IMR Press / FBE / Volume 14 / Issue 2 / DOI: 10.31083/j.fbe1402011
Open Access Original Research
Culturomics remains a highly valuable methodology to obtain rare microbial diversity with putative biotechnological potential from two Portuguese salterns
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1 Department of Biology, Faculdade de Ciências da Universidade do Porto (FCUP), Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
2 Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
3 Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
*Correspondence: (Eduarda Almeida)
Academic Editor: Yiannis Kourkoutas
Front. Biosci. (Elite Ed) 2022 , 14(2), 11;
Submitted: 30 November 2021 | Revised: 13 January 2022 | Accepted: 18 January 2022 | Published: 8 April 2022
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.

Background: The high salt concentration is the major factor limiting microbial growth at salterns, along with solar radiation, temperature, and pH. These environmental factors play key roles in the acquisition of unique genetic adaptations for the survival of microorganisms in salterns, which can result in the production of interesting secondary metabolites. The main goal of the present work was to isolate and compare the culturable microbiota from two geographically distant salterns in Portugal and access their biotechnological potential. Methods: Culturomics approaches using different culture media were applied for microbial isolation. All isolates were identified either by 16S rRNA or ITS genes sequencing, and their biotechonological potential was assessed by PCR. Results: Overall, 154 microbial isolates were recovered that were phylogenetically assigned to 45 taxa from 9 different phyla. From these, 26 isolates may represent putative new taxa. The predominant genera obtained were Penicillium (41 isolates, 26.6%), Streptomyces (13 isolates, 8.4%) and Sinomicrobium (11 isolates, 7.1%). Moreover, the polyketide synthase I gene was present in 64 isolates, the nonribosomal peptide synthethase gene in 16 isolates, and both genes in 23 isolates. Conclusions: This study adds up valuable knowledge on the culturable microbiota of Portuguese salterns and on its potential for production of secondary metabolites. In the long run, this study provides a widely diverse microbial collection for future works. Data public repository: All DNA sequences were deposited in the GenBank database at National Centre for Biotechnology Information (NCBI) web platform under accession numbers OK169439-OK169485, OK216020-OK216124, OK287059 and OK326927.

microbial isolation
microbial diversity
bioactive potential
molecular screening
polyketide synthase I
nonribosomal peptide synthethase
1. Introduction

Portugal has an extensive coastline harbouring a wide range of different environments, including solar salterns. In Portugal, the production of salt in traditional salterns, especially in the North, takes place in the summer season. During salt production, saltern waters are hypersaline environments characterized by high concentrations of NaCl, UV radiation, temperature and pH. These factors are determinant in biodiversity modulation [1], narrowing the microbial community to well adapted halophilic (extremophiles) or halotolerant microorganisms. The microbial diversity of hypersaline environments has been targeted by the scientific community along the last decade [1, 2, 3, 4, 5, 6, 7]. The abundance of microorganisms from different phyla was acknowledged on this type of extreme environment, comprising members of Euryarchaeota, Planctomycetota, Bacteroidota, Rhodothermota (previously included in Bacteroidota [8]), Pseudomonadota, Actinomycetota and Cyanobacteria [2, 3], Bacillota [7, 9], Gemmatimonadota [6] and Eukaryota as microalgae [10] and Ascomycota [11, 12].

The ability of microorganisms to produce natural products (NPs) with relevant biotechnological value is well recognised [13] and NPs obtained from extremophiles have proved their biotechnological value in a wide range of fields [14]. As extremophiles are often exposed to sudden and repeated fluctuations derived from global changes, such as temperature and water availability, they require a great physiological adaptability at different cellular levels, namely in their biological membranes, proteins and extracellular metabolites [14]. Due to these metabolic adaptations, the unexploited microbiota of salterns presents a high potential for novel NP discovery with applications in important biotechnological fields, such as medicine, pharmaceutics, cosmetics, agriculture, and the food industry [15, 16, 17, 18].

One of the currently applied molecular methodologies to assess the potential of microorganisms to produce bioactive molecules consists in Polymerase Chain Reaction (PCR) protocols. Genes of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are commonly targeted as a preliminary screen of bioactive properties, since they are well known to be responsible for the production of enzymatic complexes that are involved in the synthesis of NPs. Specifically, NRPSs are extensively associated with the production of structurally diverse peptides presenting a wide range of activities, like antibacterial, antitumour, cytostatic or immunosuppressive [19], while PKSs are engaged in the assembly of distinct NPs – the polyketides. This class of NPs includes many biotechnologically important compounds such as antibiotics, antiparasitics, anticancer, immunomodulators, antifungals and anticholesteremics [20]. In fact, the annual sales of medicines derived from polyketides had already reached 20 billion US dollars [21].

The microbial diversity of Portuguese salterns has been poorly studied. Few studies were obtained by culture independent approaches [22, 23], and a few more through culture dependent methods [24, 25, 26]. The present study targeted the culturable microbial diversity, in late summer of 2018, of two Portuguese salterns, Aveiro and Olhão salterns. These have different geographical locations: Aveiro is situated in the North, being influenced by the Atlantic Ocean, while the Olhão saltern is in the South, and is exposed to the influence of not only the Atlantic Ocean but also the Mediterranean Sea. Average temperature and pluviosity levels per year are also different at both locations: 15.6 C and 1064 mm in Aveiro and 18.2 C and 482 mm in Olhão [27]. The main goal of the present study was to assess the microbial diversity of both salterns, during salt production season, through a broad range of microbial isolation and subsequent phylogenetic analysis. The present study also aimed at isolates biotechnological prospection by molecular screening for NRPS and PKS-I genes. This work provides additional insights about the microbiota of Portuguese salterns and about its potential for NPs production. Moreover, this study contributes with a wide and diverse microbial collection for further prospections.

