Fermentation medium determination

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

Here k is the rate constant for the irreversible reaction, Ceo is the total enzyme concentration, Cs is the substrate concentration, and is the Michaelis-Menton constant. Both k and KM may be functions of pH, temperature, and other properties of the fermentation medium. From this kinetic expression, we see that at high substrate concentrations the rate of product formation is independent of Cs and is approximately equal to kCm-This is due to the presence of a limited amount of enzyme, which is required for the reaction to proceed, and adding more substrate under these conditions will not cause the reaction rate to increase further. At low substrate concentrations, the rate of product formation becomes first-order with respect to Cs- Under these conditions the substrate concentration becomes the determinant for product formation, and increasing Cs produces a proportional increase in rate. The rate is also proportional to the total enzyme concentration under all conditions of substrate concentration. 

The quantity of EPS produced after fermentation was determined through dry weight measurements. The fermentation broth was heated at 80° 1°C for 10 min, to ensure microbial inactivation. A filtration was then conducted to remove the cells. To precipitate the EPS, ethanol P.A. (3 1) was added to the fermented broth. After total precipitation of the EPS present in the medium, the mixture (broth plus ethanol) was filtered through a 0.2-pm Millipore membrane using a Gouche crucible previously weighed. The obtained product was dried at 80 1°C until constant weight. All determinations were done in triplicate. 

The fermentation medium was inoculated with Bacillus polymyxa prepared as follows 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 coli showed the presence of 1,200 units of polymyxin per cubic centimeter. 

Mass transfer into viscous fermentation media was examined by Loucaides and McManamey (1973). The rates of oxygen mass transfer into simulated fermentation medium, made up of 16 kg of paper pulp per cubic meter of aqueous sodium sulfite solution with a cupric ion catalyst, were determined in vessels of 0.187, 0.291, and 0.451 m diameter using flat-bladed turbine impellers. As was found for gas-liquid systems (Mehta and Sharma, 1971 ... 

The rifamycins were first isolated by Sensi et al.3) from Nocardia mediterranei as a complex mixture (Rifamycins A—E). Addition of diethylbarbiturate to the fermentation medium led to the sole production of rifamycin B6 which was obtained in crystalline form. Its structure has been determined by chemical7,and X-ray analysis9. The rifamycins might easily have excaped detection altogether, since rifamycin B has no antibacterial activity. However, it is spontaneously oxidized to rifamycin 0 and hydrolyzed to rifamycin S, a naphthoquinone derivative reduction yields the naphthohydroquinone derivative rifamycin SV (Fig. 4). These compounds inhibit the growth of Gram-positive bacteria at concentration as low as 0.0025 jug/ml. 

The substrate is added to the fermentation medium at the time of inoculation or during a later phase of microbial growth1. The optimum time of substrate addition must be determined, whereafter incubation is continued until maximum yield of transformation has been reached. The level of the enzyme responsible for the desired hydroxylation may be enhanced by induction if the substrate is added during active growth of the microorganism. On the other hand, if the substrate inhibits cell growth, substrate addition must be delayed until maximum cell mass is obtained. 

Whereas the specific growth rate is determined by the rate of addition of the growth-limiting substrate, the cell concentration in the fermenter is determined by the concentration of the limiting substrate in the feed medium. At steady state the cell yield (Y) on the growth-limiting substrate can be represented by ... 

The type of fermenter used will be determined by whether the fermentation is to be conducted aerobically or anaerobically, as well as other factors. However, even anaerobic fermentations such as those used for alcohol production, can be conducted in open-topped fermenters when located inside a building. To do this, the top of the fermenting medium is protected from contact with air by the blanket of coproduced carbon dioxide, which is heavier than air. However, closed top fermenters are usually preferred for aerobic or anaerobic fermentation to reduce the risk of contamination of the process by unwanted organisms. 

Initially, tests were performed to verily the best supplementary carbon source for lipase production, using the conditions described by Penha et al. [19]. The experiments were carried out in Erlenmeyer flasks (250 ml), and the fermentation medium consisted of 100 g of powdered wheat bran (60% moisture adjusted adding a 0.91% (m/v) ammonium sulfate solution, pFl=7.0) and 2% mim of vegetable oil (castor, soybean, olive, com, and palm oils). The medium was mixed and sterilized at 1.0 atm for 15 min, and afterwards, inoculated with 10 spores/g substrate. All flasks were closed and incubated in a biochemical oxygen demand (BOD) environment, keeping the moisture and ventilation conditions constant, at 32 C for 96 h. Then, the samples were analyzed to determine the glycosamine content and the lipase activity. 

The content of glycosamine was determined hy the method revised by Penman et al. [20], in which a solution composed of 5 ml of the dried fermented medium previously digested in 70% v/v sulfuric acid for 24 h and sterilized for 1 h at 120°C and 1 atm, was neutralized and mixed with acetyl acetone, ethanol and p-dimethylaminobenzaldehide. After 45 min, the reaction was completed, and the absorbance data could be measured by using a Perkin-Elmer Lambda 10 speetrophotometer, at 530 nm. 

I. Lukkari, J. Ruzicka, G.D. Christian, Determination of total ammonium-nitrogen and free ammonia in a fermentation medium by sequential injection analysis, Fresenius J. Anal. Chem. 346 (1993) 813-818. 

Anaerobiospirillum succiniciproducens is a strict anaerobe and grows at an optimal temperature of 39 °C. A typical fermentation medium contains dextrose, peptone, yeast extract, and salts. The optimal pH range for this organism was determined to be between 5.8 and 6.4. This organism can produce approximately 30 g of succinate/1 fi-om a starting glucose concentration of 50 g/1. Calcium hydroxide is added to produce a calcium succinate product, which can be precipitated fi-om the broth (Datta 1992). 

---