Antoine Taly

F Nitti

Marc Baaden

S Pasquali

Molecular modelling as the spark for active learning approaches

come   L'archive ouverte pluridisciplinaire

The route toward new teaching strategies

As natural sciences professors in life science departments, we regularly see the struggle of our students with the perceived methodological rigor of the subjects we teach (mathematics, physics, chemistry) that seem to pose a much greater obstacle to the progression toward a degree than most other subjects. In our experiences we have identified at least two issues contributing to the disappointing results we often face. The first issue comes from the fact that students who choose life sciences might have done so precisely because they already have had difficulties with the related disciplines in previous years and therefore arrive at the college level with a shaky background. The second issue comes from their difficulties in seeing the purpose of studying mathematics, physics and, to a lesser extent, chemistry, as a necessary and integral part of their curriculum. Interestingly, a very recent paper studied this issue, showing that whereas mathematics literacy is crucial for biology research, researchers in biology often realize this importance only after their studies [START_REF] Marshall | Are biologists getting the mathematical training they need in college?[END_REF]. From our perspective the two issues are intertwined. We will not be successful in our teachings if we cannot clearly demonstrate the importance of our subjects to students and show them hands-on how concepts that might seem at first very abstract have a high practical use in biology. If we can get the students interested in our subjects, we then have the opportunity to address the first problem by reaching out to those who come to us with more difficulties from the start, and, by applying less conventional teaching methods, have the chance to help them fill the gaps in their knowledge.

Many previous experiences have taught us that traditional lectures do not constitute an appropriate and efficient solution to these issues, even when a lot of care is put into showing examples and applications from biology. The mere fact that our classes are named mathematics or physics may be enough to scare some students off. We then started experimenting different alternative solutions stemming from our privileged position of researchers working at the interface between biological sciences, physics and chemistry. Inspired by active teaching methods [START_REF] Crouch | Peer instruction: Ten years of experience and results[END_REF], by our own personal experiences with the Montessori method [START_REF] Montessori | Il Metodo della Pedagogia Scientifica applicato all'educazione infantile nelle Case dei Bambini[END_REF], and after having had the chance to attempt different teaching strategies throughout the years, we developed a one week workshop, named RIGOLE (a French acronym for group work around the origins of life, and that in French corresponds to a word that could be translated as laughter) aimed at first and second year Life Science bachelor students. The goal of the workshop was, and still is, to boost students' interest and motivation by showing them through unconventional teaching how all subjects matter and how the knowledge from very different disciplines is needed to be able to tackle interesting and fundamental biological (research) questions. For this purpose, biomolecular modeling constitutes an ideal playground for bringing together the various disciplines around a common challenge. Indeed, a recent study showed that bioinformatics is amenable to high school students being compatible with their knowledge and allowing students to interact with authentic research software [START_REF] Machluf | Making authentic science accessiblethe benefits and challenges of integrating bioinformatics into a high-school science curriculum[END_REF]. In order to break the barriers and the fears that students may have with respect to subjects such as chemistry, physics and mathematics, all teachings of the RIGOLE workshop are as far as possible interactive, integrating scientific games, hands-on activities, and group projects, together with short guiding lectures. Students therefore have the opportunity to see the importance of these disciplines in the context of a scientific challenge and to approach them from a new perspective, with less apprehension, thanks to the active pedagogy.

The specificity of French education

Before describing the details of the workshop implementation, it is useful to set the stage by providing a brief presentation of the French high school and college system, to put our work into context.

High school students choose an orientation in their junior and senior years. One of the options is science, which in the senior year differentiates further in the three options of math, physics and chemistry, and life sciences. The science option is the choice made by students that have an interest in pursuing a career in science-related fields, spanning from hard sciences and engineering to more soft sciences and medicine, as it is the only orientation opening to college studies in these fields. The science orientation is often perceived as more challenging than other orientations and it is suggested to students with a solid school record in the previous years. Since in college we teach to students coming from this background, our high school workshop is offered only to junior students having chosen the science option, which implies that we touch a population with a bias of interest in science and generally a good performance in this field. About half of the high school students who attended the workshop had already in mind to study physics, chemistry or engineering in college, while another half was leaning toward medicine or biology. This second category is of the kind of students that we wish to motivate in particular. The French university system is composed of two paths: regular universities, that all students with a degree opening to the specific curriculum can attend, and selective universities where students access via an exam. Top students generally choose this second option, while all others are directed to regular universities, which is where we teach mostly. This situation means that despite having a high school diploma with a science orientation, our college students do not necessarily have a solid background in the hard sciences, which are the main topics of the exam to enter selective universities for science-oriented studies. In college, students choose one major that they pursue for three years, with a main imposed curriculum and a few classes that can be chosen more freely. There are generally no minors. After the three years, they obtain a bach-elor degree and can then continue in a multitude of master programs. In fields such as biology and medicine, hard sciences are taught in the first two years and account for a non-negligible portion of the curriculum. The specific topics treated depend on the major and can vary from one university to another, but they generally involve some basic calculus, statistics, differential equations, for maths, optics and acoustics, related to perceptions of living organisms, fluids and transport, related to circulatory systems and membrane equilibrium, electrostatics, for physics, and organic and inorganic chemistry.

Interdisciplinary teachings are rare both at high school and college level, even though this situation is slowly changing in the last few years. Subjects such as computer science and, more in general, topics where the use of computer programs is predominantly present, such as molecular visualization, bioinformatics, and simulations, are completely absent from high schools and in college are offered only to specific computer science majors or in complement to other disciplines, in the most advanced years.

Design of an interdisciplinary workshop

The workshop we present here was developed in 2015 and has run several times since 2016. Overall, about 180 university students have participated. The workshop is also offered to high school students, in an adapted version, with the aim of showing them early on in their curriculum the importance of each subject. This information gives them a first idea of the research carried out at university, and ultimately offers them the chance to make a more educated choice for their future professional orientation. About 100 high school students have participated so far. Information on the logistics of the workshop organization can be found as supplementary material.

Setting goals

The workshop has two main objectives:

• Discover what scientific research is and understand the importance of interdisciplinarity through a syllabus centered around the questions of the origins of life from a molecular standpoint. Understand the interconnections between disciplines showing how each one is necessary to study the behavior of biomolecules through molecular dynamics simulation approaches.

• Motivate students for the study of scientific disciplines through an interactive and fun teaching where the student is the primary actor of the learning process.

More specifically, several sub-objectives have been identified to promote the effectiveness of the learning process. i) Discover the context of university research, different possible orientations after the second year of college, the pleasure of learning without pressure. ii) Understand the coherence of first and second-year life science curricula, concepts studied in first and secondyear courses, applying them to a specific problem, scientific reasoning, how different disciplines are interdependent. iii) Share with students coming from different backgrounds and interests, with teachers coming from various scientific disciplines working side-by-side with the students, with more advanced university students helping in the course as tutors. iv) Enjoy learning without fears, learning through games and practical activities, group work, open discussions with students and teachers.

