Fluorescence detection to determine proteins and humic-like substances fingerprints of exopolymeric substances (EPS) from biological sludges performed by size exclusion chromatography (SEC)

Abstract

Fingerprints of extracellular polymeric substances (EPS) from activated and anaerobic granular sludges were obtained by size exclusion chromatography coupled to UV (210 and 280 nm) and fluorescence (221/350 nm (protein-like molecules) and 345/443 nm (humic-like substances)) detection. The total area below the peaks obtained with fluorescence detection is linked to the protein or humic-like substances EPS content. The EPS protein fingerprints, usually recorded with UV-280 nm, change dramatically, mainly in the relative size of peaks when they were measured by a florescence detection method. It means that the apparent molecular weight (aMW) distribution of EPS chomatophores and fluorophores is different. Protein-like and humic-like substances were found to be specific fingerprints of the EPS, affected by the type and origin of the bacterial aggregate and improve EPS sample differentiation. The protein-like fraction of EPS displays a wide range of aMW (>600 kDa–<10 kDa) whereas the humic-like substances fraction is composed of molecules of low aMW (6–<1.2 kDa).

Introduction

Extracellular polymeric substances (EPS) from biofilms and biological sludges result from active bacterial secretion, cell lysis or molecules from effluents (Sheng et al., 2010). EPS are a complex mixture of high molecular weight macromolecules with ill-defined structures, variable molecular weight (MW) and chemical properties. Furthermore, the wastewater type and operating conditions of the treatment plant affect the composition of the EPS (Sponza, 2002).

EPS contain polysaccharides, proteins and humic-like substances as major constituents (Wingender et al., 1999), whereas nucleic acids and phospholipids are minor EPS constituents (Frølund et al., 1996). Dvorák et al. (2011) showed that humic-like substances are the major component of activated sludge EPS. Görner et al. (2003) analyzed the distribution of molecular weight of proteins varying from 670 kDa to 45 kDa of EPS from various activated sludges extracted by a Cationic Exchange Resin (CER). Garnier et al. (2005) found the molecular weight of proteins of EPS from various activated sludges varied from small (10 kDa) to large (600 kDa) sizes.

Many extraction methods of EPS have been described in the literature (Sheng et al., 2010). Simple centrifugation can release only soluble EPS, which are present in the surrounding of bacterial aggregates. Additional treatments are necessary to realease the bound EPS (more strongly linked to bacterial aggregates) enclosed in the inner structure of bacterial aggregates (Wingender et al., 1999). Among chemical methods, the CER method has become the most widely accepted method of EPS extraction (Sheng et al., 2010) because of its high efficiency and low cell lysis. CER extraction gives the highest concentration of the protein fraction and lower humic-like substances compared to other chemical extraction methods (D’abzac et al., 2010).

The EPS characterization is essential from different points of view (composition, size distribution, ability to link metal ions, electric charge,…) in order to understand their role in (i) bacterial aggregates and consequently in the wastewater treatment process, or (ii) the fate of metals in the environment (Guibaud et al., 2003, Sheng et al., 2010). Unfortunately, current methods like colorimetric methods give only quantitative information i.e. the total concentration of polysaccharides, proteins, humic-like substances, uronic acids, nucleic acids, etc. (Wingender et al., 1999, Liu and Fang, 2003). Qualitative informations of EPS for better characterization are then required. Size exclusion chromatography (SEC) can give access to valuable information about the fingerprints and/or the distribution of apparent molecular weight (aMW) of EPS present in a sludge or biofilm (Frølund and Keiding, 1994, Görner et al., 2003, Comte et al., 2007). UV spectrometric detection at 210 or 280 nm (Frølund and Keiding, 1994, Görner et al., 2003, Comte et al., 2007, Ras et al., 2011) is usually used after separation by SEC or asymmetrical flow field-flow fractionation (Alasonati and Slaveykova, 2011). It is assumed that a wavelength of 280 nm mainly corresponds to the protein fraction of the EPS (Görner et al., 2003). Simon et al. (2009) specified nevertheless that other conjugated macromolecules present in the EPS such as humic-like substances or nucleic acids, can also be detected at 280 nm, UV-absorbance at 210 nm corresponds to aliphatic-like compounds where the whole organic and mineral compounds of EPS are detected.

Recently, other types of detection modes were used to better characterize the qualitative composition of the EPS from sludges or bacteria. Refractometric detection gives contradictory information about polysaccharides in the EPS (Görner et al., 2003, Seviour et al., 2010). In membrane bioreactors, the characterization of foulant component partly composed of EPS is performed by organic carbon analysis (Wang and Wu, 2009). More recently, Alasonati and Slaveykova, 2011, Alasonati and Slaveykova, 2012 used multi-detection by UV–VIS spectrometry, differential refractive index and a seven angles laser light scattering system to better characterize the soluble and bound EPS from the bacterium Sinorhizobium meliloti. To identify the foulant materials of a nanofiltration membrane, Her et al. (2007) used multi-online detectors composed of UV–VIS spectrometry, dissolved organic carbon (DOC) analyses and fluorescence detection after SEC separation of molecules from the Seine River. Dissolved organic matter (DOM) characterization (Her et al., 2007) usually, done by fluorescence detection, could lead to more specific chromatograms, proteins (Ex/Em-221/350 nm) or humic-like substances (Ex/Em-345/443 nm).

Except for the work of Bourven et al. (2012) on the characterization of the protein fraction of EPS extracted by EDTA, no work has been published on fluorescence detection to record EPS fingerprints after SEC separation. Several recent works have been performed with 3-D fluorescence spectroscopy to characterize EPS (Sheng and Yu, 2006) or the foulant materials of membrane bioreactors (Liu et al., 2011) directly or on the collected fraction after SEC separation (Adav and Lee, 2011). Fluorescence is an easy and non-degradative method for monitoring protein and humic-like substances based on the association of functional groups to excitation-emission region distribution. In various natural organic matters, Chen et al. (2003) differentiate five regions corresponding to protein and humic-like substances: I (Aromatic proteins I, or tyrosine-like proteins), II (Aromatic proteins II or tryptophan-like proteins), III (Fulvic acids-like), IV (soluble microbial by-products-like) and V (humic-like substances) regions.

The main objective of this research is to differentiate protein and humic-like substances fingerprints in EPS samples by coupling SEC with two on-line detectors: UV and fluorescence spectrometers. For fluorescence detection, Ex-Em wavelengths (221/350 nm and 345/443 nm for protein-like molecules and humic-like substances, respectively) were chosen according to a preliminary study of the three-dimensional excitation-emission matrix (3D-EEM) generated by EPS samples. Two wavelengths, 210 and 280 nm, were selected for UV detection, according to the literature. These techniques were applied to EPS from different origins (i.e., activated (AS) or anaerobic granular (GS) sludges) extracted by CER.