2. Material and methods
2.1 Sampling and microbial isolation

Fresh and wet salt, sediment from under the salt and water, all sampled directly from salt production ponds, were collected from Aveiro saltern (40º38’50” N, 8º39’46” W) in September 2018 and from Olhão (371’35” N, 751’59” W) in August 2018, during salt production season. All samples were used for microbial isolation, aiming at obtaining members from different phyla, applying, therefore, different methodologies represented in Fig. 1 and specified below.

Fig. 1.

Representation of the sampling locations along with the overall culturomics methodologies applied.

2.1.1 Planctomycetota

Media M600, M607 [28] and M607SW (this study) (Supplementary Table 1), supplemented with 200 μg/mL ampicillin (Batch: 1R001084; AppliChem, Darmstadt, Germany), 1 mg/mL streptomycin (Batch: 6H015348; AppliChem) and 50 μg/mL cycloheximide (Batch: 4C016643; AppliChem/ Panreac, Darmstadt, Germany), were used to target the isolation of Planctomycetota phylum members. The antibiotic supplementation was used because Planctomycetota known members present resistance to these drugs [28], and cycloheximide presents antifungal activity.

Salt samples were dissolved in autoclave-sterilized seawater (SW) until saturation and serially diluted until 10-3 in sterile SW. All SW used in this study was collected from Estação de Zoologia Marítima “Dr. Augusto Nobre” at Molhe beach, Porto, Portugal (N: 419’49.981”; W: 841’11.820”). Both, initial suspension and dilutions, were individually used as inoculum in the different isolation media. The wet salt samples, at the bottom, accumulated a brown residual liquid, which was directly plated in the isolation media and serially diluted in sterile SW until 10-3, which were also individually plated in the isolation media.

Concerning sediment samples, 1 g of each saltern sediment was individually suspended in 1 mL of sterile SW by vigorously vortexing. From this suspension, serial dilutions until 10-3 were made. Both, initial suspension and dilutions, were individually used as inoculum in the different isolation media. The saltern water samples were directly plated in the isolation media and serially diluted in sterile SW until 10-3, which were also plated in the isolation media.

Agar media were used for direct isolation, while broth versions of the media were used for enrichments. Specifically, enrichments were carried out in 24-well plates containing 900 μL of broth isolation media per well. Enrichments were carried out by using not only the raw samples, as in the case of the water and the brown residual liquid, but also the initial suspensions of salt and sediment. In all cases, 100 μL of each sample were individually used as initial inoculum and then serially diluted in the corresponding broth medium until 10-3. All broth conditions for each sample concentration were carried out in triplicate. The inoculated 24-well plates were incubated at room temperature (RT) at 120 rpm, while inoculated agar media plates were incubated at 26 C. When visible growth was observed in the wells, 100 μL of each culture suspension were transferred to the corresponding agar medium and incubated at 26 C until axenic cultures were achieved.

2.1.2 Actinomycetota

Agar media regularly used for Actinomycetota isolation were selected, namely R2A-Agar (BD Difco™, Maryland, USA), Starch-Casein-Nitrate agar (SCN; adapted from Küster and Williams [29]), M3 [30] and a modified Nutrient-Poor Sediment extract agar (NPS; adapted from Jensen et al. [31]) (Supplementary Table 1). All agar media were supplemented with 50 mg/L cycloheximide (Batch: 9I011709; Sigma-Aldrich, Missouri, United States), 50 mg/L nalidixic acid (Batch: 8D012744; AppliChem) and 50 mg/L nystatin (Batch: 9J012880; Sigma-Aldrich).

Salt samples were dissolved in 5 mL of sterile SW until saturation, while 1 g of each sediment sample was suspended in 2 mL of sterile SW by shaking at 200 rpm for 30 min and by vortexing at maximum speed for 5 min. Two mL of the salt brown residual liquid and of the water samples were directly used as inoculum. All these samples were exposed to a heat pre-treatment at 60 C for 30 min to select for Actinomycetota [32, 33, 34]. Pre-heated samples were serially diluted until 10-4 in sterile SW, and all samples, either diluted or not, were individually plated on the above-mentioned isolation agar media (Supplementary Table 1).

Incubation of samples with calcium carbonate (CaCO3) was previously reported as a strategy that promotes the selection of Actinomycetota in isolation events [35]. So, this strategy was applied in all saltern samples. Specifically, (i) 1 g of sterile CaCO3 was macerated with 2 g of each salt sample and 6 mL of sterile SW were added to help on the maceration process; (ii) 1 g of sterile CaCO3 was added to 6 mL of each salt brown residual liquid sample; (iii) 1 g of sterile CaCO3 was macerated with 2 g of each sediment sample and 6 mL of sterile SW were added to help on the maceration procedure; and (iv) 1 g of sterile CaCO3 was added to 6 mL of each saltern water sample. All these suspensions with CaCO3 were incubated at 28 C for 5 weeks and then were serially diluted until 10-4 in sterile SW. All the pre-treated samples, either diluted or not, were individually plated in the isolation agar media (Supplementary Table 1) and incubated at 28 C.

2.1.3 Fungi

Potato Dextrose Agar (PDA; BD Difco™) is widely used for Fungi cultivation and was the medium selected for the present study, which was prepared with SW. All samples, namely salt, salt brown residual liquid, sediment and water, as well as the serial dilutions of these samples were prepared as described above for isolation of Planctomycetota. All these suspensions, either diluted or not, were individually plated in PDA.

In all methodologies previously described, bacterial colonies with different morphologies were restreaked until the achievement of axenic cultures. All pure isolates were cryopreserved in 24% glycerol at –80 C.

2.2 Microbial DNA extration

Each axenic isolate was cultivated in the corresponding broth medium of isolation and these cultures were used for DNA extraction. For isolates obtained in PDA medium, NZY Plant/Fungi gDNA Isolation kit (NZYTech, Lisbon, Portugal) was used, according to the manufacturer’s instructions. In the case of the isolates obtained in the remaining media, DNA was extracted using the E.Z.N.A. Bacterial DNA kit (Omega Bio-Tek, Norcross, Georgia, USA), according to the manufacturer’s instructions. DNA quantification was done by using the μDropTM platform (Thermo Fisher Scientific, Massachusetts, USA) and DNA quality was checked through a 30 min electrophoresis at 100 V in a 0.8% agarose gel with 1X Tris-acetate-EDTA (TAE) buffer (Bio-Rad, Hercules, California, USA) and stained with GreenSafe Premium (NZYTech).