The pedagogical and organizational approach

The workshop adopts a large variety of teaching methods including group activities, practical activities and on-line games and demos. Activities are always supervised and guided by professors and researchers from different disciplines (biology, chemistry, bioinformatics, physics and mathematics), as well as more advanced university students (from the bioinformatics curriculum, from the interdisciplinary curriculum Frontiers of Life Science, and from the pharmaceutical science curriculum) acting as tutors for practical activities and group projects. The workshop lasts a full week (5 days), for a total of 30 hours and it is organized in half-day modules (see Fig. 1). Each disciplinary module is composed of short 10-20 minute lectures, with the professor introducing and developing concepts, mixed with short practical activities, while the longerlasting activities (on-line scientific games) are presented to students at the end of the modules (biology and chemistry) in order to introduce the main ideas of the game and its relation to the workshop [START_REF] Baaden | Ten simple rules to create a serious game, illustrated with examples from structural biology[END_REF]. Students are then encouraged to continue playing in their free time. At the end of the disciplinary module of chemistry and physics a quiz (available in Supplementary Material) is proposed to the students to promote further reflection and possibly independent investigations. In the first workshop, the quiz was given as homework, while more recently it has been integrated into the modules and is used as a basis for group discussions and debriefing. Students are divided in small groups to answer the questions of the quiz. Then, groups are paired to form larger groups where the answers are reviewed and discussed in case of disagreement. Finally, the whole class comes together and the answers are once more reviewed in the presence of the instructor who steps in to redirect the discussion correcting possible mistakes.

The message and learning objectives

The central theme of the workshop is evolution, from the standpoint of molecular biology, with the hypothesis of an RNA world and the later development of DNA and proteins. Students discover a very rich molecular world that goes well beyond the DNA double helix that they are used to when considering the basis of life. They discover the existence of RNA molecules of complex architectures that can act as the cell's machinery in the absence of proteins. Each discipline contributes complementary building blocks needed for developing the central theme focusing in particular on the topics presented in Table 1.

Biology

Concepts needed to address the question of the origin of life

From organisms to cells to molecules, Definition of a living organism, Darwins natural selection, LUCA (Last Universal Common Ancestor) and the main constituents of eukaryotic and prokaryotic cells, Definition of a living organism, Different types of biomolecules (nucleic acids, proteins, lipids) and their functions, The role of water, RNA world hypothesis.

Chemistry

Structural properties of biomolecules

From atoms to biomolecules, Atomic structures and properties, Intermolecular forces, Interactions and folding of Proteins and Nucleic acids.

Physics

Concepts needed to build a molecular model and a MD simulation

From classical mechanics to molecular dynamics, Newton's equations of motion and trajectories, Building a molecular model force-field, The role of temperature.

Maths

Quantitative analysis of a force-field

How to sketch a function from calculus, Maxima and minima to find equilibrium points, Limits to understand long and short range behavior of intermolecular interactions.

Computer Science

Basic programming concepts and use of a molecular dynamics software How to provide instructions to a computer, Programming elements (variables, functions, loops), Example of simple programming languages (SCRATCH and Python), Visualization tools (VMD [START_REF] Humphrey | VMD -Visual Molecular Dynamics[END_REF] and UnityMol [START_REF] Doutreligne | UnityMol: interactive and ludic visual manipulation of coarse-grained RNA and other biomolecules[END_REF]), Interactive simulations [START_REF] Mazzanti | What Can Human-Guided Simulations Bring to RNA Folding?[END_REF].

Table 1: List of topics discussed in each disciplinary module.

The Workshop in detail

In this section we are going to present each teaching sequence, highlighting the main concepts discussed in class together with the activities proposed to the students as independent or group work. All teaching material including slides, links to online activities, instruction booklet, computer programs, and more, can be found on the workshop website where it is used as support for the class: http://rigole.galaxy.ibpc.fr (the website and some of the material are in French).

Monday morning: Biology

The goal of the Biology module is to convey to the students that the most important properties of living organisms are determined and can be explained by their molecular properties. The fact that the role of RNA cannot be captured by the central dogma is put forward. This observation allows us to point to other properties of RNAs that are related to their capability to fold in 3D. This property is both directly related to the RNA world hypothesis and the molecular structure preparing the students to understand the importance of the point of view of the other disciplines.

The basic concepts for biology are provided via a series of short videos [9] available to the students before the workshop. The session then starts with a reminder of the definition of life. Depending on the level of the students, they are either asked to build their own definition or are given one by the instructor. The students are then asked to consider a list of words and to decide whether a given word corresponds to a living organism or not. The following list has been constructed over time: bee, palm tree, bacteria, mitochondria, virus, lichen, fish, computer virus, 3D printer, prion, fire. Many of these examples do not raise questions, but others do. For example, the bee is often considered problematic because only the queen reproduces in some cases, leaving the others apparently off evolution. Lichen, being a symbiont, might also be seen as an issue. These two cases force the reasoning to occur at the level of species, not individuals. Viruses are also frequently the matter of intense debates, reflecting the ones of the scientific literature. This case is used to prepare students to see the influence of the initial definition put forward, but also the need to change their point of view, which echoes the fact that later they will have to consider RNA for its potential role in the RNA world and not just in protein production.

In order to identify the main biological molecules of interest for a minimal origin of life scenario, the students are asked to build a portrait of LUCA. They are advised to start from the observation of current living organisms and to look for their common points that might have been already present in LUCA. They usually easily arrive at a cell, surrounded by a lipid bilayer, with genetic material made of DNA and RNA, and proteins. The structure/function relationships of the molecules can be highlighted: e.g. the lipids are amphiphilic which allows them to form bilayers, which in turn allows them to separate cell interior and exterior. This property has important consequences for evolution by maintaining together DNA and proteins. The link between genotype and phenotype is also highlighted for RNA, which can both bear genetic information and act as a catalyst. It is underlined that both properties originate from RNA's ability to form hydrogen bonds, in replication and 3D folding. The citizen science game Foldit [START_REF] Cooper | Predicting protein structures with a multiplayer online game[END_REF] is used as a tool to allow students to experience the physico-chemical properties of proteins. The activity is inspired by the one described in Ref. [START_REF] Franco | Online gaming for understanding folding, interactions, and structure[END_REF].

Monday afternoon: Chemistry

The main goal of the chemistry module is to provide students with the understanding that in order to study the behavior of DNA, RNA and proteins, it is necessary to look at the physical interactions that govern these systems. We start by briefly reviewing the structure of atoms, focusing on the geometries of the orbitals and electronegativity properties, highlighting how these two factors determine the kinds of interactions forming between two atoms, ionic or covalent. For covalent bonds, we focus on the relationship between the shape of the orbitals and the geometry of the molecules that are formed when these orbitals are involved in the bond. This part is given as a short lecture that includes several schematic videos [12]. Students are then asked to turn to a practical activity where they first have to build simple molecules (water, methane, ammonia) using chemistry balland-stick kits. These same molecules are then studied in silico using the VMD software 1 , where the students can make measurements of bond lengths and angles. We then resume the lecture and present the different kinds of non-bonded molecular interactions, namely the different electrostatic terms (Coulomb and dipolar), Hydrogen bonds, and Van der Waals. These terms are presented in a qualitative fashion using movies and demos [12]. As a practical activity, students are asked to use VMD to explore the structure of an ionic crystal of liquid water and of ice.

