralocorticoid receptor antagonists in heart failure with reduced ejection fraction: an analysis of 11 215 patients from the Swedish Heart Failure Registry[END_REF][START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF][START_REF] Thorvaldsen | Use of evidence-based therapy and survival in heart failure in Sweden 2003-2012[END_REF][START_REF] Savarese | Incidence, Predictors, and Outcome Associations of Dyskalemia in Heart Failure With Preserved, Mid-Range, and Reduced Ejection Fraction[END_REF]. In the large majority of these cases, reported contraindications, a documented intolerance or concerns about potential intolerance (hyperkalaemia, worsening renal function, hypotension) were the main reasons for the nonprescription of these drugs [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF]. From this HF-LT Registry dataset, it was reported that the true rate of under-treatment for ACEi/ARB and MRAs in this well treated population was 3.2% and 5.4%, respectively (compared to others ( 4)), at least in terms of RAASi use [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF]. We also previously reported that with regard to the target dosages of these drugs, fewer than one-third of the patients were currently prescribed the target dosages suggested by current guidelines: 29.3% for ACEIs, 24.1% for ARBs, and 30.5% for MRAs [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF].

For the present study, the threshold of 50% of the recommended ACEI /ARB (and beta-blocker) was selected since in a recent European survey, patients receiving less displayed an increased risk of death and/or heart failure hospitalisation [START_REF] Ouwerkerk | Determinants and clinical outcome of uptitration of ACE-inhibitors and beta-blockers in patients with heart failure: a prospective European study[END_REF]. Altogether, these data confirm that hyperkalaemia in itself is a major barrier toward the implementation of, and importantly maintenance of RAASi and more specifically of MRA, since prescribers, in accordance with the guidelines, are not prone to use MRA [START_REF] Komajda | Physicians' guideline adherence is associated with better prognosis in outpatients with heart failure with reduced ejection fraction: the QUALIFY international registry[END_REF][START_REF] Shirazian | Underprescription of renin-angiotensin system blockers in moderate to severe chronic kidney disease[END_REF] or to discontinue them in presence of hyperkalaemia [START_REF] Trevisan | Incidence, predictors and clinical management of hyperkalaemia in new users of mineralocorticoid receptor antagonists[END_REF][START_REF] Chang | Antihypertensive Medications and the Prevalence of Hyperkalemia in a Large Health System[END_REF]. In other words, beyond its proarrythmogenic properties, hyperkalaemia could also be a risk marker that leads to suboptimal use of RAASi, in particular MRAs, which in turn is causative of poor outcomes, as also suggested by previous surveys [START_REF] Lund | Is hyperkalaemia in heart failure a risk factor or a risk marker? Implications for renin-angiotensin-aldosterone system inhibitor use[END_REF], and as shown here for the first time, being a marker of discontinuation.

Clinicians may be well served by recognizing the causes of RAASi underuse. The present analysis from the ESC-EORP-HF-LT Registry suggests that hyperkalaemia is harmful because it causes RAASi discontinuation and a similar study from the SwedeHF registry suggests that hyperkalaemia is harmful because it causes RAASi avoidance and discontinuation [START_REF] Cooper | Association between Potassium Level and Outcomes in Heart Failure wi 1 th Reduced Ejection Fraction: ACohort Study from the Swedish Heart Failure Registry[END_REF]. Together with a previous analysis from BIOSTAT suggesting that hyperkalaemia is in itself not harmful but prevents RAASi up-titration [START_REF] Beusekamp | Potassium and the use of renin-angiotensin-aldosterone system inhibitors in heart failure with reduced ejection fraction: data from BIOSTAT-CHF[END_REF] it is becoming increasingly clear that the universally poor implementation of RAASi and especially MRA drugs in HFrEF is extensively driven by actual or concerns for hyperkalaemia [START_REF] Lund | Is hyperkalaemia in heart failure a risk factor or a risk marker? Implications for renin-angiotensin-aldosterone system inhibitor use[END_REF]. Whether the vicious circle surrounding hyperkalaemia, which is an inherent risk factor for RAASi under-use, may be overcome by the availability of new potassium binders [START_REF] Pitt | Evaluation of the efficacy and safety of RLY5016, a polymeric potassium binder, in a double-blind, placebocontrolled study in patients with chronic heart failure (the PEARL-HF) trial[END_REF][START_REF] Pitt | Evaluation of an individualized dose titration regimen of patiromer to prevent hyperkalaemia in patients with heart failure and chronic kidney disease[END_REF] and/or novel MRAs less prone to hyperkalaemia warrants prospective CV outcome trials.

Limitations:

As previously acknowledged [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF], there are limitations of this and other registry analyses. All centres were cardiology centres, potentially limiting generalizability to internal medicine and primary care. To mitigate this issue, the centres in HF-LT were selected in proportion to the size of the population of the participating countries, taking into account the different technological levels of the cardiology centres invited to participate [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF].

More specifically in relation to the present analysis, observational data cannot establish causality. This is particularly relevant for the associations between RAASi use and better outcomes, where available randomized trials exist. Secondly, only baseline serum potassium and baseline target doses were considered, whereas we previously reported from this registry that, in approximately one third of the patients not achieving the target doses, a drug up-titration was still ongoing [START_REF] Maggioni | Are hospitalized or ambulatory patients with heart failure treated in accordance with European Society of Cardiology guidelines? Evidence from 12,440 patients of the ESC Heart Failure Long-Term Registry[END_REF], and neither the timing of RAASi discontinuation nor potassium levels thereafter were recorded. Third, the study population consisted of an amalgamation of HFrEF, HFmrEF and HFpEF patients (in whom target doses were derived from HFrEF recommendations). However, ACEI, ARB or MRA discontinuations were found associated with all-cause death and CV death across the ejection fraction strata (i.e. EF <40, 40-50, >50 %). Fourth, sacubutril/valsartan was not considered, not being approved at the time of data collection, while its use may be associated with less frequent hyperkalaemia in MRA recipients [START_REF] Desai | Reduced Risk of Hyperkalemia During Treatment of Heart Failure With Mineralocorticoid Receptor Antagonists by Use of Sacubitril/Valsartan Compared With Enalapril: A Secondary Analysis of the PARADIGM-HF Trial[END_REF]. However, findings related to the combined use of ACEi/ARB/MRA match the latest findings in a recently described European dataset. Indeed, the Dutch CHECK-HF registry of HFrEF patients observed that, between 2013-2016, 84% of patents were treated with ACEi/ARB and 56% with MRA, and approximately one-half of the HFrEF patients taking the prescribed medication were receiving less than the target dose of MRAs and ACEi/ARB [START_REF] Brunner-La Rocca | Contemporary Drug Treatment of Chronic Heart Failure With Reduced Ejection Fraction: The CHECK-HF Registry[END_REF].

In conclusion, in this large, contemporary, international CHF population, hyper and hypokalaemia-as well as non -use of ACEI/ARB were associated with worse outcomes. Importantly, after adjusting for discontinuation of a RAASi (ACEI/ARB or MRA), hyperkalaemia was no longer associated with increased risk, suggesting that hyperkalaemia may be a risk factor for RAASi discontinuation and through this association becoming a risk marker rather than a risk factor for worse outcomes [START_REF] Lund | Is hyperkalaemia in heart failure a risk factor or a risk marker? Implications for renin-angiotensin-aldosterone system inhibitor use[END_REF]. This was further confirmed by mediation analyses especially considering MRA discontinuation. Hyperkalaemia, also at baseline, represented a major hurdle against MRA use, thus its treatment and/or the prevention of its (re)occurrence under RAASi treatment may represent a potential therapeutic target.