2.3 Microbial identification

Bacterial DNA was PCR-amplified with the universal primers 27F and 1492R [36], while Fungi DNA was PCR-amplified by using ITS1 and ITS4 primers [37].

For the isolates which strain designation starts with “C.”, the 16S rRNA gene primers, 27F and 1492R, were used and the PCR mixture consisted of 1 × Qiagen Multiplex PCR Master Mix (Qiagen, California, USA), 0.1 μM each primer and 25 ng of DNA template, in a final volume of 10 μL. The PCR reaction was carried out on the Veriti® Thermal Cycler (Applied Biosystems, Massachusetts, USA) using the following PCR program: initial denaturation for 5 min at 95 C, followed by 30 cycles of denaturation at 95 C for 45 s, annealing at 56 C for 45 s, and extension at 72 C for 1 min, and a final extension of 10 min at 72 C. PCR products were visualized through electrophoresis in a 0.8% agarose gel with 1 × TAE buffer, which was stained with SYBR Safe (Thermo Fisher Scientific). Purification and sequencing of these amplicons was carried at i3S (Porto, Portugal).

For the remaining isolates, including Fungi, the PCR mixture (25 μL) consisted of 1 × NZYTaq 2 × Green Master Mix (NZYTech), 0.1 μM each primer and 25 ng of DNA template. PCR program was carried out in a MyCycler™ Thermo Cycler (Bio-Rad) and, for 16S rRNA gene amplification, consisted in an initial denaturation step of 5 min at 95 C, followed by 30 cycles of 1 min at 95 C, 1 min at 56 C (annealing temperature) and 1.5 min at 72 C, and a final extension of 10 min at 72 C. For amplification of the ITS region, the PCR program was the same as previously described, with exception that an annealing temperature of 55 C was applied. PCR products were visualized through electrophoresis in a 0.8% agarose gel with 1 × TAE buffer, which was stained with GreenSafe Premium. These amplicons were purified using GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, United Kingdom) and sequenced at GATC Biotech (Constance, Germany).

Sequence analyses were carried out using Geneious Prime (Biomatters Ltd, Auckland, New Zealand). Curated sequences were matched in the EzBioCloud [38] database to determine their closest relatives. Phylogenetic trees were built using MEGA 7 (Pennsylvania State University, Pennsylvania, USA) software [39]. Briefly, all sequences, either from the isolates in this study and from the closest related strains determined on EzBioCloud, were grouped by phyla. Separately, phyla-grouped sequences were aligned using the ClustalW algorithm with a Gap Open Penality of 15.00 and a Gap Extension Penalty of 6.66. The resulting multiple alignments for each phylum were used to construct phylogenetic trees, by applying the Maximum Likelihood statistical method, the phylogeny test based on the Bootstrap method considering 1000 replicates, and the Tamura-Nei substitution model. Different strains were used as outgroup depending on the phyla.

All sequences obtained on this study were submitted to the GenBank database [40] at National Centre for Biotechnology Information (NCBI) web platform under the following accession numbers: OK169439-OK169485, OK216020-OK216124, OK287059 and OK326927.

2.4 Diversity, richness, dominance and distribution of salterns microbial communities

PAST 3.22 (University of Oslo, Oslo, Norway) [41] software was used to calculate the Fisher’s α, Margalef’s and Simpson’s indexes, which allow the characterization of the microbial community in terms of diversity, richness, and evenness, respectively.

A sample rarefaction curve was estimated in PAST 3.22 by using the Mao’s Tau index [42]. Similarities among samples were calculated on PAST 3.22 by using the Sørensen coefficient and Bray-Curtis index. All results were computed taking into account a 95% confidence and 1000 iterations as bootstrap value.

2.5 PCR screening of the bioactive potential of saltern isolates

The putative potential of all isolates obtained in this study to produce NPs was assessed through PCR. Briefly, isolates DNA was screened for the presence of NRPS and PKS-I genes. NRPS genes were amplified using primers MTF2 [5- GCNGG(C/T)GG(C/T)GCNTA(C/T)GTNCC-3 (AGGAYVP, core motif I)] and MTR [5-CCNCG(AGT)AT(TC)TTNAC(T/C)TG-3 (QVKIRG, core motif V)] [43], while β-ketosynthase (KS) domain fragments within the Type I polyketide synthase genes (PKS-I) was amplified using primers MDPQQRf (5-RTRGAYCCNCAGCAICG-3) and HGTGTr (5-VGTNCCNGTGCCRTG-3) [44]. PCR reactions of 25 μL containing 1 × NZYTaq 2 × Green Master Mix, 0.8 μM of each primer and 25 ng DNA template were prepared. The PCR protocol was carried out in a MyCycler™ Thermo Cycler and consisted of an initial denaturation at 95 C for 5 min, 11 cycles of denaturation at 95 C for 1 min, annealing at 60 C for 30 s and extension at 72 C for 1 min, with the annealing temperature being reduced 2 C every cycle; followed by 30 cycles of denaturation at 95 C for 1 min, annealing at 40 C for 30 s and extension at 72 C for 1 min, and a final extension of 10 min at 72 C [45]. Amplicons were visualized in a 1.2% agarose gel prepared with 1 × TAE buffer and stained with GreenSafe Premium. NRPS and PKS amplicons have an expected size of approximately 1000 bp [43] and 700 bp [44], respectively. The Planctomycete UC49.1 was used as positive control for amplification of both genes.

