Study of diversity of mineral-forming bacteria in sabkha mats and sediments of mangrove forest in Qatar

Highlights • Microorganisms play a significant role in the formation of carbonate minerals in both evaporitic environments and mangrove forests.• The study investigated the biodiversity mineral-forming bacteria in mangrove forests and Qatari sabkha through protein level analysis using MALDI-TOF MS com protein profiles combined with PCA.• The diversity of the minerals formed in pure cultures was evidenced by SEM/EDS and XRD analysis.


Introduction
The coastline of Qatar consists of complex marine ecosystems including sabkhas and mangroves forests.Studies in both sabkha and mangrove habitats are important due to their sedimentary structure and biodiversity [1].The supratidal sabkhas are unique with their characteristics as they are also defined as marine environments [2].All these types of ecosystems exist also in the coastal regions of other areas like the Mediterranean area [3].However, along the Arabian Gulf coast, sabkhas are mostly evaporitic areas, representing specific sites of diversity due to the harsh weather conditions [4].Indeed, the average temperature of the sea surface in the Arabian Gulf Sea fluctuates around 20 -34 • C [5], while the temperature above the surface of the sedimentary structures may reach 60 • C in the summer.Consequently, the salinity can rise to 30% [6].The high salinity can contribute to the formation of minerals in sabkhas as well as to the role of mineral-forming bacteria evidenced in these evaporitic areas [7].
Various environments can be ideal for the existence of microbial mats [8].The coastal intertidal sediments and sabkhas are considered one of those environments, contributing significantly to improving the sedimentary deposits.The microbial mats in sabkhas serve as an important natural laboratory for studying the role of the mineral-forming microorganisms in the evaporitic mineralization [9][10][11].The various microorganisms living in sabkha mats can perform highly diversified metabolic activities as evidenced inside these microbial mats [12].The microbial mats found in sabkha regions are not only distinctives ecosystems, but also dynamic ones as they constitute enormous biodiversity of living microorganisms, which can shape the assembly of the sediments within the environment [13].On the other hand, sabkhas are considered vulnerable to climate change, due to the lack of freshwater supplies and rainfall as well as increase of human population [14].Although the fluctuations of the harsh conditions that characterize the Arabian Gulf areas, it was demonstrated that the marine habitats including mangroves forests, sabkhas and seagrasses still can serve as CO 2 sinks [15], and in turn, can affect the "blue carbon," which refers to carbon stored in marine environments [16].
Mangroves are a group of trees and shrubs that grow in coastal saline or brackish water habitats in tropical and subtropical regions [17].They have the ability to endure a variety of challenging conditions, including minimal rainfall, intense sunlight, significant temperature fluctuations, and extreme salinity [18].Symbiosis between microorganisms, developed in their rhizosphere, might be one of the crucial factors of their existence [19].In addition, sediments under mangrove forests are shown as great reactors for the formation of minerals [20].Their presence in tropical and subtropical areas provides all the factors necessary for the formation of minerals [21].The later work showed that the activities of mineral formation and biotransformation were performed by bacteria isolated from sediments of mangroves forests.The mangrove forest of Simaisma in Qatar is one of the mangrove-rich zones in the coastline of Qatar.The specific Qatari conditions ensure a strong adaptation within the microbial communities, leading to new biological activities, of interest [22].
MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) is a powerful analytical technique that enables the rapid and accurate identification of proteins, peptides, and other biomolecules based on their mass-to-charge ratio (m/z) [23].The  technique has a wide range of applications in fields such as proteomics, drug discovery, and clinical diagnostics [24][25][26].The MALDI-TOF MS technique has several advantages, for example, it is relatively fast and easy to use, requires minimal sample preparation, and can analyze a wide range of sample types, including proteins, peptides, lipids, and carbohydrates [27].Recently, it was shown efficient in the identification, differentiation and categorization of mixtures of bacteria living in the environment as consortia even at the species levels [7,28].It becomes a useful approach for the study of functional biodiversity in environmental studies.
Here, mineral-forming bacteria were isolated form microbial mats of Qatari sabkha and sediments of mangrove forests.By using MALDI-TOF MS to study their biodiversity, a new collection of mineral-forming bacteria from both environments could be examined.This would provide insight into the diversity of these bacteria at the genus and species levels, as well as their metabolic proteins which express their adaptive diversity.The adaptations of these bacteria are linked to their spontaneous biodiversity.By combining bacterial identification and protein profiling, with a focus on protein markers discovered during mineral formation, these bacteria can be classified as biomineral-forming bacteria.This classification represents important evidence of biodiversity related to the adaptation of mineral-forming bacteria and demonstrates that Qatari sabkhas and mangrove sediments form a continuous, dynamic bacterial system for capturing CO 2 .