The last part of the chemistry module is devoted to presenting proteins and nucleic acids and how they form structured molecules through folding. We first discuss the peptide bond that constitutes the backbone for proteins and look at the different physico-chemical properties of amino acid side chains discussing how their interactions lead the molecule from an unfolded, unstructured conformation to a well defined folded state. As a practical activity, students are asked to build two amino acids with the chemistry kit and to then form an oriented dipeptide. The dipeptide can then be visualized in VMD for a further, more quantitative analysis. In VMD the students can comparatively visualize the dipeptide as part of a full protein and start getting familiar with the fold of a protein as a whole (Fig. 2). At last, we introduce nucleic acids, focusing on the analysis of the interactions that nucleobases can form with each other. We discuss the double helix, but put the accent on how, based on the chemistry of the bases, it is possible to form many other base pairs that are non-canonical, and on how these pairs are often important in the 3D fold of single-stranded RNA. Examples are provided with the help of several videos [14,[START_REF]Pasquali[END_REF]. A group quiz is given before the prolonged practical activity. We conclude the module with the on-line citizen science game EteRNA [START_REF] Lee | RNA design rules from a massive open laboratory[END_REF], where the students can explore the secondary structures of single stranded RNA molecules. In the game, students are asked to optimize an RNA sequence to obtain a given 2D structure. After guiding them through the first levels of the game, students are introduced to a module specifically addressing the hypothesis of an RNA world. In the latest workshop the activity has been replaced by Pangu, a game in augmented reality in which the player builds molecules with chemistry kits and take pictures of the molecules to make progress in the game [17].

Tuesday and Wednesday morning: Physics and Mathematics

The physics module focuses on explaining how to give a quantitative description of the molecular interactions described in the chemistry module. The goal is to show students that in order to study the interactions of molecules, they need to understand their physical behavior in terms of equations, and therefore they need to be familiar with some basic principles of classical mechanics. In this module, we present the logical steps, and explain the main physical concepts, needed to build a molecular dynamics simulation: Newtonian dynamics, potential energies, and force fields. To present the concepts underpinning equations of motion, the relationship between potential energies and forces, as well as the concept of bound and unbound states, we recall some mathematical concepts from calculus, in particular limits and derivatives.

This module is at the heart of the workshop as it directly involves a discipline with which some students have difficulties. Here students have the chance to see first-hand the role of physics and mathematics in the interdisciplinary challenges posed by the biological problem and understand their role in the overall picture. It is therefore important to focus on a few central concepts, to allow enough time to treat the subject, and to put to use different teaching tools. Thereby we always combine the formal presentation in terms of equations and mathematical analysis, with fun, practical exercises and hands-on examples to provide the opportunity to build an intuition. The module takes two half-days, Tuesday and Wednesday morning, with computer science done on Tuesday afternoon, to allow the students a break and focus on something more practical.

The module starts with a qualitative presentation of what molecular dynamics is, in order to clearly show that the goal of the module is to be able to understand the various steps of the simulation process. By presenting MD first, teachers have a thread to which they can refer back to during the module, refocusing the students on why the different physical concepts are needed.

The first concept presented is Newton's second law, F = ma, discussing what forces, accelerations, velocities and displacements are, and what an equation of motion is. At first, we give a discrete treatment in terms of finite variations of quantities over time. Next, we move to the continuum description using derivatives to define the velocity and the acceleration. We present the concept of derivative, highlighting its role as the slope of a function, and review simple rules to compute such derivatives. The students work in groups to determine the equations of motion for a free-falling object. We then continue to present the principles of the harmonic oscillator, which will later be used extensively to model covalent bonds. An on-line exercise is proposed to explore the properties of this simple system and to visualize the behavior of the displacement as a function of time, from which students see first hand the arising of a sinusoidal function [18,19]. To conclude this part, we discuss Newton's third law of action and reaction to explain the behavior of multi-body systems and we review vector sums to combine multiple forces acting on the same particle.

The second chapter is devoted to energy: potential energy, kinetic energy and energy conservation. We first introduce potential energy and its relation to work and force. Marble roller coasters are used to familiarize students with the concept of potential energy function and equilibrium points. Together with the instructor, students build the equivalent of a harmonic potential function and of a Lennard-Jones potential and discuss the role of maxima, minima and long-range behavior. Next, we present kinetic energy, total energy and energy conservation in relation to the concepts of bound and unbound states based on the functional profile of the potential energy and the total energy provided to the system. Students further explore these concepts through an online application with examples of a roller coaster and a skateboard [20].

The next section presents the functional forms commonly used to describe intermolecular potentials, that is harmonic functions for covalent bonds, inverse power laws for electrostatic interactions and Lennard-Jones for Van der Waals interactions. We review how to sketch the profile of a function, computing the existence domain, limits and determining maxima and minima through derivatives. For each of the functional forms analyzed, we discuss the possible existence of bound and unbound states based on the presence of minima and on the limits at large distances, and put this in relation to the molecular behavior we want to capture with these models (Fig. 3). Students are asked to do some calculations to sketch these potentials themselves.

The last section is devoted to the role of temperature from a microscopic point of view in relation to bound and unbound states and the depth of potential wells. We present the roughness of a biomolecular energy landscape and the role of temperature on the ability of the system to explore it. To make this concept clearer, we ask students to shake a model of a roughed energy funnel printed in 3D with a small bead inside, representing a molecule exploring its energy landscape (Fig. 3). To conclude the section, students are asked to explore the role of temperature on various forms of molecular interactions (charged particles, neutral particles,...) using online applets, guided by some questions [21,22,23].

The module concludes with a group quiz in which the students are asked to critically review the main physical concepts presented in the module in relation to their application to molecular modeling (see supporting material).

Tuesday afternoon: Computer science

Computer science is typically not a subject in the curriculum of first-year life science students. However we considered it useful to introduce the basics of giving instructions to a computer, in order to provide students with some understanding of what a computer simulation is and to show them at least the existence of simple coding languages that can be used to perform data analysis. We approach the subject from a distance and first propose a practical activity seemingly unrelated to computers to make students understand the logic of giving unambiguous instructions to a computer. Students are spread out in the room and each one is given a small Lego abstract construction of a dozen pieces of different shapes and colors. They are given 15 minutes to write down instructions on paper on how to build the exact object they received, using only textual descriptions and no drawings. After that time they are asked to take a picture of the object with their phone and to disassemble it, leaving it on the table together with the instructions. Students swap places and in another 15 minutes they have to rebuild the object following the instructions. Reconstructed objects are then compared with the pictures of the originals. This activity is used to start a discussion session in which the successful and unsuccessful instruction strategies are analyzed and in which students are led to deduce what an effective way to give instruction to a machine would be. A short lecture is then given to describe the basic elements of a computer code: variables, loops and functions, presenting examples with the Python programming language. The rest of the module is dedicated to practical activities, first using Python and a series of exercises proposed by the Codeacademy website [24], and then presenting an enzymedocking code written using Scratch [25], a programing language developed by MIT to teach the basics of programming to children. After working on some common predefined exercises, students are instructed to choose one of the two programming languages and to either extend the enzyme-docking code to make it perform new functions, or to write a completely new code on a subject of their choice.

