Marie Aureille Socialist

Multispecies encounters in the ruins

AAA/CASCA Conference Vancouver 2019

Panel "Great Human Master Plans and its Non-Human Challengers", November 21 st 2019 Socialist development according to the Marabú. Multispecies encounters in the ruins of citrus plantations in Sandino, Cuba.

Marie Aureille EHESS, Institut Interdisciplinaire d'Anthropologie du Contemporain PhD Candidate, Paris, France marie.aureille@gmail.com "Welcome to Sandino where Cuba's sun goes down", this sign on the side of the road, marks the entrance of Sandino, the easternmost municipality of the island. Apart from this sign, there is no indication that the sea is so close. While following the road to get to the main town, the eye is caught by massifs of a thorny shrub with dark green leaves. They are all over pastures, forest and fruit tree plantations and even at the edge of the road. It's marabú, the challenger of this story. This plant, reputedly invasive, is omnipresent in the media, in official and daily speeches as soon as the issue of the countryside or agriculture is discussed. This shrub is a Legume which proliferates in agricultural wastelands, on poor and open land. It is a typical case of "farol proliferation"1 . In Cuba, it is the symbol of the crisis of agricultural production and the underutilisation the farmland. It is an extremely visible symbol since shrubs are often two or three meters high.

My first weeks of fieldwork in Sandino were filled with ambiguous accounts of the past. People's narratives were full of nostalgia for the orange tree plantations which, ten years ago, surrounded the city as far as the eye could see. They described to me the immense fields, clean and well maintained, the numerous roads, the trees loaded with fruit where they only had to stretch their hand to taste a juicy orange. Their eyes would lit up, they became talkative, proud, and enthusiastic.

The talked about abundance, the sweet taste of oranges and the beauty of the landscape. I was touched by these narratives and salivated to stories of orange treats. However, their Garden of Eden was a paradise of industrial monoculture, a huge socialist masterplan that had designed their daily lives, aspirations, the economy and the landscape for fifty years.

After these effusive descriptions, followed the tale of the fall: today the orange fields have disappeared invaded by the marabú who has closed the roads, obstructed the landscape. Only 1/10e of farmland is now cultivated in Sandino, plantations have also been reverted to pasture, forest plantations or just abandoned 2 . In Sandino's landscape, the marabú has outnumbered humans and eclipsed their masterplans. He is a real challenger to anyone who wants to put the land back into cultivation for his wood is very hard to chop down and he can sprout very quickly from the roots.

Marabú's proliferation is a "naturalcultural phenomenon" according to Donna Haraway 3 . Following marabú's ambiguous entanglements, I wish to question the transformations of a model «socialist» plantation. How does marabú participate in shaping the landscape, the economy, perceptions of the past and the aspirations of Sandino dwellers?

A Socialist masterplan

Revolutionary discourses in Cuba have insisted, as early as the 1950s, on breaking with the model of latifundio and large private land ownership. Capitalist sugar plantations owned by foreign capital were one of the first targets of the new socialist government. Yet the system of monoculture state farms that was established in the sixties in Cuba inherits in part the plantation's model and its "high modernist ideology", as James Scott puts it 4 . The orientation of the Cuban economy towards the export of raw materials, the socio-technical system of intensive monoculture and the social organization based on wage workers, favours and dependency relationships are not questioned by the regime.

Back in the 50s, Marabú, who was introduced in Cuba from Africa during the 19th century, was already a public problem. Following the sugar rush, he proliferates on deforested lands that were abandoned by planters due to the poor quality of the soil. Revolutionaries used marabú's example to point out the failure of capitalist agriculture to make rational use of the land. But they didn't take into account the plantation's "ecological simplifications" 5 which enabled Marabú to spread. They believe they could do better and they generalized this system. This is what happened in the municipality of Sandino, an area of marginal forest plain, sparsely populated and exploited to the west of Cuba. While foreign investors had failed to establish sustainable monocultures oriented towards export before the 1960s, socialist planning transformed it into a huge citrus plantation in the 1960s and an agro-industrial complex intended for export production for the Soviet Union.

Sandino became a symbol of 'socialist rural development'. In the middle of the fields, they created a city from scratch, designed to fill all the needs of the "new men" that is: access to modern housing, education, healthcare, culture, sports facilities. The replacement of small-scale family agriculture by 3 Donna J. HARAWAY, When Species Meet, Minneapolis, University of Minnesota Press, 2008. 4 James C. SCOTT, Seeing like a state: How certain schemes to improve the human condition have failed, Yale University Press, 1998. 5 Reinaldo FUNES MONZOTE, « La conquista de Camagüey por el azúcar, 1898-1926. El impacto ambiental de un milagro económico », Tiempos de América: revista de historia, cultura y territorio, 8, 2001, p. 3-28; Reinaldo FUNES MONZOTE, « Azúcar, deforestación y paisajes agroindustriales en Cuba, 1815-1926 », Varia Historia, 21-33, 2005, p. 105-128. wage labour also meant the inclusion of women and the reduction of racial inequalities. The plantation was both an infrastructure6 and an ideal model of a 'better life' allying high modernism, technological progress and significant improvement of living conditions.

Although this process is experienced as a positive transformation by most of the locals, it is based on the production of abstractions (through land and economic planning) and forms of alienation. As Anna Tsing puts it: alienation occurs when human and non-human beings are disconnected from the "entanglements of living" to which they belong and become self-contained mobile and interchangeable units7 . Indeed, Sandino prospered on forced displacement of peasants from the Escambray Mountainswhich were suspected of supporting a counter-revolution in the 60s -and planned displacement of recently graduated students to supply the municipality with a qualified workforce. It implied the importation and the selection of high yield varieties of citrus and the transformation of landscape with dams, roads and new settlements for workers and displaced dwellers. It also relied on free labour from students. 33 escuelas al campo (countryside boarding schools) were built, where thousands of young people from the age of 12 were spending half of the time studying and half of the time weeding, pruning and harvesting the citrus trees.

Crisis: disconnection and reconnection of more-than-human entanglements

The disintegration of this plantation system in the 1990s, following the end of the exchanges with the USSR, is the result of disconnection processes within «more-than-human entanglements» which made the plantation exist daily. The departure of students from the escuelas al campo who carried out most of the maintenance tasks, the disinvestment of workers due to the lack of means of production and the focus on survival strategies loosened the daily care relations with the trees. To face food shortage, workers started to focus on growing their food. They would use the inputs of the plantations or exchange them in the black market and so one. This left room for the emergence of new interspecies relationships.

The spread of the citrus greening disease from China -which affected all the main areas of citrus production including Florida -was linked to the huanglongbing virus carried by a wasp. Insecticides were scarce to control the wasp and workers say that when it reached Cuba, trees were already in bad shape due to the lack of care. Experiments were made to graft productive citrus varieties to autochthonous varieties of bitter orange tree. It worked but it meant to replace all the trees. In some areas of Cuba, an Israeli company invested and they succeeded to maintain the production. But no foreign company invested in Sandino and the plantations were gradually abandoned. Then marabú could spread without obstacles.

He even received the help of the digestive system of the cows kept by the inhabitants to ward off the threat of famine. The cows love marabú fruits but can't digest the seed, so they naturally spread them with a fair amount of organic matter. A lot of cows' owners let them graze freely in the wasteland since barbed wire is scarce in the centrally planned economy. Plus they don't have to worry about feeding them.

But cows also love marabú sprouts, so when grazing is intensive, marabú remains like lawn, although he is still there. The grip of former State plantation on human and non-human actors was hegemonic. It created and relied on ambiguous and precarious entanglements. The grip of marabú is unambiguous, it spreads everywhere if he is not contained.

Weediness

Today, State policies aiming at boosting agricultural production are clashing with the "weediness"8 of the inhabitants, the cows and the marabú who have become used to living on their own, using the resources of what remains of the plantation system.

Meet Angela and Osvaldo. They live in a small wooden cabin which used to be a prisoners dormitory.

Prisoners were sent to work in the state farm and Osvaldo used to work there as a guard. When the State farm stopped to run these lands, Osvaldo retired but he stayed there and turned the dormitory into his house. He enclosed a small plot around the house and started to grow fruit trees and to raise chicken and pigs. Osvaldo and Angela make money from making charcoal with marabú. The resource is abundant and they can sell charcoal for a good price on the black market.

They also sell it to a state enterprise that recently developed an exportation industry of "organic charcoal" to European countries and the US. It's the first commodity to be exported to the US in sixty years. Charcoal has always been a way to make quick money outside of the formal economy in the countryside since a lot of people still rely on it to cook.

Like them, a lot of former state workers are now appropriating state buildings to make houses and living from a mix of charcoal production, poaching and picking in the lands that remain under production or cultivating on small plots.

Since the 90s, the Cuban State is trying to channel and control this weediness and to externalize the responsibility of agricultural production to individuals and cooperatives. A lot of people received small plots of land in usufruct. You can make good money from agriculture but most of them are struggling to cultivate their new land.

Getting rid of the marabú is always a big issue alongside input shortages or commercialization problems.

Most of these "new peasants" just have axes and oxen to clean the land. Marabú pervasiveness favours persons that have resources and can rely on strong networks to rent private or state machinery. Marabú is also used as a disciplining tool by state bureaucrats that manage the distribution of land. When they receive a plot, farmers commit to clean the land from marabú within 6 months. Marabú is a state indicator of land underuse, inspectors are looking at it to assess the good use of the land and judge normatively the farmer's effort and goodwill to produce for country. There is no consideration of the arrangement people can have with marabú: it provides shade, and since it's a legume it fertilizes the soil.

Scientists also say it's better not to cut marabú if you are not able to make intensive use of the land because he can grow stronger and multiply and it's harder to get rid of him.

Farmers mostly don't agree with state official orders, although they rarely say it out loud. But their point is not about the best way to tackle land ecology. According to them, if the State wants them to clean the land, it has to give them tractors, bulldozers, chainsaws. After all, the State and its masterplans are responsible for this situation.

Sandino dwellers maintain ambiguous relations with the masterplan. Despite their criticism of the hierarchical system of dependence within state enterprises, they are mostly nostalgic of that time.

Although they have developed many other ways of living, they seem unable to imagine a "better life" without plantations. The prospect of the arrival of a mega-tourism project and new crop opportunities for export reinvigorates this desire and arouses a startling enthusiasm linked with the feeling of reconnecting with the global world.

