P Costa

J A Simões

A S H Costa

J P C Lemos

D Navas

J F Hocquette

C R Calkins

R J B Bessa

Repercussions of growth path on carcass characteristics, meat colour and shear force in Alentejana bulls

Keywords: Bulls, compensatory growth, meat tenderness, carcass traits Implications

The aim of this study was to evaluate the carcass and meat characteristics of eight muscles from bulls with distinct growth paths. A total of 40 Alentejana male calves were allocated to two distinct feeding regimes. In the continuous growth (CG) system, the animals were fed concentrates plus hay and were slaughtered at 18 months of age. On the other hand, in the discontinuous growth (DG) system, the animals were fed hay until 15 months of age; the cattle were then fed the same diet provided to the CG group from 15 to 24 months of age. The DG reduced hot carcass weight, fatness and dressing %, but the proportions of fat, bone and muscle tissues in the leg were not affected. In contrast, there was a positive impact of compensatory growth on supraspinatus, triceps brachii, semitendinosus, biceps femoris muscle tenderness, overcoming the negative effects of age at slaughter. The reasons for such improvement in meat tenderness were not related to intra-muscular fat content or myofibrillar protein degradation values. An association between tenderness and muscle collagen properties was not established. The results indicate that the compensatory growth has a muscle-dependent effect.

Introduction

In order to reinforce sustainable agriculture in the Mediterranean areas, it is important to study alternative strategies to the conventional cattle production based on feeding high-energy concentrates provided ad libitum in feedlot, from weaning until slaughter at 15 to 18 months of age. An alternative to these intensive programmes is the traditional system where calves born in summer are weaned at the beginning of spring and spend all this season on pasture. The cattle are then maintained on low-quality pasture, supplemented with hay until the beginning of the following spring in order to ensure at least the maintenance of live weight. This discontinuous growth (DG) production system allows farmers to take advantage of the bull's compensatory growth during the second spring when the pasture is abundant and finish the cattle on concentrates for 2 to 3 months before slaughter at 24 months of age. Compensatory growth is the animal's ability to exhibit, after feed restriction, greater growth rates than control animals of the same chronological age [START_REF] Hornick | Different periods of feed restriction before compensatory growth in Belgian Blue bulls: I. animal performance, nitrogen balance, meat characteristics, and fat composition[END_REF]. The incorporation of compensatory growth into a cattle management strategy could increase financial returns to cattle producers from enhanced meat quality. However, the positive effects of compensatory growth could be limited by a number of factors. These include the severity and duration of feed restriction, the age at which nutritional restriction occurred, the duration of the compensatory response and the nutritional plan on which † E-mail: paulocosta@fmv.ulisboa.pt a Authors contributed to the project equally.

Animal, page 1 of 9 © The Animal Consortium 2015 doi:10.1017/S1751731115000427 animal animals are re-fed [START_REF] Coleman | Effect of nutrition, age and size on compensatory growth in two breeds of steers[END_REF][START_REF] Carstens | Physical and chemical components of the empty body during compensatory growth in beef steers[END_REF]. The traditional or discontinuous production system is less expensive and contributes to the environmental sustainability and biodiversity in Mediterranean areas. However, some beef producers are reluctant to use this system mainly because in order to obtain commercially acceptable carcasses, the animals have to be slaughtered about 6 months later than those produced in intensive systems.

Among beef-eating quality attributes, tenderness is one of the most important beef quality attributes and is influenced by several factors including muscle fibre type profile, sarcomere length, pH, intra-muscular fat (IMF), rate of tenderization (Shackelford et al., 1997; Gil et al., 2001) and total amount and chemical composition of collagen [START_REF] Chriki | Cluster analysis application identifies muscle characteristics of importance for beef tenderness[END_REF]. Nutritional status before slaughter can influence collagen content and its solubility and consequently meat tenderness (Thénard et al., 2006). Several studies investigated the effects of growth path and rate on carcass characteristics or meat quality in cattle [START_REF] Jones | Myofibrillar protein turnover in feed-restricted and realimented beef cattle[END_REF][START_REF] Hornick | Different periods of feed restriction before compensatory growth in Belgian Blue bulls: I. animal performance, nitrogen balance, meat characteristics, and fat composition[END_REF], but they were restricted to evaluation of the Longissimus muscle, regardless that compensatory growth effects on carcass and meat characteristics may be muscle-dependent (Hansen et al., 2006b). Consequently, results obtained in past studies cannot be extrapolated to the rest of the carcass.

Thus, the aim of the present investigation was to evaluate the effects of production system (continuous growth (CG) v. discontinuous growth (DG)) differences on carcass characteristics and meat quality in eight major muscles from bulls produced in the Mediterranean areas.

Material and methods

Animals and meat samples

A total of 40 purebred Alentejana male calves (9.0 ± 0.46 (mean ± s.d.) months of age, 239.0 ± 45 kg live weight) were randomly allocated to four adjacent pens (two pens per experimental group) and were allocated to two distinct feeding regimes. In the CG production system, animals were fed ad libitum with concentrates plus hay throughout the trial and were slaughtered at 18 months of age. In the DG production system, animals were fed ad libitum only hay from 9 to 15 months of age, the cattle were then fed the same diet provided to the CG group (concentrates plus hay) from 15 to 24 months of age. The pens (area about 1000 m 2 ) and the feed bunks (35 m long) were large in order to reduce the animal's interactions and limitation to feed and to simulate the traditional systems where the bulls graze freely.

The animals were individually weighed every 14 days during the experimental period and were slaughtered at 18 (CG) or 24 (DG) months of age. The weight data are presented in Figure 1 according to time periods.

After being stunned with a captive bolt, the cattle were dressed with carcasses suspended by the Achilles tendon. Dressing percentage was calculated as follows: weight of cold carcass/empty BW × 100. The carcasses were split along the spine and the kidney knob and channel fat (KKCF) from both sides were removed and weighed. Subsequently, carcass fatness was assessed using a 15-point scale (1 for very limited fat cover to 15 for very extensive fat cover) based on subcutaneous fat deposition as described by [START_REF] Boer | Manual on E.A.A.P. reference methods for the assessment of carcass characteristics in cattle[END_REF]. After 24 h of chilling at 6°C, the carcasses were then stored at 4°C until 7 days postmortem (cold carcass). The muscle pH and temperature of the longissimus thoracis (Lt) muscle (12th dorsal vertebrae) were assessed at 45 min, 3 h and 24 h postmortem to control chilling rate and pH decline in order to prevent carcasses with the risk for cold shortening (pH above 6.0 at 10 to 12°C and pH 24 above 5.8) being included in the present study, as recently reviewed by [START_REF] Warner | Factors influencing the incidence of high rigor temperature in beef carcasses in Australia[END_REF].

After ageing, carcasses were jointed and two 2.5-cm-thick steaks were taken from the mid-point of the following muscles: supraspinatus (Ss), triceps brachii (Tb), semitendinosus (St), biceps femoris (Bf), rectus femoris (quadriceps femoris (Qf)), gluteus medius (Gm), semimembranosus (Sm) and from the space between the 8th and 12th dorsal vertebrae in the Lt muscle. The steaks, collected from the right carcass side, were vacuum packed and frozen at -30°C for subsequent analysis. The left leg was dissected into bone, muscle, subcutaneous fat and IMF. This joint has been suggested to be representative of the overall bovine carcass composition in the Alentejana breed [START_REF] Simões | Distribution of tissues in carcasses at the same proportion of total fat in Portuguese cattle breeds[END_REF].

Shear force measurements

After thawing for 24 h at 4°C, steaks were grilled to an internal temperature of 70°C (Lufft C120, Munchen, Germany) and 8 to 10 cores parallel to the direction of the muscle fibres were taken from each steak. Shear force, expressed in kg, was measured in a texturometer (TA-tx2i Texture Analyser; Stable Micro Systems, Surrey, UK) equipped with a Warner-Bratzler shear device (WBSF), and data were collected using a specific software (Texture Expert Exceed; Stable Micro Systems, Surrey, UK).

IMF

The IMF was determined using fresh samples from the eight muscles by hydrolysis with 4 M HCl followed by Soxhlet Costa, Simões, Costa, Lemos, Navas, Hocquette, Calkins and Bessa extraction, for 6 h, with petroleum ether (Association of Official Analytical Chemists, 2000).

Myofibril fragmentation index (MFI)

The MFI was determined using frozen samples obtained 7 days postmortem according to the method described by Culler et al. (1978).

Total and soluble collagen Total collagen (TC) from the eight muscles was estimated from the hydroxyproline content of 4 g of minced muscle as described by the International Standard-ISO 3496 (1994).

Heat-soluble collagen (SC) content was determined according to a procedure adapted from [START_REF] Hill | The solubility of intramuscular collagen in meat animals of various ages[END_REF]. Duplicate samples (4 g) were homogenized (IKA T-25 Ultra-Turrax, Staufen, Germany) for 30 s and heated at 78°C for 70 min in 0.25 strength Ringer's solution. After centrifugation, the supernatant was hydrolysed and total collagen content was determined. Insoluble collagen (IC) was determined by calculating the difference between total collagen and heat-soluble collagen. Collagen was expressed as mg/g of live weight fresh muscle.

Meat colour

At seven days postmortem, the lean meat colour of the eight muscles was measured using a Minolta CR-300 chromameter (Konica Minolta, Tokyo, Japan) in the lightness (L*), redness (a*) and yellowness (b*) system, 1 h after air exposure to allow blooming.

Statistical analysis

Meat and carcass traits were analysed using the Proc Mixed procedure of SAS software, version 9.2 (SAS Institute, Cary, NC, USA), considering the animal as the experimental unit. The model included the fixed effect of production system (CG v. DG). Differences between groups were examined for statistical significance at P < 0.05 using the PDIFF option. All data were reported as mean ± standard error. Average daily gains (ADGs) for each experimental time period were evaluated and are presented in Figure 1, using the STATISTICA software (StatSoft Inc., 2004, Tulsa, OK, USA). Pearson's correlation coefficients were calculated using the STATISTICA software in order to elucidate possible associations between meat and carcass traits, after Bonferroni correction.

Results

Daily gain and carcass traits

The ingredients and chemical composition of the experimental diets are presented in Table 1.

Regarding Figure 1, the ADG between the beginning of the trial (9 months of age) and 15 months of age was more than 1.5 kg/day greater (P < 0.0001) in CG than in DG animals. Conversely, in the period between 15 and 18 months of age, the ADG was almost 0.5 kg greater (P = 0.0004) in DG than in CG bovines. ADGs for DG bulls were ~1.3 and 1.1 kg/day from 18 to 21 months of age and from 21 to 24 months of age, respectively.

