Initial Substrate Concentration Research Articles

Enzymatic hydrolysis of highly concentrated substrates obtained from Miscanthus giganteus

This work is the first to study the enzymatic hydrolysis of four types of substrates obtained from Miscanthus giganteus of the KAMIS variety of Russian breeding. The study was conducting using the authors’ methodology based on a chemical pretreatment of substrates at atmospheric pressure with HNO3 and NaOH dilute solutions. A one-stage pretreatment of Miscanthus giganteus allows the polysaccharide content to be increased up to 90.4–90.8%, compared to 98.3–99.4% following a two-stage treatment. The experimental results of enzymatic hydrolysis of the four obtained substrates in the range of initial concentrations from 30 to 120 g/l are approximated using fractal kinetics approaches. An increase in the initial substrate concentrations in the specified range leads to an increase in the initial hydrolysis rate by 2.8–3.3 times and a decrease in the yield of reducing sugars by 12.4–13.1%. All four pretreatment types turned out to be extremely effective for Miscanthus giganteus, ensuring an increase in the reactivity to enzymatic hydrolysis by 34–36 times compared to the starting raw material. Lowered yields of reducing sugars are observed during enzymatic hydrolysis of the alkaline delignification product of Miscanthus giganteus, which is associated with the resistance of Miscanthus giganteus to treatment with sodium hydroxide. An increase in the initial concentration of substrates from 60 to 90 g/l does not lead to a significant decrease in the yield of reducing sugars. Therefore, enzymatic hydrolysis of highly concentrated substrates can be successfully used to produce biofuels and biochemicals.

A study using a semi-analytical approach to the reaction kinetics that describes the behavior of heterogeneous reaction–diffusion process with a glyoxyl-agarose immobilized enzyme (penicillin G acylase) in a batch reactor

This research article examines the reaction–diffusion process in a batch reactor by means of immobilized penicillin G acylase (PGA) with a glyoxyl-agarose matrix support. This study employs the Akbari-Ganji’s Method (AGM) when the system is under the internal (intra-particle) diffusional restrictions, and the external (film) diffusional restrictions is negligible. The semi-analytical approximations by the AGM technique are employed for computing the dimensionless steady-state solutions for a system of nonlinear differential equations to examine the impact of internal diffusional restrictions (IDR) as a function of catalyst enzyme loading and particle size. The model entails highly nonlinear components that adhere to the standard Michaelis-Menten kinetics. In addition to the proposed model, the dynamics for the mean integrated effectiveness factor (MIEF) of the dimensionless substrate concentration phenylglycine methyl ester (PGME) is presented. In light of this, there is a satisfactory level of agreement on the comparison of the semi-analytical result with the simulated numerical results across the whole concentration range where the dimensionless initial substrate and product concentrations are S1b=30,S2b=15, and P1b=60,P2b=100. The behavior of concentration profiles under the steady/unsteady state conditions for the effect of IDR on the overall reaction rates by immobilized biocatalyst PGA were examined. A sensitivity analysis is carried out to identify the effective parameter which can influence the diffusional restrictions on MIEF. An error analysis is performed on the dimensionless concentration equations to assess the amount of accuracy and the convergence of a solution.

Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N-acetylglucosamine supplementation and kinetic modeling.

Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin-an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30gL-1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N-acetylglucosamine (S2) resulted in a significant 1.6-fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi-substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.

Development of a model for the ethanol concentration limit as a function of temperature and initial substrate concentration using the yeast Saccharomyces cerevisiae.

In this study, a model was developed to simulate the effect of temperature ( ) and initial substrate concentration ( ) on the ethanol concentration limit ( ) using the yeast Saccharomyces cerevisiae. To achieve this, regressions were performed using data provided by other authors for to establish a model dependent on and capable of predicting results with statistical significance. After constructing the model, a response surface was generated to determine the conditions where reaches higher values: temperatures between 28°C and 32°C and an initial substrate concentration around 200 g/L. Thus, the proposed model is consistent with the observations that increasing temperatures decrease the ethanol concentration obtained, and substrate concentrations above 200 g/L lead to a reduction in ethanol concentration even at low temperatures such as 28°C.

