Search Results (66)

Thermophilic prokaryotic Argonaute proteins (pAgos) have emerged as powerful tools for nucleic acid manipulation, with applications in nucleic acid detection, and DNA assembly. However, their strong dependence on high-temperature catalytic activity limits their utility under moderate conditions. TcAgo, a thermophilic Argonaute nuclease from Thermogladius calderae, exhibits efficient DNA-guided target DNA cleavage above 80 °C, yet its structural basis and catalytic mechanism remain unclear. In this study, we attempted to analyze the structure of the TcAgo ternary complex and performed rational engineering based on its structure and characteristics of cold-adapted enzymes. A mutant, mTcAgo (K574G, D577G), was obtained with enhanced activity at moderate temperatures. Compared with the wild type, mTcAgo exhibited significantly improved cleavage activity toward both DNA and RNA targets at 37 °C. It utilized multiple guide types, including 5′OH- and 5′P-modified DNA and RNA guides, with a preference for 5′P-gDNA. mTcAgo displayed optimal activity at pH 7–8, broad salt tolerance, and an extended catalytic temperature range from 37 °C to 95 °C. Notably, it retained high activity after incubation at 90 °C, with a melting temperature of ~88 °C, and efficiently cleaved GC-rich targets under low Mg²⁺ conditions. These results demonstrate that rational cold-adaptation engineering can expand the functional temperature range of thermophilic pAgos, providing a promising strategy for developing versatile nucleic acid tools. Full article

We report on the isolation and comprehensive genomic and biochemical characterization of Aspergillus fumigatus VP2T, a thermophilic filamentous fungus recovered from Himalayan Forest soil with exceptional lignocellulolytic capacity. Whole-genome sequencing revealed a 32.1 Mb genome encoding 12,675 predicted genes, including an extensive repertoire of >300 carbohydrate-active enzymes (CAZymes). Notably, the genome harbors multiple auxiliary activity enzymes, including AA9-family lytic polysaccharide monooxygenases and several cellobiose dehydrogenases (CDHs), supporting oxidative–hydrolytic synergism during biomass degradation. Submerged fermentation using a cellulose–wheat bran–rice straw substrate induced high enzyme titers, including 33 U/mL endoglucanase and 131 U/mL CDH, exceeding activities commonly reported for both native and engineered fungal strains. Although exoglucanase (0.02 U/mL) and xylanase (14.22 U/mL) activities were comparatively modest, the strain VP2T demonstrated superior hydrolysis of untreated rice straw, achieving a 1.89-fold increase in saccharification efficiency relative to the commercial enzyme cocktail Cellic^® CTec2. Scanning electron microscopy confirmed extensive disruption of lignocellulosic architecture, consistent with enhanced enzyme accessibility and oxidative fiber loosening. Collectively, genomic evidence and functional assays identify A. fumigatus VP2T as a redox-optimized, moderately thermophilic biocatalyst suited for low-pH lignocellulose conversion. This study highlights the value of exploring thermophilic fungal biodiversity to discover native strains with inherent oxidative capacity, offering promising alternatives to pretreatment-intensive biorefinery processes and informing the rational development of tailored enzyme systems. Full article

Composting represents a sustainable and effective strategy for converting organic waste into nutrient-rich soil amendments, providing a safer alternative to raw manure, which poses significant risks of soil, crop, and water contamination through pathogenic microorganisms. This study, conducted under semi-arid Moroccan conditions, investigated the efficiency of co-composting green garden waste with sheep manure in an open window system, with the objective of assessing pathogen inactivation and evaluating compost quality. The process, conducted over 120 days, maintained thermophilic temperatures exceeding 55 °C, effectively reducing key pathogens including Escherichia coli, total coliforms, Staphylococcus aureus, and sulfite-reducing Clostridia (SRC), while Salmonella was not detected throughout the composting period. Pathogen reductions exceeded 3.52-log despite moderate temperature fluctuations, indicating that additional sanitization mechanisms beyond heat contributed to inactivation. Compost quality, assessed using the CQI, classified Heap 2 (fallen leaves + sheep manure) as good quality (4.06) and Heap 1 (green waste + sheep manure) as moderate quality (2.47), corresponding to differences in microbial dynamics and compost stability. These findings demonstrate that open windrow co-composting is a practical, low-cost, and effective method for safe organic waste management. It supports sustainable agriculture by improving soil health, minimizing environmental and public health risks, and providing guidance for optimizing composting protocols to meet regulatory safety standards. Full article

Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5′-SNAAAG-3′, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems. Full article

Plastic pollution, particularly from polyethylene terephthalate (PET), poses significant environmental concerns due to ecosystem persistence and extensive packaging use. Conventional recycling methods face inefficiencies, high costs, and limited scalability, necessitating sustainable alternatives. Biodegradation via PET hydrolases offers promising eco-friendly solutions, although most natural PET-degrading enzymes are thermophilic and require energy-intensive high temperatures. In contrast, psychrophilic enzymes function efficiently at extremely low temperatures but often lack stability under moderate conditions. Therefore, this study aimed to enhance the ability of the Mors1 enzyme from Moraxella TA144 to operate effectively under mesophilic conditions, which is closer to the optimal conditions for environmental application. Three strategic hydrophobic substitutions (K93I, E221I, and R235F) were introduced in loop regions, generating the mutant variant Mors1^MUT. Comparative characterization revealed that Mors1^MUT retained 98% of its activity at pH 9 and displayed greater resilience across both acidic and alkaline conditions than did the wild-type enzyme. Thermal stability assays revealed that Mors1^MUT preserved 61% of its activity at 40 °C and 14% at 50 °C, whereas the wild-type enzyme was fully inactivated at these temperatures. The enzymatic hydrolysis of PET films significantly improved with Mors1^MUT. Gravimetric analysis revealed weight losses of 0.83% for Mors1^WT and 3.46% for Mors1^MUT after a 12-day incubation period. This corresponds to a 4.16-fold increase in hydrolysis efficiency, confirming the enhanced catalytic performance of the mutant variant. The improvement was further validated by scanning electron microscopy (SEM), atomic force microscopy (AFM), and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) analysis. Optimization of the reaction parameters through response surface methodology (enzyme load, time, pH, temperature, and agitation) confirmed increased PET hydrolysis under mild mesophilic conditions. These findings establish Mors1^MUT as a robust mesophilic PETase with enhanced catalytic efficiency and thermal stability, representing a promising candidate for sustainable PET degradation under environmentally relevant conditions. Full article

Thermophilic microorganisms represent an untapped reservoir of thermostable biocatalysts and stress-resilient biomolecules for industrial biotechnology. Aeribacillus pallidus, a Gram-positive moderate thermophile, has attracted attention for its enzymatic versatility and environmental adaptability, yet its genomic potential remains underexplored. Here, we present a comparative genomic analysis of 13 A. pallidus strains to uncover conserved and strain-specific traits relevant to biotechnology. Genomes ranged from 3.24 to 4.98 Mb, with GC content largely conserved (~39%) except for GS3372 (57.4%), indicating possible horizontal gene transfer. All strains encoded complete central metabolic pathways, while carbohydrate-active enzyme profiling revealed abundant glycoside hydrolases and glycosyltransferases, with GS3372 and MHI3390 enriched for lignocellulose-degrading enzymes. Secondary metabolite mining identified diverse biosynthetic gene clusters, including terpenes, sactipeptides, and bacteriocins, with PI8, W-12, and 8m3 exhibiting the greatest biosynthetic diversity. A core set of heat shock and universal stress proteins underscored robust thermotolerance. Phylogenomic and pan-genome analyses revealed high intraspecific diversity and an open pan-genome structure. Collectively, these findings position A. pallidus as a promising thermophilic chassis organism for sustainable applications, including biomass conversion, biofuel production, bioremediation, and the synthesis of heat-stable antimicrobial agents. Full article

Chloride ions can enhance the bioleaching of copper minerals, yet most biomining microorganisms are highly sensitive to chloride and cannot survive or colonize mineral surfaces in saline environments. Chloride tolerance varies among acidophilic iron-oxidizing bacteria, but the concentrations at which they remain active are generally too low to permit the industrial use of seawater. Therefore, identifying highly chloride-tolerant leaching microorganisms and studying their bioleaching potential in chloride-containing systems is of utmost importance. This study investigated chloride tolerance and adaptability of bacteria from different genera, with a focus on Sulfobacillus thermosulfidooxidans subsp. asporogenes 41, a moderately thermophilic strain that can oxidize both Fe (II) and reduced inorganic sulfur compounds (RISCs). This dual activity makes it advantageous for bioleaching by facilitating sulfur removal, generating acidity, and preventing mineral passivation. Comparative experiments on the bioleaching of pyrite and chalcopyrite demonstrated that adaptation to 0.3 M NaCl enhanced the chloride tolerance of S. thermosulfidooxidans subsp. asporogenes 41. The adapted strain exhibited significantly improved copper extraction under saline conditions compared with the native culture. Maximum copper recovery was achieved at 0.4 M NaCl, highlighting the potential of chloride-adapted moderate thermophiles for biomining applications in saline environments. In contrast the minimal inhibitory concentration for Acidithiobacillud ferrooxidans Dr was 0.005 M (causing 41.2% inhibition), while Leptospirillum ferriphilum CC was unaffected by lower concentrations (0.01–0.02 M) and only showed severe inhibition (86.5%) at 0.1 M NaCl, defining its minimal inhibitory concentration (MIC) at 0.05 M. Full article

