Rima Ouchene
email: rima.ouchene@univ-bejaia.dz.

Laurent Intertaglia

Nawel Zaatout

Mouloud Kecha

Marcelino T Suzuki

Selective isolation, antimicrobial screening, and phylogenetic diversity of marine actinomycetes derived from the Coast

Keywords: Marine actinomycetes, Polluted marine environment, Selective isolation, Antimicrobial potential, Phylogenetic diversity

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Introduction

For decades, severe infections caused by resistant bacteria have been responsible for a major global healthcare problem, requiring longer and more complex treatments that are significantly more expensive to diagnose and treat [START_REF] Alanis | Resistance to Antibiotics: Are We in the Post-Antibiotic Era?[END_REF]. Moreover, infections generated by Methicillinresistant Staphylococcus aureus (MRSA) have emerged as a widespread cause of community infections, which is due to their ability to develop resistance [START_REF] Chambers | Waves of Resistance: Staphylococcus Aureus in the Antibiotic Era[END_REF][START_REF] Pasberg-Gauhl | A Need for New Generation Antibiotics against MRSA Resistant Bacteria[END_REF]; [START_REF] Barbosa | Targeting Antimicrobial Drug Resistance with Marine Natural Products[END_REF]. This emerging problem has been observed in many countries, including Algeria [START_REF] Mairi | Methicillin-Resistant Staphylococcus Aureus ST80 Clone: A Systematic Review[END_REF]. Thus, the search for new antibiotics remains a worldwide research priority.

Marine environments present a vast source of new bioactive natural products, but they are still either unexplored or underexplored [START_REF] Bhatnagar | Immense Essence of Excellence: Marine Microbial Bioactive Compounds[END_REF]Kim 2010, Gogineni and[START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF].

Those environments are characterized by particular conditions such as high salinity, pressure, low temperatures, and variable oxygen concentrations (Bullet al. 2000). Hence, marine organisms require distinctive metabolic capabilities to adapt and thrive in these environments [START_REF] Bose | LC-MS-Based Metabolomics Study of Marine Bacterial Secondary Metabolite and Antibiotic Production in Salinispora Arenicola[END_REF]). An increasing number of studies have been conducted on marine actinobacteria as important producers of bioactive compounds in recent years [START_REF] Maldonado | Diversity of Cultivable Actinobacteria in Geographically Widespread Marine Sediments[END_REF][START_REF] Bull | Marine Actinobacteria: New Opportunities for Natural Product Search and Discovery[END_REF][START_REF] Manivasagan | Marine Actinobacteria: An Important Source of Bioactive Natural Products[END_REF][START_REF] Kamjam | Deep Sea Actinomycetes and Their Secondary Metabolites[END_REF][START_REF] Barbosa | Targeting Antimicrobial Drug Resistance with Marine Natural Products[END_REF][START_REF] Ibrahimi | Marine Actinobacteria: Screening for Predation Leads to the Discovery of Potential New Drugs against Multidrug-Resistant Bacteria[END_REF][START_REF] Goel | Antimicrobial Resistance in Biofilms: Exploring Marine Actinobacteria as a Potential Source of Antibiotics and Biofilm Inhibitors[END_REF]. Interestingly, microorganisms living in unusual growth conditions, such as polluted environments, could be a significant source of interesting bacteria. Even though bacteria isolated from polluted environments have been instead investigated for their enzymatic activity their secondary metabolite production remains poorly understood.

Actinomycetes have long been recognized as a major source of natural products, particularly antibiotics [START_REF] Bérdy | Bioactive Microbial Metabolites: A Personal View[END_REF][START_REF] Kurtböke | Biodiscovery from Rare Actinomycetes: An Eco-Taxonomical Perspective[END_REF][START_REF] Jose | New Dimensions of Research on Actinomycetes: Quest for Next Generation Antibiotics[END_REF][START_REF] Jose | Actinobacteria in Natural Products Research: Progress and Prospects[END_REF]. Recently, rare actinobacteria have attracted more attention and are defined as strains other than the commonly known Streptomyces or strains with less frequency of isolation under conventional methods [START_REF] Baltz | Marcel Faber Roundtable: Is Our Antibiotic Pipeline Unproductive Because of Starvation, Constipation or Lack of Inspiration?[END_REF]. They have taken an increasingly important position in the production of compounds of biotechnological interest. They have yielded a wide range of molecules with medicinal, industrial, or agricultural uses [START_REF] Dhakal | Marine Rare Actinobacteria: Isolation, Characterization, and Strategies for Harnessing Bioactive Compounds[END_REF][START_REF] Ding | The Secondary Metabolites of Rare Actinomycetes: Chemistry and Bioactivity[END_REF][START_REF] Mast | Actinomycetes: The Antibiotics Producers[END_REF], notably those isolated from rare actinobacteria derived from extreme environments [START_REF] Hui | The extremophilic actinobacteria: from microbes to medicine[END_REF].

Although the isolation of rare actinomycetes in the marine environment is relatively low due to the need for appropriate isolation procedures and the application of different selection conditions, they are difficult to cultivate and maintain under conventional conditions [START_REF] Subramani | Culturable Rare Actinomycetes: Diversity, Isolation and Marine Natural Product Discovery[END_REF][START_REF] Azman | Mangrove Rare Actinobacteria: Taxonomy, Natural Compound, and Discovery of Bioactivity[END_REF]. Therefore, a high number of them are "viable but nonculturable" (VBNC) strains [START_REF] Bernard | Genetic Diversity of Total, Active and Culturable Marine Bacteria in Coastal Seawater[END_REF]. Recently, several strategies have been developed that have promoted the growth of some previously VBNC species, such as mimicking the natural environment in terms of pH, oxygen gradient, nutrient composition, etc. [START_REF] Dhakal | Marine Rare Actinobacteria: Isolation, Characterization, and Strategies for Harnessing Bioactive Compounds[END_REF].

Microorganisms derived from extreme biomes have recently attracted scientists' interest since they could house structurally unique natural products that are still unexploited. For that, the current study focuses on the selective isolation of actinomycetes from an unexplored marine environment along the coastline of Bejaia City (Algeria) and screening of their antimicrobial potential against a set of human pathogenic microorganisms.

Materials and methods

Sampling sites

The sampling was performed in the summer of 2017 from different sites (S1, S2 and S3) in the coastal area of Bejaia City, Algeria (Figure 1), and at different depths that ranged from 0.5 to 20 m with the help of a diver. S1 samples were collected at 10-20 m of depth, S2 at 1-2 m, and S3

samples were collected at 0-0.5 m of depth. The sampling area is polluted by different sources, mainly by the petroleum port (S1), industrial wastes (S2), and domestic discharges (S3). Sediment samples (S1) and water samples (S2 and S3) were taken from different points and were collected in sterile glass bottles, then transferred in a cool box to the laboratory for bacteria isolation.

