Takeshi Kunishima
email: takeshi_kunishima@jtekt.co.jp

Yasuharu Nagai

Takanori Kurokawa

Gaëtan Bouvard

Jean-Christophe Abry

Vincent Fridrici

Phillippe Kapsa

Tribological behavior of glass fiber reinforced-PA66 in contact with carbon steel under high contact pressure, sliding and grease lubricated conditions

Keywords: Polyamide, Glass fibers, Wear, Grease, Composite, Worm gear

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

Introduction

The requirements for lighter automobile resin parts to achieve energy savings and to reduce carbon dioxide emissions are increasing owing to energy issues such as global warming. In addition, the use of resin-made sliding parts in the automotive industry is increasing in response to the demand for higher levels of quiet accompanying the computerization and hybridization of automobiles [START_REF] Kurokawa | Performance of plastic gear made of carbon fiber reinforced polyether-ether-ketone: Part 2[END_REF][START_REF] Tappera | An evaluation of life cycle assessment and its application to the closed-loop recycling of carbon fibre reinforced polymers[END_REF][START_REF] Balakrishnana | Impact and damage behaviour of FRP-metal hybrid laminates made by the reinforcement of glass fibers on 22MnB5 metal surface[END_REF][START_REF] Nakata | Grease lubrication technology for sliding automobile parts[END_REF]. Among various types of resin materials, polyamide 66 (hereinafter PA66) is known as an engineering plastic, and has useful properties, such as heat resistance, high strength, toughness, and high wear resistance [START_REF] Lee | Effect of carbon-nanotube length on friction and wear of polyamide 6,6 nanocomposites[END_REF][START_REF] Lates | Temperature, pressure, and velocity influence on the tribological properties of PA66 and PA46 Polyamides[END_REF][START_REF] Gebretsadik | Friction and wear characteristics of PA 66 polymer composite/316L stainless steel tribopair in aqueous solution with different salt levels[END_REF][START_REF] Chen | The effect of double grafted interface layer on the properties of carbon fiber reinforced polyamide 66 composites[END_REF]. Therefore, PA66 is widely used for sliding parts in automobiles or industrial machines such as various types of gears [START_REF] Senthilvelan | Damage Mechanisms in injection molded unreinforced, glass and carbon reinforced nylon 66 spur gears[END_REF][START_REF] Sarita | Effects of lubricant on the surface durability of an injection molded polyamide 66 spur gear paired with a steel gear[END_REF][START_REF] Senthilvelan | Fiber reinforcement in injection molded nylon 6/6 spur gears[END_REF][START_REF] Tavčar | Accelerated lifetime testing of reinforced polymer gears[END_REF][START_REF] Zhang | A physical investigation of wear and thermal characteristics of 3D printed nylon spur gears[END_REF], bearing retainers [START_REF] Scott | Composite materials for rolling bearing cages[END_REF][START_REF] Oh | Failure mechanisms of ball bearings under lightly loaded, non-accelerated usage conditions[END_REF][START_REF] Kohout | Strength changes of moulded polyamide composite caused by thermal oxidation[END_REF], and rollers [START_REF] Harrass | Tribological behavior of selected engineering polymers under rolling contact[END_REF]. Recently, the requirements for the downsizing of automobiles and industrial machines and growing concern for the environment have increased the demand for reducing the size and weight of these plastic sliding parts and their capability to withstand high stress [START_REF] Bondy | Tensile fatigue characterization of polyamide 66/carbon fiber direct/in-line compounded long fiber thermoplastic composites[END_REF][START_REF] Fonseca | Design and numerical evaluation of recycled-carbon-fiber-reinforced polymer/metal hybrid engine cradle concepts[END_REF].

The demand for the use of plastic worm gears is particularly important in the worm reducer of the automobile electric power steering because the gears are lightweight, reduce vibration and noise, and are corrosion resistant. The plastic worm gear of the reducer used in electric power steering experiences a significantly high contact pressure (over 100 MPa) under sliding conditions. Therefore, some form of grease lubrication is necessary to reduce friction between the plastic worm gear and the steel worm shaft [START_REF] Kim | Durability characteristics analysis of plastic worm wheel with glass fiber reinforced polyamide[END_REF][START_REF] Ahn | Wear of glass fiber reinforced polyamide worm gear according to the direction of the glass fiber[END_REF][START_REF] Bormuth | Lubrication of plastic worm gears[END_REF]. In this application, the effect of sliding is greater than the effect of rolling. The recent downsizing of automotive components, coupled with the high demand for electric power steering in large vehicles owing to their impact on fuel economy, highlights the need for robust worm gear support. However, applying higher torque to worm gears will lead to large deformation of the teeth of the plastic gear (increase in the backlash), breakage at a much earlier stage, and increase of the sliding torque [START_REF] Kunishima | Clarification of tribological behavior on tooth surface of resin worm gear for electric power steering[END_REF].

As a consequence, improvements in the mechanical properties of plastic materials are required. Adding reinforcement fibers, such as glass fibers (GF), carbon fibers (CF), or aramid fibers (AF), is a common means to improve the tribological properties of polyamide [START_REF] Kukureka | Effect of fibre reinforcement on the friction and wear of polyamide 66 under dry rolling-sliding contact[END_REF][START_REF] Zhou | Effect of carbon fiber reinforcement on the mechanical and tribological properties of polyamide6/polyphenylene sulfide composites[END_REF][START_REF] Gordon | The wear and friction of polyamide 46 and polyamide 46/aramid-fibre composites in sliding-rolling contact[END_REF][START_REF] Kim | Tribological properties of short glass fiber reinforced polyamide 12 sliding on medium carbon steel[END_REF][START_REF] Shin | Friction and wear of polyamide 66 with different weight average molar mass[END_REF][START_REF] Kim | Friction and wear of monolithic and glass-fiber reinforced PA66 in humid conditions[END_REF]. Kim et al. [START_REF] Kim | Tribological properties of short glass fiber reinforced polyamide 12 sliding on medium carbon steel[END_REF] noted that adding GF can decrease the friction coefficient and wear amounts of polyamide 12 material by decreasing the adhesion between polyamide 12 and carbon steel under dry conditions; the lowest friction coefficient and a better wear resistance of polyamide 12 composite were achieved at 30 wt% addition of GF. In addition, they noted that fibers in a parallel orientation exhibited slightly higher friction levels than those in a perpendicular orientation. However, the fiber orientation had less effect on the friction and wear of the composite than the fiber content or the applied load. Shin et al. [START_REF] Shin | Friction and wear of polyamide 66 with different weight average molar mass[END_REF] found that the amount of GF in the composite and the molecular weight were strongly affected by the friction level and the wear rate because these altered the shear strength and adhesion of PA66 when performing block-steel ring sliding tests under dry conditions. They also stated that the temperature at the sliding interface was important in determining the morphology of the wear debris, which was closely related to the wear resistance. Kim et al. [START_REF] Kim | Friction and wear of monolithic and glass-fiber reinforced PA66 in humid conditions[END_REF] demonstrated that the wear resistance of unreinforced PA66 and GF reinforced PA66 became worse with water absorption because of softening caused by absorbed water and increased interfacial adhesion on the steel counter surface. In addition, the degraded wear characteristics of unreinforced PA66 resulting from chain scission of the amide functional group caused by water molecules were substantially improved by using short-GF reinforcement.

The majority of the previous studies on the tribology of fiber reinforced polyamide were performed in dry conditions, and there are very few reports on the tribological properties of fiber reinforced PA66 under grease lubrication. The reason is that polymer materials have self-lubricating properties, and it is possible to use them as sliding parts without lubrication in many cases. Kurokawa et al. [START_REF] Kurokawa | Performance of plastic gear made of carbon fiber reinforced polyamide 12[END_REF] reported that the tribological properties of spur gear made using CF-reinforced polyamide 12 material improved under grease lubrication, and that the load bearing characteristic was higher with increasing molecular weight of polyamide 12. However, the counterpart gear was made of the same polyamide 12 material. In addition, they reported that increasing the molecular weight of polyamide 12 can improve the fatigue properties of resin gear. However, few explanations were provided regarding the wear mechanism of the composite material. Kurokawa et al. [START_REF] Kurokawa | Performance of plastic gear made of carbon fiber reinforced poly-ether-ether-ketone[END_REF] further reported on the tribological properties of spur gear made from CF-reinforced PEEK (poly-ether-ether-ketone) under lithium grease lubrication, with a counterpart of CF-reinforced PEEK or steel. They stated that the affinity between PEEK and CF, the difference in the intervention of CF worn debris at the engagement region, and the characteristics of CF (strength and modulus of fibers) play a significant role in determining the wear resistance of the composite. Kunishima et al. [START_REF] Kunishima | Reactive extrusion mechanism, mechanical and tribological behavior of fiber reinforced polyamide 66 with added carbodiimide[END_REF] reported on the tribological properties of carbodiimide-added GF and AF-reinforced PA66 in contact with steel in the presence of Ba complex grease. They stated that the toughness and wear resistance of the PA66 composite were improved by the increase of the molecular weight of PA66 through the reaction extrusion between PA66 and poly-carbodiimide compounds. However, the detailed tribological mechanism of the composite and steel counterpart was not discussed.

