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Exopolysaccharides (EPSs), a constitutive part of bacterial biofilm, act as a protecting sheath to the extremophilic bacteria and are of high industrial value. In this study, we elucidate a new EPS produced by thermotolerant (growth from 34-44 °C) strain Pseudomonas alcaligenes Med1 from Medano hot spring (39.1 °C surface temperature, pH 7.1) located in the Central Andean Mountains of Chile. Bacterial growth was screened for temperature tolerance (10-60 °C) to confirm the thermotolerance behaviour. Physicochemical properties of the EPS were characterized by different techniques: Scanning Electron Microscopy- Energy Dispersive X-ray Spectroscopy (SEM-EDS), Atomic Force Microscopy (AFM), High-Performance Liquid Chromatography (HPLC), Gel permeation chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Thermogravimetric analysis (TGA). Whole genome of P. alcaligenes Med1 has also been studied in detail to correlate the structural and functional characteristics with genomic insight. The EPS demonstrated amorphous surface roughness composed of evenly distributed macromolecular lumps composed of mainly carbon and oxygen. The monosaccharide analysis has shown the presence of glucose, galactose, and mannose sugars at different ratios. TGA revealed the high thermal stability (315.3 °C) of the polysaccharide. The GPC has shown that Med1 is a low molecular weight polysaccharide (34.8 kDa) with low PI. The 2D-NMR linkage analysis suggests a diverse array of glycosidic bonds within the exopolysaccharide structure. The functional properties of the EPS were evaluated for food industry applications, specifically for antioxidant (DPPH, FRAP an H 2 O 2 ). Extracted Med1 EPS revealed significant emulsification activity against different food grade vegetative oils (Coconut oil, Corn oil, Canola oil, Avocado oil, Sunflower oil, Olive oil, and Sesame oil). The highest 33.9% flocculation activity was observed with 60 mg L -1 EPS concentration. It showed water-holding (WHC) of 107.6% and oil-holding (OHC) capacity of 110.8%. The functional EPS produced by Pseudomonas alcaligenes Med1 from Central Andean Chilean hot spring of central Chile can be a useful additive for the food-processing industry., (© 2024. The Author(s).)

Acidophiles are a group of organisms typically found in highly acidic environments such as acid mine drainage. These organisms have several physiological features that enable them to thrive in highly acidic environments (pH ≤3). Considering that both acid mine drainage and solfatara fields exhibit extreme and dynamic ecological conditions for acidophiles, it is crucial to gain deeper insights into the adaptive mechanisms employed by these unique organisms. The existing literature reveals a notable gap in understanding the multi-stress conditions confronting acidophiles and their corresponding coping mechanisms. Therefore, the current review aims to illuminate the intricacies of the metabolic lifestyles of acidophiles within these demanding habitats, exploring how their energy demands contribute to habitat acidification. In addition, the unique adaptive mechanisms employed by acidophiles were emphasized, especially the pivotal role of monolayer membrane-spanning lipids, and how these organisms effectively respond to a myriad of stresses. Beyond mere survival, understanding the adaptive mechanisms of these unique organisms could further enhance their use in some biotechnological and environmental applications. Lastly, this review explores the strategies used to engineer these organisms to promote their use in industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial adhesion and biofilm maturation is significantly influenced by surface properties, encompassing both bare surfaces and single or multi-layered coatings. Hence, there is an utmost interest in exploring the intricacies of gene regulation in sulfate-reducing bacteria (SRB) on copper and graphene-coated copper surfaces. In this study, Oleidesulfovibrio alaskensis G20 was used as the model SRB to elucidate the pathways that govern pivotal roles during biofilm formation on the graphene layers. Employing a potent reporter green fluorescent protein (GFP) tagged to O. alaskensis G20, the spatial structure of O. alaskensis G20 biofilm on copper foil (CuF), single-layer graphene-coated copper (Cu-GrI), and double-layer graphene-coated copper (Cu-GrII) surfaces was investigated. Biofilm formation on CuF, Cu-GrI, and Cu-GrII surfaces was quantified using CLSM z-stack images within COMSTAT v2 software. The results revealed that CuF, Cu-GrI, and Cu-GrII did not affect the formation of the GFP-tagged O. alaskensis G20 biofilm architecture. qPCR expression showed insignificant fold changes for outer membrane components regulating the quorum-sensing system, and global regulatory proteins between the uncoated and coated surfaces. Notably, a significant expression was observed within the sulfate reduction pathway confined to dissimilatory sulfite reductases on the Cu-GrII surface compared to the CuF and Cu-GrI surfaces.

