Search Results (156)

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small amounts of feces containing cysts or trophozoites to the culture medium. Implantation success is lower when starting from cysts, and the mechanisms and early events of excystation remain poorly understood. In this study, we describe the sequence of events involved in excystation and identify factors potentially important for culture establishment. Cysts were obtained from orangutan feces and genetically confirmed as B. coli. Only viable cysts, determined by trypan blue or methylene blue exclusion, were used. After artificial digestion with pepsin and trypsin, cysts were incubated at 28 °C for up to 72 h in DMEM supplemented with L-glutamine, yeast extract, fetal bovine serum, and starch granules. Excystation began with a fissure in the cyst wall, allowing for bacterial entry. This appeared to stimulate the trophozoites, the increased motility of which progressively weakened and ruptured the wall, allowing for their emergence. Wall rupture and bacterial entry were critical for activation., whereas starch type had no apparent influence. Excystation occurred within the first hours; otherwise, cysts degenerated. Full article

Seed microbes play crucial roles in plant health, but studying their diversity is challenging due to host DNA contamination. This study aimed to optimise methodologies for investigating seed microbiomes across diverse plant species, focusing on the efficacy of peptide nucleic acid (PNA) clamps to reduce host DNA amplification. We tested PNA clamps on three plant species: Melaleuca quinquenervia (tree), Microlaena stipoides, and Themeda triandra (grasses). The effectiveness of PNA clamps was assessed through in silico analysis, axenic tissue culture, and metabarcoding techniques. In silico analysis confirmed the specificity of PNA clamps to the 16S rRNA gene V4 region of chloroplasts in the grass species. Axenic tissue culture experiments showed that applying PNA clamps at both 1 µM and 0.25 µM concentrations significantly reduced plant DNA amplification. Metabarcoding analyses further confirmed that PNA clamps effectively suppressed host DNA, enhancing microbial diversity estimates across all three species while preserving core microbial taxa. The efficacy of the clamps varied among host species, with T. triandra exhibiting the highest blocking efficacy, and chloroplast clamps outperforming mitochondrial ones. This study demonstrates that PNA clamps are a useful for improving seed endophyte metabarcoding datasets, although they require optimisation for some plant species. This knowledge will contribute to enhancing our understanding of seed microbiome diversity and its ecological implications. Full article

Microbial decomposition of persistent natural compounds such as phenolic lignin and polysaccharides in plant cell walls plays a crucial role in the global carbon cycle and underpins diverse biotechnological applications. Among microbial decomposers, fungi from the Ascomycota and Basidiomycota phyla have evolved specialized mechanisms for efficient lignocellulosic biomass degradation, employing extracellular enzymes and synergistic fungal consortia. Fungal coculture, defined as the controlled, axenic cultivation of multiple fungal species or strains in a single culture medium, is a promising strategy for industrial processes. This approach to biomass conversion offers potential for enhancing production of enzymes, biofuels, and other high-value bioproducts, while enabling investigation of ecological dynamics and metabolic pathways relevant to biorefinery operations. Lignocellulosic biomass conversion into fuels, energy, and biochemicals is central to the bioeconomy, integrating advanced biotechnology with sustainable resource use. Recent advancements in -omics technologies, including genomics, transcriptomics, and proteomics, have facilitated detailed analysis of fungal metabolism, uncovering novel secondary metabolites and enzymatic pathways activated under specific growth conditions. This review highlights the potential of fungal coculture systems to advance sustainable biomass conversion in alignment with circular bioeconomy goals. Full article

