Sarah Coffinet
email: scoffinet@marum.de

Arnaud Huguet

Nikolai Pedentchouk

Laurent Bergonzini

David Williamson

Christelle Anquetil

Martin Jones

Amos D Majule

Thomas Wagner

Sylvie Derenne

Christine Omuombo

Evaluation of branched GDGTs and leaf wax n-alkane δ2H as (paleo) environmental proxies in East Africa

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

Introduction

The East African Rift System (EARS) is one of the best examples of an active rift system. The initial rifting in the area dates back to the Permo-Triassic and Cretaceous [START_REF] Macgregor | History of the development of the East African Rift System: A series of interpreted maps through time[END_REF]. The development of this complex system with several phases of rifting dramatically changed the East African landscape. Yet its potential influence on the vegetation and local and African climate is hardly constrained. Previous modelling [START_REF] Sepulchre | Tectonic Uplift and Eastern Africa Aridification[END_REF] postulated that the rise of the EARS should have greatly contributed to the long term aridification of East Africa. However, these simulations only compared 2 extreme scenarios: (i) no elevation and (ii) high elevationreflecting the lack of a well constrained history of the EARS rise. Moreover, paleo-climate reconstructions in East Africa have long been hampered by 2 challenges: (i) traditional hydrological proxies, such as lake level studies, are influenced not only by changes in the amount of precipitation but also by ambient temperature; (ii) temperature changes are poorly constrained because few sedimentary proxies are sensitive enough to accurately record the small scale temperature variations in tropical lakes [START_REF] Verschuren | Half-precessional dynamics of monsoon rainfall near the East African Equator[END_REF].

Recently, novel proxies were developed based on the analysis of fossil organic compounds containing environmental information in their structures. Notably, leaf wax n-alkane δ²H (δ²H wax ) was proposed as a (paleo) elevation proxy [START_REF] Jia | Soil n-alkane δD vs. altitude gradients along Mount Gongga, China[END_REF] while branched glycerol dialkyl glycerol tetraether (br GDGT) membrane lipids are increasingly used as a (paleo) temperature proxy [START_REF] Weijers | Environmental controls on bacterial tetraether membrane lipid distribution in soils[END_REF]. The combined use of these 2 proxies could thus help to better constrain the relationships between topography and climate in East Africa.

Long chain n-alkanes are produced primarily by higher terrestrial plants [START_REF] Eglinton | Leaf epicuticular waxes[END_REF] and their 2 H/ 1 H ratio, typically expressed as δ²H wax , is linked with the 2 H/ 1 H ratio of the plant water source (e.g. [START_REF] Sauer | Compound-specific D/H ratios of lipid biomarkers from sediments as a proxy for environmental and climatic conditions[END_REF]. Because n-alkanes are well preserved in the geological archives [START_REF] Eglinton | Molecular proxies for paleoclimatology[END_REF] and their hydrogen not easily exchangeable [START_REF] Schimmelmann | Hydrogen Isotopic (D/H) Composition of Organic Matter During Diagenesis and Thermal Maturation[END_REF], the δ²H wax is frequently used in paleo-climate studies to track the variability of the 2 H/ 1 H ratio of meteoric water (δ²H p ), as recently reviewed by [START_REF] Sachse | Molecular Paleohydrology: Interpreting the Hydrogen-Isotopic Composition of Lipid Biomarkers from Photosynthesizing Organisms[END_REF]. The 'altitude effect', described by [START_REF] Dansgaard | Stable isotopes in precipitation[END_REF], is one of the physical parameters impacting the δ²H p and it corresponds to the progressive ²H-depletion of precipitation along altitudinal gradients due to successive Rayleigh distillation equilibria as the air mass rises. This effect has been observed in precipitation along several mountains across the world [START_REF] Araguás-Araguás | Deuterium and oxygen-18 isotope composition of precipitation and atmospheric moisture[END_REF] including 2 equatorial African mountains: Mt. Cameroon (Cameroon; [START_REF] Gonfiantini | The altitude effect on the isotopic composition of tropical rains[END_REF] and Mt. Kilimanjaro (Tanzania; [START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF].

The ability of δ²H wax to track this altitudinal effect was first shown at Mt. Gongga (China; [START_REF] Jia | Soil n-alkane δD vs. altitude gradients along Mount Gongga, China[END_REF]. This relationship was further observed at several mountains in Asia, Oceania and America (e.g. [START_REF] Luo | Empirical relationship between leaf wax n-alkane δD and altitude in the Wuyi, Shennongjia and Tianshan Mountains, China: Implications for paleoaltimetry[END_REF][START_REF] Ernst | Biomarkers record environmental changes along an altitudinal transect in the wettest place on Earth[END_REF][START_REF] Bai | Different altitude effect of leaf wax n-alkane δD values in surface soils along two vapor transport pathways, southeastern Tibetan Plateau[END_REF][START_REF] Zhuang | Altitudinal shift in stable hydrogen isotopes and microbial tetraether distribution in soils from the Southern Alps, NZ: Implications for paleoclimatology and paleoaltimetry[END_REF].

Branched GDGTs represent another group of biomarkers increasingly used for paleo-climate reconstructions in terrestrial archives (e.g. [START_REF] Peterse | Decoupled warming and monsoon precipitation in East Asia over the last deglaciation[END_REF]. These membrane lipids (Suppl. Fig.) are ubiquitous in both terrestrial and aquatic environments [START_REF] Schouten | The organic geochemistry of glycerol dialkyl glycerol tetraether lipids: A review[END_REF] and are produced by yet unknown bacteria. In soils from across the globe, the relative abundance of the different br GDGTs was shown to depend on environmental parameters [START_REF] Weijers | Environmental controls on bacterial tetraether membrane lipid distribution in soils[END_REF], leading to the development of global calibrations between br GDGT distribution and mean annual air temperature (MAAT) and pH [START_REF] Weijers | Environmental controls on bacterial tetraether membrane lipid distribution in soils[END_REF][START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF]. These compounds can therefore be used as paleo-thermometers. Soil-derived br GDGTs have been investigated along several mountain transects (e.g.; [START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF][START_REF] Ernst | Biomarkers record environmental changes along an altitudinal transect in the wettest place on Earth[END_REF][START_REF] Anderson | Sources of local and regional variability in the MBT′/CBT paleotemperature proxy: Insights from a modern elevation transect across the Eastern Cordillera of Colombia[END_REF] with a high variability in temperature (as temperature decreases with altitude). In the previously published papers, the related br GDGT-derived MAAT were found to (i) be in agreement with local measured MAAT and (ii) relate with altitude (i.e. following the natural temperature gradient), even though a high degree of scatter was observed for some case studies (e.g. Mt. Gongga; [START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF].

The combination of δ²H wax and br GDGT provided promising results which significantly expand their potential for (paleo) climate reconstruction. In modern soils, they were found to both correlate with altitude along Mt. Gongga (China, Jia et al., 2008 and[START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF], Mt. Meghalaya (India, [START_REF] Ernst | Biomarkers record environmental changes along an altitudinal transect in the wettest place on Earth[END_REF] and the Southern Alps (New Zealand, [START_REF] Zhuang | Altitudinal shift in stable hydrogen isotopes and microbial tetraether distribution in soils from the Southern Alps, NZ: Implications for paleoclimatology and paleoaltimetry[END_REF] implying that δ²H wax was recording the altitude effect in δ²H p and br GDGTs the natural temperature gradient. In geological archives, the approach has been employed to reconstruct the early Eocene elevation and climatic history of the Sierra Nevada [START_REF] Hren | Biomarker reconstruction of the early Eocene paleotopography and paleoclimate of the northern Sierra Nevada[END_REF]. It was deduced from the analysis of δ²H wax from fossil leaves and br GDGTs from the sediment matrix that, during the Eocene Climatic Optimum, Sierra Nevada elevation was high (> 2 km) with warmer air temperature than today. In East Africa, however, altitudinal transects from the southern slope of Mt. Kilimanjaro (Tanzania) was investigated by several groups (Sinninghe [START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF][START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF][START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF], resulting in inconsistent results. While estimated air temperatures derived from the analysis of br GDGTs were consistent with the observational temperature data (Sinninghe [START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF], no clear altitudinal trend was observed for δ²H wax [START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF][START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF]. This prompts the question regarding the relative importance of site (mountain)-related and regional (East African)-related controls on the link between altitude and soil δ²H wax record.

In the present study, δ²H wax and br GDGTs were analysed in 60 surface soils along 2 East African mountains -Mt. Rungwe (n=40; Southwest Tanzania; Fig. 1) and Mt. Kenya (n=20; Central Kenya; Fig. 1) -to further test their joint applicability as (paleo) environmental proxies in this climatically sensitive area. Our results were then combined with previously published data for 2 additional East African mountains (Mt.

Rwenzori, Uganda, [START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF]Mt. Kilimanjaro, Tanzania, Peterse et al., 2009[START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF][START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] to potentially generate regional calibrations of δ²H wax variations with elevation and br GDGT variations with temperature. This could improve the precision/quality of regional scale (paleo) elevation and (paleo) temperature reconstructions in comparison with global calibrations. The ultimate goal of this work is to introduce improved tools for terrestrially-based paleoclimatic reconstructions in East Africa that take into account not only temperature but also altitude changes, and which could then be used to reconstruct the evolution of topography and climate dynamics over geological time in the EARS.

Materials and Methods

Study sites

Br GDGTs and n-alkanes were analysed in 40 surface soils collected between 500 and 2800 m along the gentle then steep slope of Mt. Rungwe in 2012 and 2014, and in 20 samples collected between 1900 and 3300 m along the steady gentle slope of Mt. Kenya in 2013 (Table 1). About 50-60 g of soil were collected in the A horizon (0-5 cm) and kept at room temperature before shipping to France by plane.

