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By immobilization in a fibrous‐bed bioreactor (FBB), we succeeded in adapting and selecting an acid‐tolerant strain of Clostridiumtyrobutyricumthat can produce high concentrations of butyrate from glucose and xylose. This mutant grew well under high butyrate concentrations (>30 g/L) and had better fermentative ability as compared to the wild‐type strain used to seed the bioreactor. Kinetic analysis of butyrate inhibition on cell growth, acid‐forming enzymes, and ATPase activity showed that the adapted cells from the FBB are physiologically different from the original wild type. Compared to the wild type, the adapted cultureapos;s maximum specific growth rate increased by 2.3‐fold and its growth tolerance to butyrate inhibition increased by 29‐fold. The key enzymes in the butyrate‐forming pathway, phosphotransbutyrylase (PTB) and butyrate kinase (BK), were also more active in the mutant, with 175% higher PTB and 146% higher BK activities. Also, the mutantapos;s ATPase was less sensitive to inhibition by butyric acid, as indicated by a 4‐fold increase in the inhibition rate constant, and was more resistant to the enzyme inhibitor N, N′‐dicyclohexylcarbodiimide (DCCD). The lower ATPase sensitivity to butyrate inhibition might have contributed to the increased growth tolerance to butyrate inhibition, which also might be attributed to the higher percentage of saturated fatty acids in the membrane phospholipids (74% in the mutant vs 69% in the wild type). This study shows that cell immobilization in the FBB provides an effective means for in‐process adaptation and selection of mutant with higher tolerance to inhibitory fermentation product.

Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three‐dimensional (3‐D) fibrous culture had better cell growth and higher metabolic rates than the two‐dimensional (2‐D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long‐term (1 month) T‐flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at ∼15%. These results could explain why the fibrous bed bioreactor had good long‐term stability and was advantageous for production of non‐growth‐associated proteins by animal cell cultures.

The fermentation kinetics of acetic acid production from fructose by Clostridium formicoaceticumwas studied at pH 7.6 and 37 °C. Recycle batch, fed‐batch, and continuous fermentations using immobilized cells in a fibrous‐bed bioreactor were studied for their potential application in producing acetic acid from fructose, a fermentable sugar commonly found in corn steep liquor and many other food processing wastes. For the immobilized cell fermentation, acetic acid yield from fructose was ∼1.0 g/g, with a final acetate concentration of ∼78 g/L and the overall reactor productivity (based on the fibrous bed bioreactor volume) of ∼0.95 g/(L·h) in the fed‐batch fermentation. For a similar fed‐batch fermentation with free cells, acetic acid yield was ∼0.9 g/g, the highest final acetate concentration was ∼46 g/L, and the overall productivity was ∼0.12 g/(L·h). In the continuous fermentation with immobilized cells, the reactor productivity decreased from 3.2 to 1.3 g/(L·h) as retention time increased from 16 to 72 h to reach 100% conversion. Compared to free‐cell fermentations, the superior performance of the fibrous‐bed bioreactor can be attributed to the high density (>30 g/L) of viable cells immobilized in the fibrous bed. The fermentation product, acetic acid, was found to be a noncompetitive inhibitor to the cells. However, the immobilized cells had a higher maximum production rate (pmax) and a higher value for the inhibition rate constant (Kp) than those for the free cells, suggesting that the immobilized cells in the fibrous‐bed bioreactor were less sensitive to acetic acid inhibition than the free cells. This improvement in kinetic behaviors for immobilized cells confirms that the fibrous‐bed bioreactor can be used as an effective tool for adapting and screening for acetate‐tolerant strains.