3. Results and discussion
3.1 Microbial isolation and identification

Aiming at a broader range of microbial isolation, 6 media with different nutritional compositions along with 2 pre-formulated media (Supplementary Table 1) were assayed. A medium selective for fungi, PDA, allowed the isolation of 45 fungal strains. The most effective media for bacteria were M600 (32 strains), R2A (32 strains) and M607 (31 strains). The remaining media used were less rich in nutrients and apparently less successful (14 strains altogether in 4 different media). Ultimately, the media used proved to be effective in the isolation of a highly heterogenous microbiota from salterns.

The microbial isolation screening carried out for samples from Aveiro and Olhão salterns allowed the isolation of 154 strains, of which 47 were affiliated with Fungi and 107 with Bacteria. Particularly, 26 fungal and 75 bacterial isolates were obtained from Aveiro, and 21 fungal and 32 bacterial isolates from Olhão. All these isolates have been putatively identified based on the ITS and 16S rRNA genes for Fungi and Bacteria, respectively. The isolates relatedness was examined by building phylogenetic trees (Supplementary Fig. 1).

Despite the high abundance of salterns in Portugal, their microbiota has been poorly explored. The existent research reports comprises (i) the description of new strains detected by metagenomics approaches of Tavira saltern (Algarve salt flats) microbiota [46], (ii) metagenomics studies of Algarve salt flats [22, 23, 47], (iii) characterization of novel isolated species from Tavira saltern [26] and from Tejo salt flats [48], (iv) an archaeal isolate genome announcement from Figueira da Foz salt flats [49] and (v) extensive isolation approaches targeting Aveiro saltern and a saltern from Olhão (Algarve salt flats) focusing on the overall bacterial microbiota [24]. In this last study, the direct comparison between the microbiota of both salterns was not possible since the nature of samples, the methodology applied and the time of sampling were different between both salterns. None of these studies have targeted the fungal community, most likely because the prevalence of fungi in marine and aquatic environments, including hypersaline ones, was overlooked for many years [50, 51, 52]. In the last decades, Fungi members have been reported in every aquatic environment explored, including marine and hypersaline ones [50, 51, 52]. The first description of fungi in salterns was reported by Gunde-Cimerman and collaborators [53], which aroused a fascinating interest in the scientific community [12, 54, 55]. Since this, saltern fungi have been studied worldwide, however the Portuguese salterns mycobiota remains unknown. In this study, the microbial diversity was assessed at genera (Fig. 2) and phyla (Fig. 3) levels. The fungal isolates obtained were phylogenetically related with the phylum Ascomycota (Fig. 3), with the exception of isolate AW17 that was affiliated to the genus Sporobolomyces (Fig. 2), specifically to the species Sporobolomyces ruberrimus, a yeast within the phylum Basidiomycota (Fig. 3).

Fig. 2.

Microbial diversity of the isolates obtained from Aveiro and Olhão salterns represented by genera.

Fig. 3.

Overall microbial biodiversity, phyla-ranked, of the isolates obtained from Aveiro and Olhão salterns, belonging to two microbial kingdoms.

Colour scheme representation by phylum: grey for Ascomycota, black for Basidiomycota, blue for Pseudomonadota, orange for Actinomycetota, purple for Bacillota, brown for Bacteroidota, green for Planctomycetota, yellow for Rhodothermota and red for Gemmatimonadota. Full filled with colour represent isolates common to both salterns and filled with pattern represents exclusivity to a specific saltern. (*) represents isolates identified only to the family level.

Overall information of the environmental prevalence of strains phylogenetically closely related with the isolates obtained with the present study is compiled in Table 1 (Ref. [1, 5, 9, 11, 24, 26, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107]), where presence or absence in salterns environments was highlighted.

