Sarah Caradec

Vincent Grossi
email: grossi@com.univ-mrs.fr

Franck Gilbert

Catherine Guigue

Madeleine Goutx

Influence of various redox conditions on the degradation of microalgal triacylglycerols and fatty acids in marine sediments

Keywords: Triacylglycerols hydrolysis, Fatty acids, Redox conditions, Nannochloropsis salina, Kinetics of degradation, Bioturbation, Superficial sediments

Sediment cakes, supplemented with microalgal cells (Nannochloropsis salina), were incubated for 35 days under permanently oxic, oscillating (5d:5d changeover oxic/anoxic) and strictly anoxic conditions of oxygenation in diffusively ''open'' sedimentary systems. Total lipids (T Lip ) and triacylglycerols (TG) concentrations were monitored by thin layer chromatography-flame ionisation detection, whereas the concentrations of the main extractable (free+ester-bound) individual fatty acids (C 16:0 , C 16:1 , C 18:1 ) were followed using gas chromatography-mass spectrometry. Under the three conditions of oxygenation, TOC, T Lip and TG showed a sharp decrease in concentration during the early days of incubation and seemed to stabilise thereafter, defining an apparent non degradable fraction (G NR ). The G NR content was systematically higher in the anoxic incubation than under the oxic and oscillating conditions. The ratio of the main hydrolysis products of TG versus TG [(Free fatty acids+Monoacylglycerols+1,2-Diacylglycerols)/TG], used as an indicator of the hydrolysis of TG, showed that the presence of oxygen in the sediments (oxic and oscillating conditions) stimulates the hydrolysis of TG and the subsequent degradation of their metabolites. Unlike TOC, T Lip and TG, individual fatty acids (FA) showed a continuous concentration decrease until the end of the experiment, which was fitted with a simple first order model [G (t) =G 0 e Àkt ] to yield apparent degradation rate constants. The values observed under oscillating conditions (k FA =0.019 AE 0.001 d À1 ) were intermediate to those observed during oxic (k FA =0.029 AE0.003 d À1 ) and anoxic (k FA =0.011 AE 0.001 d À1 ) incubations, and no significant difference between individual FA could be observed. The production of saturated and monounsaturated C 16 (and to a lesser extent C 18 ) alkanols under oscillating and anoxic redox conditions suggested that (a part of) the dominant FA were reduced to the corresponding alcohols under anoxic conditions, following their release from acylglycerols.

Introduction

Senescent (phyto)plankton sinking through the water column constitutes a major source of organic detritus for marine sediments. Among the major constituents of plankton, lipids are often used as biomarkers to study the source, transformation and fate of organic matter [START_REF] Hedges | Early diagenesis consequences for applications of molecular biomarkers[END_REF]. The decomposition of lipids in near surface sediments is influenced by various factors such as their origin (aquatic vs. terrigeneous), their inherent structures (e.g. free molecules vs. combined forms), and the redox conditions encountered [START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in lipids under oxic and anoxic conditions[END_REF][START_REF] Hulthe | Effect of oxygen on degradation rate of refractory and labile organic matter in continental margin sediments[END_REF][START_REF] Sun | Rates and mechanisms of fatty acids degradation in oxic and anoxic coastal marine sediments of Long Island Sound, New York, USA[END_REF][START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF][START_REF] Sinninghe Damste´ | The influence of oxic degradation on the sedimentary biomarker record II. Evidence from Arabian Sea sediments[END_REF].

The influence of oxygen content on organic matter (OM) preservation has been much debated and studies comparing aerobic and anaerobic degradation have reached contrasting conclusions. Some studies have suggested that oxygen has no significant impact on the rate of OM degradation, which may rather be influenced by the age, origin, chemical composition or the structure of the OM [START_REF] Kristensen | The fate of organic carbon and nitrogen in experimental marine sediment systems: influence of bioturbation and anoxia[END_REF][START_REF] Lee | Controls on organic preservation: the use of stratified water bodies to compare intrinsic rates of decom-position in oxic and anoxic systems[END_REF][START_REF] Kristensen | Aerobic and anaerobic decomposition of organic matter in marine sediment: which is fastest?[END_REF]. Other studies showed that the degradation of a variety of organic components, including lipids, is largely controlled by oxygen and that aerobic degradation is generally faster than anaerobic degradation [START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in lipids under oxic and anoxic conditions[END_REF][START_REF] Bianchi | Breakdown of phytoplankton pigments in Baltic sediments: effects of anoxia and loss of deposit-feeding macrofauna[END_REF][START_REF] Sun | Rates and mechanisms of fatty acids degradation in oxic and anoxic coastal marine sediments of Long Island Sound, New York, USA[END_REF][START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF], indicating a dominant control of oxygen on OM preservation. It was also demonstrated that organic carbon burial efficiency is inversely correlated with the length of time accumulating particles are exposed to molecular oxygen in sedimentary pore waters [START_REF] Hartnett | Influence of oxygen exposure time on organic carbon preservation in continental margin sediments[END_REF][START_REF] Hedges | Sedimentary organic matter preservation: a test for selective degradation under oxic conditions[END_REF]. At a molecular level, it seems that no single rule can be drawn as the two lines of thought may overlap. The degradation of organic substrates reflects both the influence of the chemical structure and the presence of oxygen. For instance, fatty acids can be degraded at similar rates independently of their degree of saturation in the presence of oxygen, whereas unsaturated acids are preferentially degraded under anoxic conditions [START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in lipids under oxic and anoxic conditions[END_REF][START_REF] Sun | Rates and mechanisms of fatty acids degradation in oxic and anoxic coastal marine sediments of Long Island Sound, New York, USA[END_REF][START_REF] Grossi | Anaerobic biodegradation of lipids of the marine microalga Nannochloropsis salina[END_REF].

In oxygenated sediments, bioturbation is a major process affecting redox conditions. Due to its reworking activity, the biological mixing alters aerobic/anaerobic boundaries and modifies the expression of the main bacterial processes, which influence the degradation of organic matter [START_REF] Kristensen | The fate of organic carbon and nitrogen in experimental marine sediment systems: influence of bioturbation and anoxia[END_REF][START_REF] Aller | Bioturbation and remineralization of sedimentary organic matter: effects of redox oscillation[END_REF][START_REF] Aller | Effects of particle and solute transport on rates and extent of remineralization in bioturbated sediments[END_REF]. In particular, the construction of tubes and burrows and their intermittent ventilation by benthic inhabitants allows the circulation of oxygenated water into anoxic parts of the sediment, which are thus subjected to oscillating conditions of oxygenation [START_REF] Forster | Continuously measured changes in redox potential influenced by oxygen penetrating from burrows of Callianassa subterranea[END_REF]. Although it has been demonstrated that bioturbation can have a significant impact on the burial and degradation of sedimentary lipid components (e.g. [START_REF] Gilbert | The in vitro influence of the burrowing polychaete Nereis diversicolor on the fate of petroleum hydrocarbon in marine sediments[END_REF][START_REF] Gilbert | In situ bioturbation and hydrocarbon fate in an experimental contaminated Mediterranean coastal ecosystem[END_REF][START_REF] Sun | Enhanced degradation of algal lipids by benthic macrofaunal activity: effect of Yoldia limatula[END_REF][START_REF] Ingalls | The influence of deposit-feeding on chlorophyll-a degradation in coastal marine sediments[END_REF][START_REF] Grossi | Burial, exportation and degradation of acyclic petroleum hydrocarbons following a simulated oil spill in bioturbated Mediterranean coastal sediments[END_REF][START_REF] Grossi | Burial and reactivity of sedimentary microalgal lipids in bioturbated Mediterranean coastal sediments[END_REF], the influence of redox oscillation on lipid degradation has been little studied. Using a diffusively ''open'' incubation sedimentary system that mimics varying conditions of oxygenation in bioturbated sediments, [START_REF] Sun | Laboratory studies of oxic and anoxic degradation of chlorophyll-a in Long Island Sound sediments[END_REF] showed that redox changes influenced the diagenetic pathways of chloropigments. More recently, using a similar experimental approach, these authors demonstrated that the frequency of oxic:anoxic oscillation could also alter the rates and pathways of degradation of individual cell-associated lipids [START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF].

Limited information is presently available concerning the influence of redox oscillation on the diagenesis of more complex phytoplanktonic lipids such as fatty acidcontaining macromolecular lipids (e.g. acylglycerols, phospholipids). To address this question, we used an experimental approach similar to that of [START_REF] Sun | Laboratory studies of oxic and anoxic degradation of chlorophyll-a in Long Island Sound sediments[END_REF][START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF] to study the degradation of microalgal triacylglycerols in parallel with that of individual fatty acids under varying redox conditions. Sediment cakes supplemented with microalgal cells (Nannochloropsis salina; Eustigmatophyceae) were incubated under three contrasting conditions of oxygenation: permanently oxic, oscillating (5d:5d changeover oxic/anoxic) and strictly anoxic. Using combined analytical approaches [thin layer chromatography-flame ionisation detection (TLC-FID) and gas chromatography (GC)], the fate of triacylglycerols (TG), their hydrolysis products [total free fatty acids (FFA), monoacylglycerols (MG) and diacylglycerols (DG)] and extractable (free+esterbound) individual fatty acids (FA) were monitored as a function of time and redox conditions.

