Regenerating heavily biofouled dissolved oxygen sensors using bacterial viruses

Abstract

Bacterial biofilms are aggregates of bacterial cells embedded in a self-produced extracellular polymeric matrix. Biofilm formation has always been considered a major challenge for sensors used in underwater measurements, and is a primary source of measurement error, especially when it comes to long-term in situ monitoring. We demonstrate the utility of lytic bacteriophages (bacterial viruses) as a non-invasive strategy for removing bacterial biofilms formed on the gas permeable membrane of electrochemical dissolved oxygen sensors. Our results show that a 4 day Pseudomonas aeruginosa biofilm with a fully developed matrix significantly affected the sensor signal and response time, decreasing the signal by 32% and increasing the response time by 94%. In addition, measurements with the biofouled membrane had a very low signal to nose ratio compared to a clean sensor membrane. A single dose of overnight phage treatment effectively removed the biofilm (as indicated by scanning electron micrographs and fluorescence images of the membrane), without the need for repeated treatments. Furthermore, the sensor signal that had plummeted by 32% for a fully biofouled membrane, was returned to the original value (7.96 ± 0.27 mg L^-1) after phage treatment and the signal to noise ratio (calculated as the ratio of mean to standard deviation) increased 8 folds for a phage-treated membrane compared to a biofouled membrane. Our data indicate near complete regeneration and signal recovery for the dissolved oxygen sensor, making the biofouled sensor reusable without the use of harsh chemicals that could destroy the fragile sensor membrane.

Fig. 1. (A) Schematic diagram showing the workflow for artificially fouling the membranes of DO sensors and subsequent phage treatment. After baseline measurement with the DO sensor, the membranes were incubated with a PA culture for 4 days to form a thick bacterial biofilm, then treated with phage overnight before subsequent DO measurements. (B) A simplified diagram of the phage lytic cycle that leads to lysis and destruction of the bacterial cells comprising the biofilm on the sensor membrane. First, phage attaches to specific receptors on the bacteria cell surface and injects its genome into the bacterium, thus taking over the cell reproduction machinery. Hundreds of phage particles are synthesized and assembled in each bacterium and the bacterium is subsequently lysed to release the new phages into the environment to infect more bacterial cells.

Fig. 2. (A) Amount of PA biofilm grown in a microtiter plate for the duration of one to four days, quantified as absorbance of re-solubilized crystal violet stain at 590 nm. Each data point represents the average of 20 technical replicates (n = 3). (B) SEM images of PA biofilm grown on DO sensor membranes for one, two, three, and four days. White arrows indicate some of the water channels visible in the matrix structure. (C) Schematic of a PA biofilm lifecycle: (i) initial attachment of cells to the surface; (ii) lateral spreading of cells and production of EPS, leading to irreversible attachment; (iii) early development of biofilm structure; (iv) mature biofilm architecture; (v) dispersion of cells from the biofilm and continuation of the cycle.

Fig. 3. (A) Fluorescence intensity of biofouled and phage treated membranes for 6 h and overnight phage treatments, stained with Hoechst dye and quantified with ImageJ software. Significant difference in fluorescence intensity was observed between biofouled and phage treated membranes. Overnight phage treatment resulted in a significantly lower fluorescence signal, compared to 6 h treated phage. Significant difference when comparing biofouled membranes with phage treated membranes, and overnight versus 6 h treatment (*P < 0.05, **P < 0.001). (B) Fluorescence micrographs of biofouled, 6 h- and overnight phage treated membranes. The fluorescence signal does not differentiate between intracellular and extracellular DNA. (C) SEM images depicting a clean DO sensor membrane, a biofouled membrane with a 4 day PA biofilm, and a biofouled membrane subjected to an overnight phage treatment.

Fig. 4. (A) DO measurement for clean, biofouled, phage treated, and bleached sensor membranes in DI water. (B) Response time of DO sensor measured with clean, biofouled, phage treated and bleached membranes. The data points show the average of three measurements with each membrane. Experiments were performed in triplicates, and for each replicate, 2 membranes were used, bringing the total number of membranes used for each condition to six. Different color shades have been used to differentiate the data points for each biological replicate. Significant difference when comparing phage treated membranes with biofouled and clean membranes (*P < 0.05, **P < 0.01).