Unravelling Extremophilic Microbiome Diversity and Functional Dynamics in Hypersaline Environment

Abstract

Solar salt pans are extreme hypersaline environments that represent functionally specialised microbial communities mediating essential biogeochemical transformation. Vedaranyam, a coastal region of the Bay of Bengal containing artificially constructed solar salterns for salt production. There is limited information available on the metagenome diversity and functional profiling of this saltpan, which prompted us to investigate it. Here, we report the first whole metagenome sequencing to explore the dynamics of the functional structure of microbial communities in saltpan during the preharvest and postharvest phases of salt production. Methanobacteriota and Pseudomonadota dominated both phases at the phylum level, while Halobacteria comprised the most abundant class (53.2% preharvest; 48% postharvest). A notable bloom of Dactylococcopsis salina was observed during postharvest (4.28% to 12.67%) and flock doubling of Cyanobacterota relative abundance (5.5% to 10.6%), reflecting photosynthetic primary production following salt removal. Conversely, during postharvest phase sulfur oxidising Guyparkeria halophila reduced 23 fold, while the DMSP accumulating osmolyte producer Salinibaculum marinum dominated preharvest (6.98%). However, functional classification of the metagenome revealed active participation of the microbial community across five major biogeochemical cycles. Encompassing carbon fixation by cyanobacteria and diverse haloarchaea, nitrogen cycling through diazotrophy and denitrification, a cryptic preharvest sulfur cycle coupling sulfate reduction and sulphide oxidation, phase shifted DMSP catabolism, and light driven bacteriorhodopsin through archaeal energy conservation. Metagenomic assembly yielded ten metagenomic assembled genomes (MAGs), revealing the taxonomic diversity and metabolic potential of the dominant halophilic community across biogeochemical cycles. These results provide critical insights into the ecological succession from an anaerobic, chemolithotrophy-rich preharvest microbial community to an aerobic, photosynthetically driven postharvest assemblage, advancing our understanding of microbial biogeochemistry in managed hypersaline ecosystems.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00248-026-02817-z.

Introduction

Salt pans are one of the extreme environments on Earth characterized by high salinity, intense sunlight, and extreme temperature fluctuations [1]. The microbial communities in salt pans form microbial mats with a vertically stratified structure, which hosts a diverse group of microorganisms with unique metabolic capabilities [2]. Haloarchaea are one among them, exhibiting remarkable adaptations to high-saline environments. Salinity, pH, and oxygen are three key physicochemical features that influence the microbial diversity of saltpans [3]. High salinity was often associated with increasing proportions of Archaea, especially those belonging to the Halobacteriales order within the Halobacteriota phylum [4], thereby reducing overall microbial diversity. Additionally, the microbial community structure varies in response to changes in the physicochemical parameters of their environment, including salinity, oxygen concentrations, and metabolic activity [5]. Earlier studies have focused on the isolation of culture-dependent and halotolerant enzyme-producing bacteria with recent studies focussed on documentation of bacterial diversity [6–8].

Recent research on the metagenome of microbial communities from saltpans has revealed a notable diversity of microbes with bio-prospecting potential. These communities represent important sources of salt tolerant enzymes for industrial applications [9], bioactive compounds with pharmaceutical relevance [10], and microbial rhodopsin with applications in biosensing, optogenetics, and bioenergy conversion [11]. These bacteria are resistant to harsh environments because they have stress adaptation genes linked to osmotic balance, membrane modification, and DNA repair mechanisms.

The salt production cycle comprising the pre- and post-harvest phases, alters the physicochemical environment in the saltpans, supporting varied microbial communities. The pre-harvest phase was characterized by a progressive increase in salinity levels due to evaporation, whereas, the post-harvest phase is characterized by moderate saline conditions due to salt drainage [12]. These transitions significantly influence the microbial community composition and associated metabolic pathways, particularly those involved in osmoadaptation, phototrophy, and biogeochemical cycling [13]. Insights into the microbial dynamics at each of these phases will provide critical information about the adaptability and ecological roles of halophilic communities. These investigations can also shed light on the potential role of halophilic microbial communities in nutrient turnover and possible biotechnological uses, including the synthesis of bioactive compounds and biosaline agriculture [14]. Metagenomic analyses focused on community structures and functional genes can reveal pathway-level shifts associated with bacteriorhodopsin-based phototrophy, sulfur cycling, and compatible solute biosynthesis, which are critical for ecosystem functioning in extreme salinity gradients [15].

The Vedaranyam saltpan, located along the southeast coast of Tamil Nadu state, is the second-largest producer of salt in India. These saltpans represent a unique and extreme environment characterized by high salinity and varying physicochemical parameters, making it an ideal habitat for extremophiles. In the present study, we used whole-genome metagenome sequencing to characterize the dynamics of microbial community structure between pre and post-harvest stages of salt production in Vedaranyam Salt Pan. High-throughput sequencing coupled with advanced bioinformatics analysis was employed to assemble, annotate, and classify the metagenomic data. Understanding these community and functional shifts not only advances our knowledge on microbial adaptation and survival in extreme environments but also supports the sustainable exploitation of saline habitats for potential biotechnological applications.

Materials

Study Area and Sample Collection

Surface sediment samples were collected from a Vedaranyam salt pan (10.377806°N, 79.849515°E) (Fig. 1) during two key stages of the salt production cycle: the pre-harvest period in February and the post-harvest period in June. During each phase, surface sediment samples (5 cm depth) were collected in triplicate using a sterile spatula and placed into sterile collection bags. All collected samples were immediately transported to the laboratory in dry ice containing a sampling box and subsequently stored at − 20 °C until further analysis. The collected samples were carefully screened to evaluate the physicochemical characteristics associated with different production stages of the saltpan ecosystem.

Fig. 1.

Location of the Vedaranyam sampling site. (A) Map showing the geographical location of Vedaranayam along the southeastern coast of India. (B) preharvest stage of the salt pan at Vedaranyam, showing crystallization prior to salt collection. (C) Postharvest stage of the salt pan at Vedaranyam, showing the salt pan after harvest with residual salt deposits and salt pan with fresh sea water

Physicochemical Analysis of Sediment Samples

The sediment samples were homogenized and 50 g of each sample was suspended in 100 mL of Milli-Q water (Millipore, USA). The suspensions were thoroughly stirred to ensure uniform mixing and then centrifuged at 5,000 rpm for 10 min at 4 °C to obtain the supernatant for physicochemical analysis. A digital pH meter (Eutech, Germany) was used to measure the pH, and a handheld refractometer (ATAGO, Japan) was used to detect the salinity. An automated SKALAR analyzer (Skalar, Netherlands) was used to measure the concentrations of nitrite (NO₂⁻), nitrate (NO_₃⁻), ammonia (NH_₃), phosphate (PO_₄^³⁻), calcium (Ca^²⁺), and silicon dioxide (SiO_₂) in accordance with established procedures [6]. All analyses were performed in triplicate to ensure precision and reproducibility.

Statistical Analysis

The physicochemical parameters of the preharvest and postharvest sediment samples were analysed for statistical significance using one-way analysis of variance (ANOVA). The mean values and standard deviations were calculated for each parameter, and the level of statistical significance was denoted.