This study, conducted in Mexico, evaluates the agricultural potential of three compost formulations BFS1, BFS2, and BFS3 produced from mining tailings and thermophilic microbial mats and collected from geothermal environments. The physicochemical characterization included pH, electrical conductivity (EC), macronutrients (N, P, K, Ca, Mg, and S), micronutrients (Fe, Zn, B, Cu, Mn, Mo, and Ni), organic matter (OM), and the carbon-to-nitrogen (C/N) ratio. All composts exhibited neutral pH values (7.38–7.52), high OM content (38.5–48.4%), and optimal C/N ratios (10.5–13.9), indicating maturity and chemical stability. Nitrogen ranged from 19 to 21 kg·t⁻¹, while potassium and calcium were present in concentrations beneficial for crop development. However, EC values (3.43–3.66 dS/m) and boron levels (>160 ppm) were moderately high, requiring caution in saline soils or with boron-sensitive crops. A semi-quantitative Compost Quality Index (CQI) ranked BFS3 highest due to elevated OM and potassium content, followed by BFS1. BFS2, while rich in nitrogen, scored lower due to excessive boron. One-way ANOVA revealed no significant difference in nitrogen (p > 0.05), but it did reveal significant differences in potassium (p < 0.01) and boron (p < 0.001) among formulations. These results confirm the potential of mining tailings—microbial mat composts are low-cost, nutrient-rich biofertilizers. They are suitable for field crops or as components in nursery substrates, particularly when EC and boron are managed through dilution. This study promotes the circular reuse of geothermal and industrial residues and contributes to sustainable soil restoration practices in mining-affected regions through innovative composting strategies. Full article

The purpose of this work was to obtain specialized enrichment cultures from an original extreme acidophilic consortium of extremely acidophilic microorganisms and to study their microbial community composition and biotechnological potential. At temperatures of 25, 35, 40 and 50 °C, distinct enrichments of extremely acidophilic microorganisms used in the processes of bioleaching sulfide ores were obtained using nutrient media containing ferrous sulfate, elemental sulfur and a copper sulfide concentrate as nutrient inorganic substrates, with and without the addition of 0.02% yeast extract. The microbial community composition was studied using the sequencing of the V3–V4 hypervariable region of the 16S rRNA genes. The different growth conditions led to changes in the microbial composition and relative abundance of mesophilic and moderately thermophilic, strict autotrophic and mixotrophic microorganisms in members of the genera Acidithiobacillus, Sulfobacillus, Leptospirillum, Acidibacillus, Ferroplasma and Cuniculiplasma. The dynamics of the oxidation of ferrous iron, sulfur, and sulfide minerals (pyrite and chalcopyrite) by the enrichments was also studied in the temperature range of 25 to 50 °C. The study of enrichment cultures using the molecular biological method using the metabarcoding method of variable V3–24 V4 fragments of 16S rRNA genes showed that enrichment cultures obtained under different conditions differed in composition, which can be explained by differences in the physiological properties of the identified microorganisms. Regarding the dynamics of the oxidation of ferrous ions, sulfur, and sulfide minerals (pyrite and chalcopyrite), each enrichment culture was studied at a temperature range of 25 to 50 °C and indicated that all obtained enrichments were capable of oxidizing ferrous iron, sulfur and minerals at different rates. The obtained enrichment cultures may be used in further work to increase bioleaching by using the suitable inoculum for the temperature and process conditions. Full article