Marabú acts as a physical as well as a perceptual barrier: you can't see around like before, he obstructs the view of the landscape as he seems to obstruct the future, the ability to look beyond this barrier. It participates in impeding Sandino's dwellers to imagine other forms of "good life" outside of high modernism and the plantation model. It seems to reflect their powerlessness regarding things that are too big and too pervasive such as the marabú or "the State". Marabú while taking care of the damaged soils and allowing weedy forms of life in the margins of the socialist system, is also kind of a conservative force which participates in the reproduction of State power and modernist aspirations.

Anna Lowenhaupt TSING, The mushroom at the end of the world: on the possibility of life in capitalist ruins, Princeton, Princeton University Press,

2 ONEI, Anuario estadístico 2017 -Sandino, Oficina Nacional de Estadísticas eInformación, 2018, p. 59-61.

Kregg HETHERINGTON et Jeremy M. CAMPBELL, « Nature, Infrastructure, and the State: Rethinking Development in Latin America », The Journal of LatinAmerican and Caribbean Anthropology, 19-2, 2014, p. 191-194.

A. L. TSING, The mushroom at the end of the world, op. cit.

Ashley CARSE, « Dirty Landscapes. How Weediness Indexes State Disinvestment and Global Disconnection », in Kregg HETHERINGTON (dir.), Infrastructure, environment, and life in the Anthropocene, Durham London, Duke University Press, 2019, p.


Baudouin Saintyves

Bhargav Rallabandi

Theo Jules

Jesse Ault

Thomas Salez

Clarissa Schönecker

Howard A Stone

L Mahadevan
email: lmahadev@g.harvard.edu

Rotation of a submerged finite cylinder moving down a soft incline

A submerged finite cylinder moving under its own weight along a soft incline lifts off and slides at a steady velocity while also spinning. Here, we experimentally quantify the steady spinning of the cylinder and show theoretically that it is due to a combination of an elastohydrodynamic torque generated by flow in the variable gap, and the viscous friction on the edges of the finitelength cylinder. The relative influence of the latter depends on the aspect ratio of the cylinder as well as the deformability of the substrate, which we express in term of a single scaled compliance parameter. By varying this compliance parameter, we show that our experimental results are consistent with a transition from an edge-effect dominated regime for short cylinders to a gapdominated elastohydrodynamic regime when the cylinder is very long.

I. INTRODUCTION

The interplay between lubricated flow and deformable surfaces is ubiquitous in nature and engineering in settings spanning a broad range of length scales, e.g. earthquakes [START_REF] Ma | Evidence for fault lubrication during the 1999 Chi-Chi, taiwan, earthquake[END_REF], avalanches [START_REF] Glenne | Sliding friction and boundary lubrication of snow[END_REF], landslides [START_REF] Campbell | Self-lubrication for long runout landslides[END_REF], lubrication of cartilaginous and artificial joints [START_REF] Maroudas | Balance between swelling pressure and collagen tension in normal and degenerate cartilage[END_REF][START_REF] Greene | Adaptive mechanically controlled lubrication mechanism found in articular joints[END_REF][START_REF] Grodzinsky | Electromechanical properties of articular cartilage during compression and stress relaxation[END_REF][START_REF] Mow | Fluid transport and mechanical properties of articular cartilage: a review[END_REF][START_REF] Mow | Mechano-electrochemical properties of articular cartilage: their inhomogeneities and anisotropies[END_REF][START_REF] Bouchet | Experimental study and modeling of boundary lubricant polyelectrolyte films[END_REF] or industrial bearings [START_REF] Hamrock | Fundamentals of Fluid Film Lubrication[END_REF]. Often, this elastohydrodynamic coupling is seen in the presence of confined flow where pressure gradients are likely to be large. Previous theoretical works have studied confined flows in the soft lubrication approximation and accounted for the roles of elasticity [START_REF] Sekimoto | A mechanism for shear thickening of polymer-bearing surfaces: elastohydrodynamic coupling[END_REF][START_REF] Skotheim | Soft lubrication[END_REF][START_REF] Skotheim | Soft lubrication: the elastohydrodynamics of nonconforming and conforming contacts[END_REF][START_REF] Snoeijer | Similarity theory of lubricated Hertzian contacts[END_REF][START_REF] Beaucourt | Optimal lift force on vesicles near a compressible substrate[END_REF][START_REF] Urzay | The elastohydrodynamic force on a sphere near a soft wall[END_REF], fluid compressibility [START_REF] Balmforth | Contact in a viscous fluid. part 2. A compressible fluid and an elastic solid[END_REF], the inertia of the fluid and the elastic medium [START_REF] Clarke | Elastohydrodynamics induced by a rapidly moving microscopic body[END_REF], and viscoelasticity of the substrate [START_REF] Pandey | Lubrication of soft viscoelastic solids[END_REF]. More recent works have focused on elastohydrodynamic effects for liquids confined at the micro and nano scales [START_REF] Villey | Effect of surface elasticity on the rheology of nanometric liquids[END_REF][START_REF] Karan | Small-scale flow with deformable boundaries[END_REF][START_REF] Zhang | Direct measurement of the elastohydrodynamic lift force at the nanoscale[END_REF], which has important consequences for surface mechanical characterization [START_REF] Leroy | Hydrodynamic interaction between a spherical particle and an elastic surface: a gentle probe for soft thin films[END_REF][START_REF] Wang | Elastic deformation during dynamic force measurements in viscous fluids[END_REF]. For symmetrical objects, the results show that elastic deformations lead to a non-symmetric pressure field and to the emergence of a friction-reducing lift force. Of particular importance in nature are cases of freely moving particles close to soft surfaces as seen in flows of cells in vessels [START_REF] Goldsmith | Red cell motions and wall interactions in tube flow[END_REF] or microfluidic devices [START_REF] Byun | Characterizing deformability and surface friction of cancer cells[END_REF][START_REF] Davies | Elastohydrodynamic lift at a soft wall[END_REF], the mobility of suspended or falling objects near elastic membranes [START_REF] Daddi-Moussa-Ider | Long-lived anomalous thermal diffusion induced by elastic cell membranes on nearby particles[END_REF][START_REF] Daddi-Moussa-Ider | Mobility of an axisymmetric particle near an elastic interface[END_REF][START_REF] Rallabandi | Membrane-induced hydroelastic migration of a particle surfing its own wave[END_REF][START_REF] Daddi-Moussa-Ider | Reciprocal theorem for the prediction of the normal force induced on a particle translating parallel to an elastic membrane[END_REF], the behavior of vesicles near walls [START_REF] Abkarian | Tank treading and unbinding of deformable vesicles in shear flow: Determination of the lift force[END_REF] or the collisions between suspended particles [START_REF] Davis | The elastohydrodynamic collision of two spheres[END_REF]. It is only very recently that a theoretical work [START_REF] Salez | Elastohydrodynamics of a sliding, spinning and sedimenting cylinder near a soft wall[END_REF] addressed freely moving objects and showed how a free falling cylinder can sediment, slide and spin along a soft incline. A particularly interesting result is that the elastohydrodynamic lift force can counteract sedimentation and lead to an emergent sliding steady state that has since been confirmed experimentally [START_REF] Saintyves | Selfsustained lift and low friction via soft lubrication[END_REF]. The experimental study also raised a new question associated with observations of rotational motion, which led to a recent theoretical study of the rotation [START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF] that remains untested.

In this article, we experimentally quantify the rotation of cylinders falling along a soft incline. We show that there is a steady rotation speed for finite-length cylinders that increases with substrate deformability, qualitatively consistent with a recently developed theory for an infinite cylinder near a soft substrate [START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF]. However, the latter fails to describe quantitatively our results. We show that a complete theory that takes into account both the elastohydrodynamic torque along the cylinder length and the viscous friction on the edges of the cylinder is in quantitative agreement with our experiments and that our control parameters can be combined into a single dimensionless compliance number. When this compliance increases, i.e., the thickness of the substrate increases or its stiffness decreases, the angular velocity follows a relationship that contains two regimes, a first one domi-nated by edge effects and the second by the elastohydrodynamic stresses due to the substrate deformation. In contrast with the theory for infinite cylinders developed previously, here the edge effects do not allow for the existence of simple power law behaviors in the range of our experimental parameters.

II. EXPERIMENTAL SYSTEM AND OBSERVATIONS

The experiments follow the same protocol as described previously [START_REF] Saintyves | Selfsustained lift and low friction via soft lubrication[END_REF], with a metal cylinder of either aluminum or brass (densities ρ = 2720 and 8510 kg/m 3 ) with radii a = 12.7 and 6.35 mm. For both cylinders, the length L = 12.7 mm such that their respective aspect ratios are a/L = 1 and 1/2. The cylinders are immersed in a silicone oil bath of density ρ oil = 970 kg/m 3 and viscosity µ = [0.35 -30] Pa.s. They freely move down a rigid glass incline (angle varied in the range α = [11 -45] • ) coated with a soft gel with shear moduli G in the range [100 -3 × 10 5 ] Pa (Fig. 1(a)). The coating thickness is varied in the range h e = [100 -2000] µm. The coatings are made of polydimethylsiloxane (PDMS) and polyacrylamide (PAA) in which we can change the concentrations of monomers and crosslinkers to tune the shear modulus. The latter is measured on an Anton Paar MCR501 rheometer with a CP50 cone-plate geometry, using an amplitude of 0.1% for PAA and 0.5% for PDMS, with an angular frequency of 10 rad/s. When the cylinder moves along the incline (undergoing both translation and rotation), it deforms the substrate (Fig. 1(b)) and its motion is recorded from the side with a camera.

Image analysis allows us to track the center of the cylinder and provides a direct measurement of the translation speed of the cylinder u c , and its rotation velocity u θ = aΩ, with a the cylinder radius and Ω the angular speed. Figures 2(a) and (b) show the rotation angle as a function of time for the aluminum cylinder for different coating moduli and thicknesses, respectively. We observe that the rotation speed Ω is constant, which is reminiscent of the constant sliding speed observed earlier in similar experiments [START_REF] Saintyves | Selfsustained lift and low friction via soft lubrication[END_REF]. We also observe that this rotation speed decreases when the coating becomes less deformable, i.e, when the shear modulus G increases, or when its thickness h decreases.

