Fed-batch Mode Research Articles

Yarrowia lipolytica application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L−1) and was stimulated by moderate pH (4.5) and agitation (800 rev min−1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L−1 of PA with volumetric productivity of 0.90 g L−1 h−1. The yield of PA biosynthesis reached a high level of 1.03 g g−1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.

Methane Production by Co-Digesting Vinasse and Whey in an AnSBBR: Effect of Mixture Ratio and Feed Strategy.

The most common approach to deal with vinasse (sugarcane stillage) is fertigation, but this technique compromises soil structure and surrounding water bodies. A possible solution is to transport vinasse to local cheese whey producers and perform the co-digestion of these wastewaters together, reducing their organic load and generating bioenergy. Therefore, this study investigated the application of an AnSBBR (anaerobic sequencing batch biofilm reactor) operated in batch and fed-batch mode, co-digesting vinasse and whey at 30°C. The effect of influent composition and feeding strategy was assessed. In all conditions, the system achieved high organic matter removal (approximately 83%). Increasing the percentage of vinasse from 0 to 100% in the influent resulted in a decrease in methane productivity (76.3 to 51.1 molCH4 m-3 day-1) and yield (12.7 to 9.1 molCH4 kgCOD-1), but fed-batch mode operation improved reactor performance (73.0 molCH4 m-3 day-1 and 11.5 molCH4 kgCOD-1). From the kinetic metabolic model, it was possible to infer that, at the best condition, methane is produced in a similar way from the acetoclastic and hydrogenotrophic routes. A scheme of four parallel reactors with a volume of 16,950m3 each was proposed in the scale-up estimation, with an energy recovery estimated in 28,745MWh per month.

Boosting mediated electron transfer in bioelectrochemical systems with tailored defined microbial cocultures.

Bioelectrochemical systems (BES) hold great promise for sustainable energy generation via a microbial catalyst from organic matter, for example, from wastewater. To improve current generation in BES, understanding the underlying microbiology of the electrode community is essential. Electron mediator producing microorganism like Pseudomonas aeruginosa play an essential role in efficient electricity generation in BES. These microbes enable even nonelectroactive microorganism like Enterobacter aerogenes to contribute to current production. Together they form a synergistic coculture, where both contribute to community welfare. To use microbial co-operation in BES, the physical and chemical environments provided in the natural habitats of the coculture play a crucial role. Here, we show that synergistic effects in defined cocultures of P. aeruginosa and E. aerogenes can be strongly enhanced toward high current production by adapting process parameters, like pH, temperature, oxygen demand, and substrate requirements. Especially, oxygen was identified as a major factor influencing coculture behavior and optimization of its supply could enhance electric current production over 400%. Furthermore, operating the coculture in fed-batch mode enabled us to obtain very high current densities and to harvest electrical energy for 1 month. In this optimized condition, the coulombic efficiency of the process was boosted to 20%, which is outstanding for mediator-based electron transfer. This study lays the foundation for a rationally designed utilization of cocultures in BES for bioenergy generation from specific wastewaters or for bioprocess sensing and for benefiting from their synergistic effects under controlled bioprocess condition.

Influence of environmental and genotypic factors on biofilm formation by clinical isolates of group B streptococci

Group B Streptococcus (GBS) colonizes the gastrointestinal and urogenital tracts of approximately 30% of women, and it can cause sepsis and meningitis in neonates. GBS has been shown to form biofilms in vitro, but the effects of environmental and genotypic factors upon GBS biofilm formation are unclear. The aim of the present study was to optimize culture conditions for enhanced GBS biofilm production. Furthermore, this study also investigated the influences of strain lineage, pilus profile, and isolation source on GBS biofilm formation. The results demonstrate that the fed-batch mode and acidic pH strongly enhanced GBS biofilm formation in vitro. These findings suggest that the fed-batch mode may be suitable for both screening and fundamental studies of GBS biofilm formation. Moreover, this study demonstrated a correlation between the hyper virulent clonal complex 17 and a strong biofilm phenotype.

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

BackgroundScreening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli.ResultsIn combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation.ConclusionsThe newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations.

Biochemical characterization and immunogenicity of Neureight, a recombinant full-length factor VIII produced by fed-batch process in disposable bioreactors

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce ‘Neureight’, a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ± 556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

Improved microscale cultivation of Pichia pastoris for clonal screening

BackgroundExpanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development.ResultsParallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli. The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode.ConclusionThe comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.

Proteomic analysis of micro-scale bioreactors as scale-down model for a mAb producing CHO industrial fed-batch platform

The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.

