Recognizing Dissolved Organic Matter with an Improved Fluorescence Regional Integral Method

Corresponding Author: Wang Xinyi Institute of Resources and Environment, Henan Polytechnic University, Jiaozuo 454000, China; Collaborative Innovation Center of Coalbed Methane and Shale Gas for Central Plains Economic Region, Henan Province, Jiaozuo 454000, China Email: zwwfhh321@126.com Abstract: The aim of this paper was to study transformations of Dissolved Organic Matter (DOM) in marl rock groundwater. To achieve this, we carried out a series of soil column experiments, using a DOM solution as the water sample and marl rock as the testing rock sample. Three-Dimensional Excitation-Emission Matrix Spectra (3DEEMS) were generated for DOM solutions and the Analytic Hierarchy Process (AHP) was introduced to improve the traditional method of fluorescence regional integral (TMFRI). We termed this approach the Improved Method of Fluorescence Regional Integral (IMFRI) and used it to identify the type of DOM in the marl rock. The result shows that DOM recognition using TMFRI is only 50% accurate and consequently transformations are hard to recognize. In contrast, our results show that use of IFRIM has a DOM type recognition accuracy of 83.33% and so this method can be used with confidence to describe transformation characteristics. And we find that in the marl rock, the leaching test showed a new fluorescence peak at 312 h and the humic acid was degraded into soluble microbial products by microorganisms.


Introduction
Dissolved Organic Matter (DOM) is an important solute in groundwater. These solutions commonly comprise a mixture of aliphatic and aromatic organic compounds that include oxygen, nitrogen and sulfur functional groups. DOM is usually defined as an organic matter that can pass through a membrane filter with a 0.45 μm aperture size (Leenheer and Croué, 2003;Buffle and Leppard, 1995). DOM includes tyrosine, tryptophan fulvic acid, metabolite from soluble microorganisms, humic acid substances, etc. Since there is absorption, exchange and complexation between the functional groups in DOM and the ions in the environment (Maximiliano et al., 2009), research in this area has been both a focus and area of contention in international hydro geochemistry.
Research on DOM was initiated from the late eighteenth century (Ghabbour and Davies 2001). Pallo, for example, divided humic acid and fulvic acid into different components and developed a method to subdivide the former into soluble and non-soluble components (Waksman 1938). Waksman introduced the theory of lignin protein and demonstrated its importance in nature (Pollo, 1993). Much later, Christman and Gjessing (1983) presented a definition for aquatic humic acid as opposed to its terrestrial counterpart. Zhou et al. (2010;Porcal and Kopáček, 2018;Zhou et al., 2019;Johs et al., 2019;Li et al., 2018), have done many studies on the role of transformation and migration that DOM has on polycyclic aromatic hydrocarbons, phosphorus, copper ions, mercury and cadmium.
With the appearance of three-Dimensional Excitation-Emission Matrix Spectra (3DEEMS), the study of DOM has gone a step further. The core principle of this method involves generating a 3DEEMS image by sending fluorescence spectra intensity at given excitation and emission wavelengths on a plane as abscissa that ordinate with one another in order to distinguish different kinds of DOM (Coble et al., 1990). Cui et al. (2016) researched the constitution and features of resolvable matters in the Baiyangdian District through 3DEEMS. He et al. (2015) researched the origin and constitution of resolvable organic matter in the groundwater polluted by leachate using 3DEEMS in tandem with chemical analysis. Zhu et al. (2010) conducted research on the effect of the oxidation-242 reduction condition on 3DEEMS of DOM in water and found that humid acid matter is easy to degrade in oxidation environments.
The Traditional Method of Fluorescence Regional Integral (TMFRI) has most often been utilized to determine the amount of fluorescence in different DOM partitions based on integrals from 3DEEMS. Applying the TMFRI can greatly enhance analyses based on 3DEEMS both qualitatively to quantificationly. Zhao et al. (2013) examined the fluorescence characteristics of fulvic acid in the sediment in Xingkai Lake using the TMFRI. Jiang et al. (2015) used the same approach to present a quantitative evaluation of the effects of DOM removal from different plants. Lei et al. (2015) quantitatively characterized the DOM of natural soil using a three-Dimensional (3D) matrix in combination with the TMFRI method. However, because TMFRI does not include the location of the peak appearance of 3DEEMS, the quantitative result is quite different from reality. It has therefore proved problematic to precisely describe changes in DOM using this approach.
Based on a dynamic leaching modeling experiment with marl rock distilled on the scene, this essay quantitatively analyzes the DOM features of leach ate using the combinative Analytic Hierarchy Process (Wang et al., 2017) (AHP) as well as 3DEEMS TMFRI. We expect that a reasonable confirmation of weight can be achieved to describe the transformation regularity of DOM, contributing to the mechanism study of ground water recharge, runoff and discharge in loose aquifers.