Dr. Crespo Leiro reports grants from CIBERCV, personal fees and non-financial support from Novartis, personal fees from Abbott, personal fees and non-financial support from MSD, nonfinancial support from Daichi-Sankio, Vifor-Pharma, grants from Astra-Zeneka, outside the submitted work. Dr. Savarese reports grants and personal fees from Vifor, grants and non-financial support from Boehringer Ingelheim, personal fees from Societa´ Prodotti Antibiotici, grants from MSD, grants and personal fees from AstraZeneca, personal fees from Roche, Servier, grants from Novartis, personal fees from GENESIS outside the submitted work; Baseline factors associated with all-cause death in multivariable analysis considering RAASi prescription at the baseline

Notes:

Number of patients with complete data and included in the analysis: 8173/9222.

RAASi= Renin-Angiotensin-Aldosterone System inhibitors; CI= Confidence Interval; ACEi=

Hazard ratio (95% CI)

<3 vs [4 -5) [3 -4) vs [4 -5)

  Dr. Filippatos reports other from Novartis, Servier, BI, Medtronic, Vifor, outside the submitted work; Dr. Lainscak reports personal fees from Vifor, Astra Zeneca during the conduct of the study; grants from Roche, Boehringer Ingerlheim, personal fees from Novartis, outside the submitted work. Dr. Lund reports personal fees from Merck, grants from Boehringer Ingelheim, personal fees from Sanofi, grants and personal fees from Vifor-Fresenius, AstraZeneca, Relypsa, personal fees from Bayer, grants from Boston Scientific, grants and personal fees from Novartis, personal fees from Pharmacosmos, Abbott, grants and personal fees from Mundipharma, personal fees from Medscape, outside the submitted work; Dr. Maggioni reports personal fees from Bayer, Fresenius, Novartis outside the submitted work; C. Laroche, M Piepoli, G Rosano, T McDonagh, F Ruschitzka have nothing to disclose. Dr. Rossignol reports grants and personal fees from AstraZeneca, Bayer, CVRx, personal fees from Fresenius, grants and personal fees from Novartis, personal fees from Grunenthal, Servier, Stealth Peptides, Vifor Fresenius Medical Care Renal Pharma, Idorsia, NovoNordisk, Ablative Solutions, G3P, Corvidia, Relypsa, outside the submitted work; and Cofounder: CardioRenal. Cofounder: CardioRenal, a company developing a telemonitoring loop in heart failure (including potassium measurements) Dr. Sahuquillo reports personal fees from Servier during the conduct of the study;

Figures legendsFigure 1 :Figure 2 :Figure 3 A:Figure 3 B:

 1233 Figures legends

Figure 4 :Figure 5 :Figure 1

 451 Figure 4: Baseline factors associated with all deaths in multivariable analysis considering

Figure 2

 2 Figure 2

Figure 3 AFigure 3 BFigure 4

 334 Figure 3 A

  Body Mass Index; SBP= Systolic Blood Pressure; COPD= Chronic Obstructive Pulmonary Disease; EF= Ejection Fraction; PVD= Peripheral Vascular Disease; TIA= Transient Ischemic Attack; vs= versus. Variables are the same as in the previous model but instead of RAASi treatment at baseline, RAASi treatment correspond to RAASi treatment during the 1-year follow-up with for each treatment, 3 possibilities: discontinuationno treatment at baselineno discontinuation with No discontinuation as reference).

  April 2011 and May 2017, 19,136 patients were included in the Registry. Of these,

	8,290 (43.3%) were in-patients hospitalised with a diagnosis of AHF and 10,846 (56.7%)
	were out-patients with CHF. Among out-patients, baseline potassium values were available in
	9,222 (85.0%) patients (Online supplement Figure), who represented our study population,
	and of whom 16.6% had hyperkalaemia at baseline (≥5.0 mmol/L). Over a median follow-up
	of 371 days [363-427], there were 789 deaths (8.6%), 241 (2.6%) patients lost to follow-up,
	and 410 CV deaths (4.4%, or 52% of all deaths) as well as 195 deaths (2.1%, or 25% of all
	deaths) from unknown causes.
	Patient baseline features are presented in Table

Table 1 :

 1 Patient baseline characteristics and RAASi maintenance after one year

						[5 -5.5	≥5.5
			<3 mmol/L	[3 -4 mmol/L)	[4 -5 mmol/L)
		Total				mmol/L)	mmol/L
	Variable	Modality	(N=14,	(N=1431,	(N=6249,			P-value
		(N=9222)				(N=1202,	(N=326,
			0.2%)	15.5%)	67.8%)
						13.0%)	3.5%)
	Age, Median
		67 [58; 75]	56.5 [46; 68]	67 [56; 75]	66 [57; 75]	69 [60; 76]	69 [62; 78]	<0.001
	[Q1-Q3]
		2646/9222		471/1431	1779/6249	305/1202	86/326
	Female		5/14 (35.7%)					<0.001
		(28.7%)		(32.9%)	(28.5%)	(25.4%)	(26.4%)
	Body Mass Index (kg/m²)
	Median [Q1; Q3]

  Angiotensin-Aldosterone System inhibitors; SBP= Systolic Blood Pressure; HR= Heart Rate; NYHA= New York Heart Association; TIA= Transient Ischemic Attack; ICD= Implantable Cardioverter Defibrillator; CRT-P/D= Cardiac Resynchronization Therapy with or without defibrillator; eGFR= estimated Glomerular Filtration Rate; ACEi= Angiotensin Converting Enzyme inhibitor; ARB= Angiotensin Receptor Blocker; MRA= Mineralocorticoid receptor antagonists; BB= Beta Blocker.

