Online measurement of viscosity for biological systems in stirred tank bioreactors

One of the most critical parameters in chemical and biochemical processes is the viscosity of the medium. Its impact on mixing, as well as on mass and energy transfer is substantial. An increase of viscosity with reaction time can be caused by the formation of biopolymers like xanthan or by filamentous growth of microorganisms. In either case the properties of fermentation broth are changing and frequently non-Newtonian behavior are observed, resulting in major challenges for the measurement and control of mixing and mass transfer. This study demonstrates a method for the online determination of the viscosity inside a stirred tank reactor. The presented method is based on online measurement of heat transfer capacity from the bulk medium to the jacket of the reactor. To prove the feasibility of the method, fermentations with the xanthan producing bacterium Xanthomonas campestris pv. campestris B100 as model system were performed. Excellent correlation between offline measured apparent viscosity and online determined heat transfer capacity were found. The developed tool should be applicable to any other process with formation of biopolymers and filamentous growth

Metabolic responses of Escherichia coli upon glucose pulses captured by a capacitive field-effect sensor

Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the "welfare" of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis-Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.status: publishe

Metabolic responses of Escherichia coli upon glucose pulses captured by a capacitive field-effect sensor.

Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the ‘‘welfare’’ of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis– Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed

SDS-PAGE Analysis of Enzyme Preparations.

<p>5 µg of protein were applied to each lane and separated on a 12% SDS-gel; Legend: SH_hex,wt = wildtype six-subunit SH purified from <i>Cn</i>; SH_tet,var1 = four-subunit SH purified from recombinant strain SH1F (N-terminally-StrepII tagged HoxF; HoxF*); SH_hex,var2 = six-subunit SH purified from recombinant strain SH2F (N-terminally-StrepII tagged HoxI; HoxI*).</p

Deletion experiments for <i>in vivo</i> maturation of the SH in <i>Escherichia coli</i>.

<p>Strains and values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068812#pone.0068812.s008" target="_blank">Table S3</a>; Control strains: <b>K0</b>: pSH4.wt (structural genes without maturation related genes); <b>K1A</b>: pSH4.wt and pM1 (structural genes and complete M1 set; 100%; 1.95±0.24 U·mg⁻¹). Deleted complexes or proteins omitted are indicated for the K1A deletion strains. <b>K1B</b> represents the substitution strain, in which the M1 Hyp proteins are replaced by the M2 analogs (pSH4.wt and pM2). Results are given as specific activities exhibited by extracts obtained from three independent experiments, which were normalized for control strain K1A. Error bars indicated represent standard deviations.</p

Selection of strains, which were generated in this study and used for production of recombinant SH variants.

a<p>SH variants: wt = wildtype SH, untagged; 1 = SH variant 1: four-subunit enzyme HoxFUYH with N-terminally StrepII-tagged HoxF; 2 = SH variant 2: six-subunit enzyme HoxFUYHI₂ with N-terminally StrepII-tagged HoxI.</p>b<p>All recombinant strains were generated with <i>E. coli</i> BL21Star™ (DE3) as the basic strain.</p>c<p>Specific activities were determined in extracts from cells obtained in three independent ‘autoinduction’ batches under optimized growth conditions outlined in the methods section of the paper. Given values are arithmetic means of the triplicate measurements. Statistical values indicated (±) represent standard deviations. 1 Unit is defined as the H₂-mediated reduction of 1 µmol NAD⁺per minute.</p

Purification table for endogenous SH purified from <i>Cupriavidus necator</i> cells.

<p>About 20 g of wet packed cells were used for purification.</p>a<p>H₂:NAD⁺physiological activity, measured under anaerobic conditions. 1 Unit is defined as the H₂-mediated reduction of 1 µmol NAD⁺per minute.</p

UV/Vis Spectroscopy of Recombinant SH.

<p>Main: Spectrum of purified, oxidized SH_var2 (1 mg·mL⁻¹); Inset: Difference spectrum between oxidized and dithionite (500 µM) reduced enzyme.</p

Purification table for recombinant SH variant 1 (four-subunit enzyme with 5′-Strep tagged HoxF) purified from recombinant <i>E. coli</i> strain SH1F cells.

<p>About 6 g of wet packed cells were used for purification.</p>a<p>H₂:NAD⁺physiological activity, measured under anaerobic conditions. 1 Unit is defined as the H₂-mediated reduction of 1 µmol NAD⁺per minute.</p