Carine Alcon

Toutou Cheu

Cheu Xiong✉
email: tou-cheu.xiong@inrae.fr

Calcium Live Imaging at Multi-Scales from Cellular to Organ Level in Arabidopsis thaliana

Keywords:

Plants must adapt to environmental constraints. For this, they are able to perceive several types of stress in isolation or in combination manner. At the cellular level, after the perception of stress, cell signaling is set up to allow the establishment of the specific response. The calcium ion is known to be one of the ubiquitous second messengers which is involved in most of the stresses perceived by the plant. Changes of free cytosolic calcium but also in other cellular compartments are able to activate or inactivate several mechanisms involved in the cell to cope with the changes of environmental conditions. Several calcium reporters have been intensively used to visualize calcium signals in different conditions. In this chapter, we will present only genetically encoded fluorescent reporters for calcium imaging in living plant tissues to measure variations in calcium at several scales. The FRET (fluorescence resonance energy transfer) YC3.60 and the intensiometric GCamP3 sensors will be used in this method chapter. The image analyses will be also detailed for fluorescence quantification of calcium variation.

Introduction

The calcium ion is a ubiquitous second messenger in cells and changes of free calcium concentration have been studied for several decades for its role in cell signaling. Thanks to calcium imaging, the role of calcium in cell signaling has been described in detail, and the "calcium signature" concept has emerged to encode specific information that is relayed to downstream signaling events. Calcium signals have been imaged with different fluorescentce dyes in animal cells and to some extent in plant cells. However, the use of calcium fluorescentce dyes shows some limitation of permeability due to the plant cell wall and to subcellular targeting. Later on, the genetically encoded fluorescent reporters that have been generated have made it make much easier to carry out calcium imaging. Then the introduction of genetically encoded calcium indicators (GECIs) into plants has been successfully done, thus allowing to measure free calcium changes in different tissues or whole plants. There are two types of GECIs: bioluminescent and fluorescent ones. The first type does not require excitation by light, but needs a cofactor. The wellknown bioluminescent GECI is the aequorin from jellyfish and possesses three calcium-specific EF-hand domains. Upon calcium binding, aequorin emits one single photon that can be collected for calcium quantification. The apo-aequorin sensor is the first GECI used in plants and allows to show calcium signals in response to abiotic stress [START_REF] Knight | Transgenic plant aequorin reports the effects of touch and cold-shoc k and elicitors on cytoplasmic calcium[END_REF]. Its low-light emission is a limit for visualizing easily calcium signals in plants with a microscope, and it is more dedicated for whole plant calcium monitoring with a luminometer device [START_REF] Knight | Transgenic plant aequorin reports the effects of touch and cold-shoc k and elicitors on cytoplasmic calcium[END_REF]. Improved bioluminescent sensors working by bioluminescence resonance energy transfer (BRET) have

Affiliationids : Aff1

Affiliationids : Aff1, Correspondingaffiliationid : Aff1 been successfully engineered and several bioluminescent sensors showed an up to fivefold increase when compared to aequorin [START_REF] Baubet | Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ repo rters at the single-cell level[END_REF][START_REF] Xiong | Imaging long distance propagating calcium si gnals in intact plant leaves with the BRET-based GFP-aequorin reporter[END_REF]. However, bioluminescence calcium imaging is still difficult without a dedicated camera that has the capacity to detect a single photon. The development of fluorescence-based GECIs has deeply changed calcium signaling studies in plants. The FRET (fluorescence resonance energy transfer) YC2.1 sensor has been one of the calcium reporters successfully used with microscopy. One major example of its use has been the study of the cytosolic calcium variation in stomatal guard cells induced by the plant hormone abscisic acid [START_REF] Allen | A defined range of guard cell calcium oscillation parameters encodes stomatal m ovements[END_REF]. An oscillation signal has been measured with an optimal pattern of cytosolic calcium increase and period parameters for triggering stomatal closure [START_REF] Allen | A defined range of guard cell calcium oscillation parameters encodes stomatal m ovements[END_REF]. The FRET sensor is composed of two fluorescent proteins (FPs), CFP (cyan FP), and YFP (yellow FP), linked by a calmodulin (CaM) and its M13 synthetic peptide target sequence. Upon calcium binding, conformation is changed in a way that allows FRET events between CFP and YFP. Then, the CFP fluorescent signal decreases, whereas the YFP signal increases during the FRET that reflects free calcium level increase. FRET sensors have been improved to give a better dynamic range to visualize small calcium variations by replacing the fluorescent partners CFP and YFP by cerulean FP and Venus FP, respectively. The modification improves the FRET efficiency [START_REF] Nagai | Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permute d yellow fluorescent proteins[END_REF]. Another FRET sensor, YC3.60, has a good dynamic range and has been used in diverse plant cell types such as guard cells, pollen tubes, and root hairs [START_REF] Monshausen | Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosoli c Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis[END_REF][START_REF] Iwano | Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth[END_REF][START_REF] Yang | Isolation of a strong Arabidopsis guard cell promoter and its pot ential as a research tool[END_REF] or in different subcellular compartments such as chloroplast, mitochondria, and peroxisomes [START_REF] Loro | Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmi c/mitochondrial Ca handling relationship in plant cells[END_REF][START_REF] Loro | Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent pro tein sensors reveals organelle-autonomous Ca2+ signatures in the stroma[END_REF][START_REF] Krebs | FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca 2+ dynamics[END_REF][START_REF] Grenzi | Structural insights into long-distance signal transduction pathways mediated by plant glutamate receptor-like channels[END_REF][START_REF] Costa | H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scaveng ing system[END_REF]. A low affinity sensor was also designed in order to measure calcium levels in different compartments with a high calcium concentration such as endoplasmic reticulum [START_REF] Bonza | Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arab idopsis root cells using a genetically encoded Cameleon sensor[END_REF]. Another type of fluorescent calcium sensor, the GCamP sensors, consists of circularly permuted green fluorescent protein (cpGFP), CaM, and CaM-interacting M13 peptide [START_REF] Nakai | A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein[END_REF]. These sensors thus retain CaM and the M13 peptide as calcium sensor, but they are much simpler and easier to use. The GCamP sensors are mono-excitation and monoemission and do not need sophisticated devices to detect increases of free calcium concentration. The dynamic range is also much higher than YC3.60 and contributes to the improved calcium signal detection at lower magnification for long distance calcium imaging. This type of intensiometric sensor was expanded to red and blue color to create the R-GECO-1 and B-GECO1 indicators [START_REF] Zhao | An expanded palette of genetically encoded Ca2+ indicators[END_REF]. The single fluorescent protein-based Ccalcium indicators GCamP3 and R-GECO1 were successfully introduced in plants [START_REF] Keinath | Live cell imaging with R-GECO1 sheds light on flg22-and chitin-induced transi ent [Ca(2+)]cyt patterns in Arabidopsis[END_REF][START_REF] Nguyen | Identification of cell populations necessary for leaf-toleaf electri cal signaling in a wounded plant[END_REF] and have become the most popular calcium sensors used in plants. These fluorescent reporters are able to visualize calcium changes in many conditions and contribute to highlighting the role of calcium in plant signaling. However, these intensiometric sensors show some weakness due to the absence of a reference for the fluorescence normalization over time, whereas the FRET ratiometric YC3.60 shows more accuracy. Nevertheless, the choice of calcium reporters is dependent on the magnification needed for the observation and the imaging systems available for the acquisition. Here we describe in detail the protocol to use either the ratiometric YC3.60 or the intensiometric GCamP3 for different magnification observations. The image analysis for the quantitative determination of calcium signals will be also described.

