Climate change and human activities have accelerated soil salinization, threatening global food security and economic stability. Restoring salt-affected soils is essential to expand arable land and ensure sustainable agriculture. However, few studies integrate microbial genomic traits with functional validation in crops under high salt stress. This study evaluated the plant growth-promoting and salt tolerance potential of two microbial strains, Halobacillus marinus SL2 and Halomonas elongata DM6, isolated from the hypersaline environments of Sambhar Lake and Drang Mine, respectively, under 300 mM NaCl stress. Both strains underwent genome analysis and were assessed for their ability to enhance stress resilience and promote wheat growth. They tolerated up to 20% NaCl and pH 9, and exhibited key in vitro traits including phosphate and potassium solubilization, siderophore production, and indole-3-acetic acid (IAA) synthesis. LC-MS/MS analysis revealed that SL2 produced phytohormones such as indole-3-butyric acid, indole-3-propionic acid, acetylsalicylic acid, dihydrojasmonic acid, and kinetin, while DM6 primarily synthesized salicylic acid and kinetin. Both also produced osmoprotectants contributing to salt stress tolerance. In wheat assays, SL2 increased seed germination by 50% (P < 0.001) and DM6 by 47.7% (P < 0.0001); biomass increased by 46.6% (SL2; P < 0.01) and 53.7% (DM6; P < 0.05) over controls. Genomic analysis identified genes involved in ion homeostasis, compatible solute production, and plant growth enhancement. These findings suggest SL2 and DM6 are promising biofertilizer candidates for saline soils, capable of improving crop productivity, reducing chemical inputs, and supporting sustainable agriculture

Solid waste management in the Indian Himalayan Region (IHR) is a growing challenge, intensified by increasing population and tourism, which strain non-sanitary landfills. This study investigates microbial diversity and functional capabilities within these landfills using a high-throughput shotgun metagenomic approach. Physicochemical analysis revealed that the Manali and Mandi landfill sites were under heavy metal contamination and thermal stress. Taxonomic annotation identified a dominance of bacterial phyla, including Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, with genera like Pseudomonas and Bacillus prevalent. Squeezemeta analysis generated 9,216,983 open reading frames (ORFs) across the sampling sites, highlighting diverse metabolic potentials for heavy metal resistance and degrading organic, xenobiotics and plastic wastes. Hierarchical clustering and principal component analysis (PCA) identified distinct gene clusters in Manali and Mandi landfill sites, reflecting differences in pollution profiles. Functional redundancy of landfill microbiome was observed with notable xenobiotic and plastic degradation pathways. This is the first comprehensive metagenomic assessment of non-sanitary landfills in the IHR, providing valuable insights into the microbial roles in degrading persistent pollutants, plastic waste, and other contaminants in these stressed environments.

Domestication procedures or changing habitats of medicinal plants significantly reduces the amount of plant specialized metabolites (PSMs). Previous studies have already shown the direct influence of rhizosphere microorganisms on PSMs. Hence, re-introducing plant-beneficial rhizosphere microorganisms selected from plants' natural habitats back into the rhizosphere during cultivation is expected to increase PSMs production. However, such efforts have not yet been made with Hypericum perforatum L. plants. Consequently, whether PSMs increase, decline, or remain unaffected remains elusive. Nevertheless, previous investigations have demonstrated that despite plant specificity, the PSMs increase after the inoculation of beneficial microorganisms. However, hitherto, the effectiveness of plant-specific (host plant-associated) beneficial rhizobacteria over un-specific (non-host plant-associated) rhizobacteria remains obscure. To fill these knowledge gaps, we first compared the bacterial community structure of wild H. perforatum rhizosphere with the experimental field and found significant discrepancies. We then attempted to reintegrate H. perforatum plants with plant-specific and un-specific beneficial rhizobacteria. The bacterial community assemblages significantly differed between wild and cultivated plants, even after reintegrating plants with plant-specific beneficial rhizobacteria. Nonetheless, plant-specific and unspecific beneficial rhizobacteria have distinctively tailored rhizosphere bacterial community composition. The bacterial species richness (Chao1) and diversity (Shannon index) were significantly highest in the rhizosphere of plants treated with plant-specific beneficial rhizobacteria. Ammonia-oxidizing bacteria (Nitrosomonadaceae_MND1) and Burkholderiales_SC_I_84 were differentially abundant in the rhizospheres of plants treated with un-specific rhizobacteria. Notably, complete ammonia-oxidizing bacteria (comammox Nitrospira), Gemmatimonas, and Vicinamibacteraceae were differentially abundant in the rhizosphere of plants treated with plant-specific rhizobacteria. These taxa were associated with the highest accumulation of PSMs, corroborating the higher expression profiles of pivotal genes regulating their biosynthesis. This study enhances our understanding of how plant-specific and un-specific beneficial rhizobacteria distinctively shape rhizosphere bacterial communities' composition and differentially impact PSMs' biosynthesis, guiding future cultivation of H. perforatum.

