Application of Excitation-Emission Matrix Fluorescence (EEMF) in the Wastewater Field

Abstract

Fluorescence is a versatile and useful analytical technique for the analysis of waters, both natural waters (freshwaters and marine waters) and wastewaters (urban wastewaters and industrial effluents). Among the various fluorescence techniques currently available, excitation-emission matrix fluorescence (EEMF) is the most used nowadays since it provides comprehensive information on the dissolved organic matter (DOM) present in water. EEMF spectra can be represented either in the form of a 3D-graph or a 2D-contour map and fluorescence peaks can be studied by the fast and simple peak-picking method (more suitable for routine measurements in water treatment plants, allowing a rapid response in case of potential problems in the sequence of treatment) or using mathematical tools such as PARAFAC (more suitable for research purposes and accurate identification of the fluorophores). The EEMF peaks commonly found in waters are peaks A and C (humic substances), peaks B1, B2, T1, and T2 (protein-like peaks), and peak M (microbial-like peak). EEMF was first applied to the characterization of natural waters, but in recent years, more attention is being paid to the wastewater field. Urban wastewaters have been mostly studied, whereas there are fewer studies focused on industrial effluents. This chapter provides a brief review of these EEFM applications.

1. Introduction

In recent years, the number of studies using fluorescence techniques for the characterization of dissolved organic matter (DOM) in natural and wastewaters has significantly increased [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. There are several reasons for this fact: fluorescence is a fast, sensitive, and nondestructive analytical technique that requires small volumes of the sample. Moreover, in most cases, samples just require a simple pretreatment (pH adjustment and filtration, if necessary) and fluorescence probes can be readily adapted to automated devices for in situ measurements.

Fluorescence offers several advantages over other alternative techniques often used in water analysis. For instance, global parameters such as biochemical oxygen demand (BOD) and chemical oxygen demand (COD) provide no information on the structure and properties of DOM, in addition to being time-consuming methods (5 days and 2 hours, respectively). Other more sophisticated techniques, such as gas chromatography-mass spectrometry (GC/MS), infrared spectroscopy (FTIR), and ¹H- and ¹³C-nuclear magnetic resonance (NMR), require complicated and laborious procedures for extraction-purification of the aqueous samples. Moreover, when analyzing complex matrices, such as wastewaters, FTIR, and NMR signals, usually overlap into broad and poorly resolved bands, thus making the interpretation of the spectra difficult [17].

Several fluorescence techniques can be applied to the analysis of freshwaters and wastewaters, such as the conventional emission scan fluorescence (ESF) and the more interesting synchronous fluorescence spectroscopy (SFS). However, the most useful and complete technique used at present is excitation-emission matrix fluorescence (EEMF, also known as total luminescence spectroscopy: TLS), in which a series of emission scans are collected for a range of excitation wavelengths. The generated matrix of data can be represented either in the form of a 3D-graph or a 2D-contour map, thus making it easier for quick identification of the main fluorescence peaks present in the sample. Peak coordinates are represented as (λ_ex/λ_em, in nm) and their maximum intensities of fluorescence (F_max) are representative of the relative concentration of the fluorophores [18, 19]. The interpretation of the spectra can be conducted using either the traditional “peak-picking” method or more sophisticated mathematical tools, such as the parallel factor analysis (PARAFAC). The peak-picking method is simpler, faster (an EEMF spectrum in the range λ_ex/λ_em: 220–450/300–550 nm is usually collected in 8–10 minutes), and useful for quick monitoring of the typical fluorophores present in waters, which makes it more suitable for routine measurements in water treatment plants as these plants usually demand fast and user-friendly analytical techniques. PARAFAC turns out to be more appropriate for research purposes since this tool requires a higher degree of analytical expertise.

The main EEMF peaks found in natural and wastewaters are the following and can be classified into three major groups (λ_ex/λ_em, in nm):

• Humic-like peaks: peak A (230–260/400–480 nm, fulvic-like), peak C (320–360/420–460 nm, humic-like). They are associated with humic substances (fulvic and humic acids).