Effect of recirculation on bioelectricity generation using microbial fuel cell with food waste leachate as substrate

Recirculation is one of the effective techniques used to upsurge the output of anaerobic reactors. The present study investigates the effect of recirculation of anolyte on bioelectricity generation using food waste leachate in two chamber Microbial Fuel Cell (MFC) with carbon electrodes and Ultrex as proton exchange membrane (PEM). The MFCs are operated in fed-batch mode at varying COD concentrations of 500–1250 mg/L with the hydraulic retention time of 17 h for recirculation. Maximum current density, power density and columbic efficiencies of 100.34 mA/m2, 14.42 mW/m2 and 10.25% respectively for MFC without recirculation and 150.30 mA/m2, 29.23 mW/m2 and 14.22% respectively for MFC provided with recirculation are obtained at COD of 1250 mg/L. Comparative performance analysis of the cells indicates that recirculation enhances the bioelectricity production in MFC. Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) analyses are also done to find the changes in PEM.

Hydrogen production rates with closely-spaced felt anodes and cathodes compared to brush anodes in two-chamber microbial electrolysis cells

Flat anodes placed close to the cathode or membrane to reduce distances between electrodes in microbial electrolysis cells (MECs) could be used to develop compact reactors, in contrast to microbial fuel cells (MFCs) where electrodes cannot be too close due to oxygen crossover from the cathode to the anode that reduces performance. Graphite fiber brush anodes are often used in MECs due to their proven performance in MFCs. However, brush anodes have not been directly compared to flat anodes in MECs, which are completely anaerobic, and therefore oxygen crossover is not a factor for felt or brush anodes. MEC performance was compared using flat felt or brush anodes in two-chamber, cubic type MECs operated in fed-batch mode, using acetate in a 50 mM phosphate buffer. Despite placement of felt anodes next to the membrane, MECs with felt anodes had a lower hydrogen gas production rate of 0.32 ± 0.02 m3-H2/m3-d than brush anodes (0.38 ± 0.02 m3-H2/m3-d). The main reason for the reduced performance was substrate-limited mass transfer to the felt anodes. To reduce mass transfer limitations, the felt anode electrolyte was stirred, which increased the hydrogen gas production rate to 0.41 ± 0.04 m3-H2/m3-d. These results demonstrate brush electrodes can improve performance of bioelectrochemical reactors even under fully anaerobic conditions.

Effect of extraction conditions on citrate-mediated phosphorus removal and recovery from intercepted particulate runoff

Abstract Phosphorus (P) is a nonrenewable mineral resource that is critical to achieving food security for the growing world population. However, nearly half of total annual mined P is lost through nonpoint particulate runoff streams. Previously, we demonstrated inorganic phosphate extraction from runoff particulate mineral phases using P-solubilizing fungi (PSF) generating the organic acids citrate and oxalate. The objective of the current study was to investigate the effect of extraction process conditions on P extraction efficiency and kinetics from impounded urban storm water runoff sediments using citrate as the sole P solubilization agent. Abiotic extraction experiments were conducted according to a one-factor-at-a-time approach. High P removal and solubilization were favored at low sediment loading (5 g/100 mL), low initial pH (3), and high initial organic acid loading (75 mM), with P recovery efficiencies of 75% to 100% and 240 ppm solubilized phosphate levels achieved under these conditions. Initial and equilibrium extractant pH levels between citrate pK a1 (3.08) and pK a2 (4.74) appeared conducive for citrate ligand-induced particulate P release, possibly due to optimal balance of protonated and dissociated carboxylic groups. The results suggest that operating the extraction under fed-batch or semi-continuous modes with pH control can help maximize runoff particulate P recovery efficiency in PSF-mediated bioextractions wherein citric acid is the major fungal organic acid product.

Efficient biocatalytic synthesis of nicotinic acid by recombinant nitrilase via high density culture

The constitutively expression system for P. putida nitrilase was firstly constructed to improve the nicotinic acid production and reduce the production costs. High density culture strategy was employed to enhance the biomass and nitrilase production of recombinant strain. The total nitrilase activity reached up to 654 U·mL−1 without the induction. 541 g·L−1 nicotinic acid was accumulated via fed batch mode of substrate feeding through 290 min of conversion.

Dielectric Spectroscopy and Optical Density Measurement for the Online Monitoring and Control of Recombinant Protein Production in Stably Transformed Drosophila melanogaster S2 Cells.

