Cell Recycle Batch Fermentation (CRBF)
Cell recycle batch fermentation (CBRF) is a recently developed batch fermentation method where yeast cells are separated and reused in the next batch. The cell recycling techniques can significantly reduce time and costs associated with inoculum preparation [135,136], and are also an interesting strategy for improving ethanol production in the simultaneous saccharification fermentation (SSF). However, in the case of fermentation of lignocellulosic materials, it is very difficult, or in many cases impossible, to collect only microbial cells by conventional cell collection procedures. This is because lignocellulosic residue, which is not utilized by microbes, is retained in the fermentation medium with yeast cells in the solid fraction after fermentation [137-139]. Lignin left as solid residue in the fermentation medium also inhibits cellulase activity. Therefore, the removal of lignin is required for the efficient saccharification of cellulose in the subsequent batch.
Matano and coworkers have recently developed a new approach of cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for consolidated bioprocessing [140]. In this method a two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid-liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. A schematic diagram of Matano and coworkers CRBF method for fermentation of lignocellulosic materials is shown in Figure 8.7 [140].
After the fermentation, C-5 and C-6 sugars in the fermentation medium and the broth are separated by centrifugation at low gravity (20, 50, or 300 g) for 2 min to obtain a supernatant and a pellet; this pellet contains the lignocellulosic residue. Then the supernatant is separated by decantation, and then centrifuged for 5 min at 4000 g to precipitate yeast cells. The pellet obtained at the second stage was used as the yeast cell fraction for the next batch fermentation.
According to Matano and coworkers, five consecutive batch fermentations of 200 g/L hydrothermally pretreated rice straw led to an average ethanol titer of 34.5 g/L. Moreover, they claimed that the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2 g/L. After five-cycle fermentation only 3.3 g/L sugar was retained in the fermentation medium because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. They reported that fermentation ability of the recombinant strain was successfully kept during five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass.