Deep-sea encompasses a wide diversity of microbiomes including bacteria, fungi and viruses which play crucial significant roles in nutrient biogeochemical cycling thereby imparting majorly to functional biodiversity of these hotspots. Sea mounts harboring microbes with extremophilic properties found in deep oceans could be conserved as living repository by functional metagenomics approach which is a potent source to screen bioactive compounds and novel enzymes thereby could address biological question on developing next generation plant biostimulants. This study outlines construction of fosmid metagenome library and adapted combined strategy of functional and nanopore sequence-based metagenomic screening to unveil phosphatase enzymes from Arabian Sea seamount sediment. About 9068 metagenomic clones were generated with an average insert size of 38 kb and stored in pools of 1024 clones, out of which 42 were found to be positive for phosphatase. Five clones with high phosphatase activity were further characterized and NIOT F41 showed the greatest specific activity for phosphatase (41.2 U/mg). Gluconic (1041 mg/L), oxalic (327 mg/L), and succinic acids (610 mg/L) were the predominant organic acids produced by recombinant clones. Fosmid DNA were extracted from five potential clones for nanopore-based metagenomics sequencing which generated an average of 6,00,786 reads. Taxonomic analysis revealed an abundance of Proteobacteria and Firmicutes phyla harboring phosphate-solubilising bacteria Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus warneri. Furthermore, functional annotation using phosphorus cycling database (PCycDB) predicted variation in relative abundance of phosphatase gene clusters encoding alkaline phosphatase (PhoD, PhoX and PhoA) and acid phosphatase (OlpA, PhoNand PhoC) produced by recombinant clones. In the pot assay, potential metagenomic clones exhibited positive impacts on shoot length (9.1 ± 1.1 cm, p < 0.05), root length (2.05 ± 0.05 cm, p < 0.05), wet biomass (39.3 ± 0.65 mg, p < 0.05), and dry biomass (5.1 ± 1.15 mg, p < 0.05) compared to the negative control indicating significant effect on promoting plant growth. The advanced nanopore sequencing and functional metagenomics methods employed in this study could serve as a marine biodiversity conservation approach for deep-sea microbes hidden in sea mount sediments towards harnessing potential next generation plant biostimulants with promising biotechnological application for sustainable agriculture.

The field of metagenomics has been revolutionized by technological advancements in synthetic biology, bioinformatics, and sequencing which could help to unravel microbiomes and their functional interaction in deep-sea ecosystem with significant bio-prospection perspectives. Functional and sequence-based metagenomics have been recently adopted towards understanding microbiomes of different ecological niche and bio-prospecting novel biocatalysts from uncultured microbes in environmental sources including water and soil. Recent studies proposed that sequence-based metagenomics could enable the identification of potential genes involved in biogeochemical cycling, whereas function-based screening involves the development of clones from metagenomic libraries of various environmental niche to bio-prospect enzymes with a specific function. Hence, it would be essential to combine both approaches for conservation of deep-sea microbiome through developing functional metagenome libraries of extreme environments, which would result in preserving biodiversity hotspots and better characterization of their enzymes for industrial application.

Oceans, as a habitat, provide a complex and extraordinary physical and chemical environment that is essential for all types of microorganisms, particularly marine bacteria, viruses and protists to thrive in specific ecological niche. Deep-sea sediments, within marine habitats, constitute a harsh environment for microbial populations due to high pressure, low temperature, lack of light, poor nutrient availability and are recognized as well adapted versatile environment with understudied oceanic ecosystems, which imparts the significant necessity for conservation strategies in this fragile ecosystem to ascertain their future sustainability. Seamounts are typically regarded as hotspots of species diversity and oases of biomass abundance due to their distinct ecosystems in the deep ocean. Considerable efforts have been undertaken in the last few years to investigate the diversity, ecology, and function of the prokaryotes that live in seamount habitats through the use of metagenomics, which has made it possible to record the diversity of microbes and investigate their functional potential in the ocean, where more than 99% of microbes remain yet uncultured. Recent bathymetric surveys report on many of the selected seamount region in the Laxmi basin of Arabian Sea indicating their volcanic origin, which suggests that sea mounts of Arabian Sea could be potential hotspots for bio-prospection of industrially important enzymes from marine microbes with extremophilic properties. Hence, it is proposed that metagenomic clone libraries could be used for conserving unculturable microorganisms as living repository of Indian deep-sea seamounts samples for a longer period towards addressing solutions through exploring sustainable biotechnological applications from recombinant microbes without exploiting the Oceanic environment for routine sampling and collection of samples.

