Philippe Gaudard
email: p-gaudard@chu-montpellier.fr

Hélène David

Patrice Bideaux

Pierre Sicard

Jean-Paul Cristol

Gilles Guillon

Sylvain Richard

Pascal Colson

Anne Virsolvy
email: anne.virsolvy@inserm.fr

Involvement of Vasopressin in Tissue Hypoperfusion during Cardiogenic Shock Complicating Acute Myocardial Infarction in Rats

Keywords: myocardial infarction, cardiogenic shock, vasopressin, copeptin, microcirculation

Acute heart failure (AHF) due to acute myocardial infarction (AMI) is likely to involve cardiogenic shock (CS), with neuro-hormonal activation. A relationship between AHF, CS and vasopressin response is suspected. This study aimed to investigate the implication of vasopressin on hemodynamic parameters and tissue perfusion at the early phase of CS complicating AMI. Experiments were performed on male Wistar rats submitted or not to left coronary artery ligation (AMI and Sham). Six groups were studied Sham and AMI treated or not with either a vasopressin antagonist SR-49059 (Sham-SR, AMI-SR) or agonist terlipressin (Sham-TLP, AMI-TLP). Animals were sacrificed one day after surgery (D1) and after hemodynamic parameters determination. Vascular responses to vasopressin were evaluated, ex vivo, on aorta. AHF was defined by a left ventricular ejection fraction below 40%. CS was defined by AHF plus tissue hypoperfusion evidenced by elevated serum lactate level or low mesenteric oxygen saturation (SmO2) at D1. Mortality rates were 40% in AMI, 0% in AMI-SR and 33% in AMI-TLP. Immediately after surgery, a sharp decrease in SmO2 was observed in all groups. At D1, SmO2 recovered in Sham and in SR-treated animals while it remained low in AMI and further decreased in TLP-treated groups. The incidence of CS among AHF animals was 72% in AMI or AMI-TLP while it was reduced to 25% in AMI-SR. Plasma copeptin level was increased by AMI. Maximal contractile response to vasopressin was decreased in AMI (32%) as in TLP-and SR-treated groups regardless of ligation. Increased vasopressin secretion occurring in the early phase of AMI may be responsible of mesenteric hypoperfusion resulting in tissue hypoxia. Treatment with a vasopressin antagonist enhanced mesenteric perfusion and improve survival. This could be an interesting therapeutic strategy to prevent progression to cardiogenic shock.

Introduction

Acute myocardial infarction (AMI) remains the main cause of heart failure and represents a major cause of death worldwide [START_REF] Reynolds | Cardiogenic Shock: Current Concepts and Improving Outcomes[END_REF][START_REF] Delmas | Design and Preliminary Results of FRENSHOCK 2016: A Prospective Nationwide Multicentre Registry on Cardiogenic Shock[END_REF]. According to the European Society of Cardiology guidelines, left ventricular ejection fraction (LVEF) of 40% is the threshold defining heart failure with reduced ejection fraction [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]. Occlusion of the main coronary arteries results in an acute reduction of LVEF likely to lead to cardiogenic shock (CS). In humans, CS occurs in 5 to 10% of acute coronary syndromes with ST-segment elevation but is responsible for a mortality of 40 to 50% and requires high medical resources in intensive care units [START_REF] Mebazaa | Management of Cardiogenic Shock Complicating Myocardial Infarction[END_REF]5]. CS is defined by end-organ hypoperfusion due to primary cardiac dysfunction [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] and peripheral vasoconstriction. Diagnosis criteria in humans include persistent hypotension with cardiac index less than 2.2 L/min/m2, despite adequate or elevated filling pressure, and symptoms of end-organ hypoperfusion [START_REF] Reynolds | Cardiogenic Shock: Current Concepts and Improving Outcomes[END_REF][START_REF] Hochman | Cardiogenic Shock Complicating Acute Myocardial Infarction-Etiologies, Management and Outcome: A Report from the SHOCK Trial Registry. SHouldWe Emergently Revascularize Occluded Coronaries for Cardiogenic Shock?[END_REF]. The course of cardiogenic shock usually includes two entangled hemodynamic profiles. On the one hand, the acute reduction of cardiac output (CO) induces vasoconstrictive response involving sympathetic system and vasoactive hormones (angiotensin II, vasopressin), attempting to maintain the perfusion pressure of organs. On the other hand, AMI and tissue hypoperfusion may lead to a systemic inflammatory response syndrome and an increased expression of inducible NO synthase, which are both responsible for inadequate vasodilation [START_REF] Reynolds | Cardiogenic Shock: Current Concepts and Improving Outcomes[END_REF]. Therapeutic strategies aim to restore circulation and tissue perfusion. Thus, in addition to myocardial revascularization, pharmacological treatments consisting of vasopressors and/or inotropes are administered to support coronary and organ perfusion [START_REF] Mebazaa | Management of Cardiogenic Shock Complicating Myocardial Infarction[END_REF]. Norepinephrine is recommended as first-line therapy to restore quickly the blood pressure and possibly with the addition of an inotropic agent if stroke volume is not adequately improved [START_REF] Jolly | Effect of Vasopressin on Hemodynamics in Patients with Refractory Cardiogenic Shock Complicating Acute Myocardial Infarction[END_REF]. Vasopressin is another agent utilized as second-line therapy for vasoconstriction. It is increasingly used in distributive shock and vasoplegia following cardiac surgery but not preferred in CS due to its lack of beneficial effect on cardiac output [START_REF] Jolly | Effect of Vasopressin on Hemodynamics in Patients with Refractory Cardiogenic Shock Complicating Acute Myocardial Infarction[END_REF].

Vasopressin or arginine-vasopressin (AVP) is a nonapeptide synthesized in the hypothalamus from a prohormone, pre-pro-vasopressin, along with two other peptides: neurophysin II and copeptin, stored in the posterior pituitary and released after stimulation of baroreceptors. The hemodynamic action of AVP is mediated by the V1a receptor in the vascular smooth muscle inducing vasoconstriction. With a better stability and a longer plasmatic half-life, copeptin can be used as a biomarker of AVP release [START_REF] Colson | Post Cardiac Surgery Vasoplegia Is Associated with High Preoperative Copeptin Plasma Concentration[END_REF][START_REF] Khan | C-Terminal Provasopressin (Copeptin) as a Novel and Prognostic Marker in Acute Myocardial Infarction: Leicester Acute Myocardial Infarction Peptide (LAMP) Study[END_REF]. Copeptin level at the hospital admission for AMI has a good predictive value for progression to chronic heart failure and one-year mortality [START_REF] Khan | C-Terminal Provasopressin (Copeptin) as a Novel and Prognostic Marker in Acute Myocardial Infarction: Leicester Acute Myocardial Infarction Peptide (LAMP) Study[END_REF][START_REF] Lattuca | Copeptin as a Prognostic Biomarker in Acute Myocardial Infarction[END_REF]. Copeptin is a diagnostic and prognostic biomarker of cardiovascular diseases, including the rapid detection of acute myocardial infarction (AMI) and stroke, and the prediction of mortality in heart failure (HF) [START_REF] Mu | Copeptin as a Diagnostic and Prognostic Biomarker in Cardiovascular Diseases[END_REF]. Elevated copeptin levels traducing vasopressin level increase are reported in the setting of AMI and shock [START_REF] Goldsmith | Increased Plasma Arginine Vasopressin Levels in Patients with Congestive Heart Failure[END_REF]. This increase is thought to improve perfusion pressure at the cost of increased cardiac afterload, which may further impair myocardial function and promote ventriculoarterial uncoupling [START_REF] Reynolds | Cardiogenic Shock: Current Concepts and Improving Outcomes[END_REF][START_REF] Levy | Epinephrine versus Norepinephrine for Cardiogenic Shock After Acute Myocardial Infarction[END_REF].

The model of choice for AMI in rats consists of the surgical left coronary artery ligation (LCA). This experimental model is interesting for monitoring myocardial remodelling, in particular the hemodynamic and morphological modifications which are similar to human clinical situations of acute coronary syndromes [START_REF] Pfeffer | Myocardial Infarct Size and Ventricular Function in Rats[END_REF][START_REF] Fishbein | Experimental Myocardial Infarction in the Rat: Qualitative and Quantitative Changes during Pathologic Evolution[END_REF][START_REF] Samsamshariat | A Novel Method for Safe and Accurate Left Anterior Descending Coronary Artery Ligation for Research in Rats[END_REF][START_REF] Chen | Ischemic Model of Heart Failure in Rats and Mice[END_REF]. Although infarcts ranging from 4% to 65% have been described in various rat models [START_REF] Pfeffer | Myocardial Infarct Size and Ventricular Function in Rats[END_REF][START_REF] Kainuma | Influence of Coronary Architecture on the Variability in Myocardial Infarction Induced by Coronary Ligation in Rats[END_REF], none mentions the use of this LCA ligation model to study acute hemodynamic response and CS. The twofold objective of the present study was, on the one hand, to develop a rat model of CS following LCA ligation and, on the other hand, to investigate the implication of vasopressin system on acute hemodynamic changes and vascular reactivity during CS complicating AMI in that model. Thus, using a selective agonist, terlipressin, and a selective antagonist, SR-49059, of V1a receptors, we showed that in the early phase of CS, vasopressin is involved in mesenteric tissue hypoperfusion. Additionally, treatment with SR-49059 significantly reduced animal mortality after LCA ligation.

Results

Groups Determination and Mortality Rates

The study included 103 rats spread out into 6 groups (Figure 1), depending on AMI and on treatment with either a vasopressin receptor antagonist (SR-49059, SR) or agonist (terlipressin, TLP).

Among rats with LCA ligation, 22 over 59 died at D1 after surgery in AMI without preoperative treatment (37.3%) but none in AMI-SR (p = 0.035). Thus, we observed that treatment with SR significantly reduced AMI-induced mortality.

According to our definition criteria of AHF (LVEF < 40%), those with LVEF > 40% after LCA ligation at D1 were therefore excluded from the study. Among the 60 animals finally included, 16 were preoperatively treated with SR-49059 and 44 received no preoperative treatment. Thus, this defined two groups that we compared to evaluate the intrinsic effects of SR (Table 1). As shown in Table 1, no difference was observed regardless the examined parameter whether it be weight, heart rate, cardiac function or mesenteric oxygenation. Thus, no specific preoperative effect of SR was detected.

Hemodynamic Changes Induced by Myocardial Infarction

We observed that myocardial infarction size did not differ between all AMI groups validating ligation and reproducibility of surgical procedure (Table 2). The average value of 45 •} 1% reflects a large left ventricular infarction.

