Christopher Choi

Kevin Robert

Grace Whang

Pascal Roussel

Christophe Lethien
email: christophe.lethien@univ-lille.fr

Bruce Dunn
email: bdunn@ucla.edu

Photopatternable hydroxide ion electrolyte for solid-state micro-supercapacitors

Keywords: Solid electrolytes, hydroxide-ion electrolytes, photopatternable electrolytes, on-chip energy storage, 20 micro-supercapacitors

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

Introduction

Advances in electrochemical energy storage (EES) technology have changed the landscape of the portable electronics industry and the electric vehicle market in the past decade and are on the same track for enabling the 'Internet of Things' (IoT). 1 A robust, high-energy and high-power micropower system is a key technology for the future development of micro/nano electronics, such as nanorobotics, microelectromechanical systems (MEMS), environmental sensors, and connected smart electronics. 2,3 However, the power sources for these devices often become the limitation in achieving the small formfactor required by miniaturized electronics because energy-storage components scale down poorly in size. It is well known that miniaturization of electrochemical energy storage (EES) system devices such as batteries and capacitors have lagged far behind Moore's Law and that improvements of only about 10%/year have been achieved. 4 A brief overview of miniaturized EES that use various fabrication methods is presented in the SI and summarized in Table S1. Of particular interest for the current research is the development of integrated on-chip EES to power miniaturized electronic devices. Such a technology holds significant promise towards enabling the IoT and further enhancing the distribution and utilization of sustainable energy. To achieve this goal, however, there needs to be an appropriate chemistry where EES components, i.e. the anode, cathode, and solid electrolyte, can be formed and defined spatially through the very techniques that are utilized in achieving high-resolution patterns in the semiconductor industry. 5,6 In recent years, there have been substantial efforts at fabricating EES devices using semiconductor processing fabrication methods. This research includes on-chip micro-supercapacitors (MSC) [7][8][9][10] and multi-dimensional batteries. 5,[11][12][13] Some devices have demonstrated areal energy densities (> 1 mWh cm -2 ) or power densities (> 5 mW cm -2 ) that are comparable to those of bulk-scale EES systems. However, these micropower sources use liquid electrolytes which are not compatible with on-chip integration because of the inability to spatially confine a liquid. There is, therefore, a critical need to develop solid electrolyte materials which would enable the fabrication of micropower sources using semiconductor processing methods. Typical solid electrolytes for miniaturized MSC devices consist of an ionic salt solvated in polymeric or inorganic solid host materials. The most popular solid electrolytes being explored for supercapacitors use aqueous electrolytes embedded in polymer hydrogels such as poly(vinyl alcohol) (PVA), poly(ethylene oxide) (PEO), and poly(acrylic acid) (PAA). [14][15][START_REF] Dubal | Towards flexible solidstate supercapacitors for smart and wearable electronics[END_REF][START_REF] Alipoori | Review of PVA-based gel polymer electrolytes in flexible solid-state supercapacitors: Opportunities and challenges[END_REF] A representative system is KOH in PVA, which exhibits an ionic conductivity exceeding 10 -2 S cm -1 at room temperature. Ionogel electrolytes, based on the incorporation of an ionic liquid such as 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [EMIM][TFSI] encapsulated in SiO2 or polyvinylidene fluoride, constitutes another class of solid electrolyte that is used in supercapacitors. [START_REF] Bideau | Ionogels, ionic liquid based hybrid materials[END_REF] This nonaqueous solid electrolyte exhibits an electrochemical window up to 2.5 V in MSCs, although its ionic conductivity at ambient temperatures ( 10 -3 to 10 -4 S cm -1 ) is lower than aqueous systems due to slow diffusivity of the bulky ions. [START_REF] Asbani | Reflow Soldering-Resistant Solid-State 3D Micro-Supercapacitors Based on Ionogel Electrolyte for Powering the Internet of Things[END_REF] Both of these systems represent gel-type electrolytes whose utilization in advanced devices is restricted by several concerns including poor mechanical integrity and fabrication difficulties which limit their integration with high resolution devices. [START_REF] Beidaghi | Capacitive energy storage in micro-scale devices: recent advances in design and fabrication of micro-supercapacitors[END_REF][START_REF] Ashby | Patternable, SolutionProcessed Ionogels for Thin-Film Lithium-Ion Electrolytes[END_REF] In this paper, we describe the development of a hydroxide-conducting photopatternable solid electrolyte that possesses a high room temperature ionic conductivity of 10 mS cm -1 along with excellent thermal stability and mechanical integrity. This material represents one of the very few photopatternable solid electrolytes reported to date and, to the best of our knowledge, is the first one to exhibit hydroxide ion conduction. This solid-state electrolyte, and especially its ability to be photopatterned, enables interdigitated MSC to be fully fabricated by semiconductor processing methods. We demonstrate the application of the hydroxide-conducting photopatternable solid electrolyte by integrating it with VN interdigitated electrodes, leading to the fabrication of high-resolution, high performance solid-state VN MSC devices. In addition to demonstrating individual MSC devices, we characterize tandem device architectures which lead to increased cell voltages and total currents beyond what is available with individual devices.

Results and Discussion

Our approach to creating photopatternable solid electrolyte materials involves modifying the chemistry of commercial SU-8 photoresists to achieve ion transport without compromising its photopatterning functionality. We previously demonstrated this general methodology enabling the development of lithiumion conducting photopatterned electrolytes with sub-100 μm resolution. 6 In the research reported here, the particular challenge in achieving hydroxide-ion conduction is the hydrophobic nature of the SU-8 photoresist. Our synthesis strategy for preparing the photopatternable hydroxide-conducting solid electrolyte involved two steps. First, the cationic polymerizable 1-Allyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [AMIM][TFSI] was grafted onto the SU-8 polymer matrix. This was followed by anion exchange to incorporate hydroxides (Figure 1a, S1). The resulting electrolyte system consists of the SU-8 host material that provides the photopatterning capability while hydroxides in the polymerizable ionic liquid phase provide the charge-carrying hydroxide ions required for the VN redox process (Figure 1b). Ionic liquid [AMIM][TFSI] grafted SU-8 polymer was successfully photopatterned using a photomask with cylindrical features 100 μm in diameter (Figure S1). This signifies that the photopatterned solid electrolyte material can be paired with any miniaturized EES systems that are 10 x 10 μm 2 in size. The photopatterned electrolyte was further characterized using energy-dispersive X-ray spectroscopy (EDS). The presence of ionic liquid constituents (sulfur and fluorine) in the photopatterned structure confirms that the photopatternable polymer matrix encapsulates the ionic liquid (Figure S1b). After the anion exchange process with hydroxides, only trace amounts of fluorine and sulfur were detected (Figure S1c).

The lithography-assisted crosslinking between polymerizable cations and SU-8 matrix was verified by analyzing Fourier transform infrared (FT-IR) spectra. The disappearance of the vinyl group at 1648 cm -1 (stretching vibration of C=C) upon polymerization indicates that vinyl groups are participating in the photopolymerization process (Figure S2a). The grafting of polymerizable ionic liquids was further confirmed by evaluating the solubility of the ionic liquid phase using an IL miscible solvent (i.e. acetone or ethanol). In general, for a given ionogel system, ionic liquids contained in the host network are readily soluble in certain solvent media. [START_REF] Bideau | Ionogels, ionic liquid based hybrid materials[END_REF][START_REF] Ashby | Patternable, SolutionProcessed Ionogels for Thin-Film Lithium-Ion Electrolytes[END_REF] However, it is evident in the infrared spectra in Figure S2b that polymerizable cations in the [AMIM][TFSI] ionic liquid are covalently bonded to the SU-8 matrix and are no longer soluble. Once grafting of the cation was confirmed, the mobile TFSI -anions were exchanged with OH -in 1M KOH (aq.). The approximate concentration of hydroxide anions in [AMIM][OH]/SU-8 electrolyte is calculated to be 1.8M based on the total volume of the electrolyte with the assumption that TFSI -anions are fully substituted. The anion exchange process in the ionic liquid grafted SU-8 electrolyte was assessed by observing changes in the characteristic peaks (Figure 2a) ascribed to TFSI -anion (Table S2) and OH -anion (3200-3400 cm -1 ). It is also clear that the IR absorbance bands assigned to the imidazolium cation (752, 1432, 1464 cm -1 ) are still present after the TFSI -anions are exchanged.

We validated the mobility of OH -transport in the photopatternable [AMIM][OH]/SU-8 material by using it as a solid electrolyte which leads to charge storage in NiCo2O4. This transition metal oxide is well known for undergoing redox reactions with hydroxide anions through the following mechanisms [START_REF] Umeshbabu | Urchin and sheaf-like NiCo 2 O 4 nanostructures: Synthesis and electrochemical energy storage application[END_REF][START_REF] Ding | A facile and cost-effective synthesis of mesoporous NiCo2O4 nanoparticles and their capacitive behavior in electrochemical capacitors[END_REF] :

NiCo2O4 + OH -+ H2O ↔ NiOOH + 2CoOOH + e - (1)
CoOOH + OH -↔ CoO2 + H2O + e - (2)
In this experiment, a 10 μm film of the [AMIM][OH]/SU-8 solid electrolyte was coated and polymerized on the NiCo2O4 electrode. The CV curves for this system were compared to those for the same NiCo2O4 material in 1M KOH (Figure S3). The similarity in redox potential, peak current response, and observed gravimetric capacities (89 vs 92 mAh g -1 at 10 mV s -1 ) for NiCo2O4 samples tested in patternable solid electrolyte and 1M KOH control liquid electrolyte, confirms that hydroxide ion transport occurs in the patternable electrolyte. This observation was consistent over a range of sweep rates (1, 2, 5, 10 mV s -1 ), suggesting that the [AMIM][OH]/SU-8 not only facilitates OH -transport but also provides a coherent electrode/electrolyte interface. These attributes are among the most vital electrochemical features for solid electrolytes.

