Tetanus toxin production from <Emphasis Type="Italic">Clostridium tetani</Emphasis>, using a casein-based medium in a single-use bioreactor

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials.     

Regulation of Staphylococcal Enterotoxin B

Several factors influenced the formation of enterotoxin B by Staphylococcus aureus strain S-6. In the standard casein hydrolysate medium, toxin was not produced in detectable quantities during exponential growth; it was produced during the post-exponential phase when total protein synthesis was arithmetic. The rate of toxin synthesis was much greater than the rate of total protein synthesis. The appearance of enterotoxin was inhibited by chloramphenicol; thus, the presence of toxin was dependent on de novo protein synthesis. When low concentrations of glucose (<0.30%) were added to the casein hydrolysate medium, growth was diauxic; glucose was completely metabolized during the first growth period. During the second growth period, enterotoxin was synthesized. In unbuffered casein hydrolysate medium containing excess glucose, toxin synthesis was completely repressed. The absence of toxin production under such conditions might be explained by the low (4.6) pH resulting from the acid end products of glucose metabolism. At pH <5.0, little or no toxin was produced. Toxin synthesis was initiated in the presence of glucose when the medium were buffered at any pH above 5.6. In such media, the differential rates of toxin synthesis, with respect to the rates of total protein synthesis, were lower than the differential rates in casein hydrolysate medium alone. Addition of glucose to a culture synthesizing toxin resulted in an immediate decrease in the differential rate without any change in pH. Thus, toxin synthesis appeared to be regulated by catabolite repression.     

Optimization of diphtheria toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> using a casein-based medium in a fermenter

The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of K _La in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal K _La value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Cultivation of Neisseria meningitidis serogroup A on semisynthetic liquid nutrient media]

In cultivation of meningococcus of serological group A in fluid semisynthetic medium of simple composition prepared on the basis of purified acid casein hydrolysate with profound splitting there were obtained microbial cultures with a density of 4-5 x 10(9) microbial cells per 1 ml after 20-24 hours of cultivation with shaking. Alkalinity of the medium increased (to pH 8.0-8.2 during the stationary phase) with increase of the microbial cell concentration. A study of the accumulation of group-specific thermostable polysaccharide antigen in dynamics of meningococcus cultivation on semisynthetic medium tested showed the preparations obtained by alcoholic precipitation to be colourless and to contain much antigen (by inhibition of indirect hemagglutination), particularly at the phasees of negative growth acceleration and at the stationary phase. The suggested fluid semisynthetic medium of simple composition could be used for production of diagnostic and prophylactic meningococcus preparations belonging to the serological group A.     

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.     

Mutants of Corynebacterium diphtheriae PW8 that produce toxin in medium with excess iron.

Two mutants that produce toxin in medium with excess iron were isolated from the PW8 strain of Corynebacterium diphtheriae. These mutants produced as much toxin in medium containing excess iron (3 mug of Fe2+ per ml) as did the parent PW8 strain in iron-depleted medium, and they will be very useful for easy production of toxin.     

Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines

The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4–111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l^?1 in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.     

Effect of complex nitrogen sources on the production of penicillin acylase byBacillus megaterium

The following complex nitrogen sources were evaluated for the production of penicillin acylase byBacillus megaterium: casein hydrolysate, corn steep liquor, stick water concentrate, blood meal and defatted sunflower meal. Experiments were run in shake flasks at 30‡C and pH 7.0. Best results were obtained with casein hydrolysate: 244 IU/I were produced with a productivity of 20.3 IU/l/h and yield of 717.6 IU/g of nitrogen. The lowest results correspond to sunflower meal with 39 IU/1.     

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.     

Studies on staphylococcal enterotoxin B. II. Production and regulation (author's transl)]

Enterotoxigenic strain of Staphylococcus aureus (ATCC 14458) was grown under various conditions with constant shaking to determine the requirements for maximum toxin production. It was evident that 3% tryptic soy broth, 3% NZ-Amine NAK + 3% casein hydrolysate, 3% NZ-Amine NAK + 1% yeast extract, and 3% NZ-Amine NAK + 1% yeast extract + 0.2% glucose are most available toxin production media. But concentration of glucose could strictly triggered the enterotoxin producing efficiency. When glucose concentration was less than 0.5%, although with higher yield, the toxin production was delayed for certain period of time. However, if glucose concentration was up to more than 0.5%, the enterotoxin production was almost inhibited. Some metabolites of glucose to elucidate the inhibitory effect have also investigated. Our results indicated that glycerol and citric acid inhibited the toxin production directly, while the inhibitory effect of lactic acid and acetic acid were due to those acidic metabolites, decreased the pH value of media, and adversely suppressed the bacterial growth.     

Production and characteristics of hemolysins of Escherichia coli

Snyder, Irvin S. (University of Iowa, Iowa City), and Nancy A. Koch. Production and characteristics of hemolysins of Escherichia coli. J. Bacteriol. 91:763-767. 1996.-Filterable and nonfilterable hemolysins were produced by Escherichia coli in beef-heart infusion, casein hydrolysate, and chemically defined media. Differences were found among the three media in the time of appearance and in the relationship between production of these two hemolysins. The nonfilterable hemolysin produced in the three media, as well as the filterable hemolysin produced in the beef-heart infusion medium, were destroyed in 1 hr at 56 C. The filterable hemolysin produced in the casein hydrolysate and chemically defined media was unaffected by exposure to 56 C for 1 hr. Formalin inactivated the hemolysins produced in all three media. The optimal pH for activity of the nonfilterable hemolysin varied with time of production, whereas the optimal pH for the filterable hemolysin was constant. Certain carbohydrates were required for the production of filterable hemolysin by E. coli grown in casein hydrolysate and chemically defined media.     

Production of coleonol (forskolin) by root callus cells of plant Coleus forskohlii

Summary Coleonol was produced in callus culture; the kind and level of phytohormones, glycine, casein hydrolysate and sucrose content of the medium differently influenced growth and product formation. Maximum specific growth rate was obtained in medium containing 7% sucrose. Biomass production was highest with 4 ppm of NAA. Maximum product (0.075% of dry cells) was formed in medium containing 0.5 ppm IAA and IBA each, 5 ppm glycine, 200 ppm casein hydrolysate and 7% sucrose.Abbreviations Su Sucrose - NAA naphthalene acetic acid - 2,4-D-2,4 diphenoxy acetic acid - IBA Indole-3-butyric acetic acid - IAA indole 3-acetic acid - Kn Kinetin - Gl glycine - Ch casein hydrolysate     

PR toxin production in different Penicillium roqueforti strains.

Different Penicillium roqueforti strains from the American Type Culture Collection were tested for the production of PR toxin. All four strains were able to produce the toxin on semisynthetic medium at 24 degrees C after certain periods of incubation. The yields were correlated with the pH of the medium. Timing of the harvest influenced both the yield and purification of the toxin.     

PR toxin production in different Penicillium roqueforti strains.

Different Penicillium roqueforti strains from the American Type Culture Collection were tested for the production of PR toxin. All four strains were able to produce the toxin on semisynthetic medium at 24 degrees C after certain periods of incubation. The yields were correlated with the pH of the medium. Timing of the harvest influenced both the yield and purification of the toxin.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.