Mt. Rungwe is located in the Southwest of Tanzania (9° S, 33° E; Fig. 1) and experiences a typical tropical climate, with alternations between a hot and humid (from November to May) season and a colder and drier season (from June to October; [START_REF] Delalande | Mbaka lakes isotopic ( 18 O and 2 H) and water balances: discussion on the used atmospheric moisture compositions[END_REF]. Natural vegetation along the altitudinal transect belongs to the Zambezian Miombo-type woodland at low altitude and Afromontane vegetation at higher altitude (e.g. [START_REF] Vincens | Pollen-based vegetation changes in southern Tanzania during the last 4200 years: climate change and/or human impact[END_REF]. Agriculture -including banana, rice, cocoa, tea, coffee, maize cultivations -is practiced up to 1500 m leading to partial deforestation of the mountain slopes [START_REF] Williamson | A potential feedback between landuse and climate in the Rungwe tropical highland stresses a critical environmental research challenge[END_REF]. MAAT was continuously measured with temperature loggers (weather station, DAVIS, Instruments, Hayward, CA, USA) at 540 m, 920 m and 1720 m and varied from 25.6 °C at 540 m to 22.6 °C at 920 m and 16.9 °C at 1720 m, leading to a temperature gradient of 0.7 °C/100 m.

Mt. Kenya is the highest mountain in Kenya (5199 m.a.s.l.) and is located on the equator (0° S 37° E; Fig. 1). The twice-yearly passage of the Intertropical Convergence Zone (ITCZ) leads to a bimodal rainfall pattern characterized by the onset and duration of the long rains (March to May) and short rains (October to November), the other months experiencing a drier climate [START_REF] Camberlin | Climatic gradients along the windward slopes of Mount Kenya and their implication for crop risks. Part 1: climate variability[END_REF]. Present day vegetation is marked by five distinguishable altitudinal zones, [START_REF] Bremond | Phytolith indices as proxies of grass subfamilies on East African tropical mountains[END_REF]Hamilton, 1982) from the mountain forest belt at its base (1960-2500 m.a.s.l.) to Afroalpine at its top (3400-4200 m.a.s.l.), with Bamboo and Ericaceous zones in between. Based on meteorological data from weather stations along Mt. Kenya and in Central Kenya [START_REF] Camberlin | Climatic gradients along the windward slopes of Mount Kenya and their implication for crop risks. Part 1: climate variability[END_REF]Kenya Meteorological Department, 1984;Kenya Meteorological Department;[START_REF] Smith | CLIMWAT for CROPWAT: A climatic database for irrigation planning and management[END_REF], temperature lapse rate along Mount Kenya was determined as 0.63 °C/100 m.

Sample preparation

Samples were frozen and freeze-dried at their arrival in Paris (France) and then kept at -18 °C prior further treatment. Sample preparation was identical to that detailed by [START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF]. Briefly, soils were extracted (3 × 5 min) with dichloromethane (DCM): methanol (MeOH) (9:1, v:v) using an accelerated solvent extractor (ASE 100 -Dionex; 100°C, 10 × 10 6 Pa). The extract was separated into 2 fractions through a 2 cm diam. column of Al 2 O 3 (activated overnight at 150 °C) using heptane:DCM (9:1, v:v) and DCM:MeOH (1:1, v:v) respectively. The 2 fractions were then rotary evaporated, redissolved in 1 ml heptane and centrifuged using an Eppendorf Mini Spin centrifuge (1 min, 7000 rpm) prior to further analysis.

n-Alkane analysis

n-Alkanes were analysed at UPMC, Paris, France by gas chromatography coupled to a mass spectrometer (GC-MS) using an Agilent Network 6890 GC System coupled with a 5973 Mass Selective Detector, with electron impact at 70eV. 1 µl was injected and the separation was achieved using a Restek RXI-5 Sil MS silica capillary column (30 m × 0.25 mm i.d., 0.50 µm film thickness) with He as the carrier gas at 1 ml/min flow rate.

The GC oven initial temperature was set to 50 °C and then increased to 320 °C at a 4 °C/min. Samples were injected in splitless mode and the injector temperature was 280 °C.

n-Alkane hydrogen isotopic composition was measured at Newcastle University, UK using a Delta V+ isotope-ratio mass spectrometer (IRMS, ThermoFisher) connected to a GC Ultra Trace (ThermoFisher), a Finnigan GC Combustion III (ThermoFisher) and a high temperature conversion (HTC) system set up at 1400 °C. The GC oven initial temperature was set to 50 °C and then increased to 250 °C at 15 °C/min and from 250 °C to 320 °C at 5 °C/min. The GC oven was held at 320 °C for 15 min. Every sample was analysed in duplicate and the 2 H/ 1 H ratio was reported on the VSMOW (Vienna standard mean ocean water) scale to determine compound specific δ 2 H Cn (‰). The H 3 factor was measured daily and varied between 2.48 and 2.61 during the course of analysis.

Sample n-alkane δ²H values were corrected individually using an n-alkane standard (n-C16 to n-C30; mix A5) and 5α-androstane standard (A. Schimmelmann, Indiana University), run at the beginning and at the end of each sample sequence. Standard error of the measurements of the long chain n-alkanes (C 25 -C 31 ) from this standard mix ranged between 0.3‰ and 1.1‰.

Br GDGT analysis

Br GDGT analysis was performed at UPMC, Paris, France by high pressure liquid chromatography coupled to a mass spectrometer with an atmospheric pressure chemical ionization source (HPLC-APCI-MS). Samples from Mt. Rungwe collected in 2014 were analysed with a Shimadzu LCMS-2020, as described by [START_REF] Coffinet | Occurrence and distribution of glycerol dialkanol diethers and glycerol dialkyl glycerol tetraethers in a peat core from SW Tanzania[END_REF], whereas samples from Mt. Kenya and those from Mt. Rungwe collected in 2012 were analysed with an Agilent 1100 series HPLC instrument equipped with an automatic injector and coupled to a PE Sciex API 3000 mass spectrometer, using a procedure described by [START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF]. Similar chromatographic conditions were used on the 2 machines and samples from [START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF] were re-run with the Shimadzu LCMS-2020 to assess the method reproducibility between the 2 LCMS apparatus (mean errors: 0.03 for MBT, 0.05 for CBT and 0.28 °C for MAAT).

Semi-quantification of br GDGTs was performed by comparing the integrated signal of the respective compound with the signal of a C 46 synthesised internal standard [START_REF] Huguet | An improved method to determine the absolute abundance of glycerol dibiphytanyl glycerol tetraether lipids[END_REF] assuming their response factors to be identical.

The MBT' (methylation index of branched tetraethers; eq. 1; [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] and CBT (cyclisation ratio of branched tetraethers; eq. 2; [START_REF] Weijers | Environmental controls on bacterial tetraether membrane lipid distribution in soils[END_REF] indices were determined from the following equations:

[ ] [ ] [ ] [ ] Ic Ib Ia IIc IIb IIa IIIa Ic Ib Ia MBT' + + + + + + + + = (1) [ ] [ ] [ ] [ ]         + + - = Ia IIa Ib IIb log CBT (2)
Roman numerals refer to the structures in the Supplementary Figure.

Based on triplicate injections, the maximal analytical error for the different indices was: 0.09 for MBT' and 0.03 for CBT.

MAAT was estimated from the global soil calibration developed by [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF]Eq. 3): MAAT = 0.81 -5.67 × CBT + 31.0 × MBT' (3)

Estimation of the H isotopic composition of precipitation

To characterize the H isotope composition of precipitation (δ²H p ) at the studied sites, results from previous publications were gathered (Rieti-Shati et al., 2010;[START_REF] Nivet | RESON: 3-year records of rainfall isotopic composition from 3 stations of the Rungwe volcanic province (SW Tanzania)[END_REF][START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF]. Due to the limited number of instrumental data, δ²H p was also estimated with the Online Isotopes in Precipitation Calculator (OIPC; [START_REF] Bowen | The Online Isotopes in Precipitation Calculator[END_REF][START_REF] Bowen | Interpolating the isotopic composition of modern meteoric precipitation[END_REF]. The OIPC model was developed to allow point estimation of δ²H p . It is based on a spatial interpolation from the 340 Global Network for Isotope in Precipitation (GNIP) station database. This interpolation includes the latitude and altitude effects on the isotopic composition of precipitation but in some regions, including East Africa, it is limited by additional sources of uncertainty. In East Africa, the major source of uncertainty is the small number of GNIP stations which lowers the dataset resolution [START_REF] Bowen | Interpolating the isotopic composition of modern meteoric precipitation[END_REF]West et al., 2004) while the δ²H p variability is there especially high (due to the intertropical context and the orographic complexity). In addition, [START_REF] Bowen | Spatial distribution of δ 18 O in meteoric precipitation[END_REF] pointed out 2 high altitude stations (Addis-Abeba and Entebbe) where the measured isotopic composition of precipitation was anomalously enriched in heavy isotopes, leading to an increased uncertainty in the prediction of δ²H p in this region. For the estimations along the 3 studied mountains, the 95% confidence level of the model varied from 3-4‰ at low altitudes to 6‰ at higher altitudes. Rungwe. The distribution parameters of these compounds (chain length range, C n range; average chain length, ACL; carbon preference index, CPI; cf. Supplementary Table 1) do not show any statistically relevant correlation with altitude. Along the two transects, long chain n-alkanes with odd-over-even predominance were the most abundant, as reflected by overall high ACL (28 ± 2; Suppl. Table 1) and CPI values (between 3 and 18; Suppl. varied between -166‰ and -125‰ (Table 1) along Mt. Rungwe and between -158‰ and -119‰ along the slope at Mt. Kenya.

Results

3

3.2. Br GDGT-derived mean annual air temperature (MAAT) along the altitudinal transects at Mts. Rungwe and Kenya Br GDGTs were analysed along Mt. Kenya in the same soil samples as those analysed for n-alkanes (n=20). Br GDGT proxies and corresponding temperature estimates were previously determined in 20 surface soils collected at Mt. Rungwe in 2012 [START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF]. This dataset was extended in the present study by the analysis of br GDGTs in 16 additional soil samples (same sample set as for the n-alkane analysis except 3 of them which were not analysed, see details in Table 1) collected along Mt.

Rungwe. The distribution of br GDGTs was similar in soils collected in 2012 and 2014, as shown by the comparable ranges of MBT' and CBT values in these samples (cf. Suppl.

Table 2).

