Dr A J Gross

Dr S Cosnier

Chapter 1: Buckypapers for bioelectrochemical applications

Introduction to buckypapers for bioelectrochemistry

 , buckypapers are also emerging for hybrid supercapacitors 10 , photoelectric biofuel cells 11 , and other bioelectronic systems including biosensors 4,12 , bioreactors 13 and biologic devices 14 .

 . Free-standing "lab-made" buckypaper sheets are finally obtained after washing, drying, and peeling from the underlying membrane.

"Commercial" buckypaper prepared by continuous manufacturing is a popular type of buckypaper.

.1.

Buckypaper fabrication is conceptually straightforward but factors such as dispersion homogeneity, CNT type and chemical functionality, membrane porosity, and the presence of

additives in the dispersion, all create differences with respect to material reproducibility and functionality. Several studies have focused on tuning the physical and mechanical properties of buckypaper such as porosity, Young's modulus, hardness and electrical conductivity [START_REF] Oh | Easy Preparation of Self-Assembled High-Density Buckypaper with Enhanced Mechanical Properties[END_REF][START_REF] Choi | A study on the effect of pyrene derivatives on the noncovalent sidewall functionalisation of carbon nanotube buckypapers[END_REF][START_REF] Chen | Highly conductive carbon nanotube buckypapers with improved doping stability via conjugational cross-linking[END_REF][START_REF] Li | A theoretical evaluation of the effects of carbon nanotube entanglement and bundling on the structural and mechanical properties of buckypaper[END_REF][START_REF] Yang | Tuning electrical and thermal connectivity in multiwalled carbon nanotube buckypaper[END_REF][START_REF] Young | Continuous Buckypaper Manufacturing Process: Process Investigation and Improvement[END_REF][START_REF] Wang | Nanoscale infiltration behaviour and through-thickness permeability of carbon nanotube buckypapers[END_REF][START_REF] Zhang | Roll-to-roll continuous carbon nanotube sheets with high electrical conductivity[END_REF][START_REF] Whitby | Geometric control and tuneable pore size distribution of buckypaper and buckydiscs[END_REF][START_REF] Shen | Carbon nanotube length governs the viscoelasticity and permeability of buckypaper[END_REF] . For example, Shen et al. investigated CNT length and showed how this parameter strongly governed the viscoelasticity and permeability of buckypaper [START_REF] Shen | Carbon nanotube length governs the viscoelasticity and permeability of buckypaper[END_REF] . Whitby et al. demonstrated that the porosity of buckypaper could be tuned using different casting solvents [START_REF] Whitby | Geometric control and tuneable pore size distribution of buckypaper and buckydiscs[END_REF] . Oh et al. revealed the crucial roles of CNT suspension concentration and filtration velocity for self-assembled alignment of buckypaper to enhance the mechanical properties [START_REF] Oh | Easy Preparation of Self-Assembled High-Density Buckypaper with Enhanced Mechanical Properties[END_REF] . In our recent electrochemical study, we compared the physical, chemical, electrochemical and bioelectrocatalytic properties of lab-made and commercial buckypapers [START_REF] Chen | Comparison of commercial and labmade MWCNT buckypaper: Physicochemical properties and bioelectrocatalytic O 2 reduction[END_REF] .

For bioelectrochemical applications, surfactant-free methods of producing buckypaper are highly desirable owing to the undesirable impact of residual surfactant on conductivity and biocompatibility risks such as cell lysis [START_REF] Gross | A high power buckypaper biofuel cell: Exploiting 1,10phenanthroline-5,6-dione with FAD-dependent dehydrogenase for catalytically-powerful glucose oxidation[END_REF][START_REF] Chen | Comparison of commercial and labmade MWCNT buckypaper: Physicochemical properties and bioelectrocatalytic O 2 reduction[END_REF][START_REF] Whitby | Geometric control and tuneable pore size distribution of buckypaper and buckydiscs[END_REF] . Buckypaper prepared from non-aqueous solvents such as DMF or alcohols without surfactant has been reported with success, for example, owing to improved CNTsolvent and the possibility to dissolve a wide range of chemical modifiers [START_REF] Gross | A high power buckypaper biofuel cell: Exploiting 1,10phenanthroline-5,6-dione with FAD-dependent dehydrogenase for catalytically-powerful glucose oxidation[END_REF][START_REF] Choi | A study on the effect of pyrene derivatives on the noncovalent sidewall functionalisation of carbon nanotube buckypapers[END_REF][START_REF] Roy | Formation of carbon nanotube bucky paper and feasibility study for filtration at the nano and molecular scale[END_REF][START_REF] Yang | Removal of natural organic matter in water using functionalised carbon nanotube buckypaper[END_REF][START_REF] Bourourou | Freestanding redox buckypaper electrodes from multi-wall carbon nanotubes for bioelectrocatalytic oxygen reduction via mediated electron transfer[END_REF] . However, the removal of non-aqueous solvents is crucial to minimize enzyme denaturation, microbe deactivation and material toxicity, in particular for in-vivo applications. Porous 3D-nanostructured carbon structures based on CNTs [START_REF] Cosnier | Recent advances in carbon nanotube-based enzymatic fuel cells[END_REF] , mesoporous carbons [START_REF] Funabashi | Hierarchical meso/macro-porous carbon fabricated from dual MgO templates for direct electron transfer enzymatic electrodes[END_REF][START_REF] Tsujimura | Exceptionally high glucose current on a hierarchically structured porous carbon el c d wi h "wir d" fl vin d nin dinucl id -dependent glucose dehydrogenase[END_REF] and carbon black [START_REF] Sugimoto | Effects of mesoporous structures on direct electron transfer-type bioelectrocatalysis: Facts and simulation on a three-dimensional model of random orientation of enzymes[END_REF] have become privileged conducting supports in bioelectrode design. Many of the best performing enzymatic bioelectrodes to date have been fabricated using CNTs due to their high specific surface area and exceptional electronic and mechanical properties, for example, that allow effective direct and mediated electron transfer with enzymes for bioelectrocatalysis [START_REF] Cosnier | Recent advances on enzymatic glucose/oxygen and hydrogen/oxygen biofuel cells: Achievements and limitations[END_REF][START_REF] Cosnier | Recent advances in carbon nanotube-based enzymatic fuel cells[END_REF] . Porous 3Dnanostructured carbon electrodes are particularly attractive due to the high surface/volume ratio which increases the loading of catalyst per geometric area. The size and curvature of pore and nanotube structures (e.g. nano-, micro-and meso-pores), and their surface chemistry, play a crucial role on the bioelectrocatalytic parameters of the electrode, such as mass transport, enzyme orientation and activity, and electron transfer kinetics [START_REF] Chen | Comparison of commercial and labmade MWCNT buckypaper: Physicochemical properties and bioelectrocatalytic O 2 reduction[END_REF][START_REF] Funabashi | Hierarchical meso/macro-porous carbon fabricated from dual MgO templates for direct electron transfer enzymatic electrodes[END_REF][START_REF] So | Analysis of factors governing direct electron transfertype bioelectrocatalysis of bilirubin oxidase at modified electrodes[END_REF][START_REF] So | Significance of the length of carbon nanotubes on the bioelectrocatalytic activity of bilirubin oxidase for dioxygen reduction[END_REF] .

