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 €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.     

Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N‐protected phenylalanine in near‐anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0–80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration‐induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration‐induced inactivation that takes place during the first activity measurement, (2) a phase of first‐order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:870–875, 2013     

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 7.4 pmol 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.     

Growth of Ca-D-malate crystals in a bioreactor

To develop a bioreactor for solid-to-solid conversions, the conversion of solid Ca-maleate to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes was studied. In a bioreactor seeded with product (Ca-D-malate) crystals, growth of Ca-D-malate crystals is the last step in the solid-to-solid conversion and is described here. Crystal growth is described as a transport process followed by surface processes. In contrast to the linear rate law obeyed by the transport process, the surface processes of a crystal-growth process can also obey a parabolic or exponential rate law. Growth of Ca-D-malate crystals from a supersaturated aqueous solution was found to be surface-controlled and obeyed an exponential rate law. Based on this rate law, a kinetic model was developed which describes the decrease in supersaturation due to Ca-D-malate crystal growth as a function of the constituent ions, Ca(2+) and D-malate(2-). The kinetic parameters depended on temperature, but, as expected (surface-controlled), they were hardly affected by the stirring speed.     

A multicomponent reaction–diffusion model of a heterogeneously distributed immobilized enzyme

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.     

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.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Vegetation study as a generator for population biological and physiological research on salt marshes

The paper deals with some views on the phytocoenose in relation to the functioning of vegetation and its plant-species populations in space and time. From these viewpoints the study of vegetation is seen as a field of tension between the organismic and reductionistic approaches. Both have their value, provided any dogmatism is avoided and either can be applied to the other.In the field of vegetation structure characteristic features of life-form spectra and species distribution, inversion phenomena in zonation, and community architecture in relation to production and decomposition are discussed. In this connection some remarks are made on habitat and niche differentiation with respect to the phytocoenose concept.Vegetation dynamics are discussed in relation to the introduction of Spartina anglica, the frequency of flooding by the tides, different environmental disturbances caused by heavy winter frost, rainfall and hot and dry periods, as well as to human interferences for agricultural and civil-technical purposes.It is suggested that salt-marsh plants may have found refuge areas in inland habitats as well as on more southerly coastal sites during glaciations.Nomenclature follows Tutin et al. (1964–1980).The author wishes to thank his collaborators Messrs B. P. Koutstaal and W. de Munck for much fieldwork, and Mrs M. J. van Leerdam-de Dreu and Messrs A. A. Bolsius and J. A. van den Ende for the preparation of text and figures.Communication No. 302 of the Delta Institute for Hydrobiological Research.     

Some functional aspects of canine corticotrophs

To examine the regulation and functional significance of canine pituitary pars intermedia corticotrophs, ACTH and cortisol responses to CRF were studied in healthy dogs before and after treatment with dexamethasone. In addition the effects of the dopamine agonist bromocriptine and the dopamine antagonist pimozide were investigated. In the latter two instances prolactin concentrations were also measured. Finally the pituitaries were studied immunocytochemically for ACTH and alpha-MSH. No response of ACTH or cortisol to bromocriptine was observed. Pimozide caused a slight rise in ACTH levels in some dogs. However, prolactin levels significantly decreased with bromocriptine and increased with pimozide. Injection of synthetic ovine CRF to dogs was followed by sharp increases in ACTH and cortisol values. These responses were obliterated by prior treatment with dexamethasone. In 1 of 4 dogs given dexamethasone before euthanasia, there were few pars distalis cells with ACTH(1-24) immunopositivity, although persistence of ACTH(1-24) reaction was noted within cells of the pars intermedia. The results indicate that none of the CRF-induced ACTH secretion in dogs is derived from pars intermedia corticotrophs. Dosages of bromocriptine and pimozide that clearly alter prolactin secretion do not consistently affect ACTH levels.     

Novel anaerobic gas-lift reactor (AGLR) with retention of biomass: Start-up routine and establishment of hold up

A start-up routine for a novel type of anaerobic gas-lift reactor using sand as support particles for Bacterial adhesion and involving a dilution rate shift-up is shown to result in rapid formation of mixed-culture aggregates from freely suspended cells. Formation of aggregates changed the general metabolism from acetate-butyrate production to acetate-propionate production. This change is attributed to a selection by washout, favoring propionate-producing bacteria with superior adhesive properties. Sand is shown to be essential in establishing, but not in sustaining, elevated holdup ratios. The importance of maintenance processes and cellular lysis in deeper parts of aggregates are manifest from a reduced effluent biomass concentration and a pronounced production of valeric acid.     

Specific rates of substrate oxidation and product formation in autotrophically growingChromatium vinosum cultures

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.