A microbial community as a whole can be treated as a super- or mega-organism, whose metabolic pathways and networks are accessible with the use of metagenomics approaches. Metagenome sequencing analysis has emerged as a potent technique for comprehending the biogeochemical cycling (e.g., phosphorus, nitrogen, carbon, sulphur, and metals) driven by microbes in different environments. Oxford Nanopore Technologies is a powerful metagenomics tool, producing long reads that resolve complex genomic regions like repetitive or conserved elements which effectively recovers complete metagenome assembled genomes from various samples, including sludge, water, sediments, and feces, demonstrating its versatility. Its ability to deliver fast results at a lower cost makes it an invaluable tool for diverse applications, from agricultural studies to medical research. Nanopore sequencing based method has recently emerged as significant metagenomics technique, which has been implemented in our earlier studies towards understanding microbial diversity and harnessing potential of deep-sea environment including Bay of Bengal and Arabian Sea sediment. Several detailed studies have been conducted on the cycling and fractionation of phosphate in the deep ocean of Arabian Sea. Recent research showed that the formation of a coastal upwelling zone in the Southeastern Arabian Sea during southwest and northeast monsoons results in the cycling of Phosphorus. The genes responsible for phosphorus cycling (PCGs) and the microorganisms involved in the process are not thoroughly studied. Alkaline phosphatases (EC 3.1.3.1) are the most investigated phosphoric monoester hydrolases and its genes phoD and phoX are shown to be more prevalent in marine environments than phoA. The gene encoding a new alkaline phosphatase was discovered in a metagenomic library constructed from ocean-tidal flat sediments of Korea's west coast which was identified to be a part of a newly discovered PhoX family, different from the well-researched classical PhoA family. There has been limited research on the gene clusters that play a role in the cycling of phosphorus in deep-sea sediments, leaving a significant gap in our understanding of this crucial process.

Recently, several sequencing techniques have been applied in soil metagenomes to predict the entire sequence of biosynthetic gene clusters in silico. Metagenomic methods have also been adopted to clone and sequence soil genome for screening specific enzymes where its functional analysis could lead to more accurate metagenome annotation than sequence-based analysis alone. Functional screening of enzymes through metagenomics could find potential implication to address the challenges associated with low enzyme expression and complex purification processes. The most appropriate vector must be chosen to insert the metagenomic DNA depending on the objective of functional screening wherein expression vectors like fosmids, cosmids (< 40 kb), bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs) (> 40 kb) are useful for larger biosynthetic gene clusters. Recent reports unveil that soil metagenomes could be cloned using cosmid or fosmid library to identify the biosynthetic gene clusters efficiently which is adopted in the present study for constructing metagenomic libraries from Arabian Sea seamount sediments.

In general, it is necessary to confirm functional screening of any enzyme from metagenome clone library by sequencing-based methods. Recent reports on sequencing of fosmid clone libraries with large inserts revealed that functional screening of fosmids clones with specific enzyme activity must be de novo sequenced which is more significant towards identifying specific gene targets. Earlier reports reveal that numerous novel genes that encode variant enzymes from marine environments have been discovered by functional metagenomics, which include lipase from marine sponge, cellulase from Coral, Siderastrea stellata and DNA polymerase from glacial ice. Fosmid metagenome library from ocean tidal flat sediments has been previously reported to have undergone fluorescence-based screening for lipase, cellulase, and phosphatase through GESS-FACS. Recent progress in functional metagenomics screening methods combined with advances in next generation sequencing technologies could result in efficient bio-prospection of significant enzymes and metabolites from specific environmental niche. These coupled methodologies have been adopted for mining industrial biocatalyst esterase from deep-sea samples including thermal water of alkaline hot spring at Lobios (Ourense, Spain) and Atlantis IIbrinepool, Red Sea(Indic Ocean) even amylase from Arabian Sea sediment.

Several recent reports reveal the significance of Phosphate-solubilizing bacteria (PSB) as resourceful biofertilizers for sustainable agriculture due to its ability to secrete alkaline phosphatase (phoD), acid phosphatase (phoC) and phytase enzymes that can solubilise insoluble phosphorus in the soil for efficient absorption, uptake and utilization by plants. Currently, only a small number of such plant growth-promoting bacteria are used in agriculture wherein our recent findings revealed two potential phosphate solubilising bacteria Priestia megaterium and Bacillus velezensis, from seamount sediments of Arabian Sea which promotes plant growth. Recent approaches target the use of synthetic synergistic communities, obtained from one or multiple soil microbiomes that show best growth conditions even under biotic or abiotic stress, aiming at a broader target range, higher robustness and a better plant-beneficial efficacy. Furthermore, advanced nanopore sequencing methods is emerging as a potent tool to generate plant biostimulants that could help to increase crop yield, thereby resulting in development of next generation fertilizers to overcome harmful impact of synthetic molecules on environment. However, there are no reports on bio-prospection of plant biostimulating enzymes from biodiversity hotspots including deep-sea sediment which could serve as a living metagenomic repository of extremophilic enzymes and biomolecules that could be used for sustainable agriculture with implication in environmental restoration.

This study aimed to bio-prospect phosphatase enzyme using functional metagenomics approach by constructing a metagenomics clone library using fosmid vector from seamount sediments of Arabian Sea as methodology to develop biological innovations through conservation of deep-sea microbes. Further, the potential metagenomic clones producing maximum phosphatase were sequenced by nanopore long read technology to understand the microbiome hidden in metagenomic clones of dark seamount sediment and to confirm functional potential of PSB towards identifying phosphate cycling gene expression variation conferring phosphate solubilisation. This study focused to correlate the abundance of PSB from seamount sediment with plant growth promoting properties towards understanding its implication in sustainable agriculture.