Mycelia

Tempeh. DehuUed cooked soybeans are inoculated with the mold, Thi pus oligosporus packed in perforated plastic bags, and allowed to ferment for 18 h. The mold mycelium overgrows the soybean cotyledons and forms a compact cake. When sHced and deep-fried in oil, a crisp and golden brown product is obtained. Although native to Indonesia, tempeh has become popular with vegetarians in the United States and other Western countries (93). 

Polyethers are usually found in both the filtrate and the mycelial fraction, but in high yielding fermentations they are mosdy in the mycelium because of their low water-solubiUty (162). The high lipophilicity of both the free acid and the salt forms of the polyether antibiotics lends these compounds to efficient organic solvent extraction and chromatography (qv) on adsorbents such as siUca gel and alumina. Many of the production procedures utilize the separation of the mycelium followed by extraction using solvents such as methanol or acetone. A number of the polyethers can be readily crystallized, either as the free acid or as the sodium or potassium salt, after only minimal purification. 

Polyethers such as monensin, lasalocid, salinomycin, and narasin are sold in many countries in crystalline or highly purified forms for incorporation into feeds or sustained-release bolus devices (see Controlled-RELEASE technology). There are also mycelial or biomass products, especially in the United States. The mycelial products are generally prepared by separation of the mycelium and then drying by azeotropic evaporation, fluid-bed driers, continuous tray driers, flash driers, and other types of commercial driers (163). In countries allowing biomass products, crystalline polyethers may be added to increase the potency of the product. 

Miconazole. Miconazole nitrate [22832-87-7] (Fig. 2), the 1-phenethyl-imidazole derivative first described in 1969, interferes at low doses with the cytochrome P-450 dependent ergosterol biosynthesis in yeasts and fungi. The result is accumulation of C-14 methylated sterols on the one hand and reduction of the ergosterol levels in the membranes on the other hand (12). Analogous to clotrimazole, this leads to a disturbance in the membranes it results in inhibition of ceU repHcation, mycelium development (in C. albicans) and finally, ceU death. High concentrations of miconazole, which may be achieved with topical use, disturb the orientation of phosphoHpids in the membranes, which produces leaks (13). 

Mycelium A mass of filaments composing the vegetative body of many fungi and some bacteria. 

The mold metabolite mycelianamide, isolated from the mycelium of strains of Penicillium gritieofulvum Dierckx, was first investigated by Oxford and Raistrick. The reinterpretation and extension of this work by Birch et and the revision by Bates et of the structure first proposed for the terpenoid side-chain, have led to the formulation of mycelianamide as 11. This structure has been confirmed by further degradations and by a synthesis of racemic deoxymyceli-anamide by Gallina and co-workers. The ready decomposition of the heterocyclic ring by either acid or alkali is discussed later (Section... 

Briefly stated, the production of chloramphenicol by the surface culture method involves inoculating a shallow layer, usually less than about 2 cm, of a sterile, aqueous nutrient medium with Streptomyces ver)ezuelae and incubating the mixture under aerobic conditions at a temperature between about 20° and 40°C, preferably at room temperature (about 25°C), for a period of about 10 to 15 days. The mycelium is then removed from the liquid and the culture liquid is then treated by methods described for Isolating therefrom tne desired chloramphenicol. 

This material was made up with distilled water to provide 41 g per liter, and the mixture was adjusted to pH 7.0 with potassium hydroxide solution. To the mixture were added per liter 5.0 g of calcium carbonate and 7.5 ml of soybean oil. 2,000 ml portions of this medium were then added to fermentation vessels, equipped with stirrers and aeration spargers, and sterilized at 121°C for 60 minutes. After cooling the flasks were inoculated with a suspension of strain No. ATCC 11924 of Streptomyces lavendulae, obtained from the surface of agar slants. The flasks were stirred for 4 days at 28°C at approximately 1,700 rpm. At the end of this period the broth was found to contain cycloserine in the amount of about 250 C.D.U./ml of broth. The mycelium was separated from the broth by filtration. The broth had a pH of about 7.5. Tests showed it to be highly active against a variety of microorganisms. 

Each flask was inoculated with mycelium of Streptomyces F.l. 1762 whose quantity corresponds to one-fifth of a suspension in sterile water of the mycelium of a 10 day old culture growth in a test tube containing the following ingredients dissolved in tap water. 

The culture broth (about 1,100 gallons in volume) is adjusted to pH 9.5 with 40% sodium hydroxide solution and is filtered to remove the mycelium, the filtration being assisted by use of 3% of Hyflo Super-Cel, a filter aid, (sold by Johns-IVlanville Company). The clear filtrate is extracted with amyl acetate in a Podbielniak extractor using a ratio of 1 volume of amyl acetate to 6 volumes of clarified broth. The amyl acetate extract is in turn extracted batchwise with water brought to about pH 5 by the addition of sulfuric acid. Two... 

