Search Results (246)

This study endeavors to develop an economical and user-friendly biological sulfide oxidation system and explore its mechanism for generating biological elemental sulfur under micro-aerobic conditions using psychrophilic anaerobically digested media (liquid/solid inoculums obtained from agricultural livestock wastes) for sulfide-free biogas production. With an initial hydrogen sulfide concentration of 5000 ppm, a biogas flow rate ranging from 0.9 to 1.8 L/h-L_inoculum-mix, and an air injection rate of 0.6–1% (oxygen concentration in biogas), a remarkable biodesulfurization efficiency of 99–100% was attained using solid inoculum as the biodesulfurization medium. This efficiency was achieved without compromising the methane quality in the treated biogas. Compared to liquid inoculum, solid inoculum requires less than half the volume and no mixing equipment, such as bubble column reactors. The biodesulfurization reactor requires only 1 m³, which is approximately 1.5% of the volume of a wet anaerobic digester and 3% of a dry anaerobic digester, while processing cow manure (Total Solids: 20%) at 1.03 m³ of manure per day. Moreover, it can be operated at (19–20 °C), leading to substantial reductions in cost and footprint. Full article

In the present review, we summarize genome mining of genomic data obtained from the psychrophilic Antarctic marine ciliate Euplotes focardii and its evolutionary-close mesophilic cosmopolitan counterpart E. crassus. This analysis highlights adaptation strategies that are unique to the Antarctic ciliate, including antioxidant gene duplication and distinctive substitutions that may play roles in increased drug binding affinity and enzyme reaction rate in cold environments. Enzymes from psychrophiles are usually characterized by high activities and reaction rates at low temperatures compared with their counterparts from mesophiles and thermophiles. As a rule, catalyst cold activity derives from an increased structural flexibility that may lead to protein denaturation in response to temperature fluctuation. Molecular thermolability has been a major drawback of using macromolecules from psychrophiles in industrial applications. Here, we report a case study in which the role of peculiar amino acid substitution in cold adaptation is demonstrated by site-directed mutagenesis. Combined with a rational design approach, these substitutions can be used for site-directed mutagenesis to obtain cold-active catalysts that are structurally stable. Furthermore, molecular docking analysis of β-tubulin isotypes extrapolated from E. focardii and E. crassus genomes allowed us to obtain additional insight on the taxol binding site and drug affinity. E. focardii genome mining and the comparison with the mesophilic sibling counterpart can be used as an inspiration for molecular engineering for medical and industrial applications. Full article

Cold-active lipase from the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania) was cloned and characterized as an extremophilic biocatalyst for silybin acylation. Structural analyses highlighted conserved motifs confirming a functional lipase and the presence of primary structure elements for catalysis at low temperatures. The recombinant enzyme (PSL2) heterologously expressed in Escherichia coli was purified in one step by affinity chromatography with a yield of 12.08 ± 1.72 µg L⁻¹ of culture and a specific activity of 20.1 ± 3.2 U mg⁻¹ at 25 °C. Functional characterization of PSL2 showed a neutral (7.2) optimal pH and a high thermal stability up to 90 °C. Also, this lipase was stable in the presence of different organic solvents, with 60% residual activity when using 20% DMSO. Kinetic measurements indicated performant catalytic efficiency of PSL2 for different short and long chain fatty acids, with Km in the mM range. The catalytic activity of PSL2 was assessed for silybin acylation with various fatty acids and fatty acid methyl esters, demonstrating a 90% silybin conversion when methyl decanoate ester was used. This result clearly highlights the biocatalytic capability of this new cold-active lipase. Full article

