Article pubs.acs.org/est
Characterization of Dissolved Organic Matter in Municipal Wastewater Using Fluorescence PARAFAC Analysis and Chromatography Multi-Excitation/Emission Scan: A Comparative Study Wen-Tao Li, Shi-Yu Chen, Zi-Xiao Xu, Yan Li, Chen-Dong Shuang, and Ai-Min Li* State Key Laboratory of Pollution Control and Resources Reuse, Collaborative Innovation Center for Advanced Water Pollution Control Technology and Equipment, School of the Environment, Nanjing University, Nanjing, 210023, China S Supporting Information *
ABSTRACT: Dissolved organic matter (DOM) in municipal wastewater was mainly characterized using high-performance liquid chromatography (HPLC) and size exclusion chromatography (HPSEC) with multi-excitation/emission fluorescence scan. Meanwhile, fluorescence excitation−emission-matrix combined with parallel factor analysis (EEM-PARAFAC) was also applied. Compared with chromatography fluorescence fingerprints, the EEM-PARAFAC model could not reflect the variety of DOM species with similar fluorescence but different physicochemical properties. The chromatography results showed that the protein-like species were variable among different municipal wastewater treatment plants, some of which are in combination with humic-like species; while there were two major humic-like species fractionated by hydrophilicity and molecular weight (MW), which are also the major contributors to UV absorbance at 254 nm. It was also identified that the relatively hydrophilic humic fractions were slightly larger than the relatively hydrophobic humic fractions. In all the investigated wastewater treatment plants, the relatively hydrophilic and larger MW humic fraction mainly contributed to the fluorescence intensity of humic-like EEM-PARAFAC components. As well as facilitating interpretations of EEM-PARAFAC components, the HPLC/HPSEC fluorescence fingerprints also contributed to a better understanding of fluorescent DOM species in municipal wastewater.
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INTRODUCTION
Due to its high sensitivity, good selectivity and nondestruction of samples, fluorescence excitation emission matrix (EEM) spectroscopy has been frequently used to characterize DOM in water and wastewater treatment systems.2,7−9 EEM may provide a wealth of information about DOM, but in itself it can be very difficult to interpret.10 Over the last decades, a series of methods have been developed to interpret the complex information in EEM, including ratios of fluorescence,11 fluorescence index,12,13 fluorescence regional integration1 and parallel factor analysis (PARAFAC).14,15 As a technique of multivariate data analysis, PARAFAC can mathematically decompose the complex fluorescence spectra into individual fluorescent components for both quantitative and qualitative analysis.10 And with the published tutorials,10,15 fluorescence EEM combined with PARAFAC (EEM-PARAFAC) has been extensively applied.2,9,14,16−25 For PARAFAC modeling, determination of the right number of components is
Dissolved organic matter (DOM), including humic substances, proteins, and other aromatic or aliphatic organic compounds, plays an important role in both natural and engineered water systems.1,2 In municipal wastewater treatment, the presence of DOM not only affects the current discharge standards, but also presents significant challenges in wastewater reclamation, such as membrane fouling and disinfection byproducts (DBPs) formation upon chlorination.3,4 Knowledge of the concentration and composition of effluent DOM is beneficial to the development of advanced wastewater treatment technology. A series of high resolution techniques, such as Fourier-transform ion cyclotron mass spectrometry and multidimensional nuclear magnetic resonance spectroscopy, have been applied for molecular characterization of DOM.5 However, because of its high heterogeneity, none of these high resolution techniques is as yet able to characterize the isomeric structure of a single humic molecule. In terms of frequent monitoring, it is more practical to characterize DOM on the basis of several major components as well as their physicochemical properties (e.g., polarity and molecular size).6 © 2014 American Chemical Society