It is necessary to consider not only the wear resistance of PA66 but also the wear resistance of the metallic counterpart, especially when using hard-fiber reinforced PA66 material, which may have the most aggressive effects on the metallic counterpart because the wear on both materials will lead to much earlier breakage of actual parts and to an increase of the sliding torque. However, the wear mechanism has not been clarified sufficiently. Particularly, considering an actual worm gear, the machining of the teeth and the heat treatment of the steel worm shaft affect the performance of the products and their production costs. For example, applying a heat treatment to the steel worm shaft counterpart before the formation of the teeth will make the formation of the teeth much more difficult, leading to increased production costs. On the other hand, if the heat treatment of the worm shaft is applied after the formation of the teeth, the accuracy of the dimensions of the worm shaft decreases because of the strain induced by the heat treatment, and the gear meshing accuracy will decrease, leading to undesirable effects on the product's performance. Therefore, it is necessary to understand the tribological phenomena to properly design the contact surface of the composite and to select the proper steel and grease. A previous study on the effects of the metallic counterpart on the tribological properties of fiber reinforced-PA66 by Chen et al. [START_REF] Chen | The effect of different metallic counterface materials and different surface treatments on the wear and friction of polyamide 66 and its composite in rolling-sliding contact[END_REF] found that the type of metallic counterpart (such as aluminum, brass, or steel with different surface treatment or heat treatment) has notable effects on the wear resistance of GF-reinforced PA66 and on the friction coefficient during the rolling-sliding contact. They indicated that this difference was attributed to the capability of the metal surface to form a stable polymer transfer film, and that the surface treatment for steel (such as Tufftride coating) has a significant effect on the wear rate of GF-reinforced PA66. However, all sliding tests were performed in dry conditions, and the effect of the hardness of steel was not investigated in their study.

Given these points, the present study focuses on the typical tribological properties during sliding of GF-reinforced PA66 and a steel counterpart under high contact pressure and grease lubricated conditions (i.e., the wear resistance of GF-reinforced PA66 and steel, and the associated friction properties). First, the change over time of the tribological properties is investigated, and the contribution of each parameter such as the damage in the sliding surface, the contamination by wear debris in the grease, and the increase of surface temperature on the tribological properties are elucidated. The wear resistance of the steel counterpart is discussed, considering the fiber orientation of the composite sliding surface. Furthermore, the effects of the mechanical properties of the steel counterpart on the tribological behavior are evaluated.

Experimental

Experimental set-up, measurement, and observation

Tribological properties were evaluated through sliding tests under grease-lubricated conditions using a rotating composite ring in contact with four fixed steel cylinders [START_REF] Kunishima | Clarification of tribological behavior on tooth surface of resin worm gear for electric power steering[END_REF][START_REF] Kunishima | Reactive extrusion mechanism, mechanical and tribological behavior of fiber reinforced polyamide 66 with added carbodiimide[END_REF]. Figure 1 presents the schematic view of the test specimens used for the sliding test as well as the tribometer setup. With this test setup, it is possible to perform sliding tests under high-contact pressure and sliding conditions, similar to those encountered by commercial worm gears. Intermittent sliding, in which rotation and stopping periods are alternated, was introduced to reduce excessive sliding heat generation. The sliding tests were carried out under lubricated conditions with 0.85 g of grease used for each sliding test. A normal load was applied using dead weights. The weight and height of each composite ring specimen were measured before and after each sliding test to estimate the wear and the creep separately, and to investigate the contributions of wear and creep in a sample. The total height loss htotal is the total loss caused by wear and creep, while the wear height loss hwear can be calculated from the wear rate of the composite Mwear, the density of the composite d = 1.23 g/cm 3 , and the sliding surface area s (approximately 200 mm 2 ) using the following equation.

ℎ = (1)
The creep height loss hcreep was then estimated by subtracting the wear height loss from the total height loss.

ℎ = ℎ -ℎ (2)
In addition, the vertical displacement was measured in situ during the sliding tests. However, this value was not exactly the same as the height loss of the composite ring measured before/after sliding tests because the composite ring specimens expanded because of the heat generated by sliding during the tests and then contracted because of cooling after each test. In our tests, the temperature of the steel cylinder holder was measured with a thermocouple.

The friction torque F was measured with a friction torque sensor, and the friction coefficient µ was calculated using the normal load N and rotation radius r (11.4 mm).

= (3)

Friction torque was measured every 0.01 s, and the average value and maximum value of the friction coefficient calculated in 1 s increments. One cycle in this sliding test represents one rotation of the composite ring. Table 1 lists the sliding test conditions. The sliding surfaces of the composite ring were observed before and after each sliding test with a microscope and using scanning electron microscopy (SEM). The mass losses of individual steel cylinders were measured, and wear scars were observed using interferometry. The Young's modulus of the composite sliding surface and hardness of the sliding surface of the steel cylinder were measured through micro indentation. Table 2 presents the measurement conditions. Unload: 300 mN⇒40 mN for 4 s

Composite material

Commercially available raw materials were extruded to fabricate pellets (granules). Test specimens were then fabricated through injection molding of the pellets. Unreinforced PA66 pellets were used as the raw PA66 material. GF were cut to the desired length for use (fiber diameter: 6.5 µm, length: 3 mm). The chopped GF strands were treated with an agent to ensure good adhesion between the PA66 and GF (details: see Supplementary Figure S1). The extrusion of PA66 and GF chopped strands was carried out by using a twin-screw extruder (TEM-26SX, Toshiba Machine Co., Ltd.). PA66 was fed into the top feed (i.e., fed from the root of the barrel of the twin screw extruder), while fibers were fed into the side feed (i.e., fed from the middle of the barrel of the twin screw extruder) to prevent damage of fibers during extrusion and to ensure the length of the fibers was maintained inside the pellets. The amount of GF was 15 wt%. After extrusion, injection molding was carried out with an injection molding machine (ROBOSHOT S-2000i 100B, FANUC CORPORATION), and the sliding test specimens described in Section 2.1 were prepared. The viscosity number, which is related to the molecular weight of PA66, was measured using the relative viscosity method with a formic acid solvent at 25 °C, according to the ISO307 standard. The viscosity of the test specimens was 145 ml/g, which falls within the normal level of the molecular weight of PA66.

The composite ring specimens were cut in the height direction by 3 mm with a lathe, and then the sliding surface was polished with #600 polishing paper before each sliding test. The objective of this process was to simulate the conditions of the sliding surface of actual worm gear tooth surfaces, which are formed by mechanical hob-cutting after the injection molding process to ensure dimensional stability (see Supplementary Figure S2).

Steel cylinders

Steel cylinders with a diameter of 3.5 mm and length of 30 mm were fabricated using S45C steel, which contains 0.45 % carbon, following JIS G 4051 (Japanese Industrial Standards). The roughness of the sliding surface was Ra 0.2 µm, obtained using a centerless grinding process. In addition to the raw carbon steel, cylinders with different hardness and metal structure were prepared by applying different heat treatment -7 -processes (quench hardening and tempering), as presented in Figure . 2. First, the same types of carbon steel cylinders with a diameter of 3.6 mm were prepared and heat treatments under different conditions were applied.

Afterward, each sample was machined to a diameter of 3.5 mm using the same process to obtain the same roughness (Ra of 0.2 µm). Table 3 presents the heat treatment conditions and the measured hardness of the cylinders measured in the way described in Section 2.1 (steel structure: see Supplementary Figure S3).