DNA Extraction and Illumina Sequencing

The salt pan sediment samples were thawed to room temperature, and a 200 mg sediment sample was resuspended in phosphate-buffered saline (PBS) and homogenized in a rotary shaker at 150 rpm for 5 min. The mixture was then subjected to low-speed centrifugation at 700 rpm in a centrifuge (Remi K-70, India) to remove coarse sediment particles. The collected supernatant was further centrifuged at 10,000 rpm (Eppendorf, Germany) for 10 min. The pellet was collected and used for DNA extraction according to Zhou et al. [16], . This method has been widely employed for DNA extraction from natural environments, particularly soil samples.

The collected pellet was mixed with 500 µL of extraction buffer comprising 100 mM Tris-HCl, 100 mM EDTA (pH 8.0), 100 mM sodium phosphate (pH 8.0), and 1.5 M NaCl. The mixture was centrifuged at 10,000 rpm for 5 min at 25 °C, after which the supernatant was discarded. Subsequently, 500 µL of extraction buffer, 50 µL of 10% (w/w) CTAB, 50 µL of 20% (w/w) SDS, and 10 µL of proteinase K (20 mg/mL) were added to the pellet. The reagents were mixed thoroughly and incubated at 55 °C for 2 h. After incubation, the samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was carefully transferred to a new microcentrifuge tube. An equal volume of chloroform: isoamyl alcohol (24:1, v/v) was added, mixed gently, and centrifuged at 10,000 rpm for 20 min. The aqueous phase was carefully transferred to a fresh tube, and 600 µL of isopropanol was added. The mixture was incubated at room temperature overnight to precipitate the DNA, followed by centrifugation at 14,000 rpm for 30 min. The resulting DNA pellet was washed with 70% (v/v) ice-cold ethanol, air-dried, and finally resuspended in Milli-Q water (Millipore).

Due to the high salinity and significant organic matter content characteristics of saltpan sediments, DNA extraction posed significant technical challenges, including low yield and the presence of enzymatic inhibitors. To overcome these bottlenecks and ensure sufficient high-quality DNA for deep sequencing, extractions from two independent sediment samples per production phase were pooled. This biological pooling strategy was employed to generate a representative composite metagenome for each phase to meet the input requirement for the sequencing platform. DNA quality was determined using a Qubit 4 fluorometer. Whole genome metagenome libraries were prepared using the NEBNext Ultra DNA library preparation kit. The clean fragments were indexed using limited cycle PCR to generate final libraries for paired-end sequencing on the Illumina NovaSeq platform at MedGenome Labs Pvt. Ltd., Bangalore, India.

Metagenomic data Analysis

Data Pre-Processing and Quality Assessment

The raw paired-end reads were checked for initial quality using FASTQC (v0.12.1). The raw sequences are submitted to Sequence Read Archive (SRA). Following the QC, the adapters were trimmed using fastp (v0.24.1) with a Phred quality score 20 and a minimum length filter of 50 bases for the trimmed reads.

Assembly and Binning

MEGAHIT (v1.2.9) was used for assembly with the –presets meta-sensitive option, minimum contig length of 1000 bp and multi-k-mer strategy (21, 29, 39, 59, 79, 99, 119, 141). The metagenomic contigs were assembled and mapped against the trimmed fastq sequences using BWA-MEM (V.07.17-r1188). Genome coverage was not used for relative abundance estimation in read based profiling; coverage depth (calculated using jgi_summarize_bam_contig_depths) was used exclusively for contig binning during the MAG assembly workflow. Metagenome binning was performed using two independent algorithms to maximize MAG recovery: MetaBAT2 (v2.15) was run using tetranucleotide frequency and differential coverage profiles with a minimum contig length of 2500 bp, minimum cluster size of 200 kb, and default sensitivity parameters. MaxBin2 (v2.27) was run using the 107 marker gene set with a minimum contig length of 2500 bp. Scaffold-to-bin mapping tables were generated from each binner output using the Fasta_to_Contig2Bin.sh utility. Bin outputs from all available binning algorithms were integrated and refined using DAS- Tool (v1.1.7) (Dereplication, Aggregation and Scoring Tool) with a score threshold of 0.5 and Diamond as the search engine. DAS-Tool employs a dereplication and scoring strategy based on single-copy gene content to select an optimised, non-redundant set of bins from the combined input. Bin quality was assessed using CheckM (v1.2.4) using the lineage-wf workflow. Quality metrics including completeness, contamination and strain heterogeneity were calculated for all bins. MAGs were classified according to MIMAG (Minimum Information about a Metagenome-Assembled Genome) standards: High-Quality MAGs (completeness ≥ 90%, contamination ≤ 5%) and Medium-quality MAGs (completeness ≥ 50%, contamination ≤ 10%). Bins not meeting medium quality thresholds were excluded from downstream analysis.

Taxonomic Classification

Taxonomic profiling was performed using a combination of read based classification and genome resolved approaches (MAGs). Taxonomic classification of the MAGs was performed using GTDB-Tk (v2.3.2) against GTDB release 226 database. For the read-based method, trimmed paired-end reads were classified using Kraken2 (v2.1.3) with the standard database (Build: 2/26/2026), applying a confidence threshold of 0.1. The database which includes archaea, bacteria, and viral reference genome sourced from the NCBI RefSeq Database. Relative abundance values presented in all figures were derived from Bracken-estimated read counts. Bracken redistributes reads classified by Kraken2 to the specified taxonomic level (phylum, class, or species) using a Bayesian probabilistic framework. Fractional relative abundances were calculated as the proportion of reads assigned to each taxon relative to the total classified reads at that taxonomic level, expressed as a percentage.

Functional Profiling and Metabolic Reconstruction

The trimmed paired end reads were analysed using the DiTing tool (https://github.com/xuechunxu/DiTing) to determine the relative abundance of metabolic and biogeochemical functional pathway (C/N/S) of bulk metagenome. The Transcripts Per Million (TPM) method was used to calculate the gene- level abundance, which normalizes for both gene length and sequencing depth. Relative abundance of different biogeochemical pathways were subsequently derived by aggregating the TPM values of constituent marker genes (KO numbers) using pathway specific-formulae that account for alternative enzymatic routes, as defined in the DiTing pipeline.

The metabolic potential and functional repertoire of the ten high-quality MAGs selected for MAGs were characterized using the METABOLIC-C (Metabolic and BiogeochemicaL mOdular iNteraction Chromosome-level) pipeline. This integrated workflow was employed to perform comprehensive functional annotation and biogeochemical pathway reconstruction. Core metabolic functions were identified by scanning sequences against Hidden Markov Model (HMM) databases (KOfam, TIGRFAM, and Pfam). We measured total hits (HMMHitNum) and identified specific metabolic markers (Functionhit). Metabolic modules for carbon, nitrogen, and sulphur cycles were identified using the KEGG database (KEGGModuleHit) and pathway completeness was validated by verifying each enzymatic step (KEGGModuleStepHit). The complex carbohydrate breakdown was assessed using the dbCAN2 database (dbCAN 2Hit), and proteolytic potential of peptidases and proteases was identified with the MEROPS database (MEROPSHit).