Materials

YC3.60 Transgenic Plants

The Arabidopsis thaliana (Col-0 ecotype) transgenic plants harboring the cytosolic NES-YC3.60 and sensor are described in Krebs et al. [START_REF] Krebs | FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca 2+ dynamics[END_REF]. The reporter is expressed under the ubiquitin 10 promoter. Seeds of transgenic lines are plated on 0.5× MS media complemented with 1% sucrose (w/v) and 0.7% agar (w/v). Seedlings are growing under 120 μmol/m /s for 16 h of light at 21 °C for 6 days.

The 6-day-old seedlings of YC3.60 transgenic line are mounted between two coverslips with a thin layer of agar 0.5%. The primary root has been then imaged with CFP, FRET, and bright-field channel acquisitions.

GCamP3 Transgenic Plants

The Arabidopsis thaliana (Col-0 ecotype) transgenic plants harboring the GCamP3 sensor are described in Nguyen et al. [START_REF] Nguyen | Identification of cell populations necessary for leaf-toleaf electri cal signaling in a wounded plant[END_REF].

The reporter is expressed under the ubiquitin 10 promoter. For the GCamP3 line, seeds are directly sown on soil. Condition of growth is 16 h of light, 2 °C and 120 μmol/m /s. After 4 weeks, leaves are detached and left on water for recovery. The detached leaves have been imaged under the macroscope, and 100 μL of solution were dropped on the petiole to trigger a calcium signal. Three image tiles were acquired to see the entire leaf during calcium imaging.

Solutions

1. Ferrozine (Fe iron chelator) solution is dissolved in water at 100 mM stock solution and stored at -20 °C.

2. Solutions of 10 mM of CaCl and 100 mM of NaCl were both prepared in water and kept at room temperature.

Equipment for FRET YC3.60 Microscopic Measurement

The images have been acquired with an inverted microscope Axiovert 200M (Zeiss) equipped with the following:

1. A halogen lamp with an external shutter for bright field acquisition.

Equipment for GCamP3 Microscopic Measurement

1. AxioZoom V16 macroscope (Zeiss) with objective PlanNeoFluar Z 1.0x/0.25.

LED source Xylis (Excellitas technology).

3. LED source CL9000 (Zeiss) with gooseneck light guide for bright-field illumination.

4. Filter wheel with filters set for excitation at 470/40 nm, BS 495 nm, and emission at 525/50 nm.

5. Camera Orca Flash 4.0LT (Hamamatsu).

6. Zen blue 3 software.

Methods

FRET YC3.60 Microscopic Measurement

Acquisition Settings

The setup is designed for single excitation and dual emission recording. A time lapse is done with a sequential acquisition between the FRET, CFP, and BF (bright-field) channels.

1. Channel 1 (FRET channel) was imaged with excitation at 438/24 nm and emission at 535/30 nm with 295 ms exposure time (see Note 1).

2. Channel 2 (CFP donor channel) was imaged with excitation at 438/24 nm and emission at 480/40 nm with 300 ms exposure time (see Note 1).

3. Channel 3 for BF was imaged with 100 ms exposure time.

The image sequences are acquired with the cool snap Coolsnap camera (Subheading 2.4 ) set at 12 bits with a binning of 2 (see Note 2). Binning combines adjacent pixels that are read out together as a super pixel rather than reading out the data of each individual pixel. Pictures have been taken every 10 s. Metafluor 7.5 software was used for acquisition.

During the acquisition, 100 μL of treatment solution was added gently to induce calcium signals (Figs. 1 and2 ) (see Note 3).

Fig. 1

Ferrozine induces an increase of both free calcium level and speed of root growth. The primary root of YC3. Correlation between root growth rate and increase of free calcium level can be seen in (e) after application of ferrozine at 3 min (ee, black arrow). Scale bar = 50 μm Figure 1 shows that iron chelation in the medium induces an increase of the fluorescent signal along the root . The low magnification used in this experiment (objective 5×) allows to correlate variation and root growth. Figure 2 shows a second example taken at higher magnification (20×). The increase of the resolution is suitable for analyzing calcium oscillation induced by CaCl in the medium at the level of root cells. one stack image. This step is done for all the channels. The images are opened with the FIJI open-source image processing package [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] by using the import image sequence process. Every channel (CFP and FRET channels) is opened separately with FIJI, using the following commands:

File → import image sequence

Background Correction

A background region of interest (ROI) is selected from non-plant pixels with square tool selection. Backgrounds are subtracted with the plug-in "Background subtraction from ROI" (see Note 4). The median filter is then applied with radius pixel = 2 (see Note 5), using the following commands:

Process → filters → median

Registration

If the sample is moving (or growing) during the acquisition, a registration has been processed to align the images with the plug-in Registration:

Register → Register Virtual Stack Slice (see Note 6)

Select the original image sequence and create a new folder for saving the new images.

Use "Rigid" as Registration model.

Save the transforms to re-apply it on the two other channels (CFP and FRET) prior to doing the ratio. Image registration has been performed in Fig. 2 to avoid stack images shift during calcium measurement.

Ratio Image

The ratio of FRET/CFP is performed with the plug-in "Ratio Plus" (see Note 7). The thresholds of each channel are first estimated prior to ratio calculation. A "Ratio Calculator" window is opened when the plug-in "Ratio Plus" is launched. Select the two images (or stacks) as "image 1" and "image 2." Fill the "Clipping Value1" and "Clipping Value2" with the threshold values of each corresponding image or stack and press "OK." The ratio will be performed as image 1 over image 2.

Save the stack ratio file as "Tiff" file.

Quantification

Calcium signal is quantified by first making a kymograph (or time-space plot). A segmented line or straight line is used to select the region to be measured. Use the Reslice tool to create the kymograph of the ROI, using the following commands

Image → Stacks Reslice [/]
Save the kymograph as "Tiff" file.

On the kymograph picture, select the ROI to be quantified and plot the profile, using the following commands:

Analyze → Plot Profile

GCamP3 Macroscopic Measurement

Acquisition Settings

The detached leaf is placed under the 1× objective with a zoom of 7×. Three image tiles were necessary to see the entire leaf.

Sequential images have been acquired for the two channels.

1. Channel 1 (470/40 nm) was imaged with 200 ms exposure time, with light source intensity set at 30%.

2. Channel 2 (BF) was imaged with 2 ms exposure time.

The images are acquired in 16 bits with the Orca Flash 4 camera, and a binning of 4 was set (see Note 2).

Example of acquisition is shown at the macroscopic scale to visualize the calcium waves in the entire leaf of Arabidopsis thaliana in response to salt stress (Fig. 3 ). A mosaic acquisition of three image tiles is taken every 10 s during 16 min.

Image Processing

Stitching

When a mosaic picture is required for a very large sample, stitching is performed with Zen blue software (see Note 8) to combine all the image tiles with a partial overlapping field of view.

Tiles are fused with the following settings: 5% of minimal overlap, maximal shift 10% (Fig. 3 ).

Background Correction

For macroscopic image, the background is subtracted in Fiji as follows:

Process → Subtract Background → Rolling ball radius = 50 pixels This tool is appropriate for correcting an uneven background. Calcium signal is quantified by measuring the fluorescence on the entire leaf with Fiji.

1. Select the leaf with "Polygone selections," and add it as ROI in ROI manager, using the following commands:

Analyze → Tools → ROI manager 2. Select the leaf ROI in the ROI manager and measure mean values of each time point, using the following command:

More → Multi measure 3. The average of the mean gray value (a.u. = arbitrary units) is plotted vs time (see Note 9) (Fig. 3c ).

