Single-stage Culture Research Articles

Simplified bioreactor processes for recombinant butyrylcholinesterase production in transgenic rice cell suspension cultures

Metabolically regulated transgenic rice cell suspension culture has been developed aiming to be an efficient and cost-effective platform for production of rice-recombinant butyrylcholinesterase (rrBChE) that could be used as a bioscavenger enzyme against organophosphate nerve agents. In two-stage batch cultures, switching between growth and induction phases by exchanging media, the maximum active rrBChE levels were comparable among the bioreactor runs with controlled dissolved oxygen (DO) concentrations from 10–40% DO and without controlled DO at constant aeration rate. For a single-stage batch operation with no media exchange at 40% DO, production of rrBChE is triggered simply through sugar depletion due to the uptake of sugar by the cells, showing similar maximum active cell-associated rrBChE level compared to two-stage batch operations. However, the bioreactor process using uncontrolled DO, no media exchange, and half-concentration of sucrose resulted in a 1.4-fold increase in the maximum total active rrBChE (77 μg/g fresh weight) and 1.6-fold increase of total active rrBChE specific productivity (86 μg (g dry weight)−1 day−1) compared to the two-stage batch cultures. We found that monitoring the culture pH is a simple non-invasive technique that can help identify the start and progress of the induction phase in a single-stage culture. Altogether, we demonstrate a simplified, efficient, economical, and scalable bioreactor process for rrBChE production in metabolically regulated transgenic rice cell cultures.

A method to simplify bioreactor processing for recombinant protein production in rice cell suspension cultures

Transgenic plant cell suspension culture is a promising platform for recombinant protein production. Rice cell suspension culture is one of the systems that has been developed due to its unique metabolically-regulated promoter, rice alpha-amylase 3D (RAmy3D), that is up-regulated in sugar-deprived medium. Using the RAmy3D promoter system in transgenic rice cell suspensions results in two phases of the culture, the growth phase and the induction phase. Conventionally, medium exchange is performed to remove residual sugar and induce recombinant protein. In this work, a simplified production process is demonstrated in a 5-L bioreactor, including reduction of sugar concentration in the initial culture medium, elimination of the media exchange operation, and uncontrolled dissolved oxygen (DO) with constant aeration. The simplified method significantly improves the accumulation level of a recombinant protein, protein purity, and productivity compared to the conventional method. This method also reduces costs associated with material and labor.• The method of simplified bioreactor processing includes single-stage culture, uncontrolled dissolved oxygen (DO) but controlled inlet air flowrate, and lower (50% reduction) initial sucrose concentration in the culture medium.• This method improves recombinant protein production level and productivity compared to the conventional method.• This method reduces material and labor costs.

Improvement of biomass and fatty acid productivity in ocean cultivation of Tetraselmis sp. using hypersaline medium

In this study, hypersaline media were used for ocean cultivation of the marine microalga Tetraselmis sp. KCTC12432BP for enhanced biomass and fatty acid (FA) productivity. Hypersaline media (55, 80, and 105 PSU) were prepared without sterilization by addition of NaCl to seawater obtained from Incheon, Korea. The highest biomass productivity was obtained at 55 PSU (0.16 g L−1 day−1) followed by 80 PSU (0.15 g L−1 day−1). Although the specific growth rate of Tetraselmis decreased at salinities higher than 55 PSU, prevention of contamination led to higher biomass productivity at 80 PSU than at 30 PSU (0.03 g L−1 day−1). FA content of algal biomass increased as salinity increased to 80 PSU, above which it declined, and FA productivity was highest at 80 PSU. Ocean cultivation of Tetraselmis was performed using 50-L tubular module photobioreactors and 2.5-kL square basic ponds, closed- and open-type ocean culture systems, respectively. Culturing microalgae in hypersaline medium (80 PSU) improved biomass productivities by 89 and 152% in closed and open cultures, respectively, compared with cultures with regular salinity. FA productivity was greatly improved by 369% in the closed cultures. The efficacy of salinity shift and N-deficiency to enhance FA productivity was also investigated. Lowering salinity to 30 PSU with N-starvation following cultivation at 80 PSU improved FA productivity by 19% in comparison with single-stage culture without N-deficiency at 30 PSU. The results show that salinity manipulation could be an effective strategy to improve biomass and FA productivity in ocean cultivation of Tetraselmis sp.

