Miguel Lopez-Ferber
email: miguel.lopez-ferber@mines-ales.fr

Virus-based biological control facing climate change

Keywords: pests, diseases, integrated control, climatic change, adaptation

Climate change will probably induce changes in the use of virus-based biocontrol agents at various levels. Climatic changes will affect host development, indirectly modifying virus behaviour. Invasive species will constitute potential new niches for virus populations, but also may reduce their traditional hosts. Introduction of new viruses adapted to such invasive species in their new geographic areas might be an appropriate strategy. Variation in UV radiation, in rain or wind will directly affect the survival or the availability of the virus for the appropriate hosts. Variation in temperatures influence insect host development, but also plant development, and will indirectly affect virus/host relationships.

Instructions of use, timing and doses should be re-evaluated in function of new climate parameters. In addition, formulation changes might be required to optimise the persistence of virus-based products in new environmental conditions.

Introduction

Climate change is now well established and is affecting us in many different ways. Abundant literature has been devoted to the effects of climate changes on agriculture, mainly concentrating on the ability of crops to cope with increasing temperatures, or with drought, and the ways to avoid production losses, by changing varieties or crop species by others more adapted to the new climatic conditions. These adaptations often imply changes in the pest affecting the crops, and thus, in the way to control them.

Another consequence of the climate change is the colonisation of new areas by invassive species, that might acquire the pest status in the new area. Fighting these invasive species may require the adaptation of native pathogens or predators. Alternatively, a solution could be the introduction of the agents that regulate the invader in its original distribution area, where often it does not represent a problem.

In temperate regions, increasing the temperature will accelerate the development of insects, leading to a higher generation number in multivoltine insect species in temperate regions. Similarly, increasing the period of appropriate temperature will have similar consequences. This effect will of cours have an upper limit that is the maximal temperature supported by the insect.

However, the most limiting factor for larvae survival seems to be the lowest temperature that larvae can stand during diapause. The work by Causse (1976) showed that the density of first generation is clearly influenced by the lowest temperature during the winter. Climatic changes, by inducing higher extreme might also have this effect. Whatever situation, climatic change will affect the life cycle of the insect host, and thus, that of the virus pathogen. From an agronomic perspective, that probably means adapting the treatments, maybe having more treatment per season.

When adressing specifically the virus based biocontrol agents, all these concerns should be taken into account. In addition, there are other points to consider, due to the specificity of the virus. In this work we will adress more specifically the insect pathogenic viruses that are or could be used to control insect pests. Even if at the present time baculovirus are almost the only virus group used for as biocontrol agents, I'll try to depinct a more general view, that could be applied to other virus families.

Invasive insect species: adaptation of virus populations

Adaptation of a virus to a new host species has been described both in laboratory conditions and in fields. Determination of baculovirus host range determinants was a hot reseach topic in the 90's. Using the model baculoviruses AcMNPV and BmNPV, Maeda's group in Japan and Croizier's group in France demonstrate that AcMNPV could be adapted to replicate in silkworm and silkworm cells by modifying the virus helicase gene [START_REF] Kondo | Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus[END_REF]Crozier et al., 1994;[START_REF] Argaud | Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae[END_REF]. Other works revealed that there was often possible to adapt(baculo)viruses to new hosts, but that the genes involved are not allways the same, preventing a systematic approach [START_REF] Thiem | Baculovirus host range[END_REF][START_REF] Williams | Nucleopolyhedrovirus Coocclusion Technology: A New Concept in the Development of Biological Insecticides[END_REF].

Field observations confirm the ability of a local virus population to adapt to a new host. The guatemale tuber moth, Tecia solanivora progressively invaded south America and recently was found in the Canary Islands [START_REF] Puillandre | Resistance of the alfalfa caterpillar, Colias eurytheme, at high temperatures to a cytoplasmic-polyhedrosis virus and thermal inactivation point of the virus[END_REF]. This pest displaced the potato tuber moth, Phthorimaea operculella, but the two species coexist in many locations. The Phthorimaea operculella granulovirus is found associated to all P. operculella populations. The isolates of this virus collected were or when no T. solanivora is present do not replicate in this species. However, soon after invasion, granuloviruses able to replicate in T. solanivora larvae were isolated in Venezuela (Niño de Gualdron and Notz, 2000). The process of invassion of T. solanivora in Colombia has been analysed in the 2000's [START_REF] Zeddam | Responses of different geographic populations of two potato tuber moth species to genetic variants of Phthorimaea operculella granulovirus[END_REF]. Sampling for viruses was carried out and revealed that the genetic composition of virus isolates changed to adapt to the presence of this new potential host (and probably to the reduction of the original host). This adaptation process will probably take place for every invassion, although the succes cannot be granted.

A complementary or alternative way is the introduction of a virus already adapted to this species. This approach has often been taken both for invassive species or for already established pests. As exemple, the mexican isolate of Cydia pomonella granulovirus (CpGV-M), the first discovered, was used for developping virus based insecticides to control codling moth in european orchards.

Sensibility to environmental conditions

The primary infection is a key point that may be heavily affected by climatic change. Tranmission will not be affected for viruses that are propagated by close contact of individuals. This can be the situation for social insects living in colonies (bees, termites, …), gregarious insects, or those in high density situation. A well know exemple is Oryctes virus used for the control of the rynoceros beetle, Oryctes rhynoceros [START_REF] Huger | The Oryctes virus: Its detection, identifcation, and implementation in biological control of the coconut palm rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae)[END_REF]. Two main ways of contamination have been proposed. Virus replicate actively in the midgut epithelium, and particles are released in high numbers on the feces, contaminating feeding burrows of palm crows. The Oryctes virus is a non occluded virus, whos particle and replication remembers that of baculoviruses. It was classified as a "non occluded baculovirus" before an independent family was proposed in the 2000's. This virus is highly susceptible to environnmental conditions, heat and UV radiation, but the hughe amounts released and the shield offered by palm leaf certainly attenuate such limitations. In addition, the second way of transmission of the infection, that is supposed to be the most important, is by direct contact during mating or coocupation of the same palm. In such circunstances, the delai on the external environment is very reduced. The same constraints will apply to other viruses with similar transmission strategies.

Viruses that need to survive into the environment between infection cycles will be affected by changes in climate. If the virus is supposed to survive in the environment between infections, variation in the climatic conditions may affect its persistance. Increasing temperature is probably not a direct limiting factor for survival of these viruses. Baculovirus OBs can stand temperatures up to 50 °C without significantly loosing infectivity [START_REF] Mcleod | Inactivation of Baculovirus heliothis by ultraviolet irradiation, dew, and temperature[END_REF]. The same applies for other occluded insect viruses, like cytoplasmic polyhedrosis viruses (Reoviridae) [START_REF] Puillandre | Resistance of the alfalfa caterpillar, Colias eurytheme, at high temperatures to a cytoplasmic-polyhedrosis virus and thermal inactivation point of the virus[END_REF]. Baculovirus can survive as OBs to low temperatures without significantly loosing pathogenicity. Accordingly, stronger or mildler winter temperatures should not greatly influence the virus survival. However, when transmission between years is mainly dependent on a diapausing stage, like in vertical transmission, or in chronically infecting viruses, the survival of the insect may be affected, and indirectly, that of the virus. In addition, increasing the average temperatures will induce faster insect development, and thus an increasing number of generations per year. From an agronomic perspective, this means expanding the treatment period.

Increasing the sun light irradiance implies a higher UV dosis, that affects virus survival. Increasing rainfall intensity may result on virus wash off from the aerial parts of the plant. Similarly, strong winds may also affect the disponibility of the virus to the insect pest. Baculovirus inactivation by UV light has been studied both in laboratory and in field conditions. It is one of the key point that limit the development of this crop protection biocontrol agents in the actual climatic conditions. To increase the survival time, commercial products include UV protection, like flurescent brigtners or other UV protective substances (Çakmak et al., 2021). If the changes on UV radiation are significant, it may be necessary to reevaluate the level of UV protection required, by increasing the concentration of UV protector, or by changing the substance used. Brassel and Bentz (1979) repported a 5.6 increase in the resistance to UV inactivation on CPGV after selection. In their work they do not specify which isolate they used, but it was probably the CpGV-M, that is know to contain extremely low genetic diversity, which may explain the relatively low response to the selection process. Similar approaches were taken for other baculoviruses, confirming a genetic variability for the resistance to UV light, that could be exploited when developping biocontrol agents, and that the virus could mobilize for its adaptation to evolving climatic conditions.