Notes

1. Exposition times for the channels have to be adjusted depending on the magnification lens in order to have a ratio equal to 1.

2. Binning will increase the signal, but decrease the resolution. Then, exposure time could be reduced. Choose a good balance between signal and resolution.

3. Stress application has to be done without moving the sample. This is a critical step to have a good quantification. Then, the volume to apply could be adjusted with slow application with the help of the micropipette.

4. Use the same ROI for the Background subtraction for each channel (FRET and CFP). The Background subtraction plug-in could be applied several times to make sure that baseline is the lowest and is constant over time.

5. Application of median filter decreases the noise, but will blurry the picture. Alternative filters like "Gaussing Blur" could be used.

6. Use first the bright-field channel for registration and save the transform file to apply it on the two fluorescentce channels. Registration with the fluorescentce channel is not properly performed due to the fluctuation of the fluorescentcte signal.

7. Alternative solution is to use the image calculator tool in Fiji. Select the two channels and divide. Tick the "32-bit (float) result" box to get decimal values, using the following commands:

Process → Image Calculator 8. Stitching is performed with Zen blue. If the stitching is not accurate, use edge detector to improve the stitching. Minimum overlapping and maximal shift could also be changed to improve the stitching. An alternative solution is to use Fiji freeware to perform the stitching manually with the plug-in "Mosaic J" [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. 9. To get the average values, make sure that measurement parameter is set properly, using the following commands:

Analyze → set measurement → tick the relevant setting (mean gray value)

2+

  60 plant was exposed to 500 μM iron chelator Ferrozine at 3 min (black arrow) after the beginning of calcium measurement was assessed. Images are acquired every 10 s during 18 min. Pictures of FRET/CFP ratios every 3 min are shown in (a). A kymograph on the root is shown in (b) to highlight root growth and calcium signals along the root (b). Bright-field pictures of the root and its corresponding kymograph are shown in (c) and (d). Calcium variation on the entire root is shown in (e,) (red curve) and averages of root growth every 3 min are plotted on the same chart (ee, box plot, e).

Fig. 2

 2 Fig. 2 External calcium application induces calcium oscillation in the root. The primary root of YC3.60 plant is exposed to 10 mM of CaCl and changes of calcium levels are measured with the FRET sensor every 10 s during 40 min. Standard deviation projection over time shows the localization of calcium changes in (a). Three regions of interest (ROI) are selected for kymograph representation in (b). The oscillation signal is then observed after 1 min for 40 min. The quantification of the signal for the three ROI is shown in (c). Scale bar = 20 μm

Fig. 3

 3 Fig.3

  eProofing https://eproofing.springer.com/ePb/printpage_bks/thMVrDhrQu7D44dlvjVqA3_OApUn620f-tS-Z... 5 sur 8 30/01/2023, 14:28 NaCl induces calcium waves in detached leaves. The entire leaf is imaged by taking three image tiles every 10 s for 16 min (a). Pictures at different times are shown after adding 100 μL of 100 mM of NaCl at the petiole at time 0 s. Time (min) is indicated in each image (a). A pseudo color fire Look-Up Table (LUT) is used to reflect differences in intensity (see the calibration color bar at the right). Panel (b) shows a synthetic picture corresponding to the standard deviation projection (STD) of the signal intensity during the time lapse (right) and the bright-field (left) acquisition at time 0 s (left). A quantification of the signal (A.u. = arbitrary units) is done by drawing a region of interest (ROI) of the entire leaf (c). Scale bar = 1 cm

sur 8 30/01/2023, 14:28

Acknowledgments

We thank the PHIV La Gaillarde Imaging facilityPlatform. We thank Prof. Jörg Kudla (University of Münster, Germany) for the proUBI10::NES-YC3.60 line and Prof. Edward Farmer (University of Lausanne, Switzerland) for the proUBI10::GCamP3 line.


Tetyana Kyrpel

Vita Saska

Anne De Poulpiquet

Mathieu Luglia

Audrey Soric

Magali Roger

Oksana Tananaiko

Marie Thérèse Giudici-Orticoni

Elisabeth Lojou

Ievgen Mazurenko

Hydrogenase-based electrode for hydrogen sensing in a fermentation bioreactor

Keywords: Bioelectrode, hydrogenase, enzymatic sensor, amperometric sensor, hydrogen detection, fermentation bioreactor

The hydrogen-based economy will require not only sustainable hydrogen production but also sensitive and cheap hydrogen sensors. Commercially available H2 sensors are limited by either use of noble metals or elevated temperatures. In nature, hydrogenase enzymes present high affinity and selectivity for hydrogen, while being able to operate in mild conditions. This study aims at evaluating the performance of an electrochemical sensor based on carbon nanomaterials with immobilised hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus for H2 detection. The effect of various parameters, including the surface chemistry, dispersion degree and amount of deposited carbon nanotubes, enzyme concentration, temperature and pH on the H2 oxidation are investigated. Although the highest catalytic response is obtained at a temperature around 60 °C, a noticeable current can be obtained at room temperature with a low amount of protein less than 1 µM. An original pulse-strategy to ensure H2 diffusion to the bioelectrode allows to reach H2 sensitivity of 4 µA cm -2 per % H2 and a linear range between 1-20 %. Sustainable hydrogen was then produced through dark fermentation performed by a synthetic bacterial consortium in an up-flow anaerobic packed-bed bioreactor. Thanks to the outstanding properties of the A. aeolicus hydrogenase, the biosensor was demonstrated to be quite insensitive to CO2 and H2S produced as the main co-products of the bioreactor. Finally, the bioelectrode was used for the in situ measurement of H2 produced in the bioreactor in steady-state.

Introduction

Global climate change urges scientists to look for alternative CO2-neutral and less polluting energy sources and carriers. Hydrogen shows high expectations as a neutral energy vector and energy storage solution (Staffell et al., 2019). The transitional storage of large hydrogen amounts generated during the production peaks and used upon need is however a cornerstone of the hydrogen economy. Moreover, being a light and explosible gas (flammability range 4 -75 %) hydrogen represents non-negligible risks when stored in large quantities. An example of this is an explosion at the hydrogen fuel station in Norway in 2019 caused by a leak ('Nel ASA:

Status update #5 regarding incident at Kjørbo', 2019). Furthermore, hydrogen is a colourless and odourless substance which makes impossible its detection by human senses and these are the points of attention that require the installation of reliable sensors to detect possible leaks.

The production of hydrogen in a sustainable way is another requirement for the hydrogen economy. Among others, (photo)electrochemical water splitting (Tee et al., 2017) but also biological methods are envisioned [START_REF] Akhlaghi | [END_REF]Hidalgo and Martín-Marroquín, 2022). In particular, hydrogen production in the dark fermentation process by a bacterium or a consortium of several bacteria from a wide range of food and agricultural feedstocks could become one of the eco-friendliest processes (Sarangi and Nanda, 2020;Xu et al., 2022). This process is developed in an anaerobic reactor due to the oxygen-sensitivity of most fermentative bacteria and H2 production is associated to the acetate or butyrate pathways, while CO2 is released as a co-product. Despite multiple fundamental challenges still present (Brar et al., 2022;Ghimire et al., 2015;RenNanqi et al., 2011), numerous types of bioreactors producing hydrogen have been built (Barca et al., 2015). Yet, the level of H2-production in such bioreactors remains unstable and depends, among other factors, on the substrate used, pH and bacteria fitness. Hence, a hydrogen detector installed at the gas outlet of reactors and providing the information about the current production rate in real time seems to be an indispensable device.