Selective isolation of actinomycetes isolates

To isolate rare genera of actinomycetes, on should avoid the growth of non-actinomycetes bacteria that affect the isolation of actinomycetes known for their slow-growing. Therefore, different selective isolation media were used as previously recommended in several studies, such as: ISP2 [START_REF] Claverías | Culturable Diversity and Antimicrobial Activity of Actinobacteria from Marine Sediments in Valparaíso Bay, Chile[END_REF], Starch-Nitrate [START_REF] Ghanem | Isolation and Enumeration of Marine Actinomycetes from Seawater and Sediments in Alexandria[END_REF], A4 [START_REF] Gontang | Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments[END_REF]), M2 [START_REF] Jensen | Distribution of Actinomycetes in Near-Shore Tropical Marine Sediments[END_REF], and Chitin-agar [START_REF] Pisano | Application of Pretreatments for the Isolation of Bioactive Actinomycetes from Marine Sediments[END_REF]). In addition, the isolation media were combined with different isolation methods that are detailed in Table 1. The isolation media were inoculated with 100 µL aliquots of each sample previously pre-treated with methods 1, 2, and 3, as well as method 4, which consisted of the inoculation of untreated samples onto media with added antimicrobial compounds. All inoculated plates were incubated at 28 °C until up to 5 months (with wetted paper towels under the plates to avoid desiccation after 2 months) and examined daily, and all actinomycetes colonies were streaked into new plates containing the same media of isolation until a pure culture was obtained. It should be noted that all isolation media were adjusted to pH 7.2 ±0.2 and prepared with 100% of natural seawater.

Preliminary screening of antimicrobial activity

Preliminary screening for antimicrobial production was estimated by the conventional agar diffusion assay described by [START_REF] Shomura | Studies on Actinomycetales producing antibiotics only on agar culture[END_REF] and [START_REF] Engelhardt | Production of a New Thiopeptide Antibiotic, TP-1161, by a Marine Nocardiopsis Species[END_REF]. Briefly, agar discs (8 mm in diameter) recovered from the actinomycetes culture (M2 medium, 14 days of incubation at 28 °C) were placed on the surface of Muller Hinton agar pre-seeded with X µl of the target microorganism (Table 2) at 10 7 UFC/mL, _Plates were kept for 2 h at 4 °C for good diffusion of the active compounds. Activity was evaluated by measuring the diameters of the inhibition zones around the agar disc after 24 h of incubation at 37 °C. The M2 medium was composed of 10 g/L of starch, 1 g/L of casein bovine milk, 18 g/L of microbiological agar [START_REF] Jensen | Distribution of Actinomycetes in Near-Shore Tropical Marine Sediments[END_REF], and 1 mL (per liter of final medium) of trace salt solution prepared according to [START_REF] Shirling | Methods for Characterization of Streptomyces Species[END_REF]. All experiments were performed three times independently.

Secondary screening of antimicrobial activity

The previously selected isolates were screened a second time by the well diffusion method to improve the production of antibacterial substances in the broth medium using the supernatant of the culture prepared on M2 medium [START_REF] Patil | Selective Isolation, Evaluation and Characterization of Antagonistic Actinomycetes against Rhizoctonia Solani[END_REF]. Positive activity was estimated by the inhibition zone around the culture supernatant well (8 mm in diameter) of actinomycetes isolates.

All experiments were performed three times independently and the results were shown as means with the standard error as error bars.

Molecular identification and phylogenetic analyses of the active isolates

All active isolates were cultured on M2 medium for 14 days at 28 °C. Genomic DNA was extracted from the isolates using the Wizard® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's instructions. PCR and sequencing were performed as previously described by [START_REF] Fagervold | Pleionea Mediterranea Gen. Nov., Sp. Nov., a Gammaproteobacterium Isolated from Coastal Seawater[END_REF], using universal primers recommended for bacteria 27F mod: 5'AGRGTTTGATCMTGGCTCAG 3' and 1492R mod: 5'TACGGYTACCTTGTTAYGACTT 3'. PCR products were purified using a purification kit (Promega, USA) and sequenced using the Sanger-dideoxy-termination method on the Bio2Mar platform with an automated sequencer (3730XL). The 16S rRNA sequences have been deposited in the GenBank database under accession numbers that are given in parentheses in Figure 5.

The 16S rRNA gene sequences were submitted to the EZBiocloud database for identification [START_REF] Yoon | Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and wholegenome assemblies[END_REF]), available at: https://www.ezbiocloud.net. The sequences were imported into the SILVA_138.1_SSUref_NR99_12_06_20_opt.arb database and aligned using the arb fast aligner tool. Sequence positions (662 total), excluding ambiguous and missing, and present in a majority of sequences, were exported and used in phylogenetic reconstruction. Subsequently, a phylogenetic tree was constructed using MEGA X software [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF]) by the Neighbor-Joining (NJ) method using the p-distance model [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF], and the robustness of the tree was estimated by 1000 bootstrap random resampling of the original dataset.

Phenotypic characterization of the selected strain (P21)

The micro-morphology of the selected strain (P21) was established by light microscopy to visualize mycelia organization and sporulation. The strain was grown on M2 medium at 28 °C for two weeks before being observed under an optical microscope (Euromex). Furthermore, morphological and physiological characteristics were carried out as described in the International Streptomyces Project (ISP) recommendations by [START_REF] Shirling | Methods for Characterization of Streptomyces Species[END_REF], as well as on three "no ISP" media, including nutrient agar, Bennet medium [START_REF] Yokota | Catenuloplanes japonicus gen. nov., sp. nov., norn. rev., a New Genus of the Order Actinomycetales[END_REF]) and GYEA medium [START_REF] Athalye | Selective Isolation and Enumeration of Actinomycetes Using Rifampicin[END_REF]. Melanoid pigment production was determined on tyrosine agar medium (ISP7) recommended by [START_REF] Shirling | Methods for Characterization of Streptomyces Species[END_REF]. In addition, the utilization of carbohydrates was tested on a minimal medium (ISP9) containing different carbon sources (D-glucose, sucrose, xylose, inositol, mannitol, D-fructose, rhamnose, and cellulose) at 1% concentration. Basal medium without a carbon source served as a control. Moreover, the conditions of growth, such as the effect of pH (4-14), temperature of growth (4-45 ˚C), and NaCl tolerance were evaluated [START_REF] Aouiche | Activité antimicrobienne de Streptomyces sp. PAL111 d'origine saharienne contre divers microorganismes cliniques et toxinogènes résistants aux antibiotiques[END_REF]. Resistance to different antibiotics was carried out using the disc diffusion test as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2016). The tested antibiotics were: Penicillin G (6 µg), Ciprofloxacin (5 µg), Clindamycin (2 µg), Tetracycline (30 µg), Gentamicin (10 µg), and Chloramphenicol (30 µg). Other tests on the selected strain were carried out according to (Gordon and Mihm 1956), such as the ability of the strain to decompose substrates including casein, starch, and gelatin, and resistance to lysozyme at 0.005% (W/V), while the tyrosine assimilation was performed according [START_REF] Kurup | A scheme for the identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis[END_REF].

Statistical analyses

All experiments were carried out in triplicate. Firstly, the selective isolation data were analyzed using different statistical tests. The normality of the distribution in the studied groups was confirmed by the Shapiro-Wilk test. To find out the effect of the isolation methods, the used media, and the sampling depths on the number of actinomycetes isolated (note, we did not correct for the dilution in Method 1), isolation data were compared by three-way ANOVA (Analysis Of Variance) (3WA), followed by the post hoc Tukey test without correction for multiple comparisons. The test was considered statistically significant if (p<0.05) and was conducted using XLSTAT software (version 2018.1).

