Utility of metagenomics for bioremediation: a comprehensive review on bioremediation mechanisms and microbial dynamics of river ecosystem.

Global industrialization has contributed substantial amounts of chemical pollutants in rivers, resulting in an uninhabitable state and impacting different life forms. Moreover, water macrophytes, such as water hyacinths, are abundantly present in polluted rivers, significantly affecting the overall water biogeochemistry. Bioremediation involves utilizing microbial metabolic machinery and is one of the most viable approaches for removing toxic pollutants. Conventional techniques generate limited information on the indigenous microbial population and their xenobiotic metabolism, failing the bioremediation process. Metagenomics can overcome these limitations by providing in-depth details of microbial taxa and functionality-related information required for successful biostimulation and augmentation. An in-depth summary of the findings related to pollutant metabolizing genes and enzymes in rivers still needs to be collated. The present study details bioremediation genes and enzymes functionally mined from polluted river ecosystems worldwide using a metagenomic approach. Several studies reported a wide variety of pollutant-degrading enzymes involved in the metabolism of dyes, plastics, persistent organic pollutants, and aromatic hydrocarbons. Additionally, few studies also noted a shift in the microbiome of the rivers upon exposure to contaminants, crucially affecting the ecological determinant processes. Furthermore, minimal studies have focused on the role of water-hyacinth-associated microbes in the bioremediation potentials, suggesting the need for the bioprospecting of these lesser-studied microbes. Overall, our study summarizes the prospects and utilities of the metagenomic approach and proposes the need to employ it for efficient bioremediation.

Genome sequencing and protein modeling unraveled the 2AP biosynthesis in Bacillus cereus DB25.

2-Acetyl-1-pyrroline (2AP) is an important and major flavor aroma compound responsible for the fragrance of basmati rice, cheese, wine, and several other food products. Biosynthesis of 2AP in aromatic rice and a few other plant species is associated with a recessive Betaine aldehyde dehydrogenase 2 (BADH2) gene. However, the literature is scant on the relationship between the functional BADH2 gene and 2AP biosynthesis in prokaryotic systems. Therefore, in the present study, we aimed to explore the functionality of the BADH2 gene for 2AP biosynthesis in 2AP synthesizing rice rhizobacterial isolate Bacillus cereus DB25 isolated from the rhizosphere of basmati rice (Oryza sativa L.). Full-length BcBADH2 sequence was obtained through whole genome sequencing (WGS) and further confirmed through traditional PCR and Sanger sequencing. Then the functionality of the BcBADH2 gene was evaluated in-silico through bioinformatics analysis and protein docking studies and further experimentally validated through enzyme assay. The sequencing and bioinformatics analysis results revealed a full-length 1485 bp BcBADH2 coding sequence without any deletion or premature stop codons. Full-length BcBADH2 was found to encode a fully functional protein of 54.08 kDa with pI of 5.22 and showed the presence of the conserved amino acids responsible for enzyme activity. The docking studies confirmed a good affinity between the protein and its substrate whereas the presence of BcBADH2 enzyme activity confirmed the functionality of BADH2 enzyme in B. cereus DB25. In conclusion, the findings of the present study suggest that B. cereus DB25 is able to synthesize 2AP despite a functional BADH2 gene and there may be a different molecular mechanism responsible for 2AP biosynthesis in bacterial systems, unlike that found in aromatic rice and other eukaryotic plant species.

Genomic surveillance reveals early detection and transition of delta to omicron lineages of SARS-CoV-2 variants in wastewater treatment plants of Pune, India.