Wednesday afternoon: Molecular dynamics lab

Half of a day is fully dedicated to a computer lab where students use an interactive simulation software developed for our research activities [START_REF] Cragnolini | Coarse-Grained HiRE-RNA Model for ab Initio RNA Folding beyond Simple Molecules, Including Noncanonical and Multiple Base Pairings[END_REF]27] to explore DNA double strand assembly and single-stranded RNA folding. After the comparatively more abstract physics and chemistry modules, before the computer lab, we propose a physical activity in order to bring the students back to the central theme of the RNA world. In the human folding game, each student receives a T-shirt of one of four different colors, each color representing one of the RNA bases. The class is then asked to form chains of a given sequence and attempt to form contacts to reproduce given RNA secondary structure motifs such as the double helix, hairpin and pseudoknots, as well as to optimize the sequence for a given structure, following up on what was done in the earlier EteRNA exercises. After the game, lasting usually about 20 minutes, students start working at the computer lab using the UnityMol [START_REF] Lv | Game on, science-how video game technology may help biologists tackle visualization challenges[END_REF] software and entering the HiRE-RNA folding competition [29] (Fig. 4). The software performs a molecular dynamics simulation using a simplified, coarse-grained representation for nucleic acids, which is visualized in real time on the computer screen. By selecting a particle of the system it is possible to apply an external pulling force, which is integrated in the Newtonian dynamics, allowing the user to pull the molecule toward a desired conformation 2 . Pop-up graphs monitor different energy terms from which one can deduce, for example, when a base pair is formed. Students first familiarize themselves with the software performing simple interactive simulation tasks on the DNA double helix, such as unwinding a preformed helix. Then they have to solve four RNA folding puzzles with increasing complexity, from hairpins, to a pseudoknot, to a triple helix, starting from a completely unfolded configuration. By observing the stability of the conformations they generate and by analyzing the graphs of the energy terms, the students select plausible native-like folded candidates and submit them to the HiRE-RNA folding contest web server to be automatically compared to the known experimental structures, receiving a score and a ranking in the class. As a side note, this student experiment brought up particularly interesting observations. We found that humans (e.g. students) explore phase space in a very different way compared to automated computational approaches, as discussed in more detail elsewhere [START_REF] Mazzanti | What Can Human-Guided Simulations Bring to RNA Folding?[END_REF].

In the 2018 workshop with junior high school students, a parallel session was added to the MD lab to present ligand-biomolecule interaction through interactive simulations and virtual reality3 . This hands-on demonstration allowed us to show yet another aspect of molecular modeling where the interplay between biology, chemistry and physics is essential for conducting an in silico experiment addressing biomolecules.

Thursday and Friday morning: Posters group work and presentation

Students have 1.5 days to prepare a poster to be presented to the rest of the class on the last half day of the workshop.

A number of pre-selected topics ranging from more biological to more physical ones are proposed to groups of 3 or 4 students. Groups are chosen to be as much as possible heterogeneous and complementary in the disciplinary strengths of their members. Topics include the role of clay in the formation of the first "cellular" compartments, the Urey and Miller's experiment for the creation of the first amino acids, the life cycles of phages comparing systems based only on RNA with those also using DNA, RNA thermometers, Gquadruplexes, and ribozymes. Students are encouraged to use the knowledge and tools explored in the disciplinary modules to address the subject from different angles. On purpose, we do not provide more than the title of each subject, to allow each group to develop its study creatively and critically. A sample of posters produced by high school students in 2018 can be found in SI.

The poster presentation is organized as a rotation, so that each group member has the chance to present the work in front of a group of participants and a professor. For the workshops addressed to college students as a course, the poster session contributes to the students' grades. Through the rounds of presentations, each student is evaluated both by his/her peers and by a professor, and acts themselves as evaluator for other participants. More information on the organization of this kind of poster presentation can be found in a separate manuscript [START_REF] Lopez | Poster session with peer grading[END_REF].

Goals Assessment

The workshop was offered so far to three high school groups of students, one of seniors and two of juniors (about 100 overall), two 45-student groups from the first and second year biology degree, and three 30-student groups from the first year of the interdisciplinary degree of "Frontiers of life science".

Unfortunately, long-term follow-up of students has not been feasible for us neither for high school nor for college. Because of the articulation of the French education and its administration, we were not able to seize students' records and track how they performed in their studies after the workshop. We have tried to contact students one or two years after the workshop, but we collected only a few replies. Therefore, we could only perform an assessment of our initial goals on the short-term, by observing the evolution of the students' attitudes and performances during the week of the workshop.

We analyzed several factors which included a survey at the end of each workshop, results of the quizzes when these were given as homework, results of the folding challenge, quality of the posters produced in the group work, and the impressions of all instructors (eleven all together) on the students' attitude, participation and reflection. Even though the overall number of participants is not large enough to perform a statistical analysis of the impact of the workshop and given that some appreciations could only be qualitative, we nonetheless are able to draw some overall conclusions. The full results of the survey are presented as supplementary material.

Students of all categories showed a great enthusiasm for the workshop. They very much appreciated the innovative teaching methods giving them a more active role than traditional teachings, as well as the learning environment in a more relaxed and fun setting. Their active participation grew over the days. While at first they were relatively shy, not being acquainted with a system where they were asked to participate in open discussions with professors and with classmates, they rapidly found their place building constructive interactions. This more direct relationship with the teachers opened the grounds for facilitated communication, essential to address the harder topics among the proposed ones, thereby promoting a genuine and deep understanding of the subjects. The productivity of the students in the group work was satisfactory, with a good portion of posters reflecting the interdisciplinarity of the teachings of the week.

To further investigate the impact on high school students, in 2018 we proposed a new, additional, survey to gauge the initial interest of participants in the different subjects, their assessment of the importance of each subject to address biological problems, their perception about how easy or hard each subject is, and to what extent they would like to further study the subject in their future curriculum. The same questions were posed at the end of the workshop to see to what extent the program changed the students' point of view. Questions and results are available in supplementary material.

Overall, what emerges from this inquiry is that the workshop helped giving participants a more realistic perception of the four disciplines, and, as was our initial goal, helped them seeing the connections between them and the need for pursuing some knowledge of the hard sciences in order to make progress in biology. As discussed above, this is an important result given that students, and even researchers, in biology typically realize that they need other disciplinary knowledge, in particular math [START_REF] Marshall | Are biologists getting the mathematical training they need in college?[END_REF], only very late in their education. Interestingly, this insight seems to motivate students to pursue studies in biology even further than initially assessed.

Another criterion to assess the impact of the workshop was through the feedback received by colleagues and institutions. The workshop has been an opportunity to spread innovative teaching methods among university professors. At first only three of the eleven teachers involved in the project were active users of unconventional teaching methods, and through the conception and the realization of the workshop the other teachers enthusiastically discovered the new methods and some of them applied them in other contexts as well. The workshop was presented in various teaching councils and faculty meetings, gathering significant interest also at the institutional level. As a consequence, after the first year when funds to cover all expenses, including teaching hours, were provided by an outside source (Idex Paris Sorbonne Cité University), the various disciplinary departments took charge of the teaching load allowing the workshop to continue to be offered both as a college course and as an extracurricular activity for high school students.

The workshop also received important institutional recognition being selected for the initial financial support by the "Paris Sorbonne Cité University" (USPC), and, more recently, from the joint Sao Paolo University -USPC grant to offer the workshop in Sao Paolo University in March of 2018.

Perspectives and conclusions

Convinced of the effectiveness of the teaching method we have developed within this original workshop, in the three years since its first conception the workshop has evolved and parts of its content and teaching strategies have been used as blocks for other courses.

