Consecutive Batch Fermentation For Very High Gravity Ethanol Production Using Flocculating Yeast

Fuel ethanol, both environmentally friendly and renewable, is the mostly widely used biofuel in the world. Cost analysis of ethanol production from starch-based feedstocks reveals that energy consumption contributes about 30% of the total cost, the second largest only after that from feedstocks consumption. Very high gravity (VHG) fermentation, aiming at achieving more than 15% (v/v) ethanol, can significantly save energy consumption, not only in the distillation, but also in the treatment of the distillage with the DDGS (distilled dry grain with solubles) process, making it much more promising. However, the biggest challenge of VHG fermentation comes from the strong inhibition of ethanol. Batch and plug flow reactors are two ideal models to alleviate product inhibition, but biomass conconcentration is usually low within a batch reactor, which correspondingly needs longer fermentation time. The multistage fermentation system that can alleviate backmixing to some extent and produce 12-13% (v/v) ethanol is still problematic for VHG ethanol fermentation.Yeast flocculation is a phenomenon that is controlled by FLO genes and affected by bivalent cations, particularly Ca2+, through which yeast cells adhere together and form flocs. In the brewing industry, yeast flocculation benefits the separation of biomass at the end of the fermentation. Recently, ethanol fermentation with the self-flocculating yeast SPSC01 immobilized within bioreactors was developed for fuel ethanol production. In this thesis, VHG ethanol fermentation by SPSC01 was studied, with the following focuses:1. Development of consecutive batch VHG ethanol fermentation strategyConventional batch mode using the flocculating yeast SPSC01 under VHG conditions needs much longer fermentation time, and thus its ethanol productivity is very low, which confers no advantages over free yeast cells. Moreover, without a seed culture system to supplement viable yeast flocs into the fermentation system periodically, continuous ethanol fermentation using SPSC01, whether a single fermentor or tanks in series, produced high ethanol concentration for a while, but was unstable thereafter due to significant viability loss of yeast flocs suffered from severe ethanol inhibition. Consecutive batch fermentation with SPSC01 at high biomass density was then developed to shorten fermentation time. Taking advantage of yeast flocs to separate from fermentation broth by self-sedimentation, the inhibition of ethanol in yeast cell growth was significantly alleviated. Compared to other VHG fermentation technologies, the consecutive batch mode produced more than 15% (v/v) ethanol, with an average productivity as high as 8.62 g/L/h.2. Investigation of the settling performance of SPSC01Within the duration of the consecutive batch fermentation, yeast flocs experienced significant change in their settling performance, from free settling of intact flocs to hindered settling of snowflake-like incompact flocs, followed by compression settling, which was slow and undesirable for separating yeast flocs from fermentation broth, exacerbating ethanol inhibition in the yeast flocs. The impact of the density and size of yeast flocs and the apparent viscosity of fermentation broth on their settling performance was thus investigated. It was found that the decrease of the size of yeast flocs was the main reason for this phenomenon.3. Mechanism of the deflocculation of SPSC01 and process modificationPrevious studies indicated that the gene FLO1 is crucial to the flocculation of SPSC01, and its expression confers both flocculation and hydrophobicity to yeast cells. It was found that the hydrophobicity of yeast flocs was not the main factor affecting their flocculation performance. Neither were the physiochemical effect of ethanol on yeast cell wall and its physiological effect on intracellular mitochondrial functions. Moreover, no significant change in the gene intergrity was detected, since the flocculating ability of the de-flocculating yeast flocs could be restored by the regeneration of them in flask culture. The analysis of the FLO1 transcription indicated that compared with flask culture, its expression level decreased sharply at the end of the consetutive batch fermentation, which was the underlying reason for the deflocculation of SPSC01.The process was modified by purging yeast cells at the end of each batch within the duration of the consecutive batch fermentation, which lowered the density of yeast flocs within the fermentor, and thus simulated their growth. The flocculationd settling performance of yeast flocs were improved, and the fermentatioin system was run reliably.4. Effect of ethanol concentration and production rate on FLO] transcriptionEthanol affects the flocculating ability of yeast flocs by inhibiting FLO1 transcription. It was found that the higher the ethanol concentration and the faster the production rate, the more severe was the gene transcription inhibited. The high ethanol production rate change not only impaired FL01 transcription, but also induced pseudohyphal development, a significant morphology change.In summary, consecutive VHG batch fermentation using SPSC01 that could improve ethanol productivity was established. The deflocculation of the yeast flocs under VHG conditions and subsequent impact in their settling performance were contributed to the decrease in the FLO1 transcription. The effect of ethanol concentration and production rate on the FLO1 transcription and morphology of SPSC01 were observed. With these understandings, the consecutive VHG batch fermentation process was modified and the performance was greatly imporved.