Chilling stress significantly impairs seed germination, crop growth, and yield, posing a significant agricultural challenge. This study aimed to evaluate the biostimulant properties of the psychrotrophic bacterium Pseudomonas putida IRS13 and its exopolysaccharide (EPS) in promoting wheat growth under chilling stress (6 degrees C +/- 2 degrees C). Their biostimulant efficacy was compared with chemical growth regulators, melatonin and acetylsalicylic acid. Additionally, key genes and metabolites of strain IRS13 involved in stress tolerance and plant growth promotion were identified. Isolated strain IRS13 was characterized, and it possesses plant growth-promoting activities such as indole acetic acid (IAA) production, nitrogen fixation, potassium mobilization, phosphate solubilization, siderophore production, and 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Wheat seeds and seedlings were treated with strain IRS13, its EPS, melatonin, and acetylsalicylic acid at 6 +/- 2 degrees C. Strain IRS13 and its EPS treatment significantly enhanced plant growth, including morphological parameters (root and shoot length, root and shoot weight), physiological parameters (chlorophyll a, b and carotenoid content) and wheat germination efficiency, compared to chemical treatments under chilling stress. Genomic analysis revealed genes involved in alginate EPS biosynthesis, oxidative stress alleviation, cold tolerance and plant growth promotion. Metabolomic profiling identified osmolytes (ectoine, betaine), phytohormones (zeatin, indole-3-acetic acid), melatonin, and acetylsalicylic acid. This is the first report demonstrating that purified EPS confers plant cold tolerance. The findings highlight the potential of Pseudomonas putida IRS13 and its EPS as sustainable biostimulants for improving crop resilience in cold agroecosystems.

Hypersaline ecosystems are distributed all over the globe. They are subjected to poly-extreme stresses and are inhabited by halophilic microorganisms possessing multiple adaptations. The halophiles have many biotechnological applications such as nutrient supplements, antioxidant synthesis, salt tolerant enzyme production, osmolyte synthesis, biofuel production, electricity generation etc. However, halophiles are still underexplored in terms of complex ecological interactions and functions as compared to other niches. The advent of metagenomics and the recent advancement of next-generation sequencing tools have made it feasible to investigate the microflora of an ecosystem, its interactions and functions. Both target gene and shotgun metagenomic approaches are commonly employed for the taxonomic, phylogenetic, and functional analyses of the hypersaline microbial communities. This review discusses different types of hypersaline niches, their residential microflora, and an overview of the metagenomic approaches used to investigate them. Various applications, hurdles and the recent advancements in metagenomic approaches have also been focused on here for their better understanding and utilization in the study of hypersaline microbiome.

ABSTRACT Climate change is causing unpredictable seasonal variations globally. Due to the continuously increasing earth’s surface temperature, the rate of water evaporation is enhanced, conceiving a problem of soil salinization, especially in arid and semi-arid regions. The accumulation of salt degrades soil quality, impairs plant growth, and reduces agricultural yields. Salt-tolerant, plant-growth-promoting microorganisms may offer a solution, enhancing crop productivity and soil fertility in salinized areas. In the current study, genome-resolved metagenomic analysis has been performed to investigate the salt-tolerating and plant growth-promoting potential of two hypersaline ecosystems, Sambhar Lake and Drang Mine. The samples were co-assembled independently by Megahit, MetaSpades, and IDBA-UD tools. A total of 67 metagenomic assembled genomes (MAGs) were reconstructed following the binning process, including 15 from Megahit, 26 from MetaSpades, and 26 from IDBA_UD assembly tools. As compared to other assemblers, the MAGs obtained by MetaSpades were of superior quality, with a completeness range of 12.95%–96.56% and a contamination range of 0%–8.65%. The medium and high-quality MAGs from MetaSpades, upon functional annotation, revealed properties such as salt tolerance (91.3%), heavy metal tolerance (95.6%), exopolysaccharide (95.6%), and antioxidant (60.86%) biosynthesis. Several plant growth-promoting attributes, including phosphate solubilization and indole-3-acetic acid (IAA) production, were consistently identified across all obtained MAGs. Conversely, characteristics such as iron acquisition and potassium solubilization were observed in a substantial majority, specifically 91.3%, of the MAGs. The present study indicates that hypersaline microflora can be used as bio-fertilizing agents for agricultural practices in salinized areas by alleviating prevalent stresses. IMPORTANCE The strategic implementation of metagenomic assembled genomes (MAGs) in exploring the properties and harnessing microorganisms from ecosystems like hypersaline niches has transformative potential in agriculture. This approach promises to redefine our comprehension of microbial diversity and its ecosystem roles. Recovery and decoding of MAGs unlock genetic resources, enabling the development of new solutions for agricultural challenges. Enhanced understanding of these microbial communities can lead to more efficient nutrient cycling, pest control, and soil health maintenance. Consequently, traditional agricultural practices can be improved, resulting in increased yields, reduced environmental impacts, and heightened sustainability. MAGs offer a promising avenue for sustainable agriculture, bridging the gap between cutting-edge genomics and practical field applications.