Grease

The base oil of the evaluated grease was PAO8 (Polyalphaolefin 8) and the thickener consisted of urea compounds. The grease contained zinc carboxylate acting as a low-friction agent and contained a sulfur type anti-oxidation agent. At the inflection point, the slope of the temperature increased, which is related to the increase of the friction coefficient. Afterward, a steady state displacement was observed, and then the displacement began to increase proportionally to the number of cycles. The sliding tests were performed under the same conditions using the same materials seven times, and the first inflection point of the vertical displacement ranged between 19,000 cycles and 23,000 cycles. Figure 5 presents each test specimen and grease before and after the sliding test of 61,500 cycles, as observed by microscope. The entire sliding surface of the composite ring was damaged, and the sliding surface was expanded in both inner and outer directions; this indicated that both wear and creep occurred during sliding. The sliding surface of the steel cylinder was worn severely (exhibiting scratches) in the sliding direction, indicating that 2-body abrasive wear occurred. The total wear volume of the four steel cylinders after 61,500 cycles was 0.158 mm 3 and was less than 112 mm 3 , which was the wear volume of the composite ring after 61,500 cycles. In addition, the color of the grease changed from a light color to black.

Results and Discussion

Detailed tribological behavior of the contact and damage of the composite

Figure 6 shows the wear debris inside the grease after a sliding test. The image was obtained by centrifugation of the grease to separate the base oil and then heating the residue to evaporate any organic agents (such as thickeners). The presence of fibers can be observed in the wear debris. In addition, the elements related to GF (Si, Al) and carbon steel (Fe) were detected by SEM-EDX analysis of the wear debris in the grease (images in Supplementary Fig. S5). To clarify the reasons for the first inflection point of the vertical displacement and to estimate the ratio of wear and creep in each stage, eight individual tests with different cycle numbers were performed. Figure 7 shows the absolute value of the wear and the creep represented by the total height loss, and the calculated ratio of wear and creep for each cycle. There was good correlation between the creep as estimated by the calculation explained in Section 2.1 and the surface area after the sliding tests (measured with the optical microscope), which is supposed to be directly related to the creep of the composite ring (see Supplementary Figure S6). In the initial stage of the sliding test (in other words, in the first inflection point of the vertical displacement), the height decrease was mainly due to creep; however, the effects of wear started to increase after the first inflection point, and in the last stage of the sliding test, the main factor contributing to height loss was wear. The process involving wear and creep during the sliding tests was clarified by detailed observations as follows.

Figure 8 shows the optical microscope observation of the sliding surface of the composite ring at different cycles. At 10,000 and 17,000 cycles, partial peeling of the sliding surface has already occurred, especially in the center region. By contrast, at 22,531 cycles (just before the inflection point of the vertical displacement for this test), the peeling of the center area of the sliding surface was advanced. The reason why the peeling of the composite was promoted especially in the center area will be explained in Section 3.2 by considering the fiber orientation at the sliding surface of the composite ring. In the sample tested for 19,995 cycles (just after the inflection point for this test), peeling of the center area of the sliding surface was observed. From then on, peeling spread across the entire sliding surface. To discuss the damage of the sliding surface quantitatively, the mechanical properties of the sliding surface were measured by micro indentation. Using this method, the size of the indentation after a measurement was approximately 100 µm. Therefore, the mechanical properties of not only the fibers and the PA66 itself, but also the composite itself, can be discussed. Figure 9 presents the obtained Young's modulus in an area without peeling (no-peel area) and a peeled area for each cycle. In the no-peel area, the value was almost the same as a new sample until 17,000 cycles, and decreased slightly up to 22,531 cycles. By contrast, the Young's modulus decreased compared to the initial conditions even at 10,000 and 17,000 cycles in the peeled area, and by 22,531 cycles, the value had decreased significantly. Considering these results, the damage to the sliding surface was related to the mechanical properties in a quantitative manner.

Figure 10 presents the SEM observation of the sliding surface of the composite ring at different cycles. SEM observations were performed under the BSE (backscattered electron) mode, where the white area is the GF and the gray area is the PA66. After 10,000 cycles, small holes in the PA66 are observed. However, GF adhered to PA66 correctly and severe damage is not observed. At 17,000 cycles, additional damage is observed; holes where the GF had fallen off the sliding surface were observed, and scratches of the sliding surface related to the missing GF are observed. At 22,531 cycles (just before the inflection point of the vertical displacement in this test), even in an area where severe peeling was not present (observed with the optical microscope), peeling off of GF, damage to GF, and wear debris from the steel are confirmed. In addition, some scratches are observed in the sliding direction.

Considering these mechanical properties and the observations made using a microscope and SEM, the friction coefficient is considered to have increased owing to the damage incurred by the sliding surface, and initial creep -11 -was induced by the decrease in the mechanical properties of the composite due to the peeling off of GF.

Afterward, the GF dropped off continuously from the sliding surface and further wear was promoted as the sliding cycles increased in number.

Wear behavior of counterpart steel and effect of the fiber orientation in the composite ring

The wear behavior of the counterpart steel cylinder was investigated. Figure 11 shows the wear volume of the steel cylinder for different cycles under the test conditions discussed in Section 3.1. Wear was measured by interferometry and corresponds to the total wear volume of the four cylinders. The wear volume increased proportionally with the number of cycles.

Figure 12 presents an optical microscope observation of a steel cylinder after 22,531 cycles (just before the first inflection point of the displacement), interferometric images of the cylinder before and after removal of the cylindrical shape for a better visualization of the wear, and a central longitudinal 2D profile measured through interferometry. The left area corresponds to the inner diameter side of the composite ring, and the right area corresponds to the outer diameter side of the composite ring. Severe wear of the steel cylinder, which appears to be 2-body abrasive wear, occurred even before the inflection point of the vertical displacement. This is related to the hardness of the steel and of the GF. The hardness of GF, which was measured by nano indentation, was approximately 5.7 GPa, and this value is much higher than the hardness of steel without heat treatment (4.50 GPa). Furthermore, the wear of the steel cylinder in the center area was much lower compared to the surface sides. This exhibits an inverse tendency to the wear of the composite ring, as discussed in Section 3.1. These results can be explained by the orientation of the GF in the composite as follows. To better understand the differences in wear for each area of the steel, the differences in fiber orientation in the composite ring were evaluated quantitatively with an image analysis of the sliding surface of a composite ring. First, the polished sliding surface of the composite ring was prepared using #4000 polishing paper, and three BSE images of the inner/center/outer areas at a magnification of x300 were obtained, as shown in Figure 13. Afterward, the GF and PA66 areas were separated by binarization using the software package imageJ (produced by Wayne Rasband) as shown in Figure 14, and the total area of GF and the total perimeter of GF (corresponding to the total length of the contact between GF and PA66) were calculated. Furthermore, the distribution of the circularity of each GF was calculated. Circularity indicates the orientation of GF in the sliding surface, and a value of circularity close to 1 means that the GF have a circular shape and are oriented in the perpendicular direction to the sliding surface. On the other hand, GF have a fibrous or oval shape when the value of the circularity decreases, indicating that the GF are oriented parallel to the sliding direction. Table 4 lists the calculated total area and total perimeter of GF in each area. Both values are similar in each area, therefore it is confirmed that the differences of the wear in the steel in each area are not attributed to the total area or perimeter of GF. Figure 15 presents the relationship between the circularity of the GF and cumulative area of GF, and Table 5 lists the average value of C50 in each area (C50 is the value of the circularity when the cumulative area of GF is 50%). The results indicate that the circularity in the center area is much lower than in the inner/outer areas, meaning that the majority of GF in the center area are aligned in parallel with the sliding direction. Therefore, the number of edges of GF which have much higher aggressive effects on the steel is lower, and wear in this area of the steel cylinder was much lower. In contrast, the GF can be easily peeled off because of the sliding shear stress; therefore, peeling of the composite material easily occurred. On the other hand, the circularity of the inner/outer areas is much higher than in the center area, therefore the GF are perpendicular to the contact surface. As a consequence, the wear on the steel cylinder is promoted because the number of edges of GF is much higher compared to the center area, and the wear of the composite ring is reduced.

The reason why the differences in GF orientation occurred can be explained as follows: inside the metal die during the injection molding process, the solidification speed is much higher in the inner and outer areas of the composite ring because these parts are in contact with the surface of the metal die, therefore the GF are oriented to the direction of resin flow. On the other hand, in the center area of the composite ring, the solidification speed is much lower because this area is not in contact with the metal die, therefore the GF flows before solidification occurs in the metal die.

