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      Real‐time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO 2 removal in shake flasks and mini‐bioreactors leads to superior growth and recombinant protein yields

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          Abstract

          Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O 2 (DO) and dissolved CO 2 (dCO 2) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small‐scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real‐time monitoring of DO and dCO 2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO 2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.

          Abstract

          Surface aeration intensification was implemented in shake flasks enabled with in house developed monitoring sensors for pH, dissolved oxygen and dissolved CO 2. Efficient removal of CO 2 from the shake flasks and enhanced O 2 supply resulted in increased biomass growth and protein yield. Improved surface aeration in shake flasks can translate into better scale down models and smoother scale up activities, and thus will bridge the gap that exists in process development.

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          Most cited references 31

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          Introduction to advantages and problems of shaken cultures.

           Jochen Büchs (2001)
          Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.
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            Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals.

            Harnessing lipogenic pathways and rewiring acyl-CoA and acyl-ACP (acyl carrier protein) metabolism in Yarrowia lipolytica hold great potential for cost-efficient production of diesel, gasoline-like fuels, and oleochemicals. Here we assessed various pathway engineering strategies in Y. lipolytica toward developing a yeast biorefinery platform for sustainable production of fuel-like molecules and oleochemicals. Specifically, acyl-CoA/acyl-ACP processing enzymes were targeted to the cytoplasm, peroxisome, or endoplasmic reticulum to generate fatty acid ethyl esters and fatty alkanes with tailored chain length. Activation of endogenous free fatty acids and the subsequent reduction of fatty acyl-CoAs enabled the efficient synthesis of fatty alcohols. Engineering a hybrid fatty acid synthase shifted the free fatty acids to a medium chain-length scale. Manipulation of alternative cytosolic acetyl-CoA pathways partially decoupled lipogenesis from nitrogen starvation and unleashed the lipogenic potential of Y. lipolytica Taken together, the strategies reported here represent promising steps to develop a yeast biorefinery platform that potentially upgrades low-value carbons to high-value fuels and oleochemicals in a sustainable and environmentally friendly manner.
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              Dual excitation ratiometric fluorescent pH sensor for noninvasive bioprocess monitoring: development and application.

              The development and application of a fluorescent excitation-ratiometric, noninvasive pH sensor for continuous on-line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion-exchange resin, which was subsequently entrapped into a proton-permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6-9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on-line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off-line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed-loop feedback control systems.
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                Author and article information

                Contributors
                grao@umbc.edu
                Journal
                Biotechnol Bioeng
                Biotechnol. Bioeng
                10.1002/(ISSN)1097-0290
                BIT
                Biotechnology and Bioengineering
                John Wiley and Sons Inc. (Hoboken )
                0006-3592
                1097-0290
                09 January 2020
                April 2020
                : 117
                : 4 ( doiID: 10.1002/bit.v117.4 )
                : 992-998
                Affiliations
                [ 1 ] Department of Chemical, Biochemical and Environmental Engineering Center for Advanced Sensor Technology, University of Maryland Baltimore Maryland
                Author notes
                [* ] Correspondence Govind Rao, Chemical, Biochemical & Environmental Engineering Director, Center for Advanced Sensor Technology (CAST), University of Maryland Baltimore County (UMBC), Baltimore, MD 21250.

                Email: grao@ 123456umbc.edu

                Article
                BIT27252
                10.1002/bit.27252
                7078866
                31840800
                © 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 5, Tables: 2, Pages: 7, Words: 4696
                Product
                Funding
                Funded by: Bill and Melinda Gates Foundation , open-funder-registry 10.13039/100000865;
                Categories
                Article
                ARTICLES
                Bioprocess Engineering and Supporting Technologies
                Custom metadata
                2.0
                April 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.7.8 mode:remove_FC converted:18.03.2020

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