Karen E Assmann
email: k.assmann@eren.smbh.univ-paris13.fr

Indunil Ruhunuhewa
email: indunil_ruhunuhewa@yahoo.com

Moufidath Adjibade
email: m.adjibade@eren.smbh.univ-paris13.fr

Zhen Li

Raphaëlle Varraso
email: raphaelle.varraso@inserm.fr

Serge Hercberg
email: s.hercberg@eren.smbh.univ-paris13.fr

Pilar Galan
email: p.galan@uren.smbh.univ-paris13.fr

Emmanuelle Kesse-Guyot

The mediating role of overweight and obesity in the prospective association between overall Dietary Quality and Healthy Aging

Keywords: aging, diet, nutrition, body mass index (BMI), overweight obesity, prevention

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

Introduction

The burden of age-related health decline and the dependence of the elderly population result in major societal implications [1]. To address these challenges, various public health promotion interventions for the aging population have been implemented [START_REF] Heller | Interventions to promote health: Crossing networks of intellectual and developmental disabilities and aging[END_REF]. Moreover, in geriatric research, multidimensional concepts of "successful aging" or "healthy aging" (HA) have emerged that have made it possible to consider different chronic diseases, as well as physical, cognitive, mental, and social health during aging in a combined, holistic manner [START_REF] Martinson | Successful aging and its discontents: A Systematic Review of the Social Gerontology Literature[END_REF]. The most well-known multidimensional concept of "successful aging" or HA is the framework proposed by Rowe and Kahn [START_REF] Rowe | Successful aging[END_REF][START_REF] Rowe | Successful Aging 2.0: Conceptual Expansions for the 21st Century[END_REF], which proposes three main dimensions of HA: avoiding disease and disability, maintaining high levels of physical and cognitive functioning, and having an active engagement with life [START_REF] Rowe | Successful aging[END_REF]. The work of Rowe and Kahn notably underlines that there is a large heterogeneity in aging trajectories, and that premature overall health-decline is not inevitable, but is related to a diversity of parameters that include modifiable environmental factors [START_REF] Rowe | Successful aging[END_REF]. In terms of the specific period during which such factors may have a particularly important role, midlife appears to be of particular interest. This period is considered a crucial time-window, since previous research has highlighted that age-related diseases and disabilities appear to be strongly related to risk factors present at midlife [START_REF] Franzon | Modifiable midlife risk factors, independent aging, and survival in older men: Report on long-term follow-up of the Uppsala Longitudinal Study of Adult Men cohort[END_REF][START_REF] Reed | Predictors of health aging in men with high life expectancies[END_REF][START_REF] Willcox | Midlife risk factors and healthy survival in men[END_REF][START_REF] Singh-Manoux | Obesity phenotpes in midlife and cognition in early old age: The Whitehall II cohort study[END_REF].

Several specific modifiable environmental factors have been proposed as determinants of overall health during aging, including physical activity [START_REF] Daskalopoulou | Physical activity and healthy ageing: A systematic review and meta-analysis of longitudinal cohort studies[END_REF], avoiding smoking [START_REF] Sabia | Influence of individual and combined healthy behaviours on successful aging[END_REF], moderate alcohol consumption [START_REF] Sabia | Influence of individual and combined healthy behaviours on successful aging[END_REF], and air pollution [START_REF] Cohen | Air pollution and successful aging: Recent evidence and new perspectives[END_REF]. In addition, nutrition has been suggested to play a fundamental role in modulating healthy life expectancy [START_REF] Jong | Nutrition and healthy ageing: The key ingredients[END_REF]. Specifically, multiple observational studies have observed that individuals with a high overall dietary quality are more likely to age healthily [START_REF] Akbaraly | Does overall diet in midlife predict future aging phenotypes? A cohort study[END_REF][START_REF] Assmann | Dietary scores at midlife and healthy ageing in a French prospective cohort[END_REF][START_REF] Assmann | A healthy dietary pattern at midlife, combined with a regulated energy intake, is related to increased odds for healthy aging[END_REF][START_REF] Gopinath | Adherence to dietary guidelines and successful aging over 10 years[END_REF][START_REF] Hodge | Dietary patterns as predictors of successful ageing[END_REF][START_REF] Samieri | The association between dietary patterns at midlife and health in aging: An observational study[END_REF][START_REF] Assmann | Association between adherence to the mediterranean diet at midlife and healthy aging in a cohort of french adults[END_REF].

In addition, much attention of both epidemiological [START_REF] Franzon | Modifiable midlife risk factors, independent aging, and survival in older men: Report on long-term follow-up of the Uppsala Longitudinal Study of Adult Men cohort[END_REF][START_REF] Reed | Predictors of health aging in men with high life expectancies[END_REF][START_REF] Willcox | Midlife risk factors and healthy survival in men[END_REF][START_REF] Bell | Late-life factors associated with healthy aging in older men[END_REF][START_REF] Singh-Manoux | Association of body mass index and waist circumference with successful aging[END_REF][START_REF] Tyrovolas | Skeletal muscle mass and body fat in relation to successful ageing of older adults: The multi-national MEDIS study[END_REF][START_REF] Yates | Exceptional longevity in men: Modifiable factors associated with survival and function to age 90 years[END_REF] and mechanistic [START_REF] Frasca | Aging, obesity, and inflammatory age-related diseases[END_REF] research has been paid to the role of overweight and obesity for age-related chronic diseases and age-related functional decline. A main biological mechanism that appears to underlie the detrimental role of excess body fat, and especially central obesity, is adipose tissue inflammation. This process is characterized by an increased secretion of pro-inflammatory cytokines by adipocytes and the infiltration of immune cells (including macrophages and T-cells) into the adipose tissue. This adipose tissue inflammation appears to mediate insulin resistance [START_REF] Kohlgruber | Adipose tissue inflammation in the pathogenesis of type 2 diabetes[END_REF], and has also been suggested to be involved in the etiology of cardiovascular disease [START_REF] Berg | Adipose tissue, inflammation, and cardiovascular disease[END_REF], depression [START_REF] Shelton | Inflammation in depression: Is adiposity a cause? Dialogues[END_REF] and cognitive decline [START_REF] Nguyen | Obesity and cognitive decline: Role of inflammation and vascular changes[END_REF][START_REF] Dye | The relationship between obesity and cognitive health and decline[END_REF]. In line with this, various observational studies have revealed a detrimental role of an elevated body mass index (BMI) or an increased body fat percentage with respect to multidimensional concepts of HA [START_REF] Franzon | Modifiable midlife risk factors, independent aging, and survival in older men: Report on long-term follow-up of the Uppsala Longitudinal Study of Adult Men cohort[END_REF][START_REF] Reed | Predictors of health aging in men with high life expectancies[END_REF][START_REF] Willcox | Midlife risk factors and healthy survival in men[END_REF][START_REF] Bell | Late-life factors associated with healthy aging in older men[END_REF][START_REF] Singh-Manoux | Association of body mass index and waist circumference with successful aging[END_REF][START_REF] Tyrovolas | Skeletal muscle mass and body fat in relation to successful ageing of older adults: The multi-national MEDIS study[END_REF][START_REF] Yates | Exceptional longevity in men: Modifiable factors associated with survival and function to age 90 years[END_REF][START_REF] Ruhunuhewa | Prospective association between body mass index at midlife and healthy aging among French adults[END_REF], including a previous article based on data from the French 'Supplementation with Vitamins and Mineral Antioxidants' (SU.VI.MAX-'SUpplémentation en VItamines et Minéraux AntioXydants') study [START_REF] Ruhunuhewa | Prospective association between body mass index at midlife and healthy aging among French adults[END_REF].

Obesity is a condition with multiple presumed determinants, including genetics, physical activity and sedentary time, unfavorable sleeping patterns, as well as diet [START_REF] Hruby | Determinants and consequences of obesity[END_REF]. Diet and physical activity/sedentary time are particularly important factors, since they are directly related to a disturbed energy balance, and thus a storage of excess energy as fat within the adipose tissue [START_REF] Hill | Energy Balance and Obesity[END_REF]. Multiple studies indicate that it is not only overall energy intake but also the quality of dietary patterns that is related to weight gain and obesity [START_REF] Millen | The 2015 dietary guidelines advisory committee scientific report: development and major conclusions[END_REF]. Given the importance of diet for avoiding overweight and obesity, and the key role of obesity as a determinant of health during aging [START_REF] Franzon | Modifiable midlife risk factors, independent aging, and survival in older men: Report on long-term follow-up of the Uppsala Longitudinal Study of Adult Men cohort[END_REF][START_REF] Reed | Predictors of health aging in men with high life expectancies[END_REF][START_REF] Willcox | Midlife risk factors and healthy survival in men[END_REF][START_REF] Bell | Late-life factors associated with healthy aging in older men[END_REF][START_REF] Singh-Manoux | Association of body mass index and waist circumference with successful aging[END_REF][START_REF] Tyrovolas | Skeletal muscle mass and body fat in relation to successful ageing of older adults: The multi-national MEDIS study[END_REF][START_REF] Yates | Exceptional longevity in men: Modifiable factors associated with survival and function to age 90 years[END_REF], it is of interest to better characterize how the relationship between diet and overall HA is mediated by indicators related to body fatness.

Of note, the relationship between dietary quality and health is often modeled by adjusting for (amongst other things) BMI or BMI category. This approach makes it possible to isolate the "direct" role of diet for health outcomes, but it is questionable whether this is generally desirable or whether adjustment for mediating factors should be considered as overadjustment [START_REF] Schisterman | Overadjustment bias and unnecessary adjustment in epidemiologic studies[END_REF]. Most importantly, simply adjusting for BMI-related indicators does not make it possible to obtain specific insight into the direct and indirect association between diet and health outcomes. In the context of a potential mediation by BMI status, the "direct" association can be characterized as the part of the relationship that is mediated by mechanisms that are independent of overweight and obesity. On the other hand, the "indirect" association can be conceptualized as the part of the relationship that is mediated by mechanisms related to excess body fat.