The initial weight was similar between production systems (P = 0.41). On average, the CG bulls had about 37 kg heavier (P = 0.08) slaughter weights than DG animals (Table 2). Compared with DG bulls, the CG bovines had 25 kg The fatness score was evaluated using a scale ranging from 1 (very low fat cover) to 15 (very high fat cover).

Growth path effects on meat quality in bulls heavier hot carcass (HCW) (P = 0.023), 1.2% greater dressing (P = 0.001), 0.5% of KKCF (P = 0.06) and a 1.6 greater fatness score.

The production system had no effect (P > 0.05) neither on the leg joint dissection tissues nor on the muscle/total fat and muscle/bone ratios (Table 3).

Meat quality traits

The Lt temperature was 2.8°C higher in DG than in CG animals at 45 min (P = 0.061), similar at 3 h and 3.6°C higher in CG than in DG animals at 24 h (P < 0.001) (Table 4). Longissimus muscle pH values were consistently greater during all three measurements in CG compared with DG carcasses (P < 0.01).

Meat traits of the Ss, Tb, St, Bf, Qf, Gm, Sm and Lt muscles are presented in Table 5. The effect of production system on meat traits greatly depended on the muscle considered. The production system had no noticeable influence (P > 0.06) neither on IMF content nor on MFI 7 days values across muscles. In contrast, the total (TC) and insoluble (IC) collagen contents were greater in Qf (P < 0.01) and lower in Bf (P < 0.05) in DG and CG systems, respectively. The DG group had greater SC level in Tb (P = 0.041), St (P = 0.0023) and Qf (P = 0.041) muscles than the CG bulls. As a consequence, Muscle growth impetus according to [START_REF] Berg | New concepts of cattle growth[END_REF].

Costa, Simões, Costa, Lemos, Navas, Hocquette, Calkins and Bessa the SC/TC was greater in St (P = 0.011) and Bf (P = 0.044) muscles in the DG group compared with their counterparts.

The WBSF values for Ss, Tb, St and Bf muscles were lower (P < 0.03) for DG compared with CG bulls. There were no production system differences (P > 0.09) in WBSF for Qf, Gm, Sm and Lt muscles.

The colour parameters were widely affected by the production system.

L* values were lower (P < 0.03) in Tb, St, Gm and Sm muscles from DG bulls.

A similar trend was observed for the a* parameter, DG bulls had lower values (P < 0.01) for the Ss, St, Bf, Qf and Sm muscles than bulls on the CG system. The b* value was higher (P < 0.05) in DG bulls for Tb, Qf and Gm muscles compared with the CG system. Interestingly, the converse was observed for the Ss muscle with the higher value in the CG group (P = 0.0004).

Correlations between carcass and meat traits

Correlations between carcass and meat traits are presented in Table 6. The muscles are presented whenever the correlation between carcass and meat traits was found significant (P < 0.05) within muscles.

For the CG production system, positive correlations were recorded between HCW and the following: IMF for Tb (r = 0.57, P = 0.03) and St (r = 0.51, P = 0.04); MFI 7 days for Qf (r = 0.52, P = 0.04); and WBSF for Lt (r = 0.50, P = 0.04) muscles. In contrast, the HCW was negatively correlated with SC/TC for Lt (r = -0.50, P = 0.04) muscle. Furthermore, the increase in KKCF was positively correlated with IMF in St (r = 0.59, P = 0.009) and Gm (0.62, P = 0.007); MFI 7 days for Bf (0.50, P = 0.04); and WBSF for Ss (r = 0.49, P = 0.04) muscles. Moreover, positive correlations were found between IMF and MFI 7 days for Bf (0.61, P = 0.009); and WBSF for Ss (r = 0.50, P = 0.04); and Gm (r = 0.56, P = 0.02) muscle. The SC/TC ratio was positively correlated with MFI 7 days for Gm muscle (r = 0.58, P = 0.01) and negatively correlated with WBSF for St (r = -0.50, P = 0.04), Qf (r = -0.49, P = 0.04) and Lt (r = -0.51, P = 0.04).

Concerning the DG production system, a noticeable relationship was observed between the carcass traits HCW and KKCF (r = 0.50, P = 0.04). An increase in HCW was related to a lower SC/TC ratio in Bf (r = -0.62, P = 0.008) and St (r = -0.67, P = 0.004) muscles. Moreover, positive correlations between HCW and MFI 7 days were recorded for Ss (r = 0.49, P = 0.04) and Gm (r = 0.53, P = 0.03) **significant at 1% (P < 0.01), *significant at 5% (P < 0.05), §: non-muscle dependent correlation.

Growth path effects on meat quality in bulls muscles. In contrast, a negative correlation was observed between HCW and MFI 7 days in Lt (r = -0.50, P = 0.04) muscle. A direct correlation was observed between HCW and WBSF in Ss (r = 0.56, P = 0.02) muscle. Furthermore, increasing KKCF proportions in Ss (r = 0.71, P = 0.002), Tb (r = 0.60, P = 0.006) and Qf (r = 0.60, P = 0.007) muscles were associated with greater levels of IMF proportion and lower MFI 7 days in Lt (r = -0.51, P = 0.04) muscle. Moreover, increasing the KKCF amount was associated with a greater WBSF in Ss (r = 0.50, P = 0.04) and with lower WBSF values in Tb (r = -0.66, P = 0.004) and Bf (r = -0.50, P = 0.04) muscles. In addition, the IMF level was positively correlated with SC/TC in St (r = 0.54, P = 0.03) and was negatively correlated with MFI 7 days in Lt (r = -0.51, P = 0.04) muscle. A negative correlation was observed between IMF and WBSF in Tb (r = -0.49, P = 0.04) muscle.

Discussion

Carcass traits

Regarding carcass traits, at 18 months of age, the bulls of the CG production system had similar (P > 0.05) slaughter weights compared with bulls of the DG system and slaughtered at 24 months of age. However, the carcasses from the latter group were lighter, which could be due to a lower dressing %. The compensatory growth in beef cattle is characterized by high growth rates [START_REF] Carstens | Physical and chemical components of the empty body during compensatory growth in beef steers[END_REF] due to improved efficiency in growth and fattening, as a result of an increased feed intake, an increased efficiency in the utilization of protein and energy and an adjustment in energy partitioning (Allingham et al., 1998). Depending on restriction length and intensity, the reduction in the level of energy and protein intake generally leads to a decrease in growth rate with a more pronounced effect on fat deposition [START_REF] Robelin | Body composition of cattle as affected by physiological status, breed, sex and diet[END_REF].

Regarding the period of re-feeding (15 to 18 months of age), the daily gain was more than 0.45 kg greater in the DG group than in the CG group, reflecting the high restriction level used in the experiment. Indeed, under certain limits, the duration of the compensatory growth is proportional to the level of restriction [START_REF] Coleman | Effect of nutrition, age and size on compensatory growth in two breeds of steers[END_REF]. The 15 to 18 months of age period in DG bulls was followed by a sharp decline in ADG, which could be explained by the completion of compensatory growth, and, therefore, an increase in fat deposition. This result is in line with the knowledge that compensatory growth is the highest when the period of growth restriction is relatively short (about 3 months in cattle) [START_REF] Hornick | Mechanisms of reduced and compensatory growth[END_REF].

In general, ad libitum-fed animals have greater dressing percentages than those fed the same diet on a restricted scale. Moreover, the content of the digestive tract differs widely between animals fed different diets [START_REF] Béranger | Estimation du poids du contenu digestif des bovins à partir du poids du contenu du rumen[END_REF]. Thus, significant modifications in the diet, when cattle change from concentrate feeding to roughage diets, which increases the weight of both the digestive tract and gut contents, could negatively influence dressing percentage [START_REF] Kempester | Carcass characteristics and quality[END_REF]. When compared with CG bulls, the lower dressing proportion found in DG animals reveals that the finishing period (9 months) on ad libitum-fed concentrates provided to the DG group, in order to obtain an acceptable commercial carcass, could not have been sufficient to neutralize the effects of the hay-fed phase on the digestive system and some fat depots (including fat cover), which are known to determine dressing percentage [START_REF] Kempester | Carcass characteristics and quality[END_REF].

Tissues react differentially to feed restriction and some are more depleted than others (viscera > adipose tissue > muscle) [START_REF] Hornick | Mechanisms of reduced and compensatory growth[END_REF]. Fatness score depends on subcutaneous fat deposition, which can be more easily depleted than other fat depots during periods of low energy input [START_REF] Yambayamba | Compensatory growth of carcass tissues and visceral organs in beef heifers[END_REF], as ocurred in DG bulls. During recovery, the gross efficiency of weight gain (weight gain/kg of feed intake) in DG is greater than in CG animals at the same BW, because the weight gain in the former is mainly due to less fat accumulation and more water and protein [START_REF] Robelin | Patterns of growth of cattle[END_REF]. The divergent tissue composition and weight increases between continuous and compensatory growth animals could help explain the differences observed in KKCF and fatness score between CG and DG bulls. The DG had 1.6 points lower fatness score than CG bulls, but this difference probably could have only minor commercial implications. According to the European Union grid method (European Economic Community Regulations (EEC) no. 1208/81 and no. 2930/81), the value corresponds to fat class 2 in DG and 3 in CG carcasses. In contrast to carcass fatness score, the finishing period on concentrates was sufficient to reverse the negative effects of feed restriction on leg muscle, bone and fat contents, as no differences were observed on leg joint dissection between CG and DG bulls (Table 3). The leg joint dissection is a rough estimation of overall carcass tissue composition. However, due to its economic relevance, this finding should be considered by cattle producers when they choose the production system in Mediterranean areas.

Meat traits Despite the lower temperature at 45 min (P = 0.06), there was a slower decrease of meat temperature between 45 min and 24 h after slaughter in CG than in the DG group (data not shown), which may be explained by the greater proportion of carcass fat cover in CG bulls, preventing heat escape. A slower drop in temperature is expected to be associated with a quicker decrease in muscle pH, because higher meat temperature stimulates enzymatic activity and accelerates the rate of pH decline in muscles. Beef carcasses undergoing rapid pH fall while the loin muscle temperature is still high are described as heat shortened, with subsequent negative effects on quality traits [START_REF] Warner | Factors influencing the incidence of high rigor temperature in beef carcasses in Australia[END_REF]. The temperature at which the muscle enters rigour will impact on the degree of muscle shortening. Cold shortening occurs at temperatures below 15°C, and heat shortening occurs above 20°C [START_REF] Devine | Effect of rigor temperature on muscle shortening and tenderisation of restrained and unrestrained beef M. longissimus thoracicus et lumborum[END_REF], and each is known to influence the quality characteristics of meat, to varying degrees, particularly the tenderness [START_REF] Warner | Factors influencing the incidence of high rigor temperature in beef carcasses in Australia[END_REF].