The thermal stability of the electrolyte was determined using thermogravimetric analysis (TGA) as shown in Figure 2b. In view of possible heating effects associated with high power operation, thermal stability of the electrolyte can be a potential concern. The photopatterned electrolyte is quite stable as it showed no appreciable weight loss to temperatures above 300 o C in the TG profile. In contrast, the unmodified SU-8 photoresist begins to undergo thermal decomposition below 200 o C (Figure S4a). One other feature of the solid-like behavior of [AMIM][OH]/SU-8 is that there is only a nominal change in the mechanical integrity after heat treatment (Figure S4b). The noticeable increase in thermal stability is attributed to the grafting of the thermally stable [AMIM]-based ionic liquid with the SU-8 matrix. Overall, the results showed excellent thermal stability with about 5 % weight loss by 320 o C.

The mechanical properties of the patternable solid electrolyte films were evaluated using nanoindentation measurements (Figure 2c). The mechanical integrity of the solid electrolyte is important for maintaining good contact with the electrodes and preventing electrical shorts. Prior to the ionic liquid grafting, the SU8 film demonstrates a high elastic modulus of 5.1 GPa. Upon incorporation of the polymerized ionic liquid phase, the elastic modulus value was reduced to 230 MPa. Nonetheless, the [AMIM][OH]/SU-8 ionogel electrolyte is far more rigid and dimensionally stable than that of the PVA/KOH gel electrolyte (3.75 MPa) and other gel electrolytes including PAA/KOH and polyacrylamide(PAM)/KOH. [START_REF] Wu | Preparation and characterization of PVA/PAA membranes for solid polymer electrolytes[END_REF][START_REF] Tran | Effects of Crosslinker Concentration in Poly(Acrylic Acid) KOH Gel Electrolyte on Performance of Zinc Air Batteries[END_REF][START_REF] Cao | Planar all-solid-state rechargeable Zn-air batteries for compact wearable energy storage[END_REF] To put this into perspective, the elastic modulus of 230 MPa (compressive) exceeds the 100 MPa elastic modulus requirement (tensile) identified for commercial Li-ion battery separators to possess sufficient mechanical integrity during battery manufacturing. [START_REF] Zhang | A review on the separators of liquid electrolyte Li-ion batteries[END_REF][START_REF] Kalnaus | Mechanical behavior and failure mechanisms of Liion battery separators[END_REF] It is also important to reiterate that none of these other electrolytes have demonstrated photopatterning and thus, it is not possible to use these electrolytes to achieve the high spatial resolution that is required for on-chip integration.

One of the most favorable hydroxide-redox electrode materials for MSCs is VN. The material possesses high electronic conductivity (1.6 x 10 4 S cm -1 ) with a high rate surface redox electrochemical response (Figure S5). [START_REF] Choi | Fast and Reversible Surface Redox Reaction in Nanocrystalline Vanadium Nitride Supercapacitors[END_REF] The preparation strategies and electrochemical characterization of interdigitated VN MSCs have been reported recently. [START_REF] Robert | Novel insights into the charge storage mechanism in pseudocapacitive vanadium nitride thick films for high-performance on-chip micro-supercapacitors[END_REF] These MSC devices, however, were based on using a KOH aqueous electrolyte which is not compatible with on-chip integration because of the inability to spatially confine a liquid. The high-resolution photopatterning capability of [AMIM][OH]/SU-8 combined with interdigitated VN devices enables the integration of a solid-state MSC at the point-of-load and leads to in-series and/or in-parallel device configurations in a defined area.

The high electronic conductivity of the VN film allows it to be used as a blocking electrode in characterizing the impedance of the [AMIM][OH]/SU-8 ionogel electrolyte. Two-and three-electrode cells were used to compare the impedance of the ionogel electrolyte with that of 1M KOH. For the former, a 10 μm film of the ionogel electrolyte was patterned directly on VN electrodes. This thickness is comparable to the interlayer gap spacing distance used in the MSC interdigitated devices (vide infra). The Nyquist plot (Figure 3a, S8a) indicates that both electrolytes display a low series resistance with a low frequency capacitive tail. Despite the relatively low ionic conductivity of the patternable solid electrolyte compared to that of 1M KOH liquid electrolyte (σ ~ 0.5 S cm -1 ), the near-identical trend in impedance spectra highlights the effectiveness of high-resolution spatial control that can be achieved by [AMIM][OH]/SU-8 patterning. The room temperature ionic conductivity at 25 °C for the hydroxide conducting patternable solid electrolyte is calculated to be 10 mS cm -1 which represents a comparable or higher ionic conductivity than that of previously reported hydroxide-conducting electrolytes, such as PVA/KOH, PEO/KOH, and PAM/tetraethylammonium hydroxide. [START_REF] Yang | All solid-state electric double-layer capacitors based on alkaline polyvinyl alcohol polymer electrolytes[END_REF][START_REF] Lewandowski | Supercapacitor based on activated carbon and polyethylene oxide-KOH-H2O polymer electrolyte[END_REF][START_REF] Gao | Alkaline quaternary ammonium hydroxides and their polymer electrolytes for electrochemical capacitors[END_REF][START_REF] Li | Investigation of polyacrylamide based hydroxide ion-conducting electrolyte and its application in all-solid electrochemical capacitors[END_REF] Although PVA/KOH is the most widely used hydroxideconducting electrolyte for solid supercapacitors, 15,[START_REF] Alipoori | Review of PVA-based gel polymer electrolytes in flexible solid-state supercapacitors: Opportunities and challenges[END_REF] there has been only modest improvement in its electrochemical and physical properties since the adoption of PVA hydrogel chemistry. Additionally, the ionic conductivity of the [AMIM][OH]/SU-8 ionogel electrolyte is as much as 5 times higher than another polymer based ionogel, [EMIM][TFSI]/PVDF, which was incorporated in 3D MSCs with MnO2 pseudocapacitive electrodes. [START_REF] Asbani | Reflow Soldering-Resistant Solid-State 3D Micro-Supercapacitors Based on Ionogel Electrolyte for Powering the Internet of Things[END_REF][START_REF] Yang | Physically CrossLinked Homopolymer Ion Gels for High Performance Electrolyte-Gated Transistors[END_REF] It is also interesting to note that the room temperature conductivity value for the hydroxide ion conducting electrolyte is comparable to that of conventional nonaqueous liquid electrolytes based on quaternary ammonium salts and lithium-ion compounds. [START_REF] Ue | Electrochemical Properties of Organic Liquid Electrolytes Based on Quaternary Onium Salts for Electrical Double Layer Capacitors[END_REF] Figure 3b compares the Bode plots for the [AMIM][OH]/SU-8 solid electrolyte and the 1M KOH. The characteristic time constants for the two samples were about 8.0 s for the liquid electrolyte and 12.6 s for the patterned ionogel electrolyte, signifying that shorter charge/discharge times may lead to a kinetically limited electrochemical response. Nonetheless, the fact that it is possible to fabricate high spatial resolution patterns of this solid electrolyte with only a small difference in transient response time compared to standard 1M KOH, is very impressive.