To the best of our knowledge, no previous study has specifically focused on the mediating role of BMI status in the relationship between overall dietary quality and multidimensional healthy aging. Thus, our objective was to quantify the extent to which the association between adherence to nutritional recommendations at midlife and later healthy aging is mediated through BMI status, while distinguishing 3 categories: (a) underweight/normal weight (BMI < 25 kg/m 2 ), (b) overweight (BMI ≥ 25 and <30 kg/m 2 ), and (c) obesity (BMI ≥ 30 kg/m 2 ). Adherence to the French official nutritional guidelines for the general population was measured by the Programme National Nutrition Santé-Guidelines Score (PNNS-GS).

Materials and Methods

Study Population

The randomized, double-blinded placebo-controlled trial 'Supplementation with Vitamins and Mineral Antioxidants' (SU.VI.MAX-'SUpplémentation en VItamines et Minéraux AntioXydants') was conducted in 1994-2002 to test the effect of a daily supplementation with antioxidant vitamins and minerals at nutritional doses on the incidence of cancer, cardiovascular disease and overall mortality [START_REF] Hercberg | MAX Study: A randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF]. This trial included 12,741 men and women. Next, a post-supplementation observational study 'SU.VI.MAX 2' was conducted in 2007-2009 and 6850 subjects were recruited on a voluntary basis.

The SU.VI.MAX and SU.VI.MAX 2 studies were conducted according to the Declaration of Helsinki, and were approved by the ethics committee for studies with human subjects of Paris-Cochin Hospital (CCPPRB number 706 and number 2364, respectively) and the "Commission Nationale de l'Informatique et des Libertés" (CNIL number 334641 and number 907094, respectively). All participants provided written informed consent. The SU.VI.MAX trial was registered at www.clinicaltrials.gov/ct2/show/NCT00272428.

The eligibility criteria for the present analysis were: being a participant of the SU.VI.MAX 2 study with available and accurate (at least three 24 h dietary records) information on adherence to the French dietary guidelines (N = 4048), an age of 45-60 years at recruitment (1994)(1995), the absence of chronic diseases (cancer, cardiovascular disease or diabetes) at recruitment in 1994-1995 (N = 3083), and the absence of missing values for baseline BMI and for variables necessary to determine HA status in 2007-2009 (N = 2351). Individuals with missing data for baseline covariables-such as sociodemographic factors (gender, age, educational level, occupation, and family situation), smoking status and number of 24 h records-were also excluded, leaving a final sample of 2249 individuals (Figure 1).

Data Collection

Timeline of Data Collection

All participants included in our analyses were aged 45-60 years at inclusion in the initial SU.VI.MAX study (1994)(1995). Moreover, they were free of cancer, cardiovascular disease and diabetes at recruitment (1994)(1995). The dietary data used for the current analyses were collected in 1994-1996, and the anthropometric data used were collected during a clinical examination in 1995-1996. Participants were followed for a median of 13 y, until the SU.VI.MAX 2 observational follow-up point in 2007-2009.

Of note, from inclusion (1994)(1995) until 2002, participants either received a placebo or a daily dose of antioxidant nutrients for approximately 8 years (1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002), according to the design of the initial SU.VI.MAX trial. In the present analyses, this antioxidant supplementation was only considered as an adjustment variable.

Healthy Aging (HA) Status (2007-2009)

The main outcome measure of our study was HA status (yes/no), defined on the basis of a multidimensional concept that included the following eight criteria [START_REF] Assmann | Healthy aging 5 years after a period of daily supplementation with antioxidant nutrients: A post hoc analysis of the french randomized trial SU.VI.MAX[END_REF]: absence of incident major chronic disease (cancer, cardiovascular disease or type II diabetes) during follow-up, good cognitive functioning, good physical functioning, absence of limitations in instrumental activities of daily living, absence of depressive symptomatology, absence of health-related limitations in social life, good overall self-perceived health, and absence of function-limiting pain. Participants were characterized as aging healthily when all eight HA components were met. HA status was determined between 2007 and 2009. More details on this HA definition and on each specific component are provided in Table 1. Additional information has been published elsewhere [START_REF] Assmann | Healthy aging 5 years after a period of daily supplementation with antioxidant nutrients: A post hoc analysis of the french randomized trial SU.VI.MAX[END_REF]. The SPPB includes 3 tests (administered by trained physicians): Repeated chair stands, balance testing, gait speed testing [START_REF] Guralnik | A short physical performance battery assessing lower extremity function: Association with self-reported disability and prediction of mortality and nursing home admission[END_REF]. Higher scores on the SPPB indicate better performance.

Good cognitive functioning (yes/no)

Mini Mental State Examination (MMSE) score ≥27/30 and rappel indicé-48 items (RI-48) score ≥19/48 and Delis-Kaplan Trail-making test (DK-TMT) scaled score ≥5.5

The MMSE [START_REF] Folstein | The Mini-Mental State Examination[END_REF] tests global cognitive functioning, the RI-48 tests verbal episodic memory [START_REF] Adam | Optimization of encoding specificity for the diagnosis of early AD: The RI-48 task[END_REF], and the DK-TMT tests mental flexibility [START_REF] Swanson | The Delis-Kaplan Executive Function System A Review[END_REF][START_REF] Homack | Test review: Delis-Kaplan executive function system[END_REF] To meet this criterion, participants had to declare that their health was generally "good" to "excellent" Participants' responses were collected via the SF-36 questionnaire [START_REF] Leplège | The French SF-36 Health Survey: Translation, cultural adaptation and preliminary psychometric evaluation[END_REF].

No function-limiting pain (yes/no)

To meet this criterion, participants had to report having experienced no more than "mild" physical pain during the previous month or that such pain had only limited or no impact on their daily activities Participants' responses were collected via the SF-36 questionnaire [START_REF] Leplège | The French SF-36 Health Survey: Translation, cultural adaptation and preliminary psychometric evaluation[END_REF].

No incident major chronic disease (yes/no)

No incident cancer, cardiovascular disease, or diabetes during follow-up Events of cancer and of cardiovascular disease were recorded during follow-up and validated by an independent external committee of medical doctors. Cancer was defined as cancer of any kind, except for basal cell carcinoma. Cardiovascular disease was defined as codes I20-I25, I63, I65, I66, I70, I71, and I74 from the 10th International World Health Organization Classification of Diseases). Incident diabetes was defined as having a fasting blood glucose value ≥ 1.26 g/L, anti-diabetic medication use, or self-reported diabetes at the end of follow-up.

Overall healthy aging

Participants meeting all of the above criteria were considered to be aging healthily, while participants for which at least one criterion was = "no" were not considered to be aging healthily → Thus, a binary indicator of overall healthy aging was obtained (yes/no).

1 All criteria were assessed at follow-up (2007-2009), apart from events of major chronic disease, for which incidence during follow-up (1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) was assessed.

Baseline Dietary Data (1994-1996)

Participants of the SU.VI.MAX trial were asked to complete one 24 h dietary record every 2 months. Accordingly, computerized questionnaires and an instruction manual including validated photographs of more than 250 foods were provided. Seven possible portion sizes were available to choose from [START_REF] Moullec | Validation du manuel-photos utilisé pour l'enquête alimentaire de l'étude SU[END_REF]. A detailed description of dietary data treatment in the SU.VI.MAX study has been published previously [START_REF] Kesse-Guyot | Higher adherence to French dietary guidelines and chronic diseases in the prospective SU.VI.MAX cohort[END_REF]. Briefly, dietary records reporting energy intakes of <100 kcal/d or >6000 kcal/d were considered as invalid. To further account for energy under-reporting, men reporting < 800 kcal/d and women reporting <500 kcal/d across ≥1/3 of valid records were excluded. Baseline questionnaires were used to collect data on alcohol and seafood consumption, as these food groups tend to be consumed less frequently than others. Alcohol consumption was estimated by a short, validated, semi-quantitative dietary questionnaire [START_REF] Lasfargues | Validity of a short self-administered dietary questionnaire compared with a dietetic interview[END_REF]. The average number of dietary records available per participant was 10.2 (interquartile range: 8-13). All eligible 24 h records collected during the first 2 years following inclusion were used to compute food and nutrient intakes. Details on the computation of the PNNS-GS have been published previously [START_REF] Estaquio | Adherence to the French programme national nutrition santé guideline score is associated with better nutrient intake and nutritional status[END_REF]. Briefly, the maximum number of points is 15, and the score includes 13 components: eight concern food serving recommendations (fruit and vegetables, starchy foods, whole grain food, dairy products, meat poultry seafood and eggs, seafood, vegetable fat and non-alcoholic beverages), 4 concern moderation (added fat, salt, sweetened foods, alcohol), and 1 concerns physical activity. Points are deducted for overconsumption of salt and sweets and for elevated energy intakes. Data (1995Data ( -1996) ) Weight was measured to the nearest 0.5 kg using an electronic scale (Seca, Hamburg, Germany), with participants wearing indoor clothing and no shoes. Height was measured to the nearest 0.5 cm with a wall-mounted stadiometer under the same conditions. A "BMI status"-variable of 3 categories was created, which distinguished: (a) underweight/normal weight (BMI < 25 kg/m 2 ), (b) overweight without obesity (BMI ≥ 25 and <30 kg/m 2 ), and (c) obesity (BMI ≥ 30 kg/m 2 ).

Anthropometric

Baseline Covariates (1994-1996)

Self-administered questionnaires were completed at recruitment (1994)(1995)(1996), including information on gender, date of birth, educational level (primary/secondary/university or equivalent), occupation (homemaker/manual worker/office employee/intellectual profession or managerial staff), family situation (living alone/cohabiting), smoking status (non-smoker/former smoker/current smoker), number of 24 h records, and antioxidant supplementation (yes/no).

Statistical Analysis

Descriptive Statistics and Results on the Simple Association between Dietary Quality and HA

Baseline characteristics of participants were compared according to HA status (yes/no), using Mann-Whitney U tests and Chi 2 -tests.

In order to describe the relationship of the PNNS-GS with HA, we used robust-error-variance Poisson regression models, which make it possible to estimate relative risks (RRs) and 95% confidence intervals in the presence of a frequent binary outcome [START_REF] Zou | A modified poisson regression approach to prospective studies with binary data[END_REF].

