Introduction

Water is typically purified by passing it through layers of porous materials, each specially selected for its ability to remove specific forms of contamination. All porous materials can filter particles by size, many have extra capabilities thanks to their chemical properties. For example, charcoal - or activated carbon - is a porous component of household and industrial water filters that can also bind large or electron-rich molecules via the van der Waals forces, and catalyse the breakdown of other chemicals such as molecular chlorine. While there are many different types of filters, we can categorise and compare them using their measurable physical and chemical properties. The key physical properties are:

• Pore size - average or maximum size of pores in the material
• Porosity - volume of the filter not occupied by solid material
• Tortuosity - length of paths through the filter compared with a straight line
• Adhesion - the strength of hydrogen bond interactions between the fluid and filter
• Kinetic rate constants - parameters defining how the filter material affects chemical reactions in the fluid

These properties contribute to the filter’s flow rate, measured in units of fluid filtered per unit surface area of the filter per unit time (e.g. Ga m^-2 h^-1). Altering the filter’s flow rate generally affects the filter’s efficiency at removing its targets, as increasing the flow rate typically involves increasing the pore size or reducing the surface area of the filter in contact with the fluid. This trade-off is the main hurdle in industrial water filtration, where demands for high-quality filtration require very low flow rates. Industrial processes are further complicated by blockage of the pores, fouling of the filter by organisms and the lack of a complete set of materials that will exhaustively filter all contaminants - heavy metal ions, small organic molecules and non-polar compounds remain difficult to filter without prior chemical treatments that themselves need to be filtered out.

Turbidity

Ultrafiltration membranes remove particulates by size exclusion. Our bacterial cellulose membranes completely removed turbidity from input water outputting clean, potable water. Input water containing 1% w/v organic solids from Imperial College Queens lawn was vortexed then 3ml passed through filters of 25cm3 area. Turbidity of flowthrough was measured in a spectrophotometer, absorbance at OD₅₀₀ as recommended by Epa (1993).

Cellulose sheets produced by G. xylinus iGEM were dried to thin filters around 20 g/m³. Coffee press filtration with these filters was compared to similar filtration by tissue paper and VWR qualitative medium filter paper (5-13 um pore size).

Average OD₅₀₀ values from three replicates:

• Water unfiltered: 0.072.
• Tissue Paper filtered: 0.039.
• Filter paper filtered: 0.023.
• Bacterial Cellulose filtered: 0.00.
• Distilled water: 0.00.

Cellulose functions as a water filter and completely removes suspended particles.

Phytochelatin-dCBD metal binding assay

Metal binding proteins fused to CBDs were bound onto cellulose in 96-well plates and tested against 3 different metals (Nickel, Copper, Zinc). First, the fusion protein lysate was incubated overnight in the cellulose wells. Following this, the metal salt solutions are added in excess into the wells. Finally, an EDTA step removes the bound metal ions into solution, and the metal concentration in the EDTA solution is quantified by mass spectrometer. Multiple washes with PBS and water were done between each binding step, ensuring that metal ions read were released from the metal binding proteins. Then as mentioned in Xu (2002), the assay was repeated using the same cellulose discs, to test if the phytochelatin was re-useable.

If the functionalised cellulose has bound the metal in question then the final EDTA solution that is measured using the mass spectrometer should be found to have a high concentration. EDTA is a powerful chelator and will bind any metals in the sample preferentially to other organic molecules. The highest concentrations were found within the cellulose only tests for all metals, however due to the large number of hydroxyl groups, it isn’t too surprising that the cellulose is capable of chelating metals. All samples that had cell lysate applied were much lower, even the non-CBD phytochelatin controls, which may be caused by the phytochelatin and other molecules in the cell lysate reducing the natural chelating power of the cellulose. For Zinc, the metal that phytochelatin most successfully binds, the PC-CBD fusion tests all resulted in roughly double the concentration of Zinc. We propose that the reduction in concentration due to blocking of the cellulose by lysate is still there and that the extra concentration recorded is purely from Zinc that was bound to PC-CBD. However, this pattern is not observed in the Copper or Nickel tests.

The results from the re-used functionalised cellulose assay demonstrated much lower concentrations of metals than the first wash. However the cellulose samples in this part of the experiment have been in contact with EDTA which, due to its carboxyl group, has been shown capable of binding to cellulose (Tsukinoki, 2008). Since the non-functionalised cellulose samples showed much lower concentration of all metals, it can be concluded that the cellulose binding to metals is disrupted. However, one of the PC-CBD fusions had consistently higher concentrations of metal; the PC-dCBD. This suggests that even with the interference with cellulose that is observed when adding the EDTA, the PC-dCBD stays bound to the cellulose and works as a functioning phytochelatin. In fact, PC-dCBD showed the only increase in metal concentration in the second wash compared to the first, seen in its nickel sample. The other PC-CBD fusions have results that match the PC alone samples more suggesting that they do not bind the cellulose as strongly under these conditions. This concept can be observed in the graph that shows the difference in concentration between the two washes; the PC-dCBD samples are the only samples that change little between the two experiments. The PC-CBDcipA copper samples are quite similar but the values are very low for both. Note that sfGFP-dCBD fusions showed high results in the sfGFP characterisation of CBD assay.

Time constraints have prevented repetition of the experiment, but clearly more work is needed to fully characterise the binding of these different CBDs. The assay should ideally be repeated with different concentrations of the PC-CBDs, though same total concentration of lysate by combining with wild type lysate, to investigate the impact of other proteins blocking the binding of the cellulose. We conducted the experiment with lysate as being able to apply lysed cells expressing the protein of interest would be cheaper than the purifying the protein first. However, in the interest of characterisation of the CBDs it would be beneficial to purify the protein to remove any interaction from background molecules and proteins found in the lysate. Another adaptation would be to, after the binding of the metals has occurred and the full wash cycles completed, dissolve the cellulose membranes in Nitric acid and run those samples on the mass spectrometer. This would allow us to confirm that the EDTA does indeed fully chelate any metal found within the functionalised cellulose (be the metals bound to the cellulose or the CBD-PC). Finally, testing with different concentrations of metal is vital to show the range that the functionalised cellulose is useful for.

Nickel filtration assay

The nickel ions are an example of heavy metals, poisonous even in relatively small concentrations in water and notoriously difficult to filter with current filtration methods. Therefore our filtration concept was tested against a concentration of nickel in water that far exceeds the safe limits. We have attempted to filter high amount of nickel (250 μM) through the cellulose filters grown by the G. Xylinus ATCC53582 strain (K1321305). The phytochelatin-dCBD fusion (K1321110) was coated on the surface of the cellulose to make a nickel specific functionalised ultra filtration membrane. To test the two membranes we have used coffee press. As a control measure we have also attempted to the filter the nickel solution through the cellulose that was not further functionalised. Obtained filtrates have been quantified and analysed using Mass Spectrometry.

Results