-16 -

Contribution of each parameter to the increase of friction and damage

The individual contributions of four parameters: the evolution of the sliding surface, the contamination of grease by wear debris, the wear of the steel cylinder, and the increasing sliding surface temperature were investigated by performing sliding tests under different conditions. Steel cylinders without heat treatment (hardness: 4.50 GPa) were used, and the same normal load and sliding speed as discussed in Section 3.1 were applied in all tests to clarify the contribution of each parameter separately.

First, the effect of the sliding surface damage was investigated. Pre-damaged composite rings after 17,000 cycles, 20,000 cycles, and 30,000 cycles were prepared, and sliding tests were performed using these pre-damaged rings under the same test conditions, using new steel cylinders and grease. Figure 16 presents the sliding surfaces before the sliding tests and the results of the sliding tests. The average friction coefficient becomes much higher with increased pre-damaged cycles. The temperature depends on the value of the friction coefficient, and a higher friction coefficient induces a greater increase of the temperature. In addition, the inflection point of the vertical displacement occurs much earlier with increased pre-damaged cycles.

Considering these results, it is worth mentioning that the initial surface condition of the composite ring has a significantly strong effect on the increase in friction and therefore on the ultimate damage. The effect of the contamination of grease by wear debris on the friction and damage was investigated. After a sliding test of 61,500 cycles under the standard test conditions, grease was collected. This grease was contaminated by wear debris consisting of steel and GF, as explained in Section 3.1 (Figure 6). A new sliding test was then performed using this contaminated grease to compare the results when using new grease. Figure 17 presents the sample holder of a steel cylinder with the tested grease and the results of sliding tests. The sliding time evolution of the friction coefficient, temperature, and displacement were the same in both greases. In addition, the wear amount and morphology of the counterpart steel was also at the same level (with using new grease: 0.156 mm 3 , using the contaminated grease: 0.151 mm 3 in the total wear amount of four cylinders measured by interferometry) as presented in Figure . 18. Given these results, there is no effect of wear debris on the friction and wear of composite and steel. In addition, the wear mode of the steel is not 3-body abrasion; rather it is 2-body abrasion, because the wear debris of dropped off GF and steel which were present in the contaminated grease had no negative effects on the tribological properties of the contact, and wear of the steel is affected by the GF embedded in the PA66 when considering the wear scar of steel presented in Figure 5.

Furthermore, the wear mode of the composite was also not 3-body abrasion, and the wear resistance of the composite is dominated by the damage to its sliding surface, as mentioned above. The effect of the worn steel cylinder was also investigated. Pre-damaged steel cylinders after a 17,000 cycles sliding test were prepared, and a new sliding test was performed using these pre-damaged cylinders with new composite rings and grease. Figure 19 presents the steel cylinders before and after the sliding tests. The friction coefficient tends to decrease when using the pre-damaged steel cylinders compared to the results with new cylinders, and the temperature decreased. In addition, when using the pre-damaged steel cylinders, the inflection point of the vertical displacement occurs much later than when using new cylinders. The reason why using the pre-damaged steel cylinders improved the tribological properties appears to be a decrease of the contact pressure when using the worn cylinders. Therefore, the friction coefficient and temperature decreased, and wear resistance was improved. Additionally, the effect of the increase of temperature was investigated by performing sliding tests in which the test was stopped after 17,000 cycles and restarted once the temperature fell to room temperature. Figure 20 presents the test conditions: Test No. Consequently, the wear of the composite and particularly the linear increase in the vertical displacement are mainly dependent on the number of contact cycles and the contact pressure, and is less dependent on the contamination of the wear debris in grease or wear of the steel cylinder. Therefore, it would be possible to be concluded that the wear of the composite could be induced by contact fatigue, generating the maximum von-Mises stress below the contact surface, which can induce the peeling observed.

Effects of the hardness of the steel counterpart

The effects of the hardness of the counterpart steel cylinder on the tribological properties were investigated.

Tests were performed with cylinders having different values of hardness (from 4.50 GPa to 9.89 GPa, measured by micro indentation). Total sliding cycles were 33,000 cycles. the counterpart steel cylinders for different values of hardness (total of the wear of four cylinders). Figure 22 presents the charts for each sliding test. Figure 23 presents microscope observation images of the sliding surface of a composite ring after the tests. It is observed that wear and creep of the composite increase with increasing hardness of the counterpart steel cylinders from 4.50 GPa to 5.36 GPa. On the other hand, wear is at the same level and the slope of the increase in creep decreases for hardness values ranging from 5.36 GPa to 7.81 GPa.

Moreover, the timing of the inflection point of the friction coefficient and the vertical displacement occurs much earlier when using harder steel cylinders (from 4.50 GPa to 7.18 GPa), and sliding surface temperature is also increased. The evolution of the temperature, the friction coefficient, and the vertical displacement is the same between 7.18 GPa and 9.89 GPa. On the other hand, the wear of the counterpart steel cylinders sharply decreases with increasing hardness of the steel cylinders from 4.50 GPa to 5.36 GPa, and very little wear of steel is observed for hardness values ranging from 5.36 GPa to 7.81 GPa. A change in the color of the grease was not observed in this range.

These results are explained by the difference between the hardness of the GF and the counterpart steel cylinders. The hardness of GF was measured using the following process. First, the GF were embedded in epoxy resin in the horizontal or perpendicular direction, as presented in Figure 24(a), and the epoxy resin was cured.

Then, the surface of the epoxy resin was polished and the hardness of the GF in each direction was measured using a nano indentation process; the normal load used for the measurements was 4,000 µN. Figure 25 shows the load displacement curve for the measurements and the obtained hardness of the GF in each direction. No difference in hardness was observed between the horizontal and perpendicular directions, and the value was close to the hardness of steel with a hardness of 5.36 GPa.

Considering these results, it can be stated that when the hardness of the counterpart steel is significantly lower than the hardness of the GF, the wear of the composite and the damage to GF are reduced, while the aggressive effects of GF on the softer counterpart steel increase. In addition, the wear and creep of the composite decrease owing to the effects of the decreased contact pressure between GF and steel, which are related to the lower hardness of the steel. Furthermore, the large amount of wear on the counterpart steel induces a further decrease of the contact pressure, and wear and creep of the composite are also reduced. By contrast, wear and creep of the composite increased with increasing hardness of the counterpart steel because the aggressive effects of GF on the steel decreased, and the aggressive effects of the steel on the composite increased, as presented in Figs. 21(a) and (b). The contact pressure remains at a higher level because the wear on the steel cylinders decreased. In addition, the higher contact pressure between GF and steel related to much higher hardness of the steel increases the wear and creep of the composite. -25 -

Conclusions

The tribological mechanisms of glass fiber-reinforced polyamide 66 material sliding in contact with a steel counterpart under high contact pressure in grease-lubricated conditions were clarified. The following points should be highlighted.

1) Detailed tribological behavior of the contact area and damage to the composite

An initial inflection point of the vertical displacement during sliding tests was observed and it was related to the creep of the composite ring. After this inflection point, the wear increased linearly with increasing number of cycles. The mechanical properties of the sliding surface change over time according to the damage inflicted on the sliding surface of the composite, and the damage to the sliding surface induces the increase of creep and friction coefficient.

2) Wear behavior of counterpart steel and the effect of the fiber orientation of the composite

An important effect regarding the fiber orientation on the sliding surface of the composite was observed on the wear resistance of the counterpart steel and composite. The aggressive effects observed on the counterpart steel were much greater when fibers were oriented in the perpendicular direction. By contrast, the wear of the composite increased when the fibers were aligned parallel to the sliding direction.

3) Contribution of each parameter on the increase of friction and damage

The damage to the sliding surface of the composite ring has an important effect. The contamination of the grease has no effect on the friction coefficient and damage. Friction and damage of the composite were reduced when worn steel was used, which is attributed to a decrease of the contact pressure. Damage on the composite ring decreased with decreasing temperature, which is attributed to the recovery of the mechanical properties of the composite once it cools .

4) Effects of the hardness of the counterpart steel

The relative hardness between the steel and fibers is important in understanding the wear induced in the steel and composite. The wear resistance of the composite improves when the hardness of the counterpart steel is lower, because aggressive effects on a composite that has harder fibers are reduced when the steel is softer, while the wear on a steel cylinder reduces the contact pressure.

