Unveiling the Diversity and Biotechnological Potential of Halophilic Actinobacteria from the Sebkha of Lake Naïla, Morocco

Abstract

Saline ecosystems, including saline lakes, are indeed major hotbeds of microbial novelty, harboring diverse and largely unexplored microbes. The sebkha of Lake Naïla (Morocco), an ecologically protected area registered under the Ramsar Convention in 1998, remains largely unexplored. Isolation using three different selective media enabled seven phenotypically distinct actinobacterial isolates to be obtained. Molecular characterization, based on 16S RNA gene sequencing, was used to identify strains as members of the genera Streptomyces, Nocardiopsis, and Prauserella. Three strains showed antimicrobial potential against pathogenic microorganisms, with Streptomyces sp. strain 43 exhibiting the most potent effects. Additionally, all isolates displayed plant-growth-promoting (PGP) traits, including phosphate solubilization, auxin (IAA) synthesis, siderophore secretion, and ammonia production. Notably, Nocardiopsis sp. strain 42 produced the highest IAA levels (282 μg/mL), while Streptomyces sp. strain 39, Streptomyces sp. strain 43, and Streptomyces sp. strain 48 excelled in phosphate solubilization. GC-MS profiling of Streptomyces sp. strain 43 revealed a complex metabolite repertoire, including 1,2-propanediol and nonanal, highlighting the strain’s versatile secondary metabolism. These findings highlight that the sebkha of Lake Naïla represents a rich source of halophilic actinobacteria with promising dual potential for antimicrobial and biofertilizer applications. The findings provide a solid basis for new perspectives on biotechnology applications and sustainable agriculture.

1. Introduction

Extreme environments represent a valuable source of novel microbial species due to the unique conditions that shape their evolutionary adaptations [1,2]. In these habitats, microorganisms are exposed to harsh conditions—such as extreme temperatures, high salinity, extreme pH, or radiation—that require specialized survival mechanisms [2,3]. To survive and thrive under such stressors, these microbes have naturally evolved unique metabolic pathways, enhanced stress tolerance, and efficient resource utilization strategies, which may offer significant biotechnological and environmental applications. The high mutation rates driven by environmental stress have been identified as a potential key mechanism underlying evolutionary change [1]. Exploring these extreme environments may provide fertile ground for isolating previously undiscovered microbial species with plant-growth-promoting traits and novel bioactive compound production, offering biotechnological and industrial applications [4]. The actinobacteria inhabiting extreme environments exemplify not only considerable taxonomic diversity but also substantial genetic heterogeneity in their biosynthetic pathways, which are responsible for the production of novel biological compounds [5].

Among these extreme environments, Khenifiss lagoon, also known as Dayet Naïla or Lac Naïla, is a saline coastal lake located in the Khenifiss National Park in southern Morocco [6]. Lake Naïla forms part of an extensive wetland system that includes sand dunes, mudflats, and a lagoon system along the Atlantic coast [6]. The lagoon is particularly important for biodiversity as it serves as a critical stopover and wintering site for migratory birds, including flamingos, herons, and various shorebirds. The lake and its surrounding area are recognized for their high ecological value, hosting rich flora and fauna that are adapted to the unique coastal desert environment [6]. As a Ramsar wetland of international importance, Lake Naïla is protected to ensure the conservation of its biodiversity, particularly given the challenges of arid conditions and human pressures on local ecosystems. The lake is also a site of interest for researchers studying wetland and saline ecosystems and migratory patterns in North Africa.

Saline, moderately saline, and hypersaline environments are recognized for their remarkable microbial diversity, where selective pressures such as salinity, extreme temperatures, and nutrient limitations drive the evolution of microorganisms with specialized metabolic capabilities [7]. These microorganisms, particularly actinobacteria, are considered promising candidates for the discovery of novel bioactive compounds because of their ability to produce a wide array of secondary metabolites, including antibiotics, antifungals, and anticancer agents [8]. Actinobacteria are widely distributed in saline lakes and marine environments, particularly in sediments, and have also been reported in association with various marine organisms, including fish, sponges, and macroalgae [9,10]. Isolates obtained from saline sediments have been shown to produce a broad spectrum of bioactive metabolites, including antibiotics, antitumor compounds, and anti-inflammatory agents [11,12]. Despite the significant potential of such environments, the microbial diversity of Lake Naïla has remained largely uncharacterized. To fill this gap, the present study investigates the actinobacterial diversity of the sebkha of Lake Naïla and assesses their biotechnological potential.

