Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase

High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ. In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge

On bubble clustering and energy spectra in pseudo-turbulence

3D-Particle Tracking (3D-PTV) and Phase Sensitive Constant Temperature Anemometry in pseudo-turbulence--i.e., flow solely driven by rising bubbles-- were performed to investigate bubble clustering and to obtain the mean bubble rise velocity, distributions of bubble velocities, and energy spectra at dilute gas concentrations ($\alpha \leq2.2$%). To characterize the clustering the pair correlation function $G(r,\theta)$ was calculated. The deformable bubbles with equivalent bubble diameter $d_b=4-5$ mm were found to cluster within a radial distance of a few bubble radii with a preferred vertical orientation. This vertical alignment was present at both small and large scales. For small distances also some horizontal clustering was found. The large number of data-points and the non intrusiveness of PTV allowed to obtain well-converged Probability Density Functions (PDFs) of the bubble velocity. The PDFs had a non-Gaussian form for all velocity components and intermittency effects could be observed. The energy spectrum of the liquid velocity fluctuations decayed with a power law of -3.2, different from the $\approx -5/3$ found for homogeneous isotropic turbulence, but close to the prediction -3 by \cite{lance} for pseudo-turbulence

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting

Numerical study of the coupling between reaction and mass transfer for liquid-liquid slug flow in square microchannels

While the benefits of miniaturisation on processes have been widely demonstrated, its impact on microfluidics and local mechanisms such as mass transfer is still little understood. The aim of this work is to simulate coupling between reaction and mass transfer in microchannels for liquid-liquid slug flow. First, the extrapolation to confined flow of the classical model used to calculate interfacial mass fluxes in reactive infinite media was studied. This model consists in estimating transferred fluxes between two phases as a function of the enhancement factor E. Its expression depends on the model used to represent interfacial mass transfer. In infinite media, Lewis and Whitman’s stagnant film theory is generally preferred for its simplicity and its reliability. In the case of confined slug flow, the limitation of such a model to predict interfacial fluxes is highlighted. Secondly, the case of liquid-liquid competitive consecutive reactions in microchannels is considered. This work emphasizes the unfavourable impact of the length between droplets on selectivity. This is a direct consequence of mass transport mechanisms in microchannels

Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification. 2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium. For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

BACKGROUND: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. RESULTS: For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. CONCLUSION: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations

Thermodynamic and Kinetic Aspects of Mercury Sorption on Activated Carbon in the Process of Mercury Bioreduction

A unique biotechnological method for remediation of industrial wastewater contaminated by toxic mercury,based on the enzymatic reduction of ionic mercury by live bacteria,has been developed by prof.Deckwer and co-workers at GBF (at present HZI), Germany,and implemented in a pilot-plant scale.The experience gained during operation of this installation led to the idea,that the process of bioremediation may be integrated in one bioreactor with the adsorption of mercury by immobilization of the bacteria onto the activated carbon.For this it was necessary to define several significant parameters of the activated carbon selected for the process and the adsorption process itself. The paper presents results of the equilibrium and kinetics investigations of the process of ionic mercury sorption from aqueous solutions onto 8 different types of activated carbon.The effective diffusion coefficients in the carbon particles were obtained from the transient-state experiments using a mathematical model of the process and the sorption isotherms as well as the saturation capacity of the sorbents in relation to ionic and metallic mercury were identified.From the temperature dependence of adsorption con- stants the values of adsorption enthalpy for both metallic and ionic mercury on activated carbon impregnated with sulfur were estimated.The obtained results enabled selection of the optimal sorbent for the fixed-bed activated-carbon bioreactor which will be applied for the modified,integrated process of biological detoxification of mercury in industrial wastewaters

Gas-liquid-solid flow modelling in a bubble column

An alternative approach to the modelling of solid-liquid and gas-liquid-solid flows for a 5:1 height to width aspect ratio bubble column is presented here. A modified transport equation for the volume fraction of a dispersed phase has been developed for the investigation of turbulent buoyancy driven flows (Chem. Eng. Proc., in press). In this study, a modified transport equation has been employed for discrete phase motion considering both solid-liquid and gas-liquid-solid flows. The modelling of the three-phase flow in a bubble column was achieved in the following case: injecting a slug of solid particles into the column for 10 s at a velocity of 0.1 m s-1 and then the gas phase flow was initiated with a superficial gas velocity of 0.02 cm s-1. © 2003 Elsevier B.V. All rights reserved