An important aspect which is frequently overlooked in bioelectrode design is the development of porous electrodes with practical physical properties for their target application. For example, for wearable biofuel cell and biosensor applications, "skin-like" electrodes which are soft, bendable, and even stretchable are required [START_REF] Bandodkar | Wearable biofuel cells: A Review[END_REF][START_REF] Bandodkar | Non-invasive wearable electrochemical sensors: A review[END_REF] . For implantation, compact and lightweight electrodes are required with flexible geometries and superior stability and biocompatibility [START_REF] Gross | Buckypaper bioelectrodes: emerging materials for implantable and wearable biofuel cells[END_REF][START_REF] Katz | Implanted biofuel cells operating in vivo[END_REF] . Many bioelectrodes, including 3D-nanostructured electrodes, are prepared via the simple adsorption of nanocarbons (MgOtemplated carbon [START_REF] Funabashi | Hierarchical meso/macro-porous carbon fabricated from dual MgO templates for direct electron transfer enzymatic electrodes[END_REF] , mesoporous nanoparticles [START_REF] Trifonov | Enzyme-Capped Relay-Functionalized Mesoporous Carbon Nanoparticles: Effective bioelectrocatalytic matrices for sensing and biofuel cell applications[END_REF] , and Ketjen black [START_REF] Sugimoto | Effects of mesoporous structures on direct electron transfer-type bioelectrocatalysis: Facts and simulation on a three-dimensional model of random orientation of enzymes[END_REF] , for example) onto robust but bulky and non-flexible glassy carbon supports. The resulting thin films can also be fragile and prone to delamination from the electrode. Bulk CNT "pellet" electrodes formed by compression in the presence of enzyme emerged as a more practical solution to glassy-carbon-based electrodes implantable biofuel cells; however, the pellet electrodes were still quite cumbersome, fragile and brittle [START_REF] Cinquin | A glucose biofuel cell implanted in rats[END_REF][START_REF] Zebda | Single glucose biofuel cells implanted in rats power electronic devices[END_REF] . For wearable applications, carbon electrodes based on printed CNT inks and pellets supported on a flexible substrate have been developed [START_REF] Jia | Epidermal biofuel cells: Energy harvesting from human perspiration[END_REF][START_REF] Bandodkar | Soft, stretchable, high power density electronic skin-based biofuel cells for scavenging energy from human sweat[END_REF] .

Buckypaper is an alternative form of 3D-nanostructured electrode which is the electrode itself (no support is required) and has attractive qualities for interfacing biological and electrochemical systems for bioelectrochemical applications [START_REF] Gross | Buckypaper bioelectrodes: emerging materials for implantable and wearable biofuel cells[END_REF] . Compared to the existing CNT pellet bioelectrodes, for example, buckypapers offer a higher density of CNTs per surface area and improved stability owing to their more compact structure. A comparison of the quantity of enzymes used per electrode also reveals that buckypapers are more economical than CNT pellet electrodes [START_REF] Gross | A high power buckypaper biofuel cell: Exploiting 1,10phenanthroline-5,6-dione with FAD-dependent dehydrogenase for catalytically-powerful glucose oxidation[END_REF][START_REF] Abreu | Assembly and stacking of flow-through enzymatic bioelectrodes for high power glucose fuel cells[END_REF] . Nevertheless, buckypapers do suffer from being brittle and fragile in several cases. In addition, lab-made fabrication of buckypapers can also require the use of significant amounts of organic solvent.

Biological fuel cell devices

One of the important applications of buckypaper is in the field of biofuel cells [START_REF] Gross | Buckypaper bioelectrodes: emerging materials for implantable and wearable biofuel cells[END_REF] . Buckypaper electrodes have been used in implanted bioelectronic devices since 2012. Notably, enzymatic biofuel cells based on commercial buckypaper have been implanted in different animals and used for in-vivo energy harvesting and device powering via the organism [START_REF] Halámková | Implanted biofuel cell operating in a living snail[END_REF][START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF] . Biofuel cells are emerging power sources which can generate clean electrical energy from chemical substrates present in various media such as biofluids and environmental waters using enzymes (enzymatic biofuel cells) or microorganisms (microbial biofuel cells) as the catalysts [START_REF] Cosnier | Recent advances on enzymatic glucose/oxygen and hydrogen/oxygen biofuel cells: Achievements and limitations[END_REF] . Power is generated from bioelectrochemical reactions via the oxidation of fuels such as hydrogen and sugars (at the anode), and the reduction of oxidants such as oxygen (at the cathode). Biological fuel cells offer advantages compared to conventional batteries and fuel cells including the use of renewable and non-toxic catalysts and fuels. Enzymatic biofuel cells are considered as promising power sources for low-power electronic devices for short periods (e.g. 3 months to 1 year) whereas microbial biofuel cells are better suited for large-scale powering and industrial applications over longer periods (e.g. years). A short discussion highlighting some of the latest innovations in biofuel cell research, as well as some of the hype, can be found in our recent opinion article [START_REF] Cosnier | Beyond the hype surrounding biofuel cells: Wh ' h fu u f nzym ic fu l c ll ? Cu Opin El c ch m[END_REF] .

Implantable enzymatic biofuel cells

Katz and coworkers were the first to report the use of buckypaper for the construction of enzymatic biofuel cells. In their pioneering research, the buckypaper-based biofuel cells were implanted in snails, 17 clams 51 , rats [START_REF] Castorena-Gonzalez | Biofuel cell operating in vivo in rat[END_REF] and lobsters [START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF] and operated for durations ranging from a few hours to several weeks. The open circuit voltages (OCVs) and maximum power outputs of the biofuel cell devices ranged from 0.14 V to 0.54 V (up to 1.2 V for anode-cathode pairs connected in series) [START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF] and ca. 0.5 μW to 160 μW, respectively. The implanted biofuel cells exploited commercial MWCNT buckypaper (Buckeye Composites) modified with adsorbed pyrroloquinolinequinone-dependent glucose dehydrogenase (PQQ-GDH) at the anode, for glucose oxidation, and laccase from Trametes versicolor (TvLc) at the cathode, for oxygen reduction. The glucose/O 2 biofuel implanted in the shell of the snail which operates from the hemolymph is depicted in Figure 1.2. Buckypaper proved to be an effective choice due to its bioelectrocatalytic performance as well as its physical properties such as sub-mm (typically 5-200 µm) thickness, flexibility, and shapeability, which facilitated their insertion via small incisions into the organisms (e.g. in lobsters) [START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF] or their conformal contact with internal muscle tissues (e.g. in rats) [START_REF] Castorena-Gonzalez | Biofuel cell operating in vivo in rat[END_REF] .

In an early fascinating experiment, Katz and coworkers demonstrated that feeding the snail, or allowing it to rest for up to 1 hour, allowed the power output of the biofuel cell to be restored via substrate diffusion and metabolism [START_REF] Halámková | Implanted biofuel cell operating in a living snail[END_REF] . The stable operation of the implanted biofuel cell over two weeks highlighted practical stability and limited inhibition and fuel cell deactivation. Katz and coworkers later demonstrated the connection of implanted biofuel cells in series and the possibility to produce sufficient energy to turn an electric motor or an electronic watch [START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF][START_REF] Szczupak | Living batterybiofuel cells operating in vivo in clams[END_REF] . In another breakthrough, the connection of five buckypaper biofuel cells in series generated an OCV of 2.8 V in human serum, highlighting the potential of buckypaper biofuel cells to meet the ca. 1.4 V requirement to power a range of low-power microelectronic devices without a voltage boost convertor [START_REF] Lámk Vá | cyb " l b p c m k powered by implantable biofuel cells[END_REF] .

Wearable enzymatic biofuel cells

Enzymatic buckypaper electrodes have great potential in the emerging field of wearable biofuel cells owing to their attractive properties, depending on the fabrication methods used, such as their flexibility and skin-conformability under bending [START_REF] Gross | Buckypaper bioelectrodes: emerging materials for implantable and wearable biofuel cells[END_REF][START_REF] Bandodkar | Wearable biofuel cells: Past, present and future[END_REF] . Compared to implantable biofuel cells that require surgical procedures, wearable biofuel cells can be mounted on (i.e. non-invasive) or inserted in (i.e. minimally-invasive) the body without the need for surgical intervention. Wearable devices are therefore somewhat easier to develop and more user friendly compared to implantable devices. Skinbased wearable biofuel cells which exploit human sweat are popular as sweat is easily accessible, can be produced in high quantities, and contains attractive fuels such as lactate and glucose. Lactate is particularly attractive owing to its high concentrations of ca. 10 to 100 mmol L -1 compared to ≤ 1 mmol L -1 for glucose, for example [START_REF] Lee | Wearable/disposable sweat-based glucose monitoring device with multistage transdermal drug delivery module[END_REF][START_REF] Onor | Potentiometric sensor for non invasive lactate determination in human sweat[END_REF] .

The first lactate-oxidizing buckypaper bioanode was developed by Atanassov and coworkers.

Commercial MWCNT buckypaper (Buckeye Composites) was modified with methylene green as an electrocatalyst to regenerate dissolved NAD + cofactor, chitosan as a biocompatible stabilizing matrix, and adsorbed lactic dehydrogenase (LDH) from Lactobacillus leichmannii as the selective catalyst [START_REF] Villarrubia | Biofuel cell anodes integrating NAD(+)dependent enzymes and multiwalled carbon nanotube papers[END_REF] .