Experimental

Materials

The sediment was collected using a multiple sediment corer (i.d. 15 cm) at the SOFI Station in the Gulf of Lion, Mediterranean Sea (43 04N, 5 08E; 170 m depth). At this station, the total organic carbon content of the sediment varies from 0.36 to 0.99% throughout the year [START_REF] Schaaff | Erosion of particulate inorganic and organic matter in the Gulf of Lion[END_REF]. The station is continuously oxygenated and the sediment has an oxic layer of ca. 2.5 mm. The sediment, preserved in isothermic bags, was transported to the laboratory where it was immediately stored at the in situ temperature (15 C). Subsequently, the top layer of sediment (0-1 cm) was passed through a 0.250 mm mesh sieve to remove macrofauna and debris.

The marine microalga N. salina (Eustigmatophyceae) was chosen because of its high lipid and TG contents [START_REF] Volkman | The biochemical composition of marine microalgae from the class Eustigmatophyceae[END_REF][START_REF] Schneider | Radiolabeling studies of lipids and fatty acids in Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga[END_REF]. It was grown non-axenically to stationary phase at 15 C in f/2 medium (20 l) under continuous light conditions. Cells were harvested by centrifugation after the stationary phase had been reached for several days. Cells were freeze dried and mechanically mixed (30 min) with 600 ml of sieved sediment. This increased the total organic carbon content (TOC) of the initial sediment from 0.37 to 1.6% and the total lipid content by ca. 54%.

Experimental set-up

The experimental set-up was based on the diffusively ''open'' incubation method developed by [START_REF] Aller | Open-incubation, diffusion methods for measuring solute reaction rates in sediments[END_REF]. The mixture of sediment and microalgal cells was poured into 44 polyvinylchloride (PVC) ring holders (68 mm i.d.Â2 mm thick). Two sediment plugs were immediately frozen (time zero) and 42 sediment plugs were distributed among nine containers (each containing 14 l of 0.2 mm-filtered seawater) and were incubated for 35 d (darkness; 15 C) under different conditions of oxygenation (Fig. 1a). The overlying water in two sets of three containers was continuously purged with either air or a mixture of N 2 -CO 2 (ca. 2.3% CO 2 ) to establish aerobic or strictly anaerobic conditions, respectively. The overlying water in the third set of three containers was purged alternatively with air or N 2 -CO 2 (5 d changeover), creating oscillating conditions of oxygenation in the containers (Fig. 1b). The thickness of the plugs (2 mm) and their TOC content ( <2%) allowed the complete penetration of oxygen into the sediment during the aerated conditions [START_REF] Gilbert | The influence of macrofaunal burrow spacing and diffusive scaling on sedimentary nitrification and denitrification: an experimental simulation and model approach[END_REF]. Every 2-10 d, duplicate plugs were removed from each set of containers and kept frozen until analysis.

TOC analysis

TOC was analysed with a NA 1500 NC Carlo Erba elemental analyser (Fisons) on samples pretreated with HCl to remove inorganic carbon.

Extraction of total lipids

The lipids of the initial N. salina cells as well as those of each sediment plug were extracted ultrasonically with methanol (Â2), methylene chloride-methanol (1:1, v/v; Â2) and methylene chloride (DCM; Â2). The combined extracts were concentrated by rotary evaporation and evaporated to dryness under nitrogen. Extracts were then split into sub-samples to analyse lipid classes by TLC-FID on one hand and individual fatty acids by GC and GC-MS on the other.

Analysis of lipid classes by TLC-FID

Lipid classes were separated on Chromarods SIII (0.9 mm diameter, 150 mm length, 75 mm silica thick) and quantified using a thin layer chromatograph-flame ionisation detector (TLC-FID) Iatroscan TH10 apparatus model MK-IV (Iatron, Japan); (hydrogen flow, 160 ml min À1 ; air flow, 2000 ml min À1 ). The separation scheme involved five elution steps in solvent systems of increasing polarity as described by [START_REF] Striby | Improvement in the Iatroscan thin-layer chromatographic-flame ionisation detection analysis of marine lipids. Separation and quantitation of monoacylglycerols and diacylglycerols in standards and natural samples[END_REF]. Lipid classes were quantified with an external calibration using a mixture of standard lipids.

Analysis of individual compounds by GC and GC-MS

Sub-samples of the total lipid extracts were saponified with 1N KOH in MeOH/H 2 O (1:1, v/v, reflux 2 h) to analyse free and ester-bound individual compounds. After extraction of the neutral lipids from the basic solution (n-hexane, 3Â20 ml), acids were extracted (DCM, 3Â20 ml) following addition of HCl (pH=2). Alcohols and fatty acids were converted to trimethylsilyl ether derivatives by reaction with bis-trimethylsilyl-trifluoroacetamide (BSTFA, Supelco) in pyridine (1:1 v/v; 30 min at 60 C), and were identified with a HP 5890 Series II Plus gas chromatograph coupled to a HP 5972 mass spectrometer [START_REF] Grossi | Anaerobic biodegradation of lipids of the marine microalga Nannochloropsis salina[END_REF]. Individual compounds were quantified with an external standard calibration (squalane and nonadecanoic acid for alcohols and acids respectively) by gas chromatography using a HP4890 GC/FID system equipped with a HP-5MS capillary column (30 mÂ0.25 mm i.d.Â0.25 mm film thickness; [START_REF] Grossi | Burial and reactivity of sedimentary microalgal lipids in bioturbated Mediterranean coastal sediments[END_REF].

Isolation and identification of TG fatty acids in N. salina

A total lipid extract of the initial culture of N. salina was separated by thin layer chromatography (silica gel G plates; thickness 0.25 mm) with n-hexane-diethylether-formic acid (85:15:1, v/v) as eluant. On a separate plate, standard lipids (triolein and olive oil, Sigma) were eluted and revealed using iodine to determine the response factor of triacylglycerols (R f =0.39). The microalgal TG band [not revealed with iodine in order to avoid the degradation of polyunsaturated fatty acids (PUFAs)] was then scraped off the former TLC plate and TG were ultrasonically extracted from the silica before being saponified as described above. The released fatty acids were silylated and identified by GC-MS. Another total lipid extract of N. salina was directly saponified to determine the total free plus esterified fatty acid composition of the initial culture.

Kinetics of decay

For those classes of organic components whose concentrations stabilised before the end of the experiment (TOC, T Lip , TG), we defined a simple 1-G model in which a reactive fraction (G 01 ) follows an exponential decrease in concentration with time (i.e. first-order decay, k 1 ) and a resistant fraction which is not degraded during the experiment (G NR ; cf. [START_REF] Westrich | The role of sedimentary organic matter in bacterial sulfate reduction: the G model tested[END_REF]. Attempts to use a 2G-model did not allow us to distinguish two fractions having distinct reactivity (G 01 and G 02 ; [START_REF] Westrich | The role of sedimentary organic matter in bacterial sulfate reduction: the G model tested[END_REF][START_REF] Sempe´re´ | Microbial decomposition of large organic particles in the Northwestern Mediterranean Sea: an experimental approach[END_REF]. The decrease in concentration was fitted using a least square minimization method (Solver function of Excel) of concentration versus time using the equation: G T (t)=G 01 [exp(Àk 1 t)]+G NR , where G T (t)=total concentration at time t.

Since individual fatty acid (FA) concentrations did not stabilise over the 35 d incubation, no G NR fraction could be considered. The decrease in concentration of these compounds was fitted using the equation G (t) =G 0 e Àkt (where G (t) =concentration of a component at time t and G 0 =original concentration). Apparent degradation rate constants (k) were obtained from the slopes of log-transformed lipid concentrations versus time for each condition of incubation [START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in lipids under oxic and anoxic conditions[END_REF].

Results

Triacylglycerols and fatty acids of N. salina in the initial plugs

Iatroscan-analysed total lipids of the initial culture of N. salina revealed that TG were the major lipid class, representing 84% of the total lipid pool, whereas free fatty acids (FFA) accounted only for 3% of the total lipids. The mixing of the sediment with N. salina cells induced an enrichment of ca. 100% for both classes of compounds. However, the proportion of TG relative to the total lipid fraction in the initial sediment plugs was 46% due to the presence of other lipid classes (hydrocarbons, pigments, phospholipids) in the original sediment.

The fatty acid composition of the isolated TG of the initial culture of N. salina was dominated by C 16:0 , C 16:1(n-7) and C 18:1(n-9) components (32, 37 and 15% respectively), together with smaller amounts of C 14:0 , C 18:2 and C 18:0 fatty acids (4, 3 and 5% respectively). Other fatty acids (C 12:0 , C 15:0 , C 15:1 , C 17:0 , C 17:1 , C 20:0 , C 22:0 , C 24:0 ) had a minor contribution whereas, surprisingly, C 20 PUFAs (C 20:4 and C 20:5 ) were not detected in TG and accounted for less than 2% of the fatty acids present in the total lipids. Due to the strong predominance of TG in N. salina cells and of C 16:0 , C 16:1 and C 18:1 fatty acids in TG, we focussed our analysis of individual fatty acids on these latter three compounds. These FA were also present in the initial sediment but, for each component, the contribution from the sediment always accounted for < 4% of the amount detected in the plugs.