The modelling of buoyancy driven flow in bubble columns

Using the analogy between lateral convection of heat and the two-phase flow in bubble columns, alternative turbulence modelling methods were analysed. The k-ε turbulence and Reynolds stress models were used to predict the buoyant motion of fluids where a density difference arises due to the introduction of heat or a discrete phase. A large height to width aspect ratio cavity was employed in the transport of heat and it was shown that the Reynolds stress model with the use of velocity profiles including the laminar flow solution resulted in turbulent vortices developing. The turbulence models were then applied to the simulation of gas-liquid flow for a 5:1 height to width aspect ratio bubble column. In the case of a gas superficial velocity of 0.02 m s-1 it was determined that employing the Reynolds stress model yielded the most realistic simulation results. © 2003 Elsevier B.V. All rights reserved

Dynamic optimization of a gas-liquid reactor

The final publication is available at Springer via http://dx.doi.org/10.1007/s10910-011-9941-1A dynamic gas-liquid transfer model without chemical reaction based on unsteady film theory is considered. In this case, the mathematical model presented for gas-liquid mass-transfer processes is based on mass balances of the transferred substance in both phases. The identificability property of this model is studied in order to confirm the possible identifiable parameters of the model from a given set of experimental data. For that, a different modeled of the system is given. A procedure for the identification is proposed. On the other hand, the aim of this work is to solve the quadratic optimal control problem, using an explicit representation of the model. The problem includes some results on controllability, observability and stability criteria and the relation between these properties and the parameters of the model. Using the optimal control problem we study the stability of the system and show how the choice of the weighting matrices can improve the behavior of the system but with an increase of the energy control cost. © 2011 Springer Science+Business Media, LLC.This work has been partially supported by PAID-05-10-003-295 and by MTM2010-18228.Cantó Colomina, B.; Cardona Navarrete, SC.; Coll, C.; Navarro-Laboulais, J.; Sánchez, E. (2012). Dynamic optimization of a gas-liquid reactor. Journal of Mathematical Chemistry. 50(2):381-393. https://doi.org/10.1007/s10910-011-9941-1S381393502Bayón L., Grau J.M., Ruiz M.M., Suárez P.M.: Initial guess of the solution of dynamic optimization of chemical processes. J. Math. Chem. Model. 48, 28–37 (2010)Ben-Zvi A., McLellan P.J., McAuley K.B.: Ind. Eng. Chem. Res. 42, 6607–6618 (2003)Cantó B., Coll C., Sánchez E.: Structural identifiability of a model of dialysis. Math. Comp. Model. 50, 733–737 (2009)Cantó B., Coll C., Sánchez E.: Identifiability of a class of discretized linear partial differential algebraic equations. Math. Probl. Eng. 2011, 1–12 (2011)Craciun G., Pantea C.: Identifiability of chemical reaction networks. J. Math. Chem. 44, 244–259 (2008)Dai L.: Descriptor Control Systems. Springer, New York (1989)Deckwer W.D.: Bubble Column Reactors. Wiley, Chichester (1992)Kantarci N., Borak F., Ulgen K.O.: Bubble column reactors. Proc. Biochem. 40(7), 2263–2283 (2005)Kawakernaak H., Sivan R.: Linear Optimal Control Systems. Wiley-Interscience, New York (1972)Kuo B.C.: Automatic Control Systems, 6th edn. Prentice-Hall, Englewood Cliffs (1991)Navarro-Laboulais J., Cardona S.C., Torregrosa J.I., Abad A., López F.: Practical identifiability analysis in dynamic gas-liquid reactors. Optimal experimental design for mass-transfer parameters determination. Comp. Chem. Eng. 32, 2382–2394 (2008)Navarro-Laboulais J., López F., Torregrosa J.I., Cardona S.C., Abad A.: Transient response, model structure and systematic errors in hybrid respirometers: structural identifiabilit analysis based on OUR and DO measurements. J. Math. Chem. 44(4), 969–990 (2007)Patel R., Munro N.: Multivariable Systen. Theory and Design. Pergamon Press, New York (1982)Sondergeld K.: A generalization of the Routh–Hurwitz stability criteria and a application to a problem in robust controller design. IEEE Trans. Automat. Contr. AC-28(10), 965–970 (1983