In Chrysodeixis chalcites nucleopolyhedrovirus, two genes homologous to photolyases were found by when sequencing the entire virus genome, phr1 and phr2. The DNA repair activity was demostrated for phr2, but not for phr1 [START_REF] Van Oers | Active DNA photolyase encoded by a baculovirus from the insect Chrysodeixis chalcites[END_REF]. However, homology searches have detected homologous genes in group II alphabaculoviruses infecting plusiine insects [START_REF] Xu | Conservation of DNA photolyase genes in group II nucleopolyhedroviruses infecting plusiine insects[END_REF], but not in other baculoviruses.

Overexpression of the Bm65 virus gene results in a higher UV tolerance. Bm65, homologous to Ac79, is predicted to be an endonuclease, that could be involved in UV-induced DNA repair [START_REF] Tang | Overexpression of Bm65 correlates with reduced susceptibility to inactivation by UV light[END_REF]. [START_REF] Tang | BmNPV infection correlates with the enhancement of the resistance of Bombyx mori cells to UV radiation[END_REF] observed that BmN cells infected with BmNPV show higher resistance to high UV radiation than non infected cells, suggesting than the baculovirus activate UV repearing pathways, either by producing UV repair enzymes or by complementing cellular pathways.

Other virus families also encode photolyases. The activity of the Amsacta moorei entomopoxvirus gene 25 (AMV025) is able to rescue photolyase deficient E. coli cells, confirming its ativity as a functional photolyase [START_REF] Nalcacioglu | Amsacta moorei entomopoxvirus encodes a functional DNA photolyase (AMV025)[END_REF].

A recent paper by a sudafrican team [START_REF] Mwanza | Selection for and Analysis of UV-Resistant Cryptophlebia Leucotreta Granulovirus-SA as a Biopesticide for Thaumatotibia leucotreta[END_REF], describes selection for UV resistance on a betabaculovirus local isolate, the Cryptophlebia Leucotreta Granulovirus-SA. These authors obtain a more than 1000 fold improved pathogenicity after only five passages on insects after UV exposure. The authors did not conclude on the genes involved in UV tolerance or resistance, but address the point that UV resistance probably has a fitness cost, as it is not generally distributed in the virus population.

As increasing the resistance to UV radiation in baculovirus is a key point in the development of biocontrol agents, research was done by inserting DNA repair enzymes from other viruses (Petrick et al., 2003). These authors did not obtain a significant decrease in the inactivation rate for OBs.

Increasing rainfall or wind intensity will wash off the virus from the leaves, but may also mobilize the virus present in the soil. Most probably, the adhesion of the virus particle to a surface is a pasive mechanism. Crude virus preparations of Heliothis zea nucleopolyhedrovirus strongly adhere to surfaces [START_REF] Ignoffo | Sunlight Stability and Rain-Fastness of Formulations. of Baculovirus heliothis[END_REF], probably due to the soluble components of the dead larva tissues, but this does not seem to be a general feature. Virus-based insecticides can be supplemented with adjuvants that increase the adhesion of particles to the surfaces. Soil is a reservoir for virus, protecting it from UV. If the target passes trhough soil, it can be contaminated. Alternatively, heavy rain or wind can transport virus from the soil to the aerial parts of plants [START_REF] Jacques | Stability of entomopathogenic viruses[END_REF]. Andrews and Sikorovski (1973), cite in [START_REF] Jacques | Stability of entomopathogenic viruses[END_REF], noted that deposits of HNPV on cotton leaves were inactivated during the night when dew caused the deposits to be wet. Plants secrete substances that degrade the virus, maybe combined with humidity. Secretion is a response to stress, that can be climatic (higher temperatures, lower water availability), and accordingly, it might be neccesary to adjust formulations to varying exterior temperatures. A more subtle interaction is the response of plants by making the upper parts of the leaves thicker, and sclerotised, and thus, not eateble to larvae. If the virus is located on this upper part, the probability of ingestion will be lower.

Tritrophic interactions

Conclusion

Climatic change will affect the use of viruses as biocontrol agents for the regulation of insect pests. It will offer opportunities to develop new approaches, but direct transposition from what we know to work in one place could not be done to new locations. A careful analysis of the local climate will be necessary. If introduction of a imported virus is the preferred solution, the similarity of climatic conditions between the virus original region and the intended location should be taken into account.


Aurélie Hinsberger

Benoit Graillot

Christine Blachère-Lopez

Aurelie Hinsberger

Miguel Lopez-Ferber
email: miguel.lopez-ferber@mines-ales.fr

Stability over passages on genotype frequencies and pathogenicity of CpGV mixed genotype populations

Keywords: Cydia pomonella granulovirus, synergy, codling moth control

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

Introduction

Codling moth, Cydia pomonella (L.), is a major pest of apples and pears distributed worldwide in temperate climates. Massive use of chemicals against this pest progressively led to the emergence of resistance to most of them. In parallel, other methods were developed to control this pest.Among them, the Cydia pomonella granulovirus (Baculoviridae, Betabaculovirus). In Europe, CpGV use developped since the first registration in 1988, reaching more than 100 000 ha. All commercial formulations before 2010 were derived from the first discovered Cydia pomonella granulovirus (CpGV) isolate, CpGV-M [START_REF] Lacey | Codling moth granulovirus: A comprehensive review[END_REF].

Resistance to CpGV-M products has been reported in Germany and France from 2005. All resistant populations examined appear to harbor similarly inherited resistances [START_REF] Schmitt | The occurrence and distribution of resistance of codling moth to Cydia pomonella granulovirus in Europe[END_REF]. This resistance -called type I resistance-is specific against CpGV-M. It is controlled by a single locus located on the Z chromosome of the moth [START_REF] Asser-Kaiser | Sex linkage of CpGV resistance in a heterogeneous field strain of the codling moth Cydia pomonella (L.)[END_REF]. The highly resistant (type I) RGV insect colony was obtained from a natural population of south France by selection to near allele fixation and adaptation to laboratory conditions. It presents a caracteristic resistant ratio of 10,000 when challenged with CpGV-M, compared to susceptible colonies [START_REF] Berling | Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts[END_REF].

Various CpGV isolates are able to control type I resistant insects. Among them, CpGV-R5 [START_REF] Graillot | Progressive adaptation of a CpGV isolate to codling moth populations resistant to CpGV-M[END_REF]. [START_REF] Gebhardt | Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38[END_REF] analyzed representative virus isolates differing in their ability to overcome resistance. They demonstrated the link between an allele of the p38 virus gene and the ability to replicate in resistant insects, a 24bp insertion in the pe38-M allele compared to the resistance breaking allele, pe38-R.

Fitness analysis were carried out both at the host and at the virus levels. No detectable fitness cost was found in laboratory conditions for insects carrying type I resistance [START_REF] Undorf-Spahn | High stability and no fitness costs of the resistance of codling moth to Cydia pomonella granulovirus (CpGV-M)[END_REF]. From the virus perspective, no statistically significant differences were found on the OB production levels between CpGV-M and CpGV-R5 [START_REF] Graillot | Progressive adaptation of a CpGV isolate to codling moth populations resistant to CpGV-M[END_REF]. In addition, an analysis of the stability of CpGV-R5, during successive passages on susceptible larvae could complement such studies.

To that aim, we replicated CpGV-R5 for 20 cycles on susceptible insects at high multiplicity of inoculation, mimiking epizootic conditions. Then, we analyzed both their genetic composition at the level of the p38 virus gene, and their ability to replicate and control the resistant laboratory colony RGV.

Materials and methods

Insects and viruses

Two laboratory colonies of Cydia pomonella were used in this study, CpNPP and RGV. The colony CpNPP is susceptible to both CpGV-M and CpGV-R5, while the RGV colony harbours the major genetic determinant for resistance present in resistant European populations. Both insect colonies are reared on artificial diet as previously described [START_REF] Berling | Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts[END_REF] Virus isolates used in this work, CpGV-M and CpGV-R5 have been described previously. CpGV-R5 is able to overcome the resistance of the RGV colony and results in a productive lethal infection in resistant insects. No polymorphisms were detected by restriction enzyme length polymorphism analysis in this virus isolate [START_REF] Graillot | Progressive adaptation of a CpGV isolate to codling moth populations resistant to CpGV-M[END_REF]). An experimental genotype mixture was made by mixing 1 % of CpGV-M and 99 % of CpGV-R5 OBs (1M:99R), and used to start a virus lineage by amplifiying on susceptible CpNPP larvae.

