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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

Title: Engineered biosealant producing inorganic and organic biopolymers

Authors
item Bergdale, Terran -
item Pinkelman, Rebecca -
item Hughes, Stephen
item Ciurli, Stefano -
item Zambelli, Barbara -
item Bang, Sookie -

Submitted to: Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 2, 2012
Publication Date: N/A

Interpretive Summary: In this study, mucoid microorganisms were genetically engineered to create a recombinant strain producing a potentially stronger biosealant. This is important in the repair of structural cracks in granite and concrete, and could more strongly bond the solid to the cracks. The construction of the recombinant strain is a significant contribution to genetic engineering of extremely mucoid microorganisms, overcoming specific difficulties during transformation. This result will benefit the construction and masonry industries.

Technical Abstract: Microbiologically induced calcium carbonate precipitation (MICCP) is a naturally occurring biological process that has shown its potential in remediation of a wide range of structural damages including concrete cracks. MICCP involves sequential microbiological and chemical reactions, such as urea hydrolysis by microbial urease to ammonia and hydrogen carbonate, followed by calcium carbonate (CaCO3) precipitation due to the pH increase of the milieu in which the biochemical reaction occurs. In this study, genetically engineered microorganisms, capable of producing extracellular polymeric substances (EPS) as well as inducing MICCP, were developed based on the assumption that the complex of inorganic CaCO3 and organic EPS would provide a stronger matrix than MICCP alone as biosealant. In order to develop a recombinant biosealant microorganism, the entire Sporosarcina pasteurii urease gene sequences including ureA, ureB, ureC, ureD, ureE, ureF, and ureG from plasmid pBU11 were sub-cloned into the shuttle vector, pUCP18. The newly constructed plasmid, pUBU1, was transformed into two Pseudomonas aeruginosa strains, 8821 and PAO1, to develop recombinants capable of inducing calcite precipitation in addition to their own ability to produce EPS. Nickel-dependent urease activities were expressed from the recombinant P. aeruginosa 8821 (pUBU1) and P. aeruginosa PAO1 (pUBU1), at 99.4% and 60.9% of the S. pasteurii urease activity, respectively, in a medium containing 2 mM NiCl2. No urease activities were detected from the wild type P. aeruginosa 8821 and P. aeruginosa PAO1 under the same growth conditions. The recombinant Pseudomonas strains induced CaCO3 precipitation at a comparable rate as S. pasteurii, maintaining similar amounts of extracellular alginate produced by their wild types. Scanning electron microscopy evidenced the complex of CaCO3 crystals and EPS layers surrounding the recombinant cells.

Last Modified: 11/24/2014
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