Correction to: Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus.

Unfortunately, in the "How to Cite as" section, the given and the family name of the author was incorrectly published, the correct name is Lladó Maldonado. S.

Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus.

Bioreactors at the microliter scale offer a promising approach to accelerate bioprocess development. Advantages of such microbioreactors include a reduction in the use of expensive reagents. In this study, a chemostat operation mode of a cuvette-based microbubble column bioreactor made of polystyrene (working volume of 550 µL) was demonstrated. Aeration occurs through a nozzle (Ø ≤ 100 µm) and supports submerged whole-cell cultivation of Staphylococcus carnosus. Stationary concentrations of biomass and glucose were determined in the dilution rate regime ranging from 0.12 to 0.80 1/h with a glucose feed concentration of 1 g/L. For the first time, reaction kinetics of S. carnosus were estimated from data obtained from continuous cultivation. The maximal specific growth rate (µmax = 0.824 1/h), Monod constant (KS = 34 × 10- 3gS/L), substrate-related biomass yield coefficient (YX/S = 0.315 gCDW/gS), and maintenance coefficient (mS = 0.0035 gS/(gCDW·h)) were determined. These parameters are now available for further studies in the field of synthetic biology.

Cellular uptake of coumarin-6 under microfluidic conditions into HCE-T cells from nanoscale formulations.

UNLABELLED: In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. RESULTS: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.

Improving 2-phenylethanol and 6-pentyl-α-pyrone production with fungi by microparticle-enhanced cultivation (MPEC).

Trichoderma atroviride IMI 206040 synthesizes the coconut lactone 6-pentyl-α-pyrone (6-PAP) de novo and Aspergillus niger DSM 821 produces the rose-like flavour compound 2-phenylethanol (2-PE) from the precursor l-phenylalanine. Here, microparticles of different chemical composition and nominal particle diameter in the range 5-250 µm were added to shake-flask cultures of both fungi to investigate the particles' effect on product formation. Maximum 2-PE concentration increased by a factor of 1.3 to 1430 mg/l with the addition of 2% w/v talc (40 µm diameter). Maximum 6-PAP concentration increased by a factor of 2 to 40 mg/l with the addition of 2% w/v iron (II, III) oxide. The influence of ions leaching out of the particles was investigated by cultivating the fungi in leached particle medium. For the first time, the positive effect of the microparticle-enhanced cultivation (MPEC) technique on the microbial production of volatile metabolites, here flavour compounds from submerged fungal cultures, is demonstrated. The effect is strain- and particle-specific.

Disposable parallel poly(dimethylsiloxane) microbioreactor with integrated readout grid for germination screening of Aspergillus ochraceus.

In this work a disposable, parallel microbioreactor (MBR) suitable for screening in batch or continuous mode is presented. The reactor consists of five parallel microchambers made of poly(dimethylsiloxane) bonded to a glass substrate. A grid structure is engraved on each chamber, allowing subsequent morphology imaging. Measurements are recorded over the entire cultivation period with constant parameters, namely, position and focus in the z-axis. The microdevice may be used for either parallel, uni- or multiparametric screening, and overcomes the drawback of gridless microwell plates which require expensive equipment such as an inverted microscope with an automatic stage. To validate the scalability from laboratory scale to microscale, and thus the cultivation protocol in the MBR, the germination of fungal spores (A. ochraceus) is evaluated for two different key magnitudes (pH and temperature) and compared to the results obtained from conventional laboratory scale systems (flasks and agar plates). Information on germination capacity with regard to interspecies' variability allows for optimization of industrial processes as optimal pH and temperature matched to the mesoscopic cultivation systems. The germination conditions therefore remain unaffected inside the MBR, while providing the following advantages: (i) dramatic reduction of medium consumption, (ii) submerged cultivation with constant oxygen supply, (iii) assured low cost and disposability, and (iv) possibility of a continuous cultivation mode.

Morphology and rheology in filamentous cultivations.

