Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.     

Antigenic characteristics and various biological properties of glycoprotein from Neisseria meningitidis of serological group A]

It was shown that the antigen determining the group specificity of meningococcus belonging to serological group A was of mixed polysaccharide-protein nature. Carbohydrate component is responsible for the interaction with the group-specific antibodies in this antigen. Glycoprotein can be isolated both from the cells and from the culture fluid where it passes during the N. meningitidis cultivation in fluid nutrient medium. The described antigen possesses no properties of endotoxin.     

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.     

Changes in phospholipid composition of Thiobacillus neapolitanus during growth

Summary During the stationary growth phase, the phospholipids of Thiobacillus neapolitanus consisted of phosphatidyl glycerol (PG), diphosphatidyl glycerol (DPG), phosphatidyl-N-monomethylethanolamine (PME) and phosphatidyl ethanolamine (PE) in increasing amounts. In general, the phospholipids increased to a maximum concentration during the stationary phase and then decreased in concentration. Individually, PG and PE increased to a maximum in late lag or early exponential phase and then decreased in concentration. DPG and PME increased during the transition between the exponential and the stationary phase and reached a maximum concentration in the stationary phase. In older cultures, a quantitative interconversion between PG and DPG and PE and PME was observed. A lyso-phospholipid compound also appeared in the late stationary phase.The phospholipid composition of the culture supernatant fluid was essentially similar to that of the cells at all stages of growth. No excessive secretion of these products into the medium was observed at any growth stage of the culture.Abbreviations used PG Phosphatidyl glycerol - DPG Diphosphatidyl glycerol - PME Phosphatidyl-N-monomethylethanolamine - PE Phosphatidyl ethanolamine - GPGPG Glycerophosphoryl glycerophosphoryl glycerol - GPG Glycerophosphoryl glycerol - GPE Glycerophosphoryl ethanolamine - GPME Glycerophosphoryl-N-monomethylethanolamine     

Detection of the etiologic structure of generalized and localized forms of meningococcal infection using the passive hemagglutination test

Preparations of formalin-treated erythrocytes sensitized with meningococcus polysaccharides of serological groups A, C, X, Y, and Z were used for the purpose of examination of patients with meningococcus infection; these preparations were highly specific in the tests of precipitation, hemagglutination and hemagglutination inhibition. Indirect hemagglutination test with the sera of 99 patients suffering from generalized forms of meningococcus infection was conducted with the mentioned preparations in Moscow and Novosibirsk in 1974--1975 when a stable morbidity decline was noted in these towns after an epidemic rise. The diagnostic value of this test was confirmed: it permitted to diagnose meningococcus etiology beginning from the 5th day of the disease and to decipher it from the aspect of individual serological groups. As shown, the incidence of cases caused by serological group A, reaching 87% at the height of the epidemic rise, fell to 49.5% at the stage of decline. Cases caused by group Y which was not encountered formerly were revealed in 16.2% of the patients. Among 127 patients with miningitis of nonmeningococcus etiology meningococcus antibodies to groups A and Y were revealed with the same frequency (in titres of not over 1 : 20--1 : 80), but the leading role of serological group A in the etiology of the manifest forms permitted to draw a conclusion on the presence of a higher invasiveness in the strains of group A.     

The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.     

A new Escherichia coli strain producing human tumor necrosis factor

An Escherichia coli strain producing human tumor necrosis factor (TNF-alpha) was obtained using a semisynthetic gene partially optimized in respect of codon composition and a phage T7 promoter. The expression product was accumulated in cells as inclusion bodies in a yield of 50-70 mg/l of culture medium. The recombinant TNF-alpha in the form of inclusion bodies was used for immunization of rats to give a polyclonal antiserum. The resulting antibodies were specific under the immunoblotting conditions to the antigen used for the immunization. A dilution-based refolding procedure was developed; it provided a yield of soluble protein exceeding 85%.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Behaviour ofPropionibacterium shermanii in alkaline medium

Physiological and morphological properties ofPropionibacterium shermanii were estimated in a liquid semisynthetic medium of initial pH between 7 – 10. Prolongation of the lag phase and a shift of the stationary phase occurred at higher initial pH values of the medium, The growth response of the strain depended on the way of glucose addition (either sterilized in the medium or added aseptically after sterilization). At pH 8.5 and higher the cells begin to form slime and capsules. The strain exhibited growth activity even at initial pH values of the medium 11.5.     

Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile,Bacillus pallidus

The aerobic biodegradation of high-concentration, to 24 g l(-1), 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus, was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60 degrees C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate (micro) was about 0.2 h(-1) for both systems, with a maximum cell density of 2.4 x 10(8) cells ml(-1) obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l(-1) h(-1), respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l(-1) h(-1) were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2-2.3 g l(-1) after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate ( micro ) obtainable was 0.247 h(-1) at 4 g l(-1) acetone and 0.202 h(-1) at 8 g l(-1) IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth.     

Microbial lipids as a source of biofuel

This review presents the main directions and experimental data aimed at searching for active producers of lipids among different species of microorganisms and ways to optimize the lipidogenesis process in the most promising stains. It was shown that enzymatic processes can be directed by maintaining the necessary cultivation conditions. The influence on the growth, development, and biochemical activity of the microbial medium composition and temperature and the aeration and oxidation reduction conditions was considered. Changes in these factors affected the biosynthetic activity of microorganisms and lipidogenic yeasts and the composition of synthesized lipids. The ability of lipidogenic yeasts, as well as the relatively rapid ability of changing the amount and composition of lipids by direct cultivation, leads to the conclusion that lipids obtained by microbial synthesis can be a source of commercial raw materials for biofuel.     

Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.     

Environmental and Nutritional Factors Affecting the Production of Rubratoxin B by Penicillium rubrum Stoll

Rubratoxin B can be produced in a semisynthetic medium by Penicillium rubrum under varying environmental and nutritional conditions. Maximum production (552.0 mg/500 ml) was obtained with P. rubrum NRRL A-11785 grown in stationary cultures of Mosseray's simplified Raulin solution supplemented with 2.5% malt extract broth at ambient temperature. Zinc is required at levels of at least 0.4 mg per liter. In the absence of iron sulfate, there was a 50-fold reduction in rubratoxin B production but not in growth. No toxin was produced by this isolate in 5- or 7-liter fermentors.     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.     

Changing extracellular matrix of cells during development of suspension culture of Triticum timopheevii Zhuk

A study was made of the contents of the main polysaccharide fractions in the cell wall, and extracellular polysaccharides, and of the activity of cell wall enzymes during cultivation of suspension culture of cells of the winter wheat Triticum timopheevii Zhuk. It was shown that within 3 days of cultivation (a phase enriched in dividing cells), on the background of increased callose contents in plant cells, amounts of pectins and hemicelluloses extracted by 4N alkali decreased. The content of polysaccharides reached its initial level by the end of culturing. A parallel analysis of glycosidase activity in cell walls has shown their considerable activation at the stage enriched by dividing cells, which decreased at a transition of culture into the stationary level. The increased activity of hydrolyzing enzymes was combined with an increased efflux of extracellular polysaccharides into culture medium. The detected changes in polysaccharide composition of the cell wall at the first phase indicate its qualitative changes during cell wall reconstruction at the beginning of cytokines, whereas extensive expansion of cell wall was seen on the phase of elongation.