Amplicon-Based 16S rRNA Inventory of a Laboratory-Cultivated Hypersaline Halophilic Biofilm from Indonesian Salt Ponds: A Descriptive Pilot of Bacterial–Archaeal Composition

Hypersaline environments such as salt lakes, solar salterns, and saline soils represent some of the most extreme ecosystems on Earth, where salinity levels exceed the tolerance limits of most organisms. Despite their harshness, these habitats sustain diverse halophilic and halotolerant microorganisms that have evolved remarkable physiological and molecular strategies to cope with osmotic stress, high ionic strength, and desiccation. Their activities contribute to global biogeochemical cycles through transformations of carbon, nitrogen, and sulfur, thereby linking hypersaline ecosystems to planetary processes [1, 2]. Because of these unique attributes, hypersaline habitats are often regarded as natural laboratories for investigating the limits of life and the resilience of extremophiles. Beyond ecology, halophiles are increasingly valued for their biotechnological potential, producing salt-tolerant enzymes, Osmoprotectants, and secondary metabolites with applications in biofuels, pharmaceuticals, and food industries [3].

Within these habitats, biofilms represent a particularly critical microbial lifestyle. Unlike planktonic populations, biofilm-forming microorganisms are embedded in an extracellular polymeric substance (EPS) matrix that provides structural integrity, mediates adhesion, and buffers against extreme ionic conditions. The biofilm matrix also creates steep microscale gradients in oxygen, nutrients, and salinity, generating distinct niches that allow diverse taxa to coexist [4, 5]. In hypersaline systems, biofilms often consist of both bacteria and archaea, whose interactions enhance community stability under fluctuating conditions. Consequently, hypersaline biofilms are promising model systems for exploring how fine-scale spatial organization influences microbial resilience and ecosystem functioning.

Despite their importance, biofilms in hypersaline environments remain far less studied than planktonic communities or large microbial mats. Previous research has mainly documented dominant taxa and overall diversity patterns, while the stratified architecture of biofilms, particularly the way bacterial and archaeal members partition ecological roles across baseline inventory, remains poorly characterized. The distribution of niches within the EPS matrix, the influence of baseline inventory on functional complementarity, and the role of interdomain interactions in sustaining survival under extreme salinity are still largely unresolved. Clarifying these aspects is essential, as microscale organization is likely fundamental to the ecological persistence and evolutionary strategies of extremophile communities [1, 6, 7].

Recent advances in molecular ecology provide tools to close this gap. High-throughput 16S rRNA gene sequencing, coupled with bioinformatic approaches, has revealed unexpected microbial diversity in hypersaline environments, including novel lineages with unique strategies for osmotic adaptation, phototroph, and polymer degradation [8, 9]. However, most studies stop at taxonomic inventories. Few have integrated these data into ecological models that explicitly consider spatial structure and interdomain interactions within biofilms. As a result, our understanding of hypersaline biofilms as stratified and cooperative assemblages remains limited.

The present study addresses this knowledge gap by investigating the microbial community of a halophilic biofilm from Indonesian salt ponds. Using high-throughput 16S rRNA sequencing and operational taxonomic unit (OTU) based analysis, characterized taxonomic composition, diversity patterns, and potential ecological roles of bacterial and archaeal members. Specifically, the present a descriptive 16S rRNA amplicon inventory of a halophilic biofilm cultivated in a laboratory microcosm using waters collected along an Indonesian salt-pond salinity gradient. Due to low biomass obtained at each salinity stage, biofilm material was pooled prior to DNA extraction. Therefore, this study does not test spatial stratification or salinity-specific differences. Instead, they provide baseline community composition data from an understudied region and outline priorities for future spatially resolved sampling and functional validation.

A key finding is the unusual dominance of Limimaricola and Oceanicaulis, lineages rarely reported as major components in global hypersaline environments. Their prominence in the Indonesian system suggests biogeographic specialization driven by local environmental gradients. By documenting this baseline inventory and unusual taxonomic composition, the study contributes to broader models of extremophile ecology while highlighting region-specific features that enrich global perspectives. Beyond fundamental insights, these results also have applied relevance, as stratified halophilic biofilms may serve as reservoirs of salt-tolerant enzymes, biosurfactants, and bioremediation agents for high-salinity effluents. This research provides a novel ecological model of hypersaline biofilms as stratified, cooperative consortia where bacterial and archaeal taxa integrate complementary adaptation strategies. It not only fills a critical knowledge gap on fine-scale biofilm architecture but also positions Indonesian salt-pond biofilms as valuable systems for advancing both ecological theory and biotechnological innovation.

