Search Results (108)

The use of methane as a carbon source for producing bacterial single-cell protein (SCP) has been one of the most interesting developments in recent years. Most of these upcoming industries are using a methanotroph, Methylococcus capsulatus Bath, for SCP production using natural gas as the substrate. In the present study, we have explored the possibility of using an indigenously isolated methanotroph from a rice field in India, Methylomagnum ishizawai strain KRF4, for producing SCP from biogas [derived from cow dung]. The process was eco-friendly, required minimal instruments and chemicals, and was carried out under semi-sterile conditions in a tabletop fish tank. As the name suggests, Methylomagnum is a genus of large methanotrophs, and the strain KRF4 had elliptical to rectangular size and dimensions of ~4–5 µm × 1–2 µm. In static cultures, when biogas and air were supplied in the upper part of the growing tank, the culture grew as a thick pellicle/biofilm that could be easily scooped. The grown culture was mostly pure, from the microscopic observations where the large size of the cells, with rectangular-shaped cells and dark granules, could easily help identify any smaller contaminants. Additionally, the large cell size could be advantageous for separating biomass during downstream processing. The amino acid composition of the lyophilized biomass was analyzed using HPLC, and it was seen that the amino acid composition was comparable to commercial fish meal, soymeal, Pruteen, and the methanotroph-derived SCP-UniProtein^®. The only difference was that a slightly lower percentage of lysine, tryptophan, and methionine was observed in Methylomagnum-derived SCP. Methylomagnum ishizawai could be looked at as an alternative for SCP derived from methane or biogas due to the comparable SCP produced, on the qualitative level. Further intensive research is needed to develop a continuous, sustainable, and economical process to maximize biomass production and downstream processing. Full article

In this study, cellulose nanocrystals (CNCs) were successfully isolated through the acid hydrolysis of freeze-dried and oven-dried bacterial nanocellulose (BNC) recovered from the floating pellicle generated during Kombucha tea production. The influence of the BNC drying method and its concentration on the yield and main characteristics of the CNCs obtained were studied. Additionally, selected CNC suspensions at various pH levels were subjected to freeze-drying and oven-drying, followed by an assessment of their dispersibility in water after undergoing different mechanical treatments. Results demonstrate the potential of utilizing byproducts from the expanding Kombucha industry as an alternative cellulose source for CNC production. Furthermore, the drying method applied to the BNC and its initial concentration in the hydrolysis medium were found to significantly impact the properties of the resulting CNCs, which exhibited diverse size distributions and Z-potential values. Finally, the redispersion studies highlighted the beneficial effect of drying CNCs from neutral and alkaline dispersions, as well as the requirement of ultrasound treatments to achieve the proper dispersion of dehydrated CNC powders. Full article

Background: Animal-associated antimicrobial-resistant staphylococci pose a One Health concern, as they can spread into the environment and cause serious infections. Yet, donkeys in Nigeria have been largely overlooked as potential reservoirs of these pathogens. Aim/Objectives: To isolate Staphylococcus aureus from donkeys in Obollo-Afor, southeast Nigeria, assess their antimicrobial resistance profiles, and evaluate their virulence potential. Materials and Methods: Staphylococci were isolated from the nasal swabs of 250 donkeys, using mannitol salt agar, confirmed biochemically, with Staphylococcus aureus identified via a latex agglutination test and mass spectrometry. The resistance profiles of the isolates, including in regard to methicillin, inducible clindamycin, and β-lactamase production, were determined using disc diffusion, while vancomycin resistance was assessed through the use of agar dilution. The virulence factors were evaluated phenotypically. Results: Of the 250 samples, 11 (4.4%) contained S. aureus and 239 (95.6%) grew other Staphylococcus species. The resistance rates of the 11 S. aureus isolates to gentamicin, penicillin, tigecycline, cefoxitin, linezolid, and chloramphenicol were 45.5%, 66.7%, 54.5%, 27.3%, 36.4%, and 18.1%, respectively. The phenotypic methicillin-resistant S. aureus prevalence was 1.2%. Additionally, 23.5% of the S. aureus isolates were multidrug resistant, with a mean antibiotic resistance index of 0.25. All the S. aureus isolates exhibited virulence factors like clumping factor expression, catalase, caseinase, lecithinase, and gelatinase activity, while the occurrence of haemagglutinin, biofilm, pellicle, and hemolysin occurred in 27.3%, 54.5%, 36.4%, 72.2%, respectively. Conclusion: Although a small percentage of donkeys in Nigeria may harbor S. aureus, these animals are potentially spreading antimicrobial resistance, including multidrug and methicillin resistance, to humans and the environment. Full article