The COVID-19 pandemic has emphasized the urgency for rapid public health surveillance methods to detect and monitor the transmission of infectious diseases. The wastewater-based epidemiology (WBE) has emerged as a promising tool for proactive analysis and quantification of infectious pathogens within a population before clinical cases emerge. In the present study, we aimed to assess the trend and dynamics of SARS-CoV-2 variants using a longitudinal approach. Our objective included early detection and monitoring of these variants to enhance our understanding of their prevalence and potential impact. To achieve our goals, we conducted real-time quantitative polymerase chain reaction (RT-qPCR) and Illumina sequencing on 442 wastewater (WW) samples collected from 10 sewage treatment plants (STPs) in Pune city, India, spanning from November 2021 to April 2022. Our comprehensive analysis identified 426 distinct lineages representing 17 highly transmissible variants of SARS-CoV-2. Notably, fragments of Omicron variant were detected in WW samples prior to its first clinical detection in Botswana. Furthermore, we observed highly contagious sub-lineages of the Omicron variant, including BA.1 (~28%), BA.1.X (1.0-72%), BA.2 (1.0-18%), BA.2.X (1.0-97.4%) BA.2.12 (0.8-0.25%), BA.2.38 (0.8-1.0%), BA.2.75 (0.01-0.02%), BA.3 (0.09-6.3%), BA.4 (0.24-0.29%), and XBB (0.01-21.83%), with varying prevalence rates. Overall, the present study demonstrated the practicality of WBE in the early detection of SARS-CoV-2 variants, which could help track future outbreaks of SARS-CoV-2. Such approaches could be implicated in monitoring infectious agents before they appear in clinical cases.

Comparative evaluation of advanced oxidation processes (AOPs) for reducing SARS-CoV-2 viral load from campus sewage water.

Presence of SARS-CoV-2 in wastewater is a major concern as the wastewater meets rivers and other water bodies and is used by the population for various purposes. Hence it is very important to treat sewage water in an efficient manner in order to reduce the public health risk. In the present work, various advanced oxidation processes (AOPs) have been evaluated for disinfection of SARS-CoV-2 from sewage water collected from STP inlet of academic institutional residential. The sewage water was subjected to ten AOPs, which include Ozone (O3), Hydrodynamic cavitation (HC), Ultraviolet radiation (UV), and their hybrid combinations like HC/O3, HC/O3/H2O2, HC/H2O2, O3/UV, UV/H2O2, UV/H2O2/O3, and O3/H2O2 to reduce SARS-CoV-2 viral load. Further, AOP treated sewage water was subjected to total nucleic acid isolation followed by RT-qPCR for viral load estimation. The sewage water treatment techniques were evaluated based on their viral concentration-reducing efficiency. It was found that ozone and ozone-coupled hybrid AOPs showed the most promising result with more than 98 % SARS-CoV-2 viral load reducing efficiency from sewage water. Interestingly, the best six AOPs used in this study significantly reduced both the SARS-CoV-2 and PMMoV (faecal indicator) viral load and improved water quality in terms of increasing DO and decreasing TOC.

Spatio-temporal variation of the microbiome and resistome repertoire along an anthropogenically dynamic segment of the Ganges River, India.

Aquatic ecosystems are regarded as a hub of antibiotic and metal resistance genes. River Ganges is a unique riverine system in India with socio-cultural and economic significance. However, it remains underexplored for its microbiome and associated resistomes along its anthropogenically impacted course. The present study utilized a nanopore sequencing approach to depict the microbial community structure in the sediments of the river Ganges harboring antibiotic and metal resistance genes (A/MRGs) in lower stretches known for anthropogenic impact. Comprehensive microbiome analyses revealed resistance genes against 23 different types of metals and 28 classes of antibiotics. The most dominant ARG category was multidrug resistance, while the most prevalent MRGs conferred resistance against copper and zinc. Seasonal differences dismally affected the microbiota of the Ganges. However, resistance genes for fosmidomycin and tetracycline varied with season ANOVA, p < 0.05. Interestingly, 333 and 334 ARG subtypes were observed at all the locations in pre-monsoon and post-monsoon, respectively. The taxa associated with the dominant ARGs and MRGs were Pseudomonas and Burkholderia, which are important nosocomial pathogens. A substantial phage diversity for pathogenic and putrefying bacteria at all locations attracts attention for its use to tackle the dissemination of antibiotic and metal-resistant bacteria. This study suggests the accumulation of antibiotics and metals as the driving force for the emergence of resistance genes and the affiliated bacteria trafficking them. The present metagenomic assessment highlights the need for comprehensive, long-term biological and physicochemical monitoring and mitigation strategies toward the contaminants associated with ARGs and MRGs in this nationally important river.