Antarctic yeasts represent a poorly explored source of novel bioactive compounds with antineoplastic activity and a favorable toxicological profile. The present paper presents the newest data on the antiproliferative and antimicrobial potential of extracts obtained from the psychrophilic strain AL₁₀₃ of the species Sporobolomyces roseus. The capacity of AL₁₀₃ to grow under different cultivation conditions, including in a bioreactor system with optimal biomass quantities of approximately 6.0 g/L, was demonstrated. A comparative examination of the metabolic profiles (GC-MS-based) of yeast extracts revealed a wide variety of synthesized molecules responsible for the different levels of antineoplastic activity depending on the tissue origin of the malignant cell lines. Concentration response curves were generated by the MTT dye reduction test. The respective IC₅₀ values were extrapolated and found between 35.3 and 163 µg/mL. The antibacterial potential of both extracts was evaluated with the broth microdilution test against four referent pathogenic bacterial strains. The estimated minimal inhibitory concentrations revealed a moderate antibacterial activity. According to the GC-MS results, both extracts are rich in long-chain fatty acids which are known for their antibacterial properties. In conclusion, the Antarctic strain AL₁₀₃ possesses promising potential for further pharmacological investigations aiming to elucidate its application as a health-promoting food additive or/and as a source of biologically active compounds. Full article

Fresh-cut salads are highly appreciated by consumers due to their healthy and convenient nature. Fresh-cut (FC) lettuce is one of the best-selling FC products due to its freshness and low preparation time for salads. However, FC lettuce is very perishable and, in addition, has been recently associated with severe foodborne illness alerts. Alternative natural sanitizing treatments to conventional sodium hypochlorite are needed. The antimicrobial activity of vinegar has been well-known since ancient times. In addition, bioactive compounds from plant byproducts are widely recognized for their antioxidant properties. This work aimed to evaluate the use of a novel and encapsulated vinegar powder enriched with bioactive compounds from fruit byproducts with high antimicrobial and antioxidant properties to preserve the physicochemical (titratable acidity, total soluble solids, weight loss, and color), microbial (psychrophiles, enterobacteria, lactic-acid bacteria, molds, and yeasts), and sensory quality of FC lettuce at 4 °C for up to 10 days. Small to no differences were observed in terms of physicochemical quality (≈0.1% titratable acidity; 2.3–3.3% total soluble solids; <1% weight loss) in comparison to control samples through storage. Vinegar treatments reduced by 2–4 log CFU g⁻¹ the microbial loads after 10 days. In addition, a synergistic sensory antibrowning effect (greener and less yellowish appearance, showing −a* and −b*) between organic acids and the released polyphenols from the encapsulated plant byproduct extracts was observed. Hence, the enriched encapsulated vinegar represents an effective green alternative to conventional sanitizers to maintain the quality of FC lettuce through storage time. Full article

Deschampsia antarctica and Colobantus quitensis are the only two vascular plants colonized on the Antarctic continent, which is usually exposed to extreme environments. Endophytic bacteria residing within plant tissues can exhibit diverse adaptations that contribute to their ecological success and potential benefits for their plant hosts. This study aimed to characterize 12 endophytic bacterial strains isolated from these plants, focusing on their ecological adaptations and functional roles like plant growth promotion, antifungal activities, tolerance to salt and low-carbon environments, wide temperature range, and biofilm formation. Using 16S rRNA sequencing, we identified several strains, including novel species like Hafnia and Agreia. Many strains exhibited nitrogen-fixing ability, phosphate solubilization, ammonia, and IAA production, potentially benefiting their hosts. Additionally, halotolerance and carbon oligotrophy were also shown by studied bacteria. While some Antarctic bacteria remain strictly psychrophilic, others demonstrate a remarkable ability to tolerate a wider range of temperatures, suggesting that they have acquired mechanisms to cope with fluctuations in environmental temperature and developed adaptations to survive in intermediate hosts like mammals and/or birds. Such adaptations and high plasticity of metabolism of Antarctic endophytic bacteria provide a foundation for research and development of new promising products or mechanisms for use in agriculture and technology. Full article

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography–Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol–sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation. Full article

