Figure - available from: Frontiers in Microbiology
This content is subject to copyright.
Taxonomic affiliation of 16S rRNA metagenomic fragments. The phylogenetic distribution for (A) the overall population, (B) Firmicutes, (C) Proteobacteria, and (D) Archaea.
Source publication
The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total or...
Similar publications
Metagenomic analyses of microbial communities from aquatic sediments are relatively few, and there are no reported metagenomic studies on sediment from inland ponds used for aquaculture. Catfish ponds in the southeastern U.S. are eutrophic systems. They are fertilized to enhance algae growth and encourage natural food production, and catfish are fe...
Citations
... Microbial profiling by the 16 S rRNA amplicon sequencing and shotgun metagenomic sequencing provides a comprehensive picture of the hot spring microbial community [4,5] and leads to discovering the many novel and rare species, their metabolites, and biocatalysts [6]. ...
Objectives:
Indonesia's location at the convergence of multiple tectonic plates results in a unique geomorphological feature with abundant hot springs. This study pioneers the metagenomic exploration of Indonesian hot springs, harbouring unique life forms despite high temperatures. The microbial community of hot springs is taxonomically versatile and biotechnologically valuable. 16s rRNA amplicon sequencing of the metagenome is a viable option for the microbiome investigation. This study utilized Oxford Nanopore's long-read 16 S rRNA sequencing for enhanced species identification, improved detection of rare members, and a more detailed community composition profile.
Data description:
Water samples were taken from three hot springs of the Bali, Indonesia (i) Angseri, 8.362503 S, 115.133452 E; (ii) Banjar, 8.210270 S, 114.967063 E; and (iii) Batur, 8.228806 S, 115.404829 E. BioLit Genomic DNA Extraction Kit (SRL, Mumbai, India) was used to isolate DNA from water samples. The quantity and quality of the DNA were determined using a NanoDrop™ spectrophotometer and a Qubit fluorometer (Thermo Fisher Scientific, USA). The library was created using Oxford Nanopore Technology kits, and the sequencing was done using Oxford Nanopore's GridION platform. All sequencing data was obtained in FASTQ files and filtered using NanoFilt software. This dataset is valuable for searching novel bacteria diversity and their existence.
... This spring is rich in organic matter due to its forested environment and constant supply of plant litter, forming a unique ecosystem with various thermophiles. In 2015, we generated extensive sequence data using an Illumina HiSeq 2500 sequencer, which was then used to predict metabolic diversity in the hot spring, but only at a low resolution as bioinformatics pipelines were still limited at that time [ 1 ]. This report therefore aims to reprocess the Illumina raw shotgun data and create MAGs so that the scientific community interested in thermophiles or the hot spring microbiome can benefit from this data. ...
... Readers can use the fasta files for further analyzes that are not limited to those mentioned above under 'Value of the data'. It should be noted that this current data was analyzed based on the earlier water sample taken from SK hot spring that was initially published in 2015 [ 1 ]. There is another geothermal feature a few meters away from SK, which we named SKY, and MAGs of biofilms from SKY were reported recently [ 10 ]. ...
... The metagenome library was prepared using Illumina Nextera DNA Sample Preparation Kit, following the manufacturer's protocols. Shotgun sequencing was performed using the Illumina HiSeq 2500 sequencer (San Diego, CA, USA), utilizing a 150 bp paired-end approach with dual indexing [ 1 ]. Earlier reports presented data on microbial diversity and gene functional analyses but did not include MAGs due to the limitations of bioinformatic pipelines at the time. ...