3.1 Sequencing output, OTU generation, and annotation

High-throughput sequencing of hypersaline biofilm samples produced over 1.6 million effective reads, with nearly all sequences successfully assigned to known taxa. Clustering at 97% similarity generated 196 OTUs, reflecting substantial microbial diversity. Although dominant taxa shaped most of the community, the presence of singletons contributed to a long-tail distribution, indicating coexistence of abundant and rare members within the biofilm (Figure 1). This pattern is typical of microbial ecosystems where a few lineages dominate, yet low-abundance taxa maintain additional diversity.

Figure 1. Sequencing tags and OTUs from hypersaline biofilm

3.2 Taxonomic composition of the microbial community

Taxonomic classification at the phylum level showed that the biofilm community was strongly structured by Proteobacteria and Halobacterota, which together comprised over two-thirds of the total relative abundance (Figure 2). This dominance reflects the dual bacterial–archaeal foundation of hypersaline biofilms, where Proteobacteria contribute to heterotrophic metabolism and EPS production, while Halobacterota represent the major archaeal lineage adapted to salt-saturated conditions through “salt-in” osmotic strategies.

Figure 2. Relative abundance of dominant microbial phyla in the biofilm

At finer resolution, the cladogram (Figure 3) revealed clear clustering of archaeal taxa from the class Halobacteria (notably Haloferax, Halorubrum, and Haloplanus) alongside bacterial groups such as Halomonas (Proteobacteria) and Salinibacter (Bacteroidota). These lineages are consistent with complementary ecological strategies previously reported for hypersaline biofilms: haloarchaea are typically associated with phototrophy and osmotic balance, while bacterial members often contribute to organic matter degradation and EPS reinforcement. The observed enrichment of these taxa suggests potential metabolic complementarity, although this inference is based solely on taxonomic composition.

Figure 3. Cladogram of bacterial and archaeal genera in the biofilm

Table 2. Top 15 species identified in the hypersaline biofilm

The relatively high abundance of Oceanicaulis and Bacillus (Table 2) distinguishes this community from most previously described hypersaline systems, where these genera are usually minor components. Their prominence may reflect local environmental characteristics of Indonesian salt ponds, indicating a possible biogeographic trend that merits further comparative investigation.

3.3 Alpha diversity and richness patterns

Alpha diversity analysis revealed a moderately rich and balanced community, with 196 observed OTUs and diversity indices indicating even distribution among dominant and minor taxa. Richness estimators (Chao1 and ACE) closely matched observed values, suggesting that most taxa were successfully captured. Importantly, Good’s coverage of 0.9997 confirmed near-complete sequencing depth, providing confidence in the representativeness of the dataset (Table 3). In contrast, the Indonesian biofilm demonstrated both substantial taxonomic richness and balanced representation of bacterial and archaeal lineages, underscoring the ecological uniqueness of its stratified structure.

Table 3. Alpha diversity of halophilic biofilm

Rarefaction analysis (Figure 4) further confirmed that sequencing depth was sufficient, with the curve plateauing near 196 OTUs, while the rank abundance distribution (Figure 5) reflected a community shaped by a few abundant lineages supported by a diverse rare biosphere. Such a pattern mirrors other hypersaline environments where cooperative interactions between dominant taxa and low-abundance specialists are thought to enhance ecological resilience under fluctuating salinity. Thus, the Indonesian biofilm not only fits within global patterns of halophilic diversity but also expands them by demonstrating a more balanced bacterial–archaeal coexistence than many previously studied systems.

Figure 4. Rarefaction curves of hypersaline biofilm sequences

Figure 5. Rank-abundance distribution of biofilm taxa

3.4 Comparative insights with other hypersaline

Comparative analysis of halophilic microbial communities across hypersaline environments revealed consistent dominance of both Bacteria and Archaea, with taxonomic variation depending on location and physicochemical gradients (Table 4). In the current study, biofilm samples from Indonesian salt ponds were dominated by bacterial genera such as Limimaricola, Bacillus, Oceanicaulis, and Halomonas, alongside archaeal taxa including Halohasta and Haloferax.

Table 4. Comparative dominant taxa in global hypersaline environments

3.5 Discussion

3.5.1 Ecological structure and comparison to global systems

The ecological structure of halophilic biofilms revealed in this study demonstrates a highly stratified and functionally diverse microbial consortium. The bacterial domain by Oceanicaulis sp. (28.6%) and Bacillus horikoshii (24.7%), while archaeal representatives such as Haloferax and Halohasta were also consistently detected. This taxonomic pattern indicates that both bacterial and archaeal lineages contribute to the biofilm structure, with dominance shared rather than restricted to a single group. The presence of a long-tail rare biosphere further demonstrated coexistence between abundant and low-abundance taxa within the EPS matrix.