The presence of hydrogen detection systems thus appears essential in the critical nodes of the future hydrogen infrastructure, at the production, use and storage points. Such systems relate This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 more to the sensor technology having low cost, small size and fast response than to the classical large and high-tech instruments such as gas chromatography and mass-spectrometry. Further requirements include suitable detection range, safe performance, robustness and low crosssensitivity. It should be noted that despite an active development, no hydrogen sensors were demonstrated satisfying all the requirements postulated by the US Department of Energy (DoE) (Darmadi et al., 2020;Multi-Year Research, Development, and Demonstration Plan 2011-2020. Section 3.7 Hydrogen Safety, Codes and Standards, 2015). Main sensing principles have been extensively reviewed and corresponding devices commercialized (Hübert et al., 2011). They can be classified into catalytic, thermo-conductive, electrochemical, resistive, work function, mechanical, optical and acoustic types. Whatever the transducer principle, the sensing element often includes a material that changes its properties upon contact with hydrogen. Traditionally, palladium, platinum and other noble metals are used since they are efficient catalysts, have high affinity and can absorb large quantities of hydrogen. Many sensor types, e.g. catalytic and resistive, additionally require elevated temperatures (>100 °C) to function, which not only leads to high power consumption, but also poses security risks.

Catalytic sensors also require oxygen to be present in the atmosphere and thus are not suitable for the hydrogen determination in anaerobic conditions. On the other hand, the humidity seems to affect adversely the response of the sensors too, although only few studies have investigated its impact (Darmadi et al., 2020).

As it is for the hydrogen production, an alternative to the expensive rare metals catalysts for the hydrogen detection may be found in nature. The ability to catalyse hydrogen oxidation is an intrinsic property of hydrogenases, one of the most studied redox enzymes (Lubitz et al., 2014). There were few attempts to explore this ability of hydrogenases to detect hydrogen. In one of the first works, Qian et al developed an electrochemical mediated sensor by entrapping hydrogenase from T. roseopersicina and poly(butylviologen) mediator between two clay layers.

A sensitivity in the order of 1 µA cm -2 per % H2 was achieved, but a slow response time of tens of minutes was also noted (Qian et al., 2002). Lutz et al used an O2-tolerant hydrogenase from R. eutropha to develop a mediated chronoamperometric biosensor capable to detect H2 in the 1-100% range with currents in the range of 1-2 µA cm -2 per % H2 (Lutz et al., 2005). The method involved however toxic benzyl viologen as a dissolved mediator. Alonso-Lomillo et al manufactured multiwalled carbon nanotubes (MWCNT) modified gold electrodes, and employed them as a platform for the immobilisation of the O2-sensitive hydrogenase from D. gigas allowing high currents for hydrogen oxidation with sensitivity of 2 µA cm -2 per µM H2 This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 when the electrode was rotating at 2500 rpm (Alonso-Lomillo et al., 2007). Besides an artificial convection required to achieve such activity, the O2-sensitivity of the hydrogenase from D. gigas limits considerably the application scope of such a sensor. This is also most probably one of the reasons why so few reports on the enzymatic hydrogen sensors can be found in the literature, the majority of hydrogenases requiring strict anaerobic conditions for manipulations.

Membrane-bound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus (Aa Hase) has been shown previously to possess promising features for biotechnological applications, such as thermostability, H2 affinity, O2-and CO-tolerance (Brugna-Guiral et al., 2003;Luo et al., 2009). Several generations of H2/O2 fuel cells have been notably reported featuring Aa Hase anode (Ciaccafava et al., 2012b;Mazurenko et al., 2017;de Poulpiquet et al., 2014). The outstanding properties of Aa Hase combined with the already acquired knowledge of its functioning at electrochemical interfaces suggest the enzyme could be advantageously used as a sensing element.

In this work we developed an immobilisation platform and explored the prospects of Aa Hase as a key element of an amperometric hydrogen biosensor, suitable in particular for the H2 quantification at the biogas outlet of an anaerobic bioreactor. We optimised the bioelectrode fabrication based on a simple procedure of CNT-modification and enzyme adsorption. This bioelectrode was further tested at different conditions, and calibration curves were obtained in the forced-convection and stationary conditions. The impact on the detection of main interferents known to be produced in the reactor was studied. Finally, the developed biosensor was employed for the quantification of the biohydrogen produced in a dark fermentation bioreactor while the results were compared with gas chromatography.

Experimental part

Materials and reagents

Potassium hydrogen phosphate (99%), potassium dihydrogen phosphate (99%), potassium hydroxide (KOH), sodium sulphide (Na2S) were purchased from Fisher Scientific, N-methyl-2-pyrrolidone (NMP, 99.5%) was from Acros Organics.

This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios. 2023.115106 Non-functionalized (MWCNT) CNTs were from Nanocyl, Belgium (Nanocyl 3100). NH2functionalized CNTs (MWCNT-NH2) were from Dropsens, Spain (DRP-MWCNTNH2).

COOH-modified CNTs were purchased from NanoLab, USA (PD15L1-5 COOH).

N2 and CO2 high purity gases were supplied from Linde, France. Hydrogen with declared purity > 99.99995% was produced in a generator Parker Domnick Hunter 110H-MD, UK.

Membrane-bound [NiFe]-hydrogenase from Aquifex aeolicus (Aa Hase) was purified according to the procedure described previously (Brugna-Guiral et al., 2003).

Electrode preparation

Carbon nanotubes were dispersed in NMP (MWCNT and MWCNT-NH2) or MilliQ water (MWCNT-COOH) at the concentration 1 mg mL -1 by ultrasonication for 4 h (NMP) or 1 h (water). Pyrolytic graphite (PG) electrode (Ø 3 mm) was masked with a Parafilm o-ring mask leaving only the electrode surface available for the dispersion. 5 µL of the CNT-dispersion was dropped on the electrode surface and dried under the vacuum for at least 5 min until the solvent was completely evaporated. In the case of water-based dispersion, the electrode was dried for 15 min at 60 °C. The last step was repeated several times if necessary. Then, 5 µL of Aa Hase solution was deposited on the electrode surface, covered with a microtube to avoid evaporation and placed in the fridge at 4 °C for the adsorption. Before electrochemical study the electrode was rinsed with MilliQ water and immediately placed in the electrochemical cell.

Electrochemical experiments

Electrochemical measurements (Cyclic voltammetry (CV), and chronoamperometry) were performed in a standard 3-electrode cell (comprising a modified PG as a working electrode, a Hg/Hg2SO4 reference electrode (sat. K2SO4) and a Pt-wire auxiliary electrode) using a potentiostat from Metrohm-Autolab PGSTAT101 controlled by Nova 2.1 software (Eco Chemie). All potentials are quoted vs normal hydrogen reference electrode (NHE) by adding 620 mV to the measured potential. Scan rate was 10 mV s -1 for all CV experiments. Current densities are reported to the geometrical surface of the electrode (0.071 cm 2 ). All experiments with electrode rotation were performed on the Metrohm-Autolab rotating ring-disk system with a home-made glassy carbon electrode (GC) tip with similar surface area.

Phosphate buffer with pH 7.0 and concentration 0.1 M and temperature 25±1 °C was used as an electrolyte, unless mentioned otherwise. The solution in the electrochemical cell was This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 continuously bubbled with pure gases or mixtures with various ratios. The necessary gas ratios were created using two flow meter controllers (Sierra Instruments) with different scales allowing to cover the range 0.04 -50 %.

Scanning electron microscopy

The electrode samples were observed by a Vega3 Tescan scanning electron microscope (SEM).

An accelerating voltage of 10 keV with a working distance of 5 mm was used.

