Sebastien Elgue

Annelyse Conte

Alain Marty

Jean-Stephane Condoret

Two-phase enzymatic reaction using Process Intensification technologies

Keywords: Esterification, biocatalysis, lipase, Process Intensification, flow chemistry, two-phase reaction

Developing continuous enzymatic process, especially when a two-phase medium is required, remains nowadays a great challenge. So, there is a need to provide a technical solution for generating highly controlled interfacial area. In this study, three types of reactors, conventionally used for chemical process intensification, were tested for continuous lipasecatalysed esterification in two-phase medium. On the single criterion of mass transfer performance that is considered here, the Nitech oscillating reactor proved to be the more efficient to transform the input mechanical power into mass transfer while proposing easily adjustable residence time. Nevertheless Corning and Chart flow reactors were shown to be very suitable candidates even if mass transfer performances are dependant on the flow-rate and therefore are not uncoupled from the residence time. This results in a less convenient choice of operating conditions. Also, it has been shown here that the model reaction could also be a simple way to characterize mass transfer performance of these technologies. KEyWORDS ABSTRACT overcome this technical difficulty and make the more efficient and economic use of the biocatalyst, this latter is used as an immobilized biocatalyst packed in a tubular reactor (1,4,5). Therefore, 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 opens the field to the so-called "intensified" reactors which is now an identified target for successful industrial development of new processes. 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. The assessment of the candidates will be done using a model reaction, the conventional lipase-catalysed esterification of oleic acid with ethanol in order to focus on the reactor performances only.

MATERIAL AND METHODS

Description of the reaction

The chosen reaction is a model reaction, the lipase-catalysed esterification with ethanol of a fatty acid, the oleic acid, to yield ethyloleate and water (Figure 1). To catalyse this esterification, the commercial liquid form of lipase B from Candida antarctica, namely Lipozyme CalB L (Novozymes, Bagsvaerd, Denmark), was chosen. Its concentration is 5000 LU/g. To obtain an enzyme activity in agreement with the residence times envisaged in the continuous process, it was necessary to dilute from 50 to 100 times the initial solution in a phosphate buffer 0.1M, pH 7.2. Esterifications are equilibrated reactions, nevertheless the reaction is shifted towards esterification because the hydrophobic ester accumulates in the organic solvent which indeed plays the role of a reservoir for the most hydrophobic reactant, the ester. In this work, cyclohexane has been used to play this role.

INTRODUCTION

O peration of enzymatic reactions in two-phase medium, i.e. oil-water dispersions, is very common when hydrophobic reactants are concerned, as for instance in the case of hydrolysis of lipids (1-2). Moreover, enzymatic reactions, such as esterification, can be effective in two-phase media using free enzymes (3). Indeed, even in a two-phase system with a water activity close to unity, esterification can be effective due to a shift of the thermodynamic equilibrium by maintaining a low ester activity. After preliminary studies, when industrial development is envisaged, continuous operation must be considered in order to increase productivity and economic viability. But, at present time, it is not possible to propose a realistic continuous liquid-liquid reactor where controlled contact area and steady operation is insured, while this would greatly simplify the development of the process. So, usually, to 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 to the fluidic module to control the process temperature. A sampling valve is located at reactor outlet to estimate the final conversion by HPLC chromatography analysis.

RESULTS AND DISCUSSION

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. 90.4 % conversion was obtained within 30 minutes. In addition, to make the comparison as complete as possible, the esterification has also been carried out in a standard glass reactor at lab scale (1L, double jacketed with impeller stirring device) at optimal stirring velocity (360 rpm). In that case, the operating conditions have been chosen to be comparable to those applied in the different continuous reactors. Batch study shows that kinetics (estimated from conversion rate) is 6 times lower in the batch laboratory scale reactor than in small Eppendorf tubes. Such behaviour demonstrates the influence and the limitation of the 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 (14-15): an increase of flow-rate favours the generation of interfacial area and therefore mass transfer. Therefore, as reactor volume is fixed, increasing flow-rate reduces the residence time. Consequently, in the case of two-phase reactions, depending of the prominent 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 global flowrate influence has been carried out in Corning and Chart flow reactors (figure 3). Other operating conditions are respectively: volumic phase ratio equal to 1, temperature of 30°C, lipase concentration of 100 LU/g and molar ratio between ethanol and oleic acid equal to 3. The evolution of conversion in respect to global flow-rate in the Corning reactor exhibits a flat bell-shaped curve, with 25% conversion at maximum fixed by the reactor volume (120 mL).