Fig. 1

 1 Fig. 1 Schematic view of test specimens and sliding test setup

Fig. 2

 2 Fig. 2 Heat treatment process of steel cylinder.

Figure 3

 3 Figure3shows the evolutions of the vertical displacement, the temperature, and the average friction coefficient (µ) during 61,500 cycles for a normal load of 350 N and a stopping time of 1 s (after every 10 s of sliding), with the steel cylinders (hardness: 4.50 GPa). The temperature of the sliding surface was increased from 25 °C to 102 °C. The displacement did not increase in the initial stage of the test (indicating that neither wear nor creep occurred) and the average friction coefficient was 0.035-0.045. Example of the evolution of the friction coefficient for 2 s from the restart of the sliding after stopping phase are shown in Supplementary FigureS4. After the initial stage (around 21,000 cycles), both the maximum friction coefficient and average friction

Fig. 3 Fig. 4

 34 Fig. 3 Test chart over 61,500 cycles.

Fig. 5

 5 Fig. 5 Test specimens and grease before/after the sliding test (observed by optical microscope).

Fig. 6

 6 Fig. 6 Optical microscope image of wear debris in grease.

Fig. 7 (Fig. 9 Fig. 10

 7910 Fig. 7 (a) Height loss representing wear and creep at different cycles and (b) height loss ratio due to wear and creep

Fig. 11

 11 Fig. 11 Wear volume of steel cylinder for different sliding cycles.

Fig. 12

 12 Fig. 12 Observation of steel cylinder after 22,531 cycles by (a) optical microscope and (b) 3D images of interferometry; (c) central longitudinal 2D profile measured through interferometry.

Fig. 13 SEM

 13 Fig. 13 SEM images of sliding surface of composite in each area (a) Global view; (b) Inner area; (c) Center area; (d) Outer area

Fig. 15

 15 Fig. 15 Example of the relationship between the circularity of GF and cumulative area of GF

Fig. 16 (

 16 Fig. 16 (a) Sliding surface of pre-damaged composite rings before sliding tests; (b) evolutions of temperature; (c) evolutions of friction coefficient; (d) evolutions of displacement

Fig. 17

 17 Fig. 17(a) Grease in sample holder before sliding tests; (b) Evolutions of temperature;

  Fig. 17(a) Grease in sample holder before sliding tests; (b) Evolutions of temperature; (c) Evolutions of friction coefficient; (d) Evolutions of displacement

Fig. 19 (

 19 Fig. 19 (a) Sliding surface of a pre-damaged steel cylinder before sliding tests; (b) Evolutions of temperature; (c) Evolutions of friction coefficient; (d) Evolutions of displacement

2 and No. 4 ,

 4 Fig. 20 (a) Test conditions; (b) Evolutions of temperature; (c) Evolutions of friction coefficient; (d) Evolutions of displacement

1 .

 1 Figure 21(a) presents the estimated wear, creep, and total height loss of the composite ring for different values of steel cylinder hardness. The estimation of wear and creep was performed in the same way as presented in Section 2.Figure 21(b) shows the wear volume of

Figure 24 (

 24 b) shows the AFM images after the hardness measurements, and the indentations are observed on the surface of the GF.

Fig. 21 (

 21 Fig. 21 (a) Wear, creep, and total height loss of composite ring for different hardness values of steel cylinders, as measured by micro indentation (average values of 2 measurements); (b) Wear volume of counterpart steel cylinder for different hardness values of steel cylinders, as measured by micro indentation (average values of 2 measurements).

Fig. 22 Fig. 23

 2223 Fig. 22 Test charts for different values of hardness of steel cylinders; (a) evolution of temperature; (b) evolution of friction coefficient; (c) evolution of displacement

Table 1

 1 Test conditions for the sliding tests

		Diameter	3.5 mm
	Steel cylinders	Length	30 mm
	(x4)	Material	Carbon steel
		Young's modulus	205 GPa
		Outer diameter	25.6 mm
	Composite ring (JIS K 7218)	Inner diameter	20 mm
		Height	12 mm
		Mean rotation speed	790 rpm (Radius of rotation: 11.4 mm)
		Mean sliding speed	1 m/s
		Normal load	350 N
	Test	Contact pressure	198 MPa (at 25 °C)
	conditions		12-88 min: intermittent process
		Test time	(10 s sliding and 1 s stopping)
		Cycles	1,900 -61,500
		Environment temperature

Table 2

 2 Micro indentation test conditions.

	Measured sample	Composite ring	Steel cylinder
	Type of indenter	Vickers	Vickers
	Maximum normal load	1,500 mN	300 mN
		Load: 0→1,500 mN for 10 s	Load: 0→300 mN for 10 s
	Indentation test procedure	Creep: 1,500 mN for 5 s	Creep: 300 mN for 5 s
		Unload: 1,500 mN⇒40 mN for 4 s

Table 3 Heat

 3

	Quench hardening	Tempering	Hardness measured by micro indentation
	-(Raw steel)	-	4.5 +/-0.1 GPa
		412 °C×60 min. Air cooling	5.4 +/-0.2 GPa
	890 °C × 30 min.	300 °C×60 min. Air cooling	7.2 +/-0.1 GPa
	Oil cooling
		120 °C×60 min. Air cooling	9.9 +/-0.2 GPa

treatment conditions for steel cylinders. Values of the hardness are the average values for 5 measurements.

Table 4

 4 Calculated total area and total perimeter of GF in each area (Average value of different 3

	positions).

Table 5

 5 Values of C50 (the circularity when the cumulative area of GF is 50%, the average value of different 3

	positions).
	Area	Values of C50
	Inner area	0.63 +/-0.09
	Center area	0.25 +/-0.04
	Outer area	0.73 +/-0.07

  1 used the normal test conditions without stopping after 17,000 cycles. In test No. 2, the sliding test was intentionally stopped after 17,000 cycles (before the inflection point of the vertical displacement) and the temperature decreased to room temperature (the normal load was removed to avoid creep during the cooling period), and the sliding test was restarted without changing the grease or steel cylinders. In test No. 3, the sliding test was stopped at the same number of cycles and the temperature was allowed to decrease under the same conditions, but the grease was replaced with new grease before restarting the sliding test. In test No. 4, steel cylinders were replaced with new ones; however, the grease was not changed before the restart of the sliding test after 17,000 cycles. Furthermore, in test No. 5, the grease and cylinders were both replaced after 17,000 cycles and the sliding test was restarted. Figs. 20 (b), (c), and (d) present these test results. Comparing the results of tests No. 1 and No. 2, the inflection point of the friction coefficient and verticaldisplacement occurs much later when the test is stopped after 17,000 cycles than when the test is not stopped. This is attributed to the recovery of the surface mechanical properties of the composite ring resulting from the decrease of the temperature; the tribological properties were improved compared to the continuous sliding condition. Thus, it can be stated that the increase in temperature deteriorates the tribological properties.Comparing the results of tests No. 2 and No. 3, very similar results were obtained, even if the grease was replaced after the stop at 17,000 cycles in test No. 3, further verifying that there are no adverse effects due to contamination of wear debris in the grease, as mentioned previously. Comparing the results of tests No.


I Feurgard
email: ivan.feurgard@imt-lille-douai.fr

C Lors

R Gagné

D Damidot

Use of Colloidal Thickeners to Inject and Retain Bacterial Growth Media to Repair Cracked Concrete

Keywords: bacteria, cracks, concrete, thixotropy, thickeners, CaCO3

This study aimed at developing a bacteria-based method to repair cracks in concrete (crack opening < 1 mm) that would be suitable for fullscale operations and economically viable. CaCO3-producing bacteria were injected into calibrated cracks with bioavailable calcium and nutrients in a suspension thickened with a combination of Welan gum and Attagel® 50. These thickeners have synergistic effects, enhancing viscosity and shear-thinning behavior. These rheology characteristics made injection without drainage possible for crack widths ranging from 150 to 800 µm. Bacterial growth and the associated CaCO3 biomineralization were enhanced in the presence of thickeners.