Br GDGT-derived MAATs were estimated using the global soil calibration [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] and varied from 9.3 °C at 3270 m.a.s.l. to 16.9 °C at 1900 m.a.s.l.

along Mt. Kenya and from 12.2 °C at 2055 m.a.s.l. to 22.5 °C at 529 m.a.s.l. (Table 1).

Discussion

Variation of δ²H of n-alkanes in East Africa

In East Africa, the altitude effect on the hydrogen isotopic ratio of precipitation (δ²H p ) was reported along Mt. Kilimanjaro above 2000 m.a.s.l. (1.4‰/100 m) while it tends to be much weaker at lower altitudes [START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] and Fig. 2). As [START_REF] Gonfiantini | The altitude effect on the isotopic composition of tropical rains[END_REF] observed at Mt. Cameroon (Cameroon), the altitudinal gradient in the δ²H p seems to increase with altitude. Along Mt. Rungwe, the establishment of a long term monitoring of the isotopic composition of precipitation is ongoing [START_REF] Nivet | RESON: 3-year records of rainfall isotopic composition from 3 stations of the Rungwe volcanic province (SW Tanzania)[END_REF].

Preliminary data indicate values ranging from -16.9‰ at 1720 m.a.s.l. to -12.9‰ at 920 m.a.s.l. and -11.3‰ at 540 m.a.s.l. (Fig. 2) but more data are needed at higher altitudes to compare with Mts. Kilimanjaro and Cameroon trends. Along Mt. Kenya, Rieti-Shati et al.

( 2000) suggested the altitude effect to be absent based on precipitation events collected between 3000 and 4000 m.a.s.l. and groundwater collection above 4500 m.a.s.l. However

this assumption is based on a very limited dataset from high altitude sampling sites on the western side of the mountain whereas sampling for the present study was performed on the North-East slope of Mt. Kenya. Long-term monitoring of precipitation along a wide elevation range of this slope side at Mt. Kenya is needed to precisely assess the variation of its isotopic composition.

Because of this limited number of measured isotopic values of precipitation along the studied mountains, δ²H p had to be estimated using the OIPC (the Online Isotopes in Precipitation Calculator; [START_REF] Bowen | The Online Isotopes in Precipitation Calculator[END_REF][START_REF] Bowen | Interpolating the isotopic composition of modern meteoric precipitation[END_REF]Fig. 2). A 2 Hdepletion lapse rate of 1.5 ‰/100 m was computed for Mts. Rungwe and Kenya and of 1.4‰/100 m for Mt. Kilimanjaro, similar to the one measured by [START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] above 2000 m.a.s.l. (Fig. 2). Nevertheless, along the latter, the OIPC data were observed to be shifted from the measured ones, particularly at higher altitudes, towards more depleted values and to a higher extent than the 95% confidence level estimated by the model (ca. 11‰ difference; Fig. 2). Such a shift was already observed and discussed in Kenya by [START_REF] Soderberg | Using atmospheric trajectories to model the isotopic composition of rainfall in central Kenya[END_REF] and is likely due to the low number of GNIP stations whilst the rain composition exhibits a high spatial variability in the region. Therefore the absolute values derived from the OIPC must be treated with caution. However, they should depict the likely trend of δ²H p related to the altitude effect and allow comparison between the three studied sites. Notably, the δ²H p varies from one mountain to another (Fig. 2). At a given altitude, 870 m.a.s.l. for example, δ²H p equals -22‰, -12‰, -8‰ along Mts. Rungwe, Kilimanjaro and Kenya, respectively. More specifically, the more northern the latitude, the more enriched the δ²H p . This illustrates that, in addition to altitude, a latitudinal effect is also likely to impact the δ²H p distribution in East Africa, as previously noted at the global scale by [START_REF] Dansgaard | Stable isotopes in precipitation[END_REF]. No calibration of δ²H p with altitude could thus be deduced at the regional, East African, scale and the altitudinal effect in δ²H p could only be tracked at the mountain scale. Consequently, in the following, the ability of δ²H of n-alkanes (δ²H wax ) to track the altitudinal effect on δ²H p was solely investigated at the mountain scale for each study site.

The concentration-weighted mean values of δ²H C 27 -C 31 alkanes (δ²H wax , Table 1) were plotted against altitude for Mt. Kenya and Mt. Rungwe (Fig. 3A-B). A statistically significant, though scattered, 2 H-depletion trend was observed along Mt.

Kenya (-1.2‰/100 m; Fig. 3A; p < 0.05) close to the gradient observed for precipitation along Mt. Kilimanjaro (-1.4‰/100 m, Fig. 2). This result indirectly suggests the existence of an altitude effect in δ²H p along Mt. Kenya, unlike what was previously reported by [START_REF] Rieti-Shati | Stable isotope composition of tropical high-altitude fresh-waters on Mt. Kenya, Equatorial East Africa[END_REF]. The δ²H wax values along Mt. Kenya support the hypothesis that n-alkanes are able to record the altitude effect in their 2 H/ 1 H ratio, consistent with evidence from several other mountain systems in Asia (e.g. [START_REF] Jia | Soil n-alkane δD vs. altitude gradients along Mount Gongga, China[END_REF][START_REF] Bai | Different altitude effect of leaf wax n-alkane δD values in surface soils along two vapor transport pathways, southeastern Tibetan Plateau[END_REF] and New Zealand [START_REF] Zhuang | Altitudinal shift in stable hydrogen isotopes and microbial tetraether distribution in soils from the Southern Alps, NZ: Implications for paleoclimatology and paleoaltimetry[END_REF]. In contrast, no trend was noticed at Mt. Rungwe (Fig. 3B, p> 0.05), as previously observed along Mt. Kilimanjaro [START_REF] Peterse | Assessment of soil n-alkane delta D and branched tetraether membrane lipid distributions as tools for paleoelevation reconstruction[END_REF][START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF].

For these two mountains (Rungwe and Kilimanjaro), we suggest that the altitude effect observed in the precipitation may have been overprinted during the biosynthesis of n-alkanes and/or their diagenetic reworking in the soil. As reviewed by [START_REF] Sachse | Molecular Paleohydrology: Interpreting the Hydrogen-Isotopic Composition of Lipid Biomarkers from Photosynthesizing Organisms[END_REF], lipid biosynthesis comprises several steps where 2 H/ 1 H fractionation can occur.

The magnitude of fractionation on the end-product δ 2 H wax signal is still poorly constrained for most plant species. Notably, [START_REF] Sachse | Molecular Paleohydrology: Interpreting the Hydrogen-Isotopic Composition of Lipid Biomarkers from Photosynthesizing Organisms[END_REF] highlighted several parameters that could have a high impact on the δ 2 H wax fractionation, such as vegetation changes, deposition processes and leaf physiology. Several studies pointed out large variability in the leaf-derived δ 2 H wax of different species within a same site (e.g. Chikaraishi and Naraoka, 2007;[START_REF] Pedentchouk | δ 13 C and δD compositions of n-alkanes from modern angiosperms and conifers: An experimental set up in central Washington State, USA[END_REF]. Thus, the important vegetation changes with altitude documented along Mts. Kilimanjaro and Rungwe [START_REF] Bremond | Phytolith indices as proxies of grass subfamilies on East African tropical mountains[END_REF][START_REF] Williamson | A potential feedback between landuse and climate in the Rungwe tropical highland stresses a critical environmental research challenge[END_REF] could have influenced the δ 2 H wax signal, leading to an overprinting of the altitude effect on the δ²H of the precipitation. Moreover, Zech et al.

(2015) postulated that along Mt. Kilimanjaro the altitudinal effect should be outcompeted by the increasing evaporative ²H-enrichment of the leaf water due to changes in humidity with increasing altitude. The impact of these potential additional effects could be enhanced in East Africa, where the altitude effects on δ²H p (Fig. 2) are in the lower range of the gradients measured across the world (between -1 and -4‰/100 m, Araguás- et al., 2000).

Araguás
The results from this study highlight the complexity of the H isotopic signal recorded in n-alkanes from East African soils. The altitude effect on δ²H p was recorded in the δ²H wax signal at only one altitudinal transect (Mt. Kenya). This suggests that the use of δ²H wax as a (paleo) elevation proxy might be site-dependent. This study also points to the competitive impact of different hydroclimatic and biogeochemical factors on the soil δ²H wax record. Unfortunately the lack of extensive environmental monitoring in these remote locations prevented us from determining which of these factors are overprinting the δ²H wax signal at the other two mountains. Notably, more instrmental data are needed to improve the estimation of the δ²H p in East Africa. These observations probably also hold for other mountainous regions across the world and should therefore be taken into consideration in regional scale (paleo) elevation studies. The application of δ²H wax for (paleo) elevation reconstructions must therefore be limited to the mountain scale and should always be accompanied by detailed surveys of the environmental setting of the study site.

East African regional br GDGT calibration

Linear altitudinal gradients of measured mean annual air temperatures (MAAT)

were observed along all the investigated East African mountains: 0.56 °C/100 m in Mt.

Kilimanjaro (Tanzania, Sinninghe [START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF],, 0.53 °C/100 m in Mt. Rwenzori (Uganda, [START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF], 0.73 °C/100 m in Mt. Rungwe (Tanzania, Coffinet et al., 2014, Williamson et al., 2014) and 0.63 °C/100 m in Mt. Kenya (Kenya, Camberlin et al., 2014;Kenya Meteorological Department, 1984;Kenya Meteorological Department;[START_REF] Smith | CLIMWAT for CROPWAT: A climatic database for irrigation planning and management[END_REF]. The goal of this study was to investigate whether it is possible to establish a regional East African framework for the use of soil derived-br GDGTs as a paleothermometer. Thus, the measured MAAT data from all the local East African transects mentioned above were combined to calculate a regional temperature lapse rate (Fig. 4).

This rate, determined as 0.65 °C/100 m, with 97% of the MAAT variance being explained by altitudinal variations (Fig. 4), is similar to the one commonly used in low precision studies [START_REF] Rolland | Spatial and Seasonal Variations of Air Temperature Lapse Rates in Alpine Regions[END_REF]Grab, 2013 and references therein) and will allow us to assess the effect of temperature changes on br GDGT distribution at the regional scale.

