Particle-tracking microrheology is an in situ technique that allows quantification of biofilm material properties. It overcomes the limitations of alternative techniques such as bulk rheology or force spectroscopy by providing data on region specific material properties at any required biofilm location and can be combined with confocal microscopy and associated structural analysis. This article describes single particle tracking microrheology combined with confocal laser scanning microscopy to resolve the biofilm structure in 3 dimensions and calculate the creep compliances locally. Samples were analysed from Pseudomonas fluorescens biofilms that were cultivated over two timescales (24 h and 48 h) and alternate ionic conditions (with and without calcium chloride supplementation). The region-based creep compliance analysis showed that the creep compliance of biofilm void zones is the primary contributor to biofilm mechanical properties, contributing to the overall viscoelastic character.

The role of the physicochemical and surface properties of NF/RO membranes influencing bacterial adhesion has been widely studied. However, there exists a poor understanding of the potential role membrane topographical heterogeneities can have on bacterial adhesion. Heterogeneities on material surfaces have been shown to influence bacterial adhesion and biofilm development. The purpose of this study was therefore to investigate whether the presence of membrane topographical heterogeneities had a significant role during bacterial adhesion as this could significantly impact on how biofouling develops on membranes during NF/RO operation. An extensive study was devised in which surface topographical heterogeneities from two commercial membranes, NF270 and BW30, were assessed for their role in the adhesion of two model organisms of different geometrical shapes, Pseudomonas fluorescens and Staphylococcus epidermidis. The influence of cross-flow velocity and permeate flux was also tested, as well as the angle to which bacteria adhered compared to the flow direction. Bacterial adhesion onto the membranes and in their surface topographical heterogeneities was assessed using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), fluorescence microscopy and image analysis. Results showed that up to 30% of total adhered cells were found in membrane defect areas when defect areas only covered up to 13% of the membrane surface area. This suggests that topographical heterogeneities may play a significant role in establishing environmental niches during the early stages of biofilm development. Furthermore, no noticeable difference between the angle of cell attachment in defect areas compared to the rest of the membrane surface was found.

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PublisherElsevierItem record/more information http://hdl.handle.net/10197/4556 Publisher's statement þÿ T h i s i s t h e a u t h o r s v e r s i o n o f a w o r k t h a tAbstract 22 It is imperative that nanofiltration membranes are disinfected before they are used for 23 laboratory-scale bacterial adhesion or biofouling experiments, yet currently no suitable 24 disinfection protocol exists. This study aimed to determine if an ethanol treatment at a 25 minimum inhibitory concentration (MIC) could be used to effectively disinfect nanofiltration 26 membranes without altering membrane properties which could affect research. Two strains of 27 bacteria, Pseudomonas fluorescens and Staphylococcus sp., were exposed to a range of 28 ethanol concentrations to determine the MIC required for a 4log 10 reduction in bacteria. In 29 parallel, ethanol's effects on the filtration, surface and mechanical properties of a Dow 30 Filmtec NF90 membrane were analysed. A 1.5 hour treatment with 40% ethanol was shown 31 to effectively disinfect the membrane without significantly affecting any of the membranes 32 properties tested. This treatment protocol can now be safely used to disinfect the studied 33 membrane prior to bacterial adhesion or biofouling experiments. This study also acts as a 34 guideline for researchers using other membranes to determine a suitable disinfection protocol 35 for their needs.36 37 38 39 40 41 42 43 44 45 Keywords 46 Disinfection, ethanol, pre-treatment, MIC, nanofiltration 47 51Nanofiltration research in these areas has focused on two main objectives: 1) maximising 52 permeation while achieving a high level of rejection, and 2) maintaining the longevity of 53 filtration properties, primarily through the mitigation of fouling [4, 5]. 55Of the several types of fouling that occur, the adhesion and proliferation of microorganisms 56 on the membrane known as biofouling is particularly problematic. It is of utmost importance 57 that fundamental research in which bacterial pure cultures are used, for adhesion and 58 biofouling experiments, be conducted on sterile surfaces. Biofouling experiments performed 59 on non-sterile surfaces are at risk of experimental bias, resulting in unspecified multispecies 60 biofilms. Virgin membranes received from manufacturers may be initially clean but are non-61 sterile, representing a potential contamination source for controlled adhesion and biofouling 62 studies in which pure cultures are utilised. 63 64 Sterilisation can be achieved by chemical, thermal, and irradiation means. There is a major 65 emphasis on sterility in microbiological research and yet there is little research into 66 sterilisation techniques for NF and reverse osmosis (RO) membranes. Although a number of 67 different research groups have reported the use of thermal [6, 7] and chemical [8-11] 68 sterilization procedures on membranes, few have considered the damaging effects that such 69 treatments could potentially have. This comes at notable risk considering the available 70 information showing the th...

35It was found that instead of adhering on or into the organic fouling layer, the bacterial cells 36 penetrated the thin fouling layer and adhered directly to the membrane surface underneath. 37Contrary to what surface energy measurements of the fouling layer would indicate, bacteria 38 adhered to a greater extent onto clean membranes (24 ± 3 % surface coverage) than onto 39 those fouled with humic acid (9.8 ± 4 %) or alginic acid (7.5 ± 4 %). These results were