Introduction

Cementitious materials are prone to developing cracks. Cracks narrower than 100 µm can be closed by natural self-healing, mainly due to the formation of calcium carbonate (CaCO3) [START_REF] Gagné | A study of the natural self-healing of mortars using air-flow measurements[END_REF]. Larger cracks, however, can dramatically reduce the lifespan of a structure by allowing aggressive compounds to penetrate deeper layers of materials and accelerate deterioration. Fine cracks (<1 mm) are currently mostly repaired with synthetic resins, which are easy to use, inexpensive, and watertight. These compounds are, however, pollutants. In addition, the treatment has to be repeated every 10 to 20 years due to chemical degradation of the polymers, resulting in additional costs and emissions [START_REF] Pacheco-Torgal | Biotech cementitious materials: Some aspects of an innovative approach for concrete with enhanced durability[END_REF]- [START_REF] Sargent | Respiratory Effects of Occupational Exposure to an Epoxy Resin System[END_REF]. Supplemental CaCO3 (mostly calcite) formed through bacterial activity [START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF] could provide an environmentally friendly alternative to synthetic resins: calcite is stable and longlived in most concrete service environments. Consequently, a growing number of studies are focusing on producing self-healing concrete or repairing cementitious structures through biomineralization [START_REF] De Muynck | Microbial carbonate precipitation in construction materials: A review[END_REF]- [START_REF] Gupta | Autonomous healing in concrete by biobased healing agents -A review[END_REF].

When used as repair agents, calcite-producing bacteria have to be injected into cracks. Maintaining the bacteria alive and able to produce enough calcium carbonate remains a challenge, as cracks are hostile environments. Moreover, conventional liquid bacterial cultures cannot keep bacteria, nutrients, and water inside the cracks. Therefore, a specific growth medium has to be developed. The literature has already reported on several bacteria immobilization techniques already in use, such as polyurethane foam [START_REF] Bang | Calcite precipitation induced by polyurethane-immobilized Bacillus pasteurii[END_REF], glass beads [START_REF] Bang | Microbial calcite, a bio-based smart nanomaterial in concrete remediation[END_REF], sand [START_REF] Ramachandran | Remediation of Concrete Using Micro-organisms[END_REF], [START_REF] Annamalai | Production and characterization of Bio Caulk by Bacillus pasteurii and its remediation properties with carbon nano tubes on concrete fractures and fissures[END_REF],

silica gel [START_REF] Van Tittelboom | Use of bacteria to repair cracks in concrete[END_REF]- [START_REF] De Belie | Crack repair in concrete using biodeposition[END_REF], and agar gel [START_REF] Qian | Bio-mineralization on cement-based materials consuming CO2 from atmosphere[END_REF]. Natural colloidal thickeners, however, seem to have the most potential for designing an injectable medium to repair cracked concrete. Indeed, these compounds are nonpolluting and inexpensive as well as being well suited for bacterial immobilization [START_REF] Martins | Immobilization of microbial cells: A promising tool for treatment of toxic pollutants in industrial wastewater review[END_REF]. Besides, most colloidal thickeners have highly shear-thinning and/or thixotropic behavior [START_REF] Picout | Rheology of Biopolymer Solutions and Gels[END_REF], which enhances injection efficiency. Clays, in particular, can entangle bacteria in biofilm-like networks providing protection against protozoa predation, desiccation, and washout [START_REF] Marshall | Clay Mineralogy in Relation to Survival of Soil Bacteria[END_REF]- [START_REF] Lünsdorf | Clay hutches': A novel interaction between bacteria and clay minerals[END_REF]. Used in combination, clays and polysaccharides often show synergistic effects, increasing the stability of the suspension and the effect of both thickeners, making highly shear-thinning and thixotropic suspensions used for various applications [START_REF] Ciullo | Rheological properties of magnesium aluminum silicate / xanthan gum dispersions[END_REF]- [START_REF] De Melo | Properties of Extruded Xanthan-Starch-Clay Nanocomposite Films[END_REF].

The aim of this study was to use the synergistic effect of two colloidal thickeners-Welan gum and Attagel® 50 palygorskite clay-to create a thickened medium suitable for injection into concrete cracks from 150 to 800 µm in width.

This study focuses first on the rheological characterization of the thickened growth medium using different Welan and Attagel concentrations to determine the most promising composition. Second, the behavior during injection and subsequent drying of the selected growth medium was tested on calibrated cracks in mortar samples. Lastly, the bacterial activity and the associated amounts of bioprecipitated calcite were assessed.

Materials and Methods

Suspension Preparation

All assays were performed in a solution containing 75 g.L -1 calcium Llactate hydrate (Sigma-Aldrich), 47 g.L -1 calcium nitrate tetrahydrate (Fisher Scientific), 20 g.L -1 calcium gluconate (Sigma-Aldrich), and 3 g.L -1 yeast extract (Difco), dissolved in ultrapure water (referred to below as LNG medium).

Welan gum (Kelco) and Attagel® 50 (BASF) powders were sterilized separately at 120°C in an autoclave for 15 minutes. Welan or Attagel suspensions were prepared by dispersing the powder at 22°C in the LNG medium, followed by homogenization with a magnetic stirrer at 1000 rpm for 24 hours. To prepare suspensions containing both thickeners, each thickener was separately dispersed in the LNG medium at 22°C at twice the concentration (ensuring better dispersion), then mixed together, and homogenized with a magnetic stirrer at 1000 rpm for 24 hours. The concentration of Welan and Attagel is expressed as a weight percent of the total weight of the suspension (w:w).

Rheological Assays

The suspensions were prepared 24 hours before the rheological measurements in order to allow the suspensions to rest after homogenization.

Rheological characterizations were carried out with a rheometer with coaxial cylinders (MCR302, Anton Paar) equipped with a CC27/P6 (grooved) inner cylinder (Anton Paar). After being poured into the cylinder, the suspensions were allowed to rest for 30 minutes with the inner cylinder in lower position.

The first measurement program applied an increasing, then decreasing rotational shear to the fluid. The measuring program set shear rates at 0,1, 1, 10, 50, 100, 250, 500, 250, 100, 50, 10, 1, and 0,1 s -1 . Each stage lasted 20 seconds and included 50 measurements of shear stress. The second program, starting immediately after the first, was divided in three steps: first, negligible oscillatory shear (oscillation; amplitude: 0,001%; angular frequency: 10 s -1 ) for 100 seconds, second, high rotational shear at 500 s -1 for 60 seconds; and lastly, negligible oscillatory shear (oscillation; amplitude: 0,001%; angular frequency: 10 s -1 ) for 10 minutes. The negligible oscillatory shear simulates rest in order to characterize the thixotropic behavior of the suspension.

The rheological characterization of the LNG medium thickened with Welan and Attagel followed a two-step experimental design. A first step was to determine the boundaries of the rheological characterization with a Hadamard matrix (Table 1). Based on the experimental results, the response of the medium to changes in thickener concentration was determined according to a composite experimental design. This design had two factors-Welan and Attagel concentrations-and three variables for each. As a result, 9 experiments had to be performed (Table 2).

Two additional suspensions were characterized to complete the surface graphs:

WA34, containing 0,3% Welan and 4% Attagel, and WA38, containing 0,3% and 8% Attagel.

Study of Pseudoplastic Behavior

The pseudoplastic behavior of each suspension was quantified with the Ostwald-deWaele law for viscoelastic fluids, also referred to as the power law:

= (1)
where η = viscosity, K = consistency index, = shear rate, and n = power-law index.

The power-law index is defined by the shear-thinning behavior of the suspension.

It is equal to 1 for Newtonian fluids and decreases as the shear-thinning behavior increases. The consistency index K defines the overall viscosity and is equal to the viscosity of the fluid at 1 s -1 . In this study, a custom indicator-the "viscosity drop factor"-was used to illustrate the power-law index. This factor is equal to the viscosity at 0,1 s -1 divided by the viscosity at 500 s -1 . It represents the loss of viscosity of the suspension under shear.

Injection Assays

Injection assays were performed on annular cracked mortar samples made as described by Ducasse-Lapeyrusse [START_REF] Ducasse-Lapeyrusse | Traitement de mortiers fissurés par biocicatrisation : vers une évaluation quantitative de l'efficacité bactérienne[END_REF] (height: 50 mm, outer diameter: 150 mm, inner diameter: 55 mm, reinforced with a 95 mm steel ring). Mortars were cast according to ASTM C109M [START_REF]C 109/C 109 M : Standard Test Method for Compressive Strength of Hydraulic Cement Mortars[END_REF] with a water/cement ratio of 0,485 and a sand/cement ratio of 2,75 with standard pure silica Ottawa sand and ordinary Canadian Portland cement (GU, Blaine fineness 381 m 2 .kg -1 ), Curing lasted for 28 days at 23°C and 100% RH. Crack openings between 150 and 500 µm were made by forcing a steel expansive core inside the mortar, as described by Gagné

and Argouges [START_REF] Gagné | A study of the natural self-healing of mortars using air-flow measurements[END_REF]. Samples were then carbonated for one month at 4% CO2, 20°C, 40% RH, allowing crack self-healing. Crack openings were measured with a videomicroscope on 24 points evenly distributed on both sides of the mortar.

