Search Results (82)

Phosphofructokinase 1 (PfkA) mediates the ATP-dependent phosphorylation of fructose-6-phosphate and is a key, controlling enzyme in glycolysis for Escherichia coli and other organisms. In this study, 22 chromosomally expressed PfkA variants were constructed in E. coli C. These variants, the wild-type strain, and the ∆pfkA strain were compared for growth rates using glucose as the sole carbon source. The majority of variants (14 of 22) attained a growth rate less than 20% of the growth rate of the wild-type strain (0.94 h⁻¹) and thus similar to the knockout strain (0.12 h⁻¹). Three variants (R171S, F76Y, and R77A), representing a range of growth phenotypes, and strains expressing the wild-type PfkA and the ∆pfkA deletion strain were additionally examined for key intracellular metabolites and gene expression under nitrogen-limited steady-state conditions. These five strains could be distinguished by two groupings: strains with relatively high growth rates under batch conditions (wild-type and R77A variant) showed the greatest glucose consumption rate and formed acetate, whereas strains with low growth rates (F76Y, R77A, and ∆pfkA) exhibited low glucose consumption and did not accumulate acetate. As the PfkA mutation severity increased, the intracellular concentrations of acetyl-CoA and fructose-1,6-bisphosphate and the sum of dihydroxyacetone and glyceraldehyde-3-phosphate greatly decreased. Although the mutation severity had a limited effect on the expression of maeB and icd genes expressing malic enzyme and isocitrate dehydrogenase, it correlated with reduced expression of zwf and pta genes expressing glucose-6P-dehydrogenase and phosphotransacetylase, respectively. The results highlight the great sensitivity of the enzyme to substitutions and the key role it plays in controlling glycolytic flux. Full article

Cupriavidus necator is a well-studied microorganism with potential application in bioregenerative life support systems for single-cell protein and bioplastic production. Most studies have been carried out in autotrophy or heterotrophy, requiring O₂ as the final electron acceptor. In the context of inhabited missions, access to O₂ will primarily be limited to the crew. In this study, we investigated the capacity of C. necator to carry out nitrate respiration as a strategy to limit oxygen supply to the cultures by providing nitrate from another compartment of the Bioregenerative Life Support System (BLSS). Batch bioreactor experiments were carried out to determine the best conditions for nitrate utilization in terms of pH and aeration. Continuous cultures were then performed under two carbon sources (glucose vs. acetic acid) and two substrate limitations (nitrate vs. carbon). The optimal conditions were found to be pH 7.5 under anaerobiosis. They were applied in chemostats, where three steady-states were obtained at a low dilution rate. In all cases, the biomass consisted of a mixture of protein (from 29 ± 1% Cell Dry Weight (CDW) to 39 ± 2% CDW) and polyhydroxybutyrate (from 45 ± 2% CDW to 57 ± 3% CDW), which was found to be a key component for nitrate respiration metabolism. Microaerobic conditions were also tested in batch culture, reporting for the first time aerobic nitrate respiration in C. necator. Under these conditions, growth parameters improved during the nitrate phase; however, the specific growth rate during the nitrite phase was lower than that observed under strictly anaerobic conditions. Full article

This work develops and analyzes a novel fractional-order chemostat system (FOCS) with a Caputo fractional derivative (CFD) featuring a sliding memory window and periodic boundary conditions (PBCs), designed to model microbial pollutant degradation in sustainable water treatment. By incorporating the Caputo fractional derivative with sliding memory (CFDS), the model captures time-dependent behaviors and memory effects in biological systems more realistically than classical integer-order formulations. We reduce the two-dimensional fractional differential equations (FDEs) governing substrate and biomass concentrations to a one-dimensional FDE by utilizing the PBCs. The existence and uniqueness of non-trivial, periodic solutions are established using the Carathéodory framework and fixed-point theorems, ensuring the system’s well-posedness. We prove the positivity and boundedness of solutions, demonstrating that substrate concentrations remain within physically meaningful bounds and biomass concentrations stay strictly positive, with solution trajectories confined to a biologically feasible invariant set. Additionally, we analyze non-trivial equilibria under constant dilution rates and derive their stability properties. The rigorous mathematical results confirm the viability of FOCS models for representing memory-driven, periodic bioprocesses, offering a foundation for advanced water treatment strategies that align with Sustainable Development Goal 6 (Clean Water and Sanitation). This work establishes a comprehensive mathematical framework that bridges fractional calculus with sustainable water treatment applications, providing both theoretical foundations and practical implications for optimizing bioreactor performance in environmental biotechnology. Full article

