Pierre Weill

Claire Plissonneau

Philippe Legrand

Vincent Rioux

Ronan Thibault
email: ronan.thibault@chu-rennes.fr

Ronan Pr

Thibault

May omega-3 fatty acid dietary supplementation help reduce severe complications in Covid-19 patients?

Keywords: α-linolenic acid, docosopentaenoic acid, eicosapentaenoic acid, immunity, lipid, virus ALA, α-linolenic acid, ARA, arachidonic acid, ARDS, Acute Respiratory Distress Syndrome, DHA, docosahexaenoic acid, DPA, docosapentaenoic acid, EPA, eicosapentaenoic acid, LC-PUFA, long-chain polyunsaturated fatty acid, SPM, specialized pro-resolving lipid mediators, SREBP, Sterol Regulatory Element Binding Protein

In around 10% of SARS-CoV-2 infected patients, coronavirus disease-2019 symptoms are complicated with a severe lung damage called Acute Respiratory Distress Syndrome (ARDS), which is often lethal. ARDS is mainly associated with an uncontrolled overproduction of immune cells and cytokines, called "cytokine storm syndrome"; it appears 7-15 days following the onset of symptoms, leading to systemic inflammation and multiple organ failure. Because they are well-known metabolic precursors of specialized pro-resolving lipid mediators (SPMs), omega-3 long-chain polyunsaturated fatty acids (omega-3 LC-PUFAs) could help improve the resolution of the inflammatory balance, limiting therefore the level and duration of the critical inflammatory period. Omega-3 LC-PUFAs may also interact at different stages of the viral infection, notably on the virus entry and replication. In the absence of demonstrated treatment and while waiting for vaccine possibility, the use of omega-3 LC-PUFAs deserve therefore to be considered, based on previous clinical studies suggesting that omega-3 supplementation could improve clinical outcomes of critically ill patients at the acute phase of ARDS. In this context, it is crucial to remind that the omega-3 PUFA dietary intake levels in Western countries remains largely below the current recommendations, considering both the omega-3 precursor α-linolenic acid (ALA) and long chain derivatives such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). An optimized omega-3 PUFAs status could be helpful to prevent infectious diseases, including Covid-19.

Introduction

The viral epidemic caused by the new Coronavirus SARS-CoV-2 is responsible for the Coronavirus-2019 disease . Upon binding via its Spike (S) protein, SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as an entry receptor in several cell types, including lung alveolar epithelial cells (type 1 and type 2 pneumocytes). This receptormediated endocytosis is followed by the activation of the S protein in the viral envelope by the transmembrane serine protease 2 (TMPRSS2), a membrane-bound enzyme localized near the ACE2 receptor [START_REF] Ballout | The lysosome: A potential juncture between SARS-CoV-2 infectivity and Niemann-Pick disease type C, with therapeutic implications[END_REF]. Alternatively, direct proteolytic cleavage of the viral S protein by TMPRSS2 on the surface of host cells has been described to induce the fusion of the viral and plasma membranes, leading to the release of the viral single stranded-RNA into the cytoplasm [START_REF] Mahmoud | SARS-CoV-2 entry in host cellsmultiple targets for treatment and prevention[END_REF]. Once in endosomes or cytoplasm, the viral RNA activate nucleic acid sensing systems like Toll-like receptors (TLR7 and 9) which initiate the anti-viral response of the innate immune system by triggering first the expression of type 1 interferons, followed by activation of NF-kB and proinflammatory cytokines.

In 10-15% of all cases, Covid-19 is complicated by severe pneumonia requiring hospitalization, with a high risk of developing an Acute Respiratory Distress Syndrome (ARDS), which can lead the patient to the intensive care unit (ICU) and is often lethal. ARDS is mainly associated with an uncontrolled overproduction of immune cells and cytokines, called "cytokine storm syndrome", which appears 7-15 days following the onset of symptoms, leading to systemic inflammation and multiple organ failure. Covid-19 general mortality rate is about 2% and hospital mortality rate is about 10% [START_REF] Guo | The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak-A n update on the status[END_REF]. To date, there is no vaccine and only the antiviral prodrug Remdesivir has been approved as an effective drug therapy to treat the disease [START_REF]Coronavirus (COVID-19) Update: FDA Issues Emergency Use Authorization for Potential COVID-19 Treatment | FDA[END_REF][START_REF]First COVID-19 treatment recommended for EU authorisation | European Medicines Agency[END_REF]. Additionally, little is known to prevent its progression to a serious condition.

The global burden of infection being high and worldwide, all kind of treatments, including J o u r n a l P r e -p r o o f dietary supplementation and pharmaconutrition trials, deserve to be tested [START_REF] Rogero | Potential benefits and risks of omega-3 fatty acids supplementation to patients with COVID-19[END_REF]. Background from other coronaviruses, like SARS-CoV-1 and MERS-CoV are also important to remind.

In this context, it is well admitted that adequate supply and balance of nutrients are required for proper functioning of both the innate and adaptive immune system. Essential PUFAs coming from the diet have a very special role in this process, because they participate to control chronic and acute inflammations. Because they are well-known metabolic precursors of specialized pro-resolving lipid mediators (SPMs), omega-3 long-chain polyunsaturated fatty acids (omega-3 LC-PUFAs) could notably help improve the resolution of the inflammatory balance, limiting therefore the level and duration of the critical inflammatory period [START_REF] Calder | Optimal nutritional status for a well-functioning immune system is an important factor to protect against viral infections[END_REF]. Omega-3 LC-PUFAs may also interact at different stages of the viral infection, notably on the virus entry and replication. Therefore, the nutritional status for PUFAs is particularly important in tissue inflammatory status and overall immune response [START_REF] Calder | Optimal nutritional status for a well-functioning immune system is an important factor to protect against viral infections[END_REF][START_REF] Messina | Functional role of dietary intervention to improve the outcome of COVID-19: A hypothesis of work[END_REF]. In this context, it is crucial to remind that the omega-3 PUFA dietary intake levels in Western countries remain largely below the current recommendations, considering both the omega-3 precursor α-linolenic acid (ALA) and long chain derivatives such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [START_REF] Anses | Avis relatif à la 3ème étude individuelle nationale des consommations alimentaires[END_REF][START_REF] Anses | Actualisation des apports nutritionnels conseillés pour les acides gras[END_REF].

In this Hypothesis article, we suggest that omega-3 PUFA dietary supplementation may be beneficial to reduce the risk of coronavirus complications, progressing to serious outcomes like ARDS, with the need for artificial ventilation in ICU. The rational for such Hypothesis is based on the following data. An increased intake level (long-term supplementation for prevention against infection or short-term supplementation in acute inflammation phase) of omega-3 PUFAs, including both α-linolenic acid (ALA) as precursor and long-chain derivatives (EPA, DPA docosapentaenoic acid and DHA) may increase their overall tissue storage. This higher cell and tissue omega-3 status could lead to a better conversion to SPMs, leading in turn to a more rapid resolution of inflammation, improving J o u r n a l P r e -p r o o f the severity of the cytokine storm, and improving finally the outcome of Covid-19 infected patients.

To support these hypotheses, we first present the demonstrated and putative molecular mechanisms involving omega-3 PUFAs in the prevention from viral infection and in the inflammation resolution. Then, we review the data obtained in animal experiments, human clinical trials and epidemiological studies supporting an interesting role of omega-3 PUFA supplementation against viral infection-associated inflammation.