Crack width was determined with air permeability measurements according to the method described by Gagné and Argouges [START_REF] Gagné | A study of the natural self-healing of mortars using air-flow measurements[END_REF]. The samples were dried at ambient temperature for 48 hours before the air permeability measurements.

For the injection tests, 10 mL of suspension were injected inside the cracks at low pressure with a custom injection device (Fig. 1). The suspension was slowly injected inside the crack, then the injection device was removed and the excess levelled off. If the crack had not been completely filled, the suspension was considered to be too thick. Conversely, the suspension was too fluid if drainage was observed after injection. In total, 52 cracks were tested for injection of thickened suspensions, including 18 with the WA36 suspension to take crack diversity into account. Six cracks were selected to test repeated injections of WA36.

Fig. 1. From left to right -cross-sectional view of the injection device setup on a mortar sample; upper side view; cracked mortar sample after injection of a colored solution

After injection, the sample was dried at ambient temperature for 48 hours, and then sawn to observe the cross section of the crack under scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. A small part of the inner surface of each crack was directly observed, and referred to as the "raw surface." All samples were metallized with carbon thread prior to observation.

Bacterial Growth Assays

In order to assess the effect of the thickeners on the production of calcium carbonate by the bacteria, the bacterial strain Bacillus pseudofirmus (DMS 2516, issued by DSMZ) was cultivated at 20°C in LNG medium versus in thickened LNG (referred to as LNGW) medium (Table 3). Stock bacteria were stored at -85°C in cryotubes containing 1 mL of bacteria in NBN medium (Table 3) and 0,5 mL glycerol (30%) at a bacterial concentration of 5.10 9 to 1.10 10 bacteria/mL. During the pre-culture, the bacteria were cultivated for 30 hours in NBN medium at 30°C on an orbital shaker (150 rpm). After 30 hours, the pre-culture was centrifuged at 4000 rpm for 20 minutes at 20°C. One pellet from 100 mL of pre-culture was used to inoculate each 100 mL batch of the growth assay.

The growth assays were performed in 250 mL Erlenmeyer flasks containing 100 mL LNG or LNGW medium (Table 3). Each condition was performed in triplicate. The bacteria were cultivated for 35 days at 20°C on an orbital shaker (150 rpm). Three milliliters of bacterial suspension were periodically sampled in order to monitor pH (Sensitix 41 probe, WTW), bacterial concentration, and lactate concentration during the assay. After 35 days, the assay was stopped and the cultures were centrifuged at 4000 g for 20 minutes at 20°C, rinsed twice with 40 mL ultrapure water, then dried at 40°C and weighed to determine the concentration of solids. A fraction of the dry pellet was used to determine the calcium carbonate concentration by TGA (Jupiter STA 449 F3, Netzch). To monitor bacterial concentration, the samples were diluted in saline suspension (c = 9 g.L -1 ), stained with acridine orange (Sigma-Aldrich), and fixed on nuclepore filters (Whatman). The bacterial cells were then counted under an epifluorescence microscope (Leica, France) at 490 nm. Lactate concentration was measured by ion chromatography (ICS-3000, Dionex). First, suspensions defined by the Hadamard matrix were studied (Table 4).

Results and Discussion

LNG medium containing 4% of Attagel did not show a strong viscosity recovery at rest. These suspensions were stable and presented no segregation or loss of consistency after being stored for three days at ambient temperature. The LNG medium containing 8% Attagel, however, was prone to segregation. The optimal range of concentration of Attagel was higher than 4% to ensure a significant effect of the thickener, but lower than 8% to prevent its destabilization.

Welan did not have a strong impact on the stability or on the thixotropic behavior of the suspension, but did affect viscosity. Indeed, suspensions containing 0,2% Welan had low viscosity and might therefore be prone to drainage after injection into cracks. Increasing the Welan concentration to 0,4% dramatically increased the viscosity of the studied suspensions. This concentration was sufficient to avoid post-injection drainage at any Attagel concentration.

Indeed, suspension WA48, containing 0,4% Welan and 8% Attagel, was pastelike under high shear and formed a weak gel after 4 hours at rest. The nine suspensions in the experimental design described in Table 2 yielded flow curves accurately fitting the Ostwald law (R² > 0,99) and had strong shear-thinning behavior, with power-law indexes lower than three (Table 5). Figure 2 provides the effect of each thickener on the consistency index K.

Both thickeners increased the value of K and, therefore, the viscosity of the suspensions proportionally to their concentrations.

Fig. 2. Evolution of the consistency index of the studied suspensions as a function of Welan and

Attagel contents

Fig. 3. Evolution of the power law index of the studied suspensions as function of Welan and Attagel contents

In all samples, when shear stress is displayed as a function of time, the modification of shear rate was followed by a progressive response of shear stress (Fig. 4: for clarity, only one sample (WA36) is displayed as an example). The suspensions therefore have short-term thixotropic behavior. The time needed for viscosity stabilization after a change in shear is approximately of 15 seconds, which is similar for destructuration (following an increase in shear rate) and recovery (following a drop in shear rate).

. Flow curve of suspension WA36 containing 0,3% Welan and 6% Attagel, displayed as shear stress / shear rate as a function of time

Moreover, the complex viscosity and storage modulus of all the samples progressively increased when subjected to 10 minutes of oscillatory measurements, following destructuration at 500 s -1 (Fig. 5). The Welan-Attagel suspensions therefore had long-term thixotropic behavior. After 10 minutes, the complex viscosity of the suspension was still increasing, suggesting a greater buildup in viscosity. The most concentrated suspensions (WA46, WA37, WA38, and WA48; see Table 5) were able to form weak gels after 6 hours at rest, which confirms that viscosity recovery continued to greatly increase viscosity.

Fig. 5 (A) complex viscosity and (B) dynamic modulus of the suspension WA36 containing 0,3% Welan and 6% Attagel under oscillatory shear after 30 s., stirring at 500 s -1

Combining Welan and Attagel yields shear-thinning, thixotropic, and settling-resistant suspensions for injection into cracked mortar. The rest of the experiments using thickened solutions were performed with suspension WA36, which contained 0,3% Welan and 6% Attagel dispersed in the LNG medium (referred to hereafter as LNGW).

Injection Assays with the LNGW Suspension

The LNGW suspension was successfully injected into all the cracks (150 to 500 µm) and filled at least 80% of the cracks with no post-injection drainage.

Fig. 6. Film formed inside a 800 µm crack by the thickened suspension (LNGW) after injection and drying

Fig. 7 Cross-sectional SEM observation of a 580 µm crack after injection of the thickened suspension (LNGW)

The film was made of interlaced 5 µm long needles with some particles, such as dolomite cubes, mortar debris, and precipitates (Fig. 8). This observation is similar to SEM observations of Attagel described in the literature [START_REF]Attagel rheology modifiers : guidelines[END_REF], showing that the film is mainly composed of clay. Indeed, the composition of the solid deposit, as determined by EDS analysis (Table 6), was close to the composition of the Attagel as provided by the supplier [START_REF]Attagel rheology modifiers : guidelines[END_REF], although SiO2 concentrations varied in the sample. shows that the deposit of clay polymer film in the crack significantly reduced the air permeability (Fig. 9). This led to a reduction in the apparent width of the crack by more than 50 µm after each injection of thickened suspension. The fact that the film itself was mostly thinner than 50 µm confirms the important role of the crest network in air permeability reduction. Repeated injections led to an accumulation of layers, producing a proportional reduction of the apparent crack width up to 200 µm.

Fig. 8. SEM observations of the inner surface of a crack after injection of thickened suspension. (A) dolomite cube covered with clay needles; (B) clay deposit

Bacterial Growth Assays with the LNGW Suspension

Figure 10 shows the evolution of the bacterial population during the growth assay. As concentrated inoculum was used, the initial population already ranged between 3.10 9 and 4.10 9 bacteria/mL at day 0. In the non-thickened medium (LNG), the bacterial population rapidly increased and stabilized at 1.10 10 bacteria/mL during the rest of the assay. The bacteria were in the form of small cocci, mostly assembled in tetrads or small clusters (Fig. 11). In the thickened medium (LNGW), the bacterial population also initially increased, but to a lesser extent than in LNG. Then, the bacterial population decreased slightly during the assay, but this might be attributed counting bias. Indeed, the cocci tended to form massive clusters with time, hampering counting accuracy.