Cardiac and hemodynamic parameters at D0, before surgery, were not different between groups (Table 2). At D1, differences were observed for left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), cardiac output (CO) and mean arterial pressure (MAP). These differences, predominantly significant between Sham and AMI whatever the treatment, are consistent with coronary ligation and myocardial ischemia. Averaged and compared to Sham groups, all AMI groups had higher LVEDD (7.4 •} 0.2 vs. 6.5 •} 0.1 mm; p = 0.0008), lower CO (109 •} 7 vs. 137 •} 7 mL/min; p = 0.007). Concerning LVEF, the reduced value observed in all AMI groups reflected our selection criteria, i.e., exclusion of animals with LVEF > 40% after ligation. However, if AMI induced a huge decrease of LVEF, we observed no difference between treatments. Concerning MAP, a lowering was observed in all AMI groups versus Sham groups (91 •} 5 vs. 123 •} 7, t-test: p < 0.001). When compared to AMI group, no change of MAP was demonstrated in AMI-SR (p = 0.251), while a hypertensive effect of terlipressin is highlighted (p = 0.003). Of note, a slight hypotensive tendency of SR was observed in Sham-SR compared to Sham (101 •} 6 vs. 118 •} 6, t-test: p = 0.101, ANOVA: p = 0.422).

Assessment of Tissue Perfusion

SmO2 under anesthesia at D0 before surgery was identical in all groups with an average value of 39.0 •} 0.6% (p = 0.368). Immediately after surgery, we observed a similar drop in SmO2 in all groups (p = 0.141) (Figure 2a), with an average value of 30.0 •} 0.5%, different from D0 value before surgery (p < 0.0001). The drop tended to be more severe in averaged AMI groups compared to Sham groups (-24.9 •} 1.9% vs. -19.1 •} 2.4%, t-test: p = 0.065). At D1, SmO2 increased and recovered in Sham, Sham-SR and AMI-SR while it remained low in AMI and in TLP-treated animals (Figure 2a). SmO2 values were lower in AMI compared to Sham (p = 0.032), in Sham-TLP compared to Sham (p = 0.005) and to Sham-SR (p = 0.009), and in AMI-TLP compared to AMI-SR (p = 0.003), (Figure 2b). No difference in SmO2 was noticed between Sham-SR and AMI-SR, with values in these groups similar to that of Sham.

Blood lactate levels are frequently used to assess impaired tissue oxygenation. Here, the highest plasma lactate levels were measured in AMI, AMI-TLP and also in Sham-TLP, with mean values greater than that of Sham (Figure 2c). In Sham-SR and in AMI-SR a moderate but not significant elevation of lactate level was noticed. Both high lactate level and low SmO2 determined tissue hypoperfusion. A blood lactate level higher than 2.2 mmol/L indicated anaerobic metabolism and was therefore a marker of tissue hypoperfusion. Similarly, mesenteric hypoxia was evidenced when SmO2 was <35%. The incidence of mesenteric hypoxia was reduced in AMI-SR compared to AMI and to AMI-TLP (respectively 13%, 90% and 80%, p = 0.003). Combining those two parameters, we observed on Figure 2d that the incidence of tissue hypoperfusion was notably different between all groups (p = 0.001). Thus, tissue hypoperfusion was evidenced in nine AMI (69%), two AMI-SR (25%) and four AMI-TLP (80%) indicating a potential protecting effect of SR after AMI. Additionally, AMI did not increase the incidence of tissue hypoperfusion compared to Sham in SR-treated animals. Tissue hypoperfusion was also detected in Sham animals, with different incidence between groups, four Sham (19%), two Sham-SR (25%) and five Sham-TLP (100%) confirming the deleterious effect of TLP in animals without LCA ligation.

Vasopressin System Stimulation: Copeptin

Copeptin is considered as a reliable surrogate marker of AVP secretion. Thus, in order to evaluate AVP system stimulation, we assayed copeptin in plasma samples. Under physiological conditions, vasopressin and copeptin circulating levels are low as confirmed by the copeptin concentration of 40 •} 6 pmol/L measured in control non operated rats (n = 3). In all operated Sham animals, we observed plasma copeptin concentrations at D1 higher than the physiological basal value of controls, with an average value of 361 •} 42 pmol/L (Figure 3). No difference was detected in Sham between treated or not treated groups. Overall, copeptin level in AMI groups was higher than in Sham groups with significant differences between AMI and Sham (p = 0.034) and between AMI-SR and Sham-SR (p = 0.006). In AMI-SR, copeptin level even tended to be more important than in AMI (p = 0.057). In contrast, no difference was observed between Sham-TLP and AMI-TLP with a copeptin concentration in AMI-TLP lower than in AMI-SR (p = 0.002) and in AMI (p = 0.054). Of note, no increase of copeptin level was induced by AMI in TLP-treated animals.

Effect of Myocardial Infarction on Vascular Reactivity

To evaluate AVP sensitivity and efficiency in vascular bed, we analyzed the ex vivo contractile responses of rat aortic rings to cumulative doses of AVP normalized to the effect of a maximally active KCl concentration (60 mM). AVP induced concentration-dependent contractions in all groups (Figure 4). However, in AMI, the maximal contractile response and the sensitivity to AVP were decreased when compared to Sham with a 32% lowering of contraction (p = 0.011) and a slightly increased EC50 value (Table 3). In SR-treated animals, and similarly in Sham-SR and in AMI-SR, both contractile response and sensitivity were substantially decreased when compared to either Sham or AMI. In these groups, the strong increase in the EC50 values reflects the occupancy of the receptors by the competitive agonist SR 49059 [START_REF] Gal | Biochemical and Pharmacological Properties of SR 49059, a New, Potent, Nonpeptide Antagonist of Rat and Human Vasopressin V1a Receptors[END_REF]. Moreover, in TLP-treated animals, and similarly in Sham-TLP and in AMI-TLP, only a decrease of maximal contraction to AVP was observed, with no change of EC50 value. Of note, KCl-induced contraction and vasorelaxant effect of acetylcholine were identical in all groups. Only the response to PE differed with an increased contraction in TLPtreated animals either Sham or AMI (Table 3). Those results are in line with AVP receptors desensitization. In both SR-and TLPtreated animals this phenomenon is partially due to the respective presence of an antagonist and an agonist of AVP. Thus, in AMI, desensitization is to be linked to the increase in copeptin levels and therefore in AVP circulating levels.

Discussion

AMI with AHF represented a high-risk for CS (69% with tissue hypoperfusion) in the nontreated population. Increased plasma AVP levels have been observed in patients with AHF and have been shown predictive of severe HF development [START_REF] Francis | Comparison of Neuroendocrine Activation in Patients with Left Ventricular Dysfunction with and without Congestive Heart Failure. A Substudy of the Studies of Left Ventricular Dysfunction (SOLVD)[END_REF][START_REF] Rouleau | Prognostic Value of Neurohumoral Activation in Patients with an Acute Myocardial Infarction: Effect of Captopril[END_REF]. AVP could be involved in the onset of CS complicating AHF. In this study, we demonstrated for the first time that, in a rat model of CS, increased AVP blood level and vasopressinergic system activation contributed to mesenteric hypoperfusion. We showed that treatment with the AVP antagonist SR-49059 prevented tissue hypoperfusion and improved prognosis of CS while, in contrast, the agonist terlipressin aggravated them. These results suggested a critical role of vasopressinergic system in AMI-induced CS.

Experimental myocardial infarction in rats induced by coronary artery ligation provides a clinically relevant model for the consequences of myocardial infarction. However, CS animal models are scarce and exploring CS in animal is challenging. We have developed such a model and defined the criteria that allowed assessing CS following coronary ligation. CS is generally defined as a state of end-organ hypoperfusion and hypoxia due to primary cardiac disorders. In humans, common CS criteria are reduced blood pressure and evidence of hypoperfusion assessed by end-organ dysfunction (neurologic disorder, oliguria, clod extremities, mottling) or elevated serum lactate (>2.2 mmol/L) [5]. In rats, no criteria are defined. In the study, we have set a LVEF threshold of 40% to define AHF after MI.With this selection criterion, we observed in AMI group, without vasopressin antagonist or agonist treatments, hypotension and reduced CO associated with higher lactate levels and lower SmO2 values. These data are consistent with some criteria of CS defined in human and allowed us to validate our model.

Higher plasma copeptin levels were found in AMI. Copeptin is the C-terminal fragment of preprovasopressin which is co-secreted with AVP in the circulation. It is much more stable than AVP and could be used as a surrogate biomarker reflecting AVP production [START_REF] Khan | C-Terminal Provasopressin (Copeptin) as a Novel and Prognostic Marker in Acute Myocardial Infarction: Leicester Acute Myocardial Infarction Peptide (LAMP) Study[END_REF][START_REF] Morgenthaler | Assay for the Measurement of Copeptin, a Stable Peptide Derived from the Precursor of Vasopressin[END_REF]. The increased copeptin level indicates a substantial vasopressin release in response to myocardial infarction or AHF in line with previous findings [START_REF] Francis | Comparison of Neuroendocrine Activation in Patients with Left Ventricular Dysfunction with and without Congestive Heart Failure. A Substudy of the Studies of Left Ventricular Dysfunction (SOLVD)[END_REF][START_REF] Rouleau | Prognostic Value of Neurohumoral Activation in Patients with an Acute Myocardial Infarction: Effect of Captopril[END_REF]. However, we noted that the plasma copeptin values were higher in the Sham groups (360 pmol/L) compared to the non-operated rats (40 pmol/L). This reflected the activation of the vasopressinergic system induced by the surgery, as indicated previously [START_REF] Finnerty | The Surgically Induced Stress Response[END_REF]. It is also known that, following an acute myocardial infarction, the arginine-vasopressin system as the renin-angiotensin and the sympathetic nervous systems, is activated [START_REF] Rouleau | Prognostic Value of Neurohumoral Activation in Patients with an Acute Myocardial Infarction: Effect of Captopril[END_REF][START_REF] Mcalpine | Neuroendocrine Changes in Acute Myocardial Infarction[END_REF][START_REF] Mcalpine | Neuroendocrine Activation after Acute Myocardial Infarction[END_REF][START_REF] Cohn | Neuroendocrine Activation after Acute Myocardial Infarction[END_REF][START_REF] Riegger | Neuroendocrine Excitation in Heart Failure[END_REF]. We further observed an even higher copeptin level in AMI compared to Sham, revealing an additional release of AVP that can be interpreted as secondary to heart injury. Although such compensatory responses may be beneficial initially, epidemiological studies showed that sustained neurohumoral activation is associated with increased mortality [START_REF] Hartupee | Neurohormonal Activation in Heart Failure with Reduced Ejection Fraction[END_REF]. Unlike the renin-angiotensin system and the sympathetic nervous system, the role of the vasopressin system in the development of heart failure is understudied. In our CS rat model, the activation of the vasopressin system is corroborated not only by the increase in AVP secretion but also by the decrease in the contractile response to AVP on isolated artery ex vivo. Indeed, in AMI group, we observed a lower Emax value and a slightly higher EC50 value which reflected a decreased sensitivity and efficiency of exogenous AVP consistent with receptor desensitization. This phenomenon, generally occurring after more or less prolonged exposure to the agonist [START_REF] Holmes | Science Review: Vasopressin and the Cardiovascular System Part 1-Receptor Physiology[END_REF], reveals here a prior vasopressinergic stimulation following the increase in AVP release.