	(mmHg) Median [Q1; Q3] HR (bpm) Median [Q1; Q3] Last known Ejection Fraction class, (%) Variable NYHA, (%) Pulmonary rales, (%) Peripheral oedema, (%) Variable Primary Etiology, (%) Variable Stroke/TIA, (%) Diabetes class, (%) Hypertension Treatment, (%) Peripheral vascular disease, (%) Chronic kidney dysfunction, (%) Variable eGFR (ml/min/1.73m²) Median [Q1; Q3] eGFR (ml/min/1.73m²) in class <30 <40% 40-50% >50% NYHA I NYHA II Modality NYHA III NYHA IV Modality Dilated cardiomyopathy Hypertension Ischemic heart disease Other Tachycardia related cardiomyopathy Valve disease Modality Modality 30-60 >60 ACEi, (%) Variable Modality Betablockers, (%) MRA (%) ACEi/ARB and MRA, (%) Diuretics Oral, (%) Ivabradine, (%) Variable Modality ACEi target dose, (%) < 50% max dosage ≥ 50% max dosage ARB target dose, (%) < 50% max dosage ≥ 50% max dosage MRA target dose, (%) < 50% max dosage Variable Modality ACE Discontinuation, (%) ARB Discontinuation, (%) BB Discontinuation, (%) MRA Discontinuation, (%) Covariates Age (in years) Aortic stenosis Atrial fibrillation BMI at baseline Systolic BP (in mmHg) at baseline 120 [110; 138] 120 [90; 144] 120 [110; 138] 120 [110; 138] 120 [110; 139] 70 [62; 80] 89 [71; 105] 70 [63; 81] 70 [61; 80] 70 [60; 79] 5073/8369 (60.6%) 4/12 (33.3%) 749/1236 (60.6%) 3445/5685 (60.6%) 678/1128 (60.1%) 1918/8369 (22.9%) 4/12 (33.3%) 275/1236 (22.2%) 1315/5685 (23.1%) 259/1128 (23.0%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 1378/8369 (16.5%) 4/12 (33.3%) 212/1236 (17.2%) 925/5685 (16.3%) 191/1128 (16.9%) 1512/9197 (16.4%) 2/14 (14.3%) 198/1420 (13.9%) 1058/6236 (17.0%) 209/1201 (17.4%) 5213/9197 (56.7%) 7/14 (50.0%) 771/1420 (54.3%) 3549/6236 (56.9%) 695/1201 (57.9%) 2288/9197 (24.9%) 4/14 (28.6%) 420/1420 (29.6%) 1510/6236 (24.2%) 278/1201 (23.1%) 184/9197 (2.0%) 1/14 (7.1%) 31/1420 (2.2%) 119/6236 (1.9%) 19/1201 (1.6%) 1365/8934 (15.3%) 2/14 (14.3%) 250/1369 (18.3%) 894/6038 (14.8%) 166/1189 (14.0%) 1859/8945 (20.8%) 8/14 (57.1%) 354/1371 (25.8%) 1216/6047 (20.1%) 215/1191 (18.1%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 2585/9171 (28.2%) 3/14 (21.4%) 382/1421 (26.9%) 1790/6210 (28.8%) 324/1201 (27.0%) 777/9171 (8.5%) 1/14 (7.1%) 125/1421 (8.8%) 516/6210 (8.3%) 101/1201 (8.4%) 3956/9171 (43.1%) 3/14 (21.4%) 588/1421 (41.4%) 2670/6210 (43.0%) 547/1201 (45.5%) 931/9171 (10.2%) 4/14 (28.6%) 149/1421 (10.5%) 628/6210 (10.1%) 124/1201 (10.3%) 132/9171 (1.4%) 0/14 (0.0%) 30/1421 (2.1%) 85/6210 (1.4%) 13/1201 (1.1%) 790/9171 (8.6%) 3/14 (21.4%) 147/1421 (10.3%) 521/6210 (8.4%) 92/1201 (7.7%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 860/9213 (9.3%) 1/14 (7.1%) 127/1428 (8.9%) 594/6246 (9.5%) 105/1199 (8.8%) 2988/9222 (32.4%) 5/14 (35.7%) 400/1431 (28.0%) 1959/6249 (31.3%) 484/1202 (40.3%) 5523/9208 (60.0%) 9/14 (64.3%) 859/1427 (60.2%) 3703/6239 (59.4%) 730/1202 (60.7%) 1149/8936 (12.9%) 0/14 (0.0%) 161/1370 (11.8%) 793/6040 (13.1%) 144/1188 (12.1%) 1870/9213 (20.3%) 2/14 (14.3%) 259/1425 (18.2%) 1145/6246 (18.3%) 327/1202 (27.2%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 62.9 [46.6; 79.9] 64.8 [49.3; 77.6] 65.0 [49.5; 81.2] 64.6 [48.6; 81.5] 55.5 [41.0; 73.0] 604/8583 (7.0%) 1/14 (7.1%) 79/1281 (6.2%) 335/5824 (5.8%) 111/1153 (9.6%) 3278/8583 (38.2%) 5/14 (35.7%) 457/1281 (35.7%) 2122/5824 (36.4%) 546/1153 (47.4%) 4701/8583 (54.8%) 8/14 (57.1%) 745/1281 (58.2%) 3367/5824 (57.8%) 496/1153 (43.0%) 6066/9210 (65.9%) 4/14 (28.6%) 879/1431 (61.4%) 4154/6239 (66.6%) 822/1201 (68.4%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 8197/9217 (88.9%) 8/14 (57.1%) 1234/1431 (86.2%) 5582/6244 (89.4%) 1100/1202 (91.5%) 5412/9218 (58.7%) 8/14 (57.1%) 838/1431 (58.6%) 3725/6245 (59.6%) 702/1202 (58.4%) 4907/9216 (53.2%) 4/14 (28.6%) 721/1431 (50.4%) 3395/6244 (54.4%) 661/1202 (55.0%) 7684/9217 (83.4%) 13/14 (92.9%) 1242/1431 (86.8%) 5149/6244 (82.5%) 1014/1202 (84.4%) 815/8958 (9.1%) 0/14 (0.0%) 116/1376 (8.4%) 560/6052 (9.3%) 114/1192 (9.6%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 2195/5743 (38.2%) 3/4 (75.0%) 338/827 (40.9%) 1483/3937 (37.7%) 296/782 (37.9%) 3548/5743 (61.8%) 1/4 (25.0%) 489/827 (59.1%) 2454/3937 (62.3%) 486/782 (62.1%) 666/1885 (35.3%) 0/1 (0.0%) 105/290 (36.2%) 464/1283 (36.2%) 76/250 (30.4%) 1219/1885 (64.7%) 1/1 (100.0%) 185/290 (63.8%) 819/1283 (63.8%) 174/250 (69.6%) 511/5253 (9.7%) 0/8 (0.0%) 69/807 (8.6%) 310/3611 (8.6%) 104/690 (15.1%) Total (N=9222) <3 mmol/L (N=14, 0.2%) [3 -4 mmol/L) (N=1431, 15.5%) [4 -5 mmol/L) (N=6249, 67.8%) [5 -5.5 mmol/L) (N=1202, 13.0%) 914/5237 (17.5%) 1/3 (33.3%) 147/724 (20.3%) 580/3599 (16.1%) 142/726 (19.6%) 418/1962 (21.3%) 0/1 (0.0%) 69/301 (22.9%) 268/1326 (20.2%) 53/261 (20.3%) 787/7161 (11.0%) 2/7 (28.6%) 137/1040 (13.2%) 495/4890 (10.1%) 110/979 (11.2%) 890/4748 (18.7%) 2/7 (28.6%) 135/701 (19.3%) 583/3295 (17.7%) 130/618 (21.0%) Comparison HR (95% CI) Pvalue 1.03 (1.02;1.04) <0.001 Yes vs No 1.92 (1.29;2.85) 0.001 Yes vs No 1.35 (1.08;1.69) 0.009 0.96 (0.94;0.99) 0.002 0.98 (0.97;0.98) <0.001 COPD Yes vs No 1.28 (0.98;1.67) 0.073 Depression Yes vs No 1.68 (1.22;2.31) 0.001 Diabetes class Yes vs No 1.44 (1.14;1.82) 0.002 Multivariable Covariates Comparison HR (95% CI) Pvalue Primary Etiology Ischemic heart disease vs Non-ischemic heart disease 1.15 (0.91;1.45) 0.249 Hepatic dysfunction Yes vs No 1.42 (0.92;2.18) 0.110 Chronic kidney dysfunction Yes vs No 1.82 (1.44;2.32) <0.001 Peripheral vascular disease Yes vs No 1.68 (1.29;2.18) <0.001 Stroke/TIA Yes vs No 1.36 (1.00;1.84) 0.050 Sex Female vs Male 0.80 (0.61;1.05) 0.111 Covariates Comparison HR (95% CI) Pvalue Systolic BP (in mmHg) at baseline 0.97 (0.97;0.98) <0.001 COPD Yes vs No 1.19 (0.89;1.58) 0.234 Depression Yes vs No 1.70 (1.22;2.37) 0.002 Diabetes Yes vs No 1.50 (1.17;1.92) 0.001 EF (%) 40-50 vs <40 0.97 (0.72;1.32) 0.861 >50 vs <40 0.79 (0.53;1.18) 0.256 Primary Etiology Ischemic heart disease vs Non-ischemic heart 1.20 (0.94;1.54) 0.145 Multivariable Covariates Comparison HR (95% CI) Pvalue Chronic kidney dysfunction Yes vs No 1.380 (1.062;1.79 3) 0.016 Peripheral vascular disease Yes vs No 1.755 (1.327;2.32 2) <0.001 Stroke/TIA Yes vs No 1.211 (0.871;1.68 4) 0.256 Sex Female vs Male 0.852 (0.634;1.14 5) 0.288 RAASi= Renin-Multivariable 40-50 vs <40 0.94 (0.70;1.25) 0.651 Beta blockers (Prior) Yes vs No 0.61 (0.46;0.80) <0.001 0.622 (0.462;0.83 disease Beta blockers (Prior) Yes vs No 0.002 Atrial fibrillation 3545/9221 (38.4%) 8/14 (57.1%) 604/1430 (42.2%) 2350/6249 (37.6%) 448/1202 (37.3%) ICD, (%) 1468/9203 (16.0%) 1/14 (7.1%) 223/1425 (15.6%) 1018/6237 (16.3%) 180/1201 (15.0%) ARB, (%) 2234/9218 (24.2%) 1/14 (7.1%) 349/1431 (24.4%) 1510/6245 (24.2%) 294/1202 (24.5%) Digitalis, (%) 1982/9214 (21.5%) 6/14 (42.9%) 337/1430 (23.6%) 1340/6242 (21.5%) 223/1202 (18.6%) ≥ 50% max dosage 4742/5253 (90.3%) 8/8 (100.0%) 738/807 (91.4%) 3301/3611 (91.4%) 586/690 (84.9%) EF (%) >50 vs <40 0.75 (0.51;1.09) 0.129 Mitral regurgitation Yes vs No 1.16 (0.91;1.47) 7) 0.232 Hepatic dysfunction Yes vs No 1.45 (0.94;2.26) 0.096 1.158 (0.891;1.50 CRT -P/D, (%) 1236/9203 (13.4%) 2/14 (14.3%) 202/1425 (14.2%) 828/6237 (13.3%) 165/1201 (13.7%) ACEi/ARB, (%) 8129/9210 (88.3%) 5/14 (35.7%) 1201/1431 (83.9%) 5559/6239 (89.1%) 1089/1201 (90.7%) Statins, (%) (61.2%) (28.6%) (55.6%) (61.6%) (67.1%) ACE/ARB Discontinuation, (%) (13.5%) (25.0%) (15.9%) (12.4%) (14.6%) Peripheral vascular disease Yes vs No 1.66 (1.26;2.18) <0.001 Notes: 6) 5646/9218 4/14 796/1431 3845/6245 806/1202 949/7045 1/4 159/1001 600/4830 141/963 Chronic kidney dysfunction Yes vs No 1.78 (1.38;2.29) <0.001 Mitral regurgitation Yes vs No 0.273	120.5 [110; 140] 70 [63; 81] 197/308 (64.0%) 65/308 (21.1%) ≥5.5 mmol/L (N=326, 3.5%) 46/308 (14.9%) 45/326 (13.8%) 191/326 (58.6%) 76/326 (23.3%) 14/326 (4.3%) 53/324 (16.4%) 66/322 (20.5%) ≥5.5 mmol/L (N=326, 3.5%) 86/325 (26.5%) 34/325 (10.5%) 148/325 (45.5%) 26/325 (8.0%) 4/325 (1.2%) ≥5.5 mmol/L (N=326, 3.5%) 33/326 (10.1%) 140/326 (42.9%) 222/326 (68.1%) 51/324 (15.7%) 137/326 (42.0%) ≥5.5 mmol/L (N=326, 3.5%) 43.6 [29.9; 61.4] 78/311 (25.1%) 148/311 (47.6%) 85/311 (27.3%) 207/325 (63.7%) ≥5.5 mmol/L (N=326, 3.5%) 273/326 (83.7%) 139/326 (42.6%) 126/325 (38.8%) 266/326 (81.6%) 25/324 (7.7%) ≥5.5 mmol/L (N=326, 3.5%) 75/193 (38.9%) 118/193 (61.1%) 21/61 (34.4%) 40/61 (65.6%) 28/137 (20.4%) ≥5.5 mmol/L (N=326, 3.5%) 44/185 (23.8%) 28/73 (38.4%) 43/245 (17.6%) 40/127 (31.5%) 27/325 (8.3%) 46/326 (14.1%) 80/326 (24.5%) 76/326 109/137 (79.6%) (23.3%) 135/326 (41.4%) 39/326 (12.0%) 275/325 (84.6%) (59.8%) (19.4%) 195/326 48/247	<0.001 0.580 P-value <0.001 0.015 <0.001 P-value 0.053 P-value 0.853 <0.001 0.034 0.139 <0.001 P-value <0.001 <0.001 <0.001 P-value <0.001 <0.001 <0.001 0.001 0.496 P-value 0.254 0.446 <0.001(S) P-value 0.002 0.006 <0.001 <0.001 0.525 0.680 0.007 0.006 0.823 <0.001 <0.001 <0.001