Sampling sites
The sampling locations in two sabkhas were chosen based on prior research that confirmed the existence of various types of precipitates and forecasted the likelihood of dolomite formation [10,11].Khor Al-Adaid sabkha is a settlement existing in southeast region of Qatar in Alwakrah municipality (GPS coordinates 24 • 38′45.870″N51 • 19′35.760″E).Dohat Faishakh sabkha is a bay located in Al-Rayyan municipality (GPS coordinates 25 • 38′8.17″N50 • 57′36.48″E).Four samples of the microbial mats (DFC, DFD, DFL and DFM) were collected aseptically from intertidal zone of Dohat Faishakh sabkha (10 m apart) and one microbial mat (KA1) was collected from Khor Al-Adaid sabkha.Another sampling location was in Simaisma mangrove forests existing in Al Daayen municipality, eastern region of Qatar, (GPS coordinates 25 • 34′41.1″N51 • 29′19.1″E).Sediment samples were collected in a sterile 50 mL Falcon tube.The samples were collected from a depth of 20 cm under two mangrove trees (MC1 and MC2) and at 1 m far from each mangrove tree (MC1F and MC2F).Upon collection, all samples were temporarily kept in an icebox at 4 • C. The samples were transferred to the laboratory and then preserved at − 20 • C for further analysis.The three sampling locations are illustrated in Fig. 1.

Culture media
The liquid MD1 medium is composed of (g/L): 10 yeast extract, 5 peptone, 1 glucose, 12 magnesium acetate, 1.5 calcium acetate and 35 sodium chloride.Solid MD1 medium was prepared by adding 15 g/L agar.The pH was adjusted to 7.0 before sterilization at 121 • C for 20 min.Luria-Bertani (LB) medium is composed of (g/L): 10 tryptone, 5 yeast extract, 10 sodium chloride added to 1 L of distilled water.Solid LB contains 15 g of agar in addition to the former for 1 L. LB media were autoclaved at 121 • C for 20 min.

Isolation and preservation of the mineral-forming bacterial strains
Mineral-forming bacterial strains were isolated from all the sabkhas and mangrove sediments by the process of enrichment cultures.The medium MD1 was used for the growth of this category of bacteria [10].One g of sample was suspended in 20 mL MD1 and incubated for 72 h in a rotary shaker set at 30 • C and 150 rpm.Four successive enrichment cultures were then proceeded under the same conditions, each

Table 1
List of the bacterial strains isolated from the decaying mats of Dohat Faishakh sabkha, the living mats from Khor Al-Adaid sabkha and the sediments of Simaisma mangroves and their identification (by MALDI-TOF MS and ribotyping).inoculated with 2 mL from the former culture.Serial dilutions were plated from the last culture on solid MD1 and the dilutions allowing for selection of separate colonies.Distinct colonies were transferred to a new MD1 plates using the streaked plate method.Then, five successive sub-cultures of a separate colony from each, allowed purification of each strain.The isolated strains were given codes and preserved at − 80 • C in 60% glycerol in LB medium.Before being used to inoculate pure cultures, each strain was plated in LB medium and separate colonies were used.