The bioanode was not tested in a fuel cell setup but could potentially be exploited. However, the catalytic current of 53.4 ± 5.1 μA cm -2 in buffer solution is low and the need to add the enzyme's cofactor are major limitations.

A practical lactate-oxidizing bioanode integrated into a hybrid photoelectric biofuel cell was recently reported by Dong and coworkers. In this work, MWNCT buckypaper (Buckeye Composites) was modified with Meldola blue, chitosan, and lactate oxidase (LOx) from Pediococcus sp [START_REF] Yu | Single wearable sensing energy device based on photoelectric biofuel cells for simultaneous analysis of perspiration and illuminance[END_REF] . In addition to serving as a redox mediator, the pi-pi stacked Meldola blue molecules on the CNTs shifted the buckypaper surface wettability from hydrophobic (˂100°) to hydrophilic (˂30°). The hydrophilicity is attractive to promote enzyme adsorption, during modification, and contact with electrolytic solution, during operation. The bioanode delivered high catalytic current densities of ca.

2.1 mA cm -2 in buffer solution and was successfully integrated into a wearable epidermic lactate/O 2 biofuel cell capable of powering a Bluetooth module and transmitting data to a smartphone (Figure 1.3).

Figure 1.3: Principle of a wearable hybrid biofuel cell with a polyterthiophene-coated indium tin oxide photocathode and a lactate-oxidizing buckypaper bioanode.

Buckypaper bioelectrodes have also been developed for energy harvesting from tear fluid to power 'smart' electronic contact lenses for applications in human vision and sensing [START_REF] Reid | Contact lens biofuel cell tested in a synthetic tear solution[END_REF] . It is envisioned that such biofuel cells could be used to power contact lenses worn by type I diabetics for non-invasive glucose monitoring [START_REF] Bruen | Glucose sensing for diabetes monitoring: Recent developments[END_REF] . Minteer and coworkers exploited commercial MWNCT buckypaper (Buckeye Composites) immobilized on an elastomeric contact lens [START_REF] Reid | Contact lens biofuel cell tested in a synthetic tear solution[END_REF] . Buckypaper was used to benefit from its high surface area, laser cuttability, durability and mechanical properties such as flexibility. For example, the authors reported that the buckypaper exhibited better mechanical properties compared to other conductive papers such as Toray paper and evaporated metal electrodes. The bioanode was constructed with NAD-dependent lactate dehydrogenase (NAD-LDH) from Escherichia coli modified with methylene green electrocatalyst and a cross-linked polymeric hydrogel matrix to entrap the NAD + cofactor and enzyme on the electrode surface. The use of immobilized NAD + is in principal more practical than the classical approach that requires cofactor to be added to the solution. A further advantage is that this strategy could be applied to a wide range of NAD-dependent enzymes. In addition to lactate oxidation, electrocatalytic ascorbic acid oxidation was also observed, although this had parasitic effects on the fuel cell. Lisdat and coworkers also observed parasitic effects due to ascorbic acid oxidation at buckypaper electrodes in human biofluids [START_REF] Göbel | Operation of a carbon nanotube-based glucose/oxygen biofuel cell in human body liquids-Performance factors and characteristics[END_REF] . For the biofuel cell developed by Minteer and coworkers, the lactate-oxidizing bioanode was combined with an oxygen-reducing biocathode prepared via oriented adsorption of bilirubin oxidase (MvBOx) on pyrenemethyl anthracene modified buckypaper. The biofuel cell delivered an OCV of 0.41 V and a small power output up to 8 μW cm -2 at 0.2 V in tear solution with stability for several hours [START_REF] Reid | Contact lens biofuel cell tested in a synthetic tear solution[END_REF] . The low power output and stability revealed that there is plenty of room for improvement. Improved performance could be achieved by removing the ascorbic acid interference, increasing the amount and stability of the immobilized enzymes and cofactor, and by reducing biocatalyst leaching.

The mechanical and chemical properties of buckypaper can also be improved for wearable and implantable applications. The strength and Young's modulus of buckypaper electrodes can be enhanced by incorporating functional polynorbornenes [START_REF] Aj | bifunc i n l buckypapers from carbon nanotubes and polynorbornene copolymers for flexible engineering of enzymatic bioelectrodes[END_REF] and molecular cross-linkers such as pyrene derivatives [START_REF] Choi | A study on the effect of pyrene derivatives on the noncovalent sidewall functionalisation of carbon nanotube buckypapers[END_REF][START_REF] Bourourou | Freestanding redox buckypaper electrodes from multi-wall carbon nanotubes for bioelectrocatalytic oxygen reduction via mediated electron transfer[END_REF] . The introduction of polynorbornene copolymers, as illustrated in Figure 1.4, revealed the possibility to introduce multiple functionalities to the buckypaper, such as pyrene groups, for cross-linking, and a reactive N-hydroxysuccinimide ester, for covalent tethering of molecules for enzyme wiring or the enzymes themselves [START_REF] Aj | bifunc i n l buckypapers from carbon nanotubes and polynorbornene copolymers for flexible engineering of enzymatic bioelectrodes[END_REF] . Alternatively, the strength, properties and hardness of buckypaper can be significantly enhanced by aligning the CNTs in the buckypaper with a high packing density [START_REF] Oh | Easy Preparation of Self-Assembled High-Density Buckypaper with Enhanced Mechanical Properties[END_REF] . Lab-made rather than commercial buckypapers may hold the key to improved buckypaper biofuel cell performance, for example, due to (i) the ability to better tune the chemical and physical properties of the bulk materials, (ii) their superior larger surface areas, and (iii) their apparently improved enzyme wiring for bioelectrocatalysis [START_REF] Gross | A high power buckypaper biofuel cell: Exploiting 1,10phenanthroline-5,6-dione with FAD-dependent dehydrogenase for catalytically-powerful glucose oxidation[END_REF][START_REF] Chen | Comparison of commercial and labmade MWCNT buckypaper: Physicochemical properties and bioelectrocatalytic O 2 reduction[END_REF] .

Portable enzymatic biofuel cells

Enzymatic glucose/O 2 biofuel cells have been designed for powering of portable electronics such as music players and toy cars [START_REF] S Ny C P I N S Ny D V L P ; Bi B Y | G n in El c ici y f m Su[END_REF] . One potentially exciting application is the development of eco-friendly paper-based power sources. Atanassov and coworkers initiated the development of MWCNT buckypaper-based paper biofuel cells that delivered up to 1.8 V (3 biofuel cells in series) and power outputs up to 13.1 mW under quasi-steady flow [START_REF] Villarrubia | Practical electricity generation from a paper based biofuel cell powered by glucose in ubiquitous liquids[END_REF][START_REF] Villarrubia | Self-feeding paper based biofuel cell/selfp w d hyb id μ-supercapacitor integrated system[END_REF] . In one example, a digital clock was powered using the commercial soft drink, Gatorade, which was supplied via cellulose filter paper. In addition to demonstrating the portable paper-based biofuel cell concept (using buckypaper from Buckeye Composites), this work nicely demonstrated the possibility to use commercially-available liquids rather biofluids or environmental matrices to generate power. The need to add NAD + cofactor for glucose oxidation at the NAD-dependent glucose dehydrogenase (NAD-GDH) anode is nevertheless a significant inconvenience [START_REF] Villarrubia | Practical electricity generation from a paper based biofuel cell powered by glucose in ubiquitous liquids[END_REF] . In later work by the authors, the same bioanode was combined with an air-breathing MWCNT buckypaper/Toray paper composite modified with MvBOx as the oxygenreducing biocathode [START_REF] Villarrubia | Self-feeding paper based biofuel cell/selfp w d hyb id μ-supercapacitor integrated system[END_REF] . This paper biofuel cell also benefited from a supercapacitive element that facilitated the production of mW power outputs, under pulse operation, and 4200 discharge/recharge cycles over a period of 3 days [START_REF] Villarrubia | Self-feeding paper based biofuel cell/selfp w d hyb id μ-supercapacitor integrated system[END_REF] .

