Search Results (87)

Diploid Vaccinium fuscatum is a wild blueberry species with a low chilling requirement, an evergreen growth habit, and soil adaptability to southeast US growing regions. Regardless of its potential to improve the abiotic and biotic resilience of cultivated blueberries, this species has rarely been used for blueberry breeding. One hurdle is the ploidy barrier between diploid V. fuscatum and tetraploid cultivated highbush blueberries. To overcome the ploidy barrier, vegetative shoots micro-propagated from one genotype of V. fuscatum, selected because it grew vigorously in vitro and two southern highbush cultivars, ‘Emerald’ and ‘Rebel,’ were treated with colchicine. While shoot regeneration was severely repressed in ‘Emerald’ and ‘Rebel,’ shoot production from the V. fuscatum clone was not compromised at either 500 µM or 5000 µM colchicine concentrations. Due to the high number of shoots produced in vitro via the V. fuscatum clone shoots of this clone that had an enlarged stem diameter in vitro were subjected to flow cytometer analysis to screen for induced polyploidy. Sixteen synthetic tetraploid V. fuscatum, one synthetic octoploid ‘Emerald,’ and three synthetic octoploid ‘Rebel’ were identified. Growth rates of the polyploid-induced mutants were reduced compared to their respective wildtype controls. The leaf width and length of synthetic tetraploid V. fuscatum and synthetic octoploid ‘Emerald’ was increased compared to the wildtypes, whereas the leaf width and length of synthetic octoploid ‘Rebel’ were reduced compared to the wildtype controls. Significant increases in stem thickness and stomata guard cell length were found in the polyploidy-induced mutant lines compared to the wildtypes. In the meantime, stomata density was reduced in the mutant lines. These morphological changes may improve drought tolerance and photosynthesis in these mutant lines. Synthetic tetraploid V. fuscatum can be used for interspecific hybridization with highbush blueberries to expand the genetic base of cultivated blueberries. Full article

Bioreactors enable scalable cell cultivation by providing controlled environments for temperature, oxygen, and nutrient regulation, maintaining viability and enhancing expansion efficiency. Automated systems improve reproducibility and minimize contamination risks, making them ideal for high-density cultures. While fed-batch bioreactors dominate biologics production, continuous systems like perfusion cultures offer superior resource efficiency and productivity. The Quantum hollow-fiber perfusion bioreactor supports cell expansion via semi-permeable capillary membranes and a closed modular design, allowing continuous media exchange while retaining key molecules. We developed a multiple-harvest protocol for suspension cells in the Quantum system, yielding 2.5 × 10¹⁰ MEL-745A cells within 29 days, with peak densities of 4 × 10⁷ cells/mL—a 15-fold increase over static cultures. Viability averaged 91.3%, with biweekly harvests yielding 3.1 × 10⁹ viable cells per harvest. Continuous media exchange required more basal media to maintain glucose and lactate levels but meaningfully less growth supplement than the 2D culture. Stable transgene expression suggested phenotypic stability. Automated processing reduced hands-on time by one-third, achieving target cell numbers 12 days earlier than 2D culture. Despite higher media use, total costs for the automated were lower compared to the manual process. Quantum enables high-density suspension cell expansion with cost advantages over conventional methods. Full article

This study explores the feasibility of producing biohydrogen from winery wastewater using a dual-chamber microbial electrolysis cell (MEC). A mixed microbial consortium pre-adapted to heavy-metal environments and enriched with Geobacter sulfurreducens was anaerobically cultivated from diverse waste streams. Over 5000 h of development, the MEC system was progressively adapted to winery wastewater, enabling long-term electrochemical stability and high organic matter degradation. Upon winery wastewater addition (5% v/v), the system achieved a sustained hydrogen production rate of (0.7 ± 0.3) L H₂ L⁻¹ d⁻¹, with an average current density of (60 ± 4) A m⁻³, and COD removal efficiency exceeding 55%, highlighting the system’s resilience despite the presence of inhibitory compounds. Coulombic efficiency and cathodic hydrogen recovery reached (75 ± 4)% and (87 ± 5)%, respectively. Electrochemical impedance spectroscopy provided mechanistic insight into charge transfer and biofilm development, correlating resistive parameters with biological adaptation. These findings demonstrate the potential of MECs to simultaneously treat agro-industrial wastewaters and recover energy in the form of hydrogen, supporting circular resource management strategies. Full article

