H.H. Beeftink

Optimization of the dilute maleic acid pretreatment of wheat straw.

BackgroundIn this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.ResultsWhen costs are disregarded, an almost complete glucan conversion to glucose can be reached (90% from solids, 7%-10% in liquid), after enzymatic hydrolysis. During the pretreatment, up to 90% of all xylan is converted to monomeric xylose. Taking cost factors into account, the optimal process conditions are: 50 min at 170°C, with 46 mM maleic acid, resulting in a yield of 65 €/Mg (megagram = metric ton) dry straw, consisting of 68 €/Mg glucose benefits (from solids: 85% of all glucan), 17 €/Mg xylose benefits (from liquid: 80% of all xylan), 17 €/Mg maleic acid costs, 2.0 €/Mg heating costs and 0.68 €/Mg NaOH costs. In all but the most severe of the studied conditions, furfural formation was so limited that associated costs are considered negligible.ConclusionsAfter the dilute maleic acid pretreatment and subsequent enzymatic hydrolysis, almost complete conversion of wheat straw glucan and xylan is possible. Taking maleic acid replenishment, heating, neutralization and furfural formation into account, the optimum in the dilute maleic acid pretreatment of wheat straw in this study is 65 €/Mg dry feedstock. This is reached when process conditions are: 50 min at 170°C, with a maleic acid concentration of 46 mM. Maleic acid replenishment is the most important of the studied cost factors.

Thermodynamically controlled synthesis of β-lactam antibiotics. Equilibrium concentrations and side-chain properties

For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mm from 20 mm starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling. Copyright (C) 1999 Elsevier Science Inc. All rights reserved. | For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mM from 20 mM starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2-3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling.

Bioreactors in series: An overview of design procedures and practical applications

Abstract “If one fermenter gives good results, two fermenters will give better results and three fermenters better still. This is sometimes true, but often false.” Herbert, D. 9

A generic model for glucose production from various cellulose sources by a commercial cellulase complex

The kinetics of cellulose hydrolysis by commercially available Cellubrix were described mathematically, with Avicel and wheat straw as substrates. It was demonstrated that hydrolysis could be described by three reactions: direct glucose formation and indirect glucose formation via cellobiose. Hydrolysis did not involve any soluble oligomers apart from low amounts of cellobiose. Phenomena included in the mathematical model were substrate limitation, adsorption of enzyme onto substrate, glucose inhibition, temperature dependency of reaction rates, and thermal enzyme inactivation. In addition, substrate heterogeneity was described by a recalcitrance constant. Model parameters refer to both enzyme characteristics and substrate-specific characteristics. Quantitative model development was carried out on the basis of Avicel hydrolysis. In order to describe wheat straw hydrolysis, wheat straw specific parameter values were measured. Updating the pertinent parameters for wheat straw yielded a satisfactory description of wheat straw hydrolysis, thus underlining the generic potential of the model.

Enzyme distribution derived from macroscopic particle behavior of an industrial immobilized penicillin-G acylase

The macroscopic kinetic behavior of an industrially employed immobilized penicillin‐G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct d‐(–)‐phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100‐fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial‐rate d‐(–)‐phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active‐site titration. These experiments revealed that the loading of penicillin‐G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass‐transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 μm) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.

Thermodynamically controlled synthesis of cefamandole

The direct enzymatic synthesis of the antibiotic cefamandole is presented as an alternative for the current processes based on either chemical synthesis or kinetic enzymatic synthesis. The influence of pH and temperature on the apparent equilibrium constant was measured; from these data, the maximum cefamandole concentration was predicted. In spite of a beneficial apparent equilibrium constant at pH 3.75, less product was formed as compared to the concentration at the optimum pH (4.25). This effect is caused by the low solubility of one of the substrates at pH 3.75.The maximum product concentration (at the optimum pH of 4.25) was found to be 22 mM which is a significant concentration. The thermodynamically controlled synthesis may prove to be an alternative for the currently used processes if it can be combined with an effective in-situ cefamandole removal step.

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

Kinetics of D-malate production by permeabilized Pseudomonas pseudoalcaligenes.

Abstract The kinetics of d -malate production from maleate by permeabilized Pseudomonas pseudoalcaligenes was described by assuming competitive product inhibition in addition to biocatalyst inactivation. The kinetic parameters for conversion and inactivation were determined from a simultaneous fit of two independent data sets of substrate concentration vs. time. In order to select appropriate conditions, the effects of pH and temperature on the initial substrate conversion rate (excluding inactivation) were studied first. The pH optimum ranged from 7–8. The temperature optimum was approximately 45°C. The conversion parameters increased with pH and/or temperature. The first-order inactivation rate constant depended on temperature only; it increased strongly between 25–35°C.

Process Design for Enzymatic Adipyl-7-ADCA Hydrolysis

Adipyl‐7‐ADCA is a new source for 7‐aminodeacetoxycephalosporanic acid (7‐ADCA), one of the substrates for antibiotics synthesis. In this paper, a novel process for enzymatic 7‐ADCA production is presented. The process consists of a reactor, a crystallization step, a membrane separation step, and various recycle loops. The reactor can either be operated batch‐wise or continuously; with both types of processing high yields can be obtained. For batch reactors chemical degradation of 7‐ADCA can be neglected. For continuous reactors, chemical stability of 7‐ADCA is a factor to be taken into account. However, it was shown that the reaction conditions and reactor configuration could be chosen in such a way that also for continuous operation chemical degradation is not important. Downstream processing consisted of crystallization of 7‐ADCA at low pH, followed by a nanofiltration step with which, at low pH, adipic acid could be separated from adipyl‐7‐ADCA and 7‐ADCA. The separation mechanism of the nanofilter is based on size exclusion combined with charge effects. Application of this filtration step opens possibilities for recycling components to various stages of the process. Adipic acid can be recycled to the fermentation stage of the process while both adipyl‐7‐ADCA and 7‐ADCA can be returned to the hydrolysis reactor. In this way, losses of substrates and product can be minimized.

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics.

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.