The production of recombinant proteins in bioreactors requires real-time process monitoring and control to increase process efficiency and to meet the requirements for a comprehensive audit trail. The combination of optical near-infrared turbidity sensors and dielectric spectroscopy provides diverse system information because different measurement principles are exploited. We used this combination of techniques to monitor and control the growth and protein production of stably transformed Drosophila melanogaster S2 cells expressing antimicrobial proteins. The in situ monitoring system was suitable in batch, fed-batch and perfusion modes, and was particularly useful for the online determination of cell concentration, specific growth rate (µ) and cell viability. These data were used to pinpoint the optimal timing of the key transitional events (induction and harvest) during batch and fed-batch cultivation, achieving a total protein yield of ~25 mg at the 1-L scale. During cultivation in perfusion mode, the OD880 signal was used to control the bleed line in order to maintain a constant cell concentration of 5 × 107 cells/mL, thus establishing a turbidostat/permittistat culture. With this setup, a five-fold increase in productivity was achieved and 130 mg of protein was recovered after 2 days of induced perfusion. Our results demonstrate that both sensors are suitable for advanced monitoring and integration into online control strategies.

Process Analytical Technology for Advanced Process Control in Biologics Manufacturing with the Aid of Macroscopic Kinetic Modeling.

Productivity improvements of mammalian cell culture in the production of recombinant proteins have been made by optimizing cell lines, media, and process operation. This led to enhanced titers and process robustness without increasing the cost of the upstream processing (USP); however, a downstream bottleneck remains. In terms of process control improvement, the process analytical technology (PAT) initiative, initiated by the American Food and Drug Administration (FDA), aims to measure, analyze, monitor, and ultimately control all important attributes of a bioprocess. Especially, spectroscopic methods such as Raman or near-infrared spectroscopy enable one to meet these analytical requirements, preferably in-situ. In combination with chemometric techniques like partial least square (PLS) or principal component analysis (PCA), it is possible to generate soft sensors, which estimate process variables based on process and measurement models for the enhanced control of bioprocesses. Macroscopic kinetic models can be used to simulate cell metabolism. These models are able to enhance the process understanding by predicting the dynamic of cells during cultivation. In this article, in-situ turbidity (transmission, 880 nm) and ex-situ Raman spectroscopy (785 nm) measurements are combined with an offline macroscopic Monod kinetic model in order to predict substrate concentrations. Experimental data of Chinese hamster ovary cultivations in bioreactors show a sufficiently linear correlation (R2 ≥ 0.97) between turbidity and total cell concentration. PLS regression of Raman spectra generates a prediction model, which was validated via offline viable cell concentration measurement (RMSE ≤ 13.82, R2 ≥ 0.92). Based on these measurements, the macroscopic Monod model can be used to determine different process attributes, e.g., glucose concentration. In consequence, it is possible to approximately calculate (R2 ≥ 0.96) glucose concentration based on online cell concentration measurements using turbidity or Raman spectroscopy. Future approaches will use these online substrate concentration measurements with turbidity and Raman measurements, in combination with the kinetic model, in order to control the bioprocess in terms of feeding strategies, by employing an open platform communication (OPC) network—either in fed-batch or perfusion mode, integrated into a continuous operation of upstream and downstream.

High biobutanol production integrated with in situ extraction in the presence of Tween 80 by Clostridium acetobutylicum

Abstract This study investigates the improvement of butanol production in the presence – Tween 80 by Clostridium acetobutylicum NJ4. With supplementation of 15% Tween 80 (v/v), the final butanol titer could be significantly enhanced to 18.65 g/L from 70 g/L glucose, which is 38% higher than that in the control batch (13.47 g/L). With further in situ extraction using biodiesel, the butanol titer was finally improved to 33.90 g/L in the fed batch mode. The supplementation of Tween 80 could improve butanol tolerance of strain NJ4 up to 18 g/L compared to 10 g/L of control. Meanwhile, butanol dehydrogenase activity was improved to 3.89 U/mg, which is 80.93% higher than that of the control. These factors may attribute to the high butanol production. The finding of this study thus offers fundamental knowledge for the future development of alternative ABE production strategies.

Evaluation of anammox biocathode in microbial desalination and wastewater treatment

This study presents the use of an autotrophic microorganism, Anammox bacteria, as a sustainable biocatalyst/biocathode in microbial desalination cells (MDCs) for energy-positive wastewater treatment. We report the first proof of concept study to prove that anammox mechanism can be beneficial in MDCs to provide simultaneous removal of carbon and nitrogen compounds from wastewater while producing bioelectricity. A series of experiments were conducted to enrich and evaluate the anammox mechanism and the process performance in continuous, fed-batch mode conditions. Coulombic efficiency of MDCs and nitrite and ammonium removal of wastewater increased in successive batch studies. A maximum power density of 0.092 Wm−3 (or a maximum current density of 0.814 A m−3) with more than 90% of ammonium removal was achieved in this system. We calculated the Nernst potential for the nitrite reduction in the anammox biocathode chamber and compared with experimental values. Sequential removal of carbon and nitrogen compounds in anode and cathode chambers respectively, was also evaluated. Further, the inhibition effect of high nitrogen concentrations and the variations in microbial community profiles, especially, anammox presence was studied at different carbon and ammonia concentrations. Experimental studies and microbial community analysis are presented in detail.

