Growth and carotenoid production by pH-stat cultures of Phaffia rhodozyma

To optimize biomass and carotenoid production by Phaffia rhodozyma in pH-stat cultures, two methods of feeding glucose were studied. In the first method, which is comparatively simple to operate, the glucose feeding set point (pH 5.02) was higher than the culture pH (5.00) and P. rhodozyma grew at a low specific growth rate (=0.055 h^–1). In the second method, the glucose feeding set point (pH 4.98) was lower than the culture pH (5.00) and the yeast grew at a specific growth rate of =0.095 h^–1. With the second method of glucose feeding, which is more complex and in order to prevent overfeeding of glucose, a time interval was added to the control strategy of the glucose pump and allowed to expire before the next dose of glucose was added. The length of the time interval affected biomass and carotenoid production. A critical time interval (T_c) was defined. In pH-stat cultures of P. rhodozyma, it was found that if the time interval was set longer than the critical time interval, the yeast did not grow.     

Isolation of astaxanthin-overproducing mutants of Phaffia rhodozyma

We isolated mutants of Phaffia rhodozyma strain NRRL Y-17269 that overproduced astaxanthin when grown on corn-based fuel ethanol stillage (thin stillage, TS, or fuel ethanol byproducts). Ten ml cultures of mutant strain JB2 produced 1.54 ± 0.21 mg carotenoid/mg dry weight when grown on 70% thin stillage at pH 5.2, compared with 0.38 ± 0.04 g/mg produced by the parental strain. Furthermore, JB2 produced similar astaxanthin concentrations when grown in either thin stillage or yeast malt broth. By comparison, previously described astaxanthin overproducing strain NRRL Y-17811 yielded 1.08 ± 0.07 g/mg in yeast malt broth but only 0.67 ± 0.03 g/mg in thin stillage. Five liter fermentation experiments using JB2 grown on 70% thin stillage at pH 5.2 yielded 2.01 ± 0.17 g/mg astaxanthin. Thus, JB2 is uniquely suited for astaxanthin production from low cost thin stillage.     

Batch cultivation and astaxanthin production by a mutant of the red yeast,Phaffia rhodozyma NCHU-FS501

Phaffia rhodozyma cells were treated with the mutagenic agent NTG several times and plated on yeast-malt agar containing -ionone as a selective medium. This mutagenesis of the yeast yielded a mutant (NCHU-FS501) with a total carotenoid content of 1454 g g^–1 dry biomass. Temperature and pH had only a slight effect on the volumetric pigment production by the red yeast, however astaxanthin yield and specific growth rate were influenced more significantly by temperature and pH. The optimum inoculum size, temperature and air flow rate for astaxanthin formation by the mutant in a bench-top fermentor were 7.5% (v/v), 22.5°C and 3.6 vvm, respectively. Glucose (1%, w/v) as carbon source yielded the highest volumetric astaxanthin production (6.72 g ml^–1). Peptone (15.8% total nitrogen) was the best nitrogen source for astaxanthin production (6.72 g ml^–1). Pigment formation by the mutant was further improved by increasing the glucose concentration to 3.5%, where the astaxanthin concentration was 16.33 m ml^–1. At 4.5% glucose or above astaxanthin formation was inhibited. Control of the pH of the fermentation broth did not improved pigment production.     

Astaxanthin production by Phaffia rhodozymaenhanced in fed-batch culture with glucose and ethanol feeding

Of various carbon sources, examined for the cultivation of Phaffia rhodozyma, ethanol enhanced the astaxanthin content but severely decreased growth. Therefore, high cell mass was obtained by glucose fed-batch culture with pH-stat, and the ethanol feeding was performed based on DO-stat. As a result of this two-stage fed-batch cultivation, 30 g dry cells per liter were obtained, and the astaxanthin content reached 0.72 mg/g, which was 2.2-fold higher than that without ethanol feeding.     

Feeding in the rotifer Brachionus calyciflorus

Summary The laboratory feeding behavior of Brachionus calyciflorus varies depending upon the type of food cell available in suspension. When feeding on the yeast Rhodotorula glutinis, rotifers show a continuous increase in ingestion with increased cell density between 0.01 and 1000 g dry weight ml^-1. Effective clearance rates drop from ca. 50 l animal^-1 h^-1 to less than 0.5 l animal^-1 h^-1 over this food density range. When feeding on Englena gracilis, B. calyciflorus ingestion rates are constant between 1.0 and 100 g ml^-1 of available food, averaging close to 25 ng animal^-1 h^-1. The decrease in clearance rate is more striking than with R. glutinis, dropping from 45 l animal^-1 h^-1 at 0.1 g ml^-1 to 0.13 l animal^-1 h^-1 at 100 g ml^-1. Differences between the patterns obtained with the two food types indicate fundamental dissimilarities in the feeding behavior of this rotifer species when presented with these different foods.     