Dynamic light scattering

Dynamic light scattering (DLS) to determine the size of dispersed particles was performed using Malvern ZetaSizer Nano ZS, UK. Due to the high optical absorbance of CNT-dispersions, a 10-times dilution was performed before the measurement. Back scattering detection at an angle 173° was used and three replicate measurements were systematically made.

Bioreactor setup

An up-flow anaerobic packed-bed bioreactor of 5 L (Minifors 1, Infors), working volume of 3 L, was set up for the experiments. The bioreactor was packed with Kaldnes K1 as biofilm carriers and the porosity of the packed-bed was 84%.

The bioreactor was operated according to a continuous up-flow of about 0.78 L h -1 of medium, thus leading to a hydraulic retention time of about 3.4 h. An internal recirculation of the culture from the top to the bottom of the bioreactor was applied at a flow of 15 L h -1 . The bioreactor was stirred at the bottom with Rushton impeller at 300 rpm. N2 gas was continuously sparged at the bottom of the bioreactor at a flow of 3 L h -1 . The temperature was controlled at 37 °C and the pH was regulated at 5.8 by addition of 1.5 N NaOH in the headspace.

The medium used for the experiments consisted of (for 1 L): K2HPO4 (0.5 g), NH4Cl (2 g), yeast extract (1 g), D-glucose (2.5 g), and trace element stock solution 1000X (1 mL). The bioreactor was inoculated with a synthetic consortium of two strains: Clostridium acetobutylicum ATCC824 and Desulfovibrio vulgaris Hildenborough DSM644 at 1:1 ratio (O.D.).

After inoculation step, the bioreactor was operated in batch mode during 22 h. Then a D-glucose stock solution was added to a final concentration of 2.5 g L -1 in the bioreactor, thus operating in fed-batch mode during 20 h. After this period the bioreactor was turned in continuous mode.

This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106

At the gas outlet of the bioreactor, the gas flow rate was recorded every 5 min using a volumetric gas meter (drum-type gas meter TG0.5, Ritter). Then downstream the gas composition was measured each hour by gas chromatography (MicroGC 490, Agilent), previously calibrated and operating in continuous flow. The MicroGC was equipped with thermal conductivity detector with two modules consisted of: a module A with a backflush injector heated at 95 °C, a 5 m PBQ + 10 m MS5A column heated at 145 °C with Ar as the gas carrier, and a module B with a variable injector heated at 95 °C, a 10 m PPU column heated at 70 °C with He as the carrier gas.

Results and Discussions

Electrocatalytic response as a function of CNT type. The electrode surface plays an important role in enzyme electrochemistry. Since the rate of interfacial electron transfer decays in an exponential manner with the increase of the distance to the electrode, only a monolayer of adsorbed redox proteins can be practically electroactive. On the other hand, enzyme molecules are rarely of spherical shape with an active centre in the middle, so that the configuration in which the enzyme molecule is adsorbed on the electrode surface plays a role by modifying the electron tunnelling distance. The surface of the electrode has to be tuned to favour the adsorption in the configuration with the shortest distance between enzyme and the active centre. In line with our previous findings of the importance of electrostatic interactions (Mazurenko et al., 2016;Monsalve et al., 2016), we have tested different types of MWCNTs as a support for Aa Hase immobilisation. Pristine non-modified MWCNTs, COOH-modified and NH2-modified MWCNTs were notably tested. The properties of all MWCNTs, notably the zetapotential, elemental composition and size, were reported earlier (Hitaishi et al., 2020;Mazurenko et al., 2016). While pristine MWCNTs and COOH-modified MWCNTs were already evaluated as surface modifiers for Aa Hase immobilisation (Monsalve et al., 2016), MWCNTs covalently-modified with NH2-groups have not been yet used with this enzyme. To This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 facilitate the comparison, the same quantity of all MWCNT types was deposited on the PGelectrode. from 25-30 µA cm -2 in the cases of MWCNT and MWCNT-COOH to 90 µA cm -2 in the best case of MWCNT-NH2. Such difference between positively-and negatively-charged CNTs has been already observed earlier for Aa Hase, except that positively-modified CNTs were prepared by non-covalent modification by Pyrene-NH2 molecules (Monsalve et al., 2016). Interestingly, the current is enhanced by the same factor of ca. 3 irrespective of the way the positive charge is introduced on the CNT-walls. This can be explained by the weak fluctuating dipole moment of Aa Hase that displays large direction variations but still points predominantly away from the distal [FeS]-cluster [START_REF] Oteri | [END_REF]. This property allows Aa Hase to display a higher catalytic current on positively-charged CNTs while also being somewhat active on negativelycharged CNTs. In consequence, MWCNT-NH2 were used for all further studies. The process of electrode modification was also characterised step-by-step by cyclic voltammetry and electrochemical impedance spectroscopy (Figure S1, Figure S2) using K3[Fe(CN)6] / K4[Fe(CN)6] couple as a redox probe. When PG is modified with MWCNT-NH2, an increase of the apparent electron transfer constant is observed, which doesn't change significantly upon Aa Hase immobilisation (Table S1) suggesting that the large enzyme molecules cannot block completely the CNT-surface from small diffusing probes.

Optimization of Aa Hase adsorption. In order to maximise the catalytic current while keeping the amount of the adsorbed enzyme as low as possible, the optimal concentration of the deposited enzyme solution was investigated. The dependence of the catalytic current on the enzyme concentration should have a form of Langmuir isotherm with a saturation in the region where an electroactive monolayer of adsorbed enzymes is completed. In our case, this dependence had a peak-shaped form with both low and high concentrations yielding to the drop of the catalytic current (Figure 2A). The optimum was obtained with 0.5 µM Aa Hase, highlighting that a very low amount of protein is required for H2 detection. While unusual, such dependence was already observed e.g. for M. verrucaria bilirubin oxidase on planar gold electrodes where it was explained by the formation of more crowded and sterically-hindered adlayer at higher concentrations (McArdle et al., 2015). While the same reasoning can be applied here, in the particular case of Aa Hase such dependence can have two additional possible explanations: a) after purification, Aa Hase sample contains a non-negligible amount of surfactant (DDM) which helps the membrane-bound enzyme to maintain its structure and functions (Ciaccafava et al., 2012a). The surfactant itself should interact with the partially hydrophobic MWCNTs. With sample dilution the concentration of the surfactant drops and this may lead to the micelle restructuration and, in turn, to the change of the adsorption mechanism;

This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 b) since Aa Hase was purified without affinity tags, the purity of the final sample remains lower than 90% (Figure S3). Some low-concentration impurities might have higher affinity for the MWCNT-NH2 surface than Aa Hase. Even if their amount is not sufficient to block the electrodes surface when the diluted samples are used, they may hinder the adsorption of the enzyme from the concentrated solutions, possibly leading to the catalytic current drop as observed on the Figure 2A. The fact that a catalytic current can be observed even from the enriched protein samples should be considered as an advantage for biotechnologies since high purity proteins can hardly be obtained after a native enzyme purification. It must be however emphasized that the induced catalytic current may vary as a function of the enzyme fraction. quantities didn't enhance the response and even led to its decrease. The explanation might be found in the increased thickness of the porous layer that prevents hydrogen from the solution to reach the enzymes immobilised deep inside. This is coherent with the observation that the current stays almost the same for 30 min of adsorption irrespective of the total layer thickness since the enzyme molecules have time to penetrate only to a certain depth during this time. It should be noted that although 60 min of adsorption showed higher current, some further experiments were performed with 30 min of adsorption to mitigate a higher dependence on the CNT quantity observed at 60 min.

