Daniel W. Karl1, Kris M. Roth2’3, Frederick J. Schendel4, Van D. Gooch5,

and Bruce J. Jordan2

Daniel Karl Scientific Consulting, 430 Saratoga Street South,

St. Paul, MN 55105

2Morris Ag-Energy, Inc., P. O. Box 111, Morris, MN 56267
department of Chemistry, University of Minnesota, Morris, MN 56267
4ENCORE Technologies, 111 Cheshire Lane, Suite 500,
Minnetonka, MN 55305

department of Biology, University of Minnesota, Morris, MN 56267

High levels of carbon dioxide are known to be inhibitory to yeast growth, at least at the low temperatures prevailing in the brewing industry, and have also been suggested to favor increased diversion of carbon to glycerol. Since it was not clear whether the inhibitory effects depend on the bulk concentration of CO2 or on its partial pressure, it was not clear whether the same results would be obtained under the higher temperatures employed in fuel alcohol fermentation.

We first determined the conditions prevailing in an industrial com-to — ethanol fermentation plant employing relatively small fermentors, then carried out laboratory fed-batch fermentations with glucose feed with CO2 partial pressures of 0.5, 1.5, 2.5, and 3.5 atm absolute.

Elevated carbon dioxide slowed the fermentation, particularly at the later stages, decreased the maximum number of viable cells obtained and increased cell death rates slightly. High carbon dioxide also decreased overall glycerol production. Low-level aeration also decreased glycerol productivity on a per-cell basis but stimulated cell growth to a compensating extent so that the final level was comparable to the control

Carbon dioxide has both stimulatory and inhibitory effects on the metabolism of living cells. It is known to be required, at relatively low concentrations, by several essential biochemical pathways. For yeast, carbon dioxide concentrations up to 5% in the gas phase have been found to be stimulatory (1,2). Inhibition of various functions begins in at higher concentrations. Aerobic metabolism is significantly inhibited at 0.5 atm CO2 (3), but fermentation per se is not inhibited at 3.5 atm (4) and only begins to be inhibited at 10 atm (5). Anaerobic yeast growth is inhibited

© 1997 American Chemical Society

by lower concentrations, with effects apparent as low as 1.5 atm, depending on the temperature at which the yeast are growing and the strain of yeast (6-9), some selected strains have been propagated at elevated CC>2 concentrations (10). Both rate and extent of growth are affected by inhibitory levels of CO2 under conditions of the brewing industry; the presence of abortive buds and enlargement of the cells suggests interference at specific steps in the cell cycle. Rice et al. present evidence that it is the concentration of dissolved carbon dioxide, and not its partial pressure, which determines the extent of inhibition (6). Thus a given partial pressure of CO2 became less inhibitory as the temperature increased, within the temperature range encountered in brewing. Whether this trend continues into the substantially higher temperature range employed in fuel alcohol production is not known.

The mechanisms involved in CO2 inhibition are unclear, although there are many candidates (11). Carbon dioxide is believed to partition freely into and through biological membranes (12,13), so a purely osmotic mechanism seems unlikely. Yeast cells employ ion-transport mechanisms to maintain their internal pH in spite of the perturbing effect of membrane-permeating weak acids such as CO2, acetic acid, or propionic acid; this is effective in limiting the intracellular pH change to about one unit for a four-unit change in the external pH (14), but carries a cost in energy expenditure. It is not known whether yeast cells have a mechanism for expelling the resulting bicarbonate ion; if they do not, intracellular bicarbonate concentrations could become high enough to inhibit cytoplasmic enzymes (15). Carbon dioxide is similar to other weak acids in inducing potassium uptake by yeast (14). Action at or within the plasma membrane may also account for some or all of the inhibition, similar to mechanisms postulated for ethanol inhibition (16,17).

Oura in 1977 argued that carbon dioxide can also have a substantial influence to increase production of the fermentation coproducts glycerol and succinic acid (18). Glycerol, which is produced to maintain redox balance within the cell, can account for a substantial diversion of carbon away from ethanol production. A major part of the glycerol production occurs to correct a redox imbalance due to production of succinic acid. Decreased carbon dioxide partial pressure and increased available nitrogen were suggested as means of minimizing succinate-associated glycerol production. The available evidence, however, suggests that high carbon dioxide partial pressure decreases rather than increases glycerol production, at least under semi-aerobic conditions in continuous fermentation (19,20).

Although hydrostatic pressures of a few atmospheres have no detectable effects on yeast, hydrostatic pressure increases the saturation concentration of carbon dioxide. Tall fermentors may engender hydrostatic pressures of two atmospheres or more; adding atmospheric pressure and supersaturation may result in local CO2 partial pressures of 3.5 atmospheres near the bottom of a tall industrial fermentor. Indeed, adoption of the use of tall tanks prompted much of the brewing industry’s interest in carbon dioxide effects. We sought to determine whether carbon dioxide effects on fermentation and carbon diversion to glycerol under conditions of the fuel alcohol industry should be a consideration in fermentor design and plant operation.

This investigation had three parts. First, we determined the conditions prevailing during ethanol fermentation in a commercial ethanol plant. Second, we conducted controlled laboratory fermentations at various carbon dioxide pressures. We did not attempt an exact duplication of the industrial process, but rather to simulate certain of the biologically relevant conditions in a more controllable fashion. Thus we used a steady glucose feed to simulate the continuous release of glucose from starch in the industrial fermentation, and used yeast extract to provide the complex nitrogen compounds provided in the industrial proceed by recycled stillage (backset). The remainder of the medium was based on a, well known defined medium to insure nutritional adequacy. Finally, based on what we observed in the laboratory runs, we attempted to decrease carbon dioxide levels and control glycerol production in the industrial process by operation at reduced pressure or with slow air sparge.