Identification of the isolated strains by MALDI-TOF MS
For identifying and generating the protein profile of each of the isolated strains by MALDI-TOF MS, they were cultured on LB solid medium and incubated overnight at 30 • C. MALDI-TOF MS was used as described by Abdel Samad et al. (2020).It generates a specific mass spectrum related to the proteins of each strain, then compared with the database entries.Proteins having a m/z between 2000 and 20,000 are utilized to identify the bacterial strain based on individual mass peaks matching to specific ribosomal proteins, available in a database provided in the software.The data are presented as log (scores) by the Biotyper software which generates them by default.For each strain, a log scale ranging from 0.000 to 3.000 was attained.The identification is at the extremely probable species level with high confidence if the score falls between 2.300 and 3.000.The scores between 2.000 and 2.299 offer highly accurate genus-level identification and probable correct species-level identification.Probabilistic genus level identification is provided by scores between 1.700 and 1.999.Analysis and protein profiles were performed with the MALDI Biotarget-48 sample spots using MALDI Biotyper Real time classification software.The manual process of protein profiles was performed using the Flex Analysis Software which is necessary for smoothing and subtraction of baseline from the profile.

Differentiation and diversity study of the isolated strains by MALDI-TOF MS and PCA
Principle component analysis (PCA) was used in this study in order to reduce the effect of the dimensionality of the data set maintaining thus the original information.PCA is a statistical technique used to transform a dataset into a lower-dimensional space by identifying the principal components.Each principal component is a linear combination of the original variables and represents a specific pattern or direction of variability in the data.The components are ordered in terms of the amount of variance they explain, with the first component explaining the most variance, the second component explaining the second most, and so on.The PCA used the peaks obtained in each protein profile as generated by MALDI-TOF MS.In fact, the peaks which are not matching to specific ribosomal proteins, are of not identified proteins and peptides The results of PCA are interesting since the profiles are clustered into groups of similar variation characteristics allowing the visualization of the differences between the strains.Here, the data are represented in 3D coordinate system offering at least 50% of the total variance between the samples.This study was based on the standard operating procedure of the MALDI Biotyper instrument.

Identification of the isolated strains by ribotyping (16S rRNA)
DNA was extracted from cells that had grown overnight on LB plates at a temperature of 30 • C. In a sterile environment, a single colony of bacterial biomass was stripped from the surface of culture plates.The bacterial biomass was suspended in 500 μL distilled water, placed in water bath at 100 • C for 10 min, and then transferred immediately to − 20 • C for 10 min [29].After centrifugation of 5 min at 10,000 rpm, the supernatants were transferred into new tubes.PCR mixtures with total volume of 25 μL were prepared by adding 3 μL of DNA for each sample, 2.5 μL of the two selected universal primers (RibS74sp The PCR reactions were performed using an Applied Biosystems instrument.The genomic DNA of the isolates were used as template for the PCR reactions.To initiate the PCR reactions, the first step involved an initial denaturation at 94 • C for three minutes.This was followed by 35 cycles consisting of denaturation at 94 • C for 45 s, annealing at 50 • C for 45 s, and elongation at 72 • C for 45 s.A final extension step was then performed at 72 • C for two min.Afterward, the DNA amplicons were purified with the Thermo Scientific GeneJET PCR Purification Kit.The Sanger sequencing was carried out in laboratories of Weill Cornell Medicine -Qatar.The obtained DNA sequences were compared to the most closely sequences available at NCBI Blast server.

Screening of the studied isolates for the mineral formation capabilities
The potential of the isolates to form minerals was evaluated using the MD1 and two modified solid media namely MD1P and MD1Y, as evidenced by Abdelsamad et al. (2022).The two modified media contain the same components of MD1 media but MD1P is made up without yeast extract and MD1Y without peptone.Each isolate was grown on the solid media and incubated at 30 • C for 3 weeks.The growth areas were monitored regularly with a light microscope to observe the formation of crystals.

