Inoculum tanks

Fermenter contaminated[ [inoculum tank contaminated] /inoculum line con-taminated/procedure wrong/tank dirty/air leak/leak from the coil or jacket/faulty sensors/antifoam is not sterile/dirty gaskets, bottom valve, sample line and valve, vent line valve, vacuum breaker/nutrient feed tank or line not sterile/all lines wee not up to sterilization temperature/steam condensate left in lines/the humidity of the fermenter air upstream of the sterile filter is > 90%/pH and DO probes were not deaned between runs/probe holders were not brushed and deaned with a hypochlorite or formaldehyde solution/for a previously contaminated vessel the valves and gaskets were not replaced, instrument sensors were not removed and cleaned high boiling germicide, such as sodium carbonate or sodium phosphate was not used. 

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. 

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. 

The above sterilized medium was inoculated with 11 liters of seed inoculum having a bacterial count of approximately 20 billion per cc. The tank was fermented at 37°C without pH adjustment, aeration, or other modification for 14 hours at the end of which time 320 cc of 50% dextrose was added. After this the pH was adjusted to 7.0 at 15 minute intervals with 5.0 N sodium hydroxide. The volume of sodium hydroxide required for neutralization was noted and 115% of this volume of 50% dextrose solution added after each pH adjustment. At the end of about 8 hours the bacterial count had ceased to increase and the fermentation was terminated. At this time the fermentation medium contained approximately 1,000 units of streptokinase per cc. 

Laboratory-Scale Reactors. Laboratory-scale sterns consisted of small continuously stirred tank reactors (CSTR) with 3.5-liter worlong volumes and were constructed and operated as previously described (62). The digester inoculum for these systems was obtained from the anaerobic digestion of municipal sewage sludge from the Denver... 

Production. The inoculum grew vigorously in the rich yeast extract containing media and produced a thick viscous dispersion in the stirred tank bioreactor. No lignin peroxidase activity could be detected at this stage. When transferred to the 1000 1 production bioreactor, the mycelium of P.chrysosporium attached completely to the nylon wool sheets within a few hours after inoculation and the medium remained completely clear throughout the cultivation. The enzyme had to be harvested immediately after the maximal activity level was reached due to its... 

Remove residual water from filling trays, pipes, tanks, pumps, etc. Water left in the equipment will support microbial growth and can serve as an inoculum for fresh materials. If water cannot be drained, treat with an effective biocide. 

Calculate the output glycerol concentration (/iM) after the microorganisms have increased their biomass to a steady-state level. Also calculate what the steady-state biomass level is (cells m-3). Assume you have a tank with V= 10 m3, a waste water flow Q = 50 m3 d l, and a microbial inoculum with growth properties like those shown in Table 17.6 for the Aerobacter sp., a die off coefficient b of 0.1 d-1, and a glycerol-to-biomass yield of 1014 cells -mol-1. 

Note that this result is independent of the concentration of glycerol in the input stream as long as the other necessary nutrients for growth are sufficient. This result is also independent of the original size of the inoculum. The bacteria simply increase in number until their use of glycerol in the tank (100 /iM - 26 /iM = 74 /iM) corresponds to the bacterial biomass they form during a tank detention time. To obtain an estimate of this steady-state bacterial biomass, we use the glycerol mass balance equation ... 

Fig. 3. Process flowsheet for cellulase production in wheat bran culture [25]. A Submerged seed culture of T. viride, B oil-free compressed air C air filter D inoculum E exhaust air F sample collection G centrifugal pump H automatic wheat bran culture of T. viride I water spray I ammonium sulfate, alcohol and water J conveyor belt J screw conveyor K hopper L extraction column M storage tank centrifuge 0 precipitation P mixing tank Q ion-exchange column R membrane concentrator S spray dryer T filter press U rotary dryer V mixer W cellulase preparation and salt stabilizer... 

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