The elegant concept of using cellulose paper as a transport medium to induce passive flow via wicking has also been explored for construction of a paper-based "enzyme cascade"-based biofuel cell. This novel type of biofuel device was developed to improve bioanode performance by exploiting sequential catalytic reactions and multiple fuels (Figure 1.5). Multiple NAD(H)-dependent enzymes were used at the MWCNT buckypaper (Buckeye Composites) anode for the multi-step oxidation of ethanol to acetate, and complete oxidation of methanol to CO 2 [START_REF] Lau | Paper based biofuel cells: Incorporating enzymatic cascades for ethanol and methanol oxidation[END_REF] . The ability to use air-breathing cathodes is a major advantage for portable biofuel cells compared to in-vivo biofuel cells due to the possibility to enhance oxygen delivery at the electrode interface.

The testing and optimization of MWCNT buckypaper-based air-breathing biocathodes with immobilized MvBOx has been reported [START_REF] Babanova | Design of experiments and principal component analysis as approaches for enhancing performance of gas-diffusional airbreathing bilirubin oxidase cathode[END_REF][START_REF] Santoro | Bilirubin oxidase based enzymatic air-breathing cathode: Operation under pristine and contaminated conditions[END_REF] . As well as air-breathing cathodes, the use of glucoseoxidizing anodes which exploit the emerging enzyme, FAD-dependent glucose dehydrogenase (FAD-GDH), is considered to be one of the most practical biofuel cell setups currently available. The use of FAD-GDH, which is oxygen insensitive (unlike glucose oxidase) and can now be obtained commercially, has only very recently reported in combination with quinone-modified buckypapers, and promisingly, gave high catalytic currents up to 1.97 mAcm -2 65 and 5.4 mA cm -2 6 . The integration of an FAD-GDH-based buckypaper anode in biofuel cells was reported but not yet in combination with an air-breathing cathode.

The integration of energy storage elements such as supercapacitors together with biofuel cell stacks, assembled in series or parallel, is expected to become increasingly important for the powering of portable miniaturized electronic devices [START_REF] Hou | An integrated device of enzymatic biofuel cells and supercapacitor for both efficient electric energy conversion and[END_REF] . Hou and Liu reported a flexible lab-made buckypaper biofuel cell device connected in series based on a FAD-GDH modified anode and a laccase modified cathode. In addition, a supercapacitor was incorporated based on polyvinylalcohol (PVA)-H 3 PO 4 and a MWCNTs-polyaniline composite [START_REF] Hou | An integrated device of enzymatic biofuel cells and supercapacitor for both efficient electric energy conversion and[END_REF] . The self-charging device delivered a charging voltage of 0.8 V and a power density up to 326 μW cm -2 (a two-fold increase in power compared to the biofuel cell without the supercapacitor). The capacitance of the MWCNTs/polyaniline electrode was observed to be 329 F g -1 at 10 mVs -1 . For comparison, Tran et al. reported a potentially better-performing supercapacitor based on the in-situ electropolymerization of polyaniline at buckypaper (397 Fg -1 at 10 mV s -1 ) [START_REF] Tran | High-performance supercapacitor electrode based on buckypaper/polyaniline composite[END_REF] . Furthermore, Gross et al. observed clear performance differences with FAD-GDH based bioanodes with different mediators [START_REF] Gross | A high power buckypaper biofuel cell: Exploiting 1,10phenanthroline-5,6-dione with FAD-dependent dehydrogenase for catalytically-powerful glucose oxidation[END_REF] . For example, it was demonstrated that around a 5-fold improvement in catalytic current density can be achieved by changing from a naphthoquinone (NQ) Enzymatic biofuel cells have also been explored as portable, low-temperature and sulfurinsensitive alternatives to solid oxide fuels cells that run on JP-8 jet fuel, for example, for military applications. Minteer and coworkers exploited an enzyme cascade of alkane monooxygenase (AMO) and alcohol oxidase (AOx) at a MWCT buckypaper (Buckeye Composites) anode for mediated bioelectrocatalytic oxidation of hexane, octane, and jet fuel [START_REF] Ulyanova | Bioelectrocatalytic oxidation of alkanes in a JP-8 enzymatic biofuel cell[END_REF] . This study demonstrated the possibility to exploit alkanes as fuels for biofuel cells for the first time, and additionally, showed the possibility to produce power densities up to 3 mW cm -2 without pre-processing of alkane-based fuels.

Microbial biofuel cells

Microbial biofuel cells which exploit electroactive microbes such as bacteria are an eco-friendly technology for applications such as energy-generating wastewater treatment, self-powered sensors, and large scale electricity production from biomass [START_REF] Santoro | Microbial fuel cells: From fundamentals to applications. A review[END_REF][START_REF] Pasternak | Self-powered, autonomous biological oxygen demand biosensor for online water quality monitoring[END_REF] . Compared to enzymatic biofuel cells, microbial biofuels typically give lower catalytic efficiencies but are better suited to large-scale energy generation and therefore larger raw power output. Bioelectrodes based on bacteria are also generally more durable under harsh and polluted conditions compared to enzymatic electrodes. A typical microbial biofuel cell consists of an anode with acidophilic bacteria on the electrode surface, for oxidation via metabolic processes, and a cathode, typically Pt or activated carbon, for oxygen reduction. The anode and cathode are classically placed in individual chambers and separated by an ion selective membrane.

In 2013, Santoro, Atanassov, Ieropoulos and coworkers reported for the first time the use of a microbe-based carbon cloth anode together with a MWCNT buckypaper (Buckeye Composites)carbon cloth enzymatic air-breathing cathode in a compartmentless hybrid biofuel cell [START_REF] Santoro | High power generation by a membraneless single chamber microbial fuel cell (SCMFC) using enzymatic bilirubin oxidase (BOx) air-breathing cathode[END_REF] . The biofuel cell operated in buffer with additional sodium acetate and wastewater solutions. A key observation was that the biofuel cell exploiting the oxygen-reducing BOx modified cathode exhibited a ca. 200 mV improvement in OCV compared to the biofuel cell with a Pt cathode under equivalent conditions.

A maximum power density of 2 W m -2 (200 μW cm -2 ) was achieved in 125 mL of buffer solution.

A hybrid microbial biofuel cell was also developed and tested in both laboratory and marine environments. The carbon felt microbial anode was prepared with silica-encapsulated Shewanella Towards real-world applications, the biofuel cell device was also tested in a marine environment using a floating watercraft. To improve power performance for portable wastewater applications, Santoro, Atanassov and coworkers reported a "smart" supercapacitive microbial biofuel cell [START_REF] Santoro | Self-powered supercapacitive microbial fuel cell: The ultimate way of boosting and harvesting power[END_REF] . The fuel cell was designed with an air-breathing MWCNT buckypaper (Buckeye Composites) cathode modified with BOx, and an activated carbon/carbon black microbial anode with a mixed cultures bacteria. In addition, a carbon brush supercapacitor electrode was short-circuited to the microbial anode. The supercapacitive fuel cell delivered up to 19 mW (84.4 W m -2 , 152 W m -3 ) at pulse currents as high as 45 mA, which is one of the highest power values reported for a microbial fuel cell. The authors highlighted that the integrated capacitor exhibited shorter recharging times (seconds to minutes) compared to the use of external supercapacitors (hours). Having shorter recharge times is attractive due to the possibility to increase the power output frequency. A further advantage is that the supercapacitor size can be modulated to de-couple energy and power depending on the target application.