Acknowledgements

EORP Oversight Committee, Registry Executive Committee and Steering Committee of the EURObservational Research Programme (EORP). Data collection was conducted by the EORP department from the ESC by Emanuela Fiorucci as Project Officer, Gérard Gracia and Maryna Andarala as Data Managers. Statistical analyses were performed by Cécile Laroche. Overall activities were coordinated and supervised by Doctor Aldo P. Maggioni (EORP Scientific Coordinator). All investigators listed in the Supplemental Appendix.

Funding

Since the start of EORP, the following companies have supported the programme: Abbott Vascular Int. (2011-2021), Amgen Cardiovascular (2009-2018), AstraZeneca (2014-2021), Bayer AG (2009-2018), Boehringer Ingelheim (2009-2019), Boston Scientific (2009-2012), The Bristol Myers Squibb and Pfizer Alliance (2011-2019), Daiichi Sankyo Europe GmbH (2011-2020), The Alliance Daiichi Sankyo Europe GmbH and Eli Lilly and Company (2014-2017), Edwards (2016-2019), Gedeon Richter Plc. (2014-2016), Menarini Int. Op. (2009-2012), MSD-Merck & Co. (2011-2014), Novartis Pharma AG (2014-2020), ResMed (2014-2016), Sanofi (2009-2011), SERVIER (2009-2018), Vifor (2019-2022).