Copper is known to have toxic effects on bacterial growth. This study aimed to determine the influence of copper ions on Oleidesulfovibrio alaskensis G20 biofilm formation in a lactate-C medium supplemented with variable copper ion concentrations. OA G20, when grown in media supplemented with high copper ion concentrations of 5, 15, and 30 µM, exhibited inhibited growth in its planktonic state. Conversely, under similar copper concentrations, OA G20 demonstrated enhanced biofilm formation on glass coupons. Microscopic studies revealed that biofilms exposed to copper stress demonstrated a change in cellular morphology and more accumulation of carbohydrates and proteins than controls. Consistent with these findings, sulfur ( dsrA , dsrB , sat , aprA ) and electron transport ( NiFeSe , NiFe , ldh , cyt 3) genes, polysaccharide synthesis ( poI ), and genes involved in stress response ( sodB ) were significantly upregulated in copper-induced biofilms, while genes ( ftsZ , ftsA , ftsQ ) related to cellular division were negatively regulated compared to controls. These results indicate that the presence of copper ions triggers alterations in cellular morphology and gene expression levels in OA G20, impacting cell attachment and EPS production. This adaptation, characterized by increased biofilm formation, represents a crucial strategy employed by OA G20 to resist metal ion stress.

A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications., Importance: Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity., Competing Interests: The authors declare no conflict of interest.

Noncoding RNAs (ncRNAs) play key roles in the regulation of important pathways, including cellular growth, stress management, signaling, and biofilm formation. Sulfate-reducing bacteria (SRB) contribute to huge economic losses causing microbial-induced corrosion through biofilms on metal surfaces. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation. This study aimed to identify ncRNAs in the genome of a model SRB, Oleidesulfovibrio alaskensis G20 (OA G20). Three in silico approaches revealed genome-wide distribution of 37 ncRNAs excluding tRNAs in the OA G20. These ncRNAs belonged to 18 different Rfam families. This study identified riboswitches, sRNAs, RNP, and SRP. The analysis revealed that these ncRNAs could play key roles in the regulation of several pathways of biosynthesis and transport involved in biofilm formation by OA G20. Three sRNAs, Pseudomonas P10, Hammerhead type II, and sX4, which were found in OA G20, are rare and their roles have not been determined in SRB. These results suggest that applying various computational methods could enrich the results and lead to the discovery of additional novel ncRNAs, which could lead to understanding the "rules of life of OA G20" during biofilm formation.

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., (© 2024. The Author(s).)

In the present study, a thermophilic strain designated CamBx3 was isolated from the Campanario hot spring, Chile. Based on 16S rRNA gene sequence, phylogenomic, and average nucleotide identity analysis the strain CamBx3 was identified as Bacillus paralicheniformis . Genome analysis of B. paralicheniformis CamBx3 revealed the presence of genes related to heat tolerance, exopolysaccharides (EPS), dissimilatory nitrate reduction, and assimilatory sulfate reduction. The pangenome analysis of strain CamBx3 with eight Bacillus spp. resulted in 26,562 gene clusters, 7,002 shell genes, and 19,484 cloud genes. The EPS produced by B. paralicheniformis CamBx3 was extracted, partially purified, and evaluated for its functional activities. B. paralicheniformis CamBx3 EPS with concentration 5 mg mL -1 showed an optimum 92 mM ferrous equivalent FRAP activity, while the same concentration showed a maximum 91% of Fe 2+ chelating activity. B. paralicheniformis CamBx3 EPS (0.2 mg mL -1 ) demonstrated β -glucosidase inhibition. The EPS formed a viscoelastic gel at 45°C with a maximum instantaneous viscosity of 315 Pa.s at acidic pH 5. The present study suggests that B. paralicheniformis CamBx3 could be a valuable resource for biopolymers and bioactive molecules for industrial applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Narsing Rao, Singh, Sani and Banerjee.)

