Inocula

A commercial technology (69), the SABRE process, treats contaminated water and soil ia a two-stage process by adding a readily degradable carbon and an inoculum of anaerobic bacteria able to degrade the contaminant. An initial aerobic fermentation removes oxygen so that the subsequent reduction of the contaminant is not accompanied by oxidative polymerization. 

Table 8. Biodegradation of Propylene Glycols With Standard Municipal Inoculum...

Superior penicillin producing cultures ate capable of producing in excess of 30 mg/mL of penicillin G (154). Cephalosporin producing strains, however, generally grow poorly and cephalosporin C production is not as efficient as is that of penicillin. Factors such as strain maintenance, strain improvement, fermentation development, inoculum preparation, and fermentation equipment requkements ate discussed in the hterature (3,154). 

Low and high refer to the number of colony forming units (CPU) used to inoculate the media. Comparing MICs at low (10 ) and high (10 ) CPU, a large increase in MIC at the high inoculum level vs a P-lactamase producer is an indicator of instability via enzymatic hydrolysis. 

Most methods used for the production of the commercially important a-amino penicillins, such as ampicillin and amoxicillin, are based on modifications of an enamine process employing the appropriate phenylglycine and methylacetoacetate followed by coupling with 6-APA (64). Other aspects of the fermentation, strain maintenance, equipment, inoculum development, media, and procedures used in the production of penicillin are well covered in previous editions of the Enyclopedia. Developments in these areas have been reviewed (65). 

The converted mash is pumped to a clean sterilised fermentor and the yeast inoculum is added. The set temperature range for whiskey fermentation of 72 hours is usually 17—21°C. At the beginning, the mash converted composition is approximately 80% sugars, mainly maltose and some (<1%) dextrose (primary conversion). The pH is adjusted to reduce initial bacterial growth. Grain neutral spidts are usually set at 27—29°C to expedite fermentation. Temperatures above 35°C inhibit yeast reproduction and promote rapid bacterial growth. Above 40°C actual yeast kill occurs. 

Specific procedures exist for storing (16,17) and propagating microorganisms to obtain reproducible fermentations. The stock culture is stored frozen (<—80°C) or freeze-dried. To prepare the inoculum (seed) mixture, an aUquot is taken and grown in consecutive soHd or Hquid cultures of increasing volume. The volume of the last step, the seed fermentor, is typically 4—12% of the main fermentor volume. 

To alleviate this "cementing", the microbes were grown above-ground in a 500-gal container. They were then allowed to percolate down through the soil. This inoculum had a neutral pH and did not react with the soil. 

Inoculum A batch fermentation dial starts with an initial charge of cells. 

Impf-fliissigkeit, /. inoculation lymph, vaccine lymph, -kristall, w. seed crystal, -mittel, n, inoculating agent, inoculum, inoculant vaccine, -stift, m> inoculating pencil, -stoff, m. inoculating material, inoculum vac-... 

The following process description is taken from U.S. Patent 2,987,449. An appropriate S. aureofaciens strain such as mutant S1308 (ATCC No. 12,748) is grown aerobically in a suitable inoculum medium. A typical medium used to grow the primary inoculum is prepared according to the following formula sucrose, 20.0 g corn steep liquor, 16.5 ml ammonium sulfate, 2.0 g calcium carbonate, 7.0 g and water to 1,000 ml. 

Ten percent of the resulting inoculum is then transferred to a 250 ml Erlenmeyer flask containing 50 ml of the medium employed above and the flask agitated a further 72 hours under the same conditions. One ml of the resulting inoculum is then employed for the inoculation of 10 ml of an aqueous medium containing, per 1,000 ml, 30 grams extraction... 

An inoculum broth is prepared having the following composition 32 pounds starch 32 pounds soybean meal 10 pounds corn steep solids 10 pounds sodium chloride 6 pounds calcium carbonate and 250 gallons water. 

Inoculum Preparation Stage Two batches of inoculum of about 50 gallons each are prepared by the following method A 25 ml inoculum (from the germination stage) is transferred to each of four 2-liter flasks, each containing 500 ml of the sterile medium utilized for germination. The flasks and contents are incubated for 5 days at 28°C on a rotary shaker (280 rpm, 2 inch stroke). 

The experiment was carried out on the 1,000 gallon scale. Three impellers 1 8" diameter at 220 rpm were employed. The air rates were 0 to 5 hours, 40 cfm, 5 to 10 hours, 80 cfm and after 10 hours, 125 cfm. The inoculum rate was 10% v/v. It was prepared by the standard inoculum development technique on the following medium ... 

This was inoculated with a spore suspension of P. patu/um (1 liter containing 3-5 x 10 spores/ml) and grown at 25°C in 100 gallon tank. The inoculum is transferred at 40 hours or when the mycelial volume (after spinning 10 minutes at 3,000 rpm) exceeds 25%. The fermentation is conducted as near to the ideal pH curve as possible by addition of crude glucose, according to U.S. Patent 3,069,328. 

The broth culture so obtained is employed as an inoculum (1%). Into each of ten flasks containing 100 ml of sterile nutrient broth is added 1 ml of the inoculum. The flasks are agitated on a rotary shaker for 8 hours at 28°C at 240 strokes per minute. After this growth period, a solution of 25 mg of 16/3-methy(cortisone in 0.5 ml of methanol is aseptically added to each flask which in turn is reshaken and incubated for an additional 24 hours. The final pH is 7.8. 

At the end of this period, this 600-ml volume was used as an inoculum for ten liters of the same glucose-corn steep liquor medium which in addition contained 10 ml of an antifoam (a mixture of lard oil and octadecanol). The fermentor was placed into the water bath, adjusted to 28°C, and the contents stirred (300 rpm) and aerated (0.5 liter air/10 liters beer). After 17 hours of incubation, when a good growth developed and the acidity rose to pH 6.7, 2 g of 6a-methylhydrocortisone plus 1 g of 3-ketobisnor-4-cholen-22-al, dissolved in 115 ml of dimethylformamide, was added and the incubation (conversion) carried out at the same temperature and aeration for 24 hours (final pH 7.9). 

After the pH was adjusted, 5 g of calcium carbonate was added. This inoculum medium was then subjected to heat sterilization. The medium was then cooled and 2 ml of a spore sus-... 

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. 

A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27 C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coll showed the presence of 1,200 units of polymyxin per cubic centimeter. The polymyxin was extracted and purified by removing the mycelia, adsorbing the active principle on charcoal and eluting with acidic methanol. 

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