This data report presents prokaryotic metagenome-assembled genomes (MAGs) from a hot spring stream with temperatures between 64 and 100°C. The stream water was filtered and the extracted total DNA was sequenced using the Illumina HiSeq 2500 platform. Approximately 80 Gb of raw data were generated, which were subsequently assembled using MEGAHIT v1.2.9. The MAGs were generated using MetaWRAP with binning approaches of MetaBAT2, CONCOCT and MaxBin2. We constructed 25 medium-quality and 24 high-quality archaeal MAGs, and 152 medium-quality and 112 high-quality bacterial MAGs. The fasta files of these MAGs are available in the NCBI database as well as Mendeley Data. Major phyla identified include Bacteroidota, Chloroflexota, Desulfobacterota, Firmicutes, Patescibacteria, Proteobacteria, Spirochaetota, Verrucomicrobiota, Armatimonadota, Nitrospirota, Acidobacteriota, Elusimicrobiota, Planctomycetota, Candidate division WOR-3, Aquificota, Thermoproteota, and Micrarchaeota. This dataset is valuable for studies on thermophilic genomes, reconstruction of biochemical pathways and gene discovery.
... Conversely, Archaea remained below 1% in all samples. This aligns with previous research on hot springs in Malaysia, Eritrea, and Algeria (Chan et al. 2015;Ghilamicael et al. 2017;Adjeroud et al. 2020). The observed bacterial dominance suggests better adaptation to the specific environmental conditions within these hot springs. ...
Northeastern Algeria boasts numerous hot springs, yet these hydrothermal sites remain largely unexplored for their microbial ecology. The present study explores the bacterial abundance and diversity within two distinct Algerian hot springs (Hammam Saïda and Hammam Debagh) and investigates the link between the prevailing bacteria with geochemical parameters. High-throughput 16S rRNA gene sequencing of water and sediment samples revealed a bacterial dominance of 99.85–91.16% compared to Archaea (0.14–0.66%) in both springs. Interestingly, Saïda hot spring, characterized by higher temperatures and sodium content, harbored a community dominated by Pseudomonadota (51.13%), whereas Debagh, a Ca–Cl-SO4 type spring, was primarily populated by Bacillota with 55.33%. Bacteroidota displayed even distribution across both sites. Additional phyla, including Chloroflexota, Deinococcota, Cyanobacteriota, and Chlorobiota, were also present. Environmental factors, particularly temperature, sodium, potassium, and alkalinity, significantly influenced bacterial diversity and composition. These findings shed light on the interplay between distinct microbial communities and their associated geochemical properties, providing valuable insights for future research on biogeochemical processes in these unique ecosystems driven by distinct environmental conditions, including potential applications in bioremediation and enzyme discovery.
... and 101°23′35.51′′E). The park has a main shallow main stream, featuring temperatures of 60-100 °C and a pH range of 7-9, possesses minimal plant litter and we have previously described microbial diversity in water and sediment of these microhabitats [18]. Alongside the main stream at the SKY site, one encounters submerged leaves and woody plant litter in various stages of decomposition. ...
Background
Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58–74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism.
Results
Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures.
Conclusions
This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.
... With metagenomics and advanced sequencing tools, untapped phylogenetic diversity has been expanded from hot springs. For example, Aquaficales, a bacterial division, Crenarchaeota and Korarchaeota, archaeal phyla, have been explored from the hot springs using metagenomics approaches (Chan et al., 2015;Narsing Rao et al., 2021). Nanoarchaeota equitants, a thermophilic symbiont, have also been characterized from the hot submarine vent (Huber et al., 2002). ...
... Cyanobacteria, Chloroflexi, Methanocella, and Methanomassiliicoccus, which have been the source of several small bioactive compounds, have also been characterized by metagenomics studies harboring in hot springs Sharma et al., 2020;Wani et al., 2021). In a metagenomics study on the water and sediment samples of Sungai Klah hot spring in Malaysia and based on 16S rRNA sequencing, Proteobacteria and Firmicutes were found in abundance (Chan et al., 2015). In other alkaline hot springs around the globe, Leptolyngbya, Geitlerinema, Aquaspirillum, Stenotrophomonas, and Fischerella were among the dominant taxa (Podar et al., 2020). ...
