Search Results (67)

Hypersaline soils are poly-extreme terrestrial habitats characterized by high salinity, in some cases heavy-metal contamination, temperature fluctuations, and nutrient limitation. These conditions impose strong selective pressures, and many prokaryotic inhabitants still remain uncultured. Here, we conducted an extensive culturomics study of 549 isolates from the hypersaline soils of the Odiel Saltmarshes Natural Area (Southwest Spain) and compared the results with previously generated shotgun metagenomic datasets from the same environment in order to evaluate taxonomic composition, functional potential, and ecological representativeness. Cultivation across media containing 7.5%, 15%, and 25% (w/v) total salts yielded microorganisms belonging to three major phyla: Pseudomonadota, Bacillota (Bacteria) and Halobacteriota (Archaea). At the genus level, bacterial isolates were dominated by Marinobacter, Halomonas, and Aquibacillus at 7.5% (w/v) salinity, whereas extremely halophilic archaea, including Halorubrum, Halogeometricum, and Haloarcula, were predominantly recovered from media containing 25% (w/v) salts. Among the isolates, 57 strains displayed identity values < 98.65% for 16S rRNA gene sequence comparison, suggesting their putative status as new taxa. Comparison with metagenomic datasets showed that culture-dependent approaches successfully recovered the dominant haloarchaeal groups but missed some abundant bacterial phyla, such as Gemmatimonadota. Conversely, culturomics enabled the isolation of unknown species from the rare biosphere, including representatives of the novel genus Terrihalobacillus, which are typically detected at low abundance in metagenomic datasets. Together, these results demonstrate the complementarity of culturomics and metagenomics and provide an insight into the microbial communities inhabiting the hypersaline soils of the Odiel Saltmarshes. Full article

Optimizing the dietary nitrogen-to-sulfur (N:S) ratio is crucial for enhancing ruminal microbial fermentation and nutrient digestibility. This study investigated the effects of different dietary N:S ratios on ruminal antioxidant indices, digestive enzyme activities, immune parameters, volatile fatty acid (VFA) profiles, microbial communities, and metabolomic profiles in plateau-type Tibetan sheep. Ninety 2-month-old male Tibetan sheep (initial body weight, 15.55 ± 0.20 kg) were randomly assigned to three dietary treatments formulated with N:S ratios of 9.5:1 (HP-H), 8.5:1 (HP-M), and 7.5:1 (HP-L). Sheep fed the HP-H diet exhibited improved ruminal antioxidant indices compared with the other treatments, with CAT and T-AOC activities being higher than those of both the HP-M and HP-L groups, while SOD and GSH-Px activities were significantly higher only compared with those in the HP-L (p < 0.05). Furthermore, ruminal cellulase activity, as well as propionate and butyrate levels, were significantly higher in the HP-H group (p < 0.05). Microbiome analysis revealed that the HP-H diet enriched the relative abundances of Candidatus Saccharimonas, the Rikenellaceae RC9 gut group, and uncultured rumen bacteria. Correspondingly, untargeted metabolomics identified higher signal intensities of 3-hydroxyphenylacetic acid, nervonic acid, arachidonic acid (peroxide-free), and calciferol in the ruminal fluid of the HP-H group relative to the other treatments. In conclusion, when the nitrogen-to-sulfur ratio was 9.5:1, ruminal antioxidant enzyme activities were increased, and the concentrations of propionic acid and butyric acid were increased. The differential microorganisms and metabolites were mainly associated with lipid metabolism, as well as cofactor and vitamin metabolism. Full article

Microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity. However, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds. While conventional techniques such as selective media and enrichment culture remain foundational, they are inherently limited by community biases and the inability to support low-abundance, oligotrophic species. To address these bottlenecks, a diverse array of innovative isolation strategies has emerged. This review systematically categorizes and evaluates these methodologies, ranging from in situ cultivation to high-resolution single-cell manipulation. We first examine membrane diffusion-based cultivation (e.g., iChip), which mimics natural microenvironments to resuscitate recalcitrant microbes. Subsequently, we explore high-throughput single-cell technologies, including microfluidics for physicochemical separation, optical tweezers for precise manipulation, and fluorescence-activated cell sorting (FACS). Special attention is given to Raman-activated cell sorting (RACS) as a label-free functional screening tool and reverse genomics for targeted capture. By synthesizing the strengths and limitations of these approaches, we propose integrated workflows designed to accelerate the mining of untapped microbial resources. Full article

