Integrating multi-platform assembly to recover MAGs from hot spring biofilms: insights into microbial diversity, biofilm formation, and carbohydrate degradation.

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.

Microbial community dynamics and functional potentials in the conversion of oil palm wastes into biomethane.

Oil palm processing generates substantial waste materials rich in organic content, posing various environmental challenges. Anaerobic digestion (AD), particularly for palm oil mill effluent (POME), offers a sustainable solution, by converting waste into valuable biomethane for thermal energy or electricity generation. The synergistic activities of the AD microbiota directly affect the biomethane production, and the microbial community involved in biomethane production in POME anaerobic digestion has been reported. The composition of bacterial and archaeal communities varies under different substrate and physicochemical conditions. This review discusses the characteristics of POME, explores the microbial members engaged in each stage of AD, and elucidates the impacts of substrate and physicochemical conditions on the microbial community dynamics, with a specific focus on POME. Finally, the review outlines current research needs and provides future perspectives on optimizing the microbial communities for enhanced biomethane production from oil palm wastes.

Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1.

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.

Impact of sterilization and chemical fertilizer on the microbiota of oil palm seedlings.

Soil nutrients and microbiota are known as essential components for healthy plant growth and crop productivity. However, limited studies have been conducted on the importance of soil microbiota in the early growth of oil palm seedlings (Elaeis guineensis Jacq.) under the influence of nitrogen, phosphorus and potassium (NPK) compound fertilizer (nitrogen, phosphorus, and potassium). In this study, we analyzed the root microbial community associated with seedlings grown under normal and sterilized soil conditions to ascertain the microbial strains potentially associated with soil, plant health and chemical fertilizer efficiency. Oil palm seedlings were grown under four treatments: (i) fertilized normal soil (+FN), (ii) unfertilized normal soil (-FN), (iii) fertilized sterilized soil (+FS) and (iv) unfertilized sterilized soil (-FS). Our findings revealed that chemical fertilizer promoted the growth of the copiotrophs Pseudomonadota and Bacteroidota in the control +FN, which are known to degrade complex polysaccharides. After autoclaving, the soil macronutrient content did not change, but soil sterilization reduced microbial diversity in the +FS and -FS treatments and altered the soil microbiota composition. Sterilized soil with a depleted microbial population adversely affected crop growth, which was exacerbated by fertilizer use. In the rhizosphere and rhizoplane compartments, a total of 412 and 868 amplicon sequence variances (ASVs) were found depleted in the +FS and -FS treatments, respectively. Several genera were identified in the ASVs with diminished abundance, including Humibacter, Microbacterium, Mycobacterium, 1921-2, HSB OF53-F07, Mucilaginibacter, Bacillus, Paenibacillus, and several unclassified genera, suggesting their possible roles in promoting the plant growth of oil palm seedlings. Soil sterilization might remove these beneficial microbes from the bulk soil pool, affecting the colonization ability in the rhizocompartments as well as their role in nutrient transformation. Therefore, this study provides useful insights concerning the benefits of a soil microbiome survey before making fertilizer recommendations.

Text-Mining to Identify Gene Sets Involved in Biocorrosion by Sulfate-Reducing Bacteria: A Semi-Automated Workflow.

A significant amount of literature is available on biocorrosion, which makes manual extraction of crucial information such as genes and proteins a laborious task. Despite the fast growth of biology related corrosion studies, there is a limited number of gene collections relating to the corrosion process (biocorrosion). Text mining offers a potential solution by automatically extracting the essential information from unstructured text. We present a text mining workflow that extracts biocorrosion associated genes/proteins in sulfate-reducing bacteria (SRB) from literature databases (e.g., PubMed and PMC). This semi-automatic workflow is built with the Named Entity Recognition (NER) method and Convolutional Neural Network (CNN) model. With PubMed and PMCID as inputs, the workflow identified 227 genes belonging to several Desulfovibrio species. To validate their functions, Gene Ontology (GO) enrichment and biological network analysis was performed using UniprotKB and STRING-DB, respectively. The GO analysis showed that metal ion binding, sulfur binding, and electron transport were among the principal molecular functions. Furthermore, the biological network analysis generated three interlinked clusters containing genes involved in metal ion binding, cellular respiration, and electron transfer, which suggests the involvement of the extracted gene set in biocorrosion. Finally, the dataset was validated through manual curation, yielding a similar set of genes as our workflow; among these, hysB and hydA, and sat and dsrB were identified as the metal ion binding and sulfur metabolism genes, respectively. The identified genes were mapped with the pangenome of 63 SRB genomes that yielded the distribution of these genes across 63 SRB based on the amino acid sequence similarity and were further categorized as core and accessory gene families. SRB's role in biocorrosion involves the transfer of electrons from the metal surface via a hydrogen medium to the sulfate reduction pathway. Therefore, genes encoding hydrogenases and cytochromes might be participating in removing hydrogen from the metals through electron transfer. Moreover, the production of corrosive sulfide from the sulfur metabolism indirectly contributes to the localized pitting of the metals. After the corroboration of text mining results with SRB biocorrosion mechanisms, we suggest that the text mining framework could be utilized for genes/proteins extraction and significantly reduce the manual curation time.