Polar habitats offer excellent sites to isolate unique bacterial strains due to their diverse physical, geochemical, and biological factors. We hypothesize that the unique environmental conditions of polar regions select for distinct strains of lactic acid bacteria (LAB) with novel biochemical properties. In this study, we characterized ten strains of psychrotrophic LAB isolated from hitherto poorly described sources—High Arctic and maritime Antarctic soils and soil-like materials, including ornithogenic soils, cryoconites, elephant seal colonies, and postglacial moraines. We evaluated the physiological and biochemical properties of the isolates. Based on 16S rRNA and housekeeping genes, the four LAB strains were assigned to three Carnobacterium species: C. alterfunditum, C. maltaromaticum, and C. jeotgali. The remaining strains may represent three new species of the Carnobacterium genus. All isolates were neutrophilic and halophilic psychrotrophs capable of fermenting various carbohydrates, organic acids, and alcohols. The identified metabolic properties of the isolated Carnobacterium strains suggest possible syntrophic interactions with other microorganisms in polar habitats. Some showed antimicrobial activity against food pathogens such as Listeria monocytogenes and human pathogens like Staphylococcus spp. Several isolates exhibited unique metabolic traits with potential biotechnological applications that could be more effectively exploited under less stringent technological conditions compared to thermophilic LAB strains, such as lower temperatures and reduced nutrient concentrations. Analysis of extrachromosomal genetic elements revealed 13 plasmids ranging from 4.5 to 79.5 kb in five isolates, featuring unique genetic structures and high levels of previously uncharacterized genes. This work is the first comprehensive study of the biochemical properties of both known and new Carnobacterium species and enhances our understanding of bacterial communities in harsh and highly selective polar soil ecosystems. Full article

In the context of global climate changes, it is important to assess the sustainability perspective of aquatic ecosystems based on marker organisms. In this work, we analyzed seasonal dynamics of silica-scaled chrysophytes in freshwater communicating environments which have considerable differences in water temperature between two ecosystems: the deep and cold oligotrophic Lake Baikal versus the shallower and warmer downstream Irkutsk Reservoir having mesotrophic features. During three seasons of the open water period of 2023, 38 species of silica-scaled chrysophytes were observed at 17 stations using scanning and transmission microscopy. The distribution of silica-scaled chrysophytes was shown to correlate with the water temperature. The greatest species richness was observed in the spring season in a large bay of the Irkutsk Reservoir (23 species), the smallest in the cold spring waters of Southern Baikal (up to 7 species). Widespread species living in Southern Baikal continued to grow in warmer waters of the reservoir. Using the example of silica-scaled chrysophytes, the stability of the high-latitudinal freshwater ecosystems affected by climate change is discussed. Continuous increment of the water temperature can lead to an increased abundance of widespread species and the displacement of psychrophilic species, affecting the overall biodiversity in such ecosystems. Full article

This study investigates the impact of water faecal contamination on highly threatened European brook lamprey larvae (Lampetra planeri). Water samples and the midgut contents of lampreys collected from a small lowland river upstream (site 1) and downstream from a wastewater treatment plant (WWTP) discharge (site 2) were analysed to check how the faecal microbial load of the habitat is reflected in the intestines of larval lampreys. The counts of viable mesophiles, psychrophiles, Escherichia coli and faecal streptococci as bacterial indicators of microbial (including faecal) water contamination were estimated. Microbial composition and abundance in larval midgut contents depended on the numbers of various microorganisms in the water environment. At site 2, the water was heavily microbiologically contaminated throughout the year by sewage inflow from the WWTP, and the amounts of studied bacteria were also high in the midgut of lampreys inhabiting site 2 regardless of the season. At site 1, water quality was better, and the levels of tested microbial indicators were lower in the intestines of the lampreys living there. The numbers of bacteria dependent on water temperature were growing in warmer seasons both in water and in intestines. Sewage pollution negatively influenced the condition of lampreys in site 2, where they exhibited lower body condition than in site 1. Full article