Table 1.Environmental occurrence of the species isolated in this study from the two Portuguese salterns, Aveiro and Olhão, taking into consideration their phylogenetic rank.
Kingdom Phylum Class Order Family Closest related microorganism Isolates from this study Environments reported
Fungi Ascomycota (Supplementary Fig. 1a) Dothideomycetes Pleosporales Pleosporaceae Alternaria alternata ASBR4 Salterns [56]
Dothideales Dothioraceae Aureobasidium pullulans ASED7 Salterns [53, 57, 58]
Capnodiales Davidiellaceae Cladosporium herbarum AW2 Salterns [54, 59]
Cladosporium perangustum AS110 Marine [60]
Cladosporium phaenocomae AS102 Plants [61], deteriorated wood [62]
Eurotiomycetes Eurotiales Trichocomaceae Penicillium brevicompactum ASED17, ASED27, ASED28, ASBR9, AS103, AS104, AS108, AS111, OSBR109, OS1, OS3, OS4, OS6, OS7, OS8, OW12 Salterns [54]
Penicillium brocae ASED21, ASED25, ASED29, OSBR107, OSBR117, OS2, OS5, OS10, OW1, OW2, OW3, OW8, OW17 Marine [63]
Penicillium chrysogenum ASED16, ASED18, ASED19, ASBR1, ASBR2, ASBR5, AW1, AW11, OSBR108, OW5 Salterns [11, 54, 64]
Penicillium dierckxii ASED8 Hypersaline [65]
Penicillium sp. OSBR118 Plants (NCBI: EF694632)
Basidiomycota (Supplementary Fig. 1b) Microbotryomycetes Sporidiobolales Incertae sedis Sporobolomyces ruberrimus AW17 Psychrophilic [66, 67, 68]
Bacteria Actinomycetota (Supplementary Fig. 1c) Actinobacteria Micrococcales Dermabacteraceae Brachybacterium paraconglomeratum C.OS8 Hypersaline [69, 70]
Brevibacteriaceae Brevibacterium sediminis C.AS1, C.AS9 Salterns [24]
Microbacteriaceae Microbacterium aerolatum C.ASBR1, C.OS5 Air [71]
Microbacterium amylolyticum C.AS10, C.AW5 Salterns [24]
Microbacterium ginsengiterrae C.AW1 Soil [72]
Micrococcaceae Micrococcus luteus C.OS4 Salterns [73]
Rothia kristinae OSBR105, OSBR106 Human commensal and opportunistic pathogen [74]
Mycobacteriales Gordoniaceae Gordonia oryzae AW6 Plants [75]
Tsukamurellaceae Tsukamurella tyrosinosolvens OW4 Human commensal and opportunistic pathogen [76]
Propionibacteriales Nocardioidaceae Nocardioides salarius OSBR100 Marine [77]
Streptomycetales Streptomycetaceae Streptomyces intermedius C.AS7, C.ASBR4, Members of this genus have been associated with Salterns [9, 78, 79, 80]
Streptomyces rubrogriseus C.AS5, C.ASBR6, C.ASBR8, C.ASBR9, C.ASBR10, C.ASBR11, Members of this genus have been associated with Salterns [9, 78, 79, 80]
Streptosporangiales Nocardiopsaceae Nocardiopsis lucentensis C.ASBR7, C.ASED1, Salterns [79, 81]
Nocardiopsis prasina C.ASED4, C.AW6 Members of this genus have been associated with Salterns [9, 78, 79, 80]
Acidimicrobiia Acidimicrobiales Iamiaceae Uncultured bacterium OSBR104 Air [82]
Bacteroidota (Supplementary Fig. 1d) Flavobacteriia Flavobacteriales Flavobacteriaceae Psychroflexus tropicus AW7 Hypersaline [83]
Sinomicrobium oceani C.AS2, C.AS3, C.AS4, Members of this genus have been associated with Salterns [1]
Kingdom Phylum Class Order Family Closest related microorganism Isolates from this study Environments reported
Bacteria Bacillota (Supplementary Fig. 1e) Bacilli Bacillales Bacillaceae Alkalihalobacillus hwajinpoensis ASED10, ASED13 Salterns [24, 73, 84]
Bacillus safensis AW4 Salterns [85]
Bacillus sinesaloumensis AW3, AW5 Human commensal [86]
Cytobacillus luteolus OSBR113 Salterns [24, 84]
Mesobacillus sp. AW16, OSBR115 Salterns [24]
Metabacillus litoralis OSBR101, OSBR112, OSBR114, OSBR116, OW6 Salterns [84]
Rossellomorea sp. AW18, OW14 Salterns [24]
Thalassobacillus cyri OSBR110, OSBR111 Hypersaline [87]; Members of this genus have been associated with Salterns [73]
Paenibacillaceae Paenibacillus pabuli ASED15, ASED30 Members of this genus have been associated with Salterns [84]
Planococcaceae Paenisporosarcina quisquiliarum OS9 Soil [88]; Members of this genus have been associated with Salterns [84]
Gemmatimonadota (Supplementary Fig. 1f) Longimicrobia Uncultured Uncultured Uncultured bacterium AW12 Members of this phylum have been associated with Salterns [73, 89]
Planctomycetota (Supplementary Fig. 1g) Planctomycetia Planctomycetales Planctomycetaceae Alienimonas californiensis ASED1 Algae [90]
Maioricimonas rarisocia ASED14 Marine [91]
Pirellulales Pirellulaceae Rhodopirellula pilleata ASED26 Marine [92]
Rhodopirellula rubra ASBR8 Algae [93]
Phycisphaerae Phycisphaerales Phycisphaeraceae Uncultured bacterium ASED31 Members of this class have been associated with Salterns [26]
Pseudomonadota (Supplementary Fig. 1h) Alphaproteobacteria Hyphomicrobiales Aurantimonadaceae Aureimonas glaciistagni OW13 Members of this genus have been associated with Salterns [84]
Caulobacterales Caulobacteraceae Brevundimonas sp. ASED5 Members of this genus have been associated with Salterns [94]
Rhodospirillales Rhodovibrionaceae Rhodovibrio sodomensis OW16, OW18, ASBR14, ASBR15, ASBR16, AS100, AS106, AS114, AW21, AW23, OW9 Salterns [95]
Rhodobacterales Rhodobacteraceae Roseibacterium elongatum AW9 Hypersaline [5]
Hyphomicrobiales Stappiaceae Stappia stellulata ASED24 Marine [96, 97]
Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax dieselolei C.AW4 Members of this genus have been associated with Salterns [24]
Alcanivorax sp. C.ASED6 Members of this genus have been associated with Salterns [24]
Alteromonadales Alteromonadaceae Marinobacter confluentis AW10 Marine [98]
Marinobacter sp. ASED12 Marine [99]
Oceanospirillales Halomonadaceae Halomonas fontilapidosi AW8, AW13 Salterns [24, 100, 101]
Halomonas ventosae ASED11 Salterns [100, 101]
Salinicola zeshunii OW15 Members of this genus have been associated with Salterns [102, 103, 104]
Lysobacterales Lysobacteraceae Luteimonas padinae ASBR3, ASBR7, ASBR10 Salterns [105]
Cellvibrionales Microbulbiferaceae Microbulbifer halophilus C.ASBR5, C.OW1, C.OW2 Members of this genus have been associated with Salterns [106]
Chromatiales Aquichromatiaceae Uncultured bacterium ASBR12, AW14, AW19, AW20 Members of this genus have been associated with marine habitat [107]
Rhodothermota (Supplementary Fig. 1i) Balneolia Balneolales Balneolaceae Uncultured bacterium ASBR13, AS101, OSBR103 Members of this family have been associated with Salterns [1, 24]
Those associated with saltern environment are highlighted in bold.

Within Fungi, members of genus Penicillium were the only ones detected in both salterns and the most abundant ones (Figs. 2 and 3; Supplementary Table 2).