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that plays a crucial role in regulating biofilm development, yet the role in Gram-positive bacteria remains elusive. Here, we demonstrated that dispersed cells from biofilms of Bacillus velezensis FZB42 exhibit a unique phenotype and gene expression compared to planktonic cells. Transcriptomic analysis revealed 1327 downregulated and 1298 upregulated genes, among which the c-di-GMP phosphodiesterase coding yuxH gene was remarkably upregulated. Deletion of the yuxH gene led to elevated c-di-GMP levels accompanied by reduced amounts of “actively dispersed cells” from the pellicle and the capacity of motility. Deletion of spoIIIJ, spo0J, and kinA resulted in increased c-di-GMP levels and reduced biofilm dispersal ability. Also, the level of c-di-GMP was increased when adding the cues of inhibition biofilm dispersal such as glucose and calcium ions. Collectively, these present findings suggest the c-di-GMP level is negatively correlated with biofilm dispersal in Bacillus velezensis FZB42, which sheds new light on biofilm regulation in Bacillus velezensis FZB42. Full article

This study investigates the differences in the lithographic impact of particles on the pellicle surface depending on the type of extreme ultraviolet (EUV) mask pattern. Using an EUV ptychography microscope, we analyzed how mask imaging performance is affected by locally obstructed mask diffraction caused by a 10 μm × 10 μm patterned tin particle intentionally fabricated on the pellicle surface. The resulting critical dimension variations were found to be approximately three times greater in line-and-space patterns than in contact hole patterns. Based on these findings, we recommend defining the critical size of particles according to the mask pattern type to optimize lithographic quality. Full article

The pellicle is a coproduct of kombucha beverage production without economic value. This material is based on cellulose produced from bacteria and has better physical properties than cellulose isolated from plants. This review systematically analyzed the research literature about pellicle (KBC—kombucha bacteria cellulose) valorization. In general, KBC has been used in food applications, especially as a packaging ingredient to improve the physical properties of biopolymer-based films, as well as to manufacture packaging materials based on KBC. In addition, some studies have investigated the potential of KBC to encapsulate food ingredients or as a food additive. Furthermore, KBC has been used in nonfood applications with a special interest in the development of materials for textile and medical applications and as a substitute for disposable materials (e.g., spoons). Although the literature shows promising results, it is necessary to increase the production scale of this material, as well as to analyze its economic viability. It is also necessary to establish quality standards and international regulations for KBC with respect to its different food and nonfood applications. Full article

Legumes consumption is still low in Western countries, and their incorporation into bakery products could be a solution. However, a minimally processed legume-based product is still a challenge because of its negative impact on acceptance by consumers. Here, an oven-baked chip recipe, based on lentil flour, was fortified with 5% hazelnut skin (HS), a byproduct of hazelnut industrial processing, to improve the nutritional, antioxidant, and sensory features of this innovative food. Indeed, HS addition allows a nutritional profile improvement, increasing the fibers from 11.71% to 15.63%, and maintaining a high protein content (24.03 g/100 g). Furthermore, HS fortification increased total phenolic compounds and total antioxidant capacity by 1.6- and 2-fold, respectively, compared to the control. Finally, HS significantly improved the overall judgment score by 1.2 points (from 5.6 to 6.8 in control and experimental chips, respectively) halving the pulse-like aroma from 8.6 to 4.3 due to the strong decrease in the dodecane compound and due to HS volatile composition, rich in hexanal. Therefore, HS could be a valuable ingredient in improving the nutritional and functional features of bakery products as well as the sensory profiles of less palatable but healthy legume-based foods. Full article