Cyanobacteria are ubiquitous in freshwater environments, but their role in aquatic resistome remains unclear. In this work, we performed whole genome sequencing on 43 cyanobacterial strains isolated from Portuguese fresh/wastewaters. From 43 available non-axenic unicyanoabacterial cultures (containing only one cyanobacterial strain and their co-occurring bacteria), it was possible to recover 41 cyanobacterial genomes from the genomic assemblies using a genome binning software, 26 of which were classified as high-quality based on completeness, contamination, N50 and contig number thresholds. By using the comprehensive antibiotic resistance database (CARD) on the assembled samples, we detected four antibiotic resistance gene (ARG) variants, conferring resistance in pathogenic bacteria to tetracyclines, fluoroquinolones (adeF-type) and macrolides (ermF-type, mefC-type and mphG-type). Among these, adeF-type was the most prevalent gene, found across 11 cyanobacterial genomes from the Nostocales order. Planktothrix presented the highest variety of close ARG matches, with hits for the macrolide resistance genes ermF-type, mefC-type and mphG-type. An analysis of the genomic assemblies also revealed an additional 12 ARGs in bacteria from the phyla Firmicutes, Proteobacteria and Bacteroidetes, present in the cyanobacterial cultures, foreseeing the horizontal gene transfer of ARGs with cyanobacteria. Additionally, more than 200 partial ARGs were detected on each recovered cyanobacterial genome, allowing for future studies of antibiotic resistance genotype/phenotype in cyanobacteria. These findings highlight the importance of further efforts to understand the role of cyanobacteria on the aquatic resistome from a One Health perspective. Full article

Wastewater (WW) treatment using biofilms harboring bacteria and microalgae is considered a promising polishing solution to improve current treatment technologies present in wastewater treatment plants (WWTPs), but their interaction in a sessile community remains to be understood. In this work, multi-species biofilms of Chlorella vulgaris, Chlorella sorokiniana, or Scenedesmus obliquus were selected as representative microalgae species of interest for WW bioremediation, and Rhodococcus fascians, Acinetobacter calcoaceticus, or Leucobacter sp. were selected as the bacteria for co-cultivation in a synthetic WW since they are normally found in WW treatment processes. The attached consortia were developed in specific carriers (K5 carriers) for 168 h, and their biofilm formation ability was evaluated in a profilometer and via scanning electron microscopy (SEM) imaging. From the selected microorganisms, C. sorokiniana was the microalga that adapted best to co-cultivation with R. fascians and A. calcoaceticus, developing a thicker biofilm in these two consortia (3.44 ± 0.5 and 4.51 ± 0.8 µm, respectively) in comparison to the respective axenic cultures (2.55 ± 0.7 µm). In contrast, Leucobacter sp. did not promote biofilm growth in association with C. vulgaris and C. sorokiniana, while S. obliquus was not disturbed by the presence of this bacterium. Some bacterial clusters were observed through SEM, especially in A. calcoaceticus cultures in the presence of microalgae. In some combinations (especially when C. vulgaris was co-cultivated with bacteria), the presence of bacteria was able to increase the number of microalga cells adhered to the K5 carrier. This study shows that biofilm development was distinctly dependent on the co-cultivated species, where synergy in biofilm formation was highly dependent on the microalgae and bacteria species. Moreover, profilometry appears to be a promising method for biofilm analyses. Full article

Furfural is a relevant industrial product, but its presence in water and soil generates contamination and health risks. Moving bed biofilm reactors (MBBRs) are an increasingly used alternative to eliminate contaminants with the advantage of occupying small spaces, despite their high dependence on support and the microorganisms involved in the process. This work proposes furfural elimination through a laboratory-scale MBBR using Bacillus licheniformis GTQ1, Microbacterium sp. GISTAQ2, and Brevundimonas sp. GISTAQ1 isolated from an industrial effluent and agroforestry waste (rice husks, pine sawdust, and quebracho chips) as supports. The biofilm development was tested with both axenic and mixed cultures, confirming high coverage by Scanning Electron Microscope (SEM) images, especially in triple-mixed cultures. Biodegradation tests were carried out in the MBBR with 15 g rice husks or quebracho chips as supports and a 4000 mg L⁻¹ initial furfural concentration for 72 h. The mixed culture achieved almost a 100% furfural removal in three days with a rate of 3.97% per hour with rice husks and 2.61% per hour with quebracho chips. This laboratory-scale MBBR development is a promising first step ready for a scale-up for its implementation in industries to significantly reduce the environmental impact of the discharge of this type of effluent. Full article

