Method for visualizing and quantifying biofilm on solid surfaces

Abstract

To provide a method for visualizing a biofilm on solid surfaces.SOLUTION: The method comprises steps of: (a) providing a solid surface in contact with an aqueous medium, where at least a portion of the solid surface is potentially covered with biofilm; (b) maintaining the solid surface in a horizontal position and covering the solid surface with an aqueous dispersion of carbon particles; (c) tilting the solid surface at an angle of at least 5 from the horizontal position to allow an excess of the aqueous dispersion of carbon particles to flow from the surface; and (d) detecting any biofilm present on the solid surface by determining whether there are areas not covered by the aqueous dispersion of carbon particles.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to methods for visualizing and quantitating biofilms on solid surfaces, particularly polymeric surfaces. [Background technology]

[0002] Biofilm growth (biofouling) on ​​reverse osmosis (RO) and nanofiltration (NF) membrane elements remains one of the key challenges in the RO / NF market. Blockage by bacterially produced biofilms can cause increased pressure drop across the RO module and can lead to hydraulic imbalance and possible damage to the module. Furthermore, biofilms can affect membrane transport properties and can cause transmembrane pressure (TMP) reduction, reducing flux. Each of these effects increases the operating energy but also leads to frequent cleaning (CIP) to restore membrane performance. Typical methods require staining the biofilm with dyes, which can be irreversible and detrimental to the membrane. For example, N. Sreedhar et al., Desalination, 2018, 425:12-21, reports a method using crystal violet to stain the membrane. The present invention aims to provide a tool for enhanced visualization and quantification of biofilm structure without permanently affecting the biofilm structure or the membrane. Summary of the Invention [Means for solving the problem]

[0003] The present invention is a method for visualizing a biofilm on a solid surface, the method comprising: (a) providing a solid surface in contact with an aqueous medium, at least a portion of the solid surface potentially being covered by a biofilm; (b) maintaining the solid surface in a horizontal position and coating the solid surface with an aqueous dispersion of carbon particles; (c) tilting the solid surface at an angle of at least 5° from the horizontal to allow the excess aqueous dispersion of carbon particles to flow off the surface; (d) detecting any biofilm present on the solid surface by determining the presence or absence of areas not covered by the aqueous dispersion of carbon particles; Includes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] Unless otherwise stated, all percentages are weight percentages (wt%) and all temperatures are in °C. Unless otherwise stated, averages are arithmetic means. All operations in the examples were carried out at room temperature (18-25°C) unless otherwise stated. Preferably, the process of the present invention is carried out at a temperature range of 3°C to 45°C, preferably 10°C to 35°C.

[0005] Preferably, the aqueous dispersion of carbon particles is "India Ink". This is an aqueous dispersion of carbon particles under which the product is sold. Preferably, the carbon particles have an arithmetic mean diameter of 2 microns or less, preferably 1 micron or less, preferably 0.7 microns or less. Preferably, the particles have an arithmetic mean diameter of at least 0.01 microns, preferably at least 0.05 microns, preferably at least 0.1 microns. The upper and lower limits are combinable. Preferably, the carbon content of the aqueous dispersion is 0.5-10% by weight, preferably 1-9% by weight, preferably 2-8% by weight. The aqueous dispersion may contain small amounts of other substances, such as binders, surfactants, etc.

[0006] Preferably, the solid surface is a polymeric surface, preferably a membrane, such as a reverse osmosis, nanofiltration or hyperfiltration membrane. Preferably, the polymer forming the polymeric surface is a polyamide (e.g., comprising polymerized units of m-phenylenediamine or piperazine and trimesoyl chloride), a polyester (e.g., polyethylene terephthalate) or a polyolefin, preferably a polyamide.

[0007] Preferably, the aqueous dispersion is applied to a solid surface in a horizontal position and then the surface is tilted to remove excess ink. Preferably, a small amount of ink is placed in one corner of the polymer surface in a horizontal position. Preferably, the amount of ink added to the surface is at least 0.1 mL / cm based on the area of ​​the surface. 2 , preferably at least 0.11 mL / cm 2 , preferably at least 0.12 mL / cm 2 , preferably at least 0.13 mL / cm 2 The maximum amount of ink is not important, since any amount greater than required will simply run off the surface when it is tilted. Typically 0.2 mL / cm 2 The following is required: Preferably, the surface is tilted so that the ink flows in the direction that the liquid will flow over the surface in use. Preferably, the surface is tilted at an angle of at least 20°, preferably at least 30°, preferably no more than 80°, preferably no more than 60°, preferably no more than 50°, preferably no more than 40° from the horizontal. Preferably, the surface is tilted for 2 to 40 seconds, preferably at least 5 seconds, preferably at least 10 seconds, preferably no more than 30 seconds, preferably no more than 20 seconds. Preferably, the area of ​​the surface bearing the biofilm is determined by visual observation, preferably by calculation using digital photography and digital image processing.