The effect of vasopressin on hemodynamic state and tissue perfusion during CS, was further investigated by treating animals either with a selective V1a receptor antagonist, SR-49059, or with an agonist, terlipressin. SR-49059, with commercial name Relcovaptan®, is a potent and selective non-peptide vasopressin V1a receptor antagonist devoid of agonist activity and displaying high affinity and efficacy for rat and human V1a receptors [START_REF] Gal | Biochemical and Pharmacological Properties of SR 49059, a New, Potent, Nonpeptide Antagonist of Rat and Human Vasopressin V1a Receptors[END_REF]. Terlipressin is a vasopressin analogue with potent vasoconstrictive properties involving V1a receptors [START_REF] Colson | a Vasoactive Prodrug Recommended in Hepatorenal Syndrome, Is an Agonist of Human V1, V2 and V1B Receptors: Implications for Its Safety Profile[END_REF]. To date, the effect of vasopressin receptor antagonists has not been described in the setting of CS. Several antagonists (vaptan) have been studied mainly focusing on chronic heart failure or on renal effect via V2 receptor inhibition [START_REF] Izumi | Therapeutic Potential of Vasopressin-Receptor Antagonists in Heart Failure[END_REF][START_REF] Gilotra | Arginine Vasopressin as a Target in the Treatment of Acute Heart Failure[END_REF]. In our study, the preoperative administration of SR-49059 had no incidence on the various measured parameters suggesting no effect on cardiac function and mesenteric oxygenation at baseline before surgery. At D1, a rather weak, decrease of MAP was observed in the Sham-SR group that could reflect a slight vasodilation with SR-49059 probably consecutive to an antagonism of the vasopressinergic system otherwise activated by surgery-induced stress [START_REF] Finnerty | The Surgically Induced Stress Response[END_REF]. This activation of the vasopressinergic system was also suggested by the high copeptin level compared to that of the non-operated animals. MAP was also decreased in AMI group. This hypotension at D1 in AMI group, despite a raise of copeptin level, is in agreement with the clinical observations and may be explained mainly by the decrease of CO insufficiently compensated by the vasoconstriction response. On the contrary, the increased MAP in animals treated with TLP is to be related to the high dose of agonist used, adapted to ensure a maximum vasoconstrictor effect. Since CO and systemic vascular resistance are the determinants of arterial blood pressure, a rise of MAP can be observed despite poor LV function and low CO in cases of major vasoconstriction. This hypertensive effect is also well documented [START_REF] Morelli | Terlipressin in the Treatment of Septic Shock: The Earlier the Better?[END_REF]. It is known that shock state first induces an increase in AVP level causing vasoconstriction that helps to maintain end-organ perfusion. A fall in AVP level, probably due to depletion of stores, follows that initial increase and may contribute to hypotension and should be implicated in secondary vasoplegia and distributive shock [START_REF] Colson | Post Cardiac Surgery Vasoplegia Is Associated with High Preoperative Copeptin Plasma Concentration[END_REF][START_REF] Levy | Epinephrine versus Norepinephrine for Cardiogenic Shock After Acute Myocardial Infarction[END_REF][START_REF] Leone | Decreased Vasopressin Responsiveness in Vasodilatory Septic Shock-like Conditions[END_REF]. Copeptin, due to its long half-life, could fail to follow rapid variations in AVP secretion and especially in decreases. This could certainly be a limitation in our model. Additionally, as shown by the lower copeptin level in TLP groups, treatment with terlipressin appears to have inhibited vasopressin release. The existence of such negative feedback through baroreceptors stimulation of the GABAergic pathway has been described from decades [START_REF] Renaud | CNS Pathways Mediating Cardiovascular Regulation of Vasopressin[END_REF]. Furthermore, both agonist and antagonist treatments showed a competitive profile on aortic contractile responses to AVP consistent with the high affinity of SR-49059 and of terlipressin for the V1a receptor [START_REF] Gal | Biochemical and Pharmacological Properties of SR 49059, a New, Potent, Nonpeptide Antagonist of Rat and Human Vasopressin V1a Receptors[END_REF][START_REF] Colson | a Vasoactive Prodrug Recommended in Hepatorenal Syndrome, Is an Agonist of Human V1, V2 and V1B Receptors: Implications for Its Safety Profile[END_REF]. An interesting observation is that treatment with SR-49059 had a positive impact on the evolution of AHF in AMI with improvement of tissue hypoperfusion and CS occurrence and outcome. In contrast, terlipressin treatment worsened the course of heart failure as shown by decreased SmO2 and increased serum lactate level in the TLP-treated groups. This is in line with a recently published study showing that ultra-low dose of exogenous AVP increased gastric microcirculatory oxygenation (μHbO2) while high dose of AVP reduced it [START_REF] Truse | Exogenous Vasopressin Dose-Dependently Modulates Gastric Microcirculatory Oxygenation in Dogs via V1A Receptor[END_REF]. The effects of AVP on μHbO2 were otherwise completely abolished by V1a receptor inhibition. Our finding supports a deleterious effect related to endogenous AVP release following AMI, involving prolonged mesenteric ischemia and anaerobic metabolism. This is the first description of vasopressin implication in microcirculation alteration in the context of post-ischemic CS. These findings are consistent with a preliminary prospective human cohort of patients with CS complicating AMI [37]. In this short series, patients had both severe impairment of microvascular reactivity and high copeptin level and early reversal of microcirculation impairment in response to hemodynamic support was associated with survival. The potential clinical implications of these results are to assess vasopressinergic system stimulation by measuring the level of plasma copeptin in the first hours after AMI and to find a way to prevent excessive vasopressin release. The potential protective effect of antagonist treatment must be explored and confirmed in case of administration after the onset of coronary occlusion to be transposable for clinical use. In the context of quick reduction of maximal contractile response to AVP, the agonist treatment appears unsound to restore arterial pressure. In case of vasoplegic syndrome following AMI, therapeutic compensation of vasopressin relative deficiency should be considered only once arterial blood flow is well restored, microcirculation closely monitored, and this treatment is quickly eliminated if not tolerated.

In summary, these results highlight the critical role of vasopressinergic response in the very early phase of ischemic AHF and its potential impact on time course of CS. Indeed, the acute release of vasopressin participates in the vasoconstrictive response of CS which induces, as a salvage response, a redistribution of blood flow to autoregulated organs but which is also responsible for side effects in particular mesenteric ischemia. This tissue damage may lead to organ dysfunction and contribute to severe complications, such as bacteria translocation from the gut, and to the secondary systemic inflammatory response observed in CS [5].

Materials and Methods

Animal Ethics

All experimental procedures were conducted in accordance with the European Union Laboratory Animal Care Rules (2010/63/EU Directive) and NIH Guidelines. The project (APAFIS#9808) was approved by the local committee for Animal Care of Montpellier-Languedoc-Roussillon (N• CEEA-LR-9808). Healthy male Wistar rats (350-400 g) were maintained in our animal facility one week before experiments with free access to food and water.

Experimental Design

Animals were randomly assigned for various surgical procedures and treatments. AMI was surgically induced by left coronary artery ligation. Fake surgery was performed in Sham. Six groups were studied: Sham and AMI without or with treatment with either terlipressin (Sham-TLP and AMI-TLP) or SR-49059 (Sham-SR and AMI-SR). Animals were sacrificed one day after surgery (D1). In order to study AMI-induced AHF, a LVEF ≤ 40% was a prerequisite for the selection of animals in AMI groups. To assess CS, a tissue hypoperfusion (lactate > 2.2 mmol/L) or mesenteric hypoxia (SmO2 < 35%) was required.

Surgical Procedure

Animals were anaesthetized by intraperitoneal injection of ketamine 130 mg/kg and xylazine 8.5 mg/kg and intubated with a 14-gauge catheter. They were mechanically ventilated with 2.5 mL of tidal volume by 70 cycles per minute. The heart was exposed for ligation after a left para-sternal thoracotomy and incision of the pericardium. AMI was induced by permanent ligation (supplemental Figure S1A, at 1 to 2 mm from the left atrial appendage line, of the LCA and the efficacy of the ligation was assessed by changes in electrocardiogram and in color of anterior myocardium turning white. At the end of surgery, pneumothorax was drained off, local analgesia was achieved with ropivacaine 2 mg/mL 0.5 mL/kg, skin was closed with Premio 5-0 stitches (Peters Surgical®) and 1 mL of isotonic saline solution was injected in peritoneum for volume compensation. Sham surgery was performed according to the same protocol except closing of the ligation. After surgery, rats were allowed to recover in individual cages. They were regularly checked and had free access to food and water. They were housed under conditions of controlled temperature (22-24 •C), humidity (45-50%) and light cycle (12 light hours/12 dark hours). One day after surgery (D1), full hemodynamic evaluation was performed under the same protocol of general anesthesia and animals were then sacrificed after exsanguination. Infarct size was determined after sacrifice and according to previously validated method [START_REF]Histological Quantification of Chronic Myocardial Infarct in Rats-Video[END_REF]. The heart was quickly excised and sliced into four 2.0mm-thick sections perpendicular to the long axis. Sections were incubated in 1% triphenyltetrazolium chloride at 37 •C for 10 min and then imaged using a colour flat-bed scanner at 600 dpi resolution (see illustration on supplemental Figure S1B). Healthy myocardium appeared in red and dead tissues in white. The infarcted area, colored in white, was determined by computerized planimetry with ImageJ® 1.52v software in each slice. The infarcted area/total LV myocardial area ratios for each slice were calculated. Results are expressed in average of percentage of infarcted area on total area on the 4 slices.

Heart Rate and Blood Pressure Measurement

The heart rate was determined by electrocardiography monitoring at D0 and D1 during anesthesia. The arterial pressure was measured invasively at D1 via cannulation of the right carotid artery using a pressure transducer connected to a monitor (ADInstruments, Sidney, Australia). The pressure signal was analyzed by a data acquisition software (LabChart Reader 8.1.5, ADInstruments, Sidney, Australia) and the mean arterial pressure (MAP) was determined.