Investigation of the minerals composition by X-Ray diffraction (XRD), screening electron microscopy and energy dispersive X-Ray (SEM/ EDS) analysis
The sediment samples collected from sabkhas, and mangrove forest had been analyzed initially to ensure the existence of high magnesium carbonate as a bulk in these sites.One g from each sediment sample was manually grounded and homogenized with a mortar and pestle, mixed evenly, and left to dry at 37 • C overnight.The dried samples were analyzed by XRD and SEM/EDS.
The minerals formed in pure bacterial cultures were extracted from the solid media.The bacterial biomass was stripped from the surface of the solid medium by slightly scraping the top layer with a sterile scalpel.The mineral crystals were washed three times with 15 mL distilled water.This process does not alter the shape of the crystals, as confirmed by optical microscopy before and after recovery [10].The overnight dried samples at 37 • C, were analyzed by SEM/EDS and XRD techniques.
The SEM analysis was conducted utilizing a Nova Nano Scanning Electron Microscopy equipped with a Bruker EDX Detector with a magnification of 200,000X and a resolution of 5 nm.The EDS was obtained in accordance with the "ASTM standard method E1508-12a", using a spot size of 5 and an accelerating voltage of 20 kV with an error rate of 4%.The PANalytical-multipurpose Empyrean X-ray diffractometer was used to determine the bulk mineralogical composition of the retrieved minerals.

Investigation of the occurrence of carbonate minerals in the mangrove sediments by XRD and SEM/EDS analysis
The occurrence of a variety of carbonate minerals, including calcium carbonates, magnesium carbonates and dolomite, was demonstrated earlier by XRD in the living mats and the decaying mats of both Sabkhas; Dohat Faishakh and Khor Al-Adaid sabkhas [10,11].Here, these minerals were evidenced in sediments sampled from Sabkha and Simaisma mangroves.Fig. 2 shows the occurrence of different carbonate mineral phases in these samples.Indeed, the XRD analysis confirms the presence of a mixture of carbonate minerals including, aragonite, calcite, high-Mg calcite, and dolomite.The SEM/EDX was performed on all the samples from both sabkhas and mangrove sites, and the images of the bulk sediments are shown in Fig. 3.The results show the existence of carbonate minerals with different Mg +2 : Ca +2 ratios in all the samples, including Khor Al-Adaid sabkha sediment KA1, Dohat Faishakh sediment DFM and Simaisma mangrove sediment MC23.

Identification of the isolated strains by MALDI-TOF MS and by ribotyping
Thirty-nine bacterial strains were isolated: five strains from living mats of Khor Al-Adaid, fourteen strains from decaying mats of Dohat Faishakh and twenty strains from Simaisma mangrove sediments.The identification of the thirty-nine isolated strains was performed using MALDI-TOF MS using the Bruker database entries [30].Only nineteen strains were given a MALDI score higher than 1.7, which allowed their identification at the genus level and for certain ones, at the species level.The MALDI-TOF MS protein profiles of the others were not matching to the database entries.Indeed, the database of the used MALDI machine comprises the data related to bacteria, which are mostly searched in the health and environmental field, including those belonging to genera: Shewanella, Staphylococcus, Vibrio, Micrococcus, Bacillus and Salinivibrio.Out of the nineteen isolates identified by MALDI-TOF MS, five were identified as Salinivibrio proteolyticus and five as Vibrio alginolyticus, two of each Bacillus subtilis, Bacillus lichenformis and Shewanella putrefaciens and one of each Bacillus seohaeanensis and Staphylococcus epidermidis and Micrococcus luteus.Previously, strains belonging to these genera were isolated from different environments and identified by both techniques (MALDI-TOF MS using the same database and ribotyping) [7,24,27,28,[31][32][33][34].The identification by ribotyping of the twenty strains which were not identified by MALDI-TOF MS was performed and the sequences were compared to the most closely sequences available at NCBI Blast server.Among these twenty isolated, five were   high sensitivity of the MALD-TOF MS procedure in considering the specific proteins in the MALDI-scores determination.A score below 1.7 out 2.3 is not considered for the accurate identification.The use of MALDI-TOF in the differentiation of very close strains is efficient [31].The two strains were identified by ribotyping.Table 1 indicates the isolates that were isolated from the three studied site and their identifications.