Patterns of TOC and total lipids decay

TOC showed a similar pattern of loss under the different conditions of incubation (Fig. 2a). TOC concentrations decreased exponentially (by ca. 80%) and seemed to reach a steady level by the end of the experiment. This suggests that only a reactive fraction of TOC (G 01 ) was degraded and that a refractory fraction (G NR ) remained after 35 days. Decay constants calculated by the 1G-model (see Experimental) indicated that TOC was degraded slightly faster under anoxic conditions than under oxygenated conditions, the lowest decay constant being observed for fully oxic conditions (Table 1). A larger proportion of refractory material remained, however, by the end of the anoxic incubation whereas oscillating redox conditions resulted in the more extensive degradation of TOC (Table 1). Total lipid concentrations also showed a similar pattern of decay under the three redox regimes. However, their concentration rapidly dropped during the first 10 days before reaching a threshold value below which no further degradation could be observed (Fig. 2b). This plateau value (corresponding to the refractory fraction G NR ) varied from one condition to another: after 35 days of incubation, 44, 49 and 59% of total lipids remained under oxic, oscillating and anoxic redox conditions, respectively (Table 1). It is noteworthy that the 1G-model fitted to the data could not calculate reliable decay constants of total lipids for oscillating and anoxic conditions (Fig. 2b, Table 1), which might be due to the lack of measurements over the first five days.

Patterns of TG and individual FA decay

The patterns of microalgal TG degradation under the different redox conditions resembled those of TOC and T Lip , showing a sharp decrease in concentration during the early days of incubation (10 d for oxic and oscillating conditions and 5 d for the anoxic incubation), followed by a steady level until day 35 (Fig. 2c). Like TOC and T Lip , the refractory fraction (calculated with the 1G-model) by the end of the incubation period was higher under anoxic conditions (Table 1). The initial rate of TG hydrolysis however appeared higher under anoxic conditions.

Free fatty acids (FFA), monoacylglycerols (MG) and diacylglycerols (1,2-DG and 1,3-DG) are the main acyl lipid breakdown products of TG through enzymatic or chemical hydrolysis [START_REF] Parrish | Dissolved and particulate marine lipid classes: a review[END_REF][START_REF] Goutx | Triacylglycerol biodegradation experiment in marine environmental conditions: definition of a new lipolysis index[END_REF]. FFA, MG and DG were not detected or were present in small amounts in the initial plugs, but were formed during the experiment. Although 1,3-DG could not be reliably quantified due to partial co-elution with other lipids during TLC-FID analysis, the ratio (FFA+ MG+1,2-DG)/TG could be used as an indicator of the hydrolysis of TG in the sediment plugs (Fig. 3). This ratio peaked at days 10 and 20 under oxic and oscillating conditions respectively, whereas it remained roughly stable in anaerobiosis.

Parallel to TG and their hydrolysis products, the fate of the major extractable (free+ester-bound) individual fatty acids (C 16:0 , C 16:1 and C 18:1 ) was followed using GC-FID and GC-MS. Unlike the global compound classes (i.e. TOC, T Lip , TG), individual FA showed a steady decrease in concentration under all conditions (Fig. 4). The highest extents of degradation of individual FA after 35 days were observed during the oxic incubation (70-71%) and the lowest for the anoxic incubation (36-44%; Table 2). It is possible, however, that a longer incubation time would have resulted in further degradation and/or in the characterisation of a non-degradable fraction for individual FA. For each condition of oxygenation, individual FA showed similar apparent degradation constants (calculated using a model in which no G NR is considered, see Experimental), but a 2).

Formation of new compounds

During the experiment, C 15 and C 17 branched chain bacterial acids were formed, but they never accounted for more than 2% of the total fatty acids present in the plugs, suggesting a relatively low bacterial contribution to the fatty acid pool. On the other hand, a production of saturated and monounsaturated C 16 (and to a lesser extent C 18 ) alkanols was observed in the plugs incubated under oscillating and anoxic redox conditions (Fig. 5). In both conditions of incubation, the concentration of these alcohols started to increase around day 5 and reached a maximum or plateau value around day 20 before dropping. The maximum concentrations of these newly produced alcohols varied in duration and magnitude depending on the redox conditions, the greatest accumulation being observed under anoxic conditions (Fig. 5).

Discussion

Lipid composition of N. salina

The large amount of TG in the initial culture of N. salina is consistent with previous observations of high concentrations of these reserve lipids in Nannochloropsis cells. [START_REF] Volkman | The biochemical composition of marine microalgae from the class Eustigmatophyceae[END_REF] and [START_REF] Schneider | Radiolabeling studies of lipids and fatty acids in Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga[END_REF] observed that TG could represent 43-50% of the total lipids in Nannochloropsis species. Moreover, nitrogen deficient and light saturating conditions of growth of Nannochloropsis sp. resulted in very high proportions (50-79%) of TG in total lipids [START_REF] Suen | Total lipid production of the green alga Nannochloropsis sp. QII under different nitrogen regimes[END_REF][START_REF] Sukenik | Regulation of fatty acid composition by irradiance level in the Eustigmatophyte Nannochloropsis sp[END_REF]. As cells were harvested during the stationary phase, it is most likely that nutrient deficiency was responsible for the high TG content (84% of total lipids) of N. salina cells used for the present study.

Also, C 16:0 , C 16:1 and C 18:1 FA are classically reported as the major fatty acids of Nannochloropsis species, but they are generally accompanied by a large amount of eicosapentenoic acid C 20:5 ; [START_REF] Volkman | The biochemical composition of marine microalgae from the class Eustigmatophyceae[END_REF][START_REF] Zhukova | Fatty acid composition of 15 species of marine microalgae[END_REF][START_REF] Grossi | Anaerobic biodegradation of lipids of the marine microalga Nannochloropsis salina[END_REF]. [START_REF] Schneider | Radiolabeling studies of lipids and fatty acids in Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga[END_REF] did not detect this polyunsaturated fatty acid (PUFA) in the TG of a Nannochloropsis sp. culture, but found it in more polar lipid classes (monogalactosyl-and digalactosyl-diglycerides, phosphatidylglycerol). [START_REF] Sukenik | Regulation of fatty acid composition by irradiance level in the Eustigmatophyte Nannochloropsis sp[END_REF] also showed that cells of Nannochloropsis sp. grown under saturating light exhibited high contents of C 16:0 and C 16:1 FA and a low proportion of C 20:5 FA. In the present case, like the high proportion of TG, the absence of C 20:5 FA in TG as well as its very low concentration in the total lipids were likely due to nutrient limiting conditions at stationary phase and/or to saturating light exposure.

Degradation of total lipids and TG vs. TOC in the sediment plugs

In marine sediments, the relative proportions of labile and refractory components largely control the consumption rate of the OM pool and a wide range of decay rates have been reported for total pools and individual components [START_REF] Westrich | The role of sedimentary organic matter in bacterial sulfate reduction: the G model tested[END_REF][START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in lipids under oxic and anoxic conditions[END_REF][START_REF] Sempe´re´ | Microbial decomposition of large organic particles in the Northwestern Mediterranean Sea: an experimental approach[END_REF][START_REF] Hoefs | The influence of oxic degradation on the sedimentary biomarker record I: evidence from Madeira Abyssal Plain turbidites[END_REF][START_REF] Panagiotopoulos | Bacterial degradation of large particles in the southern Indian Ocean using in vitro incubation experiments[END_REF]. The different patterns of decay observed for TOC and T Lip (Fig. 2a andb; Table 1) might be due to different proportions of labile organic matter within the two pools. Indeed, the addition of fresh microalgal cells to the sediment induced a significant input of labile molecules (i.e. sugars, proteins and lipids) which strongly contributed to the overall degradation pattern of TOC. On the other hand, this enrichment represented only a 54% increase in T Lip due to the presence of (presumably refractory) lipids in the original sediment. Since T Lip concentration rapidly reached a threshold value representing 44-59% of the initial amount in the plugs (Table 1; Fig. 2b), it is likely that after the degradation of most reactive lipids, the original background lipids contributed significantly to the apparent non-degradable fraction (G NR ).

The rapid decrease in TOC and T Lip concentrations is in good agreement with the trends observed during the experimental bacterial decomposition of natural phytoplanktonic communities [START_REF] Westrich | The role of sedimentary organic matter in bacterial sulfate reduction: the G model tested[END_REF][START_REF] Harvey | Kinetics of phytoplankton decay during simulated sedimentation: changes in biochemical composition and microbial activity under oxic and anoxic conditions[END_REF][START_REF] Teece | Microbial degradation of the marine prymnesiophyte Emiliania huxleyi under oxic and anoxic conditions as a model for early diagenesis: long chain alkadienes, alkenones and alkyl alkenoates[END_REF]. Calculated degradation rate constants for TOC (k 1 =0.072-0.097 d À1 ; Table 1) were also in the same order of magnitude as those determined previously, using a 2G-model, for the most reactive fraction of TOC in marine sediments [START_REF] Westrich | The role of sedimentary organic matter in bacterial sulfate reduction: the G model tested[END_REF][START_REF] Rabouille | Recycling of organic matter in Antarctic sediments: a transect through the polar front in the southrn Ocean (Indian Sector)[END_REF]. Although initial TOC and TG decay rate constants appeared higher under anoxic conditions, the proportion of material that could be degraded throughout the 35 d period (G 01 ) was systematically higher under oxygenated conditions, suggesting a stronger capacity of aerobic processes to achieve low background concentrations of organic carbon in our experimental system.