A series of experiments was carried out in which the photopatternable [AMIM][TFSI]/SU-8 served as the electrolyte for sputtered VN films, thus enabling the electrochemical properties to be determined. The asprepared VN films exhibit strong preferential crystalline orientation in the (111) direction (Figure S6a) and faceted growth (Figure S6b,c). The intercolumnar porosity from this faceted morphology enables electrolytes in their liquid phase to penetrate throughout the structure (Figure S6d,e, S7). After the [AMIM][TFSI]/SU-8 ionogel electrolyte was patterned directly on top of the planar VN and polymerized, the uniform penetration of the electrolyte was verified by an EDS elemental mapping image (Figure S7), showing that fluorine and sulfur signals originating from the ionic liquids are present throughout the VN layer. The cross-sectional SEM image in Figure S6c indicates that the as-prepared VN electrode thickness was about 5 μm. The surface composition of the VN film, probed by the X-ray photoelectron spectroscopy (XPS) analysis (Figure S6f), is consistent with other VN systems reported in the literature. [START_REF] Choi | Fast and Reversible Surface Redox Reaction in Nanocrystalline Vanadium Nitride Supercapacitors[END_REF][START_REF] Robert | Novel insights into the charge storage mechanism in pseudocapacitive vanadium nitride thick films for high-performance on-chip micro-supercapacitors[END_REF] The charge storage properties of sputtered VN films using the photopatternable solid electrolyte were evaluated in both planar and interdigitated configurations. Linear sweep voltammetry experiments displayed no evidence of electrolyte decomposition between -1.2 and 0.2 V (vs. Hg/HgO) for the photopatternable [AMIM][OH]/SU-8 electrolyte (Figure S4c). The cyclic voltammogram (CV) responses of solid electrolyte patterned VN exhibits the ideal quasi-rectangular capacitive response between 5-20 mV s -1 sweep rates over 0.6 V window (Figure 3c). As discussed in previous studies, this pseudocapacitive charge storage behavior arises from redox properties associated with the reduction and oxidation of vanadium between V 3+ and V 4+ occurring at the surface or near surface of the VN. [START_REF] Robert | Novel insights into the charge storage mechanism in pseudocapacitive vanadium nitride thick films for high-performance on-chip micro-supercapacitors[END_REF] Because the electrolyte penetrates the intercolumnar pore network of VN prior to polymerization, the resulting solid electrolyte is in contact with the redox active VN material throughout the entire film and not just at the surface of the film. At a sweep rate of 5 mV s -1 , the VN coated with the solid-state electrolyte achieves an areal capacitance of about 0.43 F cm -2 which corresponds to 860 F cm -3 based on the electrode volume. This areal capacitance is comparable to those reported for CDC films and Ti3C2Tx electrodes tested in liquid electrolyte (1M H2SO4). 7,[START_REF] Ghidiu | Conductive twodimensional titanium carbide 'clay' with high volumetric capacitance[END_REF] That is, the solid-state VN electrode exhibited an areal capacitance that exceeds or is on par with high performance supercapacitor materials tested in liquid electrolytes. Moreover, up to a sweep rate of 50 mV s -1 , the areal capacitance for VN electrodes with either KOH or solid-state electrolytes are comparable (Figure 3d). Above this sweep rate, the capacitive signature starts to deviate from the ideal shape for both the liquid and solid electrolytes (Figure 3c andS8b). This response can be associated with the fact that the sweep rate of 50 mV s -1 corresponds to a 12s charge / discharge cycle time, which matches the characteristic time constant obtained from the Bode plot for the solid electrolyte. The VN electrode paired with the [AMIM][OH]/SU-8 patternable electrolyte also demonstrates excellent long-term cyclability, retaining about 80% of the initial capacitance at 10 mA cm -2 by the 10,000 th cycle (Figure 3e).

VN interdigitated microdevices were fabricated at the wafer-level as described in the SI (Figure S9a). Figure S9b and Table S3 illustrate the important device parameters for interdigitated cells and fabrication approaches to achieve solid-state microdevices. CV profiles (Figure 4c) of a solid-state interdigitated MSC showed well-defined capacitive behavior over a wide range of scan rates (20 to 200 mV s -1 ). The areal capacitance of the cell was calculated to be almost 250 mF cm -2 at a scan rate of 20 mV s -1 and an individual solid-state MSC device was capable of delivering about 10 μWh cm -2 in the range of 1-10 mW cm -2 . This level of areal capacitance for the solid-state VN MSC is comparable to other interdigitated MSCs tested in liquid electrolytes, including both carbon-based 7 and pseudocapacitive systems (Table S4). 9,[START_REF] El-Kady | Engineering three-dimensional hybrid supercapacitors and microsupercapacitors for highperformance integrated energy storage[END_REF][START_REF] Tian | Multifunctional Nanocomposites with High Strength and Capacitance Using 2D MXene and 1D Nanocellulose[END_REF][START_REF] Li | Cladding nanostructured AgNWs-MoS2 electrode material for high-rate and long-life transparent in-plane microsupercapacitor[END_REF] The capacitive behavior of this solid-state microdevice was further studied through galvanostatic measurements at current densities from 2 to 50 mA cm -2 (Figure 4d). The galvanostatic profiles at all current densities demonstrated linear behavior, which is a defining feature of a pseudocapacitive material. [START_REF] Choi | Achieving high energy density and high power density with pseudocapacitive materials[END_REF] Figure 4h shows a Ragone plot comparing the performance of solid-state VN MSC to other solid-state MSC devices paired with solid electrolytes such as PVA based hydrogels and silica ionogels. [START_REF] Asbani | Reflow Soldering-Resistant Solid-State 3D Micro-Supercapacitors Based on Ionogel Electrolyte for Powering the Internet of Things[END_REF][START_REF] El-Kady | Scalable fabrication of high-power graphene microsupercapacitors for flexible and on-chip energy storage[END_REF][START_REF] Wang | High-performance all solid-state microsupercapacitor based on patterned photoresist-derived porous carbon electrodes and an ionogel electrolyte[END_REF][START_REF] Wang | Silicon-Based 3D All-Solid-State Micro-Supercapacitor with Superior Performance[END_REF][START_REF] Brousse | Facile and Scalable Preparation of Ruthenium Oxide Based Flexible Micro Supercapacitors[END_REF] The results show that VN MSC with the [AMIM][OH]/SU-8 electrolyte achieves better performance in terms of areal energy and power densities compared to previously reported solid-state MSC systems. Moreover, it is important to emphasize that all components of the solid-state VN MSC are defined spatially via lithography, thus enabling miniaturized arrays to be formed in a tiny form factor.

To demonstrate the advantages of semiconductor-based processing, we tested 'tandem' MSC microdevices. These interdigitated structures are connected in series and/or parallel configurations using VN layers (without any external wires or deposition of metals) to increase the working cell voltage and/or total current response of an integrated microdevice (Figure 4 e-g). The ability to integrate multiple MSCs in a small footprint area is achieved because spatial control of the photopatternable hydroxide-conducting solid electrolyte effectively prevents leakage current between individual MSC devices. The tandem microdevice in a series configuration demonstrated reliable cycling over a 1.2 V window, twice the voltage of a single MSC device. Accordingly, the total capacitance for the tandem device increased to over 11 mF. As expected, a parallel configuration provided twice the current response compared to a single MSC device. Capacitive behavior was maintained throughout a 0.6 V window in both cyclic voltammetry and galvanostatic experiments, producing a total current above 1 mA. Unfortunately, the circuit layout for the microfabricated wafer did not allow us to evaluate more than four devices in either parallel or series configuration. Nonetheless, these electrochemical measurements made on tandem VN-MSC microdevices confirm that the cell voltage range and total current output of MSC devices can be tailored easily by modifying the device configurations. Considering that all components in this solid-state tandem energy storage system are fully processed with semiconductor-based fabrication techniques, the approach shown here provides the basis for achieving miniaturized energy-storage systems that can be incorporated with densely packed integrated circuits without compromising their electrochemical properties.

It is interesting to consider the prospect of using miniaturized EES systems to power IoT devices. In particular, to power a sensor node platform (MICA2 and IMote2), as an example, an average power consumption for these sensor nodes are 3 and 12 mW with the peak power requirement of 60 and 250 mW, respectively. [START_REF] Hancke | The Role of Advanced Sensing in Smart Cities[END_REF] It becomes clear that a single 4 mm 2 solid VN MSC device that is capable of delivering 110 mW cm -2 is still far below the power requirements for commercial sensor nodes, especially with regard to the limited energy density it provides (~ 10 μWh cm -2 ). However, the demonstrated tandem approach to MSCs suggests a viable solution as the footprint area of these sensor nodes is around 18 cm 2 . Thus, fabricating MSCs at wafer-scale offers the prospect of providing the energy and power requirements for this basic IoT component. To be sure, there are still a number of challenges that need to be addressed in order to realize this micropower system, but the basic elements of using a single VN material to be both redox-active electrodes and interconnects, coupled with the photopatternable, hydroxide ion conducting solid electrolyte provide the basis for enhancing micropower technology.

Conclusion

Among the few hydroxide ion conducting solid electrolytes reported to date, none offer the combination of high room temperature ionic conductivity (10 mS cm -1 ), excellent thermal stability, mechanical integrity, and the photopatterning capability of the [AMIM][OH]/SU-8 solid electrolyte presented here. The ion transport properties of this electrolyte and ability to carry out charge transfer at the electrolyte/electrode interface were validated using hydroxide-based redox-active NiCo2O4 and VN. For device-level demonstration, [AMIM][OH]/SU-8 hydroxide-conducting solid electrolyte was directly patterned onto VN MSC electrodes to create both individual and tandem devices. The performance of individual MSC devices of nearly 250 mF cm -2 at 20 mV s -1 sweep rate goes beyond what has been reported for interdigitated MSC devices with liquid electrolytes and underscores the significance of this solid electrolyte. Electrochemical testing of tandem micro-supercapacitor devices has shown that the potential range and total current output of micro-supercapacitor devices can be tailored by modifying the device arrangements in appropriate series or parallel configurations. Finally, the semiconductor processing methods demonstrated here hold promise for achieving the power and energy density demands for IoT technology.

Experimental Procedures

Resource availability

Lead contact: Further information and requests for resources and materials should be directed to and will be fulfilled by the lead contact, Bruce Dunn (bdunn@ucla.edu).

Materials availability: This study did not generate new unique materials.

Data and Code Availability: Much of the data presented in this work is available by request from the lead contact. This study did not generate any code for analysis.