Buckypaper has also been explored as a high surface area conductive support for the preparation of microbial anodes in biofuel cells [START_REF] Kipf | Systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1[END_REF][START_REF] Kipf | Systematic investigation of anode materials for microbial fuel cells with the model organism G. sulfurreducens[END_REF] . Kerzenmacher and coworkers reported a comparison of bioanodes modified with Shewanella oneidensis MR-1 and prepared using different carbon materials; namely, activated carbon felt and cloths, graphite felt and foil, Toray paper, and lab-made MWCNT buckypaper. The high surface area activated carbon cloth with a Brunauer-Emmett-Teller (BET) specific area of ca. 800 m 2 g -1 outperformed the other carbon materials in terms of catalysis per geometric area and also OCP due to factors such as improved mass transport, electron transfer (e.g. mediated electron transfer via self-secreted flavins), and bacterial migration into the porous structure (e.g. enhanced catalyst loading). Nevertheless, the buckypaper exhibited the largest catalytic current in terms of the volumetric density. Volumetric density is arguably a more accurate representation of current density due to the 3D nature of the electrodes. Lower ohmic losses were also observed using buckypaper, attributed to the enhanced conductivity of CNTs. The lab-made MWCNT buckypaper reported in this work had a BET surface area of 70 m 2 g -1 which compares to values of ca. 30 and 260 m 2 g -1 reported recently by us for commercial (Buckeye Composites) and lab-made MWCNT buckypaper electrodes [START_REF] Chen | Comparison of commercial and labmade MWCNT buckypaper: Physicochemical properties and bioelectrocatalytic O 2 reduction[END_REF] . In a 2018 study, Kerzenmacher and coworkers reported a systematic comparison of bioanodes with the prominent model organism, Geobacter sulfurreducens [START_REF] Kipf | Systematic investigation of anode materials for microbial fuel cells with the model organism G. sulfurreducens[END_REF] . Similar catalytic current densities were observed at different carbon and metal-based anodes, which included lab-made MWCNT buckypaper. The highest limiting current density of up to 756 μA cm -2 was observed with graphite foil, which compares to the 613 μA cm -2 observed when buckypaper was used. In terms of the time until the limiting current is reached, buckypaper anodes took ca. 319 hours whereas graphite foil took ca. 1134 hours (including the one week initial growth phase) using stepwise galvanostatic characterization.

Biosensors

Biosensors which employ glucose-oxidizing enzyme electrodes are devices that have already shown success in the form of electrochemical glucose biosensors for diabetes management [START_REF] Wang | Glucose biosensors: 40 Years of advances and challenges[END_REF] . Enzymes are attractive for biosensing owing to their exceptional substrate selectivity, high specific activity per active site, and inherent biocompatibility. The development of implantable and wearable biosensors offers the ability to continuously monitor physiological analytes with minimal patient intervention and to provide real-time health information [START_REF] Wang | Special issue for wearable electrochemical sensors[END_REF][START_REF] Kotanen | Implantable enzyme amperometric biosensors[END_REF] . Future wearable biosensors will be non-invasive or minimally-invasive devices and therefore (i) avoid the need for surgery and (ii) reduce complications associated with biocompatibility. The realization of wearable sensors is therefore more tangible than implantables. To date, wearable sensors have been almost exclusively developed for monitoring physical physiological parameters such as skin temperature, heart rate and pressure [START_REF] Wang | Special issue for wearable electrochemical sensors[END_REF] . For the development of wearable electrochemical sensors, electrode materials are required that are soft, flexible and stretchable (e.g. mechanical properties similar to human skin), as well as being lightweight and having large surface areas. Buckypapers and their composites can offer these types of properties.

Minteer and coworkers developed a self-powered amperometric lactate biosensor based on LOx from Pediococcus sp cross-linked onto a MWCNT buckypaper modified with a Fc-based polyethylenimine polymer hydrogel [START_REF] Hickey | A self-powered amperometric lactate biosensor based on lactate oxidase immobilized in dimethylferrocene-modified LPEI[END_REF] . The authors reported that the flexibility of buckypaper and its ability to adhere to non-planar surfaces during repeated bending was attractive for the envisaged lactate skin patch and contact lens sensors. The amperometric lactate biosensor based on mediated electron transfer exhibited a detection limit of 1 μmol L -1 , a calibration range up to 40 mmol L -1 , and storage stability up to 21 days. In addition, the biosensor was stable in the presence of ascorbate, a common interference present in biofluids. Chatterjee and Chen subsequently reported a buckypaper-based biosensor for the determination of H 2 O 2 in urine for monitoring of oxidative stress in-vivo [START_REF] Chatterjee | Functionalization of carbon buckypaper for the sensitive determination of hydrogen peroxide in human urine[END_REF] . The same buckypaper-titanium substrate was used as reported previously [START_REF] Ahmadalinezhad | Mediator-free electrochemical biosensor based on buckypaper with enhanced stability and sensitivity for glucose detection[END_REF] , followed by electrochemical surface oxidation, and immobilization of HRP, chitosan, and the mediator methylene blue. A low detection limit of 7.5×10 -8 M, a dynamic range of 0.1 to 500 μmol, and a sensitivity of 54 μA mmol L -1 cm -2 were reported. Spike recovery analysis revealed recoveries of 95-105% in human urine, demonstrating the validity of the method as an in-vivo biosensor.

Decoration of CNT buckypapers with nanoparticles can be used to improve conductivity, [START_REF] By N | Gun'k YK P p i n nd p p i f buckyp p -gold nanoparticle composites[END_REF] mechanical [START_REF] Byrne | p i n f buckypaper-copper composites and investigation of their conductivity and mechanical properties[END_REF] and catalytic performance [START_REF] Tan | A RuO 2 nanoparticle-decorated buckypaper cathode for non-aqueous lithium-oxygen batteries[END_REF][START_REF] Holade | Pacemaker activated by an abiotic biofuel cell operated in human serum solution[END_REF] , and enzyme wiring via electronic bridging or electron mediation [START_REF] Gutiérrez-Sánchez | Gold nanoparticles as electronic bridges for laccase-based biocathodes[END_REF][START_REF] Gross | Redox-active glyconanoparticles as electron shuttles for mediated electron transfer with bilirubin oxidase in solution[END_REF] . Papa et al. developed a glucose biosensor based on lab-made acid-treated SWCNT and MWCNT buckypapers prepared in the presence of citrate-capped gold nanoparticles [START_REF] Papa | Fabrication of functionalized carbon nanotube buckypaper electrodes for application in glucose biosensors[END_REF] . A schematic showing the construction of the sensor mechanism is shown in Figure 1.8. GOx enzyme, HRP and chitosan were subsequently cast onto the buckypaper to generate the bioelectrode. The SWCNT BP with embedded gold nanoparticles exhibited the best performance with a dynamic range of 0.02 to 7 mmol L -1 , sensitivity of 21.5 μA mmol L -1 cm -2 , and no significant response from physiological interferents (ascorbic acid and uric acid). More recently, the same authors expanded the development of the gold nanoparticle-modified SWCNT and MWCNT buckypapers for the electrochemical detection of biologically important molecules such as tryptophan, L-carnitine, tyrosine, and myoglobin with 1000-fold increase in the signal in buffer compared with classical glassy carbon electrode sensors [START_REF] Chatterjee | Engineered carbon nanotube buckypaper: A platform for electrochemical biosensors[END_REF][START_REF] Chatterjee | Nanomaterial based electrodes and methods[END_REF] . For the myoglobin sensor, methylene blue was integrated into the buckypaper to facilitate electron transfer between the electrode and the protein's active site. The reported patent covers methods for the construction of functionalized buckypaper biosensors as an alternative to the use of screen-printed and glassy carbon based electrodes [START_REF] Chatterjee | Nanomaterial based electrodes and methods[END_REF] . It is noted that the specificity of the GOxgold nanoparticle-based biosensors in the presence of interferences was not demonstrated.

Biosynthesis

An exciting and largely unexplored application of buckypaper electrodes is in the field of electroenzymatic synthesis. Enzymes offer attractions compared to chemical catalysts such as high selectivity and reaction rates under eco-friendly conditions, and are therefore recognized as powerful catalysts in industrial biotechnology, for example, for the generation of pharmaceuticals and fine chemicals including enantiomerically pure compunds [START_REF] Choi | Industrial applications of enzyme biocatalysis: Current status and future aspects[END_REF] . Etienne, Walcarius and coworkers have made progress on an electroenzymatic "reactor" concept, for example, for the regioselective conversion of D-sorbitol into D-fructose at an appreciable rate [START_REF] Mazurenko | Enzymatic bioreactor for simultaneous electrosynthesis and energy production[END_REF] . An activated carbon/MWCNT bioanode was modified with polymethylene green and NAD-dependent D-sorbitol dehydrogenase (DSDH) for catalytic sorbitol and NADH oxidation. The substrate and NAD + cofactor were flowed continuously into the reactor during operation. More recently, Etienne and coworkers developed a new catalytic bioelectrode design based on buckypaper for electroenzymatic synthesis [START_REF] Zhang | Electrocatalytic biosynthesis using a bucky paper functionalized by [Cp*Rh(bpy)Cl] + and a renewable enzymatic layer[END_REF] . Lab-made MWCNT buckypaper was elegantly modified with a Rh complex catalyst via diazonium surface chemistry and used for efficient NADH regeneration during sorbitol synthesis via the enzyme DSDH (Figure 1.9).