Results

Physicochemical Analysis of the Sediments

The physicochemical properties of the sediment samples taken from the Vedaranyam salt pan at the preharvest and postharvest stages are displayed in Table 1. During the preharvest phase, the salt pan exhibited higher salinity (3.5 M NaCl) compared to postharvest phase (1.5 M NaCl), due to the evaporation of salt. The calcium concentration in the postharvest sample was 1070 µg/L, which was higher than the 787 µg/L in the preharvest sample. Additionally, the postharvest phase had higher silica (SiO₂) contents (6.88 µg/L) than did the preharvest phase (5.59 µg/L). Conversely, the phosphate concentrations were marginally greater in the preharvest phase sample (6.56 µg/L) than in the postharvest sample (6.39 µg/L). Postharvest had comparatively greater levels of ammonium (NH_₄) (3.04 µg/L) than did preharvest phase (2.33 µg/L). In both samples, the levels of nitrite (NO_₂) remained at 0.07 µg/L. The concentrations of combined nitrate and nitrite (NO_₃ + NO_₂) varied slightly, ranging from postharvest (1.65 µg/L) to 1.78 µg/L preharvest phase.

Table 1.

Physicochemical analysis of sediment samples from the Vedaranyam salt pan during preharvest and postharvest phase. The statistical significance was determined using one way ANOVA, and Post hoc Tukey’s Honest Significant Difference (HSD) test was applied to identify the pairwise difference between sites. Significance levels are denoted as p < 0.05(*), p < 0.01(**), p < 0.001(***), and ns indicates no significance

Sediment parameters	Production phase	Mean	SD	Significance
Ca (µg/L)	Postharvest	1073.00	3.00	***
	Preharvest	784.33	2.30	***
NO₂ (µg/L)	Postharvest	0.50	0.37	Ns
	Preharvest	0.09	0.01	Ns
NO₃+NO₂ (µg/L)	Postharvest	1.65	0.01	***
	Preharvest	1.77	0.01	***
NH₄ (µg/L)	Postharvest	3.06	0.03	***
	Preharvest	2.32	0.00	***
SiO₂ (µg/L)	Postharvest	6.89	0.01	***
	Preharvest	5.57	0.01	***
Phosphate	Postharvest	6.37	0.01	***
	Preharvest	6.60	0.04	***

Taxonomic Classification

Read Based Classification

The salt pan microbial communities’ taxonomic composition revealed distinct phase dependent shifts at phylum, class and species levels. The phylum-level distribution showed a distinct trend in the microbial composition of both the preharvest and the postharvest samples, with notable variations in relative abundance (Fig. 2). Methanobacteriota was the most dominant phylum in both samples, accounting for 53.5% of the total abundance in preharvest and 48.4% in postharvest. This was followed by Pseudomonadota, which contributed 38.4% in preharvest and 38% in postharvest. In contrast, Cyanobacteriota showed a notable increase from 5.5% in preharvest samples to 10.6% in postharvest samples. Other phyla, including Rhodothermota, Bacillota, and Actinomycetota were present in low abundance (< 2.0%) and exhibited minimal differences between the phases.

Fig. 2.

Relative abundance (%) of bacterial and archaeal phyla in preharvest and postharvest salt pan sediment samples from Vedaranyam. The stacked bar chart displays the top ten dominated phyla identified through metagenome sequencing

Halobacteria predominated in both phases at the class level, accounting for 53.2% in preharvest phase and 48.2% in postharvest samples (Fig. 3). In the postharvest phase, Alphaproteobacteria decreased slightly from 17.5% to 16%, whereas Betaproetobacteria increased from 10.4% to 13.3%. A notable increase was exhibited in Cyanophyceae, which ranged from 5.5% in preharvest to 10.6% in postharvest phase samples. Other classes like, Gammaproteobacteria, Rhodothermia, Actinomycetes, and Bacilli remained at modest abundance with slight variations.

Fig. 3.

Relative abundance (%) of microbial classes in preharvest and postharvest salt pan sediment samples from Vedaranyam. The stacked bar chart displays the top twelve dominated classes identified through metagenome sequencing

The comparative heat map analysis (Fig. 4) revealed notable phase dependent shifts in microbial community structure between the preharvest and postharvest salt pan sediments. In the preharvest phase higher abundance of phototrophic and extremophilic haloarchaea species were observed, which includes Salinibaculum marinum, Halosegnis rubeus, Halorubrum salinarum, Haloarcula sp. CBA1115 and the carbohydrate-utilizing, sulfur-reducing haloarchaeal isolates Halapricum desulfuricans were constantly abundant in both phases but showed reductions in the postharvest phase. The cyanobacterial representative Dactylococcopsis salina revealed a substantial increase in the postharvest phase, becoming one of the dominant taxa. In addition, bacterial species such as, Sulfitobacter faviae, Roseovarius indicus, and Halorarius halobius exhibited increased abundance in the postharvest phase, whereas species like Guyparkeria halophila showed a marked decrease. Furthermore, a large number of archaea and bacterial species present in the preharvest phase, entirely disappeared in the postharvest phase. Overall, the results signify the shift from archaeal dominance toward increased bacterial and cyanobacterial representation following salt harvesting (postharvest).

Fig. 4.

Heatmap showing the relative abundance (%) of the top 30 dominant microbial species in preharvest and postharvest salt pan sediment samples from Vedaranyam. Color intensity represents relative abundance, ranging from low (purple/blue) to high (yellow/ red)

Genome Based Classification

To complement read based classification and overcome the limitations of unclassified reads, genome based resolved metagenomics was employed. We could recover 13 bins from the preharvest phase and 2 bins from postharvest samples. Based on DAS Tools and CheckM refinement, 9 bins from preharvest and 1 bins from postharvest samples were further taken for downstream analysis as High quality (HQ) and medium quality (MQ) MAGs (Completeness > 90%, contamination < 10%). The preharvest MAGs for high quality 9 bins represent the phylum Patescibacteriota, Verrucomicrobiota, Babelota, Bacteroidota, Alphaproteobacteria and halophilic, Halobacteriota. In the postharvest sample obtained a single MAGs dominated by Patescibacteriota representatives. Overall, the recovered MAGs exhibit successful genome reconstruction and reveal taxonomic shifts between preharvest and postharvest microbial communities. The GTDB-tk classification of 10 MAGS (9 from preharvest and 1 from postharvest) is given in Table 2. One MAG (Bin 01) from both preharvest and postharvest are classified under the same phylum Patescibacteriota and 8 other phyla in Preharvest samples.

Table 2.

