Search Results (106)

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalizations in future sustainable economies and can be efficiently immobilized on the cell surface of their heterologous production yeast. This immobilization has versatile uses, ranging from the mL to m³ scale; but the production of the yeast surface displayed UPOs, and their handling has yet to be optimized to advance sustainable industrial processes in light of the UN’s sustainable development goals. Here, we present optimized production protocols for surface-displayed UPOs for shaken and stirred systems in different scales and describe suitable storage conditions and a sterilization method. We utilized one-factor-at-a-time and design of experiments approaches. We were able to streamline published protocols for shaken flask cultivations to achieve a 60% increase in volumetric activity, using reduced amounts of media. We also show at least a doubling of final activity for bioreactor cultivations by utilizing a different medium than the industry standard. Finally, we present a novel, robust protocol for parallelized methanol-induced enzyme production in Komagataella phaffii in a BioLector XT^® reactor. Enzyme activity did not decrease and even increased by our recommended sterilization method and during storage over 87 days. This study aims to advance the yeast surface display immobilization method by providing methods for efficient production, storage and utilization of this promising biocatalyst. Full article

The sustainable production of healthy structured lipids (SLs) using oils extracted from agro-industry by-products or non-conventional lipid sources is of utmost importance in the framework of a circular bioeconomy, toward a zero-waste goal. In this study, low-calorie triacylglycerols (TAGs) containing a long-chain (L) fatty acid (FA) at position sn-2 and medium-chain (M) FAs at positions sn-1,3 (MLM type SL) were obtained from virgin cold-pressed milk thistle (51.55% linoleic acid; C18:2), grapeseed (66.62% C18:2), and apricot kernel (68.61% oleic acid; C18:1) oils. Lipase-catalyzed acidolysis with capric acid (C10:0) or interesterification with ethyl caprate (C10 Ethyl) in solvent-free media were performed. In batch reactions, immobilized Rhizomucor miehei lipase (Lipozyme RM) was used as a biocatalyst. For all tested oils, new TAG (SL) yields, varying from 61 to 63%, were obtained after 6 h of interesterification. Maximum new TAG yields were reached after 6, 24, and 30 h of acidolysis with grapeseed (64.7%), milk thistle (56.1%), or apricot kernel (69.7%) oils, respectively. Continuous acidolysis and interesterification of grapeseed oil were implemented in a packed-bed bioreactor, catalyzed by immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). Throughout 150 h of continuous operation, no lipase deactivation was observed, with average SL yields of 79.2% ± 4.1 by interesterification and 61.5% ± 5.91 by acidolysis. Full article

The valorization of cheese whey, a rich by-product of the dairy industry that is rich in lactose (approx. 70%), proteins (14%), and minerals (9%), represents a promising approach for microbial fermentation. With global whey production exceeding 200 million tons annually, the high biochemical oxygen demand underlines the important need for sustainable processing alternatives. This review explores the biotechnological potential of whey as a fermentation medium by examining its chemical composition, microbial interactions, and ability to support the synthesis of valuable metabolites. Functional microorganisms such as lactic acid bacteria (Lactobacillus helveticus, L. acidophilus), yeasts (Kluyveromyces marxianus), actinobacteria, and filamentous fungi (Aspergillus oryzae) have demonstrated the ability to efficiently convert whey into a wide range of bioactive compounds, including organic acids, exopolysaccharides (EPSs), bacteriocins, enzymes, and peptides. To enhance microbial growth and metabolite production, whey fermentation can be carried out using various techniques, including batch, fed-batch, continuous and immobilized cell fermentation, and membrane bioreactors. These bioprocessing methods improve substrate utilization and metabolite yields, contributing to the efficient utilization of whey. These bioactive compounds have diverse applications in food, pharmaceuticals, agriculture, and biofuels and strengthen the role of whey as a sustainable biotechnological resource. Patents and clinical studies confirm the diverse bioactivities of whey-derived metabolites and their industrial potential. Whey peptides provide antihypertensive, antioxidant, immunomodulatory, and antimicrobial benefits, while bacteriocins and EPSs act as natural preservatives in foods and pharmaceuticals. Also, organic acids such as lactic acid and propionic acid act as biopreservatives that improve food safety and provide health-promoting formulations. These results emphasize whey’s significant industrial relevance as a sustainable, cost-efficient substrate for the production of high-quality bioactive compounds in the food, pharmaceutical, agricultural, and bioenergy sectors. Full article