Echocardiography

High resolution echocardiography (VisualSonics/Fujifilm, Toronto, ON, Canada with a MS250D ultrasound probe 20 MHz) was performed at D0 before surgery and at D1 before sacrifice, under anesthesia by 2% isofluorane inhalation at 37 •C, and under ECG and respiratory rate monitoring, as previously described [START_REF] Rouhana | Early Calcium Handling Imbalance in Pressure Overload-Induced Heart Failure with Nearly Normal Left Ventricular Ejection Fraction[END_REF]. Transthoracic echocardiographic recordings are illustrated in supplemental Figure S2. Left ventricular (LV) parasternal long axis 2D view in B-mode and M-Mode was performed at the level of papillary muscle to assess LV wall thicknesses and internal diameters, allowing the calculation of LVEF and left ventricular end diastolic diameter (LVEDD). The velocity time integral of blood flow in the ascending aorta was used to calculate CO.

Mesenteric Haemoglobin Oxygen Saturation

Tissue oxygenation was assessed by a regional measurement of mesenteric haemoglobin oxygen saturation (SmO2) with a transcutaneous NIRS device (INVOS™ 5100C cerebral/ somatic oximeter, Medtronic, Brampton, ON, Canada), reflecting the oxygen content in microcirculation (vessels diameter < 1 mm) through a sensor applied on the abdominal area between the xiphoid appendix and bladder, analyzing the tissue at a depth of 1.5 cm as illustrated in supplemental Figure S3. Determinations of SmO2 were performed at D0 just before and immediately after the surgery and at D1, by the average of the acquisitions obtained from two positions of the NIRS sensor.

Lactate and Copeptin Assay

Blood sample collected in an EDTA tube (8 mL) was immediately centrifuged for 12 min at 1300× g, 4 •C and plasma was frozen at -80 •C for further assays. Lactate concentration was determined with Cobas 8000 analyzer (colorimetric assay, reference interval [0.5-2.2] mM, Roche Diagnostics, Meylan, France). Copeptin was assayed using ELISA kit (Cusabio Technology, Houston, TX, USA) according to manufacturer instructions.

Vascular Reactivity

Experiments were performed as previously described [START_REF] Fort | New Insights in the Contribution of Voltage-Gated Na(v) Channels to Rat Aorta Contraction[END_REF] on isolated aortic rings mounted onto thin stainless-steel holders and placed in organ chambers filled with a saline solution (PSS) maintained at 37 •C and continuously bubbled with O2. Changes in isometric tension were measured using a force transducer (IT1-25) and recorded by an IOX computerized system (EMKA Technologies, Paris, France). After an equilibration period of 1 h at a resting tension of 2 g, functional integrity of each arterial segment was evaluated by the contractile response to a single dose of phenylephrine (PE, 10 μM) and then by the relaxant effect of acetylcholine (ACh, 1 μM) which validated endothelium function. After a wash-out and stabilization period of 20 min, the contractile capacity of vascular smooth muscle cells was evaluated with cumulative dose responses to the different agonists: AVP, KCL, or PE. The relaxation was studied with cumulative doses of ACh on arterial rings previously contracted with a submaximal dose of PE (10 μM).

Drugs

SR-49059 (Relcovaptan) is a selective nonpeptidic V1a receptor antagonist. It was obtained from Sanofi-Aventis (Sanofi-Aventis, Toulouse, France). SR-49059 was dissolved in DMSO (48 mg/mL) then extemporaneously emulsified in Cremophor® RH40 (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and diluted in 9% sodium chloride solution before injection. Treatment consisted of three intraperitoneal injections of SR-49059 solution performed, 1 h before anesthesia (4.5 mg/kg), 8 h after the surgery (6 mg/kg) and 1 h before sacrifice (4.5 mg/kg). Terlipressin is a well-documented vasopressin receptor agonist inducing intense and prolonged vasoconstriction. Commercial terlipressin, Glypressine®, was obtained from Ferring GmbH (Kiel, Germany). Its purity has been previously evaluated by mass spectrometry [START_REF] Colson | a Vasoactive Prodrug Recommended in Hepatorenal Syndrome, Is an Agonist of Human V1, V2 and V1B Receptors: Implications for Its Safety Profile[END_REF]. Terlipressin solution (20 μg/mL) was prepared by solubilization in 9% sodium chloride. Treatment consisted of three intraperitoneal injections of terlipressin solution performed 1, 12 and 23 h after surgery and corresponding to a total dose of 80 μg/kg/24 h. To reduce the risk of bleeding due to the increase of blood pressure, terlipressin was not administered either preoperatively or immediately after surgery.

Statistical Analysis

Results, expressed as mean and standard error of the mean (SEM), were compared between non treated groups (Sham and AMI), antagonist treated groups (Sham-SR and AMI-SR) and agonist treated groups (Sham-TP and AMI-TP) by an ordinary one-way ANOVA or a Kruskal-Wallis test according to the variable distribution. An appropriate post-hoc multiple comparisons test was used to determine which groups are different. Paired t-test or Wilcoxon test were used for comparisons between D0 and D1 within each group. Distribution of outcome endpoints were compared by a Fisher's exact test. All probability values are 2-sided, and values of p < 0.05 were considered to be significant. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA).

Conclusions

CS after AMI was characterized by a sustained decrease in mesenteric oxygenation and by lactate production. Increased vasopressin secretion seems involved in the early phase of AHF, which may result in worsening mesenteric hypoperfusion despite quick reduction in vascular reactivity to vasopressin. High vasopressin or agonist stimulation following AHF may explain the alteration of arterial contractile response to vasopressin and the implication of this mechanism in secondary vasodilation syndrome needs to be further investigated.

Figure 1 .

 1 Figure 1. Experimental design and group selection for analysis at postoperative day 1 (D1). AMI, acute myocardial infarction; SR-49059, vasopressin receptor antagonist; LVEF, left ventricular ejection fraction; TLP, Terlipressin. a Fisher exact test between the three AMI groups: p = 0.057. § Chi-2 test between preoperatively non-treated AMI vs. SR-treated AMI groups: p = 0.035.

Figure 2 .

 2 Figure 2. Tissue hypoperfusion assessment by mesenteric oxygenation saturation (SmO2) and lactate level. (a) Variation of SmO2 measured for all groups at D0 before surgery, at D0 immediately after surgery and at D1. Values were normalized to the initial value at D0 and data are mean •} SEM. £ p < 0.05, ££ p < 0.01 for paired measures D1 vs. D0 after surgery in each group. (b) Box and whisker plots of SmO2 values at D1 (Kruskal-Wallis test, p < 0.0001). * p < 0.05 for AMI versus Sham with the same treatment. § § p < 0.01 for treated versus non-treated group for the same coronary intervention. $$ p < 0.01 AMI-SR vs. AMI-TLP and Sham-SR vs. Sham-TLP. (c) Box and whisker plots of plasma lactate level at D1 (Kruskal-Wallis test, p = 0.034). * p < 0.05 for AMI versus Sham with the same treatment. § p < 0.05 for treated versus non-treated group for the same coronary intervention. (d) Distribution of animals with tissue hypoperfusion, defined as lactate > 2.2 mmol/L or SmO2 < 35% at D1, and expressed as percentage (Chi-square: p = 0.001). No: no hypoperfusion; Yes: with hypoperfusion.

Figure 3 .

 3 Figure 3. Copeptin levels measured at D1 before sacrifice in all groups. Data are presented as box and whisker plots. Analyses were performed using an ordinary one-way ANOVA (p = 0.001) followed by Holm-Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01 for AMI versus Sham with the same treatment; § § p < 0.01 for the effect of treatment in the same coronary intervention.

Figure 4 .

 4 Figure 4. Vascular reactivity: ex vivo vasopressin-induced contractile responses of rat aortic rings.

Acknowledgments:

We thank the small animal imaging platform of Montpellier (IPAM; http://www.ipam.cnrs.fr (accessed on 1 January 2019)) for access to the echocardiography and the network of animal facilities RAM.

Funding:

This work was funded by the French Institute for Health and Medical Research (INSERM) and without specific grant.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Author Contributions:

Conceptualization, methodology, formal analysis and data curation P.G. and A.V.; validation, G.G., P.C., J.-P.C. and S.R.; investigation, P.G., H.D., P.B. and P.S.; data curation P.G., P.S. and A.V.; writing-original draft preparation, P.G. and A.V.; writing-review and editing, S.R., G.G. and P.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:

All animal experimental procedures were conducted in accordance with the European Union Laboratory Animal Care Rules (2010/63/EU Directive) and NIH Guidelines. The study protocol was approved by the local committee for Animal Care of Montpellier-Languedoc-Roussillon (N• CEEA-LR-9808) with the authorization number APAFIS#9808 (13 June 2017).

Informed Consent Statement:

Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Omar Alrehaili
email: oalrehaili@ksu.edu.sa

Ana S Fajardo

Paul Westerhoff

Microfluidic Flow-By Reactors Minimize Energy Requirements of Electrochemical Water Treatment Without Adding Supporting Electrolytes

Keywords: Microfluidics, Electrochemical treatment, energy, mass transport, diffusion, water treatment

While electrode material advances show how electrochemical processes can degrade wide range of environmental pollutants, less research exists on how to lower energy requirements for electrolysis through innovation in reactor geometry that control mass transfer. Microfluidic devices with high electrode surface area to solution volume ratios offer unique insights since they can minimize mass transfer limitations in electrochemical reactors and decrease the energy requirements. Shortening interelectrode distances hold promise to overcome charge transfer limitations, which otherwise requires electrolytes addition for low conductivity drinking waters.

To examine the interconnected effects of electrode separation distances and solute concentrations on mass transport and energy efficiency, electrochemical treatment using a model water pollutant (nitrite ion) was evaluated in a flow-by microelectromechanical system (MEMS) reactor where the electrodes were ~ 40 µm apart in microfluidic reactors. The performance was compared to conventional completely-mixed batch reactors with interelectrode distance of 1 cm. The microfluidic reactors showed decrease of electrical energy per order (EE/O) in one order of magnitude with the ability to degrade contaminants with concentrations as low as 5 mg/L without increasing the energy consumption with the absence of supporting electrolytes. This paper presents a promising approach to study and ultimately develop cost-effective electrochemical technologies by optimizing electrode separation distances, instead of increasing salinity which is often required to achieve sufficient conductivity in water but is counter producing low-salt potable drinking water.