In addition to Mts. Kenya and Rungwe investigated in the present study, br GDGT distribution was previously investigated along two additional East African mountains, Mt. Rwenzori (Uganda; [START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF] and Mt. Kilimanjaro (Tanzania, Sinninghe [START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF]. Br GDGT-derived MAATs were estimated using the global soil calibration [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF][START_REF] Weijers | Environmental controls on bacterial tetraether membrane lipid distribution in soils[END_REF] and were found to successfully record the decrease in MAAT with altitude along the slopes of these two previously studied mountains. However, the soils sampled at the highest elevations in Mt.

Rwenzori exhibited a significant cold bias [START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF]. The authors suggested that the water saturation of these soils may additionally impact the br GDGT distribution.

An impact of extreme soil water content (either aridity or saturation) on br GDGT distribution and corresponding temperature estimates has indeed been reported in several publications (e.g. [START_REF] Huguet | Occurrence and distribution of glycerol dialkyl glycerol tetraethers in a French peat bog[END_REF][START_REF] Dirghangi | Distribution of glycerol dialkyl glycerol tetraethers in soils from two environmental transects in the USA[END_REF][START_REF] Menges | Influence of water availability in the distributions of branched glycerol dialkyl glycerol tetraether in soils of the Iberian Peninsula[END_REF][START_REF] Dang | Evidence of moisture control on the methylation of branched glycerol dialkyl glycerol tetraethers in semi-arid and arid soils[END_REF].

Br GDGT-derived MAATs calculated using the soil calibration by [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] were also shown to decrease linearly with altitude along Mts. Rungwe and Kenya (R² = 0.79 and 0.66 respectively; figures not shown). Combined together, these four studies highlight the robustness of br GDGTs as a paleo-temperature proxy, at least for East Africa (MAAT lapse-rate = 0.4 °C/100 m, R² = 0.63; Fig. 5C). Despite this positive assessment, the application of the global soil calibration still leads to a substantial uncertainty in temperature reconstruction (Root Mean Square Error (RMSE) = 4.2 °C, [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF]; R² = 0.56, Fig. 5A).

Combination of the new and previously published (Sinninghe [START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF][START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF][START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF] This regional soil calibration (Fig. 5B) strongly improves both the R² and RMSE of br GDGT-reconstructed MAAT over the global soil calibration derived from globally distributed soils (Fig. 5A, [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF]. The new East African calibration produces robust quantitative temperatures from br GDGT distributions in soils as it takes into account the regional specificities in soil water content, soil type, biome type, all of which that could potentially impact br GDGT abundance and distribution (e.g. [START_REF] Dirghangi | Distribution of glycerol dialkyl glycerol tetraethers in soils from two environmental transects in the USA[END_REF][START_REF] Menges | Influence of water availability in the distributions of branched glycerol dialkyl glycerol tetraether in soils of the Iberian Peninsula[END_REF]. This highlights the necessity for regional determinations of the relationship between temperature and br GDGT distribution in order to improve past-temperature estimates. But it also points to the question of defining coherent regions over the globe with a relevant scale.

The East African soil calibration developed in this study was applied to the MAAT reconstruction in modern soils along all four altitudinal transects (Fig. 5D). The resultant br GDGT-derived MAAT lapse rate (0.52 °C/100 m; Fig. 5D) is closer to the measured one (0.65 °C/100 m; Fig. 5D) than when using the global soil calibration (0.41 °C/100 m; 5C), but still underestimates the actual lapse rate by ca. 20 %. This points to the unsuitability of using br GDGTs alone to reconstruct past elevation changes.

Independently of that, the correlation between MAAT estimates and altitude is higher when using the new regional soil calibration (R 2 = 0.80; Fig. 5D) than the global one (R² = 0.63; Fig. 5C), and closer to the correlation found between instrumental MAAT and altitude (R 2 = 0.97; Fig. 4).. Therefore, the consistency and increase in quality of this new regional calibration for temperature reconstruction calls for its wider application in paleosol-based paleo-climatic reconstructions in East Africa. So far, in this region, br GDGT-derived proxies have only been used in lacustrine sedimentary records (e.g. [START_REF] Tierney | Environmental controls on branched tetraether lipid distributions in tropical East African lake sediments[END_REF][START_REF] Damsté | Distribution of tetraether lipids in the 25-ka sedimentary record of Lake Challa: extracting reliable TEX 86 and MBT/CBT palaeotemperatures from an equatorial African lake[END_REF]). This regional but valuable climatic information can also be deduced from paleosol sequences (e.g. [START_REF] Hatté | δ 13 C of Loess Organic Matter as a Potential Proxy for Paleoprecipitation[END_REF][START_REF] Gocke | Introducing an improved multi-proxy approach for paleoenvironmental reconstruction of loess-paleosol archives applied on the Late Pleistocene Nussloch sequence (SW Germany)[END_REF], which are abundant in East Africa due to the high rate of volcanic activity (e.g. in the Rungwe volcanic province, [START_REF] Fontijn | Holocene explosive eruptions in the Rungwe Volcanic Province, Tanzania[END_REF]. First applications of br GDGTs in such soil settings from across the world have revealed promising results (e.g. [START_REF] Peterse | Decoupled warming and monsoon precipitation in East Asia over the last deglaciation[END_REF][START_REF] Zech | Branched glycerol dialkyl glycerol tetraethers in Pleistocene loess-paleosol sequences: Three case studies[END_REF].

Conclusions

Figure captions

Fig [START_REF] Bowen | The Online Isotopes in Precipitation Calculator[END_REF][START_REF] Bowen | Interpolating the isotopic composition of modern meteoric precipitation[END_REF] along Mt. Rungwe, Tanzania (red segment), Mt. Kenya, Kenya (green segment) and Mt. Kilimanjaro, Tanzania (blue segment) together with on field δ²H p measurements along Mt. Kilimanjaro (blue circles; [START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] and Mt. Rungwe (red diamonds; [START_REF] Nivet | RESON: 3-year records of rainfall isotopic composition from 3 stations of the Rungwe volcanic province (SW Tanzania)[END_REF]. Note that the delimitations of each segment corresponds to the altitudinal range of the sample set investigated in the present study for each mount. [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] and (B) the new East African regional MBT'/CBT calibration developed in this study, the 1:1 line being represented in dashed grey as a reference. Application of these 2 calibrations to reconstruct br GDGT-derived MAAT variation with altitude in East Africa: (C) the global soil calibration [START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF], (D) the new East African soil calibration where the black solid line is the derived linear regression obtained from br GDGT-derived MAAT and the dashed black line is the measured MAAT altitudinal gradient (as obtained in Fig. 3). 2012) calibration and from the regional calibration developed in this study. Mt. Kilimanjaro [START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] Mt. Rungwe [START_REF] Nivet | RESON: 3-year records of rainfall isotopic composition from 3 stations of the Rungwe volcanic province (SW Tanzania)[END_REF] Linear (Mt. Kenya

  .1. δ²Hp and δ²Hwax along the altitudinal transects at Mt. Kenya and Mt. Rungwe The δ²H p values along Mts. Kenya, Rungwe and Kilimanjaro obtained using the OIPC (the Online Isotopes in Precipitation Calculator; Bowen, 2016; Bowen and Revenaugh, 2003; Fig. 2) span from -42‰ at 3270 m.a.s.l. to -23‰ at 1900 m.a.s.l. along Mt. Kenya and from -49‰ at 2800 m.a.s.l. to -16‰ at 520 m.a.s.l. along Mt. Rungwe and from -45‰ at 3245 m.a.s.l. to -23‰ at 1727 m.a.s.l along Mt. Kilimanjaro. n-Alkanes were analysed in 20 soil samples collected between 1900 to 3160 m along Mt. Kenya and in 40 soil samples collected between 500 and 2800 m along Mt.

δ²H

  wax and br GDGT distributions were determined in 60 surface soils collected along altitudinal transects of two East African mountains (Mt. Rungwe in Tanzania and Mt. Kenya in Kenya). The obtained values were combined with data from altitudinal gradients of previous studies in the region to assess the applicability of integrated δ²H wax and br GDGTs data sets as coupled paleo-topography and paleo-temperature proxies, respectively, in East Africa. Variations in δ²H wax do not systematically document altitudinal changes, with only one out of the three investigated mountains showing a positive relationship -even though the altitude effect was present in the δ²H of precipitation. At the regional (East African) scale, coupling of estimated and observed δ²H p data highlights their spatial heterogeneity. The use of δ²H wax as a paleo-elevation proxy must therefore be restricted to site (mountain)-specific studies. In contrast, br GDGT-derived MAAT reliably track measured MAAT gradients along the four studied mountains (Mts Rungwe, Kenya, Kilimanjaro and Rwenzori). The large dataset of both measured and br GDGT-derived MAAT in the study area enabled to establish a new regional East African soil calibration between br GDGT distribution and MAAT. This new calibration improves the accuracy of MAAT reconstruction for the studied region, in comparison to other global soil calibrations, and highlights the potential of br GDGTs as paleo-thermometers in recent and ancient East African soils.

Fig. 3 .