Milking methods have significant impacts on the microbiological composition, which could affect the quality of raw buffalo milk. Hence, the current study was conducted on the impact of milking methods on microorganisms in buffalo tank raw milk from 15 farms in Guangxi, China. The farms were divided into two groups based on the milking method: mechanical milking (MM, n = 6) and hand milking (HM, n = 9). Somatic cell counts, bacterial cell counts and nutrients of the raw buffalo milk samples were analyzed. The comparison of raw buffalo milk samples was analyzed using metagenomic sequencing to detect any differences between the two groups. There was no significant difference in the basic nutritional compositions and somatic cell count of raw buffalo milk between the two milking methods. However, the HM samples had significantly higher bacterial counts and diversity compared to the MM samples. The results showed that Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the major microbes present in canned raw buffalo milk. However, the differences between the two milking methods were the relative abundance of core microorganisms and their potential mastitis-causing genera, including the content of antibiotic-resistance genes and virulence genes. Our study revealed that Staphylococcus spp. and Streptococcus spp. were significantly more abundant in the MM group, while Klebsiella spp. was more abundant in the HM group. Regardless of the milking method used, Pseudomonas spp. was identified as the primary genus contributing to antibiotic resistance and virulence genes in canned raw buffalo milk. These findings affirm that there are differences in the microbial and genomic levels in canned raw milk. To prove the functional roles of the discovered genes and how these genes affect milk quality, further research and experimental validation are necessary. Full article

Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s⁴U), 7-methyl-guanosine (m⁷G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s⁴U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies. Full article

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K₄[Fe(CN₆)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments. Full article

At psychrophilic temperatures (<20 °C), anaerobic digestion produces less methane (CH₄). For psychrophilic anaerobic digestion (PAD) to be successful, investigation of cold-adapted microbial consortia involved in methane production is critical. This study aimed to investigate the microbial community driving enhanced methane production from the cold-adaptation process and bioaugmentation of PAD with cold-adapted inoculum (BI). Microbial consortia in cattle manure (CM) and food waste (FW) were adapted and applied during batch PAD of CM and FW to bioaugment methane production at 15 °C. Cold adaptation and PAD with BI resulted in cumulative specific methane yields of 0.874 ± 0.231 and 0.552 ± 0.089 L CH₄ g⁻¹ volatile solids, respectively, after 14 weeks, while the absence of BI (control) led to acidification and no methane production during PAD. Following 16S rRNA V4–V5 amplicon sequencing and metagenomic analyses, Methanosarcina was revealed as a key driver of methanogenesis during cold adaptation and PAD bioaugmentation. Furthermore, based on the predictive functional and metabolic analysis of the communities, possible synergies were proposed in terms of substrate production and utilization by the dominant microbial groups. For instance, during methane production, Bacteroides and Methanobrevibacter were possibly involved in a syntrophic relationship, which promoted methanogenesis by Methanosarcina. These findings provide insight into the prospective microbial synergies that can be harnessed and/or regulated in cold-adapted inoculum for the improvement of methane production during PAD. Full article

It has been documented that the shelf life of fishery products is extremely reduced due to microbial development and its endogenous biochemistry. For this reason, food technologists around the world are researching how to reduce the main processes that lead to spoilage. Recently, high-intensity ultrasound (HIU) has had different applications in the food industry because the cavitation effect can inhibit or reduce microbial development as well as cause conformational changes in muscle enzymes. Therefore, in this study, HIU was applied for 30, 60, and 90 min to the tilapia (Oreochromis niloticus) fillet, and subsequently, it was stored on ice for 20 days. During this period, samples were taken every 5 days (day 0, 5, 10, 15, and 20), and moisture content, pH, total volatile base (TVB-N), non-protein nitrogen (NPN), texture, electrophoresis, color, and microbiological analyses (mesophiles and psychrophiles) were determined. No significant changes (p ≥ 0.05) were observed in the moisture content, pH, and the L* parameter, while a significant decrease (p < 0.05) in TVB-N (from 29.67 to 15.09), NPN (from 0.39 to 0.27%), and texture (from 4.88 to 2.69 N) were found. On the other hand, an increase (p < 0.05) in a* (from 2.02 to 4.27) and b* (from 10.66 to 12.45) parameters, as well as total mesophile count (from 2.48 to 6.52 log CFU/g) were detected due to the application of ultrasound. The results suggest that the application of this treatment represents a viable alternative to increase the shelf life and quality of tilapia fillets stored on ice. Full article