Virus amplification and successive passages on the susceptible host colony

Virus amplifications were performed as previously described [START_REF] Graillot | Biological Characteristics of Experimental Genotype Mixtures of Cydia pomonella Granulovirus (CpGV): Ability to Control Susceptible and Resistant Pest Populations[END_REF]. Larvae inoculated with 1M:99R showing clear signs of infection at 4 dpi were collected and placed in groups of three into microfuge tubes and incubated one more day at 25 °C, then frozen (-20 °C). For OB extraction, larvae were thawed, pooled and homogenized in distilled water, then filtrated through nylon cloth to eliminate debris. The suspension was then centrifuged 5 min at 8,000 × g, and the resulting pellet was re-suspended in distilled water. This suspension constituted the first amplification (R5-S1) of the virus. The purified OB concentrations were evaluated after each passage, adjusted to 800 OB/µl and used as inoculum for the following passage. Successive amplifications have been performed up to the 20th generation, called R5-S20.

Bioassays

Quantitative bioassays were performed using established protocols using neonate larvae of both insect colonies CpNPP and RGV [START_REF] Berling | Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts[END_REF]. Virus induced mortality was recorded after seven days. Mortality data were subjected to probit analysis using the POLO+ software [START_REF]Polo-pc software. A user's manual for Probit or Logit analysis[END_REF]. Each bioassay was repeated three times. The results for each treatment were pooled after control of their homogeneity.

DNA extraction, PCR amplification and HRM analysis

Lysis of OBs was performed in order to liberate viral DNA from 10 of the OB suspensions obtained previously (R5, R5-S1, R5-S3, R5-S5, R5-S8, R5-S10, R5-S13, R5-S15, R5-S18, R5-S20) and the 99M:R1 experimental mixture. qPCR was carried out using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories), followed by HRM as previously described [START_REF] Hinsberger | A Combination of Real-Time PCR and High-Resolution Melting Analysis to Detect and Identify CpGV Genotypes Involved in Type I Resistance[END_REF]. Biorad CFX maestro™ and Precision Melt Analysis™ were used for data analysis.

Results and discussion

CpGV genotypes able to control type I resistant insects were not present in CpGV-M isolate. This could be the reflection of a cost for the virus to maintain the ability to bypass resistance or just be the consequence of a founder effect as CpGV-M comes from a limited number of larvae. Once resistance became a problem, resistance-breaking genotypes were identified both by sampling for new isolates in various locations, or by selection from other stored isolates. Among them, the CpGV-R5 isolate.

Assays of mixed infections in the same host suggested a synergistic effect at least in some conditions [START_REF] Graillot | Biological Characteristics of Experimental Genotype Mixtures of Cydia pomonella Granulovirus (CpGV): Ability to Control Susceptible and Resistant Pest Populations[END_REF]. The frequency of each genotype over generations depends both on the host genotype, and on the epizootic conditions [START_REF] Graillot | Importance of the Host Phenotype on the Preservation of the Genetic Diversity in Codling Moth Granulovirus[END_REF].

To identify a putative selection of virus genotypes, twenty successive passages have been carried out on susceptible insects CpNPP (R5-S1 to R5-S20), starting with a virus population obtained by mixing 1 % CpGV-M and 99 % CpGV-R5. The evolution of this virus lineage was followed by analysing the genetic composition at the level of the pe38 gene by PCR followed by HRM, using a couple of primers allowing differentiating between pe38-M and pe38-R alleles [START_REF] Hinsberger | A Combination of Real-Time PCR and High-Resolution Melting Analysis to Detect and Identify CpGV Genotypes Involved in Type I Resistance[END_REF].

The HRM analysis identifies 7 different clusters (Figure 1 Amplicons of different passages were checked by electrophoresis and sequencing to verify if recombination occurs. The sizes and sequences correspond to those expected for CpGV-M and CpGV-R5.

The pathogenicity of this lineage has been determined at passages 0 (corresponding to 1 % M:99 % R5), R5-S5, R5-S10, R5-S15 and R5-S20 on RGV and CpNPP. A limited variation in the ability to control the resistant insect colony is observed, far lower than expected if the resistance trait was being lost (Figure 2). These results suggest that in conditions without selective pressure for maintaining the ability to replicate in resistant insects, the CpGV-M genotype presents a higher fitness. However, this advantage is low, as CpGV-R5 genotype is still present after 20 generations, and the ability to control resistant populations did not significantly drop during the whole experiment. This maintain may be due to the observed synergy when both viruses infect the same larvae, that is, in conditions of high prevalence. However, in conditions of low prevalence, the probability of such mixed infections drops, resulting in a progressive increase of the small differences in the final proportion of each genotype at each generation. The fact that in the Mexican isolate no genotypes able to control type I resistant insects were found is likely the result of a founder effect or of replication at low multiplicity conditions.

  a). Only sample R5-S1 clusters with the parental population. From passage 3, the virus lineage tends to CpGV-M, the melting profile adopting a two peak profile (R5-S10) or a single intermediate profile R5-S15 to R5-S20 (Figure 1 b).

  Figure 1. Melting curves analysis of amplicons from an experimental CpGV population composed of two genotypes, CpGV-M and CpGV-R5, amplified on susceptible insect for the number of passages indicated. (a) Normalized RFU (b) First derivative of fluorescence vs temperature profile (c) Plot of deviations on RFU relative to the (CpGV-R5) (in red), used as reference.

Figure 2 .

 2 Figure 2. Changes of efficacy of the virus lineage on neonate resistant larvae RGV after passages on susceptible larvae CpNPP. The efficacy is measured as LC50 or LC90.

Acknowledgements

This study received funding from the European Commission Seventh Framework Programme (FP7/2007-2013), under grant agreement FP7-265865. Benoit Graillot received an Agence Nationale de la Recherche Technologique doctoral scholarship. Aurelie Hinsberger received a Region Occitanie doctoral scholarship. The authors want to thank A. Bezier for critical comments on the manuscript.


Ibrahim Bel-Hadj
email: ibrahim.bel-hadj@univ-lille.fr

Zahia Bougrioua
email: zahia.bougrioua@univ-lille.fr

Katir Ziouche
email: katir.ziouche@univ-lille.fr

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

Keywords: metallic thermoelectrics, planar thermopile, micro-thermoelectric generators, CMOS-compatible technology, thermal modeling, membranes

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

Introduction

The recent progress of the internet of things (IoT) is leading to an increasing need for energy because of the large amount of connected sensors, which is estimated to reach more than 25 billion by 2030 [START_REF]IoT connected devices worldwide 2019-2030[END_REF]. Most of these sensors have traditionally been powered by batteries that require regular maintenance or replacement, raising their environmental impacts [START_REF]Lithium-ion batteries need to be greener and more ethical[END_REF][3][4]. The energy harvesting technology development, for the powering of autonomous devices, is gradually becoming an absolute necessity. Micro-thermoelectric generators (µTEGs) are micro-devices that convert waste heat into useful electrical power directly without an intermediate process. Generally speaking, the active part of a typical thermoelectric (TE) device consists of an assembly of n-type and p-type semiconductor materials, connected electrically in series and thermally in parallel [START_REF] Rowe | Thermoelectrics handbook: macro to nano[END_REF][START_REF] Snyder | [END_REF]. Their working principle is based on the Seebeck effect [7], which allows the direct conversion of temperature gradient into electrical voltage.

The conversion efficiency of µTEGs can be related to the so-called dimensionless figure of merit = ⁄ , where is the absolute temperature, is the Seebeck coefficient, is the electrical resistivity and is the thermal conductivity [START_REF] Rowe | Thermoelectrics handbook: macro to nano[END_REF]. This figure of merit concept makes it possible to assess the ability of such TE devices to convert heat into electrical power. Thus, in thermoelectricity, great efforts have been paid on the optimization of the electronic and thermal transport properties of TE materials [8][9][10][11][12][13]. For a large variety of TE materials, values are rarely exceeding unity over a very wide temperature range [14]. This makes the efficiency of µTEGs lower than some other energy harvesting techniques such as photovoltaics [15]. Nevertheless, the abundance of heat in our environment makes them a good candidate for thermal energy harvesting. Furthermore, for many applications where the heat source is available, the conversion efficiency is not necessarily the most important criterion for evaluating the performance of the device: the output power of the µTEG is more relevant [16].