With the recent involvement of a Pharmacy department supporting the project, we have developed an alternative workshop based on the theme of the interactions of biomolecules with drugs. With respect to the original theme of the origins of life, this extension involves changes in the biology module, in the practical lab, and in the subjects given for the poster group work. Instead of folding an RNA molecule, in the new lab, students analyze the results of a molecular dynamics simulation of a protein interacting with a small ligand in its native sequence and structure and of a protein where mutations have been introduced near the ligand binding site. The subject of the posters are now focused on the binding of small ligands to proteins. For example, students are asked to investigate how caffeine and nicotine work at the molecular level. As a further development, for the summer of 2018, an experimental wet lab was introduced in which students investigated the structure of biomolecules using biophysical techniques.

As an example of how the workshop can be easily be adapted to a different context, we illustrate our experience with a course for pharmacy students in an early elective research program, Pharmascience. The one-week setup of the workshop was proposed as a biophysics/modeling course for students in their second year, adapting and changing certain modules to the higher level of these students and course requirements. The modules of chemistry and physics of RIGOLE were used as starting points to familiarize students with the structural analysis of biomolecules and modeling. For both subjects additional, more in depth, concepts were introduced. For example, for physics we devoted one half-day to normal mode analysis and related applications to proteins and nucleic acids. We developed more in depth the concepts from calculus on functional studies and asked students to perform comparisons of different functional forms to describe molecular interactions and to investigate the consequences of their possible choices. The computer lab on protein-ligand interactions was expanded to lead the students to make more quantitative observations and students were asked to write a short report.

Instead of the poster group work, a paper on modeling DNA at different scales was chosen to be analyzed and presented by the students, with different groups being assigned a different scale (quantum description of bases interactions, atomistic description, coarse-grained description, and mesoscale description).

A second example comes from the workshop offered in Brazil, addressed to master and Ph.D students in pharmaceutical science. The biology, chemistry and physics modules were preserved but adapted to the higher degree of experience of the students, as was the case for Pharmascience, but they were discussed in less time than for the RIGOLE workshop (two full days). The computer labs were expanded and a module on docking was introduced in addition to the analysis of molecular dynamics simulations on protein-ligand systems. The group work on drugs was maintained, but instead of posters, the groups had to prepare an oral presentation.

Taking a wider perspective, the concept, format and methods developed here can be easily exported to other scientific disciplines and subjects beyond the realm of biology/biochemistry. Indeed, the problems with traditional pedagogical methods which motivated us, and which we have outlined in the introduction, are common to the vast majority of the first-year curricula in the hard sciences. The main key to developing similar workshops in other contexts is the choice of an appropriate subject: on the one hand, it should be specific enough to be introduced and explored satisfyingly in a short time frame without the need of too much pre-acquired knowledge; on the other hand, the subject chosen should lend itself to the investigation from different angles and methodologies, to promote the idea that science is not a compartmentalized endeavor and that each single aspect of a university curriculum contributes organically. Interesting examples of this kind can be found in all disciplines.

One example which is particularly well suited in the domain of the physical sciences is cosmology. In this context, an interdisciplinary workshop focused on the theory and observation of the basic features of the evolution of the Universe and its driving forces could be developed along similar lines as the one described in this article. The basic mathematical laws which describe the universe evolution are very simple to write down and understand even by first-or second-year undergraduates with no prior knowledge in the field. Furthermore, this subject involves many different physics sub-domains (astrophysics, nuclear physics, optics, fluid dynamics...), and features the interplay of phenomena governed by different characteristic scales (like it is the case for macromolecules). Also, computer simulations are an important tool in this subject area, and one can easily envision the development of simplified toy versions of the software which is used in real-life research. Finally, interactive on-line pedagogical tools centered on many different aspects of cosmology (Hubble expansion, cosmic microwave background, etc.) are already available (e.g. [33,34]) and can be integrated in the workshop.

In conclusion, our experience has shown us that offering an interdisciplinary approach to the study of biomolecular systems is effective at all levels of education, from high-school to graduate, especially when this teaching is carried out with a mixture of theoretical and practical activities. Given that many different subjects are addressed in the workshop from various disciplines, this combination of theory and activities becomes essential to lead the students to the understanding of all concepts4 . Offering a relaxed teaching environment, where fun activities are proposed, is interesting also for more advanced students. Indeed, proposing less academic activities forces them to think out of the schemes they are used to, helping them to acquire a more critical view even toward subjects they have previously studied in disciplinary courses.

We hope the present article will allow others to replicate and adapt the workshop in even more contexts.

Figure 1 :

 1 Figure 1: Schedule of the typical week for the workshop.

Figure 2 :

 2 Figure2: left: building amino acids using ball-and-stick chemistry kits, right: analysis of the same amino acids inside a peptide using the VMD software.

Figure 3 :

 3 Figure 3: top left: energy conservation presented with the roller coaster applet, top right: marbles roller coaster to explain the concept of potential energy function, bottom left: the equation for the Lennard-Jones potential is discussed at the board, bottom left: the concept of binding energy and its relation to temperature is presented in a lecture (T indicates the thermal energy due to a heat bath and E 0 indicates the depth of the energy well of the potential, defining bound states).

Figure 4 :

 4 Figure 4: top left: human folding activity, top right: interactive RNA folding simulation on the computer, bottom left: interactive docking (for high school students in 2018), bottom right: example of lego structure used in the computer science module.

As noted by Burgin and colleagues, VMD offers the advantage of being used in research labs, which makes it an authentic activity[START_REF] Burgin | High school biology students use of visual molecular dynamics as an authentic tool for learning about modeling as a professional scientific practice[END_REF]

As noted by O'Connor and colleagues, an interactive simulation set-up is very intuitive making it an appealing tool in an education context[START_REF] Connor | Sampling molecular conformations and dynamics in a multi-user virtual reality framework[END_REF]

More details on the freely available virtual reality implementation and experiments are provided elsewhere[START_REF] Baaden | [END_REF]

One aspect of the activities offered in the workshop that should probably not be neglected is the use of research software that is attractive to students perceiving the authentic nature of the activity they perform[START_REF] Burgin | High school biology students use of visual molecular dynamics as an authentic tool for learning about modeling as a professional scientific practice[END_REF][START_REF] Machluf | Making authentic science accessiblethe benefits and challenges of integrating bioinformatics into a high-school science curriculum[END_REF]

Acknowledgements

We wish to thank the many actors of this work, starting from the funding sources that have allowed us to set up and continue the project: IDEX USPC grant "RIGOLE", LABEX Dynamo (Grant "DYNAMO", ANR-11-LABX-0011-01 ), IDEX Brazil USP-USPC grant, the Biology and Physics Departments of Paris Diderot University, the Pharmacy department of Paris Descartes University, and the "Frontières du Vivant" undergraduate program. A special thanks is given to the pedagogical coordinator Eva-Coralie Fryde who worked with us in 2015-2016 to set up RIGOLE, as well as to Geoffrey Letessier and Sébastien Doutreligne who helped set up the related websites. Their combined role was essential in getting the workshop started. We also thank all professors who, with us, contributed to teaching: Anne Badel, Miguel Bermudez, Nathalie Caulet-Demont, Sylvain Chaty, Delphine Flatters, Gautier Moroy, Julien Serreau, Magali Blaud, Nicolas Leulliot, Elisa Frezza as well as all the college students that helped as tutors for the workshops for high school students. And, of course, we thank all the students who enthusiastically participated to the workshop and who motivated us to keep this project going through the years.