... Chan et al. studied the thermophilic diversity of Sungai Klah hot spring in Malaysia using 16S rRNA and shotgun metagenomics. They revealed that Proteobacteria and Firmicutes accounted for 57% of the microbiome with Hydrogenobacter spp., one of the major phyla (Chan et al., 2015). Ward et al. performed metagenomic characterization of a proterozoic-analog hot spring known as Jinata Onsen in Japan and revealed thermophilic lithotrophs, iron-tolerant phototrophs, and some previously unknown microorganisms. ...
Hydrothermal sources are of paramount importance for the exploration of small molecules carrying important biochemical and genetic information. They harbor thermophilic microbial communities that are useful in developing commercially useful products for pharmaceutical, agriculture, cosmetics or aquaculture industries. South Asian countries are rich in their hot-spring sources on the Himalayas, Pamir, and Karakoram mountains. The majority of the microbial diversity associated with these hot springs has been missed by traditional genomics due to limitations in culture-based methods. Metagenomics technique provides an insightful lens to reveal the potentially valuable microbe-derived molecules that can revolutionize microbial investigation. Various research works have been carried out on hot springs, a substantial variety of valuable biomolecules such as Taq DNA polymerase and hydrolases have been derived besides discovering numerous previously unknown microorganisms. This chapter provides detailed insight about the hot springs of the Asian subcontinent, including India, China, Pakistan, Bhutan, etc., and a comprehensive outlook about the metagenomic studies done on these hydrothermal sources. Moreover, the significance of metagenomics in discovering novel molecules and new microorganisms will also be highlighted in greater detail.
... Proteobacteria (19.26%), Aquificae (8.12), Verrucomicrobia (5.48%), and Thermotogae (4.11%), respectively (Chan et al., 2015). ...
... Our study categorized the bacteria into 8% mesophiles, 48% thermophiles, and 44% hyperthermophiles. Temperature is an important growth parameter for bacterial diversity [15], and these two factors exhibit a positive linear correlation [16]. An increase in temperature, especially in harsh environments such as hot springs, remarkably decreased bacterial diversity and species richness, allowing only dominant extremophiles to grow. ...
Owing to their eccentric thermostable ability, thermophiles are among the most utilized extremophiles in various industries, such as manufacturing, and clinical research. Researchers believe that many unknown thermophiles are yet to be discovered. This study aimed to genotypically characterize the diversity of thermophiles and screen them for the potential production of enzymes in the recreational hot springs located at Northwest Coast of Borneo. Water samples were collected at 45 °C-50 °C from Annah Rais and Panchor hot springs during the sampling period from January 2018 to January 2019. Three samples (water and sediment) were collected twice in a 3-week interval from each pool of the sampling sites. Each water sample was diluted up to 10 −3 and plated on thick nutrient agar at 55 °C for 24 h. Customized nutrient agar plus Bacto-agar plates were used for the optimum growth analysis of the isolates at 40 °C-90 °C for 24 h. The thermophiles were isolated, characterized biochemically, and amplified molecularly using DNA fingerprinting and 16S rRNA gene sequencing. Lipase, protease, gelatinase, amylase, catalase, and nitrate reductase enzymatic production was examined. Twenty-one thermophilic isolates were successfully characterized into seven clusters of Amnoxybacillus spp. and Geobacillus spp. by studying their phylogenetic dendrograms. Isolates AR10 and AR15 could produce most of the tested enzymes. All the isolates showed negative results in gelatinase and lipase production. PC14 was the only isolate that did not produce any of the enzymatic reactions in this experiment. The results showed that most of the thermophiles isolated from the two Borneo hot springs can synthesize enzymes and have potential to be thermostable. In conclusion, the search for the thermophilic producers of novel enzymes in Borneo is successful; further research must focus on their applications.
... Metagenome screening is an invaluable technique for exploring the vast biodiversity of nature and uncovering novel enzymes, as it allows for direct analysis without the limitations in cultivation-based methods. The mining of a metagenomic library has facilitated the identification of microbial diversity and novel enzymes (cellulase and xylanase) from a variety of environmental samples, including soil, hot spring, termite's gut, rumen of dairy cow [48][49][50][51]. Earlier study identified that experimental warming and the resultant decrease in soil moisture has a significant impact on microbial biodiversity by reducing the richness of bacteria (9.6%). ...
