Optimization of rifamycin B fermentation in shake flasks via a machine-learning-based approach

Rifamycin B is an important polyketide antibiotic used in the treatment of tuberculosis and leprosy. We present results on medium optimization for Rifamycin B production via a barbital insensitive mutant strain of Amycolatopsis mediterranei S699. Machine-learning approaches such as Genetic algorithm (GA), Neighborhood analysis (NA) and Decision Tree technique (DT) were explored for optimizing the medium composition. Genetic algorithm was applied as a global search algorithm while NA was used for a guided local search and to develop medium predictors. The fermentation medium for Rifamycin B consisted of nine components. A large number of distinct medium compositions are possible by variation of concentration of each component. This presents a large combinatorial search space. Optimization was achieved within five generations via GA as well as NA. These five generations consisted of 178 shake-flask experiments, which is a small fraction of the search space. We detected multiple optima in the form of 11 distinct medium combinations. These medium combinations provided over 600% improvement in Rifamycin B productivity. Genetic algorithm performed better in optimizing fermentation medium as compared to NA. The Decision Tree technique revealed the media-media interactions qualitatively in the form of sets of rules for medium composition that give high as well as low productivity.     

Application of a new medium supplement for propagation and storage of human cytomegalovirus

A new medium supplement, NU-SERUM, was evaluated for cultivation of human embryonic lung fibroblasts (HEL) and for propagation and storage of human cytomegalovirus (HCMV). NU-SERUM was comparable to fetal bovine serum (FBS) in promoting rapid growth of HEL if they were seeded at a sufficient density. HCMV replicated quite satisfactorily in HEL cultured with media supplemented with NU-SERUM as well as FBS. Inactivation of HCMV at 37 C occurred similarly when the medium contained FBS or NU-SERUM. However, at -70 C, HCMV was less stable in NU-SERUM-containing medium than in FBS-containing medium. Sorbitol added to the NU-SERUM-containing medium improved the unstableness of HCMV at -70 C, and HCMV was storable with such medium. Thus, NU-SERUM is useful as an alternative to FBS not only for growth of HEL but also for propagation and storage of HCMV.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

Microbiological studies on the formation of gramicidin S synthetases

Rapid and extensive growth of Bacillus brevis ATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol-ammonium sulfate-Tris-salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappearance.     

Utilization of seawater-urea as a culture medium for Spirulina maxima.

The possibilities of utilization of seawater enriched with ureas as the culture medium for a blue-green alga, Spirulina maxima, were investigated. Pretreatment by precipitation with NaHCO3 and (or) Na2CO3 was found essential to remove the excess amounts of Ca2+ and Mg2+ present in seawater prior to cultivation. A culture medium as good as the synthetic medium reported in the literature for the growth of S. maxima was obtained after treating seawater with NaHCO3 (19.2 g/L) at pH 9.2 and 35 degrees C for 2 h, filtering to remove precipitates, and enriching with K2HPO4 (0.5 g/L), NaNO3 (3.0 g/L), and FeSO4 (0.01 g/L). The same results were obtained by substituting a small amount (0.2 g/L or less) of either crystalline or polymerized urea for the NaNO3 in the above medium. Growth of S. maxima was inhibited at higher concentration of urea in the culture medium. The inhibition effect was due to the partial decomposition of urea into ammonia in alkali medium. Tests conducted on the 130-L cultivation open pond also confirmed that the seawater-urea medium supports growth of S. maxima as well as the best known synthetic medium.     

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.     

Effect of medium of lowered NaCl concentration on virus release and protein synthesis in cells infected with reticuloendotheliosis virus.

When cultures producing reticuloendotheliosis virus were incubated for 24 h in medium of lowered NaCl concentration, virus production was inhibited. The extent of inhibition increased as the salt concentration of the medium was decreased. The inhibition was rapidly reversed by replacement of low-salt medium with normal medium. During the first hour after the inhibited cultures were returned to normal medium, virus was released at an accelerated rate, making the total amount of virus released by inhibited and control cultures the same. After 1 h in normal medium, the rate of virus production in the previously inhibited cultures was the same as in the control cultures. Incubation of infected cells in low-salt medium resulted in a 60% decrease in the overall rate of protein synthesis. Although returning the cells to normal medium rapidly reversed the inhibition of virus production, it did not rapidly increase the rate of protein synthesis. These results suggest that host cell-directed protein synthesis is preferentially inhibited by the low-ionic-strength medium, whereas that required for virus production continues.     

The development of a medium supplemented with egg extract for the maintenance of Drosophila cell lines.

A saline extract was prepared from Drosophila eggs. When diluted to a concentration of 1% with Drosophila tissue culture medium, it did not support growth of cells from the Drosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time four Drosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonic Drosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells.     