III. SCALING ARGUMENTS AND THE FINITE SIZE EFFECT

To capture the main features of the experimental observations, we first revisit the scaling arguments for an immersed infinite cylinder sliding along a soft incline [START_REF] Salez | Elastohydrodynamics of a sliding, spinning and sedimenting cylinder near a soft wall[END_REF]. Due to the confinement of the flow under the cylinder within a fluid gap of thickness h f a, the typical transverse length scale of contact scales as = √ 2ah f , so that we can invoke the lubrication approximation [START_REF] Reynolds | On the theory of lubrication and its application to Mr. Beauchamp tower's experiments, including an experimental determination of the viscosity of olive oil[END_REF]. For 0 small deformations δ of the soft layer satisfying |δ| and assuming a localized linear response of the elastic layer to the applied normal stress p (Winkler approximation), the deformation can be expressed as δ = he 2G+λ p, where λ denotes Lamé's first parameter of the substrate and where the hydrodynamic pressure in the gap scales from the Stokes equation as p ∼ µu c /h 2 f . This identifies the dimensionless compliance of the elastic layer Λ as

0 1 2 3 4 -1 -2 -3 -4 0.2 -0.2 V z (mm) x (mm) (a) (b)
Λ ≡ µu c h e a 1/2 (2G + λ)h 5/2 f ∼ δ h f , (1)
which measures the scale of the substrate deformation relative to the fluid gap thickness [START_REF]The definition of Λ used here differs from the definition in[END_REF]. In this framework, previous theoretical studies [START_REF] Sekimoto | A mechanism for shear thickening of polymer-bearing surfaces: elastohydrodynamic coupling[END_REF][START_REF] Skotheim | Soft lubrication[END_REF][START_REF] Skotheim | Soft lubrication: the elastohydrodynamics of nonconforming and conforming contacts[END_REF][START_REF] Snoeijer | Similarity theory of lubricated Hertzian contacts[END_REF][START_REF] Beaucourt | Optimal lift force on vesicles near a compressible substrate[END_REF][START_REF] Urzay | The elastohydrodynamic force on a sphere near a soft wall[END_REF] have shown that for Λ 1 and for given u c and h f the motion of an infinite cylinder is accompanied by the emergence of an elastohydrodynamic lift force F ∼ Λµu c 2 L/h 2 f , which was confirmed experimentally [START_REF] Saintyves | Selfsustained lift and low friction via soft lubrication[END_REF]. Since the cylinder also rotates with negligible inertia, the sum of torques due to elastohydrodynamics (induced by the substrate's deformation due to sliding) τ s and viscous damping of the rotational motion τ Ω vanish [START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF]: τ Ω +τ s = 0. The sliding torque scales as τ s ∼ µu c a L/(h f + δ), where h f + δ is the typical gap size between the cylinder and the deformed substrate. Invoking (1), we expand the previous expression in powers of Λ for Λ 1 and recognize that the contributions proportional to Λ 0 and Λ 1 are zero for an infinite cylinder [START_REF] Salez | Elastohydrodynamics of a sliding, spinning and sedimenting cylinder near a soft wall[END_REF][START_REF] Jeffrey | The slow motion of of a cylinder next to a plane wall[END_REF] to find that τ s ∼ µu c a δ 2 L/h 3 f . As the rotational damping torque scales as τ Ω ∼ µΩa 2 L/h f , balancing it with the sliding contribution yields the scaling relationship:

aΩ u c ∼ Λ 2 ∼ µ 2 u 2 c h 2 e a (2G + λ) 2 h 5 f (infinite cylinder).
(

) 2
For infinite cylinders, no rotation occurs when Λ = 0 (a rigid substrate), a consequence of a vanishing sliding torque in this limit [START_REF] Jeffrey | The slow motion of of a cylinder next to a plane wall[END_REF]. This feature is modified for compact bodies such as spheres, where translation and rotation are coupled even when all boundaries are rigid.

For the finite-sized cylinders in experiments, we generically expect a nonzero rotation rate Ω 0 (h f /a, a/L) even as Λ → 0 due to three-dimensional flows near the cylinder ends. These flows penetrate a width into the fluid gap from the ends of the cylinder. A rough estimate of the resulting sliding torque (for a rigid substrate) is µu c a 2 /h f ∼ µu c a 2 , which is independent of h f . In lubrication flows with gap-independent scaling estimates for torque, detailed calculations typically reveal logarithmic corrections [START_REF] Goldman | Slow viscous motion of a sphere parallel to a plane wall-I motion through a quiescent fluid[END_REF][START_REF] Claeys | Lubrication singularities of the grand resistance tensor for two arbitrary particles[END_REF][START_REF] Kim | Microhydrodynamics: principles and selected applications[END_REF]. Including a log-corrected end torque µu c a 2 log(a/h f ) in the torque balance above suggests

aΩ u c = k 2 Λ 2 + k 1 a L h f a 1/2 log a h f . (3)
The second term on the right side is identified with aΩ 0 /u c and the first with (2), with constants of proportionality k 1 and k 2 . Thus, we expect two independent sources of rotation, one due to end effects and another due to the elastohydrodynamic torque over the length of the cylinder. As we will show, there is indeed a crossover from end-dominated to softness-dominated rotation in our experiments as Λ increases.

IV. THEORY

As discussed above, two-dimensional theory predicts zero hydrodynamic torque on a non-rotating (infinite) cylinder sliding along rigid walls (Λ = 0). We show below that three-dimensional end-effects qualitatively modify this result for a cylinder. End effects are confined to a penetration depth into the lubrication gap, so both ends are hydrodynamically isolated in our experiments since L = O(a)

. We focus on the flow near one of the ends, which we place at y = 0 so that the gap lies in y > 0. It is convenient to introduce dimensionless coordinates (X, Y ) = (x, y)/ , and a dimensionless lubrication pressure P (X, Y ) = p(x, y)/(µu c /h 2 f ). Since the gap thickness abruptly diverges at the ends of the cylinder, P must vanish at Y = 0. Invoking the parabolic approximation of the gap profile H(X) = 1 + X 2 and 10 2 10 3 10 4 10 5

G(Pa) focusing on Λ → 0 (the limit of a rigid substrate), the pressure in the gap satisfies the Reynolds equation

∇ • (H 3 ∇P + 6He X ) = 0 , subject to (4a) P (X, 0) = ∂ P ∂Y (X, ∞) = P (±∞, Y ) = 0, (4b)
where ∇ = e X ∂ X + e Y ∂ Y .

We seek a solution P (X, Y ) = P 2d (X) + P (X, Y ), where P 2d (X) = 2X/(1 + X 2 ) 2 is the pressure due to an sliding infinite cylinder, which satisfies (4) except for the condition at Y = 0. As we discuss below, it is sufficient to analyze the large-X behavior of P . Defining η = Y /X (the tangent of the angle in the XY plane), we seek an asymptotic solution inverse powers of X with the form P (X 1, Y ) ∼ n X -n f n (η). From the boundary condition at Y = 0 and the asymptotic behavior P 2d (X 1) ∼ 2X -3 , it is clear that the leading term of the expansion introduced above is

P (X 1, Y ) ∼ -2X -3 Q(η)
. Substituting this expression into (4a) and retaining the most slowly decaying terms at large X yields

(1 + η 2 ) d 2 Q dη 2 + 2η dQ dη -6Q = 0, subject to (5a)
Q(0) = 1 and dQ dη η→∞ → 0, (5b)
which admits the solution

Q(η) = 3η 2 + 1 1 - 2 π arctan η - 6η π . (6)
This determines the asymptotic behavior P (X 1, Y ) ∼ -2X -3 Q(Y /X). The perturbation scheme can developed further to obtain corrections to P [the next term is of the form X -5 f 5 (η)] although the leading term suffices for our purposes.

The dimensionless horizontal velocity in the reference frame of the sliding cylinder, expressed in units of

u c is V = 1 2 Z(Z -H)∇P + Z-H H e X .
The component of the shear stress responsible for its rotation, in units of µu c /h,

is σ XZ = ∂ V X ∂Z Z=H = H 2 ∂ P ∂X + 1
H , whose integral over the area of the lubrication gap yields the hydrodynamic sliding torque on the cylinder. Noting the symmetry of σ XZ about X = 0, including both (hydrodynamically noninteracting) ends of the cylinder, and recalling that the torque generated by the two-dimensional case is identically zero, the dimensionless torque can be expressed (in units of µu c a 2 /h f ) as 4

X∞ 0 ∞ 0 H 2 ∂ P ∂X dY dX, where X ∞ = O(a/ ) [corresponding to x = O(a)
] is the outer "edge" of the lubrication gap. An estimate of the previous integral at large X shows that it diverges as log X ∞ . Formally, we make a change of variables in the integral from (X, Y ) to (X, η) and isolate the divergence to obtain the dimensional sliding torque

τ c = 4µu c a 2 h f X∞ ∞ 0 1 X 3Q + η dQ dη dη dX = 32 3π µu c a 2 log a h f + c . (7)
The constant c absorbs the ambiguity in defining X ∞ , nonsingular contributions from the lubrication flow (i.e. from terms of P decaying as X -5 or faster) and the torque due to end-effects outside the fluid gap. The latter contribution includes the torque on the flat faces of the cylinder, which is generated by stresses of O(µu c /a) acting over an area of O(a 2 ) with a moment arm of O(a).

Evaluating c requires a matched asymptotic approach that we do not pursue here; instead we will estimate it from a fit to our experiments. The result ( 7) is reminiscent of the torque on a translating sphere of radius a, for which the factor of 32/(3π) is replaced by 4π/5 and the constant c ≈ -1.895 [START_REF] Goldman | Slow viscous motion of a sphere parallel to a plane wall-I motion through a quiescent fluid[END_REF].

Since the cylinder is free to rotate and has negligible inertia, the sum of the sliding torque and the rotational torque [START_REF] Jeffrey | The slow motion of of a cylinder next to a plane wall[END_REF] vanishes, yielding the rotation rate of a translating finite cylinder near a rigid wall

τ Ω = -2 √ 2πµa 2 LΩ 0 (a/h f ) 1/2 [
aΩ 0 u c = 8 √ 2 3π 2 a L h f a 1/2 log a h f + c . ( 8
)
This result is expected to dominate for stiff substrates (Λ 1) in our experiments. The leading contribution to Ω due to the softness of the substrate (denoted Ω 2 ) was shown for an infinite cylinder to be aΩ 2 /u c = (21/128)Λ 2 [START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF]. Modifications to Ω 2 due to end effects scale as /L 1 and will be neglected here. Thus, the angular speed of a translating finite cylinder is Ω ≈ Ω 0 + Ω 2 , or

aΩ u c = 8 √ 2 3π 2 a L h f a 1/2 log a h f + c + 21 128 Λ 2 . ( 9
)
This theoretical prediction makes precise the estimate (3) and reduces to the infinite-cylinder and the rigid-wall results in the respective limits a/L → 0 and Λ → 0. For gravity-driven motion along a soft incline, the gap thickness is not an independently controlled quantity but is instead set by a balance of the cylinder's buoyant weight, the elastohydrodynamic lift force and the hydrodynamic drag on the cylinder. Introducing the Poisson ratio ν [so that λ = 2Gν/(1 -2ν)], this balance yields for a thin, compressible, elastic layer [START_REF] Salez | Elastohydrodynamics of a sliding, spinning and sedimenting cylinder near a soft wall[END_REF][START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF]

h f a = 3 8 Λ tan α 2 with (10a) Λ = 2 21/10 3 4/5 1 -2ν 1 -ν 1/5 κ (10b)
where κ = ρ * ghe cos α 2G tan 3 α 1/5

and ρ * = ρ-ρ oil . In (10b), the quantity in braces is a dimensionless constant with values 1.78-0.93 for ν in the range 0-0.49, while all parameters involved in κ are either known or directly measured. Substituting (10a) into (9) yields the angular speed for gravity-driven motion near a thin, compressible coating on an incline of angle α;

aΩ u c = √ 2 a π 2 L (Λ tan α) 2 log 8 3Λ tan α + c + 21 128 Λ 2 . ( 11
)
End effects dominate the rotation rate at small Λ, although the gap thickness is still set by elastohydrodynamic stresses. The term quadratic in Λ becomes important when Λ > ∼ (a/L) tan α. In the limit of very stiff substrates, we expect aΩ/u c ∝ (h e /G) 1/5 log (G/h e ), in contrast with the two-dimensional prediction aΩ/u c ∝ (h e /G) 2/5 .

V. COMPARISON BETWEEN EXPERIMENTS AND THEORY

We now compare the prediction of the theory with the results of the experiments. The evaluation of the compliance Λ in ( 1) and (10)(a) requires us to know the value of the Poisson ratio ν. Since we did not measure it directly, we choose ν = 0.47 based on experimental studies found in the literature for polyacrylamide gels and thin films of PDMS [START_REF] Dogru | Poisson's ratio of PDMS thin films[END_REF][START_REF] Boudou | Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces[END_REF][START_REF] Takigawa | Poisson's ratio of polyacrylamide (PAAm) gels[END_REF]. The theoretical prediction for the scaled rotational speed aΩ/u c in (9) includes a constant c that is expected to be independent of the compliance Λ. As /L 1 we assume the end flows to be decoupled from each other and we thus expect c to be independent as well of the aspect ratio of the cylinder. In order to compare the theory to the experiments we force c to be the same for experiments involving different cylinders (and thus aspect ratios). In Fig. 3, we show the behavior of the scaled angular speed aΩ/u c as a function of the coating film's shear modulus G (Fig. 3a) and thickness h e (Fig. 3b). We observe that the finitesize theory, which includes both the cylinder edge-effect term and an elastohydrodynamic term (the latter corresponding to an infinite soft-lubricated cylinder) predicts remarkably well the experimental results with a single constant c = -0.715, with increasing scaled angular velocities for decreasing stiffness G and increasing coating thickness h e (increasing κ). The value for c is consistent with the typical value obtained for a sphere near a rigid wall (c ≈ -1.895 [START_REF] Goldman | Slow viscous motion of a sphere parallel to a plane wall-I motion through a quiescent fluid[END_REF]).

Combining all these experimental results allows us to plot a master curve for aΩ/u c as a function of the modified scaled compliance κ = ρ * ghe cos α 2G tan 3 α 1/5 , as shown in Fig. 4(a). We choose to plot the data as a function of κ 0. rather than Λ as the former only accounts for the experimental parameters that we can directly measure, and not ν or h f . The values of κ are very similar to those of Λ for ν = 0.47 (Λ ≈ 1.15κ). In fact the factor between Λ and κ is rather insensitive to ν (e.g. about 0.93 for ν = 0.49), and so κ is a good physical estimate of the scaled compliance Λ for our experimental conditions. We observe that, with a unique constant c = -0.715, the experimental results are very consistent with the theoretical master curves. In Fig. 4(b), we have plotted the values measured for aΩ/u c as a function of its theoretical prediction from ( 9), for the same c constant and the same data as in Fig. 4(a), but also with experiments where all parameters were varied, including the inclination angle. This unique master curve for both cylinders confirms the good agreement between theory and experiments over more than a decade.

VI. DISCUSSION

We have also plotted separately the contributions of both terms in [START_REF] Bouchet | Experimental study and modeling of boundary lubricant polyelectrolyte films[END_REF], namely the contribution of end effects for a finite-length cylinder, and the elastohydrodynamic contribution for an infinite cylinder, as shown in Fig. 4(a). Our experimental data lie in the crossover region between these two limiting behaviors. At high values of the compliance i.e., for soft or thick substrates, the experimental data for both aspect ratios appear to collapse together and converge toward the infinite cylinder theory, consistent with a regime where edge effects (and thus cylinder length) do not affect the rotation behavior. We note that at intermediate values of the compliance, edges effects tend to increase the scaled angular velocity with respect to the infinite-cylinder prediction. Finally, at small compliances, the elastohydrodynamic torque does not affect the rotation anymore, and the latter is solely generated by end effects (near a rigid wall). The crossover location depends on the aspect ratio. We can indeed see that, for the brass cylinder with a/L = 1/2, the rotation behavior is closer to the infinite cylinder one than in the case of the aluminum cylinder, with a/L = 1, where end effects play a more significant role.

It is also interesting to note that the theory predicts an angular velocity either smaller or larger than in aΩ/u c ∼ 1. The latter regime corresponds to the rolling of a cylinder in no-slip dry contact with a rigid incline and should be reached in our system typically for κ ∼ 2. However, the range of parameters explored in our experiments could not allow us to verify the existence of "super-rolling" behaviors for higher compliances.

VII. CONCLUSION

Our experiments on the rotation of an immersed finitesize cylinder moving down and near a soft incline have shown that there is a steady-state rotation with an angular speed that increases with the compliance of the substrate. While this observation is qualitatively consistent with a recent theoretical prediction for an infinite cylinder [START_REF] Rallabandi | Rotation of an immersed cylinder sliding near a thin elastic coating[END_REF], this earlier infinite cylinder (2D) theory fails to describe our experimental observations quantitatively. A modified theoretical description for a finite-length cylinder that takes into account the additional torque created by viscous friction on both its edges does allow for a quan-titative agreement with our experiments, which are typical of many applications. In particular, we have shown that for small compliances and small cylinder lengths, the contribution of the elastohydrodynamic torque to the rotation becomes small relative to those contributions from end effects, even when the gap thickness is still set by a finite elastohydrodynamic lift force. This result gives more realistic insights on the behaviors of finite-size objects in motion or in interaction close to soft interfaces, and pave the way for new theoretical development accounting for geometric and mechanical properties that are relevant to more specific biological, geophysical and engineering processes.

FIG. 1 .

 1 FIG. 1. (a) Sketch of the experimental setup: a negativelybuoyant rigid cylinder immersed in a viscous bath slides down a tilted wall that is coated with a thin elastic layer. (b) Experimental image showing a side view of the soft substrate deformation (red) by using a laser sheet with fluorescent particles placed at the surface. The white dashed line represents the cylinder contour, centered at x = 0. The black dashed line corresponds to the interface of the undeformed substrate. The white solid line follows the center of the fluorescent particles' emission, obtained by using a Gaussian fit, showing the asymmetric deformation of the substrate-fluid interface. The experimental parameters are G = 65 kPa, he = 1.5 mm, µ = 1 Pa.s, a = 12.7 mm, ρ = 8510 kg/m 3 , and α = 11 • . Figure adapted and modified from [35].

FIG. 2 .

 2 FIG. 2. (a) Evolution of the rotation angle of the cylinder as a function of time for different shear moduli of the coating with the aluminium cylinder of radius a = 12.7 mm. These experiments were conducted at constant coating thickness he = 600 µm. (b) Evolution of the rotation angle as a function of time for different coating thicknesses with the aluminum cylinder. These experiments were made at constant shear modulus G = 31 × 10 3 Pa. For both panels, the viscosity and the incline angle are fixed at µ = 1 Pa.s and α = 11 • respectively.

eFIG. 3 .

 3 FIG. 3. (a)Dimensionless angular velocity aΩ/uc as a function of the elastic substrate's shear modulus G, for the aluminum cylinder of radius a = 12.7 mm (a/L = 1), and the brass cylinder, of radius a = 6.35 mm (a/L = 1/2). The thickness of the elastic substrate is he = 600 µm. The solid lines correspond to the theoretical prediction of (9) for a/L = 1 (red) and a/L = 1/2 (blue) with c = -0.715. (b) Dimensionless angular velocity aΩ/uc as a function of the elastic substrate's thickness he, for the aluminum cylinder of radius a = 12.7 mm (a/L = 1), and the brass cylinder of radius a = 6.35 mm (a/L = 1/2). The shear modulus of the elastic substrate is G = 31×10 3 Pa. The solid lines correspond to the theoretical prediction (9) for a/L = 1 (red) and a/L = 1/2 (blue) with c = -0.715 as a fit parameter. For both studies, the viscosity and incline angle are µ = 1 Pa.s and α = 11 • respectively. The standard deviation obtained from the angle measurements as a function of time, and averaged on all the experimental points, is 0.05.

FIG. 4 .3 α 1 / 5 .

 415 FIG. 4. (a) Experimental dimensionless angular velocity aΩ/uc as a function of the modified scaled compliance κ = ρ * ghe cos α 2G tan 3 α 1/5

ACKNOWLEDGMENTS

HAS acknowledges support from the National Science Foundation via award CMMI-1661672. CS acknowledges support from the German Research Foundation (DFG) -Project-ID 172116086-SFB 926.