A mathematical model was developed for the dynamic and static simulation of a continuous ethanol production process in a tower bioreactor packed with yeast cells immobilized in citrus pectin gel. To avoid accumulation of CO₂ gas during the bioprocess, a vertical fixed bed bioreactor with a working volume of 0.245 L, divided into four stages and equipped with external gas–liquid separators was used. The performance of the bioreactor was evaluated through continuous fermentations using feed medium (sugarcane juice) with substrate concentrations of 161.4 and 312.5 g/L, temperature of 30 °C, pH 4.0 and hydraulic residence times of 5 and 6 h. The developed mathematical model takes into account mass flow by convection and dispersion axial, external and internal mass transfer to/within particle, Contois kinetics for cell growth with inhibition terms, cell death, and substrate consumption for cell maintenance. The partial differential equations regarding cell, substrate and product mass balances in the solid and fluid phase were solved by numerical methods. The calculated profiles of state variables in the fluid phase agreed satisfactorily with the experimental data. The diffusional resistances within particles concerning the substrate consumption rate were not significant, resulting in calculated values of the effectiveness factor close to one. Full article

Plumbagin is an important naphthoquinone with potent anticancer properties besides multitudinous uses in healthcare. It is produced in a limited number of species and families but mostly in the roots of Plumbaginaceae family members. The biosynthetic pathway and the genes that regulate plumbagin synthesis are not completely known, but details of these are being revealed. Several species, including Plumbago, Drosera, and others, are being uprooted for the extraction of plumbagin by pharmaceutical industries, leading to the destruction of natural habitats. The pharmaceutical industry is therefore facing an acute shortage of plant material. This necessitates enhancing the accumulation of plumbagin using suspensions and hairy roots to meet market demands. Many factors, such as the aggregate size of the inoculum, stability of the culture, and the sequential effects of elicitors, immobilization, and permeabilization, have been demonstrated to act synergistically and markedly augment plumbagin accumulation. Hairy root cultures can be used for the large-scale production, growth, and plumbagin accumulation, and the exploration of their efficacy is now imperative. The secretion of compounds into the spent medium and their in situ adsorption via resin has remarkable potential, but this has not been thoroughly exploited. Improvements in the quality of biomass, selection of cell lines, and production of plumbagin in bioreactors have thus far been sporadic, and these parameters need to be further exploited. In this review, we report the advances made relating to the importance of stable cell line selection for the accumulation of compounds in long-term cultures, hairy root cultures for the accumulation of plumbagin, and its semicontinuous production via total cell recycling in different types of bioreactors. Such advances might pave the way for industrial exploitation. The steps in the biosynthetic pathway that are currently understood might also aid us in isolating the relevant genes in order to examine the effects of their overexpression or heterologous downregulation or to edit the genome using CRISPR-Cas9 technology in order to enhance the accumulation of plumbagin. Its potential as an anticancer molecule and its mode of action have been amply demonstrated, but plumbagin has not been exploited in clinics due to its insolubility in water and its highly lipophilic nature. Plumbagin-loaded nanoemulsions, plumbagin–silver, or albumin nanoparticle formulations can overcome these problems relating to its solubility and are currently being tried to improve its bioavailability and antiproliferative activities, as discussed in the current paper. Full article

The extensive use of pharmaceuticals in human and veterinary medicine has led to their persistent environmental release, posing ecological and public health risks. Major sources include manufacturing effluents, excretion, aquaculture, and improper disposal, contributing to bioaccumulation and ecotoxicity. Mycoremediation is the fungal-mediated biodegradation of pharmaceuticals, offers a promising and sustainable approach to mitigate pharmaceutical pollution. Studies have reported that certain fungal species, including Trametes versicolor and Pleurotus ostreatus, can degrade up to 90% of pharmaceutical contaminants, such as diclofenac, carbamazepine, and ibuprofen, within days to weeks, depending on environmental conditions. Fungi produce a range of extracellular enzymes, such as laccases and peroxidases, alongside intracellular enzymes like cytochrome P450 monooxygenases, which catalyze the transformation of complex pharmaceutical compounds. These enzymes play an essential role in modifying, detoxifying, and mineralizing xenobiotics, thereby reducing their environmental persistence and toxicity. The effectiveness of fungal biotransformation is influenced by factors such as substrate specificity, enzyme stability, and environmental conditions. Optimal degradation typically occurs at pH 4.5–6.0 and temperatures of 20–30 °C. Recent advancements in enzyme engineering, immobilization techniques, and bioreactor design have improved catalytic efficiency and process feasibility. However, scaling up fungal-based remediation systems for large-scale applications remains a challenge. Addressing these limitations with synthetic biology, metabolic engineering, and other biotechnological innovations could further enhance the enzymatic degradation of pharmaceuticals. This review highlights the enzymatic innovations, applications, and challenges of pharmaceutical mycoremediation, emphasizing the potential of fungi as a transformative solution for sustainable pharmaceutical waste management. Full article