Secondly, the preliminary antimicrobial activity screening data were analyzed using principal component analysis (PCA) and hierarchical cluster analysis (HCA). The statistical analyses were performed using R software version 4.0.2 (https://www.r-project.org/https://www.rproject.org/), the RProject for Statistical Computing, with the plugin FactoMineR included in R.

The PCA plot is used to reduce the dimensionality of such datasets, increasing interpretability while minimizing information loss.

Results

Samples collection and isolation of actinomycetes

Three polluted marine sites were sampled at different depths from the coastal areas of Bejaia City (Algeria), using different methods and media. A total of 103 colonies of presumptive actinomycetes were observed and isolated. The number of isolates varied with the used methods, media, and sampling depths. As a result of the selective methods that were used, Method 1 showed the highest number of isolates with 46, while 12, 20, and 25 isolates were obtained when Methods 2, 3, and 4 were applied (Figure 2A), respectively. Based on the isolation media used, we found that M2 medium, ISP2 and Chitin-agar medium gave the highest number of isolates with 32, 27 and 22 isolates, respectively. On the other hand, only eleven isolates were obtained with Starch-Nitrate and A4 (Figure 2B). According to the results of sampling depths (Figure 2C), 56 isolates were obtained at D1 (10-20 m), 33 isolates at D2 (1-2 m), and only 14 isolates were obtained at D3 (0-0.5 m).

Furthermore, the isolation variables (methods, media, and depths) showed a significant effect on the number of actinomycetes isolated (p<0.05) based on the three-way ANOVA test. The M2 medium, Method 1 and D1 affected were the best combination for the recovery of Actinomycetes (p<0.05). Moreover, the post hoc Tukey test revealed a significant interaction between the different depths/isolation methods (p<0.05), but no significant interaction was found between the different depths/isolation media, or the isolation methods/isolation media.

Preliminary screening assay

The isolates were screened for their antimicrobial activity by the agar diffusion method against various pathogenic microorganisms (Gram-positive and Gram-negative bacteria, and a yeast). The results showed that 10 out of 103 isolates exhibited antibacterial activity against at least one of the pathogenic tested strains, particularly against Gram-positive bacteria including S. aureus, MRSA, and B. subtilis, while no activity was detected against the yeast and Gram-negative bacteria.

The results of preliminary antimicrobial screening were subjected to a PCA analysis, which was performed on the active isolates (10 isolates) using nine variables. Results showed that 100% of the total variation was distributed in three dimensions; the horizontal axis explained 65.64% of the total variance, and the vertical explained 32.65% (Figure 3A). The results demonstrated substantial differences between active strains. Moreover, it was found that only three variables contributed to the construction of this PCA, which are MRSA, S. aureus, and B. subtilis. Figure 3B presents the projection of individuals (actinomycetes isolates). Following that, the PCA data were used for Hierarchical Cluster Analysis (HCA). Therefore, the isolates were grouped into four clusters of active actinomycetes (Figure 3C) identified by their specific antimicrobial activity against targeted bacteria, according to the results of HCA analysis. The first cluster was presented by two isolates (S3 and S5) that showed activity only against B. subtilis with a 15 mm inhibition zone, while no activity was shown against MRSA and S. aureus. The isolates P19, P24, E, and C15 formed the second cluster, which showed significant activity against MRSA and S. aureus that varied between 11-22 mm, but no activity was observed against B. subtilis. The third cluster was presented by three isolates, S1, MP1m, and S4 that showed relatively similar activities against MRSA, S. aureus, and B. subtilis, with an inhibition zone that varied from 11-22 mm. The fourth cluster was presented by the strongest active isolate, P21, which showed 28, 32, and 25 mm of the inhibition zones against MRSA, S. aureus, and B. subtilis, respectively. Thus, cluster 4 is the most active cluster (p<0.05) based on inhibition diameter size with the highest antibacterial mean against MRSA and S. aureus.

Secondary screening

A secondary screening of the previously selected active actinomycetes was performed on M2 medium by the well diffusion method. Except for two isolates (S1 and S3), all active isolates showed positive activity (Figure 4). The P21 strain was found to be the most active isolate, with inhibition zones of 25, 33, and 27 mm against MRSA, S. aureus, and B. subtilis, respectively. The isolates C15, E, S4 and S5 showed activity against MRSA and S. aureus with inhibition zones that varied between 17-26 mm. The P24 isolate showed an inhibition zone of 9 mm against MRSA. The MP1m isolate showed 14, 22, and 8 mm of inhibition zones against MRSA, S. aureus, and B. aureus, respectively, and the P19 strain showed 25 and 17 mm against MRSA and B. subtilis. However, no activity was recorded against Gram-negative bacteria or yeast.

Molecular identification and phylogenetic analyses of the active isolates

The 16S rRNA sequences of active isolates were identified using the EZBiocloud database.

Isolates were found to belong to the Actinobacteria phylum and were grouped into two genera:

Streptomyces (8 out of 10), and Nocardiopsis (2 out of 10), with similarity percentages to known stains ranging from 98 to 100% (Table S1). Isolates P21 and S5 belonging to the Nocardiopsis genus showed 100% similarity to Nocardiopsis halotolerans DSM 44410(T) and Nocardiopsis aegyptia DSM 44442(T), respectively. Two isolates, C15 and P19, were closely similar to Streptomyces xinghaiensis S187(T) with 99.78% and 99.71% of similarity, respectively. The P24 isolate had a 99.79% similarity with Streptomyces albidoflavus DSM 40455(T). The S1 isolate showed the highest similarity (99.76%) to Streptomyces hydrogenans NBRC 13475(T). The S3 isolate was the most closely (98.44%) related strain to Streptomyces rimosus subsp. rimosus ATCC 10970(T), which could possibly indicate the presence of a new species within the Streptomyces genus. Moreover, the S4 isolate showed 99.79% similarity to Streptomyces albogriseolus NRRL B-1305(T). Both E and Mp1m isolates were similar to Streptomyces griseoflavus LMG 19344(T) with 99.62% and 99.61% of identity, respectively.

A phylogenetic tree based on the 16 rRNA gene sequences (Figure 5) was constructed using the neighbor-joining method [START_REF] Saitou | The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees[END_REF] using Micrococcus luteus NCTC 26650 as an outgroup. The P21 strain formed a monophyletic line with the S5 strain supported by 100% of the bootstrap replicates. The following strains: C15, E, Mp1m, S4, P19, P24, S3, and S1 appeared in one cluster as closest neighbors.

Phenotypic characterization of a selected strain (P21)

The observation of the P21 strain under an optical microscope showed that it has a branching substrate mycelium and produces short chains of spores (Figure 6A). On the other hand, Figure 6B shows the aerial mycelium, which has the typical zig-zag form of Nocardiopsis strains that are fragmented in older cultures. The cultural characteristics of the selected strain are presented in Table S2. The P21 strain was growing well on all media except nutrient agar, where poor growth was observed. Differences in the growth rates were observed, and the color of the substrate mycelium varied from brown-red to white. The production of soluble pigments was observed in some media (ISP2, ISP5, M2, and Bennet) but not in others (ISP3, ISP4, and Nutrient Agar).