Genomic Analysis to Elucidate the Lignocellulose Degrading Capability of a New Halophile Robertkochia solimangrovi.

Robertkochia solimangrovi is a proposed marine bacterium isolated from mangrove soil. So far, the study of this bacterium is limited to taxonomy only. In this report, we performed a genomic analysis of R. solimangrovi that revealed its lignocellulose degrading ability. Genome mining of R. solimangrovi revealed a total of 87 lignocellulose degrading enzymes. These enzymes include cellulases (GH3, GH5, GH9 and GH30), xylanases (GH5, GH10, GH43, GH51, GH67, and GH115), mannanases (GH2, GH26, GH27 and GH113) and xyloglucanases (GH2, GH5, GH16, GH29, GH31 and GH95). Most of the lignocellulolytic enzymes encoded in R. solimangrovi were absent in the genome of Robertkochia marina, the closest member from the same genus. Furthermore, current work also demonstrated the ability of R. solimangrovi to produce lignocellulolytic enzymes to deconstruct oil palm empty fruit bunch (EFB), a lignocellulosic waste found abundantly in palm oil industry. The metabolic pathway taken by R. solimangrovi to transport and process the reducing sugars after the action of lignocellulolytic enzymes on EFB was also inferred based on genomic data. Collectively, genomic analysis coupled with experimental studies elucidated R. solimangrovi to serve as a promising candidate in seawater based-biorefinery industry.

Draft genome sequence of Thermovorax subterraneus 70BT, a thermophile isolated from a geothermally active underground mine that produces hydrogen.

Thermovorax subterraneus 70BT is a thermophile found in a geothermically active underground mine. The strain 70BT belongs to the class of Clostridia, order of Thermosediminibacterales, and family of Thermosediminibacteraceae. Strain 70BT was the only type strain since the genus was discovered >10 years ago. Strain 70BT was compared to strains from other genera in terms of its phenotypics, chemotaxonomics, and phylogenetics (16S rRNA gene) in previous studies. However, the genome sequence of this strain has not been described. We herein described the genome sequence of strain 70BT. In total, the assembled genome of strain 70BT has a size of 2,451,552 bp, contributed by 44 contigs, with a coverage of 445X, a N50 of 86,294 bp, and a GC% of 43.8. A total of 2,540 genes were encoded in the genome, including 2,431 protein-coding sequences, 46 pseudogenes, and 63 RNA genes. Through the Cluster of Orthologous Groups (COGs) analysis, a total of 2,404 protein-coding genes were functionally assigned to COGs in the genome of strain 70BT. Among the members of Thermosediminibacteraceae family, strain 70BT has the closest relationship to Caldanaerovirga acetigignens JW/SA-NV4T based on the genome-to-genome comparison indexes (i.e., ANI, dDDH, AAI, and POCP). An earlier study reported that strain 70BT could produce hydrogen. We discovered genes encoding [FeFe] hydrogenase through gene mining analysis. For future research, this genome data will be used as a reference for all matters pertaining to the genus Thermovorax and family Thermosediminibacteraceae.

Draft Genome Sequence of Microaerobacter geothermalis Nad S1T, a Microaerophilic Bacterium Isolated from Tenusia Hot Spring.

Microaerobacter geothermalis Nad S1T is a rare Bacillaceae thermophile that grows optimally at 55°C and circumneutral pH. Although strain Nad S1T was discovered >10 years ago, its genome is yet to be described. The release of the Nad S1T genome sequence serves as reference genetic information for subsequent use.

Genome Analysis of Celeribacter sp. PS-C1 Isolated from Sekinchan Beach in Selangor, Malaysia, Reveals Its ß-Glucosidase and Licheninase Activities.

A halophilic marine bacterial strain, PS-C1, was isolated from Sekinchan beach in Selangor, Malaysia. The 16S rRNA gene sequence analysis indicated that strain PS-C1 was associated with the genus Celeribacter. To date, there have been no reports on enzymes from the genus Celeribacter. The present study reports on the cellular features of Celeribacter sp. PS-C1, its annotated genome sequence, and comparative genome analyses of Celeribacter glycoside hydrolase (GH) enzymes. The genome of strain PS-C1 has a size of 3.87 Mbp and a G+C content of 59.10%, and contains 3739 protein-coding genes. Detailed analysis using the Carbohydrate-Active enZYmes (CAZy) database revealed that Celeribacter genomes harboured at least 12 putative genes encoding industrially important GHs that are grouped as cellulases, ß-glucanases, hemicellulases, and starch-degrading enzymes. Herein, the potential applications of these enzymes are discussed. Furthermore, the activities of two types of GHs (ß-glucosidase and licheninase) in strain PS-C1 were demonstrated. These findings suggest that strain PS-C1 could be a reservoir of novel GH enzymes for lignocellulosic biomass degradation.

Thermophiles and carbohydrate-active enzymes (CAZymes) in biofilm microbial consortia that decompose lignocellulosic plant litters at high temperatures.

The SKY hot spring is a unique site filled with a thick layer of plant litter. With the advancement of next-generation sequencing, it is now possible to mine many new biocatalyst sequences. In this study, we aimed to (i) identify the metataxonomic of prokaryotes and eukaryotes in microbial mats using 16S and 18S rRNA markers, (ii) and explore carbohydrate degrading enzymes (CAZymes) that have a high potential for future applications. Green microbial mat, predominantly photosynthetic bacteria, was attached to submerged or floating leaves litter. At the spring head, the sediment mixture consisted of plant debris, predominantly brownish-reddish gelatinous microbial mat, pale tan biofilm, and grey-white filament biofilm. The population in the spring head had a higher percentage of archaea and hyperthermophiles than the green mat. Concurrently, we cataloged nearly 10,000 sequences of CAZymes in both green and brown biofilms using the shotgun metagenomic sequencing approach. These sequences include ß-glucosidase, cellulase, xylanase, α-N-arabinofuranosidase, α-L-arabinofuranosidase, and other CAZymes. In conclusion, this work elucidated that SKY is a unique hot spring due to its rich lignocellulosic material, often absent in other hot springs. The data collected from this study serves as a repository of new thermostable macromolecules, in particular families of glycoside hydrolases.

Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia.

Cellulomonas sp. PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Draft Genome Sequence of Roseovarius sp. PS-C2, Isolated from Sekinchan Beach in Selangor, Malaysia.

Roseovarius sp. PS-C2 is a bacterium that was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation technique. Here, we present a high-quality annotated draft genome of strain PS-C2 and suggest potential applications of this bacterium.

Metagenomics and Culture Dependent Insights into the Distribution of Firmicutes across Two Different Sample Types Located in the Black Hills Region of South Dakota, USA.

Firmicutes is almost a ubiquitous phylum. Several genera of this group, for instance, Geobacillus, are recognized for decomposing plant organic matter and for producing thermostable ligninolytic enzymes. Amplicon sequencing was used in this study to determine the prevalence and genetic diversity of the Firmicutes in two distinctly related environmental samples-South Dakota Landfill Compost (SDLC, 60 °C), and Sanford Underground Research Facility sediments (SURF, 45 °C). Although distinct microbial community compositions were observed, there was a dominance of Firmicutes in both the SDLC and SURF samples, followed by Proteobacteria. The abundant classes of bacteria in the SDLC site, within the phylum Firmicutes, were Bacilli (83.2%), and Clostridia (2.9%). In comparison, the sample from the SURF mine was dominated by the Clostridia (45.8%) and then Bacilli (20.1%). Within the class Bacilli, the SDLC sample had more diversity (a total of 11 genera with more than 1% operational taxonomic unit, OTU). On the other hand, SURF samples had just three genera, about 1% of the total population: Bacilli, Paenibacillus, and Solibacillus. With specific regard to Geobacillus, it was found to be present at a level of 0.07% and 2.5% in SURF and SDLC, respectively. Subsequently, culture isolations of endospore-forming Firmicutes members from these samples led to the isolation of a total of 117 isolates. According to colony morphologies, and identification based upon 16S rRNA and gyrB gene sequence analysis, we obtained 58 taxonomically distinct strains. Depending on the similarity indexes, a gyrB sequence comparison appeared more useful than 16S rRNA sequence analysis for inferring intra- and some intergeneric relationships between the isolates.