However, the relationships between temperature and pH values are not easily established in the present study because the pH in Lt muscle was lower in DG than in CG bulls Costa, Simões, Costa, Lemos, Navas, Hocquette, Calkins and Bessa at 45 min, 3 and 24 h, but the temperature was similar between production systems at 45 min and 3 h and was lower in DG bulls at 24 h.

In contrast to carcass subcutaneous fat, the IMF content across muscles was not affected by the production system. The muscle IMF content may affect meat palatability, particularly flavour, juiciness and tenderness of cooked beef. Low amounts of IMF can decrease the eating experience for consumers (Hocquette et al., 2010). In fact, marbling score has been reported as the carcass grade trait most highly, positively correlated with the beef palatability attributes [START_REF] May | Effect of days fed, carcass grade traits, and subcutaneous fat removal on postmortem muscle characteristics and beef palatability[END_REF]. In the present study, the negative correlations between IMF content and WBSF were limited to Tb muscle in DG group (Table 6). Unexpectedly, a positive correlation between those quality traits was observed for Ss and Gm muscles in the CG group, which were among the muscles with the highest IMF content.

Meat tenderness strongly influences the consumer's re-purchase and choices for a particular meat cut and/or brand [START_REF] Schönfeldt | Effect of age and cut on tenderness of South African beef[END_REF][START_REF] Costa | Effect of low-and high-forage diets on meat quality and fatty acid composition of Alentejana and Barrosa beef breeds[END_REF]. The two major factors determining meat tenderness are the ageing time and the quantity and characteristics of the intramuscular collagen [START_REF] Takahashi | Structural weakening of skeletal muscle tissue during postmortem ageing of meat: the non-enzymatic mechanism of meat tenderization[END_REF][START_REF] Chriki | Cluster analysis application identifies muscle characteristics of importance for beef tenderness[END_REF]. The influence of the production system on WBSF was muscledependent, which is in agreement with the findings from other authors (Hansen et al., 2006a). Interestingly, within muscles, the WBSF numeric values were greater in CG than in DG bulls. However, the differences were only statically significant (P < 0.05) for Ss, Tb, St and Bf muscles.

The Ss, Tb, St and Bf muscles are those in which the connective tissue content gives a major contribution to tenderness [START_REF] Koohmaraie | Meat tenderness and muscle growth: is there any relationship?[END_REF]. In general, muscles with greater collagen content possess higher crosslink concentrations per unit of collagen than muscles with lower collagen content [START_REF] Light | The rôle of epimysial, perimysial and endomysial collagen in determining texture in six bovine muscles[END_REF]. It is noteworthy that the WBSF, but not the MFI 7 days values, were significantly affected by the production system for these four muscles (Table 5). This figure could be, in part, associated with the effects on collagen quality, particularly the SC content in Tb and St muscles. Newly synthesized collagen could dilute the preexisting, heat-stable collagen, leading to increased collagen solubility. Positive correlations between WBSF and collagen content were previously reported [START_REF] Oury | Interrelationships between meat quality traits, texture measurements and physicochemical characteristics of M. rectus abdominis from Charolais heifers[END_REF] and were confirmed in the present study only for the St, Qf and Lt muscles within CG bulls (Table 6). These results suggest that collagen content has a different response to production systems depending on the muscle considered, which is in agreement with studies by [START_REF] Cassar-Malek | Muscle-specific metabolic, histochemical and biochemical responses to a nutritionally induced discontinuous growth path[END_REF]. These authors stated that nutrition in cattle plays a significant role in the regulation of muscle characteristics, and this regulation is highly dependent on muscle type. Individual muscles respond differently to a compensatory growth path in terms of collagen solubility. However, the contribution of collagen to beef tenderness depends on ageing time, and comparisons with results from other studies should be carried out with caution when different ageing times are used.

Meat tenderness is associated with protein degradation, influenced by the postmortem activity of proteolytic enzymes.

As these enzymes are also involved in protein turnover in the living muscles, it could be expected that feed-restriction, followed by re-alimentation, could have the potential to improve tenderness [START_REF] Therkildsen | Muscle protein degradation in bull calves with compensatory growth[END_REF]. Rapid growth, as observed in the compensatory phase, increases protein turnover and the ratio between protein synthesis and degradation in connective tissue and myofibrils, making these younger and more liable to breakdown during conditioning and cooking. Generally, the structural changes in intra-muscular connective tissue caused by compensatory growth have a positive effect on SC content, reversing the negative effects of age on solubility [START_REF] Gerrard | Collagen stability, testosterone secretion and meat tenderness in growing bulls ans steers[END_REF]. This could explain the increased amount of SC observed in Tb, St and Qf muscles from older DG bulls. As quoted before, these differences in SC content between DG and CG bulls may have contributed to explain the lower WBSF values found in the group slaughtered at an older age.

Paradoxically, the MFI 7 days value was not affected by the production system for any muscle. This contrasts with previous studies addressing the effects of feed restriction and compensatory growth in steers [START_REF] Jones | Myofibrillar protein turnover in feed-restricted and realimented beef cattle[END_REF][START_REF] Muir | Effects of growth pathway on beef quality in 18-month-old Angus and South Devon × Angus pasture-fed steers[END_REF], calves [START_REF] Therkildsen | Effect of growth rate on tenderness development and final tenderness of meat from Friesan calves[END_REF] and bulls slaughtered before 13 months of age (Therkildsen, 2005, Hansen et al., 2006a). These authors reported that compensatory growth promotes postmortem myofibrillar protein degradation as a consequence of a greater protein turnover before slaughter. However, in opposition to studies by Hansen et al. (2006a), [START_REF] Jones | Myofibrillar protein turnover in feed-restricted and realimented beef cattle[END_REF] and [START_REF] Therkildsen | Muscle protein degradation in bull calves with compensatory growth[END_REF] where the compensation period was short (⩽5 months), the DG had a long period (9 months) of re-feeding and the ADG peak occurred between 15 and 18 months of age. It is possible that, at 24 months of age, part of the effects on myofibrillar proteins were diluted and no longer observed. In addition, the lack of a production system effect on MFI 7 days may be attributed to the inhibitory effects of testosterone on protein turnover, which is known to peak at around 15 months of age [START_REF] Gerrard | Collagen stability, testosterone secretion and meat tenderness in growing bulls ans steers[END_REF], and to an enhanced calpastatin activity. Increased postmortem activity of calpastatin was reported to decrease the activity of μ-calpain, and therefore the amount of postmortem protein degradation and tenderization [START_REF] Morton | Calpaincalpastatin and toughness in M. longissimus from electrically stimulated lamb and beef carcasses[END_REF]. The inhibitory effect of testosterone on myofibrillar protein degradation was not extended to the connective tissue in the present study. This is consistent with the findings of [START_REF] Geesink | μ-Calpain is essential for postmortem proteolysis of muscle proteins[END_REF], who reported that μ-calpain is the enzyme primarily responsible for postmortem degradation of myofibrillar proteins, acting on muscle contraction structure. Research has shown that μ-calpain action is temperature-and pH-dependent, as it is modulated by calpastatin binding, which in turn is influenced by both factors [START_REF] Morton | Calpaincalpastatin and toughness in M. longissimus from electrically stimulated lamb and beef carcasses[END_REF]. These authors found that the lower the pH the faster the decline in μ-calpain activity, and both were positively related to meant tenderness at 48 h postmortem. In the present study, the pH at 45 m, 3 h and 24 h evaluated in the Lt muscle was greater in CG when compared with the DG group (Table 4). However, no differences were found between groups neither for MFI 7 days nor Growth path effects on meat quality in bulls for WBSF values in Lt muscle. Furthermore, no significant correlations were found between MFI 7 days and WBSF within CG and DG groups (Table 6), which re-inforce the lack of relationship between these two parameters in this study. Taking into account the differences in Lt pH between CG and DG groups and the role of pH in beef tenderness, it would have been useful to assess the levels of glucose, lactate, adrenaline and cortisol in blood in order to better understand the effect of growth path in muscle pH and WBSF. This remark should be considered in future studies about the effect of growth path on meat quality.

Meat colour is one of the most important characteristics according to which consumers make judgements on meat quality. It influences the consumer's purchase decisions because it is perceived as a measure of freshness and quality [START_REF] Mancini | Current research in meat color[END_REF].

The ultimate pH is highly correlated to meat colour and lightness in particular [START_REF] Mancini | Current research in meat color[END_REF]. Regarding the Lt muscle (Tables 4 and5), if the pH at 24 h is considered as ultimate pH, the difference between CG and DG bulls (0.26 pH units, P = 0.0011) was not sufficient to cause differences in colour. However, among the eight muscles studied, the Lt muscle was the only one where the production system did not affect the colour parameters. However, the ultimate pH is not the only factor responsible for differences in meat colour [START_REF] Priolo | Effects of grass feeding systems on ruminant meat colour and flavour. A review[END_REF]. In a study by [START_REF] Hornick | Different periods of feed restriction before compensatory growth in Belgian Blue bulls: I. animal performance, nitrogen balance, meat characteristics, and fat composition[END_REF], higher a* and b* values in the Lt muscle were found in groups that exhibited a more noticeable compensatory growth. The authors associated the higher colour values with the higher temperature found in Lt muscle after slaughter. In the present study, a relationship between colour parameters and carcass temperature (evaluated in the Lt muscle) was only observed in Ss, Qf and Sm muscles, where the CG bulls had the greater a* values and higher carcass temperatures at 24 h postmortem. The b* values did not follow the same pattern, and when compared with CG bulls, DG bulls had higher b* values in Tb and Qf and lower values in Ss, St and Bf muscles. In general, it was expected that colour variables would be higher in older animals (DG bulls), because muscle myoglobin increases ageing. However, other muscle-related factors including muscle allometry, chemical composition and fibre profile, all of which are known to be influenced by age and growth rate, could have contributed to the differences found in colour parameters.

Conclusions

Taking advantage of compensatory growth in bovine production could represent a relevant economic opportunity, which contributes to the environmental sustainability and biodiversity in Mediterranean areas. The compensatory growth production system produced slight negative effects on hot carcass weight, fatness and dressing percentage, but the proportions between fat, bone and muscle remained the same. In contrast, there was an overall positive impact of compensatory growth on muscle tenderness, overcoming the negative effects of age at slaughter. The reasons for such an improvement in meat tenderness are not clear because neither IMF nor myofibrillar protein degradation could explain the differences between production systems. Moreover, an association between meat tenderness and collagen properties was difficult to establish, possibly due to the short ageing time. Finally, our results indicate that compensatory growth has a muscle-dependent effect and a significant impact on several muscle and carcass characteristics.