Material

The use of several flow reactor technologies, already existing at commercial scale, is considered. In fact, 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 are 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 two-phase 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 via the use of mechanical stirrers. Three commercial solutions are considered here (figure 2). The ability to carry out two-phase reactions and the technological maturity have driven the choice of these technologies. Hence, two heat exchanger-reactors, respectively commercialized by Corning (6-9) and Chart Industries (10)(11), and the Nitech Solutions COBR TM (12-13) involving dynamic mixing by a pulsation device, were studied. It has been chosen to work on the pilot scale versions of these equipments, in order to be able to easily extrapolate the process to industrial scale. Consequently, the following versions of each reactor have been used: -Corning AFR Gen 1, with channel width of 4 mm and depth of 0.7 mm. -Chart Shimtec with channel width of 2mm and depth of 2 mm. -Nitech COBR with tube diameter of 15 mm and doughnuts diameter of 8 mm.

Methods

The lipase-catalysed esterification of oleic acid with ethanol has been studied at pilot scale, which means that the global flow-rate (i.e., the sum of the flowrates of both phases) is in the range 15 to 150 g/min. A specific fluidic module has been used for controlling 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), mass-flowmeters based on the Coriolis effect (Micromotion) and temperature and pressure sensors. PID controllers ensure constant delivering of flow-rates by acting on the motor pump regime. Special care has been taken to prevent from pulsating flow-rates, especially in the case of COBR TM . The equipment is a DN15 version of the reactor, including a piston to provide pulsation. This dimension imposes to increase the global flow-rate compared to the value required in Corning and Chart reactors. However, the length and the volume of the reactor have then been adapted to obtain the same 140 seconds residence time. From its technical principle, the velocity in the Nitech reactor is low (about 0.01 m/s) that entails a poor mixing by the static mixing effect. As a consequence, the mass transfer performances are only governed by the pulsation, through its amplitude and frequency. As it was shown that the reaction occurs in mass transfer regime, the pulsation, characterized by the product amplitude-frequency, has a direct effect on mass transfer and favours the conversion (figure 5).

Such a profile complies with the curves reported in literature (14). Nevertheless, in the present case, the profile is sluggish according to the reaction characteristics in terms of mass transfer and of kinetics. At flow-rates lower than 105 g/min, 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 global flow-rate). At higher flow rates, the conversion will decrease because reduction of the residence time is more detrimental than the gain in mass transfer which moreover will reach an upper cap. Unfortunately, because of the pressure drops involved and the limitation of dosing line pumps, it was not possible to experimentally observe such behaviour.

For the Chart reactor, the improved mixing and the residence time reduction seem to counterbalance in the range of tested flow rates. This is in agreement with the fact that the Chart reactor offers better mass transfer performances at low flowrates. As a consequence, the maximal conversion (around 25% according to the reactor volume of 120 mL) 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 also in high power consumption because pressure drop was larger. Different parameters have been investigated to compare the associated behaviours of the Corning and Chart reactors. In this way, the influence of temperature, lipase concentration and substrate ratio (molar ratio between ethanol and oleic acid) have been thoroughly studied. These studies emphasize the limitation of the considered lipase catalysed esterification by mass transfer and the possibility of process optimisation. For these two flow reactors, this parametric study has shown that, in spite of a possible optimisation, the antagonism residence time / mass transfer did not unable high conversion level to be reached in the considered reactor. To estimate the residence time required to obtain high conversion, specific experiments were carried out in both reactors with operating conditions favourable to kinetics: flow-rate of 46 g/min, temperature of 50°C, and substrate ratio of 6. Results (figure 4) emphasize clearly the possibility to reach high conversion levels with moderate increase of residence time, i.e. of reactor volume. Compared to the initial 140 s residence time, this means that such residence times can be easily obtained by using reactors with larger volumes (3 to 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 open-loop intensified flow-reactors. Note that a correlative increase of the pressure drop and therefore of the power consumption must also be taken into account. When efficient continuous operation is really desired, this implies to operate the reaction in a monophasic system in order to be able to use a continuous fixed bed of immobilized enzymes. Additional R&D studies are therefore needed, especially 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. 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 gives access to comparison of their ability to generate liquid-liquid dispersion and to a global assessment of the generated interfacial area. Therefore, in respect to this criterion, easy benchmarking can unexpectedly be proposed and may constitute an additional tool for the choice of the more suitable technology and optimal design and control of two-phase reactions performed in a continuous mode.