The Pseudomonadota genera Rhodovibrio and Microbulbifer were the only ones common to both saltern (Fig. 2). The similarity score of all 4 Microbulbifer isolates with M. halophilus (<98.50% in the 16S rRNA gene) revealed a distant relatedness, suggesting that these isolates can be members of a novel taxon (Fig. 4, Ref. [39]). Two other species of Pseudomonadota were detected as singletons in Olhão saltern, which were closely affiliated with Salinicola zeshunii and Aureimonas glaciistagni. Neither of these two species have been associated specifically with microbiota of salterns (Table 1). Furthermore, one Pseudomonadota isolate was affiliated with strains of Brevundimonas not yet related with halophilic environments (Table 1), but instead associated with humanized habitats, namely washing machines [108] and space laboratory [109]. Additionally, the Roseibacterium isolate AW9 obtained on this study showed a low similarity (97.53% in the 16S rRNA gene) with the closest related species, R. elongatum, which might be indicative of a novel species (Fig. 4). Also from Aveiro saltern, 4 strains of phylum Pseudomonadota phylogenetically related to uncultured members of Aquichromatiaceae were isolated, but showing low levels of similarity (98.08 up to 98.30% in the 16S rRNA gene), so highlighting the novelty of these strains (Fig. 4).

Fig. 4.

Phylogenetic framing of the putative novel taxa isolated from the two Portuguese Salterns. Maximum-Likelihood (ML) phylogenetic trees constructed with MEGA 7 software [39], using 16S rRNA gene sequences of the saltern isolates and of the phylogenetically closest sequences obtained in the EzBioCloud webserver. Bootstrap values were calculated based on 1000 replications and the numbers at each branch represent the bootstrap support in percentage for each cluster. The tree was constructed using representative members of each class within the phyla detected; Bacteroides fragilis (AB050106) was used as outgroup; Bar, 0.050 substitutions per nucleotide position.

Within the phylum Bacillota, Mesobacillus and Rossellomorea were the only two Bacillota genera that were detect at both Aveiro and Olhão salterns. Despite the geographical distance, the 2 strains (AW18 and OW14) closely affiliated with the novel established genus Rossellomorea [110] showed a perfect similarity between their 16S rRNA gene sequences and a close phylogenetic relationship with the isolate Rossellomorea sp. es.034 (PDIY01000001). Therefore, these strains are strong candidates for the description of a novel species (Fig. 4). Moreover, 2 isolates (AW3 and AW5) showed a similarity of up to 98.66% in the 16S rRNA gene with Bacillus sinesaloumensis, and other 2 isolates (OSBR114 and OW16) showed similarity scores of up to 98.4% in the 16S rRNA gene with Metabacillus litoralis. All of them may be representatives of novel taxa (Fig. 4).

Within Actinomycetota, 3 isolates were phylogenetically closely related with known opportunistic human pathogens (Rothia and Tsukamurella), that were obtained from Olhão saltern [74]. The presence of these species in Olhão saltern microbiota may be related with human activity, not only through salt exploitation but also through touristic related activities happening in Olhão salterns, like saltern baths (Table 1). Additionally, one isolate affiliated with Gordonia oryzae was obtained. This specie was recently described [75] and was not associated with extreme environments until this study. Furthermore, the Olhão saltern isolate OSBR104 was affiliated with the Actinomycetota family Iamiaceae, but the closest related strain described was Aquihabitans daechungensis [111] with 92.37% similarity in the 16S rRNA gene. Therefore, OSBR104 may represent a novel taxon (Fig. 4). Additionally, isolates related with Microbacterium amylolyticum (C.AS10 and C.AW5), Microbacteium ginsengiterrae (C.AW1), Micrococcus luteus (C.OS4) and Nocardioides salarius (OSBR100) showed low similarities with the mentioned strains (Supplementary Table 2) and may represent new taxa (Fig. 4).

Both classes of the phylum Planctomycetota, namely Planctomycetia and Phycispharae were isolated in this study. Planctomycetia isolates obtained were affiliated with genera Alienomonas (1 isolate), Rhodopirellula (2 isolates) and Maioricimonas (1 isolate) (Fig. 2). Four out of the 5 Planctomycetia isolates showed only up to 97.95% similarity in the 16S rRNA gene with the closest related described species (Fig. 4; Supplementary Table 2). These data are indicative of novel Planctomycetia taxa (Fig. 4). Within the less known Planctomycetota class Phycispharae, only one isolate (ASED31) was obtained, for which the species Algisphaera agarilytica was the most related one but in a far distant level (88.8% similarity in the 16S rRNA gene), being putatively indicative of a new family within Phycispharae (Fig. 4). Curiously, all planctomycetal isolates obtained in the present study were obtained from Aveiro saltern.

Within Rhodothermota, the family Balneolaceae was the only one recovered with 3 isolates (ASBR13, AS101 and OSBR103), that due to the phylogenetic distance, could not be related with any known Rhodothermota genus (Fig. 4).

Inside the phylum Gemmatimonadota, only one isolate (AW12) phylogenetically placed within the family Longimicrobiaceae was retrieved (Fig. 3). However, the 16S rRNA gene distant relatedness with the closest described species, represented by 85.82% similarity with Longimicrobium terrae, may be indicative of a novel family (Fig. 4).

3.2 Diversity, richness, dominance and distribution of the obtained isolates

The most frequent fungal genus was Penicillium, since 26.6% of the overall microbial isolates were classified within this genus (Supplementary Table 2). The most common bacterial genera retrieved from both salterns were the Actinomycetotal Streptomyces (8.4%) and the Pseudomonadota Rhodovibrio (7.1%), followed by the genus Sinomicrobium (6.5%) that is a member of phylum Bacteroidota (Supplementary Table 2).

Regarding overall phyla and genera, the isolated microorganisms associated with Aveiro saltern showed a higher diversity (Fisher’s α index) and richness (Margalef’s index), as well as a lower dominance (Simpson’s index) (Table 2). By running separated analyses of the genera diversity within the fungal kingdom and the genera diversity within the bacterial domain, one difference about these indexes was observed when compared to the overall analysis. Particularly, the diversity of bacterial genera was higher in Olhão saltern population (Table 2).