Because of their metabolic diversity, high production capacity, secretion efficiency, and capability of carrying out posttranslational modifications, filamentous fungi are widely exploited as efficient cell factories in the production of metabolites, bioactive substances, and native or heterologous proteins, respectively. There is, however, a complex relationship between the morphology of these microorganisms, transport phenomena, the viscosity of the cultivation broth, and related productivity. The morphological characteristics vary between freely dispersed mycelia and distinct pellets of aggregated biomass, every growth form having a distinct influence on broth rheology. Hence, the advantages and disadvantages for mycelial or pellet cultivation have to be balanced out carefully. Because of the still inadequate understanding of the morphogenesis of filamentous microorganisms, fungal morphology is often a bottleneck of productivity in industrial production. To obtain an optimized production process, it is of great importance to gain a better understanding of the molecular and cell biology of these microorganisms as well as the relevant approaches in biochemical engineering. In this chapter, morphology and growth of filamentous fungi are described, with special attention given to specific problems as they arise from fungal growth forms; growth and mass transfer in fungal biopellets are discussed as an example. To emphasize the importance of the flow behavior of filamentous cultivation broths, an introduction to rheology is also given, reviewing important rheological models and recent studies concerning rheological parameters. Furthermore, current knowledge on morphology and productivity in relation to the environom is outlined in the last section of this review.

Population balance modeling of the conidial aggregation of Aspergillus niger.

Numerous biotechnological production processes are based on the submerse cultivation of filamentous fungi. Process design, however, is often hampered by the complex growth pattern of these organisms. In the morphologic development of coagulating filamentous fungi, like Aspergillus niger, conidial aggregation is the first step of filamentous morphogenesis. For a proper description of this phenomenon it is necessary to characterize conidial populations. Kinetic studies performed with an in-line particle size analyzer suggested that two distinct aggregation steps have to be considered. The first step of conidial aggregation starts immediately after inoculation. Both the rate constants of formation and disintegration of aggregates have been determined by measuring the concentration of conidia at the beginning of the cultivation and the concentration of particles at steady state during the first hours of cultivation. In contrast to the first aggregation step, where the collision of conidia is presumed to be responsible for the process, the second aggregation step is thought to be initiated by germination of conidia. Growing hyphae provide additional surface for the attachment of non- germinated conidia, which leads to a strong decrease in particle concentration. The specific hyphal length growth rate and the ratio of particle concentration to the growing adhesion hyphal surface are decisive matters of the second aggregation step. Both aggregation steps can be described by population dynamics and simulated using the program package PARSIVAL (PARticle SIze eVALution) for the treatment of general particle population balances.

Morphology and productivity of filamentous fungi.

Cultivation processes involving filamentous fungi have been optimised for decades to obtain high product yields. Several bulk chemicals like citric acid and penicillin are produced this way. A simple adaptation of cultivation parameters for new production processes is not possible though. Models explaining the correlation between process-dependent growth behaviour and productivity are therefore necessary to prevent long-lasting empiric test series. Yet, filamentous growth consists of a complex microscopic differentiation process from conidia to hyphae resulting in various macroscopically visible appearances. Early approaches to model this morphologic development are recapitulated in this review to explain current trends in this area of research. Tailoring morphology by adjusting process parameters is one side of the coin, but an ideal morphology has not even been found. This article reviews several reasons for this fact starting with nutrient supply in a fungal culture and presents recent advances in the investigation of fungal metabolism. It illustrates the challenge to unfold the relationship between morphology and productivity.

Influence of mechanical stress and surface interaction on the aggregation of Aspergillus niger conidia.

Productivity of fungal cultures is closely linked with their morphologic development. Morphogenesis of coagulating filamentous fungi, like Aspergillus niger, starts with aggregation of conidia, also denominated as spores. Several parameters are presumed to control this event, but little is known about their mode of action. Rational process optimization requires models that mirror the underlying reaction mechanisms. An approach in this regard is suggested and supported by experimental data. Aggregation kinetics was examined for the first 15 h of cultivation under different cultivation conditions. Mechanical stress was considered as well as pH-dependent surface interaction. Deliberations were based on a two-step aggregation mechanism. The first aggregation step is only affected by the pH-value, not by the fluid dynamic conditions in the bioreactor. The second aggregation step, in contrast, depends on the pH-value as well as on agitation and aeration induced power input. For the given experimental set-up, agitation had much more influence than aeration. In addition, hyphal growth rate was determined to be the driving force for the second aggregation step.