Theoretical Analysis of Pre-Steady State Behaviour of Non-Linear Double Intermediate Enzymatic Reaction

This paper explores the mathematical modelling of a non-linear double intermediate enzymatic reaction, focusing on its pre-steady state behaviour. Using homotopy perturbation and Taylor's series methods, an approximate analytical solution is derived for the concentrations of substrate, the first enzyme-substrate complex, and the second enzyme-substrate complex. The model is applicable in scenarios where the initial substrate concentration dramatically exceeds that of the enzyme and when they are comparable. Comparisons between analytical and numerical solutions are made, highlighting the efficacy of the proposed model. Additionally, an improved understanding is achieved by comparing results obtained from Matlab simulations with analytical solutions. Moreover, sensitivity analysis on parameters affecting the concentrations is conducted. This mathematical model enhances comprehension of biochemical reactions in living organisms.

A robust, low-temperature, closed-loop anaerobic system for high-solid mixed farm wastes: advancing agricultural waste management solutions in Canada.

This study investigates the effectiveness of low-temperature (20 ± 1 °C) anaerobic digestion (AD) for two organic multiple farm substrate combinations: Set 1 comprising chicken manure (CM), dairy manure (DM), and waste corn silage (CS) and Set 2 comprising CM, DM, pig manure (PM), and CS. Inoculum adaptation steps were carried out using CM and CM+DM for Set 1 and Set 2, respectively. Over three consecutive operating cycles spanning 245 days with increasing organic loads, 4.3 and 2.8 g VS L-1 d-1 for Sets 1 and 2 during Cycles 1 to 5.1 and 4.6 g VS L-1 d-1for Sets 1 and 2 during Cycle 3, a closed-loop two-stage liquid-solid AD system was employed, with performance assessed via stability ratios of short-chain volatile fatty acids and alkalinity. Results demonstrate that mono-digestion of CM with adapted inoculum yielded the highest biogas production of 424 ± 4 L over 77 days, indicating superior performance by Set 1 during Phase I, whereas a similar performance was observed during Phase 2, where Sets 1 and 2 exhibited highest specific methane yields of 0.233 ± 0.028 and 0.262 ± 0.004 L g-1 VSfed, respectively, over 68 days. Analysis of heavy metal concentrations in digestates revealed a significant decrease compared to initial raw substrate concentrations, highlighting their role as nutrients for microbial growth. This study, the first of its kind, highlights the potential of low-temperature AD systems to manage diverse organic residues/byproducts and offers insights into effective performance monitoring without compromising system integrity.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

The Michaelis–Menten Reaction at Low Substrate Concentrations: Pseudo-First-Order Kinetics and Conditions for Timescale Separation

We demonstrate that the Michaelis–Menten reaction mechanism can be accurately approximated by a linear system when the initial substrate concentration is low. This leads to pseudo-first-order kinetics, simplifying mathematical calculations and experimental analysis. Our proof utilizes a monotonicity property of the system and Kamke’s comparison theorem. This linear approximation yields a closed-form solution, enabling accurate modeling and estimation of reaction rate constants even without timescale separation. Building on prior work, we establish that the sufficient condition for the validity of this approximation is s0≪K\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$s_0 \\ll K$$\\end{document}, where K=k2/k1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$K=k_2/k_1$$\\end{document} is the Van Slyke–Cullen constant. This condition is independent of the initial enzyme concentration. Further, we investigate timescale separation within the linear system, identifying necessary and sufficient conditions and deriving the corresponding reduced one-dimensional equations.

Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy.

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

Lactic acid production from different sources of organic solid waste: evaluation of the inoculum type and operational optimization

AbstractThis work aimed to evaluate the effect of the sources of organic solid waste on lactic acid (HLa) production. Organic fractions from cafeteria (CW), market (MW), and recycling plant (RW) wastes were used as substrates. HLa production during the self-fermentation of CW, MW, and RW, as well as during the fermentation of each substrate with anaerobic sludge (CW + sludge, MW + sludge, and RW + sludge), was evaluated in batch tests at 37 °C, initial pH of 6.5, and 150 rpm. Subsequently, the initial pH and substrate concentration of self-fermentation of CW were optimized using a central composite design. The highest HLa production was observed in the self-fermentation of CW, where several Lactobacillus species predominated in the microbial community. The higher value of HLa production using CW (20.7 g/L) was obtained at a pH of 7.29 and 115.5 gVS/L. In the optimization tests, Lactobacillus and Weissella spp. were identified as the dominant lactic acid bacteria; however, only Lactobacillus species were associated with the highest HLa production.