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 to perform. For instance, with a residence time of 350 seconds, conversion was found to be about 60%. This offers interesting perspective when coupled with optimisation of the reaction conditions: temperature, concentration, ethanol equivalent, volumic phase ratio, etc.

Benchmarking

The results obtained on different equipments and various operating conditions lead to the same conclusions: the considered lipase catalysed esterification is limited by mass transfer. Therefore, the operation of this model reaction using these different technologies can also be considered as an assessment of the mass transfer performances 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 characterise mass transfer performances of different devices. Indeed, a first approach of benchmarking of equipments could be performed in this way. This was done for the different equipments considered in the present work. A given 140 second global residence time was applied, with temperature of 30°C, lipase concentration of 50 LU/g and molar ratio between ethanol and oleic acid of 3. For COBR TM , optimal amplitude and frequency have been chosen as being those leading to the maximal conversion. Figure 6 illustrates the resulting comparison of mass transfer performances of the different equipments. Based on this criterion, Nitech COBR TM appears the most efficient. Moreover, in such a device the possibility to reach high residence time (up to 100 minutes) also goes along with its enhanced mass transfer performances.

CONCLUSION

The main result of this work was to demonstrate that novel technologies recently proposed for intensification of chemical processes are indeed very suitable for two-phase reactions. This was tested on a model enzymatic reaction but representing an important class of enzymatic reactions where lipases are used for organic synthesis. Three technologies of intensified reactors were tested. From the criterion of mass transfer performances, 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 reactors were shown to be very suitable candidates even if mass transfer performances depend on the flow-rate of the reacting mixture and therefore are not uncoupled from the residence time. This results in a less convenient choice of operating conditions. But such technologies are likely to be more reliable and less expensive than the Nitech reactor that counterbalances partly weaker mass transfer performance. More generally, it was shown here that the usual technical drawback for operation of these reactions in two-phase systems, i.e., the mastering of the liquid-liquid dispersion, can be circumvented. This is very interesting because such a drawback usually directs the industrialisation of such reactions towards the use of low productivity batch systems using large

Figure 1 .

 1 Figure 1. Reaction scheme

Figure 2 .Figure 3 .

 23 Figure 2. Considered Process Intensification technologies -external and internal designs respectively from left to right for Corning, Chart and Nitech reactors

Figure 4 . 5 .

 45 Figure 4. Esterification conversion according to residence time in Corning reactor Figure 5. Evolution of conversion in COBR TM in respect to pulse characteristics

Figure 6 .

 6 Figure 6. Benchmark of mass transfer performances


Florian Venel

Hiroki Nagashima

M Rankin

Christelle Anquetil

Dr Vytautas Klimavicius

G Torsten

Gerd Gutmann

Sylvie Buntkowsky

Olivier Derenne

Arnaud Lafon

Huguet

Andrew G M Rankin

Prof Torsten G Gutmann

Gerd Buntkowsky

Dr Sylvie Derenne

Olivier Lafon
email: olivier.lafon@univ-lille.fr

Arnaud Huguet

Frédérique Pourpoint
email: frederique.pourpoint@centralelille.fr

Dr H F Venel

Dr A G M Nagashima

Prof O Rankin

Dr F Lafon

Pourpoint

Dr B C Anquetil

Buntkowsky

Characterization of Functional Groups in Estuarine Dissolved Organic Matter by DNP-enhanced 15 N and 13 C Solid-State NMR

Keywords: C, 15 N, DNP, DOM, Seine estuary

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

Introduction

Dissolved organic matter (DOM) is one of the most mobile carbon pools at Earth's surface and is an important component of the global carbon cycle. Changes in the chemical or structural composition of DOM can drastically influence the aquatic ecosystem [1]. For example, DOM is a major source of energy and nutrients for bacteria in sea water. In this context, estuaries play a pivotal role in the transfer of DOM from the continents to the oceans.

