Production of diacetyl (2,3-butanedione) by continuous fermentation with simultaneous product separation

Wen-Shan Lee, Purdue University

Abstract

A study on the fermentation production of important buttery flavor compound, diacetyl, was conducted. Thirteen potential bacteria strains were selected to test their diacetyl production ability through batch fermentations. The homofermentative Lactobacillus casei ATCC 393 was found to synthesize the highest amount of diacetyl, and was chosen for future study. The effect of environmental conditions (e.g. temperature, initial pH, water activity, citrate and pyruvate concentration) on the diacetyl production and cell growth was investigated; a temperature at 30$\sp\circ$C, a initial pH between 6.0 to 5.5, and a water activity-reduced medium favor the organism's diacetyl production. During batch fermentations at 30$\sp\circ$C and initial pH 6.0, L. casei ATCC 393 was found to accumulate as much as 210 mg/L of diacetyl from a modified MRS medium during the exponential growth phase. The diacetyl production stopped after the organism had stopped growth. The diacetyl is not a final metabolite. The diacetyl was reduced to acetoin, and the accumulated diacetyl shows decrement during the organism's stationary growth phase. A kinetic model was developed to describe the diacetyl production as a function of cell growth rate, accumulated diacetyl, and cell mass concentration. In addition to the problem of diacetyl reduction to acetoin, diacetyl was also found to be a serious inhibitor of cell growth. These results suggested that simultaneous fermentation with product separation might improve diacetyl production. A new fermenter with a product stripping column was built to separate diacetyl. Compared to the fermentation without gas stripping, the diacetyl produced with product separation was about 300% higher. From pH-controlled batch fermentations, the diacetyl production was found to have an optimum at pH 5.5. Continuous fermenters with product separation were built to combine the effect of high cell growth rates, product removal, and the suppressed diacetyl reductase activity by maintaining the pH at 5.5. Diacetyl production in a 1,200 ml-scaled fermentation was 2.5 grams over 5 days. A theoretical model for diacetyl production was developed by summarizing information from diacetyl physical properties study, batch fermentations, and pH-controlled batch fermentations with product separation. The model was found to predict reactor performance well. A 50-cm height immobilized trickle bed reactor was built and its performance was tested. The reactor only got a small cell mass load, which results in a poor diacetyl, acetoin, and lactic acid production. The reason found was the quick pH drop along the bed height. A height-simulated immobilized cell reactor was then built to keep the pH at a higher value. The reactor had high cell mass load and good lactic acid and acetoin production but still had a poor diacetyl production. The cell aging in the immobilized cell reactor should be the reason.

Degree

Ph.D.

Advisors

Okos, Purdue University.

Subject Area

Agricultural engineering

COinS