Long-read single-cell transcriptomics (scRNA-Seq) is revolutionizing the way we profile heterogeneity in disease. Traditional short-read scRNA-Seq methods are limited in their ability to provide complete transcript coverage, resolve isoforms, and identify novel transcripts. The scRNA-Seq protocols developed for long-read sequencing platforms overcome these limitations by enabling the characterization of full-length transcripts. Long-read scRNA-Seq techniques initially suffered from comparatively poor accuracy compared to short read scRNA-Seq. However, with improvements in accuracy, accessibility, and cost efficiency, long-reads are gaining popularity in the field of scRNA-Seq. This review details the advances in long-read scRNA-Seq, with an emphasis on library preparation protocols and downstream bioinformatics analysis tools.

CRISPR-Cas technology has reshaped the field of microbiology. It has improved the microbial strains for better industrial and therapeutic utilization. In this chapter, we have tried to provide an overview of this technology with special reference to its associated applications in the various fields of interest. We have discussed the origin, classification, and different genome editing methods of CRISPR-Cas to understand its historical significance and the basic mechanism of action. Further, different applications in the area of agriculture, food industry, biotherapeutics, biofuel, and other valuable product synthesis were also explained to highlight the advancement of this system in industrial microbes. We have also tried to review some of the limitations offered by CRISPR and insights into its future perspective.

Numerous metagenomics studies, conducted in both full-scale anaerobic digesters and household biogas plants, have shed light on the composition and activity of microbial flora essential for optimizing the performance of biogas reactors, underscoring the significance of microbial community composition in biogas plant efficiency. Although the efficiency of household biogas plants in the sub-Himalayan region has been reported, there is no literature evidence on the microbial community structure of such household biogas plants in the sub-Himalayan region. The current study evaluated the physico-chemical properties and bacterial community structure from the slurry samples of household biogas plants prevalent in the sub-Himalayan region. The slurry samples were observed to be rich in nutrients; however, their carbon and nitrogen contents were higher than the recommended standard values of liquid-fermented organic manure. The species richness and diversity indices (Chao1, Shannon, and Simpson) of household biogas plants were quite similar to the advanced biogas reactors operating at mesophilic conditions. 16S rRNA gene amplicon sequencing reveals microbial diversity, showing a higher abundance of Firmicutes (70.9%) and Euryarchaeota (9.52%) in advanced biogas reactors compared to household biogas plants. Microbial analysis shows a lack of beneficial microbes for anaerobic digestion, which might be the reason for inefficient biogas production in household biogas plants of the sub-Himalayan region. The lack of efficient bacterial biomass may also be attributed to the digester design, feedstock, and ambient temperatures. This study emphasized the establishment of efficient microbial consortia for enhanced degradation rates that may increase the methane yield in biogas plants.

Aims Climate change is responsible for extreme cold winters, causing a significant loss in crop yield and productivity due to chilling stress. This study aims to investigate the potential of psychrotrophic plant growth-promoting rhizobacteria (PGPR) strain to promote wheat growth under cold stress and explore the adaptive responses of wheat. Methods and results Wheat seeds and seedlings were inoculated with the psychrotrophic strain IRS14 and the plants were cultivated for five weeks at 6 & DEG;C & PLUSMN; 2 & DEG;C. The genetic, biochemical, physiological, and molecular analysis of the bacterium and plant was done to evaluate the effect of the PGPR strain in alleviating chilling stress. IRS14 possesses antioxidant activity and produced multiple phytohormones, which enhanced seed germination (& SIM;50%) and plant growth (& SIM;50%) during chilling stress. Conclusions Here, we reported that the application of IRS14 helps to regulate the biochemical and metabolic pathways in wheat plants. It alleviates chilling stress and increases plant growth rate and biomass. Strain IRS14 in wheat effectively increased chlorophyll content, antioxidants, carotenoid, proline, and endogenous phytohormones compared with untreated wheat.

Some hypersaline ecosystems exist in the form of halite, the salt deposits formed due to the evaporation of salt water. Over a period of time, the salt-saturated hypersaline lakes or seas get dried up, leading to the deposition of halite and other significant minerals. The ancient halite deposits may contain halophiles from the same era as the surrounding minerals. In the current study, the taxonomic diversity of halite deposited in the Drang Mine was analysed and compared with the microbiome of similar ecosystems, i.e., a nearby brine, Pangong Lake and Sambhar Lake. The analysis showed that Proteobacteria was the most abundant phylum across all the samples. The phylum Firmicutes exhibited the second-highest abundance in the Drang ecosystem, while Bacteroidetes were more numerous than Firmicutes in other selected niches. The lowest species richness based on the Chao I index was found in Sambhar Lake, likely because of the presence of two major extreme parameters, namely high salinity and alkaline pH. The species diversity calculated by Shannon and Simpson indices was lowest in Drang Mine, possibly because of the very high salt concentration in halite ecosystems, as also corroborated by physi-ochemical analysis. The beta-diversity analysis based on the Bray-Curtis matrix showed the formation of a separate cluster of Drang Mine samples, pointing towards its unique bacterial community structure as compared to other samples. The current study not only aided in the generation of baseline information about the taxonomic diversity of halite deposited in the Drang Mine but also highlighted its different bacterial community structure when compared to other similar ecosystems. It also indicated their potential role in N, S and CH4 biogeochemical cycling.

Bacillus licheniformis is a well-known probiotic that can be found in a variety of foods. The strain Bacillus licheniformis MCC 2514 was previously characterized by our group for its bio-physiological capabilities establishing it as a promising probiotic, but information on the genetic evidence for its attributes was lacking. In the current study, whole genome analysis identified the underlying molecular determinants responsible for its probiotic potential. The circular genome of MCC 2514 was 4,230,480 bp with 46.2% GC content, 24 rRNA, and 83 tRNA genes. The pangenome analysis between B. licheniformis MCC 2514 and 12 other B. licheniformis strains revealed a pangenome of 6008 genes and core genome of 3775 genes. Genome mining revealed NRPS and bacteriocins producing gene clusters indicating its biocontrol properties. Several genes encoding carbohydrate degrading enzymes, which aid in proper food degradation in the intestine, were also observed. Stress tolerance, vitamin, and essential amino acids biosynthesis related genes were found, which are important characteristics of a probiotic strain. Additionally, vital genes responsible for gut adhesion and biofilm formation were observed in its genome. The bacterium has been shown to improve the shelf life of idli batter by preventing whey separation, CO2, and odour production while maintaining the pH of 3.96-4.29, especially at cold temperatures. It has significantly reduced coliform contamination at both room and low temperatures, demonstrating its bio-preservative ability, which is also corroborated by the presence of the NRPS and bacteriocin gene clusters in its genome. The present study helped to understand both, the ability of B. licheniformis MCC 2514 to adapt the intestinal gut environment and its probiotic functionality for food preservation.

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNASeq applications have evolved in conjunction with sequence technology and bioinformatic tool advances. In this review, we explained various computational resources, tools, and bioinformatics analyses advancement for small and large non-coding RNAs. These include non-coding RNAs (ncRNAs) such as piwi, micro, circular, and long ncRNAs. In addition, this article discusses future challenges, single-cell level sequencing for non-coding RNAs, and advantages of using long-read sequencing to annotate lncRNAs.

Fermented food and beverages constitute a significant part of the human diet (5%–40%) worldwide. Fermentation has been used for preservation and to augment the flavor, texture, and nutritional qualities of the food, since antiquity. During fermentation, the bioavailability of vitamins, minerals, and other constituents increases due to the microorganisms' metabolic activities. Besides enhancing nutritional quality, fermented foods contain live organisms reported to prevent/treat many health disorders. Types of the fermentation process are also classified based on these microorganisms. In developing countries, fermented foods were usually prepared using traditional methods without any standardized techniques. Considering the beneficial effects of fermented foods, industrial-level production requires consistent specific microorganisms, fermentation methods, evaluation of nutritional compositions, and food safety testing. This chapter discusses the fermented foods and associated organisms, different sources available for the consumption of fermented foods, and food component's effect on microorganism's efficacy.