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collected in clean polyethylene bottles and then filtered through prewashed 0.45 mm cellulose membrane filters. The filtered samples were then stored at 4 °C until analysis, which was done within three days in each sampling campaign. EEM and PARAFAC Modeling. UV254 absorbance was measured with a Shimadzu UV-1800 ultraviolet−visible (UV/ vis) spectrophotometer. No UV254 absorbance exceeded 0.3 cm−1 (SI Table S1), and all samples with no dilution were used for EEM analysis.21 The EEM fluorescence spectra were obtained with operation parameters as previously described.28 The scanning field was set at excitation from 200 to 400 nm and emission from 280 to 550 nm with 5 and 1 nm sampling interval on excitation (Ex) and emission (Em) modes, respectively. All fluorescence data were presented in arbitrary units. The PARAFAC analysis was conducted with the DOMFluor toolbox,10 which contains all of the tools used to identify outlier samples as well as to perform split-half analysis and residual errors diagnostics. The outlier occurs when a sample contains some measurement error or it is properly measured but is very different from the others.10 Therefore, the EEM of Sample 19 from anion exchange treatment process was not used for PARAFAC analysis. With an initial exploratory analysis, two outliers (EEMs of Sample 6 and 15) were further removed from the data set. After several preparative steps (i.e., data loading, scattering removal, initial explorative data analysis, and outlier identification), the PARAFAC models with two to seven components were computed. The residual analysis, visual inspection and split half analysis were applied to determine the correct numbers of components. HPLC/HPSEC Analysis. The Agilent 1200 LC Systems equipped with diode array detector (DAD) and fluorescence detector (FLD) were used in this study. HPLC equipped with Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm) was applied. The mobile phase was modified as the mixture of ammonium acetate (10 mM) and acetonitrile as shown in Table 1. The
very critical, and hence several diagnostic algorithms (e.g., residual analysis and split half analysis) have been developed for model validation.10 However, these diagnostic algorithms are statistically based on EEM itself, which prefer larger EEM data sets spanning a gradient or following a process. Notice that the fluorescence properties of DOM may be only decided by minimal fluorescent structures, whereas polarity and molecular size are the properties of the bulky molecules. Therefore, one PARAFAC component might consist of different DOM species with similar fluorescence. Additionally, PARAFAC components are spectrally independent, but whether they are also independent in their existence remains unclear. It has been critically reviewed that inconsistencies between reoccurring components across studies are also prevalent,2 indicating that when interpreting EEM, physicochemical properties of DOM, as well as fluorescence should be acknowledged. High-performance liquid chromatography (HPLC) or highperformance size exclusion chromatography (HPSEC) coupled with UV absorbance, fluorescence, or organic carbon detector have been used to estimate the polarity or molecular size of DOM components.3,4,9,26,27 However, due to the interference from other fluorescent or nonfluorescent matter, it is difficult to directly identify the EEM-PARAFAC components in chromatograms of UV, organic carbon, and even fluorescence signal at a single pair of Ex/Em wavelength. Recently, we demonstrated that HPLC/HPSEC separation of DOM followed by fluorescence multi-excitation/emission scanning of resulting DOM fractions showed promise for fluorescent DOM analysis, directly relating DOM fluorescence spectra to polarity and molecular size. 28 Furthermore, the application of UV absorbance and fluorescence detector in tandem can help to identify the major contributors of fluorescent species to UV absorbance at 254 nm (UV254). Especially, the newly developed method would be a promising tool for interpretation of EEMPARAFAC components. Although a recent study has used PARAFAC to characterize a large and diverse EEM data set from six recycled water treatment plants,21 the physicochemical properties of DOM have not been characterized along with PARAFAC modeling. Thus, a comparative study between EEM-PARAFAC and HPLC/HPSEC fluorescence fingerprints is necessary. It is also expected that some common characteristics of fluorescent DOM in municipal wastewater will be found after a systematic investigation. In this study, both EEM-PARAFAC and HPLC/HPSEC multi-excitation/emission techniques were applied to characterize the fluorescent DOM in municipal wastewater. The primary objectives of the physicochemical characterization were to (1) fit the PARAFAC model to EEM data set; (2) compare PARAFAC model with HPLC/HPSEC fluorescence fingerprints; (3) relate fluorescent DOM species to their physicochemical properties including hydrophilicity and molecular size. These results may contribute to a better understanding of fluorescent DOM species in municipal effluents as well as facilitating interpretations of EEMPARAFAC components.
Table 1. HPLC Gradient Elution Program time (min)
solvent B (%)a
solvent D (%)b
flow rate (mL/min)
0.00 3.00 5.00 10.00
20.0 40.0 70.0 80.0
80.0 60.0 30.0 20.0
1 1 1 1
a Solvent B: acetonitrile. bSolvent D: ammonium acetate solution (10 mM), theoretically pH = 7.
