Introduction

Metabolic activity of microorganisms depends on many factors, including oxidation and reduction reactions, or the “redox potential” of the culture environment. Redox reactions govern metabolism of biologically important nutrients such as carbon, hydrogen, oxygen, nitrogen and sulfur. Measuring their redox potential allows the fermentor operator to monitor the addition of reducing or oxidation agents, while ensuring that the potential is in the proper range for cell growth, especially when the dissolved oxygen (DO) level is very low.

Since free electrons never exist in any noteworthy concentration, reduction and oxidation reactions are always coupled together, and can be considered a measure of the ease with which a substance either absorbs or releases electrons. The determination of redox potential is a po-tentiometric measurement, expressed as millivolts (mV). Practically, however, no electrical current flows through the sample solution during this potential measurement.

Redox sensors are most commonly used to maintain anaerobic conditions in a fermentation broth. They can be used to measure trace amounts (<1 ppm) of dissolved oxygen, at levels that are too low for the DO sensors in various anaerobic fermentation processes. Glucose-containing feed medium can be treated as a reducing source in oxidation-reduction of the culture medium. When the oxidation capacity is increased, the Redox potential level will elevate to a higher value. On the flip side, its value will become lower when the culture broth has a higher reducing capacity.

Our study used Saccharomyces cerevisiae yeast (ATCC 20602), because Saccharomyces is widely used in industry (e.g. beer, bread and wine fermentation and ethanol production), as well as in the lab due to its ease of manipulation and growth. Additionally, yeasts are eukaryotic and comparatively similar in structure to human cells. S. cerevisiae metabolizes glucose to ethanol primarily by way of the Embden-Meyerhof pathway. However, a small concentration of oxygen can be provided to the fermenting yeast, as it is a component in the biosynthesis of polyunsaturated fats and lipids. Typical amounts of O₂ maintained in the broth are 0.05–0.10 mm Hg oxygen tension. We used redox potential measurements to maintain these special anaerobic fermentation conditions. A trace of air (oxygen) was introduced as an oxidation agent to raise the redox potential level. A short pulse of air was introduced into the vessel when redox fell below −180 mV (Figure 1).

Figure 1. (Click to enlarge)

Materials and Methods

S. cerevisiae strain ATCC 20602 was grown in a 5-liter working volume benchtop BioFlo® 310 fermentor (New Brunswick Scientific). Ethanol production and glucose concentrations were measured with a 2700 Select (YSI). A redox sensor (Mettler Toledo) was directly connected to the BioFlo 310 controller to track redox potential. Beck-man-Coulter's Vi-Cell XR Cell Viability Analyzer was used to measure cell viability and concentration throughout the entire process.

A seed culture using a 1.0 mL frozen suspension was prepared in a 1-liter Erlenmeyer flask containing 250 ml of DIFCO YM growth medium (Becton Dickinson). The culture was incubated at 29°C for 18 hours in an orbital shaker (NBS model Innova® 43R) at 240 rpm. The entire inoculum was transferred to the BioFlo 310 fermentor vessel containing 4.75 liters of medium. Medium composition was as follows, at grams per liter concentrations: glucose, 10.0; MgSO4.7H₂O, 0.6; (NH4)₂SO₄, 3.0; KH₂PO₄, 10.0; CaCl₂.2H₂O, 0.14; yeast extract, 18.0; soy peptone, 18.0; Na₂HPO₄,1.0; Thiamine, 0.01; trace metal solution, 1 mL/L; antifoam, 0.5–1.0 mL/L.

Setpoints were as follows: Temperature: 30°C, pH: 5.0, aeration rate: 2.5 L/min (0.5 vvm), and agitation speed: 200-800 rpm. pH was controlled with a 29% NH₄OH base solution. 50% glucose was used as feed medium. DO and redox potential were measured during the entire process. DO was cascade-controlled at 30% via agitation in the growth phase. Glucose feed started at 7 hours of elapsed fermentation time (EFT) after the glucose was close to 1 g/L. New Brunswick Scientific's BioCommand® with OPC bioprocessing software was used to control and log the entire process. The optical density of fermentation broth was measured at 600 nm to monitor cell growth. To determine concentrations of glucose, ethanol and the dry cell mass, samples were centrifuged and the supernatant and biomass were collected separately. Biomass samples were dried at 80°C for 48 hours.

Anaerobic ethanol production phase

After 24 hours of cell growth, the fermentation process was switched to an anaerobic condition by exposing the culture to two simultaneous perturbations: a rapid depletion of oxygen and glucose feeding regulated by redox potential measurement. Nitrogen, instead of air, was used to sparge the fermentor vessel. Gas flow rate was kept at 0.5 vvm (2.5 L/min). On-line redox potential readings of −180 mV triggered a solenoid valve of air supply to maintain the oxidation-reduction level for the ethanol production (shown in Figure 1). pH was well controlled at 5.0. Cells remained healthy; OD values were maintained around 80 and viability was 85% at 72 hours EFT. 85 g/L of ethanol were produced in 70 hours as shown in Figure 3.

Figure 2. (Click to enlarge)

Figure 3. (Click to enlarge)

Conclusion

S. cerevisiae was cultured in an aerobic fermentation, and then switched to an anaerobic process using on-line redox measurements to maintain oxidation-reduction levels for ethanol production. Our study produced 85 g/L of ethanol in 70 hours, while cell viability was maintained at levels as high as 88%. The study provides a new technique for using redox potentials to monitor and control ethanol production from yeast, and demonstrates the BioFlo 310 fermentor as a versatile fermentor for aerobic and anaerobic fermentations.