This paper investigates the stabilization of a chemostat system with biomass settling dynamics using feedback linearization and model reduction techniques. The original three-dimensional system, composed of substrate, free biomass, and settled biomass compartments, is reduced to a two-dimensional system by assuming quasi-steady-state for the settled biomass population. A nonlinear feedback control law for the dilution rate is then designed using feedback linearization, aiming to regulate the free biomass concentration around a desired set point. The proposed control strategy compensates for nonlinearities introduced by Monod-type microbial growth and biomass settling effects. To evaluate robustness, time-varying disturbances are introduced into the inflow substrate concentration. Numerical simulations in MATLAB confirm that the closed-loop system maintains stability and tracks the biomass target despite sustained inflow fluctuations. The results demonstrate the efficacy of the reduced-order feedback linearization approach in chemostat stabilization and its potential for bioreactor control under uncertain environmental conditions. Full article

This research characterized a novel Komagataella phaffii strain generated through intraspecific crossing between a wild isolate and a laboratory strain. This segregant, called S467, expressed 2.2-fold more secreted recombinant β-glucosidase than its parental strains in microtiter scale, which suggested that S467 could be an attractive host for bioprocess optimization. S467 was grown alongside the laboratory strain CBS7435 expressing β-glucosidase (CBS_BGL9), as a control, in a 1.5 L bioreactor to determine kinetics parameters, and similar cell growth rate (0.12 h⁻¹) but higher recombinant protein activity, measured as enzymatic activity, was observed in S467. The effect of specific cell growth rate was studied using continuous cultures (chemostat) at different dilution rates, identifying conditions that provided up to a twofold increase in enzymatic activity in S467. RT-qPCR was conducted on key genes associated with the genetic background of S467, in order to clarify differences at the transcriptomic level that render S467 as a potential superior host for recombinant protein production. Overall, this study provides quantitative evidence of the positive effect of the natural isolate IRA1 allele for the generation of recombinant β-glucosidase and highlights the usability of natural genetic diversity in K. phaffii. Full article

Haemophilus influenzae type b (Hib) is a Gram-negative bacterium that causes severe infections in children under five, especially in developing countries. Although vaccination using capsular polysaccharide by Hib (linear polymer 5-D-ribitol-(1→1)-β-D-ribose-3-phosphate) conjugated to tetanus toxoid is effective, its production is complex and costly. This study aimed to develop a continuous production process for PRP to increase productivity, reduce batch numbers, and simplify manufacturing. Using a 1 L bioreactor, five dilution rates (0.13 to 0.32 h⁻¹) were tested, with the best performance observed at 0.23 h⁻¹, reaching a productivity of 167 mgL⁻¹·h⁻¹. Under optimized conditions, parameters such as free and immobilized PRP, glucose consumption, acetate formation, and biomass were monitored. The process yielded 874 mgL⁻¹ of PRP after 74.4 h, with 78% in the free form and a final productivity of 165 mgL⁻¹·h⁻¹, approximately six times higher than batch processes and twice as high as fed-batch processes. The continuous process proved more efficient and required less infrastructure to meet production demands. However, further optimization is needed to enhance product quality and assess overall feasibility. Full article