Materials: Physical and Chemical Characterizations: FTIR spectroscopy was carried out using a Jasco 6100 with a 4 cm -1 spectral resolution from 4000 to 500 cm -1 . The photopatternable electrolyte was prepared on IR transparent polytetrafluoroethylene films for the measurement. Nanoindentation measurements were made using an MTS Nano Indenter XP Instrument with a Berkovich indenter to determine the elastic modulus of hydroxide-conducting SU 8 films. Samples were indented to a depth of 2 µm at 20 different locations. The elastic modulus was determined using a Poisson's ratio of 0.22 for SU 8 and the Oliver and Pharr analysis. Film thicknesses were measured using a Dektak 6 surface profilometer. TGA was performed using an Instrument Specialists thermogravimetric analyzer tool in air at a heating rate of 10 •C min -1 . Scanning electron microscope (SEM) was performed using an FEI Nova 230 Nano with attached EDS. XPS analysis was carried out using a Kratos Axis Ultra DLD with a monochromatic Al Kα radiation source. The charge neutralizer filament was used to control charging of the sample, a 20 eV pass energy was used with a 0.05 eV step size, and all scans were calibrated using the C 1s peak shifted to 284.8 eV. The integrated area of the peaks was found using the CasaXPS software.

Synthesis of NiCo2O4: NiCo2O4 was prepared using a one-step microwave synthesis. Nickel nitrate hexahydrate (0.15 g, Sigma-Aldrich) and cobalt nitrate hexahydrate (0.18 g, Sigma-Aldrich) precursors were dissolved in deionized water (5 ml), and ethylene glycol (17.5 ml) and urea (0.1 g, Sigma-Aldrich) were added in this solution with stirring. The homogeneous solution was then transferred into the 30 ml microwave reaction vessel and placed in a microwave synthesis system (Discover SP, CEM Corporation).

The sample was heated to 140 °C for 30 min. The resulting green-colored solution was centrifuged and washed with deionized water and ethanol and dried in vacuum for 24 h. The samples were then fully oxidized by annealing the as-prepared samples at 300 °C for 1 h.

Vanadium Nitride Electrode Preparation:

To prevent electrical short-circuits and chemical etching from basic electrolytes, a 500 nm-thick Si3N4 layer was deposited by low pressure chemical vapor deposition at 800 °C to protect the Si wafer. The VN thin films were then deposited by reactive magnetron sputtering using a 4-inch target of pure metallic vanadium in an Alliance Concept CT-200 reactor. Prior to deposition, the pressure of the chamber was kept below 10 -6 mbar and the target-substrate was fixed at 6 cm. The deposition parameters were fixed at 100°C for the temperature, 25 and 5 sccm for the argon and nitrogen flow rates, 7.5 x 10 -3 mbar for the deposition pressure, and 2 W.cm -2 for the power density. The deposition time was tuned to achieve 5µm-thick VN thick films. The typical loading of the VN electrode film was 2.5 mg cm -2 .

Fabrication of the symmetric interdigitated micro-supercapacitor (MSC):

After the deposition of the Si3N4 (500 nm) and the VN (5 µm) thin films, photoresist was spin-coated to form the resist mask. The VN was etched using Inductively Coupled Plasma Reactive Ion Etching (ICP RIE) process using a mixture of chlorine/argon gases (Cl2 / Ar). To avoid short-circuits between the interdigitated VN electrodes, the etching was stopped once the top 10 nm of the Si3N4 layer was etched. Due to this four-step process, 40 MSCs can be fabricated on a single 3-inch silicon wafer (7.5 cm diameter / thickness = 0.385 mm).

Electrochemical Characterization: All electrochemical measurements were using a Bio Logic VMP 3 Potentiostat. Electrochemical impedance spectroscopy (EIS) was performed on a VN/[AMIM][OH]SU8/VN 2-electrode cell between 1 MHz and 5 mHz using a 10 mV amplitude and 0 V bias. Linear-sweep voltammetry (LSV) was used to evaluate the electrochemical stability window of the [AMIM][OH]/SU-8 using a solid electrolyte coated glassy carbon as a working electrode. LSV was conducted from the open circuit voltage (OCV) to 0.7 V (vs. Hg/HgO) and from OCV to -1.4 V at a sweep rate of 10 mV s -1 . For the CV and galvanostatic cycling measurements, the planar VN film (deposited directed on a Si/Si3N4 wafer) was tested in 1M KOH using a conventional 3-electrode set-up. A Hg/HgO reference electrode and a Pt foil were used as a reference electrode and counter electrode, respectively. For the solid-state samples, 10 μm thick film of the photopatternable electrolyte was directly patterned onto the VN and tested using the same cell configuration. For the electrochemical testing of interdigitated devices, the 2-electrode measurement was carried out using a standard probe station with the sample stationed on an air table. power densities of solid-state VN MSC compared with other highperformance solid-state MSC systems. Our solid VN MSC demonstrates the highest areal energy and power densities, delivering about 10 μWh cm -2 in the range of 1-10 mW cm -2 . The legend lists the active materials/ionic species/solid host material.

Physical and electrochemical properties of pseudocapacitive electrode materials

Solid-state VN micro-supercapacitor fabrication

The central role played by the photopatternable hydroxide-conducting solid electrolyte in miniaturized EES applications is best shown by providing an overview of the vanadium nitride (VN) fabrication process in Figure S9a. Briefly, the vanadium nitride electrode material is sputtered onto a silicon (Si) / silicon nitride (Si3N4) substrate. An important benefit of VN-based MSC devices is that a separate current collector is not needed because of the high electronic conductivity for VN. The VN layer can then be patterned and selectively etched using conventional photolithography and plasma etching techniques, creating highresolution interdigitated electrodes with a minimum feature size of 10 μm. Subsequently, the liquid precursor for the solid-state electrolyte is spin-coated directly onto the interdigitated device structure to provide ion conduction between electrodes. The pseudocapacitive charge storage mechanism in VN involves a faradaic reaction between V 4+ and V 3+ through an oxy/oxyhydrate couple at the surface. 14 An important factor here is that the highest pseudocapacitive contributions to VN have been observed using basic electrolytes, with hydroxide being the dominant charge-carrying ion. This behavior indicates that fast ion transport of hydroxide in the solid electrolyte material is required in order to achieve rapid charge storage in VN materials.

The process was designed such that for each 3" wafer, 40 individual devices are fabricated and electrically connected in various series and parallel configurations without any additional metal layers. Each device has a 4 mm 2 footprint area and is composed of two VN interdigitated electrodes with a 10 μm spacing between electrode fingers. It is important to note that this spacing of 10 μm, the distance that charge carrying ions travel during charge/discharge, is much shorter than the thickness of commercial separators typically ranging between 20-50 μm. 15 The thickness of the VN layer is approximately 5 µm. Once interdigitated VN microdevices were fabricated, the hydroxide ion conducting solid electrolyte was patterned directly over, covering the entire device to provide ion conduction between the two VN electrodes upon charge/discharge. Table S3. The important design parameters of wafer-scale fabrication of interdigitated MSC devices.

Number of interdigitated devices 40

Number of fingers per device 8.5

Figure 1 f

 1 Figure 1

  Figure 1

Figure 2 f

 2 Figure 2

  Figure 2

FigureFigure 1 .

 1 Figure Captions and Legends

Figure 2 .

 2 Figure2. (a) FT-IR of the photopatterned electrolytes before (red) and after (blue) the hydroxide anion exchange. Once the anions exchanged, the infrared absorbance bands of TFSI anions disappeared as those of OH -emerged. It is should be noted that the characteristic peaks of polymerized cations are still present after this process (TableS2). (b) TGA of ionic liquid modified SU-8. The [AMIM][OH]/SU-8 patternable electrolyte exhibits excellent thermal stability up to 320 °C in air. (c) Mechanical properties of [AMIM][OH]/SU-8 films measured by nanoindentation compared with other hydroxide conducting solid electrolytes.[START_REF] Wu | Preparation and characterization of PVA/PAA membranes for solid polymer electrolytes[END_REF][START_REF] Huo | Quaternary ammonium functionalized poly(aryl ether sulfone)s as separators for supercapacitors based on activated carbon electrodes[END_REF][START_REF] Zeng | Physicochemical properties of alkaline doped polybenzimidazole membranes for anion exchange membrane fuel cells[END_REF] The elastic modulus of [AMIM][OH]/SU-8 was 230 MPa.

Figure 3 .

 3 Figure 3. (a) Impedance spectra of [AMIM][OH]/SU-8 and 1M KOH. The ionic conductivity value determined for the patterned electrolyte was 10 mS cm -1 . (b) Bode plot for the two electrolyte systems. The characteristic time constants determined from the transition frequency for two samples were about 8 s for the liquid electrolyte control and 12.6 s for the patterned electrolyte. (c) CV profile of the [AMIM][OH]/SU-8 solid electrolyte patterned VN electrode at sweep rates of 5 to 20 mV s -1 . The dashed lines of the same color represent the CV results from the 1M KOH liquid electrolyte which serves as a control. (d) Comparison of the areal capacitance vs. sweep rate for planar VN electrodes using 1M KOH and [AMIM][OH]/SU-8 patternable electrolytes. At scan rates below 50 mV s -1 , the areal capacitance values are nearly identical. (e) Longterm galvanostatic cycling of the solid electrolyte patterned VN electrode at a 10 mA cm -2 current density.

Figure 4 .

 4 Figure 4. (a) A photograph of wafer-scale fabricated interdigitated VN MSC devices. 40 individual interdigitated devices are fabricated per a 3" Si wafer. The solid-state device configurations for (b) a single MSC device and (e) two MSC units connected in parallel and in series. (c) Cyclic voltammetry of a symmetric solid-state MSC device at sweep rates of 20 to 200 mV s -1 over 0.6 V window. (d) Galvanostatic charge-discharge curves of a symmetric solid-state MSC device at various areal current densities. The solid-state MSC device exhibits a linear behavior at all current densities. (f) CV curves of MSC devices configured in various series and parallel arrangement. Various configurations show a capacitor-like rectangular feature, indicating an ideal capacitive behavior. (g) Galvanostatic profiles of tandem MSC devices connected in various series or parallel arrangement compared with a single MSC device. (h) Areal energy and

Figure 2 .Figure 3 . 8 Figure 4 .