Analyses on the relationship between the PNNS-GS and HA were presented for the overall PNNS-GS score and for modified scores, obtained by sequentially removing each of the 13 components from the PNNS-GS one by one, with further adjustment for the removed component. The aim of these analyses was to test whether any of the score components explained a major part of the association between the overall PNNS-GS and HA, or whether the association was due to a combination of all components.

Models were adjusted for age, gender, smoking status, educational level, occupational status, living situation, antioxidant supplementation, total energy intake and number of 24 h records.

Main Analysis: Mediating Role of BMI Status in the Association between Diet Quality and HA

The PNNS-GS score (modeled as quartiles) was the observed exposure of interest (A), BMI status was the mediator (M) and HA the outcome (Y). Mediation analysis was conducted according to a counterfactual-based method proposed by Lange et al., which has similarities with marginal structural models, and allows to estimate natural direct and indirect effects [START_REF] Lange | A Simple unified approach for estimating natural direct and indirect effects[END_REF]. Briefly, this method includes the following steps:

(1) Creation of a new dataset by repeating each observation 4 times, including a new variable A*, which corresponded to each of the 4 PNNS-GS quartiles. This new variable A* was equal to A (the genuinely observed PNNS-GS quartile) for one line per subject and different from A for the 3 other lines;

(2) Two multivariable (i.e., confounder-adjusted) logistic regression models were applied to the new dataset to estimate the association between PNNS-GS quartiles (A/A*) and BMI status (M), first using the original variable A, and then the new variable A*;

(3) 'Individual stabilized weights' were calculated using the predicted probabilities obtained from these two logistic regressions, as follows:

W C i = P(M = M i |A = A * i , C = C i )/P(M = M i |A = A i , C = C i ) (1)
where i denotes the individual, A the observed exposure, A* the created exposure, M the mediator, and C denotes the different potential confounders; (4) A weighted Poisson regression model was applied to estimate the association between PNNS-GS quartiles (A and A*) and HA. This Poisson model and the above-mentioned logistic regression model were all adjusted for age, gender, smoking status, educational level, occupational status, living situation, antioxidant supplementation, total energy intake and number of 24 h records. The RR associated with the variable A was interpreted as the direct effect of PNNS-GS quartiles with respect to HA, and the RR associated with the variable A* as the indirect effect. The total effect was calculated as the product of the natural direct effect and the natural indirect effect. For the total effect, 95% confidence intervals were derived from a bootstrap sample distribution (N = 1000). The proportion mediated by BMI status was computed using the following formula:

[RR NDE (RR NIE -1)] ÷ [RR NDE × RR NIE -1] × 100% (2)
where NDE is natural direct effect and NIE is natural indirect effect. The mediation analysis was conducted under the working hypothesis that the following underlying conditions were satisfied [START_REF] Lange | A Simple unified approach for estimating natural direct and indirect effects[END_REF]: (1) no unmeasured confounders for the associations between: (a) PNNS-GS quartiles and HA, (b) BMI status and HA, (c) PNNS-GS quartiles and BMI status; and (2) no association between confounders of the relation between BMI status and HA with PNNS-GS quartiles.

All analyses were performed using 9.4 version of the SAS software (SAS Institute Inc., Cary, NC, USA).

Results

Descriptive Statistics

On average, participants were aged 51.9 (SD = 4.5) years at baseline, and the average follow-up duration was 13.5 (SD = 4.3) years. The prevalences of overweight (excluding obesity) and obesity were 30.8% and 5.8%, respectively.

A quite large proportion of participants were defined as "aging healthily"; 42% and of men and 36% of women (39% in total). Table 2 shows characteristics of participants defined as "aging healthily", as compared to their counterparts who did not meet ≥ 1 of the criteria of our HA definition. As expected, these "healthy agers" were younger, more educated, and were part of more advantaged occupational categories. In addition, a larger proportion of men than of women in our sample were defined as "healthy agers", and "healthy agers" were more often cohabiting than living alone. Finally, participants that showed good overall health during aging had a higher physical activity level, showed a higher level of adherence to the French nutritional recommendations, had a lower BMI, and a higher fiber intake.

Relationship between Dietary Quality and Overall Healthy Aging (HA)

Results concerning the associations between overall dietary quality (measured by the PNNS-GS) and HA status are displayed in Table 3. There was an overall positive association between higher scores on the PNNS-GS and the probability to age healthily, and this remained true even when removing different components of this score (representing different dimensions of dietary quality). Of note, the strength of the association between the dietary scores and HA was quite similar when comparing the original PNNS-GS and versions where specific components had been removed (relative risks ranged from 1.04 to 1.07, and all tested associations were statistically significant).

The Mediating Role of Body Mass Index (BMI) Status

Table 4 shows the results of our analysis that aimed to characterize the mediating role of BMI status in the association between dietary quality and HA. This analysis indicates a quite small but statistically significant mediating role of BMI status; for the comparison of the highest vs. lowest PNNS-GS quartile, the estimated percentage of the association with HA mediated by BMI status was 5%. This corresponds to a 'natural indirect effect' relative risk estimate of 1.31 (95% confidence interval = 1.13, 1.53), as compared to a 'natural direct effect' relative risk estimate of 1.01 (95% confidence interval = 1.01, 1.02).

Discussion

The findings of the present investigation suggest that greater adherence to the French nutritional recommendations at midlife was longitudinally associated with HA, through a direct effect and to a lesser extent an effect mediated by BMI status. The role of corpulence was small but not negligible, indicating that high dietary quality may in part contribute to the preservation of good overall health via the prevention of obesity.

In analyses on the link between adherence to the French national nutrition recommendations and HA, all nutritional scores (the global PNNS-GS and the different modified scores created by removing each component one by one) were associated with HA, arguing for a role of overall adherence to the French nutritional recommendations-rather than a specific role of one or several particular guidelines.

A number of studies have investigated the relationship between the overall diet, using holistic approaches, and multidimensional concepts of HA [START_REF] Akbaraly | Does overall diet in midlife predict future aging phenotypes? A cohort study[END_REF][START_REF] Assmann | Dietary scores at midlife and healthy ageing in a French prospective cohort[END_REF][START_REF] Assmann | A healthy dietary pattern at midlife, combined with a regulated energy intake, is related to increased odds for healthy aging[END_REF][START_REF] Gopinath | Adherence to dietary guidelines and successful aging over 10 years[END_REF][START_REF] Hodge | Dietary patterns as predictors of successful ageing[END_REF][START_REF] Samieri | The association between dietary patterns at midlife and health in aging: An observational study[END_REF][START_REF] Assmann | Association between adherence to the mediterranean diet at midlife and healthy aging in a cohort of french adults[END_REF]. Although indicators of dietary quality were quite heterogeneous across studies, the observed findings overall argue for an important role of nutrition for preserving good overall health during aging.

Midlife BMI and/or overweight and obesity have been identified as major determinants of HA measured by multidimensional concepts [START_REF] Reed | Predictors of health aging in men with high life expectancies[END_REF][START_REF] Singh-Manoux | Association of body mass index and waist circumference with successful aging[END_REF][START_REF] Tyrovolas | Skeletal muscle mass and body fat in relation to successful ageing of older adults: The multi-national MEDIS study[END_REF][START_REF] Ruhunuhewa | Prospective association between body mass index at midlife and healthy aging among French adults[END_REF], amongst others in a previous investigation of data from the SU.VI.MAX cohort [START_REF] Ruhunuhewa | Prospective association between body mass index at midlife and healthy aging among French adults[END_REF]. In that previous investigation, obesity was associated with a significantly reduced probability to age healthily, while overweight was only linked to a decreased probability of HA when associated with impaired metabolic health [START_REF] Ruhunuhewa | Prospective association between body mass index at midlife and healthy aging among French adults[END_REF]. Hence, it should be noted that while the current study used a 3-class indicator of BMI status (i.e., underweight/normal weight/overweight/obesity), it can be assumed that the mediation effect observed is mainly attributable to the avoidance of obesity and less to the avoidance of overweight.

On the other hand, in the British Whitehall II cohort, both overweight and obesity (as well as a large waist circumference), at midlife were related to a lowered probability of "successful aging", defined as the absence of coronary heart disease, stroke, cancer and diabetes; good cardiovascular, metabolic, respiratory, physical and cognitive functioning; and the absence of mental health problems [START_REF] Singh-Manoux | Association of body mass index and waist circumference with successful aging[END_REF].

To the best of our knowledge, our study is the first to specifically and formally investigate the mediating effect of BMI status in the relationship between diet and healthy aging. While certain investigations on the link between diet and HA have not taken BMI into account [START_REF] Gopinath | Adherence to dietary guidelines and successful aging over 10 years[END_REF][START_REF] Hodge | Dietary patterns as predictors of successful ageing[END_REF], an investigation of data from the Nurses' Health study has presented analyses adjusted for BMI [START_REF] Samieri | The association between dietary patterns at midlife and health in aging: An observational study[END_REF]. However, since this was done in a non-sequential manner (i.e., no otherwise fully-adjusted model excluding BMI was shown in addition to the fully-adjusted model including BMI), it is not possible to specifically characterize the mediating role of BMI based on these results. On the other hand, a study based on the Melbourne Collaborative cohort has presented such sequentially adjusted models, and the conclusions that can be drawn from these data are concordant with our results [START_REF] Hodge | Dietary patterns as predictors of successful ageing[END_REF]. Indeed, after additional adjustment for BMI and waist-to-hip ratio, an only partial attenuation of the association between an a posteriori dietary pattern high in meat and fatty foods and HA was observed (OR 4th quartile compared to the 1st of 0.69 (95%CI = 0.55-0.86) versus 0.78 (95%CI = 0.62-0.98)). However, sequential adjustment for BMI does not allow to assess a mediating effect of corpulence in an exact, quantitative manner.

