Mathieu Le Goc

Pierre Dragicevic

Samuel Huron

Jeremy Boy

Jean-Daniel Fekete
email: jean-daniel.fekete@inria.fr

A Better Grasp on Pictures Under Glass: Comparing Touch and Tangible Object Manipulation using Physical Proxies

Keywords: CCS Concepts, Human-centered computing → User studies, Empirical studies in HCI, Tangible user interfaces, touch interfaces, physical proxies

We introduce a novel method based on physical proxies for investigating fundamental differences between touch and tangible interfaces. This method uses physical chips to emulate the flat, non-graspable objects that make up touch interfaces, in a way that supports direct comparison with tangible interfaces. We ran an experiment to test the effect of object thickness on participants' behavior, performance and subjective experience in spatial rearrangement tasks. We found that for the tasks tested, thick objects are faster but less accurate to operate, and that their graspability is only used occasionally. We also found that coarse manipulation of multiple thin objects is error-prone, an issue that only thick objects may allow to alleviate.

INTRODUCTION

In this article, we introduce a novel proxy-based methodology for comparing touch and tangible interaction paradigms. We use thin physical chips (0.2mm) as surrogates for the graphical elements of a direct manipulation touch interface, which we compare with thicker pucks (see Figure 1) in order to isolate the effect of object thickness on spatial rearrangement tasks.

Touch interfaces are commonly thought to support rich and engaging interactions, and are often showcased in futuristic movies and concept videos. At the same time, Bret Victor eloquently argues that touch interfaces are nothing but "pictures under glass" [START_REF] Victor | A brief rant on the future of interaction design[END_REF], and that the gestures they While such viewpoints can be inspiring, it is also important to evaluate claims rigorously, and to try to pinpoint the fundamental differences between touch and tangible interaction paradigms. Granted for many applications, both interaction styles are likely to be complementary, but understanding how they differ will inform design decisions and future research.

Many studies have suggested that people are more effective at operating tangible interfaces (TUIs) (e.g., [START_REF] Antle | Comparing motor-cognitive strategies for spatial problem solving with tangible and multi-touch interfaces[END_REF][START_REF] Lucchi | An empirical evaluation of touch and tangible interfaces for tabletop displays[END_REF][START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF][START_REF] Schneider | Benefits of a tangible interface for collaborative learning and interaction[END_REF][START_REF] Terrenghi | Affordances for manipulation of physical versus digital media on interactive surfaces[END_REF]) -presumably because TUIs support a richer manipulation vocabulary. These findings are consistent with Bret Victor's assertion that touch interfaces do not allow us to fully "use our hands" [START_REF] Victor | A brief rant on the future of interaction design[END_REF]. However, previous studies do not provide robust explanations as for these advantages -they do not investigate fundamental differences between the two interaction paradigms, i.e., differences that are technologyindependent and are likely to hold in future implementations.

Our interest is to establish technology-agnostic methods for answering precise questions regarding differences between touch and TUIs. To illustrate such methods, we used flat and thick objects in order to isolate the effect of object thickness on participants' speed and accuracy when performing spatial rearrangements of multiple objects on a 2d surface. As such, our main contributions are:

• a novel technology-agnostic method based on physical proxies for studying how (and how well) people spontaneously manipulate typical touch and tangible objects, and • an assessment of the effect of object thickness on the manipulation of collections of objects, with implications for the design of both touch and tangible interfaces.

BACKGROUND

There is no general consensus on how touch and tangible interfaces should be defined [START_REF] Shaer | Tangible user interfaces: past, present, and future directions[END_REF]. Therefore, we propose the following working definitions for the purpose of this article:

Touch interfaces are user interfaces where digital information is manipulated through skin contact with the surface of a single physical object.

Tangible interfaces are user interfaces where digital information is manipulated by changing the spatial arrangement of multiple physical objects.

These definitions are by no means authoritative: they are purely stipulative and meant to clarify upcoming discussions. Although they focus on input, they align well with what is generally understood by both terms [START_REF] Shaer | Tangible user interfaces: past, present, and future directions[END_REF], and capture a broad range of interactive systems. However, our definition of touch excludes all interfaces that employ contactless (midair) sensing, as well as all interfaces that have moving parts; and our definition of tangible excludes single-device input (e.g., regular, gyroscopic or 3D mice, or mobile phones with accelerometers), but includes space-multiplexed input device setups [START_REF] Fitzmaurice | An empirical evaluation of graspable user interfaces: towards specialized, space-multiplexed input[END_REF].

Given these definitions, how do touch interfaces compare to tangible interfaces? Although some studies have looked at differences in subjective experience (e.g, [START_REF] Alzayat | Quantitative measurement of virtual vs. physical object embodiment through kinesthetic figural after effects[END_REF]), most studies focused on performance. An early evaluation of TUIs by Fitzmaurice and Buxton [START_REF] Fitzmaurice | An empirical evaluation of graspable user interfaces: towards specialized, space-multiplexed input[END_REF] demonstrated the benefits of space-multiplexed input (one physical device per function) over time-multiplexed input (a single generic device -e.g., a computer mouse). However, tabletop systems supporting multipoint direct touch input rendered this distinction less useful [START_REF] Tuddenham | Graspables revisited: multi-touch vs. tangible input for tabletop displays in acquisition and manipulation tasks[END_REF].

Over the past years, several studies have been conducted comparing multitouch tabletop setups with tabletop TUIs on object manipulation tasks. A replication of Fitzmaurice and Buxton's study revealed that tangibles are still superior [START_REF] Tuddenham | Graspables revisited: multi-touch vs. tangible input for tabletop displays in acquisition and manipulation tasks[END_REF]. Similar results have been found for puzzle sorting tasks [START_REF] Antle | Comparing motor-cognitive strategies for spatial problem solving with tangible and multi-touch interfaces[END_REF][START_REF] Terrenghi | Affordances for manipulation of physical versus digital media on interactive surfaces[END_REF], spatial arrangement tasks [START_REF] Lucchi | An empirical evaluation of touch and tangible interfaces for tabletop displays[END_REF][START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF] and collaborative layout optimization tasks [START_REF] Schneider | Benefits of a tangible interface for collaborative learning and interaction[END_REF]. Observed benefits include faster task completion times, less errors and more user satisfaction, and are generally explained by the fact that tangibles support richer as well as more efficient gestures. For example, people spontaneously rotate physical objects using multifinger grips, which is presumably more efficient than pinch gestures [START_REF] Tuddenham | Graspables revisited: multi-touch vs. tangible input for tabletop displays in acquisition and manipulation tasks[END_REF].

Besides user performance, many of the previous studies report observational data on the gestures used. For example, North et al [START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF] identified several gesture types in their study, some of which (e.g., lift or stack) were only possible with TUIs.

While informative, previous studies have so far focused on comparing specific implementations of touch and tangible interfaces. As a result, findings are hard to interpret and generalize. In some studies, touch interfaces may have been given an unfair advantage by supporting more advanced features and interaction techniques. For example, one study [START_REF] Lucchi | An empirical evaluation of touch and tangible interfaces for tabletop displays[END_REF] provided undo, lasso selection and snap-dragging on the touch interface but not on the tangible one. Similarly, contact shape sensing is rarely implemented (but see [START_REF] Antle | Comparing motor-cognitive strategies for spatial problem solving with tangible and multi-touch interfaces[END_REF]), thus limiting the potential expressiveness of touch [START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF][START_REF] Wilson | Bringing physics to the surface[END_REF] and potentially giving tangibles an advantage. Moreover, researchers have recently discovered that the lag produced by any currently available touch system negatively impacts performance and user experience [START_REF] Jota | How fast is fast enough?: a study of the effects of latency in direct-touch pointing tasks[END_REF].

To summarize, previous studies are insightful but often suffer from experimental confounds that make it hard to understand the fundamental differences between touch and tangible interfaces -i.e., differences that will persist and remain relevant as both touch and tangible technologies mature. Thus it would seem beneficial to explore complementary methods that do not depend on currently available technology.

METHODOLOGY

Here, we explain and motivate our proxy-based method for comparing touch and tangible interfaces.

Rationale for Using Physical Proxies for Touch

The goal of a physical proxy is to help empirically investigate user interfaces that are hard to build, or that may only exist in the future. Nevertheless, we stress that a physical proxy necessarily differs from the user interface it emulates.

Physical proxies have already occasionally been used for comparing 2d and 3d visualization techniques. Dwyer has proposed hand-crafted physical models to emulate perfect 3d displays [8, pp. 39-45], while Jansen has suggested to use static physical visualizations in user studies as substitutes for future self-actuated physical visualizations [12, p. 133].