Generally, the colors of the aerial mycelium were not the same as those of the substrate mycelium.

Table S3 lists the physiological and biochemical characteristics of the P21 isolate. The selected strain was not able to assimilate the carbon sources tested. However, it showed good growth when the medium was supplemented with 10% of sodium chloride, but poor growth was observed at 3-7% and no growth was found at 0% and 15-30% of NaCl. The optimum range of pH growth was found between 7 and 10. Moderate growth was observed at pH 3-5, while no growth was observed at pH 12-14. Moreover, the temperature ranges for growth were between 28 and 40 °C, with an optimum at 28 °C. The P21 strain was found to be able to degrade a variety of substrates, including starch, tyrosine, gelatin, and casein from bovine milk. Furthermore, based on the susceptibility test results, it was found that the P21 strain was resistant to Penicillin G and Clindamycin, but sensitive to Ciprofloxacin, Tetracycline, Gentamicin, and Chloramphenicol.

Discussion

Marine environments are unexplored in Algeria in terms of their microbiota; therefore, the aim of our study was to isolate actinomycetes from those environments. A total of 103 actinomycetes isolates were obtained from the Coast of Bejaia City using different methods and media. These isolates were screened for their antimicrobial activity, and ten isolates showed activity against at least one Gram-positive bacterium (S. aureus, MRSA, and B. subtilis), while no activity was found against Gram-negative bacteria or the yeast tested.

Different selective methods and media were used in this study to promote the growth of rare genera of actinobacteria and avoid the growth of terrestrial actinomycetes while reducing undesirable flora occurring on the isolation plates, as have been reported by others [START_REF] Pisano | Application of Pretreatments for the Isolation of Bioactive Actinomycetes from Marine Sediments[END_REF][START_REF] Undabarrena | Exploring the Diversity and Antimicrobial Potential of Marine Actinobacteria from the Comau Fjord in Northern Patagonia, Chile[END_REF][START_REF] Kamjam | Deep Sea Actinomycetes and Their Secondary Metabolites[END_REF]. The application of pretreatments or the presence of antimicrobial substances influences the number of actinomycetes isolates [START_REF] Bull | Marine Actinobacteria: New Opportunities for Natural Product Search and Discovery[END_REF]. In the present study, Method 1 proved to be among the most effective methods for isolating actinomycetes, which is consistent with the observation of [START_REF] Pisano | Hudson River sediments as a source of actinomycetes exhibiting antifungal activity[END_REF] that recorded that heat pretreatment of either 55 °C or 100 °C is a suitable alternative to the higher temperature for actinomycetes isolation, the fact that we did not correct the numbers for the initial dilution makes this result more notable and could also suggest that dilution of inhibiting substances in this highly polluted samples could have also be in play. The low (103) number of total strains in 540 petri dishes supports the possibility of negative effects of the pollution on the actinomycete community.

Using a filter membrane (Method 3) or adding antimicrobial substances to the isolation media (Method 4) have been previously reported to be the best methods for actinomycetes isolation from water and sediments according to [START_REF] Hirsch | Novel Method for Selective Isolation of Actinomycetes[END_REF] and [START_REF] Bredholdt | Rare Actinomycete Bacteria from the Shallow Water Sediments of the Trondheim Fjord, Norway: Isolation, Diversity and Biological Activity[END_REF]), but in our case, these methods (3 and 4) gave fewer isolates compared to Method 1. Even though [START_REF] Bredholt | Actinomycetes from sediments in the Trondheim Fjord, Norway: Diversity and Biological Activity[END_REF] found that heating and exposure of samples to phenol increased the number of mycelium-forming actinomycetes, this method (2) yielded the lowest number of isolates. Based on the isolation media used, the M2, ISP2 and Chitin-agar seem to be the best media for isolating actinomycetes from marine environments. Nevertheless, other studies revealed that low-nutrient media were more efficient than rich-nutrient media for the isolation of rare marine actinobacteria [START_REF] Jensen | Culturable Marine Actinomycete Diversity from Tropical Pacific Ocean Sediments[END_REF][START_REF] Maldonado | Diversity of Cultivable Actinobacteria in Geographically Widespread Marine Sediments[END_REF]. Furthermore, the sampling depth influences the number of actinomycetes isolated. It was therefore found that the number of isolates increased proportionally with the depth, and the highest number of isolates was obtained at D1 (10-20 m). A similar observation was reported previously by [START_REF] Jensen | Distribution of Actinomycetes in Near-Shore Tropical Marine Sediments[END_REF]. Nonetheless, based on the statistical significance of 3WA and Tukey's post hoc test, the best procedure that gave the highest number of actinomycetes isolates involves using the M2 medium, Method 1, and sampling at 10-20 m (D1) of depth. According to these findings, we suggest that the differences in the responses to the isolation conditions between our study and other studies are very likely due to the level of pollution in the isolation area that affects the abundance of actinobacteria strains. Moreover, the designated methods to isolate actinomycetes from polluted marine environments including sediment and water samples are not optimal for such biotopes, and more effects are required to establish specific isolation methods for such environments.

The obtained isolates were screened for their antimicrobial activity against nine human pathogenic bacteria. Targeting more than one pathogenic microorganism to obtain diverse bioactive bacteria has previously been supported by [START_REF] Genilloud | Current Approaches to Exploit Actinomycetes as a Source of Novel Natural Products[END_REF]. Ten isolates were selected among 103 that showed activity against at least one targeted Gram-positive bacteria.

However, this percentage is relatively low in comparison with the results of [START_REF] Peela | Studies on Antagonistic Marine Actinomycetes from the Bay of Bengal[END_REF], who reported that the majority of marine actinomycetes isolated showed antagonistic potential, including antibacterial and antifungal activities. The low rate of active strains could have several possible reasons: 1). Is probably due to the fact that the screening of isolates was based on a specific activity that could be inappropriate for the isolates' potential, and thus we missed other biological activities. 2). The isolates might possess biosynthesis gene clusters that encode for bioactive compounds but are, "silent" or "cryptic", which means they are not expressed under laboratory conditions. 3). The method or medium used is inappropriate for the production of metabolites. For these reasons, further investigations are being envisaged. Interestingly, the active isolates only showed activity against Gram-positive bacteria, suggesting that the isolates have developed specific marine adaptation mechanisms that likely affect secondary metabolite biosynthetic pathways.