Poly-gamma-glutamic acid biopolymer: a sleeping giant with diverse applications and unique opportunities for commercialization.

Poly-gamma-glutamic acid (γ-PGA) is a biodegradable, non-toxic, ecofriendly, and non-immunogenic biopolymer. Its phenomenal properties have gained immense attention in the field of regenerative medicine, the food industry, wastewater treatment, and even in 3D printing bio-ink. The γ-PGA has the potential to replace synthetic non-degradable counterparts, but the main obstacle is the high production cost and lower productivity. Extensive research has been carried out to reduce the production cost by using different waste; however, it is unable to match the commercialization needs. This review focuses on the biosynthetic mechanism of γ-PGA, its production using the synthetic medium as well as different wastes by L-glutamic acid-dependent and independent microbial strains. Furthermore, various metabolic engineering strategies and the recovery processes for γ-PGA and their possible applications are discussed. Finally, highlights on the challenges and unique approaches to reduce the production cost and to increase the productivity for commercialization of γ-PGA are also summarized.

Metagenomic mining of Indian river confluence reveal functional microbial community with lignocelluloytic potential.

Microbial carbohydrate-active enzymes (CAZyme) can be harnessed for valorization of Lignocellulosic biomass (LCB) to value-added chemicals/products. The two Indian Rivers Ganges and the Yamuna having different origins and flow, face accumulation of carbon-rich substrates due to the discharge of wastewater from adjoining paper and pulp industries, which could potentially contribute to the natural enrichment of LCB utilizing genes, especially at their confluence. We analyzed CAZyme diversity in metagenomic datasets across the sacred confluence of the Rivers Ganges and Yamuna. Functional annotation using CAZyme database identified a total of 77,815 putative genes with functional domains involved in the catalysis of carbohydrate degradation or synthesis of glycosidic bonds. The metagenomic analysis detected ~ 41% CAZymes catalyzing the hydrolysis of lignocellulosic biomass polymers- cellulose, hemicellulose, lignin, and pectin. The Beta diversity analysis suggested higher CAZyme diversity at downstream region of the river confluence, which could be useful niche for culture-based studies. Taxonomic origin for CAZymes revealed the predominance of bacteria (97%), followed by archaea (1.67%), Eukaryota (0.63%), and viruses (0.7%). Metagenome guided CAZyme diversity of the microflora spanning across the confluence of Ganges-Yamuna River, could be harnessed for biomass and bioenergy applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03190-7.

High throughput sequencing based direct detection of SARS-CoV-2 fragments in wastewater of Pune, West India.

Given a large number of SARS-CoV-2 infected individuals, clinical detection has proved challenging. The wastewater-based epidemiological paradigm would cover the clinically escaped asymptomatic individuals owing to the faecal shedding of the virus. We hypothesised using wastewater as a valuable resource for analysing SARS-CoV-2 mutations circulating in the wastewater of Pune region (Maharashtra; India), one of the most affected during the covid-19 pandemic. We conducted study in open wastewater drains from December 2020-March 2021 to assess the presence of SARS-CoV-2 nucleic acid and further detect mutations using ARTIC protocol of MinION sequencing. The analysis revealed 108 mutations across six samples categorised into 39 types of mutations. We report the occurrence of mutations associated with Delta variant lineage in March-2021 samples, simultaneously also reported as a Variant of Concern (VoC) responsible for the rapid increase in infections. The study also revealed four mutations; S:N801, S:C480R, NSP14:C279F and NSP3:L550del not currently reported from wastewater or clinical data in India but reported worldwide. Further, a novel mutation NSP13:G206F mapping to NSP13 region was observed from wastewater. Notably, S:P1140del mutation was detected in December 2020 samples while it was reported in February 2021 from clinical data, indicating the instrumentality of wastewater data in early detection. This is the first study in India to demonstrate utility of sequencing in wastewater-based epidemiology to identify mutations associated with SARS-CoV-2 virus fragments from wastewater as an early warning indicator system.