Although both touch interfaces and TUIs will keep improving, touch will only ever allow flat objects to be operated from above, while TUIs allow objects with volume to be pushed sideways, grabbed, lifted, etc. Therefore, we use thin discs or "chips" as proxies for touch-operated objects, and cylinders or "pucks" as proxies for tangible objects (Figure 1).

The proxy for the complete touch interface consists of a collection of chips placed on a planar surface to emulate GUI icons that can be freely dragged around. This setup necessarily differs from today's touch interfaces in several respects, while sharing several characteristics with an "idealized" touch interface. More specifically, the differences are: Df 1. with enough effort, the proxies can be grasped and lifted, something that is impossible on actual touch interfaces; Df 2. the proxies can slide and fall off the surface if it is inclined-which means that our particular proxy implementation is inappropriate for hand-held and mobile touch devices; Df 3. the proxies' collision behavior is unusual for traditional touch interfaces: the proxies can both push each other and overlap-unlike graphical objects which either interpenetrate or collide, depending on the chosen metaphor; and Df 4. the proxies do not support abstract gestures or advanced commands (e.g., selection, grouping, undo), which are common in touch interfaces. Nevertheless, this proxy setup can help us learn more about how touch interfaces may differ from tangible interfaces by making the comparison fairer in other respects, since the setup:

• has zero-lag: lag on any current touch interface has been found to be noticeable and detrimental [START_REF] Jota | How fast is fast enough?: a study of the effects of latency in direct-touch pointing tasks[END_REF]; • has infinite resolution, both in terms of input and output; • provides physically realistic direct manipulation, both for single and multiple objects [START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF][START_REF] Wilson | Bringing physics to the surface[END_REF];

• provides realistic rendering: in particular, no light is emitted by the surface (similar to, e.g., an e-ink display); • provides realistic touch haptics [START_REF] Bau | Teslatouch: electrovibration for touch surfaces[END_REF]: object edges can be felt, and no skin friction occurs when an object is dragged. Currently, only TUIs have all these characteristics. However, none of them are fundamentally incompatible with touch interfaces, for which both display and sensing technology is quickly improving: a lag of 1 ms has been recently achieved in research prototypes [START_REF] Jota | How fast is fast enough?: a study of the effects of latency in direct-touch pointing tasks[END_REF], as well as tactile feedback [START_REF] Bau | Teslatouch: electrovibration for touch surfaces[END_REF].

Research Questions

Our goal is to compare how (and how well) chips and pucks are spontaneously manipulated, in order to assess the sole impact of object thickness, a fundamental (i.e., implementationindependent) difference between touch and tangible interaction paradigms. Our two major research questions are:

1. Does the extra thickness of pucks present any manipulation benefits? That is, do people spontaneously make use of the extra spatial manipulation strategies offered?

2. How good is our physical proxy implementation? That is, to what extent do Df 1-Df 4 affect the way in which chips are manipulated vs. the way we expect them to be manipulated in an idealized touch interface?

Rationale for the Choice of Setup and Tasks

For the comparison between chips and pucks to be fair, and to address Df 2 at the outset, we chose to simulate tabletop touch and tabletop tangible interfaces, which is what has been compared in the vast majority of previous studies [START_REF] Antle | Comparing motor-cognitive strategies for spatial problem solving with tangible and multi-touch interfaces[END_REF][START_REF] Lucchi | An empirical evaluation of touch and tangible interfaces for tabletop displays[END_REF][START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF][START_REF] Schneider | Benefits of a tangible interface for collaborative learning and interaction[END_REF][START_REF] Terrenghi | Affordances for manipulation of physical versus digital media on interactive surfaces[END_REF][START_REF] Tuddenham | Graspables revisited: multi-touch vs. tangible input for tabletop displays in acquisition and manipulation tasks[END_REF]. To address Df 4, we limited our study to manipulative gestures [START_REF] Kammer | Towards a formalization of multi-touch gestures[END_REF], which both setups support. Direct manipulation is the main interaction style in TUIs and it is also dominant in modern touch devices; and while in principle TUIs could support symbolic gestures, most currently do not.

Finally, to understand how users manipulate collections of objects (chips and pucks), we chose to use repetitive spatial rearrangement tasks-in a similar fashion to previous studies such as North et al [START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF]. Such tasks are reasonably complex in the motor domain (compared to, e.g., target acquisition tasks), while at the same time devoid of high-level cognitive components that are typically present in real-world tasks, but that could interfere with motor and manipulative behavior. Our study focuses not on high-level cognition, but on how people naturally and spontaneously "use their hands" [START_REF] Victor | A brief rant on the future of interaction design[END_REF].

EXPERIMENTAL PROCEDURE

We now describe in more detail our setups, tasks, procedure, and experiment design.

Physical Proxy Setup

The touch and TUI proxies shared the same setup (see Figure 2). The setup included a white plastic support surface, with 3 mm-thick edges to prevent objects from sliding off.

We digitally-fabricated forty 0.2 mm thick disks (chips), and forty 12 mm thick cylinders (pucks); all had a diameter of 25.75 mm (the size of a 2-euro coin). The chips were made of transparent laser-cut mylar sheets, and weighed roughly 0.2 g (see Figure 1 left). We chose mylar as it remains rigid, even when very thin. This way, chips would be difficult to bend and therefore difficult to grasp. The pucks were made of several layers of laser-cut transparent acrylic sheets glued together, and weighed roughly 7.5 g (Figure 1 right). We informally verified that mylar and acrylic had similar friction properties. Finally, all chips and pucks were spray-painted, half of them blue and the other half red. Chips were only painted on top, and pucks were painted on top and on the sides. Thus both object types had an unpainted bottom.

Tasks

An experimental task consisted of moving all objects from an initial configuration to a final configuration. There were 3 different types of configurations, all involving the same 40 objects (20 blue and 20 red, see Figure 3):

• Random-packed : all objects are packed within a disc of diameter 24 cm at the center of the workspace. They fill 46% of the disc area, with an inter-object spacing of ∼0.5 cm. • Random-spread : same arrangement but within a larger disc of diameter 40 cm (density 17%, separation ∼2.5 cm). • Sorted : all red objects are placed on one side of the table, while all blue objects are placed on the other. Object location within the area is not important. From these three configurations we derive four different tasks:

• Packed sorting: from random-packed to sorted.

• Spread placement: from sorted to random-spread.

• Spread sorting: from random-spread to sorted.

• Packed placement: from sorted to random-packed. Since the final configuration of a task is also the initial configuration of the next task, these tasks could be performed in sequence and looped over without the need for actuation or experimenter intervention. Object colors in placement configurations were randomly shuffled, and sorting areas were randomly swapped. We henceforth refer to a sequence of four successive tasks as a block. A block can start with any of the four tasks above, thus four different block designs are possible.

Tasks were given to subjects by top-projecting the desired final configuration on the workspace, during the entire task duration. Visual targets were made dim enough so as not to interfere with physical manipulation. For placement tasks, individual object targets were shown with red and blue semicircles 120% the size of objects (30.9 mm) (see Figure 3). For sorting tasks, a red line and a blue line were displayed on each side of the table. Objects simply had to be moved across the lines.

The use of semicircles instead of circles is due to limitations of the top projection setup. Since the projector could not be oriented perpendicularly to the table, pucks casted a shadow, which pilots suggested might impair puck placement tasks. The use of semicircles addressed this issue, at the cost of rendering placement tasks less well-specified. This was addressed by instructing participants not to focus on accuracy, and by later analyzing both completion times and placement accuracy.

Task Instructions

We wanted to observe spontaneous manipulation behavior, thus no instruction was provided in the way objects were to be manipulated. In particular, the touch proxy was not presented as such, and no reference was made of touch or tangible interfaces. Objects were referred to using the neutral names chips and pucks. At no point did the experimenter manipulate chips or pucks in front of participants.

Participants were instructed to be as fast as possible without committing any obvious placement error, i.e., all objects were required to be approximatively placed within their respective targets (or beyond lines for sorting) with no color inversion. Emphasis on speed was used as an incentive for participants to converge to optimal motor strategies. It is reasonable to assume that similar optimal strategies will be developed in natural settings after extended use of such user interfaces.

The system did not check for correct object placement. Instead we told participants that a photo snapshot will be taken each time they complete a trial, and trials will be later examined for correctness. Correctness was also enforced by the experimenter during practice trials.

PROCEDURE

The participant was asked to sit comfortably at the table and was given an instruction sheet. All 80 objects had been previously placed in storage areas ( 3 in Figure 2), with chips and pucks stored in their respective areas.