Ruth Vanderschueren
email: ruth.vanderschueren@kuleuven.be

Vincent De Mesmaeker

Sandra Mounicou

Marie- Pierre Isaure

Emmanuel Doelsch

Daniela Montalvo

Jan A Delcour

Eduardo Chavez

Erik Smolders

The impact of fermentation on the distribution of cadmium in cacao beans

 Cadmium concentrations are highest in cacao testa followed by nibs and mucilage  LA-ICP-MS was used successfully to visualize the distribution of Cd in cacao  X-ray absorption spectroscopy showed Cd bound to O/N-ligands in cacao nib and testa  Extensive fermentation can reduce the Cd concentration in the final product  Nib pH controls Cd migration to outer tissues during cacao fermentation Graphical

INTRODUCTION

Although cacao-derived products are generally consumed in small quantities compared to staple foods, they can be an important source of dietary Cd because of potentially high Cd concentrations.

The European Food Safety Authority estimated that cacao-derived products account for 4.3% of the total dietary Cd exposure in the European population [START_REF] Efsa | Cadmium dietary exposure in the European population[END_REF]. Therefore, the European Commission approved threshold limits for Cd in cacao-derived products, which were enforced in January 2019 (European Commission, 2014) Similar limits were adopted by the Codex Alimentarius (Codex Alimentarius Commission, 2018). This new regulation on Cd in cacao will impact South-American cacao farmers, as Cd concentrations in South-American cacao are generally higher than those in cacao from other origins. [START_REF] Bertoldi | Multielemental fingerprinting and geographic traceability of Theobroma cacao beans and cocoa products[END_REF] reported Cd concentrations in South-American cacao beans more than tenfold larger than those in West-African cacao. These findings have prompted researchers to explore potential techniques to lower Cd in the final product, focusing mostly on the relation between cacao bean Cd and soil Cd. For example, [START_REF] Argüello | Soil properties and agronomic factors affecting cadmium concentrations in cacao beans: A nationwide survey in Ecuador[END_REF] has mapped the concentrations of Cd in cacao beans and corresponding soils in Ecuador and was able to identify soil parameters to predict cacao bean Cd. [START_REF] Ramtahal | The effectiveness of soil amendments, biochar and lime, in mitigating cadmium bioaccumulation in Theobroma cacao L[END_REF] investigated potential soil amendments and showed that both biochar and lime application may reduce Cd accumulation in cacao.

A recent survey by [START_REF] Vanderschueren | The elemental composition of chocolates is related to cacao content and origin: A multi-element fingerprinting analysis of single origin chocolates[END_REF] showed that the Cd concentration in cacaoderived products is correlated to the cacao content of the product, indicating that Cd originates from the raw material (cacao nibs) rather than other ingredients or contamination during processing. Similar results have also been reported by [START_REF] Abt | Cadmium and leas in cocoa powder and chocolate products in the US Market[END_REF], [START_REF] Villa | Cadmium and Lead in Chocolates Commercialized in Brazil[END_REF] and [START_REF] Yanus | Trace elements in cocoa solids and chocolate: An ICPMS study[END_REF]. The cacao fruit (Theobroma cacao L.) consists of an outer pod husk containing 20-50 cacao beans (seeds), surrounded by a sugary mucilage and attached to a central tissue (cacao placenta). Each cacao bean comprises of a seed coat or testa and two cotyledons known as the nib [START_REF] Beckett | Industrial Chocolate Manufacture and Use[END_REF]. Chocolate production requires extensive post-harvest processing, starting with fermentation. The pod husk and placenta are discarded, and the cacao beans with surrounding mucilage are fermented for two to ten days depending on the cultivar and local practices.

Fermentation is anaerobic during the first one to two days and the microbial population mostly consists of yeasts. Pectinolytic enzymes produced by the yeasts liquefy the mucilage and cause fermentation sweatings, which are drained through holes at the bottom of the fermentation boxes.

This results in an increase in oxygen levels, allowing growth of lactic acid and acetic acid bacteria.

The concentrations of lactic acid and acetic acid in the mucilage start to increase after two days of fermentation [START_REF] De Vuyst | The cocoa bean fermentation process: from ecosystem analysis to starter culture development[END_REF]. The fermentation process results in a temperature increase from ambient temperature to 45-50 °C; an increase in mucilage pH from 3-4 to 4.5-5 due to conversion of citric acid to ethanol; and a decrease in nib pH from 6.5-7 to 4.5-5 due to penetration of lactic and acetic acid [START_REF] De Vuyst | The cocoa bean fermentation process: from ecosystem analysis to starter culture development[END_REF][START_REF] Thompson | Cocoa and Coffee[END_REF]. After fermentation, cacao beans are dried to reach a moisture content of maximum 6-8% [START_REF] Afoakwa | Chocolate Science and Technology[END_REF]. During the second more industrial stage of the chocolate manufacturing process, the cacao beans are roasted and the testa is removed. The roasted nibs are then ground to cacao liquor and further processed to obtain consumer products (i.e. chocolate and cacao powder).

Post-harvest strategies that lower the Cd concentration in the cacao nib may offer viable options to lower the Cd concentration in the final product because the nib is the only part of the cacao fruit retained during processing. Developing such strategies requires better understanding regarding the distribution and speciation of Cd in the different cacao tissues, as well as the influence of conventional post-harvest processes on this distribution. Most previous work reports on the cacao nib, testa and pod husk with little attention to the mucilage and placenta. The Cd concentration in the testa is generally reported higher than in the nibs. For example; [START_REF] Lewis | Genetic variation in bioaccumulation and partitioning of cadmium in Theobroma cacao L[END_REF] found more than twofold higher Cd concentrations in the testa compared to the nib, and [START_REF] Ramtahal | Relationships between Cadmium in Tissues of Cacao Trees and Soils in Plantations of Trinidad and Tobago[END_REF] reported higher Cd concentrations in the testa compared to the nib, in cacao from Trinidad and Tobago. The reverse has also been observed. [START_REF] Chavez | Concentration of cadmium in cacao beans and its relationship with soil cadmium in southern Ecuador[END_REF] measured the Cd concentrations in cacao from 19 Ecuadorian farms and found generally higher Cd concentrations in the nib compared to the testa. Sample treatment often differs, cacao samples were provided by chocolate manufacturers with little information regarding sample processing [START_REF] Lee | Determination of Cadmium, Lead, Copper and Arsenic in Raw Cocoa, Semifinished and Finished Chocolate Products[END_REF], samples were either or not washed with water which can affect Cd concentrations in the outer tissues [START_REF] Gramlich | Soil cadmium uptake by cocoa in Honduras[END_REF][START_REF] Lewis | Genetic variation in bioaccumulation and partitioning of cadmium in Theobroma cacao L[END_REF][START_REF] Ramtahal | Relationships between Cadmium in Tissues of Cacao Trees and Soils in Plantations of Trinidad and Tobago[END_REF], or samples were washed with chelating agent solutions [START_REF] Chavez | Concentration of cadmium in cacao beans and its relationship with soil cadmium in southern Ecuador[END_REF]. To the best of our knowledge, the influence of fermentation on the Cd distribution in cacao has not been reported to date. [START_REF] Thyssen | Bioimaging of the elemental distribution in cocoa beans by means of LA-ICP-TQMS[END_REF] mapped the 2D distribution of Cd in sections of fermented cacao beans using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and found elevated signals for Cd, Cu, K, Mg, Na, Pb and Zn in the testa compared to the nib, however they did not investigate unfermented samples. Fermentation has been reported as a possible technique for reducing Cd in rice [START_REF] Zhai | Removal of cadmium from rice by Lactobacillus plantarum fermentation[END_REF][START_REF] Zhang | Study on the Removal of Cadmium in Rice Using Microbial Fermentation Method[END_REF]. [START_REF] Zhai | Removal of cadmium from rice by Lactobacillus plantarum fermentation[END_REF] reported Cd removal efficiencies >90% for rice fermented with lactic acid bacteria and related this to a combination of the Cd binding potential of the bacteria, and the effects of the organic acid production mobilizing Cd. This phenomenon may also occur during cacao fermentation due to the lactic and acetic acid production, but has not been studied to date.

The objectives of this study were (i) to determine the distribution and speciation of Cd between and within the different cacao fruit tissues, i.e. pod husk, placenta, mucilage, testa and nibs; and

(ii) to investigate the influence of fermentation on the distribution of Cd between these different cacao bean tissues. Better understanding of the distribution and speciation of Cd in cacao and the influence of post-harvest processing may shed light on opportunities to lower Cd concentrations in the final product.

MATERIALS AND METHODS

Cacao material

Ripe cacao fruits were collected at four fields in the provinces El Oro (batch A, CCN-51 cultivar), Guayas (batches B and C, Nacional cultivar) and Sucumbíos (batch D, Nacional cultivar) in Ecuador (Table 1). Unfermented cacao beans for XANES spectroscopy were collected at different fields in the provinces Esmeraldas (Nacional cultivar), Guayas (CCN-51 cultivar) and Sucumbíos (Nacional cultivar).

Sampling and sample preparation

A minimum of three intact cacao fruits was collected for each batch, and each fruit was considered as an independent replicate. The intact fruits were manually separated to obtain pod husk, placenta, mucilage and cacao beans. Residual mucilage was removed from the cacao beans using paper towels and all cacao tissues were oven dried for 72 hours at 65 °C. After drying, subsamples of intact cacao beans of batches C and D were collected for LA-ICP-MS imaging. The remainder of the cacao beans was manually separated in nibs and testa, and all dried fractions (pod husk, placenta, mucilage, testa and nib) were ground using a coffee grinder before chemical analyses. but were subjected to different fermentation conditions (Table 1). Fermentation experiments were conducted in Ecuador following local practices using wooden boxes with perforated floors to allow drainage of the fermentation sweatings. The boxes were covered with jute bags to retain heat. For each batch, the total mass of cacao needed to fill two fermentation boxes (about 580-1180 kg, Table 1) was thoroughly mixed and divided in duplicate fermentation boxes. Different subsamples of 1 kg cacao mass were taken and placed in mesh bags (Figure S1) to facilitate bean sampling during fermentation. All mesh bag subsamples (3-7 per box, depending on the batch) were placed in the center of the fermentation boxes at the start of fermentation and relocated in the same position after mixing. Daily sampling was performed by taking out one of the mesh bag subsamples from each fermentation box. Each mesh bag subsample was considered an independent replicate for that fermentation day and the two fermentation boxes were considered as the duplicates for the batches or fermentation experiments. Fermentations A and B were performed in cascades of three wooden boxes measuring 60×60×60 cm (width×depth×height) and the fermenting masses were mixed every two days by depositing them in the next box of the cascade (Figure S2). Batches C and Cbis were fermented in single 100×100×60 cm wooden boxes. This cacao was mixed manually after one day (Cbis) or two days (C) and remained in the initial box throughout the fermentation period. Cacao beans of batch Cbis were pre-dried over night before fermentation, mimicking a common practice in some fermentation facilities. In this pre-drying method, fresh cacao was spread out on a concrete floor and left to dry overnight. This method, also referred to as bean spreading, is a common practice to prevent excessive acidity of cacao beans during fermentation [START_REF] Biehl | Bean spreading: A method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia[END_REF][START_REF] Meyer | Post-harvest pod storage: A method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia[END_REF][START_REF] Schwan | The microbiology of cocoa fermentation and its role in chocolate quality[END_REF]. The total fermentation time for each batch was determined by local practices (Table 1). The endpoint of each fermentation was based on quality assessment by local farmers. Beans were sampled daily by removing one mesh bag subsample (1 kg cacao mass) from the center of each box. The cacao beans in the mesh bags were then manually separated in mucilage and beans and oven dried at 65 °C for 72 hours, with beans split in nibs and testa and further ground as described above.

