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Engineering Nanoporous Bioactive Smart Coatings Containing Microorganisms: Fundamentals and Emerging Applications 1,2
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M. C. Flickinger , M. Fidaleo , J. Gosse , K. Polzin , S. Charaniya , C. Solheid , O. K. Lyngberg , M. Laudon , H. Ge , J. L. Schottel , D. R. Bond , A. Aksan , and L. E. Scriven 1,5
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BioTechnology Institute, Departments of Biochemistry, Molecular Biology and Biophysics, Microbiology, Mechanical Engineering, and Chemical Engineering and Materials Science, University of Minnesota, Minneapolis and Saint Paul, MN 55455 3
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Nanoporous, adhesive latex coatings and ink-jet deposited latex microstructures containing concentrated, viable, but nongrowing microorganisms may be useful smart coatings. When rehydrated, these bioactive coatings can be used for multi-step oxidations, reductions, as biosensors, in biofuel cells, or high intensity industrial biocatalysts. Engineering coating microstructure, preservation of microbe viability during drying at ambient temperature and the stability of these coatings following rehydration is investigated in 5 μm to 75 μm thick coatings of microbes concentrated 10 to 10 -fold on polyester, metals or electrode substrates. Nanoporosity is essential for preserving microbial viability in dry coatings and bioreactivity following rehydration. Non-toxic (low biocide or biocide-free) latex emulsions contain carbohydrate porogens which vitrify to arrest polymer particle coalescence during film formation generating nanopores. However, the molecular mechanism of how vitrified carbohydrates function as osmoprotectants and preserve microbial viability by formation of glasses in the pore space during film formation is unknown. Coating nanoporosity in hydrated films is estimated by tracer diffusivity and visualized by cryogenic-SEM. Emul sion composition, drying conditions and coating thickness 2
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© 2009 American Chemical Society
In Smart Coatings II; Provder, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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53 affect microbial viability, substrate adhesion, and coating reactivity following drying, storage and rehydration. The specific reactivity of the entrapped microorganisms can be induced to express enzymes for optimal reactivity prior to coating or the microbes can be "activated" by inducing gene expression following coat drying and rehydration. Laser scanning confocal microscopy is used to investigate spatial gene expression as a function of coating depth and diffusion resistance. Model microbial smart coatings investigated include: an E. coli ionic mercury biosensor, an anaerobic starch hydrolyzing coating of Thermotoga maritima at 80°C, photoreactive coatings of Rhodopseudomonas palustris for anoxic production of hydrogen, coatings of Gluconobacter oxydans which oxidizes D-sorbitol --> L-sorbose, and current-generating coatings of Geobactor sulfurreducens on conductive electrode materials.
Biocatalytic coatings are an emerging class of smart coatings (/, 2). Biocatalytic coatings include water-borne coatings engineered to stabilize, concentrate, and intensify the reactivity of enzymes or cells entrapped in a nanostructured adhesive coating formed by a polymer matrix or partially coalesced polymer particles. Biocatalytic coatings containing entrapped or chemically bound enzymes have been used as reactive smart sensors to detect environmental contaminants, as self-cleaning surfaces, textile coatings, or to catalytically decontaminate surfaces from exposure to chemical warfare agents (2). While enzyme stability and activity can be manipulated by well developed protein engineering methods and enzymes can be efficiently manufactured by microbial recombinant D N A technology, smart coatings containing enzyme-based additives are capable of only single-step hydrolytic reactions (2-9); only recently have nanoporous materials containing more than one immobilized enzyme been reported for multi-step biocatalysis (10). A n additional limitation of enzyme-containing smart coatings is that many isolated enzymes cannot carryout oxidizations or reductions because they require expensive cofactors (such as A T P for phosphorylation or release of energy by hydrolysis) and biological electron donors (reduced co-enzymes N A D H , F A D H , or reduced ferredoxin). While in vitro biological cofactor regeneration systems including cofactor "tethering" have been reported for immobilized enzyme systems (10% it is more cost effective and robust to use living cells to regenerate cofactors and therefore industry still uses slowly growing, entrapped, or "resting" microbes for selective oxidations and reductions to produce chemical intermediates, for environmental decontamination and waste treatment.
In Smart Coatings II; Provder, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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Why Develop Smart Coatings of Nongrowing Microorganisms Preserved at Ambient Temperature? Much effort has been expended in the coating industry to eliminate or inhibit the growth of microorganisms in latex emulsions in order to extend paint shelf-life and functionality following coating and film formation. Microbial smart coatings are the opposite - adhesive latex emulsions that are non-toxic to a very high concentration of microbes in order to preserve microbial viability following film formation. Why would living microbes be useful reactive additives in smart coatings? Living microbes are highly selective catalysts capable of a vast array of multi-step chiral reactions (//, 12) and surprisingly many of these reactions can be carried out by nongrowing cells. Microorganisms can detoxify environmental pollutants, produce a variety of useful products (peptides, proteins, alcohols, organic acids, biopolymers, pharmaceutically active compounds such as antibiotics, or useful gases such as H or methane), or transport electrons to an electrode surface. Extremeophile microorganisms are capable of carrying out these reactions in harsh chemical environments (extreme pH, salinity, pressure, temperatures >100°C) and therefore are attractive "green" biocatalysts. Microbes can be genetically manipulated using recombinant D N A technology to alter their capabilities as biocatalysts. However, living cells as biocatalysts are seldom available as "off-the-shelf (stabilized) highly reactive (concentrated) reagents. Most microbes cannot be stored in a partially desiccated state without loss of activity unless frozen, are used in dilute suspension, and have insufficient stability (active half-life) and specific reactivity (intensity) to replace existing chemical catalysts in industrial processes. In contrast to dilute suspensions, most chemical catalysis is carried out in the pore space of robust catalytic media. The rate of biocatalysis could be significantly increased if living microorganisms also could be immobilized at high density on surfaces with minimal diffusion resistance in coatings that remain adhesive when continuously irrigated with water. Using coating technology to preserve non-growing living bacteria in thin (