The naphthazarins shikonin and alkannan are strongly chromogenic, dark red enantiomers, each of which has biological activity, that are found primarily in the plant family Boraginaceae. These compounds and their many chemical metabolites, derivatives, oligomers, and analogs (“shikonoids”) are an important group of phytochemicals, utilized since antiquity as components of dyes, traditional medicines, and food and cosmetics. They are now recognized for their potent anti-inflammatory and regulatory activity on a variety of molecular signaling pathways in humans. Since many Boraginaceae species are overly exploited or endangered, we developed a pilot-scale in vitro shikonoid production system using Plagiobothrys arizonicus (Gray) Greene ex A.Gray, the Arizona popcorn flower, native to the southwestern USA and the Sonoran floristic province in the Madrean region of Mexico. Aseptic root cultures were initiated from fresh leaf tissue and stimulated to continuously produce shikonoids in liquid shake cultures layered under paraffin oil from which the shikonoids were extracted and concentrated. The crude, red extracellular product from these rapidly expanding root masses was also fractionated by Centrifugal Counter-Current Chromatography (CCC) into its component shikonin derivatives. A number of these shikonoids profoundly up-regulated detoxification and antioxidant proteins (phase 2 enzymes) and inhibited inflammation in mammalian cell bioassay systems. This prototype shikonoid production methodology can be readily scaled to either batch or chemostat culture. Full article

Azotobacter vinelandii OP is a bacterium that can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HBV), a biodegradable and biocompatible polymer with applications in the biomedical field. This study aimed to evaluate P3HBV production and its 3-hydroxyvalerate (3HV) fraction under different agitation rates and oxygen uptake rates (q_O2) in chemostat cultures of A. vinelandii OP. Steady-state conditions with either oxygen or carbon limitation were established by modulating the agitation rates. Under oxygen-limited conditions (low q_O2 values) biomass and P3HBV concentrations increased to 3.3 g L⁻¹ and 2.1 g L⁻¹, respectively. At higher q_O2 values, the chemostat cultures were limited by carbon, and P3HBV content decreased from 62% to 33% (w w⁻¹). The highest 3HV molar fractions, 33.7 and 36.4 mol %, were observed at both the lowest and highest q_O2 levels, possibly linked to comparable valeric acid consumption rates. An elevated NAD(P)H/NAD(P)⁺ ratio was also observed under oxygen limitation, favoring polymer accumulation by indicating a more favorable intracellular redox state. These findings highlight the impact of nutrient limitation and respiratory activity on the biosynthesis of P3HBV and the 3HV composition by Azotobacter vinelandii OP. Such insights can support the development of tailored bioprocesses to modulate polymer characteristics, enabling a broader range of potential biomedical applications for P3HBV. Full article

The three-tiered microbial food chain without maintenance under leachate recirculation is the subject of a mathematical seven-dimensional dynamical system that is proposed in this work. This model captures the complex interactions between chlorophenol degraders, phenol degraders, and methanogens in the presence of hydrogen inhibition. The implementation allows for investigation of how hydrogen levels affect the overall system dynamics and phenol production. There is a thorough qualitative analysis provided. A stability analysis of equilibrium points is performed. It is demonstrated that the persistence of the three bacteria is correlated with the existence of the positive equilibrium point, assuming some monotonicity properties on the growth rates. Asymptotic coexistence is satisfied, although periodic orbit possibilities are not ruled out. In order to decrease the amount of organic materials within the reactor, we suggest an optimal strategy on the rate of leachate recirculation in the second stage. Lastly, we offer a few numerical investigations that support and strengthen the theoretical conclusions. Full article