Fermentation

Temperature and pH

The mucilage pH was measured immediately after sampling or after opening of the cacao fruits.

Cacao beans with mucilage attached were vigorously shaken for 2 min in a 1:10 solid to deionized water ratio and the pH of the suspension was measured. To determine nib and testa pH, dried and ground material was treated likewise in a 5:10 solid sample to deionized water ratio and filtered (F2040 filter paper, retention 7-9 µm, CHMLAB GROUP, Barcelona, Spain) to obtain a clear supernatant for pH measurement. The temperature in the fermentation experiments was measured daily in the center of the fermentation boxes using a digital thermometer (VWR International, Darmstadt, Germany).

Determination of the elemental composition of cacao beans

Duplicates of 100 mg dry material were digested in 3 mL concentrated Suprapur® nitric acid (HNO3, 65% w/w; Merck, Darmstadt, Germany) in an open digestion block for 8 hours at a maximum temperature of 130 °C. Digests were diluted five times with Milli-Q water (18.2 MΩ cm -1 ) and the Cd concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Agilent Technologies, Santa Clara, USA). The ICP-MS analysis was performed in helium (He) collision cell mode monitoring the 111 Cd isotope, using 103 Rh as an online internal standard. The limit of quantification (LOQ) for the ICP-MS analysis was 0.02 µg Cd L -1 which corresponded to a solid sample LOQ of 0.006 mg Cd kg -1 dry matter. Blank samples (in quadruplicate) and certified reference material NIST 2384 baking chocolate (in triplicate) were included in all digestions and treated the same way as the cacao tissue samples. Recoveries of the certified reference material ranged 98-108% and the coefficient of variation (CV) for the duplicate digestions ranged 0.1-23% (average CV 5%). The concentrations of several other elements were also determined, i.e. 27 Al, 75 As, 44 Ca, 59 Co, 52 Cr, 63 Cu, 39 K, 24 Mg, 55 Mn, 95 Mo, 60 Ni, 31 P, 208 Pb and 66 Zn.

Visualization of the elemental distribution by LA-ICP-MS in unfermented cacao beans

Unfermented cacao beans for LA-ICP-MS imaging were sampled from batches C and D. For each LA-ICP-MS sample (which was a single bean), the Cd concentration was determined from other cacao beans obtained from the same fruit using the digest method and ICP-MS analysis as described previously. Transverse cacao bean cross-sections with a thickness of 65 µm were made following the method described by [START_REF] Lombi | Speciation and distribution of arsenic and localization of nutrients in rice grains[END_REF] for rice grains. The cacao beans were sliced with a vibrating microtome (Microm HM 650 Vibratome, Thermo Scientific, Walldorf, Germany) using diamond blades (GFD Gesellschaft für Diamantprodukte, Ulm, Germany). The crosssections were made at approximately 80% of the length of the cacao bean, measured from the cacao radicle side. Once a flat surface was obtained, the surface of the cacao bean was defatted using hexane (HiPerSolv Chromanorm 97%, VWR, Leuven, Belgium) to enable sticking of the tape on the cacao surface. Then, a piece of Kapton polyimide tape was pressed on the surface and the diamond blade cut underneath, leaving a cacao bean cross-section glued on the Kapton tape.

For elemental detection, a quadrupole 7700cs ICP-MS (Agilent Technologies, Santa Clara, USA) was used, mounted with platinum (Pt) cones. The sensitivity and operational conditions (stability, background and mass calibration) were checked using a 1 µg L -1 Y, Tl, Li, Ba and Ce tuning solution. The ICP-MS was then coupled to a 213-nm laser ablation system equipped with a TV2 cell (NWR213, ESI, Freemont, CA) and coupling was optimized using a NIST 612 glass by monitoring 238 U and 232 Th for maximum sensitivity and a U to Th ratio as close as possible to the unit. Imaging of cacao cross-sections was performed by subsequent line scans with a 20 Hz laser shot repetition rate, a fluency maintained between 5-5.3 J cm -2 , a laser beam of 20 µm 2 and 50 µm 2 , a scan speed of 23 and 50 µm s -1 , and a distance between each line of 40 and 100 µm for high and low resolution images, respectively. The ablated cacao particles were transported with 800 mL min -1 He and mixed with Ar gas from ICP-MS before the ICP torch inlet. The ICP-MS was used in He mode, allowing monitoring of 111 Cd (0.2 s), 114 Cd (0.2 s), 31 P (0.1 s), 39 K (0.005 s), 44 Ca (0.15 s), 60 Ni (0.1 s) and 64 Zn (0.1 s as integration time). The acquisition time was set according to the ablation time needed for one line. One data file recording the intensity of each element versus time was acquired for each line, and a homemade program under Python was used to generate 2D images of element intensities per pixel with a colour code.

X-ray absorption near-edge structure spectroscopy

The speciation of Cd in unfermented cacao was investigated using X-ray absorption near-edge structure spectroscopy (XANES). The samples comprised of cacao beans from CCN-51 and Nacional cultivars. The cacao testa and nib were separated manually and both materials were dried, ground and pressed into pellets for XANES analysis. The XANES spectra were collected at the Xray absorption spectroscopy beamline (Australian synchrotron, ANSTO). The Cd K-edge (26711 eV) was scanned rather than the Cd LIII-edge (3538 eV) to avoid interference with the K K-edge (3608 eV), as K is expected to be abundant in cacao tissues. Sample spectra were measured in fluorescence mode with a 100-elements solid-state Ge detector, at 10 °K to prevent beam damage.

One spectrum represents the average of 2-26 scans, depending on the concentration of Cd. Each scan was measured on a different spot on the pellet to limit beam damage. Reference X-ray spectra for Cd metal foil were collected simultaneously with the sample spectra and were used for both energy calibration and spectral alignment. Several Cd reference compounds were also measured: Cd-chloride, Cd-phosphate, Cd-sulfate, Cd-oxide, Cd-acetate, Cd-lactate, Cd-citrate, Cd-histidine, Cd-phytate, Cd-malate, Cd-cysteine and Cd-glutatione (details about reference compound preparation are given in the supplementary information). Data extraction was performed using Sakura (https://sakura.readthedocs.org) while background subtraction, normalization and linear combination fitting (LCF) were done using Athena software [START_REF] Ravel | ATHENA, ARTEMIS, HEPHAESTUS: data analysis for Xray absorption spectroscopy using IFEFFIT[END_REF]. For each sample spectrum, LCF was performed by fitting regions between -25 and 70 eV using the library of Cd reference compounds. Satisfactory fits were obtained with a combination of two reference compounds. Three compound LCFs were not retained as the residual factor (R-factor) used to assess the goodness of fit was not significantly smaller compared to the R-factor of the two compound LCFs. Linear combination results were normalized to 100% to compare the relative speciation between samples.

Statistical analysis

All statistical analysis was executed using JMP ® Pro version 14.0.0 (SAS Institute 2018). The differences in Cd concentration between the different cacao tissues were tested using Tukey's Honestly Significant Difference test (P-value≤0.05) using the mean data of sampling replicates (e.g. the two fermentation boxes), as the independent replicates. The effect of fermentation time on the elemental composition of the different tissues was tested using Pearson's correlation test (P-value≤0.05).

RESULTS AND DISCUSSION

Distribution of Cd in unfermented cacao beans

Cacao beans from batch D showed the highest Cd concentrations in nib and testa, followed by batch C, and finally batches A and B (Table 2). The coefficients of variation (CV) of nib Cd between cacao fruits of the same batch ranged between 20 and 37%, indicating variation in cacao bean Cd between fruits from the same plantation. In a large survey of 159 Ecuadorian fields, [START_REF] Argüello | Soil properties and agronomic factors affecting cadmium concentrations in cacao beans: A nationwide survey in Ecuador[END_REF] observed that the average CV in bean Cd concentration among fruits of different trees within the same field was 39%. They related this variation in bean Cd to the large spatial variation in soil Cd. The Cd concentrations were overall highest in the testa, followed by the nibs, placenta and pod husk (all similar in Cd content) and finally the mucilage. No information was found in literature regarding the Cd concentration in the placenta or the mucilage. [START_REF] Gramlich | Soil cadmium uptake by cocoa in Honduras[END_REF] measured the Cd concentration in cacao from 55 farms in Honduras and did not find a significant difference between the Cd concentration in the pod husks (1.1 ± 0.2 mg kg -1 ) and that in the nibs (1.1 ± 0.1 mg kg -1 ). Conversely, [START_REF] Ramtahal | Relationships between Cadmium in Tissues of Cacao Trees and Soils in Plantations of Trinidad and Tobago[END_REF] reported higher Cd concentrations in the pod husks (0.53-4.49 mg Cd kg -1 ) compared to the nibs (0.35-3.82 mg Cd kg -1 ) for cacao from 45 farms in Trinidad and Tobago.