Metagenome recovered MAGs from the preharvest phase of the sediment sample

MAG ID	Taxonomic assignment	Completeness (%)	Contamination   (%)	BioSample accessions
Preharvest
Bin 01	Bacteria Patescibacteriota; Minisyncoccia;	86.05	3.45	SAMN58069190
Bin 06	Bacteria Patescibacteriota; Minisyncoccia;	81.9	1.72	SAMN58069191
Bin 12	Bacteria Verrucomicrobiota; Verrucomicrobiia; Opitutales; Puniceicoccaceae; Puniceicoccus	94.43	1.62	SAMN58069192
Bin 15	Archaea; Halobacteriota; Halobacteria; Halobacteriales	88.78	8.26	SAMN58069193
Bin 16	Bacteria; Babelota; Babeliae; Babelales; Babelaceae	83.15	0	SAMN58069194
bin 19	Bacteria; Bacteroidota; Rhodothermia; Rhodothermales; Salinibacteraceae; Salinibacter	65.68	2.05	SAMN58069195
Bin 20	Archaea; Halobacteriota; Halobacteria; Halobacteriales; Haloarculaceae; Halosegnis	95.04	2.21	SAMN58069195
Bin 24	Bacteria; Pseudomonadota; Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae; Rhodosalinus	91.83	2.21	SAMN58069197
Bin 25	Bacteria; Bipolaricaulota; Bipolaricaulia	86.44	1.69	SAMN58069198
Postharvest
Bin 01	Bacteria : Patescibacteriota; Minisyncoccia	86.05	3.45	SAMN58069199

Alpha Diversity

Alpha diversity metrics revealed that both the preharvest and the postharvest phases are composed of diverse and well-balanced microbial communities (Table 3). The Shannon diversity index, which considers both richness and evenness, was 4.63 for preharvest and 4.12 for postharvest, indicating greater taxonomic diversity prior to harvesting. Similarly, Simpson’s index, which emphasizes lower dominance of taxa, yielded higher community diversity in preharvest samples (0.979) relative to postharvest (0.967). These results indicate that both production phases support complex microbial communities with high evenness (Table 3). In contrast, evenness was slightly higher in postharvest sediment (0.809) compared to preharvest sediment (0.738), revealing a more uniform distribution of taxa despite reduced species richness following harvesting activities.

Table 3.

Alpha diversity indices of the preharvest and postharvest microbial communities from the Vedaranyam salt pan

Sediment Sample	Observed Species	Shannon index	Chao1	Simpson index	Inverse Simpson	Pielou’s evenness
preharvest	538	4.63	537	0.97	48.36	0.73
postharvest	164	4.12	164	0.96	31.01	0.80

Functional Potential of the Saltpan Communities Differs in two Phases of Salt Production

Biogeochemical (C/N/S/DMSP) Pathways of Bulk Metagenome

Glycolysis associated genes (glk, pfk, pyk) showed the highest abundance in the postharvest sample, while the preharvest sample exhibited comparatively lower glycolytic gene representation (Fig. 5). The Entner-Doudoroff (ED) pathway followed a similar trend, with slightly elevated counts in postharvest relative to preharvest. Anaplerotic genes (pyruvate → oxaloacetate) were exclusively enriched in preharvest, while postharvest samples showed nearly zero abundance for this pathway. Photosystem I (psaABCDEF), and the cytochrome b6/f complex (petABCDGLMN) were detected at low but slightly higher levels in preharvest. Among fermentation pathways, fermentation to acetate via acetyl-coA (ach1, eutD, pta, acyp) was absent in postharvest but present in low levels in preharvest. Fermentation to lactate (LDH), formate (pf1D), formate → CO₂ and H₂ (fdh), ethanol, and succinate were detected comparatively at higher levels in the preharvest sample. The overall preharvest sample showed diverse carbon cycling related pathways.

Fig. 5.

Relative abundance of the pathways involved in the carbon cycle. The pie chart indicates the relative abundance of each pathway in each metagenomic sample. The size of pie charts represents the total relative of each pathway, CBB, Calvin Benson Basshom cycle: rTCA, reductive citric acid cycle: WL, Wood Ljungdahi pathway: 3HB, 3-hydroxypropionate bicycle; DHC, dicaroxylate hydroxybutyrate cycle

Nitrogen cycling pathways were notably higher in preharvest samples compared to postharvest (Fig. 6). Pathways involved in dissimilatory nitrate reduction, nitrate → nitrite (narGHI or napAB), assimilatory nitrate reduction, nitrite → ammonia (NIT-6 or nirA), denitrification, nitrite → nitric oxide (nirK or nirS), denitrification, nitric oxide → nitrous oxide (norBC), denitrification, nitrous oxide → nitrogen (nosZ), Nitrification, nitrite → nitrate (nxrAB) were higher in preharvest. Postharvest sample showed markedly reduced representation across all nitrogen cycling pathways, with dissimilatory nitrate reduction, nitrate → ammonia (narGHI or napAB) and nitrification, nitrite → nitrate (nxrAB) are the only pathway with comparatively higher abundance in postharvest.

Fig. 6.

Relative abundance of the pathways involved in the nitrogen cycle. The pie chart indicates the relative abundance of the pathway in each metagenomic sample. The size of pie charts represents the total relative abundance of each pathway. ANRA, Assimilatory nitrate reduction to ammonium; DNRA, Dissimilatory nitrate reduction to ammonium; Anammox, anaerobic ammonium oxidation

Sulfur cycling pathways (Fig. 7) exhibited marked phase dependent compositional shifts between the two production phases. Preharvest phase was dominated by assimilatory and dissimilatory sulphate reduction and thiosulfate oxidation, supporting sulphur incorporation into biomass. Conversely, the postharvest phase was dominated mainly by reductive sulphur processes, particularly dissimilatory sulphate and sulphate reduction, while oxidation pathways were reduced or absent. Sulfur cycling pathway gene abundances were higher in the post-harvest sample compared to the pre-harvest sample for the majority of pathways. Among sulfur cycling pathways, assimilatory and dissimilatory sulfate reduction (sat and aprAB) were the only pathway showing elevated gene abundance in the postharvest samples, while all other sulfur oxidation and transformation pathways were more abundant in the preharvest. DMSP demethylation(DMSP→MMPA →MeSH;dmdBCD or acuH) exhibited the highest gene abundance pathway in preharvest sample followed by dissimilatory sulfate reduction, (sat and aprAB), alternative thiosulfate oxidation (tsdA)and Sulfide oxidation, (fccAB).

Fig. 7.

Relative abundances of the pathways involved in the sulfur cycle. The pie chart indicates the relative abundance of each pathway in each metagenomic sample. The size of pie charts represents the total relative abundance of each pathway. ASR, assimilatory sulphate reduction; DSR, dissimilatory sulphate reduction

Metagenomic analysis revealed significant differences in the distribution and abundance of genes associated with the DMSP (dimethyl sulfoniopropionate) cycle (Fig. 8) between the preharvest and postharvest phases. The preharvest phase environment showed comparably higher abundance of DMSP associated genes than postharvest phase.

Fig. 8.

Relative abundances of the pathways involved in the DMSP cycle. The pie chart indicates the relative abundance of each pathway in each metagenomic sample. The size of pie charts represents the total relative abundance of each pathway

Metabolic Potential of MAGs

The metabolic potential of all 10 MAGs was assessed using the METABOLIC-C pipeline, which examined encoded enzymatic functions across the biogeochemical cycles of carbon, nitrogen, sulfur, and trace elements. The results revealed a broad and balancing biogeochemical cycle distributed unevenly across phyla and harvest stages. Preharvest community exhibiting markedly greater functional diversity than the single postharvest MAG. The recovered bin from the postharvest phase suggests a significant shift in microbial community structure or depletion in biomass or diversity following harvest activities, which warrants further investigation into the resilience of these sediment ecosystems.

CAZYme profiling from METABOLIC-C pipeline revealed presence of 32 carbohydrate active enzyme families, among which 29 belonged to Glycosyl Hydrolase (GH) and 3 belonged to Polysaccharide Lyase (PL) families (Table S1). bin 19 and bin 25 from the preharvest phase demonstrated the highest CAZyme family richness. Of the 32 families observed, 29 were exclusive to preharvest bins, with only GH001, GH013 and GH104 shared across both phases.