Plasma-treated water (PTW) recently entered science as a sanitizing agent, which possess the capability for on-demand production. It offers interesting possibilities for sustainable and resource-saving applications in healthcare and food production. The present study monitors the impact of PTW on suspended cells before the biofilm formation of the putrefactive bacterium B. subtilis. Light and electron microscope imaging captures the maturing of growing biofilms within the first 24 h. Microbiological assays (proliferation, LIVE/DEAD, and XTT), which mirror the proliferation of the bacterium, the metabolic activity, and the integrity of the cell membrane, underpinning the metabolic response of still-suspended cells. B. subtilis cells without any treatment build up a resistive biofilm within the 24 h. Cells that remain in the supernatant predominantly appear as monomers or dimers. Treated B. subtilis cells have hampered biofilm formation and were not able to build up a confluent growing biofilm within the first 24 h. Moreover, the microscopic observation of PTW-treated suspension showed cellular aggregates with an unusually high connectivity of the individual cells. The findings suggest this cellular reaction as a counter measure against the adverse impact of PTW treatment. The experiments show the adverse impact of PTW on B. subtilis–biofilm formation and the phenomenological reaction of B. subtilis. Full article

Bacterial cellulose (BC) is a versatile biopolymer with significant potential across biomedical, food, and industrial applications. To remove bacterial contaminants, such as protein and DNA, BC pellicles undergo purification, which traditionally relies on harsh alkali treatments, such as sodium hydroxide or strong surfactants, which present environmental concerns. In response, this study evaluates the efficacy of various non-conventional surfactants—both non-biodegradable and biodegradable—as alternatives for BC purification. Among the surfactants tested, sodium cocoyl isethionate (SCI), a mild anionic and biodegradable surfactant, emerged as particularly effective, achieving an 80.7% reduction in protein content and a 65.19% reduction in double-stranded DNA (dsDNA) content relative to untreated samples. However, these advantages were not without additional challenges, such as the appearance of residual surfactants. Given SCI’s promising performance and biodegradability, it was further examined in two-step treatment protocols; additionally, sodium dodecyl sulfate (SDS) was also examined as a more traditional anionic surfactant as well as NaOH. For the two-step treatment protocol, BC pellicles were treated with one reagent for 3 h, followed by a second reagent for an additional 3 h. Notably, by using NaOH as the final step in the two-step treatment protocol, residual surfactant was not detected in the FTIR analysis. Overall, this work demonstrates that SCI, in addition to subsequent NaOH treatment, can be used as a surfactant-based approach for BC purification, representing a potential environmentally friendly alternative to traditional surfactant-based approaches for BC purification. Full article

Food reformulation is a strategy to make healthier foods by using food waste matrices that are still nutritionally valid. A shortbread cookie was reformulated replacing hazelnut skin (HS) of the Tonda Gentile Romana variety (5% and 10%) to refined flour and proportionally decreasing the butter amount. This resulted in significant, two- and five-fold, increases in the antioxidant capacity compared with the control, in the 5% and 10% fortified recipes, respectively. Among the most important antioxidants, gallic acid, catechin, phloridzin, and protocatechuic acid were found. Moreover, here we found, for the first time, that HS from the Romana variety had a high total fiber content (44.13 g/100 g), most of which was insoluble fiber. Therefore, HS 10% addition to the shortbread cookie recipe caused a significant increase in fiber content, making the experimental cookie earn the nutritional claim of “high fiber content”. Finally, preliminary evidence demonstrated that 10% HS, in comparison to 5%, following in vitro upper gastrointestinal digestion, conferred significant prebiotic activity in an in vitro culture of L. rhamnosus. Therefore, from the perspective of the circular economy, HS could be a valuable ingredient to increase the antioxidant and prebiotic activities of conventional foods. Full article

