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2021 ◽  
Vol 12 ◽  
Jianrong Wang ◽  
Xiaoming Li ◽  
Hao Chen ◽  
Bilian Lin ◽  
Liangzhong Zhao

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Fermentation ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 288
Andreea Cristina Dobrescu ◽  
Henrique César Teixeira Veras ◽  
Cristiano Varrone ◽  
Jan Dines Knudsen

An economically viable production of second-generation bioethanol by recombinant xylose-fermenting Saccharomyces cerevisiae requires higher xylose fermentation rates and improved glucose–xylose co-consumption. Moreover, xylose-fermenting S. cerevisiae recognises xylose as a non-fermentable rather than a fermentable carbon source, which might partly explain why xylose is not fermented into ethanol as efficiently as glucose. This study proposes propagating S. cerevisiae on non-fermentable carbon sources to enhance xylose metabolism during fermentation. When compared to yeast grown on sucrose, cells propagated on a mix of ethanol and glycerol in shake flasks showed up to 50% higher xylose utilisation rate (in a defined xylose medium) and a double maximum fermentation rate, together with an improved C5/C6 co-consumption (on an industrial softwood hydrolysate). Based on these results, an automated propagation protocol was developed, using a fed-batch approach and the respiratory quotient to guide the ethanol and glycerol-containing feed. This successfully produced 71.29 ± 0.91 g/L yeast with an average productivity of 1.03 ± 0.05 g/L/h. These empirical findings provide the basis for the design of a simple, yet effective yeast production strategy to be used in the second-generation bioethanol industry for increased fermentation efficiency.

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7173
Tao Cheng ◽  
Xiuhong Liang ◽  
Yaqun Wang ◽  
Ningning Chen ◽  
Dexin Feng ◽  

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2097
Georges Bastin ◽  
Véronique Chotteau ◽  
Alain Vande Wouwer

Although the culture of VERO cells in bioreactors is an important industrial bioprocess for the production of viruses and vaccines, surprisingly few reports on the analysis of the flux distribution in the cell metabolism have been published. In this study, an attempt is made to fill this gap by providing an analysis of relatively simple metabolic networks, which are constructed to describe the cell behavior in different culture conditions, e.g., the exponential growth phase (availability of glucose and glutamine), cell growth without glutamine, and cell growth without glucose and glutamine. The metabolic networks are kept as simple as possible in order to avoid underdeterminacy linked to the lack of extracellular measurements, and a unique flux distribution is computed in each case based on a mild assumption that the macromolecular composition of the cell is known. The result of this computation provides some insight into the metabolic changes triggered by the culture conditions, which could support the design of feedback control strategies in fed batch or perfusion bioreactors where the lactate concentration is measured online and regulated by controlling the delivery rates of glucose and, possibly, of some essential amino acids.

Brian James Kirsch ◽  
Sandra V. Bennun ◽  
Adam Mendez ◽  
Amy S. Johnson ◽  
Hongxia Wang ◽  

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Catherine Boy ◽  
Julie Lesage ◽  
Sandrine Alfenore ◽  
Stéphane E. Guillouet ◽  
Nathalie Gorret

AbstractIt is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a system. In this work, robustness of an engineered strain is defined as plasmid expression stability, cultivability, membrane integrity and macroscopic cell behavior and was assessed in response to implementations of sugar feeding strategies (pulses and continuous) and two plasmid stabilization systems (kanamycin resistance and Post-Segregational Killing hok/sok). Fed-batch bioreactor cultures, relevant mode to reach high cell densities and higher cell generation number, were implemented to investigate the robustness of C. necator engineered strains. Host cells bore a recombinant plasmid encoding for a plasmid expression level monitoring system, based on eGFP fluorescence quantified by flow cytometry. We first showed that well-controlled continuous feeding in comparison to a pulse-based feeding allowed a better carbon use for protein synthesis (avoiding organic acid excretion), a lower heterogeneity of the plasmid expression and a lower cell permeabilization. Moreover, the plasmid stabilization system Post-Segregational Killing hok/sok, an autonomous system independent on external addition of compounds, showed the best ability to maintain plasmid expression level stability insuring a greater population homogeneity in the culture. Therefore, in the case of engineered C. necator, the PSK system hok/sok appears to be a relevant and an efficient alternative to antibiotic resistance system for selection pressure, especially, in the case of bioprocess development for economic and environmental reasons.

Polymers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 3950
Anna Żywicka ◽  
Daria Ciecholewska-Juśko ◽  
Radosław Drozd ◽  
Rafał Rakoczy ◽  
Maciej Konopacki ◽  

The aim of this study was to demonstrate the applicability of a novel magnetically assisted external-loop airlift bioreactor (EL-ALB), equipped with rotating magnetic field (RMF) generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200 × higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible water-related properties, mechanical strength, nano-fibrillar structure and total crystallinity index. The lack of any negative impact of inoculum preparation method using RMF-assisted EL-ALB on BC properties is of paramount value for its future applications, including use as a biomaterial in tissue engineering, wound healing, and drug delivery, where especially BC liquid capacity, nanostructure, crystallinity, and mechanical properties play essential roles.

Amélie Mahé ◽  
Alexandra Martiné ◽  
Séverine Fagète ◽  
Pierre-Alain Girod

AbstractIn the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (qP), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the qP with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.

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