Omega-3 PUFAs may interfere with virus entry and replication

Omega-3 PUFAs may modulate membrane rafts where ACE2 and TMPRSS2 are mainly expressed

ACE2 and TMPRSS2 are described to be mainly present in lipids rafts [START_REF] Ballout | The lysosome: A potential juncture between SARS-CoV-2 infectivity and Niemann-Pick disease type C, with therapeutic implications[END_REF], which are cholesterol and sphingolipid-enriched membrane microdomains within the lipid bilayer. It was previously shown that lipid rafts were involved in SARS-CoV entry into Vero E6 cells [START_REF] Lu | Lipid rafts are involved in SARS-CoV entry into Vero E6 cells[END_REF]. In addition, Glende et al. [START_REF] Glende | Importance of cholesterol-rich membrane microdomains in the interaction of the S protein of SARS-coronavirus with the cellular receptor angiotensin-converting enzyme 2[END_REF] suggested that lipid raft modulation may be an option to reduce ACE2-mediated virus infection.

As constitutive part of the membrane phospholipids, omega-3 PUFAs can regulate membrane properties, such as membrane fluidity and protein complex assembly in lipid rafts.

The number and size of raft and non-raft domains may also modulate the expression, stability and enzymatic activities of ACE2 and TMPRSS2. Among omega-3 LC-PUFAs, DHA is described to directly regulate the formation of lipid rafts [START_REF] Wassall | Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation[END_REF].

Omega-3 PUFAs may counteract the viral activation of SREBP1/2

Viruses are known to manipulate cell metabolism to facilitate their replication cycle. A described virus strategy is to reprogram host lipid metabolism, in order to provide enough J o u r n a l P r e -p r o o f lipid molecules required for the synthesis of the virion replication membranes. The isomers of the transcription factor SREBP (Sterol Regulatory Element Binding Protein) are involved in this metabolic reprogramming. For instance, MERS-CoV has been shown to manipulate host cellular lipid metabolism and reprogram the de novo SREBP-dependent lipogenesis pathway, to ensure its replication [START_REF] Yuan | SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target[END_REF]. SREBPs has also been shown to be activated during flavivirus infection [START_REF] Pombo | Perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections[END_REF]. Therefore, SREBP1/2 was defined as a potential broad-spectrum antiviral target for therapeutic intervention.

In this context, one can speculate that, since omega-3 LC-PUFAs are well-known intracellular inhibitors of SREBP transcription and maturation, a higher cellular impregnation level may counteract the activating effect of the virus and lower its replication duration. Indeed, EPA and DHA inhibit SREBP1 conversion from its inactive form to its active mature form and increase the degradation of SREBP-1c mRNA. This inhibition results in overall decrease in cellular concentration of the mature form of SREBP and less transcription of lipogenic genes coding notably for Acetyl-CoA Carboxylase, Fatty Acid Synthase and Stearoyl-CoA Desaturase [START_REF] Deckelbaum | n-3 Fatty acids and gene expression[END_REF]. It was suggested that the cholesterol and fatty acid biosynthesis pathways play a role in Hepatitis C Virus (HCV) RNA replication and infection [START_REF] Kapadia | Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids[END_REF]. In connection with the previous hypothesis on lipid rafts, omega-3 LC-PUFAs may also inhibit SREBP2-induced cholesterol synthesis, mainly via the transcription level of HMG-CoA reductase.

Omega-3 PUFAs may help improve the resolution phase of inflammation

LC-PUFAs play a critical role in inflammatory processes and innate immune response, thereby affecting human health and diseases. As a first step of the immune response, inflammation is a biological defense against physical, biological or infectious injury to body cells. It involves a series of processes, like leucocyte recruitment, whose first purpose is to J o u r n a l P r e -p r o o f eliminate the causes and aggressors. This first phase is called the "promotion" phase. Then, the second phase tries to restore the affected cell and tissue homeostasis and is called the "resolution" phase.

Inflammation begins in particular with the release of PUFAs in phospholipid sn-2 position from the cell membranes, under the action of the enzyme phospholipase A2 (PLA2) [START_REF] Murakami | Regulatory functions of phospholipase A2[END_REF]. Arachidonic acid (ARA) is the main precursor for this synthesis, leading to the production of cellular mediators promoting inflammation, like leukotrienes (LTB4 and LTC4) and prostaglandins (PGE2 and PGD2), through the action of cyclooxygenases (COX1 and 2), lipoxygenases (LOX) and cytochrome P450 [START_REF] Choque | Linoleic acid: Between doubts and certainties[END_REF][START_REF] Serhan | Resolvins and protectins in inflammation resolution[END_REF]. Therefore, PUFAs of the omega-6 series are described to activate the promotion phase of inflammation.

Omega-3 PUFAs are now described to activate the resolving phase, directly in the site of inflammation [START_REF] Calder | Omega-3 polyunsaturated fatty acids and inflammatory processes: Nutrition or pharmacology?[END_REF][START_REF] Ortega-Gómez | Resolution of inflammation: An integrated view[END_REF][START_REF] Serhan | Resolving inflammation: Dual antiinflammatory and pro-resolution lipid mediators[END_REF]. EPA and DHA are the main precursors for those resolving inflammation [START_REF] Calder | Omega-3 polyunsaturated fatty acids and inflammatory processes: Nutrition or pharmacology?[END_REF]. Indeed, the specialized pro-resolving mediators (SPMs) issued from EPA and DHA, known as resolvins, maresins and protectins inhibit the synthesis of proinflammatory cytokines through the down-regulation of the NF-κB pathway [START_REF] Basil | Specialized pro-resolving mediators: Endogenous regulators of infection and inflammation[END_REF]. So, they play an efficient role for inflammation resolution processes. E-series resolvins from EPA, Dseries resolvins and protectins from DHA have a significant anti-inflammatory effect by limiting leucocytes infiltrations in damaged tissues [START_REF] Serhan | Resolving inflammation: Dual antiinflammatory and pro-resolution lipid mediators[END_REF][START_REF] Serhan | Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their endogenous aspirintriggered epimers[END_REF]. Maresins derived from DHA can also induce macrophages phagocytosis of neutrophils to resolve inflammation [START_REF] Rius | Resolution of inflammation in obesity-induced liver disease[END_REF]. Recently, another minor omega-3 LC-PUFA, the docosapentaenoic acid (C22:5 n-3, omega-3-DPA) has also been suggested to be important in inflammatory resolution in the lung [START_REF] Morin | Docosapentaenoic acid monoacylglyceride reduces inflammation and vascular remodeling in experimental pulmonary hypertension[END_REF].

An optimal resolution of acute inflammation, as well as the control of chronic lowgrade inflammation [START_REF] Hotamisligil | Inflammation and metabolic disorders[END_REF], is therefore based on the cellular availability of these omega-3 fatty acids exclusively provided by the diet and in particular on the dietary intake level of ALA, EPA and DHA.

J o u r n a l P r e -p r o o f

Omega-3 PUFA dietary intake levels are largely below the recommendations

In this context, it is crucial to remind that the omega-3 PUFA dietary intake levels in Western countries remains largely below the current recommendations, considering both the omega-3 precursor α-linolenic acid (ALA) and long chain derivatives such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [START_REF] Legrand | Update of French nutritional recommendations for fatty acids[END_REF]. Linked to great changes in agricultural production and consumption habits these last 50 years, today's consumption of omega-6 PUFAs is at least equal to the current recommendation and often excessive (depending on the countries) while the omega-3 PUFA's intake is almost always in deficit in western countries [START_REF] Ailhaud | Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity[END_REF][START_REF] Blasbalg | Changes in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century[END_REF]. This leads to a high (10 and more) omega-6/omega-3 ratio since the 80s [START_REF] Ailhaud | Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity[END_REF].

