Optimization of the dilute maleic acid pretreatment of wheat straw.

BACKGROUND: In 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 euro/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables. RESULTS: When 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 degrees C, with 46 mM maleic acid, resulting in a yield of 65 euro/Mg (megagram = metric ton) dry straw, consisting of 68 euro/Mg glucose benefits (from solids: 85% of all glucan), 17 euro/Mg xylose benefits (from liquid: 80% of all xylan), 17 euro/Mg maleic acid costs, 2.0 euro/Mg heating costs and 0.68 euro/Mg NaOH costs. In all but the most severe of the studied conditions, furfural formation was so limited that associated costs are considered negligible. CONCLUSIONS: After 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 euro/Mg dry feedstock. This is reached when process conditions are: 50 min at 170 degrees C, with a maleic acid concentration of 46 mM. Maleic acid replenishment is the most important of the studied cost factors.

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.

Virtual parameter-estimation experiments in Bioprocess-Engineering education.

Cell growth kinetics and reactor concepts constitute essential knowledge for Bioprocess-Engineering students. Traditional learning of these concepts is supported by lectures, tutorials, and practicals: ICT offers opportunities for improvement. A virtual-experiment environment was developed that supports both model-related and experimenting-related learning objectives. Students have to design experiments to estimate model parameters: they choose initial conditions and 'measure' output variables. The results contain experimental error, which is an important constraint for experimental design. Students learn from these results and use the new knowledge to re-design their experiment. Within a couple of hours, students design and run many experiments that would take weeks in reality. Usage was evaluated in two courses with questionnaires and in the final exam. The faculties involved in the two courses are convinced that the experiment environment supports essential learning objectives well.

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 microm) 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.