Factors affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica

Biomass production and oil productivity in microalgae culture are the most important key factors for algal biodiesel production. However, proper culture condition for the biomass production of microalgae is different from that for the oil production of microalgae. A study on the biomass production of Tetraselmis suecica using various light intensities and nitrate concentrations as growth factors was carried out to evaluate proper culture conditions in 20-L batch culture. The effect of nitrate depletion on the oil accumulation was also evaluated with two-stage culture. It took 5 days to reach the stationary phase for the cultures of T. suecica on the light intensities of 108.9 and 133.1 μmol m(-2 )s(-1) with biomass of 0.89 and 0.88 g dcw L(-1), respectively. Biomass productions of 1.07 and 1.00 g dcw L(-1) were obtained with the nitrate concentrations of 18.6 and 24.7 mg L(-1), respectively. The two-stage culture increased oil contents from 7.6 to 17.3% (w/w) and contents of C(16)-C(18) fatty acids from 540.2 to 720.5 mg g(-1) oil. The predominant fatty acid was palmitic acid (C(16:0)) in nitrate depletion group, however, oleic acid (C(18:1)) was predominated in nitrate added groups. The two-stage culture enhanced overall oil productivity of 18.7 mg g(-1) day(-1) which is higher than that of 12.2 mg g(-1) day(-1) in single-stage culture.

Optimization of cell growth and carotenoid production of Xanthophyllomyces dendrorhous through statistical experiment design

This study was to identify and optimize the major culture factors for both cell growth and carotenoid biosynthesis in Xanthophyllomyces dendrorhous through statistical experiment design and data analysis. A screening test was first conducted on seven culture factors including pH and six medium components using a Plackett–Burman design, from which glucose, ammonium sulfate and pH were identified as the significant factors affecting both cell growth and carotenoid biosynthesis. These significant factors were optimized by central composite design of experiments and response surface methodology, as 34.3 g/L glucose, 2.95 g/L ammonium sulfate and pH 5.85 for cell growth in the culture, and 19.3 g/L glucose, 0.81 g/L ammonium sulfate and pH 5.19 for carotenoid accumulation in the yeast cells, respectively. Then a two-stage culture process was exercised with these optimal conditions tailored for cell growth (first stage) and carotenoid accumulation (second stage), achieving 95.8% higher volumetric yield (18.6 mg/L versus 9.5 mg/L in the single-stage culture) and 22.6% higher volumetric productivity (2.33 mg/(L day) versus 1.90 mg/(L day)) of total carotenoid. The medium optimization results suggest that carotenoid biosynthesis in X. dendrorhous is promoted by high C/N ratio, low carbon and nitrogen concentrations, and a slightly acidic condition when the cell growth is suppressed.

Development of the Two-Stage Culture System for Production of Useful Compounds Through Simultaneous Gene-Amplification

We have constructed the recombinant phage harboring the genes that code for enzymes for cysteine biosynthesis with acetylCoA regeneration system by using the Sleeper system that we have developed originally.We designed a two-stage culture system in which the stage between for growth and for enzyme production can be separated through a heat exchanger and the culture conditions for each stage can be controlled independently, combined with the Sleeper system. Fermentation performance of the lysogen that harbors the genes for cysteine biosynthesis in the two-stage culture system was compared with the single-stage culture system.In the two-stage culture system we have designed the serine acetyltransferase(SAT) activity reached to 2.5U/mg-protein while lU/mg-protein in case of the single-stage culture.

Development of anin vitroContinuous Flow Culture Model of the Murine Intestinal Tract

Enzyme activities and bacterial populations were compared in single stage, two stage (pH=5 and 7), and two stage with cell recycling continuous cultures of mouse faecal microbiota. Results were compared to in vivo findings in the mouse caecum and faeces in order to choose the system which best models the ecology of the murine lower intestinal tract. Fecal homogenates had higher numbers of total aerobes and lactose fermenting enterics than caecal homogenates. The microbial balance of the continuous culture systems more closely resembled the faecal microbiota (the culture inoculum) than the caecal microbiota. Lactobacilli numbers were significantly lower in single stage cultures than in two stage cultures or in vivo samples. Enzyme activities were comparable between culture types, with the exception of β-glucuronidase, which was closest to the in vivo level in single stage cultures. Because no one in vitro system proved superior with regards to all the parameters studied, the choice of which system to use may be based largely on the study purpose. Keywords: continuous culture, intestinal microbiota, enzymes.

Peroxidase production from carrot hairy root cell culture

Carrot hairy roots have shown vigorous growth and extensive lateral root branching in the absence of phytohormone. The production of peroxidase which was not detected in suspension cells was observed in high amounts in carrot hairy root. High peroxidase activity (19.2 Ug −1 fresh cell weight) was obtained by increasing the total phenolics concentration in the media. Peroxidase synthesis could be increased using elicitors such as yeast extract, V 2O 5, and Hg 2Cl 2, which are known to induce the synthesis of PAL (phenylalanine ammonia lyase). As the initial sucrose concentration in the medium increased, peroxidase activity and total phenolics increased while final cell mass decreased. A high level of sucrose also affected the elevation of the phenolics concentration in the cell. By adopting a two-stage culture method, the activity of peroxidase (55 U g −1 fresh cell weight) was ten times higher than that of a single-stage culture (5 U g −1 fresh cell weight) and about three times higher than that of field-grown horseradish on an activity per weight basis.