HPSEC (Agilent 1200 series) application and molecular weight (MW) calibration was according to our previously described operation methods.28 DAD with multiabsorption scan was conducted from 200 to 300 nm. In tandem with DAD, FLD was conducted with multi-excitation scan or multi-emission scan for the same sample. According to results of EEM, HPLC with multi-excitation scan was setup at Em = 340/Ex = 220− 300 for protein-like species and Em = 430/Ex = 220−400 for humic-like species. Both HPLC and HPSEC were setup as Ex = 230/Em = 300−500 nm for multi-emission scan. Because of the similar emission spectra of different humic fractions in HPLC or HPSEC, regression analysis was conducted on the fluorescence intensity of 18 samples (Sample 1−18) in order to relate their polarity and molecular weight together. First, the fluorescence peaks scanned at Ex230/ Em430 nm in HPLC or HPSEC chromatograms were integrated, which represent the intensity of humic-like peaks.
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MATERIALS AND METHODS Sampling. Nineteen water samples were collected from different points along the process trains of five municipal wastewater treatment plants (WWTPs), of which the details are as shown in Supporting Information (SI) Table S1. The municipal WWTPs were selected to represent a range of treatment process and treatment capacity. Samples were 2604
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Figure 1. EEM contours, excitation (red dash lines) and emission (black solid lines) loadings of the four components identified by the DOMFluorPARAFAC analysis.
corresponding to the same emission maxima (at 440 nm), which has been identified as the humic-like component.9,24 Component 4 (C4) also exhibited shoulder emission peaks. The left shoulder peaks (Ex225, 280/Em300 nm) were similar to tyrosine-like component while the right shoulder peaks (Ex225, 280/Em330 nm) were similar to tryptophan-like component.22 In summary, C1, C4, and left shouder peaks in C2 were protein-like components, whereas C3 and right shoulder peaks in C2 were humic-like components. Notice that both C2 and C4 had shoulder emission peaks with parallel excitation maxima. Due to intramolecular relaxation, the fluorescent structure of multi-emission is minimal.31 Besides split half analysis and residual analysis, PARAFAC modeling should be further constrained by unimodality in emission wavelength, that is, emission spectra should only exhibit one maximum.10 However, these shoulder emission peaks could not be further separated by increasing the number of components (SI Figure S5). As a concern regarding the size of EEM data set, another 21 EEM spectra of effluent samples were further added for PARAFAC modeling. There were no apparent changes in the components’ number and spectral characteristics (SI Figure S6). Such phenomena were also observed in previous PARAFAC models for different size of EEM data sets (SI Table S2).9,21,23−25 Therefore components with shoulder emission peaks might not arise from the data set size. The secondary emission peaks occurred at times possibly because PARAFAC had difficulty in distinguishing two components.21 Hence the PARAFAC model will be further assessed by HPLC fluorescence fingerprints with regard to the components’ spectral features as well as relationships among components. Revaluation of EEM-PARAFAC Components. Both HPLC and HPSEC fluorescence fingerprints can be used for the interpretation of EEM-PARAFAC components. However, HPLC is more suitable in terms of its higher separation capacity and shorter elution time. The HPLC multi-excitation/emission chromatography can present an informative fingerprint of the online isolated DOM species. Coinciding well with PARAFAC model, the HPLC Excitation-Time Maps (SI Figure S7) verified the prevalence of multi-excitation properties of fluorophores, which has already been discussed in our previous study.28 Since humic-like and protein-like components can be distinguished by their emission spectra, it is supposed that the
Then the integration value was normalized into percentage, that is, each value was divided by the total sum in each chromatogram. And finally the percentages of humic-like peaks in HPLC chromatograms were regressed against those in HPSEC chromatograms. For further verification, humic substances were extracted from WLK effluent with the XAD-8 resin procedure,29 and then fractioned by HPLC. The collected humic fractions were lyophilized, redissolved for EEM analysis.