In France for instance, the advised dietary intake recommends 2.2 g of ALA daily intake (1% of energy intake per day) and 500 mg for EPA + DHA (in which 250 mg of DHA minimum) [START_REF] Anses | Actualisation des apports nutritionnels conseillés pour les acides gras[END_REF]. However, the French INCA 3 consumption study reported in 2017 the mean omega-3 consumption as follows: 1 g/day of ALA instead of the 2.2 recommended, and 286 mg/day of EPA + DHA instead of 500 recommended. Therefore, only 15% of the French population meet DHA intake recommendation, only 8% for EPA and only 1.6% for ALA [START_REF] Anses | Avis relatif à la 3ème étude individuelle nationale des consommations alimentaires[END_REF][START_REF] Dubuisson | The Third French Individual and National Food Consumption (INCA3) Survey 2014-2015: Method, design and participation rate in the framework of a European harmonization process[END_REF]. Breast milk composition is also a good biomarker for PUFAs intake levels. As expected, it exhibits a lack of omega-3 in most of the western countries [START_REF] Tressou | Very low inadequate dietary intakes of essential n-3 polyunsaturated fatty acids (PUFA) in pregnant and lactating French women: The INCA2 survey[END_REF].

In a large range of other countries (38 countries) [START_REF] Hibbeln | Healthy intakes of n-3 and n-6 fatty acids: estimations considering worldwide diversity[END_REF], the omega-3 LC-PUFA intake varied from 0.02% of total energy in Bulgaria (corresponding to about 40 mg/day) to 0.44% of total energy in Iceland (corresponding to 900 mg/day). For the authors of this study, only a few number of countries have an optimal intake of omega-3 LC-PUFAs over 0.2% of total energy (corresponding to about 410 mg/day): Portugal (0.203%, i.e. 420 mg/day), South Korea (0.218%, i.e. 450 mg/day), Finland (0.228%, i.e. 470 mg/day), Norway (0.244%, i.e.

J o u r n a l P r e -p r o o f 10 500 mg/day), Malaysia (0.341%, i.e. 700 mg/day), Japan (0.374%, i.e. 760 mg/day) and Iceland (0.435%, i.e. 900 mg/day).

These abundant and consensual deficit data deserve our attention in the context of this pandemic and its inflammatory complications. Indeed, countries with a high omega-3 LC-PUFA intake (0,2% of total energy intake) have a very low number of victims related to the population: more than 4 times lower than the average of the 31 other countries (36 versus 175 deaths per million in June 2020) [START_REF]Coronavirus COVID-19[END_REF].

Taking into consideration (i) the role of omega-3 LC-PUFAs in the virus entry, replication and in the resolution phase of inflammation; (ii) the inflammatory processes leading to ARDS; (iii) the large and general deficit in omega-3 LC-PUFAs in the western population, it is possible to raise the hypothesis of a link between omega-3 LC-PUFA consumption deficiency and Covid-19 excessive inflammatory complication (Figure 1) .

Animal data, clinical trials and epidemiological studies supporting an interesting role of omega-3 LC-PUFA supplementation in ARDS

Omega-3 PUFA supplementation in animal models

In animal studies, omega-3 LC-PUFA supplementation clearly led to increased levels of tissue omega-3, including the lung. For instance, rats fed semi-synthetic diets supplemented with 1% total lipids of EPA, DPA and DHA in ethyl esters form from weaning to 6 weeks clearly increased the omega-3 LC-PUFA level in all studied tissues, including the lung [START_REF] Drouin | Comparative effects of dietary n-3 docosapentaenoic acid (DPA), DHA and EPA on plasma lipid parameters, oxidative status and fatty acid tissue composition[END_REF].

Similarly, maternal omega-3 LC-PUFA (ALA, EPA and DHA) supplementation during the perinatal period (from embryonic day 15 to day 20 after parturition) increased the level of total omega-3 in breast milk as well as the lung tissue of the rat pups and protected them against hyperoxia-induced pulmonary hypertension [START_REF] Zhong | Maternal omega-3 PUFA supplementation prevents hyperoxiainduced pulmonary hypertension in the offspring[END_REF].

J o u r n a l P r e -p r o o f

Animal experiments have also shown a beneficial impact of high omega-3 diets against acute pneumonia when animals were exposed to microbial infection. Sharma et al. [START_REF] Sharma | Dietary supplementation with omega-3 polyunsaturated fatty acids ameliorates acute pneumonia induced by Klebsiella pneumoniae in BALB/c mice[END_REF] used mice infected by Klebsiella pneumoniae with control or omega-3 PUFA enriched diets. They reported that the dietary supplementation of omega-3 LC-PUFAs can exert an overall beneficial effect through the upregulation of the host's specific and nonspecific immune defenses. It is interesting to underline that the beneficial effect exists whatever the type of omega-3 LC-PUFA supplementation (ALA, EPA or DHA diets). Hinojosa et al [START_REF] Hinojosa | Omega-3 fatty acids in contrast to omega-6 protect against pneumococcal pneumonia[END_REF] used mice intranasally infected with Streptococcus pneumoniae. Survival rate increased significantly in omega-3 diet's group when bacterial burden decreased. In omega-3 LC-PUFA-fed animal tissues, it was possible to measure a significant decrease in the levels of pro-inflammatory cytokines.

Omega-3 PUFA supplementation in humans

In 2018, the European Society for clinical nutrition and metabolism (ESPEN) expert group recommended the use of omega-3 rich fish oil in enteral and parenteral nutrition, for its general clinical benefits due to its anti-inflammatory and immune-modulating effects [START_REF] Calder | Lipids in the intensive care unit: Recommendations from the ESPEN Expert Group[END_REF]: reduction in infection rate, and length of hospital stay in medical and surgical patients admitted to the ICU. Indeed, in humans, supplementation with EPA and DHA was shown to lead to the incorporation of both omega-3 LC-PUFA in body lipid pools [START_REF] Browning | Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish1-4[END_REF]. The rate of incorporation varied between sample types, with the time to maximal incorporation ranging from days (plasma phosphatidylcholine) to months (mononuclear cells), and higher for adipose tissue [START_REF] Browning | Incorporation of eicosapentaenoic and docosahexaenoic acids into lipid pools when given as supplements providing doses equivalent to typical intakes of oily fish1-4[END_REF]. In addition, several plasma EPA-and DHA-derived SPMs were shown to respond linearly with the increased intake of EPA and DHA [START_REF] Ostermann | Plasma oxylipins respond in a linear dose-response manner with increased intake of EPA and DHA: results from a randomized controlled trial in healthy humans[END_REF].

J o u r n a l P r e -p r o o f

In connection with these interesting results, various SPMs synthesized from EPA and DHA have already shown protection and resolution of acute lung lesions in ARDS [START_REF] Hamilton | Lipid Mediators in the Pathogenesis and Resolution of Sepsis and ARDS[END_REF].

Omega-3 PUFA, in the form of fish oil, mostly rich in EPA and DHA, have also been used as a treatment in several clinical trials in enteral and parenteral nutrition with ARDS patients.

Recent meta-analyses [START_REF] Langlois | Omega-3 polyunsaturated fatty acids in critically ill patients with acute respiratory distress syndrome: A systematic review and meta-analysis[END_REF][START_REF] Dushianthan | Immunonutrition for acute respiratory distress syndrome (ARDS) in adults[END_REF][START_REF] Dushianthan | Immunonutrition for adults with ARDS: Results from a cochrane systematic review J o u r n a l P r e -p r o o f and meta-analysis[END_REF] demonstrated a beneficial impact of these omega-3 supplementation, especially when the supplementation was limited to enteral nutrition [START_REF] Langlois | Omega-3 polyunsaturated fatty acids in critically ill patients with acute respiratory distress syndrome: A systematic review and meta-analysis[END_REF].