Table 2.Diversity, richness and dominance indexes of microbial communities isolated from Aveiro and Olhão salterns.
Fisher’s α Margalef’s Simpson’s
Aveiro Olhão Aveiro Olhão Aveiro Olhão
General by phylum 2.389 1.74 1.733 1.259 0.1983 0.2624
(2.04–2.389) (1.74–1.74) (1.517–1.733) (1.259–1.259) (0.1756–0.2365) (0.2218–0.3471)
General by genus 17.06 12.85 6.934 5.037 0.07597 0.1826
(12.82–17.06) (6.969–12.85) (5.85–6.934) (3.526–5.037) (0.05715–0.1071) (0.1136–0.3001)
Fungi 1.841 0.2185 1.228 0 0.6095 1
(0.8763–1.841) (0.2185–0.2185) (0.6139–1.228) (0.4172–0.855)
Ascomycota 1.344 0.2185 0.932 0 0.6576 1
(0.5116–1.344) (0.3107–0.932) (0.4752–0.8528)
Basidiomycota 0 0 1
Bacteria 16.21 22.82 6.254 5.482 0.06453 0.07031
(11.32–16.21) (9.49–22.82) (5.096–6.254) (3.751–5.482) (0.05209–0.08656) (0.06641–0.1309)
Bacillota 4.632 5.403 1.82 2.085 0.2346 0.2727
(1.576–4.632) (1.359–5.403) (0.9102–1.82) (0.8341–2.085) (0.2099–0.5062) (0.1901–0.5702)
Pseudomonadota 5.949 3.878 2.762 1.542 0.1627 0.3061
(3.143–5.949) (0.9354–3.878) (1.842–2.762) (0.5139–1.542) (0.1302–0.2899) (0.2653–0.7551)
Bacteroidota 0.7972 0.7959 0.4551 0 0.8025 1
Actinomycetota 1.968 13.19 1.276 2.919 0.2968 0.157
(1.399–1.986) (2.261–13.19) (0.9568–1.276) (1.251–2.919) (0.2401–0.4669) (0.1405–0.3554)
Planctomycetota 9.284 1.864 0.28
(1.235–9.284) (0.6213–1.864) (0.28–0.52)
Rhodothermota 0.7959 0 0 0 1 1
Gemmatimonadota 0 0 1

In Aveiro, the highest diversity and richness were registered within Planctomycetota and Pseudomonadota, while the lowest value of dominance was observed in Pseudomonadota, highlighting the heterogeneity of the Pseudomonadota isolates community (Table 2). At Olhão saltern, Actinomycetota and Pseudomonadota showed the highest diversity, while the lowest diversity was observed within Ascomycota and Rhodothermota (Table 2). Additionally, in Olhão, the highest values of diversity and richness along with the lowest value of dominance were observed within Actinomycetota (Table 2), demonstrating the high heterogeneity of Actinomycetotal isolates obtained.

Despite the high isolated diversity, the Mao’s Tau rarefaction curve presented a continuous rise without achieving an asymptote, confirming that not all microbial diversity was recovered (Fig. 5).

Fig. 5.

Mao’s Tau rarefaction curve (red) obtained on the genera diversity analysis of microbial isolated communities from Aveiro and Olhão salterns and representing the species accumulation with 95% confidence intervals (in blue).

Concerning the fungal biodiversity, 3 singletons were observed in Aveiro, while none was detected in Olhão. These singletons represent 6.4% of the whole fungal diversity (Supplementary Material). The overall bacterial diversity was comprised by 40 different genera, with 25 singletons, that represent 23.4% of the overall bacterial diversity (Supplementary Table 2). These may be rare members of the isolated bacterial communities of salterns. Additionally, from the 28 bacterial genera recovered from Aveiro saltern, 12 occurred as singletons, representing 16.0% of the overall diversity of isolates, while Olhão taxa biodiversity consisted in 20 different bacterial genera, where 13 occurred as singletons, representing 37.1% of the overall diversity of isolates (Supplementary Table 2). From all these singletons, only 2 were common to both salterns, namely, the Bacillota genera, Mesobacillus and Rossellomorea (Supplementary Table 2).

The Sørensen and Bray-Curtis indexes were determined between the microbial populations of both salterns as 0.33 and 0.43, respectively. These parameters are measures of the similarity between both microbial populations, therefore highlighting the dissimilarities between them. Taking into account that sampling dates were close and that all the procedures undertaken were the same for all samples, these differences between the two microbial populations studied may be a result of the different geographical locations of the two salterns (Aveiro at the North of Portugal vs. Olhão at the South of Portugal).

3.3 Molecular assessment of NRPS and PKS-I genes

The biotechnological potential of all isolates obtained in this study was screened by the PCR analysis of PKS-I and NRPS genes (Table 3; Supplementary Table 2). From the 154 microorganisms isolated in this study, 51 (33.1%) did not present any PKS or NRPS genes and 80 (52%) presented one (64–41.6% PKS positive and 16–10.4% NRPS positive) or both genes (23 bacteria, 14.9%) (Table 3; Supplementary Table 2). Overall, this screening revealed that 66.9% of the isolates may present at least one of the genes, revealing their putative biotechnological potential (Table 3; Supplementary Table 2). Although microbial ability to produce bioactive metabolites is not strictly linked to PKS and NRPS genes, because many other genes have been associated with bioactive NPs production [112], the genomic presence of these genes already proved to be a good indicator of the production of antimicrobial compounds [113]. Furthermore, NPs derived from these genes, as polyketides (PK), nonribosomal peptides (NRP) or even PK/NRP hybrids, have demonstrated their high economical and pharmacological value [20, 114]. These gene clusters were previously detected in ascomycetes and in a high number, which may be associated with the production of several different NRPS- and PKS-derived NPs [115].