Figure 1

 1 Figure 1 Average daily gain (ADG) of bulls produced according to continuous growth (CG) and discontinuous growth (DG) systems during different time periods (mean ± s.e.). a-c: Different letters differ significantly (P < 0.05).

  carcass weight (kg); KKCF = kidney knob and channel fat (kg); IMF = intra-muscular fat (g/100 g of fresh muscle); IC = insoluble collagen (mg/g of fresh muscle); SC = soluble collagen (mg/g of fresh muscle); MFI 7 days = myofibril fragmentation index; WBSF = Warner-Bratzler shear force (kg); Ss = supraspinatus ; Tb = triceps brachii ; St = semitendinosus ; Bf = biceps femoris ; Qf = quadriceps femoris ; Gm = gluteus medius ; Sm = semimembranosus (Sm); Lt = longissimus thoracis muscles. Correlation values for each muscle are presented in brackets.

Table 1

 1 Ingredients and chemical composition of experimental diets

	Concentrate feed (n = 2) Hay (n = 1)
	Ingredient (g/kg fresh weight basis)
	Maize	325
	Wheat	201
	Barley	197
	Soya bean meal	135
	Sunflower meal	80
	Hydrogenated fat	13
	Calcium carbonate	20
	Sodium bicarbonate	10
	Calcium phosphate	9
	Salt	8
	Vitamin-mineral premix †	2
	Chemical composition (g/kg DM)
	CP	139	65
	Crude fat	30	9.0
	Crude fibre	58	367
	Total-N	22	10
	NDF	188	622
	ADF		475
	ADL		59
	Starch	483	24
	Gross energy (MJ/kg DM)	15.4	17.3
	Ash	60	91
	Calcium	13	8.7
	Phosphorus	5.0	2.9
	Silica	3.3
	Sodium		1.3
	Potassium		22
	Magnesium		1.4
	Dry matter (DM, %)	88.5	90.9
	In vitro DM Digestibility (%)		55.1

n = number of diet samples. † Vitamin-mineral premix contained per kg: 5000 IU vitamin A, 1000 IU vitamin D3, 20 IU vitamin E, 0.16 mg citric acid, 9.5 mg Fe, 110.6 mg Zn, 52.5 mg Mn, 0.3 mg Mo, 0.53 mg Co, 0.11 mg Se, 6.75 mg I, 25.0 mg BHT and 0.16 mg ethoxyquin.

Table 2

 2

	Initial weight (kg)	249	237	11	0.41
	Slaughter weight (kg)	643	606	14	0.08
	Hot carcass weight (kg)	369	339	9	0.02
	Dressing (%)	57.3	56.0	0.4	0.03
	KKCF (%)	2.7	2.2	0.2	0.06
	Fatness score †	7.2	5.6	0.3 <0.001

Carcass traits for bulls produced according to continuous (CG) and discontinuous growth (DG) systems CG (n = 17) DG (n = 17) s.e.m. P-value KKCF = kidney knob and channel fat. †

Table 3

 3 Leg joint dissection variables for bulls produced according to continuous (CG) and discontinuous growth (DG) production systems CG (n = 17) DG (n = 17) s.e.m. P-value

	Leg dissection (g/100 g)
	Muscle	77.4	76.6	0.7	0.38
	Bone	11.7	12.1	0.4	0.46
	Subcutaneous fat	5.80	5.92	0.25	0.37
	Inter-muscular fat	4.76	5.07	0.22	0.34
	Total fat	10.6	11.0	0.4	0.50
	Others	0.34	0.31	0.02	0.48
	Ratios
	Muscle/total fat	7.67	7.06	0.35	0.22
	Muscle/bone	6.80	6.47	0.30	0.42
	Total fat = subcutaneous + inter-muscular fat.
	Others = lymphatic ganglia, large blood vessels and nerves, tendons and joint
	capsules.

Table 4

 4 Longissimus thoracis muscle temperature and pH values of

	bulls produced according to continuous (CG) and discontinuous growth
	(DG) production systems
		CG (n = 17)	DG (n = 17)	s.e.m.	P-value
	Temperature (°C)
	45 m	32.8	35.6	0.9	0.06
	3 h	26.9	27.8	0.7	0.35
	24 h	14.7	11.1	0.5	<0.001
	pH
	45 m	6.63	6.29	0.06	<0.001
	3 h	6.36	5.99	0.05	<0.001
	24 h	5.67	5.41	0.05	0.001

P-value CG DG s.e.m. P-value CG DG s.e.m. P-value CG DG s.e.m.

Table 6

 6 Pearson correlation coefficients between carcass and meat quality in bulls produced according to continuous (CG) and discontinuous growth (DG) production systems

	HCW	KKCF	IMF	SC/TC	MFI 7 days	WBSF
	Continuous growth (CG)
	HCW		Tb (0.57)*	-Lt (-0.50)*	Qf (0.52)*	Lt (0.50)*
			St (0.51)*
	KKCF		St (0.59)**		Bf (0.50)*	Ss (0.49)*
			Gm (0.62)**
	IMF				Bf (0.61)**	Ss (0.50)*
						Gm (0.56)*
	SC/TC				Gm (0.58)*	-St (-0.50)*
						-Qf (-0.49)*
						-Lt (-0.51)*
	MFI 7 days

Acknowledgements

This research was supported by the Fundação para a Ciência e a Tecnologia (FCT) through financial support from FCT grants (PTDC/CVT/65707/2006 and PTDC/CVT/111744/2009) and individual fellowships to Paulo Costa (SFRH/BPD/46135/2008) and to Ana S. H. Costa (SFRH/BD/61068/2009). We are grateful to António Sequeira for his decisive role in animal management during the trial. We are grateful to the EZN slaughterhouse staff, particularly to Paula Menezes for her help with carcass data and meat sample collection.


Aleksandar Karajić

Pascal Merzeau

Emmanuel Suraniti

Sébastien Gounel

Christèle Jaillet

Alexander Kuhn

Nicolas Mano

Enzymatic Glucose-Oxygen Biofuel Cells for Highly Efficient Interfacial Corrosion Protection

Keywords: Glucose-oxygen biofuel cell, Corrosion protection, Bioelectronics, Boost converter, Bioelectrochemistry

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

Introduction

Biofuel cells (BFC) [START_REF] Mano | O 2 Reduction in Enzymatic Biofuel Cells[END_REF][START_REF] Xiao | Tackling the Challenges of Enzymatic ( Bio ) Fuel Cells[END_REF][START_REF] Rasmussen | Enzymatic Biofuel Cells: 30 Years of Critical Advancements[END_REF] are low power electrochemical devices capable of transforming chemical into electrical energy either with immobilized enzymes [START_REF] Rasmussen | Enzymatic Biofuel Cells: 30 Years of Critical Advancements[END_REF] , microorganisms [START_REF] Kaiser | Electrogenic Single-Species Biocomposites as Anodes for Microbial Fuel Cells[END_REF][START_REF] Higgins | Hybrid Biofuel Cell: Microbial Fuel Cell with an Enzymatic Air-Breathing Cathode[END_REF] or living organelles [START_REF] Arechederra | Organelle-Based Biofuel Cells: Immobilized Mitochondria on Carbon Paper Electrodes[END_REF] . Optimized over several decades by implementing new materials, [START_REF] Mazurenko | Recent Developments in High Surface Area Bioelectrodes for Enzymatic Fuel Cells Recent Developments in High Surface Area Bioelectrodes for Enzymatic Fuel Cells[END_REF][START_REF] Pinyou | Enzyme-Modified Electrodes for Biosensors and Biofuel Cells[END_REF][START_REF] Mano | Recent Advances in High Surface Area Electrodes for Bioelectrochemical Applications[END_REF] fabrication technologies and strategies, BFCs nowadays are well-adapted to allow various practical applications, such as self-powered biosensors [START_REF] Liu | Miniature Amperometric Self-Powered Continuous Glucose Sensor with Linear Response[END_REF][START_REF] Bandodkar | Highly Stretchable Fully-Printed CNT-Based Electrochemical Sensors and Biofuel Cells: Combining Intrinsic and Design-Induced Stretchability[END_REF][START_REF] Katz | Self-Powered Enzyme-Based Biosensors [8[END_REF][START_REF] Jeerapan | On-Body Bioelectronics: Wearable Biofuel Cells for Bioenergy Harvesting and Self-Powered Biosensing[END_REF][START_REF] Gai | Ultrasensitive Self-Powered Cytosensors Based on Exogenous Redox-Free Enzyme Biofuel Cells as Point-of-Care Tools for Early Cancer Diagnosis[END_REF][START_REF] Zhang | Self-Powered Sensor for Hg 2+ Detection Based on Hollow-Channel Paper Analytical Devices[END_REF] , wearable electronics [START_REF] Jia | Wearable Textile Biofuel Cells for Powering Electronics[END_REF] and ''bio-batteries'' for powering small electronic devices [START_REF] Jia | Wearable Textile Biofuel Cells for Powering Electronics[END_REF][START_REF] Katz | Implanted Biofuel Cells Operating in Vivo-Methods, Applications and Perspectives-Feature Article[END_REF][START_REF] Zebda | Single Glucose Biofuel Cells Implanted in Rats Power Electronic Devices[END_REF] . In enzymatic biofuel cells, those that rely on the biocatalytic oxidation of glucose and reduction of oxygen are among the most abundant examples reported in the literature due to the fact that glucose and oxygen are readily available in living organisms and can be easily replenished in biological fluids [START_REF] Slaughter | Enzymatic Glucose Biofuel Cell and Its Application[END_REF] .

The main drawbacks of biofuel cells are their low power/voltage output and limited lifetime, rendering them less suitable for continuous powering of small electronic devices than other energy storage/supply systems such as batteries. Many strategies have been explored in the past to boost the power output to the levels required for electronic circuits. This includes the implementation of different materials with high electrochemical active surface area (nano- [START_REF] Xiao | Nanoporous Gold-Based Biofuel Cells on Contact Lenses[END_REF] and macroporous [START_REF] Zhang | Rational Design of Enzyme-Modified Electrodes for Optimized Bioelectrocatalytic Activity[END_REF] materials, carbon-nanotubes [START_REF] Zebda | Mediatorless High-Power Glucose Biofuel Cells Based on Compressed Carbon Nanotube-Enzyme Electrodes[END_REF][START_REF] Xiao | A Biofuel Cell in Non-Aqueous Solution[END_REF][START_REF] Macvittie | Cyborg" Lobsters to a Pacemaker Powered by Implantable Biofuel Cells[END_REF] , graphene [START_REF] Liu | Membraneless Enzymatic Biofuel Cells Based on Graphene Nanosheets[END_REF] , etc.), the design of new enzyme mutants [START_REF] Luz | Enzyme Biofuel Cells : Thermodynamics , Kinetics and Challenges in Applicability[END_REF] with improved activity and stability or the use of boost converters able to enhance the output voltage generated by a BFC [START_REF] Katz | Implanted Biofuel Cells Operating in Vivo-Methods, Applications and Perspectives-Feature Article[END_REF][START_REF] Hanashi | BioRadioTransmitter: A Self-Powered Wireless Glucose-Sensing System[END_REF] . However, the latter is only possible at the expense of the globally available power [START_REF] Katz | Implanted Biofuel Cells Operating in Vivo-Methods, Applications and Perspectives-Feature Article[END_REF][START_REF] Suraniti | Uphill Production of Dihydrogen by Enzymatic Oxidation of Glucose without an External Energy Source[END_REF] (i.e. biofuel-cell output), partially consumed by the boost converter for its proper functioning.

