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.

Integration of text mining and biological network analysis: Identification of essential genes in sulfate-reducing bacteria.

The growth and survival of an organism in a particular environment is highly depends on the certain indispensable genes, termed as essential genes. Sulfate-reducing bacteria (SRB) are obligate anaerobes which thrives on sulfate reduction for its energy requirements. The present study used Oleidesulfovibrio alaskensis G20 (OA G20) as a model SRB to categorize the essential genes based on their key metabolic pathways. Herein, we reported a feedback loop framework for gene of interest discovery, from bio-problem to gene set of interest, leveraging expert annotation with computational prediction. Defined bio-problem was applied to retrieve the genes of SRB from literature databases (PubMed, and PubMed Central) and annotated them to the genome of OA G20. Retrieved gene list was further used to enrich protein-protein interaction and was corroborated to the pangenome analysis, to categorize the enriched gene sets and the respective pathways under essential and non-essential. Interestingly, the sat gene (dde_2265) from the sulfur metabolism was the bridging gene between all the enriched pathways. Gene clusters involved in essential pathways were linked with the genes from seleno-compound metabolism, amino acid metabolism, secondary metabolite synthesis, and cofactor biosynthesis. Furthermore, pangenome analysis demonstrated the gene distribution, where 69.83% of the 116 enriched genes were mapped under "persistent," inferring the essentiality of these genes. Likewise, 21.55% of the enriched genes, which involves specially the formate dehydrogenases and metallic hydrogenases, appeared under "shell." Our methodology suggested that semi-automated text mining and network analysis may play a crucial role in deciphering the previously unexplored genes and key mechanisms which can help to generate a baseline prior to perform any experimental studies.

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.

Graphene as Thinnest Coating on Copper Electrodes in Microbial Methanol Fuel Cells.

Dehydrogenation of methanol (CH3OH) into direct current (DC) in fuel cells can be a potential energy conversion technology. However, their development is currently hampered by the high cost of electrocatalysts based on platinum and palladium, slow kinetics, the formation of carbon monoxide intermediates, and the requirement for high temperatures. Here, we report the use of graphene layers (GL) for generating DC electricity from microbially driven methanol dehydrogenation on underlying copper (Cu) surfaces. Genetically tractable Rhodobacter sphaeroides 2.4.1 (Rsp), a nonarchetypical methylotroph, was used for dehydrogenating methanol at the GL-Cu surfaces. We use electrochemical methods, microscopy, and spectroscopy methods to assess the effects of GL on methanol dehydrogenation by Rsp cells. The GL-Cu offers a 5-fold higher power density and 4-fold higher current density compared to bare Cu. The GL lowers charge transfer resistance to methanol dehydrogenation by 4 orders of magnitude by mitigating issues related to pitting corrosion of underlying Cu surfaces. The presented approach for catalyst-free methanol dehydrogenation on copper electrodes can improve the overall sustainability of fuel cell technologies.

Photosynthetic microbial fuel cells for methanol treatment using graphene electrodes.

Photosynthetic microbial fuel cells (pMFC) represent a promising approach for treating methanol (CH3OH) wastewater. However, their use is constrained by a lack of knowledge on the extracellular electron transfer capabilities of photosynthetic methylotrophs, especially when coupled with metal electrodes. This study assessed the CH3OH oxidation capabilities of Rhodobacter sphaeroides 2.4.1 in two-compartment pMFCs. A 3D nickel (Ni) foam modified with plasma-grown graphene (Gr) was used as an anode, nitrate mineral salts media (NMS) supplemented with 0.1% CH3OH as anolyte, carbon brush as cathode, and 50 mM ferricyanide as catholyte. Two simultaneous pMFCs that used bare Ni foam and carbon felt served as controls. The Ni/Gr electrode registered a two-fold lower charge transfer resistance (0.005 kΩ cm2) and correspondingly 16-fold higher power density (141 mW/m2) compared to controls. The underlying reasons for the enhanced performance of R. sphaeroides at the graphene interface were discerned. The real-time polymerase chain reaction (PCR) analysis revealed the upregulation of cytochrome c oxidase, aa3 type, subunit I gene, and Flp pilus assembly protein genes in the sessile cells compared to their planktonic counterparts. The key EET pathways used for sustaining CH3OH oxidation were discussed.

