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Abstract 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 H2O2). 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.

ABSTRACT 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.IMPORTANCEMethylotrophs 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.

Over the past decade, copper (Cu) has been recognized as a crucial metal in the differential expression of soluble (sMMO) and particulate (pMMO) forms of methane monooxygenase (MMO) through a mechanism referred to as the “Cu switch”. In this study, we used Methylosinus trichosporium OB3b as a model bacterium to investigate the range of Cu concentrations that trigger the expression of sMMO to pMMO and its effect on growth and methane oxidation. The Cu switch was found to be regulated within Cu concentrations from 3 to 5 µM, with a strict increase in the methane consumption rates from 3.09 to 3.85 µM occurring on the 6th day. Our findings indicate that there was a decrease in the fold changes in the expression of methanobactin (Mbn) synthesis gene (mbnA) with a higher Cu concentration, whereas the Ton-B siderophore receptor gene (mbnT) showed upregulation at all Cu concentrations. Furthermore, the upregulation of the di-heme enzyme at concentrations above 5 µM Cu may play a crucial role in the copper switch by increasing oxygen consumption; however, the role has yet not been elucidated. We developed a quantitative assay based on the naphthalene–Molisch principle to distinguish between the sMMO- and pMMO-expressing cells, which coincided with the regulation profile of the sMMO and pMMO genes. At 0 and 3 µM Cu, the naphthol concentration was higher (8.1 and 4.2 µM, respectively) and gradually decreased to 0 µM naphthol when pMMO was expressed and acted as the sole methane oxidizer at concentrations above 5 µM Cu. Using physical protein–protein interaction, we identified seven transporters, three cell wall biosynthesis or degradation proteins, Cu resistance operon proteins, and 18 hypothetical proteins that may be involved in Cu toxicity and homeostasis. These findings shed light on the key regulatory genes of the Cu switch that will have potential implications for bioremediation and biotechnology applications.

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 Fe2+ 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.

Abstract Life on Earth has displayed remarkable adaptability to the harshest environments, spanning polar regions, scorching deserts, abyssal oceans, lightless caves, noxious lakes, boiling hot springs, and nuclear waste sites. These resilient organisms, known as extremophiles or polyextremophiles, owe their survival due to their unique genetic adaptations. This collection, titled ‘Genomics of Extreme Environments’, comprises several articles published in the esteemed journal Scientific Reports. Each article within this collection investigated genetic signature and adaptation in different extreme environments, including the cold polar region, arid desert, oxygen-deprived Tibetan mountains and others. These studies provide invaluable understanding of how life thrives and evolves under extreme conditions, shedding light on genetic mechanisms and adaptation strategies.

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.

Methane, a potent greenhouse gas, has gained significant attention due to its environmental impact and economic potential. Chemical industries have focused on specialized catalytic systems, like zeolites, to convert methane into methanol. However, inherent limitations in selectivity, irreversibility, and pore blockages result in high costs and energy requirements, thus hindering their commercial viability and profitability. In contrast, biological methane conversion using methanotrophs has emerged as a promising alternative, offering higher conversion rates, self-renewability, improved selectivity, and economically feasible upstream processes. Nevertheless, biological methane oxidation encounters challenges including the difficulty in cultivating methanotrophs and their slow growth rates, which hinder large-scale bioprocessing. Another highlighted limitation is the limited mass transfer of methane into liquid in bioreactors. Practical strategies to enhance methane oxidation in biological systems, including optimizing reactor design to improve mass transfer, altering metal concentrations, genetic engineering of methane monooxygenases, enzyme encapsulation, and utilizing microbial consortia are discussed. By addressing the limitations of chemical approaches and highlighting the potential of biological methods, the review concluded that the utilization of genetically engineered methanotrophic biofilms on beads within a biotrickling reactor, along with enhanced aeration rates, will likely enhance methane oxidation and subsequent methane conversion rates.

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.

Scanning electron microscopy (SEM) techniques have been extensively performed to image and study bacterial cells with high-resolution images. Bacterial image segmentation in SEM images is an essential task to distinguish an object of interest and its specific region. These segmentation results can then be used to retrieve quantitative measures (e.g., cell length, area, cell density) for the accurate decision-making process of obtaining cellular objects. However, the complexity of the bacterial segmentation task is a barrier, as the intensity and texture of foreground and background are similar, and also, most clustered bacterial cells in images are partially overlapping with each other. The traditional approaches for identifying cell regions in microscopy images are labor intensive and heavily dependent on the professional knowledge of researchers. To mitigate the aforementioned challenges, in this study, we tested a U-Net-based semantic segmentation architecture followed by a post-processing step of morphological over-segmentation resolution to achieve accurate cell segmentation of SEM-acquired images of bacterial cells grown in a rotary culture system. The approach showed an 89.52% Dice similarity score on bacterial cell segmentation with lower segmentation error rates, validated over several cell overlapping object segmentation approaches with significant performance improvement.