Few examples of biofuel cells connected to a boost converter for powering electronic devices/components such as pacemakers [START_REF] Southcott | A Pacemaker Powered by an Implantable Biofuel Cell Operating under Conditions Mimicking the Human Blood Circulatory System -Battery Not Included[END_REF] , electronic watches [START_REF] Jia | Wearable Textile Biofuel Cells for Powering Electronics[END_REF] , digital thermometers [START_REF] Zebda | Single Glucose Biofuel Cells Implanted in Rats Power Electronic Devices[END_REF] , light emitting diodes [START_REF] Zebda | Single Glucose Biofuel Cells Implanted in Rats Power Electronic Devices[END_REF][START_REF] Chen | Stretchable and Flexible Buckypaper-Based Lactate Biofuel Cell for Wearable Electronics[END_REF] or capacitors [START_REF] Baingane | Self-Powered Electrochemical Lactate Biosensing[END_REF] , were reported in the literature. Recently, our group has demonstrated the concept of temporarily storing the energy released in one system (i.e. glucose/oxygen biofuel cell) in the form of electromagnetic energy, to drive another electrochemical reaction (i.e. water splitting) with an overall positive ΔG value (ΔG glucose/oxygen < ΔG water splitting ), while sharing the same electrolyte. The system did not require any external power source and the circuit was composed of the BFC and a water electrolyzer, connected via a boost converter for the voltage increase and a flyback for galvanic separation. [START_REF] Suraniti | Uphill Production of Dihydrogen by Enzymatic Oxidation of Glucose without an External Energy Source[END_REF] In this proof-of-concept study, we examine the electrochemical corrosion protection of an iron surface in chloride containing PBS buffer by using an initially low voltage (0.3 V) generated by a single glucose/oxygen biofuel cell. The voltage generated in the BFC was modulated via a boost converter to reach a much higher value of 2.3 V, the minimum voltage level that is required for successful corrosion protection. To the best of our knowledge, this is the first study reporting total corrosion protection based on a simple glucose-oxygen BFC. These findings might open up new application opportunities for BFCs, far beyond the traditional areas of energy storage and conversion.

Experimental section

Materials

All aqueous solutions used in this work were prepared in MilliQ® water (18 MΩ cm) from analytical grade chemicals. D-glucose (99.6%), sulfuric acid (96%), sodium chloride (≥ 99.5 %), sodium hydrogen phosphate heptahydrate (≥ 98 %), sodium-dihydrogen phosphate monohydrate (≥ 98 %) and catalase (from bovine liver; activity: 14000 U mg -1 of protein) were purchased from Sigma Aldrich. Iron rods (purity: 99+ %, Ø: 2 mm) were obtained from AlfaAesar.

Agarose (electrophoresis grade) was obtained from Invitrogen TM . Electrode polishing alumina slurry (0.05 μm) was purchased from Buehler (IL, USA). Glucose oxidase from Aspergillus Niger (activity: 480 U.mg -1 ) was purchased from Sigma Aldrich and purified according to a previously published procedure. [START_REF] Courjean | Deglycosylation of Glucose Oxidase for Direct and Efficient Glucose Electrooxidation on a Glassy Carbon Electrode[END_REF] Bilirubin oxidase (BOD) from Magnaporthe Oryzae (activity: 568 U.mg -1 ) was produced and purified as previously reported. [START_REF] Durand | Bilirubin Oxidase from Magnaporthe Oryzae: An Attractive New Enzyme for Biotechnological Applications[END_REF] Gold-coated glass slides were purchased from Kerdry, France. Carboxylated multiwall carbon nanotubes (MWCNT-COOH; 0.54 wt.% in MilliQ water) [START_REF] Bartholome | Electromechanical Properties of Nanotube-PVA Composite Actuator Bimorphs[END_REF][START_REF] Bartholome | Influence of Surface Functionalization on the Thermal and Electrical Properties of Nanotube-PVA Composites[END_REF] , as well as anodic [START_REF] Mao | Long Tethers Binding Redox Centers to Polymer Backbones Enhance Electron Transport in Enzyme "Wiring" Hydrogels[END_REF] and cathodic [START_REF] Cadet | Design of a Highly Efficient O2 Cathode Based on Bilirubin Oxidase from Magnaporthe Oryzae[END_REF] Os-redox polymers were synthesized as reported earlier.

The electrochemical measurements that include the cleaning of the gold electrodes, corrosion studies and the characterization of the BFC were performed with a CH Instruments bipotentiostat (842B or 760C). Ag/AgCl (3M NaCl) reference electrodes were purchased from BASi while the platinum wire used as a counter electrode was obtained from AlfaAesar. Glassy carbon rotating disc electrodes were purchased from Pine Research Instrumentation. PDMS monomer and initiator were purchased from GE Bayer Silicones (Leverkusen, Germany).

Preparation of planar gold electrodes

The 2.25 cm 2 squared gold-coated glass slide electrodes were prepared by soldering the electric leads and selecting a geometric surface area of approximately 2.25 cm 2 (≈1.5cm x 1.5cm) with a thin layer of a commercially available nail varnish. Once the nail-varnish was completely dried, the electrochemical cleaning of the electrode surface was performed by cyclic voltammetry in an argon-saturated 100 mM H 2 SO 4 solution. 50 CVs at 100 mV s -1 were performed between -0.2 V to +1.6 V vs. Ag/AgCl (3M NaCl) until a steady shape of consecutive voltammograms is obtained. Then, the electrodes were rinsed with MilliQ water, dried in an argon stream and immediately used for the preparation of the glucose/oxygen biofuel cell.

Electrode potentials are expressed versus a Ag/AgCl (3M NaCl) reference electrode (referred to as Ag/AgCl) if not stated otherwise.

Biofuel cell preparation

The anodic hydrogel was prepared by mixing 43.5 μl of anodic Os-redox polymer (PVP-Os[(1,1dimethyl-2,2′biimidazole)2-2-[6-methylpyrid-2yl] imidazole] 2+/3+ ; γ: 8.0 mg.ml -1-in MilliQ water) with 43.3 μl of glucose oxidase solution (γ: 5.0 mg.ml -1 in MilliQ water) and 30.9 μl of polyethyleneglycol diglycidyl ether (PEGDGE ) as a cross-linker (γ: 2.0 mg.ml -1 in MilliQ water), respectively. The wt% ratio of the GOx/Os-polymer/PEGDGE in the anodic hydrogel was 35/55/10. 106.2 μl of the so-obtained hydrogel were carefully spread over the entire surface of a freshly cleaned gold electrode and dried at 4 °C for 48h. The final hydrogel loading was 250 μg.cm -2 .

For the preparation of the cathodic composite hydrogel, 80.6 μl of the cathodic Os-redox polymer (PAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine) 2 Cl] +/2+ ; γ: 10 mg.mL -1 in MilliQ water) was mixed with 38.6 μl of BOD, 84.2 μl of MWCNT-COOH suspension (w: 0.054 wt.% in MilliQ water) and 23.9 μl of PEGDGE cross-linker, respectively. 198.6 μl of this composite suspension were spread onto the surface of the Au electrode and dried at 4 °C for 48h. The obtained composite MWCNT-COOH/hydrogel loading was 416.7 μg.cm -2 . The ratio between the cathodic hydrogel and MWCNT-COOH was 60/40 while the wt% ratio of BOD/Ospolymer/PEGDGE was kept at 30/62.57/7.43.

The bioelectrodes were removed from the fridge at least 1h before the electrochemical characterization. The BFC was assembled prior to its use with the two electrodes facing each other while a 7 mm thick PDMS spacer was introduced between them. The entire assembly was wrapped with a few layers of adhesive tape to ensure the structural integrity of the BFC.

PDMS was synthesized by thermal curing of a 10 : 1 = monomer: initiator mixture at 95 °C for 2h.

Optimization of the composition of MWCNT-COOH/BOD/Os-redox polymer/PEGDGE hydrogel composite

Cyclic-voltammetry on a rotating glassy carbon (GC) disc electrode modified with the composite BOD hydrogel was performed to determine the optimal MWCNT-COOH loading for the preparation of the cathodic hydrogel. For that purpose, a glassy-carbon electrode (Ø: 5 mm) was intensively polished with alumina slurry and thoroughly rinsed with MilliQ water. The surface of the so-prepared electrode was modified by drop-casting MWCNT-COOH/BOD based hydrogelcomposite that contains different amounts of CNTs (i.e. 20, 30, 40, 50, 60 wt%). The loading of the BOD/Os-redox polymer/PEGDGE hydrogel was kept constant at 108 μg.cm -2 . The cyclic voltammograms (potential window: +0.6 V to -0.15 V) were recorded in an argon and oxygen saturated PBS solution (20 mM PB + 140 mM NaCl) at 5 mV s -1 , 37.5 °C, and 500 rpm with a Pt wire and Ag/AgCl serving as counter and reference electrodes, respectively. The cathodic 4e - oxygen reduction diffusion-limited current was measured at -0.15 V.

Electrochemical characterization of the BFC

The electrochemical characterization of bioelectrodes was performed in a standard threeelectrode electrochemical set-up with the enzymatic-hydrogel modified cathode/anode, a coiled Pt wire and Ag/AgCl serving as working, counter and reference electrodes, respectively. The platinum counter electrode was cleaned prior to every use in electrochemical experiments.

The size of a standard glass-body Ag/AgCl reference electrode was reduced by mounting a Ushaped glass capillary extension (i.e. made from a glass pipette) to its end. The opening of the extension was filled with 3M NaCl solution and sealed with a small agarose seal, serving as a salt bridge. For this purpose, a 2% agarose solution was prepared by dissolving agarose in 3M NaCl solution at 80 °C. A small amount of the so-obtained solution was introduced by suction into the capillary end before the agarose gelified. The reference electrode was placed in the confined space between biocathode and bioanode during the electrochemical characterization experiments.

The electrochemical characterization of the separate bioelectrodes was performed by cyclicvoltammetry at 5 mV s -1 , 37.5 °C in phosphate buffer saline (PBS; 20 mM phosphate buffer + 140 mM NaCl; pH: 7.2) solution by cycling the potential between -0.3 V and +0.6 V. The first part of the electrochemical characterization was performed in an argon saturated PBS solution in the absence of glucose and oxygen. This step is followed by the addition of a glucose stock solution (C final : 50 mM), 20 nM catalase and intensive oxygen purging. The oxygen was purged through a space between two parallel electrodes for at least 10 minutes before the addition of glucose and catalase to ensure its saturation in PBS solution under experimental conditions.

The polarization curve was recorded at a scan rate of 5 mV s -1 in a two-electrode system with the bioanode connected to a potentiostat via short-circuited counter/reference electrode contacts and a biocathode that was connected as the working electrode. The BFC power curve was calculated by multiplying the current with the voltage measured during the polarization experiment.

Measurement of the MWCNT-COOH zeta potential

The Zeta potential of MWCNT-COOH suspended in MilliQ water was determined by using disposable cuvettes with a capillary channel and a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd, Worcestershire, UK) in quintuplicate at 25 °C.

Preparation of iron rods for the corrosion experiment

Iron rods were cut into 5 cm long pieces and the surface oxide layer was removed with sandpaper of different grit size (400, 800 and 1200, respectively) until a macroscopically smooth and polished surface was obtained. This step was followed by 5 min ultrasonication in absolute acetone and ethanol [START_REF] Ejaz | Effects of Hydrogen on Corrosion Products Formed on Iron in Chloride Solutions[END_REF] , respectively. Finally, the samples were dried in an argon stream and used for the corrosion experiment.

Optimization of the corrosion-preventing voltage

The influence of the applied voltage on the corrosion process in PBS solution was evaluated with a CH Instruments potentiostat/galvanostat (Model: 660C) in a two-electrode electrochemical cell composed of a Fe rod as working electrode and short-circuited counter-reference platinum electrodes. Different voltages were applied for 3h followed by extensive rinsing (i.e. MilliQ water and absolute ethanol, respectively) and drying (i.e. stream of argon) steps. Samples were preserved in an inert atmosphere until used.

Electrochemical impedance spectroscopy (EIS)

EIS measurements were performed on a CH Instruments potentiostat/galvanostat (Model: 660C) in PBS buffer (20 mM PB + 140 mM NaCl; pH: 7.2) with a three-electrode system composed of the Fe rod as working, a Pt coil as counter and Ag/AgCl as reference electrode. The EIS spectra were recorded around the OCV (i.e. 0.7 V; the OCV was measured continuously for 30 minutes), frequency range from 10 -2 Hz to 10 5 Hz, and 10 mV amplitude.

The Fe rods used in EIS experiments were either in their pristine form (i.e. cleaned with sandpaper, acetone, and ethanol respectively) or after applying a corrosion-preventing voltage over a period of 4h. The control sample was immersed in PBS solution and left to corrode for 4h.

Scanning electron microscopy (SEM) characterization

SEM images were obtained by using a tabletop SEM microscope (TM-1000, Hitachi, Japan).

The EDS spectra were recorded with a field emission scanning electron microscope (JMS-6700F, JEOL Ltd., Japan).

Boost converter

A low-power BQ25505EVM-218 boost converter was purchased from Texas Instruments. To meet our specific requirements, the boost converter was modified with a 5 MΩ potentiometer in series with a 8.2 MΩ resistor, to make the output voltage tunable (Figure S1, Supplementary Information). The OCV was measured by the maximum power point tracker (MPPT) every 16 s in order to establish the voltage at which a maximum power is drained from the biofuel cell (V Pmax ≈ 45% of the OCV). The current and the voltage at the input (output of the BFC) and the output (input of the corrosion cell) of the boost converter were continuously monitored by Keithley 2000 multimeters (Keithley, USA).

Results and Discussion

Optimization of the conditions for electrochemical corrosion protection

The first evaluation of the corrosion process was performed with an iron rod working electrode immersed in PBS combined with a short-circuited counter/reference Pt coil electrode. The values of the corrosion potential obtained from the potentiodynamic polarization experiment (Figure S2) show a higher thermodynamic instability of the Fe surface in PBS (E corr. = -1.16 V vs. Pt ) in comparison with NaCl solution (E corr. = -0.83 V vs. Pt) commonly used in corrosion studies because of an intensive chloride-induced pitting corrosion. [START_REF] Wang | Effect of PH and Chloride on the Micro-Mechanism of Pitting Corrosion for High Strength Pipeline Steel in Aerated NaCl Solutions[END_REF][START_REF] Frankel | Pitting Corrosion of Metals[END_REF] Another advantage of using PBS in this proof-of-concept study is the ability of phosphate ions to chemically capture the corrosion products (i.e. Fe 2+ ions) in the form of a colored and insoluble phosphate/oxide heterogeneous layer that precipitates on the electrode surface. This has already been observed with either construction [START_REF] Yohai | Inhibiting Steel Corrosion in Simulated Concrete with Low Phosphate to Chloride Ratios[END_REF] or carbon steel [START_REF] Bastidas | Corrosion Inhibition Mechanism of Phosphates for Early-Age Reinforced Mortar in the Presence of Chlorides[END_REF] alloys. The EDS analysis of the corroded iron surface (Figure S3) confirmed the presence of a significant amount of surface oxygen and phosphorous atoms, originating from the iron oxide and phosphate.

The time-dependent evolution of the corrosion process is shown in Figure S4. The SEM images show that the corrosion process and the formation of the insoluble corrosion layer takes place soon after the substrate is immersed in PBS electrolyte (i.e. 3 min; Figure S4a). Further growth of the mixed oxide/phosphate corrosion crust occurs continuously with the formation of cracks that are present at its surface once a critical thickness is reached (Figure S4e andf). This indicates an extremely fast corrosion kinetics, expected for such a corrosion-prone, non-noble metal-based surface. The proposed mechanism for the corrosion of a pure iron surface in PBS buffer is shown in Scheme S1.

In order to optimize the voltage that needs to be applied in a Fe-Pt two-electrode system for successful electrochemical corrosion prevention, a series of chronoamperometric experiments were performed at different applied voltages. Figure 1 depicts the SEM images of the samples subjected to voltages from -1.6 V to -2.3 V (vs. Pt reference) during 3h. SEM observation shows that voltages more negative than -2.2V are required for complete corrosion protection. The presence of microscopic grooves and surface imperfections, that can be observed on the highmagnification SEM images (e.g. Fig. 1b and1f)), originate from sharp SiC crystals present in the polishing paper and are not related to the corrosion process itself. EIS measurements were used for the further evaluation of the electrochemical corrosion protection concept. The obtained Nyquist plots (Figure S5) clearly show a significant increase in the charge-transfer resistance in the case of the control sample (no voltage applied; 4 h in PBS buffer) compared to the sample that was exposed to -2.3 V for 4 h. A small difference in the semicircle radius was observed between the pristine metal and a sample that was kept at -2.3 V. This can be ascribed to the presence of a thin iron-oxide layer that was spontaneously formed in the case of the pristine Fe rod in comparison with the sample that was used immediately after the cleaning step for the corrosion protection and EIS experiments.

Biofuel cell engineering and characterization

Enzymatic bioelectrodes were prepared by modifying the selected (1.5 cm x 1.5 cm) area of gold-coated glass slides with the specially formulated enzymatic hydrogels consisting of cathodic bilirubin oxidase from Magnaporthe Oryzae 37 , anodic glucose oxidase from Aspergillus Niger, a PEGDGE cross-linker and custom-designed cathodic and anodic Os-based redox polymers. They serve as redox mediators for an efficient wiring of the redox centers of the enzymes to the electrode surface, regardless of the orientation of the enzyme molecules within the hydrogel matrix. [START_REF] Ruff | Redox Polymers in Bioelectrochemistry : Common Playgrounds and Novel Concepts[END_REF]44 The power generated by a BFC is often limited by the cathodic oxygen reduction reaction [START_REF] Cadet | Design of a Highly Efficient O2 Cathode Based on Bilirubin Oxidase from Magnaporthe Oryzae[END_REF] due to its low solubility 3 (i.e. 0.2 mM in water equilibrated with air at 37 °C) [START_REF] Heller | Miniature Biofuel Cells[END_REF] and modest diffusion coefficients [START_REF] Barton | Enzymatic Biofuel Cells for Implantable and Microscale Devices[END_REF] in aqueous media. To improve the power output of the biofuel cell by minimizing the above-mentioned cathodic limitations, MWCNT were incorporated into the cathodic hydrogel. The optimization of the ratio between BOD/Os-polymer/PEGDGE hydrogel and MWCNT-COOH was performed by cyclic voltammetry on glassy-carbon rotating disc electrodes in oxygen saturated PBS. The diffusion limited currents obtained at 37.5 °C and 500 rpm (Figure S6) show that the optimal ratio between BOD/Os-polymer/PEGDGE hydrogel and MWCNT-COOH is 60:40. Moreover, the low value of the measured zeta potential for MWCNT-COOH (ξ: -62.48 ± 2.92 mV; n: 5 measurements; 95% level of confidence) indicates the possibility for the formation of an electrostatic complex between the positively charged Os redox polymer molecules and the negatively charged deprotonated MWCNT-COOH. A similar mechanism of electrostatic interaction between the positively charged Os redox polymer and negatively charged enzyme molecules at physiological pH has been discussed in the literature. [START_REF] Heller | Miniature Biofuel Cells[END_REF][START_REF] Katakis | Electrostatic Control of the Electron-Transfer Enabling Binding of Recombinant Glucose Oxidase and Redox Polyelectrolytes[END_REF] As a result, a better interaction is obtained between the MWCNT-COOH and the enzyme-containing hydrogel resulting in a better electron-transport throughout the volume of the composite hydrogel. In addition, the higher conductivity of MWCNT-COOH, along with a beneficial increase in active surface area allows to overcome the current limitations of the cathode.

The biofuel cell was constructed by placing the two electrodes in a parallel configuration relative to each other with a 7 mm thick PDMS spacer in between them (Scheme S2). Oxygen was supplied to the confined volume between the two electrodes through a pipette tip that was placed under the opening at the bottom of the biofuel cell. In that way, it ensured constant convection and delivery of the fresh amounts of glucose and oxygen.

Both the anode and cathode were first characterized separately under argon at a scan rate of 5 mV s -1 (Figure 2). In the absence of substrates only two redox peaks (Figure 2a) corresponding to the reversible electrochemical oxidation/reduction of osmium redox centers can be observed at a formal potential of +0.30 V and -0.03 V, for the cathodic and anodic polymers, respectively.