However, the transformations of DOM in estuaries are not yet well understood. [2][3][4] The characterization of DOM is challenging owing to its structural complexity and heterogeneity. [5] It has been shown that solid-state nuclear magnetic resonance (ssNMR) spectroscopy can provide unique information on the atomic-level structure of DOM as well as the spatial and temporal changes in DOM composition. [6][7][8] Most ssNMR studies of DOM rely on the observation of 13 C nuclei, since 13 C ssNMR spectra of these samples exhibit good resolution and can be acquired within a day. Nevertheless, it has been shown that 15 N ssNMR of DOM can allow the identification of several nitrogen-containing functional groups, including amine, amide, and heterocyclic moieties, such as pyrrole and indole groups [9,10]. However, 15 N isotope suffers from a low natural abundance (0.37%) and a low gyromagnetic ratio (γ( 15 N)/γ( 1 H) ≈ 10.1%). Furthermore, atomic N/C ratios commonly range between 10 -1 and 10 -2 in DOM [11]. Consequently, conventional ssNMR detection of 15 N nuclei lacks sensitivity and requires acquisition times of several days, which often preclude the use of this technique.

Dynamic nuclear polarization (DNP) under magic-angle spinning (MAS), which is based on the microwave-driven transfer of polarization from unpaired electrons to the nuclear spins, can enhance the sensitivity of ssNMR [12,13].

We have recently shown that DNP improves the sensitivity for the detection of 13 C nuclei in DOM by a factor of 4 with respect to conventional ssNMR, i.e. an acceleration of 16 in acquisition times. [14] The sensitivity gain provided by DNP has been leveraged to detect NMR signals of 15 N nuclei in organic, hybrid and inorganic materials [15][16][17][18][19][20]. However, to the best of our knowledge, it has never been applied to record 15 N NMR spectra of DOM.

DNP-enhanced NMR is applied herein to probe the local 13 C and 15 N environments as well as the functional groups in DOM of the Seine estuary.

Experimental session

Samples

Four water samples (100 liters each) were collected in April 2015 at mid-depth along the three parts of the Seine estuary: upstream (Caudebec), in the maximum of turbidity zone (Tancarville and Fatouville) and downstream (Honfleur) (Fig. 1). [4] All samples were filtered at 0.7 μm using glass fiber filters, concentrated to a few liters and desalted using reverse osmosis and electrodialysis before being concentrated further and freezedried, as described in ref. [4] Figure 1. Map of the Seine estuary in France with the location of the sampling sites (yellow dots).

Scanning electron microscopy

The SEM images were recorded on an Itachi S-3400N equipped with a tungsten filament operating at an acceleration voltage of 15.0 kV. The powder is sprinkled on a double-sided carbon tape and then a carbon deposit is made to avoid charge effects. S1. The investigated samples were packed into a 3.2 mm sapphire rotor closed with zirconia caps. DNP experiments were performed at 9.4 T using a Bruker BioSpin Avance III DNP NMR spectrometer equipped with a triple-resonance 1 H/X/Y 3.2 mm low-temperature MAS probe and a gyrotron generating a continuous 263 GHz microwave irradiation [21]. The microwave irradiation was transmitted through a corrugated waveguide to the NMR MAS probe, and its power delivered to the sample was ca. 6 W. For all experiments, the B0 field was 9.4 T, which corresponds to 1 H, 13 C and 15 N Larmor frequencies of 400.0, 100.6 and 40.6 MHz, respectively. The samples were spun at a MAS frequency of νR = 8 kHz.