We hypothesize that bacteria isolated from free-ranging animals could potentially be useful for practical applications. To meet this objective a Gram-positive bacterium was isolated from the gastrointestinal (GI) tract of a Gray Wolf (Canis lupus) using Brucella broth with hemin and vitamin K (BBHK). By small ribosomal RNA (16S) gene sequencing the bacterium was initially identified as a novel Carnobacterium maltaromaticum strain. The bacterium could be propagated both anaerobically and aerobically and was both catalase/oxidase negative and negative by the starch hydrolysis as well as negative using lipase assays. The reference whole genome sequence (WGS) was obtained using both Illumina and Nanopore sequencing. The genome assembly was 3,512,202 bp in length, encoding core bacterial genes with a GC% content of 34.48. No lysogenic bacteriophage genes were detected, although the genome harbors genes for the expression of bacteriocin and other secondary metabolites with potential antimicrobial properties. Multilocus sequence typing (MLST), WGS phylogenetics, average nucleotide identity (ANI), and single nucleotide polymorphism (SNP) analyses of the isolate’s genome indicate this bacterium is a newly identified Carnobacterium maltaromaticum sequence type (ST). Members of the Carnobacteria have anti-listeria activities, highlighting their potential functional properties. Consequently, the isolate could be a potential probiotic for canids and this is the first report on an axenic C. maltaromaticum culture from the genus Canis. Full article

Applications of lanthanide chemistry have been successful in metallics and the petroleum industry. In the medical realm, lanthanides have shown utility in radiotherapy agents, photodynamic therapy agents, and magnetic resonance imaging (MRI) contrast agents. The lanthanide group elements have a few known biological roles, notably among some bacteria and the yeast Saccharomyces cerevisiae, which have been used as models for changes in gene expression. However, the systematic effects of lanthanide nitrates on eukaryotic cell model systems have not yet been reported. This study presents the first documented effects on cell viability, after acute incubations of various lanthanide nitrate salts, using axenic C6 glial or PC12 neuronal cells in vitro. Cultures were exposed to a 1 mM concentration of lanthanide nitrate salts for 24 h. In comparison to the saline control, several cultures demonstrated significantly lower cell viability, as measured by the MTT viability assay. Data were analyzed as an average absorbance of n = 4 replicate samples, corrected for the average absorbance of cell-free blanks. The reported results were normalized to the average of the saline control cells. Among the 13 lanthanides tested, Praseodymium, Holmium, Erbium, Thulium, and Ytterbium nitrates exhibited the most pronounced inhibitory effects, resulting in over 40% reduction in cell viability at 1 mM for either or both cell types. Recovery after lanthanide exposure also was cell-type-dependent as well as lanthanide-type-dependent, with Lutetium having the greatest effect on both cell types. PC12 cells displayed greater sensitivity for inhibition than the C6 cells with some of the lanthanides but not all. Furthermore, the controls of sodium nitrate and calcium nitrate showed only modest discernible impacts on cell viability for PC12 and C6 cells, highlighting the role of the lanthanides in influencing cell viability. Full article

Synthetic non-metallic nanoparticles (NMNPs) such as carbon nanotubes (CNTs), carbon nanodots (CNDs), and cell-penetrating peptides (CPPs) have been explored to treat harmful algal blooms. However, their strain-specific algicidal activities have been rarely investigated. Here we determined their acute toxicity to nine freshwater cyanobacterial strains belonging to seven genera, including Microcystis aeruginosa UTEX 2386, M. aeruginosa UTEX 2385, M. aeruginosa LE3, Anabaena cylindrica PCC 7122, Aphanizomenon sp. NZ, Planktothrix agardhii SB 1810, Synechocystis sp. PCC 6803, Lyngbya sp. CCAP 1446/10, and Microcoleus autumnale CAWBG635 ATX. We prepared in-house three batches of CNDs using glucose (CND-G) or chloroform and methanol (CND-C/M) as the substrate and one batch of single-walled CNTs (SWCNTs). We also ordered a commercially synthesized CPP called γ-Zein-CADY. The axenic laboratory culture of each cyanobacterial strain was exposed to an NMNP at two dosage levels (high and low, with high = 2 × low) for 48 h, followed by measurement of five endpoints. The endpoints were optical density (OD) at 680 nm (OD₆₈₀) for chlorophyll-a estimation, OD at 750 nm (OD₇₅₀) for cell density, instantaneous pigment fluorescence emission (FE) after being excited with 450 nm blue light (FE₄₅₀) for chlorophyll-a or 620 nm red light (FE₆₂₀) for phycocyanin, and quantum yield (QY) for photosynthesis efficiency of photosystem II. The results indicate that the acute toxicity was strain-, NMNP type-, dosage-, and endpoint-dependent. The two benthic strains Microcoleus autumnale and Lyngbya sp. were more resistant to NMNP treatment than the other seven free-floating strains. SWCNTs and fraction A14 of CND-G were more toxic than CND-G and CND-C/M. The CPP was the least toxic. The high dose generally caused more severe impairment than the low dose. OD₇₅₀ and OD₆₈₀ were more sensitive than FE₄₅₀ and FE₆₂₀. QY was the least sensitive endpoint. The strain dependence of toxicity suggested the potential application of these NMNPs as a target-specific tool for mitigating harmful cyanobacterial blooms. Full article