The participant was first asked to bring all objects from one storage area to the main area (chips or pucks depending on the participant) and to complete a discovery block -i.e., four tasks in sequence -where she could get acquainted with the experiment logics without time pressure. Then the participant was asked to switch object type (see procedure below) and complete a training block with time pressure but no performance recording. The participant then switched objects again and went on performing the experimental trials.

An experimental trial consisted in the administration of a single (placement or sorting) task and proceeded as follows:

1. A message invites the participant to press the timer button ( 8 in Figure 2) when she is ready to start, upon which a two-second countdown appears on the table. 2. Two seconds later the configuration to achieve is displayed, and a beep sound is played signifying that the timing started. 3. The participant moves the objects to their final destination ( 4 for sorting tasks or 2 for placement tasks). 4. As soon as the task is completed, the participant presses the timer button again and a second beep sound is played, signifying that the timing stopped. The projected configuration disappears and a message invites the participant to lean back and get her hands off the table. 5. Two seconds later, a photo snapshot of the workspace is taken (using 5 in Figure 2), accompanied with a camera shutter sound. The invite message for the next trial is then displayed (step 1 above). This trial design together with the unpredictable ordering of colors prevented participants from pre-planning their motor actions. This incentivized participants to interleave motor planning with motor execution, a strategy closer to typical UI object manipulation scenarios. Participants were however allowed to place their hands as they wished during the two-second countdown, as long as they were not touching any object. Thus hand placement, which was irrelevant to the purposes of the experiment, was not included in task times.

Every two blocks, the participant was asked to switch to the other object type (chip or puck). Object switching followed the following procedure: The participant is asked to move all current objects in one of the storage areas, then move all the objects from the other storage area in front of her. Then the final configuration from the last task is displayed and the participant is invited to place all objects, without time pressure.

All experiment instructions were given through short messages and visual annotations, all projected on the table (using 1 in Figure 2). Each participant was recorded from different angles using two cameras ( 6 and 7 in Figure 2). At the end of the experiment, the participant was asked to fill in a brief questionnaire. The experiment lasted 35 minutes on average.

Design

Our independent variables were object type Object ∈ {Chips, Pucks} and type of task Task ∈ {Packed-placement, Spread-placement, Packed-sorting, Spread-sorting}, both withinsubject factors. Each combination of Object and Task was administered four times, i.e., four Block were generated per Object condition. Our dependent variables were time-ontask Time, and placement accuracy Accuracy.

Accuracy was only measured for Packed-placement and Spread-placement tasks. For each trial, the position of all 40 objects was extracted from photo snapshots through image processing and visual inspection. Positions were then matched to the nearest slot in the placement template. Then, for each slot, a position dispersion was computed by taking the unbiased estimate of the positions' standard distance deviation (SDD). Four distance measurements were used in computing each SDD (one per repetition). SDDs were then averaged across all 40 slots, yielding an accuracy measure for each Object and Task ∈ {Packed-placement, Spread-placement} combination.

To reduce learning effects, Object was switched every two blocks. In addition, the presentation order of the first Object was counterbalanced across participants as well as the first task presented in all blocks, yielding 2 × 4 = 8 unique designs. Red and blue colors in task configurations were fully randomized.

Overall the experiment consisted in 2 Object × 4 Task × 4 Block = 32 trials, yielding 32 measures of Time and 4 measures of Accuracy per participant.

Participants

We recruited 8 participants (5 male), all naive to the purposes of the experiment, by email announcement to our lab. Ages ranged from 24 to 32 with a median of 28. All had a university degree, from Bachelor to PhD. They were all right-handed, owned a touch device and were familiar with such technologies (as reported in the post-experiment questionnaire). All participants agreed to be videotaped.

PERFORMANCE AND SUBJECTIVE EX-PERIENCE

Due to growing concerns about the limits of null hypothesis significance testing for reporting and interpreting results [START_REF] Dragicevic | Fair statistical communication in hci[END_REF], we base our analyses on simple effect sizes with confidence intervals (CIs). See [START_REF] Cumming | Inference by eye: Confidence intervals and how to read pictures of data[END_REF] on how to interpret plots with CIs.

Time

We perform our time analyses on log-transformed data [START_REF] Sauro | Average task times in usability tests: what to report?[END_REF]. We thus report geometric means, and all comparisons are expressed as time ratios. CIs assume lognormal distributions.

Completion times were averaged per participant for each combination of Object and Task. The results shown in Figure 4 confirm that pucks are overall faster than chips. There is strong evidence for placement tasks, and a possibly similar trend for sorting tasks, but the evidence is much weaker.

Accuracy

Accuracy observations were averaged per participant and per Object condition, and population means (in mm) as well as within-subject ratios (dimensionless) were estimated using 95% BCa bootstrap CI. Results are shown also in Figure 4: on average, object placement variability ranges from 6 to 8 mm, with clear signs that chips tended to be positioned more consistently than pucks on placement tasks. Thus it appears that pucks can be manipulated faster than chips under our setup, but their advantage is less apparent in sorting tasks. They also tend to be positioned slightly less accurately.

Subjective Experience and Feedback

Our post-experiment questionnaire included five items to assess participants' subjective experience, and also invited them to elaborate on their responses. Figure 5 shows participants' mean responses, given on 5-point Likert scale.

Unsurprisingly, participants strongly agreed that pucks "felt physical". However, participants were also reluctant to admit that chips felt like a touch interface. Looking at spontaneous comments, tactile feedback seems to have strongly influenced their experience. One participant commented that "the tactile feedback is different [from a touch interface]".

Responses revealed a remarkably more positive experience with pucks. Participants reported that they "became increasingly frustrated with [their] inability to pick and place the chips", while "pucks are easier to handle and [...] more comfortable to manipulate". Participants reported that pucks made them feel more efficient and more in control: "You feel more in control with the pucks, they are easier to grasp".

A participant further elaborated "Because pucks could be picked up and placed, they allowed for a wider array of placement strategies [...]. Moving the last few chips into position often required sliding them all the way around the already placed ones, which felt cumbersome. I preferred having the ability to use multiple placement strategies with the pucks and their physicality was satisfying".

Discussion

Participants' comments give us hints as to why pucks were manipulated faster and yielded a better subjective experience. Participants seemed frustrated that the chips could not be grasped and lifted. Indeed, we observed that placement tasks required more caution not to muddle up already placed chips while sliding new ones in. In contrast, pucks could be easily picked and placed anywhere. Arguably, in many real touch interfaces dragged objects go through other objects, thus emulating "pick and place" operations. This was not possible with chips. Note that it is also not possible with touch metaphors that implement collision detection [START_REF] Antle | Comparing motor-cognitive strategies for spatial problem solving with tangible and multi-touch interfaces[END_REF][START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF][START_REF] Wilson | Bringing physics to the surface[END_REF]. However, this metaphor also generally ensures that objects never occlude each other, which was not the case for our proxy.

Despite the limitations of our proxy, there is still evidence that pucks yielded benefits that even an idealized touch interface could not provide. First, the sorting task is a coarse motor task that should not have suffered from chip collision and overlap -yet, our results suggest that pucks might have been still faster. Second, as we will see with the video analysis, issues with overlap were not that common and chips appear to have suffered from problems unrelated to overlap and collision.

VIDEO ANALYSIS

We now report on a video analysis of the gestures performed by participants in the Chips and Pucks conditions.

Manipulation Gesture Typology

The first step of the coding process consisted in agreeing on an initial set of gestures. We focus on physical gestures, not gestures in the traditional HCI (input) sense. We therefore decided to identify recurrent gestures based on video footage. Doing so we noticed common manipulation errors-i.e., manipulations with unintended consequences. We thus agreed on a typology that included a total of 21 gestures and errors, of which we report 13 here (this selection will be explained later on). The manipulation gestures were: Fine drag: Move object(s) while pushing against their top surface with one or more fingertips. Coarse drag: Move object(s) while pushing against their top surface with another body part (e.g., flat hand). Grasp drag: Move object(s) by grasping and not lifting them. Lift: Move object(s) by grasping them and lifting them. Bulldozer push: Move object(s) by pushing sideways, with the body in contact with the surface. Throw: Move object(s) by grasping / pushing and releasing contact during transport. Separate: Move object(s) in order to break contact with surrounding object(s).

Whereas the manipulation errors were: Miss: Contact with object(s) is not achieved while intending to initiate a manipulation. Loss: Contact with object(s) is unexpectedly lost. Collision: Object(s) being manipulated accidentally collide with surrounding objects. Overlap: Moving several overlapping objects while intending to move less of them. Stick: Object sticks to skin when attempting to release contact. Escape: Object moves by itself instead of remaining still.