The pH progressively increased during the assay both in the LNG and in LNGW media. After 35 days, the pH increased roughly 1 point, reaching a final value of approximately 7,5 at the end of the assay, with or without thickeners.

Without thickeners, there was a steady decrease of lactate concentration in the LNG medium (Fig. 12), stopping after 20 days and leading to the consumption of 80% of the lactate. Lactate consumption was quicker in the LNGW medium, leading to complete consumption after 20 days. Thus, the presence of thickeners did not hinder the bacterial activity and even seemed to be beneficial.

Experiment performed in triplicate

In the LNG medium, the average CaCO3 production (Table 7) was 17,0 g/L (+/-0,9 g/L) after 35 days. In the LNGW medium, the average CaCO3 production after 35 days was 25,1 g/L (+/-3,0 g/L). The production of calcium carbonate was therefore more important in the thickened medium, which is consistent with the greater lactate consumption. The presence of thickeners also contributed to increasing the total dry weight at the end of the assay, as Attagel added 60 g/L to the final solid composition in the samples.

Moreover, the protective effect of clays on bacteria have been largely

documented [START_REF] Marshall | Clay Mineralogy in Relation to Survival of Soil Bacteria[END_REF][START_REF] England | Bacterial survival in soil: Effect of clays and protozoa[END_REF][START_REF] Heijnen | Improvements to the use of bentonite clay as a protective agent, increasing survival levels of bacteria introduced into soil[END_REF][START_REF] Lünsdorf | Clay hutches': A novel interaction between bacteria and clay minerals[END_REF], and might constitute a major advantage as maintaining bacterial survival and activity is a major challenge for outdoor bioprocesses. As for the scope of crack sealing, the combination of biomineralization and clay deposit is particularly interesting as both factors help reduce crack permeability.

Moreover, the clay deposit might serve as a scaffold for calcium carbonate crystals, improving their durability inside the crack. Additionally, this growth medium shows interesting properties for a pilot-scale test:

-It is nonpolluting and nontoxic, and involves inexpensive materials.

-Its rheological properties make it suitable for various sizes of cracks.

-It can be prepared by simple dispersion of dry powders and does not require complex protocols or specific precautions.

-It forms a washout-resistant film inside the crack, contributing its sealing.

Conclusion

Adding polysaccharide gum (Welan) and a palygorskite clay (Attagel) to growth medium made it possible to design stable suspensions with strong shearthinning and thixotropic behavior. The thickened suspension was efficiently injected into cracks ranging from 150 to 800 µm. Cultivated in a growth medium containing 0,3% Welan and 6% Attagel, Bacillus pseudofirmus was able to grow and precipitate larger amounts of calcium carbonate than in the non-thickened growth medium. Future studies will aim at confirming the healing potential of the thickened medium developed in this study by testing it on an outdoor pilot scale or on real-scale concrete structures.

Acknowledgment

The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada.

Figure 3

 3 Figure 3 displays the power-law index "n" as a function of Welan and

Fig. 4

 4 Fig. 4. Flow curve of suspension WA36 containing 0,3% Welan and 6% Attagel, displayed as

Fe2O3 4 5 A

 45 comparison of six mortars with different crack widths (225-350 µm)

Fig. 9

 9 Fig. 9 Apparent width of cracks in six mortar samples, initially and after repeated injections of the LNGW medium

Fig. 10 ,Fig. 11 .Fig. 12 .

 101112 Fig. 10, Evolution of bacterial population (A) and pH (B) in the LNG and LNGW suspensions at 20°C. Experiment performed in triplicate

Table 1 . Hadamard matrix for the rheological characterization of the thickened medium N Welan (% w:w) Attagel (% w:w)

 1

	1	0,2	4
	2	0,2	8
	3	0,4	4
	4	0,4	8

Table 2 . Extended experimental design for the rheological characterization of the thickened suspensions

 2

			Welan	Attagel
	N	Factor value	% (w:w)	Factor value	% (w:w)
	1	-1	0,2	-1	5
	2	0	0,3	-1	6
	3	+1	0,4	-1	7
	4	-1	0,2	0	5
	5	0	0,3	0	6
	6	+1	0,4	0	7
	7	-1	0,2	+1	5
	8	0	0,3	+1	6
	9	+1	0,4	+1	7

Table 3 . Composition of the different media used for bacterial growth

 3

	Name	Composition
	NBN medium	11,8 g/L calcium nitrate (Sigma-Aldrich)
		8 g/L nutrient broth (Difco)
		75 g/L calcium lactate (Sigma-Aldrich)
	LNG medium	20 g/L calcium gluconate (Sigma-Aldrich)
		47 g/L calcium nitrate (Fisher)
		3 g/L yeast extract (Difco)
		LNG
	LNGW medium	0,3% Welan (w/w)
		6% Attagel (w/w)

Table 4 . Hadamard matrix and rheological characterization of the thickened suspensions

 4

		0,2% Welan	0,4% Welan
		Too fluid	Correct viscosity
	4% Attagel	Slightly thixotropic	Slightly thixotropic
		Viscosity at 1 s -1 : 2,3 Pa.s	Viscosity at 1 s -1 : 4,9 Pa.s
		Correct viscosity	Excessive viscosity
	8% Attagel	Slight tendency to segregate	Slight tendency to segregate
		Viscosity at 1 s -1 : 12,1 Pa.s	Viscosity at 1 s -1 : 15,3 Pa.s

Table 5 . Fitting of suspensions containing Welan gum and Attagel with Ostwald law for viscoelastic fluids

 5

	Sample	Welan (%)	Attagel (%)	K	n	Viscosity Drop Factor	R²
	WA24	0,2	4	2,33	0,235	633	0,997
	WA25	0,2	5	3,93	0,204	829	0,998
	WA26	0,2	6	5,82	0,185	933	0,998
	WA27	0,2	7	8,57	0,173	1038	0,999
	WA28	0,2	8	11,9	0,164	1175	0,999
	WA34	0,3	4	3,22	0,239	651	0,999
	WA35	0,3	5	4,94	0,211	810	0,999
	WA36	0,3	6	7,31	0,191	958	0,999
	WA37	0,3	7	10,9	0,175	1093	0,999
	WA38	0,3	8	14,8	0,164	1134	0,999
	WA44	0,4	4	4,18	0,247	664	0,999
	WA45	0,4	5	6,34	0,214	833	0,999
	WA46	0,4	6	8,99	0,194	979	0,999
	WA47	0,4	7	12,5	0,177	1105	0,999
	WA48	0,4	8	16,7	0,171	1147	0,999

Table 6 : Average measured composition of the solid deposit and commercial Attagel

 6

		composition [32]
	Mineral (as	Average Measured Value	Attagel Concentration (mass%)
	element)	(mass%)
	SiO2	77,7 (+/-9,0)	65,2
	Al2O3	11,7 (+/-1,2)	12,7
	MgO	11,1 (+/-0,9)	12,3
	CaO	5,6 (+/-3,2)	3,9

Table 7 . Dry weight and calcium carbonate production after 35 days in the LNG and LNGW suspensions

 7

		1	2	3		1	2	3
	Dry weight (g/L)	19,7	21,2	20,3	20,4	94,3	88,3	88,5	90,3
					(+/-0,8)				(+/-3,4)
	CaCO3 in dry	83%	84%	85%	84	38%	34%	35%	36
	residue (%)				(+/-1)				(+/-2)
	CaCO3 production	16,4	17,7	17,2	17,1	28,5	23,2	23,6	25,1
	(g/L)				(+/-0,7)				(+/-3,0)

Sample

LNG-LNG-LNG-Average LNGW-LNGW-LNGW-Average

Additional injections into cracks up to 800 µm filled cracks to 100% without postinjection drainage, which suggests a wider injection range. This versatility is due to the shear-thinning and thixotropic behavior of the suspension: viscosity can be strongly lowered by shaking to facilitate injection, while recovery prevents postinjection drainage.

Upon drying, the thickened suspension formed a thin film covering most of the inner surface of the crack, with a network of crests almost joining both sides of the crack (Figs. 6 and7). The film deposited on the inner surface of the crack was irregular and less than 50 µm thick on most of the surface.

Bacillus pseudofirmus calcite-forming bacteria