酵母浸出粉在奈瑟脑膜炎双球菌培养中的比较观察

目的探索A群,C群奈瑟脑膜炎双球菌(简称流脑)多糖疫苗生产中奈瑟脑膜炎双球菌培养的最适培养基。方法在培养基配制中用增减酵母浸出粉的方法制备相应的培养基,8h收菌,通过菌体的收获量并参考多糖量来确定较好的培养基配比。结果不同培养基用于A群、C群奈瑟脑膜炎菌培养8h后均有收获,其中2号培养基(酵母浸出粉)培养的菌体的浓度明显高于1号和3号培养基,它们之间有显著性差异(P<0.05)此种培养基能提高奈瑟脑膜炎双球菌的产量。结论添加酵母浸出粉的培养基可作为A群、C群奈瑟脑膜炎双球菌培养的最适培养基。     

Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage.

A general medium named malt-yeast extract-sucrose agar (MYSA) containing oxgall was designed. The medium was intended for the enumeration and isolation of molds and yeasts in routine examinations of animal feed stuffs. In this study MYSA was tested as a general medium for mycological examination of silage. The medium was compared with dichloran-rose bengal medium (DRBC) in an examination of more than 500 specimens of big bale grass silage. Selected characteristics of known fungal species commonly isolated from feeds were examined after growth on MYSA and DRBC and on malt extract agar, used as a noninhibitory control medium. MYSA suppressed bacterial growth, without affecting the growth of fungi common in feeds. The fungi growing on MYSA were easily recognized, and the medium seemed to slow radial growth of fungal colonies, which permitted, easy counting. The number of species found was higher on MYSA than on DRBC. When we compared MYSA with DRBC for mycological examination of grass silage samples, MYSA was found to be the medium of choice.     

Control of Culex quinquefasciatus (Diptera: Culicidae) by Bacillus sphaericus and B. thuringiensis subsp. israelensis, Produced on a New Potato Extract Culture Medium

The efficacy of Bacillus sphaericus and B. thuringiensis israelensis produced on a new potato-based culture medium as well as the conventional culture medium of Luria Bertani was compared against Culex quinquefasciatus in the field. After sporulation, the spores/crystals were harvested and used. The bacterial samples controlled the larvae and pupae for three weeks. Mortality due to the bacterial toxins produced from the new culture medium was very high and comparable to that produced using conventional medium. But the cost for cultivating these bacteria using potato extract was much lower as compared with that of the conventional medium.     

光照、琼脂和碳源对离体培养中水曲柳根尖生长的影响

研究了光照有无、琼脂添加与否和不同碳源对离体培养的水曲柳根尖生长的影响。结果表明,不添加琼脂的液体培养基对根尖的生长较添加琼脂的固体培养基和固—液培养基好,原因是增加琼脂的量会抑制根的生长;暗培养比光培养更有利于水曲柳离体根尖的培养;蔗糖作为碳源的效果较果糖和麦芽糖好,其中以3%的蔗糖对离体根尖的生长效果最好。     

Selective Culture Medium to Survey the Incidence of Haemophilus Species

A culture medium for the selective isolation of Haemophilus species is described. Bacitracin and nutritional supplements were incorporated in a rich basal agar medium to which rabbit blood was added to distinguish hemolytic species. Colony counts of seven typed strains of H. influenzae on this medium were within practical limits of counts on other media tested for clinical use. The bacitracin medium was as reliable as hemoglobin-agar for detecting H. influenzae and more sensitive for detecting other Haemophilus species in a clinical survey with the advantage of selectivity.     

草莓试管苗分化培养基优化的研究

本研究采用三种培养基（A3、F、MS）进行草莓分化对比试验．结果表明我们研制的A3基对草莓试管苗分化有明显促进作用．连续培养11周,A3的草莓苗总分化率是F基的1．25倍,是MS基的1．51倍。而且,A3基成本低于其它两基。A3、F和MS无机盐含量分虽为：1782．58mg／L、2868．33mg／L和4633．33mg／L。无机盐种类的合理配比及其低浓度有利于草莓试管的分化培养。     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.     

Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

Intensification of the growth and development of Bacillus thuringiensis H14 266/2-1 by optimizing the nutrient medium

The mathematical method of experimental design was used to develop a new enzymic medium for cultivation of Bacillus thuringiensis H14 266/2-1, a bactoculicide producer. The optimized medium based on corn flour enzyme lysate as a carbon source and fodder yeast enzyme lysate as a source of nitrogen amine made it possible to increase twice the titre biomass yield for 24 h cultivation as compared to the initial medium. The above medium does not yield to the initial production medium in the insecticide activity.     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable.Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast.Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×10⁸/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day.The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.     

The medium cycle bioreactor (MCB): Monoclonal antibody production in a new economic production system

The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system. Eighty percent of the spent medium was re-used twice. The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances. Therefore, only 20% of the actual medium was necessary to run the recycling process. During seven days culture time in a two liter scale 5 grams of IgG₁ type monoclonal antibody was produced. During that period the cell specific productivity was constant. Renewal of proteins was omitted because the protein content in the system persisted at a high level. Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function). Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step. Only cheap metabolites that are consumed by the cells had to be supplemented. Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium.