Probing protein-induced membrane fouling with in-situ attenuated total reflectance fourier transform infrared spectroscopy and multivariate curve resolution-alternating least squares

Highlights

• Protein-induced membrane fouling was probed by in-situ ATR-FTIR spectroscopy.

• 2DCOS was integrated with MCR-ALS analysis to elucidate the mechanism.

• A two-step protein-induced membrane fouling process was revealed.

• Hydrophobic effect plays a main role in protein-induced PVDF membrane fouling.

Abstract

Proteins are one of the major contributors to membrane fouling. The interaction between proteins and the polymer membrane at the molecular level is essential for the alleviation/prevention of membrane fouling, but remains unclear. In this work, time-dependent in-situ attenuated total reflectance Fourier transform infrared spectroscopy is applied to investigate the interaction process between two model proteins, bovine serum albumin and lysozyme, and the poly(vinylidene fluoride) (PVDF) membrane. Multivariate curve resolution-alternating least squares is integrated with two-dimensional correlation spectroscopy analysis to resolve the membrane-induced conformational changes of proteins. The multivariate curve resolution-alternating least squares analysis reveals a two-step process in the protein-membrane interaction and provides the kinetics of the conformational transition, which aids the segmentation of the spectral dataset. By applying two-dimensional correlation spectroscopy analysis to different groups of the time-dependent spectra, the sequential order of the secondary structural changes of proteins is determined. The proteins initially undergo unfolding transition to a more open, less structured state, which appears to be triggered by the hydrophobic membrane surface. Afterwards, the proteins become aggregated with the high anti-parallel β-sheet content, aggravating the membrane fouling. The conformational transition process of proteins was also confirmed by the atomic force microscopic images and quartz crystal microbalance measurement. Overall, this work provides an in-depth understanding of the interaction between proteins and the membrane surface, which is helpful for the development of membrane anti-fouling strategies.

Introduction

The implementation of membrane technology continues to increase in the field of water and wastewater treatment, owing to its unique advantages such as the compact area of occupation, high effluent quality, and less residue (Chen et al., 2018; Yu et al., 2019). However, membrane fouling results in a significant decrease in membrane permeability, selectivity and service lifetime, and has become the primary problem for more widespread and large-scale applications of membrane technology (Chen et al., 2018; Gao et al., 2018). Proteins are abundant in water and wastewater, and are recognized as one of the major contributors to membrane fouling (Gao et al., 2018). For instance, proteins take up around 53% of the foulants after a four-week operation of the seawater reverse osmosis membrane reactor (Khan et al., 2013). They also play an important role in the irreversible fouling within a membrane bioreactor (Zhang et al., 2017). Therefore, it is of great significance to investigate the fouling process caused by proteins.

Numerous techniques have been used to explore the interaction mechanism between proteins and membranes, and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy is an attractive tool owing to its characteristics such as non-invasive, surface-sensitive and the capability to provide rich chemical information. It has been widely used to characterize the membrane materials, determine the thickness of the coating layers on the membranes, identify the chemical state of water in the fouling process, and examine the fouling layers on the membrane surface (Bass and Freger, 2015; Belfer et al., 2000; Guo et al., 2019). Despite the extent and depth of these studies, the microscopic mechanisms of protein fouling remain unrevealed. On the one hand, in these previous studies the ex-situ ATR-FTIR spectroscopy was used to monitor membrane fouling, but failed to give comprehensive understanding of the entire fouling process. Since the membranes have to be moved out from the reactor and air-dried, the fouling layer might be damaged and the properties of the proteins might have been changed. Therefore, an in-situ, online characterization is needed to monitor the reaction process at the solution-membrane interface. On the other hand, the conformational changes of the proteins should be given attention, as the conformational details of proteins are likely to significantly influence the extent and rate of membrane fouling (Langdon et al., 2015; Wang et al., 2010; Wei et al., 2009). For instance, thermal-denaturing of BSA would expose more hydrophobic residues and result in an obvious flux decline, compared to the native BSA (Liu et al., 2018). However, the potential of ATR-FTIR spectroscopy for ascertaining the nature of proteins on the membranes is still limited owing to the severe spectral superposition.

To resolve the extensively overlapping spectral features, curve fitting methods, e.g., the Gaussian-Lorentzian function and polynomial or linear least square procedures, are generally used. However, the curve fitting results depend heavily on the artificially selected parameters such as the numbers, positions, shapes and width of the peaks (Li et al., 2006). Therefore, sufficient prior knowledge about these parameters is required to accomplish the curve fitting analysis. More recently, multivariate curve resolution-alternating least squares (MCR-ALS) and two-dimensional correlation spectroscopy (2DCOS) have been introduced to analyze the ATR-FTIR spectra and investigate the protein-involved interactions (Schmidt and Martinez, 2016; Shashilov and Lednev, 2010). MCR-ALS allows for the mathematical resolution of concentration and spectral profiles of pure components from the raw dataset without a priori knowledge about the studied system. Hence, it has been proven to be very useful to interpret the reaction processes (Alcaraz et al., 2017; del Rio et al., 2009; Li et al., 2006). For instance, MCR-ALS has been successfully applied to resolve the intermediates in protein folding and the kinetic concentration profiles from the reaction-based chemical sensors (Alcaraz et al., 2017; Shashilov and Lednev, 2010). 2DCOS, which is based on covariance and/or correlation analysis of the external perturbation-induced variations, is able to improve the spectral resolution (Chen et al., 2019; Schmidt and Martinez, 2016). Most importantly, 2DCOS can provide the sequence of the peak intensity changes and the relationship between bands, which aids the band assignment (Chen et al., 2019; Shashilov and Lednev, 2009). However, the 2DCOS methods are only suitable for the monotone changes, otherwise the reaction sequence might be misinterpreted (Shashilov and Lednev, 2009). Therefore, segmentation of the spectral dataset into small blocks is required to capture the individual processes and assign the sequential orders of intensity changes without any ambiguity, which could be accomplished on the basis of the MCR-ALS results.

Address these challenges, we established an online and in-situ ATR-FTIR spectroscopic platform to monitor the membrane fouling process caused by proteins in this work. An integrated approach of MCR-ALS with 2DCOS was developed to resolve the complex time-dependent ATR-FTIR spectra. MCR-ALS was used to extract the spectra and kinetic curve profiles of pure species of interest involved in the reaction process. The kinetic profiles served as an important basis for the segmentation of perturbation ranges. With the aid of 2DCOS analysis, the variation of the secondary structure of proteins and the sequential order of the conformational changes could be obtained in each perturbation range. As a consequence, a mechanistic insight into the complex protein-membrane interaction could be provided, which will be helpful for understanding the membrane fouling and developing anti-fouling strategies. The molecular behaviors of two model proteins, BSA and lysozyme (LYS), were examined at the interface of poly(vinylidene fluoride) (PVDF) membrane. Under neutral pH conditions, BSA and LYS carry negative and positive net charges respectively. PVDF is a widely used membrane material, and its intrinsic hydrophobicity causes easy adsorption of biomacromolecules and organic matter on the membrane surface, leading to membrane fouling (Hashino et al., 2011).