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.

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.

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.

The increasing global population will require sustainable means to sustain life and growth. The continuous depletion and increasing wastage of the energy resources will pose a challenge for the survival of the increasing population in the coming years. The bioconversion of waste generated at different stages of the food value chain to ethanol can provide a sustainable solution to the depleting energy resources and a sustainable way to address the growing food waste issue globally. The high carbohydrate and nitrogen content in the food waste can make it an ideal alternative substrate for developing a decentralized bioprocess. Optimizing the process can address the bottleneck issues viz. substrate collection and transport, pretreatment, fermentative organism, and product separation, which is required to make the process economic. The current review focuses on the opportunities and challenges for using the food loss and waste at different stages of the food value chain, its pretreatment, the fermentation process to produce bioethanol, and potential ways to improve the process economics. The impact of substrate, fermentative organisms’ process development, downstream processing, and by-product stream to make the bioethanol production from the waste in the food value chain a commercial success are also discussed.

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.

Historically, research disciplines have successfully operated independently. However, the emergence of transdisciplinary research has led to convergence methodologies, resulting in groundbreaking discoveries. Despite the benefits, graduate programs face challenges in implementing transdisciplinary research and preparing students for real-world collaboration across diverse disciplines and experience levels. We propose a convergence training framework integrating project-based learning, training modules, and collaborative teaming to address this. This approach, tested in a multi-institutional workshop, proved effective in bridging expertise gaps and fostering successful convergence learning experiences in computational biointerface (material-biology interface) research. Here, biointerface research focuses on control of biomolecular interactions with technologically relevant material surfaces, which is a critical component of biotechnology and engineering applications. Positive outcomes, including conference presentations and published models, endorse the framework's application in graduate curricula, particularly for students engaging in transdisciplinary collaboration.

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.

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.

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.

The aim of the current study was to optimize the production of xylanase, and its application for ethanol production using the lignocellulosic biomass. A highly thermostable crude xylanase was obtained from the Geobacillus sp. strain DUSELR13 isolated from the deep biosphere of Homestake gold mine, Lead, SD. Geobacillus sp. strain DUSELR13 produced 6 U/mL of the xylanase with the beechwood xylan. The xylanase production was improved following the optimization studies, with one factor at a time approach, from 6 U/mL to 19.8 U/mL with xylan. The statistical optimization with response surface methodology further increased the production to 31 U/mL. The characterization studies revealed that the crude xylanase complex had an optimum pH of 7.0, with a broad pH range of 5.0–9.0, and an optimum temperature of 75 °C. The ~45 kDa xylanase protein was highly thermostable with t1/2 of 48, 38, and 13 days at 50, 60, and 70 °C, respectively. The xylanase activity increased with the addition of Cu+2, Zn+2, K+, and Fe+2 at 1 mM concentration, and Ca+2, Zn+2, Mg+2, and Na+ at 10 mM concentration. The comparative analysis of the crude xylanase against its commercial counterpart Novozymes Cellic HTec and Dupont, Accellerase XY, showed that it performed better at higher temperature, hydrolyzing 65.4% of the beechwood at 75 °C. The DUSEL R13 showed the mettle to hydrolyze, and utilize the pretreated, and untreated lignocellulosic biomass: prairie cord grass (PCG), and corn stover (CS) as the substrate, and gave a maximum yield of 20.5 U/mL with the untreated PCG. When grown in co-culture with Geobacillus thermoglucosidasius, it produced 3.53 and 3.72 g/L ethanol, respectively with PCG, and CS. With these characteristics the xylanase under study could be an industrial success for the high temperature bioprocesses.

Nonfood lignocellulosic biomass is an ideal substrate for biohydrogen production. By avoiding pretreatment steps (acid, alkali, or enzymatic), there is potential to make the process economical. Utilization of regional untreated lignocellulosic biomass by cellulolytic and fermentative thermophiles in a consolidated mode using a single reactor is one of the ways to achieve economical and sustainable biohydrogen production. Employing these potential microorganisms along with decentralized biohydrogen energy production will lead us towards regional and national independence having a positive influence on the bioenergy sector.