The relationship between Kombucha fermentation process parameters and microbial composition was investigated. Green tea Kombucha fermented slower, producing a higher sugar/acid ratio, residual sucrose, and sweeter taste compared to black tea Kombucha. No known probiotic bacteria were identified in Kombucha since Zygosaccharomyces, Brettanomyces, Komagataeibacter, and Gluconacetobacter were the dominant genera observed based on 16s rRNA sequencing analysis. Green tea epigallocatechin and epigallocatechin gallate inhibited bacterial growth in black tea Kombucha broth in a concentration-dependent manner. Green tea Kombucha produced from a 10-day inoculum had a higher sugar/acid ratio, residual sucrose, acetic acid, gluconic acid, and a pleasant flavor compared to a 15-day inoculum. In blended green and black tea Kombucha, a solid bacterial pellicle 9-day inoculum increased the amounts of glucose, fructose, acetic acid, gluconic acid, and ethanol, yet there was a lower sugar/acid ratio and residual sucrose, but no change in the catechin profile, compared to a completely liquid inoculum. Increasing the surface area/volume ratio to 0.16 during Kombucha production increased levels of glucose, fructose, and gluconic acid, while acetic acid and ethanol levels remained the same by favoring aerobic vs. anaerobic fermentation. These Kombucha fermentation process parameter results will help producers devise strategies for better quality control. Full article

The biosynthesis of bacterial cellulose (BC) is significantly influenced by the type of carbon source available in the growth medium, which in turn dictates the material’s final properties. This study systematically investigates the effects of five carbon sources—raffinose (C₁₈H₃₂O₁₆), sucrose (C₁₂H₂₂O₁₁), glucose (C₆H₁₂O₆), arabinose (C₅H₁₀O₅), and glycerol (C₃H₈O₃)—on BC production by Komagataeibacter hansenii. The varying molecular weights and structural characteristics of these carbon sources provide a framework for examining their influence on BC yield, fiber morphology, and network properties. BC production was monitored through daily measurements of optical density and pH levels in the fermentation media from day 1 to day 14, providing valuable insights into bacterial growth kinetics and cellulose synthesis rates. Scanning electron microscopy (SEM) was used to elucidate fibril diameter and pore size distribution. Wide-angle X-ray scattering (WAXS) provided a detailed assessment of crystallinity. Selected BC pellicles were further processed via freeze-drying to produce a foam-like material that maximally preserves the natural three-dimensional structure of BC, facilitating the incorporation and release of lidocaine hydrochloride (5%), a widely used local anesthetic. The lidocaine-loaded BC foams exhibited a sustained and controlled release profile over 14 days in simulated body fluid, highlighting the importance of the role of carbon source selection in shaping the BC network architecture and its impact on drug release profile. These results highlight the versatility and sustainability of BC as a platform for wound healing and drug delivery applications. The tunable properties of BC networks provide opportunities for optimizing therapeutic delivery and improving wound care outcomes, positioning BC as an effective material for enhanced wound management strategies. Full article

The natural remineralization of enamel is of major importance for oral health. In principle, early erosions (demineralization) induced by acidic beverages and foods as well as initial caries lesions can be covered and remineralized by the deposition of calcium phosphate, i.e., tooth mineral. This remineralization effect is characterized by the presence of calcium and phosphate ions in saliva that form hydroxyapatite on the enamel surface. Although it is apparently a simple crystallization, it turns out that remineralization under in vivo conditions is actually a very complex process. Calcium phosphate can form a number of solid phases of which hydroxyapatite is only one. Precipitation involves the formation of metastable phases like amorphous calcium phosphate that convert into biological apatite in a number of steps. Nanoscopic clusters of calcium phosphate that can attach on the enamel surface are also present in saliva. Thus, remineralization under strictly controlled in vitro conditions (e.g., pH, ion concentrations, no additives) is already complex, but it becomes even more complicated under the actual conditions in the oral cavity. Here, biomolecules are present in saliva, which interact with the forming calcium phosphate mineral. For instance, there are salivary proteins which have the function of inhibiting crystallization to avoid overshooting remineralization. Finally, the presence of bacteria and an extracellular matrix in plaque and the presence of proteins in the pellicle have strong influences on the precipitation on the enamel surface. The current knowledge on the remineralization of the enamel is reviewed from a chemical perspective with a special focus on the underlying crystallization phenomena and the effects of biological compounds that are present in saliva, pellicle, and plaque. Basically, the remineralization of enamel follows the same principles as calculus formation. Notably, both processes are far too complex to be understood on a microscopic basis under in vivo conditions, given the complicated process of mineral formation in the presence of a plethora of foreign ions and biomolecules. Full article