2. Materials and Methods

2.1. Study Site Characterization and Sample Collection

The Khenifiss lagoon, extending over 20 km in length and covering an area of 65 km², is situated along the southern Atlantic coast of Morocco, approximately 170 km north of Laayoune, within the boundaries of Khenifiss National Park [6] (Figure 1). The lagoon is connected to the Atlantic Ocean through a narrow inlet known as “Foum Agouitir”, which measures approximately 100 m in width. The lagoon’s climate falls within the Saharan bioclimatic stage, characterized by a prevailing trade wind blowing from the north–west to north–east, which drives the region’s upwelling phenomenon. Due to the region’s arid conditions, annual precipitation averages only 41 mm, and freshwater inflows are virtually absent, with the only inputs into the lagoon being oceanic. The lagoon’s water is saline, with salinity levels ranging from 35 to 41 g L⁻¹, and exhibits low phosphorus and nitrate concentrations.

Sediment samples were collected from the study site using a multibeam echosounder. Three samples were collected at a depth of 2.78 m and mixed to form one composite sample. These samples were subsequently stored at 4 °C for microbiological use.

Water and sediment physicochemical parameters, including pH, temperature, electrical conductivity (EC), dissolved oxygen (DO), turbidity, and organic matter content, were measured either in situ or under laboratory conditions using calibrated multiparameter probes and standardized analytical protocols. Electrical conductivity was recorded using a field-calibrated conductivity meter (Hanna Instruments, Woonsocket, RI, USA) and served as a proxy for ionic strength. Salinity was subsequently estimated from EC values using widely accepted empirical conversion models applicable to saline aquatic systems [13]. Dissolved oxygen was measured using electrochemical sensors following standard in situ protocols [14], while turbidity was determined using nephelometric methods. Organic matter content in sediments was quantified in the laboratory using standard loss-on-ignition procedures [15].

2.2. Isolation of Halophilic Actinobacteria

Actinobacteria were isolated from sediment samples following the method described by Ribeiro et al. [16]. Briefly, ten grams of sediment were homogenized in 90 mL of sterile saline solution (0.9% NaCl, w/v). Serial 10-fold dilutions were prepared, and 0.1 mL from each dilution was subsequently spread onto selective media, including modified Bennett agar, chitin–vitamin B agar supplemented with 30 g L⁻¹ of NaCl, and marine coral agar prepared using seawater. Nalidixic acid (100 μg/mL) and cycloheximide (50 μg/mL) were incorporated into the media to selectively inhibit the growth of Gram-negative bacteria and fungi, respectively. The inoculated plates were incubated at 28 °C for three weeks. Emergent colonies were examined under light microscopy, and selected actinobacterial isolates were purified and stored in 25% (w/v) glycerol stocks at −20 °C for further analysis.

2.3. Molecular Identification of Actinobacteria

Genomic DNA was extracted from fresh single colonies using an Invitrogen PureLink™ Genomic DNA Isolation Kit (Waltham, MA, USA) according to the manufacturer’s instructions. DNA purity and concentration were checked and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) following standard procedures. Then, 16S rDNA gene amplification was performed by polymerase chain reaction (PCR) using two universal primers: 27F and 1525R. PCR amplification was conducted in a thermocycler programmed as follows: an initial denaturation at 95 °C for 2 min, followed by 30 cycles of amplification at 95 °C for 1 min, annealing at 55 °C for 1.5 min, extension at 72 °C for 3 min, and final extension at 72 °C for 10 min. The amplified product was detected by 1% (w/v) agarose gel electrophoresis and purified using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The same primers were used for 16s rRNA sequencing (about 1.5 kb) at Macrogen Inc. (Amsterdam, The Netherlands), and the obtained sequences were searched against the EzBioCloud genomic database to assess the similarity percentage with known strains. MEGA11 software (Version 11.0.13) was used to construct the phylogenetic relationship of the seven isolated actinobacteria using the neighbor-joining method and the Kimura 2-parameter model with 1500 bootstrap replications.