Achnanthidium minutissimum is a widely distributed benthic freshwater diatom. The alga can produce stalks that stick the cell to the surface and subsequently extracellular capsules developing into biofilms. Extracts of the diatom-associated bacterium Dyadobacter sp. 32 have been shown previously to induce stalk and capsule formation by the diatom. Here, we studied the impact of macronutrients on the generation of stalks induced by bacterial extracts with respect to the frequency of stalk generation and stalk lengths, using axenic cultures to avoid any additional impact of bacteria on the nutrient availability. We found that nitrate deprivation inhibited cell division of A. minutissimum within four days, but it did not initially affect stalk production or elongation. Silica limitation instead inhibited both stalk production and elongation. Similarly, sulfate was required for stalk formation, which was supported by the energy-dispersive X-ray spectroscopy of A. minutissimum cells showing that sulfur was abundant in the stalks. Full article

In vitro cultivation of arbuscular mycorrhizal fungi (AMF) is challenging due to their biotrophic symbiosis. The principal aim of this study was to demonstrate the effect of establishing in vitro dual cultures of arbuscular mycorrhizal fungi (AMF) inoculated on Swietenia macrophylla (mahogany) roots on plant growth. Furthermore, it was sought to demonstrate that plant colonization by Glomeromycota can be achieved with a replicable protocol. This study established monoxenic cultures of carrot (Daucus carota) Ri T-DNA ROC inoculated with Glomus sp. on two-compartment plates. At 75 days, hyphal growth reached 223.93 mm in the root compartment and 103.71 mm in the hyphal compartment. Spores produced in vitro measured 26.14 ± 1.70 µm, smaller than ex vitro spores (101.2 ± 4.22 µm). Rhodotorula mucilaginosa was isolated from cultures and appeared to stimulate hyphal growth and root–fungal contact. From these cultures, a dual culture of mahogany inoculated with Glomus sp. was established. No significant differences were observed between inoculated and non-inoculated plants in stem length, root length, root number, or leaf number at 30 days. Spore production ranged from 10,166 to 27,696 per plate, averaging 14,795 ± 3301, with hyphal lengths of 3655.46 ± 308.75 mm. Hyphal development included running and branching patterns, with solitary and clustered spores. Spore diameter averaged 27.68 ± 3.85 µm. Arbuscular colonization reached 41.49% at 30 days and 52.13% at 75 days, exceeding rates reported for other culture systems. Monoxenic cultures are a reliable, aseptic source of high-quality inoculum, supporting biofertilizer production and biotechnological applications. These methods provide valuable tools for studies involving AMF, such as those demonstrated with mahogany. Full article

Microorganisms in high-salinity environments play a critical role in biogeochemical cycles, primary production, and the biotechnological exploitation of extremozymes and bioactive compounds. The main challenges in current research include isolating and cultivating these microorganisms under laboratory conditions and understanding their complex adaptive mechanisms to high salinity. Currently, universally recognized protocols for isolating microalgae and cyanobacteria from salt pans, salterns, and similar natural habitats are lacking. Establishing axenic laboratory cultures is essential for identifying new species thriving in high-salinity environments and for exploring the synthesis of high-value metabolites by these microorganisms ex situ. Our ongoing research primarily focuses on photosynthetic microorganisms with significant biotechnological potential, particularly for skincare applications. By integrating data from the existing literature with our empirical findings, we propose a standardized pipeline for the isolation and laboratory cultivation of microalgae and cyanobacteria originating from aqueous environments characterized by elevated salt concentrations, such as solar salterns. This approach will be particularly useful for researchers working with microorganisms adapted to hypersaline waters. Full article