The emergence of new manufacturing technologies offers to µTEGs the possibility of having different shapes and sizes, built up with different materials and addressing new applications [17][18][19]. In general, µTEGs can be classified into two main categories depending on the thermocouple (TC) orientation relative to the surface: there are vertical and planar architectures. In the vertical structure, the TCs are arranged perpendicular to the flat surface of the substrate in contact with the heat source (or heat sink) [START_REF] Kishi | 18th Int. Conf. on Thermoelectrics. Proceedings ICT'99[END_REF][START_REF] Li | [END_REF][START_REF] Bottner | 26th Int. Conf. on Thermoelectrics[END_REF][START_REF] Venkatasubramanian | 6th Int. Workshop on Micro and Nanotechnology for Power Generation and Energy Conversion Applications[END_REF][START_REF] Snyder | [END_REF][25][26][27][28]. This configuration has large heat-absorbing surfaces that allows providing more electrical output power. However, the large surface area of these vertical TE modules gives them a very low thermal resistance compared to the one of planar structures. Also, these configurations are complex to manufacture and require significant technological resources which greatly limit their development [29]. On the other hand, the planar configuration of TE miniature modules generally uses thin-film thermoelectrics deposited by microfabrication processes, allowing to exploit temperature gradients that are generated within the very thin suspended structures [30][31][32][33][34][35][36][37][38][39]. The planar configuration is beneficial to achieve high thermal resistance that can be adjusted with the TC length. This allows adapting the µTEG to the thermal environment, in order to maximize the collected heat. Moreover, the use of TE thin films results in a small amount uptake of material and thus reduces the costs, which is important in the case of expensive and/or rare materials. Yet, their main disadvantage is related to the thermal and electrical contact resistances at the interfaces, which require a particular control in the fabrication of this type of structures [40].

In this article, we report on the design, the modeling, the fabrication and the characterization of a new family of planar µTEGs for thermal energy harvesting. The implemented technology is a CMOS-compatible microfabrication process, using low cost, abundant and environmentally friendly materials. It is based on an original topology of the thermopile with a high integration density and this thanks to a particular arrangement of the TE materials: each individual TC has a stacked configuration of its two thermoelements. To our knowledge this kind of stack have been used only by S. M. Yang et al. [39,41]. In order to reduce the electrical resistance of the µTEGs, we fabricate a thermopile that combines series and parallel electrical integration of the TCs. Also this resistance is further minimized by the use of p and n type metallic alloys, respectively Ni90Cr10 and Cu55Ni45, as both thermoelements, instead of the polySilicium/metal couple that we employed in our anterior work [33,42,[START_REF] Bougrioua | 17th Europ. Conf. on Thermoelectrics[END_REF].

Thermopile and module topologies

Folded thermopile

The thermopile structure developed in this work is schematized in Fig. 1. It is made on a Silicon substrate passivated with a SiO 2/SixNy bilayer. The surface of the module is chosen to be 1/3 cm² ( = 6×5.7 mm²). The topology is based on a stack of three levels. The first level consists of metallic strips of Ni90Cr10, so-called Chromel, representing the first TE material (Fig. 1(a)). The second level is a thin SiO 2 layer, which will be embedded between both TE layers to insulate them in the verticality, except at two corners of each TC (Fig. 1(b)) where the electrical connection takes place at the TE junctions. The third level contains the second TE material, which is Constantan Cu55Ni45, periodically deposited on the Ni90Cr10 strips, as illustrated by the final shape of the thermopile presented in Fig. 1(d). In between both TEs junctions, a thin Au layer is used to ensure a good electrical contact (Fig. 1(c) and (f)). The vertical assembly of the two thermoelements of each TC gives the "periodical folded" character of the thermopile. Also, each TC has a smaller footprint area, equivalent to the area of a single thermoelement. This increases the integration density of TCs compared to our former classical planar µTEG configuration [33,42,[START_REF] Bougrioua | 17th Europ. Conf. on Thermoelectrics[END_REF]. Furthermore, the selected TE materials, Ni90Cr10 and Cu55Ni45, present a good compromise between their elaboration costs, their Seebeck efficiency and their advantage of being non-toxic and abundant.

Benefit of series/parallel association in the thermopile

We also designed a specific pattern for the thermopile where several TCs electrically associated in series, form multiple strips (or branches) connected in parallel (see Fig. 1(d) and a zoom in Fig. 1(e)). This series/parallel association of the TCs is implemented in order to reduce the total electrical resistance of the thermopile compared to the one of a purely series association, as explained in Fig. 2.

Indeed, if each TC is modelled by its equivalent electrical schema (with its electrical resistance), then a thermopile with a purely series association of N thermocouples (on the left of Fig. 2), and a thermopile with a series/parallel association of the same number of TCs (on the right of Fig. 2, new thermopile topology) will have i/ a different internal electrical resistance and ii/ a different output voltage delivered by the whole thermopile . This latter is a function of the voltage at each TC and of the number of TCs in one strip, : = and = ∆ , where ∆ is the temperature gradient at each TC. However, the expression of the maximum output power, ! , corresponding to the electrical power thermogenerated to a matched load resistance:

! = 4 ⁄
, will be identical. Both quantities and ! can be expressed as a function of the individual TC parameters as given by the relations in Fig. 2 and its caption. So, in the case of the novel series/parallel TCs association, can be tuned by fixing the number of branches connected in parallel, # (it is # ² lower than in a series thermopile) while kipping the same value for ! . Moreover, the parallel association ensures electrical continuity (in at least one of the strips) in the case of an accidental cut in the connection between the thermocouples within one or more branches, which is a major problem in the purely series thermopile configuration.

Series association of TCs

Series/parallel association of TCs

$%& %$ = %& %$/# & ((%( = $ # = $)& %$ # ! $%& %$ = * + 4 = 4 ! %& %$/# & ((%( = * $ + 4 , $ # - = # $ 4 = 4 = ! $%& %$

Fig. 2 : Equivalent electrical schematic of a thermopile, comparing the internal electrical resistance (

) and the maximum output power ( ! ) between the case of a purely series association and the case of a combined series/parallel association of TCs;

: total number of TCs; $ : number of TCs connected in series; # : number of strips connected in parallel;

: electrical resistance of a single TC. : Seebeck voltage of a single TC.

The 2.5D module

The Si template that holds the folded thermopile is periodically etched to delimitate membranes that will enable the build-up of a temperature gradient at each TC from any incident multidirectional heat from the environment. Owing to our series/parallel thermopile design, this implies that the number of membranes (noted M) will be equal to # /2. The incident heat will be periodically concentrated on the top of the hot junctions of the thermopile via the pillars of a "Heat Concentrator" tailored within another Si wafer, as for our first family of µTEG modules which included polysilicon/gold TCs (see for instance fig. 2 in [42], the case of a 3-membranes based module).

The µTEG final topology, integrating the described thermopile, is shown with various views in Fig. 3 in the case of 2 membranes ( # =4). The overall module has a so-called 2.5D architecture. Indeed the bottom part of the module consists of a set of multilayered membranes periodically distributed on an etched Si substrate, which will be a natural "evacuator" of the heat. As shown in Fig. 3(a), these membranes contain a SiO2/SixNy bilayer (with compensated stress using thicknesses of 800 nm and 600 nm respectively [START_REF] Haffar | These de doctorat[END_REF]), the planar thermopile and a thick Polyimide (PI) layer (~ 12 µm), that is used as a passivation and electrical isolation layer between the thermopile and the heat concentrator. The PI layer also contributes to the membranes robustness. This whole µTEG bottom part is covered by an upper cap as shown in Fig. 3(b): the Si-based heat concentrator (surface = 5.7×5.2 mm²) is build up with as much pillars as membranes (ie. # /2), and it will allow to canalize the harvested heat flow through half of the thermopile junctions (contacts are located at the hot junctions of the thermopile as shown in Fig. 3(c)). One can remark that the choice of TC length will fix the number of membranes. The air cavities located on both sides of membranes (230µm and 380 µm thick resp. on top and on the bottom), allow the thermal insulation of the hot and cold junctions of the thermopile (with the heat sink and the concentrator, respectively). Mechanical supports, called bosses, placed at the 4 corners of the concentrator (visible in Fig. 3(a) and (b)), are used to ensure its rigid support onto the Si substrate and avoid membranes breaking. However, these 4 direct contacts on the module template lead to predictable lateral heat losses from the total heat flux collected onto the concentrator. These lateral losses do not contribute to the hot junction heating.

Finite element modeling of the µTEG

Basic considerations of the model

The µTEG structure we just described before, that integrates the folded thermopile architecture, was modelled using COMSOL Multiphysics® software based on the finite element methods (FEM). Indeed, this permits predicting the temperature distribution and thermal resistance of the new planar µTEG for a heat flux injected into the heat concentrator. Generally speaking, using Heat Transfer Module in COMSOL Multiphysics ® , the temperature distribution is calculated by solving the heat equations in both solid and fluid domains, which can be expressed respectively as follow [START_REF] Comsol | Heat Transfer Module User's Guide[END_REF]:

. # / /0 -2 34 • 6 2 34 7 = 8 (1)
.