Even if the doses were different and if various other supplementation (such as antioxidants) occurred in the different trials, the authors highlighted significant benefits obtained in only a few days (28 days) on mortality (-36%), as well as on the duration of ventilation, the length of stay in ICU [START_REF] Langlois | Omega-3 polyunsaturated fatty acids in critically ill patients with acute respiratory distress syndrome: A systematic review and meta-analysis[END_REF] with enteral supplementation of omega-3 [START_REF] Calder | Omega-3 polyunsaturated fatty acids and inflammatory processes: Nutrition or pharmacology?[END_REF][START_REF] Langlois | Omega-3 polyunsaturated fatty acids in critically ill patients with acute respiratory distress syndrome: A systematic review and meta-analysis[END_REF][START_REF] Dushianthan | Immunonutrition for acute respiratory distress syndrome (ARDS) in adults[END_REF]. In these trials, omega-3 PUFA was provided at high doses to patients with ARDS (from 5 to 20 times the recommended nutritional intake for EPA and DHA).

Considering all these results, administering omega-3 PUFAs appears a reasonable strategy in ARDS. A recent review suggested the systematic use of omega-3 LC-PUFAs if enteral or parenteral nutrition is indicated in the Covid-19 patients admitted in the ICU [START_REF] Thibault | Nutrition of the COVID-19 patient in the intensive care unit (ICU): a practical guidance[END_REF].

In addition to EPA and DHA, recent epidemiological and mechanistic data also suggest a specific role for omega-3 DPA in lung tissue [START_REF] Morin | Docosapentaenoic acid monoacylglyceride reduces inflammation and vascular remodeling in experimental pulmonary hypertension[END_REF][START_REF] Drouin | Comparative effects of dietary n-3 docosapentaenoic acid (DPA), DHA and EPA on plasma lipid parameters, oxidative status and fatty acid tissue composition[END_REF][START_REF] Leng | Dietary nutrients associated with preservation of lung function in hispanic and Non-Hispanic white smokers from New Mexico[END_REF]. Indeed, little studied so far, omega-3 DPA seems to be the starting point for specific routes of synthesis of pro-resolution mediators at the level of the lung [START_REF] Morin | Docosapentaenoic acid monoacylglyceride reduces inflammation and vascular remodeling in experimental pulmonary hypertension[END_REF].

Conclusion: on the way to clinical trials

Although scientific research on drug treatment are progressing, the ARDS experts recommend now, not only for Covid-19 derived ARDS, to give more attention on prevention in addition to treatment [START_REF] Spragg | Beyond mortality: Future clinical research in acute lung injury[END_REF]. In this context, we can raise the hypothesis that increasing the level of omega-3 LC-PUFAs in the diet or as a pharmaconutrient in addition to artificial Some questions are still opened. What should be the quantity of total omega-3 and LC-PUFA supplementation/intake? For the general population and in primary prevention, the data presented in this article, in addition to all the arguments previously developed concerning the prevention of other pathologies, support the crucial and urgent need to meet (at least) the nutritional recommendations (2.2 g/day for ALA and 500 mg/day for EPA+DHA). In secondary prevention, and for patients with symptoms and/or which are tested positive for Covid-19, the optimal doses for both ALA and omega-3 LC-PUFAs are not known. Meeting the nutritional recommendation of 2.2 g/day of ALA appears crucial, as well. Concerning omega-3 LC-PUFAs, it has been suggested that enteral nutrition enriched with 3.5g/day EPA and DHA can be administered in Covid-19 patients, not in a bolus [START_REF] Thibault | Nutrition of the COVID-19 patient in the intensive care unit (ICU): a practical guidance[END_REF]. Higher amounts of omega-3 LC-PUFAs, up to 9 g/day in the form of fish oil, have been administered safely [START_REF] Kristine) Koekkoek | Current evidence on ω-3 fatty acids in enteral nutrition in the critically ill: A systematic review and meta-analysis[END_REF].

What is the relative impact of the different fatty acids of the omega-3 family? Probably all the fatty acids of the omega-3 family seem have a role to play. EPA, omega-3 DPA and DHA are direct substrates for pro-resolution mediator synthesis. ALA is the initial dietary precursor of these LC-PUFAs by successive elongation and desaturation. Although it is well known that the overall conversion of ALA to DHA is weak [START_REF] Burdge | Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults[END_REF], ALA may have its own effect on inflammation resolution, as seen above, in ARDS animal trial in case of microbial infection [START_REF] Sharma | Dietary supplementation with omega-3 polyunsaturated fatty acids ameliorates acute pneumonia induced by Klebsiella pneumoniae in BALB/c mice[END_REF]. Other authors compared the effect of ALA or DHA diets on heart J o u r n a l P r e -p r o o f function [START_REF] Ayalew-Pervanchon | Long-term effect of dietary α-linolenic acid or decosahexaenoic acid on incorporation of decosahexaenoic acid in membranes and its influence on rat heart in vivo[END_REF][START_REF] Brochot | Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short-or long-term dietary exposure[END_REF] and found no differences. They additionally observed an accumulation of omega-3 DPA in the membranes of the cardiac cells in the ALA-fed rats.

To conclude, two clinical trials are on their way in Covid-19 patients and in older people with the aims to evaluate whether omega-3 PUFA diet enrichment could protect patients against severe forms of Covid-19, and to increase the awareness of the scientific community towards the beneficial role of good nutrition as a "barrier diet" against future pandemic. It is a limiting step for the synthesis of lipid mediators from PUFAs. EPA is a substrate for COX enzymes and also lipoxygenases (LOX). PGE3 and E-series resolvins are produced from EPA and have anti-inflammatory properties. DHA is a substrate for LOX and produces D-series resolvins, protectins and maresins that have anti-inflammatory properties. These specialized pro-resolving mediators from EPA and DHA, in a case of acute inflammation by activation of NF-κB, will activate peroxisome proliferator-activated receptor (PPAR)-γ. PPAR-γ inhibit NF-κB. Consequently, the production of pro-inflammatory cytokines is reduced and may prevent cytokine storm.

J o u r n a l P r e -p r o o f

  J o u r n a l P r e -p r o o f nutrition, could reduce the impact of inflammation caused by viral infectious diseases, such as the Covid-19. The biochemical mechanisms described here, reinforced by clinical, animal and epidemiological data suggest that the effect of nutritional supplementation could accelerate recovery, reduce hospitalization, duration of stay in ICU and finally reduce mortality due to the new Coronavirus SARS-Cov-2. Omega-3 LC-PUFAs could play a central role in prevention of the cytokine storm (Figure 1), at least maybe by decreasing the intensity of inflammation and risk of mortality for patients with Covid-19.

Figure 1 -

 1 Figure 1 -Expected mechanisms of the anti-inflammatory effect of omega-3 long chain

Figure 1 J

 1 Figure 1

Author contribution

PW, CP, PL, VR, RT contributed to literature search, analysis of data, writing and editing the manuscript. PW, PL, RT contributed to the conception of the clinical trial. All authors have approved the final version of the article.

Conflict of interest

Pierre Weill was former president of Valorex, a feed and food company dedicated to protein and oleaginous seeds processing.

Ronan Thibault received consulting and conference fees: Aguettant, Baxter, BBraun, Fresenius-Kabi, Nutricia, Roche; conference fees: Astra-Zeneca, Homeperf, Lactalis, Nestlé, Shire; research grants: Valorex, Bleu-Blanc-Coeur.

The other authors declare no conflict of interest.


Hélène Celerier

Jenny Jouin

Ameni Gharzouni

Virginie Mathivet

Isabel Sobrados

Nicolas Tessier-Doyen

Sylvie Rossignol
email: sylvie.rossignol@unilim.fr

Relation between working properties and structural properties from 27 Al, 29 Si and 31 P NMR and XRD of acid-based

Keywords: Metakaolin, Phosphoric acid, Thermal resistance, Water resistance, Amorphous network, NMR, XRD

Highlights: (maximum 85 characters, including spaces, per bullet point).