 2384 Figure 2. (a) 40wt% [AMIM][OH]/SU-8; 40wt% [AMIM][TFSI]/SU-8; SU-8 Reference (b) 40wt% [AMIM][OH]/SU-8 (c) SU-8; 40wt% [AMIM][OH]/SU-8; Poly (Aryl Ether Sulfone); PVA-KOH; PBI Figure 3. (a) 40wt% [AMIM][OH]/SU-8; 1M KOH (b) 40wt% [AMIM][OH]/SU-8; 1M KOH (c) 20 mV/s; 10 mV/s; 5 mV/s; 1M KOH Liquid Electrolyte Control (d) 1M KOH; 40wt% [AMIM][OH]/SU-8 Figure 4. (c) 200 mV/s; 100 mV/s; 50 mV/s; 20 mV/s (d) 50 mA cm -2 ; 20; 10; 5; 2 (f) Single Device; 2 Devices in Series; 2 Devices in Parallel; 2 x 2 Series and Parallel (g) Single Device; 2 Devices in Series; 2 Devices in Parallel (h) This work: VN/AMIM-OH/SU-8; [43]: Graphene/BMIM-TFSI/SiO2; [19]: MnO2/EMIM-TFSI/PVDF; [44]: C/EMI-TFSI/SiO2; [45]: MnO2/LiCl/PVA; [46]: RuO2/H2SO4/PVA

Figure S5 .

 S5 FigureS5. Schematic representation of a pseudocapacitive charge storage device. Pseudocapacitive materials such as RuO2, MnO2, and VN, store electrochemical energy primarily via a faradaic process involving surface or near-surface redox reactions. Due to this characteristic, pseudocapacitive materials exhibit capacitor-like electrochemical features.12,13

2 [

 2 Figure S9. (a) Schematic representation of the process for fabricating solid-state VN MSCs using the photopatternable [AMIM][OH] electrolyte. Both electrode and electrolyte materials are formed through microfabrication techniques. (b) A fabrication approach to form solid-state MSC devices. Both electrode and electrolyte materials can be patterned to high-resolution features using microfabrication techniques.

  All sample preparation was carried out in class 100 clean room with yellow lighting to reduce the exposure of the materials to shorter wavelengths. [AMIM][TFSI] SU 8 was prepared by mixing 40 weight percent [AMIM][TFSI] and SU 8 prior to deposition. Films prepared for electrochemical measurements were spin coated onto flat substrates (i.e. Si/SiO2 or VN) at 500 rpm for 5 s followed by 3000 rpm for 30 s. Samples were then soft baked at 95 °C for 5 min for solvent removal prior to patterning. Features were patterned using a Karl Suss MA6 Contact Aligner and photomask. The [AMIM][TFSI] SU 8 samples were subjected to a total of 1600 mW of UV exposure at 365 nm. The films were then postexposure baked at 95 °C for 5 min to crosslink the material

	1-Allyl-3H-imidazolium bis(trifluoromethanesulfonyl)imide ([AMIM][TFSI], 99% purchased
	from Solvionic), SU-8 3000 (purchased from MicroChem), KOH (purchased from Alfa-Aesar), and
	dimethyl sulfoxide (DMSO, 99.9% purchased from Sigma Aldrich) were used as received.
	Photopatternable Hydroxide-Conducting Solid Electrolyte Preparation:

(40% [AMIM][TFSI]/SU-8). UV exposed samples were then developed. For the anion exchange process, photopatterned [AMIM][TFSI]/SU-8 electrolytes were soaked in DMSO to swell the crosslinked polymer for a minimum of 1 hr. Subsequent soaking in 1M KOH replaced TFSI -in the structure with OH -(40% [AMIM][OH]/SU-8).

Table S2

 S2

	photopatternable electrolyte was shown to have a decent electrochemical stability window with no residual
	current between -1.2 and 0.2 V (vs. Hg/HgO).
	. Characteristic IR absorption bands of [AMIM][TFSI] and their assignments
		Frequency
		(cm -1 )	Dominant Vibrational assignment
		569	CF3 antisymmetric bend
		600	SO2 antisymmetric bend
		611	SO2 antisymmetric bend
		650	SNS bend
	Anion	789	CS stretch
	Assignment	1051	SNS antisymmetric stretch
		1132	SO2 symmetric stretch
		1182	CF3 antisymmetric stretch
		1331	SO2 antisymmetric stretch
		1348	SO2 antisymmetric stretch
	Cation	752	Ring HCCH symmetric bend

Table S4 .

 S4 Comparison of interdigitated MSCs based on solid and liquid electrolytes * Energy density at a given power density. Power density is in parentheses. Energy density given at low 130 and high values of power density.

	Solid Electrolyte
	Electrode	Electrolyte Materials	Energy Density* [ Wh/cm2]	Cycle Life (Retention) Main Text
	Materials		(Power Density in [mW/cm2])		Reference
	VN	[AMIM][OH]/SU-8	7.6 (9.1)	10,000 (80%)	This Work
	MnO2	[EMIM][TFSI]/PVDF	12.8 (0.8) 3.2 (3.8)	30,000 (96.2%)	19
	Graphene		4.5 (0.7)
		[BMIM][TFSI]/Fumed	4.4 (45.4)	10,000 (96%)	43
		Silica	4.92 (0.4)
	Pyrolyzed	[EMIM][TFSI]/Fumed	3.1 (3.1)	10,000 (94%)	44
	Carbon	Silica	4.2 (0.8)
	MnO2	LiCl/PVA	0.8 (3.8)	4,000 (92%)	45
			2.2 (0.6)
	RuO2	H2SO4/PVA	0.6 (2.2)	10,000 (90%)	46
			3.3 (1.2)
	Liquid Electrolyte
	Electrode	Electrolyte Materials	Energy Density [uWh/cm2]	Cycle Life (Retention) Main Text
	Materials		(Power Density in [mW/cm2])		Reference
	VN	1M KOH	10.6 (0.2)	50,000 (80%)	9
	CDC	1M H2SO4	0.9 (4.1) 1.6 (0.3)	10,000 (100%)	7
			1.2 (8.9)

Acknowledgements

The research was supported by the Office of Naval Research (N00014-19-1-2113). This project has received financial support from the CNRS through the MITI interdisciplinary programs. The authors also want to thank the ANR STORE-EX and the French network on electrochemical energy storage (RS2E) for the financial support. The French RENATECH network is greatly acknowledged for the use of microfabrication facilities.

The authors declare no competing interests.

Author contributions

Declaration of interests

Supplemental Text and Figures

Survey of miniaturized electrochemical energy storage systems

Miniaturized electrochemical energy storage (EES) systems, in general, have great design flexibility and have been manufactured by a range of fabrication techniques, leading to various device form factors with multiple functionalities without deterioration in performance. [1][2][3] Recent advances in the community have included the demonstration of novel fabrication approaches and unique battery designs, and the incorporation of solid electrolytes (Table S1). [4][5][6][7][8][9][10][11] Table S1 Ring in-plane antisymmetric stretch, CH3(N) stretch Figure S3. The nickel cobalt oxide material has been prepared by the microwave assisted synthesis. The corresponding XRD (a) shows low crystallinity of the material that did not affect the electrochemistry. CV curves of NiCo2O4 that were obtained using (b) OH -conducting patternable electrolytes and (c) 1M KOH control liquid electrolytes. The redox behavior is similar in the two electrolytes, confirming that there is hydroxide conduction through the solid electrolyte. (d) Graphical diagram highlighting differences between the sample preparation of a solid-state working electrode versus a control electrode tested in liquid electrolytes.