# / /0 + . # : 34 • 2 34 -2 34 • 6 2 34 7 = 8 (2)
where , . # , , , 8, and : 34 represent the density, specific heat capacity at constant pressure, temperature, thermal conductivity, heat sources, and fluid velocity, respectively. To simplify the model and reduce the computational time, some assumptions have been considered in this study, while ensuring there is no significant difference from real conditions. First, the materials used in the model are considered isotropic and homogeneous; their thermal conductivities are assumed independent of temperature. The temperature of the heat sink is assumed to be constant and equal to the ambient temperature, ie. 298K (perfect heat sink). We assume that the steady state is reached, ie. the temperature does not change with time (/ /0 ⁄ = 0).

Therefore, the first term of the equations ( 1) and ( 2) disappears. Moreover, considering the low height of the top

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators and the bottom air cavities around the membranes (as seen before only a few hundred microns thick), we will assume that the velocity of the air enclosed in these cavities is zero (: 34 = 0 34 ). Under these conditions, the equations ( 1) and ( 2) are reduced to a single simplified heat transfer equation, -2 34 • 6 2 34 7 = 8, to be solved in all domains of the geometry. The complex design of the thermopile, that can integrate a large number of TCs (up to several hundred TCs), does not allow a crude modeling of the geometry, which is by far exceeding the computational capabilities of our machines. Moreover, owing to the very low thickness of the layers (case of Ni90Cr10/SiO2/Cu55Ni45 and SiO2/SixNy, each sublayer being less than 1µm), inherent difficulties occur in the generation of the modeling mesh and it limits the calculation convergence. For all these reasons, we consider the sub-layers, associated to the thermopile and the bilayer SiO2/SixNy, as a continuous and thermally uniform "equivalent layer". This essential concept of the equivalent layer is detailed in the following paragraph.

Equivalent layer model

Taking into account i/ the symmetry of heat propagation directions across the whole module and its membranes (Fig. 3(c)), and ii/ the periodicity of the thermopile structure observed in the two transverse directions (x and y), we define the equivalent layer as shown in Fig. 4. Each membrane, whose constitution is complex, is assimilated to two continuous layers: the first one corresponds to the Polyimide layer and the second one is defined as the "thermal equivalent" layer to the other sub-layers constituting the membrane, ie. the Ni90Cr10/SiO2/Cu55Ni45 stripes and the SiO2/SixNy bilayer. This unique second material will be defined by an equivalent thermal conductivity %< and its thickness = (this latter being the sum of the thicknesses of all the sub-layers). In order to calculate the temperature distribution along the membrane using COMSOL Multiphysics ® , we must first calculate %< . Considering an elementary cell made up of the sub-layers constituting the equivalent layer, of length >? and width @ A + B, seen between two consecutive hot and cold junctions (Fig. 4(c)), the total thermal conductance C %< of the elementary equivalent layer is given by the sum of the thermal conductance of each material constituting the multilayer as:

C %< = C D E + C F A G + C A & + C A (3)
where

C D E , C F A G , C A & et C A
are the elementary thermal conductance of the SiO2, SixNy, Ni90Cr10 and Cu55Ni45 layers given respectively by: Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

C D E = D E >? *= D E + = D E I +*@ A + B+ (4)
C F A G = F A G = F A G *@ A + B+ >? (5)
C A & = A & = A & @ A & >? (6)
C A = A = A @ A >? (7)
where , =, @ et B are the thermal conductivity, the thickness, the width and the spacing between Cu55Ni45 strips (Fig. 4c) respectively. On the other hand, the equivalent thermal conductance C %< is given by (Fig. 4(c)):

C %< = %< = *@ A + B+ >? (8)
with

= = = D E + = F A G + = A & + = D E I + = A (9)
By identification between the two expressions of C %< given by ( 3) and ( 8) and taking into account the expressions given by ( 4), ( 5), ( 6), (7), and ( 9), the equivalent thermal conductivity can be written as:

%< = D E *= D E + = D E I + + F A G = F A G + A & = A & @ A & @ A + B + A = A @ A @ A + B = D E + = F A G + = A & + = D E I + = A (10)
The Table 1 summarizes an example of typical values of the geometrical dimensions and thermal conductivities of the materials constituting the equivalent layer. Using such values, the calculated equivalent thermal conductivity is %< = 8.58 W.m -1 .K -1 .

Parameter Value Unite = D E 800 nm = D E I 150 nm = F A G 600 nm = A & 150 nm = A 450 nm @ A & 200 µm @ A 150 µm B 55 µm D E 1.25 [46] W.m -1 .K -1 F A G 12 [47] W.m -1 .K -1 A & 17 [48] W.m -1 .K -1 A 23 [48] W.m -1 .K -1
Table 1 : Typical geometrical dimensions and thermal conductivities of the materials constituting the equivalent layer.

Modeling analysis

In practical conditions, the µTEG performance will be evaluated experimentally using a calibrated injected heat flux. To be able to do so, a gold-based heater, meander shaped, is realized on the top surface of the concentrator (will be presented in section 4.1). In order to simplify the calculation, this heater is modeled as a heat source uniformly distributed over the concentrator surface (Neumann boundary condition). As the µTEG is also exposed to the ambient, a part of the heat dissipated in the heater will be exchanged by convection and radiation with the outside environment. Boundary conditions were applied between the top surface of the concentrator and the ambient environment to take into account of all these exchanges. The heat transfer coefficient is set to be 25 W.m -2 .K -1 (which corresponds to natural convection), the emissivity is set at 1 (ie. very conservative case; in reality it is likely around 0,5 to account for both large emissive nature of SiO2 coated Si, and quasi-zero emissivity of the area covered by the Au heater), and the ambient temperature is 298 K. On the other hand, the bottom surface of the substrate, in contact with the heat sink, is also set to the ambient temperature (Dirichlet boundary condition). Moreover, the µTEG structure has a symmetry plane introduced by the equivalent layer model, which allows us to consider just one half of the module by adding an extra symmetry boundary condition as shown in Fig. 5. This consideration reduces the calculation time and the computer memory requirements.

Finally, the two thermal contacts i/ between the concentrator pillars (and bosses) and the part of the thermopile held on membranes (see Fig. 4.a) and ii/ between the thermopile template (ie. etched Si substrate) with the heat sink, are not perfect and cannot be neglected. Henceforth, in the modelling, a thermal contact boundary condition is applied to both those contact boundaries. The experimental results from the characterizations of µTEGs allow estimating the range of the thermal contact resistance, and this by confronting the experimental results with those of simulation. For instance, this one is estimated to be around 3.7 K.cm 2 /W for a µTEG with 3 membranes.

In Fig. 6 is given the COMSOL ® calculated temperature distribution on a half module, in the case of µTEGs with 2 and 3 membranes when a heat input power of 1 W is applied on the concentrator surface (which corresponds to a flux density of 3.3 W/cm²). So one can see how this heat flux injected is canalized through the membranes by the concentrator pillars, and creates periodic temperature gradients between the concentrator and substrate pillars. A part of the heat flux is canalized to the heat sink through the bosses (heat losses). = 137 K and 120 K (ie. decrease with the membrane number), and that those between the surface of the concentrator and the heat sink (noted ∆ J ) are obviously higher: 142 K and 126 K, respectively for the 2-and 3membranes µTEGs. The gap between ∆ and ∆ J values is mainly due to the thermal contact resistances between the concentrator pillars and the membranes, and the heat losses across the bosses. To be precise, the percentages of the fluxes exchanged by convection, radiation and through the bosses are respectively evaluated to be of 11 %, 5 % and 37 % for the 2-membranes µTEG and of 9 %, 4 % and 33% for the 3-membranes µTEG. If the two first fluxes can be considered small (especially radiation and this would be even lower if we considered an emissivity ε<1), this is clearly not the case of the heat flow lost through the bosses.

Fig. 7(a) illustrates the isothermal distribution of the temperature field obtained on the half structure of a 2membranes µTEG. The isothermal line distribution across the membranes, determined by simulation, shows that the planar temperature profile (x and y directions) depends only on the x direction. In contrast, at the proximity of the concentrator pillars and the edge of the membranes, there is naturally a slight deformation of the isothermal temperature profile along the y axis. Fig. 7(b) displays the transverse temperature profile (x and z directions), seen along two adjacent cold/hot junctions. We notice an homogeneous mapping of the temperature at the level of the concentrator and of the substrate (heat sink) due to the high thermal conductivity of the major material used, ie. Silicon (~ 140 W.m -1 .K -1 ). This mapping shows the important role of the air cavities located on both sides of each membrane, which allow an efficient insulation of the hot and cold junctions of the thermopile. Metal-based folded-thermopile for 2.5D micro-thermoelectric generators .So, this numerical calculation of the temperature distribution across the 2.5D µTEG, permits to predict the relation between the two gradients ∆ and ∆ J , this latter being experimentally difficult to measure while only ∆ is accessible, as by definition it is related to the experimental output voltage, = ∆ .