Lactic Acid Production by Lactiplantibacillus plantarum AC 11S-Kinetics and Modeling.

Lactic acid is a versatile chemical with wide application in many industries. It can be produced by the fermentation of different sugars by various lactobacilli and investigations on lactic acid production from different substrates and by different strains are still in progress. The present study aimed to study lactic acid production from lactose by Lactiplantibacillus plantarum AC 11S and to choose a mathematical model describing in the best way the experimental data obtained. The influence of initial substrate concentration was investigated, and optimal pH and temperature were determined. An unstructured mathematical model was developed comprising equations for bacterial growth, substrate consumption, and product formation. The model was solved with different terms for specific growth rates considering substrate and/or product inhibition. The best bacterial growth and lactic acid production were achieved at pH = 6.5 and 30 °C. Production of lactic acid was mainly growth-associated, and at initial substrate concentration over 15 g/L, a considerable product inhibition was observed. The parameters of different models were determined and compared. The modified Gompertz equation gave the best fit when solving only the equation for biomass growth at different initial substrate concentrations. Solving the entire set of differential equations for bacterial growth, substrate consumption, and product formation, the best results were obtained when using a variant of the logistic equation for biomass growth. This variant included a term for product inhibition and described in the best way all experimental data. Solving the model for different biomass concentrations showed that an increase in biomass led to a shorter lag phase and the stationary phase was reached faster. The results obtained, optimum conditions and the kinetic model, are good bases for studying pH-controlled fermentation, as well as a continuous process.

Use of interactive mathematical simulations in Fundamentals of Biochemistry, a LibreText online educational resource, to promote understanding of dynamic reactions.

Biology is perhaps the most complex of the sciences, given the incredible variety of chemical species that are interconnected in spatial and temporal pathways that are daunting to understand. Their interconnections lead to emergent properties such as memory, consciousness, and recognition of self and non-self. To understand how these interconnected reactions lead to cellular life characterized by activation, inhibition, regulation, homeostasis, and adaptation, computational analyses and simulations are essential, a fact recognized by the biological communities. At the same time, students struggle to understand and apply binding and kinetic analyses for the simplest reactions such as the irreversible first-order conversion of a single reactant to a product. This likely results from cognitive difficulties in combining structural, chemical, mathematical, and textual descriptions of binding and catalytic reactions. To help students better understand dynamic reactions and their analyses, we have introduced two kinds of interactive graphs and simulations into the online educational resource, Fundamentals of Biochemistry, a LibreText biochemistry book. One is available for simple binding and kinetic reactions. The other displays progress curves (concentrations vs. time) for simple reactions and complex metabolic and signal transduction pathways. Users can move sliders to change dissociation and kinetic constants as well as initial concentrations and see instantaneous changes in the graphs. They can also export data into a spreadsheet for further processing, such as producing derivative Lineweaver-Burk and traditional Michaelis-Menten graphs of initial velocity (v0) versus substrate concentration.

The unreasonable effectiveness of the total quasi-steady state approximation, and its limitations

In this note, we discuss the range of parameters for which the total quasi-steady-state approximation of the Michaelis–Menten reaction mechanism holds validity. We challenge the prevalent notion that total quasi-steady-state approximation is “roughly valid” across all parameters, showing that its validity cannot be assumed, even roughly, across the entire parameter space — when the initial substrate concentration is high. On the positive side, we show that the linearized one-dimensional equation for total substrate is a valid approximation when the initial reduced substrate concentration s0/KM is small. Moreover, we obtain a precise picture of the long-term time course of both substrate and complex

The influence of various chemical oxygen demands on microbial fuel cells performance using leachate as a substrate.