Influence of oxygen on TG hydrolysis and FA degradation

The absence of MG and DG in the initial culture and their appearance together with FFA during oxic incubation (Fig. 3) demonstrated that hydrolysis of TG occurred [START_REF] Goutx | Triacylglycerol biodegradation experiment in marine environmental conditions: definition of a new lipolysis index[END_REF]. The increase in the ratio (FFA+MG+1,2-DG)/TG observed during the oxic and the oscillating incubations showed that the presence of oxygen in sediments stimulates the hydrolysis of TG (Fig. 3). This might also be influenced by the oxygen exposure time (OET) as suggested by the time-lag between the peaks observed under oxic and oscillating conditions. In this multi-component system, hydrolysis products of TG (FFA, MG and DG) did not accumulate as shown by the fluctuating hydrolysis ratio (Fig. 3). Moreover, since FFA could be produced simultaneously from MG, DG and TG, a decrease in the above-defined ratio implied that the released FFA were further degraded, presumably at faster rates than the hydrolysis of acyl groups. This was supported by the steady decrease in concentration observed for individual (free+ esterified) FA (Fig. 4). These observations are in good agreement with the results of [START_REF] Goutx | Triacylglycerol biodegradation experiment in marine environmental conditions: definition of a new lipolysis index[END_REF] who observed the concomittant hydrolysis of tripalmitin and FFA production/degradation during aerobic TG decay in bacteria-inoculated seawater. Also, high rates of hydrolysis of FA-containing lipids have been reported during aerobic incubation conducted either with soils [START_REF] Hita | Triglyceride degradation in soil[END_REF] or with archaeological ceramic potsherds [START_REF] Dudd | Assessing microbial lipid contributions during laboratory degradations of fats and oils and pure triacylglycerols absorbed in ceramic potsherds[END_REF], and an accumulation of FFA was never observed. This reflects the capacity of aerobic bacterial communities from varied environments to easily hydrolyse FA-containing lipids and to subsequently mineralise the released FA. Conversely, the low values of the (FFA+MG+1,2-DG)/TG ratio observed during the anoxic incubation (Fig. 3) suggested a low hydrolysis of TG to be likely due to a low efficiency of microorganisms to perform the hydrolysis of acyl groups in spite of their capacity to consume FFA. This agrees with the higher proportion of TG that remained in the plugs by the end of the anoxic incubation (ca. 62% of the initial amount), compared to oxic conditions (Table 1). The inefficiency of anaerobic bacteria to hydrolyse complex organic material has already been suggested [START_REF] Kristensen | Aerobic and anaerobic decomposition of organic matter in marine sediment: which is fastest?[END_REF][START_REF] Hulthe | Effect of oxygen on degradation rate of refractory and labile organic matter in continental margin sediments[END_REF][START_REF] Kristensen | Decomposition of plant materials in marine sediment exposed to different electron acceptors (O 2 , NO 3 and SO 4 2À ), with emphasis on substrate origin, degradation kinetics, and the role of bioturbation[END_REF].

The degradation rate constants of individual (free+ esterified) FA were consistent with those calculated for individual lipids in previous studies performed under either experimental (Grossi et al., 2003, and references therein) or natural [START_REF] Canuel | Reactivity of recently deposited organic matter: degradation of lipid compounds near the sediment-water interface[END_REF] conditions. They were, however, one to three orders of magnitude higher than those generally observed in strictly anoxic sediments [START_REF] Sun | Molecular evidence for degradation and preservation of organic matter in the anoxic Black Sea Basin[END_REF][START_REF] Grossi | Anaerobic biodegradation of lipids of the marine microalga Nannochloropsis salina[END_REF][START_REF] Grossi | Burial and reactivity of sedimentary microalgal lipids in bioturbated Mediterranean coastal sediments[END_REF]. Also, for the same FA under permanently oxic conditions [START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF] observed higher (3-4 times) degradation rates compared to the present case. Differences in degradation rates from one study to the other may be due to varied bacterial communities/ metabolism (e.g. facultative vs strict anaerobes) and/or to different experimental systems involving distinct diffusion characteristics. It should also be emphasized that differences in incubation time can significantly influence the calculation of apparent degradation rate constants and that rate constants obtained using different kinetic models (e.g. taking into account a G NR fraction or not, see Experimental) cannot be compared.

The extent and rates of degradation of individual (free+esterified) FA under oscillating conditions were within the interval of values calculated for oxic and anoxic incubations (Table 2). This is consistent with previous trends obtained either with similar open incubation systems [START_REF] Sun | Laboratory studies of oxic and anoxic degradation of chlorophyll-a in Long Island Sound sediments[END_REF][START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF] or with bioturbated sediment cores [START_REF] Grossi | Burial and reactivity of sedimentary microalgal lipids in bioturbated Mediterranean coastal sediments[END_REF]. Obviously, redox oscillation corresponds to an intermediate OET in comparison with oxic and anoxic exposure, likely resulting in an intermediate state of organic matter decomposition. Previous studies have shown, however, that redox oscillation can lead to a more complete decomposition of organic matter compared to oxic or anoxic conditions alone [START_REF] Hulthe | Effect of oxygen on degradation rate of refractory and labile organic matter in continental margin sediments[END_REF] or result in diagenetic properties more characteristic of completely oxidized conditions [START_REF] Aller | Bioturbation and remineralization of sedimentary organic matter: effects of redox oscillation[END_REF][START_REF] Grossi | Burial and reactivity of sedimentary microalgal lipids in bioturbated Mediterranean coastal sediments[END_REF]. In the present study, this latter feature was essentially verified for TG (and to a lesser extent for TOC and T Lip ) which showed a reactive fraction (G 01 ) under oscillating conditions much closer to the one observed for oxic incubations, independently of their apparent degradation rates (Table 1). The intermediate degradation observed for individual FA might be due to the the fact that their concentrations were still decreasing after 35 days of incubation (Fig. 4). Using different oscillation frequencies, [START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF] demonstrated that individual planktonic lipids degrade faster when the oscillation was more frequent and exposure time to oxygen was longer in duration. These observations together with our results for TG degradation support the theory, at the level of specific (class of) organic molecules, that organic carbon degradation/preservation in continental margin sediments is largely controlled by OET [START_REF] Hartnett | Influence of oxygen exposure time on organic carbon preservation in continental margin sediments[END_REF][START_REF] Hedges | Sedimentary organic matter preservation: a test for selective degradation under oxic conditions[END_REF].

Possible degradation pathways

During the incubation of a 13 C-labelled green alga (Chlorella) under varying conditions of oxygenation, [START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF] observed the production of uniformlylabelled hexadecanol under anoxic and oscillating redox conditions. The authors suggested that it could be derived from reduction of labelled C 16:0 fatty acid, which was the most abundant lipid component in the 13 C-labelled alga. The noticeable production of saturated and monounsaturated C 16 and C 18 fatty alcohols during our oscillating and anoxic incubations (see Fig. 5 for C 16 ) might also be explained by the reduction of a proportion of the corresponding fatty acids under anoxic conditions, following the hydrolysis of acylglycerols (TG, DG and MG). The production of fatty alcohols coincided with a high level of TG hydrolysis (Figs. 2 and5), which supports a precursor-product relationship between FA esterified to TG and the alcohols detected. The greater accumulation of C 16 alcohols observed under anoxic conditions (Fig. 5) might reflect a lower efficiency of anaerobic bacteria for mineralising these compounds. For the oscillating conditions, it is possible that alcohols were produced under anoxia and consumed during oxic periods. This suggests that aerobic and anaerobic bacteria in the plugs used different assimilation pathways.

Conclusions

Incubation of microalgal cells in an experimental sedimentary system demonstrated that changes in redox condition can significantly affect the fate of fatty acid-containing macromolecular lipids such as TG. The presence of oxygen, either continuous or periodic, stimulates TG hydrolysis and the subsequent degradation of released metabolites (e.g. FFA), and leads to lower residual concentrations of such lipids than under anoxic conditions. This further emphasizes the importance of oscillating redox conditions induced by benthic macro-and meio-fauna in bioturbated sediments in regulating the degradation of oxygen-sensitive components.

The varying patterns of degradation observed under controlled conditions for both bulk parameters (TOC, T Lip ) and specific lipids (TG, individual FA) reinforces the idea that bulk parameters used to approximate the degradation of major biochemicals may be misleading as they integrate varying rates for individual compounds. On the other hand, this further highlights the necessity of using appropriate kinetic models in order to calculate reliable diagenetic parameters.

Fig. 1 .

 1 Fig. 1. (a) Open incubation experimental system (redrawn from Aller and Mackin, 1989); (b) Overlying water oxygen saturation under oscillating conditions.

Fig. 2 .

 2 Fig. 2. Concentrations of (a) Total Organic Carbon (TOC), (b) Total lipids and (c) Triacylglycerols (TG) in N. salina-enriched sediment plugs incubated under oxic (*), oscillating (&) and anoxic (~) redox conditions (each data point represents the average of duplicate samplesAEstandard deviation, except for TOC). The solid lines are curves fitted to a 1-G model of the data (see Experimental).