These findings extend previous work by demonstrating that hypersaline biofilms are not homogeneous but organized into a baseline inventory shaped by gradients of oxygen, nutrients, and salinity [2, 23, 24]. The high abundance of Firmicutes such as Bacillus horikoshii may indicate a potential role in EPS production and osmoprotectant synthesis, functions that have been frequently reported for related taxa in hypersaline systems [25], while Alphaproteobacteria such as Oceanicaulis are often reported to degrade complex organic substrates, and their presence here may suggest similar ecological roles [26]. These complementary activities collectively stabilize the biofilm matrix and support resilience under osmotic stress. The co-occurrence of Haloferax and Halohasta illustrates the salt-in strategy of osmotic adaptation [27, 28], contrasting with the compatible solute approach of their bacterial partners. Similar bacterial–archaeal partitioning has been documented in hypersaline lakes and soils [11, 24], but the simultaneous prominence of Oceanicaulis in this system highlights a biogeographic distinction from other global sites.

The EPS matrix, composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA), formed the structural foundation of the biofilm. EPS promotes adhesion to substrates such as the HDPE panels used in this study, while also buffering cells against desiccation and ion toxicity [29-31]. In line with previous studies, eDNA appears to act both as a scaffold and a nutrient reservoir [23]. The presence of rare taxa within this matrix may reflect specialization within micro-niches; however, their roles remain speculative without direct functional validation. This emphasizes the need for cautious interpretation of rare taxa in taxonomic datasets.

The ecological structure observed here also illustrates how environmental filters and microbial interactions jointly shape hypersaline communities. Laboratory simulations across salinity gradients (2–40%) demonstrated shifts in community composition consistent with prior reports: Firmicutes and Proteobacteria are favored at intermediate salinities, whereas archaeal lineages such as Euryarchaeota dominate under extreme brine conditions [13, 24]. Comparative studies from Guerrero Negro and Israeli solar salterns [4, 5, 22] support the view that hypersaline biofilms are governed by a balance of deterministic factors (e.g., salinity, pH, substrate availability) and stochastic processes (e.g., immigration of rare taxa). The Indonesian biofilm is characterized by the dominance of Oceanicaulis and Bacillus within the bacterial domain, together with Haloferax and Halohasta among Archaea. This combination is consistent with baseline inventory functional models proposed in previous studies, although the present data derived from pooled biofilm samples do not directly demonstrate spatial stratification. This structure extends our understanding of how microbial communities persist in extreme niches and highlights regional variations that enrich global perspectives on hypersaline biofilm ecology.

3.5.2 Functional implications and metabolic potential

The taxonomic composition of the hypersaline biofilm suggests important functional roles in carbon turnover, nutrient cycling, and ecological stability under extreme osmotic stress. Dominant bacterial lineages such as Oceanicaulis and Bacillus are taxa commonly associated with the degradation of complex organics and EPS production in other hypersaline systems. While such functions cannot be confirmed from 16S rRNA data alone, the observed taxa imply potential ecological roles contributing to matrix stability and nutrient cycling within the biofilm [21, 32].

Archaeal taxa, particularly Haloferax and Halohasta, add another dimension to metabolic potential. These haloarchaea employ the salt-in strategy of osmotic balance and are frequently associated with rhodopsins and retinal-based proton pumps that support phototrophic energy capture [33]. Light-driven proton transport has been shown to enhance resilience in hypersaline lakes when organic substrates are scarce [2]. In addition, haloarchaea often encode nitrogen cycling pathways, including denitrification, which further contributes to nutrient turnover in extreme saline ecosystems [24]. The coexistence of heterotrophic bacteria and phototrophic/denitrifying archaea thus exemplifies metabolic complementarity, enhancing overall ecological stability.

Rare but persistent taxa such as Roseivivax, Loktanella, and Bifidobacterium pseudolongum were detected at low abundance. Their ecological roles in this system remain unclear; however, their presence highlights possible niche specialization and should be considered as observations for future research rather than confirmed functions. Because 16S rRNA sequencing provides only taxonomic inference, the specific metabolic contributions of these rare members cannot be directly established. Their presence, however, hints at possible niche specialization and redundancy that may reinforce community resilience [11, 22].

Taken together, the inferred functional structure of the halophilic biofilm reflects a consortium where bacteria dominate EPS production and organic matter degradation, archaea contribute to phototrophy and osmotic balance, and minor taxa potentially expand metabolic flexibility. This integrated network may allow the biofilm to act both as a biogeochemical reactor and as a stability reservoir in extreme environments. Beyond ecological significance, these traits could be harnessed for biotechnological applications such as bioremediation of saline effluents, enzymatic production under high ionic strength, and bioenergy generation via light-driven proton pumps.

Nevertheless, these functional interpretations are necessarily putative, as they are based solely on 16S rRNA taxonomic profiles rather than direct genomic or transcriptomic evidence. While such data provide valuable insight into community structure and potential metabolic strategies, they cannot confirm gene content or expression. Future studies employing shotgun metagenomics and metatranscriptomics are therefore essential to validate these predictions, uncover the genetic basis of observed adaptations, and resolve the active metabolic processes underpinning hypersaline biofilm stability.