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 β-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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rohit Rai reports financial support was provided by Science and Engineering Research Board. Rajesh K Sani reports financial support was provided by National Science Foundation. Rohit Rai reports financial support was provided by Lovely Professional University. Kian Mau Goh reports financial support was provided by University of Technology Malaysia., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bacteria are capable of producing a specific type of biopolymer, termed exopolysaccharides (EPSs). EPSs from thermophile Geobacillus sp. strain WSUCF1 specifically can be assembled using cost-effective lignocellulosic biomass as the primary carbon substrate in lieu of traditional sugars. 5-fluorouracil (5-FU) is an FDA-approved, versatile chemotherapeutic that has yielded high efficacy against colon, rectum, and breast cancers. The present study investigates the feasibility of a 5% 5-fluorouracil film using thermophilic exopolysaccharides as the foundation in conjunction with a simple self-forming method. The drug-loaded film formulation was seen to be highly effective against A375 human malignant melanoma at its current concentration with viability of A375 dropping to 12% after six hours of treatment. A drug release profile revealed a slight burst release before it settled into an extended and maintained release of 5-FU. These initial findings provide evidence for the versatility of thermophilic exopolysaccharides produced from lignocellulosic biomass to act as a chemotherapeutic-delivering device and expand the overall applications of extremophilic EPSs.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Saxena, Rauniyar, Thakur, Singh, Bomgni, Alaba, Tripathi, Gnimpieba, Lushbough and Sani.)

Natural polysaccharides being investigated for use in the field of drug delivery commonly require the addition of sugars or pretreated biomass for fabrication. Geobacillus sp. strain WSUCF1 is a thermophile capable of secreting natural polymers, termed exopolysaccharides (EPSs), cultivated from cost-effective, non-treated lignocellulosic biomass carbon substrates. This preliminary investigation explores the capabilities of a 5% wt/wt amikacin-loaded film constructed from the crude EPS extracted from the strain WSUCF1. Film samples were seen to be non-cytotoxic to human keratinocytes and human skin-tissue fibroblasts, maintaining cell viability, on average, above 85% for keratinocytes over 72-h during a cell viability assay. The drug release profile of a whole film sample revealed a steady release of the antibiotic up to 12 h. The amikacin eluted by the EPS film was seen to be active against Staphylococcus aureus , maintaining above a 91% growth inhibition over a period of 48 h. Overall, this study demonstrates that a 5% amikacin-EPS film, grown from lignocellulosic biomass, can be a viable option for preventing or combating infections in clinical treatment.

Dehydrogenation of methanol (CH 3 OH) 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.

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.

Photosynthetic microbial fuel cells (pMFC) represent a promising approach for treating methanol (CH 3 OH) 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 CH 3 OH 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% CH 3 OH 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 cm 2 ) and correspondingly 16-fold higher power density (141 mW/m 2 ) 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 CH 3 OH oxidation were discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 EC 50 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 EC 50 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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bhalla, Arce, Ubanwa, Singh, Sani and Balan.)

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, β, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was -5.2, -5.7, -4.2, and -3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of -6.3, -6.7, -6.3, -6.5, and -6.5 kcal/mol, respectively, as compared to the wild type (-5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tripathi, Thakur, Saxena, Rauniyar, Gopalakrishnan, Singh, Gadhamshetty, Gnimpieba, Jasthi and Sani.)