The work constitutes significant advances in terms of reusability, good catalytic activity for NADH regeneration (turnover frequency of 1.3 s -1 ), and efficient conversion over several days (conversion rate of 87% after 95 h).

Biologic

Buckypaper bioelectrodes also offer interesting possibilities in which their biocatalytic reactions may be used for biocomputing, for example, for novel multi-signal sensor applications. The review of Katz and Privman serves as an excellent introduction to this topic [START_REF] Katz | Enzyme-based logic systems for information processing[END_REF] . Strack et al. demonstrated for the first time that buckypaper bioelectrodes could be used for sustained built-in information processing [START_REF] Strack | Bioelectrocatalytic generation of directly readable code: Harnessing cathodic current for long-term information relay[END_REF] .

A direct electron transfer biocathode configuration was used in which a laccase-based MWCNT buckypaper (Buckeye Composites) responded directly to dissolved oxygen, as a continuous input signal, and could generate an amperometric response, as the output signal. Nitrogen and oxygen were pumped into a flow system to give stable and reproducible current signals at an applied potential of 0.4 V over more than 100 cycles and a 20 day period. The current output with nitrogen served as "on"

or "1" and with oxygen served as "off" or "0". The device was used to generate a binary-based code sequence (ASCII 12-bit Code 39) that could be read by a conventional barcode scanner. This proofof-concept study ultimately demonstrated the prospect of using biocatalytic buckypapers for responding to incoming signals, processing them, and relaying them ex-vivo. Use of signals from substrates present in biological fluids such as sugars and oxygen would open up the prospects for invivo data processing.

Conclusion

Buckypaper materials based on carbon nanotubes have witnessed great success for the construction of bioelectrodes based on enzymes and microbes owing to a unique combination of properties such as their high conductivity, mechanical strength and flexibility, lightweight, and their porous high surface area structures. Both lab-made and commercial buckypapers have proved to be practical supporting frameworks for enzymes and catalytic components including nanoparticles and non-metal catalysts, as well as functional building blocks including polymers and cross-linking molecules. A wide range of functional catalytic biointerfaces have been reported over the last 5 to 7 years for applications including bioenergy conversion and storage (enzymatic and microbial biofuel cells, hybrid biofuelcapacitor systems, and photoelectric biofuel cells), chemical and biochemical detection (enzymatic and non-enzymatic biosensors), and information processing devices (biologic systems). The greatest achievement of buckypaper to date has been its use in actual implanted biofuel cells for energyharvesting from organisms such as snails and lobsters; no other electrode can boast as much success in the field of biological fuel cells. There is still a huge amount of work to be done to improve the performance of implantable and wearable biofuel cells, and we note in particular the importance of evaluating the stability, biocompatibility and toxicity of buckypaper bioelectrodes in future studies.

Until now, the majority of reported buckypaper bioelectrodes have been prepared using commercial buckypaper from Buckeye Composites, and this type of readily available buckypaper has proved to be an excellent electrode platform. We nevertheless envisage a bright future for lab-made buckypaper electrodes that can be prepared using relatively straightforward procedures in the laboratory, and which permit a higher level of control over the physical, chemical and catalytic properties of the electrode. We also recognize that a wide variety of parameters affect the performance of lab-made buckypaper and thus we take this opportunity to emphasize the importance of providing accurate details with respect to the fabrication methods and materials used for buckypaper preparation. With all of this in mind, there should be little doubt that buckypaper will continue to play an important role not only for the construction of future bioelectronic devices but also for other applications in the fields of electrochemistry, energy, and materials science.

Figure 1 . 1 :

 11 Figure 1.1: Illustration of fabrication procedures for commercial and lab-made buckypaper via mass production and lab-scale vacuum filtration, respectively.

Figure 1 . 2 :

 12 Figure 1.2: Cartoon of an implanted glucose/oxygen biofuel cell inserted via incision of two buckypaper bioelectrodes in the snail's shell.

Figure 1 . 4 :

 14 Figure 1.4: Incorporation of 'precision' polynorbornenes in buckypaper to improve the mechanical properties, such as flexibility, tensile modulus and strength, and the chemical properties, for covalent attachment of molecules to improve enzyme orientation and wiring.

Figure 1 . 5 :

 15 Figure 1.5: Schematic of a biofuel cell with a multiple NAD-dependent enzyme bioanode, for the cascade oxidation of ethanol to acetic acid, coupled with an air-breathing BOx biocathode, for oxygen reduction. The fuel ethanol is delivered to the cell via paper fluidics.

  to a phenanthroline quinone (PLQ) derivative. Nevertheless, the NQ buckypaper showed advantages such as a slightly lower onset potential and the possibility to reach the catalytic current plateau at lower potentials compared to the PLQ buckypaper. The buckypaper biofuel cell with a powerful PLQ bioanode for glucose oxidation is illustrated in Figure 1.6. Examples of the bioelectrocatalytic cyclic voltammograms recorded during half-cell characterization are also shown in Figure 1.6.

Figure 1 . 6 :

 16 Figure 1.6: Cartoon of the buckypaper-based glucose/oxygen biofuel cell using FAD-GDH and an artificial quinone mediator at the bioanode, and protoporphyrin IX-oriented BOx at the biocathode. Right: cyclic voltammograms of bioelectrocatalysis at FAD-GDH bioanodes, with different mediators (PLQ) and (NQ), and the BOx biocathode.

oneidensis DSP- 10 ,

 10 for lactate oxidation, and an oxygen-reducing MWCNT buckypaper (Buckeye Composites) cathode modified with either laccase or BOx via 1-pyrenebutyric acid Nhydroxysuccinimide ester (PBSE) cross-linking 70 . Unlike the previous report from Santoro et al., this hybrid biofuel cell used individual anode and cathode compartments separated by a polycarbonate membrane 9 . The biofuel cell, depicted in Figure 1.7, was tested in seawater and delivered 0.7 V for 9 days and 0.95 W m -3 (22.4 μW) using the best performing buckypaper biocathode based on BOx.

Figure 1 . 7 :

 17 Figure 1.7: Representation of a hybrid microbial biofuel cell for harvesting energy from sea water with an enzymatic cathode and a microbial bioanode in individual compartments.

Ahmadalinezhad

  et al. developed a buckypaper-based biosensor platform using a gold-coated titanium substrate as a mechanical support[START_REF] Ahmadalinezhad | Mediator-free electrochemical biosensor based on buckypaper with enhanced stability and sensitivity for glucose detection[END_REF] . Buckypaper was electrochemically functionalized with carboxylic acid groups then modified by adsorption of GOx from Aspergillus niger, horseradish peroxidase (HRP) and chitosan to complete the sensor. Amperometric glucose detection was indirectly monitored at 0.1 V vs AgAgCl based on the reduction of H 2 O 2 produced by the enzymatic oxidation of glucose. The mediatorless glucose biosensor demonstrated high selectivity in the presence of common interferents (ascorbic acid, uric acid and acetamidophenol), a dynamic range of 0.01 to 9 mmol L -1 , sensitivity of 20 μA mmol L -1 cm -2 , and a long lifetime of over 80 days. The excellent stability was attributed to the physical and chemical properties of the buckypaper combined with the use of chitosan for enzyme stabilization.

Figure 1 . 8 :

 18 Figure 1.8: Scheme showing the fabrication of a buckypaper-based biosensor for the indirect detection of glucose. The electrochemical signal is generated by the electrocatalytic reduction of H 2 O 2 , produced by GOx after oxidation of glucose via HRP.

Figure 1 . 9 :

 19 Figure 1.9: Schematic representation of the bioelectrocatalytic synthesis of sorbitol from fructose using a Rh complex for the regeneration of NAD and the enzyme catalyst, sorbitol dehydrogenase (DSDH).