Protopanaxadiol-type ginsenosides, the major bioactive components of Panax ginseng, exhibit diverse pharmacological activities, but suffer from low oral bioavailability due to poor water solubility and membrane permeability. Enzymatic deglycosylation has emerged as an effective strategy to enhance their therapeutic potential; however, most glucosidases lack sufficient thermostability for industrial applications. A β-glucosidase from the thermophilic bacterium Caldicellulosiruptor bescii (CbBGL) has demonstrated efficient conversion of major ginsenosides into compound K at elevated temperatures. In this study, the high-resolution crystal structure of CbBGL was determined at 1.9 Å. Structural analysis revealed that CbBGL adopts a classical (α/β)₈ TIM barrel fold and functions as a homodimer. Comparative studies with other glucosidases highlighted structural features contributing to its thermostability, including moderate B-factor distribution and a limited hydrogen bond network. Docking analyses revealed a narrow, inverted conical substrate-binding cleft, which imposes specific binding orientations and underlies the enzyme’s stepwise deglycosylation mechanism. These insights provide a structural basis for CbBGL’s thermal resilience and substrate specificity, offering a valuable platform for the rational engineering of glucosidases in ginsenoside bioconversion processes. Full article

Sulfides should be removed before the recovery of cassiterite from tin-rich minerals due to their similarity in flotation properties. However, the traditional methods used have low selectivity. Therefore, moderately thermophilic microorganisms were used to desulfurize tin ore in this study, and the success of the microbial community was investigated. The bio-desulfurization rate reached 90% on the 10th day using the mixed culture of Leptospirillum ferriphilum (L. ferriphilum), Sulfobacillus thermosulfidooxidans (S. thermosulfidooxidans), and Acidithiobacillus caldus (A. caldus), while the pure culture needs at least 14 days. The results of X-ray Diffraction (XRD) and Inductively Coupled Plasma show that the sulfides were nearly fully solubilized. XRD results showed no pyrite in the residue, indicating that pyrite was almost fully removed while cassiterite was enriched compared with the original minerals. The high-throughput sequencing analysis showed that S. thermosulfidooxidans were the predominant species during the early bioleaching period, and L. ferriphilum were the predominant species in the following period. A. caldus is consistently detected and accounts for 30–50% of the different growth stages. This study supplied a potentially practical application for the desulfurization in tin ore. Full article

Peach is one of the most common stone fruit crops, but it is also the most thermophilic. One of the main problems in peach cultivation is frost up to −8 °C in spring during pollen development, budding, and flowering. The adaptation of the plant to low temperatures could be related to the activation of the antioxidant system under cold stress. The aim of this work was to test the hypothesis of distinct adaptation mechanisms to cold stress in Prunus persica L. cultivars with various cold tolerances. The difference between this study and the previous ones is that previously, only contrasting varieties (resistant and sensitive) were studied. For the first time, we studied the effect of cold stress on cold-resistant varieties but with different degrees of resistance, such as “Loadel” and “Springold” (medium resistant) and “Podarok Like” and “Temisovskij” (highly resistant). The experiment was designed to simulate the effects of short-term cold snaps, which are a common occurrence during February and March in the south of Crimea. A series of tests were conducted on annual shoots that were frozen at −12 °C. The activity and gene expression of two major antioxidant enzymes, catalase and peroxidase, were studied by spectrophotometry and RT-qPCR, respectively. The experiment showed that these enzymes responded differently to cold stress in varieties with different cold tolerances. Catalase responded similarly in all four varieties. After frost, there was an increase in activity (7-fold in “Temisovskij” and 3-fold in “Podarok Like”) and a decrease in expression. In contrast to catalase, peroxidase showed an opposite response to cold stress in medium-tolerant and highly tolerant cultivars. Peroxidase activity after exposure to low temperatures increased in highly tolerant cultivars (1.5-fold in “Temisovskij”), while it decreased in medium-tolerant cultivars (1.5–2 fold in “Springold” and “Loadel”, respectively). The change in peroxidase expression was the opposite. It decreased in highly resistant varieties and increased in medium-resistant varieties. Thus, our results revealed the opposite response of one of the major antioxidant enzymes (peroxidase) in moderately resistant and highly resistant cultivars. The data obtained show that varieties with a high degree of resistance could have other adaptation mechanisms involved, which may be useful for selecting resistant varieties. Full article