 Acid-based geopolymers display different processing-dependent working properties.  Acid-based geopolymers are mostly composed of Al-O-P and Si(OH)4 networks.  The quantities of Al-O-P and unreacted metakaolin govern the thermal resistance.  The increase of the Young's modulus is related to a higher thermal resistance.  Ordered structures and low amounts of physisobed water lead to water resistance.

Introduction

Alkali-based geopolymers are well-known materials since many decades [i]. They result from the dissolution of an aluminosilicate source in an alkaline silicate solution [ii]. Recently, acid-based geopolymers are gaining interest from the scientific community for different applications, due to their promising properties. For instance, such materials exhibit a higher temperature resistance (up to 1450°C) [iii] and greater mechanical properties [iv] than alkalibased geopolymers. On a structural point of view, acid-based geopolymers consist of threedimensional networks of AlPO4 and [Si-O-P]. It has indeed been reported that during synthesis, the dissolution of the aluminosilicate source in phosphoric acid leads to the release of Al 3+ which react with H3PO4. The reaction between these entities later results in the formation of AlPO4 as observed by several authors [iv, v]. Moreover, the formation of [Si-O-P] bonds was also observed [vi]. Finally, the amount of available acid can affect the structure and the final properties of the material. On this subject, Douiri et al. [vii] demonstrated that the decrease of Si/P ratio leads to a higher content of amorphous phase in the geopolymer structure due to a better dissolution of the precursors.

A previous work [viii] has shown that acid-based geopolymers may have different resistance properties (thermal or water resistance), governed both by the chemical composition and the setting temperature of materials [ix, x].. However, their structure before and after thermal treatment was seldom exposed in the literature. A combination of spectroscopic techniques such as Fourier Transform Infrared spectroscopy (FTIR) [xi], Raman [xii] and Nuclear Magnetic Resonance (NMR) can be useful to study these materials due to the presence of amorphous phase. X-Ray Diffraction (XRD) patterns can also be used to study the amorphous humps and the crystalline phases formed after a heat treatment. Moreover, the publications dealing with amorphous materials such as glasses or gels can also be helpful to understand the structure and the different networks formed in acid-based geopolymers and will be developed in the next sections.

Firstly, in the case of the system Al2O3-P2O5, published works on Al2O3 doped phosphate glasses [xiii, xiv] proved the strong affinity between P 5+ and Al 3+ ions. The addition of Al 3+ ions induces a modification of the phosphate glassy networks as demonstrated by NMR experiments. The original P-O-P bonds are indeed partly replaced by Al-O-P bonds by the insertion of Al VI into the network. This fact was evidenced by the formation of Q 1 (1Al), as observed on 31 P NMR spectra. Also, an heterogeneous distribution of Al 3+ in the glassy network, causing a localized high concentration of Al 3+ , led to the formation of Q 1 (2Al).

Moreover, it was shown that the addition of aluminum ions in phosphate glasses improves their chemical stability, and in particular, their water resistance. The dehydration and the temperature dependent phase transitions of AlPO4 based molecular sieves was studied by Varlec et al. [xv].

Based on an NMR study, they demonstrated that the hydrothermally stable AlPO4-34 contains hydrated aluminophosphate below 200°C. The crystallization of triclinic phases from the amorphous aluminophosphate occurred during the synthesis, a greater occurrence being observed with an increase of the dwell time. Furthermore, XRD and NMR experiments conducted on AlPO4-5 molecular sieves [xvi] highlighted their transition from hexagonal to orthorhombic phases before calcination. The different phases observed (hexagonal, triclinic and orthorhombic) correspond to the different polymorphs of AlPO4 (assigned berlinite, phosphotridymite [xvii] and phosphocristobalite [xviii] respectively).

Then, several authors have studied the SiO2-P2O5 glass system, showing that the addition of P2O5 in silicate glasses results in the formation of Si-O-P bonds [xix, xx]. PO43 - and SiO4 4-groups indeed copolymerize, leading to the easy incorporation of P 5+ in the silicate network and the formation of the more covalent Si-O-P bonds [xxi]. However, the presence of the P=O bond in the PO4 3-group limits the formation of the Si-O-P network. This effect leads to a partial conservation of the Si-O-Si bonds. Finally, Weeding et al. [xxii] studied the devitrification of a SiO2-P2O5 glass at 850, 1000 and 1300°C. Different crystalline compounds are formed in this system: SiP2O7 regardless of the temperature and Si5O(PO4)6 at 850 and 1000°C. An amorphous compound is also found in these materials at 850 and 1000°C.

Concerning the SiO2-Al2O3 system, Zagrajczuk et al. [xxiii] presented the structure of their gel-derived materials. On XRD patterns, a broad hump, located between 20 and 30°, is characteristic of aluminosilicate amorphous materials. The main bonds present in the resulting structure are Si-O-Si, Al-O-Al and Si-O-Al. Si-OH silanol groups can also be present due to non-bridging oxygen atoms in the silica networks. After a heat treatment, the crystallization of mullite was observed, as it occurs in the solid state synthesis of SiO2 + Al2O3 powders [xxiv].

The structure of this compound corresponds to a random distribution of tetrahedrally coordinated Al and Si.

Finally, glasses belonging to the SiO2-Al2O3-P2O5 system were also studied by many authors [xxv, xxvi, xxvii, xxviii] and the reported bonds are similar to the ones formed in the different binaries. The strong affinity between Al 3+ and P 5+ leads to the predominant formation of Al-O-P bonds, followed by Si-O-P bonds. Moreover, free amorphous silica is present in the glass and is characterized by the presence of Si-OH and -P=O units in its network. The AlPO4 units formed are randomly distributed in this silica network. It is also reported that, when Al is in excess in comparison to P, it is associated with Si to form Si-O-Al bonds.

The structural evolution of acid-based geopolymers after thermal treatment is reported in literature. In all the cases, AlPO4 polymorphs were observed, although their temperature dependent distribution is different. Le-ping et al. [xxix] have shown that berlinite appears up to 900°C, then cristobalite from 1150°C and finally mullite. Gualteri et al. [xxx] confirmed the presence of berlinite from 900°C. Moreover, they observed tridymite and cristobalite type polymorphs of AlPO4 and mullite after a heat treatment at 1000°C. From 1300°C, no amorphous phase was detected [xxxi].

The present study is a structural investigation focused on four acid-based geopolymers exhibiting different resistance properties [viii]. This study, based on 27 Al, 29 Si, 31 P NMR and XRD experiments, aims to highlight the structural origin of thermal and water resistance of these materials.

Materials and methods

Raw materials

The aluminosilicate source was a kaolin supplied by Imerys (Limoges, France), with a Si/Al ratio of 1, which was calcined at 750°C for 4 hours at a heating rate of 5°C/min to obtain the metakaolin noted M3. The characteristics and reactivity of this source were previously studied by Gharzouni et al. [xxxii]. The ACS grade solution of 85 wt.% of phosphoric acid was supplied by VWR (Radnor, USA) and corresponds to a concentration of 14.7 mol.L -1 . The synthesis of acid-based geopolymer is described in Figure 1. Metakaolin M3 was mixed with diluted phosphoric acid solution in order to synthesize different compositions of geopolymers.

Samples with two Al/P molar ratios (1 and 4) as well as three consolidation temperatures (20, 40 and 70 °C) were prepared. The concentration of the diluted phosphoric acid solution varied for the two Al/P ratio, i.e. 8.9 mol.L -1 for Al/P = 1 and 2.2 mol.L -1 for Al/P = 4. According to a previous work [viii], four samples, characteristics of different properties (thermal resistance (TR), water resistance (WR1 and WR2) and none resistance (N)), were chosen. These samples' names, properties and synthesis parameters are presented in the Table 1.