Table S2 presents the MEROPS peptidase annotations identified across all 10 metagenomic bins from the Vedaranyam salt pan using the METABOLIC-C pipeline. A total of 68 peptidase families were detected with 301 combined gene hits. Metallopeptidases (class M) were the most abundant, accounting for 26 families and 136 hits, followed by serine peptidases (class S; 19 families, 81 hits) and cysteine peptidases (class C; 6 families, 47 hits). The top three families by hit count were C26 (38 hits), S33 (34 hits), and M38 (29 hits). Peptidase diversity was predominantly enriched in preharvest bins, with bin 24, bin 15, and bin 20 contributing the highest hits (75, 68, and 55, respectively), while the postharvest bin bin01 detected only 6 hits across 6 families. Only 6 families I87, M24A, M48B, S16, S66, and T02 were shared between preharvest and postharvest phases, with the remaining 62 families exclusive to preharvest bins.

Carbon Cycling

All MAGs across both harvest stages encoded pathways for organic carbon oxidation and fermentation. It indicates that heterotrophic carbon remineralisation was a core metabolic function of the Vedaranyam salt pan microbes regardless of harvest stages. However, the ability for intensive carbon transformation was confined to specific preharvest lineages. The Woo-Ljungdahl pathway (WLP) for carbon fixation was identified in preharvest bin 15 (Halobacteriales) and bin 25 (Bipolaricaulota), suggesting that autotrophic CO₂ assimilation under anoxic conditions may contribute to primary production in the brine sample (Fig. 9). Additionally, bin 25 from the preharvest phase determines the complete acetogenesis pathway positioning this lineage as a potential acetogenes in the anaerobic carbon economy of the salt pan.

Fig. 9.

Heatmap depicting the presence and absence of key carbon cycling genes across metagenome assembled genomes (MAGs) recovered from preharvest and postharvest salt pan sediment samples of Vedaranyam. Pre, preharvest: post, postharvest

Complex carbon degradation genes, including cellulose degrading, amylolytic, and chitin degradation enzymes were detected in bin 19 (Salinobacter), bin 12 (Puniceicoccus), bin 24 (Rhodosalinus), and bin 25 demonstrating these taxa collectively mediate the breakdown of structural polysaccharides in the preharvest environment. The aerobic CO oxidation and formate oxidation was determined by bin 15, bin 20 (Halosegnis) and bin 24, further reflecting the metabolic versatility of preharvest Archaea and Alphaproteobacteria.

Acetate to acetyl-CoA conversion was detected in bin 12, bin 24 and bin 25 of preharvest phase environment. Pyruvate oxidation was detected in preharvest phase bin 12, bin 24, and bin 25, while lactate utilisation was determined by bin 19 and bin 25. Notably, the postharvest MAG bin01 (Patescibacteriota) and its preharvest counterparts (bin01, bin06) showed restricted carbon metabolic repertoires. These findings are consistent with the streamlined genomes and discovered episymobtic or fermentative lifestyle of Candidate Phyla Radiation (CPR) bacteria.

In Fig. 9, represent the heat map of the carbon cycle with associated genes for all MAGs. Bin 25 harboured all three marker subunits of the WLP (cdhD, cdhE, and cooS), suggesting the genomic potential for acetogenic CO₂ assimilation under anoxic conditions. In contrast, preharvest bin 15 (Halobacteriales) encoded only CO dehydrogenase subunit cooS, indicating a partial WLP capacity. Acetogenesis genes acdA was detected in both the harvest stages especially, postharvest bin 01 and its preharvest counterpart bin 01 as well as in preharvest bin 15, bin 19, bin 20 and bin 25. An exclusive gene ack was present in bin 12 and pta genes were noted in bin 12, bin 15 and bin 20. Preharvest phase bin 12 harboured both acetogenesis associated genes (ack and pta), suggesting the genomic potential for acetate production via this pathway. Acetate to acetyl CoA conversion gene acs was detected in preharvest bin 12, bin 24 (Rhodosalinus) and bin 25. All three subunits of the CO dehydrogenase complex (coxL, coxM, coxS) were detected in preharvest bin 24, suggesting the genomic potential for complete aerobic CO oxidation in this lineage. In contrast, only a single (coxS) was detected in preharvest bin 15 and bin 25, indicating an incomplete genomic capacity for CO oxidation in these lineages. Preharvest bins 15 and 20 detected formate oxidation genes (fdoG and fdoH), while formaldehyde oxidation genes fghA and frmA were detected exclusively in bin 24, suggesting distinct C1 compound oxidation capacities across these lineages.These findings collectively suggests the genomic potential for diverse C1 compounds oxidation among Archaea and Alphaproteobacteria. Additionally, Pyruvate oxidation genes porA were detected in preharvest bins 15, 20 and 25, while lactate utilisation gene Idh was encoded in preharvest bin 19 and bin 25. Restricted carbon metabolic repertoires were observed in postharvest bin 01, preharvest bin 01, and preharvest bin 06, with only the acetogenesis genes (acdA) across these bins.

Nitrogen Cycling

Nitrogen cycling in the Vedaranyam salt pan revealed a specialised distribution, with the preharvest archaeal MAG bin 15 (Halobacteriales) being the one MAG encoding nitrite oxidation (Fig. 10). These findings suggest that nitrification under high salinity conditions may be restricted to haloarchaeal populations in the preharvest phase. Dissimilatory nitrate reduction was determined exclusively by bin 24 (Rhodosalinus) in the preharvest phase, suggesting this Alphaproteobacterium may initiate anaerobic nitrate respiration. The pathway analysis revealed that complete nitrogen metabolism encompassing nitrogen fixation, ammonia oxidation, nitrite oxidation, nitrite/nitrate reduction, nitric oxide and nitrous oxide reduction, anammox and nitrite ammonification was encoded across all the recovered MAG. In preharvest bin 01, 06 and postharvest bin 01 encoded several nitrogen transformation steps consistent with a role for Patescibacteriota. A nitrate oxidation gene (nxrB) was detected exclusively in preharvest bin 15, while no other nitrification associated genes were identified across any of the recovered MAGs. Dissimilatroy nitrate reduction genes (narG, narH) were detected exclusively in preharvest bin 24.

Fig. 10.

Heatmap illustrating the presence and absence of key nitrogen cycling genes across metagenome assembled genomes (MAGs) recovered from preharvest and postharvest salt pan sediment samples of Vedaranyam. Pre, preharvest: post, postharvest

Sulfur Cycling

According to the bin result from preharvest samples, the microbial community represents the richest sulfur cycling potential (Fig. 11). In the preharvest phase suggesting a key driver of oxidative sulfur transformation with respect to bin 24 (Rhodosalinus) encoded the broadest dissimilatory sulfur repertoire. It includes sulfur oxidation, thiosulfate oxidation (multiple sox gene groups), sulphate reduction, and the driver of oxidative sulfur transformation. In preharvest bin 25 encoded thiosulfate disproportionation, enabling simultaneous oxidation and reduction of thiosulfate, an important pathway for sulfur cycling under fluctuating redox conditions.

Fig. 11.