Background
Goat rumen microbial communities are perceived as one of the most potential biochemical reservoirs of multi-functional enzymes, which are applicable to enhance wide array of bioprocesses such as the hydrolysis of cellulose and hemi-cellulose into fermentable sugar for biofuel and other value-added biochemical production. Even though, the limited understanding of rumen microbial genetic diversity and the absence of effective screening culture methods have impeded the full utilization of these potential enzymes. In this study, we applied culture independent metagenomics sequencing approach to isolate, and identify microbial communities in goat rumen, meanwhile, clone and functionally characterize novel cellulase and xylanase genes in goat rumen bacterial communities.
Results
Bacterial DNA samples were extracted from goat rumen fluid. Three genomic libraries were sequenced using Illumina HiSeq 2000 for paired-end 100-bp (PE100) and Illumina HiSeq 2500 for paired-end 125-bp (PE125). A total of 435gb raw reads were generated. Taxonomic analysis using Graphlan revealed that Fibrobacter , Prevotella , and Ruminococcus are the most abundant genera of bacteria in goat rumen. SPAdes assembly and prodigal annotation were performed. The contigs were also annotated using the DOE-JGI pipeline. In total, 117,502 CAZymes, comprising endoglucanases, exoglucanases, beta-glucosidases, xylosidases, and xylanases, were detected in all three samples. Two genes with predicted cellulolytic/xylanolytic activities were cloned and expressed in E. coli BL21(DE3). The endoglucanases and xylanase enzymatic activities of the recombinant proteins were confirmed using substrate plate assay and dinitrosalicylic acid (DNS) analysis. The 3D structures of endoglucanase A and endo-1,4-beta xylanase was predicted using the Swiss Model. Based on the 3D structure analysis, the two enzymes isolated from goat’s rumen metagenome are unique with only 56–59% similarities to those homologous proteins in protein data bank (PDB) meanwhile, the structures of the enzymes also displayed greater stability, and higher catalytic activity.
Conclusions
In summary, this study provided the database resources of bacterial metagenomes from goat’s rumen fluid, including gene sequences with annotated functions and methods for gene isolation and over-expression of cellulolytic enzymes; and a wealth of genes in the metabolic pathways affecting food and nutrition of ruminant animals.
... However, the fact that omicsbased methods also detected much more bacterial taxa at genus and even phylum level shows that either the taxonomic resolution of omics-based methods outperformed that of 16S sequencing for bacteria or that these methods detected a high number of false-positive bacterial taxa. There is no clear consensus in the literature as to which of those methods detect more taxa, with some studies showing that amplicon sequencing detects more taxa than omics-based methods (Stat et al., 2017;Tessler et al., 2017), while others show that both methods detect equal amounts of taxa (Chan et al., 2015;Obiol et al., 2020) or that omics-based methods outperform amplicon sequencing in terms of biodiversity coverage (Shakya et al., 2013;Laudadio et al., 2018;Yan et al., 2018;Brumfield et al., 2020). Biodiversity coverage also depends on how well an environment is represented in reference databases, and for lessstudied environments that are poorly represented in reference databases, it is possible that the majority of omics-based sequences cannot be taxonomically annotated, resulting in low overall taxonomic resolution (Stat et al., 2017). ...
Introduction
Microbes are increasingly (re)considered for environmental assessments because they are powerful indicators for the health of ecosystems. The complexity of microbial communities necessitates powerful novel tools to derive conclusions for environmental decision-makers, and machine learning is a promising option in that context. While amplicon sequencing is typically applied to assess microbial communities, metagenomics and total RNA sequencing (herein summarized as omics-based methods) can provide a more holistic picture of microbial biodiversity at sufficient sequencing depths. Despite this advantage, amplicon sequencing and omics-based methods have not yet been compared for taxonomy-based environmental assessments with machine learning.