Production integrated treatment of textile wastewater by closing raw material cycles.

A method for the in-house treatment of partial wastewater flows and the recycling of treated process water into the textile finishing process was developed in order to recycle effluents from textile finishing industry and feed them back into the production process. The method is based on a two-stage biological anaerobic-aerobic process to split colouring wastewater agents and to degrade organic substances contained in the water as well as a chemical stage to remove the remaining color of the water with the help of ozone. In the framework of a research and development project a demonstration plant for a treatment capacity of 1440 m3 per working day was installed and started in a textile finishing company. At the plant, a wastewater flow and a recycling flow are treated separately in two different treatment lanes. Approximately 40% of the total wastewater flows, i.e. 576 m3/d are treated in the wastewater lane, and a maximum of 60% of total wastewater, i.e. 864 m3/d are treated in the recycling lane. Thanks to the preliminary treatment of wastewater flows, which are discharged into the municipal sewage works, a reduction of average COD levels in the sewage works effluents could be achieved.

Kinetic studies on the aggregation of Aspergillus niger conidia.

Morphology has a crucial effect on productivity and the supply of substrate for cultures of filamentous fungi. However, cultivation parameters leading to the desired morphology are often chosen empirically as the mechanisms governing the processes involved are usually unknown. For coagulating microorganisms like Aspergillus niger the morphological development is considered to start with the aggregation of conidia right after inoculation. To elucidate the mechanism of this process, kinetic studies were carried out using an in-line particle size analyzer. Based on the data obtained from these experiments a model for conidial aggregation is proposed in this article. It consists of two separate aggregation steps. The first one takes place immediately after inoculation, but only leads to a small decrease of total particle concentration. Most suspended conidia aggregate after a second aggregation step triggered by germination and hyphal growth. Aggregation velocity of this second phase is linearly dependent on the particle growth rate.

Recycling of dyehouse effluents by biological and chemical treatment.

A combined biological and chemical process of purification and recycling of residual dyehouse effluents was developed, investigated and installed at a textile finishing company which produces 330,000 m3 colored wastewater effluents per year. The process divided effluent into two streams. Both streams were subjected to anaerobic dye-cleavage, aerobic mineralization of cleavage-products and biomass separation. One stream was also membrane filtered and treated with ozone, which made possible the recycling of 60% of the total discharge. By these means it was possible to increase the quality of the treated streams for recycling purposes, as well as the dye capacity of the textile mill, and to minimize the operating costs. Furthermore, the municipal wastewater treatment plant into which the textile finishing mill's water is discharged, did not need to enhance its capacity.

Treatment of dyehouse liquors in a biological sequencing batch reactor with recursive chemical oxidation.

A new developed sequencing batch process for the purification of residual water containing concentrated azo dye was investigated. Within a treatment cycle the biological anoxic decolorization, followed by an aerobic mineralization of organic metabolites in combination with the biodegradability-achieving partial oxidation with ozone are carried out sequentially. The split flow can be destructively purified to 90% with respect to the parameter DOC. It was decolorized to an extent of 98% and the toxicity measured by the bioluminescence test decreased up to 99%. With an unspecific facultative anaerobic bacterial mixed culture anoxic decolorization of the residual liquor (20 gdye/L) without addition of an external auxiliary substrate was observed. In the first phase of the treatment cycle, the azo dye-molecules are cleft at the azo bond by biochemical reduction which leads to the corresponding sulfonated aromatic amines. In the following aerobic phase the cleft products were mineralized by the same microorganisms in the same reactor. Because of the recalcitrant and respectively toxic character of a part of the remaining metabolites, further aerobic mineralization was initialized by partial oxidation with ozone. The recursive ozonization in a recircled stream with biological post-treatment of the transformed substances led to an increased reaction selectivity and lower consumption of ozone. The results have shown that the chosen sequencing batch reactor with the ozonization bypass is suitable for an effective treatment of high concentrated dyehouse liquors.

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II.

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.

