Continuous cultivation of microorganisms.

This review presents selected studies on continuous cultivation including the genetics and selection of microorganisms, production of biomass, liquid fuels and other products, published in 1982-1984 and bearing a relationship to biotechnology.

Selection of Escherichia coli K12 1EA mutants with increased synthesis of ribitol dehydrogenase.

Selection of an interspecific hybrid Escherichia coli K12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56). Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase and D-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity of D-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis of the two dehydrogenases and growth parameters under the conditions of batch cultivation.

Production of L-aspartic acid from fumaric acid by Alcaligenes metalcaligenes CCEB 312.

Among 23 microbial strains, Alcaligenes metalcaligenes CCEB 312 was found to be most suitable for the conversion of fumaric acid to L-aspartic acid. In a growth medium containing 4% peptone, the strain produced as much as 50 g L-aspartic acid per litre.

Simultaneous and successive induction of synthesis of beta-galactosidase and tryptophanase in Escherichia coli K 12 in the chemostat.

beta-Galactosidase and tryptophanase were induced either simultaneously or successively during continuous cultivation of the inducible strain Escherichia coli K 12 in the chemostat. Growth was limited by glycerol and the dilution rate was 0.1 h-1. During both the simultaneous and successive induction specific rates of synthesis, as well as maximum enzyme levels, were identical with those obtained after independent induction of individual enzymes. As compared with batch cultivation, beta-galactosidase reached the same specific rate of synthesis in the chemostat, whereas the specific rate of synthesis of tryptophanase in the chemostat was up to five times higher.

Simultaneous induction of three catabolic enzymes in Escherichia coli.

During a simultaneous induction of three enzymes which are subject to catabolite repression (beta-galactosidase, tryptophanase and amylomaltase, or beta-galactosidase, tryptophanase and D-serine deaminase) in a batch culture, the rates of synthesis of beta-galactosidase and tryptophanase decreases, while the rates of synthesis of amylomaltase and D-serine deaminase remain unaffected. The addition of cAMP brings about a considerable increase of the rate of synthesis of D-serine deaminase and a partial synthesis rate increase of beta-galactosidase whihle the synthesis rate of tryptophanase remains lowered and the synthesis rate of amylomaltase remains unaffected. In a continuous culture beta-galactosidase, tryptophanase and D-serine deaminase are synthesized simultaneously at a maximum rate without mutual influence. The addition of cAMP increases the rate of synthesis of all three enzymes.

Simultaneous and gradual induction of beta-galactosidase and tryptophanase synthesis in an Escherichia coli batch culture.

beta-Galactosidase and tryptophanase can be induced in Escherichia coli simultaneously or gradually during a batch cultivation. In the strain Escherichia coli K 12 and ML 30, in which the synthesis of the two enzymes was induced simultaneously, only the synthesis of tryptophanase partially decreased, whereas the synthesis of beta-galactosidase was not influenced. In the strains B 28 and ATCC 9637 the synthesis of both enzymes was partially decreased. On a gradual induction of these enzymes in the strain Escherichia coli K 12 only the synthesis of tryptophanase decreased. Thus, the results obtained here resemble those observed during the simultaneous induction. In addition, it was found that it is not important which of the two enzymes is induced as the first one.

Growth of Candida tropicalis 2838 cells on straw hydrolyzates.

Improved strain of Candida tropicalis 2838 grows on nonseparated straw hydrolyzates with no addition of vitamins and trace elements at a specific growth rate mu = 0.34 and 44% yield coefficient (referred to reducing substances). The reducing substances in hydrolyzates contain predominantly monosaccharides (xylose, glucose, arabinose, mannose). Cells grown in this way are rich in proteins (62%) and essential amino acids (lysine, phenylalanine, leucine, threonine and valine). The product obtained under industrial conditions by fermentation of the nonseparated hydrolyzates contains 8--9% of proteins and it is a suitable supplement of fodder mixtures for monogastric domestic animals. Nutrition tests on rats and pigs indicated that this product can substitute the hay-flour, and, partially, blood-flour barley, and that the strain used is nonpathogenic.

Effect of cyclic adenosine-3',5'-monophosphate on the simultaneous synthesis of beta-galactosidase and tryptophanase in Escherichia coli.

When inducing simultaneously beta-galactosidase and tryptophanase in a batch culture either the synthesis of tryptophanase or of both enzymes is decreased due to an insufficient cAMP concentration. The addition of this nucleotide can overcome this decrease. In a continuous culture both enzymes are synthesized at the maximum rate, as the amount of cAMP produced during carbon limitation of growth is probably sufficient for the simultaneous synthesis of both enzymes. In the beta-galactosidase hyperproduction mutant cultivated continuously the level of beta-galactosidase markedly decreases when tryptophanase is simultaneously induced. Also this decrease is caused by cAMP insufficiency and can be overcome by increasing its concentration. cAMP is thus an important regulatory factor of both enzymes and becomes a limiting factor in their simultaneous synthesis; a competition for this regulatory compound apparently occurs and probably also a different mutual affinity of the regulatory complex with the promoter site of the enzyme operons is involved.

Enzyme synthesis of L-tryptophan.

Enzyme synthesis of tryptophan from indole, pyruvate and ammonium salts was studied using Escherichia coli cells exhibiting a significant tryptophanase activity. In addition to the effect of cultivation medium composition and cultivation conditions, factors affecting the course of the conversion were investigated. Production of 32.4 g/L of L-tryptophan was reached after 48 h under optimal conditions.

[Nanobiotechnology in pharmacology and medicine]. / Nanobiotechnologie ve farmacii a medicíne.

Nanobiotechnology is concerned with the construction and use of molecular apparatuses (nanorobots), in particular in pharmacy and medicine. In the future, these nanorobots could be employed for the diagnostics, targeted drug dispensation, elimination of xenogenous particles from the body, and repair of cells and tissues, e.g. the skin and teeth.

[The history and present status of biotechnology in the Czech Republic]. / Historie a soucasnost biotechnologie v Ceské republice.

The paper outlines the historical milestones in the development of biotechnology in the Czech Republic in the fields of research, education and manufacture. The section dealing with the present state describes fundamental research carried out in the institutes of the Academy of Sciences of the Czech Republic and the universities and technical colleges, the institutes of applied research subsidized by the pertinent ministries as well as privatized institutes. The numbers of institutes and researchers before and after the political changes in 1989 are compared and the grant agencies for support of science and research are listed. The section dealing with biotechnological industry characterizes the firms manufacturing the products and devices for pharmacy, agriculture, food industry and ecology.

Influence of pH on the growth characteristics of Neisseria gonorrhoeae in continuous culture.

A series of three continuous cultures of Neisseria gonorrhoeae was performed at pH values ranging from 6.4 to 7.2, with an average duration of 3 weeks. The maximal yield obtained was 1.12 mg/ml at a pH of 6.75 and a dilution rate of 0.26 per hr. Some morphological variations were observed at lower dilution rates, but complement-fixation titers remained stable for the duration of the cultures.