The knowledge of ∆ allows to deduce analytically the predicted maximum output power, ! , generated by the µTEG. This is the key quantity to study and to optimize and ! = $ ∆ /4 as seen in section 2. For the two µTEGs configurations studied in this work, the theoretical ! values are 177.5 and 326.6 µW/cm² respectively for the 2-and 3-membranes modules, if the Seebeck coefficient and electrical conductivities of the Chromel and Constantan are optimum (ie.

= 63 μV/K and resp. equals to 190.6 or 79.9 Ω).

Finally, one can underline that ! depends on the geometrical dimensions of the thermoelements (length, width and thickness) and their number, because these are all involved in both the thermal and electrical aspects of the µTEG. So, a priori, with this modeling, we can vary all the geometric dimensions and look for one or several optimum output power as a function of several parameters, as membrane number. This will be presented in a future work, as for this first study we directly focused on technological fabrication of the new module.

Fabrication and characterization

Fabrication of µTEGs

The 2.5D µTEG realization is based on the silicon technology starting with 3-inch, 380 µm thick, double-sided polished (100) Silicon wafers, to fabricate the two parts of the device, the folded thermopile on suspended membranes and the top concentrator. Several modules can be fabricated on each wafer. The flow chart of the microfabrication processes is given in Fig. 8 for the implementation of a thermopile suspended onto 2 membranes. For simplicity of the drawing, 32 TCs are displayed. In reality, for the 2-or 3-membranes µTEGs, the thermopile cumulates respectively 100 TCs (on 4 branches) or 150 TCs (on 6 branches), for identical footprint and identical number of TCs per parallel branch ( = 25 TCs).

The process is started by the deposition of SiO2/SixNy (800/600 nm) bilayer on both sides of the substrate (Fig. 8(b)) by LPCVD (Low Pressure Chemical Vapor Deposition) using a TEMPRESS® furnace. The thicknesses of the SiO2 and SixNy layers are adjusted to compensate their respective stresses and allow the mechanical stability of the membranes (tensile stress less than 100 MPa [START_REF] Haffar | These de doctorat[END_REF]). Moreover, this bilayer is also used as a physical mask on the backside during the etching of the Silicon substrate to liberate the membrane areas.

(a) (b) (c) (d) (e) (f) (g) (h) (i) Fig. 8 : Flow chart of the suspended membrane fabrication process. (a) Double-sided polished Si substrate; (b) LPCVD growth of SiO2/SixNy on both sides of the substrate; (c) Sputtering deposition and patterning of Ni90Cr10; (d) PECVD of SiO2 and patterning; (e) Metallization of the contacts with Ti/Au layer; (f) Sputtering deposition and patterning of Cu55Ni45 via lift-off; (g) Passivation deposition of a thick Polyimide layer; (h) Backside DRIE etching for opening the cavities and release the membranes; (i) Optical photograph of the suspended membranes seen from the backside after the Silicon deep etching.

Then a 150 nm thick Ni90Cr10 layer is deposited onto the SiO2/SixNy bilayer by Radio-Frequency magnetron sputtering at room temperature, using an Alliance Concept DP 650 machine. A much thicker layer could have been

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators better but this will require further complementary technological developments to solve a delamination problem that ultimately occurs during thermopile fabrication. A mixture of hydrochloric acid and nitric acid, known as "aqua regia", was used to etch the Ni90Cr10 in order to make the TE strips (Fig. 8(c)). The chemical etching has been optimized to control the etching rate and reduce the under-etching phenomena. A thin SiO2 layer (150nm) was then deposited by PECVD (Plasma Enhanced Chemical Vapor Deposition) using an Oxford Plasmalab 80 Plus system at 300°C. The RIE (Reactive Ionic Etching) process employing a CF 4/CHF3 gas mixture was used to etch the SiO2 layer in order to get access to the thermopile contact pads, and to the areas of the Ni90Cr10 strips that will be in contact with the second thermoelement as illustrated in Fig. 8(d). A layer of metals Ti:10 nm/Au:100 nm, is deposited by evaporation and lift-off process, used at the contacts level (Fig. 8(e)) to ensure a good electrical contact between the junctions of the thermoelements. The Cu55Ni45 strips (450 nm thick), representing the second thermoelement, are then periodically deposited by sputtering and lift-off on Ni90Cr10 strips as shown in Fig. 8(f). Here the thickness of Constantan was less an issue for the technology, opposite to the first thermoelement case (Chromel). A 12 µm thick layer of photosensitive Polyimide HD4110 is deposited by spin-coating all over the thermopile and then is opened on the contact pads by photolithography means (Fig. 8(g)). Finally, a backside DRIE (Deep Reactive Ionic Etching) of the Si substrate, based on Bosch process [START_REF] Laermer | Method of anisotropically etching silicon[END_REF], was implemented to dig completely the substrate until the multilayer encapsulating the thermopile (Fig. 8(h)) on the front-side of the wafer: the suspended membranes are realized and well visible (Fig. 8(i)).

The second part of the module (concentrator) is made from another Si wafer (Fig. 9(a)). First, a 2µm thick SiO2 passivation layer is grown by LPCVD on both sides of the wafer (Fig. 9(b)). This layer is used as a physical mask on the backside during the etching of the Si substrate to build up the concentrator pillars and the mechanical holding bosses. A meander-shaped metallic heater (Ti:20 nm/Au:250 nm) is first deposited by evaporation and lift-off process on the front-side of the wafer (Fig. 9(c)). Finally, the pillars and bosses were made in backside of the wafer by DRIE of Si as shown in Fig. 9(d),(e) and (f): the etching is 230 µm deep and corresponds to the aimed height of the pillars. The resistance of the heater varies between 10 and 14.5 Ω. The transfer of the concentrator onto the suspended membranes has, in the past, exploited the implementation of a sealing technique by thermocompression [42]. However, this technique is expensive and technologically complicated, and can lead to mechanical rupture of the membranes. For practical reasons, the alignments between the concentrator and the suspended membranes are performed, in this work, under optical microscopy using alignment wedges. In addition, some thermal grease is used to ensure a good wet contact between concentrator pillars and membranes (and substrate on heat sink) in order to minimize thermal contact resistances.

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

Properties of thermoelectric materials

As mentioned in the previous section, we have integrated, in our manufacturing masks, test patterns adapted to our characterization setups (electrical resistivity and Seebeck coefficient). These test patterns are thus issued from the same wafer and the same layers of Chromel and Constantan as those used in the µTEGs. The Seebeck coefficients of Ni90Cr10 and Cu55Ni45 are measured as a function of temperature (range 20 -100°C) using a homemade set-up and a measurement protocol described in detail in [START_REF] Ziouche | [END_REF]. Fig. 11 shows the variation of Seebeck coefficient of Ni90Cr10 and Cu55Ni45 as a function of the average temperature (the lowest value of average temperature, 35°C, was imposed by the experimental real ambient temperature). For each material, several measurements have been performed on several patterns located in different areas, in order to deduce the range of variation of the Seebeck coefficient over the 3-inch wafer. In the working temperature range, we measured an almost constant value of the Seebeck coefficient for the 150nm thick Ni90Cr10 layer (with an average deviation lower than 1µV/K). On the other hand, the Seebeck coefficient of the 450nm thick Cu55Ni45 layer increases slightly (in absolute value) as a function of the average temperature with a deviation lower than 3 µV/K. So, the average values of the Seebeck coefficients at room temperature, extrapolated from measurements in Fig. 11, are about +15 µV/K for Ni90Cr10 and -33 µV/K for Cu55Ni45 (Table 2). This results in an equivalent Seebeck coefficient of a thermocouple of about αTC = 48 µV/K. In a separate optimization work, carried out on calibration wafers that cannot yet be transferred into the 2.5D µTEG microfabrication process, because of delamination and cracking issues (not shown here), we found higher values of the thermopower and electrical conductivity for both thermoelements, when using a higher deposition temperature (for Constantan) or using an annealing stage at high temperature (for Chromel). The TC Seebeck coefficient would have been 64 µV/K if both optimized layers could be used in the folded TCs.