3.5.3 Extremophile adaptation strategies

Halophilic biofilms persist in hypersaline environments by combining complementary strategies that mitigate osmotic, oxidative, and nutritional stresses. In our dataset, bacterial members such as Bacillus spp. likely adopt the compatible solute strategy, synthesizing or accumulating osmolytes including ectoine, glycine betaine, and trehalose, which stabilize proteins and maintain turgor under high salinity [21, 34]. In contrast, archaeal taxa such as Haloferax and Halohasta employ the salt-in strategy, characterized by high intracellular KCl and acidic proteomes enriched in negatively charged amino acids that preserve protein solubility in saturated brines [2]. The coexistence of these divergent mechanisms within a single biofilm highlights functional redundancy and community-level resilience under fluctuating salinity regimes.

Structural adaptations further reinforce this resilience. EPS production, prominent in Bacillus, provides hydration, ion buffering, and adhesion, while EPS enriched with eDNA may facilitate horizontal gene transfer and the rapid spread of adaptive traits [7]. Previous studies have shown that hypersaline biofilms often exhibit micro-gradients of oxygen and light that support niche specialization. In our pooled community, the coexistence of Oceanicaulis, Bacillus, and Haloferax genera suggests a similar ecological partitioning, though no direct baseline inventory or physicochemical profiling was performed [11, 22].

Additional strategies extend the biofilm’s adaptive capacity. Haloarchaeal rhodopsins (bacteriorhodopsins and halorhodopsins) enable light-driven proton pumping and ATP synthesis under nutrient limitation [2]. Stress-tolerant enzymes such as halophilic amylases, lipases, and proteases, previously reported in Firmicutes and Haloarchaea, retain catalytic activity in high ionic strength, enhancing both ecological persistence and biotechnological potential [24]. The persistence of rare taxa, including Roseivivax and Loktanella, likely reflects niche-specific adaptations that further enhance community resilience [22]. Collectively, these findings show that adaptation in halophilic biofilms is not uniform but stratified and taxon-specific, integrating bacterial compatible solutes, archaeal salt-in mechanisms, and EPS-mediated structural defenses. This dynamic configuration extends previous models of hypersaline adaptation by demonstrating how baseline inventory and taxonomic complementarity jointly sustain biofilm function in one of Earth’s most hostile environments.

3.5.4 Biotechnological potential of biofilm halophiles from Indonesia

The taxonomic composition, particularly the dominance of Bacillus and haloarchaea, indicates possible sources of salt-tolerant enzymes and metabolites. These prospective applications are discussed further in the conclusion as avenues for future research. These enzymes have been explored for industrial processes requiring high ionic strength, including food fermentation, detergent formulation, and biofuel production [21, 35]. The Indonesian salt pond context, with its fluctuating salinity gradients (2–40%), further suggests that local isolates may harbor unique enzymatic adaptations, though experimental validation is still required.

Archaea, such as Haloferax and Halohasta, may also provide resources of interest. Their rhodopsin-based proton pumps and stress-tolerant pigments have been proposed for bioenergy applications and optogenetics. For instance, haloarchaeal rhodopsins have been considered as sustainable bionanomaterials for solar energy harvesting and photonic devices, while carotenoids from halophiles are valued for their antioxidant properties and potential nutraceutical uses [2]. Although these applications remain largely conceptual in the context of this biofilm, they illustrate the biotechnological avenues that could be tested in future studies.

The detection of low-abundance but metabolically versatile taxa, including Roseivivax and Loktanella, further points to potential sources of novel biomolecules. Related lineages have been reported to produce rhodopsin-like proteins and secondary metabolites with antimicrobial activity [11]. In addition, the EPS matrix enriched with extracellular DNA (eDNA) highlights the potential of halophilic biofilms as reservoirs for biopolymer extraction. Halophilic EPS have been studied for their stability under extreme conditions and possible applications as bioflocculants, emulsifiers, or bioplastic precursors [36, 37]. Their metal-binding capacity also suggests avenues for saline wastewater remediation, though direct evidence from this study remains pending.

Positioning these findings within the Indonesian context underscores both ecological and socio-economic relevance. Salt ponds and aquaculture systems in the region face growing challenges from saline effluents and organic waste. Indigenous halophilic biofilms could, in principle, be developed as biofilm-based bioreactors or as sources of extremozymes, offering locally adapted strategies for waste management and value-added production. However, such applications remain potential and require future validation through experimental assays, metagenomic screening, and pilot-scale trials. Thus, the biofilm described here represents not only a valuable ecological model of extremophilic adaptation but also a prospective biotechnological asset for Indonesia, bridging microbial diversity exploration with industrial and environmental innovation.