Various microorganisms thrive under extreme environments, like hot springs, hydrothermal vents, deep marine ecosystems, hyperacid lakes, acid mine drainage, high UV exposure, and more. To survive against the deleterious effect of these extreme circumstances, they form a network of biofilm where exopolysaccharides (EPSs) comprise a substantial part. The EPSs are often polyanionic due to different functional groups in their structural backbone, including uronic acids, sulfated units, and phosphate groups. Altogether, these chemical groups provide EPSs with a negative charge allowing them to (a) act as ligands toward dissolved cations as well as trace, and toxic metals; (b) be tolerant to the presence of salts, surfactants, and alpha-hydroxyl acids; and (c) interface the solubilization of hydrocarbons. Owing to their unique structural and functional characteristics, EPSs are anticipated to be utilized industrially to remediation of metals, crude oil, and hydrocarbons from contaminated wastewaters, mines, and oil spills. The biotechnological advantages of extremophilic EPSs are more diverse than traditional biopolymers. The present review aims at discussing the mechanisms and strategies for using EPSs from extremophiles in industries and environment bioremediation. Additionally, the potential of EPSs as fascinating biomaterials to mediate biogenic nanoparticles synthesis and treat multicomponent water contaminants is discussed., (Copyright © 2021 Banerjee, Sarkar, Govil, González-Faune, Cabrera-Barjas, Bandopadhyay, Salem and Sani.)

The production, characterization and bioactivities of exopolysaccharides (EPSs) from a thermophilic bacterium Geobacillus sp. strain WSUCF1 were investigated. Using glucose as a carbon source 525.7 mg/L of exoproduct were produced in a 40-L bioreactor at 60 °C. Two purified EPSs were obtained: EPS-1 was a glucomannan containing mannose and glucose in a molar ratio of 1:0.21, while EPS-2 was composed of mannan only. The molecular weights of both EPSs were estimated to be approximately 1000 kDa, their FTIR and NMR spectra indicated the presence of α-type glycosidic bonds in a linear structure, and XRD analysis indicated a low degree of crystallinity of 0.11 (EPS-1) and 0.27 (EPS-2). EPS-1 and EPS-2 demonstrated high degradation temperatures of 319 °C and 314 °C, respectively, and non-cytotoxicity to HEK-293 cells at 2 and 3 mg/mL, respectively. In addition, both showed antioxidant activities. EPSs from strain WSUCF1 may expand the applications of microorganisms isolated from extreme environments and provide a valuable resource for exploitation in biomedical fields such as drug delivery carriers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Yak dung is used as fuel in Tibetan homes; however, this use is hazardous to health. An alternative use of the dung that would be profitable and offset the loss as a fuel would be very beneficial. Sweet sorghum silage with yak dung biochar as an additive was compared with a control silage with no additives and three silages with different commercial additives, namely Lactobacillus buchneri, Lactobacillus plantarum and Acremonium cellulase. Biochar-treated silage had a significantly greater concentration of water-soluble carbohydrates than the other silages (76 vs 12.4-45.8 g/kg DM) and a greater crude protein content (75.5 vs 61.4 g/kg DM), lactic acid concentration (40.7 vs 27.7 g/kg DM) and gross energy yield (17.8 vs 17.4 MJ/kg) than the control silage. Biochar-treated and control silages did not differ in in vitro digestibility and in total gas (507 vs 511 L/kg DM) and methane production (57.9 vs 57.1 L/kg DM). Biochar inhibited degradation of protein and water-soluble carbohydrates and enhanced lactic acid production, which improved storability of feed. It was concluded that yak dung biochar is an efficient, cost-effective ensiling additive. The profit could offset the loss of dung as fuel and improve the health of Tibetan people., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Given the difficulties valorizing methane (CH 4 ) via catalytic routes, this study explores use of CH 4 -oxidizing bacteria ("methanotrophs") for generating electricity directly from CH 4 . 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 CH 4 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 CH 4 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (COD i  = 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Microbial exopolysaccharides (EPSs) exhibit diverse functionalities and offer a variety of structural options that can be altered to fit a specific purpose. EPSs can degrade within the body via biological processes, and polysaccharides are regarded as generally safe. More so, microbial EPS is replicable from several known, inexpensive, and plentiful sources. Drug delivery-related research involving polysaccharides have continuously cited minimal to zero cytotoxicity and, where tested, sufficient drug release and a competent release profile. Transdermal drug delivery systems as films not only avoids first-pass metabolism, but also provides pain-free administration, assists patients with dysphagia, has increased patient compliance, can be self-administered, and can be removed at any time. Commonly used synthetic polymers in the field of drug delivery have been related to problems regarding toxicity and immunogenicity, escalating the need for an alternative. Ultimately, the risks while using synthetic polymers could result in serious negative influences involving physiological, physiochemical, and molecular events. Research involving exopolysaccharides from extremophiles is only recently gaining attention. However, commercial use of microbial polysaccharides in other areas as well as the positive results from preliminary research suggests microbial EPSs have a promising future in biomedical engineering and medicine, especially as an alternative to current synthetic polymers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