In our study, the proportion of the overall association that was estimated to be mediated by BMI status was quite low (5%). This suggests that healthy aging may be promoted by a healthy diet through specific nutritional effects in the "direct" pathway, and to a lesser extent through mechanisms related to the prevention of overweight and obesity. Scientific literature examining the role of bioactive nutritional compounds and overall dietary patterns on different components of the healthy aging is plentiful [START_REF] Clément | The gut microbiome, diet, and links to cardiometabolic and chronic disorders[END_REF][START_REF] Szarc Vel Szic | From inflammaging to healthy aging by dietary lifestyle choices: Is epigenetics the key to personalized nutrition?[END_REF][START_REF] Fardet | Associations between diet-related diseases and impaired physiological mechanisms: A holistic approach based on meta-analyses to identifys targets for preventive nutrition[END_REF][START_REF] Franceschi | Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases[END_REF]. Potential mechanistic pathways include low-grade chronic inflammation [START_REF] Franceschi | Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases[END_REF], oxidative stress [START_REF] Harman | Free radical theory of aging[END_REF], DNA methylation and other epigenetic mechanisms [START_REF] Szarc Vel Szic | From inflammaging to healthy aging by dietary lifestyle choices: Is epigenetics the key to personalized nutrition?[END_REF], vascular damage [START_REF] Ritchie | The link between abdominal obesity, metabolic syndrome and cardiovascular disease[END_REF], and dysbiosis of the gut microbiota [START_REF] Clément | The gut microbiome, diet, and links to cardiometabolic and chronic disorders[END_REF]. In particular, low-grade chronic inflammation is affected by corpulence (since adipose tissue inflammation has been suggested to mediate comorbidities of obesity such as the metabolic syndrome and cardiovascular disease [START_REF] Cancello | Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity[END_REF]), but appears to also be directly related to the consumption of specific food groups, nutrients, and overall dietary patterns [START_REF] Shivappa | Designing and developing a literature-derived, population-based dietary inflammatory index[END_REF][START_REF] Barbaresko | Dietary pattern analysis and biomarkers of low-grade inflammation: A systematic literature review[END_REF].

Some limitations of the present study should be noted, which include residual confounding due to potential confounders such as life events and psychological factors that were not taken in to account. Furthermore, participants of the SU.VI.MAX 2 prospective study were originally volunteers who took part in the SU.VI.MAX randomized trial. This may have resulted in a lack of representativeness of the general population, and particularly in a lower prevalence of obesity. Hence, the proportion of the association mediated by obesity may be greater in the general population than in our study sample. It should also be noted that it is difficult in observational research to model the natural course over which diet presumably influences BMI status, which may then be involved in the etiology of age-related diseases and functional decline. Here, we considered both BMI status and dietary intakes assessed at baseline, but it can be assumed that dietary habits at earlier ages, including childhood, may have influenced BMI status at baseline. This may be one explanation for the rather small mediating effect of BMI status observed in the current study.

In addition, part of the data that was used to define HA status was exclusively collected during the SU.VI.MAX 2 observational study. The presence and consideration of such baseline health data would have increased the robustness of our results to potential reverse causation bias.

The main strengths of this study are of the prospective study design and the large sample size. Moreover, using a multidimensional HA concept helped to better capture the broad variety of aspects related to physical and mental wellness in a global manner. Another positive feature of the present study was the use of counterfactual-based mediation analysis, which permitted the formal investigation of the mediating role of BMI in the association between diet and HA.

In conclusion, this prospective investigation of data from a large French cohort provides novel evidence that high adherence to the French nutritional recommendations at midlife contributes to the preservation of health during aging, beyond potential impacts on obesity prevention. To our knowledge, the present study is the first to formally characterize the mediating role of BMI status in the relationship between dietary quality and a multidimensional concept of healthy aging. From a public health point of view, both dietary advice and weight management may help to promote a better overall health status during aging. However, since only a small part of the association between dietary quality at midlife and HA appeared to be mediated by BMI status, it can be hypothesized that increasing overall dietary quality may be beneficial even beyond targeting weight loss or weight stabilization. Further prospective observational studies and intervention trials are required to confirm these findings.

Figure 1 .

 1 Figure 1. Sample selection of participants from the 'Supplementation with Vitamins and Mineral Antioxidants' (SU.VI.MAX-'SUpplémentation en VItamines et Minéraux AntioXydants') study, France, 1994-2009; body mass index (BMI); Programme National Nutrition Santé-Guideline Score (PNNS-GS).

Table 1 .

 1 Definition of Healthy Aging status in the trial 'Supplementation with Vitamins and Mineral Antioxidants' (SU.VI.MAX-'SUpplémentation en VItamines et Minéraux AntioXydants') study.

	Dimension 1	Definition
		Short Physical Performance Battery (SPPB) score ≥11/12
	Good physical
	functioning (yes/no)

  . Higher scores on each test indicate better performances. All three tests were administered by trained neuropsychologists or geriatric physicians.<1 limitation on the Lawton Scale of Instrumental Activites of Daily Living (IADL)The Lawton IADL scale[START_REF] Graf | Hartford Institute for Geriatric Nursing. The Lawton instrumental activities of daily living (IADL) scale[END_REF] is a self-administered questionnaire (including, amongst others, questions on the ability to travel, go shopping, and do housekeeping). Having more limitations on the scale indicates lower independence in daily life.Center for Epidemiological Studies Depression Scale (CES-D) score <16/60The CES-D[START_REF] Morin | Psychometric properties of the center for epidemiologic studies depression scale (CES-D) in French clinical and nonclinical adults[END_REF] is a self-administered questionnaire developed for the evaluation of depressive symptoms in the general population, within epidemiological studies. Higher scores indicate more depressive symptoms.

	No limitations in IADL
	(yes/no)
	No depressive
	symptoms (yes/no)
	No health-related
	limitations in social life
	(yes/no)

No/only slight and only infrequent (perceived) interference of health problems with social life Data were collected via the Medical Outcome Study Short Form-36 (SF-36) questionnaire

[START_REF] Leplège | The French SF-36 Health Survey: Translation, cultural adaptation and preliminary psychometric evaluation[END_REF]

, a very widely used, self-administered questionnaire designed to measure vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning, and mental health Good overall self-perceived health (yes/no)

Table 2 .

 2 Baseline characteristics of the study population according to healthy aging status, SU.VI.MAX 2 study (France), 2007-2009, N = 2249.

	Healthy Aging Status

Abbreviation: SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids a Values are mean (standard deviation, SD) or % as appropriate. b P-values are based on Mann-Whitney U tests (continuous variables) and Chi-square tests (categorical variables). c Or managerial staff; Programme National Nutrition Santé-Guideline Score (PNNS-GS).

Table 3 .

 3 Relative risk estimates (95% confidence interval (CI)) for the association between nutritional requirement (PNNS-GS) components and healthy aging status, SU.VI.MAX 2 study (France), 2007-2009, N = 2249 a,b.

	Nutritional Score	RR (95% CI)	p-Value
	PNNS-GS	1.06 (1.03, 1.09)	0.0001
	PNNS-GS without Physical activity component	1.04 (1.01, 1.08)	0.0109
	PNNS-GS without Seafood component	1.06 (1.02, 1.09)	0.0009
	PNNS-GS without Fruits and vegetables component	1.06 (1.03, 1.10)	0.0006
	PNNS-GS without Whole grain food component	1.06 (1.03, 1.10)	0.0001
	PNNS-GS without Total added fats component	1.06 (1.03, 1.09)	0.0002
	PNNS-GS without Salt component	1.06 (1.03, 1.09)	0.0002
	PNNS-GS without Vegetable added fats component	1.07 (1.04, 1.10)	<0001
	PNNS-GS without Alcohol component	1.06 (1.03, 1.09)	0.0002
	PNNS-GS without Meat and poultry, seafood and eggs component	1.06 (1.03, 1.09)	0.0002
	PNNS-GS without Milk and dairy products component	1.06 (1.03, 1.09)	0.0001
	PNNS-GS without Sweetened foods component c	1.06 (1.03, 1.09)	0.0002
	PNNS-GS without Bread, cereals, potatoes and legumes	1.06 (1.03, 1.09)	0.0001
	PNNS-GS without Beverages component d	1.06 (1.03, 1.09)	0.0002

a Robust-error-variance Poisson regression models were used to model healthy aging status. b Models were adjusted for age, gender, smoking status, educational level, occupational status, living situation, antioxidant supplementation, total energy intake, number of 24 h records and the removed component, except for the overall PNNS-GS. c Points for the sweetened foods-component were calculated based on added sugar from sweetened foods (including food items such as sweet cakes, cookies, and chocolate), as a % of daily overall energy intake (% EI/d): (a) ≥17.5% EI/d → -0.5 points; (b) 17.5-12.5% EI/d → 0 points; (c) <12.5% EI/d →1 point. d Points for beverages-component were calculated as follows: (a) <1l water and >250 ml soda/d → 0 points; (b) ≥1l water and >250 ml soda/d → 0.5 points; (c) <1l water and ≤250 ml soda/d → 0.75 points; (d) ≥1l water and ≤250 ml soda/d → 1 point; Programme National Nutrition Santé-Guideline Score (PNN-GS); Relative risk (RR).

Table 4 .

 4 Relative risk estimates for the direct and the indirect effect a for the mediating role of BMI status on the association between the PNNS -Guidelines Score and healthy aging, SU.VI.MAX 2 study (France), 2007-2009, N = 2249. Based on the method of mediation analysis proposed by[START_REF] Lange | A Simple unified approach for estimating natural direct and indirect effects[END_REF]. Adjusted for age, gender, smoking status, educational level, occupational status, living situation, antioxidant supplementation, total energy intake and number of 24 h records.b The natural direct and indirect effects were obtained from the weighted mediation model of robust-error-variance Poisson regression. c Corresponds to the value of the exposure relative to the indirect path. d Confidence intervals for the total effect were assigned by bootstrapping. e The proportion mediated was computed as (OR NDE (OR NIE -1)) ÷ (OR NDE × OR NIE -1) × 100%. NDE-natural direct effect; NIE-natural indirect effect; Programme National Nutrition Santé-Guideline Score (PNNS-GS).

			PNNS-GS Quartiles RR (95% CI) b
		Q1 <6.55	Q2 (6.55-7.80)	Q3 (7.80-9.05)	Q4 >9.05	P trend
	Direct effect a	1.00 (ref)	1.08 (0.92, 1.26)	1.20 (1.03, 1.40)	1.31 (1.13, 1.53)	0.0002
	Indirect effect c	1.00 (ref)	1.01 (1.00, 1.01)	1.01 (1.00, 1.01)	1.01 (1.01, 1.02)	0.0003
	Total effect d	1.00 (ref)	1.09 (0.92, 1.26)	1.21 (1.03, 1.41)	1.33 (1.13, 1.55)
	Mediation (%) e		7%	5%	5%

a

Acknowledgments:

We thank Younes Esseddik, Thi Hong Van Duong, Frédéric Coffinieres, Régis Gatibelza, Maithyly Sivapalan and Paul Flanzy (computer scientists); Nathalie Arnault, Véronique Gourlet, Fabien Szabo, Julien Allègre and Laurent Bourhis (data-managers/biostatisticians); and Cédric Agaesse and Anne-Elise Dussolier (dieticians) for their technical contributions The authors also thank Stéphane Raffard, who was responsible for standardization of the cognitive evaluation. Sources of support: Indunil Ruhunuhewa was supported by an internship grant (MSc) from the University of Paris 13; Karen E. Assmann and Moufidath Adjibade were supported by doctoral fellowships from the Ecole Doctorale Galilée, University of Paris 13, Sorbonne Paris Cité. None of the funders had any role in study design, data collection and analysis, decision to publish or writing of the manuscript.

Author Contributions: Serge Hercberg, Pilar Galan and Emmanuelle Kesse-Guyot designed the study (development of the concept, design and protocol of the SU.VI.MAX/ SU.VI.MAX 2 studies and coordination of data collection); Indunil Ruhunuhewa, Karen E. Assmann and Emmanuelle Kesse-Guyot conducted the research, performed the statistical analysis and wrote the article; Zhen Li provided methodological guidance; Indunil Ruhunuhewa, Karen E. Assmann, Moufidath Adjibade, Zhen Li, Raphaëlle Varraso, Serge Hercberg, Pilar Galan and Emmanuelle Kesse-Guyot were involved in interpreting the results and editing the manuscript for important intellectual content; Emmanuelle Kesse-Guyot had primary responsibility for the final content. All authors have read and approved the final manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.


Sophie Joimel

Christophe Schwartz

Mickael Hedde

Sayuri Kiyota

Paul Henning Krogh

Johanne Nahmani

Guenola Peres

Alan Vergnes

Jérôme Cortet

Mickaël Hedde

Urban and industrial land uses have a higher soil biological quality than expected from physicochemical quality

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


Audrey Magnin

Lucie Hoornaert

Eric Pollet

Stéphanie Laurichesse

Vincent Phalip

Luc Avérous
email: luc.averous@unistra.fr

St Ephanie Laurichesse

Luc Av Erous

Isolation and characterization of different promising fungi for biological waste management of polyurethanes

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

Introduction

Material longevity associated with massive scale production makes synthetic polymers among the most abundant pollutants of our century. Studies attesting of their negative impacts on marine and terrestrial ecosystems are published at an increasing rate [START_REF] Jambeck | Plastic waste inputs from land into the ocean[END_REF][START_REF] Cao | Effects of polystyrene microplastics on the fitness of earthworms in an agricultural soil[END_REF][START_REF] Chae | Effects of micro-and nanoplastics on aquatic ecosystems: current research trends and perspectives[END_REF]. Polyurethanes (PU) as the 6th polymer with 18 Mt produced in 2016 [START_REF] Furtwengler | Synthesis and characterization of polyurethane foams derived of fully renewable polyester polyols from sorbitol[END_REF], are among the most pollutant polymers [START_REF] Reddy | Description of the small plastics fragments in marine sediments along the Alang-Sosiya ship-breaking yard, India[END_REF]. For instance, among the 70 plastic samples collected on a beach 39 were identified as PU [START_REF] Turner | Elemental concentrations and bioaccessibilities in beached plastic foam litter, with particular reference to lead in polyurethane[END_REF]. PU are intended for long-term applications: foams for construction and automotive industries, elastomers for sportswear or medical devices, coatings and sealants [START_REF] Reulier | Elaboration, morphology and properties of renewable thermoplastics blends, based on polyamide and polyurethane synthesized from dimer fatty acids[END_REF]. They are thus designed to resist environmental conditions. Management of PU products end-of-life is currently inappropriate with most of PU wastes going to landfill and existing solutions such as incineration are not economically and environmentally satisfying. Thus, more high value added and sustainable solutions for PU waste treatment are urgently needed.

Bioremediation is a general term to qualify the action of depollution using a biological entity while biological recycling implies obtaining valuable products from waste also using biological means. Polymer biodegradation is the basis for both processes [START_REF] Wierckx | Plastic waste as a novel substrate for industrial biotechnology[END_REF]. Briefly, microorganisms secrete extracellular enzymes able to depolymerize the macromolecules leading to the formation of oligomers and monomers. Then, these molecules can be assimilated by microorganisms and metabolized for biomass and energy production [START_REF] Lucas | Polymer biodegradation: mechanisms and estimation techniques[END_REF]. Degradation can also be performed by a single enzyme from microbial, vegetal or mammalian origins [START_REF] Marchant | Degradation of a poly (ether urethane urea) elastomer: infra-red and XPS studies[END_REF][START_REF] Tokiwa | Two types of lipases in hydrolysis of polyester[END_REF][START_REF] Brzeska | Chemical and enzymatic hydrolysis of polyurethane/polylactide blends[END_REF]. Bioremediation and biological recycling are attractive ways to fight against plastic pollution because of their low costs and environmental impacts [START_REF] Azubuike | Bioremediation techniques-classification based on site of application: principles, advantages, limitations and prospects[END_REF].

The biodegradation of PU materials is increasingly studied. PU are mainly synthesized by the reaction between polyols and polyisocyanates. The polyol is generally a polyester or a polyether. The micro-organization of PU with soft and rigid segments is one of the main key towards high-performance materials [START_REF] Carr E | Synthesis and characterization of advanced biobased thermoplastic nonisocyanate polyurethanes, with controlled aromatic-aliphatic architectures[END_REF]. The building blocks involved in PU synthesis control the final macromolecular architecture, the crystallinity and the molar mass, which are parameters highly impacting the final PU biodegradability. For instance, polyester-based PU are known to be more biodegradable than polyether ones due to the hydrolysable ester bonds [START_REF] Cregut | New insights into polyurethane biodegradation and realistic prospects for the development of a sustainable waste recycling process[END_REF].

Degradation of PU with filamentous fungi is described as more efficient than with bacteria due to outstanding enzymatic arsenals and abiotic features linked to filaments formation [START_REF] Barratt | Fungi are the predominant microorganisms responsible for degradation of soil-buried polyester polyurethane over a range of soil water holding capacities[END_REF][START_REF] Lucas | Polymer biodegradation: mechanisms and estimation techniques[END_REF]. Several types of PU materials such as polyether urethane foam [START_REF] Alvarez-Barragan | Biodegradative activities of selected environmental fungi on a polyester polyurethane varnish and polyether polyurethane foams[END_REF], thermoplastic polyester [START_REF] Khan | Biodegradation of polyester polyurethane by Aspergillus tubingensis[END_REF] and PU waterborne dispersion of polyester PU [START_REF] Russell | Biodegradation of polyester polyurethane by endophytic fungi[END_REF] have already been described as sensitive to fungal biodegradation.

Bottlenecks in PU biodegradation are the evaluation of the degradation with efficient techniques and the understanding of the degradation mechanisms. For instance, the majority of the microorganisms were tested on commercial model PU such as Impranil â , a waterborne polyester PU dispersion [START_REF] Rowe | Growth of Bacillus subtilis on polyurethane and the purification and characterization of a polyurethanase-lipase enzyme[END_REF][START_REF] Gautam | Candida rugosa lipase-catalyzed polyurethane degradation in aqueous medium[END_REF]. This polymer is a milky solution presenting the particularity of becoming clearer when hydrolysed. Although Impranil â is largely studied because convenient for screening approaches, its chemical architecture is very specific and far to be representative of most of the common PU. Then, Impranil â is not a perfect model. When more complex PU substrates are used, a significant lack of robust methods of analysis is noticeable [START_REF] Urgun-Demirtas | Laboratory investigation of biodegradability of a polyurethane foam under anaerobic conditions[END_REF][START_REF] Ibrahim | Polyester-polyurethane biodegradation by Alternaria Solani, isolated from Northern Jordan Ibrahim N. Ibrahim[END_REF]. Degradation analysis must couple overall degradation evaluation with, for example weight loss measurement and polymer surface analysis. A sufficient number of repetitions must also be considered.

In this publication, we achieved a screening of fungal strains isolated from PU-containing environments on several tailor-made TPU substrates, different models with different macromolecular architectures which are much more representative of the conventional PU, compared to Impranil â . Degradation of these model TPU represents a step towards the degradation of real and largely produced PU such as foams. Strains of fungi presenting the best degradation capacities were then identified. Analysis of the degraded polymers and enzymatic assays on the culture supernatants were then performed to suggest degradation mechanisms.

Results

Screening and identification of the fungi

Strains isolated were named according to the type of agar plate used for isolation (I = Impranil + PDA, P = PDA, MMI = Impranil + MM and MMP = MM) followed by a digit indicating the origin of the sample (see materials and methods). Twenty-seven strains were isolated. Three were yeast shaped while the others were filamentous fungi.