The P21 isolate was shown to be the most active strain against Gram-positive bacteria and belongs to the Nocardiopsis genus, which has been described as a producer of a wide range of bioactive compounds [START_REF] Fu | α-Pyrones from the Marine-Derived Actinomycete Nocardiopsis Dassonvillei Subsp. Dassonvillei XG-8-1[END_REF][START_REF] Hatem Ibrahim | Natural Product Potential of the Genus Nocardiopsis[END_REF][START_REF] Tatar | Isolation, Phylogenetic Analysis and Antimicrobial Activity of Halophilic Actinomycetes from Different Saline Environments Located near Çorum Province[END_REF]. Although this genus has received less attention than Streptomyces, which has been extensively investigated [START_REF] Lazzarini | Rare genera of actinomycetes as potential producers of new antibiotics[END_REF][START_REF] Takahashi | Actinomycetes, an Inexhaustible Source of Naturally Occurring Antibiotics[END_REF]. Previous studies have shown the anti-MRSA activity of the Nocardiopsis genus, such as the activity of the angucyclines (nocardiopsistins A-C) isolated from a marine sponge-derived Nocardiopsis sp. [START_REF] Xu | Nocardiopsistins A-C: New angucyclines with anti-MRSA activity isolated from a marine sponge-derived Nocardiopsis Sp. HB-J378[END_REF]. The P21 strain does not show any activity against Gram-negative bacteria even though previous studies have provided the antagonistic potential of Nocardiopsis strains against Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae [START_REF] Rajivgandhi | Molecular characterization and antibacterial investigation of marine endophytic actinomycetes Nocardiopsis sp. GRG 2 (KT 235641) compound against isolated ESBL producing bacteria[END_REF], Enterococcus faecium and Candida albicans [START_REF] Engelhardt | Production of a New Thiopeptide Antibiotic, TP-1161, by a Marine Nocardiopsis Species[END_REF]. Hence, this difference is likely due to the specific activity of the secreted molecules by the P21 strain against Gram-positive bacteria, especially MRSA (Figure 7).

Marine sediments harbor a large diversity of culturable actinobacteria [START_REF] Bredholdt | Rare Actinomycete Bacteria from the Shallow Water Sediments of the Trondheim Fjord, Norway: Isolation, Diversity and Biological Activity[END_REF][START_REF] Gontang | Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments[END_REF][START_REF] Claverías | Culturable Diversity and Antimicrobial Activity of Actinobacteria from Marine Sediments in Valparaíso Bay, Chile[END_REF]. In the current work, the phylogenetic diversity analysis of the active strains showed that the isolates belonged to two genera (Streptomyces and Nocardiopsis), which are widely distributed in marine environments worldwide [START_REF] Claverías | Culturable Diversity and Antimicrobial Activity of Actinobacteria from Marine Sediments in Valparaíso Bay, Chile[END_REF][START_REF] Jose | New Dimensions of Research on Actinomycetes: Quest for Next Generation Antibiotics[END_REF]. The low recovery observed both in number and diversity could be due to the influence of the contaminants that are present in the isolation region, which can reduce the biological diversity of actinobacteria. In addition, the results of the phylogenetic analysis showed that the homology of the sequences with the closely related species ranged from 98 to 100%. In particular, the S3 isolate showed 98.44% similarity to the closest strain, which is below the 98.65% threshold for this genus [START_REF] Kim | Towards a Taxonomic Coherence between Average Nucleotide Identity and 16S RRNA Gene Sequence Similarity for Species Demarcation of Prokaryotes[END_REF]. This isolate can thus be considered a presumptive new Streptomyces species. Moreover, several studies in Algeria have recorded the Nocardiopsis genera from soil samples, such as the desert soil [START_REF] Zitouni | Nocardiopsis and Saccharothrix genera in Saharan soils in Algeria: Isolation, biological activities and partial characterization of antibiotics[END_REF][START_REF] Meklat | Isolation, Taxonomy, and Antagonistic properties of halophilic actinomycetes in Saharan soils of Algeria[END_REF], the salt pan of the Kenadsa region (located in southwest Algeria) [START_REF] Messaoudi | Kenalactams A-E, Polyene Macrolactams Isolated from Nocardiopsis CG3[END_REF], a salt lake in the Algerian Sahara [START_REF] Rabia-Boukhalfa | Isolation, Purification and Chemical Characterization of a New Angucyclinone Compound Produced by a New Halotolerant Nocardiopsis Sp. HR-4 Strain[END_REF], and from the rhizosphere-associated with semi-arid soil in the North-East of Algeria [START_REF] Aouar | Taxonomic Identification of Rhizospheric Actinobacteria Isolated from Algerian Semi-Arid Soil Exhibiting Antagonistic Activities against Plant Fungal Pathogens[END_REF]. However, Nocardiopsis stains had never been isolated from marine environments in Algeria.

Morphological characteristics, mainly based on the substrate mycelium, the aerial hyphae, pigment production, spore appearance, and chaining, have been proven to be an effective and widely used reference for the preliminary identification of actinobacteria [START_REF] Taddei | Isolation and Identification of Streptomyces Spp. from Venezuelan Soils: Morphological and Biochemical Studies[END_REF]. The P21 isolate showed the typical microscopic appearance of most species of the Nocardiopsis genus.

It grew well on several media and showed various cultural characteristics that are related to the available substrate and growth conditions. The P21 strain was susceptible to four antibiotics and displayed resistance to two antibiotics tested (Penicillin G and Clindamycin). According to previous reports, antibiotic resistance may reveal an isolate's ability to produce the antibiotic(s) to which it is resistant (Basu and Paul 1992;[START_REF] Jain | Antibiotic Resistance and Chromium Reduction Pattern Among Actinomycetes[END_REF]. Furthermore, biochemical characteristics showed the potential of the P21 strain to degrade several substrates, which is in line with previous reports that described the Nocardiopsis genus as a potential source of extracellular enzymes [START_REF] Bennur | Nocardiopsis Species as Potential Sources of Diverse and Novel Extracellular Enzymes[END_REF]. Further experiments should be carried out to confirm the enzymatic abilities of this strain.

In conclusion, the Coast of Bejaia City was an interesting source of bioactive actinobacteria strains with the possibility of the presence of new Streptomyces species. Nocardiopsis sp. P21 was found to be the most active strain, with potential for new antimicrobial therapies to fight MRSA infections. Further studies are required to understand the biosynthetic pathways involved in the antibacterial activity of the active strains, including genome mining and metabolomics analyses.

Method Description

Method 1 Samples were diluted at 1:4 (v/v) with sterile sea water, then vigorously shaken.

They were then heated for 6 min at 55 °C [START_REF] Gontang | Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments[END_REF]. A 100 µL volume of the treated sample was inoculated into each media.

Method 2 Samples were heated at 120 °C for 1 h, then exposed to 1.5% phenol for 30 min [START_REF] Bredholt | Actinomycetes from sediments in the Trondheim Fjord, Norway: Diversity and Biological Activity[END_REF]. A 100 µL volume of the treated sample was inoculated into each media.

Method 3

The agar plates of all media were overlaid with a cellulose ester membrane filter (0.45 µm pore size) and then inoculated with a 100 µL volume of the untreated sample. After 2 weeks, the filters were removed and the plates were reincubated [START_REF] Hirsch | Novel Method for Selective Isolation of Actinomycetes[END_REF].

Method 4

The isolation media were supplemented with filtered (0.2-µm pore size)

Nalidixic acid (10 µg/mL), Nystatin (50 µg/mL) [START_REF] Bredholdt | Rare Actinomycete Bacteria from the Shallow Water Sediments of the Trondheim Fjord, Norway: Isolation, Diversity and Biological Activity[END_REF], and

Cycloheximide at (50 µg/mL) as recommended by [START_REF] Bredholt | Actinomycetes from sediments in the Trondheim Fjord, Norway: Diversity and Biological Activity[END_REF]. A 100 µL volume of the untreated sample was inoculated into each media.