Fig. 3 .

 3 Fig. 3. Time-dependent ratio of the main TG hydrolysis products (FFA+MG+1,2-DG) vs TG during oxic (*), oscillating (&) and anoxic (~) incubations of N. salina-enriched sediment plugs.

Fig. 4 .

 4 Fig. 4. Concentrations of individual ''free+esterified'' fatty acids during oxic (*), oscillating (&) and anoxic (~) incubations of N. salina-enriched sediment plugs (each data point represents the average of duplicate samplesAEstandard deviation). The solid lines are curves fitted to a first-order degradation model of the data (see Experimental).

Fig. 5 .

 5 Fig. 5. Concentrations of (C 16:0 +C 16:1 ) alcohols during oxic (*), oscillating (&) and anoxic (~) incubations of N. salinaenriched sediment plugs (each data point represents the average of duplicate samplesAEstandard deviation).

Table 1

 1 Coefficients of the 1G-model (G T (t)=G 01 Â[exp(Àk 1 t)]+G NR ) adjusted to TOC, total lipid and triacylglycerol concentrations (least-square minimization, Newton method) in sediment plugs incubated for 35 days under varying redox conditions a

	Lipid	Coefficients	Oxic	Oscillating	Anoxic
	TOC	G 01		82	86	77
		k 1		0.072	0.086	0.097
		G NR		18	14	23
	Total lipids	G 01		56	51	41
		k 1		0.58	n.d.	n.d.
		G NR		44	49	59
	TG	G 01		70	65	38
		k 1		0.35	0.30	0.50
		G NR		30	35	62
	n.d.: the 1G-model did not afford reliable k 1 values.
	a G 01 : effective reactive pool; k 1 : exponential decay rate
	constant of G 01 (d À1 ); G NR : refractory pool. G 01 and G NR are
	expressed in percentage of the initial amounts.
	Table 2
	Apparent first-order degradation constants (k, day À1 ), deter-
	mination coefficients (r 2 ; n=15) and extent of degradation
	(E.D.,%) of the dominant extractable (free+esterified) indivi-
	dual fatty acids in sediment plugs incubated for 35 days under
	varying redox conditions
	Fatty Acid		Oxic	Oscillating	Anoxic
	C 16:0 FA	k (r 2 ) 0.030 (0.89) 0.018 (0.77) 0.011 (0.87)
		E.D.	70	58		44
	C 16:1 FA	k (r 2 ) 0.031 (0.87) 0.019 (0.83) 0.011 (0.64)
		E.D.	71	54		36
			70	59

C 18:1 FA k (r 2 ) 0.026 (0.79) 0.020 (0.85) 0.012 (0.71) E.D.

Acknowledgements

This work was funded by the program ATI 1998 (CNRS-INSU; grants to V.G. and F.G.). We thank Ms D. Raphel for help with microalgal culture, Dr. F. Van Wambeke for help in the calculation of 1G-model parameters and Ms C. Griffin for careful English reading. We are grateful to Dr. Mark Teece, Dr. J.K. Volkman and two anonymous reviewers for constructive suggestions about an earlier version of the paper. This is Nereis Park contribution number 004.

Associate Editor -J.K. Volkman


Camille Teruel
email: camille.teruel@gmail.com

Stéphane Ducasse
email: stephane.ducasse@inria.fr

Marcus Denker
email: marcus.denker@inria.fr

Toward a modularization of Pharo : Analysis of the design space for a new module system

Keywords: object oriented programming, Pharo, modularization, local class extensions, dependency resolution

Pharo est un langage de programmation orienté objet et réflexif descendant de Smalltalk. Actuellement, il ne propose pas de notion de module ou d'espace de noms. Toutes les classes et les variables globales sont visibles depuis l'ensemble du système ; de nombreuses dépendences peuvent donc apparaître et rendre le système monolithique. Pharo doit donc fournir un nouveau concept pour permettre sa modularisation. Transformer Pharo en système modulaire n'est pas une tâche facile : pour permettre la migration, ce nouveau concept doit supporter les extensions locales de classes et les dépendances circulaires. De plus, l'espace des solutions est large : non seulement le modéle doit définir la sémantique des extensions locales de classes, mais il doit également faire de même pour la visibilité et pour la paramétrisation des modules. Dans ce contexte, cet article présente une analyse de l'espace de conception pour un système de module au travers de trois axes de conception : les extensions locales de classes, la résolution des dépendances et la paramétrisation.

Introduction

Modularity is considered to be a desirable feature for a software system. It aims to improve reuse, maintenance and independent development, making development more efficient. The main goal is to be able to build new software systems by combining reusable subsystems together. Most programming languages come with some built-in modular construct that aims to support modularity at the language level. These constructs bear miscellaneous names and support various sets of features accordingly to their own concerns. Because most of these names are widely used in computer sciences and software engineering (like component and package for example), we will refer to these constructs via the generic term module.

Definition

All successful programming languages need to cope with huge code bases at some point. Therefore, all modules are born because of the same need : to put related things together. The first concern of a module is thus to bundle correlated programming entities into a new one. The nature of these entities depends mostly on the language paradigm : in functional languages like OCaml these entities are usually types and values [START_REF] Leroy | A modular module system[END_REF] whereas in object-oriented languages they are mostly classes or objects. The following definition of module conforms to this concern :

A module is a capsule containing (definitions of) items. The module draws a strong (syntactic) boundary between items defined inside it and items defined outside in other modules [START_REF] Szyperski | Import is not inheritance -why we need both : Modules and classes[END_REF].

Modules should also be concerned with visibility in order to control the access of the entities they contain. This feature is called information hiding. The granularity and the modalities of the control can vary from one module concept to another. In most languages, information hiding is limited to a set of fixed policies 1 . Other modules can provide more sophisticated control means. For example, a single module can expose different views of itself 2 , helping the developer to produce a clean interface segregation that leads to narrow role-oriented APIs. Even more advanced mechanism that involves users, groups and permissions may be conceivable. This second concern is also found in another definition of module :

Modules are program units that manage the visibility and accessibility of names. A module defines a set of constant bindings between names and objects [START_REF] Wirfs | An overview of modular Smalltalk[END_REF].

Information hiding and bundling of related entities are orthogonal concerns. This report discuss the latter. But this concern implies another one : dependency management. Indeed, even if it is possible to design self-sufficient sets of related entities, in practice most of them have dependencies. Consequently, we state that a module is a language construct that at least permits to bundle, control the visibility and manage the dependencies of a set of related software entities. In addition, a modular construct can provide additional features. For example, some module have been seen acting like mixins [START_REF] Matsumoto | Ruby in a Nutshell[END_REF] (as in Ruby), others provide dynamic linking mechanisms [START_REF] Flatt | Units : Cool modules for hot languages[END_REF] (as in Racket) while others got unified with objects [START_REF] Bracha | Modules as objects in newspeak[END_REF]. Even more specific features have been proposed like support for late bound classes [START_REF] Nystrom | Scalable extensibility via nested inheritance[END_REF] and class extensions [START_REF] Bergel | Classbox/J : Controlling the scope of change in Java[END_REF][START_REF] Wirfs-Brock | Subsystems -proposal[END_REF]. Since class extensions are widely used in Pharo, this latter feature will be discussed in details later.

Vocabulary

In this section, we define the vocabulary used in the rest of the paper to avoid confusion. -A binding is the association of a name and a value.

-An environment is a structure that maps names to values, it consists of a list of bindings. The term namespace is also common. -A module is a programming unit that provides an environment for a set of immutable objects.

These objects can either be classes or base objects that represent constants. The classes defined by a module may contain methods that refer to names that are not bound in the enclosing module. These names are called dependencies. -A configuration for a given module is a structure that maps its dependencies to object defined in other modules. -A module together with a suitable configuration is called a concrete module. Making a clear separation between the concepts of module, configuration and concrete module is mandatory to support generic programming. Indeed, it permits to parametrize a module with classes and base objects to give a new concrete module.

Context

Smalltalk is a fully reflective object-oriented language and platform. It has been inspiring a lot of object oriented languages over the years and has evolved into many variants. Pharo is one of these variants forked from another Smalltalk implementation called Squeak. Pharo inherits from the qualities from its ancestor, but also from their flaws : it has no notion of namespace nor of class encapsulation. After a quick glance at Smalltalk, we describe the current Pharo problems and presenting class extensions. Finally, we state a set of requirements that a module construct for Pharo must support.

A small Smalltalk overview

Smalltalk is an object-oriented programming language and platform that have the following properties : -Reflection : Smalltalk is said reflective because it reifies its own meta-model, a feature that permits to extend the language easily. -Purity : Smalltalk is a pure object-oriented language : everything is represented as an object, even primitive types and classes. -Image-based : Contrary to most programming language, Smalltalk does not rely on an underlying file structure to store the code. Instead, it uses an image i.e., a full snapshot of all the objects that exist in a given system. An image is thus an alive system that one can modify incrementally in order to develop a new software. -Late binding : Like most dynamic object-oriented languages, Smalltalk uses message passing and late-binding (sometimes called duck-typing). The syntax for message passing differs from the more famous Java one. The next example shows the differences.

bob send: letter to: alice. bob.sendTo(letter,alice);

Problematic The unique environment problem

In Pharo, all classes and globals are referenced via a unique global environment (i.e., a namespace), an instance of the class SystemDictionary. This environment is accessed via Smalltalk globals (see Figure 1). Note that each class has a back-pointer to the system dictionary. This pointer is a good extension point to introduce environments. Since a single name can refer to different objects in different software projects, name clashes can appear when one tries to integrate independently developed projects. In Pharo, the practice is to prefix class names with acronyms acting as poor's man namespaces. While such name clashes look problematic, relying on environments only defer the problem to module names. Indeed, programmers still have to pay attention that module names do not clash. Introducing environments in the form of modules is needed not only to manage name clashes, but more importantly to provide an abstraction supporting encapsulation for a set of related classes. This will help identifying dependencies and will serve as a basis for providing the mandatory information hiding principles.