The collection of fungi was then screened on four TPU. No significant weight loss was detected for the ethylene glycol and phthalic anhydride-based TPU which is a poly(ester ether)-based TPU and for the PTHFbased TPU (polyether-based TPU; Fig. 1A). Evident weight losses were measured for the PCL-based TPU and the fatty acid dimer-based TPU incubated with 16 strains from the collection (weight loss >1% for at least one of the two polymers). The best strains were selected for further analysis. The strains P2a1 and MMP3b (Fig. 1) were selected because of weight loss higher than 3% on the PCL-based TPU after 2 months of incubation. MMP3c1 was chosen because of the similar weight loss measured on the PCL-based TPU and on the fatty acid dimer-based TPU. The degradation efficiency determined with five replicates was confirmed for these three strains. They displayed weight losses coherent with those from the screening (Fig. 1B). Weight losses of 3.2%, 8.9% and 1.5% were measured for P2a1, MMP3b and MMP3c1, respectively, for the PCLbased TPU. Weight losses of 1.7%, 1.3% and 1.8% were measured for the fatty acid dimer-based TPU. Fungal growth was observed on the two polymers with the three strains (Fig. S1 andS2). P2a1 which was isolated from a floor mat collected in a junkyard and MMP3b and MMP3c1, isolated from a yellowish PU foam collected in a PU production plant, were thus selected as strains of interest for PU biodegradation.

The selected fungi were then identified using molecular and morphological tools. Molecular identification allows to the definition of the genus and also an orientation towards a section within the genus. The strain P2a1 was identified as member of the genus Alternaria (CABI deposit number: 506837). Using FASTA from EBI, the ITS sequence (NCBI accession: MH410558) matched at 98-99% identity to members of species of the genus Alternaria [START_REF] Hou | Comparative genomics of pathogens causing brown spot disease of tobacco: Alternaria longipes and Alternaria alternata[END_REF]. Using the BLAST algorithm with the 'Others' database from NCBI, limited to sequences from type strains, this sample showed matches at 99% identity to type strains from species of Alternaria. There was no clear distinction between species from the sequence results; hence, identification was given at genus level. Morphological observation tends to support such identification since P2a1 conidia morphologies and growth aspects on PDA shown in Fig. 2 are similar to those of the strains of Alternaria sp., described in Matsumyia et al., [START_REF] Matsumiya | Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp[END_REF]. The strain MMP3b ITS (NCBI accession: MH410559) sequence matches at 97.7-99.6% identity to sequences from members of Penicillium section Lanata-Divaricata. This result was confirmed by partial beta-tubulin sequencing (NCBI accession: MH594856) which also gave top matches to members of this group including 98.2% identity to sequence GU981629 from the type strain of P. brasilianum, which have been published in the ICPA List of Penicillium names and in [START_REF] Visagie | Identification and nomenclature of the genus Penicillium[END_REF]. Morphological similarities of the conidia can be observed between Fig. 2 and the P. brasilianum observation from [START_REF] Cho | First report of Penicillium brasilianum and P. daleae isolated from soil in Korea[END_REF] and [START_REF] Inokoshi | Spirohexalines, new inhibitors of bacterial undecaprenyl pyrophosphate synthase, produced by Penicillium brasilianum FKI-3368[END_REF]. This strain was deposited in CABI collection (CABI deposit number: 506838). The ITS sequence of MMP3c1 showed 100% identity to sequence AF027863 from the type strain of A. flavus and to sequence EF661560 from the type strain of A. oryzae, both of which have been published in the ICPA list of Aspergillus names and in [START_REF] Peterson | Phylogenetic analysis of Aspergillus species using DNA sequences from four loci[END_REF]. No distinction can be made between these two species. Identification has thus been made to the section at genus level (CABI deposit number: 506839).

Materials analysis

Several techniques were used to deepen the characterization of the degraded TPU. One replicate of each experiment was examined by SEM (Figs 3 and4). PCLbased TPU incubated without fungi displayed a smooth and intact surface (Fig. 3). Incubation with the strain of Alternaria showed deep holes disseminated in few places of the polymer surface. Incubation with the strain of Penicillium revealed a regular network of excavations. Finally, the strain of Aspergillus led to an irregular surface with shallow cracks. Fatty acid dimer-based TPU incubated without fungi showed also an undamaged surface (Fig. 4). Whitish zones were observed when incubated with the three strains. Higher magnification on these zones revealed superimposed patches for the three degraded samples. Cracks were shallow. Only the superficial layer of the polymers was impacted.

FT-IR was performed on all the samples to characterize the chemical modifications on TPU surfaces. Spectra, available on supplementary data (Fig. S3 andS4), allowed the identification of three zones of interest corresponding to ester or urethane bonds. A wide band between 3325 and 3340 cm À1 corresponds to OH and NH stretching without differentiation between these two moieties [START_REF] Oprea | Dependence of fungal biodegradation of PEG/castor oil-based polyurethane elastomers on the hard-segment structure[END_REF][START_REF] Spont On | Biodegradation study by Pseudomonas sp. of flexible polyurethane foams derived from castor oil[END_REF]; Fig. S5). Peaks at 1720 cm À1 are attributed to C=O of an ester bond while signal at 1700 cm À1 can be attributed to C=O of a carboxylic acid [START_REF] Elzein | FTIR study of polycaprolactone chain organization at interfaces[END_REF][START_REF] Schmidt | Degradation of polyester polyurethane by bacterial polyester hydrolases[END_REF][START_REF] Osman | Degradation of polyester polyurethane by Aspergillus sp. strain S45 isolated from soil[END_REF]. The band at 1530 cm À1 is generally attributed to urethane/amide NH bending vibration in the secondary amide [START_REF] Oceguera-Cervantes | Characterization of the polyurethanolytic activity of two Alicycliphilus sp. strains able to degrade polyurethane and N-methylpyrrolidone[END_REF][START_REF] Oprea | Dependence of fungal biodegradation of PEG/castor oil-based polyurethane elastomers on the hard-segment structure[END_REF][START_REF] Spont On | Biodegradation study by Pseudomonas sp. of flexible polyurethane foams derived from castor oil[END_REF]. PCL used for the PCL-based TPU synthesis was also separately analysed (Fig. S6). No signal appeared on this zone for this polyester. In order to perform a semi-quantitative study, the intensity of peaks of interest was normalized by the intensity of the stable peak at 2 865 cm À1 corresponding to -CH 2 stretching (Fig. 5).

For the PCL-based TPU, an increase of the signal intensities at 1530 and 3330 cm À1 and a decrease of the signal intensity at 1720 cm À1 were noticed for TPU incubated with fungi, compared to the control. A band at 1700 cm À1 appeared for TPU incubated with Penicillium.

For the fatty acid dimer-based TPU, signals intensities increase at 1530 and 3330 cm À1 and decrease at 1720 cm À1 were also observed but to a lesser extent. The peak at 1700 cm À1 appeared for all the TPU incubated with fungi.

All the degraded polymers were then analysed by SEC to evaluate the evolution of the molar masses distributions. Chromatograms are available on Fig. S7 and S8. Two zones can be distinguished as follows: the main polymer chain consisting in the main peak (between 4 and 7 min of retention time) and the oligomers corresponding to smaller peaks (between 7 and 8.5 min of retention). Number average molar mass (Mn), mass average molar mass (Mw), dispersity ( D) and the percentage area of the main peak were collected in Table 1. For the PCL-based TPU, variability between the replicates increased after fungal degradation (up to 116 000 AE 13 000 g mol À1 ) while high repeatability was observed for the negative control (125 000 AE 1000 g mol À1 ). No significant variation of Mn, Mw or D was noticed between PCL-based TPU incubated with and without fungi (P-value >0.05). Only a slight decrease of dispersity was observed for the strain of Penicillium (Pvalue = 0.044). A decrease of the relative area of the main polymer chains was noticed for the three strains. For the fatty acid-based TPU, a significant increase of the Mn from 35 000 for the control to 37 000, 40 000 and 38 000 was observed for the strains of Alternaria, Penicillium and Aspergillus respectively (P-value <0.05). A significant decrease of the Mw was noticed from 120 000 g mol À1 for the negative control to 104 000, 108 000 and 101 000 g mol À1 , for the strains of Alternaria, Penicillium and Aspergillus respectively (Pvalue <0.05). The variation of Mn and Mw involved a high decrease of the corresponding dispersities. As for the PCL-TPU, the relative area of the main peak also decreased significantly.

Enzymatic assays

Esterase activity was determined on two substrates, pNitrophenyl acetate (pNPA) and pNitrophenol hexanoate (pNPH), on the supernatant media of fungal cultures on PCL-based TPU and fatty acid dimer-based TPU (Fig. 6). Both chemicals release pNitrophenol after hydrolysis of the ester bonds. Measured activities were significantly higher for the fatty acid dimer-based TPU than for the PCL-based TPU. Enzymes of Alternaria and Penicillium produced during the degradation of the fatty acid dimer-based TPU showed a better affinity for the substrate with six aliphatic carbons. Esterase activity of the supernatants of culture on PCL-based TPU was the highest for Alternaria and null for Penicillium.

Discussion

To be as close as possible to conventional PU wastes, the synthesized polymers were based on 4,4 0 MDI, thus providing highly resistant urethanes groups [START_REF] Sabbioni | Synthetic approaches to obtain amino acid adducts of 4,4 0 -methylenediphenyl diisocyanate[END_REF]. Screening on TPU only showed degradation of the two polyester PU after 2 months of incubation at 30°C with the polymers as the sole carbon source (Fig. 1). The poly(ester ether) PU and the polyether PU were already described as not degradable using efficient PU-degrading enzymes (A. Magnin., E. Pollet., R. Perrin., C. Ullmann., C. Persillon .,V. Phalip and Av erous L. unpublished results). For the poly(ester ether) PU, it seems that the macromolecular structure makes the ester bonds not easily accessible for enzymes. The degradation of the polyester PU was also confirmed by the evaluation of the fungal growth (Fig. S1 andS2). Degradation ability of the fungi thus depends on the bonds of the soft segment of the TPU but also on the macromolecular structure. Usually, additional carbon sources such as glucose [START_REF] Matsumiya | Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp[END_REF][START_REF] Khan | Biodegradation of polyester polyurethane by Aspergillus tubingensis[END_REF] or PDB (Alvarez-Barragan et al., 2016) are added to promote the microbial growth and thus, the biodegradation. Regarding the development of an efficient remediation or bio-recycling process, working without additional carbon source such as glucose, as in our case, is a major financial gain. Identifications of the strains were done at the genus level for the strain P2a1 which is Alternaria sp. and to the section for MMP3b and MMP3c1 which are respectively Penicillium section Lanata-Divaricata and Aspergillus section flavi. Alternaria species have already been described for PU degradation. For example, a strain of A. tenuissima was reported as active on pyridine-based polyether PU [START_REF] Oprea | Biodegradation of pyridine-based polyether polyurethanes by the Alternaria tenuissima fungus[END_REF]. Alternaria solani, isolated from soil, showed degradation activity towards a polyester PU [START_REF] Ibrahim | Polyester-polyurethane biodegradation by Alternaria Solani, isolated from Northern Jordan Ibrahim N. Ibrahim[END_REF]. Alternaria sp. was described for the degradation of a polyether PU foam and on model molecules with a urethane bond as a sole cleavable bond [START_REF] Matsumiya | Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp[END_REF]. Aspergillus flavus has been described as capable of degrading a polyester PU as sole carbon source [START_REF] Mathur | Degradation of polyurethane by Aspergillus flavus (ITCC 6051) isolated from soil[END_REF] while A. oryzae has never been described on PU.