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RESULTS AND DISCUSSION EEM-PARAFAC Model. The EEMs of samples taken throughout the treatment trains of five municipal WWTPs can be found in SI Figure S1. With the peak-picking function of the instrument’s FL solution software, four major peaks were found in this study, which are roughly at Ex235/Em340, Ex280/Em310, Ex240/Em430 and Ex340/Em435 nm. Despite minimal shifts in peaks’ locations, similar EEM spectra have also been reported by previous research of wastewater effluents.8 With the residual analysis and split half analysis (SI Figures S2 and S3), four fluorescent components were identified by the EEM-PARAFAC modeling (Figure 1). Generally, the fluorescence peaks with Em > 380 nm are supposed to base on polycyclic aromatic structure; while fluorescence peaks with Em < 380 nm are due to fluorophores containing benzene ring with electron denoting groups like hydroxyl and amino groups.28,30 In natural and engineered water systems, the EEM-PARAFAC components with Em > 380 nm are generally identified as humic substances, and components with Em < 380 nm are generally ascribed to protein-like (tyrosine or tryptophan-like) components.2 Component 1 (C1) was composed of two excitation peaks at Ex230, 280/Em310, which can be ascribed to tyrosine-like components.20,21 Component 2 (C2) consisted of shoulder emission peaks, and similar phenomena have also been observed in previous literature.24 The dual-excitation peaks on the left shoulder (Ex235, 290/Em340 nm) could be ascribed to tryptophan-like fluorescence. There were also dual-exciation peaks on the right shoulder (Ex235, 290/Em475 nm), which are in parallel with the left shoulder peaks. However, the upper peak was very minimal as shown in the 3D-EEM spectra (SI Figure S4). If the right shoulder peaks exist alone, they might be ascribed to a humic-like component.2,24,25 Component 3 (C3) displayed two excitation maxima (at 250, 350 nm) 2605
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Fluorescence of proteins is mainly dominated by tryptophan, which absorbs at the longest wavelength and displays the largest extinction coefficient. Energy absorbed by phenylalanine and tyrosine is often transferred to the tryptophan residues in the same protein. When tryptophan residue is coated in protein, blue shift may occur on its emission wavelength.31 As shown in Figure 2, the emission maxima of protein-like species also shifted significantly. Therefore the tyrosine-like component might be affected by proteins with coated tryptophan. For dualemission peaks in C4, it is not appropriate to explain it as indicating the same origin of both tyrosine-like and tryptophanlike components. Inferred from the split half analysis (SI Figure S3), the PARAFAC model was not well fitted in the region of protein-like components. The various protein-like species with different emission maxima might result in the failed separation of dual-emission peaks in C4. Despite different samples, there were two major humic-like species fractionated by hydrophilicity (Figure 2). The EEM spectra of HPLC humic fractions from WLK effluent are also shown in Figure 3. With visual inspection, their spectral features in the region of Em > 380 nm were very similar, which can be recognized as the same PARAFAC component. Thus C3 was actually the layer stacks of two different humic-like species. It is noteworthy that some of protein-like peaks were eluted together with humic-like peaks, indicating the possible combination through electrostatic attraction or hydrophobic effect between them.32 Recent study has shown that some polyphenolic compounds (e.g., lignin) also exhibit fluorescence like tyrosine or tryptophan.33 It is also possible that the humic species contain the critical structure like phenol or aniline, which also exhibit tyrosine-like or tryptophan-like fluorescence spectra.28 Therefore the spectrally independent PARAFAC components might not independently exist. In C2, the tryptophan-like fluorescence was also linked with the humiclike fluorescence. However, C2 could not reflect the relationship between protein-like and humic-like components. In terms of excitation and emission maxima, the right shoulder peaks did not coincide with HPLC fluorescence fingerprints and EEM spectra of the two humic fractions (Figures 2 and 3). Notice that the peak at Ex235/Em475 was located nearby the secondary Raman and Raleigh scattering, indicating it might arise from instrument artifacts and insufficient elimination of scattering in the EEMs during post processing.25 Fluorophores’ Hydrophilicity and Molecular Weight. In the HPLC with reverse phase column, the relatively hydrophilic components will be eluted with shorter retention time.3 Shown in Figure 2 and SI Figure S8, it is noticeable that the hydrophilicity of humic substances was quite similar among different WWTPs. The relatively hydrophilic humic fractions were eluted roughly at 1.2 min, whereas the relatively hydrophobic humic fractions were eluted at about 1.7 min. However, all humic substances were relatively hydrophilic, considering that they can be easily eluted with mobile phase (pH 7). Humic substances contain both aromatic and aliphatic components with acid groups, and their polarity could be highly affected by the protonation and deprotonation process.34 Thus the HPLC results of humic substances do not contradict with their conventional definition of hydrophobic acid in XAD resin procedure, because these two procedures are operated at different pH conditions.28 Some protein-like fluorophores are possibly combined with humic substances, resulting in the similar hydrophilicity. However, there exist some relatively