Testa Cd concentrations were higher than nib Cd concentrations in all batches (ratio testa Cd to nib Cd 1.8 for A, 1.7 for B, 1.5 for C and 1.7 for D). Considering the average weight fractions for nib (0.93) and testa (0.07), 91% of the total bean Cd was located in the nib and 9% in the testa in unfermented cacao beans. In accordance to the present work, [START_REF] Ramtahal | Relationships between Cadmium in Tissues of Cacao Trees and Soils in Plantations of Trinidad and Tobago[END_REF] reported significantly higher Cd concentrations in testa (0.44-4.41 mg Cd kg -1 ) compared to nibs (0.35-3.82 mg Cd kg -1 ) for unfermented cacao beans from Trinidad and Tobago. [START_REF] Lewis | Genetic variation in bioaccumulation and partitioning of cadmium in Theobroma cacao L[END_REF] reported more than two-fold higher Cd concentrations in the testa (average 1.83 mg kg -1 ) compared to those in the nibs (average 0.88 mg kg -1 ) for unfermented cacao beans from the same genetic group and grown in a common garden. Similarly, [START_REF] Lee | Determination of Cadmium, Lead, Copper and Arsenic in Raw Cocoa, Semifinished and Finished Chocolate Products[END_REF] determined the Cd concentrations in raw cacao beans from two different sources and reported higher Cd concentrations in the testa (1.32 ± 0.06 and 2.05 ± 0.01 mg Cd kg -1 ) compared to Cd concentrations in the nibs (0.76 ± 0.02 and 1.01 ± 0.01 mg Cd kg -1 ) for the two sources, respectively. Conversely, [START_REF] Chavez | Concentration of cadmium in cacao beans and its relationship with soil cadmium in southern Ecuador[END_REF] analyzed the Cd concentration in unfermented cacao nibs and testa from 19 different small-scale farms in the south of Ecuador and consistently found higher Cd concentrations in the cacao nibs compared to the testa. However, the cacao beans in that study were washed with a hypochlorite solution before peeling which may have removed some of the Cd in the outer testa.

Imaging of elemental distribution in unfermented cacao beans with LA-ICP-MS

The elemental imaging maps obtained by LA-ICP-MS for 111 Cd and 114 Cd isotopes showed close agreement, which indicates that there were no interferences affecting the Cd measurement (Figure 1, and Figures S3 andS4). There were no zones found with consistently elevated intensities for the measured isotopes ( 44 Ca, 111 Cd, 114 Cd, 39 K, 60 Ni, 31 P and 64 Zn, Figures S3 andS4) in the obtained elemental maps, indicating that the samples were sufficiently planar for reliable image interpretation and comparison. Regions with consistently lower signal intensity corresponded to inherent cracks in the cacao bean samples as visible on the sample pictures (Figure 1). The overall Cd concentrations in the nib and testa from batch D were nearly seven times higher than the Cd concentrations in the tissues of batch C. When LA-ICP-MS imaging is performed at a higher resolution, a smaller surface area of the sample is ablated and thus less sample material is transported to the ICP-MS detector. Therefore, higher resolution imaging requires samples with larger elemental concentrations (or very long measurement times). Because of this, the batch D sample could be scanned at higher resolution (20 µm² laser beam size) than the cross-section of batch C (laser beam size 50 µm²). The ICP-MS integration time was equal in both scans to limit the measurement duration. Therefore, the signal intensity (expressed as counts per second, cps) of the batch D sample was lower than that of the batch C sample even though the overall Cd concentrations in D were larger than those in C.

The testa layer was clearly distinguishable from the cacao nib and showed elevated Cd intensities for both samples (Figure 1), which is in line with the measured Cd concentrations in these tissues (Table 2). Apart from Cd, only Ca displayed clearly elevated intensities in the testa (Figures S3 andS4). The distribution of K was approximately uniform between nib and testa; and P, Ni and Zn were more abundant in the nib compared to the testa (Figures S3 andS4). Cadmium and zinc were not co-located within the cacao bean tissues even though they are considered similar in chemical properties and are often transported in plants through similar mechanisms [START_REF] Smolders | Heavy Metals in Soils[END_REF]. Dissimilar Cd and Zn distribution patterns have also been visualized in rice [START_REF] Meharg | Speciation and distribution of arsenic and localization of nutrients in rice grains[END_REF]. This may indicate a difference in transport mechanisms for Cd and Zn into the cacao seed, possibly related to a defense mechanism of the plant as Zn is an important micronutrient while Cd has no known function in plant growth. The distribution of Cd within the cacao nib was not homogeneous throughout the cross-section. Further identification of nib zones with higher Cd intensities may shed light on the way Cd is translocated into the cacao bean during plant growth. [START_REF] Thyssen | Bioimaging of the elemental distribution in cocoa beans by means of LA-ICP-TQMS[END_REF] created elemental maps of longitudinal cross-sections of fermented cacao beans using LA-ICP-MS and observed elevated signal intensities for Cd in the testa. However, they reported accumulation of P, K and Zn in the testa, which was not observed in this study. These differences compared to the present work may be explained by the influence of cacao fermentation or by possible differences in cultivars and overall different elemental concentrations between these samples.

Speciation of Cd in unfermented cacao beans

The LCF procedure identified two reference compounds to describe the speciation of Cd in all samples (Figure 2 and Figure S5). Optimal fits were obtained with a combination of Cd-histidine, where Cd is bound to amino and carboxyl groups, and Cd-citrate, where Cd is bound to alcohol and carboxyl groups. The proportions of both ligands were similar in most samples (39-58% Cdhistidine and 42-61% Cd-citrate), except for nib 3 (84% Cd-histidine and 16% Cd-citrate) and testa 1 (22% Cd-histidine and 78% Cd-citrate). These results indicate that Cd in the cacao nib and the testa is bound to O/N-ligands. In hyperaccumulator plants, Cd was found with both S-ligands and O-ligands, and the association with O-ligands was reported as a detoxification strategy in contrast to non-hyperaccumulating plants where S-ligands were predominant [START_REF] Huguet | Cd speciation and localization in the hyperaccumulator Arabidopsis halleri[END_REF][START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and Xray spectromicroscopy[END_REF][START_REF] Efsa | Scientific opinion on the risks to public health related to the presence of nickel in food and drinking water[END_REF][START_REF] Vogel-Mikuš | Localisation and quantification of elements within seeds of Cd/Zn hyperaccumulator Thlaspi praecox by micro-PIXE[END_REF]). In the present work, no evidence was found for complexation with S-ligands (thiols) in the cacao nib or testa.

Changes in pH and temperature during fermentation

The nib pH in batches A and B decreased with fermentation from 6.2 to about 4.5, the mucilage pH increased from about 3.7 to 4.5, and the testa pH increased from 4.3 to 5.0 (Figure 3). Changes in pH were less pronounced in batches C and Cbis (final nib pH 5.2 and 6.0, mucilage pH 3.8 and 4.0, and testa pH 4.8 and 4.4 for the two batches, respectively). The fermentation times for C and Cbis were shorter than in A and B (3-4 versus 5-7 days, Table 1), suggesting a lower extent of fermentation. The temperature profile was similar in all batches and increased from the start of fermentation reaching 45 °C after three to four days of fermentation. The pH and temperature values are in line with values reported in literature, which state that the temperature of the fermenting cacao bean mass increases from ambient temperature to about 45-50 °C and nib pH decreases from 6.3-7.0 to 4.0-5.5 during fermentation [START_REF] Belitz | Food Chemistry[END_REF][START_REF] De Vuyst | The cocoa bean fermentation process: from ecosystem analysis to starter culture development[END_REF][START_REF] Papalexandratou | Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria[END_REF][START_REF] Schwan | The microbiology of cocoa fermentation and its role in chocolate quality[END_REF][START_REF] Thompson | Cocoa and Coffee[END_REF].

The influence of fermentation on the distribution of Cd in cacao beans

One replicate sample of mucilage (fermentation day 3, batch B) showed an extreme Cd concentration (6.6 mg Cd kg -1 ) and was excluded from analysis. The concentration of Cd within the different cacao bean tissues before the start of fermentation (day zero, Figure 4) was in line with the values observed in intact fruits (testa > nib > mucilage, Table 2). The nib Cd concentration in batches A and B decreased with fermentation time by a factor 1.3 (Figure 4). The final nib Cd concentration in B was lower than 0.60 mg kg -1 , which is commonly considered the maximum allowed Cd concentration in cacao beans destined for export to the EU. The mucilage and testa Cd concentrations in A increased with fermentation time (factor 2.1 in testa and 7.8 in mucilage) reaching a similar plateau concentration after four days of fermentation. The same was true for the mucilage Cd in B (increased by factor 2.5) but no significant trend in testa Cd was observed. The decreasing concentrations in the nibs during fermentation are unlikely related to a change in the ability to remove the high Cd testa from the low Cd nib. Budget analysis using concentration and weight fractions of the tissues showed that the nib Cd content (expressed in mg nib Cd kg -1 total cacao bean) decreased and testa Cd content (mg testa Cd kg -1 total cacao bean) increased for A and B (Figure S6). This suggests that Cd migrates from the nib to the testa and the mucilage during fermentation, resulting in lower Cd concentrations in the final cacao based product because the outer tissues (testa and mucilage) are removed at later stages of the post-harvest process. At the end of fermentations A and B, approximately 80% of the total cacao bean Cd was located in the nibs whereas 20 % was found in the testa.

As stated previously, batches C and Cbis were fermented less extensively than batches A and B. This may explain why no change in nib Cd was observed by the end of fermentation in these batches. The mucilage Cd concentration in C increased significantly with fermentation time (factor 6.2) in a similar pattern as observed for batches A and B. But in contrast to A and B, the testa Cd concentration in C decreased with time. Fermentation of batch Cbis had no significant influence on the Cd concentrations in the testa. The mucilage Cd concentration did increase by factor 1.7 but this change was not of the same magnitude as observed in the other batches. The overall Cd concentrations in C and Cbis were higher than those in A and B. However, results from a pilot scale fermentation (5 kg) using the same high Cd cacao, showed that Cd concentrations in the nibs of this cacao did decrease with fermentation time if the cacao was fermented more extensively, i.e. four days with a decrease in nib pH from 6.1 to 4.8 (data not shown). This demonstrates that the pH, rather than the total Cd concentration, explains Cd migration. Mass balance calculations of bean Cd showed that the total bean Cd concentrations reduced with 15% by the end of fermentation in batches A, B and C (Figure S7). This may be related to the loss of mucilage through fermentation sweatings. No Cd loss was observed over the course of fermentation for batch Cbis which had been air-dried prior to fermentation. Farmers estimate that the cacao loses approximately 25% of its fresh weight during pre-drying as the mucilage liquid runs off and evaporates. As a result, sweatings during the fermentation may be much smaller in such fermentation practices and the total Cd mass may remain constant over the course of fermentation.