This work investigated the electrocatalytic activity of a thermophilic methanogenic consortia (TMC) for developing a bioelectrosynthesis process to convert food and paper wastes to methane. Electroanalytical techniques were used to analyze the electrocatalytic activity of the TMC biofilm formed onto the electrodes. The developed electromethanogenesis process enhanced the yield of methane by 54.7% than control experiments. Scanning electron micrographs of the TMC bioelectrodes showed that the electrosynthesis process accelerates biofilm formation onto the electrodes leading to enhanced direct electron transfer reactions at electrode-electrolyte interface. This study will help in developing a novel approach for valorization of food and paper waste to biofuels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

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, β-xylosidase, and β-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.

The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.

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.

Microbial fuel cell (MFC) biosensors are self-sustainable device for monitoring of various substrates; however, for heavy metals detection are still scarce. In this study, E. coli BL21 was engineered to express the zntR, ribB, and oprF genes with P zntA promoter, which could sense zinc (Zn 2+ ) for riboflavin and porin production. The engineered strain produced high levels of riboflavin (2.4-3.6 μM) and improved cell membrane permeability, with a positive correlation of Zn 2+ (0-400 μM). The strain was then employed in MFC biosensor under the following operational parameters: external resistance 1000 Ω, pH 9, and temperature 37 °C for Zn 2+ sensing. The maximum voltages (160, 183, 260, 292, and 342 mV) of the constructed MFC biosensor have a linear relationship with Zn 2+ concentrations (0, 100, 200, 300, and 400 μM, respectively) (R 2  = 0.9777). An Android App was developed for the biosensor system that could sense Zn 2+ in real-time and in situ. The biosensor was applied to wastewater with different Zn 2+ concentrations and the results showed that the detection range for Zn 2+ was 20-100 μM, which covers common Zn 2+ safety standards. The results obtained with developed MFC biosensor were comparable to conventional methods such as colorimetric, flame atomic absorption spectroscopy (FAAS), and inductively coupled plasma optical emission spectroscopy (ICP-OES). In summary, MFC biosensor with biosynthetic strain is an efficient and affordable system for real-time monitoring and sensing of heavy metals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Investigations on microbial electrocatalysis as a strategy for enhancing the rates of substrate utilization leading to enhanced yield of biomass and enhanced biofilm formation are reported. A thermophilic Geobacillus sp. strain WSUCF1 (60 °C), a potential lignocellulose degrading microorganism was used as the electrocatalyst. Glucose, cellulose, and corn stover were used as the feedstocks. The results of this investigation showed that applying the oxidation potential of -0.383 mV (vs PRE) increased the glucose utilization and COD removal by 25.5% and 29.7% respectively. The bioelectrocatalysis strategy also increased the biomass yield by 81.2, 42.1, and 49.5% in the case of systems fed with glucose, cellulose, and corn stover, respectively, when compared with the systems without applied oxidation potential. This is the first work reporting the effects of applied oxidation potential on increasing the rates of degradation of lignocellulosic biomass and enhanced biofilm formation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 (t 1/2 ) 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Successful and efficient extraction of high quality, high