Heatmap represents the presence and absence of key sulfur and DMS pathway genes across metagenome assembled genomes (MAGs) recovered from preharvest and postharvest salt pan sediment samples of Vedaranyam. Pre, preharvest: post, postharvest

Sulfur oxidation and the conversion of DMSO (dimethyl sulfoxide) to methanesulfonate were detected in preharvest bin 20 (Halosegnis), suggesting archaeal participation in volatile organosulfur cycle, a biologically important process in evaporitic marine environments. The detection of DMS (dimethyl sulfide) to methanethiol interconversion in preharvest bins 15, 19, 20, 24, and 25 indicates a wide range of volatile sulfur compound cycling throughout several lineages. The sulfate adenosine-5′-phosphosulfate (APS) pathway encoding assimilatory sulfate reduction genes were present in the preharvest phase bins 06, 12 and 19, suggesting that sulfur assimilation for amino acid biosynthesis was distributed across the microbial community.

In preharvest phase MAGs (bin 12, bin 15, bin 19, bin 20, bin 24, bin 25) observed organic metabolism, including cysteine, methionine interconversion pathways were widely distributed.

Preharvest bin 24 harboured the most diverse dissimilatory sulfur gene repertoire among the recovered MAGs, including a sulfur sulfur oxidation gene (sdo), thiosulfate oxidation genes (soxA, soxC, soxD, soxX, soxY, soxZ) and a sulfate reduction gene (sat). Thiosulfate disproportionation gene (phsA) was detected in Preharvest bin 25. Sulfur oxidation genes were additionally detected in preharvest bin 20, which also encoded DMSO to DM conversion (msuD). The interconversion of DMSO to DMS was encoded by bin 15, while DMS to methanethiol conversion (dmoB) was broadly distributed across preharvest bins 15, 19, 20, 24, and 25.

Assimilatory sulfate reduction genes (crysD and crysN) were detected in preharvest bins 06, 12 and 19, with bin 19 additionally harbouring cysNC and cysH, indicating a broader sulfur assimilation gene repertoire in this lineage. Organic sulfur and sulfur amino acid metabolism harbouring genes metE, metY, ahcY, aspB, megL were widely distributed across preharvest bin12, bin15, bin 19, bin20, bin 24, and bin 25.

Discussion

In this study, we analysed the taxonomic and functional diversity of the salt pan prokaryotic community during the pre- and post-harvest stage using whole-metagenome sequencing approach. To the best of our knowledge, this is the first report on the metagenomic diversity and functional structure of microbial communities from Vedaranyam saltpan. Our results revealed notable changes in the microbial community structure during the pre- and post-harvest phases with significant implications for the production of bioactive compounds.

Earlier studies by Sørensen et al. [17] showed the dominance of bacterial communities in the hypersaline soils and sediments, especially from the environments with a low to moderate salinity. In contrast, archaeal communities were dominant over the bacterial communities during both pre- and post-harvest phases of samples collected in this study. The higher phosphate levels in the preharvest sample suggest early microbial mobilization prior to peak evaporation or nutrient input from nearby runoff. Increased nitrification activity was observed during the preharvest phase by early colonizing microbial taxa [18]. The increased calcium indicates accumulated residual salts and concentrates brines upon falling water levels toward the end of the harvesting cycle. Elevated postharvest ammonium levels are consistent with increased microbial mineralization, as Pearl et al. [18], reported that organic matter accumulation in microbial mats during late salt pan phases favours ammonification.

Irrespective of the salt production phase, Archael communities dominated the overall microbial community composition, though their % contribution decreased slightly during the post-harvest phase. In parallel, the % contribution of Cyanophyceae and Betaproteobacteria increased during the post-harvest phase. The observed taxonomic changes between preharvest and postharvest phases reflect the strong influence of salinity dynamics on microbial community structure and functions in a hypersaline environment. Changes in salinity, food availability, and ionic composition are known to trigger microbial community assembly in hypersaline environments [19]. The dominance of methanobacteriota during the preharvest phase is characteristic of stable, high salinity conditions typical of salt crystallization stages [20]. In this phase, extreme halophiles are known to outcompete other microbial groups due to their specialized osmoadaptation strategy. Notably, with increasing salinity, the number of archaeal phylotypes increased in brine, whereas the bacterial abundance decreased [21]. Furthermore, studies from Guerrero Negro solar salterns, Taiwanese solar salterns, and the Odiel salt marshes of Spain ; reported Bacteroidetes represent up to 90% of the total Prokaryotes [22, 23, 24], contrasting with the results of the present study archaeal dominance (48–53%) observed in Vedaranyam. In fact, with increase in salinity, archaea may outcompete bacteria and establish themselves more rapidly [25].

Following salt harvesting, a slight decline in archaeal abundance was noticed, indicating the disruption of stable hypersaline conditions during the pre-harvest phase. The post-harvest phase is usually linked to increased nutrient availability and partial dilution from fresh seawater input, which collectively reduce the competitive advantages of strict halophiles. This environmental shift likely facilitates the proliferation of metabolically versatile bacterial taxa, as indicated by the relative stability of Pseudomonadota and the increase in Betaproteobacteria, which are frequently linked to organic matter degradation and nutrient cycling [21]. These findings are broadly consistent with [26], who reported dominance of Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in Goa solar salterns following similar salinity driven microbial community shifts.

The key finding of the study was the pronounced increase in Cyanobacteriota, particularly Cyanophyceae and the species Dactylococcopsis salina, during the postharvest phase. This pattern implies that postharvest conditions create a more favourable environment for phototrophic organisms due to reduced osmotic stress, improved light penetration and the availability of dissolved nutrients released during sediment disturbance. Such conditions enhance primary productivity and may represent an early stage of ecological succession following environmental disturbance. Halorarius halobius and Halosegnis rubeus are the most abundant species in the Vedaranyam salt pan, and these results are broadly comparable to the previous findings of [26], who similarly reported the dominance of these species in a comparable hypersaline environment. Halomicroarcula, although not often reported in metagenomic studies [27], was identified in the salt pan of Vedaranyam suggesting the Vedaranyam salt pan may harbor novel or understudied haloarchaeal diversity.

The comparative heat map analysis (Fig. 4) revealed phase dependent shifts in microbial community structure between the sediment samples. Transitions from archaea to bacteria dominated communities have been widely reported in saline sediment and salt pan environments experiencing decreasing salinity and nutrient depletion [20]. However, in this study, archaeal communities remained dominant during both the phases, despite the decrease in the salinity. At species level, the ability of Haloarchaea taxa such as Haloarcula and Halorubrum to survive both phases indicates their resilience and wide tolerance within hypersaline environments. However, their reduced abundance postharvest emphasized their preference for stable, extreme salinity conditions. In contrast, the increased availability of organic matter during the postharvest phase enhances the abundance of taxa such as Sulfitobacter faviae and Roseovarius indicus suggesting active heterotrophic and sulfur cycling roles in the postharvest microbial community. Several taxa, such as, Halorientalis spp. and Haloplanus were observed in both production phases but showed slight abundance shifts, indicating functional persistence with quantitative restructuring. Salinibacter, one of the most frequently detected bacterial genera in hypersaline environments worldwide [28], was also found in Vedaranyam salterns predominantly during the postharvest phase. Although Salinibacter is typically associated with prolonged and elevated salinity conditions, its presence during the lower salinity postharvest phase (1.5 M NaCl) may reflect its broad halotolerance and capacity to persist through salinity fluctuations rather than requiring peak crystallisation conditions. The occurrence of Salinibaculum marinum in the Vedaranyam salt pan is noteworthy, as this taxon remains poorly documented in Indian solar saltern ecosystems [29]. Detection of S. marinum during both pre-and postharvest phases reveals a broad tolerance of this species to salinity fluctuations (0.9–4 M NaCl) and environmental disturbance. Overall, the non-uniform distribution and distinct clustering patterns observed in the heatmap emphasize spatial and temporal heterogeneity in microbial community composition and support the concept of environment driven microbial succession in saltpan sediments. These results demonstrate that salt harvesting acts as a significant microbial shift, leading to a transition from a stable, archaeal dominated hypersaline community in the preharvest phase to a more dynamic and functionally diverse assemblage in the postharvest phase. Alpha diversity analysis of salt pan sediment samples revealed that both production phases support complex microbial communities with high evenness. Hypersaline environments experiencing constant salinity fluctuations tend to promote a diverse microbial community, including non-halophilic, halotolerant and extremely halophilic members [30].