 3 Fig. 1.Map of East Africa. The different sites mentioned in this study are represented with a star. A zoom-in is made for Mt. Kenya and Mt. Rungwe, the 2 sites specifically investigated in this study, and altitudinal profiles of the corresponding transects are shown. Fig.2. δ²H p estimations from the OIPC (the Online Isotopes in Precipitation Calculator;[START_REF] Bowen | The Online Isotopes in Precipitation Calculator[END_REF][START_REF] Bowen | Interpolating the isotopic composition of modern meteoric precipitation[END_REF] along Mt. Rungwe, Tanzania (red segment), Mt. Kenya, Kenya (green segment) and Mt. Kilimanjaro, Tanzania (blue segment) together with on field δ²H p measurements along Mt. Kilimanjaro (blue circles;[START_REF] Zech | Do n-alkane biomarkers in soils/sediments reflect the δ2H isotopic composition of precipitation? A case study from Mt. Kilimanjaro and implications for paleoaltimetry and paleoclimate research[END_REF] and Mt. Rungwe (red diamonds;[START_REF] Nivet | RESON: 3-year records of rainfall isotopic composition from 3 stations of the Rungwe volcanic province (SW Tanzania)[END_REF]. Note that the delimitations of each segment corresponds to the altitudinal range of the sample set investigated in the present study for each mount. Fig. 3. δ²H wax values (A) along the altitudinal transects at Mt. Kenya, Kenya and (B) along the altitudinal transect at Mt Rungwe, Tanzania. Fig. 4. Measured mean annual air temperature (MAAT) along altitudinal gradients in East Africa: Mt Rungwe (red diamonds; Coffinet et al., 2014); Mt. Kenya (green triangles; Camberlin et al., 2014), Mt. Rwenzori (purple squares; Loomis et al., 2011), Mt. Kilimanjaro (blue circles; Sinninghe Damsté et al., 2008) Fig. 5. Comparison of the accuracy and precision of (A) the global soil MBT'/CBT calibration[START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] and (B) the new East African regional MBT'/CBT calibration developed in this study, the 1:1 line being represented in dashed grey as a reference. Application of these 2 calibrations to reconstruct br GDGT-derived MAAT variation with altitude in East Africa: (C) the global soil calibration[START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF], (D) the new East African soil calibration where the black solid line is the derived linear regression obtained from br GDGT-derived MAAT and the dashed black line is the measured MAAT altitudinal gradient (as obtained in Fig.3). Colours correspond to each mountain of the region: Mt Rungwe (red diamonds; Mt. Rungwe, this study and[START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF]; Mt. Kenya (green triangles; this study), Mt. Rwenzori (purple squares;[START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF], Mt. Kilimanjaro (blue circles; Sinninghe[START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF]; Table1. Weighted average δ²H of C 27 , C 29 and C 31 n-alkanes (denoted δ²H wax ) together with br GDGT-derived mean annual air temperature (MAAT) from[START_REF] Peterse | Revised calibration of the MBT-CBT paleotemperature proxy based on branched tetraether membrane lipids in surface soils[END_REF] calibration and from the regional calibration developed in this study.

  Figure 1.

  Figure 1.

  Figure 2.

  Figure 3.

Table 1 )

 1 Suppl. Table1). The weighted mean δ²H wax of the long chain n-alkanes (C 27 , C 29 , C 31 )

. These results indicate that n-alkanes present in the soils originate predominantly from higher plants. The δ²H values are comprised between -109‰ and -177‰ (mean -143‰) at Mt. Rungwe, and between -116‰ and -167‰ at Mt. Kenya (mean -141‰;

  data from East Africa, leads to a br GDGT dataset from in total 105 soils. Because of the potential additional impact of water saturation on brGDGT distribution and thus associated temperature estimates, the high elevation samples from[START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF] were not used in this dataset. Based on this integrated sample set, a least square multiple linear regression was performed between br GDGT distribution (MBT' and CBT indices) and the available MAAT for the four sites (Mt. Kenya, Rwenzori, Kilimanjaro and Rungwe). The following equation was obtained:

	ܶܣܣܯ = -8.76 × ܶܤܥ + 24.24 × ′ܶܤܯ + 9.60 (R² = 0.77, RMSE = 2.4 °C) (4)

  . 1. Map of East Africa. The different sites mentioned in this study are represented with a star. A zoom-in is made for Mt. Kenya and Mt. Rungwe, the 2 sites specifically investigated in this study, and altitudinal profiles of the corresponding transects are shown. Fig.2. δ²H p estimations from the OIPC (the Online Isotopes in Precipitation Calculator;

Table 1 .

 1 Colours correspond to each mountain of the region: Mt Rungwe (red diamonds; Mt. Rungwe, this study and[START_REF] Coffinet | Potential of GDGTs as a temperature proxy along an altitudinal transect at Mount Rungwe (Tanzania)[END_REF]; Mt. Kenya (green triangles; this study), Mt. Rwenzori (purple squares;[START_REF] Loomis | Distributions of branched GDGTs in soils and lake sediments from western Uganda: Implications for a lacustrine paleothermometer[END_REF], Mt. Kilimanjaro (blue circles; Sinninghe[START_REF] Damsté | Altitudinal shifts in the branched tetraether lipid distribution in soil from Mt. Kilimanjaro (Tanzania): Implications for the MBT/CBT continental palaeothermometer[END_REF]; Weighted average δ²H of C 27 , C 29 and C 31 n-alkanes (denoted δ²H wax ) together with br GDGT-derived mean annual air temperature (MAAT) fromPeterse et al. (

b not analysed for GDGT content. 726

Acknowledgements

The authors would like to thank the RESON (Rungwe Environmental Science Observatory Network) and those who help them during field trips, notably Stephen Kajula, Winne Mosena, Matokeo Arbogast, Stephen Warui, Marcel Hale and the inhabitants of Masoko. Paul Donohoe and Bernard Bowler are warmly acknowledged for their technical support at Newcastle University. The authors are indebted to Dr. Pierre Camberlin (University of Bourgogne, France) who shared his temperature dataset from Mt. Kenya. This research benefited from a travel grant awarded by the EAOG and a PhD scholarship from UPMC to S. Coffinet as well as support from the EC2CO program (CNRS/INSU) to A. Huguet. The work under permit NCST/RCD/17/013/02 on Mt. Kenya (C. Omuombo) was supported from French AIRD and Campus France through the French embassy in Nairobi.


S Ébastien Elgue
email: sebastien.elgue@ensiacet.fr

Annelyse Cont

Alain Marty

Jean-St Éphane Condoret

S Elgue

Continuous lipase esterification using process intensification technologies

Keywords: esterification, biocatalysis, lipase, process intensification, continuous reactor, two-phase reaction

BACKGROUND: Nowadays, developing continuous enzymatic processes, especially when a two-phase medium is required, remains a great challenge. So, there is a need to provide a technical solution for generating highly controlled interfacial area in a small volume. In this study, four types of reactors conventionally used for chemical process intensification and marketed by Corning, Chart Industries, NiTech Solutions and AM Technology, were tested for continuous lipase-catalysed esterification in two-phase medium.

RESULTS:

The NiTech oscillating reactor proved to be the more efficient to transform the input mechanical power into mass transfer performance while proposing easily adjustable residence time. Nevertheless, Corning and Chart heat-exchanger reactors were shown to be very suitable candidates even if mass transfer performances depend on the flow-rate and therefore are not uncoupled from the residence time. This results in a less convenient choice of operating conditions. The Coflore system was not so convincing but it may be due to the injection device not well adapted to this system. CONCLUSION: This work demonstrates that novel technologies, recently proposed for intensification of chemical processes, are indeed very suitable and provide a relevant alternative to conventional processes for enzymatic reactions and therefore for two-phase reactions limited by the interfacial area value. Also, it has been shown here that the model reaction considered could be a simple way to characterize mass transfer performances of these technologies.

INTRODUCTION

Operation of enzymatic reactions in two-phase medi, i.e. oil-water dispersions, is very common when hydrophobic reactants are concerned, as, for instance, in the case of hydrolysis of lipids. [START_REF] Slotema | Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia[END_REF][START_REF] Mbatia | Enzymatic enrichment of omega-3 polyunsaturated fatty acids in Nile perch (Lates niloticus) viscera oil[END_REF] Moreover, reactions of synthesis, such as esterification, can also be effective in two-phase medium using free enzymes. [START_REF] Tweddell | Esterification and interesterification activities of a lipase produced by Rhizopus niveus in 3 different organic media: a comparative study[END_REF] Indeed, even in a two-phase system with a water activity close to unity, a shift of the thermodynamic equilibrium is obtained by maintaining a low ester activity. Initial laboratory studies are usually done in twophase media because enzymes are produced in aqueous phases. In this case, to assess the influence of operating conditions, such as reactant and catalyst concentrations, optimal temperature and pH, batch experiments are done in a stirred vessel. Special care is then necessary to generate reproducible and well characterized liquid-liquid dispersion, in order to prevent erroneous conclusions due to uncontrolled changes in the contact area between phases. This is a crucial point, especially when reactions are limited by the interfacial area, which is the case when very active lipases are operated at the liquid-liquid interface. Note that such a problem is also encountered during liquid-liquid chemical reactions. When industrial development is envisaged, continuous operation must be considered in order to increase productivity and economic viability. But, at the present time, it is not possible to propose a realistic continuous liquid-liquid reactor where controlled contact area and steady operation are ensured. So, usually, to overcome this technical difficulty and make the most efficient and economic use of the enzyme, this latter is used as an immobilized biocatalyst packed in a tubular reactor. [START_REF] Slotema | Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia[END_REF][START_REF] Dossat | Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the production of glycerol[END_REF]5 This implies correlative studies to provide an efficient immobilization method and is a handicap in terms of catalyst price and time for development. So, there is a need for a technical solution which makes it possible to directly use aqueous enzyme solutions in a continuous reactor, where the two-phase system is well controlled in terms of interfacial area and steadiness of operation. This kind of reactor will be efficient and attractive as it is able to provide a large controlled interfacial area in a small volume, i.e. a dense dispersion of small droplets. This opens the field to the so-called 'intensified' reactors which is now an identified target for successful industrial development of new processes. Note that, if the interfacial area is made non-sensitive to the flow-rates of the two phases, then the reactor technology becomes very attractive.

The aim of this study is to consider the use of several intensified reactor technologies, already existing at commercial scale, to operate enzymatic two-phase reactions. In order to focus on the reactor performance assessment of candidate technologies will be done using a model reaction, the lipase-catalysed esterification of oleic acid with ethanol. In addition to provide a technical solution, such an approach could also be considered as benchmarking for the different intensified reactor technologies.

For multi-phase applications, flow reactors allow enhanced mass transfer and controlled interfacial area, providing reproducible and reliable operation. From a technical viewpoint, flow reactors can be classified into two broad categories:

• Static mixing devices rely on motion of the fluid through the reactor to generate mixing effects and phase dispersion (in twophase systems). The most common type is the tubular reactor. Heat-exchanger reactors also belong to this category. • Dynamic mixing devices where, by contrast, mixing is carried out using mechanical energy input done by different systems like in the most common type, the continuous stirred tank reactor, where mixing is obtained by the use of mechanical stirrers.

A detailed technological description of the different reactors is given in the Material section.