2.4. Antimicrobial Activity

The antimicrobial activity of the isolated actinobacterial strains was assessed using the agar diffusion method [17]. The strains were first cultured on ISP2 agar plates for 7 days at 28 °C to promote metabolite production. Then, a calibrated agar cylinder (6 mm in diameter) was cut out and placed onto Mueller–Hinton agar plates (for bacteria) and Sabouraud agar plates (for fungi), previously seeded with the tested human and pathogenic strains: Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Klebsiella quasipneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Klebsiella aerogenes ATCC 13048, Aspergillus brasiliensis ATCC 16404 and Candida albicans ATCC 10231. Antimicrobial activity was determined by measuring the diameter of the inhibition zones around the agar plugs after 48 h of incubation at 37 °C and categorized as weak, moderate, or strong. Discs of ciprofloxacin (5 μg) and amphotericin B (10 μg) were used as positive controls against bacteria and fungi–yeasts, respectively.

2.5. Plant-Growth-Promoting Potential

2.5.1. Phosphate Solubilization

The phosphate solubilization potential of the isolated actinobacterial strains was tested using modified NBRIP broth supplemented with 5 g L⁻¹ tricalcium phosphate (Ca₃(PO₄)₂) as the inorganic phosphate source. Briefly, 250 mL flasks containing 100 mL of NBRIP were incubated with 1 mL of bacterial seed culture to achieve 10⁸ CFU/mL. The flasks were incubated on a rotary shaker at 140 rpm for 96 h. The content of solubilized P was quantified using the colorimetric methods as described by Olsen and Sommers [18]. Uninoculated culture media served as a control.

2.5.2. Auxin Production

Bacterial isolates were cultured at 28 °C for 96 h in a shaking incubator using Luria–Bertani (LB) broth supplemented with 1.02 g L⁻¹ of L-tryptophan. Indole-3-acetic acid (IAA) production was measured by mixing the culture supernatant of actinobacterial isolates with Salkowski reagent. Salkowski reagent was prepared as follows: 15 mL of H₂SO₄, 25 mL of distilled water, and 0.75 mL of FeCl₃∙6H₂O (0.5 M). In a microplate, 100 µL of cell-free supernatant was mixed with 100 µL of Salkowski reagent and incubated in the dark for 30 min. The absorbance was measured at 530 nm. The IAA concentration was calculated using a calibration curve, as described by Oubaha et al. [19].

2.5.3. Siderophore Production

The isolates were tested for their siderophore production capacity following the methodology described by [20]. Actinobacterial isolates were grown in Bennett broth for 10 days under continuous shaking at 140 rpm. After incubation, the cultures were centrifuged, and 100 µL of the supernatant was mixed with chrome azurol S (CAS)-Fe colorimetric solution. A control was prepared using the medium without inoculation. The production of siderophores was evidenced by a color transition from blue to orange. Quantification was performed by measuring the optical density (OD) at 630 nm and determining the degree of color reduction compared to the control [21].

2.5.4. Ammonia Production

Ammonia production by the isolates was assessed according to the method described by Cappuccino and Sherman [22]. One milliliter of actinobacteria culture from TSB liquid medium was inoculated into 10 mL of a peptone–water mixture (4%) and then incubated at 28 °C for 7 to 14 days. After incubation, Nessler’s reagent was added to the samples, and the formation of a yellow to brownish color was considered a positive result for ammonia production.

2.6. Fermentation, Extraction and GC-MS Analysis of Crude Extract

Based on the obtained results, isolate 43 was selected for secondary metabolite characterization by gas chromatography–mass spectrometry (GC–MS).

For metabolite production, fresh colonies were inoculated into 100 mL of liquid Bennett medium in sterile Erlenmeyer flasks and incubated at 28 °C for 10 days under continuous agitation at 120 rpm. After incubation, the cultures were centrifuged to separate the biomass from the culture supernatant. The supernatant was extracted with an equal volume of ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure using a rotary evaporator to obtain the crude extract.

GC–MS analysis was carried out using a Thermo Scientific Trace 1300 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a TR-5MS capillary column (30 m × 0.25 mm internal diameter, 0.25 µm film thickness) coupled to an ISQ single quadrupole mass spectrometer operating at 70 eV in electron ionization mode. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector and transfer line temperatures were maintained at 250 °C. The samples were injected in split mode. The oven temperature was initially set to 50 °C (held for a few minutes), increased to 150 °C at a rate of 8 °C/min, and then ramped to 300 °C at 15 °C/min with a final hold of 5 min.

Compound identification was performed by comparing the obtained mass spectra with those in the National Institute of Standards and Technology (NIST) mass spectral library.