Emission-Time-Maps (Ex230 nm, Em 300−500 nm) can reflect all the fluorescent DOM species within detection limit. Similarly, the fluorescence peaks with Em > 380 nm represent the humic-like species while the fluorescence peaks with Em < 380 nm represent the protein-like species. As shown in HPLC emission-time-maps (Figure 2 and SI Figure S8), the protein-like species varied to a great extent
Figure 2. HPLC emission-time-maps of 5 secondary effluents: (a) KXY, (b) KFQ, (c) QT, (d) AZ, and (e) WLK. The Em-axis denotes the emission wavelength, by which humic-like peaks (Em > 380 nm) and protein-like peaks (Em < 380 nm) can be distinguished. The timeaxis represents the elution time of DOM species, which can reflect their hydrophilicity. The color variation represents the fluorescence intensity in arbitrary units.
across samples. In the PARAFAC modeling, the protein-like components could be separated into subsets of the tryptophanlike and tyrosine-like components (C1 and the left shoulder peaks in C2), which might further consist of various species. Although protein-like components with various emission maxima have been reported,9,20−22,25 it is obvious that PARAFAC model could not reflect the variety of the underlying protein-like species. As is well-known, there are two major fluorophores in proteins, that is, tyrosine and tryptophan. 2606
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Figure 3. EEM spectra of the HPLC humic fractions from WLK secondary effluent. The color variation represents the fluorescence intensity in arbitrary units.
hydrophobic protein-like substances, which were eluted later than 4 min. With the same FLD multi-emission scan parameters, HPSEC emission-time-maps were obtained in Figure 4 and SI Figure S9, in which the Em-axis denotes the emission wavelength and the Time-axis represents the elution time, that is, the apparent MW distribution of fluorescent DOM species. With the size exclusion column, DOM species with shorter retention times are of higher apparent molecular weight.26 For humic-like component, there were two major humic species eluted at about 8.8−9.2 and 9.2−9.6 min, of which the apparent MWs are approximately in the range of 21.3−7.83 and 7.83−3.87 kDa according to the polyethylene glycol calibration kit.28 In previous characterization of wastewater effluents, humic substances were also reported with apparent MWs lower than 20 kDa.27 The molecular size of protein-like species varied across different WWTPs, but remained relatively similar in the same WWTPs. The apparent MWs of protein-like species mostly distributed in the range of calibration kit (i.e., lower than 21.3 kDa), coinciding well with previous study.26 Because of the similar fluorescence spectra of humic fractions, the HPLC/HPSEC emission-time-maps are not fine enough to combine together the hydrophilicity and molecular weight of each humic fraction. To this end, the normalized percentage of humic-like peaks’ intensity in HPLC and HPSEC chromatograms were calculated for correlation analysis. The positive correlation was observed between normalized percentage of the first humic-like peaks in HPLC and HPSEC (Figure 5). Since there were only two humic fractions, the correlation analysis indicates that relatively hydrophilic humic fractions were slightly larger than the relatively hydrophobic humic fractions. Aquatic humic substances contain only humic acids (HA) and fulvic acids (FA).29 HA generally showed higher molecular size than FA.26,35 Herein, the relatively hydrophilic and larger MW humic fractions could be assumed as HA-like species and the relatively hydrophobic and smaller MW humic fractions could be ascribed to FA-like species. Notice that all the plots distributed in the upper right corner, that is, the normalized percentages of the HA-like species were larger than those of the FA-like species. That is to say that the HA-like species contributed more than FA-like species to the humic-like component (C3) in EEM-PARAFAC model. Environmental Implications. The interpretation of EEM is limited by its complex and overlapped spectra. In theory, PARAFAC can mathematically separate the spectrally overlapped fluorescent components.10 In the past decade, the extensively applied EEM-PARAFAC models have provided an
Figure 4. HPSEC emission-time-maps of five secondary effluents: (a) KXY, (b) KFQ, (c) QT, (d) AZ and (e) WLK. The Em-axis denotes the emission wavelength, by which humic-like peaks (Em > 380 nm) and protein-like peaks (Em < 380 nm) can be distinguished. The Time-axis represents the elution time of DOM species, which can reflect their molecular weight distribution. The color variation represents the fluorescence intensity in arbitrary units.