MATERIAL AND METHODS

Materials

Commercial liquid form of lipase B from Candida antarctica, namely Lipozyme CALB L, was a gift from Novozymes (Bagsvaerd, Denmark). Its concentration is 5000 LU g -1 . 90% pure oleic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents (ethanol, cyclohexane, and heptane) were of the highest purity and were purchased from Acros Organics (Geel, Belgium).

Description of the reactors

For each type of flow reactor two commercial solutions were considered here. The ability to carry out two-phase reactions and the technological maturity has driven the choice of these technologies. Hence, two heat-exchanger reactors commercialized by Corning and Chart Industries, and two examples of dynamic mixing devices the NiTech Solutions COBR TM and the AM Technology Coflore ® were studied. It has been chosen to work on pilot scale versions of these equipments in order to be able to easily extrapolate the process to industrial scale. The four technologies tested are described below.

Corning advanced-flow TM reactor (AFR)

The Corning Advanced-Flow TM glass reactor is an assembly of glass fluidic modules connected together using appropriate piping and connectors (Fig. 1). These fluidic modules are based on the plate heat-exchanger concept, in the form of a plate with fluidic channels that have a hydraulic diameter of around 2 mm and a volume around 10 mL per plate (at pilot scale: Gen 1). The plate is sandwiched between two heat exchange plates, thus providing efficient heat transfer performance. [START_REF] Barthe | Continuous multi-injection reactor for multipurpose production -Part I[END_REF][START_REF] Zhang | Intensified liquid/liquid mass transfer in Corning ® Advanced-Flow™ reactors[END_REF] This configuration has been proved to enable operation with multiphase systems, as described in the literature. [START_REF] Chevalier | Microreactors for industrial multi-phase applications: test reactions to develop innovative glass microstructure designs[END_REF][START_REF] Brocklehurst | Nitration chemistry in continuous flow using fuming nitric acid in a commercially available flow reactor[END_REF] To ensure good mixing and enhanced mass transfer performance, the fluidic channels exhibit specific designs and are adapted for the targeted application with respect to flow-rates and physical properties of fluids The efficiency of such complex designs inevitably induces significant pressure drop. Therefore, and because the stacking of plates is only able to withstand around 20 bar, this maximum pressure drop value limits the length of the reactor and therefore the residence time. So, in this kind of reactor, 15 to 20 min residence time can be considered a maximum (corresponding to a few hundred millilitres reactor volume). Thus, reaching high conversion level for the reaction could be challenging with regard to this limitation.

Chart Industries Shimtec ® reactor

Chart Industries Shimtec ® reactors are also based on the plate heat exchanger concept. The reactor is composed of three plates: one process plate sandwiched between two utility plates. The typical module presents similar characteristics to the Corning Advanced-Flow TM reactor, with hydraulic diameter of about 2 mm and total process volume of around 15 mL. Fluidic channels exhibit a specific design to ensure significant heat and mass transfer performance, [START_REF] Anxionnaz | Transposition of an exothermic reaction from a batch reactor to an intensified continuous one[END_REF] particularly in the case of multi-phase applications. [START_REF] Jia | Plate fin fluid processing device[END_REF] Shimtec ® reactors are manufactured as a metallic complex frame made either with aluminium, stainless steel or special metals, depending on temperature, pressure and corrosiveness (Fig. 1). Assembly by diffusion bonding strengthens the heat transfer efficiency and also provides good resistance to pressure (of about 70 bar). Thus, these modules are able to operate at high temperature and pressure conditions where significant residence time and conversion levels can be reached.

NiTech Solutions Continuous Oscillatory Baffled Reactor TM (COBR TM )

NiTech Continuous Oscillatory Baffled Reactor TM (COBR TM ) technology consists of a tubular reactor including annular-baffles (Fig. 1). Based on the principle of dynamic mixing devices, the performance is governed by the combination of flow-rate and fluid oscillations across these baffles via a piston system. The mixing mechanism in a baffled cell occurs in two steps (upstroke and downstroke) creating very efficient mixing and generation of a high interfacial area. [START_REF] Mackley | Mixing and dispersion in a baffled tube for steady laminar and pulsatile flow[END_REF][START_REF] Oliveira | Effect of hydrodynamics on mass transfer in a gas-liquid oscillatory baffled column[END_REF] The COBR TM used for this study is a pilot scale version of the reactor, based on 15 mm diameter jacketed glass tubes for 51 m total length and about 9 L total volume. As performance is decoupled from fluid velocity (i.e. flow-rate) thanks to the oscillation system, this reactor makes it possible to reach significantly longer residence times (in the range of hours) and so appears to be an interesting candidate for performing moderately fast reactions.

AM Technology Coflore ® Coflore ® reactors are multi-stage flow reactors that deliver good mixing and transfer performance through the concept of dynamic mixing. In fact, the Coflore ® design implements a mixing technique where free moving agitators within each reaction cell promote mixing when the reactor body is subjected to lateral shaking (Fig. 1). This generates intense mixing without high flow velocity or the need for rotating shafts with mechanical seals or The Coflore ® used during this study is a pilot scale version of the reactor: the ACR. It uses high shear agitators in reaction cells and has a working capacity of 100 mL. The enhanced mixing and transfer performances of this reactor for multi-phase systems have been demonstrated. [START_REF] Ómez-Quero | Liquid phase catalytic hydrodechlorination of 2,4-dichlorophenol over Pd/Al2O3: Batch vs Continuous operation[END_REF] Also work on biocatalysis application [START_REF] Jones | Biocatalytic oxidase: batch to continuous[END_REF] has been reported, offering interesting perspectives for lipase catalyzed esterification.

Description of the reaction

The chosen reaction is a model reaction, the lipase-catalysed esterification with ethanol (300 mmol L -1 ) of a fatty acid, oleic acid (100 mmol -1 ), to yield ethyloleate and water (Equation ( 1 (1)

To catalyse this esterification, the commercial liquid form of lipase B from Candida antarctica, namely Lipozyme CALB L, was chosen. To obtain an enzyme activity in agreement with the residence times envisaged in the continuous process, commercial enzyme solution was 50 or 100 times diluted in a phosphate buffer 0.1 mol L -1 , pH 7.2 to reach an activity of 100 or 50 LU g -1 .

Esterifications are equilibrated reactions and the equilibrium constant is given by the following equation: Keq = a ester eq .a weq a acid eq .a alcohol eq (2) where a ieq are the thermodynamic activity of component i in the fluid phases at equilibrium. To shift the reaction towards synthesis, it is necessary to maintain the numerator of the equation as low as possible and the denominator as high as possible. In a twophase system, water activity is close to unity (a w =1) which is very unfavourable for esterification reactions. Nevertheless, the reaction is shifted towards esterification because the hydrophobic ester accumulates in the solvent which plays the role of a reservoir for the most hydrophobic reactant, the ester. In other words, the solvent maintains at a low value the thermodynamic activity of the ester. In this work, cyclohexane was used to play this role. The lipase-catalysed esterification of oleic acid with ethanol has been studied at pilot scale, which means that the total flow-rate (i.e. the sum of the flow-rates of both phases) is in the range 15 to 150 g min -1 . A specific fluidic module has been used to control the feeding of raw solutions and the temperature of the process.

The fluidic module is constituted of two dosing lines composed of micro-annular gear pumps (HNP Mikrosysteme -mzr series), mass-flow meters based on the Coriolis effect (Micromotion -elite series) and temperature and pressure sensors. PID controllers ensure constant delivery of flow-rates by acting on the motor pump regime. Special care has been taken to prevent pulsating flow-rates, especially in the case of NiTech COBR TM . This is done by using a combination of check and safety valves set on the dosing lines to prevent piston pulsation from disturbing the pump flowrates. A process-thermostat (Lauda integral XT 150) is associated with the fluidic module to control the process temperature. All operating parameters (temperature, pressure, flow-rate) are online, recorded using a computer.

Whatever flow-reactor is considered, the process is based on the flow-sheet of Fig. 2. The organic phase composed of cyclohexane, oleic acid and ethanol and the aqueous phase composed of lipase diluted in a phosphate buffer solution are respectively introduced by dosing lines 1 and 2. To obtain isothermal profiles and then make accurate comparisons, feed flows are pre-heated or pre-cooled, depending on the operating temperature, thanks to the first set of Corning DTR microstructures. Then both phases are introduced inside the selected reactor (Corning Advanced-Flow TM reactor in the case of Fig. 2). Temperature sensors are set along the reactor path and allow measurement of temperature profiles. Because of the absence of thermal effects from the enzymatic reaction, we never observed temperature variation in the temperature profile throughout the reactor for all our experiments, whatever the technology tested. A sampling valve is located at the reactor outlet to estimate the final conversion using HPLC chromatography analysis.

Analysis of reaction evolution

Samples collected at the output of the reactor are centrifuged to separate the heavy enzyme aqueous phase from the organic one containing non-reacted substrate and product. Then, organic solution is evaporated to eliminate the solvent, and the concentrated mixture is diluted with HPLC mobile phase.

Analysis of oleic acid and ethyloleate concentrations is done using a THERMO Accela 600 apparatus, equipped with a Refractive Index Detector. A Waters C18 column (250 × 4.6 mm, 3.5 µm particle size) is used. The mobile phase is Methanol (HPLC quality) with 0.02% vol. of pure acetic acid. An isocratic flow-rate of 1mLmin -1 is applied.

RESULTS

Study of the esterification using batch reactors

In order to have a reference value, batch esterification reactions were performed in small Eppendorf tubes agitated in a Vortex. The reaction was carried out at room temperature in 1.5 mL Eppendorf tubes containing 0.5 mL of organic phase and 0.5 mL of aqueous enzymatic solution. The mixture was shaken in a Vortex Genie 2 (D. Dutscher, Brumat, France). The progress of the reaction was followed at regular time intervals by taking samples from the organic phase. 90.4% conversion was obtained within 30 min (data not shown). In addition, to make the comparison as complete as possible, the esterification was carried out in a standard glass reactor at lab scale (1 L, double jacketed with impeller stirring device) at optimal stirring velocity (360 rpm). In that case (Fig. 3), the operating conditions were chosen to be comparable with those applied in the different continuous reactors.