Investigation of the biodiversity of the bacterial strains isolated from
Qatari sabkhas and mangrove sediments using PCA and dendrogram analysis MALDI-TOF MS allowed obtaining the protein profile for each identified and not identified strain.Fig. 4 shows the protein profiles of several strains.The considered profiles are composed of proteins between 2000 and 20,000 m/z.Each peak from protein profiles is presenting one of the proteins that have been produced within the bacterial cell during growth.
Additional insights into the links between closely related isolates was performed by combining MALDI-TOF MS and PCA.PCA allowed the creation of linear combinations of variables to represent the objects being studied by reducing the dimensions of the objects.The percentage variance explained for the combined isolates of sabkhas, and mangroves revealed a total of 40% with contributions of PC1 (18%), PC2 (12.5%), and PC3 (9.5%).Consequently, the first three components were not considered to be sufficient to explain the variability of data.As the general rule, the PCA total variance should be at least 50% as recommended by Streiner (1994) [35].The PCA was not performed for Khor Al-Adaid isolates because the number of isolated strains was only 5 strains enclosing only two species of bacteria.Therefore, the PCA clustering and the dendrogram were performed for data from Dohat Faishakh sabkha and Simaisma mangrove, individually.The PCA clustering for sabkha isolates demonstrated significant protein-level diversity among the analyzed strains.The variability of the principal components is shown in Fig. 5B, with PC1 (25%), PC2 (16%), and PC3 (13%) contributing to a combined 54% of the data variability.As for the principal component analysis, the differences at group level were demonstrated by the distance between clusters, whereas the variations in protein profiles were highlighted by the distance between the individual strains within clusters.Four clusters were established based on the PCA for Dohat Faishakh sabkha samples as shown in Fig. 5A.Cluster I covers V. chiguensis; cluster II contains Bacillus spp., while Cluster III is formed of V. marismortui and cluster IV of V. alginolyticus.
To investigate the hierarchical relationship between the isolates, the PCA clusters and the dendrogram for Dohat Faishakh sabkha and mangroves strains were established.The dendrogram for Dohat Faishakh sabkha isolates, shown in Fig. 6 Fig. 7B shows the variation of the principal components for the mangroves samples, with PC1, PC2, and PC3 accounting for 28%, 15%, and 12% of the data variability respectively, resulting in a total of 55%.According to Fig. 7A, the PCA analysis of the mangroves samples resulted in the identification of four clusters.Cluster I is composed of the V. alginolyticus strains.Cluster II comprises the starins of Sh.Putrefaciens, while Cluster III is formed by strains of Virgibacillus spp., O. baumannii and Halomonas sp.Cluster IV is composed of Virgibacillus sp strains.However, one of each of P. agglomerans, V. dokdonensis, and H. Hydrothermalis are not included in the groups as they are found in distance from other strains.Fig. 8 shows the dendrogram of mangroves isolates, revealing that the three main clusters (I, II, and III) are further divided into multiple clades.Specifically, Cluster I is composed of five isolates, Cluster II contains eight isolates, and Cluster III is comprised of seven strains.Cluster I is divided into two clades Ia and Ib.Ia clade contains one strain of each V. pantothenticus and Virgibacillus sp.Ib clade is divided into Ib1 and Ib2 sub-clades.Ib1 holds V. chiguensis strain and Ib2 has two Halamonas sp.strains.Cluster II is composed of two clades namely, IIa and IIb.The clade IIa is further divided into two subclades (IIa1 and IIa2).The subclade IIa1 comprises two V. alginolyticus strains, while the subclade IIa2 is composed of one P. agglomerans strain.The clade IIb contains two subclades IIb1 and IIb2.The subclade IIb1 is composed of two Sh.putrefaciens strains and one strain of S. Epdiermidis.IIb2 covers two strains M. luteus and O. baumannii.Cluster III presents two clades IIIa and IIIb.IIIa clade shows two sub-clades IIIa1 and IIa2.IIIa1 has one strain of V. chiguensis, whereas IIIa2 is divided to two sub-clades IIIa2a and IIIa2b.IIIa2a divides more to IIIa2a1 containing one V. chiguensis and one V. dokdonensis, as well as IIIa2a2 which is composed of one V. chiguensis and one Virgibacillus sp.IIIa2b contains one V. dokdonensis strain.However, IIIb is formed of one H.Hydrothermalis strain.