Table 3.Number of isolates, grouped by genera within each phylum that potentially amplified positively the bioactivity-related genes, NRPS e PKS-I, through PCR screening.
Phylum Genus Number of Isolates Only NRPS Only PKS Both
Actinomycetota Streptomyces 13 0 9 1
Nocardiopsis 6 0 6 0
Micrococcus 1 0 0 1
Microbacterium 5 1 0 2
Brevibacterium 2 0 1 0
Rothia 2 0 1 0
Gordonia 1 0 1 0
Brachybacterium 1 0 0 0
Nocardioides 1 0 0 0
Tsukamurella 1 0 0 1
Iamiaceae* 1 0 0 0
Pseudomonadota Rhodovibrio 11 1 3 1
Wenzhouxiangella 4 1 2 1
Microbulbifer 4 0 1 3
Luteimonas 3 1 0 1
Halomonas 3 0 3 0
Marinobacter 2 1 0 0
Alcanivorax 2 0 0 0
Brevundimonas 1 1 0 0
Stappia 1 0 0 1
Roseibacterium 1 0 0 0
Aureimonas 1 0 1 0
Salinicola 1 0 1 0
Bacillota Bacillus 3 3 0 0
Metabacillus 5 0 0 0
Alkalihalobacillus 2 0 2 0
Paenibacillus 2 0 0 2
Thalassobacillus 2 0 0 1
Paenisporosarcina 1 1 0 0
Mesobacillus 2 1 0 0
Cytobacillus 1 0 0 0
Rossellomorea 2 2 0 0
Bacteroidota Sinomicrobium 10 0 8 0
Psychroflexus 1 0 0 1
Planctomycetota Alienimonas 1 0 0 0
Maiorcimonas 1 1 0 0
Rhodopirellula 2 0 1 1
Algisphaera 1 0 1 0
Rhodothermota Balneolaceae* 3 0 1 2
Gemmatimonadota Longimicrobiaceae* 1 0 0 0
Ascomycota Penicillium 41 1 21 4
Cladosporium 3 0 1 0
Aureobasidium 1 1 0 0
Alternaria 1 0 0 1
Basidiomycota Sporobolomyces 1 0 0 0

The high percentages and widespread distribution of NRPS and PKS genes observed in the phyla Pseudomonadota, Actinomycetota, and Bacillota was already reported by Wang et al. [115]. In contrast, both Bacteroidota and Planctomycetota are less explored phyla in what concerns their potential for the production of bioactive molecules, being research fields that are being launched with encouraging results obtained from a preliminary screening of NRPS and PKS genes [116, 117, 118]. Concerning Rhodothermota, since this phylum was recently branched out from Bacteroidota [8], no studies targeting their overall genomic potential for production of bioactive NPs are yet published. Regardless of the only 3 representatives obtained, since, up to the present moment, the phylum Rhodothermota has only 13 described species from 4 different families of only 1 class, the presence of at least one of these genes in all isolated members of this phylum pave the way for future deeper studies on this poorly known phylum. As detected in the present work, NRPS and PKS-I gene clusters were previously detected in ascomycetes and in a high number, which may be associated with the production of several different NRPS- and PKS-derived NPs [115]. No NRPS or PKS-I amplicons were detected either in Gemmatimonadota or Basidiomycota (Table 3; Supplementary Table 2). In fact, Gressler et al. [119] reported that PKSs and NRPSs are not the main contributors for basidiomycetes diversity of NPs. On the other hand, the poorly known phylum Gemmatimonadota was firstly described less than two decades ago and presently only has 6 described species. This scarce number of described species is due to the difficulties in successfully isolating Gemmatimonadota members in laboratory, despite being widely distributed in the environment [120]. Therefore, most of the research made on its bioactive potential relies on metagenomes, which demonstrated the presence of not only NRPS, PKS and hybrid PKS/NRPS gene clusters [121], but also a widespread and high prevalence of bacteriocin-related biosynthetic gene clusters [122].

Overall, the microbiota isolated from both salterns showed a great biotechnological potential. Curiously, although with lower number of isolates, the phyla that showed higher bioactive potential were the Rhodothermota, Bacteroidota and Planctomycetota, and not the well-known bioactive top producers, Actinomycetota [123].

4. Conclusions

This work provides evidence that salterns remain an understudied extreme environment and that culturomic methods are still an important approach for the study of microbial diversity, since a high number of novel taxa from different phyla was obtained. In fact, although metagenomics is a fundamental approach for novel microbial diversity detection, pure cultures are needed to enable the biological characterization of many species yet unknown. Furthermore, the isolated microbiota from salterns showed a substantial underexplored bioactive potential providing data and biological material that encourages further research works as bioactivity screenings.

In general, the overall microbial diversity obtained has been previously associated with salterns or with other hypersaline environments, however our results also pointed out genera not yet linked to these environments.


NP, natural product; PKS-I, polyketide synthase I; NRPS, nonribosomal peptide synthethase; SW, seawater; PCR, polymerase chain reaction; NCBI, National Centre for Biotechnology Information; RT, room temperature; SCN, starch-casein-nitrate agar; NPS, nutrient-poor sediment extract agar.

Author contributions

EA, MFC and OML designed the research study. EA performed the research. EA, MFC and OML analyzed the data. EA, MFC and OML wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.


Not applicable.


The authors EA, MFC and OML thank to national funds provided by the Fundação para a Ciência e Tecnologia (FCT; Foundation for Science and Technology) at Portugal, and European Regional Development Fund (ERDF) and COMPETE under the project INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR. EA was supported by FCT for the Ph.D. Grant SFRH/BD/125527/2016. MFC wishes to acknowledge CEEC program supported by FCT (CEECIND/02968/2017), Fundo Social Europeu and Programa Operacional Potencial Humano. This work was supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT and ERDF. This research was partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by FCT and ERDF, in the framework of the program PT2020, and by the Project Funding NORTE-01-0145-FEDER-000035, designed ATLANTIDA – Plataforma para a Monitorização do Oceano Atlântico Norte e Ferramentas para a Exploração Sustentável dos Recursos Marinhos, provided by FCT and ERDF.

Conflict of interest

The authors declare no conflict of interest.

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