Sample characterization

The samples were calcined up to 1000°C in a furnace at 5°C.min -1 heating rate, followed by a two hours dwelling time and a natural cooling. The Young's modulus of each sample was measured by contact echography technique [xxxiii] on cylindrical samples (28 mm in diameter and 6 mm tall). Two couples of longitudinal and transversal transducers were used. Then the sample was placed between each couple of transducers in order to measure the time delay of the waves. From this delay, the speed of longitudinal and transversal waves and then the Young's modulus value were determined.

The amount of physisorbed water present in the samples after a 7 days immersion in water was determined both by differential thermal analysis (DTA) and thermogravimetric analysis (TGA) performed with an SDT Q600 apparatus from TA Instruments. The experiment was conducted up to 800°C at a heating rate of 5°C.min -1 , in an atmosphere of flowing dry air experiments at room temperature. NMR spectra were recorded after π/2 pulse irradiation (2 µs) using a 10 kHz filter to improve the signal/noise ratio. 120 scans were collected for each crushed sample. The time between acquisitions was set to 10 s to minimize saturation effects. DMFIT software was used to obtain the relative area of components. Different models employed were "quad 1 st model", "Gaussian/Lorentzian distribution" and "CSA model" for 27 Al, 29 Si and 31 P NMR data respectively. Estimated errors for chemical shifts and relative area are of 0.2 ppm and 2% respectively.

The crushed samples were characterized by X-ray diffraction (XRD) on a Bruker D8 Advance diffractometer using CuKα radiation. The data were collected over the 2θ angular range of 5-60° with a step size of 0.02° and an equivalent measured time per step of 50 s. The crystalline phases were identified from the experimental patterns using the powder diffraction file (PDF) database of the International Center for Diffraction Data. Peakoc software [xxxiv] was used to obtain the positions and intensities of the amorphous contributions observed. The refinement was performed from 5 to 60° using a Voigt function that takes into account Kα1 and Kα2 wavelengths. The continuous background was fitted by a 2 nd order polynomial.

Results

Working properties

Young's modulus of the consolidated samples were measured for the four different resistances, as defined in [viii], namely TR, WR1, WR2 and N. As seen in Table 2, the Young's modulus decreases, both with the increase of the setting temperature and the Al/P ratio.

Moreover, 1-M3-20 sample, which is the only thermal resistant sample, possesses the highest Young's modulus with a value of 21GPa. This is probably due to the presence of stronger bonds in the material, which is in agreement with the thermal resistance of the sample.

The amount of physisorbed water present in the samples after the water resistance test was measured using thermal analysis. As an example, Figure 2 represents the weight loss of the 1-M3-20 sample. These losses, measured between 20°C and 70°C, are reported in Table 2.

Although they are quite close, they are slightly different for each sample, with the highest value of 5.7% for 4-M3-40 and the lowest of 3.8% for 4-M3-20. Moreover, 1-M3-70 and 4-M3-20 are water resistant. This means that water resistant samples absorb less than 5% of water during a prolonged immersion.

Structural properties -NMR

Consolidated geopolymers

Structural informations about raw samples were obtained by MAS-NMR experiments.

The measured spectra, showing the identified contributions, are given in Figure 3 and all the deconvolution data are gathered in the Table 3.

As a reminder, NMR of metakaolin M3, which is also given in Table 3, has already been studied by Gharzouni et al. [xxxii] concerning 27 Al and Autef et al. [xxxv] for 29 Si. In the case of 27 Al spectra, three contributions were identified at 56, 30 and 1 ppm, corresponding to Al IV , Al V and Al VI in the metakaolin respectively. Silicium entities observed at -96 and -107 ppm could be attributed to Q 4 (2Al) of Si-O-Al bonds in metakaolin and Q 4 (0Al) of quartz.

As shown in Figure 3, whatever the geopolymer, 27 Al, 29 Si and 31 P MAS-NMR spectra present relatively broad peaks, revealing a disordered structure. Firstly, on 27 Al spectra of 1-M3-20 (TR) sample (Figure 3A), the signal includes two contributions at -13 and -22 ppm corresponding to Al VI of Al-O-P bonding [xxvi, xxxvi]. The relative area of these two contributions are quite similar (58 and 42% respectively). When the consolidation temperature is higher, i.e. 70°C (1-M3-70 (WR1) sample), the same contributions are visible but the relative area of the first peak varies from 58% to 49%, the second one remaining constant (Table 3).

Moreover, two additional contributions are observed at 43 and 28 ppm which can be attributed to Al IV of Al(OP)4 entities [xxxvi, xxxvii, xxxviii] and Al V of metakaolin [ii] respectively.

When the Al/P ratio increases (4M3-20 (WR2) and 4-M3-40 (N) samples), the contributions at -10 and -23 ppm are still observed. However, the area of the first one decreases while the second remains similar, as compared to samples with Al/P = 1. The three other indentified contributions for 4-M3-20 (WR2), at 50, 28 and 9 ppm, correspond to Al IV , Al V and Al VI of metakaolin respectively [ii, 36]. In the case of Al/P = 4, the metakaolin is in excess in the reactive mixture and its dissolution is incomplete [ii]. Therefore these contributions coincide with the quantity of unreacted metakaolin in the sample. 4-M3-40 (N) sample exhibits the same contributions as 4-M3-20 (WR2) sample, except for the Al VI which disappears. This trend is the consequence of a better dissolution of the metakaolin at this temperature.

In the case of 29 Si MAS-NMR spectra (Figure 3B), five contributions were identified for all samples at -90, -94, -102, -106 and -111 ppm, and attributed to Q 4 (3Al), Q 4 (2Al) and Q 4 (1Al) of metakaolin, Q 4 (0Al) of quartz [v, vi, xxxvi] and silicic acid [xxxix] respectively. 1-M3-20 (TR) mostly contains silicic acid. With an increase of the consolidation temperature, the relative area of each contribution slightly evolves, as seen for 1-M3-70 (WR1) (Table 3). For 4-M3-20 (WR2) sample, the curve areas vary significantly and Q 4 (1Al) becomes the major contribution with 36%. Silicic acid is again the major contribution in the case of 4-M3-40 (N) sample. This can be explained by the better dissolution of the metakaolin due to the thermal activation already supposed from 27 Al measurement. Indeed, similarly to the alkali activation [ii], the increase of the setting temperature promotes the dissolution of metakaolin as well as the polycondensation reactions. This fact also explains the absence of Al VI of metakaolin in this sample.

Finally, whatever the Al/P ratio, the same environments were identified on 31 P spectra (Figure 3C). The three contributions at -1, -7 and -16 ppm correspond to Q 1 (0Al), Q 1 (1Al) and Q 1 (2Al), respectively [xl, xli]. For all the samples, the predominant entity, with an intensity value of at least 75%, is Q 1 (2Al), which confirms the presence of Al-O-P bonds as observed in the 27 Al spectra. The minor contribution is Q 1 (0Al) (less than 4%) which is in agreement with the presence of a low quantity of unreacted PO4 3-tetrahedra from phosphoric acid. This fact confirms that almost all of the phosphoric acid reacts with the metakaolin. 1-M3-20 (TR) sample exhibits 80% of the main contribution (Q 1 (2Al)) (Table 2). When the setting temperature increases (1-M3-70 (WR1)), the percentage of the curve area of Q 1 (2Al) increases (90%) at the expense of those from Q 1 (1Al) and Q 1 (0Al). In the case of 4-M3-20 (WR2), a new contribution at -11 ppm (9%) corresponding to amorphous Al-O-P [xxxviii] is visible.

Moreover, the amount of the contribution corresponding to Q 1 (2Al) decreases when the Al/P ratio increases (around 77%). For 4-M3-40 (N) sample, the contribution of amorphous Al-O-P disappears with the increase of consolidation temperature. This leads to a higher percentage of the curve area of the contribution of Q 1 (1Al) from 13 to 19%.