Microorganisms tested References

Gram + Bacteria

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 Actinomycetes counts in sampling sites collected from the Coast of Bejaia (Alegria)

Figure 3

 3 Figure 3 Principal component analysis (PCA) and hierarchical clustering analysis of active

Figure 4

 4 Figure 4 Average inhibition zones (mm) of antimicrobial activity of active actinomycetes isolates

Figure 5

 5 Figure 5 Neighbor Joining-phylogenetic tree showing the evolutionary relationship of bioactive

  Figure 1

Figure 5 Figure 6 Figure 7

 567 Figure 5

Table 1

 1 Descriptions of the applied isolation methods

Table 2

 2 Targeted organisms tested to select bioactive Actinomycetes

Acknowledgements:

We thank the Bio2Mar platform (http://bio2mar.obsbanyuls.fr) for providing technical support and access to instrumentation. This work benefited from access to the Observatoire Oceanologique de Banyuls-sur-Mer, an EMBRC-France and EMBRC-ERIC site.

Data Availability Statement: The data are not yet available on line.

All authors have given approval to the final version of the manuscript.

Conflict of interest:

The authors declare no conflict of interest.

sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF]. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The accession number is shown in parentheses following the strain name. The scale bar corresponds to 0.01 substitutions per nucleotide position.

Legends for the supporting information


Julien C Vantourout

From Bench to Plant: An Opportunity for Transition Metal Paired Electrocatalysis

Keywords:

Electrochemistry and transition metal catalysis are gigantic fields from where several methodologies have found applications in both medicinal and process chemistry. Surprisingly, the transfer from bench to plant is much more difficult for transition metal electrocatalysis.

While a plethora of those transformations have emerged in the landscape of medicinal chemistry since more than 50 years, they were not further developed on larger scale. Of course, extra parameters have to be considered when running an electrocatalytic reaction, but they cannot justify the absence of transition metal electrocatalysis examples in process chemistry. This perspective aims to demonstrate that intrinsic characteristics related to the different electrocatalytic modes are responsible for the absence of such processes on large scale. It will also highlight why paired electro-catalyzed reactions have the potential to fill the gap between medicinal and process chemistry. Finally, a strategy to develop unprecedented and process oriented paired electro-catalyzed methods will be disclosed, and hopefully will motivate electropractitioners to study challenging and applicable transformations.

INTRODUCTION

For years, synthetic organic electrochemistry has been valued for the redox advantages it offers compared to purely chemical methods. [START_REF]For a selection of books and reviews: (a) Organic[END_REF] When it comes to manipulation of electrons, electrochemistry is certainly the easiest and most economical method to use. 2 However, due to misconceptions and categorization of electrochemistry as a "black box", it took years -and the development of intuitive and standardized equipmentfor the scientific community to appreciate the potential electrochemical processes can offer, especially in the context of sustainability and scalability. 3 Similarly, over the past 40 years, transition metal-catalyzed crosscouplings have become a mainstream, reliable and indispensable tool for carbon-carbon and carbonheteroatom bond formation. [START_REF]For a selection of books and reviews: (a) Metal Catalyzed Cross-Coupling Reactions and More[END_REF] These reactions allow for the synthesis of complex pharmaceutical ingredients including several marketed drugs or drug candidates under development in laboratories around the world. Both electrochemical and transition metal-catalyzed processes have been employed in the synthesis of important materials and are usually run-on process scales ranging from kilograms to thousands of metric tons. The BASF process to synthesize Lysmeral 1, a prevalent fragrance molecule, is one of the most famous examples that perfectly demonstrates the scalability of electrochemical processes (Figure 1A). [START_REF] Botte | Electrochemical Manufacturing in the Chemical Industry[END_REF] In the synthetic route, key intermediate 2 is obtained using an over 10,000 ton per year scale double anodic oxidation of 4tert-butyl toluene. From a transition metal catalysis perspective, many processes can provide kilogram quantities of pharmaceuticals (Figure 1A

MeO

OMe

Despite those striking examples, the development and the use of transition metal electrocatalysis in the pharmaceutical and agrochemical industries is rare and even no reported examples could be identified. This is certainly not due to a lack of motivation to develop such methods but mainly due to the difficulty to transfer them from bench to process scale. Indeed, out of the many useful transition metal electro-catalyzed methods that flourishes in the literature, none have been applied so far in a process chemistry context. This can be explained by the number of parameters one has to consider when transposing an electrochemical reaction from bench to process scale (Figure 1B). In addition to usual parameters such as yield, cost, scalability and sustainability, electrochemistry adds on several variables (electrode materials, electrolyte, sacrificial additives, current density…) which can be seen as a serious impediment compared to classic redox processes (Figure 1B). However, the recent enthusiasm and interest for synthetic organic electrochemistry provides the scientific community with the unique opportunity to develop transition metal electro-catalyzed methodologies that could be more easily transferred from bench to plant. This perspective aims to describe the similarities and distinctions between the different transition metal electro-catalyzed processes. It will also highlight their limitations when transitioning to process chemistry applications. Finally, it will attempt to demonstrate that paired electrolysis in addition to offering a smoother transition from bench to scale compared to purely oxidative or reductive methods, is also the perfect platform for reaction discovery.