Methods
In this study, we applied 16S and ITS-2 sequencing, metagenomics, and total RNA sequencing to samples from a stream mesocosm experiment that investigated the impacts of two aquatic stressors, insecticide and increased fine sediment deposition, on stream biodiversity. We processed the data using similarity clustering and denoising (only applicable to amplicon sequencing) as well as multiple taxonomic levels, data types, feature selection, and machine learning algorithms and evaluated the stressor prediction performance of each generated model for a total of 1,536 evaluated combinations of taxonomic datasets and data-processing methods.
Results
Sequencing and data-processing methods had a substantial impact on stressor prediction. While omics-based methods detected a higher diversity of taxa than amplicon sequencing, 16S sequencing outperformed all other sequencing methods in terms of stressor prediction based on the Matthews Correlation Coefficient. However, even the highest observed performance for 16S sequencing was still only moderate. Omics-based methods performed poorly overall, but this was likely due to insufficient sequencing depth. Data types had no impact on performance while feature selection significantly improved performance for omics-based methods but not for amplicon sequencing.
Discussion
We conclude that amplicon sequencing might be a better candidate for machine-learning-based environmental stressor prediction than omics-based methods, but the latter require further research at higher sequencing depths to confirm this conclusion. More sampling could improve stressor prediction performance, and while this was not possible in the context of our study, thousands of sampling sites are monitored for routine environmental assessments, providing an ideal framework to further refine the approach for possible implementation in environmental diagnostics.
... Malaysia is home to numerous hot springs where the water arises from groundwater sources is heated geothermally as compared to volcanic hot springs located in Yellowstone National Park (United States), Japan, India, New Zealand, Indonesia and the Philippines (Papke et al. 2003;Chan et al. 2015;Prihantini et al. 2016;Masaki et al. 2016;Martinez-Goss et al. 2019). The presence of cyanobacterial community in hot springs of Peninsular Malaysia has been discovered using the culture independent 16s rRNA metagenome sequencing approach (Chan et al. 3 2015;Chan et al. 2017;Lee et al. 2017;Halim et al. 2020). ...
... Nonetheless, the diversity of cyanobacteria in these hot springs remains unclear since most of these studies emphasised on the elucidation of the thermophilic microbial community in general. It was briefly mentioned that Sungai Klah hot spring from Sungkai, Perak, Malaysia comprised of cyanobacteria from the genera of Cyanobacterium, Synechococcus, Gloeobacter and Oscillatoria (Chan et al. 2015). In addition, the morphological and molecular features of culturable cyanobacteria isolates from the Malaysian hot springs are not well-characterised. ...
Malaysia is home to a number of hot springs that are rich in microbial diversity including the photosynthetic cyanobacteria. Although this microbial community has been characterised based on metagenomics approach, the culturable thermophilic isolates have not been isolated and characterised extensively. Compared to the mesophiles, information on plant growth promoting (PGP) properties of these thermophiles remain largely untapped. As the amount of arable land for microbial bioprospecting is decreasing due to extensive human activities, the search for alternative source for microbial strains with PGP properties is important for the development of potential biofertilizers. This study sought to isolate and characterise culturable cyanobacteria strains from two local hot springs – Sungai Klah (SK) and Lubuk Timah (LT) located in Perak using morphological and molecular methods. The IAA production from the axenic cultures were measured. The PGP properties were also measured by priming the rice seeds with cyanobacterial water extracts. A total of six strains were isolated from both hot springs. Strains LTM and LTW from LT were identified as Leptolyngbya sp. whereas strains SEM, SEH, STH and STM were identified as Thermosynechococcus elongatus. All six strains produced IAA ranged from 670.10 pg/ ?L to 2010 pg/?L. The water extracts were found to increase the seed amylase activity of the rice seeds from 5th day of germination (DAG) to 10th DAG. In general, the IAA production and increased seed amylase activity might have contributed in enhancing the longest root length, shoot length and root-to-shoot (RS) ratio. To conclude, the thermophilic cyanobacteria from hot springs can be further exploited as a novel source of PGP microbes for the development of biofertilisers.
