Uenishi, M. | bioRxiv Science

bioRxiv2025

Modulation of gluconic acid metabolism enhances nanofibrillated bacterial cellulose production from agro-industrial by-products

Efficient and robust bacterial cellulose production is essential for advancing the sustainable bioeconomy. In this study, we investigated the impacts of 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 3-fold increase in NFBC yield (from 2.45 to 7.59 g/L). In cultures of a glucose dehydrogenase gene-deficient ({Delta}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. ImportanceIn this study, we investigated the interplay between organic acid metabolism and nanofibrillated bacterial cellulose (NFBC) production in stirred-tank reactor (STR) 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. Key PointsO_LIGA accumulation inhibited growth and cellulose production by Komagataeibacter C_LIO_LIEnhanced central metabolism elevated NFBC yield but reduced its production rate C_LIO_LIConsistent NFBC properties were achieved in STRs using various by-product sources C_LI

bioengineering↗

bioRxiv2022

Genetic differentiation of water penny beetles may be associated with the formation process of the ancient Lake Biwa.

BackgroundWater flow is one of several factors that determine the habitats of aquatic insects. The conversions of stagnant (lentic) and running (lotic) water are physiologically and ecologically constrained. In general, aquatic insect species only occur in one of these environments. Though Eubrianax ramicornis (Coleoptera: Psephenidae) usually resides in rivers, however, it is also known to occur near the shores of Lake Biwa in Japan. Here, we investigated the genetic differences between the two Eubrianax species of Lake Biwa and its inflowing rivers, and other regions of Japan for the first time. Lake Biwa is an ancient lake and about four million years old. Though over 60 endemic species were described from Lake Biwa, they are mostly fish and molluscs, with only two species of aquatic insects. There are few reports of endemic aquatic insects in other ancient lakes, too. Discussion of the endemism of Eubrianax and its promoting factors is expected to provide insights into the evolution of aquatic insects in ancient lakes. MethodsWe surveyed and morphologically identified psephenid larvae in Lake Biwa and its inflowing rivers during the summers of 2017 and 2018 (Hayashi, 2009: Hayashi & Sota, 2009). We conducted phylogenetic analyses of two regions of COI and estimated the ages of population divergence based on fossil records of Eubrianax beetles in Lake Biwa as well as those of previously reported populations in other regions. We also analyzed two COI regions of the E. ramicornis haplotypes in Lake Biwa. ResultsFour psephenid species were identified among the samples collected in Lake Biwa and its inflowing rivers. Of these, E. ramicornis was collected only at the Lake Biwa shore whereas its congeneric E. granicollis was collected only from the rivers inflowing the lake. The phylogenetic analyses showed that E. ramicornis of Lake Biwa were clearly differentiated from those in previously reported regions and this divergence was estimated to have occurred 1.40 MYA. At that time, there was no lake at the location of present-day Lake Biwa. It is thought that rivers and their dammed flood plains to the east, with gravel and sand supplied by the rising Suzuka Mountains further to the east. Such an environments is preferable for E. ramicornis and they were thought to have become isolated from the surrounding rivers during the formation of Lake Biwa. On the contrary, there was no genetic differentiation between the E. granicollis population at the Lake Biwa inflow rivers and those elsewhere. Hence, E. granicollis might have migrated to the Lake Biwa watershed later than E. ramicornis and maintained gene flow over a wide area. The genetic differentiation of E. ramicornis reported here for the first time suggests that a species with a wide distribution may rather be evolving endemic to Lake Biwa. Reports on the endemism of aquatic insects in ancient lakes are scarce. This study is expected to bring new perspectives to the study of insect evolution in ancient lakes.

ecology↗