Morris Ag-Energy operates a dry-milling ethanol plant at Morris, Minnesota. At the time of these experiments the capacity of this plant had been increased from 4.5 million to about 6 million gpa, primarily through debottlenecking and improved operation. The plant employs conventional batch fermentation and primary distillation systems and a molecular-sieve dehydration system. The fermentors are relatively small, of shallow-tank design, about 14,500 gallons (58000 1) capacity with a working depth of about 8 ft (2.5 m). They are fitted with top-drive slow — speed agitators and internal cooling coils. One fermentor was used for all the in­plant runs; it was modified for pH monitoring and sparging with air or other gasses. A bilobe rotary compressor (Roots blower) of about 60 cfm free air capacity was used for vacuum or headspace gas recirculation.

In this operation, com is ground in a hammermill, then mixed with water (48 gpm, 182 1/min) and recycled thin stillage (39 gpm, 148 1/min) to make a mash of 20 0 Brix. The starch is gelatinized and digested at 90 °С with 120 ml/min of commercial bacterial alpha amylase (IBIS) in a series of stirred tanks; the pH is controlled at 6.5 with ammonia. Further saccharification with glucoamylase (Alltech, 1 volume per 2900 volumes mash) occurs in the fermentor after cooling to 32 °С and pH adjustment. Yeast is produced continuously in a semiaerobic yeast propagator fed the same mash; all of the glucoamylase is added through the yeast propagator so that the yeast see a high initial glucose concentration. The yeast propagator operates at pH 3.5 (adjusted with sulfuric acid) with a cell count typically 0.5×10? to 1.5×10? ml"* and viability 75%. The metabolism of the yeast in the propagator is primarily fermentative but they retain the ability to quickly consume added oxygen. For these experiments the yeast suspension from the propagator was 1/8 the total fermentor charge, resulting in an initial pH of 5.6 which declines during the course of the fermentation to a limiting value of about 3.8.

After fermentation the beer is pumped to a series of beer wells where fermentation is completed and then to the distilling column. Although the normal residence time in the fermentor is 40 h, some of the experimental runs were kept at least 48 h to monitor the completion of the fermentation.

Methods

Analytical. Cell viability was determined using methylene blue (21) , a microscope, and a hemocytometer; total and viable cell counts were determined from the same data. Glucose and glycerol were determined enzymatically using prepared commercial reagents (Sigma 315-100 and 337-40A). Total glucose was determined after acid hydrolysis (0.25M H2SO4, 30 min, 100 °С); for most runs this analysis was employed only as a check of the total conversion. FAN (free alpha-amino nitrogen) was determined by the EBC ninhydrin method (22).

Ammonia was determined by a modified Berthelot reaction (23). Ethanol was determined by gas chromatography using a Hayesep R column (Alltech Instruments). Ethanol values from the industrial runs were considered as relative values only and no interpretation was made of the absolute levels, due to possible handling losses. Carbon dioxide was determined by a modification of the Martin manometric method (24) using a commercial differential pressure sensor (Omega PX26-005DV) instead of a manometer. The reference side of the pressure sensor was connected to the vacuum pump via a 2 1 flask which acted as ballast. Linear calibration curves were obtained through at least 60 mM CO2 Concentration was related to partial pressure by the general approach of Schumpe (25); the effect of ethanol was specifically included based on literature data at low concentration (26). The effect of ethanol on CO2 solubility was judged to be sufficiently linear and reproducible to permit use of this approach over the limited range of ethanol concentrations encountered in these experiments. Fermentor pH was determined in situ with commercial instruments (Omega) and electrodes (Phoenix).

Laboratory Fermentations. These fed-batch runs employed a Biolafitte Fermentor at the BioProcess Institute, University of Minnesota, St. Paul. The medium is listed in Table I; this was based originally on the medium of Oura (27) with NH4CI and yeast extract added to simulate the ammonia and FAN levels prevailing in the industrial fermentation, but it was necessary to increase the yeast extract to even approach the rates and cell counts prevailing in the industrial fermentation. Concentrations were figured on the basis of a 16 1 final volume. The initial glucose concentration was 60 g/1; additional glucose was added to the fermentor as a concentrated solution during the run. The inoculum was Alltech alcohol-production yeast grown in 1 1 of the same medium in a 2 1 unsealed flask shaken at 200 rpm. This is the same yeast source employed by the plant to inoculate their yeast propagator. The fermentor was agitated vigorously and continuously sparged with a mixture of nitrogen and carbon dioxide. Gas mixtures were prepared by continuous metering through rotameters (Cole Parmer), using the manufacturer’s calibration graphs. A back pressure regulator was used to maintain constant pressure and thus constant CO2 partial pressure, calculated from the back pressure and the CO2 content of the sparge gas. Volumetric productivity of ethanol and glycerol was calculated as the increase in concentration since the previous point, divided by the intervening time. Productivity per cell was calculated by dividing the volumetric productivity by the geometric mean of the cell counts at the beginning and end of the interval. Exponential growth and death rates were calculated from those parts of the growth curve which were linear on semi-logarithmic plots. In the air — supplemented laboratory run, oxygen uptake was monitored by continuous mass spectrometry of the inlet and outlet gas streams. An absolute calibration was not performed but the data serve to estimate the relative level and percentage consumption of supplied oxygen. Total oxygen consumption was calculated from these data and the known flow rates.