This SERDP project was a collaborative effort among scientists at CB&I Federal Services, the University of Wisconsin-Madison, and the Naval Air Warfare Center Weapons Division-China Lake. Key objectives were to apply the tools and techniques of synthetic biology to create a biocatalyst for the production of high purity biocellulose (BC), and to develop a scalable process to produce this material for use in critical Department of Defense (DoD) applications. Biocellulose was formulated into military grade nitrocellulose (NC) and evaluated for characteristics and conformity against specifications.

This project used a multi-pronged approach to increase the production of high purity biocellulose. Synthetic biology was used to construct a bacterial strain that had controllable cellulose producing machinery. The process involved genome sequencing and manipulation of the key cellulose synthesis genes. Metabolic modeling was used to identify pathways in the strain that could be manipulated to optimize carbon and energy flow to enhance biocellulose yields, including both anabolic and catabolic pathways to allow the strain to utilize low cost feedstocks (e.g., waste glycerol from biodiesel production) as carbon and/or energy sources to support cellulose synthesis. Batches of biocellulose produced at various stages during the project were analyzed for key parameters (e.g., crystallinity, purity), and used to make and nitrocellulose.

The key results of this project were as follows:

• One of the first reported inducible promoter systems for Gluconacetobacter spp. was identified and validated.
• Isoforms of the key biocellulose synthesis enzyme AcsA that were insensitive to the metabolic control molecule c-di-GMP (CDG) were engineered and should to result in greater cellulose production than the native AcsA.
• A method to use attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to detect bacterial impurities (e.g., nucleic acids, lipids, protein) in biocellulose was developed.
• Detailed metabolic models of the cellulose producing strains used in this research were developed and used to identify key genes/pathways that could be modified to increase biocellulose production.
• Proof-of-concept synthesis of nitrocellulose from biocellulose was successfully performed, and preliminary characterization indicated that the material was comparable to military grade nitrocellulose with respect to FTIR spectra and nitrogen content.

This research has added to the scientific knowledge base regarding cellulose producing bacteria and the cellulose synthesis control. With further development, these findings could lead to the ability to produce biocellulose at an industrial scale, for use in other DoD and non-DoD purposes, including functional papers and textiles, medical implants, and food additives.