• Protein-like peaks: tyrosine-like peaks B1 (275–310/305–320 nm) and B2 (220–237/305–320 nm), tryptophan-like peaks T1 (275–285/320–350 nm), and T2 (215–237/340–381 nm). They are mainly associated with the presence of proteinaceous material (proteins and peptides) containing the amino acids tyrosine and tryptophan. However, this fluorescence might not be due exclusively to proteins, since recent studies have reported that humic substances can encapsulate proteins under certain circumstances, indicating a potential combination between them. Additionally, some polyphenolic compounds, such as lignin, have also been reported to exhibit tryptophan-like fluorescence [20].

• Microbial-like peak M (290–310/370–420 nm): this peak is associated with the release of organic compounds from recent microbiological activity.

Unfortunately, neither lipids (oil and grease) nor carbohydrates (both of them usually present in wastewaters) can be detected by EEMF, which constitutes a drawback when a comprehensive characterization of the water is required.

A location of these peaks in a typical EEMF spectrum is shown in Figure 1. In addition to the aforementioned peaks, several fluorescence indices are also used in some studies for specific purposes (see Figure 1), such as:

Figure 1.

Location of the main EEMF peaks and fluorescence indices in waters.

Fluorescence index (FI), first introduced by McKnight [21], is calculated as the ratio of emission intensity at 450/500 nm measured at λ_ex = 370 nm:

$$\mathrm{FI}={\mathrm{I}}_{\mathrm{Em}450}/{\mathrm{I}}_{\mathrm{Em}500},\mathrm{at}{\lambda }_{\mathrm{ex}}=370\mathrm{nm}$$E1

This index has been mostly used to elucidate the origin of fulvic acids in freshwaters (FI values around 1.9 denote fulvic acids of microbial origin, whereas values around 1.4 indicate terrestrially derived fulvic acids [22]. FI has been also reported to show a negative correlation with the aromaticity of humic substances [23].

Humification index (HIX): this index was proposed by Zsolnay [24] and is determined as the ratio of fluorescence intensities of the integrated emission region of λ_em = 435–480 nm divided by that of λ_em = 300–345 nm, measured at λ_ex = 254 nm.

$$\mathrm{HIX}=\sum {\mathrm{I}}_{\mathrm{Em}435–480}/\sum {\mathrm{I}}_{\mathrm{Em}300–345},\mathrm{at}{\lambda }_{\mathrm{ex}}=254\mathrm{nm}$$E2

Later on, a modification of the original HIX was introduced, calculated as the emission intensity in the 435–480 nm region divided by the sum of total intensities in the (300–345 + 435–480) nm regions. This index is denoted as “normalized HIX” (HIX_norm), as it ranges from 0 to 1.

$${\mathrm{HIX}}_{\mathrm{norm}}=\sum {\mathrm{I}}_{\mathrm{Em}435–480}/\left(\sum {\mathrm{I}}_{\mathrm{Em}300–345}+\sum {\mathrm{I}}_{\mathrm{Em}435–480}\right),\mathrm{at}{\lambda }_{\mathrm{ex}}=254\mathrm{nm}$$E3

HIX is related to the degree of humification of the organic matter in waters and is strongly correlated with DOM (dissolved organic matter) aromaticity [25].

Biological index (BIX), first introduced by Huguet [26], is determined by dividing the fluorescence intensities at the emission wavelengths of 380 and 430 nm, measured at λ_ex = 310 nm:

$$\mathrm{BIX}={\mathrm{I}}_{\mathrm{Em}380}/{\mathrm{I}}_{\mathrm{Em}430},\mathrm{at}{\lambda }_{\mathrm{ex}}=310\mathrm{nm}$$E4

As shown in Figure 1, BIX is strongly correlated with peak M, indicating the presence of organic matter recently released by microorganisms in water (autochthonous DOM from biological origin) [23].