Conflict of interest

Dr. Anker reports grants and personal fees from Vifor Int, personal fees from Bayer, Boehringer Ingelheim, Novartis, Servier, grants and personal fees from Abbott Vascular, personal fees from Impulse Dynamics, SJM, outside the submitted work; Dr. Coats reports personal fees from Astra Zeneca, Bayer, Menarini, Novartis, Nutricia, Servier, Vifor, Actimed, Cardiac Dimension, CVRx, Enopace, Faraday, Gore, Respicardia, Stealth Peptides, V-Wave, outside the submitted work; Supplementary figure 1


Hongbo Liu

Yi Zhang

Yanhong Zhou

Zhongbing Chen

Eric Lichtfouse

Self-provided microbial electricity enhanced wastewater treatment using carbon felt anode coated with amino-functionalized Fe 3 O 4

Keywords:

Bioelectricity can be produced from wastewater using microbial fuel cells (MFCs) that produce electricity during electrochemical and biochemical reactions, yet actual applications of wastewater MFCs are limited; a selfprovided microbial electricity enhanced wastewater treatment method is proposed producing microbial power and enhanced pollutants removal simultaneously. A carbon felt coated with amino-functionalized Fe 3 O 4 particles was synthesized; power generation, removal of chemical oxygen demand (COD) and NH 4 + -N were evaluated with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), cell voltage and treatment efficiency. Results showed that the amino-Fe 3 O 4 coated anode led to higher NH 4 + -N removal efficiency of 97.7 % than the Fe 3 O 4 coated anode of 48.9 %. Moreover, the maximum power density of the amino-Fe 3 O 4 anode is 208.67 mW/ m 2 , 35.1 % higher than that of the Fe 3 O 4 anode. This finding was explained by the presence of protonated amino groups NH 4

+ that favored the attachment of negatively charged bacteria. Amino-functionalization of the anode promotes a promising, novel technology to treat wastewater while producing electricity.

Introduction

Wastewater treatment using microbial fuel cells (MFCs) is an emerging technology converting organic compounds into electrical energy using microorganisms as biocatalysts [START_REF] Logan | Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies[END_REF]. MFC induces simultaneous electrochemical and biochemical reactions that are controlled by characteristics of the microorganisms [START_REF] Zhang | Improved performances of E. Colicatalyzed microbial fuel cells with composite graphite/PTFE anodes[END_REF][START_REF] Liu | Simultaneous pollutant removal and electricity generation in a combined ABR-MFC-MEC system treating fecal wastewater[END_REF]. Though MFC technology is theoretically promising, applications are actually limited by the low power generation [START_REF] He | Electricity generation from artificial wastewater using an upflow microbial fuel cell[END_REF]. Therefore, improving electricity production of MFC with stable output has been under intense research during the past decade [START_REF] Gajda | Self-sustainable electricity production from algae grown in a microbial fuel cell system[END_REF][START_REF] Guo | Influence of packing material characteristics on the performance of microbial fuel cells using petroleum refinery wastewater as fuel[END_REF].

Specifically, the low efficiency of electron transfer through the cell membrane to the anode is a key limiting factor [START_REF] Kumar | Exoelectrogens: recent advances in molecular drivers involved in extracellular electron transfer and strategies used to improve it for microbial fuel cell applications[END_REF]. Indeed, the modification of the anode surface may facilitate the electron transfer as a result of better anode morphology and surface chemistry [START_REF] Choudhury | Performance improvement of microbial fuel cell (MFC) using suitable electrode and Bioengineered organisms: a review[END_REF]. Thus, anode materials and modification materials have been developed to enhance power performance. For instance, Liao et al. [START_REF] Liao | Enhancement of power production with tartaric acid doped polyaniline nanowire network modified anode in microbial fuel cells[END_REF] showed that an anode made of polyaniline-coated carbon cloth modified with tartaric acid enhanced power density 4.1 times compared to the plain carbon cloth anode. Song et al. [START_REF] Song | Effect of surface modification of anode with surfactant on the performance of microbial fuel cell[END_REF] observed that the surface of graphite modified with 10 mM of anionic surfactant enriched the bacterial attachment compared to 5 mM of surfactant. Hence, anode modification may favor microorganism attachment on the anode surface and, in turn, increase the rate of electron transfer reactions [START_REF] Wang | Micro-sized microbial fuel cell: a mini-review[END_REF]. Since acids and surfactants are charged compounds at suitable pH, this study suggested that the presence of charged organic groups could favor bacterial attachment.

The anode could be improved by having a high biocompatibility, high specific surface area and low-electrode resistance in order to obtain better power generation [START_REF] Watson | Influence of chemical and physical properties of activated carbon powders on oxygen reduction and microbial fuel cell performance[END_REF]. MFC anodes have been designed using various modified carbon-based materials for their high biocompatibility and electro-conductivity, such as carbon felt, carbon cloth, carbon paper, carbon fiber, and graphite felt [START_REF] Mu | A microfabricated PDMS microbial fuel cell[END_REF], yet anodes made of pure carbon cannot bear higher power density [START_REF] Mehdinia | Multi-walled carbon nanotube/SnO2 nanocomposite: a novel anode material for microbial fuel cells[END_REF]. Alternatively, Fe 3 O 4 coating appears as suitable anodic modifier due to its good biocompatibility, low cost, high specific surface area, high bacterial affinity and simple synthesis [START_REF] Sun | Synthesis and characterization of biocompatible Fe3O4 nanoparticles[END_REF]. In addition, Fe 3 O 4 nanoparticles favor the formation of a multi-layered network by magnetic attraction, and thus accelerate electron transfer [START_REF] Peng | Enhanced performance and capacitance behavior of anode by rolling Fe3O4 into activated carbon in microbial fuel cells[END_REF]. For instance, higher power densities were observed with a carbon paper anode coated with Fe 3 O 4 /carbon nanotubes (CNT) [START_REF] Park | Enhanced electrical contact of microbes using Fe3O4/CNT nanocomposite anode in mediator-less microbial fuel cell[END_REF]. Song et al. also assumed that adding Fe 3 O 4 nano-spheres in graphene anodes may induce excellent bacterial affinity [START_REF] Song | Bacteria-affinity 3D macroporous graphene/MWCNTs/Fe3O4 foams for highperformance microbial fuel cells[END_REF].

Functionalization of Fe 3 O 4 particles with amino (-NH 2 ), sulfhydryl (-SH) and carboxyl (-COOH) groups may also improve energy output by changing surface properties [START_REF] Meng | Preparation of amino-functionalized Fe3O4@ mSiO2 core-shell magnetic nanoparticles and their application for aqueous Fe3+ removal[END_REF][START_REF] Shi | Preparation and the adsorption ability of thiolated magnetic core-shell Fe3O4@ SiO2@ C-SH for removing Hg2+ in water solution[END_REF][START_REF] Luong | Design of carboxylated Fe3O4/poly (styrene-co-acrylic acid) ferrofluids with highly efficient magnetic heating effect[END_REF] and electron transfer [START_REF] Saito | Effect of nitrogen addition on the performance of microbial fuel cell anodes[END_REF]. These investigations suggested that the presence of charged groups may improve bacterial attachment and electron transfer. Therefore, this study hypothesized that amino groups, which are protonated at neutral pH (-NH 4 + ) could favor the attachment of negatively charged bacteria.

