Enhanced production of nanofibrillated bacterial cellulose through regulation of gluconic acid metabolism

ABSTRACT Efficient and robust bacterial cellulose production is essential for advancing the sustainable bioeconomy. In this study, we investigated the impacts of the metabolism of organic acids, mainly gluconic acid (GA), on nanofibrillated bacterial cellulose (NFBC) production by Komagataeibacter intermedius NEDO-01 under various culture conditions in aerated stirred-tank reactors. In cultures of the wild-type strain in a standard medium, rapid GA production decreased the medium pH and depleted glucose, inhibiting cell growth and reducing the NFBC yield. However, proper pH control and continuous feeding reversed these effects, resulting in a threefold increase in NFBC yield (from 2.45 to 7.59 g/L). In cultures of a glucose dehydrogenase gene-deficient (Δ gcd ) strain, lack of a pH drop and glucose depletion facilitated better cell growth, yielding 1.85 times more NFBC than that in wild-type cultures under pH-uncontrolled no-feed conditions (4.53 g/L). Notably, GA supplementation accelerated cell growth but significantly inhibited NFBC synthesis, suggesting that GA uptake redirects the carbon flux toward central metabolism. In the corn steep liquor (Csl)-based medium, cell growth was significantly enhanced, and NFBC yield was equivalent to or higher than that obtained with the Hestrin–Shramm medium. GA accumulation was markedly reduced, suppressing pH fluctuation. Under these optimized conditions, three molasses types were tested with Csl, yielding relatively high NFBC. Structural analysis of NFBC produced using these alternative media revealed slight differences in the fiber width distribution, with crystallinity and fiber width remaining constant. Overall, NFBC of consistent quality can be produced in stirred-tank reactors using Komagataeibacter spp. from various agricultural by-products. IMPORTANCE In this study, we investigated the interplay between organic acid metabolism and nanofibrillated bacterial cellulose (NFBC) production in stirred-tank reactor cultures of Komagataeibacter intermedius NEDO-01. While it is well known that gluconic acid production competes with cellulose biosynthesis in Komagataeibacter , the quantitative relationship between these pathways under varying culture conditions has not been fully elucidated. By applying optimized feeding strategies and employing a glucose dehydrogenase knockout mutant, we demonstrated that suppressing gluconic acid accumulation significantly enhances NFBC yield. Furthermore, we explored the use of agro-industrial by-products, including molasses and corn steep liquor, as alternative, low-cost feedstocks. Structural characterization confirmed that NFBCs produced under these conditions maintained consistent quality. These findings contribute to the development of scalable, cost-effective microbial production processes for nanocellulose, which is essential for advancing the sustainable bioeconomy.