Testing Samples
In Pingdingshan coalfield, underground water in the Cambrian limestone aquifer is the main threat to coal seam mining. Most of the shallow buried Cambrian limestone is covered by Neogene marl rock. Precipitation and surface water supply to the aquifer limestone percolates through the marl. The permeation characteristics of the marl rock obtained in Pingdingshan coalfield are studied in this paper.
The sampling depth of the marl rock is 15 m and its physical properties are presented in Table 1. We used a self-confected DOM solution, with an original concentration of 70.9 mg/L, was chosen as the infiltration solution.

Experimental Devices
The simulated experimental column used in this experiment had a height of 50cm and internal diameter of 7 cm. This column was placed inside the thermostat, as shown in Fig. 1, with the injection port and sampling port positioned at the top and bottom of the column, respectively, connected to each other via a rubber hose.
Leachate was provided by a stable fluid supply device composed of a high position bucket and a peristaltic pump.
Marl samples were placed into the column alongside coarse grain quartz sand, with a particle size of 3cm, which was placed at both ends of the column to distribute the leachate smoothly and keep its homogeneous flow. DOM quality concentration was calculated in terms of Chemical Oxygen Demand measured by potassium dichromate, mg/L (CODcr), while a titration test was held following disinfection through a WMX-III microwave device. 3DEEMS detection is carried out by a Fluorescence Spectrophotometer (HITACHI F-7000, Tokyo, Japan) and the data are processed through Software Origin and characterized by the intensity contour on the fluorescence spectrum. The 3DEEMS of the origin DOM solution is shown in Fig. 2.

Experimental method
Under room temperature (25C) conditions, distilled water was pumped into the bottom of the soil column to expel the gas and prevent any penetrative effect on the rock sample. DOM solution at a concentration of 70.9 mg/L was then pumped into the rock column at 2.5 ml/h and fluid was removed from the bottom of the column. We then measured the 3DEEM and plotted the spectral intensity contours.

Improved Fluorescence Regional Integral
Traditional Method of Fluorescence Regional Integral (TMFRI) Chen et al. (2003), as well as other researchers, divided the 3DEEMS into five sections, Fluorescence peaks fall in different areas to represent different substances as shown in Fig. 3. For example, When Fluorescence peaks fall in Region I symbolizing tryptophan. Regional boundaries are as follows: Region I: EX ≤ 250 nm, EM < 330 nm, symbolizing tyrosine; Region II: EX ≤ 250 nm, 330 nm ≤E M ≤ 380 nm, symbolizing tryptophan; Region III: EX ≤ 250 nm, EM > 380 nm, symbolizing fulvic acid; Region IV: EX > 250 nm, EM ≤ 380 nm, symbolizing soluble microbial metabolites; and Region V: EX > 250 nm, EM > 380 nm, symbolizing humic acid substances.
TMFRI can be used to identify each of these five regions, as well as to calculate the regional integrals i,n of the 3DEEMS intensity in each region (He et al., 2011): I(EX/EM) is used to state the 3DEEMS intensity corresponding to the excitation-emission wavelength; λEX and λEM represent excitation wavelength and emission wavelength, respectively.
Integral proportion of the fluorescence spectra in each section can be calculated by the following two formulas: i,n is the fluorescence spectra quantity in each region (i.e., integral of fluorescence spectra intensity)