2.7. Statistical Analysis

The 16S rRNA gene sequences were analyzed using EzBioCloud, and similarity scores were used to identify the closest known type strains. Antimicrobial activity was determined based on the size of inhibition zones, and PGP traits were quantitatively compared using descriptive statistics. Experiments were conducted in triplicate, and quantitative data were subjected to statistical analysis via analysis of variance (ANOVA). Statistical significance was assigned at p ≤ 0.05.

3. Results

3.1. Site Characterization and Molecular Identification of Actinobacteria

The physicochemical parameter analysis revealed an aquatic environment with a neutral to slightly alkaline pH (7.80 ± 0.07) and moderate temperature (21.50 ± 0.33 °C). The high electrical conductivity (42.83 ± 0.38 mS/cm) was associated with elevated salinity (36.37 ± 0.78 g/L). The dissolved oxygen value was 7.03 ± 0.16 mg/L, while turbidity was around 9.67 ± 0.44 NTU. For the sediments, salinity was 30.43 ± 0.96 g/kg, with an electrical conductivity of 39.3 ± 0.85 mS/cm and a 1.13 ± 0.15% organic matter content (

Table 1. Physicochemical characteristics of sediments and water collected from the studied site.

	Water Sample		Sediment Sample
	Mean	SD	Mean	SD
pH	7.8	0.07	-	-
Salinity g/L	36.37	0.78	30.43	0.96
T °C	21.5	0.33	-	-
Electrical conductivity (mS/cm)	42.83	0.38	39.3	0.85
Dissolved oxygen (DO) (mg/L)	7.03	0.16	-	-
Turbidity (NTU)	9.67	0.44	-	-
Organic matter content (%)	-	-	1.13	0.15

SD: Standard Deviation.

).

Seven isolates were successfully recovered from composite sediment samples (n = 3) of the saline sebkha lagoon using three different selective media, revealing diverse colony morphologies and suggesting the presence of diverse actinobacterial species. Sequencing of the 16S rRNA gene revealed that the isolates belonged to known actinobacterial species, particularly within the genera Streptomyces, Nocardiopsis and Prauserella. As shown in

Table 2. The 16S rDNA sequence similarity of bacterial strains to closest type strains.

				Molecular Characteristics
Site of Isolation	Isolate Code	Medium of Isolation	Accession Number	Number of bp	Similarity %	Closest Type Strain
Lac Naïla	39	Marine coral agar	PQ838283	1460	99.65	Streptomyces marokkonensis Ap1(T)
	40	Marine coral agar	PQ838284	1482	100.00	Streptomyces silvae For3(T)
	41	Modified Bennett agar	PQ838285	1481	99.93	Streptomyces thermocarboxydus DSM 44293(T)
	42	Chitin–vitamin B agar	PQ838286	1418	100.00	Nocardiopsis dassonvillei DSM 43111(T)
	43	Chitin–vitamin B agar	PQ838287	1471	99.79	Streptomyces mutabilis NBRC 12800(T)
	44	Modified Bennett agar	PQ838288	1497	99.79	Prauserella isguenensis H255(T)
	48	Modified Bennett agar	PQ838289	1470	99.65	Streptomyces marokkonensis Ap1(T)

and Figure 2, the 16S rRNA genetic similarity of the isolates to reference strains ranged from 99.65% to 100%. Strains 39 and 48 exhibited 99.65% similarity to Streptomyces marokkonensis, while strains 43 and 44 exhibited 99.73% similarity to Streptomyces mutabilis and Prauserella isguenensis, respectively. Strain 41 displayed 99.93% similarity to Streptomyces thermocarboxydus, whereas strains 40 and 42 were identical to Streptomyces silvae and Nocardiopsis dassonvillei subsp. dassonvillei (100% similarity), respectively. These high similarity values suggest that the isolated strains are closely related to previously characterized species.

3.2. Antimicrobial Activity of Actinobacterial Isolates

The results showed significant variation in the antimicrobial activities among the different strains. Streptomyces sp. strain 43 exhibited the broadest spectrum of activity, effectively inhibiting both Gram-positive and Gram-negative bacteria. This strain showed potent antimicrobial effects, with inhibition zones reaching up to 20 mm in diameter against E. faecalis ATCC 29212, B. subtilis ATCC 6633, S. aureus ATCC 25923, and E. aerogenes. This finding suggests that Streptomyces sp. strain 43 produces highly effective antimicrobial compounds capable of targeting a diverse range of bacterial species. Similarly, Streptomyces sp. strain 39 and Streptomyces sp. strain 48 also demonstrated significant antimicrobial activity, particularly against Gram-positive pathogens such as B. subtilis, S. aureus, and E. faecalis. Both strains displayed inhibition zones with a maximum size of 20 mm, indicating strong bacteriostatic effects; however, these did not exhibit activity against Gram-negative pathogens like K. pneumoniae and P. aeruginosa.