Introduction

Electrochemical water treatment processes are promising technologies for the advanced removal of targeted pollutants from drinking water resources and wastewater treatment plants effluents, including advanced oxidation and electrocatalytic reduction processes [1][2][3][4] . Heterogeneous hydroxyl radicals (  OH) are electrogenerated from water at the anode's surface during electrochemical oxidation (EO) for indirect electrochemical oxidation process [START_REF] Panizza | Direct and Mediated Anodic Oxidation of Organic Pollutants[END_REF] . In electrocatalytic reduction, electroactive species gain electrons through reduction reactions that occur at the cathode surface. In the case of nitrite reduction for example, the reduction mechanism can involve direct charge transfer process and indirect reduction from adsorbed hydrogen (Hads) formed during the reduction of water. However, both electrochemically-driven reactions occur at the electrode surface making electrochemical treatments mass transfer limited processes. Limited mass transport might lead to high energy consumption when scaling-up electrified water treatment [START_REF] Martínez-Huitle | Single and Coupled Electrochemical Processes and Reactors for the Abatement of Organic Water Pollutants: A Critical Review[END_REF][START_REF] Pérez | A Micro Fluidic Fl Ow-through Electrochemical Reactor for Wastewater Treatment : A Proof-of-Concept[END_REF] . Different electrocatalysts can control selectivity of nitrite reduction towards nitrogen gas (N2) or ammonium (NH4 + ) [START_REF] Flores | Outlining Key Perspectives for the Advancement of Electrocatalytic Remediation of Nitrate from Polluted Waters[END_REF][START_REF] Cerrón-Calle | Strategic Tailored Design of Electrocatalysts for Environmental Remediation Based on Density Functional Theory (DFT) and Microkinetic Modeling[END_REF] . While preferential formation of N2 is the most desired outcome when considering applications for drinking water, recent studies highly value NH4 + as an added value product when considering water reuse for agriculture [START_REF] Van Langevelde | Highly Reactive Cu-Pt Bimetallic 3D-Electrocatalyst for Selective Nitrate Reduction to Ammonia[END_REF]11 .

The efficiency of an electrochemical reaction is controlled by charge or mass transfer [START_REF] Mart | Single and Coupled Electrochemical Processes and Reactors for the Abatement of Organic Water Pollutants : A Critical Review[END_REF][START_REF] Kapałka | Basic Principles of the Electrochemical Mineralization of Organic Pollutants for Wastewater Treatment[END_REF] .

Charge transfer is referred to the interfacial reaction at the electrode surface that is responsible of the redox reaction involving an electroactive species. When the reaction is governed by charge transfer, kinetics is determined by the number of electrons delivered and not by the target pollutant transport to the electrode surface. Charge transfer can be controlled by current intensity in the electrochemical process, higher current intensity leads to higher delivery of electrons per second which may accelerate the charge transfer rate. Hence, higher charge transfer can lead to a faster abatement of target pollutant. However, increasing current intensity may decrease the total efficiency of the process determined by Faradaic efficiency (FE), the efficiency of charge (electron) transfer to a system facilitating an electrochemical reaction.

Mass transfer involves the transport of the target pollutant from/towards the electrode surface. This process is mainly governed by diffusion and migration of chemicals from the bulk solution to the electrodes surfaces where the electrochemical reactions occur [START_REF] Walker | Mass Transfer and Residence Time Distribution in an Electrochemical Cell with an Air-Diffusion Electrode: Effect of Air Pressure and Mesh Promoters[END_REF][START_REF] Coñizares | Measurement of Mass-Transfer Coefficients by an Electrochemical Technique[END_REF] . Mass transfer by diffusion is controlled by diffusivity of individual species and thickness of diffusion layer that is influenced by the Reynold's number and solute concentration gradients between bulk solution and electrode surface [START_REF] Srisurichan | Mass Transfer Mechanisms and Transport Resistances in Direct Contact Membrane Distillation Process[END_REF][START_REF] Vakhrushev | Simulation of Nanocomposite Coating Created by Electrocodeposition Method[END_REF] . Mass transfer by migration is driven by electrical potential gradients in the cell. High resistance in an electrochemical cell hinders mass transport where energy consumption increases for solutions with low conductivity due to ohmic drop. Solution conductivity has a direct impact on electrocatalytic activity [START_REF] Garcia-Segura | Disparities between Experimental and Environmental Conditions: Research Steps toward Making Electrochemical Water Treatment a Reality[END_REF] . The addition of supporting electrolyte decreases the resistance and diminishes the overall energy consumption [START_REF] Herrmann | Heterogeneous Photocatalysis: Fundamentals and Applications to the Removal of Various Types of Aqueous Pollutants[END_REF][START_REF] Wang | Microfluidic Photoelectrocatalytic Reactors for Water Purification with an Integrated Visible-Light Source[END_REF][START_REF] Wang | Microfluidic Reactors for Photocatalytic Water Purification[END_REF][START_REF] Mousset | Advanced Electro Oxidation with Boron Doped Diamond for Acetaminophen Removal from Real Wastewater in a Microfluidic Reactor: Kinetics and Mass Transfer Studies[END_REF] . However, adding salt could be expensive and may require additional removal of added chemicals. Furthermore, most research add > 50 mM of ionic strength (> 3000 μS/cm) to achieve sufficient background electrolyte concentrations. The secondary drinking water standard in the United States of America for total dissolved solids (TDS) is 500 mg/L (~ 750 μS/cm or ~12 mM). Therefore, most electrochemical studies have utilized conductivity levels far in excess of salt concentrations recommended for drinking water for aesthetic or health reasons.

Advances in micro and nanofluidics can tackle the issues of high energy consumption in water treatment processes [START_REF] Shahbabaei | Advances in Nanofluidics for Water Purification and Filtration: Molecular Dynamics (MD) Perspective[END_REF] . Microfluidic reactors with micro inter-electrode distance could overcome electrochemical limitations of mass transfer and decrease energy consumption even with the absence of supporting electrolytes at low cell voltage [START_REF] Wang | Microfluidic Reactors for Photocatalytic Water Purification[END_REF][START_REF] Kuleshova | The Methoxylation of N-Formylpyrrolidine in a Microfluidic Electrolysis Cell for Routine Synthesis[END_REF][START_REF] Kuleshova | A Simple and Inexpensive Microfluidic Electrolysis Cell[END_REF][START_REF] Amemiya | Self-Supported Paired Electrosynthesis Using a Microflow Reactor Without Intentionally Added Electrolyte[END_REF][START_REF] Ziogas | Electrochemical Microstructured Reactors: Design and Application in Organic Synthesis[END_REF][START_REF] He | Electrolyte Free Electro-Organic Synthesis: The Cathodic Dimerisation of 4-Nitrobenzylbromide in a Micro-Gap Flow Cell[END_REF] . The small distance between the two electrodes accelerates and intensify the kinetic of mass transfer rate inside the reactor leading to the ability of achieving a continuous flow mode where pollutants are converted in a single passage of solution [START_REF] Scialdone | Oxidation of Organics in Water in Microfluidic Electrochemical Reactors: Theoretical Model and Experiments[END_REF][START_REF] Scialdone | Electrochemical Conversion of Dichloroacetic Acid to Chloroacetic Acid in Conventional Cell and in Two Microfluidic Reactors[END_REF] . The ability to achieve a continuous flow reaction with short residence time allows flexibility on changing applied conditions as compared to a batch reactor. Most microfluidic reactors reported on the literature are based on building a commercially available filter press cell (flow-by-cell) made of polytetrafluoroethylene PTFE spacer (100-500 µm) to separate the two electrodes [START_REF] Pérez | A Micro Fluidic Fl Ow-through Electrochemical Reactor for Wastewater Treatment : A Proof-of-Concept[END_REF][START_REF] Scialdone | Oxidation of Organics in Water in Microfluidic Electrochemical Reactors: Theoretical Model and Experiments[END_REF][START_REF] Scialdone | Electrochemical Conversion of Dichloroacetic Acid to Chloroacetic Acid in Conventional Cell and in Two Microfluidic Reactors[END_REF] . Other devices were made by micro-milled adhesive spacer for microchannel between two electrodes [START_REF] Wang | Microfluidic Reactors for Photocatalytic Water Purification[END_REF][START_REF] Scialdone | Electrochemical Conversion of Dichloroacetic Acid to Chloroacetic Acid in Conventional Cell and in Two Microfluidic Reactors[END_REF] .

In this work, microfluidic devices were fabricated as a cross flow system between two electrodes based on microelectromechanical system (MEMS) fabrication techniques, where the interelectrode distance was 40 µm. The objective is to evaluate the impact of engineering reactor design on the electrochemical systems performance. As a model reaction, nitrite conversion was studied in electrochemical microfluidic reactors in a single pass as compared to bulk batch reactors without or with supporting electrolytes (variable salinity concentrations) and benchmarked using engineering figures of merit. Nitrite is regulated by the United States of America Environmental Protection Agency (EPA) with a maximum contaminant level (MCL) of 1 mg NO2 --N/L. The influence of main parameters in electrochemical reactors such as current intensity and solution conductivity are considered. Electrical energy per order and electrical consumption were reported and compared for the electrical requirements between the two types of reactors.

Material and Method

Electrodes Preparation and Microfluidic Device Fabrication

All electrodes used in this work were made of platinum (Pt) and prepared in the lab using silicon (Si) wafers as substrates for all devices. Due to its high corrosion resistance and ability to prevent poisoning by reduction by-products, platinum electrodes prove to be excellent electrocatalysts for nitrate/nitrite reduction 2,[START_REF] Cao | Recent Advances in the Stabilization of Platinum Electrocatalysts for Fuel Cell Reactions[END_REF][START_REF] Kettler | Platinum Group Metals in Catalysis: Fabrication of Catalysts and Catalyst Precursors[END_REF] . Platinum has been reported to have high selectivity for nitrate/nitrite reduction towards ammonium [START_REF] Fajardo | Earth-Abundant Elements a Sustainable Solution for Electrocatalytic Reduction of Nitrate[END_REF] . A Platinum layer of 100 nm were coated on wafers using thermal evaporation techniques. Preparing electrodes by thermal evaporation technique results in a homogeneous and smooth electrodes surface to avoid friction forces that might increase in microchannel as reported in other works [START_REF] Pérez | A Micro Fluidic Fl Ow-through Electrochemical Reactor for Wastewater Treatment : A Proof-of-Concept[END_REF] .

Electrochemical microfluidic devices were fabricated based on microelectromechanical system (MEMS) techniques where the interelectrode distance (the distance between electrodes) is less than 50 µm (~40 µm) (Figure 1). Briefly, the platinum coated electrodes were further coated with epoxy polymer (SU-8) by a spin coater acting as a separation layer between the two electrodes in the device. The thickness of the epoxy layer determines the distance between the two electrodes in the microfluidic device and it is controlled by the viscosity of the polymer and the spin coat speed. The final result shows a thickness layer of ~40 µm. A channel was etched on the epoxy layer all the way to Pt surface on the wafer where solution pass though between the electrodes. Lastly, in order to connect the microchannel to macro reservoirs, a microfluidic delivery device fabrication (stamp) is needed. The delivery device was made of polydimethylsiloxane (PDMS) and connected to a syringe pump through needles and luer connector (silicon tubes). More details in the device fabrication were reported in the supporting information (Figure S1).