Umami is a key determinant of pork flavor, but the association between the intestinal microbial community and umami differences remains unclear. Here, we used the taste-sensing electronic tongue system to divide the Duroc × Landrace × Yorkshire pigs into high, medium and low groups. We combined 16S rRNA gene and shotgun metagenomic sequencing to study the differences in the microbial community composition and functional genes. The results showed that the microorganisms in the cecum of different groups had a similar core microbial community. The Shannon diversity analysis showed that there were no significant differences among the different groups. The Bray–Curtis distance indicated that there were differences in the bacterial communities between the high umami group and the other two groups. The LEfSe analysis and Spearman correlation analysis revealed that the uncultured species CAG-632 sp900539185 maintained a high abundance in the high umami group and was significantly correlated with umami. Metagenomic functional analysis revealed distinct functional signatures among umami groups, with enrichment of genes related to carbohydrate transport and metabolism, butanoate and other short-chain fatty acid pathways, nitrogen utilisation, cell-surface structures, adhesion and RNA metabolism in high umami groups. These research findings indicate that the differences in the delicious flavor of pork are more likely to be associated with specific microbial species and the functional characteristics of the cecal microbial community, rather than the overall situation of the entire microbial community. Full article

The presence of substantial amounts of heavy metals in the environment can result in various significant ecological issues and human health risks. Currently, bioremediation employing microorganisms is garnering significant interest due to its effectiveness. The present investigation aimed to isolate actinobacterial strains from an Italian mine and to characterise them for heavy metals resistance and plant growth-promoting characteristics. The different samples were processed for DNA extraction and 16S rRNA gene metabarcoding to investigate the bacteria and archaea communities. Cultivable microbiota were isolated and evaluated for heavy metals tolerance and different PGP traits. The most pertinent strains were tested for compatibility, merged into a consortium, and tested on Lemna minor L. Metabarcoding analysis revealed that amplicon sequence variants (ASVs) at the phylum level were mostly assigned to proteobacteria and bacteroidota. Uncultured and unknown taxa were the most prevalent in the samples at the genus level. A total of ten strains were obtained from the culture-dependent approach exhibiting interesting heavy metals tolerance and plant growth-promoting traits. The best strains (MTW 1 and MTW 5) were selected and further characterised by 16S barcoding. These strains were identified as Streptomyces atratus (99.57% identity). An in planta experiment showed that the metal-tolerant consortium MTW 1-5 improved plant physiology by significantly optimising plant growth and tolerance to heavy metals. The experiment conducted provided evidence for the possibility of using actinobacteria as bioaugmentation agents to improve the phytoextraction abilities of L. minor. Full article

Photocatalytic oxidation of microorganisms is a powerful alternative to established disinfection approaches, applicable to a variety of water matrices. Bacterial vegetative cells, spores, fungi, and viruses, represent potential biopathogens and photocatalysis targets. Inactivation efficiency is usually evaluated by assessing viability through culture. However, additional inactivation assessment approaches are needed, as some microbes, despite being unculturable, remain metabolically active and pathogenic. Nucleic acid quantification approaches (qPCR) can assess nucleic acid release and degradation during photocatalysis. We developed a novel multiplex qPCR assay for simultaneous detection/quantification of genomic DNA from different bacterial and fungal species and of MS2 bacteriophage load. Following small-scale solar titanium dioxide photocatalysis on a microbial suspension mixture containing different biopathogen classes, we assessed photocatalytic efficiency by conventional microbiological assays (culture) and our novel molecular assay. Microbiological assays show a significant reduction in microbe viability within one hour of processing, following previously reported patterns of microbial species resistance. Molecular analysis data show that nucleic acids released in solution due to microbial oxidative damage were significantly reduced due to oxidative degradation within six hours. Through targeting different biopathogen classes, our assay could be a useful tool for assessment of photocatalytic microbe inactivation both in laboratory and real-wastewater applications. Full article

