Intracellular and extracellular alkaloids of Penicillium roqueforti

The alkaloid composition of mycelium and culture liquid filtrate of the fungus Penicillium roqueforti IBPM-F-141 was studied. The new metabolite--3,12-dihydroroquefortine, a derivative of roquefortine, the main component of the alkaloid fraction of this culture, has been isolated for the first time. The structure of 3,12-dihydroroquefortine was determined by chemical and physico-chemical methods. In addition to roquefortine and 3,12-dihydroroquefortine, representatives of a new alkaloid group, the clavine alkaloids, e. g. isofumigaclavine A, isofumigaclavine B and festuclavine, were also isolated and identified. The data on the content of these compounds in mycelium and culture medium are presented.     

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.     

Potentiation effect of corn extract on the production of eremofortin C, EC oxidase, and PR toxin by Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Their structures are similar and differ only by an alcohol and an aldehyde group at the C-12 position. EC has been demonstrated to be the precursor of PR toxin, and EC is transformed to PR toxin by EC oxidase. These two compounds and EC oxidase are secreted by P. roqueforti in the culture medium, which is usually composed of 15% sucrose and 2% yeast extract. Recently, we discovered that the addition of corn extract to this medium increased the production of EC and PR toxin and the activity of EC oxidase in a coordinative manner. In a time-course study, we found that the peak yield of EC and PR toxin and the maximum activity of EC oxidase in the culture medium containing 7.5% sucrose, 1% yeast extract, and 20% corn extract were increased 6.2, 4.6, and 4.7-fold, respectively, as compared with those obtained in the medium without corn extract. Moreover, corn extract increased the production of EC and PR toxin and the activity of EC oxidase by P. roqueforti in a dose-dependent manner. On the other hand, when the concentrations of sucrose and yeast extract were increased while fixing the ratio of corn extract, we found that the levels of EC and PR toxin and the enzyme activity were decreased concomitantly. We thus conclude that corn extract can enhance the production of EC, PR toxin and EC oxidase by P. roqueforti when grown in a minimal medium and that the potentiation effect of corn extract is suppressed when the fungi are grown in a rich medium.     

Measurement of mercury methylation in lake water and sediment samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Secondary metabolites resulting from degradation of PR toxin by Penicillium roqueforti.

PR toxin is a secondary metabolite of the fungus Penicillium roqueforti. It is lethal to rats, mice, and cats. Usually, the amount of PR toxin in the culture medium decreases from its maximum on day 15 to zero within 3 to 4 days. We found that two were secondary metabolites produced in the culture medium of this fungus while the production of PR toxin was decreasing. We isolated and purified the two compounds in pure and colorless crystalline form. On the basis of elemental analysis and mass, 1H and 13C nuclear magnetic resonance, infrared, and UV spectroscopies, the two compounds were identified as PR-imine (C17H21O5N) and PR-amide (C17H21O6N). The structures of both compounds and of PR toxin (C17H20O6) were closely related, and the peak production of PR toxin appeared earlier than those of PR-imine and PR-amide. Moreover, PR toxin was transformed to PR-imine when PR toxin was incubated with the culture medium on a given culture day. Thus, we propose that PR toxin is degraded into PR-imine and PR-amide in the culture medium of P. roqueforti.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains.

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Penicillic acid production in submerged culture.

Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.     

Penicillic acid production in submerged culture.

Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.     

Isolation and Some Properties of the Enzyme That Transforms Eremofortin C to PR Toxin

PR toxin and eremofortin C are secondary metabolites of Penicillium roqueforti. The chemical structures of these two compounds are closely related to each other and differ only by an aldehyde and an alcohol group at the C-12 position. In an effort to better understand the biosynthesis of PR toxin, we discovered the enzyme of P. roqueforti that is responsible for the transformation of eremofortin C to PR toxin. The maximum activity of the enzyme in the culture medium was found to occur on day 13, which corresponded to the maximal production of PR toxin in the medium. The enzyme was isolated and purified from the culture medium and the mycelium of the fungus, respectively, through a procedure involving ammonium sulfate fractionation and DEAE-cellulose chromatography. The specific activity increased 20- and 8-fold, respectively, and the yield was 33.3 and 21.6%, respectively, for the enzyme from the medium and mycelium. The optimal pH for the enzyme reaction was ca. pH 5.6. The enzyme reaction was temperature dependent. The rates followed a linear time course when it catalyzed the transformation at 30°C and decayed with time when reacted at higher temperatures. At 100°C, the enzyme activity was completely lost. The K_m and V_max of the enzyme as determined at 30°C were 0.02 mM and 4.0 μmol/min per mg, respectively. The molecular weight of the enzyme was estimated by gel filtration on a high-pressure liquid chromatography I-250 protein column to be ca. 40,000.     

Production of eicosapentaenoic acid by marine bacteria

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.     

New process for T-2 toxin production.

Strains of Fusarium produced high levels of T-2 toxin when cultured on certain media absorbed into vermiculite. Modified Gregory medium was nutritionally complex (2% soya meal, 0.5% corn steep liquor, 10% glucose) and, when inoculated with the appropriate fungal strain, yielded maximum T-2 toxin within 24 days of incubation at 19 degrees C. On Vogel synthetic medium N (H. J. Vogel, Microb. Genet, Bull. 13:42-43, 1956) supplemented with 5% glucose, optimal toxin levels were synthesized after incubation for 12 to 14 days at 15 degrees C. Fusarium tricinctum T-340 produced 714 and 353 mg/liter on modified Gregory medium and Vogel synthetic medium N plus 5% glucose, respectively. Improved analytical procedures were developed and involved aqueous methanol extraction, purification by liquid-liquid partitions, and gas-chromatographic quantitation.     

Viriditoxin Production by Aspergillus viridi-nutans and Related Species

Bioproduction of viriditoxin on various substrates by strains of the Aspergillus fumigatus group was determined under several incubation conditions. Aspergillus viridi-nutans strains NRRL 4365 and 576 produced the largest quantities of toxin, A. brevipes gave reduced yields, and there was no detectable synthesis by isolates of four related species. After 30 days in static culture at 20 C on various autoclaved agricultural commodities, optimal yields of 440 and 380 mg of toxin were observed per kilogram of sorghum and rice. Toxin levels were reduced on corn, rye, and wheat (40-200 mg/kg); yields were low on cottonseed, barley, and oats. Incubation at 10 C restricted biosynthesis of viriditoxin, and no toxin accumulated on substrates maintained at 5 C for 120 days. In a liquid, yeast extract-sucrose medium, maximal mycotoxin production developed in shake flasks; after 156 h, 10 mg of toxin accumulated per gram of mycelium. Viriditoxin produced in submerged culture was associated with the mycelium; less than 1% was detected in the filtered broth after 156 h of incubation.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.