To summarize these results, a graphic representation is given in Figure 4, which represents the relative areas of the different contributions, plotted as a function of the chemical shifts for the species 31 P, 29 Si and 27 Al, for all the samples. It was shown that an increase in the ratio Al/P tends to enhance the contributions due to metakaolin, as it can be seen by the presence of these contributions in Figure 4A. Indeed, samples synthetized with Al/P = 4 contain an excess of metakaolin, reaching its dissolution limit. This induces, in Figure 4B, the presence of less silicic acid and Q 4 (1Al) corresponding respectively to silicon bonded to surface OH (Si(OH)4) and to Si-O-Al bonds. We can note that these bonds are formed due to the affinity of the released Al and the silicon coming from the de-aluminated metakaolin. Concerning, the reaction between Al and P, the contributions of Al VI -O-P bonds are dominant for Al/P of 1, which confirms the strong affinity of these species. The presence of these bonds allows to assume the formation of AlPO4 crystals during a high temperature thermal treatment. The increase of the setting temperature for the samples with Al/P =1 causes a decrease of Al VI -O-P replaced by Al IV . This could lead to the formation, of different types of AlPO4 crystals during the calcination, this point will be developed later. Moreover, Al V of metakaolin are not fully consumed for this sample. The increase of the amount of silicic acid is explained by the decrease of Si-O-Al bonds (Q 4 (1Al)) due to a lower quantity of free aluminum able to form these bonds.

In the case of Al/P = 4, the increase of the setting temperature does not affect the quantity of Al VI -O-P. However, the Al VI from metakaolin disappears (Figure 4A). This absence causes a decrease of Al-O-Si bonds from Q 4 (1Al), leading to more silicon being available to react with surface OH to form silicic acid.

Geopolymers thermally treated at 1000°C

After a calcination at 1000°C (Figure 2.b), the NMR peaks of the samples, represented in Figure 5, are narrower than the ones measured on consolidated samples, indicating a higher degree of structural order [xlii]. 27 Al MAS-NMR spectra (Figure 5A) show that 1-M3-20 (TR) and 1-M3-70 (WR1) samples exhibits a unique contribution at 39 ppm corresponding to AlPO4 entities of berlinite [xxvi] and phosphotridymite [xliii]. With a ratio Al/P = 4, two other contributions at 57 and 1 ppm, corresponding to Al IV and Al VI of mullite respectively, were identified [xxiv]. This mullite crystallized from the unreacted metakaolin present in these samples. On 29 Si spectra (Figure 5B), three contributions at -104, -114 and -124 ppm were observed for 1-M3-20 (TR). These contributions correspond to quartz impurity in the raw metakaolin M3, disordered free silica [xliv, xlv] and Si-O-P bonds [xxvi, xlvi] respectively with a majority of free silica (97%) (see the deconvoluted values in Table 4). The same contributions can be seen for 1-M3-70 (WR1) with quite similar proportions. In addition to these contributions, 4-M3-20 (WR1) presents three additional ones at -86, -89 and -94 ppm, which all correspond to mullite [xliv, xlv]. When the setting temperature increases for this composition (4-M3-40), the contribution of Si-O-P bonds disappears, while contributions corresponding to free silica (-110 and -115 ppm), quartz (-99 ppm) and mullite (-89 and -96 ppm) are still present.

Finally, in the case of 31 P MAS-NMR spectra (Figure 5C), the three contributions at -28, -31 and -38 ppm for 1-M3-20 (TR) sample correspond to AlPO4 entities, namely phosphotridymite [xliii, xlvii] and phosphocristobalite [xlviii] and -P=O in vitreous silica [xxviii]. Others samples contain also the same contributions except 4-M3-40 for which the contribution of -P=O in vitreous silica was not observed.

As for consolidated materials, a graphic representation is given in Figure 6 to summarize these results. It represents the relative areas of the different contributions, plotted as a function of the chemical shifts for the species 31 P, 29 Si and 27 Al, for all the samples. From these informations, it can be concluded that regardless the setting temperature for Al/P =1, all aluminum reacts with the phosphorus ions to form AlPO4 polymorphs (berlinite and phosphotridymite, see Figure 6A). Concerning silicium ions, Figure 6B, the predominant entity is free amorphous silica, which is formed from silicic acid observed in the consolidated samples before calcination. Indeed, silicic acid is completely and irreversibly deshydroxylated from 900°C [xlix]. Therefore, the identified Si-O-P bonds are formed from unreacted PO4 3-and unreacted silicon. When Al/P increases from 1 to 4, the same AlPO4 polymorphs and free amorphous silica can be observed. Al/P = 4 consolidated samples also contained an excess unreacted metakaolin leading to an excess of aluminum species which had not reacted. This aluminum combined to silicon forms mullite at high temperature. At a setting temperature of 40°C, Si-O-P bonds and P=O in amorphous silica are not formed because the increase of setting temperature allowed a better dissolution of metakaolin as explained previously. All phosphorous thus reacts with aluminum, leaving no phosphorous to react with silicon.

Structural properties -XRD

The XRD patterns of the samples before and after calcination at 1000°C are shown in the Figure 4. Before calcination, all the diagrams present some broad amorphous humps, with peaks corresponding to crystallized anatase and quartz impurities. These crystalline phases are already present in the raw metakaolin [ii, l]. The amorphous contributions, located at around 8°, 22° and 27°, correspond to silicic acid [li], amorphous silica [lii] and geopolymeric networks [liii] respectively, as observed in NMR. Regardless of the composition, the positions at 22 and 27° do not vary. However, the position of the silicic acid contribution, which is very sensitive to the amount of water, shifts towards lower angles when the setting temperature and/or the Al/P ratio increase. Moreover, for 4-M3-20 (WR1), another hump was detected at 41° and could be assigned to an amorphous Al-O-P network according to NMR results.

After a heat-treatment at 1000°C, the samples are mostly crystallized. However, samples with Al/P = 1 are better crystallized, and exhibit different AlPO4 polymorphs such as berlinite (hexagonal), phosphotridymite (triclinic) and phosphocristobalite (orthorhombic). Anatase originating from the raw metakaolin is also observed. In these samples, phosphotridymite phase predominates over the berlinite. Moreover, there are more AlPO4 phosphotridymite polymorphs in 1-M3-20 (TR) than in 1-M3-70 (WR1). The calcined samples synthetized with ratio Al/P = 4 (4-M3-20 (WR2) and 4-M3-40 (N)) are not so well crystallized and exhibit broader peaks.

Only a few peaks from AlPO4 orthorhombic and triclinic polymorphs were observed. The quartz and anatase which were present in the raw metakaolin are clearly visible. Finally, the presence of an excess of unreacted metakaolin leads to the formation of poorly crystallized mullite.

Discussion

Structure

In this study, it was observed that with an Al/P ratio of 1, an Al-O-P network combined with Si(OH)4 is formed regardless the setting temperature. The formation of Si(OH)4 is however favored by the increase of the setting temperature. When the Al/P ratio increases to 4, two main networks are formed (Si-O-Al and Al-O-P). With the increase of the setting temperature, the proportion of Al-O-P increases and different secondary networks are observed such as amorphous silica, Si-O-P and -P=O in amorphous silica.

The amount of Al-O-P bonds decreases when an increase of the setting temperature occurs in the case of an Al/P ratio of 1. As the temperature increases, consolidation-induced thermal agitation indeed makes the material more unstable. The reactions are inhibited and the species are fixed such as observed in glasses [liv].These facts are in agreement with the crystallization of different AlPO4 polymorphs and the presence of free amorphous silica observed by XRD. Moreover, the amount of phosphotridymite decreases significantly when the setting temperature increases with Al/P=1. With a ratio Al/P of 4, the excess of metakaolin leads to a higher dispersion of the reactive species. In this case, the increase of the setting temperature also favors the dissolution of the metakaolin. Thus, more free-silica is dispersed in the geopolymeric network. With an Al/P ratio of 1, XRD patterns confirms the tendency as there are slightly less geopolymeric contribution as compared to silica's one. For Al/P = 4, the presence of unreacted metakaolin changes

I G I S
, leaving some free aluminum in the system which cannot react with phosphorous. Consequently, an increase of the setting temperature leads to a drastic decrease of the intensity of the geopolymer's contribution to the benefit of silicon, which can be the reason of the poor resistance of this sample.