Improved growth of the red yeast, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), in the presence of tricarboxylic acid cycle intermediates

Catabolites related to tricarboxylic acid cycle affected growth and carotenogenesis in Phaffia rhodozyma. Glutamate, glutamine, aspartate, asparagine and proline at 75 mM of N increased biomass from 2 g l^–1 to 2.9–4.7 g l^–1 but decreased carotenoid from 420 g g^–1 yeast to 200–260 g g^–1 yeast in strain 67-385. However, simple nitrogen sources did not decrease carotenoid formation. Tricarboxylic acid intermediates repressed carotenogenesis to a less degree than the corresponding amino acids. Carotenoid hyper-producing mutants were impaired in nitrogen utilization. These results indicated that nitrogen assimilation and the concentrations of tricarboxylic acid cycle intermediates are involved in regulation of carotenoid biosynthesis.     

Astaxanthin production by a Phaffia rhodozyma mutant on grape juice

During fermenter cultivation of Phaffia rhodozyma on a grape juice medium, the presence of glucose initially delayed fructose utilization, although fructose was consumed before glucose depletion. Total pigment and astaxanthin production were growth associated and reached maximum values of 15.9 g/ml and 9.8 g/ml, respectively, after depletion of the carbon source. The total cellular pigment and astaxanthin content increased during the stationary growth phase due to a decrease in biomass, reaching final values of 2120 g/g and 1350 g/g, respectively, without the volumetric concentration in the culture changing. The final cell yield was 0.33 g/g sugar utilized. High sugar concentrations in shake-flasks as well as O₂ limitation decreased the astaxanthin content of the cells. Addition of yeast extract to a grape juice minimal medium markedly increased the maximum specific growth rate, total pigment and astaxanthin content of the cells. An excess of ammonia decreased the intracellular astaxanthin content, which reached a maximal value in cultures with no residual glucose or ammonia.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa;     

Growth inhibition of biomass adapted to the degradation of toluene and xylenes in mixture in a batch reactor with substrates supplied by pulses

A biomass adapted to degrade toluene and xylenes in mixture was grown in a batch reactor with substrates supplied by pulses. The inhibition of biomass growth in the course of substrate degradation was investigated. The maximal biomass concentration of 7 g l^–1 was obtained using 150 l of toluene and 15 l of a mixture of xylenes in one litre of liquid medium, and the maximal biomass productivity and yield were 53 mg l^–1 h^–1 and 0.32 g_DW g _s ^–1 , respectively. Higher quantities of substrate added by pulses, that is 200 l of toluene with 20 l of xylenes and 300 l of toluene with 30 l of xylenes, caused an accumulation of metabolites. These higher quantities of substrates caused inhibition of microbial growth. Among the metabolites produced, 4-methyl catechol was found in large quantities in the culture medium and in the cells.     

Amplification of phleomycin induced death and DNA breakdown by caffeine in Escherichia coli

Summary Caffeine enhanced the degradation of DNA to acid soluble fragments in cultures of Escherichia coli exposed to Phleomycin (2 g/ml). Enhancement was particularly striking with stationary phase cultures, which normally exhibit negligible DNA breakdown when treated with 2 g/ml of Phleomycin. There is little DNA breakdown or death in UVR strains treated with phleomycin (2 g/ml) during exponential growth but when caffeine was present as well as Phleomycin, the kinetics of DNA breakdown and the amounts of DNA degraded were identical in all cultures tested including those of UVR, EXR, B/r type and B strains and equal to the maximum rate observed (with an EXR strain) in the absence of caffeine (ca. 1.7 % per min). High concentrations of Phleomycin (10 g/ml) had the same effect as the caffeine+Phleomycin (2 g/ml) combination and produced a uniform pattern of DNA breakdown in all strains tested. Caffeine did not seem to increase permeability of the bacterial coat. Given to the cells before exposure to Phleomycin it was ineffective in enhancing DNA breakdown. On the other hand, exposure of the bacteria to Phleomycin for a period of 40 min at 37° followed by caffeine was as effective as adding the two drugs together.Caffeine increased the efficiency of Phleomycin as an antibiotic for both growing and stationary phase cultures of e. coli B. It is suggested that caffeine aids the cooperative denaturation of DNA initiated by the attachment of Phleomycin molecules to thymine bases. This would allow single strand-specific endonucleases to attack the DNA and initiate DNA breakdown and cell death.This paper is dedicated to charlotte Auerbach on the occasion of her official retirement.     

High frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis

Culture conditions are described for high frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis Max. Petiole explants formed embryogenic calluses at a frequency of 53% when cultured on B5 medium supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Leaf explants formed embryogenic calluses at a frequency of 21% when cultured at a combination of 4.52 M 2,4-D and 2.22 M 6-benzyladenine. Cell suspension cultures were established with petiole-derived embryogenic calluses using liquid B5 medium with 4.52 M 2,4-D. Upon plating onto B5 basal medium, cell suspension cultures produced numerous somatic embryos, which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Constant fed-batch culture of methanol-utilizing corynebacterium producing vitamin B 12

Summary A fed-batch culture of methanol-utilizing microorganism (Corynebacterium sp. XG), a vitamin B 12 producer, was carried out with constant feed of substrate. Experimental results agreed with the theorical model proposed in the literature. Using this feeding system, the final biomass and vitamin B 12 concentration reached 16.3 g/l and 880 g/l respectively after a 53 h incubation period at 30 °C.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Fed-batch culture of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> based on motile intensity

The operation of a fed-batch culture is more complicated than that of batch or continuous culture. Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed. In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on motile intensity is described. The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells. In addition, the motile intensity of B. thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition. Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition. The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control. A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.     

Thidiazuron-induced in vitro shoot formation from roots of intact seedlings of Albizzia julibrissin

Seedlings of silktree (Albizzia julibrissin Durrazz.) were grown in vitro on MS-media containing B5 vitamins, 3% sucrose, 0.25% phytagel and various concentrations (0.1–10 M) of thidiazuron (TDZ). Addition of TDZ to the culture medium greatly reduced shoot and root elongation but did not influence shoot production from the cotyledonary node or apex. Within 8–10 days the seedling roots split open, formed large masses of callus, and developed green patches which eventually grew into normal shoots while still within the culture medium containing TDZ at 0.1–1.0 M. Such callus and shoot formation did not occur in control cultures lacking TDZ. At higher TDZ concentrations (2.5–10 M), the green patches formed in the callus did not further develop into shoots. Addition of other cytokinins (kinetin, benzylaminopurine, zeatin) to the culture medium also induced some shoot formation from the roots, but higher concentrations than TDZ were required to induce regeneration. Isopentenyladenine failed to induced shoot formation. Following excision and transfer to MS media with or without 4.9 M IBA, the shoots induced by kinetin or benzylaminopurine rooted 4–7 days earlier than those induced by TDZ, but all excised shoots developed into normal rooted plantlets within 3 weeks.Abbreviations TDZ thidiazuron     

Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

Summary The present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (_max) of 0.1 h^-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the _max was 0.13 h^-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (_max liquid=0.13 h^-1, _max vapour=0.12 h^-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h^-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO₂ and unidentified byproducts.     

Linear growth in microbial cultures limited by accumulated substrate

Summary The linear growth phase in cultures limited by intracellular (conservative) substrate is represented by a flat exponential curve. Within the range of experimental errors, the presented model fits well the data from both batch and continuous cultures ofEscherichia coli, whose growth is limited in that way.List of symbols D dilution rate, h^–1 - K_S saturation constant, g.L^–1 - S concentration of the limiting substrate, g.L^–1 - S_i concentration of the limiting substrate accumulated in the cells, g.g^–1 - S_o initial concentration of the limiting substrate, g.L^–1 - t time of cultivation, h - t₁ time of exhaustion of the limiting substrate from medium, h - t_o beginning of exponential phase, h - X biomass concentration, g.L^–1 - X₁ biomass concentration at the time of exhaustion of the limiting substrate from the medium, g.L^–1 - X_o biomass concn. at the beginning of exponential phase, g.L^–1 - biomass concn. at steady-state, g.L^–1 - Y growth yield coefficient (biomass/substrate) - specific growth rate, h^–1 - _m maximum specific growth rate, h^–1     

Factors influencing secondary somatic embryogenesis inMalus x domestica Borkh. (cv ‘Gloster 69’)

A procedure for regeneration of apple plants through secondary somatic embryogenesis (SSE) was developed in apple Gloster 69. Primary somatic embryos were produced from cotyledon-derived cultures of immature zygotic embryos. These somatic embryos were multiplied by secondary somatic embryogenesis (SSE) on media with different Plant Growth Regulator (PGR) combinations. The highest SSE rate (55.5%) was obtained with a combination of NAA (5.3 M), BAP (0.9 M) and KIN (0.9 M) or with TDZ alone (10 M). In addition, effects of explant source, somatic embryo size, type and concentrations of carbohydrates and gelling agents on SSE were investigated. The optimum SSE (>73%) was obtained by the culture of large size somatic embryos or cotyledon-like structures on medium containing a combination of NAA/BAP/KIN or TDZ (10 M) alone, maltose (175 mM) and Phytagel (2.8 g/1).