Materials and methods

Synthesis of Fe 3 O 4 particles

The precipitation method was used to prepare Fe3O4 particles [START_REF] Yang | Preparation and characterization of Fe_3O_4 nano particles with oxygen[END_REF]. Typically, a 200 mL solution was prepared with 0.1 M FeSO 4 ⋅7H 2 O and 0.1 M FeCl 3 ⋅6H 2 O. 1.5 M NaOH was then added slowly to the solution, magnetically-stirred at 50 ± 1 • C. When pH reaches 12.0, the addition of NaOHwas stopped and the solution was stirred for 30 min. The formation of Fe 3 O 4 particles was indicated by a color change from brown (solution) to black (precipitation). The black precipitation solution was water bathed at 80 ± 1 • C to allow crystallization within a hour. The black solid precipitation was collected by a magnet and washed repeatedly with distilled water and pure ethanol, then dried at 60 • C for 12 h. The size of Fe 3 O 4 particles were roughly identified using Sherrer's equation as D = Kλ/(βCosθ).

Synthesis of amino-Fe 3 O 4 particles

The synthetic procedure of amino-Fe 3 O 4 particles was: 0.75 g of Fe 3 O 4 particles were dissolved in 100 mL of a 50 % aqueous acetic acid solution; then the solution pH was adjusted to 4.0 with anhydrous acetic acid, followed by ultrasonic dispersion for 10 min at 40 KHz to yield the ferrofluid; 1 mL 3-aminopropyl triethoxysilane was added to the ferrofluid and purged with dinitrogen gas to remove oxygen [START_REF] Yamaura | Preparation and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles[END_REF]. This solution was transferred into a 250 mL three-neck round-bottom flask, and stirred for 3 h in a water bath at 60 • C. The product was collected by a magnet and washed successively with distilled water, pure ethanol and anhydrous ether, and dried at 60 • C for 12 h.

Electrode fabrication

Two carbon felt anodes of 4(cm)× 3(cm)× 0.3(cm), from Beijing Sanye carbon Co., LTD China which were modified by coating. Prior to coating, the carbon felt was cleaned with pure ethanol at room temperature for 12 h, then with concentrated nitric acid at room temperature for 12 h. The treated carbon felt was thoroughly washed with distilled water and dried at 60 • C. The composite was mixed with 0.0080 g acetylene black, 0.0080 g polyvinylidenefluoride (PVDF) and 0.0625 g amino-Fe 3 O 4 particles or Fe 3 O 4 particles using N-methyl-2-pyrrolidone (NMP) as dispersant, stirring for 4 h [START_REF] Hao | Hierarchical porous carbon aerogel derived from bagasse for high performance supercapacitor electrode[END_REF]. The composite was brush-coated onto carbon felt substrates evenly, then dried at 60 • C under vacuum for 12 h.

Configuration and operation of microbial fuel cells

Two 150 mL MFC batteries made of plexiglass were coupled into a dual-chamber MFC configuration [START_REF] Fan | Enhanced Coulombic efficiency and power density of aircathode microbial fuel cells with an improved cell configuration[END_REF], one battery with a Fe 3 O 4 -coated carbon felt anode and the other with an amino-Fe 3 O 4 -coated carbon felt anode. Both batteries use 4(cm)×3(cm)×0.005(cm) stainless steel mesh from China Pingzhen Hardware Wire Mesh Co., Ltd. as the cathode. In the anode chamber, 60 mL synthetic domestic wastewater or 60 mL anaerobic sludge were used as anolyte and stirred by a magnetic stirrer. The sludge concentration in the anode chamber was about 3980-4150 mg/L. In the cathode chamber, 120 mL 0.1 M phosphate buffer solution (PBS) was used as catholyte and aerated continuously. The anode chamber and cathode chambers were separated by a 16 cm 2 of proton exchange membrane (PEM) Nafion 117, as described by Liu et al. [START_REF] Liu | Electricity generation using an air-cathode single chamber microbial fuel cell in the presence and absence of a proton exchange membrane[END_REF]. The distance between the anode and the cathode was 6 cm; the anode and cathode were connected with an external load external resistance (1000 Ω). All experiments were performed at room temperature of 25-35 • C.

The anaerobic sludge originates from the Shanghai Eastern District wastewater plant. At the start-up phase, the synthetic wastewater had a chemical oxygen demand (COD) of 500 mg/L, ammonium (NH -N of 30 mg/L and TP of 10 mg/L, that is twice compared to the start-up phase, while the operating parameters of the catholyte remained unchanged.

Analytical techniques and characterization

Cell voltages were recorded every minute by a digital multi-meter from PicoLog Technology, UK. The power density and polarization curve of the MFC were obtained by varying the external resistance from 100 to 100,000 Ω with stable voltages. The power densities P in mW/m 2 were calculated using the P = UI equation(U is voltage, I is electric current). Liquid samples were taken from the supernatant at each operational cycle and measured after filtration. COD, NH 4 + -N, nitrate (NO 3 --N) and nitrite (NO 2 --N) were measured according to standard methods from APHA [START_REF] Gilcreas | Standard methods for the examination of water and waste water[END_REF]. Dissolved oxygen (DO) was measured with a dissolved oxygen meter from JPB-607, Leici, China. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of the anode were measured by the electrochemical workstation CHI660E, Chenhua, China, in a single chamber with the working electrode (anode), a counter Pt electrode and a reference electrode made of Ag/AgCl and saturated KCl. The CV test was done from -0.3 V to 0.6 V at a scanning rate of 10 mV/s. EIS analysis was measured from 100 kHz to 700 kHz in an open circuit. The synthetic Fe 3 O 4 particles and amino-Fe 3 O 4 particles were characterized by X-ray diffraction (XRD) using a D8-Advance from Germany, and by Fourier transform infrared spectroscopy (FTIR) from ThermoFisher Scientific, Germany; the surface morphology of the biofilm on anodes was carried out by scanning electron microscopy (SEM) from Carl Zeiss EVO LS10, UK.

Results and discussion

Characterization of Fe 3 O 4 and amino-Fe 3 O 4 coatings

This study investigated the structure, properties and power generation of MFC made of carbon felt anodes coated with either Fe [START_REF] Yamaura | Preparation and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles[END_REF]. The broad bands at 999 cm -1 and 2910 cm -1 are ascribed to Si-O-Si groups and NH stretching vibration of the grafted amino material, respectively [START_REF] Javidparvar | Effect of various spinel ferrite nanopigments modified by amino propyl trimethoxy silane on the corrosion inhibition properties of the epoxy nanocomposites[END_REF][START_REF] Jiang | Preparation and application of separable magnetic Fe3O4-SiO2-APTES-Ag2O composite particles with high visible light photocatalytic performance[END_REF]. Therefore, Si-O groups and NH bonds confirm the successful addition of amino group to the surface of Fe 3 O 4 particles.

Composition, structure and performance of the surface amino modification Fe 3 O 4 particles indicated that the particle size of the magnetic particles after the surface amination was slightly increased, with better adsorption performance and better retention capacity than the original magnetic features. The results implied that MFC with aminofunctionalized Fe 3 O 4 as anode can better bind and fix the load with biomolecules in sewage, which makes MFC generate stronger current so as to achieve better sewage treatment efficiency.

Performance of microbial fuel cells 3.2.1. Performances of start up and operational runs

Fig. 2 depicted the voltage profiles during the start-up and operational stages of MFC using the amino-Fe 3 O 4 and the Fe 3 O 4 coated anodes. Inoculate and adapt the anode in MFCs containing standard Pt/ C carbon felt anodes, using an external resistance of 1000Ω, the voltage generated by MFC at this time was in a stable and easy-to-detect working state, which had good reference and practicality. Results showed that both anodes yield repeatable voltages of 530 ± 10 mV with 1000 Ω external resistors after successful start-up. The output voltage of the reactor was lower than the reactor with amino-Fe 3 O 4 anode about 350 mV using Fe 3 O 4 anode in the first five days; then it reached 520 mV after the fifth day, at which time the maximum output voltage was closer to the amino-Fe 3 O 4 anode reactor (Fig. 2a). Using the amino-Fe 3 O 4 anode, voltage output of the reactor rose to 520 mV after 12 h, then reached a maximum voltage about 620 mV after 5 days. The output voltage cycle of the reactor using Fe 3 O 4 anode was the same as the output voltage cycle of the amino-Fe 3 O 4 anode reactor, but the output voltage was always lower than that of the amino-Fe 3 O 4 anode reactor, which showed that the amino-Fe 3 O 4 anode had better and more stable power generation performance (Fig. 2b).

The start-up time of the amino-Fe 3 O 4 anode was much faster than the Fe 3 O 4 anode (left arrow in Fig. 2a). This finding implied that amino groups set more rapidly a bacterial-electrode system that favors electron transfer. This acceleration effect was supported by findings of Jiang et al. who observed that the start-up time of a MFC system with a carbon cloth anode covered by polypyrrole, a polymer containing aromatic -NHgroups, was lower than that of the plain carbon cloth anode [START_REF] Jiang | Novel electrode materials to enhance the bacterial adhesion and increase the power generation in microbial fuel cells (MFCs)[END_REF]. Therefore, this study implies that amino groups create positive charges on the anode surface and, in turn, increase the adhesion rate of negatively charged bacteria.

Fig. 2b showed the operational voltage generated within 12 days. Results showed that the maximum voltage generation was higher, of 641 mV, using the amino-Fe 3 O 4 anode, than for the Fe 3 O 4 anode of 589 mV. After 6 cycles, the voltage generation started to drop, though less rapidly for the amino-Fe 3 O 4 anode, which displays better stability. The voltage drop could be explained by microbial inhibition following addition of external substrate, and by limited energy required for microbial growth [START_REF] Liu | Modifying proton exchange membrane in a microbial fuel cell by adding clay mineral to improve electricity generation without reducing removal of toluene[END_REF]. As a consequence, the higher mass transfer and ohmic resistances limited the diffusion of protons and electrons, which decrease the output voltage. Overall, the findings of this study show that the amino-Fe 3 O 4 anode induces a higher and more stable voltage output, which could be explained by easier bacterial attachment to protonated amino groups.

Power density

Power output and power density curves of the study wastewater treatment MFC were illustrated in Fig. 3. Current generation showed a decreasing trend with increase in the resistance along with lower potential drop. Using the polarization curve in Fig. 3a, the amino-Fe 3 O 4 anode cell had a lower internal resistance than for the Fe 3 O 4 anode cell. The power density curves showed that the maximum power density using the amino-Fe 3 O 4 anode, of 208.67 mW/m 2 , was 35.1 % higher than that of the Fe 3 O 4 anode, as 154.41 mW/m 2 (Fig. 3b). The observation strengthens the previous finding, which suggested that modifying Fe 3 O 4 anode with amino groups improves power generation. This increase of power density was also in line with power density increases observed following the modification of carbon nanotubes and carbon cloth. The findings demonstrated that the addition of amino groups on the Fe 3 O 4 coated carbon felt enhances electron transfer and power output. [START_REF] Peng | Enhanced performance and capacitance behavior of anode by rolling Fe3O4 into activated carbon in microbial fuel cells[END_REF]. Results showed that, the average COD removal efficiency using amino-Fe 3 O 4 coated anode and Fe 3 O 4 coated anode are similar during one operational cycle, of 91.5 % and 90.8 % respectively. This absence of difference could be explained by the fact that the carbon sources, glucose and sodium acetateare rapidly degraded in the anode chamber, thus rapidly reach the degradation limit, which is very helpful to confirm hypothesis of a previous finding [START_REF] Penteado | Energy recovery from winery wastewater using a dual chamber microbial fuel cell[END_REF].

Wastewater treatment

This study further investigated the removal efficiency of NH 4 + -N. These findings suggested a much higher biological nitrification using the amino-Fe 3 O 4 anode. In a similar way, ammonia loss of the singlechambered MFC had been explained by volatilization due to pH increase at the cathode surface, by biological nitrification and denitrification [START_REF] Kim | Analysis of ammonia loss mechanisms in microbial fuel cells treating animal wastewater[END_REF] and by nitrification through air cathodes [START_REF] Zhang | Continuous electricity production from leachate in a novel upflow air-cathode membrane-free microbial fuel cell[END_REF]. In the amino-Fe 3 O 4 MFC, a part of NH 4 + -N reached anolyte through the proton exchange membrane, and then transformed to NO 3 --N in the presence of dioxygen. This finding reinforced hypothesis of the study by which protonated amino groups favor the attachment of bacteria that is in line with studies on amino functionalization of other materials [START_REF] Aelterman | The anode potential regulates bacterial activity in microbial fuel cells[END_REF]. The results show that the nitrification efficiency for amino-Fe 3 O 4 anode MFC was much higher than that of Fe 3 O 4 anode MFC.

Electrochemical performance of electrodes

CV measurement for the fabricated anodes was performed in the solution of 0.1 M potassium ferricyanide (Fig. 5a). Results showed that the amino-Fe 3 O 4 coated anode induced a much higher current response and redox reversibility than for the Fe 3 O 4 coated anode. In addition, both coated anodes showed higher current responses versus the bare carbon anode. These findings were explained by the higher conductivity of Fe 3 O 4 versus other materials. Moreover, the results confirmed that the addition of amino groups favors the involvement of electrochemically active bacteria, strengthens the hypothesis of bacterial attachment to the anode by positive-negative charge attraction.