Transcriptomics and Functional Analysis of Copper Stress Response in the Sulfate-Reducing Bacterium Desulfovibrio alaskensis G20.

Copper (Cu) is an essential micronutrient required as a co-factor in the catalytic center of many enzymes. However, excess Cu can generate pleiotropic effects in the microbial cell. In addition, leaching of Cu from pipelines results in elevated Cu concentration in the environment, which is of public health concern. Sulfate-reducing bacteria (SRB) have been demonstrated to grow in toxic levels of Cu. However, reports on Cu toxicity towards SRB have primarily focused on the degree of toxicity and subsequent elimination. Here, Cu(II) stress-related effects on a model SRB, Desulfovibrio alaskensis G20, is reported. Cu(II) stress effects were assessed as alterations in the transcriptome through RNA-Seq at varying Cu(II) concentrations (5 µM and 15 µM). In the pairwise comparison of control vs. 5 µM Cu(II), 61.43% of genes were downregulated, and 38.57% were upregulated. In control vs. 15 µM Cu(II), 49.51% of genes were downregulated, and 50.5% were upregulated. The results indicated that the expression of inorganic ion transporters and translation machinery was massively modulated. Moreover, changes in the expression of critical biological processes such as DNA transcription and signal transduction were observed at high Cu(II) concentrations. These results will help us better understand the Cu(II) stress-response mechanism and provide avenues for future research.

Gene Sets and Mechanisms of Sulfate-Reducing Bacteria Biofilm Formation and Quorum Sensing With Impact on Corrosion.

Sulfate-reducing bacteria (SRB) have a unique ability to respire under anaerobic conditions using sulfate as a terminal electron acceptor, reducing it to hydrogen sulfide. SRB thrives in many natural environments (freshwater sediments and salty marshes), deep subsurface environments (oil wells and hydrothermal vents), and processing facilities in an industrial setting. Owing to their ability to alter the physicochemical properties of underlying metals, SRB can induce fouling, corrosion, and pipeline clogging challenges. Indigenous SRB causes oil souring and associated product loss and, subsequently, the abandonment of impacted oil wells. The sessile cells in biofilms are 1,000 times more resistant to biocides and induce 100-fold greater corrosion than their planktonic counterparts. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation and corrosion. Here, we examine the critical genes involved in biofilm formation and microbiologically influenced corrosion and categorize them into various functional categories. The current effort also discusses chemical and biological methods for controlling the SRB biofilms. Finally, we highlight the importance of surface engineering approaches for controlling biofilm formation on underlying metal surfaces.

Bioleaching of metals from waste printed circuit boards using bacterial isolates native to abandoned gold mine.

In the present study, native bacterial strains isolated from abandoned gold mine and Chromobacterium violaceum (MTCC-2656) were applied for bioleaching of metals from waste printed circuit boards (WPCBs). Toxicity assessment and dose-response analysis of WPCBs showed EC50 values of 128.9, 98.7, and 90.8 g/L for Bacillus sp. SAG3, Bacillus megaterium SAG1 and Lysinibacillus sphaericus SAG2, respectively, whereas, for C. violaceum EC50 was 83.70 g/L. This indicates the viable operation range and technological feasibility of metals bioleaching from WPCBs using mine isolates. The influencing factors such as pH, pulp density, temperature, and precursor molecule (glycine) were optimized by one-factor at a time method (OFAT). The maximum metal recovery occurred at an initial pH of 9.0, a pulp density of 10 g/L, a temperature of 30 °C and a glycine concentration of 5 g/L, except for L. sphaericus which showed optimum activity at initial pH of 8.0. Under optimal conditions the metals recovery of Cu and Au from WPCBs were recorded as 87.5 ± 8% and 73.6 ± 3% for C. violaceum and 72.7 ± 5% and 66.6 ± 6% for B. megaterium, respectively. Kinetic modeling results showed that the data was best described by first order reaction kinetics, where the rate of metal solubilization from WPCBs depended upon microbial lixiviant production. This is the first report on bioleaching of metals from e-waste using bacterial isolates from the gold mine of Solan, HP. Our study demonstrated the potential of bioleaching for resource recovery from WPCBs dust, aimed to be disposed at landfills, and its effectiveness in extraction of elements those are at high supply risk and demand.