Biodesulfurization (BDS) is a clean technology that uses microorganisms to efficiently remove sulfur from recalcitrant organosulfur compounds present in fuels (fossil fuels or new-generation fuels resulting from pyrolysis and hydrothermal liquefaction). One of the limitations of this technology is the low desulfurization rates. These result in the need for greater amounts of biocatalyst and lead to increased production costs. To mitigate this issue, several approaches have been pursued, such as the use of alternative carbon sources (C-sources) from agro-industrial waste streams or the co-production of high-added-value products by microorganisms. The main goal of this work is to assess the potential of strawberry tree fruit residue (STFr) as an alternative C-source for a BDS biorefinery using Gordonia alkanivorans strain 1B, a well-known desulfurizing bacterium with high biotechnological potential. Hence, the first step was to produce sugar-rich liquor from the STFr and employ it in shake-flask assays to evaluate the influence of different pretreatments (treatments with 1–4% activated charcoal for prior phenolics removal) on metabolic parameters and BDS rates. Afterwards, the liquor was used as the C-source in chemostat assays, compared to commercial sugars, to develop and optimize the use of STFr-liquor as a viable C-source towards cost-effective biocatalyst production. Moreover, the high-market-value bioproducts simultaneously produced during microbial growth were also evaluated. In this context, the best results, considering both the production of biocatalysts with BDS activity and simultaneous bioproduct production (carotenoids and gordofactin biosurfactant/bioemulsifier) were achieved when strain 1B was cultivated in a chemostat with untreated STFr-liquor (5.4 g/L fructose + glucose, 6:4 ratio) as the C-source and in a sulfur-free mineral-minimized culture medium at a dilution rate of 0.04 h⁻¹. Cells from this steady-state culture (STFr L1) achieved the highest desulfurization with 250 mM of dibenzothiophene as a reference organosulfur compound, producing a maximum of ≈213 mM of 2-hydroxibyphenil (2-HBP) with a corresponding specific rate (q_2-HBP) of 6.50 µmol/g(_DCW)/h (where DCW = dry cell weight). This demonstrates the potential of STFr as a sustainable alternative C-source for the production of cost-effective biocatalysts without compromising BDS ability. Additionally, cells grown in STFr L1 also presented the highest production of added-value products (338 ± 15 µg/g_(DCW) of carotenoids and 8 U/mL of gordofactin). These results open prospects for a future G. alkanivorans strain 1B biorefinery that integrates BDS, waste valorization, and the production of added-value products, contributing to the global economic viability of a BDS process and making BDS scale-up a reality in the near future. Full article

In this work, we investigated a simple mathematical model describing the consumption of virus-infected phytoplankton by zooplankton in a chemostat. The system was studied by calculating the basic reproduction number, the equilibrium points, and their local and global stability. A sensitivity analysis was used to identify key chemostat factors that significantly affected the aquatic system. Additionally, we considered an optimal strategy based on the use of the dilution rate as an operating parameter that helps maintain the ecological balance of the aquatic food web. Full article

Riboflavin is biosynthesized and excreted extracellularly by the novel yeast Hyphopichia wangnamkhiaoensis. The steady-state kinetics of cell growth, substrate consumption, and riboflavin production by H. wangnamkhiaoensis were studied in a chemostat continuous culture at different dilution rates. The unstructured Monod and Luedeking–Piret models were used to describe cell growth, substrate consumption, and riboflavin production, and crucial kinetic parameters were estimated. The experimental data fitted the proposed models well. The maximum specific growth rate, substrate affinity constant, maintenance energy coefficient, and maximum biomass yield values were 0.1378 h⁻¹, 0.4166 g of glucose L⁻¹, 0.1047 g of glucose g⁻¹ of biomass h⁻¹, and 0.172 g of biomass g⁻¹ of glucose, respectively. The maximum yield from glucose and volumetric and specific productivities of riboflavin were 0.7487 mg of riboflavin g⁻¹ of glucose, 0.5593 mg of riboflavin L⁻¹ h⁻¹, and 0.6547 mg of riboflavin g⁻¹ of biomass h⁻¹, respectively. The estimated growth-associated riboflavin production constant (4.88 mg of riboflavin g⁻¹ of biomass) was much higher than the non-growth-associated riboflavin production constant (0.0022 mg of riboflavin g⁻¹ of biomass h⁻¹), indicating that riboflavin production by H. wangnamkhiaoensis is a predominantly growth-associated process. The chemostat continuous culture offers a promising strategy for efficiently and sustainably producing riboflavin using H. wangnamkhiaoensis. Full article