Temperature influence. Aa Hase being extracted from a hyperthermophilic organism is known to demonstrate an activity enhancement at elevated temperatures that may go up to 85-90 °C (Brugna-Guiral et al., 2003;Lojou et al., 2005;Monsalve et al., 2016). To evaluate the performance of the sensor at higher temperatures, we performed a CV on the same electrode in the temperature range from 30 to 60 °C (Figure S4). A 4-fold activity enhancement was observed when the temperature was changed from 30 to 60 °C, in agreement with previous reports (Monsalve et al., 2016).

Influence of CNT-dispersion. Due to their aspect ratio and high hydrophobicity, pristine

CNTs are hardly dispersible in the aqueous media. The use of organic solvents and ultrasonication are necessary to separate the individual tubes and even then, the dispersions can deteriorate with time. Decrease of the magnitude of the bioelectrode response was observed when the dispersion was used repeatedly for the electrode modification for 1 month (Figure 3A). On average, the response was decreased by 33 % when a 1-month old dispersion was used compared to a fresh one. Since visually the dispersion did not change its appearance during this dispersed CNTs (peak at 20-30 nm) and formation of larger aggregates (peak at 400-600 nm) due to a partial aggregation (Figure 3B). The difference between fresh and partially aggregated CNT-dispersions is visible also on the SEM-images of the deposit (Figure S5). Indeed, more aggregates can be distinguished on the low-magnification images. It can be speculated that partial aggregation impedes the formation of pores suitable for enzyme size upon deposition. It was noticed however that such aggregation occurs only when the dispersion was repeatedly used, i.e. the recipient was opened and closed.

These changes were not observed with the MWCNT-NH2 dispersion that was tightly sealed for at least several months. Regarding the reproducibility of the modification by Aa Hase, the average bioelectrode current density was 75.6 µA cm -2 with standard deviation 8 % for 6 electrodes. Michaelis constant. The sensitivity and the detection limit of a biosensor depend on the intrinsic properties of the enzymatic component, notably activity and Michaelis constant. As a general rule, higher activity and lower Michaelis constant should lead to a better catalytic efficiency and thus higher biosensor sensitivity. At the same time, the response can be considered linear in the substrate concentration range between 0 and KM. It should be noted however that in practice multiple other factors play a role. KM of 3 µM H2 for Aa Hase at 25 °C was reported before based on unpublished data. We refined this value by performing triplicate chronoamperometric measurements using a method described before (Léger et al., 2004). The measured apparent KM of the immobilised Aa Hase to H2 at -0.15 V (vs NHE) was found to be 9.8 ± 0.6 µM (Figure S7), which corresponds to 1.3 % H2. This means that Aa Hase theoretically is suitable for the determinations of H2 quantities much below the lower explosion limits (4 %) with a limit of detection discussed hereafter.

Reproducibility

Calibration. Besides Michaelis constant, mass transfer is another important factor which determines the sensitivity and the linear range of the enzymatic biosensor. Since equilibrium hydrogen concentration in an aqueous solution in contact with a gas mixture at the lower explosivity limit (4% H2) is only 31.2 µM at 25° C, the enzymatic activity will result in its fast depletion near the electrode surface and thus signal drop. Hence, the mass transfer should be enhanced in order to improve the sensitivity of the sensor to low concentrations. To facilitate this process, we first explored the performances of two different usual approaches, gas bubbling into the solution with a stationary electrode and electrode rotation.

The CVs obtained on the stationary electrode under gas bubbling in the cell were rather noisy and characterised by low reproducibility (Figure S8), especially in the range 2.5-10 % H2. This can be explained by the inhomogeneous solution stirring by the gas bubbles, and as a result concentration fluctuation near the electrode. Despite the noise, a linear dependence of the current at -0.1 V (vs NHE) on H2 content was observed in the range 1 -10% H2 with a sensitivity of 6.6 ± 0.6 µA cm -2 per % H2 (Figure 5B). When a rotating disk electrode (RDE) was used, the CVs demonstrated much lower noise level (Figure 5A) and higher sensitivity. The response was linear in the 0.5 -5 % H2 range with sensitivity 20.0 ± 0.9 µA cm -2 per % H2. This can be explained by enhanced mass transfer and well-defined diffusion profile characteristics of RDE. It should be also noted that half of the maximal response on RDE is observed at H2 concentrations (ca. 2.5 %) which are much closer to previously determined KM (1.3 %).

This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106

Pulsed detection at stationary electrode. Despite enhanced sensitivity and lower detection limit obtained on the RDE, it is difficult to imagine the employment of such cumbersome and energy-consuming systems in real sensors. A flow-method should give a similar diffusion enhancement but requires an additional energy input as well. We therefore tried to change the detection method to avoid any mechanical forced convection while maintaining the sensor characteristics. For this, we had recourse to pulsed detection with applied potential changing periodically from the value where the reaction takes place to the one where there is no reaction that allows to restore the diffusion layer. Two potentials were chosen: -0.1 V at which the activity is reasonably high but strong potential deactivation is not observed yet, and -0.4 V at which the H2 oxidation doesn't take place thus allowing the diffusion layer to be restored.

Furthermore, the lower potential allows it to reactivate any potentially inactivated fraction of Aa Hase. Then, the duration of the pulse was optimised by performing long 30 s-pulses and sampling the current at different time points from the pulse start (Figure 6). The recorded current reveals the contribution of two processes: the charging current which depends on the active surface area and exponentially decays with time; and the faradaic current which depends on the concentration of the dissolved hydrogen and decays with square root of time. This dependence has a complex nature dictated by the enzymatic activity and hydrogen diffusion. Two cases can be identified depending on the current sampling moment. At shorter sampling time less than 0.5 s, the bioelectrode has higher sensitivity to the hydrogen ca. 4 µA cm -2 per % H2 but smaller linearity range 1-10 % (Figure 6, Figure S9). The levelling of the current response above 10 % can be explained by concentrations of the hydrogen significantly exceeding the Michaelis constant of the hydrogenase. At longer sampling times more than 1 s the sensitivity of the bioelectrode gradually decreases and levels at ca. 1.3 µA cm -2 % -1 while the linearity range extends towards higher concentrations (Figure S9). The longer the oxidative potential is applied to the bioelectrode the more hydrogen gets depleted in the vicinity of the electrode and the more important contribution of the diffusion becomes. Thus, the duration of the pulses can be adjusted depending on the expected hydrogen content: the shorter pulses can be used to detect traces of H2 with high sensitivity, while longer pulses allow to extend the linear range and quantify higher H2-content.

Interferences study. Various gaseous substances can be found together with hydrogen in real samples. Some of them can cause cross-selectivity and/or poisoning of the noble metal-based sensors, e.g. CO, SO2 and H2S. Aa Hase is known to be resistant to CO due to its weak binding with [NiFe]-centre (Luo et al., 2009;Pandelia et al., 2010). In the dark fermentation process, traces of H2S are more likely to be produced than SO2, and CO2 is additionally co-produced together with H2. The mechanism of H2S inhibition of [FeFe]-hydrogenases was recently elucidated (Felbek et al., 2021;Rodríguez-Maciá et al., 2018). Sulphide-inhibition at oxidative potentials was also demonstrated for several [NiFe]-hydrogenases (Vincent et al., 2006). We therefore investigated the influence of sulphide on the catalytic signal of the biosensor by introducing Na2S into the electrochemical cell. Since the second pKa of H2S lies between 12 and 17 and the first pKa is equal to 7, Na2S is completely hydrolysed in the aqueous solutions with formation of H2S and HS - (Li and Lancaster, 2013). We have chosen a more acidic pH 6 for the interference experiments because at this pH 90 % of the added sulphide is found in the This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 form of solubilized H2S (aq) immediately after the injection. The current of the modified bioelectrode itself remains stable between pH 6 and 7 (Figure S10). The chronoamperogram of the Aa Hase modified electrode doesn't demonstrate clear response to the sulphide additions for concentrations up to 5 mM (Figure 7A). At 5 mM of Na2S a 10 % current increase was observed which can be probably attributed to the beginning of a sulphide oxidation process. We conclude therefore that sulphides and H2S do not interfere with hydrogen detection by the developed bioelectrode at sub-millimolar concentrations and that Aa Hase itself is less sensitive to sulphides than other [NiFe]-hydrogenases.