The characterization of the electrical resistivity of Ni90Cr10 and Cu55Ni45 is carried out using a four-probe Accent ® probing station HL5500 PC. This allows semi-automatic measurements of the electrical resistivity using the Van Der Pauw configuration (clover-leaf-shaped samples). The electrical resistivities of Ni90Cr10 and Cu55Ni45 measured at room temperature and the calculated values of power factor, α²/ρ, are summarized in Table 2. These obtained TE properties show that the quality of the deposited layers is moderate compared to the published values of the state of the art [51][START_REF] Sato | ICT 2005. 24th Int. Conference on Thermoelectrics[END_REF][START_REF] Sharma | [END_REF][54][55][56] and compared to our own optimum layers as stated earlier. Obviously, in future work we will have to implement an adaptation of our technological process of "folded thermopile" to be compatible with the required high temperature extra steps needed to have high quality Constantan and Chromel.

Performance of the µTEGs

In this work, each fabricated 2.5D µTEG based on Constantan/Chromel thermopile was characterized using the four-probe set-up shown in Fig. 12. The measurement principle consists in injecting a thermal power by Joule effect in the heater with two probes, and measuring with two other probes the Seebeck voltage generated by the thermopile. The module under characterization is mounted on a large Aluminum block used as a heat sink to evacuate heat and keep the backside of the µTEG at room temperature. The thermal power dissipated in the concentrator is deduced from the direct measurements of the current flowing through the heater and the voltage at its terminals. The injected current is adjusted between 0 and 290 mA, which corresponds to a thermal power dissipated ranging from 0 to 1.2 W. At the output of the µTEG, the Seebeck voltage generated is measured (using a high impedance voltmeter) as a function of the thermal power injected into the concentrator. The characteristics of both µTEG configurations realized and characterized in this work are presented in the Table 3. As stated earlier, the 2-or 3-membranes based modules have respectively 100 or 150 TCs with a series/parallel assembly. The thermoelement length and the strip width are also given in the table. We note that as expected, the internal electrical resistance of these new metal-based µTEGs, is clearly lower than the one of our first generation of µTEGs, based on a polySilicon/metal thermopiles: in this former case, resistances were of the order of a hundred kΩ to a few MΩ for modules with 2 membranes [33,[START_REF] Bougrioua | 17th Europ. Conf. on Thermoelectrics[END_REF], while for the new family it is one to three hundred Ω. This drastic reduction of resistances results from the use of TE metals (Ni90Cr10 and Cu55Ni45) with very low electrical resistivity, as well as from the new topology of the thermopile in which one part of the TCs are associated in parallel. ) that was generated, as a function of the Joule input power (P ) injected into the concentrator. Each module is characterized several times to reduce the error related to any possible misalignment of the concentrator. Only the best reproducible results are presented in this figure. To avoid overheating of the modules, we have limited the maximum input power to harvest to about 1.2 W. For each characterization, the temperature of the heat sink is measured with a probe placed close to the µTEG: it shows a quasi-no heating over the duration of the measurement thanks to the efficient heat sink dissipator. We assumed that for each characterization, P was fully injected into the concentrator and was dissipated to the heat sink through the µTEG (neglected the part exchanged with the ambient air). Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

Incidentally, one can deduce the internal thermal resistance from this relation: is 137 K/W and 120 K/W respectively for the 2-and 3-membranes µTEGs. So the thermal resistance of the novel µTEG is pretty high, nevertheless it depends on the number of membranes (ie. fixed by the TCs length) so is likely to decrease if we fabricate modules with even more membranes (ie. much shorter TCs). The highest delivered (about 201.6 mV) is obtained for the µTEG with 2 membranes for a maximum injected thermal power of 1.2 W while it is some 11% lower for the 3-membranes µTEG (consistent with a lower ∆ for the same P , due to shorter TCs).

The maximum output power generated by the µTEG, ! , can be calculated as stated earlier, using the measured Seebeck voltage and the experimental internal electrical resistance (305 Ω and 140 Ω respectively for 2-and 3-membranes µTEGs, Table 3

): ! = 4 ⁄
. The evolution of ! generated as a function of P injected into the concentrator is presented in Fig. 14 for the two µTEG configurations. These results show that the 3-membranes µTEG configuration has the best performance as was expected from our modelling. Its highest experimental value is about 125 µW/cm 2 for P ~1.07 W. Normalized to 1W of injected heat, this corresponds to an output power of 108.3 µW/cm². For the 2-membranes based µTEG, ! is as expected smaller: it is about 64 µW/cm 2 . This is still higher than the best ! obtained for the 2-membranes polySi-based µTEGs, with a standard thermopile (50 -62 µW/cm² [START_REF] Bougrioua | 17th Europ. Conf. on Thermoelectrics[END_REF]) which is encouraging. However, these both experimental performances are lower than the predicted values (see section 3.3, it is 50% lower for the 3-membranes µTEG) in part because the experimental Seebeck and electrical properties of the Chromel and Constantan layers used in the fabricated modules, are not the optimized ones. Anyway, as the thermogeneration gets better with the increase of membrane number (ie with the shortening of TCs), we expect, as for our former µTEG geometry (polySi based), to obtain an optimum value of ! for an even higher number of membranes. This will be explored in a forthcoming work.

The above-mentioned best experimental µTEG performance has been compared to the ones of the state-of-theart modules also based on metallic thermoelectrics: these are listed in Table 4. In this table we reported a factor used in the literature for comparing the performance of µTEGs, regardless of their topology or configuration: it is defined by the ratio S % = ! * ⁄ ∆ %! ), where is the area of the µTEG and ∆ %! is the temperature difference between the external surfaces of µTEG (∆ J in our work). This factor S % is often called "efficiency factor" (see for instance [33,57]). The use of this factor was first proposed by Strasser et al. [58] almost two decades ago.

In this comparison, our 3-membranes µTEG is the one that integrates the largest number of TCs despite its very small area, compared to other µTEGs. This is one of the main advantages of the proposed folded thermopile topology, that presents a high integration density of TCs. This results into better values for ! and for the efficiency factor: S % is about 6.82×10 -3 µW.cm -2 .K -2 (∆ %! =∆ J = 126 K, deduced from the experimental ∆ = 120 K and using our simulation presented in section 3.3). This factor is pretty good when compared to the other literature values (modules with metal TCs) and even compared to our own former µTEG topology (polySi-based standard thermopile), where S % was ranging from 1.3×10 -3 to 9×10 -3 µW.cm -2 .K -2 in 2-membrane based modules.

Of course, one can find in literature larger values if considering µTEGs that integrate other kind of TE compounds (not metals) in bulk, thin or thick layer form, as shown in Selvan et al. [57] who proposed a methodological survey on efficiency factors published from 1989 to 2017. If one assumes this study is still valid today, our 3-membrane metal-based µTEG would be in the average. Indeed owing to this survey: "44.1% of thick and thin film thermoelectric generators are realizing in between 0.001 and 4 µW.cm -2 .K -2 , while 43.1% of thick and thin films are earning among 10 -6 to 0.001 µW.cm -2 .K -2 " [57]. In addition, another comprehensive review has been proposed very recently by Yang et al [64], however it is focused on polySi-based miniature modules.

Anyway, those first performance results of our new µTEG validate the interest of our folded series/parallel planar architecture. It also deserves to be improved further by i/ on one hand, optimizing the quality of the two TEs (Chromel and Constantan) as stated before, and ii/ on the other hand, by looking for the optimum dimension in terms of membrane number (ie TC length and number). We already know from our studies on our former µTEG geometry (polySi/metal series thermopile) that the best efficiency factor we could experimentally obtain (Fe=0.117 µW.cm -2 .K -2 [START_REF] Bougrioua | 17th Europ. Conf. on Thermoelectrics[END_REF]) was when using shorter TCs, in a 10-membranes based module. All these points constitute the next steps in our technological roadmap.

Conclusions

We report on a novel 2.5D µTEG integrating a planar thermopile with periodically folded Ni90Cr10 /Cu55Ni45 thermocouples and distributed on 2-or 3-membranes. Our new topology has an increased TC integration density and a reduced module internal electrical resistance, due to the electrical association in "series and parallel" of the TCs and the use of metallic thermoelectrics. The two µTEG configurations, with 2 and 3 membranes, have been successfully modeled, manufactured, and characterized. The best maximum output power density is about 108.3 µW/cm 2 for 1W heat injected on the concentrator and as predicted is delivered by the 3-membranes µTEG. This corresponds to a 126 K temperature difference between the external surfaces of the module and so to an efficiency factor of 6.82×10 -3 µW.cm -2 .K -2 . This performance is better than in the state-of-the-art modules using metallic thermoelectrics. Also, the new modules thermal resistance are pretty high: 120 to 137 K/W. In upcoming work the focus will be on improving the performances of the novel µTEG by i/ optimizing the geometrical parameters as the membrane number and ii/ by solving technical issues as thermal budget in order to be able to employ Ni90Cr10 and Cu55Ni45 layers with optimum thicknesses and TE properties.

