Amyloid hybrid membranes for bacterial & genetic material removal from water and their anti-biofouling properties

Water scarcity and contamination by biological pollutants are global challenges that significantly affect public health. Reverse osmosis, nanofiltration and ultrafiltration technologies are very effective for the elimination of pathogens and most contaminants but associated with considerable capital and operating costs, high energy consumption and the use of chlorinated chemicals to suppress membrane fouling. Additionally, the pressure needed by these techniques may disrupt the pathogenic microbial cell membranes, causing the release of genetic material (fragments of DNA, RNA and plasmids) into the water. Here, we introduce the simultaneous removal of both bacteria and associated genetic material using amyloid hybrid membranes, via a combined adsorption and size exclusion mechanism. Amyloid hybrid membranes can remove upto and beyond 99% of the genetic material by adsorption, where amyloid fibrils act as the primary adsorbing material. When the same membranes are surface-modified using chitosan, the anti-biofouling performance of the membranes improved significantly, with a bacterial removal efficiency exceeding 6 log.

dried into powder and stored at -20°C. Amyloid fibrils were prepared by heat denaturation of the 2 wt% monomer solution at pH 2 at 90°C for 5 hours.

Preparation of amyloid hybrid membranes (AHM)
AHMs were prepared by adding different proportions of cellulose pulp, carbon and amyloid respectively. Giluton was added as a wet strengthening agent to the above mixture. To prepare a 4 cm diameter and 2 mm thickness membrane, 6.6 g of cellulose paper was added to the 1300 ml of tap water and soaked for 15 min. This mixture was then ground for 2 min to prepare a homogenous mixture of cellulose fibers, followed by the addition of 4 g of activated carbon, 11 ml of amyloid fibrils (2 wt% solution) and 325 µl of giluton. The above mixture was passed through a sieve having a mesh size of 0.35 mm by slow pressing. The membrane with 2 wt% amyloid fibrils composition was finally dried in an oven at 90°C overnight.
To investigate the relationship between pore size, filtration efficiency, and flux, Cellulose fibrils were ground. The increase in grinding time resulted in the shortening of the fibril length.
Additionally the membranes were pressed to remove extra water. We observed that the decreased pore size and flow rate increased the bacterial removal efficiency from 99.565% to 99.99%.

Specific Genetic Material Adsorption Capacities of Individual Components of AHM
To determine the specific adsorption capacity of each component of AHM, 3 membranes were prepared separately using the above procedure. One was with cellulose, the second one with cellulose and carbon and the third one was with cellulose, carbon and amyloid (AHM). 10 ml of genetic material with the concentration of 14.2 ppm was passed through these membranes and the adsorption capacities of carbon, cellulose and amyloid were determined.

Preparation of E. coli and Salmonella Cultures
Initially, bacterial cultures were revived from -20 °C by streaking on an LB agar plate and kept overnight at 37°C. A single colony from each of the bacterial strains was freshly grown in 6 ml of LB medium and incubated at 37 °C. The optical density of the cells was measured using a Biochrom Libra S22 UV-Vis spectrophotometer. Cells were harvested by centrifugation under specific conditions (6000 rpm, 10 min) and cell pellets of Salmonella and E. coli were suspended in 5 ml of PBS and then diluted with 45 ml PBS (pH 7) respectively. Filtration tests were performed using these bacterial solutions.

Preparation of Legionella Cultures
Legionella cultures from -20 °C were grown on BCYE agar plates for 3 to 4 days at 37 °C. A single colony from an agar plate is inoculated into 6 ml of BYE medium and grown at 37 °C. The optical density of the bacterial culture was measured after 3 to 4 days of incubation. Cells were harvested by centrifugation under specific conditions (6000 rpm, 10 min) and the cell pellet was suspended in 5 ml of PBS and then diluted with 45 ml of PBS. The resulting solution was used for filtration experiments.

Surface Modification of the AHM
The surface coating of the AHM was performed by immersing the membrane into the chitosan solution. Firstly, 1 w/v % chitosan solution was prepared in a 2 wt% aqueous acetic acid solution under constant stirring for 3 hours at room temperature. The pre-wetted hybrid membrane was immersed in the chitosan solution for 5 min. The chitosan-coated membrane was then placed in an oven at 50º C for 3 hours to vaporize the solvent from the coated layer completely. After that, the coated membrane was rinsed with NaOH solution (0.1 M in 50 v/v % water/ethanol mixture) for few hours to neutralize the chitosan film, followed by thorough washing with 50 %v/v ethanol for 10 min and distilled water for 30 min. Finally, the membrane was dried at room temperature.

Anti-biofouling Performance
The anti-biofouling behavior of the pristine and coated AHM was evaluated by a cyclic filtration experiment, in which the E. coli K12 strain was used as a model biofoulant. A cell filtration system with suction pressure and volume capacity of 100 ml was used to characterize the filtration performance of the prepared AHM. All supplies were sterilized in an autoclave before the filtration.
The changes of the flux during pure water and during bacteria solution filtration were recorded to estimate the biofouling progress, which was carried out in three steps: 1) the pure water flux of the membrane was quantified; 2) the membrane was exposed to the bacteria solution for 2 hours and (2) Where Nf and Np are the numbers of live bacteria in the feed solution and permeate solution, respectively.

Scanning Electron Microscopy (SEM)
For SEM imaging pieces of the filter were cut with a blade and fixed on SEM aluminum stubs with conductive carbon (Plano GmbH, Germany). After drying, the samples were sputter-coated with 4 nm of platinum/palladium in a CCU-10 sputter-coater (Safematic, Switzerland). Inlens and secondary electron images were recorded at 2 kV with a Zeiss Leo 1530 FE-SEM (Zeiss, Germany).

Transmission Electron Microscopy (TEM)
For TEM imaging 5µl of a 0.1wt% dispersion of BLG-fibrils were spread onto a glow discharged (Emitech K100X, Great Britain) carbon-coated Cu grid (Quantifoil, Germany). After 1 min, excess liquid was drained off with filter paper, washed twice with double distilled water and 5µl of a 2% aqueous uranyl acetate solution was placed on the grid for 1s followed by a second step with 5µl uranyl acetate for 15s. The dried specimens were examined with a TEM Morgagni 268 (Thermo Fisher Scientific, USA) operated at 100kV.

S1 Adsorption isotherms
Adsorption isotherms of the membrane were determined by filtering genetic material through a 0.0002 m2 AHM membrane and the concentration of the adsorbed genetic material after each cycle is measured by using a Nano Drop spectrophotometer. From the adsorption isotherm, 1200 g of the hybrid membrane can remove 124 g of genetic material.

Contact angle measurements
Contact angles of both CCAHM and AHM were studied. The direct contact angle measurements of the AHM was not possible to be measured as the membranes are highly porous and the water droplet adsorbs immediately on contact.
The hydrophilicity of the Chitosan membrane was studied by contact angle measurements.
Theoretically, the surface having contact angle lower than 90° is considered as hydrophilic. Water droplet was placed on the membrane surface and pictures were taken to measure both receding contact angle and advanced contact angles. For chitosan membranes, the receding contact angle is 0° and advance contact angle is 75 ° therefore the contact angle lays between 0° to 75°, indicating an hydrophilic surface.