Decoupling freshwater consumption from microbial cellulose production

Bacterial cellulose (BC), a nanoscale biopolymer produced primarily by Komagataeibacter xylinus, is valued for its exceptional mechanical strength, purity, and biocompatibility. It enables diverse applications in biomedical engineering, food processing, cosmetics, electronics, water purification, and other industrial sectors. However, conventional BC production relies almost exclusively on freshwater-based nutrient media, creating both scalability and sustainability challenges. To address this issue, the research investigates the use of ocean water (OW) as a sustainable alternative medium for BC production. OW is abundant
and naturally mineral-rich, but its high salt content shows that it negatively impacts microbial growth and product yield.

Our initial investigations elucidate BC yield, structural changes, and functional quality when K. xylinus is cultivated in 50% OW versus conventional freshwater media. A production system was developed employing Komagataeibacter xylinus cultivated in a 50% ocean water medium, achieving a cellulose dry weight concentration of 1 g L⁻¹ under optimized conditions. Scanning electron microscopy revealed a reduction in nanofiber diameters from 58 nm under freshwater conditions to 47 nm in 50% ocean water. Additionally, X-ray fluorescence microscopy demonstrated increased elemental incorporation within the cellulose matrix produced in the ocean water-based system. A techno-economic analysis was conducted to evaluate the production process, highlighting a trade-off between preserving cellulose material properties and reducing production costs. Life cycle assessment was performed specifically on comparing conventional cotton-based cellulose production which indicated that, microbial cellulose production achieved a 31% reduction in water consumption-related environmental impacts.

Furthermore, this thesis investigates microbial co-culture strategies as a means of enhancing BC yield and material performance under 50% OW conditions. Two distinct co-culture configurations were established: K. xylinus with Pseudomonas putida and K. xylinus with Vibrio natriegens, to systematically assess their effects on cellulose yield, crystallinity, and mechanical characteristics. Co-cultivation of K. xylinus with Pseudomonas putida under 50% OW conditions increased BC yield by 1.5-fold, while Vibrio natriegens improved yield by 1.23-fold, compared to monoculture. P. putida co-culture restored crystallinity to 85.65% and achieved a tensile strength of 264.7 ± 15.6 MPa, whereas V. natriegens reached 68.84% crystallinity and 218.6 ± 12.1 MPa tensile strength. Holographic X-ray computed tomography (HXCT) showed that monoculture BC had relatively homogeneous matrix with defined high-density boundaries, while P. putida co-culture produced vertically aligned fibrillar domains, localized high-density striations, and scaffold-like organization. These hierarchical structural features, along with selective acetate metabolism and synergistic cultivation may have influenced fiber alignment, likely underpin the superior mechanical and thermal properties of P. putida-enhanced BC.

Transcriptomic profiling integrated with genome-scale metabolic modelling (GIMME, iMAT, EFlux) revealed that K. xylinus DSM 2325 responds to OW by rapidly engaging osmoadaptive and NADPH-generating pentose phosphate pathway (PPP) activity (~60% flux share) while reducing peripheral biosynthesis and reallocating resources toward stress adaptation and cellulose production. Differential expression patterns indicated early induction of proteostasis and compatible-solute pathways, transient activation of cellulose synthase genes, and sustained expression of PQQ-dependent glucose dehydrogenases, the latter contributing to carbon diversion and medium acidification. Flux scanning identified amplification targets (pgi, gnd, galU, bcsAB) and repression candidates (PQQ-GDH, acetan biosynthesis) as a coordinated metabolic engineering strategy to enhance BC yield under both freshwater and OW conditions. Recognizing the critical role of reactor design in scaling BC production, the thesis also presents the development and evaluation of a novel biofilm reactor suitable for 50% OW-based processes are also presented. PET/poplar emerged as the most effective support material, and K. xylinus outperformed K. rhaeticus and K. sucrofermentans under 50% OW, producing the thickest and most stable pellicles. Across designs, drip-flow reactors were modified to incorporate air–liquid interfaces and solid supports. This enhanced cellulose dry concentration however, nutrient gradients in stacked systems created productivity differences between the lower and upper tiers.

Overall, the work advances industrial BC production through a rational process design that integrates TEA and LCA to ensure feasibility, viability, and alignment with sustainable biotechnology and the circular bioeconomy.