# Probing the fouling induced by biomolecules of a polymer microfiltration membrane using 3D cryo-FIB/SEM

Hélène Roberge, Philippe Moreau, Estelle Couallier, Patricia Abellan

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Hélène Roberge, Philippe Moreau, Estelle Couallier, Patricia Abellan. Probing the fouling induced by biomolecules of a polymer microfiltration membrane using 3D cryo-FIB/SEM. World Filtration Congress (WFC14), Jun 2025, Bordeaux, France. hal-05343641

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Submitted on 3 Nov 2025

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# Probing the fouling induced by biomolecules of a polymer microfiltration membrane using 3D cryo-FIB/SEM.

Hélène ROBERGE¹,², Philippe MOREAU¹, Estelle COUALLIER², Patricia ABELLAN¹

Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel, IMN, F-44000 Nantes, France

Nantes Université, CNRS, ONIRIS, Laboratoire de Génie des Procédés, Environnement et Agroalimentaire, GEPEA, F-44600 Saint Nazaire

Estelle.couallier@univ-nantes.fr

## Introduction

Fouling is the main technical limitation to membrane filtration processes aimed at effectively separating valuable biomolecules from complex mixtures, microalgae solutions for example. Direct visualization both inside and outside the membranes, enabling the characterization of the smallest pores of the membranes and different biomolecules, would allow to demonstrate the structure of the fouling that is usually difficult to image, and help developing innovative fractionation processes. The aim of this work is to describe fouling inside microfiltration membranes by solutions of proteins, lipids and a mixture of both with a few nanometer resolution. Detailed analysis of 3D reconstructions of fouled membranes, obtained by focused ion beam coupled with scanning electron microscopy (FIB-SEM) at cryogenic temperatures combined with deep learning segmentation methods, provides such nanometre scale information.

## Material and methods

Unstained model solutions of lipids, proteins and their mixtures were filtrated using a flat polythiersulfone microfiltration membranes (0.1µm cut off, 130cm2) in crossflow conditions (0.45bar, 0.8m.s-1). After filtration and membrane gentle rinsing, a sample of each was cut and cryofixed by HPF (High pressure freezing) before its observation. Then the whole process of FIB/SEM characterization was performed in cryogenic conditions (-140°C). Dragonfly® software (Comet Technologies Canada Inc.) was used to pre-process, align, segment and analyze the different stacks of images.
## Results

The fouling mechanisms, including 'cake' formation, pore blocking, and pore diameter reduction were revealed but the fouling structure strongly depended on the complex mixture composition (figure). A quantitative analysis of the pores that were fouled, indirectly blinded or blocked, and open pores that can actively contribute to filtration was performed. Various impacts that can not be directly probed by other means were discussed, such as the number of blind or blocked pores no longer contributing to filtration.

## Conclusion

The first 3D characterisation at the nanoscale of the internal and external fouling of MF membranes using FIB/SEM in cryogenic conditions are presented. This methodology has broad applications in other filtration processes within industry and biotechnology research.

Funding: e-BRIDGE project supported by NExT initiative (reference ANR-16-IDEX-0007), Pays de la Loire region and Nantes Métropole. FIB/SEM reconstructed volumes were collected in the CIMEN Electron Microscopy Center in Nantes, part of the PLASSMAT characterization platform of IMN.

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

Filtration of the model mixtures containing lipids, proteins or both in crossflow conditions
Characterization by cryo FIB/SEM: indentification of the external and internal fouling

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

Data volume

<figure><img src="image_6.png" /></figure>

Blocked pore

<figure><img src="image_7.png" /></figure>

Pores

<figure><img src="image_8.png" /></figure>

Membrane

<figure><img src="image_9.png" /></figure>

Fouling

3D reconstructions of PES membranes fouled with (4a) lipids, (4b) proteins and (4c) a mix of lipids and proteins. The different phases of the volumes were segmented by the SW technique and highlighted by light grey for the blocked pore (in figure 5a only), dark blue for the pores, light blue for the membrane and red for the fouling. The original data stack is shown in grey and the black dotted line indicates the membrane surface