Cereal-based traditional fermented beverages (TFBs) are prevalent among India’s ethnic community, and lugri is one such TFB popular among the tribal people of the Lahaul valley in North-Western Himalaya. Previous studies have reported that lugri harbors probiotics and contains amino acids and vitamins but comprehensive substrate-specific exploration of lugri for probiotic attributes is unexplored. The present study selected three substrate-based lugri (wheat, rice, and barley) to study their biochemical properties and explore potential probiotics. This study screened the best probiotic strains for antioxidant studies and the fermentative process. A biochemical analysis determined that rice-based lugri had a higher alcohol content, electric conductivity, crude protein, and lower pH than barley and wheat-based lugri. A total of 134 distinct morphotypes were screened, and 43 strains were selected based on their qualitatively superior acid and bile tolerance. Rice-based undistilled lugri harbored the most probiotics, with 22 out of 43 strains isolated. All 43 bacterial isolates exhibited properties like cell surface hydrophobicity, cell-auto aggregation, β-galactosidase, and exopolysaccharide production, supporting them as possible probiotics. Based on antibiotic susceptibility, hemolytic activity, and biofilm formation, all the bacterial strains were found to be non-pathogenic. Taxonomically, they ranged among eight distinct genera and 10 different species. Statistically, 12 isolates were found to be the most promising probiotic, and eight strains were isolated from rice-based undistilled lugri. Furthermore, the antioxidant activity of the promising isolates was tested, based on free-radical scavenging ability toward 2,2-diphenyl-1-picrylhydrazyl (4.39–16.41%) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (15.29–57.74%). The strain Lacticaseibacillus paracasei LUL:01 showed the best antioxidant activity and probiotic attributes, and hence was used for the production of fermented milk. The strain LUL:01 fermented the sterile milk within 18 h, and the viable count remained above the legal requirement of 6 log10 CFU/ml during 28 days storage at 4°C. The strain represents a suitable candidate for applying probiotic functional food formulation with several health benefits.

The rising population and industrialisation produce a huge amount of waste, in particular from agro-industries, thus inducing environmental pollution. Therefore, waste should be better recycled into materials and energy, e.g. by incineration, gasification, and pyrolysis. Valuable by-products include enzymes, pigments, biofertilizers and edibles. Here we review the conversion of agro-waste, with focus on fermentation, composting, anaerobic digestion, enzymes, bioethanol, biofertilizers, microbial treatment and greenhouse gases.

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.

The melting of glaciers due to global warming leads to the formation of proglacial water bodies such as streams and lakes in their downstream environment. Triloknath glacier in the North-Western Himalayas has retreated unprecedently at a rate of 180 m /decade, forming similar water bodies. Glacier meltwater is considered the primary source of microbial diversity in downstream water bodies. Documentation of such diversity is crucial to retrieve the specialized microbial population because of the shift in the aquatic system due to deglaciation. In the current study, bacterial community structure based on V3-V4 region amplicons of 16S rRNA gene of two proglacial lakes and the glacial stream of Triloknath glacier was compared. Proglacial lake I site represents the ice-contacted proglacial lake while the proglacial lake II represents an ice-distal proglacial lake generally said to be evolved from the former. Proteobacteria was the most abundant phylum across the three sites. The prevalence of Proteobacteria in the proglacial lake I, proglacial lake II and glacial stream was 43.58%, 49.91%, and 40.08%, respectively. The highest species richness based on Chao I index (656) was observed in glacial stream while, the highest species diversity based on the Shannon index (8.95) was observed in proglacial lake I. The physiochemical conditions of glacial stream and proglacial lake II were closer than the lake I. These findings were corroborated with microbial diversity results as beta diversity analysis based on the Bray-curtis matrix indicated the closeness of glacier stream and proglacial lake II ecosystem and these sites also shared maximum bacterial genera.16S rRNA gene-based functional analysis showed the occurrence of xenobiotics degrading genes exposing the presence of pollutants in glacier ecosystem. The current study generated baseline information about the change in bacterial diversity due to deglaciation and hydrological connectivity of downstream water bodies with the glacier. The study documented residential microbial population of sensitive ecological niche of proglacial water bodies of Triloknath glacier ecosystem.