EIS measurement for the fabricated anodes was performed in the solution of 0.1 M potassium ferricyanide (Fig. 5b). Results showed that the semicircle of Fe 3 O 4 coated anode was larger than that of the amino-Fe 3 O 4 coated anode. In a typical Nyquist plot, the diameter of a semicircle yields the charge-transfer resistance which represented the resistance of electrochemical reactions at the electrode [START_REF] Claye | ChemInform Abstract: Solid-State Electrochemistry of the Li Single Wall Carbon Nanotube System[END_REF]. The finding revealed that interfacial charge-transfer resistance of the amino-Fe 3 O 4 coated anode, of 26 Ω/cm 2 , was lower than that of the Fe 3 O 4 coated anode, of 34 Ω/cm 2 . This lower resistance confirmed again the higher showed that the modification of the surface of the electrode by the amino Fe 3 O 4 coating was positive, which would optimize the electrochemical performance of the MFC and remove sewage pollutants. The materials used for experimental testing were mainly used as anode materials in the MFC experimental device. In the process of sewage treatment, biofilms were formed near the anode and cathode materials of the MFC. The microorganisms in the biofilms adsorb and degrade pollutants in the sewage. Therefore, after the device had been operating stably for a period of time, we took out the carbon felt anode coated with amino functionalized Fe 3 O 4 without cleaning it, and keep the microorganisms attached to the carbon felt for direct detection. According to related information and experimental practice, the carbon felt anode coated with amino-functionalized Fe 3 O 4 had a larger particle pore size than the carbon felt anode coated with Fe 3 O 4 and ordinary carbon felt anode, which was conducive to the attachment of more microorganisms. The micro-currents formed between MFCs promote each other and had a better treatment effect.

Overall, the results of this study indicate that the increase in the surface area of the Fe 3 O 4 anode modified with amino groups is more conducive to the attachment of bacterial flora on the anode surface, which induces a higher and more stable voltage output and enhances the electron transfer of the system. The amino-modified Fe 3 O 4 anode is combined with a microbial fuel cell using high electron transfer capacity. Since amino-functional polymer magnetic electrodes could also act as adsorbent media [START_REF] Zhao | Study on adsorption of Cr(VI) in wastewater by amino functionalized Nano-Fe3O4 magnetic polymer adsorbent[END_REF], bio-adsorption process could be another contributor to better performance of this study.

Conclusions

The size of the magnetic particles did not change after surface amination, and the particle size increases slightly, which had better adsorption performance and better retention than the original magnetic characteristics. The output voltage of the amino-Fe 3 O 4 anode reactor increased to 520 mV after 12 h, and reached a maximum voltage of about 620 mV and a maximum power density of 208.67 mW/m 2 after 5 days. This data was far better than Fe 3 O 4 anode reactor. This finding indicated that the amino group can quickly establish a bacterial electrode system and promote electron transfer.

COD removal efficiency of the amino Fe 3 O 4 coated anode and the Fe 3 O 4 coated anode were simila, which indicates that in the amino-Fe 3 O 4 MFC, a part of NH 4 + -N reached the anolyte through the proton exchange membrane, and then was converted into NO 3 --N in the presence of hydrogen peroxide. This finding reinforced the hypothesis of this study that protonated amino groups were beneficial to bacterial attachment, which was consistent with the functionalization of amino groups.

  3 O 4 or amino-Fe 3 O 4. XRD patterns of amino-Fe 3 O 4 particles and Fe 3 O 4 particles were illustrated with Fig. 1a. Results showed that both amino-Fe 3 O 4 and Fe 3 O 4 particles show identical diffraction peaks typical of magnetite Fe 3 O 4 (PDF No.75-0449), at 30.647 • , 35.473 • , 43.277 • , 54.155 • , 57.050 • and 62.667 • , corresponding to (220), (311), (400), (422), (511) and (440) crystal face of Fe 3 O 4, respectively [29]. Calculation results of Sherrer's equation (D = Kλ/(βCosθ)) indicated that the size was 15 nm both for amino-Fe 3 O 4 particles and Fe 3 O 4 particles. Overall, Fe 3 O 4 particles modified with amino keep original physical properties. Fig. 1b showed the FT-IR spectra of amino-Fe 3 O 4 particles and Fe 3 O 4 particles. The peak at 565 cm -1 corresponds to Fe-O bonds in the spectra of amino-Fe 3 O 4 particles and Fe 3 O 4 particles

Fig. 1 .Fig. 2 .

 12 Fig. 1. a) X-ray powder diffraction (XRD) of amino-Fe 3 articles (A); Fe 3 O 4 particles (B); standard peaks of magnetite Fe 3 O 4 (C). b) Fourier-transform infrared (FTIR) spectra of Fe 3 O 4 particles and amino-Fe 3 O 4 particles.

  Fig. 4a depicted the chemical oxygen demand (COD) of the influent and effluent of two microbial fuel cells (amino-Fe 3 O 4 -NPs MFC and Fe 3 O 4 MFC) during the stable operation period. Under the same operating conditions and working conditions, compare the COD removal effects of the amino-Fe 3 O 4 -NPs MFC and Fe 3 O 4 MFC devices. Among them, Fe 3 O 4 MFC was the MFC with Fe 3 O 4 activated carbon felt as the anode material. Fe 3 O 4 nanoparticles facilitate the formation of a multilayer network through magnetic attraction, thereby accelerate electron transfer

4 +

 4 Fig. 4b showed the concentration of NH 4 + -N, NO 3 --N and NO 2 --N in the anolyte and catholyte of the cells. Results show that the influent NH 4 + -N concentrations for amino-Fe 3 O 4 anode MFC, of 26.87 mg/L, was lower than that of the Fe 3 O 4 anode cell, of 29.10 mg/L. During one operational cycle, the average NH 4 + -N removal efficiency reached 97.7 % using the amino-Fe 3 O 4 anode, versus 48.9 % for the Fe 3 O 4 -coated anode. The results also show that in the amino Fe 3 O 4 MFC, NO 3 --N is almost the only form of N; while in the Fe 3 O 4 anode MFC, the ratio of NO 3 --N to NH

Fig. 3 .Fig. 4 .

 34 Fig. 3. Polarization (a) and power density curves (b) of microbial fuel cells (MFC) with the amino-Fe 3 O 4 coated anode and the Fe 3 O 4 coated anode.

Fig. 5 .Fig. 6 .

 56 Fig. 5. a) Cyclic voltammetry analysis of amino-Fe 3 O 4 coated anode, Fe 3 O 4 coated anode and bare carbon felt anode; b) Nyquist plots of amino-Fe 3 O 4 coated anode and Fe 3 O 4 coated anode.

4 +

 4 -N) of 15 mg/L, total phosphorus (TP) of 5 mg/L, and contained per L of distilled water: 0.253 g glucose, 0.583 g C 2 H 3 NaO 2 ⋅3H 2 O, 0.110 g NH 4 Cl, 0.022 g KH 2 PO 4 , 0.005 g MgSO 4 ⋅H 2 O, 0.055 g NaHCO 3 , 2 mL trace elements and 2 mL 0.1 M phosphate buffer solution (PBS). The composition of trace elements (mass of trace elements per liter) are as follows: 0.900 g FeCl 3 , 0.120 g ZnSO 4 ⋅7H 2 O, 0.060 g Na 2 MoSO 4 ⋅2H 2 O, 0.060 g MnCl 2 ⋅4H 2 O, 0.180 g KI, 0.03 g CuSO 4 ⋅5H 2 O, 0.150 g CaCl 2 ⋅6H 2 O and 0.150 g H 3 BO 3 . The phosphate buffer solution (PBS) contained per liter (L) of distilled water: 8 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , and 2.9 g Na 2 HPO 4 ⋅12H 2 O. At the operational phase, the water distribution concentration of the anolyte contained COD of 1000 mg/L, NH 4

	+

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2018YFD1100502-01) and the Bill & Melinda Gates Foundation, Seattle, WA (RTTC-China 2014).

Declaration of Competing Interest

The authors have declared no conflict of interest.