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Micro-mechanism of fatigue crack growth: comparison between carbon black filled natural and styrene butadiene rubbers

Jean-Benoit Le Cam, Erwan Verron, Bertrand Huneau, Laurent Gornet, F.

Perocheau

To cite this version:

Jean-Benoit Le Cam, Erwan Verron, Bertrand Huneau, Laurent Gornet, F. Perocheau. Micromechanism of fatigue crack growth: comparison between carbon black filled natural and styrene butadiene rubbers. ECCMR IV, Jun 2005, Stockholm, Sweden. pp.115-120, 10.1201/9781315140216. hal-01094746

1I NTRODUCTION

Most of the studies dealing with fatigue crack propagation in elastomers are based either on purely mechanical theories [START_REF] Rivlin | Rupture of rubber. I.Characteristic energy for tearing[END_REF][START_REF] Gent | Engineering with Rubber[END_REF] or on fractographic analyses of fracture surfaces [START_REF] Bhowmick | Fractography of Rubber Materials[END_REF]. In spite of these researches, it is not possible to predict satisfactorily the crack paths because none of them allow to determine physical phenomena involved in fatigue crack propagation. Previous works showed that the observation of stretched crack tips can be an interesting way to investigate [START_REF] Gent | Micromechanics of fracture in elastomers[END_REF][START_REF] Bascom | Scanning electron microscopy of rubber tear[END_REF]. Indeed, using an original "micro-cutting" method applied to stretched crack tips, we recently identified microscopic phenomena involved in the growth of the crack in NR during SEM observations [START_REF] Le Cam | Mechanism of fatigue crack growth in carbon black filled natural rubber[END_REF]).

In the present study, we recall the results obtained for carbon black filled NR and present similar experiments performed for carbon black filled SBR, i.e. a noncrystallisable elastomer. The aim of experiments is firstly to determine if physical phenomena involved in crack propagation are similar for both compounds and secondly to propose and compare the two mechanisms of fatigue crack growth. In the next Section, the experimental protocol is presented and the in-situ "microcutting" method is described. Observation results and mechanisms of fatigue crack growth are given in Section 3. Finally, concluding remarks close the paper.

EXPERIMENTAL SECTION

Material and sample

In order to compare fatigue crack growth in both SBR and NR, SBR was filled with 34 phr of carbon black, i.e. the same amount of fillers as in NR compound used in our previous study [START_REF] Le Cam | Mechanism of fatigue crack growth in carbon black filled natural rubber[END_REF]. ABSTRACT: The present paper investigates the fatigue crack growth in both crystallisable and non-crystallisable elastomers under relaxing loading conditions. The study focuses on the determination of the mechanism of fatigue crack growth. For this purpose, an original "micro-cutting" method developed in a previous study for carbon black filled natural rubber (NR) is employed to observe microscopic phenomena involved in the growth of the crack during SEM observations. In crystallisable NR, the cavitation induced by the decohesion between oxides and rubber matrix is the major fatigue damage and it generates heterogeneous deformation and ligaments (more crystallized zones which resist to crack propagation). In carbon black filled styrene butadiene rubber (SBR), cavities containing zinc oxides fail when the crack propagates and the heterogeneous deformation only induces filaments which do not resist to crack propagation. Based on SEM observation, a fatigue crack growth mechanism in non-crystallisable SBR is proposed. Results are compared with those obtained with NR. 1 mold temperature was set to 160°C. To overcome aging problems, samples were frozen at Ϫ18°, 48 h after their molding. The are thawed out 24 h before testing. Samples geometry presented in Figure 1 is similar to the one proposed by Beatty [START_REF] Beatty | Fatigue of rubber[END_REF]; it is axisymmetric and is usually called "diabolo sample". Its significant radius of curvature leads to a higher local deformation state in its middle than the enforced nominal deformation. Consequently, the predominant fatigue crack is initiated in the lowest diameter section, i.e. at the center of the sample.

Fatigue loading conditions

In order to generate microstructural fatigue damage in the crack tip vicinity, NR and SBR samples are beforehand tested under uniaxial cyclic enforced displacement conditions. The stretch ratio varies between 1 and 1.33 for the NR compound, and between 1 and 1.5 for the SBR compound. The strain rate was set to limit the rise in temperature of the surface under 20°C, to not superimpose an additional thermal damage to the mechanical damage. In practice, it corresponds to loading frequencies below 5 Hz. These tests are carried out at 23°C regulated temperature until a self-initiated fatigue crack reaches several millimeters long at the surface of the undeformed samples. It takes place at the middle of the sample beside the mould halves joint. Moreover, additional fatigue tests were carried out until fracture in order to connect features of fracture surfaces to crack tips observations.

Scanning electron microscopy

Photomicrographs were performed with a HITACHI S-3200N model SEM using secondary electrons. In order to determine the chemical elements of observed surfaces, an Energy Dispersive Spectrometer of X-rays (EDSX) coupled with the SEM is used. Before observation, elastomer surfaces should be cleaned: here, the specimens are submitted to ultrasounds in a neutral solution. Moreover, as considered materials are not sufficiently conductive, charge-up can locally occur when it is irradiated with the electron beam. Thus, samples can be coated with a 90 nm thick gold layer by vapour deposition to ensure electronic conduction.

Observation method

Samples are fixed in a rudimentary static tensile apparatus (see Fig. 2(a)) and they are statically stretched at an elongation equal to 1.67 to open the crack (see Fig. 2(b)). Then, this apparatus is placed in the SEM to observe crack growth. Considering results of [START_REF] Bascom | Scanning electron microscopy of rubber tear[END_REF], specimens preparation influence observation results. Indeed, Bascom observed that, in static failure and without coating, the stretched crack tip of nitrile, natural and polybutadiene rubbers have a totally different appearance if observations are performed after that the stretched crack tip was allowed to stand in the laboratory atmosphere about 24 h. In fact, a fibrous and nodular structure appears at crack tip. So long as the specimens were held in the evacuated chamber of the SEM, there was no change in the extent or appearance of the tearing zone. To avoid the effect of ozone attack on the crack tip, Bascom recommends to examine the stretched sample within 0.5 h. Moreover, in this study, it is suspected that the gold layer evaporated on the initial cut gave some protective action against ozone and oxygen attack on rubber. For these reasons, our specimens were observed within 0.5 h after having been stretched, and were retracted after observations. In order to ensure that only fatigue damage is observed, we verify that fibrous and nodular structure does not appear the crack tip. Moreover, we also verify that observations performed with a gold layer the crack tips lead to similar results than those obtained without gold layer.

As mentioned previously, the fatigue test led to initiation and propagation of a crack in the sample, and it involved a fatigue damage in the crack tip vicinity. Thus, the crack tip should be notched to reveal the fatigue damaged zones and to determine their origin.

During SEM observations, it is obviously irrelevant to use a laboratory cutting tool to notch the crack. Nevertheless, it is possible to "micro-cut" the crack tip by imparting in-situ a sufficient amount of energy to break cross-links between macromolecules. Two energy sources are used simultaneously: the first one is mechanical and consists in statically stretching samples and the second one consists in locally concentrating the electron beam in the observation zone. In fact, the amount of energy needed to break cross-links cannot easily be determined since the number and nature of cross-links as well as the number of macromolecules in the irradiated zone are not well-known. However, the acquired experience enables us to correctly set the power of the electron beam and to repeat experiments.

RESULTS

In this section, results of the microcutting of the SBR crack tip are presented. Then, a mechanism of fatigue crack growth is proposed for non-crystallisable SBR.

Finally, this mechanism is compared to the one previously exhibited for NR.

Description and comparison of crack tips

As a summary of our recent study on NR, Figure 3 presents different zones of NR crack tip. It is composed by a few hundred micrometers long ligaments which are highly stretched and surround smooth elliptical surfaces (see Figure 3a). Figure 3b shows the rough surface located at ligaments extremities. It corresponds to retracted broken ligaments. As shown in Figure 3c, zinc oxides which size varies between a few hundred nanometers and 5 m are embedded in the relief of smooth elliptical areas and can be considered like spherical particles. Figure 4 presents SBR crack tip which is plane and seems to be exclusively composed by ligaments. They are issued from a linear zone in the middle of the crack also called tear tip by Gent and Pulford during their observations of the crack tip in a precut carbon black filled polybutadiene sample [START_REF] Gent | Micromechanics of fracture in elastomers[END_REF]. The fact that crack tip only corresponds to a linear zone in which the crack grows is the first major difference with NR crack tip. In the following, we will use the term tearing line instead of tear line as used by Gent and Pulford. Figure 5 shows zinc oxides between ligaments and the stretched cavity which contained them. As in NR, oxides size varies between a few hundred nanometers and 5 m. Figure 6 shows the bottom of the crack tip in SBR. It corresponds with ellipsoidal zones which shrunk when the crack grew. Here, these ellipsoidal zones are not similar to those encountered in NR. Their formation process will be discussed next.