After addition of 50 mM glucose and O 2 , characteristic sigmoidal voltammograms were obtained, corresponding to the reduction of oxygen at the cathode [START_REF] Cadet | Design of a Highly Efficient O2 Cathode Based on Bilirubin Oxidase from Magnaporthe Oryzae[END_REF] (black curve, Figure 2b)

and glucose oxidation at the anode (blue curve, Figure 2b). To extend the lifetime of the anode and the durability of the biofuel cell, 20 nM catalase was added to the PBS buffer to decompose hydrogen peroxide, a detrimental side product that may be formed at the anode when using osmium based redox polymers with redox potentials lower than + 0.07 V vs. Ag/AgCl (3M NaCl). [START_REF] Prévoteau | Oxygen Reduction on Redox Mediators May Affect Glucose Biosensors Based on "Wired" Enzymes[END_REF] Polarization and power curves of the biofuel cell are present in the Figure 2c. The electrode potential was cycled between the open circuit voltage (OCV) (i.e. +0.75 V) and 0 V and the current was measured. The obtained results were used to plot the power curve that is the most important feature of biofuel cells. The maximum power was 500 μW at 0.3 V with a short-circuit current value of 2 mA. The obtained power was limited by the cathodic reaction, but it might be further improved by implementing gas-diffusion electrodes in order to overcome the relatively low oxygen solubility and mass transport constraints on the cathodic side. [START_REF] Szczesny | A Gas Breathing Hydrogen/Air Biofuel Cell Comprising a Redox Polymer/Hydrogenase-Based Bioanode[END_REF][START_REF] Marković | An Air-Breathing Carbon Cloth-Based Screen-Printed Electrode for Applications in Enzymatic Biofuel Cells[END_REF]

Bioelectronic interface for corrosion protection

The results of the optimization of the voltage needed between the iron rod serving as cathode and the coiled platinum anode show that a minimum of 2.2 V is necessary for the successful corrosion protection in PBS buffer. This voltage cannot be delivered by a single glucose-oxygen BFC which usually only can provide a maximum of 0.3 V under the present operating conditions. Therefore, a low-power boost converter (BC) was used to step-up the BFC voltage to the required value of 2.3 V that was supplied to the corrosion cell. The schematic set-up used in this study is shown in Figure 3. provide a stable voltage output of 2.3 V for 2 h until the voltage dropped to 2.2 V, a threshold value for successful corrosion protection (Figure 1). While the voltage was kept at a constant value, the output power of the BFC and BC was constantly declining. This can be ascribed to a gradual deactivation of the enzymes and the concomitant loss in the bioelectrocatalytic current.

The power output of the BC is around 50% lower than the power generated by the BFC because part of the power is used to ensure its own functioning. The results of the cathodic corrosion protection experiment (Figure 5) show that the surface of the sample subjected to 2.3 V for 2 h in PBS buffer remained clean (Figure 5a) and free of any corrosion products which can be observed for the control sample that was left to corrode spontaneously in the electrolyte. This clearly demonstrates the possibility to use the very low voltage of such an electrochemical device for successful corrosion protection.

Although most of the current methods for corrosion protection are focusing on the development of new corrosion-resistant materials [START_REF] Farmer | Iron-Based Amorphous Metals: High-Performance Corrosion-Resistant Material Development[END_REF][START_REF] Shi | Corrosion-Resistant High-Entropy Alloys: A Review[END_REF] or organic/inorganic coatings, [START_REF] Montemor | Functional and Smart Coatings for Corrosion Protection: A Review of Recent Advances[END_REF] the implementation of bioelectrochemical systems to perform such a demanding task, even for a limited amount of time, is a novel approach that has not been demonstrated earlier. It is worth mentioning that pure iron is the most corrosion prone of all iron-based materials/alloys, which sets up a very demanding task for the BFC-BC system. Nevertheless, a successful corrosion protection is achieved and maintained while the BFC delivers its maximum power output.

The concept presented in this work can be further extended not only to power electronic devices with relatively low power/voltage requirements and the design of various self-powered electrochemical biosensors, but also to electrochemical systems that will for example synthesize in situ physiologically active molecules.

Conclusion

In this proof-of-concept study, a new and unconventional way of electrochemical corrosion protection is demonstrated with a model system consisting of a pure iron rod in a chloride rich electrolyte (i.e. PBS buffer). The described strategy is based on using a low-voltage (i.e. 0.3 19 V)/power (500 μW) bioelectrochemical device such as an enzymatic glucose/oxygen biofuel cell, modulating its output voltage to higher values via a well-adapted electronic circuit (low power boost converter) in order to obtain a final voltage (min. 2.2 V) that fulfills the requirements for successful corrosion protection. A voltage of 2.3 V completely prevents the corrosion process at the surface of the iron rod, as observed by SEM imaging. This clearly validates the present concept as well as the versatility of systems where biofuel cells are efficiently integrated into more complex bioelectronics devices. The Supporting Information is available free of charge on the ACS Publications website at DOI:

Figure 1 .

 1 Figure 1. SEM images of iron samples that were exposed to different voltages: a) -1.6 V; b) -1.7 V; c) -

Figure 2 .

 2 Figure 2. Cyclic voltammograms of BOD/Os-polymer/MWCNT-COOH/PEGDGE modified Au cathode (black curve) and GOx/Os-polymer/PEGDGE modified anode (blue curve) recorded at 5 mV s -1 , 37.5 °C

Figure 3 . 4 .

 34 Figure 3. Schematic illustration of the bioelectronic-electrochemical system used in the corrosion

Figure 4 .

 4 Figure 4. The BFC (black curve) and the BC (red curve) output voltage a) and power b) curves recorded

Figure 5 .

 5 Figure 5. The SEM images of the unprotected control (a-c) and the sample that was connected to the

  ASSOCIATED CONTENT Supporting information: Corrosion mechanism, Construction of the BFC, Circuit diagram, Potentiodynamic polarization experiment, EDS and SEM analysis, Corrosion evolution study, Electrochemical impedance spectroscopy, Optimization of the CNT-enzymatic hydrogel composite.

Funding Sources -Laboratory of Excellence AMADEus (ANR-10-LABX-0042-AMADEUS) -Agence Nationale de la Recherche" under the program "Initiative for Excellence IdEx Bordeaux" (ANR-10-IDEX-0003-02). ACKNOWLEDGMENT This work, realized within the framework of the Laboratory of Excellence AMADEus with the reference ANR-10-LABX-0042-AMADEUS, has benefitted from aid by the state operated "Agence Nationale de la Recherche" under the program "Initiative for Excellence IdEx Bordeaux" holding the reference ANR-10-IDEX-0003-02. The authors thank Isabelle Ly for performing the EDS experiments.

AUTHOR INFORMATION

Corresponding Author

* E-mail: nicolas.mano@crpp.cnrs.fr * E-mail: kuhn@enscbp.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


Nicolas Baghdadi

Mohammad El Hajj

Mehrez Zribi

Soil moisture retrieval algorithm using Sentinel-1 images

Keywords: Soil moisture, Sentinel-1&2, SAR, C-band, Neural networks

The aim of this communication is to present an operational approach for mapping soil moisture at high spatial resolution (plot scale) in agriculture areas by coupling S1 and S2 images. The proposed approach is based on the inversion of the Water Cloud Model (WCM) combined with the modified Integral Equation Model (IEM). Neural networks were developed and validated using synthetic SAR C-band database. The results showed that the soil moisture could be estimated in agricultural areas with an accuracy of approximately 5 vol.%.

INTRODUCTION

Soil moisture content and surface roughness play an important role in meteorology, hydrology, agronomy, agriculture, and risk assessment. These soil surface characteristics can be estimated using synthetic aperture radar (SAR) [START_REF] Baghdadi | Potential of Sentinel-1 images for estimating the soil roughness over bare agricultural soils[END_REF][START_REF] Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF][START_REF] Baghdadi | Coupling SAR C-band and optical data for soil moisture and leaf area index retrieval over irrigated grasslands[END_REF][START_REF] Hajj | Soil moisture retrieval over irrigated grassland using X-band SAR data[END_REF][START_REF] Baghdadi | Toward an operational bare soil moisture mapping using TerraSAR-X data acquired over agricultural areas[END_REF][START_REF] Baghdadi | Sensitivity of polarimetric parameters of multifrequency polarimetric SAR data to soil moisture and surface roughness over bare agricultural soils[END_REF][START_REF] Dong | Retrieving surface soil moisture using radar imagery in a semi-arid environment[END_REF][START_REF] Baghdadi | Estimation of soil parameters over bare agriculture areas from C-band polarimetric SAR data using neural networks[END_REF][START_REF] Baghdadi | Use of TerraSAR-X data to retrieve soil moisture over bare soil agricultural fields[END_REF][START_REF] Baghdadi | Estimating surface soil moisture from TerraSAR-X data over two small catchments in the sahelian part of western Niger[END_REF][START_REF] Baghdadi | Analysis of TerraSAR-X data sensitivity to bare soil moisture, roughness, composition and soil crust[END_REF][START_REF] Baghdadi | Analysis of TerraSAR-X data and their sensitivity to soil surface parameters over bare agricultural fields[END_REF][START_REF] Baghdadi | Operational performance of current synthetic aperture radar sensors in mapping soil surface characteristics: application to hydrological and erosion modelling[END_REF][START_REF] Baghdadi | Operational mapping of soil moisture using synthetic aperture radar data: application to Touch basin (France)[END_REF][START_REF] Baghdadi | Soil moisture estimation using multi-incidence and multi-polarization ASAR SAR data[END_REF][START_REF] Holah | Potential of ASAR/ENVISAT for the characterisation of soil surface parameters over bare agricultural fields[END_REF][START_REF] Baghdadi | Potential of ERS and RADARSAT data for surface roughness monitoring over bare agricultural fields : application to catchments in Northern France[END_REF]. SAR data in the C-and X-bands were widely and primarily used to estimate surface soil moisture (SSM) over bare soils and soils covered with vegetation. The arrival of free and open access Sentinel-1 "S1" (10 m spatial resolution and a 6-day revisit period) and Sentinel-2 "S2" (10 m spatial resolution and a 6-day revisit period) satellites have encouraged the development of operational algorithms for soil moisture mapping over agricultural areas with high revisit time (6 days with the two Sentinel-1 satellites) and high spatial resolution (up to plot scale).

The retrieval of soil moisture content and surface roughness requires the use of radar backscatter models capable of correctly modeling the radar signal for a wide range of soil parameter values. This estimation from imaging radar data implies the use of backscattering electromagnetic models, which can be physical, semi-empirical or empirical [START_REF] Baghdadi | Calibration of the Integral Equation Model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | An empirical calibration of IEM model based on SAR data and measurements of soil moisture and surface roughness over bare soils[END_REF][START_REF] Baghdadi | A new empirical model for radar scattering from bare soil surfaces[END_REF].