The amount of CO2 produced during dark fermentation can be the same or even exceed the quantity of H2 (Barca et al., 2015). Even though CO2 is not supposed to be reduced directly on the electrode unless poised at very negative potentials, it might represent an interfering action by lowering the solution pH and thus influencing enzyme activity due to the solubilization and reaction with water: CO2 + H2O  H + + HCO3 -. A sufficient buffer concentration is required to compensate for this possible pH decrease. We simulated the conditions of the bioreactor outlet by mixing-in higher and higher CO2-amounts to the H2. Chronoamperometric response is given in Figure 7B, the current was nearly stable before 20% CO2. Current increase around 5 % after each step was observed from 20 % CO2 that was partially restored after the return to 100 % H2. It should be noted that the concentration of H2 in this experiment was decreasing This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios. 2023.115106 with an increase of CO2 percentage. Thus, the observed current drop should rather originate from the decrease of the substrate concentration than CO2-inhibition. pH decrease might be another reason but the pH in the cell was lowered from 6 to only 5.86 after almost 2 hours of the experiment and such little pH change is unlikely to influence significantly the response.

Detection of the hydrogen in a lab-scale dark fermentation bioreactor. To test the feasibility of the hydrogen detection by the developed biosensor we connected the electrochemical cell at the gas outlet of the 3 L packed-bed bioreactor. This bioreactor employs the syntrophic association of two bacteria which are naturally involved in the process of biological hydrogen production from organic matter in anoxic environments: Clostridium acetobutylicum and Desulfovibrio vulgaris. The former carries out dark fermentation using glucose as substrate while producing volatile fatty acids and hydrogen (maximum yield of 4 H2 produced per mole of glucose utilised) and carbon dioxide. While C. acetobutylicum is thought to be the main hydrogen producer in this system, previous studies demonstrated that hydrogen production is improved in the presence of D. vulgaris (Benomar et al., 2015). Whether D. vulgaris directly participates in hydrogen production (through the activity of its periplasmic and membranebound hydrogenases) or indirectly contributes to improve hydrogen yields by rewiring C. acetobutylicum metabolism remains unclear. Nevertheless, it was shown that anaerobic packedbed bioreactor inoculated with a co-culture of C. acetobutylicum and D. vulgaris continuously fed with glucose give a continuous and stable hydrogen production (Barca et al., 2016).

The electrochemical cell was connected to the gas outlet in parallel after a 3-way valve allowing to direct the gas flow either through the cell or to the gas chromatograph omitting the cell (Figure 8A, Figure S11). Such configuration allowed to perform biosensor and cell manipulations without disturbing the gas flow.

This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 The measurements were started 7 days after the inoculation step, when the bioreactor was in steady-state. The gas chromatography measurements right before the biosensor connection and immediately after demonstrated similar gas composition (Table S2) proving that the electrochemical cell connection did not alter the bioreactor behaviour. Nitrogen carrier gas was constantly flushed through the reactor to ensure H2 gas stripping but also resulting in an approximately 4 times dilution of the biogas. This allowed notably to reach the H2 concentrations falling within the biosensor linear range.

After the initial solution saturation during 15 min, the CV was recorded. The CV of the bioelectrode in the biogas-saturated buffer solution demonstrated a shape of the catalytic current 20 This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106 similar to those obtained in the cell saturated with 100 % H2 but with lower magnitude consistent with lower H2-content in the biogas (Figure S12). The only difference was the appearance of a reduction peak at -0.22 V on the backward scan. This peak persisted during all the experiments but did not change its magnitude nor interfere with Aa Hase catalytic current.

Further experiments are required to identify the biogas component leading to this peak appearance.

To quantify the H2-content in the bioreactor gas, the detection was performed in the pulsed mode without any convection. 13 10s-pulses were performed with 60s-intervals between the pulses to permit the diffusion layer renewal (Figure 8C). Since the expected hydrogen concentration exceeded 10 %, the current was sampled at 7.5 s after the pulse beginning to extend the linear range. The pulse current remained stable over more than 15 min of the detection experiment, and the standard deviation of the current calculated over all 13 pulses was 0.7%. Using this current value and the calibration curve (Figure 8B) the hydrogen concentration found in the biogas was estimated to be 11.8 ± 0.5 %. This value correlates well with 12.1 ± 0.1 %, an average of four points measured before and after the bioelectrode connection (Table S2) found using the gas chromatography just before the beginning of the bioelectrode measurement. This proves the feasibility of the developed bioelectrode use for the hydrogen detection in the bioreactor outlet.

Conclusions

We reported the fabrication of an amperometric enzymatic bioelectrode based on a thin CNTlayer on which [NiFe]-hydrogenase from A. aeolicus is immobilised. The fabrication process was kept simple and consisted in the drop-coating of the electrode by MWCNT-NH2 and adsorption of the enzyme from the enriched protein sample. Both the adsorption time and the amount of the deposited enzyme were optimised. The amount of Aa Hase as low as 35 pmol/cm 2 was enough to achieve the optimal current densities in these conditions. The unique properties of this hydrogenase, such as thermophilicity and oxygen-tolerance, not only permit to omit the glovebox usage but also make it possible to construct a robust bioelectrode and envision real applications. The thermophilic Aa Hase is one the most stable enzymes in its class that allows to envision use of the biosensor even at elevated temperatures.

An original pulsed method developed here can ensure the detection of low amounts of H2 with high sensitivity and without additional energy input required to enhance the hydrogen masstransfer. The detection of even smaller hydrogen traces below 1% is also possible with the Aa This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios. 2023.115106 Hase, which has a low KM of 1.3 %, but it will require an artificial convection to enhance the flux of H2 towards the electrode to produce a significant current density. To solve this issue and to improve the general biosensor design, the developed bioelectrode can be replaced by a gasdiffusion bioelectrode featuring the same enzymatic catalyst. This should permit to achieve a higher sensitivity thanks to the proximity of the gas-phase to the catalyst. In addition, the gasdiffusion based design will eliminate the need to saturate the electrolyte solution with the analyte gases thus improving the response time of the biosensor.

Even in the current state, the limit of detection of the developed bioelectrode was below the lower explosion limit of the hydrogen that allows to consider it for the leak detection applications. Nevertheless, in this work we decided to target the hydrogen quantification at the outlet of an anaerobic fermentation bioreactor allowing to imagine a fully sustainable system.

We demonstrated that the main co-products of the anaerobic fermentation, CO2 and H2S do not interfere with the hydrogen quantification within the range of concentrations expected in the bioreactor gas mixture. Thanks to this and for the first time, we were able to employ the developed biosensor for the quantification of the hydrogen produced in a sustainable way directly at the outlet of the bioreactor. The results of the detection correlated well with the data obtained by an independent method of gas chromatography which proves that the developed biosensor can be successfully used for such complex tasks.

Figure 1 .

