Nitric Oxide Treatment for the Control of Reverse Osmosis Membrane Biofouling Robert J. Barnes, a, * Jiun Hui Low, a,b,c,d Ratnaharika R. Bandi, a Martin Tay, c Felicia Chua, a Theingi Aung, a Anthony G. Fane, b Staffan Kjelleberg, d,e,f Scott A. Rice d,e,f Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, Singapore a ; Singapore Membrane Technology Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, Singapore b ; Interdisciplinary Graduate School, Nanyang Technological University, Singapore c ; Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore d ; Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia e ; School of Biological Sciences, Nanyang Technological University, Singapore f Biofouling remains a key challenge for membrane-based water treatment systems. This study investigated the dispersal potential of the nitric oxide (NO) donor compound, PROLI NONOate, on single- and mixed-species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis (RO) membranes. The potential of PROLI NONOate to control RO membrane biofouling was also examined. Confocal microscopy revealed that PROLI NONOate exposure induced biofilm dispersal in all but two of the bacteria tested and successfully dispersed mixed-species biofilms. The addition of 40 M PROLI NONOate at 24-h intervals to a laboratory-scale RO system led to a 92% reduction in the rate of biofouling (pressure rise over a given period) by a bacterial community cultured from an industrial RO membrane. Confocal microscopy and extracellular polymeric substances (EPS) extraction revealed that PROLI NONOate treatment led to a 48% reduction in polysaccharides, a 66% reduction in pro- teins, and a 29% reduction in microbial cells compared to the untreated control. A reduction in biofilm surface coverage (59% compared to 98%, treated compared to control) and average thickness (20 m compared to 26 m, treated compared to control) was also observed. The addition of PROLI NONOate led to a 22% increase in the time required for the RO module to reach its maximum transmembrane pressure (TMP), further indicating that NO treatment delayed fouling. Pyrosequencing analysis re- vealed that the NO treatment did not significantly alter the microbial community composition of the membrane biofilm. These results present strong evidence for the application of PROLI NONOate for prevention of RO biofouling. M embrane technology, for the conversion of seawater and wastewater through desalination and reclamation processes into potable water, is vital for sustainable water management. However, membrane fouling by bacterial biofilms remains a key challenge for these technologies (1–3). Initially, the adsorption of organic species and suspended particles on the wetted membrane surface form a conditioning film. This enables attachment of planktonic cells to the membrane surface, followed by the forma- tion of microcolonies and biofilm maturation, where bacterial cells are embedded in a self-produced matrix of extracellular poly- meric substances (EPS) (1, 4, 5). The EPS is typically composed of polysaccharides, proteins, and nucleic acids. The attachment of microorganisms to the membrane surface is affected by factors such as the membrane material, the roughness of the membrane surface, hydrophobicity, and membrane surface charge (6). Bio- film bacteria have several advantages over single planktonic cells, including optimization of growth and survival, improved acqui- sition of nutrients, and increased protection against environmen- tal stresses, including shear forces (2, 5, 7). Biofilm formation on membrane surfaces results in a severe decline in flux, or an increase in transmembrane pressure (TMP; defined as the pressure gradient of the membrane, or the average feed pressure minus the permeate pressure) to maintain flux, higher energy consumption, and a deterioration of system perfor- mance and product water production (3, 8, 9). As the adhesive and cohesive matrix of biofilms, EPS has been suggested to be the predominant culprit for biofouling of water treatment mem- branes (1, 10, 11). The EPS is composed mainly of polysaccharides and proteins, which form hydrogel matrices (12). Common tech- niques to reduce membrane fouling include membrane cleaning and pretreatment of the feed water. However, microorganisms may survive pretreatment processes such as coagulation, floccula- tion, sand filtration, ultrafiltration, and cartridge filtration to sub- sequently colonize and foul the system (3). Membrane cleaning by physical or chemical methods is used to regenerate the function of fouled membranes, and the methods used and the frequency of cleaning depend on the type of foulant as well as the resistance of the membrane to chemical cleaning agents (13). However, these cleaning methods frequently shorten membrane life, further in- creasing operational costs (11, 14). Despite the widespread use of such chemicals, they are ineffective in removing or killing the membrane biofilms (1), and regrowth quickly occurs, resulting in Received 17 October 2014 Accepted 20 January 2015 Accepted manuscript posted online 30 January 2015 Citation Barnes RJ, Low JH, Bandi RR, Tay M, Chua F, Aung T, Fane AG, Kjelleberg S, Rice SA. 2015. Nitric oxide treatment for the control of reverse osmosis membrane biofouling. Appl Environ Microbiol 81:2515–2524. doi:10.1128/AEM.03404-14. Editor: H. Nojiri Address correspondence to Scott A. Rice, rscott@ntu.edu.sg. * Present address: Robert J. Barnes, Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.03404-14. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.03404-14 April 2015 Volume 81 Number 7 aem.asm.org 2515 Applied and Environmental Microbiology