In contrast, other strains, such as Streptomyces spp. strains 40 and 41, Prauserella sp. strain 44, and Nocardiopsis sp. strain 42 exhibited little to no detectable antimicrobial activity against the tested pathogens, as indicated by the absence of significant bacteriostatic rings in Figure 3. This may suggest that these strains either do not produce antimicrobial compounds or their activity is limited to specific conditions not reproduced in the assay to exhibit activity.

The antimicrobial activity results revealed a clear contrast among the isolates, highlighting the particularly promising bioactive potential of some strains (Figure 3). Of all the isolates evaluated, only isolates 39, 43, and 48 showed inhibitory activity, whereas isolates 40, 41, 42, and 44 were completely inactive against the test microorganisms. Isolate 39 exhibited activity mainly against Gram-positive bacteria, with marked inhibition of S. aureus (23 mm), whereas isolates 43 and 48 were distinguished by a broader and more attractive spectrum of activity compared with standard antibiotics. Indeed, isolate 43 inhibited both Gram-positive bacteria, namely, E. coli and K. aerogenes, and the fungus A. brasiliensis, while isolate 48 showed the strongest antibacterial activity against S. aureus (24 mm) and simultaneous antifungal activity against A. brasiliensis and C. albicans. Taken together, these results highlight the promising potential of isolates 39, 43, and 48 as producers of broad-spectrum antimicrobial metabolites, fully justifying their selection for further investigations aimed at identifying the bioactive compounds they produce. The phylogenetic tree (Figure 2) reveals that isolates 39 and 48 were closely related, supported by high bootstrap values, whereas isolate 43 was positioned in a distinct lineage, indicating that antimicrobial activity is not strictly associated with close phylogenetic relatedness among the strains.

3.3. Phosphate Solubilization

The phosphate solubilization capacity of the seven isolated actinobacterial strains (39, 40, 41, 42, 43, 44, and 48) was evaluated by measuring their ability to dissolve tricalcium phosphates. The solubilization capacity varied significantly among the strains, with values ranging from approximately 2 mg/L to 23 mg/L (Figure 4). Streptomyces sp. strains 39, 43, and 48 exhibited the highest phosphate solubilization activity, with values exceeding 20 mg/L, with no statistically significant differences among these three strains. In contrast, strain 40 demonstrated the lowest solubilization capacity, with values below 3 mg/L. Intermediate phosphate solubilization was observed for Streptomyces sp. strain 41, Prauserella sp. strain 44 and Nocardiopsis sp. strain 42.

3.4. IAA Production

The quantification of IAA production by the different bacterial strains, as presented in Figure 5, demonstrated significant variation in auxin production among the tested strains. Nocardiopsis sp. strain 42 produced the highest amount of IAA (282 ± 20.5 μg/mL), which was significantly greater (p < 0.05) than the other strains. This level of production was nearly threefold higher than that of the next highest producers, Streptomyces sp. strain 41 (78 ± 10.3 μg/mL) and Streptomyces sp. strain 40 (69 ± 9.8 μg/mL). In contrast, strain 39 (14 ± 3.2 μg/mL) and Streptomyces sp. strain 48 (8 ± 2.1 μg/mL) produced the lowest levels of IAA. Intermediate IAA production was observed in Streptomyces sp. strain 43 and Prauserella sp. strain 44, with values of approximately 62 and 55 μg/mL, respectively. The significant variation in IAA production among the strains suggests differences in auxin biosynthesis capabilities, which may contribute to distinct plant-growth-promoting effects.

3.5. Siderophore Production

A significant difference in siderophore production was observed among the strains (Figure 6). Streptomyces sp. strain 41 showed the highest production recorded (83 ± 5.2%). This strain produced approximately twice the amount of siderophore compared to the next-highest producer, Nocardiopsis sp. strain 42 (45 ± 4.7%). In contrast, Prauserella sp. strain 44 exhibited the lowest siderophore production (6 ± 1.9%), significantly lower than all other strains. Streptomyces sp. strain 43 (36 ± 3.8%) and Streptomyces sp. strain 40 (25 ± 4.1%) showed moderate siderophore production. This variation suggests differences in the iron-chelating abilities of the bacterial strains, which may influence their potential for enhancing plant growth under iron-limited conditions.