R 3 R 4 R 1 R 2 R 3 R 4 R 1 R 2 N 3 N 3 MnBr 2 •4H 2 O ( 5

TRANSITION METAL ELECTRO-CATALYZED PROCESSES: SIMILARITIES, DISTINCTIONS AND LIMITATIONS

Transition metals can lend or withdraw electrons from a reagent depending on the nature of the reaction which makes them excellent catalysts. They have the ability to access and interchange variety of oxidation states, to form complexes with the reagents and to be a good source of electrons making them highly useful in the context of electrochemical transformations. Once those inherent properties and subtleties associated to them are controlled, transition metal electro-catalyzed reactions can mediate oxidative or reductive pathways with better selectivity and functional group tolerance than pure electrochemical processes. 8 In addition, transition metal can activate challenging substrates or bonds (e.g., C-H activation, oxidative addition…) increasing the landscape of electrochemical reactions one could envision. [START_REF] Ma | Recent Advances in C-H Functionalization Using Electrochemical Transition Metal Catalysis[END_REF] Transition metal electro-catalyzed processes can be divided into three categories: oxidative, reductive and paired (Figure 2). [START_REF]For a selection of books and reviews: (a) Organic[END_REF] Those processes share common features but own separate characteristics allowing the practitioner to access the desired reactivity. They also suffer from specific limitations with regards to their application in process chemistry. The following examples were selected to describe the key features of the different transition metal electro-catalyzed regimen and how these characteristics can impact the transfer from bench to plant. Other examples could have been chosen and are cited in the reference section. 10 Oxidative transition metal electrocatalysis (Figure 2A). Recently, Lin and co-workers reported a mild oxidative manganese electro-catalyzed diazidation of alkenes using an undivided cell, equipped with a RVC anode and a platinum cathode, at a constant potential of 2.3 volts (Figure 2A.1). [START_REF] Fu | Metalcatalyzed electrochemical diazidation of alkenes[END_REF] A variety of useful vicinal diazide compounds that can be converted to the corresponding diamines after simple palladium-catalyzed reduction were obtained in moderate to high yields. The authors reported more than 60 examples including terminal, 1,1and 1,2-disubstituted, trisubstituted, and tetrasubstituted alkenes. Substrates bearing alcohol, aldehyde, enolizable ketone, carboxylic acid, amine, sulfide, and alkyne moieties were tolerated under the reaction conditions. It was also noted that functional groups predisposed to nucleophilic displacement by N3 -, such as, ester, alkyl chloride, epoxide and alkyl bromide, were also compatible, likely due to the use of acetic acid as a cosolvent, which attenuates the nucleophilicity of the azide anion. The unanticipated broad substrate scope of the method motivated the authors to start investigating its mechanism (Figure 2A.2). The combination of radical clock experiments with cyclic voltammetric and spectrophotometric studies confirmed the Mn-assisted dual radical group transfer to the alkene. In this purely oxidative process the Mn(II)-N3 is anodically oxidized to afford a Mn(III)-N3 species able to first induce a C-C pbond homolysis. The resulting radical then reacts with another Mn(III)-N3 to afford the desired vicinal diazide. To close the electrochemical circuit the proton reduction of acetic acid occurs at the cathode. Following this first report, the Lin group decided to adapt their electrocatalytic process to a standardized electrochemistry kit -ElectraSyn (Figure 2A.1). [START_REF] Fu | electrocatalytic approach to the synthesis of vicinal diamines[END_REF] In this new protocol, constant potential was switched to constant current and the platinum cathode was replaced by nickel foam providing a more accessible protocol. The reactions were conducted on a 0.3 mmol scale and afforded compounds such as 4 in excellent yields. For larger scale experiments (3 mmol), a 3-neck round bottom flask was equipped with RVC (anode) and platinum (cathode) electrodes and a constant potential of 2.3V was applied. These changes point out the complications one can encounter when trying to scale an electrochemical reaction. In addition, from a process chemistry anode cathode

M 1 (n) M 1 (n+p) M 2 (x) M 1 (x-y) C A B [optimal regime] [new reactivites & mechanisms] [new metal complexes]
perspective, the use of lithium perchlorate, platinum electrode, and sacrificial acetic acid is an extra barrier that would limit the uptake of this reaction on a larger scale (even if these parameters could be optimized). Reductive transition metal electrocatalysis (Figure 2B). In 2017, Hansen and co-workers disclosed the electrochemical nickel-catalyzed reductive coupling between aryl bromides and alkyl bromides (Figure 2B.1). [START_REF] Perkins | Electrochemical Nickel Catalysis for sp 2 -sp 3 Cross-Electrophile Coupling Reactions of Unactivated Alkyl Halides[END_REF] They observed that the control of the reduction rate via the current passed is a critical feature of the reaction to achieve decent conversion. After optimization of the reaction parameters, the best yields were obtained when a RVC cathode was paired with a sacrificial zinc anode in an undivided cell at a constant current of 10 mA. The scope contains 10 examples bearing important functional groups including amide, ketone, trifluoromethyl, and protecting amines as well as heterocyclic compounds such as pyridine, pyrimidine and aza-indoles. It is worth mentioning the yields are comparable to or sometimes higher than those attained using Zn powder as the reductant demonstrating electrochemistry can broaden the applicability of nickel-catalyzed reductive crosscouplings. The proposed mechanism operates similarly to the one reported by Weix and co-workers for metal powder reductive coupling (Figure 2B.2). First, Ni(0) enters the catalytic cycle via reduction of Ni(II) at the cathode. Then, oxidative addition of the aryl bromide affords a Ni(II) species that reacts with the radical issued from the alkyl bromide after single electron transfer from Ni(I). Finally, the resulting Ni(III) species delivers the desired cross-coupled product after reductive elimination. In parallel to this reductive electro-catalytic cycle, the sacrificial zinc anode is oxidized to close the circuit. Two years after this report, the same group reported a new set of conditions that addresses the challenges associated with cross-electrochemical coupling: the use of amide solvent and the need for sacrificial anode (Figure 2B.3). [START_REF] Perkins | MetalReductant-Free Electrochemical Nickel-Catalyzed Couplings of Aryl and Alkyl Bromides in Acetonitrile[END_REF] The reaction occurs in a divided cell using acetonitrile as the solvent, N,Ndiisopropylamine as sacrificial reductant and tri-tertbutyl-terpyridine and di-tertbutyl-bipyridine as a combination of ligands affording the desired crosscoupled products in moderate to high yields. As an example, the reaction between ethyl 4-bromobenzoate and (3-bromopropyl)benzene was conducted on a 6.5 mmol scale delivering compound 5 in 80% yield. This new set of conditions is therefore more amenable to scale-up but still suffers from serious limitations such as the use of a divided cell setup [START_REF]Waldvogel and co-workers in collaboration with Novartis reported a divided cell scale-up (25.9g) of a key intermediate for NS5A Inhibitors. However, a flow system was used for the upscaling[END_REF] and the need for a sacrificial reductant. Transition metal paired electrocatalysis (Figure 2C). In 2019, Baran et al. reported an optimized set of conditions of their electrochemically driven nickelcatalyzed aryl amination, initially reported in 2017 (Figure 2C.1). [START_REF] Li | Electrochemically Enabled, Nickel-Catalyzed Amination[END_REF][START_REF] Kawamata | Electrochemically Driven, Ni-Catalyzed Aryl Amination: Scope, Mechanism, and Applications[END_REF] The reaction uses RVC (anode) and Nifoam (cathode) electrodes in an undivided cell at room temperature under constant current conditions. An indepth mechanistic study including several electrochemical, analytical and spectroscopic techniques highlighted the importance of the nickel/ligand ratio as well as the need for the amine coupling partner to be used in excess. Then, an increase of the amount of ligand and the addition of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) as an external base afforded a much broader scope including amino acids, heteroaryl halides, nucleoside analogs, DNA, natural products, and oligopeptides. The mechanism of the reactions involves several nickel species with disparate oxidations state (Figure 2C.2). The electrocatalytic cycle starts with the reduction of Ni(II) to a Ni(I) species at the cathode. Then, the rapid oxidative addition of Ni(I) to an aryl halide affords a transient Ni(III) species that is readily reduced at the cathode resulting in a stable Ni(II) aryl intermediate. After amine coordination and deprotonation, this Ni(II) complex is oxidized at the anode generating a Ni(III) specie which undergoes rapid reductive elimination to produce the desired arylamine product while regenerating the active catalyst. It is worth noting that when a reaction requires both oxidation and reduction of the catalyst, electrochemistry positions itself as the method of choice. Indeed, under paired electrolysis regime, redox events can take place separately at the anode and the cathode. From a large-scale perspective, relevant intermediate 6 for the synthesis of vilazodonea drug recently approved by FDA for the treatment of major depressive disorder -was obtained in 64% on a 37.2 mmol scale using a flow system equipped with carbon felt electrodes. [START_REF] Li | Electrochemically Enabled, Nickel-Catalyzed Amination[END_REF] Importantly, various reaction scales could be envisioned simply by increasing the number of frames. However, reactions components such as the amide solvent (DMA), the electrolyte (N-tetrabutylammonium bromide), and the base (DBU) would have to be evaluated and/or changed before being used on a pilot plant scale.