Investigation on the potential of mineral formation by the isolated strains
MD1 medium employed in this work allows the enrichment of the successive cultures with bacteria, which are halophilic, heterotrophic and aerobic [10].In this study, the potential of the isolated strains to form minerals was performed using MD1, MD1P and MD1Y.The Results of their potential to mediate mineral formation are shown in Table 2.
Based on the data presented in Table 2, it can be inferred that the use of MD1P medium resulted in the highest mineral formation capability for the studied strain compared to other tested media.Therefore, MD1P can be considered as the most recommended medium for investigating the mineral-forming ability of the strain initially isolated from Sabkhas and mangrove sediments in Qatar, which is a great novelty in this field.
Most of the mineral forming bacterial strains belong to the genera Salinivibrio, Virgibacillus and Vibrio, confirming earlier findings [7,31,36,37].Remarkably, two strains of Shewanella putrefaciens (MC113 and MC2F31) obtained from Smeisma mangroves have been found to possess the ability to facilitate the formation of carbonate minerals.This finding adds to the previous reports, including the study by Chubar et al. (2015) [38], which demonstrated that Shewanella putrefaciens can mediate the formation of both manganese phosphate and manganese carbonate.Bacteria possess diverse mechanisms to actively influence and induce mineral formation [39].One such mechanism involves the production of extracellular polymeric substances (EPS), which act as a matrix for mineral nucleation and growth [40].Within the EPS, the presence of the carbonic anhydrase enzyme facilitates the localized conversion of carbon dioxide to bicarbonate ions [31].This enzymatic activity increases the availability of bicarbonate ions, promoting mineral precipitation and contributing to the controlled formation of carbonate minerals [41].
The SEM/EDX analysis of the minerals recovered from the pure cultures allowed the visualization of the formed mineral, as clear evidence and rough estimation of their elemental composition.The results of the analysis showed the formation of various types of carbonate minerals in the pure bacterial cultures, including calcium carbonates, hydromagnesite, and magnesium calcites.These minerals were found to have variable incorporation of magnesium into their crystal structure, as demonstrated in Fig. 9.
The potential of the corresponding strains to incorporate magnesium is then clearly evidenced.However, the variability in the incorporation of magnesium into the carbonate crystals observed in the SEM/EDX analysis suggests that the isolates may exhibit different mechanisms for mineral formation.Some of them, may lead to formation of precursors of dolomite at the ambient conditions.This variability could be explained by variability in the metabolic pathways or enzymes involved in carbonate precipitation, as well as differences in the environmental conditions under which the isolates were cultured.Further studies could be conducted to investigate these mechanisms and their implications for biomineralization [42].

Conclusion
Demonstrating the diversity of the mineral-forming bacteria is actually a necessity in biogeochemistry studies in sabkhas and sediments because these bacteria play a key role in the cycling of minerals and nutrients in these environments.In this study, a collection of aerobic bacterial strains isolated form decaying mats and living mats of Qatari sabkhas as well as from one Qatari mangroves site demonstrated a high diversity in their protein profiles, even within the same genus or species.Their close or far similarities should be attributed to their adaptation developed with time at specific environmental conditions.In pure cultures, the composition of the medium was found to be essential for formation of the minerals.Hence the composition of growth media should be considered for investigation the potential of mineral forming bacteria.The present work evidenced the biodiversity and the relationships with the adaptation of mineral-forming bacteria in the microenvironment of the cells.The use of MALDI-TOF MS approach was shown efficient to demonstrate the diversity of this type of bacteria and the diversity of their adaptation in the harsh conditions of Qatar environments, showing that the continuous adaptation dynamics would

Fig. 7 .
Fig. 7. PCA classification of the isolates from Simaisma mangroves, A) PCA, B) percentage of variance explained by PCA.
, displays that the three main clusters I, II, and III are subdivided into several clades.Cluster I is made up of seven isolates, cluster II contains three isolates while cluster III consists of four strains.Cluster I consists of two main clades, Ia and Ib.The clade Ia is further divided into two sub-clades, Ia1 and Ia2.Within the sub-clade Ia1, there are three strains of B. lichenformis and Ia1b has one B. subtilis strain.Ia2 includes two V. chiguensis strains.The Ib clade covers B. sochaeanensis strain.Cluster II involves two clades IIa containing two B. subtilis strains and IIb having one B. swezeyi strain.Cluster III contains two clades: IIIa and IIIb.IIIa clade encloses two V. marismortui strains and IIIb involves two V. alginolyticus strains.