The vast majority of microorganisms in the environment remain uncultured using conventional laboratory techniques, representing an immense untapped reservoir of genetic and chemical diversity. Recent innovations in cultivation strategies, combined with advances in metagenomics, single-cell genomics, and synthetic biology, have opened new avenues for accessing and harnessing bioactive natural products from these previously inaccessible microorganisms. This review highlights recent methodological and technological advancements in the cultivation and identification of novel microorganisms, and showcases the resulting discoveries of new natural products, demonstrating their potential for drug development. Full article

Microorganisms exhibit remarkable diversity, making their comprehensive characterization essential for understanding ecosystem functioning and safeguarding human health. However, traditional culture-based methods entail inherent limitations for resolving microbial heterogeneity, isolating slow-growing microorganisms, and accessing uncultivated microbes. Conversely, droplet-based microfluidics enables a high-throughput and precise platform for single-bacterium manipulation by physically isolating individual cells within microdroplets. This technology presents a transformative approach to overcoming the constraints of conventional techniques. This review outlines the fundamental principles, recent research advances, and key application domains of droplet-based microfluidics, with a particular focus on innovations in single-bacterium encapsulation, sorting, cultivation, and functional analysis. Applications such as antibiotic susceptibility testing, enzyme-directed evolution screening, microbial interaction studies, and the cultivation of novel bacterial species are discussed, underscoring the technology’s broad potential in microbiological research and biotechnology. Full article

Bacterial endophytes have emerged as critical components of plant microbiomes, offering multifaceted benefits ranging from growth promotion to stress resilience. This review synthesizes two decades of research, from 2004 to 2024, on bacterial endophyte identification and applications, highlighting advances in both traditional culture-based techniques and modern omics approaches. The review also focuses on interactions between these microorganisms and their host plants, emphasizing their roles in biocontrol, phytoremediation, and nanoparticle biosynthesis. While significant progress has been made in characterizing cultivable bacterial endophytes, challenges persist in accessing unculturable species and understanding strain-specific functional mechanisms. The integration of metagenomics, metatranscriptomics, and metabolomics has begun unraveling this hidden diversity, revealing novel metabolic pathways and plant–microbe communication systems. There have been limitations in endophyte isolation protocols and field applications, and therefore a need exists for standardized frameworks to bridge lab-based discoveries with agricultural practices. Cutting-edge multi-omics techniques, such as genomics, transcriptomics, metabolomics, proteomics, and phenomics, should be used more in future research to clarify the mechanistic underpinnings of plant–endophyte interactions to thoroughly profile the microbial communities and unlock their functional potential under diverse environmental conditions. Overall, bacterial endophytes present viable paths toward sustainable farming methods, supporting food security and crop resilience in the face of environmental difficulties by providing a transformative opportunity for next-generation agriculture, mitigating climate-related agricultural stressors, reducing dependence on synthetic agrochemicals, and enhancing crop productivity. Full article

Dermacentor is the most widely distributed tick genus in China. Dermacentor nuttalli, a predominant tick species in Inner Mongolia, can carry and transmit pathogenic microorganisms. Here, D. nuttalli were collected from Ordos (O-D) and Hinggan League (H-D) in the Inner Mongolia. D. nuttalli specimens at different developmental stages were subsequently reared under identical laboratory conditions. Sample processing, nucleic acid extraction, high-throughput sequencing, and microbial community analyses were conducted. Bacterial communities in O-D and H-D were annotated to 8 phyla, 145 genera and 16 phyla, 141 genera, respectively, with Proteobacteria showing the highest relative abundance. Differences in dominant bacterial genera were observed across developmental stages between the two regions. The most abundant bacterial species were Arsenophonus_uncultured_bacterium in O-D and Rickettsia japonica in H-D. Viral communities were annotated to 4 orders, 25 families, 61 genera, and 126 species in O-D and 6 orders, 28 families, 49 genera, 135 species in H-D. Notable difference in the viral genera with >1% abundance were identified at different developmental stages in the two regions. To our knowledge, this is the first study to compare microbial community compositions of D. nuttalli across developmental stages in two Inner Mongolian regions under under identical rearing conditions and to report the presence of R. japonica, Tacheng Tick Virus-2, and bovine viral diarrhea virus in D. nuttalli. Full article