3.6. Ammonia Production

The results revealed significant variability in ammonia production capacity among the studied isolates. Isolates 40 and 44 exhibited low production (+), suggesting limited metabolic activity, while isolates 39 and 41 showed moderate production (++). In contrast, isolates 42, 43, and 48 were characterized by high production (+++), indicating a strong capacity for ammonia generation, which could guide their selection based on nitrogen enrichment objectives (Figure 3).

3.7. Chemical Composition of Crude Extract

The analysis of the chromatogram obtained by GC-MS of the crude extract of strain 43 revealed the presence of more than one hundred compounds. Comparison of the different peaks with the NIST and WILEY databases allowed the identification of two major compounds, namely, 1,2-propanediol and nonanal, exhibiting retention times of 27.45 min and 27.68 min, respectively (Figure 7).

4. Discussion

The successful isolation and identification of actinobacteria from the sebkha lagoon underscore the potential of saline environments as reservoirs of microbial diversity. The genetic identification of isolates closely related to Streptomyces, Prauserella, and Nocardiopsis species suggests that these bacteria are well adapted to the extreme conditions of the sebkha, where salinity and nutrient limitations create a strong selective pressure for microbial survival. Several previous studies [23,24] have reported the presence of actinobacteria affiliated with these rare genera in hypersaline environments, such as marine salterns, suggesting the tolerance of these taxa to high-salinity conditions similar to those observed in the sebkha lagoon. For example, the genus Prauserella comprises 14 species, including six newly identified strains isolated from hypersaline habitats such as soil and marine sediments. Among them are Prauserella halophila and Prauserella marina, respectively [25,26]. In addition, four other novel Prauserella strains were isolated from a saline lake in China and named Prauserella salsuginis, Prauserella flava, Prauserella aidingensis and Prauserella sediminis [27]. The first report on the genus Prauserella in Morocco was published by Nafis et al. [4]. In their study, they isolated rare actinomycetes from mining soils.

Furthermore, 23 halophilic species among the 45 currently valid species of the genus Nocardiopsis have been isolated from saline ecosystems [28], including Nocardiopsis mwathae, which was recovered from the haloalkaline Lake Elmenteita in the African Rift Valley, Kenya [29]. Zhang et al. [30] described two additional species, Nocardiopsis ganjiahuensis and Nocardiopsis fragmentaris, isolated from the alkaline soils of a Ganjia Lake in Xinjiang Province, China. To date, this study represents the first report of the genus Nocardiopsis in Morocco.

Concerning the genus Streptomyces, one of the most abundant bacterial genera in nature, approximately ten studies have investigated actinobacteria in hypersaline niches. Akhwale et al. [31] identified a novel strain, named Streptomyces alkaliphilus, from sediments of an alkaline saline lake in Kenya. In addition, Turkish saline sediments have revealed several Streptomyces species, including Streptomyces iconiensis, Streptomyces seymenliensis, and Streptomyces smyrnaeus [32,33].

The antimicrobial activity exhibited by several isolates, particularly against S. aureus, underscores their biotechnological potential. For instance, strain 43, affiliated with Streptomyces mutabilis, demonstrated significant inhibitory activity, suggesting the production of potent antimicrobial compounds with potential applications against antibiotic-resistant pathogens. These results are consistent with previous studies reporting the antagonistic ability of actinobacterial strains isolated from hypersaline environments [34,35,36]. Hamed et al. [31] extracted a novel bioactive compound, N-Acetylborrelidin B (among seven more metabolites), from Streptomyces mutabilis collected from Red Sea sediments in Egypt, which showed antimicrobial activity against similar pathogens. These findings are particularly relevant in the context of increasing global antibiotic resistance.

The absence of activity against Gram-negative bacteria such as K. pneumoniae and P. aeruginosa suggests that the antimicrobial compounds produced are more effective against Gram-positive bacteria, a pattern commonly observed in actinobacterial secondary metabolites [37,38,39].

In addition to their antimicrobial activity, the actinobacterial isolates demonstrated promising PGP traits. Inorganic phosphate solubilization is a key mechanism by which microorganisms enhance plant growth [40,41]. In our study, all seven actinobacterial isolates exhibited phosphate solubilization activity, with strains 39 and 43 belonging to S. marokkonensis and strain 48 belonging to S. mutabilis, showing the highest levels of tricalcium phosphate solubilization. These findings are consistent with previous reports highlighting the phosphate solubilization potential of Streptomyces mutabilis isolated from various environments [42]. Notably, this study presents the first evidence of phosphate solubilization by Streptomyces marokkonensis, which may act as a valuable tool for enhancing plant growth.