The biotrophic fungus Ustilago maydis, which causes smut disease in maize, secretes numerous proteins upon plant colonization. Some of them, termed effectors, help to evade plant defenses and manipulate cellular processes within the host. The function of many proteins specifically secreted during infection remains elusive. In this study, we biochemically characterized one such protein, UMAG_00027, that is highly expressed during plant infection. We show that UMAG_00027 is a secreted protein with a lectin-like fold and therefore term it Llp1 (lectin-like-protein 1). Llp1 decorated the fungal cell wall of cells grown in axenic culture or proliferating in planta, which is in agreement with its potential sugar-binding ability. We were unable to identify the precise sugar moieties that are bound by Llp1. CRISPR/Cas9-mediated deletion of llp1 reveals that the gene is not essential for fungal virulence. A structural search shows the presence of several other lectin-like proteins in U. maydis that might compensate for the function of Llp1 in ∆llp1 mutants. We therefore speculate that Llp1 is part of a family of lectin-like proteins with redundant functions. Full article

Chaetoceros muelleri and Isochrysis zhanjiangensis, known for their rapid reproduction, small size, and rich nutritional content, are commonly used as feed microalgae in aquaculture. This study aimed to sterilize these microalgal species and assess the effects of antibiotics on their algal cell density. Phycospheric bacteria were isolated and identified using the spread plate method and 16S rDNA sequencing, and antibiotic susceptibility tests were conducted using four antibiotics: ampicillin, streptomycin, kanamycin, and gentamicin sulfate. A sterile system was established for C. muelleri using ampicillin, streptomycin, and gentamicin, and for I. zhanjiangensis using kanamycin, ampicillin, and streptomycin. Based on the results, antibiotics with sterilization effects were selected and added to the algal cultures. Their effects on cell density were evaluated during a six-day co-culture. Ampicillin and streptomycin effectively inhibited bacteria associated with C. muelleri, initially increasing algal cell density but later causing a decline. For I. zhanjiangensis, kanamycin and ampicillin were effective, with kanamycin significantly promoting growth throughout the cycle, achieving a 36.92% higher cell density on day six (p < 0.05). Full article

Contaminations are challenging for monocultures, as they impact the culture conditions and thus influence the growth of the target organism and the overall biomass composition. In phycology, axenic cultures comprising a single living species are commonly strived for both basic research and industrial applications, because contaminants reduce significance for analytic purposes and interfere with the safety and quality of commercial products. We aimed to establish axenic cultures of Limnospira fusiformis, known as the food additive “Spirulina”. Axenicity is strived because it ensures that pathogens or harmful microorganisms are absent and that the harvested biomass is consistent in terms of quality and composition. For the axenic treatment, we applied sterile filtration, ultrasonication, pH treatment, repeated centrifugation, and administration of antibiotics. For testing axenicity, we considered the most common verification method plate tests with Lysogeny Broth (LB) medium, which indicated axenicity after treatments were performed. In addition, we included plate tests with Reasoner’s 2A (R2A) agar and modified Zarrouk+ medium, the latter comparable to the biochemical properties of L. fusiformis’ cultivation medium. In contrast to LB plates, the other media, particularly Zarrouk+, indicated bacterial contamination. We conclude that LB-agar plates are inappropriate for contamination screening of extremophiles. Contamination was also verified by cultivation-independent methods like flow cytometry and 16S rRNA genome amplicon sequencing. We detected taxa of the phyla Proteobacteria, Bacteriodota, Firmicutes and to a lesser extent Verrucomicrobiota. Contaminants are robust taxa, as they survived aggressive treatments. Sequencing data suggest that some of them are promising candidates for in-depth studies to commercially exploit them. Full article