TUNGSTEN RECOVERY FROM SULFIDE-CONTAINING W-TAILINGS:

Beneficiation Tests and Bioleaching for Sulphide Removal Douglas PINO-HERRERA 1 , Solène TOUZE 1 , Anne-Gwénaëlle GUEZENNEC 1 , Françoise BODENAN 1

1 BRGM, Water, Environment, Process and Analysis Division, 3 Avenue Claude Guillemin, BP36009, Orléans 45060, Cedex 2, France

Recovery of W-tailings

In the frame of the H2020 RAWMINA project (Grant Agreement nº 958252), recovery of critical raw materials such as tungsten (W) in mining waste is evaluated by various innovative (bio)hydrometallurgy techniques. First, the recovery of W from sulfidic mining waste is carried out by applying gravimetric separation techniques to concentrate W-bearing minerals (mainly sheelite). W can then be leached from the beneficiated tailings using alkaline leaching. However, arsenic-containing sulfides, such as arsenopyrite, can also be significantly deported in the W concentrate which can cause an increase of the reagents consumption in the alkaline leaching process [START_REF] Suess | Thioarsenate formation upon dissolution of orpiment and arsnopyrite[END_REF]. Moreover, the presence of As in the concentrate could negatively impact downstream processes and the final products. Bioleaching is known to be efficient in the removal of sulfidic material even at low sulfide content [START_REF] Hubau | Bioleaching to reprocess sulfidic polymertallic primary mining residues : Determination of metal leaching mechanisms[END_REF]. Therefore, after the beneficiation process, bioleaching is used to treat sulfide and reduce acidity, iron and arsenic concentrations in the beneficiated material.

Beneficiation of W-tailings by gravimetric methods

A representative sample (500 kg) was sampled on a mine site in Portugal and prepared at BRGM facilities to obtain homogenized sub-samples according to well-established procedures, i.e. drying, crushing, grinding and splitting. Beneficiation tests have been performed by gravimetric separation taking advantage of scheelite density (CaWO3, 5.9-6.1 g/cm 3 ). This process can also concentrate the pyritic material (mainly constituted by arsenopyrite that have a similar density as scheelite). The first pre-treatment step was the classification in four size fractions (< 100 µm; 100 µm -250 µm; 250 µm -500 µm; 500 µm -1 mm), since scheelite was observed at particle size up to 1 mm by UV shortwave radiation (blue spots of scheelite bright fluorescence). Then, a separation step was carried out using a gravimetric Wilfley shaking table for the coarser fractions (Figure 1). W content in the beneficiated product quantified by portable XRF technology was increased by 10 fold (from around 2000 ppm to 2-5 wt. %). Concentration using Mozley Multi Gravity Separator (MGS) is also planned to treat fine fraction (< 100 µm). In addition, iron and sulfur content in the beneficiated samples have been increased, indicating that arsenopyrite (main sulfide contained in the tailings) has also been concentrated.

Figure 1: Beneficiation tests of W-tailings using a gravimetric Wilfley shaking table

Bioleaching of the beneficiated W-tailings

Bioleaching tests are currently being performed in shake flasks to adapt a selected acidophilic microbial consortium to the bioleaching of the sulfide contained in the beneficiated W-tailings. The next steps include the inoculation of 2-L reactors with the adapted microbial consortium and a progressive increase of the solid concentration in stirred tank reactors to bioleach sulfides and produce sulfidefree samples. Preliminary bioleaching tests show that this substrate is particularly suitable for the growth and the activity of S-and Fe-oxidizing activity. The dissolution of sulfide minerals (mainly arsenopyrite) is almost complete and leads to the production of a solution of sulfuric acid and ferric iron.

Conclusion and perspectives

The beneficiation process applied to the W-tailings allowed concentrating not only scheelite but also arsenopyrite in the W-tailings. An optimal flowsheet of beneficiation and bioleaching processes will be established as a pretreatment of the tailings before alkaline leaching to dissolve tungsten. Downstream process considered in RAWMINA project include the W selective recovery using nanofibrous composite materials and electrowinning. Finally, the overall RAWMINA process would allow the reprocessing of the W-tailings, recovering the remaining W and reducing the environmental risks associated with sulfide-and arsenic-containing mining residues.


est destinée au dépôt

CO2 in Closed Loop

Pr Christophe Marvillet IFFI (Institut Français du Froid Industriel)/Cnam (Conservatoire National des Arts et Métiers)

Paris France

Carbon dioxide is used in closed circuits mainly for energy use in food processing applications and especially for cold applications that is to say at temperatures below ambient and can go down to temperatures close to -40 ° C.

The two major functions provided by this fluid CO2 in refrigeration systems are:

-Use as a secondary coolant that is to say the transport of thermal energy from a point of use (heat source) to a point of cold production (heat sink) at temperatures, according to applications, betwee n 10 ° C and -40 ° C -Use as a refrigerant which flows in the circuit of the refrigeration equipment, is compressed in the compressor in vapour phase, evaporates in the evaporator in liquid/vapour phase and allows in this equipment the effective production of cold that is to say the absorption of heat at low temperature from the medium or fluid to be cooled.

The increased use of CO2 in refrigeration systems in recent years is due not only to the specific characteristics of this fluid -in particular its low impact on the environment -but above all to the new regulatory constraints that apply to HFC refrigerants (hydroflurocarbons) which have major impacts on the environment and on secondary fluids based on alcohol, synthetic or silicone oils also concerned by environmental constraints but also by their unsuitability for agro-food uses.

In this chapter, we will discuss the following points:

-The specific properties of CO2 as a coolant and refrigerant and the advantages / limitations of this fluid in the intended applications, -The use of CO2 as a secondary coolant by recalling the architecture of energy systems, the main design rules of these systems and the precautions to be taken when using this fluid in these uses.

-The use of CO2 as a refrigerant by recalling the main refrigeration cycles in which it can be valued, the main design rules of these systems.

1/ The Carbon dioxide thermo-dynamical and thermo-physical properties

Carbon dioxide is an inorganic compound whose chemical formula is CO2, the molecule having a linear structure of the form O = C = O. Under normal conditions of temperature and pressure, it presents itself as a colourless, odourless gas with a pungent flavour.

At atmospheric pressure, it sublimates at -78.5 °C (passage from the solid state to the gaseous state), but does not melt (passage from the solid state to the liquid state). The liquid phase can only exist at pressure above the minimum pressure of 519 kPa, and in a temperature range of -56.6 ° C (triple point) to 31.1 ° C maximum at 7.38 MPa (critical point). The figure 1 shows the diagram Pressure/temperature of two-phase CO2 equilibrium data (gas/solid, liquid/solid and liquid/gas). The latent heat values are the following:

-Vaporization: at 0°C the latent heat is equal to 234.5 kJ/kg, at -16.7°C to 276.8 kJ/kg, at -28.9 °C to 301.7 kJ/kg -Fusion: at -56.6°C the latent heat is equal to 199 kJ/kg It will be noted that one of the particular points of CO2 lies, whether for secondary coolant or refrigerant uses, by the high pressure values encountered with this fluid in the temperature range of -10°C to 80°C, range of temperature of use in refrigerating equipment. In comparison, with traditional fluids, we note:

-For secondary coolant use, glycol mixtures in liquid form, for ex ample, only require pressures not exceeding 300 kPa, whereas a CO2 circuit can reach values of several MPa depending on the temperatures reached.