Smalltalk

Smalltalk Image

Point Smalltalk

System

Class Extensions

Smalltalk makes heavy use of a feature called class extensions. A class extension consists in adding methods to a class that already exists in the system. Similar features in other dynamic languages are called Monkey Patching or Open Classes. In Figure 2, when the package Network is loaded, it adds a new method asUrl to the class String. When the package Network is unloaded, the asUrl method is then removed from the class String. Class extension is a powerful mechanism because a class can be extended without subclassing it. Whereas subclassing requires that instances of the new classes are used in place of the previous ones, the same instances can be used with class extensions. However, because the effect of a class extension is global i.e., visible from everywhere in the system, this concept brings several issues : -The method dictionaries of extended classes get polluted. A client can see methods that is not supposed to use. -Conflicts can appear when two independent developed modules add the same method to a class. In this case, the last loaded module destroys the class extension from the other module. Thus loading a module can break other independent modules. -These new methods usually depend on external classes. This means that new dependencies are added in the extended classes. Moreover, most of the time these dependencies are located in the same module than the class extension and a circle appears in dependencies. The introduction of a module concept is the occasion to revisit the scope and semantics of class extensions to make them local to a module. There are two choices to express the locality of a class extension : a classboxes semantic [START_REF] Bergel | Classbox/J : Controlling the scope of change in Java[END_REF][START_REF] Bergel | Classboxes : Controlling visibility of class extensions[END_REF] or a selector namespaces one [START_REF] Wirfs-Brock | Subsystems -proposal[END_REF]. Both solutions have advantages and drawbacks as we will discuss later.

Core

Migration constraints

Because Pharo had been built within a single namespace for years, a lot of dependencies appeared and led to a monolithic system. Transforming a monolithic system into a modular one is not an easy task because a lot of circular dependencies exist. Therefore, we need to have a module concept that accepts circular dependencies in order to support the migration. Obviously, having circular dependencies in a system is not a good idea, but these dependencies already exist and we must handle them, at least temporarily.

Requirements

So far, we have identified a few requirements a module construct for Pharo must fulfill : -A module is a construct that bundle related classes and objects together in a private environment. -A module must be able to embed extensions of external classes. These extensions must be visible from this module only. -Circular dependencies between modules must be supported.

Rationales for local class extensions

A class extension permits the programmer to add new methods to a class that already exists in the system. The problem is that a class extension is globally visible : it modifies directly the method dictionary of the extended class. The consequence is that a lot of circular dependencies appear as explained in the Figure 3. This figure represents the dependencies introduced by global (top) and local (bottom) class extensions. A module Network defines a class URL and adds a new method to the class String defined in the module Core. In the two cases the module Network depends on the module Core since it extends its class String. The problem with global class extensions is that the class String depends on URL and a circular dependency between Core and Network appears. In the second case the visibility of the class extension of String is limited to Network and the circular dependency disappears. It is an application of the Dependency Inversion Principle. Note that local class extensions bring other benefits : -They support both method addition and method redefinition.

-They avoid conflicts between different class extensions on a same method name.

-Because a module cannot bring changes outside itself, this solution respects the Open/Closed Principle [START_REF] Meyer | Object-oriented Software Construction[END_REF] which states that software entities must allow extensions, but forbid modifications.

Context-sensitive vs context-free execution

Local class extensions can modify the behavior defined by a class locally. The question is to know which instances are affected by this modification. The answer to this question determines what kind of locality is intended : -In a module, a class extension can affect every instance created within this module. These instances keep the same behavior all their life, even if they get referenced from another module. This means that the behavior of an instance is static and depends on where the instance is created. This kind of locality implies a context-free evaluation because the behavior of an object is determined statically. -Otherwise, a class extension can affect every instance referenced within the corresponding mo-dule. That means that instances of a class change their behavior dynamically according to the module that refers it. This kind of locality implies a context-sensitive evaluation. The problem of context-free execution is that several instances of the same class referenced from a single module can have different behaviors. Because a module cannot know if an instance of a class it extends is affected by this extension or not, it cannot use this additional behavior and the extension becomes useless. Consequently, context-free execution is inconsistent with the intended use of class extensions. Therefore, we need context-sensitive execution that comes at the price of additional steps in the lookup algorithm. Context-sensitive execution unavoidably decreases performances.

Local rebinding

One advantage of local class extensions is their ability to locally overrides methods. The question is to determine when these methods are taken into account by the method lookup algorithm. If from the point of view of the module defining a local class extension, instances of the extended class behave as if the local class extension was global, the modular construct supports local rebinding [?, ?]. This solution is really useful to handle unanticipated changes. It corresponds to the classboxes semantics. However, this semantics come at the cost of additional steps in the method lookup algorithm. These additional steps involve a performance loss and a modification of the virtual machine. Contrarily to classboxes, selector namespaces do not support local rebinding. The implementation of this mechanism can be done by prefixing every method's name with the name of the module that contains it. The method lookup takes the current module name into account to select the correct methods. All the prefixes are hidden from the developer. All methods are installed in the same dictionary but with different prefixes. This solution has the advantage to not slow down the execution and to not require virtual machine modification.

Design Space : Dependency resolution

Most constructs found in programming languages rely on explicit resolution hard-coded inside the construct (usually via import statements). However, it is not the only way to deal with dependency resolution. We present three variants : internal explicit resolution, external explicit resolution and lookup-based resolution. Then, we discuss the granularity of imports.

Explicit resolution

Explicit resolution is a trivial solution for managing dependencies. It can be either internal or external. Internal explicit resolution is found in most mainstream programming language via import statements (e.g., Java packages). We call it internal because dependencies are expressed inside the construct definition. The problem of this solution is that it doesn't permit to reconfigure a given module without reconstructing it and so without loosing its identity. External explicit binding however permits to change the configuration of a module on-the-fly. This kind of dependency resolution is also called dynamic linking [START_REF] Flatt | Units : Cool modules for hot languages[END_REF]. A similar idea is found in dependency injection frameworks where dependencies are injected into a class instead of letting a class make internal imports. The consequence is that it is possible to change the dependencies without modifying the class. It is another application of the Open/Close Principle [START_REF] Meyer | Object-oriented Software Construction[END_REF].

Lookup-based resolution

If the resolution is done via a lookup algorithm, that means that the topology of the module network is taken into account. This solution has two main flaws. First, you must find a solution with circles : ensure that a chain of module does not contain any circles or detect circle to stop the lookup. Secondly, the lookup can cross more than one module and so does dependencies. Therefore, the modification of a modules can have unexpected effects on indirectly related modules. For example, imagine a lookup chain made of three module : A -> B -> C. Lets say that a definition in A asks for the value associated with the name foo. The lookup begins in A which does not contains any value for the name foo. The lookup continue to B, which does not contain the binding so the lookup finally goes to C that returns the value of the binding it defines for the name foo. The consequence is that a modification of C can affect A whereas C is most probably not aware that A exists.

Granularity of resolution

For the granularity, imports can be enumerated explicitly or support wild-cards i.e., the importation of all the definitions contained in a given module. While wild-cards look appealing for the users, they can lead to problems when an application evolves. For example, while an application may have no naming conflicts, if one module introduces a class with a conflicting name, the user will have to address the conflict even if he did not change his own code. In addition, explicit import allows one to understand the interface size between modules that is a valuable information for refactorisation.

Design Space : Module parametrization

On certain occasions, the programmer may want to reuse a module but be able to specify certain parameters such as the classes to be used given a scenario. For example, we can imagine that we have a module for the CollectionTest and that we would like to reuse this module to verify if an alternate library fulfills the same specification. To support module parametrization, it is useful to make a clear distinction between an module and its configuration i.e., a set of external bindings. A module together with a suitable configuration gives a concrete module that is usable. The problem with module parametrization arises when a module uses a class parameter as a superclass of a class defined in the module. Indeed, the virtual machine refers to instance variables by integers that correspond to their offset. The methods of the subclass may refer to its instance variables but their offsets are not known. It is thus mandatory to specialize these methods when the parameter is given to adapt their offsets. All classes that can be passed as a module parameter don't have the same number of instance variable. That means that the methods objects of an abstract subclass cannot be shared among all the different concrete modules. Consequently, each time an abstract module is parametrized with a class it uses as a superclass, all the methods that refers to the instance variable of the abstract subclass must be copied in the concrete module and recompiled to adapt their offsets. This mechanism is called copy-down and is already used for mixin applications [START_REF] Bak | Mixins in Strongtalk[END_REF].