Operation of Electrochemical Reactors

Experiments were conducted with solutions of 50 mg-N/L of nitrite (equivalent to 3.56 mM of potassium nitrite (KNO2) and 165 mg/L as nitrite) with initial pH 6.5. Conductivity was modified by adding Na2SO4 as supporting electrolyte with concentrations ranging from 0 up to 100 mM of Na2SO4. Nitrite was used to evaluate the reactors performance as its decay reaction by platinum electrodes is well achieved. The applied current intensity was varied from 1 mA to 160 mA using a power supply (Tenma DC 72-2720). These solutions were treated galvanostatically using the microfluidic electrochemical reactor and a batch reactor illustrated in Figure 2 100 100

(Batch reactor) ( 2
)
where Qexp is the experimental charge consumed in the reaction in Coulomb (C), Qtheor is the total charge circulated in (C), n is the amount of electrons required per mol of product (mol), F is the Faraday constant (96,487 C mol -1 ), Ni-r is the amount of product generated per volume (mole/L),

Qr is the flowrate in the microfluidic reactor (L/h), Ni is the amount (mol) of product generated during the electrolysis, I is the applied current in amperes (A), t is the reaction time in hours (h), and 3600 is a unit conversion factor (3600 s/h).

To compare batch and microfluidic reactors, the electrical energy requirements were benchmarked using engineering figures of merit. The electrical energy per order (EE/O) (kWh m - 3 order -1 ) is defined as the electric energy required to remove a contaminant by one order of magnitude in a unit volume, as estimated by Eqs. ( 3) and (4) 2,35 :

/ 0 (Flow-by reactor) (3) / 0 (Batch reactor) (4)
where Ecell is the average cell potential in volt (Vs), Vs is the solution volume treated in liter (m 3 ), Q is flowrate for the continuous flow system in cubic meter per hour (m 3 /h), and C and C0 are the final and initial concentration of pollutant (mg/L). The electrical energy consumption EC (kWh g - 1 ) required for the degradation of a unit mass of contaminants was estimated by Eqs. ( 5) and ( 6) 35,[START_REF] Lanzarini-Lopes | Electrical Energy per Order and Current Efficiency for Electrochemical Oxidation of P-Chlorobenzoic Acid with Boron-Doped Diamond Anode[END_REF] :

0 (Flow-by reactor) (5) 0 (Batch reactor) (6)
Two methods are used to compare reactors performance in terms of mas transfer coefficient: (i) experimentally based on limiting current measurements using linear sweep voltammetry [START_REF] Walker | Mass Transfer and Residence Time Distribution in an Electrochemical Cell with an Air-Diffusion Electrode: Effect of Air Pressure and Mesh Promoters[END_REF][START_REF] Coñizares | Measurement of Mass-Transfer Coefficients by an Electrochemical Technique[END_REF][START_REF] Everett | Manual of Symbols and Terminology for Physicochemical Quantities and Units, Appendix II: Definitions, Terminology and Symbols in Colloid and Surface Chemistry[END_REF] , and (ii) empirically by using dimensional analysis [START_REF] Coñizares | Measurement of Mass-Transfer Coefficients by an Electrochemical Technique[END_REF][START_REF] Trellu | Electro-Oxidation of Organic Pollutants by Reactive Electrochemical Membranes[END_REF] . In this research, dimensional analysis is considered as commonly used method to determine mass transfer coefficient rate based on the three nondimensional groups: the Sherwood number (Sh) described by Eq. ( 7) [START_REF] Coñizares | Measurement of Mass-Transfer Coefficients by an Electrochemical Technique[END_REF] ; the Reynolds number (Re) defined according to Eqs. ( 8) and ( 9) [START_REF] Tchobanoglous | Wastewater Engineering[END_REF] ; and the Schmidt number (Sc) following Eq. (10). These groups are related to the mass transfer coefficient km (m/s) which was calculated from Eq. (11) [START_REF] Coñizares | Measurement of Mass-Transfer Coefficients by an Electrochemical Technique[END_REF] for Re # < 2000:

3.33 / / / (7)
(Flow-by reactor) (8)

(Batch reactor) (9) (10)
(11

)
where dh is the equivalent diameter of the channel (m), D is the diffusivity of nitrite in water (m 2 /s),

Le is the ratio of dh over electrode length, u is the velocity of fluid (m/s), is the kinematic viscosity of the fluid (m 2 /s), Dr is impeller diameter (m), n is rotation speed (rotation/s), ρ is the density of solution kg/m 3 ), and µ is the viscosity if fluid (P.s) . Also, the diffusion layer thickness δ (m) affects mass transfer in electrochemical cell as a function of ion diffusivity and diffusion transfer coefficient. Since most of mass transfer occurs due to diffusion 34 , mass transfer coefficient assumed to be due diffusion where diffusion layer thickness was calculated by Eq. ( 12) [START_REF] Everett | Manual of Symbols and Terminology for Physicochemical Quantities and Units, Appendix II: Definitions, Terminology and Symbols in Colloid and Surface Chemistry[END_REF][START_REF] Prasad | Analysis of the Diffusion Layer Thickness, Equivalent Circuit and Conductance Behaviour for Reversible Electron Transfer Processes in Linear Sweep Voltammetry[END_REF] :

(12)

Result and Discussion

Electrolysis were performed in microfluidic and conventional batch reactors with interelectrode distance of 40 µm and 1 cm, respectively. The electrolysis performance metrics were obtained for nitrite decay, EE/O, and EC by varying current density and solution conductivity with and without adding supporting electrolytes.

Electrolysis Performance for Nitrite Conversion

Electrolysis were conducted to evaluate the two reactors for nitrite decay over time without adding supporting electrolytes. The applied current density was fixed at j= 18 mA/cm 2 , with corresponding current intensity of I ~3 mA for microfluidic reactor and I = 80 mA for batch reactor. Figure 3 shows nitrite decay in microfluidic reactor that occurs in single pass where the flowrate through the reactor channel determines the hydraulic residence time (HRT). Slower flowrates lead to longer HRT and therefore improved the percentage of nitrite reduction achieved per single pass, resulting in larger amounts of charge transferred to the solution with increase HTR.

The flowrate was varied from 0.021 to 0.5 mL/min with a HRT corresponding to 1.43 to 0.06 second (s). Triplicate measurements of nitrite abatement were conducted at each HRT experiments and the results were very consistent for the microfluidic reactors at average standard deviation (SD = ± 3 x 10 -3 mg/L).

To compare the performance of both reactors for nitrite decay, Figure 3 also shows the decay of nitrite over time in a 100 mL solution in a batch reactor. Nitrite decay improves by increasing reaction time where ~95% nitrite reduction was achieved after 3 hours runs. The decay rate in microfluidic reactor was faster than batch reactor, where 95% reduction was achieved in ~1.5 s as compared to 3 hours in batch reactor at fixed current density. Faradic efficiency (FE) was calculated to measure the efficiency of the reactors by Eq. ( 1) and ( 2) for the conversion of nitrite and the production of ammonium as expected from the reduction of nitrite by Pt cathode (Figure 3). For both reactors, FE decreased by increasing the reaction time. Even with short reaction time in microfluidic reactors, FE for the two reactors were significantly different (by t-test) at more than two times higher in microfluidic reactor at an average of ~ 9% as compared to batch reactor (~3.5%) due the high area of electrode to solution volume ratio (~ 250 cm 2 /mL) as compared to (~ 0.045 cm 2 /mL) in batch reactor. FE for converting nitrite to ammonium is consistent with literature reports ranging from 2% to 9 % [START_REF] Reuben | Efficient Reduction of Nitrite and Nitrate to Ammonia Using Thin-Film B-Doped Diamond Electrodes[END_REF][START_REF] Kuang | Comparison of Performance between Boron-Doped Diamond and Copper Electrodes for Selective Nitrogen Gas Formation by the Electrochemical Reduction of Nitrate[END_REF] .

Kinetics Reaction Rate Constant

The electrolysis rate in the conventional batch reactor follows pseudo first-order kinetics for nitrite decay (r NO2-= -k [NO2 -]) with an apparent reaction rate constant of k = 3 x 10 -3 s -1 (Figure 4a). On the other hand, rates in the microfluidic reactor exhibit a two-stage kinetic as a function of HRT. Figure 4b shows nitrite rates in the microfluidic reactor first follow zero-order kinetics (r NO2-= -k') with faster decay rates of nitrite at shorter HRT (0.06 -0.14 s) followed by pseudo first-order kinetics at longer HRTs (0.24 to 1.42 s). First, the faster kinetic rate occurs until reaching a limiting current density (the maximum current to achieve a desired reaction before to the simultaneous discharge of extraneous ions), when it then becomes mass transfer limited by diffusion as described previously in kinetic models. The zero-order kinetics (r NO2-= -k') have k'

The Effect of Current Density on Microfluidic Electrolysis Performance

The applied current density (j) is an important parameter for electrochemical processes since j controls the reaction kinetics. At high applied current, more electrons are delivered per second leading to enhanced transfer of electrons between the electrodes and the electroactive species in solution. Higher availability of electrons may lead to an increase in reaction rates.

However, applied currents above the current limit result in a transition towards mass transfer limitation, where the degradation rate becomes limited by diffusion of electroactive species (i.e., nitrite) towards the electrode surface. Then, the additional electrons transferred are used in parasitic reactions such as hydrogen evolution reaction from water reduction. When increasing current has no or minimum effect on electrolysis performance, the reactor is considered a mass transfer limited.

To evaluate the effect of current density on nitrite removal in the microfluidic reactor, Figure 5 shows the electrolysis performance for nitrite reduction at different applied current density j ranging from 18 mA/cm 2 (I = 3 mA) up to 640 mA/cm 2 (I = 80 mA). The initial concentration of nitrite was 50 mg-N/L (0.53 mS/cm) and no supporting electrolytes were added.

Increasing the HRT in the cell improves the reduction percentage attained for both applied currents. However, at j = 640 mA/cm 2 , nitrite reduction increased by an average of 20% as compared with reactors where j = 18 mA/cm 2 was applied due to the increase of the amount of charge passed through the electrodes. However, increasing the current also leads to the increase of the energy consumption in the reactor.

The Effect of Current Density on Energy Requirements

The effect of current density on electrical energy requirements is evaluated by testing a range of currents at a fixed reaction time. Figure 6 shows the increase in electrical energy per order (EE/O) (kWh m -3 order -1 ) of the nitrite reduction calculated by Eq. ( 3) and ( 4) for both microfluidic and batch reactors at applied current density ranges from j= 8 mA/cm 2 to 40 mA/cm 2 . In the microfluidic reactor, the experiment proceeds at fixed HRT of 0.24 s, whereas reaction times up to 1 hour were monitored in the conventional batch reactor. Supporting electrolytes were not added for both reactors.