Microbial fuel cells (MFCs), hailed as a promising technology, hold the potential to combat various wastewater pollutants while simultaneously converting their chemical energy into electricity through biocatalysts. This study explores the applicability of a dual compartment MFC (DC-MFC) under varying conditions, targeting the removal of chemical oxygen demand (COD) from landfill leachate and electricity generation. In this setup, anaerobic sludge from a wastewater treatment plant serves as the inoculum in the anode compartment of the MFC, with a Nafion117 membrane acting as the separator between MFC units. The cathode compartments are filled with distilled water and continually aerated for 24 h to enhance air supply. The study assesses the MFC's performance across different COD concentrations, focusing on COD removal, power generation, and Coulombic efficiency. The findings reveal that COD removal efficiency is notably enhanced at higher concentrations of organic matter. Specifically, at a COD concentration of 3325.0 mg L-1, the MFC exhibited the highest COD removal efficiency (89%) and maximum power density (339.41 mWm-2), accompanied by a Coulombic efficiency of 25.5%. However, as the initial substrate concentration increased to 3825 mg L-1, the efficiency decreased to 72%, with a Coulombic efficiency of 13.56% and a power density of 262.34 mWm-2. Optical density levels increased due to bacterial growth at ambient temperature and neutral pH, reflecting the dynamic microbial response within the system.

Xylitol fermentation characteristics with a newly isolated yeast Wickerhamomyces anomalus WA

Xylitol is an increasingly popular functional food additive, and the newly isolated yeast Wickerhamomyces anomalus WA has shown extensive substrate utilization capability, with the ability to grow on hexose (d-galactose, d-glucose, d-mannose, l-fructose, and d-sorbose) and pentose (d-xylose and l-arabinose) substrates, as well as high tolerance to xylose at concentrations of up to 300 g/L. Optimal xylitol fermentation conditions were achieved at 32 °C, 140 rpm, pH 5.0, and initial cell concentration OD600 of 2.0, with YP (yeast extract 10 g/L, peptone 20 g/L) as the optimal nitrogen source. Xylitol yield increased from 0.61 g/g to 0.91 g/g with an increase in initial substrate concentration from 20 g/L to 180 g/L. Additionally, 20 g/L glycerol was found to be the optimal co-substrate for xylitol fermentation, resulting in an increase in xylitol yield from 0.82 g/g to 0.94 g/g at 140 rpm, enabling complete conversion of xylose to xylitol.

Engineering of methionine sulfoxide reductase A with simultaneously improved stability and activity for kinetic resolution of chiral sulfoxides

Methionine sulfoxide reductase A (MsrA) has emerged as promising biocatalysts in the enantioselective kinetic resolution of racemic (rac) sulfoxides. In this study, we engineered robust MsrA variants through directed evolution, demonstrating substantial improvements of thermostability. Mechanism analysis reveals that the enhanced thermostability results from the strengthening of intracellular interactions and increase in molecular compactness. Moreover, these variants demonstrated concurrent improvements in catalytic activities, and notably, these enhancements in stability and activity collectively contributed to a significant improvement in enzyme substrate tolerance. We achieved kinetic resolution on a series of rac-sulfoxides with high enantioselectivity under initial substrate concentrations reaching up to 93.0 g/L, representing a great improvement in the aspect of the substrate concentration for biocatalytic preparation of chiral sulfoxide. Hence, the simultaneously improved thermostability, activity and substrate tolerance of MsrA represent an excellent biocatalyst for the green synthesis of optically pure sulfoxides.

Co-digestion approach for enhancement of biogas production by mixture of untreated napier grass and industrial hydrolyzed food waste.

The co-digestion of untreated Napier grass (NG) and industrial hydrolyzed food waste (FW) was carried out in the batch reactor to investigate the effect of substrate ratios on biogas production performance. Two-stage anaerobic digestion was performed with an initial substrate concentration of 5g VSadded/L and a Food to Microorganism Ratio (F/M) of 0.84. The 1:1 ratio of the NG and FW showed the optimum performances on biogas production yield with a value of 1,161.33mL/g VSadded after 60 days of digestion. This was followed by the data on methane yield and concentration were 614.37mL/g VSadded and 67.29%, respectively. The results were similar to the simulation results using a modified Gompertz model, which had a higher potential methane production and maximum production rate, as well as a shorter lag phase and a coefficient of determination of 0.9945. These findings indicated that the co-digestion of Napier grass and hydrolyzed food waste can enhance biogas production in two-stage anaerobic digestion.