High-solids ethanol fermentation with single-stage methane anaerobic digestion for maximizing bioenergy conversion from a C4 grass (Pennisetum purpereum)

High-solids simultaneous saccharification and fermentation integrating single-stage anaerobic digestion was designed for bioenergy conversion from steam-exploded Pennisetum purpereum, a perennial C4 grass. Three scenarios for ethanol production, methane production and ethanol-methane co-production were investigated and compared in terms of production yield. A terminal ethanol titer of 62.6 ± 1.9 g/L with the highest ethanol yield of 90.9% was achieved when the fed-batch mode was carried out at a final substrate loading of 35% (w/v) dry matter content. The ethanol stillage was used for methane production in a semi-continuous CSTR system operated with different organic loading rates (OLRs) at a hydraulic retention time (HRT) of 30 days. The high performance was achieved at OLR of 6.0 g VS·L−1 d−1, average yielding 358.7L CH4 per kilogram volatile solid (VS) with a total VS removal rate of 73.8%. On the basis of mass balance, the overall production yield achieved in this study was: 121.6 g ethanol + 110.6 g methane per kilogram grass with a finally 89.6% of cellulose recovery + 62.5% hemicellulose recovery. These results indicated that the high-solids ethanol fermentation with single-stage methane anaerobic digestion of lignocellulosic biomass is a valid approach to maximize the holocellulose bioconversion and improve the bioenergy production.

A pilot-scale bioprocess to produce amphidinols from the marine microalga Amphidinium carterae: Isolation of a novel analogue

Marine dinoflagellate microalgae belonging to the genus Amphidinium are a key source of an interesting group of polyketide metabolites with potent bioactivities, wide-ranging functional diversity and stereochemical complexity, but low natural availabilities. The feasibility of a microalgae dinoflagellate-based sustainable bioprocess for producing amphidinols (APDNs) by photoautotrophic culture of Amphidinium carterae in a pilot-scale LED-illuminated bubble column photobioreactor (PBR) was therefore investigated. A fed-batch culture mode with pulse feeding strategy provided a growth pattern strongly limited by the availability of phosphate content in the culture medium that stimulated the production of cellular APDNs. Since A. carterae was found to be much more shear-sensitive than other shear-tolerant non-dinoflagellate microalgae, the culture height and air flow rate were established to ensure the absence of damaging levels of hydrodynamic stress. The biomass capacity yielded by the PBR at the end of the culture (0.540 g d.w. L−1 equivalent to 1.70 × 106 cell mL−1) corresponded to that estimated stoichiometrically from the experimentally determined biomass P-molar formula (C329 O126 H732 N69 S3 P1) and the total phosphorus and nitrogen balances. The downstream processing section was initially conceived to recover APDNs excreted by cells into the supernatant. A dry APDN-enriched extract concentration of 49 mg per liter of supernatant was obtained. This purification process led to partitioning of the extract into several fractions and sub-fractions thereof. Only two sub-fractions were studied, yielding thereof highly pure (>95% pure) luteophanol D and lingshuiol A, and a new, roughly purified (>80% pure) APDN, namely amphidinol 20. The percentages of luteophanol D, lingshuiol A and amphidinol 20 by dry weight of the APDN-enriched extract obtained were 1%, 0.39% and 0.31%, respectively, thus representing a concentration in the culture supernatant of 490, 191 and 152 μg L−1, respectively.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BackgroundThe increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.ObjectivesHere, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and MethodsThe encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.ResultsIn fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.ConclusionsOur founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

In-Situ Biocatalytic Production of Trehalose with Autoinduction Expression of Trehalose Synthase.

We developed an in-situ biocatalytic process that couples autoinduction expression of trehalose synthase (TreS) and whole-cell catalysis for trehalose production. With lactose as the autoinducer, the activity of recombinant TreS in recombinant Escherichia coli was optimized through a visualization method, which resulted in a maximum value of 12 033 ± 730 U/mL in pH-stat fed-batch fermentation mode. Meanwhile, the permeability of the autoinduced E. coli increased significantly, which makes it possible to be directly used as a whole-cell biocatalyst for trehalose production, whereby the byproduct glucose can also act as an extra carbon source. In this case, the final yield of trehalose was improved to 90.5 ± 5.7% and remained as high as 83.2 ± 5.0% at the 10th batch, which is the highest value achieved using recombinant TreS. Finally, an integrated strategy for trehalose production was established, and its advantages compared to the traditional mode have been summarized.