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The advances in bioprocess design, genetic engineering, and media optimization have enabled enhanced bacterial cellulose (BC) production and its application for diverse purposes. Following the requirements of a bioeconomy, numerous approaches were conducted to investigate alternative carbon or nitrogen sources from industrial by-products for BC biosynthesis. They can, however, not only affect the BC production but also its properties. Beet molasses, vinasse, and waste beer fermentation broth (WBFB) have thus been investigated in single and combined approaches for their BC production potential and effects on structural properties using Komagataeibacter xylinus DSM 2325. Therefore, the composition of each complex component was initially analyzed for total organic carbon (TOC), total bound nitrogen (TNb), sugars, organic acids, and alcohols. The polymer properties were characterized via gel permeation chromatography and X-ray diffraction. In dynamic shake flask cultivations, the exchange of Hestrin-Schramm (HS) medium components for a combination of all three complex substrates on a TOC- or TNb-based quantity resulted in the highest BC concentration (8.2 g L−1). Comparable concentrations were achieved when combining molasses and WBFB (8.1 g L−1). Each investigated complex component led to differing degrees of polymerization (DPn: 2751−4601) and BC crystallinities (26−58%) in comparison to HS medium. Beet molasses and vinasse were found to decrease the polymer crystallinity but induce higher DPn whereas the opposite occurred for WBFB. This study thus highlights beneficial effects of food industry by-products for BC biosynthesis and elucidates concomitantly occurring structural polymer alterations to enable further studies dealing with alternative substrates for structurally tailored BC production.

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