Manimozhiyan Arumugam

Jeroen Raes

Eric Pelletier

Denis Le Paslier

Takuji Yamada

Daniel R Mende

Gabriel R Fernandes

Julien Tap

Thomas Bruls

Jean-Michel Batto

Marcelo Bertalan

Natalia Borruel

Francesc Casellas

Leyden Fernandez

Laurent Gautier

Torben Hansen

Masahira Hattori

Tetsuya Hayashi

Michiel Kleerebezem

Ken Kurokawa

Marion Leclerc

Florence Levenez

Chaysavanh Manichanh

H Bjørn Nielsen

Trine Nielsen

Nicolas Pons

Julie Poulain

Junjie Qin

Thomas Sicheritz-Ponten

Sebastian Tims

David Torrents

Edgardo Ugarte

Erwin G Zoetendal

Jun Wang

Francisco Guarner

Oluf Pedersen

Willem M De Vos

Søren Brunak

Joel Doré

Metahit Consortium

Jean Weissenbach

S Dusko Ehrlich

Peer Bork

Enterotypes of the human gut microbiome

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

Various studies of the human intestinal tract microbiome. based on the 16S ribosomal RNAencoding gene, reported species diversity within and between individuals [START_REF] Eckburg | Diversity of the Human Intestinal Microbial Flora[END_REF][START_REF] Hayashi | Phylogenetic Analysis of the Human Gut Microbiota Using 16S rDNA Clone Libraries and Strictly Anaerobic Culture-Based Methods[END_REF][START_REF] Lay | Colonic Microbiota Signatures across Five Northern European Countries[END_REF] and first metagenomics studies characterized the functional repertoire of the microbiomes of several American [START_REF] Gill | Metagenomic analysis of the human distal gut microbiome[END_REF][START_REF] Turnbaugh | A core gut microbiome in obese and lean twins[END_REF] and Japanese 6 individuals. Although a general consensus about the phylum level composition in the human gut is emerging [START_REF] Eckburg | Diversity of the Human Intestinal Microbial Flora[END_REF][START_REF] Lay | Colonic Microbiota Signatures across Five Northern European Countries[END_REF][START_REF] Zoetendal | High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota[END_REF] , the variation in species composition [START_REF] Eckburg | Diversity of the Human Intestinal Microbial Flora[END_REF][START_REF] Hayashi | Phylogenetic Analysis of the Human Gut Microbiota Using 16S rDNA Clone Libraries and Strictly Anaerobic Culture-Based Methods[END_REF] and gene pools [START_REF] Turnbaugh | A core gut microbiome in obese and lean twins[END_REF][START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF] within the human population is less clear. Furthermore, it is unknown whether inter-individual variation manifests itself as a continuum of different community compositions or whether individual gut microbiota congregate around some preferred, balanced and stable community compositions that can be classified. Studying such questions is complicated by the complexity of sampling, DNA preparation, processing, sequencing and analysis protocols [START_REF] Raes | Molecular eco-systems biology: towards an understanding of community function[END_REF] as well as by varying physiological, nutritional and environmental conditions. To analyze the feasibility of comparative metagenomics of the human gut across cohorts and protocols and to obtain first insights in commonalities and differences between gut microbiomes across different populations, we Sanger-sequenced 22 European metagenomes from Danish, French, Italian and Spanish individuals that were selected for diversity (Supplementary Notes Section 1), and combined them with existing Sanger (13 Japanese 6 , 2 American 4 ) and 454 (2 American 5 ) gut datasets -totaling 39 individuals.

Global phylogenetic and functional variation of intestinal metagenomes

The vast majority of sequences in the newly sequenced 22 European samples belong to bacteria -only 0.14% of the reads could be classified as human contamination, all other eukaryotes together only comprised 0.5%, archaea 0.8% and viruses up to 5.8% (see Supplementary Notes Section 2.1 for details).

To investigate the phylogenetic composition of the 39 samples from 6 nationalities, we mapped metagenomic reads, using DNA sequence homology, to 1511 reference genomes (Supplementary Table 3) including 379 publicly available human microbiome genomes generated through the NIH Human Microbiome Project [START_REF] Nelson | A catalog of reference genomes from the human microbiome[END_REF] and the European MetaHIT consortium [START_REF]MetaHIT draft bacterial genomes at the Sanger Institute[END_REF] (Supplementary Methods Section 4.1). To consistently estimate the functional composition of the samples, we annotated the predicted genes from the metagenomes using eggNOG [START_REF] Muller | eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced nonsupervised orthologous groups, species and functional annotations[END_REF] orthologous groups (Supplementary Methods Section 6.2). We ensured that comparative analysis using these procedures was not biased by dataset origin, sample preparation, sequencing technology and quality filtering (see Supplementary Notes Section 1). We also investigated whether the relatively low and somewhat arbitrary amounts of sequence per sample (between 53-295 Mb) bias our results: we assigned habitat information to 1368 out of the 1511 reference genomes, distinguished between orthologous groups from gut and non-gut species and conclude that our dataset captures most of the functions from gut species even though functions from 'non-gut' species still accumulated with each additional sample (Fig. 1a; see Supplementary Notes Section 1.3).

We then characterized the phylogenetic variation across samples at the genus and phylum levels, and functional variation at gene and functional class levels. As infants are known to have very heterogeneous, unstable and distinctive microbiota [START_REF] Kurokawa | Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes[END_REF][START_REF] Palmer | Development of the Human Infant Intestinal Microbiota[END_REF] , we excluded the four respective Japanese samples from the analysis. Using calibrated similarity cutoffs (Supplementary Figure 1), on average, 52.8% of the fragments in each sample could be robustly assigned to a genus in our reference genome set (ranging from 22% to 80.5%), and 80% could be assigned to a phylum (ranging from 64.9% to 91%) implying that the trends observed (Fig. 1b) represent a large fraction of the metagenome.

The phylogenetic composition of the newly sequenced samples confirms that the Firmicutes and Bacteroidetes phyla constitute the vast majority of the dominant human gut microbiota [START_REF] Zoetendal | High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota[END_REF] (Fig. 1b, inset). Bacteroides was the most abundant but also most variable genus across samples (Fig. 1b; Supplementary Notes Section 2.2), agreeing with previous observations [START_REF] Kurokawa | Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes[END_REF][START_REF] Tap | Towards the human intestinal microbiota phylogenetic core[END_REF] . Our function identification protocol led to a high functional assignment rate: 63.5% of all predicted genes in the Sanger-sequenced samples analyzed (41% of all predicted genes in two samples obtained by pyrosequencing; Supplementary Table 5) can be assigned to orthologous groups (OGs), and OG abundance patterns again agree with previous observations [START_REF] Kurokawa | Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes[END_REF][START_REF] Jensen | STRING 8--a global view on proteins and their functional interactions in 630 organisms[END_REF]

Highly abundant functions from low-abundance microbes

Microbes in the human gut undergo selective pressure from the host as well as from microbial competitors. This typically leads to a homeostasis of the ecosystem in which some species occur in high and many in low abundance [START_REF] Dethlefsen | The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing[END_REF] (the "long-tail" effect, as seen in Fig. 1b), with some low-abundance species, like methanogens [START_REF] Walker | Say hello to our little friends[END_REF] , performing specialized functions beneficial to the host. Metagenomics enables us to study the presence of abundant functions shared by several low-abundance species, which could shed light on their survival strategies in the human gut. In the samples analyzed here, the most abundant molecular functions generally trace back to the most dominant species. However, we identified some abundant orthologous groups that are contributed primarily by low abundance genera (see Supplementary Figure 2, Supplementary Table 6 and Supplementary Notes Section 3). For example, low abundance Escherichia contribute over 90% of two abundant proteins associated with bacterial pilus assembly, FimA (COG3539) and PapC (COG3188), found in one individual (IT-AD-5). Pili enable the microbes to colonize the epithelium of specific host organs; they help microbes to stay longer in the human intestinal tract by binding to the human mucus or mannose sugars present on intestinal surface structures [START_REF] Krogfelt | Bacterial adhesion: genetics, biogenesis, and role in pathogenesis of fimbrial adhesins of Escherichia coli[END_REF] . They are also key components in the transfer of plasmids between bacteria through conjugation, often leading to exchange of protective functions such as antibiotic resistance [START_REF] Krogfelt | Bacterial adhesion: genetics, biogenesis, and role in pathogenesis of fimbrial adhesins of Escherichia coli[END_REF] . Pili can thus provide multiple benefits to these low-abundance microbes in their efforts to survive and persist in the human gut. This example illustrates that abundant species or genera cannot reveal the entire functional complexity of the gut microbiota. More reference genomes will facilitate better taxonomic assignments from samples and thus the detection of more low abundance species. However, there is not much room for as yet undetected, abundant genera. Even with our limited genus assignment rate of 52.8% of all reads, we estimate that we miss another 30.7% of the already classified genera due to our strict assignment criteria (Supplementary Figure 1), i.e. only 16.5% of all reads are likely to belong to hitherto unknown genera.

Robust clustering of samples across nations: Identification of enterotypes

To get an overview of the species variation we used phylogenetic profile similarities obtained by mapping metagenomic reads to the 1511 reference genomes (Fig. 2a, see Supplementary Methods Section 4.1). We excluded the two American Sanger-sequenced samples 4 from further analysis because of an unusual, very low fraction of Bacteroidetes, and suspected technical artifacts [START_REF] Salonen | Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis[END_REF] . Multidimensional cluster analysis and Principal Component Analysis (PCA) revealed that the remaining 33 samples formed three distinct clusters which we designate enterotypes (see Supplementary Notes Section 4.1, Supplementary Figure 3a and Supplementary Table 8). Each of these three enterotypes are identifiable by the variation in the levels of one of three genera: Bacteroides (enterotype 1), Prevotella (enterotype 2) and Ruminococcus (enterotype 3; Fig. 2a and2d), which was reproduced using independent array-based HITChip 20 data in a subset of 22 European samples (Supplementary Figure 4 and Supplementary Notes Section 4.5). The same analysis on two larger published gut microbiome datasets of different origins (16S pyrosequencing data from 154 American individuals [START_REF] Turnbaugh | A core gut microbiome in obese and lean twins[END_REF] and Illumina-based metagenomics data from 85 Danish individuals 8 , Supplementary Methods Section 5) shows that these datasets could also be represented best by three clusters (Supplementary Figure 3b andc, Supplementary Table 9 and Supplementary Table 10). Two of these are also driven by Bacteroides and Prevotella, while the third cluster is mostly driven by related groups of the order Clostridiales, Blautia and unclassified Lachnospiraceae in the 16S rDNA and Illumina data, respectively (Fig. 2b and2c). This can be explained by a different reference data set in case of 16S rDNA data, different mapping behavior of short reads in case of the Illumina data or current taxonomic uncertainties in the Lachnospiraceae and Ruminococcaceae clades (see Supplementary Notes Section 4.2). The differences might also hint at community subpopulations within this enterotype, which might only be detectable with substantially more samples. Correlation analysis of the Sanger data revealed that abundances of each of the three discriminating genera strongly correlate (that is they co-occur or avoid each other) with those of other genera (Fig. 2d; see Supplementary Methods Section 11), suggesting that the enterotypes are in fact driven by groups of species that together contribute to the preferred community compositions.