Biosurfactants/bioemulsifiers (BSs/BEs) can be defined as surface-active biomolecules produced by microorganisms with a broad range of applications. In recent years, due to their unique properties like biodegradability, specificity, low toxicity, and relative ease of preparation, these biomolecules have attracted wide interest as an eco-friendly alternative for several industrial sectors, escalating global microbial BS/BE market growth. Recently, Gordonia alkanivorans strain 1B, a bacterium with significant biotechnological potential, well known for its biodesulfurizing properties, carotenoid production, and broad catabolic range, was described as a BS/BE producer. This study focuses on the characterization of the properties of the lipoglycopeptide BSs/BEs produced by strain 1B, henceforth referred to as gordofactin, to better understand its potential and future applications. Strain 1B was cultivated in a chemostat using fructose as a carbon source to stimulate gordofactin production, and different purification methods were tested. The most purified sample, designated as extracted gordofactin, after lyophilization, presented a specific emulsifying activity of 9.5 U/mg and a critical micelle concentration of 13.5 mg/L. FT-IR analysis revealed the presence of basic hydroxyl, carboxyl, ether, amine/amide functional groups, and alkyl aliphatic chains, which is consistent with its lipoglycopeptide nature (60% lipids, 19.6% carbohydrates, and 9% proteins). Gordofactin displayed remarkable stability and retained emulsifying activity across a broad range of temperatures (30 °C to 80 °C) and pH (pH 3–12). Moreover, a significant tolerance of gordofactin emulsifying activity (EA) to a wide range of NaCl concentrations (1 to 100 g/L) was demonstrated. Although with a great loss of EA in the presence of NaCl concentrations above 2.5%, gordofactin could still tolerate up to 100 g/L NaCl, maintaining about 16% of its initial EA for up to 7 days. Furthermore, gordofactin exhibited growth inhibition against both Gram-positive and Gram-negative bacteria, and it demonstrated concentration-dependent free radical scavenging activity for 2,2-diphenyl-1-picrylhydrazyl (IC₅₀ ≈ 1471 mg/L). These promising features emphasize the robustness and potential of gordofactin as an eco-friendly BS/BE alternative to conventional surfactants/emulsifiers for different industrial applications. Full article

Currently, the yeast Yarrowia lipolytica is regarded as one of the most promising producers of protein, lipids, polyols, organic acids, and other metabolites. The objective of enhancing the efficiency of the target product biosynthesis can be achieved through the improvement of the strains-producers and the optimization of the cultivation conditions. The present review assesses the potential of continuous cultivation methods (chemostat, turbidostat, pH-auxostat, changestats, etc.) in order to gain insight into the impact of strains and cultivation conditions on the productivity of the developed bioprocesses. The utilization of continuous cultivation methods enables the implementation of processes under controlled and reproducible conditions, thus stabilizing the parameters of the cultivation and the physiological state of the producer, and obtaining homogeneous samples. The review focuses on nitrogen-limited chemostat cultures, which represent the most commonly employed strategy for investigating the physiological and biochemical characteristics of the yeast Y. lipolytica and for developing the processes for the production of lipids, erythritol, citric acid, and recombinant proteins. To date, such an analysis of the literature has not been conducted in the context of the yeast Y. lipolytica. Full article

In the present work, we study a simple mathematical model that describes the competition of two bacterial species with an obligate one-way beneficial relationship for a limited substrate in a bioreactor associated with leachate recirculation. The substrate is present into two forms, insoluble and soluble substrates, where the latter is consumed by the two competing bacteria, which have two general nonlinear growth rates. The reduction of the model to a 2D one facilitates the study of the nature of the equilibrium points. The dynamic system admits multiple steady states. We provide necessary and sufficient conditions on the added insoluble and soluble substrates and the dilution rate to guarantee the existence, uniqueness, and local and global stability of such steady states. It is deduced that the coexistence of both bacteria is possible, which contradicts the competitive exclusion principle. In the second step, we propose an optimal control on the leachate recirculation rate that reduces the organic matter inside the reactor. Finally, we provide some numerical examples that corroborate and reinforce the theoretical findings. Full article