Conclusion

In this article, we proposed an analysis of the design space for a module construct that respects Pharo's specific requirements. During this analysis, we gathered a set of requirements that this construct may fulfill : -Local class extensions : the module construct must support local class extensions with method addition and method redefinition but without local rebinding. -Dependency resolution : resolution of dependencies must be explicit and external to a module.

Dependencies are fulfilled one by one : there is no wild-card for imports.

-Module parametrization : it must be possible to define abstract modules that can be parametrized with different configurations to give concrete modules. However, this set of requirements can be implemented in various ways. Our further work will focus on different implementations to measure their performances and their consequences on Pharo's maintainability. The road to the modularization of a system that consists of more than 5000 classes is not easy and will take a lot of time. Once the modular construct is ready to be used, we will have to migrate all development tools (class browser, version control manager, package management system, debugger...).

Dictionary globals Point environment FIGURE 1 -

 environment1 FIGURE 1 -Pharo single namespace named Smalltalk globals, instance of the class SystemDictionary.

FIGURE 2 -

 2 FIGURE 2 -Class extension and Package in Smalltalk. The class String is extended with a class extension asUrl.

FIGURE 3 -

 3 FIGURE 3 -Dependencies with global (top) and local (bottom) class extensions

FIGURE 4 -

 4 FIGURE 4 -Local rebinding : with local rebinding, C new foo evaluated in the context of M2 returns 'Extension'. Without local rebinding, the same expression returns 'Base class'.

  m2 := CollectionTest bind: #OrderedCollection to: MyCollection.


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apparatus and was typical of acid catalyzed aerogels. Conductivity measurements, operated on the ionogels, were carried out using an original electrical 4 electrodes set up which allowed us to study the ionic conductivity at the pore scale. The results obtained showed that one can model the electrical temporal response of the EMIMAc silica ionogel with a RLC series circuit.

234567894A63B

1 Ionic liquids (IL) are molten salt which are liquid at a temperature lower than 100°C. Since the first IL synthesis, chemists and material scientists have been highly interested in this new type of conducting liquids because of their numerous properties and potential applications like green solvent, electrolytes, sensors, catalysts or batteries … [1,[START_REF] Rakita | Ionic Liquids as green solvents: Progress and Prospects[END_REF]3].

Moreover, it is possible to synthesize nano-composites materials mixing IL with silica precursors, via a sol gel route. The first ionogel (for gel synthesized with an IL) was obtained in 2000 by S.Dai [4].

Several investigations led to a variety of materials [5,6,7,8] implying numerous characterizations: porous volume, fractal dimension, conductivity of the ionogel bulk ... [ 9,10,11,12,13,14].

Asymmetric cation, N,N'dialkylimidazolium or 1-butyl-3-methylimidazolium associated with anions such as tetrafluoroborate, were the most commonly used IL for a few years [9,15].

Here we chose to work with another imidazolium cation, 1-ethyl-2-mehylimidazolium bonded to the anion acetate (EMIMAc). This IL is known to be a good solvent for cellulose and it exhibits a very high electric conductivity (2500µS/cm) at ambient temperature. Compared to halogen anions, this IL is less corrosive and toxic.

The present study is interested in the synthesis and the characterization of EMIMAc silica ionogels and their corresponding aerogels. The first characterization is an electrical one in order to study the effect of the ionogel pore walls on the ionic conductivity: by an original 4-electrodes set up, we measured the temporal response of our ionogels to a voltage pulse and we modeled these responses by a RLC circuit.

Up to now, only two studies on ionogels conductivity were performed. One of these references dealt with two plate electrodes [10,16] and the other with a 3-electrodes set up (17). Textural and structural characterizations as nitrogen adsorption/desorption and SAXS were then carried out on the aerogels.

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The ionic liquid: 1-Ethyl-3methylimidazolium acetate (EMIM Ac) of purity 1 90%, and the silica precursors, tetramethoxysilane TMOS (98%) and methyltrimethoxysilane MTMS (98%), were from Aldrich.

Technical grade ethanol was used to extract water and IL from the gel before the CO 2 supercritical drying. Because of the hydrophilicity of IL, this last was heated under vacuum (70°C, 10 mbar) for 48h before each synthesis.

Gels were prepared via an hydrolytic one step process by mixing two solutions: solution (1) with TMOS and MTMS in different molar ratios and solution (2) containing IL and acidic water (HCl concentrations equal to 0.65N). This way was preferred in order to avoid the important shrinkage which is obtained using a non-hydrolytic process via formic acid. The molar ratio n IL /n Si was between 0.3 and 0.6 and for each sample, the hydrolysis molar ratio n water /n Si was equal to 16. After stirring, the mixture was poured into the conductivity cell and in molds for a further characterization of the ionogels. Gelling occurred after a time which depended on the HCl concentrations, on the IL ratio and the MTMS ratio. EMIMAc is a basic IL and despite of the dilution effect when the quantity of IL increased, the pH also increased and the gelation time decreased. Gels were transparent disks with a diameter of 16mm and a height of 4 mm without any cracks. All these steps were done at 22±2°C.

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The knowledge of the texture and the structure of (the) ionogels imply their drying under conditions which allows to obtain dry materials exhibiting properties very close to these(s) of (the) ionogels. For these reasons, drying must be carried out under supercritical conditions to avoid capillary stresses which would lead to dense and cracked materials. For the same reasons and to avoid the dissolution/redeposition phenomenon which occurs at high temperature and which can induce textural modifications at a very low scale [18], we chose to dry the gels under supercritical CO 2 so-called COLD process.

The wet gels were aged during 12h at 22±2°C. For a further CO 2 supercritical drying, the liquid that impregnates the wet gels must exhibit a total miscibility with CO 2 . So, gels, which contained water and the IL, were first soaked into ethanol for 15 days to obtain gels with pores mainly filled by ethanol.

Then ethanol were exchanged with liquid CO 2 at 8°C and 6 MPa during 5 hours. It is worth noting that IL is probably not totally removed from the gel during the soaking with ethanol and that one part of the IL is extracted by CO 2 . Finally, temperature and pressure were raised to 38°C and 10 MPa and after 10min, the pressure was released slowly during 12h. Samples obtained were translucent (figure 1) without any cracks and exhibited a shrinkage which depended on the composition of the gel.

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These measurements were performed on ionogels using a new set up described on figure 2.

The cylindrical cell allowing these measurements consisted of a Teflon mold containing the ionogel and of 4 Pt-electrodes partially immersed in the ionogel and passing through the sides of the mold. The 4 electrodes were Pt wires with a section of 0.5 mm. These wires were immersed in the bulk of gels along 7mm and fixed to the mold to make sure that all the measurements were done in the same conditions. The mold was cylindrical with a depth of 5 mm and a diameter of 15 mm. The 4 wires were connected to a pulse generator (Aligent 33220A, 20MHz, Arbitrary waveform generator) and to an oscilloscope (TDS2022C, 200MHz, Tektronik).

A rectangular voltage pulse was applied between 2 (channel 1) of the 4 electrodes. Then the potential difference was measured between these 2 electrodes (channel 1) and between the 2 other ones (channel 2).. First, we measured the electrochemical domain of stability of the EMIMAc. We found that for an applied potential equal to 4V and an offset equal to -1.375V, there was oxydo-reduction of the IL at room temperature. We checked that for an applied potential equal to 2.5V and an offset equal to -1.25V, the IL was stable. Straighforwardly, we chose to study the temporal response of our ionogel samples at this voltage.

The voltage pulses had an absolute value of 3.75V (applied potential 2.5V+ offset 1.25V), a frequency of 50HZ and pulse durations of either 20ns or 100ns This allowed us to measure the electrical response of the solvent contained in the porous network of the ionogels. Indeed, as the diameter of the electrodes is smaller than the depth of the set up (i.e. smaller than the height of the ionogel cylinder), these electrodes were always in contact with the IL contained in the pores. This lead to an electrical characterization of our ionogel samples at a microscopic scale.

Moreover, as we applied rectangular voltage pulses to the ionogel, we were able to analyze the temporal response of the sample.

In order to clearly identify the role of the silica network, a blank was first done on EMIMAc + HCl solution. Then electrical conductivity measurements were performed on the ionogel samples within 1 hour after gelling occurred.

The bulk densities of the samples ρ a were evaluated from their weight and linear dimensions.

For the textural characterization, nitrogen adsorption/desorption isotherms at 77K were carried out on a Micromeritic ASAP2010. Samples were outgassed under vacuum during 16h at 50°C. The specific surface area was obtained using BET theory. Its accuracy was about 4%. For rigid porous materials, when the nitrogen relative pressure P/P o gets close to 0.99, the volume of adsorbed nitrogen must correspond to the porous volume [19]. The dwell time, which is the interval of time allowing a pressure change of 0.01%, was 10 seconds. The pore size distributions was derived from BJH method from the desorption branches of the isotherm. SAXS (small angle X ray scattering) and WAXS (wide angle X ray scattering) experiments were performed with an in-house setup of the Laboratoire Charles Coulomb, using a X-ray tube GeniX 3D from Xenocs and a Schneider 2D imageplate detector prototype, in order to estimate the fractal dimension of the materials and the size of the constitutive particles and aggregates.