True Order Determination of Sonochemical Degradation Kinetics of Organic Contaminants in Water Using the Half-lives Method

In the literature, the modeling of the sonolytic decomposition kinetics of organic contaminants in water has been carried out using the pseudo-first and pseudo-second order equations. The results obtained with this method are totally biased because it requires the assumption of the order of the degradation reaction, which is a real limitation, and the sonochemical oxidation mechanism is a very complex phenomenon that cannot be described by such simple models. In this paper, for the first time, the true order of the sonochemical degradation kinetics of organic pollutants in aqueous solutions at various initial substrate concentrations was determined using the half-lives method combined with the general rate law model, i.e., the pseudo-nth order equation. Linear and nonlinear regression techniques were used to determine the order of the sonolytic destruction reaction. The half-life technique for the general rate law model can adequately describe the experimental results of the sono-destruction of the investigated pollutants. For rhodamine B and naphthol blue black, the sono-destruction mechanism for both contaminants does not change as the initial contaminant concentration varies over the range examined, while for 4-isopropylphenol and furosemide, the sono-destruction mechanism changes as the initial substrate concentration varies. A fractional order of the sonolytic destruction reaction was determined for all the pollutants tested. These fractional orders of sono-destruction reactions indicate the complex nature and behavior of the reactions, often involving multiple steps or mechanisms. They also reveal a more complex interaction between pollutant concentration and sonolytic destruction rate. In a word, the method developed in this paper should be used to determine the real order of the sono-oxidation reaction of nonvolatile organic pollutants in aqueous phase.

Isomerization and epimerization of fructose in phosphate buffer under subcritical water conditions

Isomerization and epimerization of fructose to glucose, mannose, allulose, and allose were executed using a subcritical phosphate buffer solution to effectively produce useful monosaccharides. The conversion of the substrate and the yield of products were dependent on the reaction temperature, initial pH, initial substrate concentration, and buffer concentration. A high yield of mannose was achieved under the optimal reaction conditions we identified. We subsequently performed the kinetic analysis based on the proposed reaction network, and evaluated the effects of temperature and pH on the reactions. We then estimated the apparent activation energy values for each reaction.

Investigation of RH effect on uncommon limonene ozonolysis products and SOA formation in indoor air with real time measurement techniques

Scientific interest in SOA influence on indoor air quality increases since last 20 years. It is well known, that particles of nano-sized diameter pose a threat for human health causing, among others: eye, upper airway irritation, inflammatory response in cells, worsening asthma, hypertension, diabetes, and central nervous dysfunction. Terpenes are reactive VOCs, commonly emitted in indoor air and considered to be SOA precursors by oxidation reactions. The aim of this study was to investigate the relative humidity influence on i) formation of limonene ozonolysis products and ii) SOA formation process – especially the first steps of it. We managed to determine 4 uncommon limonene ozonolysis products (m/z: 43, 83, 99 and 110) appearing in the PTR-TOF-MS mass spectra with the same frequency as well-known formaldehyde, acetone, acetic acid and formic acid. We also detected m/z 75 and m/z 115 formed under dry conditions and m/z 111 and m/z 125 formed under humid conditions. Detected masses formula is proposed with probability >70 % and the probability is derived from the deviation of the exact mass to the measured one and the isotope distribution. SMPS data analysis allowed us to conclude that RH around 40% increases particle mass concentration, regardless initial limonene concentration. Unfortunately, conclusions about RH influence on particle number concentration are inconsistent. Under low initial terpene concentration, RH around 40% decreases particle number concentration. However, for high initial limonene concentration, RH around 40% caused increase in particle number concentration. Obtained results allowed to conclude, that i) RH influences both particle number and particle mass concentration, ii) initial substrate concentration influences SOA formation altering the RH effect, iii) comparison of results and drawing conclusions is difficult due to different experimental protocols in the literature and due to the number of factors influencing SOA formation initiated by terpene oxidation.