Biofilm formation of clinical isolates of Aeromonas salmonicida subspecies salmonicida was compared using scarce (minimal M9 and ABTG w/o amino acids) and enriched nutrient media (Tryptone Soya broth) at 8 °C, 16 °C, and 25 °C using direct enumeration of viable cells in biofilm (log CFU), crystal violet staining (ODc) of the formed biofilm biomass, and liquid–air border biofilm formation (pellicle test). Whole-genome sequencing (WGS) was performed with the usage of an Oxford nanopore system by Genomics and Transcriptomics Labor Düsseldorf (Heinrich-Heine-Universität Düsseldorf, Germany). A bioinformatic analysis was performed with the usage of Geneious Prime^® 2023.0.4 (Biomatters, Inc., Boston, MA, USA). All data were analyzed using Statistica software version 13.0, and changes in biofilm production in correlation to changes in the type of nutritional medium and temperature were compared between groups using a one-way ANOVA analysis and Tukey’s test. All isolates formed biofilms in minimal M9 at 8 °C and 16 °C, and nine isolates failed to form biofilms in minimal M9 at 25 °C. In an ABTG medium, all isolates produced biofilms at 8 °C; however, three isolates at 16 °C and seven isolates at 25 °C failed to form any biofilms. Significant biofilm formation was observed in TSB at all temperatures. Some strains that formed a good biofilm in solid–liquid interface did not have the ability to form a pellicle (liquid–air border biofilm), and vice versa. In all cases of nutritional medium and temperature changes, there were statistically significant differences in the intensity of biofilm production, especially in the detected number of viable cells inside biofilms (log CFU, p < 0.005). Multiple biofilm-production genes, including polar flagella (filM) LuxR family (transcriptional regulators) and VapA family of histidine-kinase-associated genes, were sequenced from all studied isolates. Genetic differences based on geographical origin were not observed among the isolates. Significant variations in isolates’ ability to form biofilms were observed, possibly due to epigenetic factors. The optimal temperature for biofilm formation of A. salmonicida subspecies salmonicida in scarce media was 8 °C, and the majority of isolates were not capable of biofilm formation at 25 °C without enriched nutrient media. Full article

The aquatic γ-proteobacterium Shewanella oneidensis is able to form two types of biofilms: a floating biofilm at the air–liquid interface (pellicle) and a solid surface-associated biofilm (SSA-biofilm). S. oneidensis possesses the Bpf system, which is orthologous to the Lap system first described in Pseudomonas fluorescens. In the Lap systems, the retention of a large adhesin (LapA) at the cell surface is controlled by LapD, a c-di-GMP effector protein, and LapG, a periplasmic protease targeting LapA. Here, we showed that the Bpf system is mandatory for pellicle biogenesis, but not for SSA-biofilm formation, indicating that the role of Bpf is somewhat different from that of Lap. The BpfD protein was then proved to bind c-di-GMP via its degenerated EAL domain, thus acting as a c-di-GMP effector protein like its counterpart LapD. In accordance with its key role in pellicle formation, BpfD was found to interact with two diguanylate cyclases, PdgA and PdgB, previously identified as involved in pellicle formation. Finally, BpfD was shown to interact with CheY3, the response regulator controlling both chemotaxis and biofilm formation. Altogether, these results indicate that biofilm formation in S. oneidensis is under the control of a large c-di-GMP network. Full article