Improved Method of Fluorescence Regional Integral (IMFRI)
TMFRI makes use of the peak value and contour intensive degree of 3DEEMS to determine the integral value, which has the advantage of simple calculation (He et al., 2011). The identified DOM type is possibly different from the actual result as the TMFRI does not take the location of the peak value of the 3DEEMSand the effect among the five regions. Here, the AHP is introduced to confirm the weight and characterize the location of the peak value of the 3DEEMS. Formula 4 and formula 5 are used to calculate the fluorescence spectra quantity and integral proportion of fluorescence spectra: Among the three equations, Wi is used to describe the weight of the 3DEEMS in each region. i,n states the fluorescence spectra quantity calculated in each region (see formula 1). Qi,n stands for the fluorescence spectra quantity in each section. QT,n represents the total amount of the fluorescence spectra quantity. Ai,n is the integral proportion of the fluorescence spectral in each region (%).
As for a complex system, which is controlled by multi-factors and is hard to be accurate, here are the basic steps to make use of the AHP for analysis and evaluation: Confirm the affiliation among all factors, construct factor hierarchy and evaluate the factors in each hierarchy, introduce the optimal transfer matrix to construct the judgment matrix to calculate the corresponding influence coefficient of the factors in each hierarchy. This approach improves the judgment accuracy, leaving out consistency checking and increasing the operability after bringing in the optimal transfer matrix (Xu et al., 2007).

The Weight Determination with IMFRI
The experiment takes the DOM state as the example (Fig. 2), with a concentration of 70.9 mg/L and provides a statement of the IMFRI. According to the position of the peak value of the fluorescence spectra intensity and the fluorescence spectra quantity i,n calculated by the TMFRI, a judgment matrix to calculate the weight Wi is built. The basic principle is that the region with peak value occurrence is more important than the one without it and the region with large i,n calculated by the TMFRI is more important than the one with small i,n.

Features of 3DEEMS
We perform a leaching experiment on the DOM solution with a concentration of 70.9 mg/L, extract the leaching solution at 0, 48, 192, 240, 312 and 600 h and test the 3DEEMS of each sample, as shown in Fig. 4.
At 600 h, the location and type of fluorescence peaks are almost the same as those at 312 h, which are a tryptophan fluorescence peak (Region II: EX = 241 nm, EM = 372 nm) and two fluorescence peaks of soluble microbial metabolites (Region IV1: EX = 280 nm, EM = 355 nm; Region IV2: EX = 273 nm, EM = 300 nm).

TMFRI Results
We use TMFRI to do a quantitative calculation of the 3DEEMS. Table 2 shows the fluorescence spectra quantity and integral proportion.  Ⅰ Ⅱ Ⅲ Ⅳ2 Ⅴ

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From the calculated value of the integral proportion, through TMFRI, we know the main types of DOM (the two largest values of the integral proportion, the red color in Table 2

IMFRI Results
According to the weight calculating steps of the DOM state in Section 2.3, we confirm the weight of DOM leachate at different times and its results are shown in Table 3. Based on the fluorescence spectra with the TMFRI (Table 2), we use the IMFRI to calculate the fluorescence spectra quantity and integral proportion with the results shown in Table 3.
Apparently, the main types recognized with IMFRI are shown as follows: At 0 h, the main types are fulvic acid in Region III and soluble microbial metabolites in Region IV. At 48 h and 192 h, the main types are soluble microbial products in Region IV and humic acid in Region V. At 240 h, 312 h and 600 h, the main types are tryptophan in Region II and soluble microbial metabolites in Region IV.
From Table 4, compared with the identification results of the TMFRI and 3DEEMS, the results are in complete agreement at time nodes 192 h, 240 h and 312 h, while the results at 0 h and 600 h are only partially in agreement and the results at 48 h are completely different. The identification types of DOM are quite different from the TMFRI and 3DEEMS. Compared the results with the IMFRI and 3DEEMS, the results are all in complete agreement except that at 0 h and the identification types of DOM are mostly the same from the IMFRI and 3DEEMS.
According to the studies above, TMFRI simply makes use of the integral value to characterize the fluorescence spectra quantity in each section. The DOM types that it identifies are quite different from that of 3DEEMS, with an accuracy of 50%, which makes it difficult to identify the features of the DOM transformation. The IMFRI can confirm the weight by wholly considering the location of the peak value of the 3DEEMS and the effect among the five sections, calculating the fluorescence spectra quantity in each section. The DOM types identified by this method are well matched with the results from 3DEEMS, with an accuracy of 83.33%, so this method can be used to identify the transformation features of DOM.