Crack tip microcutting

In NR, natural micro-cracks occur in elliptical zones surrounded by highly stretched ligaments. When the electron beam is focused a few seconds beside these micro-cracks, artificial micro-cracks initiate at the beam concentration point and propagate until they are stopped by ligaments. During their propagation, artificial micro-cracks reveal cavities similar to the ones observed in natural micro-cracks. It is notable that if the electron beam is focused on ligaments (the SEM power settings being unchanged), they will not break even if they are exposed for a long time. This experiment highlights that cavities take place in the vicinity of the crack tip due to decohesion between oxides and rubber matrix. They weaken ellipsoidal zones and generate natural micro-cracks. This process leads to the formation of highly stretched and crystallized ligaments. These results obtained under fatigue loading conditions are quiet similar to those recently established under static conditions which demonstrate that a crystallized zone appears at crack tip of initial cut lengths 0.5 and 1 mm for stretch ratios greater than 1 ϭ 1.3 [START_REF] Trabelsi | Stress-induced crystallization around a crack tip in natural rubber[END_REF]. Moreover, interesting works has been published on the real-time evolution of crystallization in NR during one loading cycle [START_REF] Toki | Strain-induced crystallization of natural rubber as detected real-time by wide-angle x-ray diffraction technique[END_REF].

In SBR, the zone favorable to microcutting obviously corresponds to the tearing line previously described. Thus, the electron beam is concentrated on a point of this tear line (point A in Figure 4). A microcrack immediately initiates at this point and propagates towards the right side. It is notable that the time needed to initiate micro-crack is very short as compared to the NR case (electron beam energy being identical). Moreover, we observed that the crack growth rate is faster than the NR one. Figure 7 shows the path of this artificial micro-crack. Contrary to NR, ligaments do not stop the artificial micro-crack which breaks them. The fact that ligaments fail under the same energy level than the tearing line is the second major difference with NR. It indicates that SBR microstructure is homogeneous at crack tip and thus in all the sample bulk. In the following, non-crystallized ligaments observed in SBR are called filaments. Now, it is necessary to determine if these filaments are formed by the same micro-mechanism as ligaments of NR which are issued from the deformation of natural micro-cracks that initiates in the ellipsoidal zones.

We f irstly observed the neighborhood of zinc oxides in SBR. Figure 8 shows a magnified view of the encircled zone in Figure 7. It appears that the tip of this artificial micro-crack is similar to the tearing line. The assumption that the microstructure of bulk material is not strain-dependent seems to be relevant. The encircled zone in Figure 8 corresponds to the deformation of a cavity containing zinc oxide particles after being broken by the propagation of artificial micro-crack. If cracking continues in the sample depth, this artificial micro-crack will grow and walls of the cavity are transformed into filaments.

Crack growth mechanism

In NR, results revealed that cavitation induced by the decohesion between zinc oxides and rubber matrix is the major fatigue damage and that the crack tip is composed by stretched elliptical zones surrounded by ligaments. The mechanism of fatigue crack growth suggests that wrenchings located on the fracture surface of NR are formed by the failure of highly stretched ligaments [START_REF] Le Cam | Mechanism of fatigue crack growth in carbon black filled natural rubber[END_REF]). Moreover, authors explained that ligaments are the most crystallized zones at the crack tip and proposed a mechanism of fatigue crack growth in NR.

Using the previous observations, the mechanism of fatigue crack growth in non crystallisable SBR is proposed in this section. Like the mechanism of fatigue crack growth proposed for NR, chronological sketches describe the phenomenon. Figures 9 presents the front view of crack growth. Here, the mechanism is simpler than that in NR, thus the front view is sufficient to entirely describe the phenomenon.

Figure 9a is a schematic view of the crack tip and more precisely a unit surface of the crack tip. The scale of the description is the one of the encircled zone in Figure 5. As shown in this view which is the first step of crack growth, the crack tip is stretched in the directions of the black arrows on the left and it is composed of filaments and ellipsoidal zones containing zinc oxides (ellipsoidal zones 1 and 2). Cavities surrounding zinc oxides and hidden in the rubber matrix behind the crack tip appear in dotted lines in this figure (cavities 1, 2, and 3). Their shape is ellipsoidal. Figure 9b presents the second step of crack growth. The crack tip grows on both sides of the tearing line and reveals highly stretched cavities behind it (cavities 1 and 2). Cavities which are not located in front of the tearing line are not revealed (here, cavity 3). During crack growth, ellipsoidal zones 1 and 2 move away from the tearing line, i.e. they are less and less stretched, and their shape become quasi-circular. In the same way, the ellipsoidal shape of cavity 3 become spherical. Figure 9c is the third step of the mechanism and presents the evolution of revealed cavities 1 and 2. It shows that cavities 1 and 2 are more and more stretched and that the crack tip grows towards new hidden cavities (cavities 4 and 5). Highly stretched cavities walls form filaments, i.e. filaments due to the deformation of revealed cavities. Here, we focus on zinc oxides because they are the most numerous particles, but it is notable that these explanations could be applied to inclusions located in rubber matrix, i.e. mineral inclusions which size can reach 300 m diameter. Figure 9d is the last step of the mechanism. During crack growth, cavity 5 is revealed and grows. Walls of cavities 1 and 2 begin to shrink and form quasi-spherical zones similar to the ones encircled in Figure 6. Finally, the mechanism starts again at the first step of this unit sequence.

CONCLUSIONS

As a summary, an original technique of in-situ microcutting allowing to observe crack tips of stretched samples led to propose a fatigue crack growth mechanism. The mechanism in SBR, i.e. non-crystallisable elastomer, is different than the one previously proposed for crystallisable elastomers. Fatigue damage in natural rubber is mostly due to the cavitation induced by the decohesion between oxides and rubber matrix. In SBR, only the failure of cavities containing zinc oxides contributes to the formation of filaments. Filaments are not crystallized since in NR highly stretched ligaments are crystallized and can resist to crack propagation. Moreover, zones in which crack tip propagates correspond to a line in SBR instead of a surface composed of ligaments and ellipsoidal zones in NR. Moreover,w e observed that in SBR the amount of carbon black has a significant influence on crack path. Further works will be devote to the influence of the amount and nature of fillers at the microscopic scale.

Figure

  Figure 1. Diabolo sample.

Figure 3 .

 3 Figure 3. NR crack tip.

Figure 6 .Figure 7 .

 67 Figure 4. SBR crack tip.

Figure 9 .

 9 Figure 8. Zoom in encircled zone of Figure 6.

  Table 1 summarizes their chemical composition and Table 2 presents some mechanical characteristics. NR and SBR were respectively cured for 7 min and 5 min, and the Micro-mechanism of fatigue crack growth: Comparison between carbon black filled NR and SBR J.-B. Le Cam, E. Verron, B. Huneau & L. Gornet

	Institut de Recherche en Génie Civil et Mécanique, France
	F. Pérocheau
	Allevard Rejna Autosuspensions, Dovai cedex, France

Table 1 .

 1 Material formulation (phr).

	Components	NR	SBR
	Rubber	100	100
	Zinc oxide	9.85	10
	Oil	3	0
	Carbon black	34	34
	Sulfur	3	3
	Stearic acid	3	3
	Antioxidant	2	5
	Accelerators	4	4.3

Table 2 .

 2 Mechanical properties.

	Properties	NR	SBR
	Density	1.13	1.12
	Shore A hardness	58	67
	Stress at break (Mpa)	22.9	16.5
	Elongation at break	635	343

ACKNOWLEDGMENT

We would like to acknowledge Allevard Rejna Autosuspensions for its the financial support and Trelleborg for its collaboration.