In the microfluidic reactor (Figure 6), nitrite reduction of 66% was achieved at low current density of j= 8 mA/cm 2 (I = 1 mA) at the given HRT. Nitrite reduction then increased by 13% when the current density was raised to j = 40 mA/cm 2 (I = 5 mA). The EE/O in the microfluidic reactor increased four-fold when the current density increases by factor of five. On the other hand, nitrite reduction in batch reactor was at ~ 50% at lower current density j= 8 mA/cm 2 (I = 40 mA) and increased by 19% when current density increased to j= 40 mA/cm 2 (I = 160 mA). The electrical energy per order increased by nine-fold when the current density was increased by a factor of five (Figure 6). However, the microfluidic reactor achieved the same nitrite reduction and more when compared to the batch reactor with at least one order of magnitude lower electrical energy required when current density was j > 20 mA/cm 2 . This is due the increase in mass transfer rate achieved by small micrometric distance between the two-electrode that leads to low resistance in the reactor.

The Effects of Electrolyte Concentrations on Energy Requirements

The conductivity of an aqueous solution indicates the ability of the solution to conduct electric current. By increasing the concentration of ions in solution, conductivity will be increased and hence resistance in the solution is decreased between the electrodes. Thus, the addition of supporting electrolytes (non-electroactive ions) is used to increase the solution conductivity, decrease the diffusion layer on the electrode, and decrease the cell potential. Enhancing the solution conductivity, it also decreases the resistance in the medium and diminishes the overall energy consumption. Moreover, adding supporting electrolytes to the system may affect the mass transfer in the electrochemical reactors by eliminating the mass transfer by migration leading to dominant mass transfer by diffusion. However, presence in high concentrations of other electrolytes could interfere with the electrochemical reactions of interest by enacting competitive reactions. Also, the requirement of salt addition may increase the operation costs and might require additional removal of salt content after treatment.

Figure 7 shows nitrite reduction and electrical energy requirements in batch and microfluidic reactors for different initial nitrite concentrations with and without adding supporting electrolytes. The conventional batch reactor experiments were conducted by using nitrite solutions (5 -50 mg-N/L) at fixed current density (j = 18 mA/cm 2 ) and fixed reaction time of one hour of 100 mL solutions (Figure 7a). At first, electrolysis runs for solutions with nitrite concentration < 50 mg-N/L (< 1 mS/cm) without the addition of supporting electrolytes were not achieved due to the high potential in the cell (> 61 V). For other nitrite solutions, there is no significant effect on the reduction by increasing solutions conductivity as indicted in Figure 3. However, the EE/O was at highest for solutions with lower conductivity and was reduced by 80% when 50 mM Na2SO4 was added as supporting electrolyte.

The small interelectrode distance in microfluidic reactors is an alternative approach to enable and enhance the electrolysis without adding supporting electrolytes. Microfluidic reactors can also perform high nitrite reductions at low concentration without increasing energy requirements. As a comparison to the batch reactor, Figure 7b shows nitrite reduction and electrical energy requirement in microfluidic reactors using same nitrite solution (5 -50 mg-N/L) with and without adding supporting electrolytes at fixed current density (18 mA/cm 3 ) and HRT (0.24 s).

When the conductivity of the solution increased by 3 order of magnitude, the electrical energy per order increased only by 25%. Nitrite reduction was the same or higher for the solution with low conductivity. At low nitrite concentration (5 mg-N/L, 0.06 mS/cm), the microfluidic reactor achieved ~91% reduction leading to a final concentration less than the maximum contaminant level of nitrite as regulated by the US Environmental Protection Agency (EPA, MCL = 1 mg-N/L). [START_REF] Garcia-Segura | Disparities between Experimental and Environmental Conditions: Research Steps toward Making Electrochemical Water Treatment a Reality[END_REF] .

Electrical energy per order requirements in the microfluidic reactors (2.2 kWh m -3 order -1 ) represent only 5% of the energy required in the batch reactor (64 kWh m -3 order -1 ) to treat 50 mg-N/L nitrite without adding supporting electrolytes and more than 4 times lower even when supporting electrolytes were added (13 kWh m -3 order -1 with 50 mM Na2SO4) in the batch reactor.

Electrical energy per order usually ranges from 20 -60 kWh m -3 order -1 for nitrite reduction as previously reported [START_REF] Fajardo | Earth-Abundant Elements a Sustainable Solution for Electrocatalytic Reduction of Nitrate[END_REF] . Note that these results evidence the promissory performance of microfluidic devices but do not analyze the effect of other co-existing species. For instance, recent experimental research demonstrates that the presence of water hardness ionic species (i.e., Ca 2+ , Mg 2+ ) may induce the precipitation of inorganic scaling on the surface of the cathode [START_REF] Atrashkevich | Overcoming Barriers for Nitrate Electrochemical Reduction: By-Passing Water Hardness[END_REF] . Precipitation is favored by the high alkaline pH localized on the cathode surface. The formation of inorganic scaling on the cathode inhibits transport of electroactive species from/towards the electrocatalytic sites of the electrode surface and therefore decreases performance of electrochemical reduction of nitrite. Effects and challenges that may arise from the treatment of real water matrices are beyond the scope of this study, but should not be overlooked when considering technology translation. Figure 7: The effect of conductivity and the addition of supporting electrolytes at fixed current density (j= 18 mA/cm 2 ) on nitrite reduction (bars) and electrical energy per order (■) for the a) batch reactor and the b) microfluidic reactor. Initial concentration of nitrite is varied to 5, 25, and 50 mg-N/L at pH = 6.5. Supporting electrolytes were added to 1, 10, 50, and 100 mM of Na2SO4 for 50 mg-N/L N solutions where corresponding initial conductivity was reported. The flowrate of microfluidic reactor is fixed at Q = 0.125 mL/min (HRT = 0.24 s). Batch reactor was run at onehour reaction time for 100 mL solutions. Error bars represent standard deviation for triplicate measurements.

Energy Consumption in Micro and Conventional reactors

To compare the two reactors in terms of energy consumption required to remove a unit mass of contaminants, nitrite electrolysis was performed for an energy consumption level as calculated using Eq. ( 5) and (6). Figure 8 shows the energy consumption for the removal of nitrite to achieve at least 90% reduction in batch and microfluidic reactors under the same conditions with and without the addition of supporting electrolytes. The initial concentration was 50 mg-N/L N and current density was fixed at 18 mA/cm 2 . Adding supporting electrolytes had no effect on the energy required in microfluidic reactor for a reduction of nitrite > 90%. The microfluidic reactor consumed one order of magnitude of energy lower than batch reactor with the absence of supporting electrolytes. Even with the addition supporting electrolytes, the microfluidic reactor consumed half the energy required as compared to batch reactors.

Mass Transfer and Diffusion Layer Thickness

Superior mass transfer for nitrite in microfluidic reactors was achieved with lower energy as compared to batch reactor. The EE/O is a figure of merit to measure energy input and reaction.

In microfluidic reactors even with increase in current we still maintain a low cell potential (< 5 V) where kinetics is fast (k = 1-7 s -1 ). The enhanced mass transfer in microfluidic reactors can be reported by higher mass transfer coefficient (km) and the compressed diffusion layer (δ) as compared to batch reactor (Table 1). Mass transfer coefficients for microfluidic and batch reactors were calculated from equations (7-11). In microfluidic reactors, km increased with increasing flowrate where the Reynolds number (Re#) is increased. The diffusion layer thickness on the other hand decreased with increasing flowrate Eq. (12). As compared to batch reactor, km in microfluidic reactor is reported to be two orders of magnitude higher than batch reactor which is consistent with the previously reported values of km for micro channel with much higher electrode area 4 . For the batch reactor, km was estimated to be 5.9 x 10 -4 cm/s and was constant with previously reported values [START_REF] Dos Santos | Scale-up of Electrochemical Oxidation System for Treatment of Produced Water Generated by Brazilian Petrochemical Industry[END_REF] . The diffusion layer was estimated based on km to be at least one order of magnitude lower in microfluidic reactors (5-17 µm) as compared to batch reactor (290 µm). Therefore, the compressed diffusion layer in microfluidic reactors led to a higher mass transfer due to the lower resistance where lower electrical energy is required. The small distance between the two electrodes in electrochemical microfluidic reactors shows promises to overcome mass transfer limitation associated with the high energy requirements in conventional electrochemical technologies. Electrolysis was performed for nitrite reduction in a microfluidic reactor (interelectrode distance = 40 µm) and a conventional batch reactor (1 cm interelectrode distance) by the variations of three parameters: 1) current density, 2) initial concentration of contaminants, and 3) the addition of supporting electrolytes. Unlike conventional batch reactors, it was found that the performance of the microfluidic reactor with electrode separations of < 100 μm is less dependent on the addition of salt concentrations above levels suitable for drinking water. Note that addition of salt to increase conductivity is not as critical for microfluidic systems than for conventional reactors where electrodes are spaced millimeters or greater distances apart. The main conclusions from this research are:  Microfluidic reactors achieved > 95% reduction of nitrite in less than 1.5 seconds where the batch reactor required 3 hours for the same decay percentage. The reaction rate constant was 3 orders of magnitude faster with the microfluidic rector than with the batch reactor.

 Even with short time of reaction in the microfluidic reactor, the efficiency was more than two times higher at 95% reduction of nitrite as compared to batch reactors (measured by Faradaic efficiency).

 At low concentration of contaminants (5 mg/L nitrite) and low solution conductivity (0.06 mS/cm), the microfluidic reactor shows > 90% reduction of nitrite with no effect on energy requirements, whereas electrolytes were not successfully performed in conventional batch reactor due to high electrical potential (> 61 V).

 Electrical energy required to degrade nitrite by one order (EE/O) in the microfluidic reactor was one order of magnitude lower than the batch reactor without adding supporting electrolytes. Even when 50 mM of Na2SO4 was added as supporting electrolyte to the solution, EE/O was 4 time higher in batch reactors.

 Microfluidic reactors achieved > 90% reduction of contaminants by an order of magnitude lower energy consumption (EC) as compared to batch reactor without the addition of supporting electrolytes.

While individual microfluidic reactors may never treat sufficient volumes of drinking water to meet human daily needs, the purpose of this work was to demonstrate the importance of interelectrode spacing on electrocatalytic reactor designs and potential to reduce addition of salt to drinking waters. Moreover, the MEMS fabricated microfluidic reactors offers a robust and highly reproducible platform to study electrocatalytic reactions in a way that allows separation of mass transport rates (from bulk solution to electrode surfaces) from surface electrode reactions, and potentially even back diffusion of reaction by-products into the bulk solution.