Electricity from methane by Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b.

Given the difficulties valorizing methane (CH4) via catalytic routes, this study explores use of CH4-oxidizing bacteria ("methanotrophs") for generating electricity directly from CH4. A preconditioned methanotrophic biofilm on 3D nickel foam with reduced graphene oxide (rGO/Ni) was used as the anode in two-compartment microbial fuel cells (MFCs). This study demonstrates a proof of concept for turning CH4 into electricity by two model methanotrophs including Methylosinus trichosposium OB3b and Methylococcus capsulatus (Bath). Both OB3b (205 mW.m-2) and Bath (110 mW.m-2) strains yielded a higher electricity from CH4 when grown on rGO/Ni compared to graphite felt electrodes. Based on electrochemistry tests, molecular dynamics simulations, genome annotations and interaction analysis, a mechanistic understanding of reasons behind enhanced performance of methanotrophs grown on rGO/Ni are presented.

Anaerobic wastewater treatment and reuse enabled by thermophilic bioprocessing integrated with a bioelectrochemical/ultrafiltration module.

The current study aimed to develop an anaerobic wastewater treatment and reuse module enabled by thermophilic bioprocessing, a microbial fuel cell (MFC) and ultrafiltration (UF) treatment. A previously unexplored consortium based on Thermoanaerobacterium thermosaccharolyticum and Arcobacter sp. was used to remove ~73% of chemical oxygen demand (COD) from wastewater under anaerobic conditions (CODi = 200 mg/L). The subsequent MFC and UF treatment removed the COD remnants to meet the secondary treatment standards and reuse criteria. The energy efficiency of polyethersulfone UF membranes was improved by modifying their surfaces with coatings based on self-polymerized dopamine, mixtures of dopamine and poly(2-dimethylamino) ethyl methacrylate methyl, and dopamine analog norepinephrine. The resulting hydrophilic, anti-fouling layers were found to reduce interactions between rejected species and the membrane surface. Finally, this study presents a comparative treatment performance and energy efficiency of the wastewater treatment and reuse modules arranged in six different configurations.

Electricity from lignocellulosic substrates by thermophilic Geobacillus species.

Given our vast lignocellulosic biomass reserves and the difficulty in bioprocessing them without expensive pretreatment and fuel separation steps, the conversion of lignocellulosic biomass directly into electricity would be beneficial. Here we report the previously unexplored capabilities of thermophilic Geobacillus sp. strain WSUCF1 to generate electricity directly from such complex substrates in microbial fuel cells. This process obviates the need for exogenous enzymes and redox mediator supplements. Cyclic voltammetry and chromatography studies revealed the electrochemical signatures of riboflavin molecules that reflect mediated electron transfer capabilities of strain WSUCF1. Proteomics and genomics analysis corroborated that WSUCF1 biofilms uses type-II NADH dehydrogenase and demethylmenaquinone methyltransferase to transfer the electrons to conducting anode via the redox active pheromone lipoproteins localized at the cell membrane.

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.

Acetate Production from Cafeteria Wastes and Corn Stover Using a Thermophilic Anaerobic Consortium: A Prelude Study for the Use of Acetate for the Production of Value-Added Products.