Method

We wanted to focus on close-to-optimal strategies and thus only analyzed two blocks (8 trials per participant) from the video recordings: the last block involving chip manipulation and the last block involving puck manipulation. This represented about 30 minutes of video for all 8 participants.

Two coders (co-authors) independently annotated video logs using ChronoViz [START_REF] Fouse | Chronoviz: a system for supporting navigation of time-coded data[END_REF]. Each time any of the 21 gestures or manipulation errors was seen occurring, the coder annotated its exact starting time as well as the hand used. This process took each coder about 30 hours, for a total of 5000 gestures and manipulation errors reported.

Inter-Coder Agreement

In order to estimate inter-coder agreement, annotations had to be temporally aligned. We used a three-pass algorithm. The first pass searched for matching annotations. A match was defined by two annotations having the same category, the same hand and being close in time (±350 ms). A second pass then searched for non-matches, defined as two annotations having the same hand, being close in time, but having different categories. Remaining annotations were discarded.

We assessed inter-coder agreement using Cohen's Kappa (K) [START_REF] Cohen | Weighted kappa: Nominal scale agreement provision for scaled disagreement or partial credit[END_REF]. K ranges from -1 to 1, with 1 being a perfect agreement, 0 what would be expected by chance. We added the hand used (left or right) to our 21 categories, yielding a total of 42. The coder agreement was K=0.47, a "moderate" agreement [START_REF] Cohen | Weighted kappa: Nominal scale agreement provision for scaled disagreement or partial credit[END_REF]. We noticed that one coder annotated more gestures than the other (12% more), revealing that micromovements were not consistently taken into account. These micro-movements were thus discarded in the rest of the analysis, leading to a "substantial" agreement of K=0.79.

Results

Figure 6 reports gesture occurrence frequencies per Object condition and per coder. Error frequencies are relative to the total number of gestures. The last column reports per-gesture coder agreements, ranging from "fair" to "substantial".

Looking at the diversity of manipulation gestures, fine drags (typical of touch interfaces) represents the vast majority of gestures in the Chips condition. Coarse drags and bulldozer push gestures, are also relatively common. Both are possible with touch interfaces, although not often implemented [START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF][START_REF] Wilson | Bringing physics to the surface[END_REF].

Surprisingly, fine drags are also the most common gesture with Pucks: participants typically manipulated pucks as if they were using a touch interface. However, the gestures are also more diverse, with more throw gestures, and gestures like lifts and grasp drags that are impossible with touch. Lifts have been observed only very rarely with Chips, confirming that Df 1 was not a problem with our proxy setup.

Despite lower coder agreements, both coders reported much more manipulation errors with Chips. Approximately twice as many losses are reported (e.g., a few chips are left behind during bulldozer pushes), and misses (e.g., a finger lands next to a chip) also seem much more common.

These results also help us understand to what extent chip overlap (Df 3) was a problem. Overlaps caused manipulation errors in only about 3% of all gestures, and only 3-4% of all hand gestures were for separating overlapping chips. This suggests that although the effect of chip overlap is measurable, other issues such as losses or misses are at least as important.

Finally, the dominant and non-dominant hands were used about equally often in both Object conditions.

Understanding Chip Losses and Misses

Video recordings revealed that chip losses happened during bulldozer pushes and coarse drags. Figure 7 shows an instance.

We informally experimented with bulldozer pushes by lining up chips so overlap is impossible. It appeared that chips are lost because it is hard to maintain a large contact area between the body and the surface. Full contact is not required with pucks because they are too thick to slip below the arm. Similarly we repeated coarse drag gestures and found that chips are lost because it is hard to distribute body pressure on several chips. With pucks, this is much easier because the empty space around makes it possible to orient and shape the hand in different ways, and the intervening space allowed the flesh to deform in a way that distributes forces more equally.

Misses cannot be easily explained by taking a two-dimensional perspective, since chips and pucks have the same radius. However, since pucks "stick out", they likely facilitate quick establishment of lateral contact. This additional error tolerance on the third dimension may have made puck acquisition both less error-prone and less attention-demanding, considering that bimanual tasks spanning a large surface like ours require split visual attention. Also, failure or success to acquire a puck is signaled with more distinctive haptic and auditory cues, thus likely reducing the need to visually monitor for misses. As one participant mentioned, it was harder to manipulate the correct amount (of chips), especially if you do not look at them.

GENERAL DISCUSSION

We turn back to our initial research questions and attempt to answer them based on the empirical data we collected.

Are Chips a Good Proxy for Idealized Touch

Interfaces?

People spontaneously manipulated chips as if they were using direct touch interfaces, i.e., by dragging one or more with their fingertips. Coarser gestures were also used to manipulate many chips concurrently, which is also possible on touch interfaces using contact shape sensing [START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF]. A single participant used once a strategy consisting in regrouping chips, lifting them together, and placing them one by one. This can easily be avoided in future experiments through explicit instructions.

The way chips physically interacted may have been unusual for a touch interface. While touch interfaces either always allow object overlap during manipulation or never do (through collision detection), physical chips implement a mix of both. Some participants complained about both unintended overlaps and the necessity to avoid already-placed chips. This issue needs to be addressed in future proxy setups, for example by designing ultra-flat objects that consistently collide.

Concerning self-reported subjective experience, chips felt less "physical" and more "touch-like" than pucks, but they still felt much more physical than current touch interfaces. Participants were highly sensitive to the tactile feedback, while the realistic visual "rendering" and the absence of lag may have also significantly contributed to this experience. All these factors only confirm that chips are not a good proxy for today's touch interfaces, which is not what chips are trying to emulate. Device vendors have been successfully building touch devices that provide a much more physically plausible experience than old touch screens, and this trend is likely to continue.

Does Object Thickness Present Benefits?

Our study seems to suggest this was the case, but not necessarily for the reasons often mentioned in the literature.

First, while previous studies find that tangibles afford more accurate manipulation [START_REF] Jansen | Tangible remote controllers for wall-size displays[END_REF][START_REF] Tuddenham | Graspables revisited: multi-touch vs. tangible input for tabletop displays in acquisition and manipulation tasks[END_REF][START_REF] Weiss | Slap widgets: bridging the gap between virtual and physical controls on tabletops[END_REF], we found that chips are positioned more precisely than pucks. This result points out benefits of touch interfaces that may have been overlooked. In particular, because they are flat, touch interfaces can be more legible, as objects do not occlude content as much as tangible objects. This may in turn facilitate precision tasks.

Second, it has been argued [START_REF] Fitzmaurice | Bricks: laying the foundations for graspable user interfaces[END_REF][START_REF] Victor | A brief rant on the future of interaction design[END_REF] and observed [START_REF] North | Understanding multi-touch manipulation for surface computing[END_REF] that tangibles support richer gestures. Our study calls for more nuance. When repositioning objects in a 2D workspace, finger drag is a dominant gesture even with tangibles. It appears to be the best for positioning small objects. Being "graspable" thus does not necessarily lead to grasping behavior. Naturally, our findings only apply to a specific type of task, and to a particular tangible form-factor appropriate for the task.

Despite this, tangibles were often lifted and grasped, and it is clear that this extra flexibility yielded a much more positive subjective experience. However, it is not clear whether the same difference in satisfaction would have been observed had participants been able to drag chips over other chips, as is often the case in touch interfaces. This finding thus calls for further studies before we can make any strong claim about speed or subjective benefits of thick objects.

A key insight provided by our empirical method is that when multiple objects need to be manipulated at a time, touch interfaces are limited not because objects cannot be grasped, but because human hands and arms are too irregular to interact in elaborate ways with flat surfaces. Indeed, coarse manipulations yielded many unintended chip losses, a problem that is easily addressed by the extra thickness of pucks. Despite previous experimentations with contact shape sensing and coarse manipulation gestures similar to what we observed [START_REF] Cao | Shapetouch: Leveraging contact shape on interactive surfaces[END_REF][START_REF] Wilson | Bringing physics to the surface[END_REF], touch interfaces may be sub-optimal for such tasks. This issue will likely become more apparent as contact shape sensing becomes mainstream, and may require specific interaction techniques and/or mid-air (thus non-touch) sensing equipment.

Finally, we found that thick objects were easier to acquire, likely because i) they "stick out" as targets and ii) they generate haptic "error messages" when mis-acquired. This suggests that touch surfaces may be less comfortable for carrying out complex coordinated tasks over large areas, even with full support for haptic rendering [START_REF] Bau | Teslatouch: electrovibration for touch surfaces[END_REF]. Nevertheless, participant comments suggest that tactile feedback is a key component of the perceived "physicality" of a touch interface.