Next-Generation Sequencing (NGS) technology was applied to evaluate Food Safety Management System (FSMS) performance in seafood-processing factories by exploring microbiome diversity alongside traditional methods for detecting foodborne pathogens. A total of 210 environmental swabs collected from processing zones in six factories underwent 16S rRNA amplicon sequencing. FSMS-certified factories exhibited significantly higher species richness, with alpha diversity p-values of 0.0036 for observed ASVs, 0.0026 for Faith’s PD and 0.032 for Shannon. Beta diversity analysis also revealed significant differences, with p-values of 0.001 for Bray–Curtis, unweighted UniFrac and Jaccard. Pathogens like Listeria monocytogenes, Salmonella spp. and Bacillus cereus were present in “uncertified” factories but absent in the “certified” factories. The “certified” factories had a significantly higher proportion of lactic acid bacteria (LAB) genera (70.22%) compared to “uncertified” factories (29.78%). The LAB genera included Streptococcus, Lactococcus, Lactobacillus and others. NGS has demonstrated superior capability by providing comprehensive microbiome detection, including the unculturable microorganisms and insights into microbial diversity, so it lacks the limitations that come with traditional culturing. These findings highlight the potential for leveraging beneficial microbes in bioremediation and pathogen control to enhance FSMS effectiveness in seafood-processing environments. Full article

Metagenome-assembled genomes (MAGs) have revolutionized microbial ecology by enabling the genome-resolved study of uncultured microorganisms directly from environmental samples. By leveraging high-throughput sequencing, advanced assembly algorithms, and genome binning techniques, researchers can reconstruct microbial genomes without the need for cultivation. These methodological advances have expanded the known microbial diversity, revealing novel taxa and metabolic pathways involved in key biogeochemical cycles, including carbon, nitrogen, and sulfur transformations. MAG-based studies have identified microbial lineages form Archaea and Bacteria responsible for methane oxidation, carbon sequestration in marine sediments, ammonia oxidation, and sulfur metabolism, highlighting their critical roles in ecosystem stability. From a sustainability perspective, MAGs provide essential insights for climate change mitigation, sustainable agriculture, and bioremediation. The ability to characterize microbial communities in diverse environments, including soil, aquatic ecosystems, and extreme habitats, enhances biodiversity conservation and supports the development of microbial-based environmental management strategies. Despite these advancements, challenges such as assembly biases, incomplete metabolic reconstructions, and taxonomic uncertainties persist. Continued improvements in sequencing technologies, hybrid assembly approaches, and multi-omics integration will further refine MAG-based analyses. As methodologies advance, MAGs will remain a cornerstone for understanding microbial contributions to global biogeochemical processes and developing sustainable interventions for environmental resilience. Full article

Kefir-based fermentation products exhibit antioxidant and anti-inflammatory effects against oxidative stress, inflammation, platelet activation and aggregation, and other related manifestations, thereby preventing the onset and development of several chronic diseases. Specifically, water kefir, a symbiotic culture of various microorganisms used for the production of several bio-functional fermented products, has been proposed for its health-promoting properties. Thus, water kefir grains and its apple pomace-based fermentation beverage were studied for bioactive amphiphilic and lipophilic lipid compounds with antioxidant, antithrombotic, and anti-inflammatory properties. Total lipids (TL) were extracted and further separated into their total amphiphilic (TAC) and total lipophilic content (TLC), in which the total phenolic and carotenoid contents (TPC and TCC, respectively) and the fatty acid content of the polar lipids (PL) were quantified, while the antioxidant activity of both TAC and TLC were assessed in vitro, by the ABTS, DPPH, and FRAP bioassays, along with the anti-inflammatory and antithrombotic activity of TAC against human platelet aggregation induced by the thrombo-inflammatory mediator, platelet-activating factor (PAF) or standard platelet agonists like ADP.ATR-FTIR spectra facilitated the detection of specific structural, functional groups of phenolic, flavonoid, and carotenoid antioxidants, while LC−MS analysis revealed the presence of specific anti-inflammatory and antithrombotic PL bioactives bearing unsaturated fatty acids in their structures, with favorable omega-6 (n-6)/omega-3 (n-3)polyunsaturated fatty acids (PUFA), which further support the findings that the most potent antioxidant, anti-inflammatory and antithrombotic bioactivities were observed in the TAC extracts, in both water kefir grains and beverage cases. The detection of such bioactive components in both the uncultured water kefir grains and in the cultured beverage further supports the contribution of water kefir microorganisms to the bioactivity and the bio-functionality of the final fermented product. Nevertheless, the extracts of the beverage showed much stronger antioxidant, anti-inflammatory, and antithrombotic activities, which further suggests that during the culture process for producing this beverage, not only was the presence of bioactive compounds produced by kefir microflora present, but biochemical alterations during fermentation of bioactive components derived from apple pomace also seemed to have taken place, contributing to the higher bio-functionality observed in the apple pomace—water kefir-based beverage, even when compared to the unfermented apple pomace. The overall findings support further studies on the use of water kefir and/or apple pomace as viable sources of antioxidant, anti-inflammatory, and antithrombotic amphiphilic bioactive compounds for the production of novel health-promoting bio-functional fermented products. Full article

