The production of butanol, acetone and ethanol by Clostridium acetobutylicum is a biphasic fermentation process. In the first phase the carbohydrate substrate is metabolized to acetic and butyric acid, in the following second phase the product spectrum is shifted towards the economically interesting solvents. Here we present a cascade of six continuous stirred tank reactors (CCSTR), which allows performing the time dependent metabolic phases of an acetone-butanol-ethanol (ABE) batch fermentation in a spatial domain. Experimental data of steady states under four operating conditions—with variations of the pH in the first bioreactor between 4.3 and 5.6 as well as the total dilution rate between 0.042 h−1 and 0.092 h−1—were used to optimize and validate a corresponding mathematical model. Beyond a residence time distribution representation and substrate, biomass and product kinetics this model also includes the differentiation of cells between the metabolic states. Model simulations predict a final product concentration of 8.2 g butanol L−1 and a productivity of 0.75 g butanol L−1 h−1 in the CCSTR operated at pHbr1 of 4.3 and D = 0.092 h−1, while 31% of the cells are differentiated to the solventogenic state. Aiming at an enrichment of solvent-producing cells, a feedback loop was introduced into the cascade, sending cells from a later state of the process (bioreactor 4) back to an early stage of the process (bioreactor 2). In agreement with the experimental observations, the model accurately predicted an increase in butanol formation rate in bioreactor stages 2 and 3, resulting in an overall butanol productivity of 0.76 g L−1 h−1 for the feedback loop cascade. The here presented CCSTR and the validated model will serve to investigate further ABE fermentation strategies for a controlled metabolic switch.

The infection process of bacterial gastroenteritis often relies on the initial binding of toxins to carbohydrate receptors on host cells. We screened the human gut microbiota for microorganisms naturally expressing toxin-binding carbohydrate structures. Out of stool samples from four healthy adult donors, we isolated bacterial strains specifically binding the cholera toxin and the heat labile toxin. These results suggest a new mechanism by which the microbiome may shape people’s individual sensitivity to gastrointestinal infections. This study may also pave the way for the development of non-antibiotic microbiome based strategies to treat and prevent gastrointestinal infections.

Besides its role during fermentation process, yeast also has potential as biological agent because of its antagonistics characteristic. We have isolated yeast from six different locations based on Phospourus differences. Isolated yeast were then tested its antagonistics ability of Fusarium oxysporum, the pathogen causing tomato wilt disease. This research aims to find out the diversity of yeast found in the tomato rhizosphere in six different contents of phosphorus locations and to know its antagonistics ability against the pathogenic. F. oxysporum. This research was conducted in the Laboratory of Mycology, Department of Pests and Diseases, Faculty of Agriculture, Brawijaya University, Malang and in the Chemistry Laboratory, Institut Teknologi 10 Nopember Surabaya, started from January up to September 2015. Yeasts have been isolated from tomato’s rhizosphere of 6 different locations around East Java proviences. The result showed that yeasts from organic field (Lower P content) were 6 genera. They are Candida sp. 1, Pichia sp. 1, Hansenula sp., Metschnikowia sp. 1, Cryptococcus sp., and Zygosaccharomyces sp. While the yeasts from inorganic field (higher P) were 3 genera. The most potential yeast in controlling F. oxysporum is Pichia sp. 2. Lower P content showed more divers than higher P content. Yeasts from higher soil P content showed more antagonists to control F. oxysporum. Keywords: Phosphor, correlation, yeast, antagonistic, disease, soil

Acetone-Butanol-Ethanol (ABE)-fermentation with Clostridium acetobutylicum is a biphasic fermentation process. The formation of organic acids in the so called acidogenesis has to precede the economically interesting phase of solvent formation called solventogenesis. A separation of these metabolic phases in two or more stages of continuously run bioreactors has been successfully applied earlier (Bahl et al., 1982). However no comprehensive mathematical modeling was performed for these multi-stage processes. We now established a new experimental model system, consisting of a cascade of continuous-stirred tank reactors (CCSTR). This arrangement enables us to gain insight into metabolic phases of the ABE-fermentation with an unprecedented resolution. Experimental data collected at two dilution rates are used here to verify a mathematical model of the continuous ABE-fermentation process. This model takes into account subpopulation dynamics, meaning that a differentiation between cells with enzyme equipments adapted to acidogenic, transition and solventogenic metabolism, respectively, is made. Applying our model we found that with the differentiation from acidogenic cells to solventogenic cells takes places in the first bioreactor stage at a dilution rate of 0.042 h-1, while this process is shifted to the second and third bioreactor at a dilution rate of0.092 h-1. Thus we conclude that the pH alone is not sufficient to trigger the metabolic switch between acidogenesis and solventogenesis.

The present paper reports the characterization of ABE (acetone-butanol-ethanol) production by Clostridium acetobutylicum DSM 792 for sugars representative of hydrolysed lignocellulosic biomass (glucose, mannose, arabinose, xylose). The attention was focused on: the selection of an optimal medium for the simultaneous conversion of the investigated sugars; the assessment of interference-synergistic effects during the fermentation of mixtures of the investigated sugars.The synthetic medium was optimised in terms of nutritional factors: the KH2PO4 K2HPO4 concentration was increased up to 5 g/L; the MgSO4 concentration was increased up to 2 g/L; the MnSO4 concentration was increased up to 0.1 g/L; the FeSO4 concentration ranged between 0.002 and 0.01 g/L); the CaCO3 concentration was increased up to 10 g/L The optimal concentration of the investigated factors was assessed and it varied from one sugar to another.The batch fermentations of a mixture of the four sugars highlighted their synergistic effects. Once set the initial concentration of the sugars (60 g/L), the butanol and solvent concentration increased up to 14.6 and 20.6 g/L, respectively, when the four sugars were present. (C) 2015 Elsevier Ltd. All rights reserved.