Working properties and resistances

The resistance properties of the samples were considered both with the working properties (Young's modulus of untreated samples and amount of physisorbed water of saturated samples) and the local environment of the samples as characterized using NMR. These informations are reported in the Figure 9.

Firstly, in the case of the thermal resistance (Figure 9A) the amount of Al-O-P bonds formed, of unreacted metakaolin in the consolidated samples and Young's modulus display the same tendency. Indeed, the contribution of Al-O-P bonds is significantly higher when the material is thermally resistant (58% instead of less than 45% for others). Moreover, the absence of unreacted metakaolin in 1-M3-20 suggests that all aluminum must react with phosphorus to obtain a thermal resistant specimen. In such a case Al-O-P bonds acts as fillers in the material, conducting to the thermal resistance. The unreacted metakaolin prevents the creation of these fillers, deteriorating these properties. The higher proportion of Al-O-P bonds and the absence of metakaolin also lead to higher Young's modulus values confirming the presence of stronger bonds in the materials (21 GPa for 1-M3-20 against 1 GPa for 4-M3-40). These strongly correlated parameters thus allow to predict the thermal properties of these geopolymers.

The properties of water resistant samples (1-M3-70 and 4-M3-20) as well as the structural data (Q 0 (1Al) and Q 1 (2Al)) and amount of physisorbed water (Figure 9B) display also the same tendency. The low amount of Q 0 (1Al) and high amount of Q 1 (2Al) in these samples suggest that water resistant materials exhibit a more ordered local environment. The presence of Q 0 (1Al) indeed leads to a disordered structure. The presence of different formed networks also induces a poor cohesion of the material, thus letting water leach into the sample.

The study of these parameters give ways to effectively predict the water resistant of sample.

Conclusion

Acid-based geopolymers can exhibit different working properties such as thermal resistance or water resistance. This study aims to explain these properties from 27 Al, 29 Materials are thermally resistant when they exhibit a high proportion of Al-O-P bonds and no unreacted metakaolin. For water resistance, the geopolymer needs an ordered structure including a low proportion of Q 0 (1Al) and a high amount of Q 1 (2Al) ( 31 P NMR data).

[i] J. Davidovits, Geopolymer: chemistry and applications, second ed. Geopolymer institute, Saint-Quentin, France, 2008.

  proportion of Al-O-P bonds Higher degree of ordered structure

(

  100 mL.min -1 ) in platinum crucibles. The temperature signals were measured with Pt/Pt-10%Rh thermocouples. The samples were previously dryed at 40 °C during 12h and then crushed in an agate mortar. The measured weight loss corresponds to the fist mass loss of the sample and was measured from 20°C to 70°C. A Bruker AVANCE-400 spectrometer operating at 79.49 MHz ( 29 Si signal), 104.26 MHz ( 27 Al signal) and 161.98 MHz ( 31 P signal) was used for high resolution MAS-NMR

Si and 31 P

 31 NMR and XRD data. The presence of different networks in the structure of geopolymer materials with different properties was confirmed by the structural analyses. Consolidated geopolymers are mainly constituted of Al-O-P and Si(OH)4 networks. If both Al/P ratio and setting temperature increase, some unreacted metakaolin is observed in the samples. After a thermal treatment at 1000°C, the samples are essentially composed of AlPO4 polymorphs, free amorphous silica and Si-O-P networks. Moreover, for Al/P = 4, mullite phase appears.According to the Al/P ratio and the setting temperature, the proportion of networks is different which induces different final properties.

Figure 1 :

 1 Figure 1: Synthesis chart of acid-based geopolymers.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 234567 Figure 2 : Weight loss (dash) and derivate of heat flow (line) curves of 1-M3-20 (TR) sample.

Figure 9 :

 9 Figure 8: Values of

Table 1 :

 1 Nomenclature of the samples belonging to the four different resistance groups. TR: thermal resistance, WR1 and WR2: water resistance with respectively Al/P = 1 and 4 and N: none resistance.

	Samples	Group	Al/P	Setting temperature (°C)
	1-M3-20	TR	1	20
	1-M3-70	WR1	1	70
	4-M3-20	WR2	4	20
	4-M3-40	N	4	40

Table 2 :

 2 Young's modulus of consolidated samples and amount of physisorbed water in the samples after a water resistance test.

	Samples	1-M3-20	1-M3-70	4-M3-20	4-M3-40
	Resistance properties	Thermal (TR)	Water (WR)	Water (WR)	No (N)
	Young's modulus value (GPa)	21	16	1	1
	Amount of physisorbed water (%)	5.1	4.8	3.8	5.7

Table 3 :

 3 27 Al,29 Si, 31 P NMR data (chemical shift δ, relative area and full width at half maximum FWHM) for raw metakaolin M3 and 1-M3-20, 1-M3-70, 4-M3-20 and 4-M3-40 samples.

			27Al			29Si			31P
	Samples	δ	Relative	FWHM	δ	Relative	FWHM	δ	Relative	FWHM
		(ppm)	area (%)		(ppm)	area (%)		(ppm)	area (%)
	M3 [31, 32]	56 30 1	24 31 45	29 25 26	-96 -107	34 59	15 15
					-90	3	5
	1-M3-20	-13 -22	58 42	16 10	-94 -102 -106	9 41 5	6 8 6	-1 -7 -16	4 16 80	5 8 12
					-111	42	8
	1-M3-70	43 28 -12 -23	8 1 49 42	15 10 12 20	-90 -94 -102 -112 -106	4 5 35 52 4	5 5 6 8 7	1 -7 -16	1 9 90	6 8 14
	4-M3-20	50 28 9 -10 -23	8 15 4 44 29	20 25 15 13 18	-90 -95 -101 -106 -111	9 17 36 19 19	7 7 8 7 7	0 -6 -11 -16	2 13 9 76	5 7 7 13
	4-M3-40	49 27 -10 -22	10 17 43 30	20 26 13 20	-90 -95 -101 -110 -105	7 14 37 30 12	5 6 7 7 6	-2 -9 -16	3 19 78	5 8 13

Table 4 :

 4 27 Al,29 Si, 31 P NMR data (chemical shift δ, relative area and full width at half maximum FWHM) for 1-M3-20, 1-M3-70, 4-M3-20 and 4-M3-40 samples after a calcination at 1000°C.

			27Al			29Si			31P
	Samples	δ	Relative	FWHM	δ	Relative	FWHM	δ	Relative	FWHM
		(ppm)	area (%)		(ppm)	area (%)		(ppm)	area (%)
					-104	2	3	-28	37	3
	1-M3-20	39	100	4	-114	97	10	-31	54	4
					-124	1	3	-38	9	10
					-106	5	6	-28	43	3
	1-M3-70	40	100	5	-113	94	12	-30	56	4
					-122	1	3	-38	1	10
					-86	1	6
	4-M3-20	57 40 1	7 75 18	25 5 25	-89 -94 -105 -112	1 1 4 92	6 6 6 12	-28 -31 -38	65 34 1	4 4 10
					-125	1	3
					-89	5	6
	4-M3-40	57 40 1	7 76 17	25 5 25	-96 -99 -110	4 2 72	6 4 11	-28 -31	54 46	4 4
					-115	17	8

Supplementary material