TRANSITION METAL PAIRED ELECTRO-CATALYZIS: THE IDEAL PLATFORM FOR REACTION DISCOVERY AND PROCESS CHEMISTRY

All the aforementioned electrochemical modes have proven useful on bench scale but still suffer from foreign considerations preventing their use in process chemistry (Figure 2). Some of those drawbacks, related to chemical parameters, can be optimized to render the reaction conditions process friendly. On the other hand, the nature of the electrochemical transformation cannot be modified. A purely oxidative electrochemical process always requires the use of a sacrificial oxidant to be reduced at the cathode (most of the time a proton source), while a purely reductive electrochemical reaction necessitates a metal-based sacrificial anode or the use of a sacrificial reductant to be oxidized at the anode. Even if these parameters seem insignificant and can undoubtedly be addressed, still they represent extra constraints one has to consider when envisioning a process scale experiment. In a paired electrolysis transformation, both the anodic oxidation and the cathodic reduction are indispensable features of the reaction pathway. Therefore, no sacrificial counterpart is needed facilitating adoption and transfer to process scale.

The notion of anticipation is also important when designing, developing and optimizing a transition metalcatalyzed reaction. Recently, a lot of effort has been dedicated to the development of process friendly set of conditions where all the components are studied toward scalability and sustainability. [START_REF] Verschueren | Electrochemistry and Photoredox Catalysis: A Comparative Evaluation in Organic Synthesis[END_REF] In addition, several technologies such as flow chemistry have been developed to facilitate the transition from bench to plant. [START_REF] Barham | For selected reviews on synthetic photoelectrochemistry[END_REF] The same vision could and should be applied to transition metal electro-catalyzed methods to give the practitioner more options than its current toolbox contains. Based on its aforementioned characteristics, paired electrolysis places itself as the ideal mode to investigate as the optimization will be similar to the one usually performed for a standard chemical process -judicious choice of solvent, chemical variables, and simple electrochemical parameters (electrode material and electrolyte).

Recently, several research groups started to combine electrochemistry with photoredox catalysis to achieve new transformations including transition metal paired electro-catalysis. [START_REF] Verschueren | Electrochemistry and Photoredox Catalysis: A Comparative Evaluation in Organic Synthesis[END_REF][START_REF] Barham | For selected reviews on synthetic photoelectrochemistry[END_REF] However, similarly to purely reductive or oxidative electrochemical reactions, cathodic or anodic sacrificial processes cannot be avoided. In addition, merging photoredox and electrochemistry clearly complexifies the setup and increases the finesses one will have to control. Overall, these limitations will certainly limit the uptake in process chemistry. Therefore, transition metal paired electro-catalyzed methodologies provide the scientific community with the unique opportunity to develop new transformations based on unprecedented electrochemical reaction pathways (Figure 3B). Such reactions will be aligned with future process chemistry developments. As an example, dual transition metal electrocatalysis is a field that has not been deeply investigated due to the challenges associated with such a complex system. Indeed, from a mechanistic standpoint, a metal will have to be oxidized when the other will have to be reduced. Therefore, a perfect control of both oxidation and reduction events is required to ensure successful outcome. In depth optimization, ligand screening, and mechanistic investigations including techniques such as kinetic studies, cyclovoltammetry, and UV-Vis analyses will have to be conducted to ensure the success of such an approach. Keeping in mind the notion of scalability and sustainability the use of earth abundant metal should be prioritized but will undoubtably increase the level of difficulty.

CONCLUSION

The fields of electrochemistry and transition metal catalysis are in constant expansion allowing for the development of diverse and useful methodologies. While the combination of these two valuable areas, transition metal electrocatalysis, has the potential to benefit both medicinal and process chemistry projects, it will have to target meaningful reactions such as key skeleton bond forming transformations. Following this idea, paired electrocatalysis seems to be the perfect match as it provides the practitioner with a unique opportunity to develop unprecedented, applicable and sustainable methods.

5

 5

FIGURE 1 .

 1 FIGURE 1. From bench to plant: The paradox of transition metal electrocatalysis. (A) Introduction (B) An electrochemical gap between bench and process scales.

7 From

 7 ). As an example, Johnson and Johnson developed a 332.5 kg scale Heck reaction in the preparation of rilpivirine 3, a potent nonnucleoside reversed transcriptase inhibitor developed as part of their HIV program. electrochemical gap between bench and process scales [scalable] [sustainable] [cost effective] [industrial applications] No pilot plant example of transition metal electro-catalysis [electrolytes] [e-parameters] [sacrificial additives]

FIGURE 2 .

 2 FIGURE 2. Transition metal electro-catalyzed processes: Similarities, differences and limitations. (A) Oxidative transition metal electrocatalysis. (B) Reductive transition metal electrocatalysis. (C) Transition metal paired electrocatalysis.

  mol%) NaN 3 (5 equiv), LiClO 4 (1.75 equiv) MeCN/AcOH, rt, N 2 C(+)/Pt(-), E cell = 2.3V General scheme, Lin Science 2017 From bench to scale, Lin Nat. Prot. 2018 [>60 examples] [abundant metal] [scalable] [mechanistic study] General scheme, Hansen Org. Lett. 2017 From bench to scale, Hansen OPRD 2019 [10 examples] [tunable parameters] [good selectivity] [scalable] Baran JACS 2019 From bench to scale, Baran JACS 2019 [>50 examples] [improved scope] [broad applications] [scalable]

FIGURE 3 .

 3 FIGURE 3. From bench to plant: Future directions for transition metal paired electrocatalysis. (A) Anticipate the choice of parameters to facilitate the transfer to process scale. (B) Development of dual transition metal electro-catalyzed processes.

From Bench to Plant: Future Directions For Transitiion Metal Paired Electrocatalysis

	A. Anticipate the choice of parameters on small scale to faciliate
	the transfer to process scale
	TRANSITION METAL PAIRED ELECTROCATALYSIS
		BENCH SCALE
	electrodes	electrolyte	additives/solvent
	▻ accessible	▻ accessible	▻ inexpensive
	▻ inexpensive	▻ inexpensive	▻ stable and scalable
	▻ robust and reusable	▻ stable and scalable	▻ process friendly
	DIRECTLY TRANSFERABLE TO PROCESS SCALE
	B. Future directions: Development of dual transition metal
	electro-catalyzed processes

[undivided cell] [new reactions] [abundant metal] [no sacrificial processes]

ACKNOWLEDGMENT

Financial support for this work was provided by the CNRS, French Ministry of Research and the University of Lyon I.

AUTHOR INFORMATION

Corresponding Authors *julien.vantourout@univ-lyon1.fr

ORCID

Julien C. Vantourout: 0000-0002-0602-069X

NOTES

The author declares no competing financial interest.

Table of Contents