Mandelic acid (MA) is a valuable α-hydroxy acid with applications in pharmaceuticals, cosmetics, and fine chemicals. While chemical synthesis is well established, concerns over toxicity and sustainability have driven interest in microbial production. Here, we engineered Escherichia coli for de novo MA biosynthesis by integrating enzyme screening, metabolic flux optimization, and pathway regulation. We first screened and identified an efficient hydroxymandelate synthase (HMAS) homolog from Actinosynnema mirum for MA synthesis, and subsequently enhanced the shikimate pathway along with the supply of the precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). Additionally, CRISPR interference (CRISPRi) was employed to repress competing pathways and redirect flux toward MA production. High-cell-density cultivation (HCDC) in a 5 L bioreactor demonstrated the strain’s industrial potential, achieving an MA titer of 9.58 g/L, the highest reported for microbial production. This study provides a systematic metabolic engineering approach for efficient MA biosynthesis from glucose, offering a foundation for sustainable large-scale production, demonstrating not only genetic-level optimizations, but also effective process scaling through high-cell-density cultivation, highlighting the power of pathway engineering in microbial cell factories. Full article

A copolymer of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a practical biodegradable plastic, and at present, the copolymer is produced at commercial scale via heterotrophic cultivation of an engineered strain of a facultative hydrogen-oxidizing bacterium, Cupriavidus necator, using vegetable oil as the carbon source. In our previous report, we investigated PHBHHx production from CO₂ via pH-stat jar cultivation of the newly created recombinants of C. necator under autotropic conditions, feeding the inorganic substrate gas mixture (H₂/O₂/CO₂ = 80:10:10 v/v%) into a recycled-gas closed-circuit (RGCC) culture system. The dry cell weight (DCW) and PHBHHx concentration with the best strain MF01/pBPP-ccr_MeJ_Ac-emd increased to 59.62 ± 3.18 g·L⁻¹ and 49.31 ± 3.14 g·L⁻¹, respectively, after 216 h. In this study, we investigated the high-concentration production of PHBHHx with a shorter cultivation time by using a jar fermenter equipped with a basket-shaped agitator to enhance oxygen transfer in the culture medium and by continuously supplying the gases with higher O₂ concentrations to maintain the gas composition within the reservoir at a constant ratio. The concentrations of ammonium and phosphate in the culture medium were maintained at low levels. As a result, the DCW and PHBHHx concentrations increased to 109.5 ± 0.30 g·L⁻¹ and 85.2 ± 0.62 g·L⁻¹ after 148 h, respectively. The 3HHx composition was 10.1 ± 0.693 mol%, which is suitable for practical applications. Full article

Potato early dying (PED) disease complex is often called the Verticillium wilt of potato and is considered one of the most economically devastating diseases of potato worldwide. The severity of the disease greatly increases with the association of the soil-borne pathogens Verticillium dahliae and V. albo-atrum and the root lesion nematode (Pratylenchus sp.). Recently, an increase in wilt disease symptoms and a sharp decline in marketable tuber yield were observed in New Brunswick (NB), Canada. A survey of 71 fields, along with eight fumigated and eight non-fumigated fields, was carried out to determine and quantify nematodes and Verticillium in the soil. Techniques used included plate counts for Verticillium (CFU/g soil), real-time qPCR (RT-qPCR) for V. dahliae (cell/g soil), and nematode identification and counts (# of nematodes/kg of soil). The survey results of the 71 fields revealed that 55 fields had Verticillium sp. ranging from 2 to 66 CFU/g of soil by the plate method, and 68 fields had high V. dahliae ranging from 261 to 27,471 cell/g of soil by RT-qPCR method. All fields had high numbers of root lesion nematodes ranging from 560 to 14,240 nematodes/kg of soil. There was an uneven distribution of PED incidence in potato fields at various locations of NB. Fumigation with Chloropicrin significantly reduced the numbers of root lesion nematodes by 34.1–99.0%, Verticillium sp. CFU/g of soil by 50–100%, and V. dahliae cell/g soil by 38–91% in the eight fumigated fields. The management of the PED complex with various disease management products under field conditions was also studied in a field plot trial setup. The nematicide Velum applied in-furrow at the recommended label rate decreased the numbers of root lesion nematodes by up to 66% compared to other products. The combination of both Velum + Aprovia and the application of ammonium-lignosulfonate significantly reduced V. dahliae by 190.55% and 274.24%, respectively, compared to other products. The fungicide Aprovia applied in-furrow at the recommended rate for the management of Verticillium wilt significantly reduced Verticillium sp. CFU/g of soil in treated soil by 73.3% compared to Velum, Mustgrow, Senator PSPT, Vapam, ammonium-lignosulfonate, Nimitz, and the untreated control. Disease management products increased potato marketable yield by 27.38–97.74%. The results of this study suggest that the root lesion nematode and V. dahliae have a ubiquitous distribution in the fields cultivated with potatoes in NB. The co-infection of potato by both V. dahliae and the root lesion nematode can greatly increase the severity of PED. Fumigation with Chloropicrin significantly reduced the levels of root lesion nematodes and Verticillium in all fumigated fields. Management practices of PED using the fungicide Aprovia, the nematicide Velum, and a combination of both Velum + Aprovia had the greatest effect in reducing the population density of the root lesion nematode and Verticillium dahliae in soils of commercial potato fields in New Brunswick. Full article

