Bioﬁlm recruitment under nanoﬁltration conditions: the inﬂuence of resident bioﬁlm structural parameters on planktonic cell invasion
It is now generally accepted that biofouling is inevitable in pressure-driven membrane processes for water purification. A large number of published articles describe the development of novel membranes in an effort to address biofouling in such systems. It is reasonable to assume that such membranes, even those with antimicrobial properties, when applied in industrial-scale systems will experience some degree of biofouling. In such a scenario, an understanding of the fate of planktonic cells, such as those entering with the feed water, has important implications with respect to contact killing particularly for membranes with antimicrobial properties. This study thus sought to investigate the fate of planktonic cells in a model nanofiltration biofouling system.
What was the main aim of your research and why did you decide to investigate this?
In recent years, the development of antimicrobial/antifouling membranes for Nanofiltration (NF) and Reverse Osmosis (RO) has gained much attention with regards to biofilm mediated fouling of NF/RO membranes. While these developments primarily focused on the fundamental and functional mechanisms of their antifouling and antimicrobial surface attributes, few studies have looked at their performance under pressure conditions typical for NF and RO. Even more so, no studies have examined at the fate of these specialised membranes once covered by an established biofilm; in other words, would they still retain their antimicrobial properties? This question led us to identify an area that had not been adequately elucidated in the context of biofilm-mediated fouling of NF/RO membranes, namely the whole issue of biofilm recruitment of planktonic cells under pressure NF/RO conditions. Our objective was, therefore, to investigate the role of an established bioﬁlm layer in its interaction with incoming ‘fresh’ cells under nanoﬁltration processes, thereby determining whether recruited cells could, in fact, travel through the host biofilm and reach the membrane surface.
How did you go about designing your study?
Our lab has, over the past five years, built critical-mass in the fundamentals of biofilm-mediated fouling on nanofiltration membranes, covering aspects of cell adhesion and biofilm formation using a membrane fouling simulator (MFS). This achievement was made possible using fluorescently-tagged Pseudomonas fluorescens and P. putida cells and an experimental rig allowing for systematic and controlled fouling experiments. Using these elements, we were able to combine experimental biofilm routines with cell adhesion protocol to answer the question of biofilm recruitment under nanofiltration conditions. We hence decided to use P. fluorescens for engineering our biofilm models to which P. putida planktonic cells were made to adhere to.
What challenges did you face?
While the experimental plan of this study is straightforward at first glance, the successful engineering and maintaining of monoculture biofilms within an MFS system were not achieved overnight. In fact, this required some design adjustments to our MFS experimental rig and protocol revisions allowing for running the nanofiltration experiment as well as the analyses after that. Some of the encountered and overcame hurdles involved, but not limited to:
-cleaning/sanitizing/rinsing the a complex experimental system
-disinfecting NF membranes for subsequent biofilm experiments. This was not trivial because NF membrane are not amenable to steam sterilisation or clorine-based sterilisation.
-standardising our experimental fouling parameters
-designing a protocol allowing the successful recovery of fouled membranes for downstream analyses.
What were the key findings from your research?
We first demonstrate that resident biofilm structure phenotypes could be engineered by merely controlling the level of carbon loading rates, which was found to affect biofilm thickness over the course of continuous 7-day experimental runs. Slow carbon loading rates led to thinner biofilms compared to faster carbon loading rates yielding thicker biofilms. Using these biofilm phenotypes, we demonstrate that bioﬁlm cell recruitment during nanoﬁltration is affected by distinctive bioﬁlm structural parameters such as bioﬁlm depth. Here, P. putida cells were predominantly embedded within the top and bottom bioﬁlm layers grown under fast carbon loading rates. Under slower rates, the surface coverage was found to be much lower within the top regions of the bioﬁlms, which increased as one approached the bottom of the P. ﬂuorescens bioﬁlm regions.
What next? What further research is needed in this area?
The results presented in this study will be critical in developing robust experimental design methodologies for the assessment of antimicrobial/antifouling membranes; more specifically, by focusing on the efficiency of antimicrobial nanoﬁltration membranes when obstructed by fouling layers constituted of organic matter or natural bioﬁlms. As a result, this would better represent industrial-scale reality. The point of concern is the actual fate of planktonic cells following internalization into fouling layers that may provide a protective and nutrient-rich environment, hence furthering and perpetuating biofouling.
Olivier Habimana & Eoin Casey