molecular weight genomic DNA from the environmental samples is an essential primary step to understand the genetic, metabolic and evolutionary characteristics of the microbial communities. Deep mine biofilm samples that contain high amounts of mucoid exopolysaccharide often pose difficulties to obtaining refined community DNA. To circumvent this hindrance, we report our "MINES" method which we developed for optimal biofilm DNA recovery suitable for all types of high-resolution downstream applications. The method is also suitable for samples collected from landfill compost, kitchen digest (KD), and for Gram-positive Geobacillus sp. strain WSUCF1 and Gram-negative E. coli DH5α strains. In one form of the method, use of a gentle preprocessing technique to loosen the mucoid layer, combined with a multi-lytic polyzyme treatment to maximize yields from all cell types in the biofilm sample, yielded >1 μg of high molecular weight DNA (16-20 kb) per gram of the biofilm sample, with an A260/280 and A260/230 ratio of about 2. Furthermore, amplification of 16S rRNA genes as well as restriction digestion with BamHI and HindIII suggest that the newly developed method can minimize any inhibitory effects of contaminants. Results indicate that it is an appropriate methodology for the extraction of total genomic DNA for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging. IMPORTANCE: Our present knowledge of microorganisms and their enzymes from deep mine subsurfaces is based largely on laboratory studies of pure microbial cultures. These methods tend only to hit nearly 1% of the entire microbial community. In this regard, metagenomics, has emerged as a strategic approach to explore unculturable microbes through the sequencing and analysis of DNA extracted from the environmental samples. This research paper discusses our "MINES" method for genomic DNA extraction from deep biosphere biofilm samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Thermophilic bacteria able to produce exopolysaccharides (EPSs) have become attractive in recent years. EPSs synthesized by thermophiles are worth investigating due to their unexplored structural and biological properties. In this study, EPSs from thermophilic, Gram-positive bacterial isolates were purified and tested for their biological activities. A total of one hundred seven thermophilic bacteria were screened for their ability to produce exopolysaccharides. Nine isolates belonging to Geobacillus, Parageobacillus, Aeribacillus, and Anoxybacillus genera with highest EPS production were chosen, and purified EPSs (20, 61, 74, 76, 78, 89, 106, 134, and 261) were used for biological activity studies. EPS yields of selected thermophilic bacteria ranged between 117 and 419 mg/L. Among the tested EPSs, 61, 106, and 261 showed antibacterial effect against E. faecalis JH2-2 at a final concentration of 1.5 mg/mL. EPS samples had significant antioxidant capacity, especially EPS 134, with the highest DPPH radical scavenging activity of 100% at a concentration of 5 mg/mL and the strongest reducing power. EPS 20 showed the highest lipid peroxidation inhibition effect at a rate of 31%. EPSs displayed weak alpha-amylase inhibition activity when compared with standard acarbose. The prebiotic indices of EPSs 20, 61, 76, 89, 134, and 261 were found to be higher than that of inulin, a representative prebiotic carbohydrate for all tested lactic acid bacteria in the study. All examined EPSs inhibited the biofilms formed by various bacteria depending on the test strain. Results indicated that thermophilic EPSs had remarkable antioxidant, prebiotic, and antibiofilm activities. Therefore, EPSs characterized in this study may have technological applications in health and food fields., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