CONCLUSION AND FUTURE WORK

We were interested in comparing touch interfaces with tangible interfaces for repositioning collections of objects on a 2D workspace. The use of physical proxies allowed us to isolate the effects of object thickness (flat vs. thick), a fundamental difference between touch and tangibles interfaces irrespective of the available technology to implement them.

Like previous studies, we observed that graspable objects are manipulated faster than their flat counterparts. However, they were also positioned less accurately, presumably because of the occlusions they entail. Video analysis also revealed that in both conditions, participants favored finger dragging gestures typical of touch interfaces, although they grasped and lifted thick objects when it was more convenient.

Meanwhile, we raised two possible limitations of touch that may seem obvious in retrospect but should probably be given more emphasis, as they may be inherent to any touch interface: i) touch objects are more prone to acquisition errors as they are not necessarily acquired orthogonally, ii) touch surfaces are flat but human hands and arms are not, which can cause object losses when trying to drag or sweep many objects at a time. Adding thickness to objects solves this issue, making the manipulation of multiple objects more comfortable, less error-prone and less attention-demanding. All these factors may contribute to a more positive subjective experience.

The physical proxy approach definitely has limitations and will never replace traditional evaluation methods, but it can be a useful complement -it provides a totally different way of looking at comparative questions. We hope this work will encourage further research in this direction.

AVI ' 16 ,

 16 June 07-10, 2016, Bari, Italy ACM ISBN 978-1-4503-2138-9. DOI: http://dx.doi.org/10.1145/2909132.2909247

Figure 1 :

 1 Figure 1: Chips (left) and pucks (right) as used in our study.

Figure 2 :

 2 Figure 2: The experimental Setup. Colors indicate areas -the surface was entirely white except for 3 whose boundaries were marked.

Figure 3 :

 3 Figure 3: Visual targets for a spread placement task (left) and a packed placement task (right).

Figure 4 :

 4 Figure 4: Mean task completion times and accuracies per condition (left), and within-subject ratios between chips and pucks, where values > 1 indicate an advantage for pucks (right). Error bars are 95% CIs.

Figure 5 :

 5 Figure 5: Questionnaire responses regarding subjective experience. Error bars are 95% bootstrap CIs.

Figure 7 :

 7 Figure 7: Four chips lost during a bulldozer push.

ACKNOWLEDGMENTS

We would like to thank Wesley Willett, Yvonne Jansen, Evanthia Dimara and Clément Riant for their help.


Mickaël Rimboud

Alain Bergel

Benjamin Erable
email: benjamin.erable@ensiacet.fr

Multiple electron transfer systems in oxygen reducing biocathodes revealed by different conditions of aeration/agitation

Keywords:

Oxygen reducing biocathodes were formed at -0.2 V/SCE (+0.04 V/SHE) from compost leachate. Depending on whether aeration was implemented or not, two different redox systems responsible for the electrocatalysis of oxygen reduction were evidenced. System I was observed at low potential (-0.03 V/SHE) on cyclic voltammetries (CVs). It appeared during the early formation of the biocathode (few hours) and resisted the hydrodynamic conditions induced by the aeration. System II was observed at higher potential on CV (+0.46 V/SHE); it required a longer lag time (up to 10 days) and quiescent conditions to produce an electrochemical signal. The hydrodynamic effects produced by the forced aeration led to its extinction. From their different behaviors and examples in the literature, system I was identified as being a membrane-bound cytochrome-related molecule, while system II was identified as a soluble redox mediator excreted by the biofilm. This study highlighted the importance of controlling the local hydrodynamics to design efficient oxygen reducing biocathodes able to operate at high potential.

Introduction

Because it is abundantly available in the environment and because its complete reduction only produces water, molecular oxygen has been targeted as the final electron acceptor in fuel cells, including microbial fuel cells (MFCs). This particular kind of fuel cell relies on bacterial biofilms that are developed on anodes and are able to catalyze the oxidation of various kinds of organic and/or inorganic matter, which makes these devices of great combined economic and environmental interest [START_REF] Mohan | Microbial fuel cell: critical factors regulating bio-catalyzed electrochemical process and recent advancements[END_REF].

The vast majority of MFCs presented in the literature rely on abiotic oxygen reducing (OR) cathodes [START_REF] Huang | Electron transfer mechanisms, new applications, and performance of biocathode microbial fuel cells[END_REF][START_REF] Erable | Microbial catalysis of the oxygen reduction reaction for microbial fuel cells: a review[END_REF]. These cathodes often use expensive catalysts, e.g. derivatives of platinum-based chemicals, and are sensitive to poisoning and biofouling in natural media like the sludge or sediments used to feed MFCs. These drawbacks make them difficult to scale up for industrial applications and OR-biocathodes would constitute worthwhile alternatives.

Electroactive bacteria coming from many different natural environments have been reported in OR-biocathodes, the most used inocula being aerobic sludge [START_REF] Rothballer | Monophyletic group of unclassified gamma-proteobacteria dominates in mixed culture biofilm of high-performing oxygen reducing biocathode[END_REF][START_REF] Xia | Oxygen-reducing biocathodes operating with passive oxygen transfer in microbial fuel cells[END_REF][START_REF] Xia | Long-term effect of set potential on biocathodes in microbial fuel cells: electrochemical and phylogenetic characterization[END_REF][START_REF] Ter Heijne | Identifying charge and mass transfer resistances of an oxygen reducing biocathode[END_REF][START_REF] Ter Heijne | Cathode potential and mass transfer determine performance of oxygen reducing biocathodes in microbial fuel cells[END_REF], seawater or marine sediment [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF][START_REF] Erable | Marine aerobic biofilm as biocathode catalyst[END_REF][START_REF] Faimali | Electrochemical activity and bacterial diversity of natural marine biofilm in laboratory closed-systems[END_REF][START_REF] Vandecandelaere | Bacterial diversity of the cultivable fraction of a marine electroactive biofilm[END_REF][START_REF] Bergel | Catalysis of oxygen reduction in PEM fuel cell by seawater biofilm[END_REF], and, to a lesser extent, soils [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF][START_REF] Chen | A novel sediment microbial fuel cell with a biocathode in the rice rhizosphere[END_REF]. The majority of studies on ORbiocathodes have implemented multispecies environmental consortia and studies using pure cultures of a single bacterium remain scarce [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Erable | Marine aerobic biofilm as biocathode catalyst[END_REF][START_REF] Rabaey | Cathodic oxygen reduction catalyzed by bacteria in microbial fuel cells[END_REF].

Various set-ups and methodologies have been used for developing OR biocathodes but analytical investigation of electron transfer mechanisms ultimately calls upon cyclic voltammetry. Different values of oxygen reduction reaction (ORR) half-wave potential (read on the reductive scan) have been obtained by various authors using this technique. As ORR potential depends on pH, values reported in works realized at unusually acidic [START_REF] Carbajosa | Electrochemical growth of Acidithiobacillus ferroxidans on a graphite electrode for obtaining a biocathode for direct electrocatalytic reduction of oxygen[END_REF] or alkaline pH [START_REF] Rimboud | The current provided by oxygen-reducing microbial cathodes is related to the composition of their bacterial community[END_REF] that would have changed the ORR potential are omitted here. Only values obtained in studies realized at neutral pH are presented in Schematic 1.

The reported potentials can be clustered in two different groups. In a first group, ORR catalysis was observed at rather low potentials, ranging between -0.1 and 0.1 V/SHE. In a second group, signals for ORR were observed at higher potentials, ranging between 0.4 and 0.5 V/SHE. Examples of "high potential" signals were notably found by Ter Heijne et al., who compared the performances of biocathodes developed from aerated sludge using different, but deliberately high, polarization potentials: 0.255, 0.355 and 0.455 V/SHE [START_REF] Ter Heijne | Cathode potential and mass transfer determine performance of oxygen reducing biocathodes in microbial fuel cells[END_REF]. They described an identical voltammetric ORR signal centered at 0.49 V/SHE, whatever the polarization potential. The main difference between the biocathodes resided in a faster start-up time with the reactors polarized at lower potential (0.255 and 0.355 V/SHE). Xia et al. conducted a similar experiment to compare the behavior of OR-biocathodes designed at 0.142, 0.302 and 0.442 V/ SHE [START_REF] Xia | Oxygen-reducing biocathodes operating with passive oxygen transfer in microbial fuel cells[END_REF]. The cyclic voltammograms showed an ORR signal at 0.46 V/ SHE with a mature biocathode. They identified 0.302 V/SHE as the best polarization potential, resulting from a compromise between fast start-up and high current densities, and consequently used a similar potential, 0.311 V/SHE to design efficient OR-biocathodes for an MFC application in a subsequent report. Strycharz-Glaven et al. found a voltammetric signal centered on 0.48 V/SHE with an OR-biocathode in a microbial solar cell inoculated with marine sediments [START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF].