DNP-enhanced NMR

The DNP-NMR spectra were acquired at a temperature of ca. 110 K, which was stabilized using a Bruker BioSpin MAS cooling system. The 1 H→ 13 C CPMAS spectra were recorded with and without microwave (µw). The DNP enhancements, εon/off, of the NMR signals were measured as the ratios between the NMR signals intensities with and without microwave irradiations under the same experimental conditions. The 1 H π/2 pulse duration was equal to 3 μs, while the cross-polarization (CP) contact times were equal to 50 μs in Fig. 5 and3.5 ms in Figs. 2 and4. During the CP transfer, the radio-frequency (RF) nutation frequency on the 13 C channel was constant and equal to 62.5 kHz, whereas the 1 H RF nutation frequency was linearly ramped from 50 to 100 kHz. SPINAL-64 decoupling with a 1 H nutation frequency equal to 47 kHz was applied during the acquisition of the 13 C free induction decay (FID). [22] The 1 H→ 13 C CPMAS spectra result from averaging 512 transients with a recovery delay of 0.73 s, i.e. an experimental time of 7 min. The 13 C isotropic chemical shifts were referenced to tetramethylsilane (TMS) using the O=C group at 176.5 ppm of 30% 15 N- 13 C labeled glycine sample in 20 mM TOTAPol in glycerol-d8/D2O/H2O (6/3/1, w/w/w) as a secondary reference.

The 1 H→ 15 N CPMAS spectra were recorded with microwave irradiation. For 1 H→ 15 N CPMAS transfer, the 1 H π/2 pulse duration and the contact time were 2.75 μs and 3 ms, respectively. During the CP transfer, the 1 H RF nutation frequency was linearly ramped from 50 to 100 kHz, whereas the 15 N RF field amplitude was constant and equal to 45 kHz. SPINAL-64 1 H decoupling was applied during the acquisition of 15 N spectra with an RF amplitude of 60 kHz. The 1 H→ 15 N CPMAS spectra were acquired with a recycle delay of 0.73 s. The number of transients and experimental times for the various samples are given in Table S2. The 15 N isotropic chemical shifts were referenced to nitromethane using the resonance of 15 N-labeled γ-glycine (-347.6 ppm) as a secondary reference [23,24]. The 1 H→ 15 N CPMAS spectra were simulated using the DMfit software [25].

Results and discussion

The conventional 1 H→ 13 C CPMAS spectra of DOM shown in Fig. S1 were recorded in experimental times ranging from 15 to 23 h. They show resonances typical of riverine DOM [8,[26][27][28], including a broad peak ranging from 0 to 60 ppm assigned to aliphatic carbon sites, an intense signal at 72 ppm with a shoulder at 107 ppm produced by O-alkyl and anomeric groups in carbohydrates, a broad resonance with maximum at 130 ppm corresponding to aromatic and phenolic C sites and a carboxyl/amide signal at 175 ppm. The relative intensities of the various functional groups only weakly vary between the different samples. Hence, the local environments of 13 C nuclei remain mostly unchanged along the Seine estuary.

Nevertheless, a limitation of these conventional 1 H→ 13 C CPMAS experiments is the duration of these experiments.

MAS DNP was used to circumvent this issue. For that purpose, these samples were impregnated with solutions of AMUPol [29] These solvents form glasses at 100 K and are partially or fully deuterated. Using a glass-forming deuterated solvent has been shown to be an efficient approach to improve the DNP enhancement [13,[START_REF] Rosay | Sensitivity-enhanced Nuclear Magnetic Resonance of biological solids[END_REF]. 1. 13 C DNP-enhancement for the four samples impregnated with either AMUPol solutions in glycerol/water or DMSO/water. Enhancements 𝜀 !"/!$$ are calculated by a comparison of the signal intensity with and without the microwaves on the peak resonating at 180 ppm on the 13 C spectrum for a spinning speed of 8 kHz. glycerol/water (Fig. 2a). In practice, the DNP-enhanced 1 H→ 13 C CPMAS spectrum of DOM from Caudebec was acquired in 7 min, instead of 22 h for the conventional spectrum of Fig. S1a. Table 1 reports DNP enhancements of this signal for the other samples. These enhancements are lower than 6 for the DOM collected downstream of Caudebec for both samples impregnated with glycerol/water and DMSO/water mixtures. The DNP enhancement has been reported to decrease when the size of the particles increases [START_REF] Pinon | [END_REF]. The size of the DOM material resulting from the isolation and desalting by reverse osmosis/electrodialysis was investigated by SEM and we noted an increase in the particle size downward the estuary (Fig. 3), which may be responsible for the decrease in DNP efficiency. These εon/off values measured for Seine DOM samples are comparable to those previously measured for DOM of Siberian rivers (5 to 10) with DMSO/water solutions of AMUPOL [14]. Furthermore, both the εon/off factors and the mass sensitivity, defined as the signal-to-noise ratio per square root of unit of time for a constant mass of sample, were higher for glycerol/water than DMSO/water. For instance, for DNP-enhanced 1 H→ 13 C CPMAS experiments on DOM from Caudebec, the mass sensitivity was equal to 90 and 40 s -1/2 .g -1 when the sample was impregnated with glycerol/water and DMSO/water, respectively. Due to a very low amount of DOM from Tancarville (Table S1), this sample could only be impregnated with one AMUPol solution. The glycerol/water solution was chosen as it yielded higher sensitivity for DOM from Caudebec. signal of DMSO at 41 ppm is superimposed on that of the aliphatic groups. Nevertheless, the 13 C signals of carboxyl/amide, anomeric and aliphatic functionalities can be observed for both solvents, whereas the 13 C signal of the O-alkyl group is clearly resolved for the samples impregnated with DMSO/water mixture. As shown in Fig. 5, the 13 C signal of deuterated glycerol and DMSO can be reduced by using short contact time in CPMAS in order to selectively transfer the DNP-enhanced 1 H polarization to the protonated 13 C sites of the DOM, but not to the deuterated 13 C nuclei of the solvent [32,33]. Nevertheless, this approach prevents the observation of the carboxyl and amide sites, which are also not protonated.