It is important to note that functional interpretations presented in this study are based on genomic and metagenomic potential, as inferred from gene presence detected through METABOLIC-C and DiTing pipelines. The detection of functional genes and biogeochemical pathway markers indicates the metabolic capacity encoded within the microbial community but does not directly confirm active expression or in situ metabolic activity under sampled environmental conditions. Metatranscriptomic analysis, which captures active mRNA transcripts, would be required to confirm whether these pathways are actively expressed. Nevertheless, genomic potential derived from Metagenomic data represents a well-established and widely accepted proxy for functional capacity in environmental microbiology, and the pathways identified here are consistent with the geochemical characteristics of the sampled environment.

In preharvest communities, anaplerotic genes converting pyruvate to oxaloacetate (OAA) were exclusively enriched, supporting metabolic balance and compatible solute biosynthesis [31], while the postharvest shift toward glycolysis and Dactylococcopsis salina proliferation suggests a transition from carbon sequestration to remineralisation. This organism likely provides the primary source of photosynthetically fixed carbon entering the salt pan food web and available to heterotrophic consumers through organic matter [32].

Nitrogen cycling potential was markedly greater in the preharvest phase, particularly nitrification. The co-occurrence of diazotrophic and denitrifying organisms in the preharvest phase creates a coupled nitrogen fixation and denitrification loop that may buffer nitrogen availability in this nitrogen-limited hypersaline environment. Halothece sp. PCC 7418, harbouring nitrogenase genes (nifH, nifD, nifK), was identified as the primary diazotroph supporting biological nitrogen fixation under hypersaline conditions [33]. In contrast, the postharvest phase is enriched with Paracoccus spp. capable of denitrification and dissimilatory nitrate reduction pathways, indicating nitrogen loss and recycling under reduced nutrient availability in postharvest conditions. This shift aligns with increased organic carbon availability and lower redox potential favouring anaerobic nitrogen transformations.

The existence of sulfur cycling differed markedly between the two production phases; preharvest was dominated by assimilatory and dissimilatory sulphate reduction and sulphur incorporation into biomass. This suggests the coexistence of sulphur oxidizing and sulfate reducing halophile communities supporting efficient sulfur recycling under relatively favourable redox and salinity conditions. The increased salinity and lowered oxygen availability after harvesting likely restrict sulphur oxidizing taxa leading to a simplified sulphur cycle. A sulfur oxidising chemolithotroph, Guyparkeria halophila belonging to the Gammaproteobacteria was the primary organism present in preharvest phase (5.14%). The abundance was reduced 23 fold (0.22%) in the postharvest phase of the salt pan. This organism oxidises reduced sulfur compounds (H₂ S, S⁰, thiosulfate) to sulfate in the presence of sox (sulfur- oxidising) gene clusters to conserve energy for chemolithoautotrophic growth [34]. Sulfate reduction was mediated by both bacterial (Desulfohalobium retbaense, Desulfarculus baarsii, and Desulfobacter postgatei) and archaeal sulfate (Halapricum desulfuricans, Halodesulfurarchaeum formicicum, and Halanaeroarchaeum sulfurireducens) reducing organisms (SROs). Bacterial SROs harbor dissimilatory reductase genes and archaeal SROs representing haloarchaeal sulfate and elemental sulfur reduction. Salinobacter ruber contributed to aerobic sulfur oxidation and xanthorhodopsin based light energy harvesting through both phases.

The strong DMSP pathway representation in the preharvest phase, including the dominance of Salinibaculum marinum (6.98%), suggests active osmolyte linked sulfur metabolism under high salinity conditions, possibly involving uncharacterized auxiliary genes. Previous studies have shown that DMSP production and cleavage to DMS are widespread in marine and coastal systems, driven mainly by phytoplankton and bacteria carrying dddP genes and the alma1, with salinity as a key regulator of DMSP biosynthesis [35]. However, direct investigations of the DMSP cycle in hypersaline saltpan environments remain scarce. In this present study, the presence of halophilic microbial communities played an active role in DMSP cycling in saltpan ecosystems during the preharvest phase. The reduced representation of DMSP pathways in the postharvest phase likely reflect extreme salinity stress and the decline of primary DMSP producers [36, 37]. The DMSP and dimethyl sulphide (DMS) cycle was dominated by members of the Alphaproteobacteria (Roseobacter). The most abundant DMSP degrading bacteria presented in postharvest phase was Sulfitobacter faviae (3.49%) and Roseovarius indicus (3.33%) [38].These organisms harbour the DMSP lyase genes (dddD / dddP) confirms active DMSP cleavage to DMS and demethylation genes (dmdA) which helps DMSP-sulfur into methionine biosynthesis. It indicates the active sulphur turnover driven by halotolerant and phototrophic microorganisms. The dominance of DMSP cleavage and oxidation pathways suggest efficient conversion of DMSP into climate relevant sulphur compounds under less extreme salinity and more oxic conditions. The preharvest dominance of Salinibaculum marinum (6.98%) was stable with osmolyte accumulation under high salinity prior to crystallisation. The model DMSP degrading organism Ruegeria pomeroyi harbouring both dmdA and dddp genes, was present exclusively in the preharvest suggesting phase specific DMSP metabolic strategies.

Collectively, the metagenomic evidence from the Vedaranyam salt pan reveals a highly coupled biogeochemical system in which carbon, nitrogen, sulfur and DMSP cycles are interconnected through shared microbial taxa and metabolic crosslinks. The individual species may harbour multiple genes driving carbon fixation, nitrogen cycling, and oxygen production (e.g.: Halothece sp. PCC 7418). This metabolic versatility within individual taxa facilitates efficient element cycling in a nutrient scarce, energy depleted extreme environment.

This study presents the first genome resolved characterization of the biogeochemical cycling potential within microbial communities of the Vedaranyam hypersaline salt pan, Tamil Nadu using METABOLIC based profiling of ten MAGs [39]. Three major observations were identified: (i) the consistent dominance of ultrasmall Patescibacteriota with conserved metabolic limitations across both harvest stages (ii) the substantial contribution of preharvest halo archaeal and bacterial MAGs to elemental cycling (iii) a clear reduction in functional diversity in the postharvest community. This difference is likely attributable in part to the recovery of only a single postharvest MAG. However, the nature of that single MAG was an ultra-small Patescibacteriota with characteristically reduced genome. This likely reflects altered physicochemical conditions, such as elevated salinity, diminished nutrient availability following salt harvest, which leads to streamlined, metabolically dependent microbes.