2. Application of fluorescence in water analysis

2.1 Fluorescence and natural waters

Before getting into the fluorescence applications in the wastewater field, it is interesting to do a brief review of its applications in natural waters, both freshwaters (rivers, reservoirs, etc.) and marine waters since this field has been the most studied for many years. The most abundant EEMF peaks found in natural waters are humic-like peaks (both A and C), which is indicative of the presence of humic and fulvic acids in water, the latter constituting the majority fraction of the aquatic humic substances. Actually, a considerable presence of protein-like peaks in freshwaters is usually related to wastewater discharges of anthropogenic origin [18, 27]. Humic substances make up most of the NOM (around 30–50%) present in freshwaters [28] and are originated from both humification processes occurring during the decomposition of vegetable organic matter in water (autochthonous microbial origin) and elutriation of soil humic substances from the surrounding terrain (terrestrial origin).

There are several drawbacks directly related to an excessive presence of humic substances in water, such as an increased formation of disinfection by-products upon chlorination (mainly trihalomethanes), they can act as carriers for micropollutants and heavy metal ions via the formation of soluble complexes with them, they contribute to membrane fouling in membrane-based water treatments (for instance, membrane biological reactors or MBR), they contribute to the biofilm formation in water distribution pipelines and they can hinder the adsorption of micropollutants onto activated carbon.

EEMF can provide interesting information on humic substances structure and properties: the location and shift of the peaks and their fluorescence intensities are correlated to some parameters, such as the aromaticity degree, carboxylic acidity, and the degree of humification. Additionally, there are several well-established behaviors concerning the fluorescence of humic substances [29, 30], namely:

• the intensity of the fluorescence peaks (both A and C) decreases with increasing the macromolecule molecular size.

• concerning substituted aromatic moieties in the humic macromolecule: electron-donating groups (-OH, -NH₂, and -OCH₃) cause an increase in the fluorescence intensity, whereas electron-withdrawing groups (-COOH) cause the opposite effect.

• hydroxyl, alkoxyl, amino, and carbonyl-containing substituents usually cause a red-shift (fluorescence maxima shift toward longer wavelengths).

• a reduction in the aromaticity degree of the macromolecule (for instance, a reduction in the number of aromatic rings) usually causes a blue-shift (fluorescence maxima shift toward shorter wavelengths).

Figure 2 shows the EEMF spectrum (2D-contour map) of natural water (Úzquiza Reservoir, which supplies to the city of Burgos, Spain) and the EEMF spectrum (3D-graph) of a pure fulvic acid (Nordic fulvic acid, reference material from the international humic substances society). As shown in Figure 2, the reservoir water is characterized by only presence of humic-like peaks, a high-intensity peak A (fulvic-like), and a less intense peak C (humic-like). There is no presence of protein-like peaks, which is indicative of the absence of urban wastewater discharges and therefore, a clear sign of good quality water. Obviously, the 3D spectrum of the pure fulvic acid (Figure 2) only contains humic-like peaks, being the fulvic-like peak A the majority one.

Figure 2.

EEMF spectrum (2D contour plot) of a reservoir water (left) and EEMF spectrum (3D graph) of aquatic fulvic acid (right). The 3D fulvic acid spectrum also shows the first and second order Rayleigh scattering peaks.

2.2 Fluorescence and urban wastewaters

Dissolved organic matter (DOM) in wastewater comprises a great variety of organic compounds, from low-molecular weight (MW) substances (amino acids, small organic acids, simple sugars, etc.) to high-MW compounds (proteins, humic substances, carbohydrates, etc.) [23, 31, 32, 33, 34]. In the wastewater field, fluorescence has been mostly applied to the characterization of effluent organic matter (EfOM) from urban wastewater treatment plants (WWTPs) [1, 2, 3, 4, 7, 8, 35, 36].

Protein-like peaks T1 and T2 (tryptophan-like peaks) are usually the most abundant EEMF peaks found in urban wastewaters. These peaks originated from both proteinaceous material present in the influent (anthropogenic origin) and protein-like compounds released by microorganisms (soluble microbial products: SMP) during the biological treatment stage in WWTPs [19, 37]. Conversely, the presence of tyrosine-like peaks (B1 and B2) in urban wastewaters is less frequent because tyrosine fluorescence is usually quenched within high molecular weight proteins due to resonance energy transfer [1]. That is why the detection of peaks B in the EEMF spectrum is usually associated with the presence of free tyrosine or tyrosine-containing small peptides (in which tryptophan is not present) in the sample [38].