Genome sequence of an uncharted halophilic bacterium Robertkochia marina with deciphering its phosphate-solubilizing ability.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.

Global Transcriptomic Responses of Roseithermus sacchariphilus Strain RA in Media Supplemented with Beechwood Xylan.

The majority of the members in order Rhodothermales are underexplored prokaryotic extremophiles. Roseithermus, a new genus within Rhodothermales, was first described in 2019. Roseithermus sacchariphilus is the only species in this genus. The current report aims to evaluate the transcriptomic responses of R. sacchariphilus strain RA when cultivated on beechwood xylan. Strain RA doubled its growth in Marine Broth (MB) containing xylan compared to Marine Broth (MB) alone. Strain RA harbors 54 potential glycosyl hydrolases (GHs) that are affiliated with 30 families, including cellulases (families GH 3, 5, 9, and 44) and hemicellulases (GH 2, 10, 16, 29, 31,43, 51, 53, 67, 78, 92, 106, 113, 130, and 154). The majority of these GHs were upregulated when the cells were grown in MB containing xylan medium and enzymatic activities for xylanase, endoglucanase, ß-xylosidase, and ß-glucosidase were elevated. Interestingly, with the introduction of xylan, five out of six cellulolytic genes were upregulated. Furthermore, approximately 1122 genes equivalent to one-third of the total genes for strain RA were upregulated. These upregulated genes were mostly involved in transportation, chemotaxis, and membrane components synthesis.

Meta-Analysis of Microbial Communities in Hot Springs: Recurrent Taxa and Complex Shaping Factors beyond pH and Temperature.

The study of microbial communities from extreme environments is a fascinating topic. With every study, biologists and ecologists reveal interesting facts and questions that dispel the old belief that these are inhospitable environments. In this work, we assess the microbial diversity of three hot springs from Neuquén, Argentina, using high-throughput amplicon sequencing. We predicted a distinct metabolic profile in the acidic and the circumneutral samples, with the first ones being dominated by chemolithotrophs and the second ones by chemoheterotrophs. Then, we collected data of the microbial communities of hot springs around the world in an effort to comprehend the roles of pH and temperature as shaping factors. Interestingly, there was a covariation between both parameters and the phylogenetic distance between communities; however, neither of them could explain much of the microbial profile in an ordination model. Moreover, there was no correlation between alpha diversity and these parameters. Therefore, the microbial communities' profile seemed to have complex shaping factors beyond pH and temperature. Lastly, we looked for taxa associated with different environmental conditions. Several such taxa were found. For example, Hydrogenobaculum was frequently present in acidic springs, as was the Sulfolobaceae family; on the other hand, Candidatus Hydrothermae phylum was strongly associated with circumneutral conditions. Interestingly, some singularities related to sites featuring certain taxa were also observed.

Robertkochia solimangrovi sp. nov., isolated from mangrove soil, and emended description of the genus Robertkochia.

To date, there is sparse information for the genus Robertkochia with Robertkochia marina CC-AMO-30DT as the only described member. We report here a new species isolated from mangrove soil collected at Malaysia Tanjung Piai National Park and perform polyphasic characterization to determine its taxonomic position. Strain CL23T is a Gram-negative, yellow-pigmented, strictly aerobic, catalase-positive and oxidase-positive bacterium. The optimal growth conditions were determined to be at pH 7.0, 30-37 °C and in 1-2 % (w/v) NaCl. The major respiratory quinone was menaquinone-6 (MK-6) and the highly abundant polar lipids were four unidentified lipids, a phosphatidylethanolamine and two unidentified aminolipids. The 16S rRNA gene similarity between strain CL23T and R. marina CC-AMO-30DT is 96.67 %. Strain CL23T and R. marina CC-AMO-30DT clustered together and were distinguished from taxa of closely related genera in 16S rRNA gene phylogenetic analysis. Genome sequencing revealed that strain CL23T has a genome size of 4.4 Mbp and a G+C content of 40.72 mol%. Overall genome related indexes including digital DNA-DNA hybridization value and average nucleotide identity are 17.70 % and approximately 70%, below the cutoffs of 70 and 95%, respectively, indicated that strain CL23T is a distinct species from R. marina CC-AMO-30DT. Collectively, based on the phenotypic, chemotaxonomic, phylogenetic and genomic evidences presented here, strain CL23T is proposed to represent a new species with the name Robertkochia solimangrovi sp. nov. (KCTC 72252T=LMG 31418T). An emended description of the genus Robertkochia is also proposed.