We further demonstrate the robustness of the enterotypes using two distinct statistical concepts. First we used the silhouette coefficient [START_REF] Rousseeuw | Silhouettes: A graphical aid to the interpretation and validation of cluster analysis[END_REF] to validate that the three clusters are superior to clusterings obtained from various randomizations of the genus profile data, suggesting a potential role for the interactions between co-occurring genera (see Supplementary Figure 5 and Supplementary Notes Section 4.3). Second we used supervised learning and cross validation to establish that these clusters have non-random characteristics that can be modeled and subsequently used to classify new samples (learning on clusters from randomized genus profiles led to considerably worse classification performance; see Supplementary Figure 6 and Supplementary Notes Section 4.4). These consistent results suggest that enterotypes will be identifiable in human gut metagenomes also from larger cohorts.

We then clustered the 33 samples using a purely functional metric: the abundance of the assigned orthologous groups (Fig. 3a). Remarkably, this clustering also showed a similar grouping of the samples with only minor differences (5 samples placed in different clusters compared to Fig. 2a) indicating that function and species composition roughly coincide with some exceptions such as Spanish sample ES-AD-3 whose genus composition belongs to enterotype 2 while its functional composition is similar to members of enterotype 1. This individual has high levels of phage-related genes compared to the other samples (see Supplementary Figure 7), hinting at partial temporal variability and dynamics of the microbiota, and perhaps indicating phage or virus bursts.

The robustness and predictability of the enterotypes in different cohorts and at multiple phylogenetic and functional levels suggests that they are the result of well-balanced, defined microbial community compositions of which only a limited number exist across individuals. These enterotypes are not as sharply delimited as, for example, human blood groups; they are rather densely populated areas in a multidimensional space of community composition. They are nevertheless likely to characterize individuals, in line with previous reports that gut microbiota is rather stable in individuals and can even be restored after perturbation [START_REF] Vanhoutte | Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers[END_REF][START_REF] Tannock | Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing Lactobacillus rhamnosus DR20[END_REF][START_REF] Seksik | Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon[END_REF][START_REF] Costello | Bacterial Community Variation in Human Body Habitats Across Space and Time[END_REF] .

Phylogenetic and functional variation between enterotypes

To determine the phylogenetic and functional basis of the enterotypes, we investigated in detail their differences in composition at the phylum, genus, gene and pathway level as well as correlations in abundance of co-occurring genera (Figs. 2 and3; also see Supplementary Methods Sections 10, 11 and 12). Enterotype 1, containing 8 samples, is enriched in Bacteroides (p<0.01; Supplementary Figure 8), which co-occurs, for example, with Parabacteroides (see Supplementary Table 11 for enriched genera and Fig. 2e for correlation networks of co-occurring genera in each enterotype). The drivers of this enterotype seem to derive energy primarily from carbohydrates and proteins through fermentation, since these closely related genera have a very broad saccharolytic potential [START_REF] Martens | Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm[END_REF] and since genes encoding enzymes involved in the degradation of these substrates (galactosidases, hexosaminidases, proteases) along with glycolysis and pentose phosphate pathways are enriched in this enterotype (see Supplementary Table 12 and Supplementary Table 13). Enterotype 2 contains 6 samples and is enriched in Prevotella (p<0.01; Supplementary Figure 9) and the co-occurring Desulfovibrio, who can act in synergy to degrade mucin glycoproteins present in the mucosal layer of the gut: Prevotella is a known mucin-degrader and Desulfovibrio could enhance the rate-limiting mucin desulfation step by removing the sulfate [START_REF] Wright | Prevotella enzymes involved in mucin oligosaccharide degradation and evidence for a small operon of genes expressed during growth on mucin[END_REF] . Enterotype 3 is the most frequent one and is enriched in Ruminococcus (p<0.01; Supplementary Figure 10) as well as co-occurring Akkermansia, both known to comprise species able to degrade mucins [START_REF] Derrien | Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium[END_REF] . It is also enriched in membrane transporters, mostly of sugars, suggesting the efficient binding of mucin and its subsequent hydrolysis as well as uptake of the resulting simple sugars by these genera. The enriched genera suggest that enterotypes employ different routes to generate energy from fermentable substrates available in the colon reminiscent of a potential specialization in ecological niches or guilds. In addition to the conversion of complex carbohydrates into absorbable substrates, the gut microbiota is also beneficial to the human host by producing vitamins. Although all the vitamin metabolism pathways are represented in all samples, enterotypes 1 and 2 were enriched in biosynthesis of different vitamins: biotin (Fig. 3b), riboflavin, pantothenate and ascorbate in the former, and thiamine (Fig. 3c) and folate in the latter. These phylogenetic and functional differences among enterotypes thus reflect different combinations of microbial trophic chains with a likely impact on the synergistic inter-relations with the human hosts.

Functional biomarkers for host properties

Enterotypes do not seem to differ in functional richness (Supplementary Figure 11), and virtually none of several measured host properties, namely nationality, gender, age or body mass index (BMI), significantly correlates with the enterotypes (with the exception of enterotype 1 which is enriched in Japanese individuals). However, some strong correlations do occur between host properties and particular functions, at the genes or module level (a module is a part of a pathway that is functionally tightly interconnected, see Supplementary Methods Sections 6.3, 13 and Supplementary Notes Section 6). The only significant correlation between a host property and a taxonomic group is a negative one between age and the abundance of an unknown Clostridiales genus (p<0.02) containing three obligate anaerobes (Supplementary Figure 12a; see Supplementary Notes Section 6.2). It should be noted that age is not constant across the nationalities (in our dataset, Italians are relatively old and Japanese young), but that individuals did not stratify by nationality, suggesting that this is not a confounding factor. Our data did not reveal any correlation between BMI and the Firmicutes/Bacteroidetes ratio and we thus cannot contribute to the ongoing debate on the relation between this ratio and obesity [START_REF] Ley | Microbial ecology: human gut microbes associated with obesity[END_REF][START_REF] Schwiertz | Microbiota and SCFA in Lean and Overweight Healthy Subjects[END_REF] .

In contrast to the little phylogenetic signal, we found several significant functional correlations with each of the host properties studied (after correcting for multiple testing to avoid artifacts; see Supplementary Methods Section 13), suggesting that metagenomicsderived functional biomarkers might be more robust than phylogenetic ones. For example, the abundance of 10 orthologous groups (OGs) varies more between than within nationalities (Supplementary Table 14) although overall, the functional composition in total was remarkably similar among the nations (also with respect to the functional core; see Supplementary Figure 13). For gender, we find five functional modules and one OG that significantly correlate (p<0.05; e.g., enriched aspartate biosynthesis modules in males; see Supplementary Table 16). In addition, twelve OGs significantly correlate with age (Supplementary Table 17). For instance, starch degradation enzymes such as glycosidases and glucan phosphorylases increase with age (which could be a reaction to decreased efficiency of host breakdown of dietary carbohydrates with age [START_REF] Woodmansey | Intestinal bacteria and ageing[END_REF] ) and so does the secA preprotein translocase (Supplementary Figure 14). Conversely, an OG coding for the facultative sigma-24 subunit of RNA polymerase, which drives expression under various stress responses and is linked to intestinal survival [START_REF] Kovacikova | The alternative sigma factor sigma(E) plays an important role in intestinal survival and virulence in Vibrio cholerae[END_REF] , decreases with age (Fig. 4a). One explanation for this could be the reduced need for stress response in the gut due to the ageassociated decline in host immune response [START_REF] Fujihashi | Mucosal immunosenescence: new developments and vaccines to control infectious diseases[END_REF] (immunosenescence). Our analyses also identified three marker modules that correlate strongly with the hosts' BMI (Supplementary Table 19, Supplementary Figure 14), two of which are ATPase complexes, supporting the link found between the gut microbiota's capacity for energy harvest and host's obesity [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF] . Interestingly, functional markers found by a data-driven approach (derived from the metagenomes without previous knowledge) gave much stronger correlations than genes for which a link would be expected (e.g. SusC/SusD, involved in starch utilization [START_REF] Martens | Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm[END_REF] ; Fig. 4b). Linear models combining the abundance of only a few functional modules correlate even better with host properties (Fig 4c,d). It should be noted that given the possibility of many confounding variables due to the heterogeneity and size of our cohort, these observations will need to be substantiated using larger, independent cohorts in the future. Furthermore, patterns in metagenomics data can (partly) reflect indirect factors [START_REF] Raes | Molecular eco-systems biology: towards an understanding of community function[END_REF] such as genome size [START_REF] Raes | Prediction of effective genome size in metagenomic samples[END_REF] (the smaller the average genome size of a sample, the higher would be the relative fraction of single copy genes therein), which does not matter for diagnostics though.