The relative abundance of tryptophan-like peaks T1 and T2 (T1/T2 ratio) in the influent depends on the specific type of domestic wastewater and the influence of industrial discharges into the municipal WWTP. Consequently, peak T1 is reported as the most abundant in some studies from the literature [19, 34, 35] whereas peak T2 in others [1, 20, 39].

EEMF has also been proved to be useful to track changes in NOM throughout the sequence of treatment in WWTPs [40]. Protein-like peaks are more biodegradable than humic-like peaks, whereas the latter are more amenable to be removed by sedimentation. Therefore, in WWTPs protein-like peaks show greater percentages of removal at the biological treatment stage, whereas humic-like peaks at the clarification stage [37].

Figure 3 shows the EEMF spectrum of an urban wastewater influent and effluent (wastewater treatment plant of Burgos and Spain). Quenching effects caused by the presence of metal ions in the wastewater (mainly iron) are negligible due to their low concentration levels, usually found in urban wastewaters. As shown in Figure 3, tryptophan-like peak T2 is the most abundant in this wastewater and the comparison of fluorescence intensities between the influent and the effluent allows the estimation of removal percentages for each peak.

Figure 3.

EEMF spectrum of urban wastewater influent (left) and effluent (right).

2.3 Fluorescence and industrial wastewaters

In the wastewater field, most studies reported in the literature have focused on urban/domestic wastewaters, but little attention has been paid to industrial effluents. In addition to the organic compounds typically present in urban wastewaters (see Section 2.2), industrial wastewaters can contain a great diversity of organic pollutants depending on the specific industry sector (phenols, pharmaceuticals, organic solvents, surfactants coming from tank cleaning processes, etc.). For this reason and contrary to urban wastewaters (where a typical EEMF spectrum with a predominance of protein-like peaks is expected in most cases), no standard EEMF spectrum can be associated with industrial effluents. For instance, food-related industries (milk, brewery, winery, biscuit industries, etc.) do show EEMF spectra similar to those of urban wastewaters (predominance of protein-like peaks) but conversely, old landfill leachates exhibit spectra just containing humic-like peaks: the higher the landfill age (and therefore the higher the humification degree of the humic substances) the greater the humic-like peak C fluorescence intensity [23]. It is interesting to note that some kinds of industries, such as pulp and mill, textile dyeing industries, and slaughterhouses, are reported to potentially show specific fingerprints that could allow a tentative identification of their origin but more research is needed on this issue [23].

Figure 4 shows the EEMF spectrum for a food industry effluent (a cold-meat processing factory) and municipal landfill leachate. As commented earlier, the spectrum of the cold-meat industry effluent is characterized by the predominance of protein-like peaks, whereas that of the landfill leachate exhibits a dominant humic-like fluorescence (peak C), indicating leachate coming from an old landfill.

Figure 4.

EEMF spectrum of a food industry wastewater (cold-meat industry effluent) and a municipal landfill leachate.

Table 1 summarizes the different types of water frequently characterized by EEMF along with the references included in this chapter.

Type of water		References
Natural waters		[5, 6, 10, 11, 12, 14, 16, 21, 24, 25, 26, 27, 29, 30, 36, 38]
Urban wastewaters		[1, 2, 3, 4, 7, 8, 9, 15, 17, 18, 19, 20, 23, 28, 31, 33, 34, 35, 36, 37, 39]
Industrial wastewaters	Food industries	[32, 41, 42, 43, 44]
	Pulp mill industries	[22, 41, 45]
	Textile industries	[41, 46, 47, 48, 49, 50]
	Slaughterhouses	[41, 51, 52]
	Landfill leachates	[40, 41, 53, 54, 55, 56]
	Pharmaceutical industries	[13]

Table 1.

Types of waters typically analyzed by EEMF and related literature references.

3. Conclusions

Fluorescence, and particularly excitation-emission matrix fluorescence (EEMF), has been proved to be a useful and versatile analytical technique for the characterization of the organic matter present in wastewaters. Due to the fact that fluorescence is a fast and user-friendly technique, it can be easily implemented in wastewater treatment plants for routine measurements, allowing a rapid response to deal with potential problems in the treatment line. New studies in this field are being continuously released and this trend will surely continue in the future.