Caudebec

The relative intensities of the different 13 C sites in Figs. After having optimized and validated the use of DNP for the observation of 13 C nuclei in estuarine DOM, we investigated how the sensitivity gain provided by DNP can facilitate the detection of 15 N NMR signals of these samples. The NMR detection of this isotope is challenging owing to (i) the low natural abundance of 15 N isotope (0.37%), (ii) to the low nitrogen content in the DOM (< 1%) and (iii) the low amount of sample (Table S2). To the best of our knowledge, DNP has never been applied to accelerate the acquisition of 15 N NMR spectra of DOM. In DNP-enhanced 1 H→ 15 N CPMAS spectra, the cross-polarization transfers the DNP-enhanced 1 H polarization to the nearby 15 N nuclei and hence, the DNP enhancements for 1 H→ 13 C and 1 H→ 15 N CPMAS experiments are expected mass sensitivity for the glycerol/water mixture in the case of 1 H→ 13 C CPMAS spectra, 1 H→ 15 N CPMAS spectra were only acquired for DOM samples impregnated with glycerol/water mixture (see Fig. 6). Sensitivity enhancement provided by DNP allows the detection of 15 N signals of these isotopically unmodified DOM within a reasonable experimental time (≤ 64 h), in spite of the low N/C atomic ratios (Tables 3 andS2, the organic and nitrogen contents of investigated DOM samples were determined by elemental analysis). Assuming similar sensitivity enhancements due to DNP for 1 H→ 13 C and 1 H→ 15 N CPMAS experiments, the latter would require more than a year without DNP. To the best of our knowledge, these spectra are the first DNP-enhanced 15 N NMR spectra of DOM reported so far. The 1 H→ 15 N CPMAS spectra are dominated by a peak resonating at 15 N isotropic chemical shift δiso ≈ -259 ppm assigned to amide group [24]. As seen in Table 4 and in Fig. 6, the broadening of this peak for the DOM from Fatouville and Honfleur indicates a larger distribution of local environments for the amide groups near the mouth of the Seine, where a decrease of the riverine vs. marine fraction of DOM occurs 4 .

Furthermore, the 15 N spectrum of DOM from Caudebec exhibits a broad shoulder centred at -216 ppm, which could subsume the contributions of substituted pyrrole and pyrrole functional groups [24]. Nevertheless, as the signal-to-noise ratio of this broad shoulder is low, its absence for the DOM sampled at other sites may result from the lower DNP enhancements. It is noted that even though amide functional groups are dominant in soil, sediment or dissolved organic matter, pyrrole groups can also be observed. In riverine DOM, the relative abundance of pyrrolic vs. amide groups was shown to be dependent on the river [24]. The relative abundance of pyrrole groups in DOM was suggested to be dependent on diagenetic (i.e. degradation) processes. The DNP-enhanced 1 H→ 15 N CPMAS spectra provide information on the presence of amide group and their local environment, which cannot be obtain by 1 H→ 13 C CPMAS spectra since the 13 C signals of carboxyl and amide groups overlap.