The nib Cd concentration was strongly correlated to nib pH throughout fermentation in batches A and B, but not in C and Cbis (Figure 5). The nib pH in C and Cbis decreased by over one unit during fermentation but remained >5, while the pH in batches A and B dropped to 4.5. This may indicate the importance of fermenting long enough to reach nib pH values <5, in order to generate Cd migration from the nib outwards. XANES speciation analysis showed that Cd was mostly bound to O/N-ligands such as histidine and citrate. The pKa value for the dissociation of the second carboxylic group in citrate is 4.77, which can explain the increase in Cd mobility when the tissue pH drops below that value. Conversely, the pKa values for histidine are 1.82 (carboxylic group), 6.00 (N in the imidazole ring) and 9.17 (amine group). [START_REF] Zhai | Removal of cadmium from rice by Lactobacillus plantarum fermentation[END_REF] stated that rice fermentation may decrease Cd concentrations and that this Cd removal capacity is related to the acid producing abilities of lactic acid bacteria present during fermentation. They reported a pH decrease from 6 (initial pH) to <4.5 by the end of fermentation, depending on the strain of lactic acid bacteria used. Testa Cd concentrations were approximately a factor two larger than nib Cd concentrations in unfermented cacao (Table 2). The migration of Cd during fermentation thus occurred against the total Cd concentration gradient. However, the cacao testa has been identified as a heavy metal adsorbent with potential applications in the treatment of industrial effluents [START_REF] Meunier | Cocoa shells for heavy metal removal from acidic solutions[END_REF]. Because of the Cd sorption capacity of the testa and the differences in pH between the nib and the testa, the concentration gradient of mobile Cd in fermented cacao beans may be the inverse of the total concentration gradient, a key speculation that requires further validation.

The influence of fermentation on other elements in cacao

Apart from Cd, several other elements were analyzed by ICP-MS (Al, As, Ca, Co, Cr, Cu, K, Mg, Mn, Mo, Ni, P, Pb and Zn) and the concentrations of most of these elements in each tissue (nib, testa, mucilage) were correlated with fermentation time (Table S1 and Figures S8 I-X).

Concentrations of Al, As, Cr and Pb were <LOQ in relevant fractions of the nib samples (Al 98%, As 80%, Cr 72% and Pb 93%), the testa samples (Pb 22%) or the mucilage samples (As 37%) and were not further discussed. Fermentation had no significant effect on Mo in any of the cacao tissues. Elemental concentrations in the nibs generally decreased while testa and mucilage concentrations increased. The nib concentrations decreased in batches A and B for Cu (factor A 1.4, B 1.2), K (A 1.6, B 1.4), Mg (A 1.4, B 1.3), Mn (A and B 1.1), Ni (A 1.7, B 1.6) and P (A 1.04, B 1.4). No significant changes were observed in the nib elemental composition for batches C and Cbis with the exception of a significant increase in nib Ni concentration in Cbis (factor 1.1).

The testa concentrations increased with fermentation in batches A and B for Cu (A 3.2, B 1.8), K (A 2.8, B 2.6), Mg (A 4.4, B 2.9), Mn (A 3.1, B 3.0), Ni (A 3.9, B 3.4) and P (A 9.8, B). Calcium

CONCLUSION

In unfermented cacao fruits, Cd concentrations are highest in the testa, followed by nibs, placenta and pod husks which all contain similar Cd concentrations, and finally the mucilage. This study is probably first to report the fate of Cd and its distribution in cacao tissues during fermentation.

Migration of Cd from the nibs to the testa was only observed if the nib pH dropped below 5. This acidic pH resulted from longer fermentation times. More extensive fermentation can thus result in lower Cd concentrations in the final product as the testa and mucilage are removed later in the post-harvest process. After fermentation, nib Cd concentrations decreased by a factor 1.3, indicating that fermentation may be useful to comply to the new Cd requirements (0.60 mg Cd kg - 1 ) in cacao beans with initial unfermented nib Cd concentrations up to 0.78 mg Cd kg -1 . Further work is required to assess the full potential of Cd migration from the nib to the testa during fermentation. Nevertheless, it is often recommended to avoid very low nib pH as this can cause an unpleasant acidic taste in the final product [START_REF] De Vuyst | The Functional Role of Lactic Acid Bacteria in Cocoa Bean Fermentation[END_REF][START_REF] Schwan | The microbiology of cocoa fermentation and its role in chocolate quality[END_REF].

A balance must thus be found between flavor quality and Cd concentration. This acidic flavor is the main reason for pre-drying practices and results confirmed that the nib pH in pre-dried cacao decreased less extensively compared to the other fermentation experiments. Our results indicate that pre-drying and short fermentation times may reduce the extent of outward Cd migration.

Fermentation

  experiments were conducted to assess the effect of fermentation on the distribution of Cd within cacao tissues, and comprised two cultivars (CCN-51 in batch A and Nacional in batches B, C and Cbis) and three common fermentation methods (cascade fermentation in batches A and B, single box fermentation in batch C and single box fermentation with pre-drying in batch Cbis). No fermentation experiments were set up for the cacao of batch D. Batch C was split in two fermentation batches, C and Cbis, which comprised of the same cacao (same variety and plantation)

  was the only element that displayed a reverse change in concentration, nib Ca increased while testa Ca decreased with fermentation time. The elemental concentrations in the mucilage generally increased, which might be caused by microbial deterioration of the outer layers of the testa. If present, this deterioration was not strong enough to cause a significant decrease in the testa weight fraction with fermentation time. The testa weight fraction remained in the range 0.05-0.10 throughout fermentation in all batches. However, regardless of the minimal change in testa weight fraction with fermentation, changes in the morphology of nib and testa may still be possible. To confirm migration of the elements rather than changes in the morphology of the tissues during the fermentation process, the elemental concentrations in each tissue (nib and testa) were multiplied by the weight fraction of that tissue. The weight fraction corrected concentrations corroborated the migration of aforementioned elements (Cu, K, Mg, Mn, Ni and P) from the nib to the testa in batches A and B. The observed migration pattern of Ni might be of importance in the future because the European Commission mentioned cacao based products among important food sources of Ni in the European population (EFSA, 2015). Based on the similar behavior of Ni and Cd observed in this work, post-harvest strategies to lower Cd concentrations in cacao during fermentation will likely also be effective for Ni.

Figure 1 Figure 2

 12 Figure 1 Relative distribution of 111 Cd in two unfermented cacao bean transversal cross sections

Figure 3

 3 Figure 3 Change in nib pH (A), mucilage pH (B), testa pH (C) and temperature in the center of the

Figure 4

 4 Figure 4 Concentrations of Cd in the different cacao bean tissues [nib ( ), testa ( ) and mucilage

Figure 5

 5 Figure 5 Nib Cd concentration is significantly (P < 0.05) correlated to nib pH during fermentation

Table 1

 1 Origin of the different cacao batches and set-up of the fermentation experiments. For batch D, no fermentation experiment was executed.

				Box dimensions	Cacao per	Box	Times mixed in	Fermentation	Immediate
		Origin	Cultivar
				(w×d×h, cm)	box (kg)	set-up	fermentation	time (days)	fermentation
	A	El Oro	CCN-51	60×60×60	290	Cascade	2	7	Yes
	B	Guayas field 1 Nacional	60×60×60	290	Cascade	1	5	Yes
	C	Guayas field 2 Nacional	100×100×60	590	Single	1	4	Yes
	Cbis Guayas field 2 Nacional	100×100×60	590	Single	1	3	Pre-dried a
	D	Sucumbíos	Nacional	/	/	/	/	/

a Pre-dried overnight at ambient temperature before fermentation by spreading the cacao beans on concrete floor.

Table 2

 2 Distribution of Cd among the different tissues of fresh cacao fruit (unfermented). Placenta and pod husk materials were only collected for batches A and B. For batch D, no mucilage material was collected. Concentrations are means (± standard deviation of sampling replicates), different letters denote significant differences within each row (Tukey test, P < 0.05).

			Cd concentration (average ± standard deviation, mg kg -1 )
	Batch	N	Nib	Testa	Mucilage	Placenta	Pod husk
	A	3	0.52 ± 0.11 bc	0.94 ± 0.22 a	0.08 ± 0.01 d 0.18 ± 0.008 cd 0.59 ± 0.21 ab
	B	3	0.39 ± 0.07 ab	0.66 ± 0.26 a	0.09 ± 0.03 b	0.31 ± 0.12 ab	0.28 ± 0.01 b
	C	6	2.4 ± 0.88 a	3.7 ± 1.6 a	0.48 ± 0.24 b	/	/
	D	6	9.6 ± 2.3 b	16 ± 4.6 a	/	/	/

ACKNOWLEDGEMENTS

This research was financed by the Research Foundation Flanders (FWO-Vlaanderen). RV is the recipient of a predoctoral fellowship from FWO-Vlaanderen. ED received funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 795614. Aquitaine Region and Feder are acknowledged for the funding of the laser ablation system from the Aquitrace project (convention number 20131206001-13010973). We acknowledge the farmers in Ecuador who supplied their expertise and the cacao material, and allowed us to use their fermentation facilities. We also thank prof. Enzo Lombi for his help and expertise regarding the XANES work and the Australian Synchrotron Facility for allocating beam time for this research.

SUPPLEMENTARY INFORMATION

The supplementary information includes a description of the preparation of XANES Cd reference compounds, pictures of the mesh sample bags (Figure S1) and the cascade set-up (Figure S2) used in the fermentation experiments, LA-ICP-MS imaging of both cacao bean cross-sections for 44 Ca, 114 Cd, 39 K, 31 P, 60 Ni and 64 Zn (Figures S3 andS4), XANES spectra for cacao tissue samples and Cd reference compounds (Figure S5), weight fraction corrected Cd concentrations in nib and testa throughout fermentation for all fermentation batches (Figure S6), Cd mass balances of the fermentation experiments (Figure S7), Pearson correlation coefficients indicating the effect of fermentation time on the elemental composition (Ca, Cd, Co, Cu, K, Mg, Mn, Mo, Ni, P and Zn) of each cacao tissue (nib, testa and mucilage) (Table S1), and the effect of fermentation on the elemental composition (Ca, Co, Cu, K, Mg, Mn, Mo, Ni, P and Zn) (Figures S8 I-X).