Compared to high-potential signals, low-potential signals are more often encountered in the literature. Such signals were observed by Freguia et al. working with Acinetobacter calcoaceticus and Shewanella putrefaciens [START_REF] Freguia | Electron transfer pathways in microbial oxygen biocathodes[END_REF] and by Cournet et al. when testing the ORR activity of 14 different bacterial strains, among them Micrococcus luteus, a Gram positive bacterium and Pseudomonas aeruginosa, a Gram negative one [START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including gram-positive[END_REF]. ORR signals were observed at -0.05 and -0.03 V/SHE, respectively, with these bacteria [START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including gram-positive[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa[END_REF]. Marine biofilms formed with [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Erable | Marine aerobic biofilm as biocathode catalyst[END_REF] or without [START_REF] Xu | Electron transfer process from marine biofilms to graphite electrodes in seawater[END_REF] prior polarization produced low potential signals and voltammetric tests performed on pure strain bacteria isolated from such biofilms also presented low potential signals [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Parot | Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater[END_REF]. Blanchet et al. observed an ORR signal at + 0.03 V/SHE during the cathodic phase of reversible bioelectrodes designed from garden compost [START_REF] Blanchet | Protons accumulation during anodic phase turned to advantage for oxygen reduction during cathodic phase in reversible bioelectrodes[END_REF]. A similar signal, centered on + 0.04 V/SHE was observed during the straightforward formation of OR-biocathodes [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF].

In conclusion, the data, presented in Fig. 1, revealed that:

-The low and high potential signals were observed with marine [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF][START_REF] Parot | Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater[END_REF] or soil inocula [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF][START_REF] Blanchet | Protons accumulation during anodic phase turned to advantage for oxygen reduction during cathodic phase in reversible bioelectrodes[END_REF]. Both types of signal were also observed when using the same electrode material, for example carbon cloth [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF][START_REF] Blanchet | Protons accumulation during anodic phase turned to advantage for oxygen reduction during cathodic phase in reversible bioelectrodes[END_REF] or carbon paper [START_REF] Xia | Long-term effect of set potential on biocathodes in microbial fuel cells: electrochemical and phylogenetic characterization[END_REF][START_REF] Freguia | Electron transfer pathways in microbial oxygen biocathodes[END_REF]. In consequence, low or high potential signals are correlated neither with the inoculum source nor with the electrode material.

-Signals at low potential were recorded mainly with pure strain cultures [START_REF] Debuy | Oxygen-reducing biocathodes designed with pure cultures of microbial strains isolated from seawater biofilms[END_REF][START_REF] Freguia | Electron transfer pathways in microbial oxygen biocathodes[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including gram-positive[END_REF], when no polarization was applied prior to voltammetric experiments [START_REF] Freguia | Electron transfer pathways in microbial oxygen biocathodes[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including gram-positive[END_REF], or, at the most, a polarization at low potential (maximum -0.04 V/SHE in Desmond-Le Quemener et al. [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]).

-Low potential signals appeared quickly after the experiment began (a few hours up to 2 days for Desmond-Le Quemener et al., whereas high potential signals needed a higher polarization potential (minimum 0.142 V/SHE, in [START_REF] Xia | Long-term effect of set potential on biocathodes in microbial fuel cells: electrochemical and phylogenetic characterization[END_REF]) and more time to develop (up to 15 days in Ter Heijne's experiments [START_REF] Ter Heijne | Cathode potential and mass transfer determine performance of oxygen reducing biocathodes in microbial fuel cells[END_REF]).

The importance of the polarization potential was particularly highlighted by Desmond-Le Quemener et al. Using two different polarization potentials, they experienced the coexistence of both low and high potential signals when the biocathode was formed at 0.34 V/SHE, but only the low potential signal for biocathodes formed at -0.16 V/ SHE.

The poor solubility of oxygen can be an important rate limiting factor of OR-cathodes operating in aqueous media. Controlling the aeration conditions is thus a crucial element for a better understanding of ORbiocathode behavior. Accordingly, different conditions of aeration were tested in this work in order to better understand the relative developments of low and high potential signals for oxygen reduction by microbial biofilms. OR-biocathodes were designed using compost leachate as the medium, under constant polarization. In a first experimental run, OR-biocathodes were either in reactors exposed to permanent forced aeration using a vigorous air flux or had no forced aeration. In the second experimental run, biocathodes initially designed under quiescent conditions were later exposed to different aeration conditions, using air or nitrogen fluxes. The electrochemical behavior of the different biocathodes was characterized by chronoamperometry (CA) and cyclic voltammetry (CV). The structures of the biofilms developed on the electrodes were imaged by epifluorescence microscopy.

Experimental

Experimental medium: garden compost leachate

The leachate used as medium and source of electroactive microorganisms was produced from commercially available garden compost (Eco-Terre) using the method described elsewhere [START_REF] Cercado | Garden compost inoculum leads to microbial bioanodes with potential-independent characteristics[END_REF]. The leachate was supplemented with 20 mM KCl in order to insure a minimum conductivity (4 mS•cm -1 ). Its initial pH was 7.8.

Electrochemical set-up and tests

The electrochemical reactors were designed from Schott bottles, filled with 650 mL garden compost leachate, which left around 40 mL gas headspace. The reactors were closed by four-hole caps. Three holes were used to connect the electrodes and the fourth was devolved to the gas supplier. The working electrode was a 6 cm 2 piece of carbon cloth (Paxitech, Grenoble, France) connected to a platinum wire. The counter electrode was a 10 cm 2 platinum grid (Héraeus, France) connected to a platinum wire. Potential was controlled and expressed versus a saturated calomel reference electrode (SCE, E 0 = 0.24 V/SHE, Biologic, France), which was regularly checked versus another SCE throughout the experiment. The electrode saturation was maintained by addition of KCl crystals to the electrode compartment to avoid any drift of the potential. Gas supply (air or nitrogen) was achieved with a glass tube ending in a porous fritted glass tip. Gas fluxes were adjusted to produce gentle bubbling. Air supply brought the oxygen concentration up from 5.4 ± 0.3 to 6.5 ± 0.1 mg L -1 . Ten minutes of N 2 flux reduced the oxygen concentration to below 0.5 mg L -1 . Oxygen concentration was measured using an optical dissolved oxygen meter (Multi 3410, WTW GmbH, Germany). Temperature in the reactors was maintained at 40 °C using a thermostatic bath. All experiments Schematic 1. Oxygen reduction half-wave potentials recorded by voltammetry on the reductive scan at neutral pH for various biocathodes described in the literature. were performed at least in duplicate. For clarity, only one set of data per experiment is presented below.

Electrochemical experiments were carried out using a multichannel potentiostat and the EC-Lab software (Biologic, France). OR-biocathodes were designed under polarization (chronoamperometry, CA) at -0.2 V/ SCE (+0.04 V/SHE), a potential high enough to develop both low-and high-potential signals [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. Punctually, biocathode polarization was stopped for 30 min to allow the biocathodes to reach a stable open circuit potential (OCP) before cyclic voltammetry (CV) was performed. CVs were recorded from OCP up to 0.4 V/SCE and then down to -0.7 V/SCE, at a scan rate of 1 mV s -1 . Each CV recording consisted of three successive scans, which were generally identical. For clarity, only the second scans are presented here. CVs were recorded at t = 0, prior to biofilm establishment, and at the end of the experiments with new, uncolonized electrodes, which served as abiotic controls.

Epifluorescence imaging

The electrodes were extracted from the reactors and washed carefully with sterile physiological water to remove all soluble and solid materials except the attached biofilms. Bioelectrodes were stained with 0.03% acridine orange (A6014, Sigma) for 10 min, rinsed with sterile physiological water and then left to dry at room temperature. Biofilms were imaged with a Carl Zeiss Axio Imager-M2 microscope equipped for epifluorescence with an HXP 200 C light source and the Zeiss 09 filter (excitor HP450e 490, reflector FT 10, barrier filter LP520). Images were acquired with a digital camera (Zeiss AxioCam MRm) every 0.5 μm along the Z-axis and the set of images was processed with the Zen® software. The biovolume was calculated by the automated image analysis of the Zen® software on an acquisition stack that was representative of the colonization observed at various locations of the electrode surface.