While individual host properties don't explain the enterotypes, the latter might be driven by a complex mixture of functional properties, by host immune modulation or by hitherto unexplored physiological conditions such as transit time or pH of luminal contents. Furthermore, the three major enterotypes could be triggered by the three distinct pathways for hydrogen disposal [START_REF] Gibson | Alternative pathways for hydrogen disposal during fermentation in the human colon[END_REF] (Supplementary Notes Section 6.4). Indeed, despite their low abundance, Methanobrevibacter (a methanogen) and Desulfovibrio (a known sulfatereducer) are enriched in enterotypes 3 and 1, respectively.

Taken together, we have demonstrated the existence of enterotypes in the human gut microbiome and have identified three of them that vary in species and functional composition using data that spans several nations and continents. As our current data do not reveal which environmental or even genetic factors are causing the clustering, and as fecal samples are not representative of the entire intestine, we anticipate that the enterotypes introduced here will be refined with deeper and broader analysis of individuals' microbiomes. Presumably, enterotypes are not limited to humans but also occur in animals. Their future investigations might well reveal novel facets of the human and animal symbiotic biology and lead to the discovery of the microbial properties correlated with the health status of individuals. We anticipate that they might allow classification of human groups that respond differently to diet or drug intake. The enterotypes appear complex, are probably not driven by nutritional habits and cannot simply be explained by host properties such as age or BMI, although there are functional markers such as genes or modules that correlate remarkably well with individual features. The latter might be utilizable for diagnostic and perhaps even prognostic tools for numerous human disorders, for instance colorectal cancer and obesity-linked co-morbidities such as metabolic syndrome, diabetes and cardio-vascular pathologies.

Methods summary

Sample collection

Human fecal samples from European individuals were collected and frozen immediately, and DNA was purified as described previously [START_REF] Godon | Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis[END_REF] .

Sequencing

Random shotgun DNA libraries of 3kb were Sanger-sequenced using standard protocols established at Genoscope.

Sequence processing

Cloning vector, sequencing primers and low quality bases were end-trimmed from raw Sanger reads, and possible human DNA sequences were removed. Reads were processed by the SMASH comparative metagenomics pipeline [START_REF] Arumugam | SmashCommunity: a metagenomic annotation and analysis tool[END_REF] for assembly and gene prediction.

Phylogenetic annotation

Phylogenetic annotation of samples was performed by (1) aligning reads (Sanger/Illumina) against a database of 1511 reference genomes (listed in Supplementary Table 3) or ( 2) classifying 16S rDNA reads using RDP classifier [START_REF] Wang | Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[END_REF] . Genus and phylum abundance was estimated after normalizing for genome size for the former, and for 16S gene copy number for the latter.

Functional annotation

Genes were functionally annotated using BLASTP against eggNOG (v2) and KEGG (v50) databases. Protein abundances were estimated after normalizing for protein length. Functional abundance profiles at eggnog-, KEGG orthologous group-, functional moduleand pathway-level were created.

Clustering and classification

Samples were clustered using Jensen-Shannon distance and partitioning around medoid (PAM) clustering. Optimal number of clusters was estimated using Calinski-Harabasz (CH) index. We used the silhouette validation technique for assessing the robustness of clusters. Additionally, within a cross-validation scheme, we trained predictive decision tree models on clusters obtained using the same clustering method and evaluated the classification of hold-out samples by accuracy, average precision and average precision gain.

Statistics

Correlations between metadata and feature abundances were computed as described previously [START_REF] Gianoulis | Quantifying environmental adaptation of metabolic pathways in metagenomics[END_REF] , based on multiple-testing corrected pairwise Spearman correlation analysis and stepwise regression for multi-feature model building. For categorical metadata and enterotype comparisons, samples were pooled into bins (male/female, obese/lean, one enterotype/rest, specific nationality/rest etc.) and significant features were identified using Fisher's exact test with multiple testing correction of p-values.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material. Inset: phylum abundance box plot. Genus and phylum level abundances were measured using reference genome based mapping with 85% and 65% sequence similarity cutoffs. Unclassified genera under a higher rank are marked by asterisks. (c) Orthologous group (OG) abundance variation box plot for the 30 most abundant OGs as determined by assignment to eggNOG [START_REF] Muller | eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced nonsupervised orthologous groups, species and functional annotations[END_REF] . OGs are colored by their respective functional category (see inset for color key). Inset: abundance box plot of 24 functional categories.

  (e.g. histidine kinases make up the largest group; Fig 1c; Supplementary Notes Section 2.3).

Footnotes#

  Correspondence and requests for materials should be addressed to P.B. (bork@embl.de) or S.D.E. (dusko.ehrlich@jouy.inra.fr). * These authors contributed equally † Lists of authors and affiliations of the additional MetaHIT members appear at the end of the paper.

Fig. 1 .

 1 Fig. 1. Functional and phylogenetic profiles of human gut microbiome (a) Simulation of the detection of distinct orthologous groups (OGs) when increasing the number of individuals (samples). Complete genomes were classified by habitat-information and the OGs divided into those that occur in known gut-species (red) and those that have not yet associated to gut (blue). The former are close to saturation when sampling 35 individuals (excluding infants) whereas functions from non-gut (probably rare and transient) species are not. (b) Genus abundance variation box plot for the 30 most abundant genera as determined by read abundance. Genera are colored by their respective phylum (see inset for color key). Inset: phylum abundance box plot. Genus and phylum level abundances were measured

Fig. 2 .

 2 Fig. 2. Phylogenetic differences between enterotypes Between class analysis, which visualizes results from Principal Component Analysis and clustering, of the genus compositions of (a) 33 Sanger metagenomes estimated by mapping the metagenome reads to 1511 reference genome sequences using an 85% similarity threshold, (b) Danish subset containing 85 metagenomes from a published Illumina dataset 8 and (c) 154 pyrosequencing-based 16S sequences 5 reveal three robust clusters that we call enterotypes. Two principal components are plotted using the ade4 package in R with each sample represented by a filled circle. The center of gravity for each cluster is marked by a rectangle and the colored ellipse covers 67% of the samples belonging to the cluster. (d) Abundances of the main contributors of each enterotype from the Sanger metagenomes. (e) Co-occurrence networks of the three enterotypes from the Sanger metagenomes. Unclassified genera under a higher rank are marked by asterisks in (b) and (e).

Fig. 3 .

 3 Fig. 3. Functional differences between enterotypes (a) Between class analysis (see Fig. 2) of orthologous group (OG) abundances showing only minor disagreements with enterotypes (transparent circles indicate the differing samples). The blue cloud represents the local density estimated from the coordinates of OGs; positions of selected OGs are highlighted. (b) Four enzymes in the biotin biosynthesis pathway (COG0132, COG0156, COG0161 and COG0502) are overrepresented in enterotype 1. (c) Four enzymes in the thiamine biosynthesis pathway (COG0422, COG0351, COG0352 and COG0611) are overrepresented in enterotype 2. (d) Six enzymes in the heme biosynthesis pathway (COG0007, COG0276, COG407, COG0408, COG0716 and COG1648) are overrepresented in enterotype 3.

Fig. 4 .

 4 Fig. 4. Correlations with host properties (a) Pairwise correlation of RNA polymerase facultative sigma24 subunit (COG1595) with age (p=0.03, rho=-0.59). (b) Pairwise correlation of SusD, a family of proteins that bind glycan molecules before they are transported into the cell, and body mass index (p=0.27, rho=-0.29, weak correlation). (c) Multiple OGs (COG0085, COG0086, COG0438 and COG0739; see Supplementary Table18) significantly correlating with age when combined into a linear model (see Supplementary Methods Section 13 and ref. 40 for details; p= 2.75e-05, adjusted R 2 =0.57). (d) Two modules, ATPase complex and ectoine biosynthesis (M00051), significantly correlating with BMI when combined into a linear model (p= 6.786e-06, adjusted R 2 =0.82).

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  ) significantly correlating with age when combined into a linear model (see Supplementary Methods Section 13 and ref. 40 for details; p= 2.75e-05, adjusted R 2 =0.57). (d) Two modules, ATPase complex and ectoine biosynthesis (M00051), significantly correlating with BMI when combined into a linear model (p= 6.786e-06, adjusted R 2 =0.82).

Nature. Author manuscript; available in PMC 2013 July 31.

Acknowledgments

The authors are grateful to Christopher Creevey, Gwen Falony and members of the Bork group at EMBL for helpful discussions and assistance. We thank the EMBL IT core facility and Yan Yuan for managing the highperformance computing resources. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013): MetaHIT, grant agreement HEALTH-F4-2007-201052 and from EMBL. Obese/non-obese volunteers for MicroObes study were recruited from the SU.VI.MAX cohort study coordinated by S. Hercberg, and metagenome sequencing was funded by ANR; volunteers for MicroAge study were recruited from the CROWNALIFE cohort study coordinated by A. Cresci, and metagenome sequencing was funded by GenoScope. Ciberehd is funded by the Instituto de Salud Carlos III (Spain). The study was supported by grants from the Lundbeck Foundation Centre for Applied Medical Genomics in Personalized Disease Prediction, Prevention and Care (LuCAMP). JR is supported by the IWIOB and the Odysseus programme of the Fund for Scientific Research Flanders (FWO). BGI was partially funded by the International Science and Technology Cooperation Project in China (0806). We are thankful to the Human Microbiome Project for generating the reference genomes from human gut microbes and the International Human Microbiome Consortium for stimulating discussions and the exchange of data.

12 Istituto Europeo di Oncologia, 20100 Milan, Italy.