Table 3. N/C ratio for the four samples determined by elemental analysis [4] .

Table 4. 15 N isotropic chemical shift and FWHM (full width at half maximum) used to simulate the 1D 1 H→ 15 N CPMAS spectra of Fig. 5 (see the simulated spectra shown in Fig. 6).

Caudebec

Conclusion

We applied DNP-enhanced 13 C and 15 N NMR spectroscopy to probe the functional groups in DOM collected along the three parts of the Seine estuary (the upstream zone, the maximum of turbidity zone and the downstream zone). DNP experiments were optimized and validated on 1 H→ 13 C CPMAS experiments. These DNPenhanced 13 C NMR spectra indicate in agreement with conventional 13 C NMR spectra that the nature and the proportion of the different functional groups in DOM are unmodified in the different zones of the Seine estuary.

Furthermore, we demonstrated for the first time the possibility using DNP under MAS to detect 15 N NMR signals of DOM samples, which contain low fraction of nitrogen atoms and are not amenable to 15 N isotopic enrichment.

These NMR spectra show that most of nitrogen atoms in DOM are in the amide form. In addition, these NMR data indicate a broader distribution in the local environment of nitrogen atoms in DOM collected near the mouth of the Seine. The structural insights provided by DNP-enhanced 15 N NMR cannot be retrieved from the 13 C NMR spectra because of the overlap between 13 C carboxyl and amide NMR signals.

For

  DNP-NMR experiments, the DOM samples were impregnated in the air with 10 mM AMUPol solution in [ 2 H8]glycerol/[ 2 H2]O (60/40 v/v) or [ 2 H6]-dimethylsulfoxide (DMSO)/[ 2 H2]O/H2O (78/14/8 w/w/w). The mass of DOM and the volume of AMUPol solution are given in Table

  nitroxide biradicals, which is currently one of the most efficient polarizing agent for DNP-NMR experiments at 9.4 T and 100 K. We tested two different 10 mM AMUPol solutions, using either [ 2 H8]glycerol/[ 2 H2]O (60/40 v/v) or [ 2 H6]-dimethylsulfoxide (DMSO)/[ 2 H2]O/H2O (78/14/8 w/w/w) mixtures as solvents.

Figure 2 .

 2 Figure 2. 1 H→ 13 C CPMAS NMR spectra with and without µw irradiation of DOM from Caudebec impregnated with 10 mM AMUPol solutions in (a) [ 2 H8]glycerol/[ 2 H2]O and (b) [ 2 H6]-DMSO/[ 2 H2]O/H2O mixtures at 9.4 T and 110 K with a MAS frequency νr = 8 kHz, CP contact time of 3.5 ms

Fig. 2

 2 Fig. 2 shows the 1 H→ 13 C CPMAS spectra of DOM from Caudebec impregnated with AMUPol solutions. DNP enhances the 13 C NMR signals of DOM and the solvent by transferring the polarization of unpaired electrons to protons. This DNP-enhanced 1 H polarization is subsequently transferred to 13 C nuclei by CPMAS. For instance, DNP enhances the 13 C signal at 180 ppm assigned to carboxyl and amide sites by a factor εon/off = 12, i.e., an acceleration factor of 144 in acquisition time, for the DOM from Caudebec impregnated with AMUPol solution in

Figure 3 .

 3 Figure 3. SEM images of DOM from Caudebec, Tancarville, Fatouville and Honfleur. The magnification are x3000 for Caudebec, x5000 for Tancarville and Fatouville x5000 and x1500 for Honfleur.