Results and discussion

Effect of permanent air flux

Electrochemistry

Chronoamperometries at -0.2 V/SCE (+ 0.04 V/SHE) were performed in parallel in four reactors. Reactors R1 and R2 were run with permanent forced aeration, while reactors R3 and R4 remained under quiescent conditions up to day 20, when forced air flux was introduced (Fig. 1).

Similar mean current densities of -0.029 ± 0.006 and -0.030 ± 0.010 A m -2 were measured initially between day 0 and day 5 whether the reactors were exposed to forced aeration or not. In the presence of forced aeration, the reduction current density remained barely higher, around -0.038 ± 0.008 A m -2 , up to the end of experiment. In contrast, under quiescent conditions, the reduction current started to increase strongly after 6.5 days of polarization. A stable maximal current density of -0.31 ± 0.02 A m -2 was reached between day 11 and day 19. Finally, when air was forced into the electrochemical reactor on day 20, the reduction current rose back to its initial value of around -0.035 A m -2 within 10 h. Two stable but different current densities were thus experienced here, depending on the aeration of the electrochemical reactor.

The CV recorded with the biocathodes formed under quiescent conditions showed a reduction wave around +0.22 V/SCE (+0.46 V/SHE) (noted II on Fig. 2). The CV recorded with the same biocathodes after exposing them to 24 h forced aeration showed a very different reduction wave, around -0.27 V/SCE (-0.03 V/SHE) (reduction wave noted I on Fig. 2). The biocathodes formed under permanent forced aeration displayed similar CV with the reduction wave at -0.28 V/SCE (-0.04 V/SHE) (wave I). The abiotic control CV carried out before the development of the biofilm was different from voltammograms with wave I and wave II. It evidenced their biotic origin.

The shape of wave IIa reduction peak followed by a plateauwas previously observed by researchers working with enzymatic ORbiocathodes under quiescent conditions [START_REF] Bergel | Catalysis of oxygen reduction in PEM fuel cell by seawater biofilm[END_REF][START_REF] Urban | Enzymatic oxygen reduction under quiescent conditionsthe importance of convection[END_REF]. It was the signature of a mass-transport limited reaction. Similar maximal current densities were observed with waves I and II, around -0.44 A m -2 , indicating that both waves were limited by oxygen mass transport. The two different kinds of waves, I and II, corresponded to two different redox systems responsible for OR-catalysis and for the two different stable current densities evidenced under polarization at +0.04 V/SHE.

Biofilm morphology

The forced aeration of the solution bulk with continuous bubbling of fine gas bubbles induced a shear stress on the electrode surface that had significant impact on their colonization and the morphology of biofilms (Fig. 3).

Under continuous air flux (R1 and R2), electroactive biofilms were diffuse, in the form of dispersed colonies. The cell density was sparse and areas of the carbon fibers making up the carbon cloth electrode were still visible even after 3 full weeks of biofilm development. In contrast, electroactive biofilms obtained without bubbling (R3 and R4) were much thicker; the carbon fibers were no longer visible in the three-dimensional biofilm images. The determination of biomass quantity by image analysis of the z-stack acquisition reported a ratio of 40 between the volume of biofilm (bio-volume) calculated on electrodes R3 and R4 and that calculated on electrodes R1 and R2. In conclusion, the shear stress induced by the forced aeration had a considerable negative impact on the colonization of the carbon cloth electrodes.

Effects of air and N 2 fluxes

Four OR-biocathodes were developed in independent electrochemical reactors under quiescent conditions. After 12 days of polarization, when the chronoamperometric response was maximal, two reactors were exposed to air flux for 24 h and the other two to nitrogen flux for 3 h (Fig. 4). Both gas fluxes led to a fast decrease in the reduction current.

Stationary electrochemical responses of the OR-biocathodes before gas bubbling were mostly identical, each displaying -0.166 ± 0.009 A m -2 between days 10 and 12. The current was mainly due to redox system II, which was the only system detected on CV performed at this time (red CVs, Fig. 5).

When nitrogen gas was forced into the medium (Fig. 4, gray line), reductive current density immediately fell to zero and neither system I nor system II was detected by CV as long as the bubbling lasted, because oxygen was completely chased out of the medium (Fig. 5A, gray line CV). This confirmed that both systems catalyzed oxygen reduction. When the nitrogen flux stopped, oxygen reduction resumed immediately (Fig. 4, gray line), producing a current density of -0.025 A m -2 that corresponded to the redox activity of system I only (Fig. 5A, blue line CV). The presence of system II was not detected on CV immediately after N 2 flux was stopped. The chronoamperometry showed that it started to produce current again only later, after day 15.

When air was flushed through the medium (Fig. 4, black line), OR current density at + 0.04 V/SHE was immediately enhanced, to -0.35 A m -2 for a few minutes, due to oxygen concentration increase and the higher convection. This enhancement was transient and the reduction current density decreased thereafter as the forced aeration continued. It stabilized at around -0.05 A m -2 after the air flux stopped. CV recorded when quiescent conditions resumed revealed that the reduction current was due to the activity of redox system I alone (Fig. 5B, blue curve). Oxygen reducing activity of system II progressively resumed from day 15, as visible on the CV recorded at the end of the experiment (green line in Fig. 5B).

Discussion

The two redox systems evidenced here corresponded well with the two signals previously experienced by [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. Their respective characteristics fitted those of low-and high-potential signals shown in the literature (see introduction). Redox system I, observed around -0.27 V/ SCE (-0.03 V/SHE), demonstrated similar behavior to low-potential signals: fast development and a relative independence from the potential at which the biocathode was formed [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. A contrario, redox system II, observed around +0.22 V/SCE (+0.46 V/SHE), needed more polarization time to establish itself (up to 10 days) and to show an electrochemical response.

Neither of the two systems displayed an electrochemical response during nitrogen flux, which confirmed that they were both related to oxygen reduction (Figs. 4 and5A). However, they behaved differently under air flux. The air flux never altered the signal produced by redox system I, which showed OR-activity under permanent air supply (Figs. 1 and2). This system demonstrated an enduring OR-activity when air was supplied in reactors previously conditioned in quiescent conditions (Figs. 4 and5B). The OR-signal due to system II was observed only under quiescent conditions and, after a brief enhancement, it vanished when air was supplied in the reactors (Figs. 1, 2, 4 and5B). Forced aeration brought more oxygen into the reactor and enhanced signal II for a brief moment but it ultimately led to its extinction. Due to its oxidative properties, oxygen at high concentrations could be toxic for microbial cells. However, its concentration only changed from 5.4 ± 0.3 to 6.5 ± 0.1 mg L -1 during air flux, and this moderate increase is unlikely to have been responsible for the loss of signal II through oxygen toxicity.

Forced aeration induced shear forces on the electrode surface, which impacted the electrode colonization (Fig. 3). Shear forces are known to strongly influence the settlement, the structure and the morphology of biofilms [START_REF] Costerton | Introduction to biofilm[END_REF][START_REF] Ochoa | Influence of non-uniform distribution of shear stress on aerobic biofilms[END_REF]. Other studies on the formation of biofilms from natural consortia or pure microbial strains under various hydrodynamic conditions have shown that the thickness, the amount of fixed biomass, the nature and amount of exopolymeric substances produced and the distribution of bacterial colonies vary widely depending on the shear mode applied [START_REF] Celmer | Impact of shear force on the biofilm structure and performance of a membrane biofilm reactor for tertiary hydrogen-driven denitrification of municipal wastewater[END_REF][START_REF] Stoodley | Oscillation characteristics of biofilm streamers in turbulent flowing water as related to drag and pressure drop[END_REF]. It is generally agreed that the greater the physical Fig. 3. Epifluorescence microscopy of biocathodes formed under constant polarization at -0.2 V/SCE (+0.04 V/SHE) under quiescent conditions or forced aeration. Fig. 4. Chronoamperograms at -0.2 V/SCE (+ 0.04 V/SHE) recorded under quiescent conditions and exposed to air (black) and nitrogen (gray) bubbling on day 12. Cyclic voltammetries were recorded before, during and immediately after forced gas fluxes, and at the end of experiment on day 23, after gas flux had been stopped for several days. force applied to the biofilm is, the more resistant, compact and thin the biofilm becomes [START_REF] Kwok | Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor[END_REF][START_REF] Melo | Physical stability and biological activity of biofilms under turbulent flow and low substrate concentration[END_REF]. Under high shear stress, biofilm detachment could occur via erosion, resulting in thinner biofilms that are rather smooth and rich in microbial cells in the colonized areas. Here, such a phenomenon of biofilm erosion should impact redox systems I and II similarly. If erosion was responsible for the disappearance of system II observed under forced aeration, it would similarly cause the destruction of system I. Consequently, biofilm erosion cannot explain the behavior of the biocathodes under forced aeration.