-For refrigerant uses, Table 1 shows the comparison of CO2 saturation pressures with the refrigerants commonly used and highlights the very important values achieved with this fluid. The CO2 thermo dynamical properties of CO2 has important impact on fluid manipulation: prior to charging the system must be evacuated first and it's important to never brake the vacuum with liquid CO2 because the spontaneous evaporation have severe cooling effect and generate risk of brittleness and thermal shock of the materials. To avoid the formation of solid CO2, it is strongly recommended to charge the plant with gaseous CO2 up to approx 10 up to 20 bars.

The thermo-physical properties that must be analyzed are those that condition the design of the technical equipment of refrigerant fluid circuits or refrigerating machines. These properties are:

-The densities of the liquid and vapour phases which determine the size of pipes and active s elements such as pumps and compressors, -The thermal capacities and thermal conductivity of the liquid and vapour phases which determine the size of the exchangers (evaporator, condenser), -The viscosity of the liquid and vapour phases which condition the pressure losses in the fluid circuits and the dimensions of the pumps and other technical equipment.

Here again we distinguish two distinct uses:

-As a coolant, a comparison (Table 2a) of properties of CO2 and a water-MEG (mono ethylene glycol) mixture is made.

-As a refrigerant, a comparison (Table 2b) of the properties of CO2 and a "traditional" fluid HFC404a is realized. Carbon dioxide has undeniable qualities as a secondary coolant especially due to its application conditions of + 15 to -50 ° C with interesting thermo-physical properties: low viscosity in the liquid phase, high thermal capacity and conductivity in liquid phase as shown in table 2. These real qualities can moreover be valorised by a use in the form of a liquid-vapor mixture which makes it possible to use the evaporation enthalpy and to very significantly reduce the flow rate of circulating fluid. The advantage of such a device lies in the very high heat-carrying power of the fluid due to the use of the latent heat of the coolant fluid (CO2): thus, the thermal power transported per unit volume (of the liquid phase) of CO2 is more than 20 higher than that transported, with a temperature difference of 5 °C, by a performing monophasic secondary coolant, that is to say water/MEG. This results in substantial gains in terms of piping and pump dimensions, pumping energy and the elimination of the temperature variation on the exchangers which makes it possible to raise the evaporation temperature of the primary fluid by 4 at 6 K, a reduction in the size of the compressors and an improvement in system performance of about 15%.

Carbon dioxide shows also good qualities as refrigerant because of high latent heat of evaporation and high vapour density which allows a strong reduction of circuit and compressor size. The thermal conductivity of liquid CO2 are significantly higher that of HFC507A, a traditional refrigerant in low temperature refrigeration machine. The refrigeration cycle with C02, due to low value of critical temperature, may show supercritical conditions: in these conditions, as can be seen on figure 2, supercritical CO2 shows atypical evolution of thermo physical properties:

-Dramatically increase of isobaric heat capacity and thermal conductivity at pressure between 74 to 86 bars: this has to be taken into account for high pressure gas cooler design. -Strong change of viscosity and density for temperature between 31°C to 41°C; this has also great impact in the heat exchangers design.

Figure 2: Thermo physical properties of supercritical C02 against temperature and P absolute pressure

2/ The Carbon dioxide environmental impacts

From a great number of years, halogenated hydrocarbons (CFC, HCFC, HFC) are used as refrigerant in the refrigeration systems. We know that stratospheric ozone is destroyed by chlorine transported at these altitudes by halogenated hydrocarbons which have a long life in the atmosphere, mainly by CFCs but also, to a much lesser extent measured by HCFCs. We also know that it is this criterion that has decided the international community to ban these compounds chlorinated. The action of each compound on stratospheric ozone is characterized by the ODP (Ozone Depletion Potential), the potential for destroying ozone. The values of the ODP are generally given with reference to R-11, (CCl3F) one of the CFCs the most aggressive from this point of view. Only refrigerants having a zero ODP should continue. Table 3 gives ODP values reported in CO2 and HFC, HCFC.

Greenhouse gases are essential for people living in our planet which, in their absence, would have a temperature much too low to be habitable (-18 °C). However, the excess of these gases, hindering the exit (towards the cosmos) of the radiation terrestrial, can, in the long run, cause a slow warming, dangerous for our world. At aside from the well-known greenhouse gases (water vapour, CO2, methane, nitrogen oxides, etc.), the halocarbon refrigerants have a very significant action. Although unfortunately not widespread in the atmosphere, their influence is much greater than, for example, that of CO2 whose greenhouse effect is the best known. The greenhouse effect of a compound is characterized by the global warming potential GWP (Global Warming Potential). Table 3 gives GWP values reported in CO2 and CFC,HFC, HCFC; these relationships change with the time period envisaged because CO2 molecules and those of gaseous refrigerants considered do not disappear from our atmosphere to the same speed. For comparisons with CO2, we generally adopt a reference period of 100 years. The GWP translates the effect greenhouse effect (consequence of leakage or non-recovery). The compression refrigeration systems generate a greenhouse effect indirect, linked to their electricity consumption (CO2 emission during electricity production).

Many of the proposed neo-refrigerants, such as HFCs, who have GWP considered by some to be too important, which leads to the wish for the exclusive use of natural fluids: CO2, water, ammonia, propane, butane, etc.

Fluid

3/ Risks and safety instructions for CO2 use

Outside ambient air today contains about 0.04% of CO2. Nevertheless at a certain concentration in the air, this gas is dangerous or even deadly because of the risk of asphyxiation or acidosis, although CO2 is not chemically toxic. The exposure limit value is 3% over a period of fifteen minutes. This value should never be exceeded. Beyond that, the health effects are all the more serious as the CO2 content increases. Thus, at 2% of CO2 in the air, the respiratory amplitude increases. At 4%, the respiratory rate accelerates. At 10%, visual disturbances, tremor and sweating may occur. At 15%, it's the sudden loss of consciousness. At 25%, respiratory arrest results in death.

Inhalation of concentrated carbon dioxide causes a blockage of ventilation, sometimes described as a violent feeling of strangulation, a breathlessness, respiratory distress or chest tightness, which can quickly lead to death if exposure is prolonged.

Since carbon dioxide is a colourless, heavy gas accumulating in layers, it is difficult to detect by an inexperienced person.

In high concentrations approaching 50 to 100%, such as those found in artificial carbon dioxide arrays of occupational origin, there may be an effect of nervous breakdown and an immediate loss of consciousness, followed by fast death in the absence of outside help. These accidents present a high risk of an accident, witnesses can rush to help the victim without thinking about their own safety and become victims of intoxication too.

Recommendations:

The limit value of exposure to CO2 at the workplace (ELV) corresponds to "Limit values of exposure to workstations" at a concentration of 0.5% by volume. Please note that CO2 not only has an asphyxiating effect but acts directly on the metabolism in our body, even if there is still enough oxygen in the ambient air. The concentration of CO2 must be monitored by taking measurements; a control of the oxygen content gives a false impression of safety.

