Presentation Information
[2EMT-01-KL]Robust Production of Nanofibrillated Bacterial Cellulose by Komagataeibacter intermedius and its Engineered Derivative
○Ryo Takahama1,2, Yuma Ishido2, Miho Suginaka2, Kenji Tajima2, Chiaki Ogino1 (1. Kobe University (Japan), 2. Hokkaido University (Japan))
Keywords:
Bacterial cellulose,Komagataeibacter,Repeated batch culture,Agro-industrial by-products
Nanofibrillated bacterial cellulose (NFBC) is a highly dispersible form of bacterial cellulose with considerable potential as a sustainable functional biomaterial, but its efficient, stable, and scalable production remains challenging. In this study, we investigated strategies to improve NFBC production by Komagataeibacter intermedius NEDO-01 and its engineered derivative under stirred and aerated conditions in bioreactors, focusing on the effects of gluconic acid (GA) metabolism, medium composition, and repeated-batch operation.
In cultures of the wild-type strain, rapid oxidation of glucose to GA caused a decrease in pH and depletion of glucose, which suppressed cell growth and reduced NFBC production. However, appropriate pH control combined with continuous substrate feeding effectively alleviated these limitations, increasing the NFBC yield from 2.45 to 7.59 g/L. A glucose dehydrogenase-deficient mutant (Δgcd) showed improved cell growth and produced 1.85-fold more NFBC than the wild type under certain conditions, owing to reduced GA accumulation and the absence of severe pH decline.
We also evaluated the use of corn steep liquor (Csl)-based medium and agricultural by-products as alternative substrates. In the Csl-based medium, cell growth was significantly enhanced, while GA accumulation and pH fluctuations were suppressed, resulting in NFBC yields comparable to or higher than those obtained in conventional Hestrin–Schramm medium. Furthermore, three types of molasses supported relatively high NFBC production in the Csl-based medium, demonstrating the feasibility of using agricultural by-products as low-cost feedstocks.
To improve production efficiency and operational stability, repeated-batch culture was examined under stirred and aerated conditions. Reducing the cycle time from 72 h to 60 h and the inoculum rate from 20% to 10% did not substantially decrease NFBC yield, identifying 60 h and 10% as efficient operating conditions. Under these conditions, NFBC yield and cell density remained stable over eight consecutive cycles. After the second cycle, peaks of cell growth and NFBC production appeared earlier than in the first cycle, indicating faster initiation of production during repeated-batch operation. Sequence analysis of the bcs operon detected no stable mutations, supporting genetic stability during prolonged cultivation. In addition, solid-state NMR, AFM, and water-dispersibility analyses showed that the chemical composition, morphology, crystallinity, and dispersibility of NFBC were largely maintained throughout cultivation, although sugar-to-NFBC conversion efficiency gradually declined in later cycles.
Overall, these results demonstrate that control of GA metabolism, use of alternative low-cost media, and repeated-batch cultivation together provide an effective strategy for robust, efficient, and stable NFBC production by Komagataeibacter strains.
In cultures of the wild-type strain, rapid oxidation of glucose to GA caused a decrease in pH and depletion of glucose, which suppressed cell growth and reduced NFBC production. However, appropriate pH control combined with continuous substrate feeding effectively alleviated these limitations, increasing the NFBC yield from 2.45 to 7.59 g/L. A glucose dehydrogenase-deficient mutant (Δgcd) showed improved cell growth and produced 1.85-fold more NFBC than the wild type under certain conditions, owing to reduced GA accumulation and the absence of severe pH decline.
We also evaluated the use of corn steep liquor (Csl)-based medium and agricultural by-products as alternative substrates. In the Csl-based medium, cell growth was significantly enhanced, while GA accumulation and pH fluctuations were suppressed, resulting in NFBC yields comparable to or higher than those obtained in conventional Hestrin–Schramm medium. Furthermore, three types of molasses supported relatively high NFBC production in the Csl-based medium, demonstrating the feasibility of using agricultural by-products as low-cost feedstocks.
To improve production efficiency and operational stability, repeated-batch culture was examined under stirred and aerated conditions. Reducing the cycle time from 72 h to 60 h and the inoculum rate from 20% to 10% did not substantially decrease NFBC yield, identifying 60 h and 10% as efficient operating conditions. Under these conditions, NFBC yield and cell density remained stable over eight consecutive cycles. After the second cycle, peaks of cell growth and NFBC production appeared earlier than in the first cycle, indicating faster initiation of production during repeated-batch operation. Sequence analysis of the bcs operon detected no stable mutations, supporting genetic stability during prolonged cultivation. In addition, solid-state NMR, AFM, and water-dispersibility analyses showed that the chemical composition, morphology, crystallinity, and dispersibility of NFBC were largely maintained throughout cultivation, although sugar-to-NFBC conversion efficiency gradually declined in later cycles.
Overall, these results demonstrate that control of GA metabolism, use of alternative low-cost media, and repeated-batch cultivation together provide an effective strategy for robust, efficient, and stable NFBC production by Komagataeibacter strains.
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