[0008] Preferably, image processing for surface biofilm quantification comprises the following steps: (a) processing the digital image, preferably using image processing software; (b) converting the image to 8-bit grayscale, preferably by splitting the color channels into RGB space, then preferably by selecting the green color layer and converting it to 8-bit grayscale; (c) converting the image to 1 bit, preferably by adjusting the black threshold from 255 to a color range where the first derivative of the number of pixels per color range is less than or equal to 0 (a simple moving average with 10 periods); (d) The percentage biofilm surface coverage is calculated by dividing the biofilm pixels (white pixels) by the total number of pixels (PT) in a 1-bit space; Includes.

[0009] Preferably, the membrane is illuminated from below to enhance contrast and visualization of areas where biofouling is present, which appear as clear and bright spots.Preferably, the polymer surface being tested has an average brightness of at least 100 lux, preferably at least 300 lux, preferably no more than 4000 lux, preferably no more than 3000 lux. EXAMPLES

[0010] For macroscopic visual examination of the stained samples, a Leica MS5 stereo microscope with magnifications between 0.63x and 4x was used, allowing realistic visualization of the samples. This instrument uses two separate light paths, with two objective lenses and eyepieces. The result is slightly different viewing angles resulting in a three-dimensional visualization of the sample. By having a light bulb under the object, transmitted light illumination is also possible. Illumination is used to enhance contrast and visualization of areas where biofouling is present. The area of ​​the sample captured by the camera ranges from 7.6 to 510.7 mm2, depending on the magnification level used in the stereo microscope. 2 The resolution of the resulting pictures, using a 12 megapixel digital camera for image acquisition, is estimated to be about 10 μm, which corresponds to the diffraction limit of a Leica MS5 stereo microscope. The technique is based on the separation of pixels corresponding to the biofilm (white) from pixels from the background (black) on a 1-bit image.

[0011] (a) Biofouling samples used to validate this method were obtained from RO coupons undergoing biofouling. A 4 cm x 4 cm wet sample from the RO coupon was placed horizontally in a Petri dish. Double-sided tape was used to hold the membrane sample together in the Petri dish. 2 mL of PELIKAN Black Fount India Drawing Ink (Pelikan, Switzerland) (arithmetic mean particle size: 0.4 microns) was placed on the side of the membrane that was in contact with the filtered raw water. (b) After pouring the ink, the sample was moved at 30° from the horizontal for 10 seconds to remove excess ink. (c) Biofouling samples were visualized using a stereo microscope (Leica MS5). The magnification of the stereo microscope used was 2x with a light intensity of 500 lux. (d) Digital photographs were obtained using a 12.1 megapixel digital camera (Canon Digital Ixus 200 IS) and processed using ImageJ™ 1.51 software. (e) The colors were split into RGB channels. The green layer was then selected and converted to 8-bit grayscale. (f) The image was converted to a 1-bit photograph by adjusting the black threshold to 110. This produced an image in which the biofilm coverage could be visualized. The image contained 2,151,502 white pixels out of 12,000,000 pixels, representing 18% biofilm surface coverage.

Claims

1. A method for visualizing a biofilm on a solid surface, comprising: (a) providing a solid surface in contact with an aqueous medium and having at least a portion of the solid surface potentially covered by a biofilm; (b) maintaining the solid surface in a horizontal position and coating the solid surface with an aqueous dispersion of carbon particles; (c) tilting the solid surface at an angle of at least 5° from the horizontal position to allow an excess amount of the aqueous dispersion of carbon particles to flow off the surface; (d) detecting any biofilm present on the solid surface by determining the presence or absence of areas not covered by the aqueous dispersion of carbon particles; wherein the solid surface is a polymer membrane selected from the group consisting of reverse osmosis membranes, nanofiltration membranes, and ultrafiltration membranes, and the area of the surface having a biofilm is determined computationally from visual observation using digital photography and digital image processing, and a 1-bit image is obtained by the digital image processing.

2. The method according to claim 1, wherein the detection is performed with a stereomicroscope.

3. The method according to claim 1, which results in three-dimensional visualization of the solid surface.

4. The method according to claim 1, wherein the resolution of the digital photograph is about 10 μm.

5. The method according to claim 1, wherein the digital photograph captures a sample of the solid surface having an area of 7.6 to 510.7 mm2.

6. The 1-bit image contains only black and white pixels, the white pixels corresponding to the biofilm-covered regions of the solid surface, the black pixels corresponding to the regions of the solid surface not covered by the biofilm, and image processing software is used to compare the number of black pixels and the number of white pixels in the 1-bit image.

7. The method according to claim 1, further comprising calculating the percentage of white pixels in the 1-bit image or determining the coverage area of the biofilm.