Biochemical structural analysis of the lantibiotic mutacin II.

Mutacin II is a post-translationally modified lantibiotic peptide secreted by Streptococcus mutans T8, which inhibits the energy metabolism of sensitive cells. The deduced amino acid sequence of promutacin II is NRWWQGVVPTVSYECRMNSWQHVFTCC, which is capable of forming three thioether bridges. It was not obvious, however, how the three thioether bridges are organized. To examine the bridging, the cyanogen bromide cleavage products of mutacin II and its variants generated by protein engineering, C15A, C26A, and C15A/C26A, were analyzed by mass spectrometry. Analysis of the wild type molecule and the C15A variant excluded several possibilities and also indicated a high fidelity of formation of the thioether bridges. This allowed us to further resolve the structure by analysis (mass spectrometry and tandem mass spectrometry) of the cyanogen bromide cleavage fragments of the C26A and C15A/C26A mutants. Nuclear magnetic resonance analysis established the presence of one and two dehydrobutyrine residues in mutacin II and the C15A variant, respectively, thus yielding the final structure. The results of this investigation showed that the C-terminal part contains three thioether bridges connecting Cys residues 15, 26, and 27 to Ser/Thr residues 10, 12 and 19, respectively, with Thr(25) being modified to dehydrobutyrine.

Iridoid glycoside biosynthesis in Penstemon secundiflorus. Another H-5, H-9 trans-iridoid glycoside.

Isolation and characterization of the new iridoid 10-hydroxy-(5 alpha H)-6-epidihydrocornin from Penstemon secundiflorus (Scrophulariaceae) is described. In biosynthetic experiments, deoxyloganic acid was incorporated into the trans-fused iridoid glycosides (5 alpha H)-6-epidihydrocornin and 10-hydroxy-(5 alpha H)-6-epidihydrocornin in P. secundiflorus. Formation of the trans-fused compounds is therefore a late event in the biosynthesis and does not occur during iridoid formation by cyclization of the open chain monoterpene precursor. In the same plant, 8-epideoxyloganic acid was not incorporated into the trans-iridoids. Deoxyloganic acid was also incorporated into 10-hydroxyhastatoside (which bears an 8 beta-methyl group), while 8-epideoxyloganic acid was incorporated into penstemoside (with an 8 alpha-methyl group). Thus, iridoid biosynthetic pathways leading from both deoxyloganic acid and 8-epideoxyloganic acid were found in the same plant.

Trans-fused iridoid glycosides from Penstemon mucronatus.

Two new trans-fused iridoid glycosides (5 alpha H)-6 alpha-8-epidihydrocornin and (5 alpha H)-6 alpha-8-hydroxy-8-epiloganin, were isolated from Penstemon mucronatus, along with cornin, penstemoside and three hastatosides. The trans-fused iridoids are only the second and third known among over 900 described cis-fused iridoid glycosides. Two pairs of iridoids, identical except for the stereochemistry at C-8, were found. Structures were determined by spectroscopic methods.

The application of behavior modification to behavior management: guidelines for the family physician.

Treatment in ambulatory care settings depends on the patient's behavior to implement the regimen prescribed by the family physician. Behavior modification offers a means for developing behavioral programs that will improve the physician's ability to effectively influence the patient's behavior. This article outlines some of the principles of behavior modification as they can be applied in office settings to diverse behavioral problems. Some specific suggestions are made for identifying behaviors, setting goals, delineating the steps to reach these goals, monitoring progress, and developing treatment contracts that will aid the physician in the management of the behavioral aspects of treatment.

[Radiogenic disorder in sense of smell (author's transl)]. / Radiogene Riechstörungen

The influence of radiotherapy on the sense of smell was investigated in 24 patient (age 28-80 years) with Elsberg's method. Out of 19 patients, who were measured 1-15 years after radiotherapy, the thresholds exceeded the normal range in no case, i.e. no permanent disturbance of smell after radiotherapy. In 5 patients, tested before, during and after irradiation, the thresholds rised continuously to the upper margin of the normal limit between 1,000 and 6,000 rads and, after the end of therapy returned to initial levels within 6-8 weeks.