The mycelium (56 g dry weight) was filtered off and the steroidal material was extracted with methylene chloride, the methylene extracts evaporated to dryness, and the resulting residue chromatographed over a Florisil column. The column was packed with 200 g of Florisil and was developed with five 400-ml fractions each of methylene chloride, Skelly-solve 8-acetone mixtures of 9 1, 8 2, 7 3, 1 1, and methanol. The fraction eluted with Skellysolve 8-acetone (7 3) weighed 1.545 g and on recrystallization from acetone gave, in three crops, 928 mg of product of MP 210° to 235°C. The sample prepared for analysis melted at 245° to 247°C. 

The SF-837 strain, namely Streptomyces mycarofaciens identified as ATCC No. 21454 was inoculated to 60 liters of a liquid culture medium containing 2.5% seccharified starch, 4% soluble vegetable protein, 0.3% potassium chloride and 0.3% calcium carbonate at pH 7.0, and then stir-cultured in a jar-fermenter at 28°C for 35 hours under aeration. The resulting culture was filtered directly and the filter cake comprising the mycelium cake was washed with dilute hydrochloric acid. 

The mixture of broth and mycelium thus formed was then transferred under aseptic conditions to a 3-iiter fermentor containing 2 00 ml of a sterile fermentation medium having the following composition 60 g Cerelose (dextrose hydrate), 18 g soybean meal, 5 g distillers solubles, 12 g cornmeal and tap water in a sufficient amount for a 1,000-ml total volume, adjusted to pH 7.0 to 7.2 with potassium hydroxide. 

After seeding the nutrient medium with the preformed inoculum previously described, the mixture was subjected to agitation and aeration under aseptic conditions for 72 hours at 27°C to 28°C for the first 24 hours, then at 25°C to 26°C for the next 48 hours during this period, the pH was in the range of 6.4 to 6.8. Aeration was accomplished by cultivation under submerged conditions at an air flow rate of one volume of air per volume of medium per minute. After termination of the process, the mycelium was removed by filtration and the filtered broth found to contain 450 7of oleandomycin per ml of solution. 

Isolation of Antibiotic 66-40 — The whole broth is adjusted to pH 2 with 6N sulfuric acid. (For the purpose of this example, quantities are given in terms of 170 8of fermentation broth obtained by pooling acidified broth from 17 batches.) The acidified broth is stirred for about 15 minutes and then filtered. Wash the mycelium with water and combine the washings with the filtrate. Adjust the pH of the filtrate to 7 with 6 N ammonium hydroxide. 

To 17 C of a culture obtained by submerged fermentation as mentioned above, siliceous earth is added and the batch is filtered. The mixture of mycelium and the siliceous earth are agi-tatedforl hour with 2.5 Cof butanol. This treatment is repeated twice. Thebutanolic extracts are combined, washed with water, evaporated to dry ness (about 10 g) and boiled with acetone (80 ml). The residue (5.41 g of yellowish powder) is distamycin. 

The flasks are then placed on a reciprocating shaker (120 one and one-half inch cycles per minute) and mechanically shaken at 25°C for 3 days. The contents of the flasks are then pooled and, after the pH of the culture is adjusted to about 4 0.2 with sulfuric acid, filtered through Seitz filter pads to separate the mycelium from the fermented medium. 

After fermentation, large volumes of spent medium containing dtric acid and mycelium remain. 

The correct answer here is in both the medium and the mycelium. It is known that some 15% may remain in the mycelium immediately after harvesting. 

Usually the dtric add outweighs the biomass by a ratio of 5 1. The initial task is to remove the mycelium from the medium, a process usually carried out by rotary filtration. The wet biomass is crushed and rewashed to obtain most of the 15% dtric add contained within and the washings are added to the spent medium. 

A simplified scheme is shown in Figure 5.10. The fermentation mixture is filtered to remove mycelium and suspended solids. The solution is treated with hot carbon and then filtered. During the process addification is necessary to reverse the neutralisation by caldum hydroxide or ammonium hydroxide employed during fermentation. 

Either a spore suspension or mycelium can be used to inoculate the production vessel. The medium contains a maximum glucose concentration of 15%. The upper limit reflects the low solubility of caldum gluconate which is normally about 4% at 30°C, but it can form supersaturated solutions up to about 15% without risk of predpitation. 

Fermentation usually occurs in a conventional stirred vessel at 30°C (with cooling) and vigorous aeration. The process from start to finish can take as littie as 24 hours thus absolute sterilisation is not crudal. However, several processes reuse the mycelium many times and in these circumstances dean conditions are a minimum requirement... 

Penicillins, like most antibiotics, are secondary products whose synthesis is not directly linked to growth. The enzymes that produce secondary products are normally repressed or inhibited under conditions which favour rapid growth. In the early work on penicillin, Penicillium rwtatum was grown as a floating mycelium on about 2 cm depth of liquid medium. The mycelium absorbed nutrients from the medium and penicillin was excreted into the medium. The mycelium and spent medium are readily separated. 

---