The strain involving the most significant weight loss on TPU is the strain of Penicillium section Lanata-Divaricata. Isolated species of Penicillium have been described on PU degradation studies such as Penicillium chrysogenum (Alvarez- [START_REF] Alvarez-Barragan | Biodegradative activities of selected environmental fungi on a polyester polyurethane varnish and polyether polyurethane foams[END_REF], but to the best of our knowledge, no isolated strains of Penicillium belonging to the section Lanata-Divaricata has been reported to degrade PU. It is thus the first time for such a strain that a PU-degrading activity is clearly demonstrated. The best match for our strain appeared to be with P. brasilianum. This species is widely known as an enzyme factory, mainly for production of cellulases [START_REF] Jorgensen | Growth and enzyme production by three Penicillium species on monosaccharides[END_REF][START_REF] Jørgensen | Production of cellulases by Penicillium brasilianum IBT 20888-Effect of substrate on hydrolytic performance[END_REF]) and feruloyl esterases [START_REF] Panagiotou | Penicillium brasilianum as an enzyme factory; the essential role of feruloyl esterases for the hydrolysis of the plant cell wall[END_REF].

Complete analysis of the degradation systems was provided for the two polyester PU incubated with the three strains of interest. Different degradation profiles were observed and followed by weight loss, SEM, FTIR analysis and enzymatic assays for the PCL-based TPU incubated with the three strains (Figs 1B, 3 and 4). SEC data did not provide differential results (Table 1).

Surface analysis of the PCL-based TPU incubated with Penicillium by SEM revealed a specific network like a fungal mycelium's one. Moreover, Fig. 2B shows the filaments and spores of the strain of Penicillium, with filaments diameters from 5 to 10 lm. The network displayed rift widths slightly higher than 10 lm, strengthening the hypothesis of material excavation corresponding to filament growth on the polymer surface. The absence of extracellular esterase activity indicates that enzymes mainly responsible of the TPU degradation seem to be cell-bound proteins. These types of enzymes have already been described on the bacterial degradation of PU [START_REF] Akutsu | Fungi for waste biomanagement of polyurethanes 553 polyurethane-degrading enzyme from Comamonas acidovorans TB-35[END_REF].

Better affinity for the pNPH, ester substrate with six aliphatic carbons, was expected for the enzymes produced within the culture medium with the PCL-based TPU as it consists in the repetition of an ester bound to a chain of six carbons. Surprisingly, none of the three strains produced enzymes with better affinity for this substrate. However, for the fatty acid dimer-based TPU, the activity on pNPH was higher than on pNPA. This could be linked to the hydrophobic chain length of the pNPH ester group matching the one of the pendant alkyl chains of the polyester segment of this TPU.

The weight loss for the fatty acid dimer-based TPU was low (about 1.5%) and almost similar for all three strains (Fig. 1B). Surface analysis performed by SEM and FTIR displayed similar polymer surface morphologies and similar changes in surface moieties (Figs 4 and5). Enzymatic assays showed close esterase activities between the strain of Alternaria and Penicillium while a significantly lower esterase activity for the strain of Aspergillus (Fig. 6B). The mechanisms implemented by the strain of Aspergillus seemed to be different than the ones of Alternaria and Penicillium. Despite these differences, the three strains presented analogous impact on the fatty acid dimer-based TPU.

In addition to strain comparison, this study allows also discussing the techniques used to evaluate the polymer degradation. For the fatty acid dimer-based TPU, SEC analysis of the remaining solid polymer showed decreasing Mw and increasing Mn highlighting some polymers alterations. This increase in Mn values could be explained by the limited cleavage of the polymer chains along with the release of very short oligomers. These small molecules could be lost by solubilization in the surrounding medium or, in the case of fungal degradation, by their metabolization by the microorganisms. It has been reported that even a very limited loss in the lower molar mass fractions may result in a significant apparent increase in Mn values [START_REF] Mohd-Adnan | Evaluation of kinetics parameters for poly(l-lactic acid) hydrolysis under high-pressure steam[END_REF]. The loss of material is limited. This is highlighted by the decrease of main polymers and the evolution of the oligomers concentration (Table 1). These oligomers could correspond to the observed superimposed patches observed by SEM at the polymer surface (Fig. 4). As oligomers remained mainly associated with the polymer pieces, the weight loss was limited even when the observed degradation was significant. The same behaviour was observed on fatty acid dimer-based TPU after 50 days of degradation at 37°c with an esterase (Magnin et al., unpublished). For the PCL-based TPU, no significant change in molar mass distributions was observed on the remaining solid polymer and only low amount of oligomers were found, most probably due to their consumption by fungi. SEC appears thus to be a good technique for the analysis of polymers showing superficial 'damages', as a complement of the weight loss measurement, but seems less relevant for highly degraded polymers.

This work offers a clear proof of the interest of FTIR for the semi-quantitative evaluation of TPU biodegradation. For the PCL-based TPU, Penicillium showed the highest peak intensity increase at 3300 and 1700 cm À1 and the highest decrease at 1720 cm À1 while displaying the highest weight loss whereas Aspergillus showed the lowest intensity variations at these three wavelengths. For the fatty acid dimer-based TPU, comparable but lower weight losses were observed with all three strains. However, FT-IR spectra showed a slight increase in peak intensity at 3300 cm À1 and a decrease at 1720 cm À1 . The intensity variation for these two peaks is thus consistent with the weight loss and thus with overall polymer degradation. However, the interpretation of the increase of the peak intensity at 1530 cm À1 appears trickier. [START_REF] Oprea | Dependence of fungal biodegradation of PEG/castor oil-based polyurethane elastomers on the hard-segment structure[END_REF] suggested that an increase of this signal is related to urethane bond hydrolysis while others suggested that a decrease of this signal attests for urethane bond degradation [START_REF] Oceguera-Cervantes | Characterization of the polyurethanolytic activity of two Alicycliphilus sp. strains able to degrade polyurethane and N-methylpyrrolidone[END_REF][START_REF] Sarkar | Oxidative and enzymatic degradations of l-tyrosine based polyurethanes[END_REF]. The mechanism and location of the hydrolysis is most likely the reason for the contradictory statements found in the literature. An esterase activity would lead to the hydrolysis of the ester bond part of the urethane group, resulting in more carbamate groups exposed and thus increased signal of secondary amide band. In the case of protease activity, the amide bond part of the urethane group would be cleaved resulting in the decrease of the corresponding band signal. The observed increase of the secondary amide signal tends to evidence that the esterase activity is predominant. But, either an increase or a decrease of this signal would attest for changes in the polymer structure. Consequently, fungal degradations have a clear impact on the urethane bond but no definitive conclusion can be made on the exact nature of this impact. This point needs additional investigations.

Identification of these three strains, and particularly the strain of Penicillium, is the premise for the development of bioremediation and biological recycling processes. Understanding the full metabolisms involved in the biodegradation should then be fully elucidated with, for example proteomic tools. Optimization of the conditions must be carried out to improve TPU degradation. For instance, pre-incubation in rich media could be tested to further improve the biodegradation. More generally, systems optimization might allow to degrade other polyurethanes, such as cross-linked ones, for example PU foam, which represent the large majority of the commercial PU. This paper lays the foundation for an effective and green solution towards relieving the burden of polyurethane waste pollution, which is in the core of one of the major and very actual concern linked to the worldwide plastic pollution.

Experimental procedures Materials

Minimal medium preparation. Minimal medium (MM) was prepared according to Alvarez-Barragan et al., with some minor modifications [START_REF] Alvarez-Barragan | Biodegradative activities of selected environmental fungi on a polyester polyurethane varnish and polyether polyurethane foams[END_REF]. Buffer with 19 mM of NaH 2 PO 4 and 33.5 mM of K 2 HPO 4 was supplemented by agar (15 g l À1 final) and HCl (1.8 mM final) and autoclaved at 121°C for 18 min. To 1 l of this buffer was added 10 ml of each of the ) was purchased from Huntsman (USA). The 1,4-butanediol (1,4-BDO, Mn = 90.12 g mol À1 ) was purchased from Invista (USA).

The TPU syntheses were performed into a jacketed reactor at 80°C under nitrogen gas flow. Stoichiometric conditions were used with a NCO/OH ratio of 1, using an equimolar quantity of the polyol and 1,4-BDO. A 'onestep' method was performed. The 4,4 0 -MDI was warmed at 60°C and then introduced into the reactor followed by the mix of polyol and 1,4-BDO, previously heated at 80°C. Resulting TPU material was then compressed at 145°C with a plate press (Labtech Hot Press, Samutprakarn, Thailand) to obtain thin films. They were then cut into pieces of 2 9 2 cm.