IAA, a key plant growth regulator, plays a major role in nutrient uptake and plant stress tolerance [43,44]. Actinobacteria-derived auxins are well known for their plant-growth-promoting effects [45,46]. However, salinity stress may inhibit IAA synthesis in some cases [47,48]. In this study, all actinobacterial strains produced IAA when supplemented with the precursor molecule L-tryptophane. Among them, strain 42 (Nocardiopsis dassonvillei) exhibited the highest IAA production (246.48 µg ml⁻¹), significantly surpassing the other isolates. This result is consistent with previous studies, where Nocardiopsis was reported to produce IAA in the range of 62.23–222.75 µg ml⁻¹ [49,50]. Furthermore, other genera of actinobacteria, such as Streptomyces, Prauserella, and Nocardiopsis, have been shown to produce IAA in vitro and in silico when supplemented with L-tryptophan [50,51].

Iron acquisition is essential for plant growth but is limited due to the low solubility of Fe³⁺ in soils. Siderophores produced by microorganisms enhance iron availability to plants [52,53]. Plant-associated bacteria, including actinobacteria, are known to produce siderophores that enhance iron availability for plant uptake. In this study, all isolates belonging to three genera—Streptomyces, Prauserella, and Nocardiopsis—produced siderophores, with strain 41 (Streptomyces thermocarboxydus) exhibiting the highest production. This finding aligns with the literature, which describes siderophore production in actinobacteria, particularly in Streptomyces and Amycolatopsis species, isolated from hypersaline environments [54,55].

Ammonia production constitutes an alternative strategy for plant growth promotion, involving the conversion of atmospheric nitrogen (N₂) into ammonia (NH₃). Numerous actinobacteria are known to possess diverse plant-beneficial properties, notably nitrogen fixation. In particular, species of Frankia and Streptomyces have been widely investigated for their capacity to fix atmospheric nitrogen and enhance its availability to host plants [56]. Beyond these well-characterized genera, other actinobacteria have also demonstrated a strong potential for nitrogen-related plant growth promotion through ammonia release. For instance, Nocardiopsis dassonvillei strain YM12, isolated from coastal agricultural land of Khambhat, India, exhibited a relatively high ammonia production level of 7.2 µmol ml⁻¹, highlighting its effective nitrogen-assimilating metabolism [50]. Such ammonia production can directly contribute to plant nitrogen nutrition, particularly in nutrient-poor or saline environments, thereby reinforcing the role of diverse actinobacterial taxa as key contributors to sustainable plant growth promotion.

These results further emphasize the potential of these actinobacterial isolates in enhancing plant growth, particularly under iron-limited conditions. Until now, no study has been conducted to evaluate the antimicrobial activity and PGP potential of halophilic species of the genus Prauserella, except our study, for Prauserella isguenensis strain 44. In this study, Nafis et al. [4] found that Prauserella NDS-4 exhibited very notable PGP activity.

Nocardiopsis species have an incredible ability to adapt to diverse environments. This resilience is explained by their flexible genome, ability to produce specific enzymes, and capacity to synthesize compatible solutes and surfactants [57]. The literature reports that many molecules have been characterized from extracts of halophilic Nocardiopsis strains. Chemical analysis of molecules purified from extracts of the haloalkaliphilic strain Nocardiopsis sp. AJ1, isolated from the saline soil of the Kovalam salt marshes in India, showed the existence of pyrrolo(1,2-A)(pyrazine-1,4-dione, hexahydro-3-[2-methylpropyl]-) and actinomycin C2 [58]. In addition, angucyclines and angucyclinones produced by Nocardiopsis sp. HR-4, isolated from the soil of a salt lake in the Algerian Sahara, were effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 [59]. Another study characterized two isomers, borrelidines C and D, produced by Nocardiopsis sp. HYJ128, which was isolated from a topsoil saltern in the Republic of Korea. Both molecules showed antimicrobial activity against Salmonella enterica ATCC 14028 [59]. Moreover, Nocardiopsis is one of the main genera of actinobacteria whose in vitro antifungal activity has been investigated to control plant pest fungi [60]. For example, Nocardiopsis dassonvillei MB22 and Nocardiopsis sp., isolated from soil, were used to control Bipolaris sorokiniana in wheat and Fusarium sp., respectively [58,61], and Nocardiopsis alba, isolated from the air, was used to control Ganoderma boninense [62]. Likewise, Nocardiopsis baichengensis was able to produce new siderophores, nobachelins, which protect Caenorhabditis elegans from Pseudomonas aeruginosa infection [63]. Moreover, new Nocobactin derivatives, Terpenibactins A–C, with antimuscarinic activity were detected through genome mining analysis for Nocardia terpenica IFM 0406 [64].

