Hot springs support diverse and interesting groups of microorganisms adapted to extreme conditions and gaining attention in biotechnological applications. However, due to limitations of cultivation methods, a majority of such extremophiles remain uncultivated and unexplored. The advent of multiple cultivation conditions and specialized culture media could possibly aid to access the unexplored microbial portion of hot springs. In the present study, different media and isolation strategies were applied to isolate hitherto unexplored bacterial taxa in the water samples collected from Unkeshwar hot springs, India. Molecular, phylogenetic and predictive functional characterization of the isolated bacterial population was done using 16S rRNA sequencing coupled with Tax4Fun tools. Furthermore, representative isolates were screened for important enzymes (cellulase, xylanase, amylase, and protease) and heavy metal tolerance (chromium, arsenic) properties. A total of 454 bacterial isolates obtained were mapped into 57 unique bacterial genera and 4 different bacterial phyla. Interestingly, 37 genera not previously isolated from Indian hot springs, were isolated for the first time in the present study. However, most of these genera (23 out of 37) were reported only in metagenomics studies from Indian and global hot springs. Furthermore, around 14 genera not previously cultivated and not detected in metagenomics studies of hot springs are documented here. The metabolic potential was ascertained by determining the abundance of specific genes using in silico based Tax4Fun tool, which identified around 315 metabolic pathways for metabolism of carbohydrates, synthesis of secondary metabolites and degradation of xenobiotic compounds. Bioprospection study revealed that 33 and 25 bacterial genera were positive for enzyme production and resistance to the heavy metals, respectively. The present study revealed the advantages of cultivation methods using a comprehensive multiple isolation approach for exploring untapped and unique bacterial diversity, and also utilities for various biotechnological and environmental applications.
Microbial communities in freshwater streams play an essential role in ecosystem functioning via biogeochemical cycling. Yet, the impacts of treated wastewater influx into stream ecosystems on microbial strain diversity remain mostly unexplored. Here, we coupled full‐length 16S ribosomal RNA gene Nanopore sequencing and strain‐resolved metagenomics to investigate the impact of treated wastewater on a mesocosm system (AquaFlow) run with restored river water. Over 10 days, community Bray–Curtis dissimilarities between treated and control mesocosm decreased (0.57 ± 0.058 to 0.26 ± 0.046) based on ribosomal protein S3 gene clustering, finally converging to nearly identical communities. Similarly, strain‐resolved metagenomics revealed a high diversity of bacteria and viruses after the introduction of treated wastewater; these microbes also decreased over time resulting in the same strain clusters in control and treatment at the end of the experiment. Specifically, 39.2% of viral strains detected in all samples were present after the introduction of treated wastewater only. Although bacteria present at low abundance in the treated wastewater introduced additional antibiotic resistance genes, signals of naturally occurring ARG‐encoding organisms resembled the resistome at the endpoint. Our results suggest that the previously stressed freshwater stream and its microbial community are resilient to a substantial introduction of treated wastewater.
38River confluences are a hub of biodiversity with limited information with respect to the structure and the 39 functions of the microbial communities. The 'River Ganges' is the national river of India, having manifold 40 significance such as social, mythological, historic, geographic and agro-economic. It forms a sacred confluence 41 (known as Triveni Sangam) with River Yamuna at Prayagraj, India. Recent reports indicated the presence of fecal 42 coliform bacteria, an indicator recognized for water contamination in Ganges River leading to pollution. However,
43Fungi are also gaining attention as potential biological indicators of the trophic status of some rivers globally, but 44 remain under-explored in terms of diversity, ecology and functional aspects. We performed whole long read, 45 metagenome sequencing (MinION) of the sediment samples collected in December 2017 from confluence zone of 46 Ganges and Yamuna Rivers spanning the pre-confluence, confluence and post-confluence zones. Mycobiome reads 47 revealed a plethora of fungal communities, extending from saprophytes, endoparasites and edible fungi, to human 48 pathogens, plant pathogens and toxin producers. The fungal genera recognized as bio-indicators of river pollution 49 (Aspergillus, Penicillium) and eutrophication (Kluveromyces, Lodderomyces, and Nakaseomyces), were present in 50 all samples. Functional gene analyses of myco-communities uncovered hits for neurodegenerative diseases and 51 xenobiotic degradation potential, supporting bio-indicators of pollution. This study forms a foundational basis for 52 understanding the impact of various anthropogenic activities on the mycobiome, as a bio-indicator of pollution 53 across the confluence of Ganges and Yamuna Rivers and post-confluence of River Ganges, and could be useful in 54 mitigating strategies for cleaning strategies of the Ganges River.55
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