Three Patescibacteriota MAGs (class Minisyncoccia) were recovered from both harvest stages representing one of the few reports of this CPR lineage from an Indian coastal hypersaline ecosystem. In agreement with known characteristics of this phylum [40], these MAGs exhibited highly reduced metabolic capacities limited to V/A-type ATPase mediated Na⁺ energy conservation and iron reduction with no evidence for dissimilatory sulphur metabolism, nitrogen cycling or carbon fixation. This conserved metabolic profile across harvest stages is consistent with observations from groundwater [41] and hypersaline soda lake sediments [42] where CPR bacteria show minimal functional variation despite environmental gradients. This supports an episymbiotic lifestyle dependent on host interactions rather than ambient geochemistry. Their recovery from both stages suggests stable persistence in the Vedaranyam salt pan despite the disturbances of harvesting.

The most functionally diverse genome recovered from preharvest sediment was bin 15 (Halobacteriales) which harboured genes associated with nitrite oxidation, iron oxidation & reduction, dissimilatory arsenate and selenate reduction, aerobic CO oxidation, Wood- Liungdahl pathway (WLP) based carbon fixation and Ni-Fe hydrogenase activity. This metabolic diversity is comparable to haloarchaeal MAGs from the Bonneville Salt Flats where similar taxa participate in multiple elemental cycles [43]. The detection of WLP associated genes in both preharvest bin 15 (Halobacteriales) and bin25 (Bipolaricaulota) suggests genomic potential for anaerobic autotrophic carbon fixation, representing a possible mechanism of light-independent primary production in preharvest sediments. This findings is consistent with reports of dark carbon fixation in Great Salt Lake sediments [44], where similar taxa have been implicated in autotrophic production under anoxic conditions. Additionally, the association of WLP with Bipolaricaulota, typically enriched at lower salinities [45] suggests that bin 25 may represent a transitional member favoured prior to peak evaporation.

The complete absence of N₂ fixation, ammonia oxidation, nitrite reduction, nitric oxide and nitrous oxide reduction, DNRA and anammox from all the recovered MAGs, indicates that these nitrogen transformation pathways are suppressed under the high osmotic stress experienced in hypersaline environment [46].

Dissimilatory sulphur metabolism emerged as the dominant biogeochemical process in the preharvest community. Preharvest bin 24 (Rhodosalinus sediminis; Rhodobacteraceae) harbour an extensive sulphur metabolism repertoire including sulphur oxidation, multiple thiosulfate oxidation (sox) gene clusters and sulphate reduction. This sulfur metabolism, along with a complete electron transport chain, indicates activity across redox gradients in salt pan sediments. Thiosulfate disproportionate encoded by bin 25 further suggests the genomic potential of Bipolaricaulota in saline environments [45]. The widespread presence of genes involved in dimethyl sulphide (DMS) to methanethiol inter-conversion across multiple MAGs (bin 15, bin 19, bin 20, bin 24 and bin 25) highlights cooperative archaeal and bacterial involvement in volatile organosulfur cycling, a key process in coastal evaporitic systems [46]. These findings are consistent with reports from Lake Barkol [47] and Siberian soda lake sediments [48], where diverse microbial taxa collectively drive interconnected sulphur transformations.

The detection of dissimilatory arsenate reduction in bin 15 and arsenite methylation in bin 12 and bin 15 indicates active arsenic cycling within the Vedaranyam salt pan which is ecologically relevant given arsenic enrichment in coastal Indian sediments. The concurrent presence of arsenate and selenate reduction in bin 15 further suggests their role in trace element cycling in solar salterns warranting further geochemical validation. A notable contrast was observed between the functionally diverse preharvest community and the postharvest condition where only a single Patescibacteriota MAG was identified. This suggests that salt harvesting may substantially reduce the functional complexity of the microbial community and significantly simplify the microbial ecosystem. Harvesting involves mechanical disturbance, reduced brine depth and altered nutrient dynamics which may suppress metabolically complex organisms while favouring minimal and episymbiotic bacteria. While metatranscriptomics was out of scope of this study, the functional genes identified through METABOLIC-C and pathway abundances estimated by DiTing provide a robust foundation for inferring the biogeochemical roles of the microbial community. Gene based functional inference from Metagenomic data is inherently a measure of metabolic potential; however, the co-occurence of multiple genes within complete or near complete pathways, particularly within high quality MAGs, strengthens the inference that these organisms possess the genomic machinery necessary to carry out the indicated metabolic functions. Future metatranscriptomics studies targeting the environment would allow direct confirmation of actively expressed pathways under in situ conditions.

Despite the novel insights delivered by this study, several limitations must be acknowledged. Sampling was conducted at a single salt pan across two time points representing distinct production phases, which restricts the generalizability of findings across broader hypersaline habitat. Metagenomic sequencing was performed from pooled DNA libraries per phase due to technical constraints of DNA extraction from high saline, humic-rich sediments, preventing formal statistical analysis of community level differences between phases. Genome resolved cross-phase comparison was limited by uneven MAGs recovery. All functional inferences in this study are based on genomic potential derived from Metagenomic data.

Conclusion

This study provides the first metagenome characterization of the microbial communities from Vedaranyam salt pan, uncovering a highly resilient microbial ecosystem that undergoes distinct taxonomic and functional restructuring during the salt production cycle. The transition from preharvest to postharvest was marked by a shift from autotrophic nitrogen and carbon retention to a heterotrophic, fermentation driven community, dominated by extreme haloarchaea. The recovery of specialized MAGs indicates metabolic specialization among microbial community members, where diverse taxa cooperate to maintain sulfur redox stability and nutrient turnover despite extreme salinity. Ultimately, the metabolic versatility including the ability to reduce metals, detoxify chemical stressors and osmotolerant strategies demonstrates the significant biotechnological potential of these halophilic communities for bioremediation and biosaline agriculture. Furthermore, haloarchaeal communities identified in this study represent a promising reservoir of biotechnologically relevant biomolecules, including haloenzymes, bacteriorhodopsin, biodegradable polyhydroxyalkanoates (PHAs), and carotenoids, demanding further exploration for industrial and biomedical applications.

Supplementary Material

Below is the link to the electronic supplementary material.

Author Contributions

SJ: writing-review & editing, writing-original draft, visualization, validation, methodology, formal analysis, data curation and conceptualization. L SA, BR, RRM : : Review & editing, validation, funding acquisition, supervision, methodology, formal analysis, and conceptualization. KP: Writing-review& editing, visualization, validation, formal analysis, data curation and conceptualization. AVS: Manuscript: review& editing. RT: Visualization, Validation. KR: Visualization, Validation. All the authors have given their consent for the manuscript to be published. HAA, NMM, AAA, SWC : Validation and Writing - review and editing.

Data Availability

The raw data and results will be provided upon request. Raw sequences are submitted to Sequence Read Archives under BioProject _PRJNA1391684.

Declarations

Competing Interests

The authors declare no competing interests.