GC-MS analysis of the crude extract of our isolate Streptomyces mutabilis 43 revealed a complex metabolic profile, with approximately one hundred compounds detected. This finding is consistent with the extensive metabolic potential of the genus Streptomyces, recognized as one of the richest bacterial producers of secondary metabolites, including antibiotics, antifungals, antitumor compounds, and other bioactive small molecules; over two-thirds of known naturally derived antibiotics originate from this genus [65].

Among these compounds, two metabolites were reliably identified: 1,2-propanediol (RT = 27.45 min) and nonanal (RT = 27.68 min). 1,2-propanediol, also known as propylene glycol, is a three-carbon diol produced by various microorganisms, often via alternative fermentation pathways or through the reduction of glycolytic derivatives such as methylglyoxal [66]. Although this molecule is more commonly studied in species such as Lactobacillus and Clostridium and metabolically engineered strains (E. coli, Corynebacterium glutamicum), these studies demonstrate that natural or artificial microbial pathways can synthesize this diol from sugars or lactate through specific enzymatic steps [67]. Its presence in S. mutabilis may therefore indicate the activation, possibly at a low level, of a flexible metabolic pathway linked to pyruvate or methylglyoxal metabolism, known in other bacteria as a precursor of this diol [66].

Nonanal is a nine-carbon volatile aldehyde frequently detected in bacterial and fungal extracts, contributing to biological volatile profiles. Although it is not commonly reported as a major bioactive metabolite in the classical Streptomyces literature, it has been identified in various microbial and plant extracts and can contribute to aromatic properties as well as exhibit mild antimicrobial or antifungal effects depending on the context [68]. Its detection in S. mutabilis NBRC 12800T illustrates this strain’s ability to produce volatile secondary metabolites potentially involved in microbial communication or defense [69]. Overall, these results highlight the potential of S. mutabilis NBRC 12800T to produce bioactive small molecules, suggesting possible applications in biotechnology or biocontrol.

Concerning the metabolites produced by members of the genus Streptomyces, although only a limited number of strains have been isolated from hypersaline environments, research on the bioactive molecules produced by these microorganisms remains relatively scarce. Among the molecules identified, a quinone-related antibiotic was produced by Streptomyces chibaensis AUBN1/7, isolated from marine sediments collected from the Bay of Bengal, India [70]. Furthermore, the strain Streptomyces sp. B692, from sandy sediments of a coastal site in Mauritius (Indian Ocean), was identified as a producer of two new anthracycline antibiotics, himalomycin A and B [71]. Likewise, Chen et al. [72] report that genome analysis of S. marokkonensis M10, isolated from marine sediments collected in Dalian, China, revealed that more than 26 gene clusters are putatively associated with the biosynthesis of multiple secondary metabolites, including antibiotics and siderophores.

Overall, these findings highlight the strong potential of actinobacteria from sebkha environments for applications in sustainable agriculture and biocontrol. Future studies should focus on purification of bioactive compounds, genome mining for biosynthetic gene clusters, and evaluation of their agricultural and pharmaceutical applications.

5. Conclusions

The sebkha of Lake Naïla harbors an unexplored diversity of halophilic actinobacteria, mainly from the genera Streptomyces, Nocardiopsis, and Prauserella. Some strains, notably Streptomyces mutabilis 43, exhibit strong antimicrobial potential and produce diverse secondary metabolites, while others display plant-growth-promoting traits, including phosphate solubilization, auxin, siderophore, and ammonia production. The findings presented here provide important insights into the untapped microbial biodiversity of Moroccan saline ecosystems and underscore the potential of Lake Naïla as a saline environment reservoir for the discovery of new bioactive compounds. The biological and habitat diversity of Lake Naïla, combined with its status as a largely unexplored site in terms of actinobacteria diversity and associated biotechnological potential, makes this North African sebkha a promising location for bioprospecting studies.