In ethanol production processes, inhibitors are formed as by-products depending on the raw materials and pretreatments. Inhibitors negatively affect both ethanol yield and biomass growth. This study aimed to examine the influence of inhibitors, including acetic acid (AA), formic acid (FA), and phenol, on ethanol production from the glucose-based medium using immobilized Saccharomyces cerevisiae in a bioreactor. The results showed that the highest ethanol yields and productions were determined as 45.64% and 38.10 g/L, 44.8% and 36.67 g/L, and 44.46% and 39.07 g/L, by the addition of 2.5 g/L AA, 0.5 g/L FA, and 0.5 g/L phenol into the fermentation medium, respectively. Regarding mathematical modeling, the models MGM (AA) and Huang (FA-phenol) were the best models to predict experimental ethanol production. It was determined that the values forecasted with the models MMF (AA-FA) and Weibull (phenol) agreed with the actual biomass growth. Additionally, to forecast the observed values of the substrate consumption, the most suitable model was Weibull (AA-FA-phenol). Consequently, the immobilized-cell ethanol fermentations with inhibitors were successfully performed, and their limit values were determined. Full article

Transient gene expression (TGE) is commonly employed for protein production, but its reliance on plasmid transfection makes it challenging to scale up. In this paper, an alternative TGE method is presented, utilizing pseudoinfectious alphavirus as an expression vector. Pseudoinfectious viruses (PIV) and a replicable helper construct were derived from the genome of the Venezuelan equine encephalitis virus. The PIV carries a mutant capsid protein that prevents packaging into infectious particles, while the replicable helper encodes a wild-type capsid protein but lacks other viral structural proteins. Although PIV and the helper cannot independently spread infection, their combination results in increased titers in cell cultures, enabling easier scale-up of producing cultures. The PIV-driven production of a model protein outperforms that of alphavirus replicon vectors or simple plasmid vectors. Another described feature of the expression system is the modification to immobilized metal affinity chromatography (IMAC), allowing purification of His-tagged recombinant proteins from a conditioned medium in the presence of substances that can strip metal from the IMAC columns. The PIV-based expression system allows for the production of milligram quantities of recombinant proteins in static cultures, without the need for complex equipment such as bioreactors, and complies with regulatory requirements due to its distinction from common recombinant viruses. Full article

Emerging pollutants such as organophosphate flame retardants (OPFRs) pose a critical threat to environmental and human health, while conventional wastewater treatments often fail to remove them. This study addresses this issue by evaluating the bioremediation potential of white-rot fungi for the removal of two OPFRs: tris(2-chloroethyl) phosphate (TCEP) and tributyl phosphate (TBP). Three fungal species—Ganoderma lucidum, Trametes versicolor, and Phanerochaete velutina—were screened for their degradation capabilities. Among these, G. lucidum and T. versicolor demonstrated removal efficiencies exceeding 99% for TBP, while removal rates for TCEP were significantly lower, with a maximum of 30%. The exploration of the enzyme role showed that cytochrome P450 is involved in the degradation while the extracellular laccase is not involved. Continuous batch experiments were performed using a trickle-bed reactor (TBR) operating under non-sterile conditions, a setting that closely resembles real-world wastewater treatment environments. G. lucidum was immobilized on oak wood chips, and the removal efficiencies were measured to be 85.3% and 54.8% for TBP and TCEP, respectively, over 10 cycles. Microbial community analysis showed that G. lucidum remained the dominant species in the reactor. These findings demonstrate the efficacy of fungal-based trickle-bed bioreactors, offering a sustainable and efficient alternative for addressing environmental pollution caused by highly recalcitrant pollutants. 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