Fig. 4

 4 Fig. 4 shows the DNP-enhanced 1 H→ 13 C CPMAS spectra with a contact time of 3.5 ms of DOM impregnated with AMUPol solutions. Despite the use of deuterated solvents, limiting the 1 H-13 C polarization transfer, the spectra are dominated by the 13 C NMR signals of the solvent, overlapping the DOM signals. However, the regions of the DOM spectrum masked by the solvent signal depend on the solvent. For the samples impregnated with glycerol/water mixture, the 13 C signals at 75 and 62 ppm assigned to the 2-C and 1,2-C sites of glycerol overlap with those of O-alkyl signals of DOM, whereas for the samples impregnated with DMSO/water mixture, the 13 C

  4 and 5 are similar for the different DOM samples. This observation is consistent with the conventional 1 H→ 13 C CPMAS spectra of Fig. S1 and confirms that the local environment of 13 C nuclei in DOM is mostly unchanged along the Seine estuary. This agreement between conventional and DNP-enhanced NMR spectra indicates notably that the impregnation with AMUPol solution does not alter the atomic-level structure of DOM. Although DNP-enhanced and conventional 1 H→ 13 C CPMAS spectra provide similar information, DNP accelerates the acquisition of the 13 C signals by two orders of magnitude with respect to conventional NMR.

Figure 4 .Figure 5 .

 45 Figure 4. 1D 1 H→ 13 C CPMAS DNP NMR spectra of DOM from Caudebec (brown), Tancarville (red), Fatouville (blue) and Honfleur (green) impregnated with 10 mM AMUPol solutions in (a) [ 2 H8]-glycerol/[ 2 H2]O and (b) [ 2 H6]-DMSO/[ 2 H2]O/H2O mixtures at 9.4 T and 110 K with a MAS frequency nr = 8 kHz, CP contact time of 3.5 ms and microwave irradiation. Asterisks denote the spinning sidebands. It should be noticed that a spinning speed of 10 kHz was also tested [not shown] in order to check that the spinning sidebands of the carbonyl group do not overlap with the aliphatic resonances.

Figure 6 .

 6 Figure 6. Experimental (blue) and simulated (red) DNP-enhanced 1 H→ 15 N CPMAS spectra of DOM from (a) Caudebec, (b) Tancarville, (c) Fatouville and (d) Honfleur impregnated with 10 mM AMUPol solution in [ 2 H8]-glycerol/[ 2 H2]O mixture and with microwave irradiation. The spectra b, c and d were simulated with one component, whereas the spectrum a was simulated with one component. The NMR parameters of the simulated spectra are reported in Table4. The full lines correspond to the DMFit simulation of the spectrum, while the dashed lines for the Caudebec spectrum represent the two components.

  Figure 6. Experimental (blue) and simulated (red) DNP-enhanced 1 H→ 15 N CPMAS spectra of DOM from (a) Caudebec, (b) Tancarville, (c) Fatouville and (d) Honfleur impregnated with 10 mM AMUPol solution in [ 2 H8]-glycerol/[ 2 H2]O mixture and with microwave irradiation. The spectra b, c and d were simulated with one component, whereas the spectrum a was simulated with one component. The NMR parameters of the simulated spectra are reported in Table4. The full lines correspond to the DMFit simulation of the spectrum, while the dashed lines for the Caudebec spectrum represent the two components.

Table 2 .

 2 Average

	Caudebec	230	10
	Tancarville	150	6
	Fatouville	425	14
	Honfleur	2,500	27

size and standard deviation for each compound calculated by averaging the sizes of 20 particles

Average size (µm²)

Standard deviation (µm²)

Acknowledgments

The Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation, Hauts-de-France Region, and FEDER are acknowledged for supporting and funding partially this work. Financial support from the contract Isite ULNE : MOFFiN-OPE-2019-43-5400 is gratefully acknowledged. We thank the GIP Seine-Aval through the funding of the MOSAIC project (2014-2017) during which the Seine estuary samples were collected. Alexandre Thibault, Edith Parlanti and Mahaut Sourzac are thanked for the collection and preparation of the DOM samples. V.K. acknowledges financial support by the AvH society.


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  Screening and modes of action of antagonistic bacteria to control two fungal pathogens, Phaeomoniella chlamydospora and Neofusicoccum parvum, involved in grapevine trunk diseases Haidar Rana, Marc Fermaud To cite this version: Haidar Rana, Marc Fermaud. Screening and modes of action of antagonistic bacteria to control two fungal pathogens, Phaeomoniella chlamydospora and Neofusicoccum parvum, involved in grapevine trunk diseases. 10. International workshop on Grapevine Trunk diseases, Jul 2017, Reims, France.

Haidar et al., 2016; Microbiological Research 8 bacteria strains: significant reduction of necrosis length in stem cuttings between 32 and 39% Résults

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