A highly thermostable laccase from Geobacillus sp. strain WSUCF1 was cloned into Escherichia coli (E. coli) using pRham N-His SUMO expression system. The thermostable laccase with a molecular weight ~30 kDa had a t 1/2 (pH 6.0) of 120 h at 50 °C. The homology modelling for laccase structure showed the presence of Cu active centers with His and Cys residues involved in the active site and ligand binding activity of the enzyme, respectively. The K m , V max , K cat and K cat /K m values of the purified enzyme with ABTS were found to be 0.146 mM, 1.52 U/mg, 1037 s -1 and 7102.7 s -1  mM -1 , respectively. The doping of recombinant WSUCF1 laccase to commercial enzyme cocktails Accellerase® 1500 and Cellic CTec2 improved the hydrolysis of untreated, alkali and acid treated corn stover by 1.31-2.28 times and bagasse by 1.32-2.02 times. Further, in-house enzyme cocktails with laccase hydrolyzed untreated, alkali and acid treated bagasse and gave 1.44, 1.1, and 0.92 folds higher sugar, respectively, when compared with Accellerase 1500. The results suggested that thermostable laccase can aid in the improved hydrolysis of lignocellulosic biomass., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Thermophiles are promising options to use as electrocatalysts for bioelectrochemical applications including microbial electrolysis. They possess several interesting characteristics such as ability to catalyze a broad range of substrates at better rates and over a broad range of operating conditions, and better electrocatalysis/electrogenic activity over mesophiles. However, a very limited number of investigations have been carried out to explore the microbial reactions/pathways and the molecular mechanisms that contribute to better electrocatalysis/electrolysis in thermophiles. Here, we review the electroactive characteristics of thermophiles, their electron transfer mechanisms, and molecular insights behind the choice of thermophiles for bioelectrochemical/electrolytic processes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the effect of atmospheric pressure cold plasma on the microbial substrate utilization and biomass yield in a thermophilic strain. Geobacillus sp. strain WSUCF1, a thermophile capable of producing cellulolytic enzymes with higher activity was used for this investigation. Treatment with cold plasma for 4 min increased the rates of glucose utilization by 74% and biomass yield by 60% when compared with the control. WSUCF1 treated with plasma also displayed enhanced biofilm formation. This study for the first time, reports the use of cold plasma for enhancing the substrate utilization and biofilm formation in a thermophile., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.

Numerous microorganisms inhabiting harsh niches produce exopolysaccharides as a significant strategy to survive in extreme conditions. The exopolysaccharides synthesized by extremophiles possess distinctive characteristics due to the varied harsh environments which stimulate the microorganisms to produce these biopolymers. Despite many bioprocesses have been designed to yield exopolysaccharides, the production of exopolysaccharides by extremophiles is inefficient compared with mesophilic and neutrophilic exopolysaccharide producers. Meanwhile, the industrial development of novel extremophilic exopolysaccharides remains constrained due to the lack of exploration. In this review, we summarize the structure and properties of various exopolysaccharides produced by extremophiles, and also discuss potential metabolic and genetic engineering strategies for enhanced yield and modified structure of extremophilic exopolysaccharides. Special focus is given to the applications of extremophilic exopolysaccharides in the areas of biomedicine, food industry, and biomaterials via nano-techniques, casting and electrospinning., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacterial exopolysaccharides (EPS) are biopolymers of carbohydrates, often released from cells into the extracellular environment. Due to their distinctive physicochemical properties, biocompatibility, biodegradability, and non-toxicity, EPS finds applications in various industrial sectors. However, the need for alternative EPS has grown over the past few decades as lactic acid bacteria's (LAB) low-yield EPS is unable to meet the demand. In this case, rhizosphere bacteria with the diverse communities in soil leading to variations in composition and structure, are recognized as a potential source of EPS applicable in various industries. In addition, media components and cultivation conditions have an impact on EPS production, which ultimately affects the quantity, structure, and biological functions of the EPS. Therefore, scientists are currently working on manipulating bacterial EPS by developing cultures and applying abiotic and biotic stresses, so that better production of exopolysaccharides can be attained. This review highlights the composition, biosynthesis, and effects of environmental factors on EPS production along with the potential applications in different fields of industry. Ultimately, an overview of potential future paths and tactics for improving EPS implementation and commercialization is pointed out., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)