Efficient and sustainable biochemical production using low-cost waste assumes considerable industrial and ecological importance. Solid organic wastes (SOWs) are inexpensive, abundantly available resources and their bioconversion to volatile fatty acids, especially acetate, aids in relieving the requirements of pure sugars for microbial biochemical productions in industries. Acetate production from SOW that utilizes the organic carbon of these wastes is used as an efficient solid waste reduction strategy if the environmental factors are optimized. This study screens and optimizes influential factors (physical and chemical) for acetate production by a thermophilic acetogenic consortium using two SOWs-cafeteria wastes and corn stover. The screening experiment revealed significant effects of temperature, bromoethane sulfonate, and shaking on acetate production. Temperature, medium pH, and C:N ratio were further optimized using statistical optimization with response surface methodology. The maximum acetate concentration of 8061 mg L-1 (>200% improvement) was achieved at temperature, pH, and C:N ratio of 60 °C, 6, 25, respectively, and acetate accounted for more than 85% of metabolites. This study also demonstrated the feasibility of using acetate-rich fermentate (obtained from SOWs) as a substrate for the growth of industrially relevant yeast Yarrowia lipolytica, which can convert acetate into higher-value biochemicals.

Heterologous expression, purification and biochemical characterization of a new endo-1,4-ß-xylanase from Rhodothermaceae bacterium RA.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Complete genome sequence of Rhodothermaceae bacterium RA with cellulolytic and xylanolytic activities.

Rhodothermaceae bacterium RA is a halo-thermophile isolated from a saline hot spring. Previously, the genome of this bacterium was sequenced using a HiSeq 2500 platform culminating in 91 contigs. In this report, we report on the resequencing of its complete genome using a PacBio RSII platform. The genome has a GC content of 68.3%, is 4,653,222 bp in size, and encodes 3711 genes. We are interested in understanding the carbohydrate metabolic pathway, in particular the lignocellulosic biomass degradation pathway. Strain RA harbors 57 glycosyl hydrolase (GH) genes that are affiliated with 30 families. The bacterium consists of cellulose-acting (GH 3, 5, 9, and 44) and hemicellulose-acting enzymes (GH 3, 10, and 43). A crude cell-free extract of the bacterium exhibited endoglucanase, xylanase, ß-glucosidase, and ß-xylosidase activities. The complete genome information coupled with biochemical assays confirms that strain RA is able to degrade cellulose and xylan. Therefore, strain RA is another excellent member of family Rhodothermaceae as a repository of novel and thermostable cellulolytic and hemicellulolytic enzymes.

Characterization of a glucose-tolerant ß-glucosidase from Anoxybacillus sp. DT3-1.

BACKGROUND: In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme(®) CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast(®) 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional ß-glucosidase. RESULTS: In this study, for the first time, a ß-glucosidase DT-Bgl gene (1359 bp) was identified in the genome of Anoxybacillus sp. DT3-1, and cloned and heterologously expressed in Escherichia coli BL21. Phylogenetic analysis indicated that DT-Bgl belonged to glycosyl hydrolase (GH) family 1. The recombinant DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-ß-d-glucopyranoside (pNPG). The DT-Bgl was purified using an Ni-NTA column, with molecular mass of 53 kDa using an SDS-PAGE analysis. It exhibited optimum activity at 70 °C and pH 8.5, and did not require any tested co-factors for activation. The K m and V max values for DT-Bgl were 0.22 mM and 923.7 U/mg, respectively, with pNPG as substrate. The DT-Bgl displayed high glucose tolerance, and retained 93 % activity in the presence of 10 M glucose. CONCLUSIONS: Anoxybacillus DT-Bgl is a novel thermostable ß-glucosidase with low glucose inhibition, and converts long-chain cellodextrins to cellobiose, and further hydrolyse cellobiose to glucose. Results suggest that DT-Bgl could be useful in the development of a bioprocess for the efficient saccharification of lignocellulosic biomass.

Highly Thermostable Xylanase Production from A Thermophilic Geobacillus sp. Strain WSUCF1 Utilizing Lignocellulosic Biomass.

Efficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylooligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail) when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70°C, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70°C, respectively. At 70°C, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, Cellic-HTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70°C). High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.