Methods

Fungal isolation. Six samples likely to contain PUdegrading fungi were collected: (i) a landfill stagnant water from a PU production plant collected in summer (Saint Julien du Sault, France), (ii) a floor mat collected in a junkyard (Oberhausbergen, France), (iii) a yellowish, old, PU foam collected in a PU production plant (Saint Julien du Sault, France), (iv) a 2-year-old standing water in contact with concrete slab covered by PU varnish (Strasbourg, France), (v) a PU foam collected in a discharge (Strasbourg, France) and (vi) a greenish PU foam from an old office chair (Strasbourg, France).

Solid samples were ground and sieved to obtain homogeneous fragments. Liquid samples were centrifuged, and the pellets were recovered. Each sample was dispersed until adhesion on the four media used for fungal isolation: MM and Potatoes Dextrose Agar (PDA) with and without 2 g l À1 of Impranil-DLN â , kindly supplied by Covestro. Potatoes Dextrose Agar is a rich medium that allowed the recovery of a large number of fungi. On the contrary, MM provides extra sources of nitrogen and minerals to promote fungal growth while using the environmental sample or Impranil as carbon source. Growing fungi were successively plated until isolation. Strains isolated were finally plated on PDA for storage at 4°C. Degradation assays. Degradation assays on the four synthesized TPU were performed in 40 ml sterile polystyrene flasks. Each flask was weighted beforehand. TPU pieces of 2 9 2 cm were washed twice with distilled water, once with ethanol 70% and dried overnight at room temperature under a laminar flow hood. One piece of TPU was then added into each flask and weighted. 2 ml of MM without agar were added in each flask. Medium was inoculated using fungal agar plate culture and an inoculation loop of 1 ll.

After 2 months of incubation at 30°C, cultures were stopped. Supernatants were recovered for biochemical assays, if necessary. TPU pieces were washed twice with distilled water, once with ethanol 70%, dried overnight at room temperature and finally, weighted. Negative control consisted in TPU pieces incubated in liquid MM without fungal inoculation. For the initial screening, cultures were performed with only one repetition while the confirmation step was performed with at least four repetitions for each pair of fungus/TPU. Fungal identification. Selected strains were grown on PDA for 3 days at 30°C and sent to the accredited service for fungi identification of CABI (Wallingford, UK). All procedures were validated and the processing was undertaken in accordance with CABI's in-house methods for filamentous fungi and yeasts. Strains were identified by internal transcribed spacer (ITS) rDNA sequence analysis and in one case, partial beta-tubulin gene sequence analysis. Several algorithms were used for identification: the BLAST algorithm with the CABI Validated Sequence Database for Fungi, the FASTA algorithm with the Fungus database from EBI and the BLAST algorithm with the 'Others' database from NCBI, limited to type strains. Strains were deposited in CABI collection.

Nikon A1R laser confocal microscope (Nikon, Tokyo, Japan) equipped with a 1009 Plan Fluor water immersion objective was used for observation of filaments and spores of fungi.

Characterization

techniques. Scanning electronic microscopy (SEM) was used to study the evolution of the surface morphology of the degraded films. Vega-3 (Tescan, Brno, Czech Republic) apparatus in high vacuum mode with working distances in the range of 6-8 mm and an acceleration voltage of 5 kV was used. Before examination, samples were coated with a thin layer of gold with a gold coater (Quorum Q 150 RS; Quorum Technologies, Lewes, UK).

Infrared spectroscopy was performed with a Fourier transformed infrared spectrometer Nicolet 380 from Thermo Scientific (US) used in reflection mode equipped with an ATR diamond module (FTIR-ATR). An atmospheric background was collected before each sample analysis (32 scans, resolution 4 cm À1 ). FTIR analyses were performed on the washed samples after the 2 months of incubation.

Size exclusion chromatography (SEC) was used to determine the evolution of the polymers molar masses. TPU was dissolved in tetrahydrofuran (THF) at room temperature and filtrated at 0.2 lm with polypropylene membranes directly in vials. An Acquity APC (Waters, Milford, Massachusetts, USA) in THF at 40 °C was used for SEC analysis. Three columns (Acquity APC XT 450 A 2.5 lm 4.6 9 150 mm, 200 and 45) were connected. 10 ll of dissolved polymer solution were injected. A run of 11 min with a flow of 0.6 ml min À1 was applied. A calibration curve using polystyrene (PS) standards was built for molar mass determination.

Enzymatic assays. The supernatants of the degradation assays were recovered and centrifuged 10 min at 10 000 g in order to remove residues of fungi. pNitrophenyl acetate (pNPA; Sigma-Aldrich, Saint-Louis, Missouri, USA) and pNitrophenyl hexanoate (pNPH; TCI Europe, Zwijndrecht, Belgium) were prepared at 55 mM in dimethylsulfoxyde (DMSO). In a microcuvette, 10 ll of substrate in DMSO were added to 690 ll of MM and 300 ll of centrifuged supernatant. Absorbance was measured at 415 nm at room temperature. The molar absorptivity of pNitrophenol under these assay conditions was 57.4 mM À1 cm À1 . Activities were expressed as lmol of pNitrophenol s À1 ml À1 of supernatant.

Fig. 1 .

 1 Fig. 1. Evaluation of the weight losses on the different TPU (A) screening of the collection of fungi and (B) control of the activity of the three selected strains.

Fig. 2 .

 2 Fig. 2. Strains selected for TPU biodegradation (A) observation on PDA after 4 days of growth at 30°C and (B) optical microscope observations of hyphae and conidiophores/conidia (Bars represent 25 lm).

Fig. 3 .

 3 Fig. 3. Observation of PCL-based TPU pieces incubated 2 months at 30°C (A) magnification of 45 (bars represent 1 mm), (B) magnification of 500 (bars represent 100 lm) and (C) magnification of 2000 (bars represent 20 lm).

Fig. 4 .

 4 Fig. 4. Observation of fatty acid dimer-based TPU pieces incubated 2 months at 30°C (A) magnification of 45, (B) magnification of 500 and (C) magnification of 2000.

Fig. 5 .

 5 Fig. 5. Semi-quantitative analysis of FTIR (A) for the PCL-based TPU and (B) for the fatty acid dimer-based TPU.

Fig. 6 .

 6 Fig. 6. Evaluation of esterase activity with pNPA and pNPH on the supernatant of fungal cultures of (A) PCL-based TPU and (B) fatty acid dimer-based TPU.

Table 1 .

 1 SEC analysis of degraded polymers (Mn and Mw expressed as g mol À1 ).

		PCL-based TPU				Fatty acid dimer-based TPU
		Neg. Ctrl.	Alternaria	Penicillium	Aspergillus	Neg. Ctrl.	Alternaria	Penicillium	Aspergillus
	Mn
	Mean	58 000	55 000	55 000	57 000	35 000	37 000	40 000	38 000
	SD	0	2000	6000	3000	1000	1000	1000	1000
	P-value		0.116	0.419	0.366		0.003	0.000	0.010
	Mw
	Mean	125 000	122 000	116 000	121 000	120 000	104 000	108 000	101 000
	SD	1000	8000	13 000	11 000	4000	3000	6000	4000
	P-value		0.439	0.148	0.267		0.001	0.017	0.001
	Disp
	Mean	2.17	2.21	2.10	2.13	3.43	2.79	2.70	2.69
	SD	0.01	0.05	0.04	0.08	0.06	0.05	0.10	0.14
	P-value		0.258	0.044	0.193		0.000	0.000	0.000
	Area
	Mean	99.5%	99.0%	98.5%	99.2%	99.0%	96.3%	94.9%	94.8%
	SD	0.0%	0.1%	0.5%	0.1%	0.1%	0.3%	0.3%	1.0%
	P-value		0.000	0.002	0.002		0.000	0.000	0.000

ª 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology., Microbial Biotechnology, 12, 544-555

  ª 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology., Microbial Biotechnology, 12, 544-555 following solutions, filtered at 0.22 lm with polystyrene filters (in this order): (i) a solution of MgSO 4 x 7H 2 O (250 lM), (ii) a solution containing FeCl 3 x6H 2 O (147 lM), ZnCl 2 (9 lM), CoCl 2 (6.5 lM), Na 2 MoO 4 x 2H 2 O (12 lM), CaCl 2 x 2H 2 O (10 lM), CuCl 2 x 2H 2 O (13 lM), MnCl 2 x 2H 2 O (14.6 lM), H 3 BO 3 (12 lm) and finally, (ii) a solution of NH 4 SO 4 (7.6 mM). Streptomycin at 60 lg ml À1 final was added in the MM to prevent bacterial growth.

	Polyurethane substrate. The waterborne polyester PU
	dispersion used for this experiment is Impranil-DLN â ,
	kindly supplied by Covestro (Leverkusen, Germany).
	Four representative TPU substrates with different macro-
	molecular architectures (i.e. PCL-based TPU, fatty acid
	dimer-based TPU, polyether-based TPU, and poly(ester
	ether)-based TPU) were synthesized. The list of polyols
	used for TPU syntheses is presented in Table 2. The
	4,4 0 -methylene diphenyl diisocyanate (4,4 0 MDI, Suprasec
	1306 -Mn = 250.25 g mol À1

Table 2 .

 2 Polyols used for TPU synthesis. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology., Microbial Biotechnology, 12, 544-555

		Supplier of		Chemical structure of the main
	Commercial name	the polyols	Composition	components of the polyols
	Capa 2302	Perstorp	Polyester: polycaprolactone (PCL)		O
				O
				*		*
					(CH2)7 COOH
				HOOC(H2C)7 HC
	Radia 7285	Oleon	Polyester based on fatty acid dimer
			from rapeseed oil and short diol
					H 3C(H2C)5
					(CH2)5 CH 3
	Terathane 2000	Invista	Polyether: Polytetrahydrofuran (PTHF)	*	O	*
	Stepanpol PD56	Stepan	Polyester based on diethylene
			glycol and phthalic anhydride		O	O
					O
				O	O
	ª 2018 The Authors.

ª 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology., Microbial Biotechnology, 12, 544-555

Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 633962 with P4SB program.

research and innovation program under grant agreement no. 633962.

Conflict of interest

None declared.

Supporting information

Additional supporting information may be found online in the Supporting Information section at the end of the article.


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