The genome of the mildly thermophilic hot spring purple sulfur bacterium, Allochromatium (Alc.) tepidum, contains a multigene pufBA family that encodes a series of α- and β-polypeptides, collectively forming a heterogeneous light-harvesting 1 (LH1) complex. The Alc. tepidum LH1, therefore, offers a unique model for studying an intermediate phenotype between phototrophic thermophilic and mesophilic bacteria, particularly regarding their LH1 Qy transition and moderately enhanced thermal stability. Of the 16 α-polypeptides in the Alc. tepidum LH1, six α1 bind Ca²⁺ to connect with β1- or β3-polypeptides in specific Ca²⁺-binding sites. Here, we use the purple bacterium Rhodospirillum rubrum strain H2 as a host to express Ca²⁺-bound and Ca²⁺-free Alc. tepidum LH1-only complexes composed of α- and β-polypeptides that either contain or lack the calcium-binding motif WxxDxI; purified preparations of each complex were then used to test how Ca²⁺ affects their thermostability and spectral features. The cryo-EM structures of both complexes were closed circular rings consisting of 14 αβ-polypeptides. The Q_y absorption maximum of Ca²⁺-bound LH1 (α1/β1 and α1/β3) was at 894 nm, while that of Ca²⁺-free (α2/β1) was at 888 nm, indicating that Ca²⁺ imparts a Q_y transition of 6 nm. Crucially for the ecological success of Alc. tepidum, Ca²⁺-bound LH1 complexes were more thermostable than Ca²⁺-free complexes, indicating that calcium plays at least two major roles in photosynthesis by Alc. tepidum—improving photocomplex stability and modifying its spectrum. Full article

Against the backdrop of the increasing copper demand in a low-carbon economy, this work statistically forecasted the distribution of China’s copper tailings for the first time, and then characterized them as finely crushed and low-grade mining solid wastes containing copper mainly in the form of chalcopyrite, bornite, covelline, enargite and chalcocite based on available research data. China is the globally leading refined copper producer and consumer, where the typical commercial-scale bioleaching of copper tailings is conducted in the Dexing, Zijinshan and Jinchuan mining regions. And these leaching processes were compared in this study. Widely used chemolithoautotrophic and mesophilic bacteria are Acidithiobacillus, Leptospirillum, Acidiphilium, Alicyclobacillus and Thiobacillus with varied metal resistance. They can be used to treat copper sulfide tailings such as pyrite, chalcopyrite, enargite, chalcocite, bornite and covellite under sufficient dissolved oxygen from 1.5 to 4.1 mg/L and pH values ranging from 0.5 to 7.2. Moderate thermophiles (Acidithiobacillus caldus, Acidimicrobium, Acidiplasma, Ferroplasma and Sulfobacillus) and extreme thermophilic archaea (Acidianus, Metallosphaera, Sulfurococcus and Sulfolobus) are dominant in leaching systems with operating temperatures higher than 40 °C. However, these species are vulnerable to high pulp density and heavy metals. Heterotrophic Acidiphilium multivorum, Ferrimicrobium, Thermoplasma and fungi use organic carbon as energy to treat copper oxides (malachite, chrysocolla and azurite) and weathered sulfides (bornite, chalcocite, digenite and covellite) under a wide pH range and high pulp density. We also compared autotrophs in a planktonic state or biofilm to treat different metal sulfides using various sulfur-cycling enzymes involved in the polysulfide or thiosulfate pathways against fungi that produce various organic acids to chelate copper from oxides. Finally, we recommended a bioinformatic analysis of functional genes involved in Fe/S oxidization and C/N metabolism, as well as advanced representation that can create new possibilities for the development of high-efficiency leaching microorganisms and insight into the mechanisms of bioleaching desired metals from complex and low-grade copper tailings. Full article

Studies investigating the taxonomic diversity and structure of soil bacteria in areas with enhanced radioactive backgrounds have been ongoing for three decades. An analysis of data published from 1996 to 2024 reveals changes in the taxonomic structure of radioactively contaminated soils compared to the reference, showing that these changes are not exclusively dependent on contamination rates or pollutant compositions. High levels of radioactive exposure from external irradiation and a high radionuclide content lead to a decrease in the alpha diversity of soil bacterial communities, both in laboratory settings and environmental conditions. The effects of low or moderate exposure are not consistently pronounced or unidirectional. Functional differences among taxonomic groups that dominate in contaminated soil indicate a variety of adaptation strategies. Bacteria identified as multiple-stress tolerant; exhibiting tolerance to metals and antibiotics; producing antioxidant enzymes, low-molecular antioxidants, and radioprotectors; participating in redox reactions; and possessing thermophilic characteristics play a significant role. Changes in the taxonomic and functional structure, resulting from increased soil radionuclide content, are influenced by the combined effects of ionizing radiation, the chemical toxicity of radionuclides and co-contaminants, as well as the physical and chemical properties of the soil and the initial bacterial community composition. Currently, the quantification of the differential contributions of these factors based on the existing published studies presents a challenge. Full article