Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5?g·L^?1), pig bone peptone (40.0?g·L^?1), Mg²⁺ (0.05?mol·L^?1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4?U mL^?1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4?g L^?1. Through this cultivation approached 30?°C with 0?g·L^?1 initial glucose concentration, the maximum β-amylase activity reached 5371.8?U mL^?1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

In situ pH management for microbial culture in shake flasks and its application to increase plasmid yield

Shake flasks are widely used to culture microorganisms, but they do not allow for pH control without additional infrastructure. In the presence of a carbon source like glucose, culture pH typically decreases due to overflow metabolism and can limit the growth of microorganisms in shake flasks. In this study, we demonstrate the use of magnesium hydroxide-loaded pH managing hydrogels (m-pHmH) for in situ base release to counter the decrease in culture pH in shake flasks using Escherichia coli as a model organism, in both complex and mineral salts medium. Base release from m-pHmH is shown to increase with decreasing pH (22-fold increase in release rate from pH 8 to 5), thus providing feedback from culture pH. The addition of m-pHmH resulted in better pH maintenance and higher biomass yields of E. coli K12 in media containing glucose as a carbon source. The use of m-pHmH with additional buffer resulted in pH being maintained above 6.9 while pH decreases below 5 without m-pHmH. We demonstrate one application of such in situ pH management to increase the volumetric plasmid yield from E. coli in shake flask culture. In situ glucose release through a hydrogel to mimic fed-batch culture along with the addition of m-pHmH resulted in a 395 % increase in volumetric plasmid yield to 38 μg/ml in shake flask culture.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Enhanced ethanol production through selective adsorption in bacterial fermentation

To alleviate the ethanol inhibition of Escherichia coli KO11 (ATCC 55124), during fermentation, online ethanol sequestration was achieved using F-600 activated carbon. Two separate schemes were tested, one involving direct addition of activated carbon to the fermentation flask for the purpose of in-situ adsorption and a second involving an externally located activated carbon packed bed. For the in-situ ethanol adsorption experiments, varying amounts of adsorbent were added to the medium at the start of the fermentation. The addition of the activated carbon in the fermentation broth resulted in increased glucose utilization and ethanol production for all flasks containing activated carbon. For the control flasks, approximately 75% of the available substrate was utilized before the fermentation was inhibited. The entire glucose supply of flasks containing activated carbon was depleted. Ethanol production was also increased from 28 g/L for the control containing no activated carbon to nearly 45 g/L (including the ethanol in the adsorbed phase) for the flasks containing activated carbon. The implementation of an externally located packed bed adsorber for the purpose of on-line ethanol removal was tested over a number of adsorption cycles to evaluate the performance of the adsorption bed and the ethanol productivity. Results indicate that maintaining ethanol fermentation medium concentrations below 20 ∼ 30 g/L extends and enhances ethanol productivity. After 3 cycles over a period of 180 h, an additional 80% ethanol was produced when compared to the control experiments, despite the suboptimal acidic pH of the medium.     

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Haemophilus influenzae type b (Hib), a Gram‐negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl‐ribitol‐phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake‐flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (～500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α‐ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508–1519, 2017     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.     

The use of glucose to regulate pH values of culture media and increase the production of baculovirus (BmNPV) and foreign protein (HBsAg)

When BmN-4 and M-BmN cells were grown in shake flasks, the pH initially dropped and later increased. The increase in pH signaled a ‘metabolic switch’ that was used here as an indicator for initiating a supplemental glucose and glutamine feed. Using the pH-based fed-batch culture method described, the maximum cell densities of BmN-4 cells and M-BmN cells were increased from 30×10⁵ cells ml⁻¹ to 43×10⁵ and 52×10⁵ cells ml⁻¹, respectively. Correspondingly, the production of polyhedra (4·5×10⁵ OBs ml⁻¹) and HBsAg (574 ng ml⁻¹), from the infection of BmN-4 and M-BmN by wild-type and recombinant BmNPV viruses, respectively, were both significantly enhanced 50% and 100%, respectively. This feeding strategy was implemented with no advanced instrumentation yet facilitated significantly increased yield in shake flasks. The technique should benefit those in research laboratories employing the baculovirus expression system as a rapid and efficient production system.     

Effects of Octadecanoylsucrose Derivatives on the Production of Inulinase by Apergillus niger SL-09

Summary The effect of the addition of octadecanoylsucrose esters to the growth medium on the production of inulinase by Aspergillus niger SL-09 was studied in batch culture using shake flasks. The activities of inulinase in vitro and in vivo formed by Aspergillus niger SL-09 was enhanced dramatically by the addition of sucrose ester S-770 to the medium, and it was confirmed that sucrose ester acted as a very efficient inducer for inulinase production. As a result, with the addition of 6 g sucrose ester l⁻¹ at the beginning of the culture, the enzyme activities were enhanced near 7-fold higher than that obtained in the basal medium.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

Improved guggulsterone production from sugars, precursors, and morphactin in cell cultures of Commiphora wightii grown in shake flasks and a bioreactor

Cell cultures of Commiphora wightii (Arnott.) Bhandari were grown in shake flasks and a bioreactor and an increase in guggulsterone accumulation up to 18 μg l⁻¹ was recorded in cells grown in the production medium containing a combination of sucrose:glucose (4% total), precursors (phenylalanine, pyruvic acid, xylose, and sodium acetate), morphactin, and 2iP. A yield of 10 g l⁻¹ biomass and ∼200 μg l⁻¹ guggulsterone was recorded in a 3-l flask and in a 2-l stirred tank bioreactor compared with 6.6 g biomass and 67 μg l⁻¹ guggulsterone in 250-ml flasks. Increased vessel size was correlated with increased biomass and guggulsterone accumulation. 2iP alone was not effective for biomass and guggulsterone accumulation in cell cultures of C. wightii.     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.