Several studies tend to use the neural networks technique to invert backscattering models and estimate the soil moisture. To estimate soil moisture and roughness over bare soils from the C-band polarimetric radar data, Baghdadi et al. [START_REF] Baghdadi | Estimation of soil parameters over bare agriculture areas from C-band polarimetric SAR data using neural networks[END_REF] first generated a database of backscattering coefficients for a wide range of bare soil conditions (Hrms between 0.3 and 3.6 cm and SSM between 5 and 45 vol.%) using the Integral Equation Model IEM [START_REF] Baghdadi | Calibration of the Integral Equation Model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | An empirical calibration of IEM model based on SAR data and measurements of soil moisture and surface roughness over bare soils[END_REF]. Then, the neural networks (NNs) were trained using the synthetic database. Finally, the trained neural networks were validated using a real database.

The aim of this paper is to present an operational approach for mapping soil moisture at high spatial resolution (up to the plot scale) in agriculture areas by coupling S1 and S2 images. The proposed approach uses the neural network technique [START_REF] Baghdadi | Estimation of soil parameters over bare agriculture areas from C-band polarimetric SAR data using neural networks[END_REF][START_REF] Baghdadi | Retrieving surface roughness and soil moisture from SAR data using neural network[END_REF]. It is based on the inversion of the WCM combined with the modified Integral Equation Model (IEM) [START_REF] Baghdadi | Calibration of the Integral Equation Model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | An empirical calibration of IEM model based on SAR data and measurements of soil moisture and surface roughness over bare soils[END_REF].

S1-SSM ALGORITHM

The approach for soil moisture mapping uses the neural networks (NNs) technique to invert the radar backscattering signal [START_REF] Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF]. It uses the Water Cloud Model parameterized by Baghdadi et al. [START_REF] Baghdadi | Calibration of the Water Cloud Model at C-Band for Winter Crop Fields and Grasslands[END_REF] for the C-band combined with the Integral Equation model (IEM) [START_REF] Baghdadi | Calibration of the Integral Equation Model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | Semi empirical Calibration of the Integral Equation Model for SAR Data in C-Band and Cross Polarization Using Radar Images and Field Measurements[END_REF]. The total radar backscattering coefficient in the WCM is considered as the sum of the direct vegetation contribution and the soil contribution multiplied by the attenuation factor. Using a vegetation descriptor (NDVI) derived from optical images (Sentinel-2), the direct contribution of vegetation as well as the attenuation of soil scattering can be computed.

The development of the inversion method consists of the following steps [START_REF] Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF]:  Generate a synthetic database of backscattering coefficients in VV polarization (σ°VV) using a parameterized WCM combined with the modified IEM;  Noise the synthetic SAR C-band database (zero-mean Gaussian random additive noises with a standard deviation of ±0.7 and ±1.0 dB to be added to the simulated backscattering coefficients in VV, respectively) and NDVI values (zero-mean Gaussian additive relative noise of 15%) to obtain synthetic database close to the real SAR and NDVI data;  Train and validate the neural networks using the noisy synthetic dataset;  Finally, apply the trained neural networks to S1 and S2 images to map the soil moisture.

Wide range of incidence angle "θ" (20° to 45°), SSM (2 to 40 vol. %), Hrms (0.5 to 3.8 cm), and NDVI (0 to 0.75) was considered in the generation of synthetic SAR database. Each element of the database is composed of a combination of four input parameters θ, SSM, Hrms, NDVI and a simulated σ°VV. Two SAR configurations for estimating the soil moisture are considered:  Configuration 1: incidence angle, noisy radar signal at VV polarization, and noisy NDVI are the inputs of the NNs;  Configuration 2: incidence angle, both noisy radar signal at VV polarization and noisy NDVI are the inputs of the NNs.

The soil roughness (root mean square surface height "Hrms") was not used as an input parameter for the neural networks training and validation. NNs were trained in using the Levenberg-Marquardt algorithm with two hidden layers. The number of neurons for each hidden layer is 20. Linear and tangent-sigmoid transfer functions were associated with the first and second hidden layers, respectively.

Figures 1a and1b show the performance of the neural networks that take °V V , θ, and NDVI as input parameters (configuration 1). For a reference SSM lower than 25 vol.% (dry to wet soils), small bias "estimated SSMreference SSM" (1.2 vol.%) and RMSE of 4.0 vol.% are observed (Figure 1a). Similarly, for a reference SSM between 25 and 40 vol.% (very wet conditions), a bias of -2.3 vol.% and an RMSE of 5.1 vol.% are obtained (Figure 1b). The approach for the operational mapping of soil moisture was applied over a study site located in Occitanie, South France (Figure 2). S1-SSM maps are available at high spatial resolution (up to plot scale) since September 2016 as open access data via the Theia French Land Data Center (http://www.theia-land.fr/en/thematic-products). An evaluation of SMOS, SMAP, ASCAT and S1-SSM products was performed by El Hajj et al. [START_REF] El Hajj | Evaluation of SMOS, SMAP, ASCAT and Sentinel-1 Soil Moisture Products at Sites in Southwestern France[END_REF] and reveals that the S1-derived soil moisture maps provide the most accurate estimation of SSM. The higher accuracy of estimated SSM moisture is probably due to the well-calibrated IEM combined with the well parameterized WCM and the use of high spatial resolution (10 m × 10 m) land cover maps derived from S2 images to eliminate SAR scattering from forest and urban areas.

A comparison between S1-SSM maps and precipitation derived from GPM (Global Precipitation Measurement) data was carried out. For each S1-SSM map date, one cumulative rainfall map for the 6 days prior to the S1-SSM map date was computed by summing the 30 min time interval of the Late IMERG GPM product. Then, each S1-derived SSM map was overlaid with the corresponding 6 days of cumulative GPM rainfall. Starting with the initial map on 21/12/2016 (Figure 2a), departments as Tarn, Hérault, Lozère, and Aveyron of the eastern and middle parts of the region encountered high soil moisture values (more than 20 vol.%) corresponding to 10-20 mm cumulative precipitation during the past 6 days, whereas Gers and Haute-Garonne of the western part shows low soil moisture values (20 vol.% at most) corresponding to the absence of precipitation in the area for the same prior period. A map derived 6 days later on 27/12/2016 (Figure 2b) shows a decrease in soil moisture values in the Hérault and Gard due to the absence of precipitation between 21/12/2016 and 27/12/2016. Moreover, high soil moisture values are estimated for Tarn due to continuous raining episodes between 21/12/2017 and 27/12/2017, and a slight increase in soil moisture values is observed in western part due to 3 mm of precipitation recorded one day prior to the S1-SSM map according to the GPM data (Figure 2b). A general decrease in soil moisture values throughout the region is observed between 27/12/2016 (Figure 2b) and 02/01/2017 (Figure 2c) as no precipitation was recorded. A continuous lack of precipitation for 12 days from 27/12/2016 until 08/01/2017 caused soil moisture values to drop to less than 15 vol.% on 08/01/2017 in the whole region as shown in Figure 2d. Six days later on 14/01/2017 (Figure 2e), soil moisture values remained low at the east as no precipitation was recorded for an additional 6 days, whereas a moderate increase in soil moisture values is observed in the middle and western parts (Tarn, Haute-Garonne, and Gers) corresponding to precipitation between 08/01/2017 and 14/01/2017 (a cumulative rainfall increase of 5-20 mm). Twenty-four days with no precipitation recorded in the Hérault and Gard region between 27/12/2016 and 20/01/2017 caused the soil moisture values to drop more, with values less than 15 vol.% in this area on 20/01/2017 (Figure 2f).

In addition, for Gers department, low soil moisture values (less than 15 vol.%) were estimated on January 20, 2017 and high cumulative precipitation was recorded between 14 and 20 January 2017 (15 mm) (Figure 2f). This low soil moisture estimates could be linked to the presence of frozen soil in the area. In a recent study using Sentinel-1 SAR data, Baghdadi et al. [START_REF] Baghdadi | Detection of Frozen Soil Using Sentinel-1 SAR Data[END_REF] showed that a decrease of at least 3 dB in the radar backscattered signal can be observed over frozen soil conditions. Such a decrease in the SAR signal yields an underestimation of soil moisture in the S1-derived SSM maps. In our case, it seems that low soil moisture values for this area on 20/01/2017, although with the presence of rainfall, is due to frozen soil conditions. To support our assumption, the temperature curve and precipitation records for a local meteorological station in Auch city located in Gers-Occitanie were analyzed. On 19 January 2017 (S1 acquisition over Gers on January 19, 2017 17:56 UTC), the temperature varied between -8°C and 2°C with a mean value of -3°C throughout the day. Three days before (15 and 16 January) 15 mm cumulative precipitation was recorded. Thus, a precipitation event followed by a decrease in the air temperature to less than 0°C justifies the presence of frozen soil conditions as the cause of a decrease in the estimated soil moisture values in the S1-derived SSM maps.

After 29 days of dry conditions in the eastern part (Hérault and Gard between 27/12/2016 and 25/01/2017), a sudden increase in the soil moisture was observed on 26/01/2017 with the beginning of a rainfall event (Figure 2g). The mean S1-SSM mean value in this area increased from 13 vol.% in 20/01/2017 to 32 vol.% in 26/01/2017. On the other hand, the absence of precipitation in the remaining parts of the region between 20/01/2017 and 26/01/2017 caused soil moisture to attain lower values (16 vol.%) on 26/01/2017.The slight increase in soil moisture values in Gers between 20 and 26 January is probably due to the disappearance of the frozen conditions observed in the previous acquisition (20/01/2017). The temporal analysis of S1-derived SSM values over a period of time shows the direct effect of raining episodes or dry conditions on S1-SSM values.

CONCLUSION

This paper present an operational approach for soil moisture estimates in agricultural areas at a high spatial resolution (up to plot scale). Our approach is based on a synergic use of radar and optical images. With the use of Sentinel-1 and Sentinel-2 data, the developed approach will provide soil moisture mapping with a high temporal frequency. Our approach for soil moisture estimates used the neural networks technique to invert the radar signal and estimate the soil moisture.

From the synthetic database, the results showed that the soil moisture in agricultural areas could be estimated with an accuracy of approximately 5 vol.%. A good coherence was observed between the temporal evolution of the soil moisture and the precipitation records derived from the GPM data. The S1-SSM values increases following rainfall event and decreases after a period without rainfall due to evaporation.

Figure 1 .

 1 Figure 1. Estimated SSM as a function of the reference SSM. (a) VV is used alone, dry to wet soils, (b) VV is used alone, very wet soils.

Figure 2 .

 2 Figure 2. S1-derived SSM map overlaid with GPM cumulative precipitation data. (a) 21/12/2016, (b) 27/12/2016, (c) 02/01/2017, (d) 08/01/2017, (e) 14/01)2017, (f) 20/01/2017, (g) 26/01/2017.

ACKNOWLEDGMENT

This research was supported by IRSTEA (National Research Institute of Science and Technology for Environment and Agriculture) and the French Space Study Center (CNES, DAR 2019 TOSCA). The authors wish to thank the European Commission and the European Space Agency for providing the S1 images. We used Copernicus level 2A S2 data processed by Theia (https://www.theia-land.fr).