COVID-19 associated mucormycosis: evolving technologies for early and rapid diagnosis.

The post-coronavirus disease (COVID-19) mucormycosis is a deadly addition to the pandemic spectrum. Although it's a rare, aggressive, and opportunistic disease, the associated morbidity and mortality are significant. The complex interplay of factors aggravating CAM is uncontrolled diabetes, irrational and excessive use of antibiotics, steroids, and an impaired immune system. Recently, India has been witnessing a rapid surge in the cases of coronavirus disease-associated mucormycosis (CAM), since the second wave of COVID-19. The devastating and lethal implications of CAM had now become a matter of global attention. A delayed diagnosis is often associated with a poor prognosis. Therefore, the rapid and early diagnosis of infection would be life-saving. Prevention and effective management of mucormycosis depend upon its early and accurate diagnosis followed by a multimodal therapeutic approach. The current review summarizes an array of detection methods and highlights certain evolving technologies for early and rapid diagnosis of CAM. Furthermore, several potential management strategies have also been discussed, which would aid in tackling the neglected yet fatal crisis of mucormycosis associated with COVID-19.

Metagenomic exploration reveals a differential patterning of antibiotic resistance genes in urban and peri-urban stretches of a riverine system.

Antimicrobial resistance in the riverine ecosystem of urban areas is an alarming concern worldwide, indicating the importance of molecular monitoring to understand their patterning in urban and peri-urban areas. In the present study, we evaluated the influence of urban rivers on the connected peri-urban rivers of a riverine system of India in the context of antibiotic resistance genes. The rivers traversing through urban (Mula, Mutha, Pawana, and Ramnadi) and peri-urban stretches (Bhima and Indrayani) form the riverine system of Pune district in Maharashtra, India. The MinION-based shotgun metagenomic analysis revealed the resistome against 26 classes of antibiotics, including the last line of antibiotics. In total, we observed 278 ARG subtypes conferring resistance against multiple drugs (40%), bacitracin (10%), aminoglycoside (7.5%), tetracycline (7%), and glycopeptide (5%). Further, the alpha diversity analysis suggested relatively higher ARG diversity in the urban stretches than peri-urban stretches of the riverine system. The NMDS (non-metric multidimensional scaling) analysis revealed significant differences with overlapping similarities (stress value = 0.14, p-value = 0.004, ANOSIM statistic R: 0.2328). These similarities were reasoned by assessing the influence of downstream sites (sites at the outskirts of Pune city; however, directly impacted), which revealed significant differences in the ARG contents of urban and peri-urban stretches (stress value = 0.14, p-value = 0.001, ANOSIM statistic R: 0.6137). Overall, we detected the dissemination of antibiotic resistance genes from the polluted urban rivers into the peri-urban rivers located downstream in the connected riverine system potentially driven by anthropogenic activities.

Translating SARS-CoV-2 wastewater-based epidemiology for prioritizing mass vaccination: a strategic overview.

The inception of the novel coronavirus has forced the world into despair. Rapid progress has been made in addressing the situation, and various clinical diagnostic methods were developed for early detection of transmission. However, with a rapidly increasing number of infected populations worldwide, the testing of each individual was impractical. The wastewater-based epidemiology (WBE) has been implemented to evaluate disease outbreaks as an early warning system for pandemic preparedness. Numerous studies reported the presence of SARS-CoV-2 in the open drains and STPs across the globe via recovery efficiency of surrogate virus from existing virus concentration protocols. However, the such reported studies did not justify the use of WBE to identify or pinpoint the specific hotspots of transmission which could be prioritized for rapid efforts to contain or accelerate active vaccination efforts. Identifying precise locations of hotspots could be an essential aspect in controlling the outbreak and surge of wave by prioritizing the region for primary outbreak response. This article focuses on the issues relating to the primary focus for WBE that can be adapted, and its suitability for utilization in the mass vaccination program is discussed. Effective use of WBE information in terms of source tracking might be crucial as we move towards mass vaccination to control outbreaks of COVID-19 pandemic.