Sediment improvers are important mediators of aquatic animals’ growth performance and the surrounding environmental quality. However, the physiological responses of crayfish (Procambarus clarkii) to different sediment improvers remain unclear. Here, we cultivated crayfish using two chemical (potassium monopersulfate and potassium ferrate) and two biological (purple nonsulfur photosynthetic bacteria and Bacillus subtilis) sediment improvers at low and high concentrations. After 42 days, we found that the addition of chemical sediment improvers was more effective in improving water quality than biological sediment improvers (e.g., more stable pH and lower nutrient concentrations). By contrast, the application of biological sediment improvers resulted in considerably enhanced final weight, weight gains, and survival rates. In all low-concentration groups, the activity of immune-related enzymes (e.g., superoxide dismutase and glutathione peroxidase) in the hemolymph and hepatopancreas considerably increased, whereas the malondialdehyde activity and mRNA expression of AMP genes (PcALF and PcCru) considerably decreased. Crayfish exposed to low concentrations of sediment improvers exhibited enhanced intestinal and hepatopancreatic integrity, with a thickened mucosal layer and increased density of epithelial cell granules. Additionally, the composition of the gut microbiota varied after the addition of different sediment improvers. In summary, our research indicated that different types of sediment improvers not only improved the farming environment but also had varying effects on crayfish. Therefore, an appropriate sediment improver based on specific aquaculture conditions is needed. Full article

(1) Autologous chondrocyte implantation (ACI) is a prominent method for treating cartilage damage, but periosteal patches can cause chondrocyte leakage. This study evaluates the potential of a decellularized membrane derived from the cell-produced extracellular matrix of 1-day-old porcine cartilage (pcECM-DM) to act as a substitute for periosteal patches. (2) The interaction between young rabbit chondrocyte cells and pcECM-DM was assessed through cytotoxicity, differentiation, cell viability, cell migration, and adhesive ability. Rabbit chondrocyte cells, cultivated until passage two, were seeded onto a 6 mm diameter membrane. Assessments included DAPI-PKH26 staining, histological staining, live/dead assay, WST-1 assay, and proteomics analysis. (3) Results: DAPI-PKH26 staining showed successful adhesion and the uniform distribution of cells on the membrane. Safranin-O and H&E staining confirmed that the membrane supports chondrocyte adhesion and extracellular matrix production with high cell density and typical chondrocyte morphology. The live/dead assay demonstrated a high proportion of viable cells at 24 and 48 h, with increased cell proliferation over time. The WST-1 assay showed a significant increase in OD450 values, confirming cell proliferation and biocompatibility. Proteomic analysis revealed the significant enrichment of genes associated with extracellular matrix organization, cell adhesion, and cartilage development. (4) Conclusions: This novel biomaterial holds the potential to enhance cartilage regeneration and offer a viable alternative to periosteal patches. Full article

Microalgae microbial fuel cells (pMFCs) are distinguished by their ability to combine waste utilization with the simultaneous recovery of energy and valuable materials. The generation of high current density is linked to the efficient electron transfer to the anode via the anodic biofilm and the high photosynthetic activity of the microalgae cultivated in the cathode chamber. This review explores the impact of wastewater type on energy production and wastewater treatment. Additionally, it discusses the challenges related to microalgae growth in the cathode chamber, the necessity of aeration, and the sequestration of carbon dioxide from the anode chamber. The efficiency of microalgae in utilizing nutrients from various types of wastewater is also presented. In conclusion, the comparison between wastewater treatment and energy balance in pMFCs and conventional wastewater treatment plants is provided. On average, MFCs consume only 0.024 kW or 0.076 kWh/kg COD, which is approximately ten times less than the energy used by activated sludge bioprocesses. This demonstrates that MFCs offer highly efficient energy consumption compared to traditional wastewater treatment systems while simultaneously recovering energy through exoelectrogenic, bioelectrochemical processes. Full article

Background: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine α_S1-casein protein. Methods: A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine α_S1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α_S1-casein production. After increasing the molecular abundance of the recombinant α_S1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α_S1-casein. Results: Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant α_S1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg_casein/g_CDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α_S1-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of B. subtilis for the recombinant production of bovine α_S1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins. Full article