Another harmful effect of carbon dioxide on the human body is that of cold. If CO2 cooled by detente comes into contact with the skin in the form of ice / dry ice, it can cause frostbite, that is, painful "cold burns". Sensitive skin tissues, such as the cornea, are particularly at risk. Ice formation in pipes and piping or valves may also pose a mechanical hazard to people in the vicinity.

Recommendations: Please follow exactly the working instructions for handling CO2. Protect your skin by wearing appropriate protective clothing and gloves. Wearing safety glasses can effectively protect the cornea from cold burns.

The following safety measures are indicated:

-Inform your employees of the special dangers of CO2 -Employees working in the vicinity of applications using CO2 must be trained and instructed accordingly so that they can interpret correctly alarms and their own observations. Develop detailed job descriptions and risk analysis for sectors using carbon dioxide. -Ensure the tightness of installations using CO2, remedy any leakage without delay. CO2 emissions from technical equipment or safety valves must be vented to the open air.

Premises with CO2-based facilities must have efficient ventilation, especially if they are below. This ventilation equipment must be regularly maintained and checked. -A CO2 monitoring and alarm system must be installed in premises where installations using this gas are located. The surveillance and alarm systems must be checked regularly, periodic maintenance must be carried out by the company that set them up. -In the event of a sudden escape of CO2, leave the premises immediately, especially if they are located in the basement (pits, cellars), because the risk of CO2 accumulation is particularly high. -Do not enter rooms where large amounts of CO2 have accumulated with a self-contained breathing apparatus. This also applies when there are injured people in the room who need urgent rescue. Fixed CO2 extinguishing systems must be put into service for control purposes or for necessary intervention only if there is no one in the area where risks exist. If the carbon dioxide can enter other premises through pipes, openings in the wall, ventilation or air conditioning, these premises are also considered risk areas.

4/ CO2

Cooling and refrigeration loop conception and design.

4.1/ Carbon dioxide cooling loop

Carbon dioxide has undeniable qualities as a secondary coolant especially due to its application conditions of + 15 to -50 ° C with interesting thermo physical properties: low viscosity in the liquid phase, high thermal capacity also in liquid phase.

These real qualities can moreover be valorised by a use in the form of a liquid-vapour mixture which makes it possible to use the evaporation enthalpy and to very significantly reduce the flow rate of circulating fluid. Under these conditions of use, the figure 3 shows a piping diagram of a technical device in which a CO2 refrigerant circuit is associated with an ammonia, CO2 or HFC cold production unit. Thus, the fluid to be cooled yields thermal energy and thus allows vaporization of the liquid CO2 (for example at -36 ° C.). The vapour phase produced is sent to the evapo-condenser, exchanger ensuring the heat transfer between the CO2 coolant loop and the cold production unit, and then condensed at a saturation temperature close to that of -36 ° C for the example considered: the condensate -substantially sub cooled -is circulated by a pump and sent back to the exchanger in contact with the fluid to be cooled. -Transcritical cycle (figure 4) with intermediate heat exchanger is a basic cycle with a low pressure stage at subcritical conditions. The machine is composed of an evaporator at low pressure, a gas cooler at very high pressure (about 80 to 110 bar), an intermediate heat exchanger HX. The HP Regulation valve has to regulate the optimum value of high pressure (for a maximum cycle efficiency) in the gas cooler. -For low temperature applications, a cascading system when condensing CO2 (figure 5) at temperature lower than 31°C, and possibly around -10 to 0°C, CO2 systems show high energy performances due to efficient thermo-physical properties (low viscosity and high thermal conductivity) and the development of cascading systems. Those cascading systems use CO2 at evaporating temperatures varying between -50 and -35°C associated with a high temperature refrigerating system (of the cascade) using either ammonia or HFCs have led to very efficient systems in the food industry. -In parallel with this effort, a number of laboratories and industries have developed new possible solutions using ejectors in order to improve the energy efficiency. Developments of expansion turbines have also been proposed for the same purpose: limitation of expansion losses. As shown in different publications, high efficiency expansion has to be studied in parallel with heat recovery by a liquid/vapour heat exchanger. The two options are in competition for improvement of energy efficiency. -The development of heat exchangers is driven by high efficiency heat exchange and the decrease of materials quantities (for mass production components, the mass of the material is a key parameter for the price). The trends are obvious: the thickness of copper tubes has been reduced to 0.3 mm, the thickness of aluminium fins is in the range of 0.1 mm, the diameters have been constantly reduced, and high efficiency fins, groove tubes, have led to improve of a factor 2 to 3 the heat exchange coefficient of air-torefrigerant heat exchangers in the last 20 years. In parallel with those developments of copper tubes / aluminium fins, fully brazed all aluminium heat exchangers. Condensers have been designed with extruded micro-channel aluminium tubes. Those tubes are brazed with accordion louvered fins, leading to a significantly higher heat transfer coefficient. The introduction of CO2 as coolant or refrigerant modifies, due to the increased complexity of the systems, strongly the design rules of the installations as well as the modalities of driving and maintenance.

The deployment of these technologies in Europe and more widely on other continents where the prohibition rules are less draconian presupposes a mastery of the rules of design, construction, security, implementation of this fluid through a rigorous respect of the cleanliness and lack of moisture in the CO2 circuits.

Figure 1 :

 1 Figure 1 : Diagram Pressure-Temperature for Carbon dioxide

  (0,016)0,00013(0,000017) *(values for vapour are in bracket) b/ Properties of refrigerant: HFC507 vs CO2 at -10°C

Figure

  Figure 3: CO2 coolant circuit scheme

Figure 4 :Figure

 4 Figure 4: CO2 single stage transcritical cycle

Table 1 :

 1 Saturated pressure (bar) for CO2 and traditional refrigerant for refrigeration systems

		-50°C	-40°C	-35°C	-10°C	+30°C
	CO2	6,83	10	12	26,5	72,1
	NH3	0,41	0,72	0,93	2,91	11,7
	R507	0,8	1,41	1,74	4,52	14,6

Table 2 :

 2 Thermo-physical properties of saturated CO2

Table 3 :

 3 Environmental impact of various refrigerants

		Fluid composition	Normal	Critical	ODP	GWP
		and designation	boiling	Temperature	(1 for	(100
			temperature	(°C)	R11)	years)
			(°C)
	Ammonia	NH3 (R717)	-33,35	132,4	0	1
	Carbon dioxide	CO2 (R744)		31	0	1
	Water	H20 (R718)	100	374	0
	Propane	C3H8 (R290)	-42,1	96,7	0	20
	Isobutane	C4H10 (R600a)	-11,7	134,7	0	20
	Trichlorofluoromethane	CFCl3 (R11)	23,7	198	1	4750
	Chlorodifluoromethane	CClF2CF3 (R115)	-39,2	80	0,04	1790
	R502	R22/R115	-45,3	81,5	0,3	4600
		(48,8/51,2)
	R507A	R125/R43a	-46,7	70,6	0	3800
		(50/50)
	R404A	R125/R143a/R134a	-46,2	72	0	3700
		(44/52/4)
	R407C	R32/R125/R134a	-43,6	86	0	1700
		(23/25/52)