Metagenomic analysis reveals genetic insights on biogeochemical cycling, xenobiotic degradation, and stress resistance in mudflat microbiome.

Mudflats are highly productive coastal ecosystems that are dominated by halophytic vegetation. In this study, the mudflat sediment microbiome was investigated from Nalabana Island, located in a brackish water coastal wetland of India; Chilika, based on the MinION shotgun metagenomic analysis. Bacterial, archaeal, and fungal communities were mostly composed of Proteobacteria (38.3%), Actinobacteria (20.7%), Euryarchaeota (76.1%), Candidatus Bathyarchaeota (6.8%), Ascomycota (47.2%), and Basidiomycota (22.0%). Bacterial and archaeal community composition differed significantly between vegetated mudflat and un-vegetated bulk sediments. Carbon, nitrogen, sulfur metabolisms, oxidative phosphorylation, and xenobiotic biodegradation were the most common microbial functionalities in the mudflat metagenomes. Furthermore, genes involved in oxidative stresses, osmotolerance, secondary metabolite synthesis, and extracellular polymeric substance synthesis revealed adaptive mechanisms of the microbiome in mudflat habitat. Mudflat metagenome also revealed genes involved in the plant growth and development, suggesting that microbial communities could aid halophytic vegetation by providing tolerance to the abiotic stresses in a harsh mudflat environment. Canonical correspondence analysis and co-occurrence network revealed that both biotic (vegetation and microbial interactions) and abiotic factors played important role in shaping the mudflat microbiome composition. Among abiotic factors, pH accounted for the highest variance (20.10%) followed by available phosphorus (19.73%), total organic carbon (9.94%), salinity (8.28%), sediment texture (sand) (6.37%) and available nitrogen (5.53%) in the mudflat microbial communities. Overall, this first metagenomic study provided a comprehensive insight on the community structure, potential ecological interactions, and genetic potential of the mudflat microbiome in context to the cycling of organic matter, xenobiotic biodegradation, stress resistance, and in providing the ecological fitness to halophytes. These ecosystem services of the mudflat microbiome must be considered in the conservation and management plan of coastal wetlands. This study also advanced our understanding of fungal diversity which is understudied from the coastal lagoon ecosystems.

Metagenomic analysis of a mega-city river network reveals microbial compositional heterogeneity among urban and peri-urban river stretch.

The rivers in the megacities face a constant inflow of extremely polluted wastewaters from various sources, and their influence on the connected peri-urban river is still poorly understood. The riverine system in Pune consists of Rivers Mula, Ramnadi, Pawana, Mutha, and Mula-Mutha, traversing through the urban settlements of Pune before joining River Bhima in the peri-urban region. We used MinION-based metagenomic sequencing to generate a comprehensive understanding of the microbial diversity differences between the urban and peri-urban zones, which has not been explored at the meta scale until date. The taxonomic analysis revealed significant enrichment of pollution indicators microbial taxa (Welsch's t-test, p < 0.05, Benjamini-Hochberg FDR test) such as Bacteriodetes, Firmicutes, Spirochaetes, Synergistetes, Euryarcheota in the urban waters as compared to peri-urban waters. Further, the peri-urban waters showed a significantly higher prevalence of ammonium oxidising archaeal groups such as Nitrososphaeraceae (Student's t-test p-value <0.05 with FDR correction), thereby probably suggesting the influence of agricultural runoffs. Besides, the microbial community diversity assessment also indicated the significant dissimilarity in the microbial community of urban and peri-urban waters. Overall, the analysis predicted 295 virulence genes mapping to 38 different pathogenic bacteria in the riverine system. Moreover, the higher genome coverage (at least 60%) for priority pathogens such as Pseudomonas, Klebsiella, Acinetobacter, Escherichia, Aeromonas in the sediment metagenome consolidates their dominance in this riverine system. To conclude, our investigation showed that the unrestrained anthropogenic and related activities could potentially contribute to the overall dismal conditions and influence the connected riverine stretches on the outskirts of the city.

Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae.

Isoprenoids, often called terpenoids, are the most abundant and highly diverse family of natural organic compounds. In plants, they play a distinct role in the form of photosynthetic pigments, hormones, electron carrier, structural components of membrane, and defence. Many isoprenoids have useful applications in the pharmaceutical, nutraceutical, and chemical industries. They are synthesized by various isoprenoid synthase enzymes by several consecutive steps. Recent advancement in metabolic engineering and synthetic biology has enabled the production of these isoprenoids in the heterologous host systems like Escherichia coli and Saccharomyces cerevisiae. Both heterologous systems have been engineered for large-scale production of value-added isoprenoids. This review article will provide the detailed description of various approaches used for engineering of methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathway for synthesizing isoprene units (C5) and ultimate production of diverse isoprenoids. The review particularly highlighted the efforts taken for the production of C5-C20 isoprenoids by metabolic engineering techniques in E. coli and S. cerevisiae over a decade. The challenges and strategies are also discussed in detail for scale-up and engineering of isoprenoids in the heterologous host systems.Key points• Isoprenoids are beneficial and valuable natural products.• E. coli and S. cerevisiae are the promising host for isoprenoid biosynthesis.• Emerging techniques in synthetic biology enabled the improved production.• Need to expand the catalogue and scale-up of un-engineered isoprenoids. Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae.

Functional metagenomic landscape of polluted river reveals potential genes involved in degradation of xenobiotic pollutants.

Rapid industrialization contributes substantially to xenobiotic pollutants in rivers. As a result, most of the rivers traversing urban settlements are in significantly deteriorated conditions. These pollutants are recalcitrant, requiring robust catabolic machinery for their complete transformation into bioavailable and non-toxic by-products. Microbes are versatile dwellers that could adapt to such contaminants by using them as a source of nutrients during growth. However, efficient bioremediation requires an in-depth knowledge of microbial diversity and their metabolism related genes in the polluted niches. We employed MinION shotgun sequencing, to comprehend the biodegradation related genes and their function potential operating in the polluted urban riverine system of Western India. A vast number of catabolic genes were detected for the xenobiotic pollutants such as Benzoate, Nitrotoluene, Aminobenzoate, Drug metabolism, and Polycyclic Aromatic Hydrocarbons. Aerobic, and anaerobic catabolism genes, were mapped for their ability of degradation of xenobiotics. Interestingly, catabolism profiles of multiple aromatic compounds culminated into the Benzoate degradation pathway, suggesting it as a plausible central pathway for the autochthonous bacterial communities. Further mapping with RemeDB database, predicted plastic and dye degrading enzymes. Moreover, the diversity indices for the pollutant degrading enzymes suggested little variations (R2 value of 18%) between the city and non-city (outskirts of city limits) riverine stretch indicating the impact of industrialization in the outskirts of the city stretch as well. Altogether, this study would serve as a preliminary baseline for future explorations concerning river cleaning programs and also exploiting such microbes for bioremediation applications.

Probiotics in the prophylaxis of COVID-19: something is better than nothing.

The new viral pandemic of COVID-19 is caused by a novel coronavirus (SARS-CoV-2) that has brought the world at another unprecedented crisis in terms of health and economy. The lack of specific therapeutics necessitates other strategies to prevent the spread of infection caused by this previously unknown viral etiological agent. Recent pieces of evidence have shown an association between COVID-19 disease and intestinal dysbiosis. Probiotics comprise living microbes that upon oral administration benefit human health by reshaping the composition of gut microbiota. The close kinship of the gastrointestinal and respiratory tract suggests why the dysfunction of one may incite illness in others. The emerging studies suggest the capability of probiotics to regulate immune responses in the respiratory system. The efficacy of probiotics has been studied previously on several respiratory tract viral infections. Therefore, the purpose of this review is to comprehend existing information on the gut mediated-pulmonary immunity conferred by probiotic bacteria, in the course of respiratory virus infections and administration as a prophylactic measure in COVID-19 pandemic in managing intestinal dysbiosis as well.