Batch study shows that initial velocity is 6 times lower in the batch laboratory scale reactor than in small eppendorf tubes, demonstrating the influence and the limitation of mass transfer during this reaction in a two-phase medium.

Study of the esterification using static mixing flow reactors

In flow reactors based on the static mixing concept, performances are driven by flow velocity: an increase of flow-rate favours the generation of interfacial area and therefore mass transfer. Also, as reactor volume is fixed, increasing flow-rate reduces the residence time. Consequently, in the case of two-phase reactions, depending on the primary reaction regime (kinetic, mass transfer or transitional regime), there is an optimal flow-rate leading to the highest conversion level. To validate these assumptions, a study of the total flow-rate influence was carried out in Corning and Chart flow reactors. Recall that total flow-rate is defined as the sum of organic and aqueous phase flow-rates. The flow-rate of each phase can be deduced from the mass ratio (given in Table 1). The operating conditions and the associated results are detailed in Table 1 and Fig. 4, respectively.

The evolution of conversion with respect to global flow-rate in the Corning reactor exhibits a flat bell-shaped curve. At low flow-rates (lower than 105 g min -1 ), conversion is increasing due to improved mixing performances and interfacial area generation, despite the reduction of global residence time (defined as the reactor volume divided by the total flow-rate). At higher flow rates, the conversion decreases because the reduction of the residence time is more detrimental than the gain in mass transfer which moreover will reach an upper limit. Unfortunately, because of the high pressure drop involved at high flow-rates, due to the limitation of dosing line pumps, it was not possible to experimentally observe such behaviour.

In the range of flow rates tested, for the Chart reactor, mixing improvement and residence time reduction seem to counterbalance. This is in agreement with the fact that the Chart reactor offers better mass transfer performance at low flow-rates. As a consequence, the maximum conversion (around 25%) was reached for different values of flow-rate. Therefore, to determine the optimal flow-rate, additional criteria should be defined, for instance in terms of productivity or power consumption. Indeed, high flow rates resulted in better productivity but high power consumption because the pressure drop was larger.

To discriminate the reactors from an industrial viewpoint, it is relevant to represent the conversion with respect to the energy dissipation rate, as presented in Fig. 5. Such computation is easily done for reactors based on the static mixing concept where the energy dissipation rate is related to flow-rate and pressure drop, according to the following equation:

ǫ = Q × P ρ × V (3)
with ǫ the energy dissipation rate (W kg -1 ), Q the volume flow-rate (m 3 s -1 ), P the pressure drop (Pa), ρ the reactive medium density (kg m -3 ), and V the volume of the reactor (m 3 ). Again this comparison highlights the better performance of the Chart technology and its ability to reach a given conversion level with reduced energy dissipation rate, i.e. less power consumption.

Different parameters have to be investigated to compare the associated behaviours of the Corning and Chart reactors. In this way, the influence of temperature, lipase concentration and substrate ratio were studied. These studies were performed at a total flow-rate of 46 g min -1 (20 g min -1 of organic phase, 26 g min -1 of aqueous phase), which corresponds to a total residence time of 140 s.

Influence of lipase concentration

As seen in Fig. 6, the performances are rather insensitive to this parameter. It can be postulated that the reaction is taking place only at the interface where lipases are concentrated and where hydrophobic substrates in the organic phase can access their active site, which is directed towards the organic phase. Indeed, lipases are known to be active at the interface. [START_REF] Sarda | Actions of pancreatic lipase on esters in emulsions[END_REF] When saturation of the interface is reached, the concentration of the biocatalyst at the interface is no longer dependent on its concentration in the aqueous phase, and therefore the kinetics is insensitive to this parameter as is observed here. This means that the reaction is governed mainly by the interfacial area value. This is an interesting result because it enables one to define a minimal quantity of lipases to be used. Operating at minimum biocatalyst quantity is favourable for the economics when such a process is envisaged at production scale. In addition, from the point of view of comparing different technologies, this makes it possible to discriminate them in terms of creating and maintaining interfacial area.

Influence of temperature

Chart and Corning reactors exhibit high heat transfer performance [START_REF] Desp Ènes | Impact of the material on the thermal behaviour of heat exchangers-reactors[END_REF] which lead to an isothermal profile of the reactive medium temperature. Thermocouples located along the channel validated this statement. Therefore, it appears relevant to study the influence of temperature on conversion, as shown on Fig. 7.

It is seen in Fig. 7 that, up to 50 • C, temperature has a slight positive influence on conversion. Then a decrease of conversion is seen which can probably be attributed to deactivation of the enzyme. Indeed, it is known that operating at high temperature very often leads to enzyme denaturation.

The first increasing part of the curve is more difficult to understand. It is very moderate and do not correspond to a plain kinetic increase. Indeed, kinetics is very sensitive to temperature and a 30 • C temperature rise should have resulted in a more significant conversion gain (from a very conventional exponential Arrhenius law). In our previous analysis of the lipase concentration influence, it was shown that the liquid-liquid interface was probably saturated with lipases and that the apparent kinetics was dependent only on the interfacial area at a given temperature. Nevertheless, the interfacial kinetics should remain temperature sensitive. So, an explanation could be that interfacial kinetics is very fast and that the phenomenon is mass transfer limited. Indeed, in this case the moderate conversion increase is in accordance with usual mass transfer enhancement by temperature. So, the weak gain in conversion can be attributed to enhancement of the diffusion of the limiting substrate, probably oleic acid, towards the interface. Nevertheless, it can also be hypothesized that increase of temperature has favourably modified certain physical properties, such as viscosity or interfacial tension, that has led to a moderate increase of the liquid-liquid dispersion, and therefore of the interfacial area. Also, this ascertains that value of conversion for this reaction is a direct assessment of the mass transfer performances of the technology. It is not really possible to uncouple the diffusion effects from the interfacial area effect, as is often the case in chemical engineering where these effects are usually gathered in the global concept of volumetric mass transfer coefficient k L a.

Influence of substrate ratio

Substrate ratio is defined as the ratio of ethanol concentration to oleic acid concentration in the initial organic phase, this latter being fixed at 3.

Regarding substrate molar ratio (Fig. 8), two zones have to be considered. First, increasing the substrate ratio, i.e. using excess of alcohol, leads to better conversion (+5% in the Corning reactor from 3 to 6 molar eq.; +10% in the Chart reactor from 3 to 10 molar eq.) due to the increased availability of ethanol for the reaction. Then, higher values induce the opposite effect and conversion decreases. This decrease is in accordance with the mechanism of such reactions which was demonstrated to be a Ping Pong Bi Bi mechanism where ethanol is a competitive inhibitor. [START_REF] Marty | Kinetics of lipase-catalyzed esterification in supercritical CO(2)[END_REF] As was mentioned in the case of the temperature effect, the initial moderate effect is not due to a kinetic effect but here also to mass transfer enhancement. More precisely in this case, this effect is probably mainly due to interfacial area enhancement. Indeed, increasing ethanol concentration reduces the surface tension of the water-ethanol mixture and therefore droplet size. Recall that ethanol partitions very favourably into the aqueous phase.

For these two flow reactors, this parametric study has shown that, in spite of a possible optimization, the antagonism between . Volumic ratio between organic and aqueous phase is 1. Substrate ratio is 6. Lipase concentration is 50 LU g -1 and temperature is 50

• C.
residence time/mass transfer did not enable high conversion levels to be reached in open-loop operation, i.e. with only one pass in the reactor. To estimate the residence time required to obtain high conversion, recycled-loop experiments were carried out. In such experiments, the flow at the reactor outlet was instantaneously collected in a cooled stirred vessel (to quench the reaction and maintain dispersion) and reintroduced at the reactor inlet via a dedicated pump. These operations were performed with special care to avoid disturbing the reactive system and to yield accurate residence time estimation. Recycled-loop experiments were carried out in both reactors at the previously used global flow-rate of 46 g min -1 . Operating conditions were optimized according to the previous parametric studies in order to increase reaction kinetics (50 • C, substrate ratio = 6). Results shown in Fig. 9 emphasize clearly the possibility of reaching high conversion levels (almost 100%) with moderate increase of residence time (about 600 s instead of 140 s in the onepass experiment). Compared with the initial 140 s residence time, this means that such residence times can be easily obtained by using reactors with larger volumes (3-4-fold increase). Indeed, such values fully comply with residence time values usually available in Corning or Chart flow reactors. This definitely validates the possibility to operate lipase-catalysed esterification in openloop intensified flow-reactors. Note that a correlative increase of the pressure drop and therefore of the power consumption must also be taken into account. Esterification study in AM Technology Coflore ® The concept of Coflore ® reactors, based on dynamic mixing through lateral shaking and free moving agitators, appears particularly suited to two-phase applications. Literature emphasizes the intense mixing performances of such equipment [START_REF] Ómez-Quero | Liquid phase catalytic hydrodechlorination of 2,4-dichlorophenol over Pd/Al2O3: Batch vs Continuous operation[END_REF] and the related advantages for a biocatalysis application. [START_REF] Jones | Biocatalytic oxidase: batch to continuous[END_REF] In spite of all this favourable background, in the device tested here, the results of lipase-catalysed esterification were rather disappointing compared with the other continuous reactors. In fact, under the same operating conditions and particularly the same global residence time (140 s, same mass ratio), the conversion was about 15%, 5% less than in static mixing flowreactors, independently of the shaking graduation on the device (i.e. the power allocated for mixing) (Fig. 10).

Indeed, visual observation during the experiments showed that it was not possible to obtain or maintain liquid-liquid dispersion at the reactor outlet. A posteriori, the lower conversion obtained tends to demonstrate that no efficient liquid-liquid dispersion was created inside the reactor, thus limiting the mass transfer performance. In this type of flow reactor, two parameters contribute to the creation of liquid-liquid dispersion: the injection system generally based on nozzles and the specific mixing performances of the device. The injection system used in this study was probably not well-designed with respect to the characteristics of the two-phase system involved here, in terms of physical properties (density, viscosity, interfacial tension) and phase ratio (aqueous vs. organic).

In the context of this work, for this specific device, it was neither possible to test different injection systems nor possible to perform further investigations to improve the mass transfer performances. However, a parametric study focusing on the application parameters (temperature, ethanol ratio, etc.) has been realized, validating the same trends as in the previous static mixing devices. 2 for conditions.

