PhD Beyond chlorine: alternative sustainable compounds to remove biofilms in drinking water environments

Topic background - Within Drinking water distribution systems (DWDS), at least 95% of the total microorganisms grows on pipes surfaces, in the form of biofilms. In biofilms, microorganisms produce extracellular polymeric substances (EPS) to form a matrix protecting them from external stresses. Biofilm formation and its detachment into the water stream affects the taste, odour and colour of drinking water, and constitutes a risk for contamination when pathogens are present. Currently, no single practice so far appears to be sufficiently effective for the management of biofilm growth/persistence in DWDS. One of these measures includes the application of disinfectants. Chlorine, the most widely used water disinfectant in Europe, has limited penetration within the biofilm due to its reaction with organic and inorganic compounds; these reactions also produces harmful and carcinogenic disinfection by-products (DPBs). The low efficiency of such disinfection treatments warrants the investigation into novel anti-biofilm agents, which can be more effective than and with less side effects. Research into the application of natural anti-biofilm agents is at the core of this project.

Research challenges - The formation and development of biofilms is a complicated procedure involving different stages, which can be the target of natural anti-biofilm agents for the prevention of their growth. Once established, biofilms are very difficult to eradicate because the EPS, made of proteins and polysaccharides, protect bacteria from disinfectants.  Considering the applicability in drinking water environments, we focus on antimicrobial natural amino acid derivatives, because their utilization is compatible with human consumption (in water), and they can affect biofilms at different stages. An example is N-acetyl L-cysteine, an acetylated derivative of L-cysteine, which is able to inhibit cell adhesion on a surface and EPS excretion (initial phase), and can disrupt or degrade EPS, causing the dispersal of existing biofilms. The same anti-biofilm effects were observed for other molecules like D-tyrosine. A disinfection strategy applying these (tested) molecules is not expected to place any selective evolutionary pressure on microorganisms to develop antimicrobial resistance, and does not drive any DBPs production, reducing the long-term impact that is observed with the use of chlorine-based disinfectants.

Objectives and methodology - The aims of this project are to 1) identify natural, sustainable compounds applicable in drinking water environment (from pipe materials to filtration membranes), 2) understand their fundamental mechanism of action, and 3) produce a cost-effective protocol to be used as maintenance strategy or in contingency situations.

The disinfection approaches will be tested on:

• Mixed planktonic cells from drinking water sampled from both the production site and from the tap, to assess the effects on biostability.  
• Biofilms developed in DWDS simulator (already present at Wetsus) to test the effectiveness on biofilms dispersal and EPS biopolymers in a drinking water asset.
• Biofilms grown on real pipes surfaces and developed on membranes materials, in order to assess the role of support material on the disinfection effectiveness.
• Pure cultures of known microbial strains, including Legionella and (bio)corrosive microorganisms.

In search of a cost-effective strategy, additional research will be dedicated to the ex-novo synthesis of a molecule which exert the same effects of the natural products. Novel anti-biofilm compounds may be produced using organic chemistry techniques. Several molecular biology and applied microscopy techniques will be applied to assess the effectiveness of the methods and understand the exact antibiofilm mechanisms of the natural molecules in drinking water environment.