The black soldier fly, Hermetia illucens, is a voracious scavenger of various organic materials; therefore, it could be exploited as a biological system for processing daily food waste. In order to survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens. Through functional screening of the library on carboxymethyl cellulose plates, we identified a fosmid clone, the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel mannan-degrading gene, ManEM6, composed of 1185 base pairs encoding 394 amino acids, with a deduced 20-amino-acid N-terminal signal peptide sequence. The conceptual translation of ManEM6 exhibited the highest identity (78%) to endo-1,4-β-mannosidase from Dysgonomonas mossii. Phylogenetic and domain analyses indicated that ManEM6 encodes a novel mannanase with a glycoside hydrolase family 26 domain. The recombinant protein rManEM6 showed its highest activity at 40 °C and pH 7.0, and it remained stable in the range of pH 5–10.0. rManEM6 hydrolyzed substrates with β-1,4-glycosidic mannoses, showing maximum enzymatic activity toward locust bean gum galactomannan, while it did not hydrolyze p-nitrophenyl-β-pyranosides, demonstrating endo-form mannosidase activity. rManEM6 was highly stable under stringent conditions, including those of polar organic solvents, as well as reducing and denaturing reagents. Therefore, ManEM6 may be an attractive candidate for the degradation of mannan under high-organic-solvent and protein-denaturing processes in the food and feed industries. Full article

This study aimed to investigate the effect of varying levels of dietary protein on digestion, metabolism, serum biochemical indexes, and rumen microflora in Lanzhou fat-tailed sheep. A total of twenty 8-month-old healthy rams with an initial average body weight (BW 25.16 ± 1.09 kg) were selected and randomly divided into four dietary treatments with different protein levels P: 9.47%, MP: 10.53%, HP: 11.56%, and EHP: 12.61%. The rams underwent a 23 day adaptation period, and the experiment was conducted for 7 d. The results showed that with increased dietary protein levels, the apparent digestibility of dry matter (DM), crude protein (CP), acid detergent fiber (ADF), and urine energy significantly increased (p < 0.05), increasing by 8.65%, 7.31%, 6.22%, and 0.1%, respectively. In contrast, the digestibility of neutral detergent fiber (NDF) first increased and then plateaued with the increase in protein levels, and the digestibility of NDF in the HP group was the highest (p < 0.05). The Shannon, Simpson, Chao1, and Ace indices of rumen microorganisms in the four groups did not change significantly (p > 0.05). Additionally, at the phylum level, the relative abundance of Bacteroidota, Firmicutes, and Verrucomicrobiota collectively accounted for over 91% of the total phylum composition. At the genus level, there was an increase in the relative abundance of uncultured_rumen_bacterium and Rikenellaceae_RC9_gut_group in the LP group, whereas the relative abundance of Prevotella decreased. In comparison to the other three groups, the HP group exhibited an increase in the relative abundance of Firmicutes. In summary, dietary protein level had no significant effects on the rumen microflora structure and blood biochemical indexes. However, diets with a high protein level can improve the apparent digestibility of nutrients and energy use efficiency of Lanzhou fat-tailed sheep. Full article