Figure 1 :

 1 Figure 1: Schematic of the microfluidic device fabricated by soft lithography. a) 3-D of the device components. b) a cross view of the device.

  , which operation is described below. Microscale microfluidic devices. Experiments were conducted in continuous flow reactors where electrochemical reactions occur in a single pass through one channel of 500 µm wide, 40 µm interelectrode distance and 25 mm length. Solution is delivered to the microreactor by a syringe pump with flowrate ranges between 0.02 mL/min and 0.65 mL/min. The corresponding residence a) b) time to the applied flowrate determines hydraulic residence time (HRT) for the electrochemical reaction in the reactor. Samples were collected at the outlet. Macroscale batch reactors. Experiments were conducted with 100 mL solutions in a beaker under magnetic stirring at 700 rpm to ensure target pollutant transport from/towards the electrode surface in the reactor. The reactor is equipped with two 4.5 cm 2 platinum (Pt) electrodes, prepared under same conditions than the electrodes used in microfluidic set ups. The interelectrode distance was kept at 1 cm for all experiments.

Figure 2 :

 2 Figure 2: Schematics of a) a flow-by microfluidic reactor where interelectrode distance is 40 µm and b) a batch reactor in a beaker where interelectrode distance is 1 cm.2.4 Measurements and AnalysisConductivity of the solutions was measured by Thermo Scientific conductivity meter (Orion Star A322 Portable) and the pH measurements were conducted by Thermo Scientific pHmeter (Orion Star A221). Samples were withdrawn from reactors at a given time and were analyzed for conversion of nitrite by UV Spectrophotometery (Hach DR6000) at the maximum absorption

Figure 3 :

 3 Figure 3: Nitrite decay over time by Pt cathode and Faradaic efficiency (red) in batch reactors ( ▲ ) and microfluidic reactors (• ). Current density is fixed at j = 18 mA/cm 2 , initial concentration C0 = 50 mg-N/L and conductivity was 0.53 mS/cm 2 and pH = 6.5. Error bars represent standard deviation for triplicate measurements (< 1% standard deviation in percentage nitrate reduction based upon triplicate experiments for microfluidic reactor).

= 4 .

 4 4 mol L -1 s -1 at lower HRTs while first-order kinetics for nitrite decay (r NO2-= -k [NO2-]) have k = 1.1 s -1 at higher HRTs.

Figure 4 :

 4 Figure 4: Nitrite decay data for a) batch reactors (▲) as it fits first order kinetics and b) microfluidic reactor (•) as showing two trends, zero and first order kinetics. Initial concentration of nitrite C0 = 50 mg-N/L, conductivity of 0.53 mS/cm and pH = 6.5. Error bars represent standard deviation for triplicate measurements (SD = 3x10 -3 mg/L of nitrite decay for microfluidic reactors).3.3 The Effect of Current Density on Microfluidic Electrolysis Performance

Figure 5 :

 5 Figure 5: The effect of applied current density on nitrite reduction over time in microfluidic reactors. Interelectrode distance = 40 µm, initial concentration of nitrite is 50 mg-N/L (0.53 mS/cm) and pH = 6.5. No supporting electrolytes were added. Error bars represent standard deviation for triplicate measurements (SD < 1% in percentage of nitrite reduction based upon triplicate experiments).

Figure 6 :

 6 Figure 6: The effect of applied current density on nitrite reduction (a) and electrical energy per order requirement (b) for the batch reactor ( ▲ ) and for the microfluidic reactor (• ). Initial concentration of nitrite is 50 mg-N/L (0.53 mS/cm and pH = 6.5). The flowrate of the microreactor is fixed at Q = 0.125 mL/min (HRT = 0.24 s). For batch reactor, the reaction time is fixed at one hour for 100 mL solution. No supporting electrolytes were added. Error bars represent standard deviation for triplicate measurements.

Figure 8 :

 8 Figure 8: Electrical energy consumption required after 90% removal of nitrite in batch and microfluidic reactors at fixed current density (j= 18 mA/cm 2 ). Initial concentration of nitrite is C0 = 50 mg-N/L and 50 mM Na2SO4 added as supporting electrolyte. The flowrate of microfluidic reactor to achieve > 90% removal was at Q = 0.02 mL/min (HRT = 1.43 s) and the batch reactor was run for three hours for 100 mL solutions. Error bars represent standard deviation for triplicate measurements.3.7 Mass Transfer and Diffusion Layer Thickness

Table 1 :

 1 Non-dimensional groups, mass transfer coefficient and diffusion layer thickness for different flowrate in microfluidic and batch reactor.

	Flowrate Q (mL/min)	Re #	Sc #	Sh #	Le	km (cm/s)	δ (µm)
				Microfluidic Reactor
	0.02	1.30		4.32		0.010	17.2
	0.04	2.59		5.43		0.012	13.6
	0.13 0.21	7.72 12.96	588	7.78 9.23	3 x 10 -3	0.018 0.021	9.5 8.0
	0.42	25.93		11.61		0.027	6.4
	0.50	30.86		12.29		0.028	6.0
	0.63	38.89		13.27		0.030	5.6
				Batch Reactor
	700 rpm (stirring)	2037	588	73.21	0.01	5.9 x 10 -4	290
	4 Summary and Conclusions

Acknowledgement

This work was partially funded by the National Science Foundation (EEC-1449500) Nanosystems Engineering Research Center for Nanotechnology-Enabled Water Treatment. We would like to thank King Saud University for their fund through Researchers Supporting Project number (RSPD2023R529), King Saud University, Riyadh, Saudi Arabia. We would like to thank the staff of the Center for Solid State Electronics Research (CSSER) at Arizona State University (ASU) Tempe AZ, for their cleanroom and nanofabrication training and technical support. The authors also acknowledge the support of the Centre National de la Recherche Scientifique (CNRS) and the funding received from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 843870.

Supporting Information: MICROFLUIDIC FLOW-THROUGH REACTORS MINIMIZE ENERGY REQUIREMENTS OF ELECTROCHEMICAL WATER TREATMENT WITHOUT ADDING SUPPORTING ELECROLYTES

Authors: Omar Alrehaili, Ana S. Fajardo, Sergi Garcia-Segura, Paul Westerhoff

Electrodes Preparation

All electrodes used in this work were made of platinum (Pt) and prepared in the lab. Silicon (Si) wafers with the two sizes were cleaned and prepared as substrates for all devices. Due to its high corrosion resistance and ability to not getting poisoned by reduction by-products, platinum electrodes prove to be excellent electrocatalysts for nitrate/nitrite reduction [1][2][3] . Thermal evaporation technique was used to coat platinum on wafers with a Lesker #3 Thermal Evaporator.

At first, 100 nm thickness layer of platinum was coated directly to Si wafers. However, the high stress film of platinum caused a detachment of the coated layer from the wafer after cleaning with just water. Therefore, an adhesive layer (10 nm) of titanium that has a relatively lower stress film was coated first on the wafer followed by 100 nm layer of platinum to achieve stability of the metal on the wafers. Preparing electrodes by thermal evaporation technique results in a homogeneous and smooth electrodes surface to avoid friction forces that might increase in microchannel as reported in other work 4 .

Microfluidic Device Fabrication

Microfluidic devices were fabricated based on microelectromechanical system (MEMS) techniques where the interelectrode distance (the distance between electrodes) is less than 50 µm (~40 µm). The fabrication involves soft lithography for etching channels on epoxy polymer (SU-8), and building a delivery system using Polydimethylsiloxane (PDMS). Water flows in a microchannel between electrodes as a continuous flow system using a syringe pump. The microfluidic device fabrication processes were implemented in a clean room.

Each device has two electrodes, top layer wafer (25 × 10 mm) and bottom layer wafer (60 × 35 mm) coated with Pt as described previously (Figure S1a). Bottom piece wafers (60 × 5 mm) with coated platinum layer were further coated with polymer epoxy (SU-8) by a spin coater as a separation layer between the two electrodes in the device. SU-8 is a common negative photoresist that is used for MEMS fabrication. The thickness of the epoxy layer determines the distance between the two electrodes in the device and it is controlled by the viscosity of the polymer and the spin coat speed. In my microdevices, SU-8 2025 (MicroChem Inc.) was spin coated (2,000 rpm for 30 second) on Pt coated wafer (bottom) followed by a soft baking step (95 for 6 min) resulting a thickness layer of ~40 µm. A channel was etched on the epoxy layer all the way to Pt surface on the wafer. A photo mask therefore was designed where a chromium channel (500 µm wide, 3500 µm long) between to circle reservoirs (2000 µm in diameter) was printed a quartz glass. When UV light is exposed to the negative photoresist (dose = 260 mJ/cm 2 ) through the mask using an EVG 620 Automated Mask Alignment, the exposed part became solid, unexposed part (reflected by the chromium channel) will be soluble later in the developing process. The substrates were then transferred to another baking step at 95 for 6 min. An image of the mask should be visible in the SU-8 photoresist coating. Substrates were then dipped in a developer (SU-8 developer) for 6 minutes to remove the soluble photoresist, followed by a hard baking step (cure) at 180 for 10 min. The resulted substrate has a visible channel with measured thickness of 40 µm (Figure S1b-c).

In order to connect the microchannel to macro reservoirs, a microfluidic delivery device fabrication (stamp) is needed. The delivery device was made of polydimethylsiloxane (PDMS).

Briefly, PDMS (Sylgard 184 Silicone Elastomer kit, Dow Corning) was mixed with its curing agent (Dow Corning Sylgard 184 Silicone Elastomer Curing Agent) at a volumetric ratio of 10:1 under a vacuum. PDMS solution was cured for 2 h at 65 before it was cast onto the top piece of silicon wafer coated with Pt and hardened for 48 hours at room temperature ~22°C. PDMS was peeled from the mold and ready for hole interconnecting. Needles, 20 gauge, were purchased from Kontes (868280-2001) and sharpen by Dremel Multipro 395. A compressed seal is formed around the needle and oxygen plasma was used for the top and bottom parts to attach them together (Figure S1d). The process of coring involves; using a sharpened needle to make a hole, and removing it, inserting another unsharpened needle in the hole, and connecting the needle with a luer connector (silicon tubes). Figure S1e shows the final shape of device ready to be connected to a power supply.

Specific Charge for Nitrite Conversion

The specific charge, the rate of energy consumption over volume, was calculated for nitrite conversion in both reactors. The microfluidic reactor showed lower specific charge needs as it requires lower energy due to the relatively faster nitrite abatement rate. Figure S2 showed specific charge over nitrite conversion for the batch and microfluidic reactors.