Fig. 1 :

 1 Fig. 1 : Description of the new thermopile. (a) Schematic view of Ni90Cr10 strips presenting the first TE material; (b) SiO2 isolation between the thermoelements; (c) Metallic Au contacts at the junctions between thermoelements and contact pads; (d) Schematic view of Cu55Ni45 strips (second TE material); This represents the case of a thermopile with 32 TCs which series/parallel association is given by the arrows; (e) Zoom of previous figure; (f) Two views to illustrate the original topology of a single TC.

Fig. 3 :

 3 Fig. 3 : (a) 3D exploded view (not to scale) of a 2.5D µTEG, in the case of 2-membranes and 32 TCs; (b) Skinned view of the structure ; (c) Cross-section view at xx' axis depicted in (b). For a 3-, 4-or M-membranes module the substrate will display 3, 4 or M cavities, the thermopiles 6, 8 or 2M strips and the heat-concentrator 3, 4 or M pillars.

Fig. 4 :

 4 Fig. 4 : Equivalent layer. (a) Cross-section shows the materials constituting the equivalent layer; (b) Top view of a part of the thermopile illustrating the periodicity of the thermopile in the two transverse directions x and y; (c) Construction of the equivalent layer from an elementary cell of the thermopile seen between two consecutive hot and cold junctions.

Fig. 5 :

 5 Fig. 5 : Symmetry plane and half of the µTEG structure with the symmetry boundary condition.

Fig. 6 :

 6 Fig. 6 : Temperature distribution calculated for 2 and 3-membranes µTEGs. 1 W input power is injected into the concentrator and the temperature of the bottom side of the substrate is fixed at 298 K.

.

  The simulated results corresponding to the applied boundary conditions, allow to anticipate by calculation that the temperature difference generated between the hot and cold junctions, noted ∆ (see Fig. 7(b)), are ∆

Fig. 7 :

 7 Fig. 7 : (a) Isothermal field distribution obtained on the half structure of the 2-membranes µTEG; (b) Symmetrical 2D cross-section seen between consecutive hot-cold junctions of one TC.

Fig. 9 :

 9 Fig. 9 : Flow chart of the main steps of the heat concentrator fabrication process. (a) Double-sided polished Silicon wafer; (b) LPCVD SiO2 growth on both sides of the wafer; (c) Realization of the metallic (Ti/Au) heater on the wafer front-side; (d) and (e) Fabrication of the pillars and bosses on the backside of the wafer by DRIE etching; (f) Image of the backside of a 2-pillars concentrator that will serve for the 2-membranes µTEG.

Fig. 10 illustrates the 2 .

 2 Fig. 10 illustrates the 2.5D µTEG module after the assembly of the concentrator on the folded thermopile. Considering the surface chosen for each module (~1/3 cm²) it is possible to accommodate 44 modules per 3-inch Si wafer, to which are added some test patterns for the electrical and Seebeck measurements of the TE layers.

Fig. 10 :

 10 Fig. 10 : Final images of the µTEG after assembly of the concentrator and the suspended membranes.

Fig. 11 :

 11 Fig. 11 : Seebeck coefficient of Ni90Cr10 and Cu55Ni45 versus average temperature.

Fig. 12 :

 12 Fig. 12 : (a) Photos of the four-probe set-up used for the µTEG characterization; (b) Zoom on a µTEG positioned under 4 probes (2 are used to inject the current in the heater and the 2 others for the Seebeck voltage measurement).

3 :

 3 Main parameters of the 2 fabricated and characterized µTEG configurations.

Fig. 13

 13 Fig. 13 shows the measured output Seebeck voltage () that was generated, as a function of the Joule input power (P ) injected into the concentrator. Each module is characterized several times to reduce the error related to any possible misalignment of the concentrator. Only the best reproducible results are presented in this figure. To avoid overheating of the modules, we have limited the maximum input power to harvest to about 1.2 W. For each characterization, the temperature of the heat sink is measured with a probe placed close to the µTEG: it shows a quasi-no heating over the duration of the measurement thanks to the efficient heat sink dissipator. We assumed that for each characterization, P was fully injected into the concentrator and was dissipated to the heat sink through the µTEG (neglected the part exchanged with the ambient air). The experimental *P + characteristics are almost linear and this linear characteristic of the output voltage as a

  Fig. 13 shows the measured output Seebeck voltage () that was generated, as a function of the Joule input power (P ) injected into the concentrator. Each module is characterized several times to reduce the error related to any possible misalignment of the concentrator. Only the best reproducible results are presented in this figure. To avoid overheating of the modules, we have limited the maximum input power to harvest to about 1.2 W. For each characterization, the temperature of the heat sink is measured with a probe placed close to the µTEG: it shows a quasi-no heating over the duration of the measurement thanks to the efficient heat sink dissipator. We assumed that for each characterization, P was fully injected into the concentrator and was dissipated to the heat sink through the µTEG (neglected the part exchanged with the ambient air). The experimental *P + characteristics are almost linear and this linear characteristic of the output voltage as a function of P are consistent with the Seebeck effect. Indeed, i/ on one hand, = $ ∆ , where N Q =25 for a stripe width of 200µm, = * A & -A + = 48 μV/K, and ii/ on the other hand, if is the internal thermal resistance of the µTEG, then the effective temperature difference between the hot and cold junctions of each thermocouple is given by ∆ = P . At the end: = $ P , so finding linearity is obvious ( is independent of the flux).

Fig. 13 :

 13 Fig. 13 : Seebeck voltage generated in 2-and 3-membranes µTEGs versus input power injected into the concentrator.

Fig. 14 :

 14 Fig. 14 : Maximum output power density generated versus input power injected into the concentrator.

Table 2 :

 2 Thermoelectric properties of Ni90Cr10 and Cu55Ni45 at 298K.

	TE material	Thickness	Seebeck coefficient	Electrical resistivity	Power Factor α 2 /ρ
		(nm)	α (µV/K)	ρ (µΩ.cm)	(µW.cm -1 .K -2 )
	Ni90Cr10	150	15	100.3	2.24
	Cu55Ni45	450	-33	59.8	18.21

Table 4 :

 4 Comparison of the performance obtained in this work with the state-of-the-art metallic based µTEGs.

	TE material	Area of µTEG (mm 2 )	Number of TCs	T UVW (Ω)	∆X YZW (K)	[ \]W (V)	^_`Z (µW/cm 2 )	a Y	2 .K -2 ) (µW.cm -	Ref
	NiCr/CuNi	34	150	140	126	0.143	108.3	6.82 × 10 -3	This work
	NiCr/CuNi	900	33	--	22.7	0.3	0.41		8 × 10 -4	[59]
	NiCr/CuNi	768	40	392	250	0.53	19.53	3.12 × 10 -4	[60]
	Cu/CuNi	250	50	56000	70	2.18	8.5	1.73 × 10 -3	[61]
	Cu/Ni	1118	10	7.41	69	0.013	0.51	1.07 × 10 -4	[62]
	Ag/Ni	383	216	--	127	--	80.4	4.98 × 10 -3	[63]

Metal-based folded-thermopile for 2.5D micro-thermoelectric generators

Acknowledgements

This research work has been partly undertaken with the support of IEMN fabrication facility (CMNF), which is a member of the French Technology Network RENATECH.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors Biography

Ibrahim Bel-Hadj received his Ph.D. in "Micro-nano-systems and Sensors" from Lille University in mid 2022, for his work at IEMN (Institute of Electronics, Microelectronics & Nanotechnology) on the fabrication and optimization of micro-thermoelectric generators. Since September 2022 he is a postdoctoral researcher at University of Strasbourg (Fr) where his research interests are focused on nanoparticle engineering and plasmonic biosensors.

Zahia Bougrioua is a senior researcher at CNRS whose expertise is in compound semiconductors and in thermoelectrics, from elaboration to characterization and device fabrication. She received her Ph.D. in Materials Sciences from Lille University in 1994. From 1995 to 2001, she worked on III-(As,P) compounds at Montreal Ecole Polytechnique (Ca), then on wide bandgap III-N at IMEC (Be). In 2001 she was appointed at CNRS, first at CRHEA (Fr) to develop III-N for transistor and light emission applications, then, at IEMN, where she mainly focus on micro-sensors and µthermogenerators. -The novel thermopile topology is periodically folded and is based on Ni90Cr10 and Cu55Ni45 thermoelectric layers.

-The thermopile integrates a high density of thermocouples with a combined series and parallel electrical association.

-In the 3-membranes based µTEG, a maximum output power density of 108.3 µW/cm 2 is generated from 1 W heat to harvest.

-Those 2.5D µTEGs have higher efficiency and thermal resistance than in the state of the art of metal based modules.