This paper presents a kinetic dynamic model of acetone-butanol-ethanol production by Clostridium acetobutylicum DSM 792 developed with the biochemical networks simulator COPASI. This model is an evolution of previous models described in the literature, updated by including various mono-, di-, hexose and pentose sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. The kinetic relationships of uptake of substrate, butanol production, cell growth and cell death are also included.The batch fermentation tests were carried out at an initial sugar concentration ranging from 5 to 100 g/L. The data from the batch tests were used to assess the kinetic parameters of the model. This model gave satisfactory results for each sugar, both in terms of simulation of fermentation - the square correlation coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged between 0.87 and 0.925 - and of comparison with the models reported in the literature.The effects of mono-, di-, hexose and pentose sugars on the growth and production of metabolites, including acids and solvents, were reviewed according to the proposed model. The low fermentation performance measured for xylose and lactose were interpreted taking into account the sugar uptake, the acid production and the hydrolysis path. (C) 2015 Elsevier B.V. All rights reserved.

The Acetone-Butanol-Ethanol (ABE) fermentation is receiving renewed interest as a way to upgrade renewable resources into valuable base chemicals and liquid fuels. Abundance and un-competitiveness with food sources are desired features of a potential substrate, and they are met by lignocellulosic biomass. This contribution is about the characterization of the ABE fermentation by C. acetobutylicum DSM 792 using sugars representative for hydrolysis products of lignocellulosic biomass: hexoses (glucose and mannose) and pentoses (arabinose and xylose). Batch fermentations of single sugars were carried out. The conversion process was characterized as a function of time in terms of biomass, acids and solvents concentrations as well as pH and total organic compounds. C. acetobutylicum was able to convert all investigated sugars. The conversion into solvents was strongly enhanced by the presence of CaCO3 in the fermentation medium.

The time-dependent development of cell polarizability and length in Escherichia coli batch fermentations were observed at-line with electrooptical measurements. While using a measurement system with fully automated sample preparation, the development of these properties can be observed with a comparable high frequency (six measurements per hour). The polarizability as well as the mean cell length both increase soon after inoculation and then decline from the growth phase on until the stationary phase is reached. Based on the dynamic behavior of polarizability, the growth phase can be divided into four distinct stages. Changes in the cultivation temperature or the pre-cultivation conditions lead to alterations in the development of the polarizability and mean cell length. Based on the frequency disperse of polarizability measured at four different frequencies from 210 to 2,100 kHz, a prediction model is established that is based on the relation of the polarizability to the metabolic activity. Applying multi-linear partial least squares methods (N-PLS), the model is able to predict the specific acetate synthesis and uptake with a root mean square error of prediction of 0.19 (6% of the mean). The method represents a tool for characterization of different stages with respect to microbial metabolic activity and the energy balance during batch cultivations.

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park “Lower Oder,” Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Early formulations of the RCWA yield, implicated by the erroneous application of factorization rules to discrete Fourier transformations, poor convergence in certain cases. An explanation for this finding and an approach to overcome the problem for crossed gratings was first given by Li [J. Opt. Soc. Am. A 13, 1870 (1996) and 14, 2758 (1997)]. A further improvement was achieved by Schuster et al. [J. Opt. Soc. Am. A 24, 2880 (2007)], using a structure dependent normal vector (NV) field. While it is trivial to create those NV fields for simple geometrical shapes, to our knowledge an appropriate algorithm for arbitrary shapes does not exist, yet. In this work we present such an algorithm.

In Fourier modal methods like the RCWA and the Differential Method the Li-rules for products in truncated Fourier space have to be obeyed in order to achieve good convergence of the results with respect to the mode number. The Li-rules have to be applied differently for parts of the field that are tangential and orthogonal to material boundaries. This is achieved in the Differential Method by including a field of vectors in the calculation that are normal to the material boundaries. The same can be done laterally in each layer of an RCWA calculation of a 2-D periodic structure. It turns out that discontinuities in the normal vector field can disturb the computation especially when metallic materials are dominant in the structure which would make the usefulness of the normal vector method questionable. So it is of great importance to investigate how normal vector fields can be established with as few discontinuities as possible. We present various methods for the 2-D RCWA and the I-D and 2-D Differential Method and compare the respective convergence behaviors. Especially we emphasize methods that are automatic and require as few user input as possible.

The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBitrade mark-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time.

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.

Today the synthesis of oligonucleotides is a well-established process. Using automatic synthesizers even kilogram quantities can be produced in a few hours. However, the purification of the final product is still time-consuming and needs a complex apparatus. In this article, a simple and fast purification method for the large-scale syntheses of oligonucleotides is described. According to the method of Sawadago and van Dyke ([1991] Nucleic Acids Res 19:674-675) for small-scale oligonucleotide purification, oligonucleotides in mumol to mmol amounts were purified by liquid-liquid extraction using butanole as the extraction liquid. Choosing appropriate ratios of extraction liquid to oligonucleotide solution, simultaneous purification and precipitation could be achieved. It was found that the yield of the purified oligonucleotide was mainly affected by the temperature. Yield decreased with increasing temperature. The use of this improved extraction procedure allows the purification of gram to kilogram quantities of oligonucleotides in less than a day with simple equipment and high yield.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.