Highly Efficient Enzyme-Functionalized Porous Zirconia Microtubes for Bacteria Filtration Stephen Kroll,* Christoph Brandes, Julia Wehling, Laura Treccani, Georg Grathwohl, and Kurosch Rezwan Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany ABSTRACT: In contrast to polymer membranes, ceramic membranes offer considerable advantages for safe drinking water provision due to their excellent chemical, thermal, and mechanical endurance. In this study, porous ceramic micro- tubes made of yttria stabilized zirconia (YSZ) are presented, which are conditioned for bacteria filtration by immobilizing lysozyme as an antibacterial enzyme. In accordance with determined membrane pore sizes of the nonfunctionalized microtube of ≤200 nm, log reduction values (LRV) of nearly 3 (i.e., bacterial retention of 99.9%) were obtained for bacterial retention studies using gram-positive model bacterium Micrococcus luteus. Immobilization studies of lysozyme on the membrane surface reveal an up to six times higher lysozyme loading for the covalent immobilization route as compared to unspecific immobilization. Antibacterial activity of lysozyme-functionalized microtubes was assessed by qualitative agar plate test using Micrococcus luteus as substrate showing that both the unspecific and the covalent lysozyme immobilization enhance the microtubes’ antibacterial properties. Quantification of the enzyme activity at flow conditions by photometric assays reveals that the enzyme activities of lysozyme-functionalized microtubes depend strongly on applied flow rates. Intracapillary feeding of bacteria solution and higher flow rates lead to reduced enzyme activities. In consideration of different applied flow rates in the range of 0.2-0.5 mL/min, the total lysozyme activity increases by a factor of 2 for the covalent immobilization route as compared to the unspecific binding. Lysozyme leaching experiments at flow conditions for 1 h show a significant higher amount of washed-out lysozyme (factor 1.7-3.4) for the unspecific immobilization route when compared to the covalent route where the initial level of antibacterial effectiveness could be achieved by reimmobilization with lysozyme. The presented platform is highly promising for sustainable bacteria filtration. (1). INTRODUCTION The access to clean water is one of the fundamental requirements for life. Today, 884 million people lack access to safe drinking water, 2.6 billion people have little or no sanitation, and millions of people die annually from diseases transmitted by polluted water. 1-6 Basically, natural water can be polluted by chemical (e.g., organic or inorganic species), physical (e.g., color), and biological (e.g., bacteria, viruses) contamination. Particularly, pathogenic bacteria (e.g., Vibrio cholerae, Legionella pneumo- philia, Salmonella typhi) are responsible for waterborne diseases and present a direct risk to human health. 7-11 Safe drinking water is commonly provided by disinfection, for example by chlorine, ozone, and UV treatment, which has a high effectiveness in killing bacteria. However, toxic or carcinogenic disinfection by-products (DBPs) are formed, which have to be removed subsequently from the purified water samples. The utilization of membranes can overcome this problem by the retention of the bacteria cells at the filtering layer and thus providing a permeate free from bacterial contaminants and DBPs. The main advantage of membrane technology is the fact that it works without the addition of chemicals, with a relatively low energy use and straightforward process handling. 12-15 However, membrane fouling is the most critical problem in many membrane technology applications where fouling is often mediated by biomolecules (e.g., proteins) or bioorganisms (e.g., fungi, bacteria, viruses). Fouling leads to a decline of permeate flux making more frequent cleaning and replacement necessary, which then increases operating costs. 16-19 Today, membranes for bacteria filtration providing a sterile barrier with a cutoff of 200 nm are widely used for process water and wastewater treatments in biotechnology, food technology, and pharmaceutical technology. To improve the antifouling behavior, antibacterial components that either kill or prevent the growth of bacteria can be immobilized onto membrane surfaces leading to self-cleaning matrices. For example, antibacterial substances are silver-based agents (silver ions and silver nanoparticles, respectively), antimicrobial peptides (AMPs such as mellitin, cecropin A), proteins (e.g., lactoferrin), and enzymes (bacterial cell wall hydrolases such as lysozyme). 20-24 Received: February 16, 2012 Revised: July 11, 2012 Accepted: July 24, 2012 Published: July 24, 2012 Article pubs.acs.org/est © 2012 American Chemical Society 8739 dx.doi.org/10.1021/es3006496 | Environ. Sci. Technol. 2012, 46, 8739-8747