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chlorination process, and therefore the assessment of their DBP formation potentials is quite necessary in future in order to identify which humic fractions should be priorly removed.
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ASSOCIATED CONTENT
S Supporting Information *
There are 2 tables and 11 figures in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(A.-M.L.) Phone: +86 25 83318402; fax: +86 25 83318402; Email:
[email protected].
Figure 5. Correlation analysis of the relative intensity of the first humic-like peaks in HPLC and HPSEC chromatograms. The relative intensity is presented in normalized percentage.
Notes
The authors declare no competing financial interest.
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informative treasure for tracking DOM components in both natural and engineering systems. However, compared with chromatography fluorescence fingerprints, the EEM-PARAFAC model could not reflect the variety of DOM species with similar fluorescence but different physicochemical properties, which might result in some inconsistences of similar PARAFAC components across studies. Furthermore, the spectrally independent EEM-PARAFAC components could not reflect the potential combination between humic-like species and protein-like species. Such limitations of EEM-PARAFAC may arise from the intrinsic deficiency of EEM technique (i.e., overlapped spectra of fluorophores) rather than PARAFAC tools. Since the HPLC/HPSEC fluorescence fingerprints also exist as a matrix of data, PARAFAC can also be developed to analyze the HPLC/HPSEC fluorescence matrices for automatic identification. However, the shortcomings of the HPLC/ HPSEC technique are also obvious. Besides the instrument’s price, it is not as sensitive as fluorescence EEM technique due to the dilution effect from mobile phase, and as a result, it preferentially fits for water samples with high fluorophores’ concentration. The optical and physicochemical properties of fluorescent components were related together, which helps to track and elucidate their behavior in advanced wastewater treatment. For the enhanced coagulation process, it prefers to remove large and hydrophobic molecules.36,37 In the enhanced coagulation process, the protein-like components could be removed more efficiently than humic substances (SI Figure S10), which can be elucidated that some of the protein-like species are relatively hydrophobic. With regard to the humic substances, the larger species are relatively hydrophilic, while the smaller species are relatively hydrophobic, indicating there is an antagonism effect on the removal efficiency between hydrophilicity and molecular weight. In chlorination process, the formation potential of carbonaceous DBPs, highly and positively relates to UV254 absorbance.38 With FLD in tandem UV absorbance detector, the HA-like and FA-like species were further identified as the major contributor to UV254 absorbance in secondary effluents (SI Figure S11). Anion exchange process has been identified as an effective process to remove UV254 absorbance and DBPs precursors.39 Because the anion exchange resin adapts to a wide range of molecular weight,40 the removal efficiency will be dominated by the hydrophilicity, thus resulting in that it prefers to remove HA-like species (SI Figure S10). However, as the important precursors of DBPs, HA-like and FA-like species are expected to exhibit different reaction rates and routes in
ACKNOWLEDGMENTS We gratefully thank generous support provided by Program for Changjiang Scholars and Innovative Research Team in University, National Science Foundation of China (51178215, 51290282 and 51308283) and Jiangsu Nature Science Fund for Distinguished Scientists (BK2011032), P. R. China. We also thank Mr. Xin-Chun Ding, Mr. Bi-Cun Jiang, Mr. Ke Wang, and Ms. Wei-Hua Yu for their kind help in sampling campaign. We also thank the comments and suggestions from International Workshop on Organic Matter Spectroscopy 2013, 1619th July 2013, La Garde City, France.
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dx.doi.org/10.1021/es404624q | Environ. Sci. Technol. 2014, 48, 2603−2609