Bioengineered livers are currently an acceptable alternative to orthotopic liver transplants to overcome the scarcity of donors. However, the challenge of using a bioengineered liver is the lack of an intact endothelial layer in the vascular network leading to thrombosis. Heparin-modified surfaces have been demonstrated to decrease thrombogenicity in earlier research. However, in our study, we aimed to apply heparin immobilization to enhance the hemocompatibility, endothelial cell (EC) adhesion, and angiogenesis of rat decellularized liver scaffolds (DLS). Heparin was immobilized on the DLS by the end-point attachment technique. The scaffold’s hemocompatibility was assessed using ex vivo blood perfusion and platelet adhesion studies. The heparinized scaffold (HEP-DLS) showed a significantly reduced thrombogenicity and platelet aggregation. HEP-DLS was recellularized with EA.hy926 cells via the portal vein and maintained in the bioreactor for 7 days, showing increased EC adhesion and coverage within the blood vessels. The Resazurin reduction assay confirmed the presence of actively proliferating cells in the HEP-DLS. The scaffolds were implanted subcutaneously into the dorsum of mice for 21 days to evaluate cell migration and angiogenesis. The results showed significant increases in the number of blood vessels in the HEP-DLS group. Our results demonstrated that heparin immobilization reduces thrombosis, promotes re-endothelialization, and enhances angiogenesis in DLS. The research provides insight into the potential use of heparin in the formation of a functioning vasculature. Full article

The application of continuous fermentation with immobilized cells in brewing is a challenge because of problems with carrier selection and reactor design, which have economic impacts on the beer produced. Moreover, immobilization alters yeast physiology, which significantly affects beer flavor and aroma. Therefore, the aim of this study was to investigate the feasibility of a continuous fermentation system, consisting of a packed bed column bioreactor, containing lager brewing yeast, immobilized in alginate–chitosan microcapsules with a liquid core, in the primary beer fermentation. The results showed that the system entered in a stationary mode on the 3rd day and worked stably in this mode for 6 days. The “green” beer was taken at every 24 h at the output of the reactor and used for secondary fermentation with the yeast cells leaked from the capsules during the primary fermentation. The extract consumption, ethanol production, and pH change during primary and secondary fermentation were investigated. Some of the secondary yeast metabolites such as vicinal diketones, higher alcohols, esters, and aldehydes in “green” and final beers were determined and it was found that the flavor profile of the final beer was comparable to two industrially produced Bulgarian beers. Full article

Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O₂-saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s⁻¹, with a formal potential of −0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography–mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms. Full article

This study presents findings regarding the kinetics of ferrous iron oxidation in solution mediated by Acidithiobacillus ferrooxidans bacteria within a continuous-flow bioreactor employing diverse types of immobilizers. The objective is to augment the rate of ferrous iron oxidation in solutions utilizing an immobilizer for Acidithiobacillus ferrooxidans strains. Immobilization represents a promising avenue for enhancing the efficiency of Fe²⁺ oxidation via acidophilic ferrooxidizing bacteria, leading to a several-fold increase in oxidation rate. A comparative analysis was conducted to evaluate the efficacy of different types of immobilizer in facilitating iron oxidation within a continuous-flow bioreactor, including the application of wood chips coated with Fe(OH)₃. The results indicate that wood chips coated with iron hydroxide serve as effective type of immobilizer, facilitating the robust attachment of Acidithiobacillus ferrooxidans via electrostatic interactions between negatively charged bacteria and positively charged surfaces. Experimental investigations were conducted using novel immobilization matrices in pilot-scale tests simulating the underground borehole leaching (UBL) of uranium. The bioactivation of leaching solutions enhances the efficiency and environmental compatibility of UBL compared to conventional chemical oxidation methods. The relationships between redox potential and ferric iron content in bioactivated solutions during the UBL of uranium were delineated. The significance of this study lies in its elucidating the pivotal role of Fe²⁺ oxidation in uranium extraction processes, particularly in the context of UBL. By employing bioactivation mediated by Acidithiobacillus ferrooxidans, the study demonstrates not only enhanced uranium extraction efficiency, but also markedly improved environmental sustainability compared to traditional chemical oxidation methods. The findings reveal crucial correlations between redox potential and ferric iron concentration in bioactivated solutions. Full article

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed. Full article