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1 Some results of electrical characterization of the ionogels lead to figures 3 and 4. The maximum of the measured amplitudes on both channel 1 and 2 are presented in table 3 for all the samples. For an input pulse duration of 20ns, the maximum of the response on channel 1 for all the ionogel samples was close to the value of the input voltage, within experimental fluctuations For an input pulse duration of 100ns, the maximum of the response on channel 1 for all the ionogel samples was larger than the input voltage For both durations, on channel 2, all the responses were damped.

For the system EMIMAc + HCl solution, the maximum of the response on channel 1 was lower than the input pulse but with oscillations whatever the pulse duration. An electrical modeling of these responses is presented in section Discussion. The relative shrinkage which occurred during the different steps, from the gel to the aerogel, and the characteristics of the aerogels are shown in table 2.

Most of the samples exhibited low densities and high specific surface areas. The specific surface area showed a tendency to increase up to a maximum value with the higher ratio of n MTMS /n TMOS while it was quite similar whatever the IL content.

A divergence between the measured porous volume V pm and the calculated one V pc was observed. This divergence, which clearly increased when the bulk density decreased, was linked to the aerogel strength and corroborated that the pore volume could not be easily obtained by nitrogen adsorptiondesorption, as reported in the literature [19]. We must also note that some IL probably remained in the aerogels (< 5% weight), as already described in the literature [11].

Nitrogen adsorption isotherms, given in Figure 5 for three compositions are of type IV. The pore size distributions, reported in Figure 6a and b, are given as an indication as it is known that these data carry some incertitude. However, they corroborate that materials are mesoporous ones. Aerogel made without MTMS displayed a broad pore size distribution. This distribution is increasingly narrow and shifted to lower diameters when the MTMS ratio is increasing (Figure 7a). However, distributions are slightly the same with the IL ratio increase (Figure 7b).

All the ionogels synthesized are fractals ones even if the fractal domain is relatively small (one decade)(figure 5).The fractal dimension is between 2.2 and 2.5, values which are characteristics of acid catalyzed aerogels. Samples C,D and E exhibit the same behavior with a larger fractal domain than sample A or B, and a fractal value around 2.5.

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1 First, we can note, that the presence of MTMS (hydrophobic groups) in the sol postpones the hydrolysis and condensation reactions and thus increases the gelation time.

It is worth noting that for all the samples investigated except one (sample A), the shrinkage after drying is rather low (<10 %). For sample A, which is obtained via a synthesis without MTMS, the soaking governs the shrinkage amplitude. Capillary tensions induced by IL exchange are probably too high comparatively to the strength of the silica network. We can make the hypothesis that the affinity of the IL for the silica surface increases these tensions and probably prevents IL from being completely removed. For others samples, the MTMS induces a repulsive wall/liquid effect (stable ≡Si-CH 3 groups on the surface of SiO 2 particles) and then reduces the capillary tensions. Consequently shrinkage decreases slightly as the increase of the MTMS content.

Though the bulk density of the samples C, D and E varies from 0,16 to 0,2 g.cm -3 depending on the IL ratio, their specific surface area does not depend on the density. Their structure and texture are very similar (Figure 5b, Figure 7b andtable 2).

On sample A, B and C, the methyl modification obviously acts on the specific surface area which increases with MTMS ratio. For the sample without MTMS, the fractal dimension is slightly smaller and the fractal domain is narrower. MTMS reduces cross-linkage and make the clusters grow slowly [21]. Regarding the aerogel texture, it is clear that as the n MTMS /n TMOS value increases the pore size distribution decreases towards the smaller pore sizes and becomes narrower [START_REF] Neouze | Ionogels obtenus par confinement d'un liquide ionique dans une matrice à base de silice[END_REF].

EMIMAc and MTMS induce noteworthy changings in the textures or structures of the aerogels, the effects on conductivities are shown below.

The electrical response of our ionogels samples is typical of a RLC series circuit. We show here in figures 8 a few examples of RLC series circuits signals to which a rectangular voltage pulse is applied. The shape of the response which is taken at the capacity component (C) in the simulation depends on the duration of the voltage pulse: if the duration of the input pulse is too short, the sample is in the electrical transient state, this is the case for pulses durations equal to 20ns in the experiments and 0.1s in the simulations. For longer pulse durations (here 100ns in the experiments and 0.4s in the simulations), the transient state is almost finished. In the case of responses measured on channel 2 (see section Results), the damping is due to the resistance of the IL either contained in the ionogels pores or in the HCl + IL solution.

This may be seen also in the simulations: fig. 8c which corresponds to channel2 in fig. 3 and4. The resistance leading to fig.8c is 15 times higher than for the other simulations. This means that on channel 2 the measured temporal resistivity is much larger than on channel 1. Indeed, the voltage is applied perpendicularly to channel2, so the displacement of the ions is limited between electrodes of channel2.

What is worth noting, is the difference between the responses of the ionogels samples and that of the HCl+IL solution: the highest amplitudes of the ionogels electrical responses on channel 1 are always larger than the responses of the HCl+IL solution. Let us make a simple model to explain this difference: the IL molecules in the solution have a larger mean free path because they are not embedded in a silica matrix but the direction of their displacement is random (Brownian motion). The same IL molecules when embedded in a ionogel pore, have a mean free path which is smaller (due to the pore walls) but their motion is oriented (in the pore direction). So when a voltage difference is applied, the charge carriers (IL ions, H + and Cl -ions in the ionogel pores) have a collective motion which leads to an appearing smaller resistance than the HCl+IL solution (when the applied potential and the measured electrical response are parallel).

To understand all the characteristics of ionic conduction in nanometric pores it would be necessary to perform numerical simulations by Brownian dynamics or Monte Carlo simulations.

To conclude this part of the discussion, we may say that we found an original method to characterize ionogels electrically. This method can be applied to other types of ionogels (with a different IL, different IL ratios etc.).

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We applied a rectangular voltage pulse to EMIMAc silica ionogels via a 4 electrodes set up : the temporal voltage response of our samples may be modeled by RLC series circuits. By comparison with an EMIMAc+HCl solution, the resistivity of the ionogels depends on the porous texture. Indeed, the ions within the ionogels pores have a collective motion induced by the pore walls contrarily to that in the EMIMAc+HCl solution where only thermal agitation acts. Indeed, textural characterization of the corresponding aerogels shows a significant effect of the TMOS/MTMS ratio on the pore network.

Finally, the structure is typical of acid-catalyzed aerogels.

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  Samples A, B, C, D, E have the same response on channel 1 within experimental variations Samples B,C, D, E have the same response on channel 2 within experimental variations Sample A has a response on channel 2 which is damped with respect to samples B, C, D, E and EMIMAc + HCl solution.

  represents the measured voltage on channel2. Although our simulations can only be qualitatively compared to the measured voltage on our ionogels, one may compare the parameters of fig.8a and 8b which correspond to fig.3b and fig.4b on channel1, to fig.8c

Figure 1 : 1 Figure 2 :

 112 Figure 1: Photographs of aerogel synthesized, samples (A) and (C)

1 1Figure 3 :Figure 4 : 1 Figure 5 :

 13415 Figure 3: electrical temporal responses to a rectangular voltage pulse (for a total amplitude of V=2.5V and a duration of t=20 ns) : (a) for an EMIMAc + HCl solution (b) for sample A

Figure 6 :

 6 Figure 6 : Adsorption/Desorption isotherms.

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 17118 Figure 7: Pore size distributions obtained from BJH method. (a) Effect of the MTMS/TMOS ratio (b) Effect of the IL ratio.

Table 1

 1

	lists the different samples synthesized by varying proportions of IL, MTMS and HCl
	concentration.

Table 1 :

 1 Compositions and gelation times for the studied samples 1

	1
	Sample	n IL /n Si	molar ratio	Concentration	Sol pH	Gelation time
			n MTMS /n TMOS	HCl (N)
	A	0.3	0		0.65		4.5	25 min
	B	0.3	0.2		0.65		4.5	35 min
	C	0.3	0.4		0.65		4.5	2h40
	D	0.45	0.4		0.65		4.5	1h40
	E	0.6	0.4		0.65		5.5	1h
	Sample	Shrinkage	Shrinkage	Bulk	Calculated*	Desorption	V pm /V pc	Specific
		after soaking	after	density	Pore volume	BJH Pore	(%)	Surface Area
		± 0.1 (%)	drying ± 0.1 (%)	ρ a ± 0.002 ( g.cm -3 )	V pc ± 0.1 (cm 3 .g -1 )	(cm 3 .g -1 ) volume V pm	(m 2 .g -1 )
	A	13.6	14.3	0,288	3.0	2,82	95	640 ± 25
	B	3	8.1		0,192	4,7	3,14	67	885 ± 35
	C	6	9.3		0,198	4,5	2,94	65	1090 ± 40
	D	6.6	7.8		0,173	5,3	3,29	62	1040 ± 40
	E	6.4	7		0,167	5,5	3,22	59	1050 ± 40

Table 2 :

 2 Shrinkage observed after each step of the drying and characteristics of the aerogels. V p c is calculated from the bulk density and the skeletal density of silica which has been estimated in the literature to about 2 g.cm -3 in silica aerogels[16]

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Table 3 :

 3 Conductivit y results for two voltage pulse durations 20ns and 100 ns measured on channel 1 and channel 2.