The most likely explanation of the difference between the two redox system behaviors is that they constituted two different mechanisms for external electron transfer (EET) from the electrode to oxygen. Different types of EET mechanisms from cathodes to various final electron acceptors were reported in the literature reviewed by Rosenbaum et al. [START_REF] Rosenbaum | Cathodes as electron donors for microbial metabolism: which extracellular electron transfer mechanisms are involved? Spec. Issue Biofuels -II Algal Biofuels Microb[END_REF].

The CV characteristics of system I were very close to those previously reported by Blanchet et al., who worked with the same inoculum (compost leachate) and polarization potential (+0.04 V/SHE) [START_REF] Blanchet | Protons accumulation during anodic phase turned to advantage for oxygen reduction during cathodic phase in reversible bioelectrodes[END_REF]. Actually, system I characteristics were similar to many low-potential signals reported in other works performed with other media and/or inocula [START_REF] Parot | Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by Pseudomonas aeruginosa[END_REF][START_REF] Cournet | Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including gram-positive[END_REF]. The voltammograms presented in these works showed characteristics (potential, current density, fast development even without polarization) that may link them to redox system I observed here. Parot et al. demonstrated a close similarity between CVs recorded with marine bacterial strains and those of an iron porphyrin adsorbed on the electrode surface [START_REF] Parot | Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater[END_REF]. Iron porphyrins are redox molecular substructures present in numerous proteins such as cytochromes. Cytochromes have been proven to achieve direct external electron transfer between bacteria and electrodes [START_REF] Richter | Cyclic voltammetry of biofilms of wild type and mutant geobacter sulfurreducens on fuel cell anodes indicates possible roles of OmcB, OmcZ, type IV pili, and protons in extracellular electron transfer[END_REF]. They are generally anchored in cellular membranes and are consequently not removed from the biofilm by hydrodynamic effects. Therefore, all these observations led us to postulate that redox system I may be related to membrane-bound cytochromes.

Another known EET pathway relies on the secretion of soluble mediators used for shuttling electrons. A large variety of bacterial extracellular redox mediators have been identified in both bioanodes and biocathodes. Among them are phenazines such as piocyanine [START_REF] Venkataraman | Quorum sensing regulates electric current generation of Pseudomonas aeruginosa PA14 in bioelectrochemical systems[END_REF], flavin derivatives such as riboflavin [START_REF] Marsili | Shewanella secretes flavins that mediate extracellular electron transfer[END_REF], or quinones such as pirroloquinoline quinone and 2-amino-3-dicarboxy-1,4naphtoquinone [START_REF] Freguia | Electron transfer pathways in microbial oxygen biocathodes[END_REF][START_REF] Freguia | Lactococcus lactis catalyses electricity generation at microbial fuel cell anodes via excretion of a soluble quinone[END_REF]. Such molecules are usually locally concentrated inside the biofilm, which ensures proximity between bacteria and electrodes and facilitates the electrochemical transfer. The soluble mediators are subjected to mass transport; they can be dispersed in the bulk in presence of increased hydrodynamic forces and thus lose the possibility to play their electron mediator function. As redox system II behavior fitted this description in all respects, it probably corresponded to a diffusible extracellular redox mediator that was poorly linked to the biofilm and was easily released to the bulk by hydrodynamic conditions.

The nature of the soluble redox mediator postulated here for system II cannot be enlarged to all similar high-potential systems reported in the literature, because they have shown different sensitivities to the hydrodynamics. The high-potential system observed by Desmond-Le Quemener et al. using the same inoculum (compost leachate) was not sensitive to hydrodynamics [START_REF] Desmond-Le Quéméner | Biocathodes reducing O 2 at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes[END_REF]. This difference may have come from the higher polarization potential they used to form their biocathode (+ 0.34 V instead of + 0.04 V/SHE), which may have resulted in selecting other electron exchange pathways between electrode and biofilm. Xia et al. used a continuously-fed electrochemical cell with forced aeration to form the biocathode, but prevented problems caused by the hydrodynamic conditions by interposing a plastic plate in the cathodic compartment [START_REF] Xia | Long-term effect of set potential on biocathodes in microbial fuel cells: electrochemical and phylogenetic characterization[END_REF][START_REF] Xia | Oxygen-reducing biocathodes operating with passive oxygen transfer in microbial fuel cells[END_REF]. Ter Heijne et al. used an electrochemical cell in continuous mode [START_REF] Ter Heijne | Cathode potential and mass transfer determine performance of oxygen reducing biocathodes in microbial fuel cells[END_REF]. This operating mode may imply a certain degree of shear stress on the cathode surface, but it did not impact the electrochemical behavior of their system. Moreover, increasing the flow rate from 0 to 21 L h -1 led to increased current densities for OR from 40 to 270 mA m -2 . Strycharz-Glaven et al. identified the presence of immobilized cofactors inside the biofilms, which achieved a direct, non-mediated EET [START_REF] Strycharz-Glaven | Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst[END_REF].

Due to its higher redox potential, system II is of greater interest than system I if the objective is to design efficient OR-biocathodes for MFC applications. The results presented here, and the past works referred to, recall that high potential oxygen reductive systems are observed when rather high polarization potentials are applied to form the ORbiocathodes. This highlights the benefit of designing OR-biocathodes under polarization. However, as reflected by the discussion above, the nature of redox system II may hinder the development of practical applications. Its sensitivity to turbulence may restrict its application to systems in which quiescent conditions are ensured. Continuous flow MFCs, for example, may be suitable provided that high retention time and low flow rate are used to mitigate the possible release of the redox mediator from the biofilm. Precautions as implemented by Xia et al., i.e. a plastic plate interposed between the aeration source and the biocathode, would certainly be of interest [START_REF] Xia | Long-term effect of set potential on biocathodes in microbial fuel cells: electrochemical and phylogenetic characterization[END_REF][START_REF] Xia | Oxygen-reducing biocathodes operating with passive oxygen transfer in microbial fuel cells[END_REF]. The use of a small-volume cathodic compartment in order to limit the dilution effect of the mediator in the solution bulk would also merit attention.

Conclusions

Oxygen-reduction biocathodes formed from compost leachate under constant polarization at + 0.04 V/SHE showed two different redox systems linked to oxygen reduction. A low-potential system (I) was detected in all biocathodes, whatever the experimental conditions, and ensured a basal oxygen reduction activity. A high-potential system (II) developed only under quiescent conditions and vanished when the solution was stirred. In the light of the literature, system I was probably a cytochrome derived compound, firmly attached in the biofilm. System II behavior was characteristic of a soluble redox mediator, the chemical nature of which remains to be determined. Its solubility made it sensitive to turbulence inside the reactor. This work shows that implementing efficient oxygen-reducing biocathodes in MFCs requires careful control of the local hydrodynamics in order to favor the development of the high-potential redox system.

Fig. 1 .

 1 Fig. 1. Chronoamperometries at -0.2 V/SCE (+0.04 V/SHE) recorded with (black) and without (gray) forced aeration in the electrochemical reactor. Encroachments in the records were caused by interruption of polarization for CV recording.

Fig. 2 .

 2 Fig. 2. Characterization of the biocathodes by cyclic voltammetry at 1 mV s -1 . Red curve: CV of the biocathode formed under permanent forced aeration (reactors R1 and R2) at the end of experiment (day 21). Air flux was stopped just during CV recording. The two blue curves were recorded with the biocathodes formed in quiescent conditions (reactors R3 and R4) before (plain blue curve) and after (dotted blue curve) aeration had been applied for 24 h between days 20 and 21. Control voltammograms (black dotted line) were recorded at the beginning of the experiments before polarization started.

Fig. 5 .

 5 Fig. 5. Cyclic voltammograms recorded with reactors exposed to nitrogen (A) or air (B) fluxes. Red and blue CVs were recorded before and after gas fluxes respectively.Green CVs were recorded at the end of experiment. Gray CV (Fig.6a) was recorded during N 2 supply. Corresponding abiotic control CVs are available in supplementary data (Fig.S1).

Acknowledgments

This work was part of the BioCathInox project, funded by the French ANR (ANR-11-JS09-016-01).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bioelechem.2016.03.002.