 1 Figure 1. Cyclic voltammograms of different electrodes modified with Aa Hase under (black dashed line) N2 and (red line) H2 atmosphere: a) PG, b) PG-MWCNT, c) PG-MWCNT-COOH, d) PG-MWCNT-NH2. Amount of Aa Hase used for modification: 5 µL of 20 µM. Supporting electrolyte: Phosphate buffer pH 7 (0.1M), 40 °C As it is evidenced from Figure 1, when modified with Aa Hase all electrode types demonstrate a well-defined anodic catalytic current in the presence of H2 with apparent onset potential -0.3 --0.35 V vs NHE. This catalytic current is related to the H2 oxidation by [NiFe] active centre of Aa Hase which proceeds through H2 binding to Ni-SIa state and heterolytic cleavage with formation of a bridging hydride (Ni-R state). The consecutive transfer of two electrons to the proximal [FeS]-cluster and a proton release results first in a Ni-C state and then in the restoration of the initial Ni-SIa state (Lubitz et al., 2014). At higher potentials, a slope change and a momentary current increase on the backward scan at -0.05 V are observed. This corresponds to the reversible deactivation-reactivation process of Aa Hase as a function of the potential. The process is known for all characterised [NiFe]-hydrogenases and it is explainedby the formation of the inactive Ni-B state of the active centre when scanning toward higher potentials and its reduction when scanning back(Vincent et al., 2007). The MWCNTs enhance the electrode surface area, as attested by the capacitive current increase under N2. This allows more enzymes to be adsorbed and to participate in the catalysis, giving rise to the catalytic current enhancement at least 5-times compared with the non-modified electrode (Figure1, red lines). It should be noted however that despite a similar capacitive current, i.e. similar amount

Figure 2 .

 2 Figure 2. (A) Magnitude of the catalytic current of H2 oxidation by Aa Hase modified PG-MWCNT-NH2 electrode at -0.1 V vs NHE and as a function of enzyme solution concentration. Phosphate buffer pH 7 (0.1M), 25 °C, VMWCNT = 5 µL, Venzyme = 5 µL, adsorption for 30 mins at 4 °C. (B) Normalized catalytic current at -0.1 V vs NHE of H2 oxidation by Aa Hase modified PG-MWCNT-NH2 electrode as a function of MWCNT quantity and adsorption time. Phosphate buffer pH 7 (0.1M), 25 °C, Venzyme = 5 µL, Cenzyme = 0.5 µM.The optimal concentration 0.5 µM of the Aa Hase was used for all further experiments. On the next step, the amount of CNTs and the enzyme adsorption time was optimised. It is safe to suggest that more CNTs should increase the amount of the adsorbed and electroactive enzyme molecules while longer adsorption time should ensure that the enzyme molecules have diffused inside the porous layer and an adsorption equilibrium has been reached. Although this tendency is confirmed (Figure1B), the maximal current gain of 200 % only could be obtained upon the optimization. One hour was sufficient to reach equilibrium and longer adsorption time didn't lead to the signal enhancement, except for the slight increase at 10 µL of CNT. Interestingly, the maximal response was obtained when 10 µL of CNT suspension was used while larger

Figure 3 .

 3 Figure 3. Influence of the CNT dispersion quality. (A) Catalytic current density of Aa Hase modified PG-MWCNT-NH2 at -0.1 V vs NHE as a function of CNT dispersion volume and quality. Phosphate buffer pH 7 (0.1M), Venzyme = 5 µL, Cenzyme = 0.5 µM, adsorption for 30 mins at 4 °C. (B) Distribution of the particles sizes measured by DLS in (top) freshly prepared and (bottom) 1-month old CNT dispersion.

  of the bioelectrode fabrication. The reproducibility of the bioelectrode fabrication was estimated by comparing two following characteristics: a) the electroactive surface area of the CNT-deposit was calculated from the capacitive current measured at various scan rates in the absence of H2 (FigureS6) and b) the electrocatalytic current generated by bioelectrode measured in the same conditions (Figure4). The procedure of the electrode modification by CNTs has satisfactory reproducibility with relative standard deviation of electroactive area not exceeding 10 % for 9 individual electrodes. The average electroactive surface area of PG-MWCNT-NH2 modified with 10 µL of CNT-dispersion was 5.89 cm 2 and the roughness factor was 83 compared with the geometrical area of bare PG-electrode.

Figure 4 .

 4 Figure 4. Reproducibility of the bioelectrode fabrication. (A) Reproducibility of the CNT-deposit electroactive surface area upon deposition of 10 µL of dispersion at different electrodes. (B) Reproducibility of the electrocatalytic response (at -0.1 V vs NHE) at different electrodes at 100 % H2. VMWCNT = 10 µL, Venzyme = 5 µL, Cenzyme = 0.5 µM, adsorption for 30 mins at 4 °C.

Figure 5 .

 5 Figure 5. Calibration curves. (A) Averaged CVs of the Aa Hase modified RDE GC-MWCNT-NH2 under various hydrogen content on RDE (2000 rpm); Phosphate buffer pH 7 (0.1M), VMWCNT = 10 µL, Venzyme = 5 µL, Cenzyme = 0.5 µM, adsorption for 30 mins at 4 °C; (B) Dependence of the catalytic current at -0.1 V (NHE) on the H2 concentration without (blue squares) and with electrode rotation (red circles). Dashed lines indicate approximative linearity zone. Error bars represent standard deviations for four identical cycles.

Figure 6 .

 6 Figure 6. Pulsed detection. (A) One chronoamperometric pulse sequence of the Aa Hase modified PG-MWCNT-NH2 under various hydrogen content: the potential applied during the sequence is shown as blue curve; the current recorded during the reductive pulse, which is identical for any hydrogen content, is shown as a black dashed curve; the current recorded during the oxidative pulse is shown as solid coloured curves, depending on the hydrogen content; (B) Dependence of the current density sampled at different time points from the oxidative

Figure 7 .

 7 Figure 7. Chronoamperograms of the Aa Hase modified PG-MWCNT-NH2 under H2 atmosphere upon (A) Na2S additions and (B) mixing with CO2. Applied potential (A) 0.0 V vs NHE or (B) -0.1 V vs NHE, 0.1M phosphate buffer pH 6. VMWCNT = 10 µL, Venzyme = 5 µL, Cenzyme = 0.5 µM, adsorption for 30 mins at 4 °C.

Figure 8 .

 8 Figure 8. Hydrogen detection in the bioreactor. (A) Scheme of the connection of the electrochemical cell to the bioreactor; (B) Calibration curve for the pulsed hydrogen detection at 7.5 s with the value detected in the bioreactor outlet superimposed; (C) Evolution of the current and potential during the hydrogen detection in the bioreactor gas outlet at the stationary conditions.

Acknowledgements

This work was supported by National Research Agency (ANR, France) under the grants ANR-19-CE05-0017 and ANR-22-CE50-0004. This work also received support from the French government under the France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-

Marseille Université -A*MIDEX (AMX-21-PEP-001). T.K. and V.S. are grateful to the Institute of Microbiology, Bioenergies and Biotechnology (IM2B, AMX-19-IET-006) for the internship scholarships.

The authors are grateful to the fermentation (M. Bauzan), protein production (D. Byrne) and microscopy platforms (A. Kosta and H. Le Guenno) of the IMM institute (Marseille, France).

The authors thank V. Fourmond for the discussion on the CO2 equilibrium and P. Infossi for the help with the enzyme purification.

For the purpose of Open Access, a CC-BY public copyright licence has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author This document is the Author's version of a Submitted Work that was subsequently accepted for publication in Biosensors and Bioelectronics after peer review. To access the final edited and published work see https://doi.org/10.1016/j.bios.2023.115106

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