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l(-1) KH2PO4 and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.

Production of emetine and cephaeline from cell suspension and excised root cultures of Cephaelis ipecacuanha

Production of the ipecac alkaloids, emetine and cephaeline was studied in cell suspension and excised root cultures of Cephaelis ipecacuanha. A two-stage cell suspension culture was developed for enhanced accumulation of the alkaloids. In the first-stage, suspension cultures were established in Murashige and Skoog's (MS) medium containing 2,4-D and NAA which was suitable for cell growth and the second-stage culture system was composed of MS medium containing IBA, IAA and 6% sucrose which favoured alkaloid production. The production of emetine and cephaeline was greatly increased in the two-stage culture method compared to the single-stage culture. Optimal alkaloid synthesis was obtained in excised root culture of the plant in medium composed of half-strength MS salts, IBA (0.25 mgl −1) and 2% sucrose. A discernible higher accumulation of cephaeline in two-stage cell suspension culture as well as in excised root culture in comparison to that of the three-year-old roots was a

Enhanced production of Shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices

Production of shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices was investigated. The cells were effectively entrapped inside the polymer matrices prepared in a cubic form with a lateral length of 0.5 cm. They proliferated further by incubation and resulted in immobilized polymer foam cubes which were fully packed with cell aggregates. Shikonin production was observed to increase significantly when the immobilized cells were used. It was excreted by the cells and directly adsorbed, by hydrophobic interaction, to the polymer site. The product could be recovered, after the cells were separated, by direct solvent extraction from the shikonin-adsorbed polyurethane matrices. It is more interesting to note that a single-stage culture with immobilized L. erythrorhizon cells yielded higher shikonin productivity than the conventional two-stage culture system, which is considered to be of practical importance for industrial applications.

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.

The growth of Aspergillus terreus and the production of itaconic acid in batch and continuous cultures. The influence of pH

AbstractAspergillus terreus (NRRL 1960) was cultivated in batch and in continuous singlestage culture. The influence of pH on the growth of the organism, on the formation of itaconic acid and on the kinetics of fermentation was studied under phosphate limitation, both at controlled pH values and also when the pH was allowed to decrease in a natural way. In the pH range 1.7–3.5, the ratio of undissociated :half‐dissociated acid varied from 190:1 to 1.5:1. The amount of completely dissociated acid may be regarded as negligible. In batch systems operated without pH control, an initial pH of 3.1 proved to be the most effective. Product formation under such conditions started at a point where the exponential growth phase commenced and was described by a zero‐order equation. The maximum itaconic acid production rate was shifted behind maximum growth rate. The continuous single‐stage system was first order with respect to product formation. At pH > 3.1, a number of aberrant and pellet forms of the mould occurred, resulting in decreased acid production.

Lipid Accumulation in an Oleaginous Yeast ( Candida 107) Growing on Glucose Under Various Conditions in a One- and Two-Stage Continuous Culture

Lipid accumulation and fatty acid composition in Candida 107 have been studied using a two-stage continuous culture system in which the first vessel was run under carbon-limited conditions and then the entire output was passed into a second vessel, where lipid accumulation was stimulated by adding only glucose. Maximum lipid accumulation (28% of yeast [dry weight]) occurred for a volume ratio of vessel 1 to vessel 2 of 3:5, with 30 g of glucose per liter being added to vessel 2 operated at 25 degrees C with an aeration rate of between 0.1 and 1.0 volume of air/volume of medium per min. Although the maximum specific rate of lipid formation (0.05 g of lipid/g of yeast per h) was higher than in a nitrogen-limited, single-stage system, the efficiency of lipid formation was much less and never exceeded 14 g of lipid produced per 100 g of glucose consumed. The fatty acid composition was not significantly altered in either the two-stage or single-stage culture (nitrogen-limited) systems by changes in growth temperature (from 19 to 33 degrees C) or aeration rates (0.05 to 1.0 volume of air/volume of medium per min); or, in the two-stage system, by changes in the residence time of the yeast in the second vessel (from 3.2 to 24.4 h), or, in the single-stage system, by changes in pH (from 3.5 to 7.5). Only when the concentration of glucose entering vessel 2 of the two-stage system was less than 30 g/liter did significant changes in the fatty acids occur. Thus, although a two-stage continuous culture system allows lipid accumulation to be separated from the growth phase, it offers no practical advantages over a single-stage system as a means of producing microbial oils and fats.