Esterification study in NiTech COBR TM

The equipment is a DN15 version of the reactor. This dimension and the associated nominal operating conditions increase the global flow-rate compared with the value required in Corning and Chart reactors. However, the length of the reactor has then been adapted to obtain the same 140 s residence time. The resultant conditions are given in Table 2 and are mainly based on the optimal conditions determined in previous flow-reactors.

It is important to note that the COBR TM then allows higher conversion level (about 40%) to be reached for the same given 140 s residence time. From its technical principle, the velocity in the NiTech reactor is low (about 0.01 m s -1 ) by comparison with Corning and Chart equipment (from 10 to 100 times more: 0.1 to 1 m s -1 ), which entails poor mixing by the static mixing effect. As a consequence, the mass transfer performances are governed only by the pulsation, through its amplitude and frequency. The influence of such 'technological' parameters has been experimentally estimated and correlated with the product amplitude-frequency, as is usually done in the case of pulsed systems. Results are presented in Fig. 11.

As it was shown that the reaction occurs as a mass transfer regime, the pulsation, characterized by the product amplitude-frequency, has a direct effect on mass transfer and favours the conversion as observed on Fig. 11. Conversions as high as 35% are obtained, significantly better than for flow-reactors for the same residence time. In fact, this reactor, thanks to the pulsation system, improves greatly the mass transfer (mainly through greater interfacial area generation) in a range that could not be reached in static mixing based reactors, where a compromise between performance and residence time has to be considered.

It is important to note that increasing residence time in the COBR TM is easy. The total possible available length in our lab is 51 m, which corresponds to more than 1 h with the operating conditions considered. For instance, with a residence time of 350 s, conversion was found to be about 60%. This offers interesting perspective when coupled with optimization of the reaction conditions (temperature, concentration, ethanol equivalent, volumic phase ratio, etc.) Therefore, the COBR TM appears to be a very interesting solution for two-phase applications, as it allows mass transfer performance to be enhanced independently of residence time. Nevertheless, the pressure drop involved with the pulsation system must be taken into account. Indeed, high mass transfer performances induce high cost in terms of pulsation power and energy consumption. To perform a comparison with static mixing based reactors (Corning and Chart), the energy dissipation rate in the COBR TM was computed. To achieve this, the evaluation method for power dissipation in pulse columns proposed by Jealous and Johnson [START_REF] Jealous | Power requirements for pulse generation in pulsed columns[END_REF] was used (Equation ( 4)). In this approach, the energy dissipation due to the net flow in the COBR TM is neglected and the total energy dissipation depends only upon amplitude and frequency of the pulsation.

ǫ = 16π 2 N 3C 2 d 1 -T 2 T 2 (x 0 f ) 3 (4)
with ǫ the energy dissipation rate (W kg -1 ), N the number of baffled cells per unit length (m -1 ), C d the orifice coefficient for the flow through the baffle hole; T the fractional free area defined as the ratio between orifice and tube diameter, x 0 the amplitude defined centre-to-peak (m), and f the oscillation frequency (Hz). The curves of Fig. 11 are redrawn in terms of energy dissipation rate in Fig. 12, where it is seen that the NiTech reactor provides the best use of energy dissipation.

Benchmarking

The results obtained on different equipments and under different operating conditions lead to the same conclusions: lipase catalysed esterification is limited by mass transfer. So as we mentioned in the introduction, the operation of this model reaction using these different technologies can also be considered as an assessment of the mass transfer performance of each reactor, especially in terms of generation of interfacial area. As the products and the reaction involved here are easy to carry out, this constitutes an excellent test to characterize mass transfer performance of different devices. Indeed, benchmarking of equipments could be performed in this way. This was done for the different equipments considered in the present work. A given 140 s total residence time was chosen and other parameters are given in Table 3. For dynamic mixing devices, 'optimal' operating conditions have been chosen as being those leading to the maximum conversion: optimal shaking for Coflore ® , optimal amplitude and frequency for COBR TM . 3 for conditions.

Figure 13 illustrates the comparison of mass transfer performance of the different equipments operated at their optimum operating conditions and at the same 140 s residence time. As expected, NiTech COBR TM appears the most efficient. Moreover, in such a device the possibility to reach high residence times (up to 100 min) also goes along with its enhanced mass transfer performance.

CONCLUSION

The main result of this work was to demonstrate that novel technologies, recently proposed for intensification of chemical processes, are indeed very suitable to operate two-phase reactions. This was tested on a model enzymatic reaction which represents an important class of enzymatic reactions where lipases are used for organic synthesis. Four commercial intensified reactor technologies were tested. The NiTech oscillating reactor proved to be the most efficient to transform the input mechanical power into mass transfer performance while enabling an easily adjustable residence time. Nevertheless Corning and Chart reactors were also shown to be very suitable candidates even though mass transfer performance depends on the flow-rate of the reacting mixture and therefore is not decoupled from the residence time. This last drawback results in a less convenient choice of operating conditions. The Coflore ® system was not so convincing, possibly due to its injection device, which was probably not well adapted to this liquid-liquid system.

More generally, it was shown here that the usual technical drawback for operation of these reactions in two-phase systems, i.e. mastering the liquid-liquid dispersion, can be circumvented. This is very interesting because such a drawback usually directs the industrialization of such reactions towards the use of low productivity batch systems using large agitated tanks with uneasy control of the two-phase dispersion. Indeed, continuous operation using agitated tanks with two-phase systems is very difficult, especially because the phase ratio in the output flow must be carefully controlled to ensure steady operation. When efficient continuous operation is desired, this implies operating the reaction in a monophasic system in order to be able to use a continuous fixed bed of immobilized enzymes. As was mentioned in the introduction, additional R&D studies are therefore needed, especially of complex immobilisation procedures, with a possible loss of activity. Conversely, using the new intensified reactor technologies proposed here, direct extrapolation to continuous steady operation of a two-phase enzymatic reaction at industrial scale is really possible.

Nevertheless, each two-phase system is unique and has a specific behaviour, depending on its physical properties: density, viscosity, interfacial tension. Therefore, the proposed demonstration is not universal and needs to be reinforced by testing other reaction systems.

Also, in addition to this interesting result, which is an incentive for promoting industrial enzymatic catalysis, it has been shown that the model reaction chosen here could also be a simple and elegant way to characterize mass transfer performance of these technologies. More precisely, it enables comparison of their ability to generate liquid-liquid dispersion and global assessment of the generated interfacial area. So, easy benchmarking can be proposed and may be an additional tool in the choice of the most suitable technology and optimal design and control of two-phase reactions performed in continuous mode.

Figure 1 .

 1 Figure 1. Flow reactors considered, from left to right: Corning Advanced-Flow TM , Chart Shimtec ® ,NiTechCOBR TM and AM Technology Coflore ® .

Figure 2 .Figure 3 .

 23 Figure 2. Flow-sheet of the continuous esterification process, based on the use of Corning Advanced-Flow TM reactor.

Figure 6 .

 6 Figure 6. Evolution of conversion according to lipase concentration with 140 s residence time. Volumic ratio between organic and aqueous phase is equal to 1. EtOH/Oleic acid molar ratio of 3. Temperature is 30 • C.

Figure 7 .

 7 Figure 7. Evolution of conversion according to temperature with 140 s residence time. Volumic ratio between organic and aqueous phase is equal to 1. EtOH/Oleic acid molar ratio of 3. Lipase concentration 100 LU g -1 .

Figure 8 .Figure 9 .

 89 Figure8. Evolution of conversion according to substrate ratio with 140 s residence time. Volumic ratio between organic and aqueous phase is equal to 1. Lipase concentration is 100 LU g -1 and temperature is 30 • C.

Figure 10 .

 10 Figure10. Evolution of conversion according to shaking power in Coflore ® with a residence time of 140 s. Substrate ratio = 3. Volumetric ratio between organic and aqueous phase is equal to 1. Lipase concentration is 100 LU g -1 and temperature is 30 • C.

Figure 11 .

 11 Figure 11. Evolution of conversion in COBR TM with respect to pulse characteristics See Table2for conditions.

Figure 13 .

 13 Figure 13. Benchmark of mass transfer performances See Table3for conditions.

  )).

	O
	OH	+	OH
	Lipase
	Phosphate Buffer pH 7,2
	O
	O		+ H 2 O

Table 1 .

 1 Operating conditions of esterification in flow reactors Influence of total flow-rate on conversion for Corning and Chart flow reactors. See Table1for conditions.

	Global			Volumic ratio		Mass ratio	Lipase	EtOH/Oleic
	flow-rate (g min -1 )		(ϕ organic /ϕ aqueous )		(ϕ organic /ϕ aqueous )	Temperature ( • C)	concentration (LU g -1 )	acid molar ratio
	From 15 to 150		1				0.78	30	100	3
		60
			Corning		Chart
		50					Chart		Corning
	Conversion (%)	20 30 40				Pressure drops limitation	Pressure drops limitation
		10
		0
		0	20	40	60	80	100	120	140	160
					Global flow-rate (g.min -1 )
	5 10 15 20 25 30 Figure 4. 0 35
		0	4		8		1 21 62 0

Energy dissipation rate (W.kg -1 ) Conversion (%)

Corning Chart Figure 5. Conversion obtained in flow reactors according to energy dissipation rate See Table 1 for conditions.

Table 2 .

 2 Operating conditions of esterification in COBR TM Conversion vs. energy dissipation rate for the different technologies See Table 1 and 2 for conditions.

	Global	Volumic ratio	Mass ratio		Lipase	EtOH/Oleic
	flow-rate (g min -1 )	(ϕ organic /ϕ aqueous )	(ϕ organic /ϕ aqueous )	Temperature ( • C)	concentration (LU g -1 )	Acid molar ratio
	110	1	0.78	30	50	3

Table 3 .

 3 Operating conditions applied for benchmarking

					EtOH/
					Oleic
				Lipase	Acid
		Residence	Temperature	concentration	molar
	Reactor	time (s)	( • C)	(LU g -1 )	ratio
	Batch
	Corning AFR
	Chart Shimtec ®	140	30	50	3
	AMT Coflore ®
	Nitech COBR TM