Large quantities of artificial single-stranded DNA (ssDNA) with user-defined sequences are increasingly required to exploit the potential of DNA nanotechnology. Cross-contamination-free ssDNA production can be achieved using Escherichia coli with an optimized helper plasmid in high-cell-density cultivation via the secretion of phagemid particles containing ssDNA with user-defined sequences. In our study, we aimed to reduce the number of phagemid particles for the initiation of ssDNA production. We tested different infection densities, ranging from a multiplicity of infection (MOI) of 10⁻⁶–10⁻² tfu cfu⁻¹ at the start of the initial batch phase in a 2.5 L stirred tank bioreactor. A MOI of 10⁻³ tfu cfu⁻¹ was the best compromise between process time and ssDNA concentration. Early initiation of ssDNA production with low MOI reduced the number of phagemid particles by a factor of 250,000. The early infection strategy was successfully scaled up to the 25 L scale, resulting in ssDNA concentrations of >100 mg L⁻¹ within a process time of one day. Transferring the infection strategy to a 1000 L scale gained 65 mg L⁻¹ ssDNA because of incomplete initial infection. The versatility of the early infection strategy was further proven with a second prolonged, user-defined ssDNA sequence. Full article

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process. Full article

This study explored the sustainable valorization of fruit waste extracts from sugarcane bagasse (SB), banana peel (BP), and watermelon rind (WR) for Euglena gracilis biomass and β-1,3-glucan production. The extracts were prepared using water extraction (WE), high-temperature and pressure treatment (HTP), and dilute sulfuric acid treatment (DSA). The DSA-treated extracts consistently yielded the best results. E. gracilis cultured in SB-DSA showed the highest cell density with a 2.08-fold increase compared to the commercial HUT medium, followed by BP-DSA (1.35-fold) and WR-DSA (1.70-fold). Photosynthetic pigment production increased significantly, with chlorophyll a yield being highest in SB-DSA (1.90-fold increase). The chlorophyll a/b ratio and total carotenoid content also improved, indicating enhanced light-harvesting capacity and photoprotection. Photosynthetic efficiency, measured by chlorophyll fluorescence, notably improved. The maximum quantum yield of PSII (Fv/Fm) increased by up to 25.88% in SB-DSA, suggesting reduced stress and improved overall photosynthetic health. The potential photochemical efficiency (Fv/F0) showed even greater improvements: up to 40.53% in SB-DSA. Cell morphology analysis revealed larger cell aspect ratios, implying a more active cellular physiological state. β-1,3-glucan yield also increased by 23.99%, 12.92%, and 23.38% in SB-DSA, BP-DSA, and WR-DSA, respectively. This study demonstrates the potential of pretreated fruit waste as a cost-effective and sustainable medium for E. gracilis cultivation, offering the dual benefits of waste valorization and high-value compound production. These findings contribute to the development of more efficient biorefinery processes and align with the circular economy principles in food biotechnology. Full article

Lactobacillus plantarum exhibits a wide range of beneficial physiological functions, including maintaining intestinal microbiota balance, reducing serum cholesterol, and promoting digestive health. According to the specific nutrient requirements of Lactobacillus plantarum P6, we investigated the effects of various carbon sources, nitrogen sources, trace elements, growth-promoting substances, as well as the initial pH and inoculum size on the growth of Lactobacillus plantarum P6 under fermentation conditions. The optimal growth conditions for Lactobacillus plantarum P6 were identified to facilitate high-density fermentation in small-scale fermenter production, achieving a cell concentration of 1.03 × 10¹¹ CFU/mL. This resulted in a 2.5-fold increase in bacterial wet weight, and fermentation time was reduced to 12 h when utilizing a specific medium enriched with 0.2% sodium alginate. It is hypothesized that sodium alginate forms a protective film around the bacterial cells, promoting cell aggregation and enhancing self-coalescence, potentially triggering a bacterial community effect. These results provide a basis for the industrial-scale high-density cultivation of Lactobacillus plantarum, offering potential for enhanced biotechnological applications. Full article

The evaluation of the analytical capabilities of a novel disposable flow cell for spectroscopic bioprocess monitoring is presented. The flow cell is presterilized and can be connected to any kind of bioreactor by weldable tube connections. It is clamped into a reusable holder, which is equipped with SMA-terminated optical fibers or an integrated light source and detection unit. This modular construction enables spectroscopic techniques like UV-Vis spectroscopy or turbidity measurements by scattered light for modern disposable bioreactors. A NIR scattering module was used for biomass monitoring in different cultivations. A high-cell-density fed-batch cultivation with Komagataella phaffii and a continuous perfusion cultivation with a CHO DG44 cell line were conducted. A high correlation between the sensor signal and biomass or viable cell count was observed. Furthermore, the sensor shows high sensitivity during low turbidity states, as well as a high dynamic range to monitor high turbidity values without saturation effects. In addition to upstream processing, the sensor system was used to monitor the purification process of a monoclonal antibody. The absorption module enables simple and cost-efficient monitoring of downstream processing and quality control measurements. Recorded absorption spectra can be used for antibody aggregate detection, due to an increase in overall optical density. Full article