- Email: [email protected]
Fluorescent and molecular weight dependence of THM and HAA formation from intracellular algogenic organic matter (IOM)
Accepted Manuscript Fluorescent and molecular weight dependence of THM and HAA formation from intracellular algogenic organic matter (IOM) Lap-Cuong Hua, Shu-Ju Chao, Chihpin Huang PII:
S0043-1354(18)30848-0
DOI:
https://doi.org/10.1016/j.watres.2018.10.051
Reference:
WR 14162
To appear in:
Water Research
Received Date: 25 August 2018 Revised Date:
17 October 2018
Accepted Date: 20 October 2018
Please cite this article as: Hua, L.-C., Chao, S.-J., Huang, C., Fluorescent and molecular weight dependence of THM and HAA formation from intracellular algogenic organic matter (IOM), Water Research (2018), doi: https://doi.org/10.1016/j.watres.2018.10.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Fluorescent and molecular weight dependence of THM and HAA
SC
RI PT
formation from intracellular algogenic organic matter (IOM)
M AN U
Lap-Cuong Hua, Shu-Ju Chao, Chihpin Huang*
EP
TE D
Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwan
AC C
Submitted to Water Research
*Corresponding author: Prof. Chihpin Huang TEL: +886-3-5712121-55507, FAX: +886-3-5725958 Email: [email protected] 1
ACCEPTED MANUSCRIPT Abstract
2
This study (i) examined the formation of two major carbonaceous disinfection by-products
3
(C-DBPs), trihalomethanes (THMs) and haloacetic acids (HAAs), during the chlorination of
4
intracellular algogenic organic matter (IOM) extracted from two commonly blooming algae M.
5
aeruginosa (MA) and Chlorella sp. (CH), and (ii) investigated the roles and relationships of
6
fluorescent and molecular weight (MW) properties on/with IOM-derived THMs and HAAs. The
7
extracted IOM samples were separated into different MW fractions by centrifugal devices with
8
membrane support with MW cut-offs of 100, 30, 10, 3, and 1 kDa. We observed an overall
9
reduction of C-DBPs with a decrease of IOM-MW from >100 kDa to <1 kDa. Of six fractionated
10
IOM, a large fraction (>100 kDa) contributed the largest amount to the MW distribution of IOM,
11
accounting for 33 and 42% of the total dissolved organic carbon (DOC) of MA and CH, respectively.
12
It also had the highest-yielding potential to produce significant levels of THMs and HAAs, and total
13
C-DBPs over other small MW fractions. Although small MW fractions (>10 kDa) contributed
14
around 50% of the total DOC, they made an insignificant contribution (>20%) to the THMs, HAAs,
15
and overall C-DBPs. Furthermore, the decrease of IOM MW caused a shift from the domination of
16
HAA formation to THM formation, especially when MW was <10 kDa. By canonical correspondent
17
analysis, the relationship of IOM-derived THMs and HAAs with IOM properties was examined. In
18
particular, large fractions of IOM, exhibiting aromatic protein- (AP) and soluble microbial product-
19
(SMP) like fluorescence, are favorable for the formation of HAAs, whereas small MW fractions of
AC C
EP
TE D
M AN U
SC
RI PT
1
2
ACCEPTED MANUSCRIPT IOM with HA- and FA-like fluorescence preferentially tends to form THMs. Our findings evidently
21
show the strong dependence of IOM-derived THMs and HAAs on the fluorescent and MW
22
properties. Therefore, the characterization of MW and fluorescent properties can provide the
23
advantages in the control of algae-derived DBPs upon the chlorination of eutrophic water.
24
Keywords: algogenic organic matter; DBPs; chlorination; fluorescence; molecular weight
25
1. Introduction
26
Bloom of algae in an aquatic environment is a critical problem in water treatment utilities. It
27
frequently causes a dramatic deterioration of water quality by increasing turbidity, taste and odor,
28
and algal toxins in the water column (Henderson et al., 2008a; Tomlinson et al., 2016).
29
Importantly, algal bloom releases a significant content of algogenic organic matter (AOM) that
30
has been proved to be a major precursor of disinfection by-products (DBPs) upon
31
chlor(am)ination or ozonation (Fang et al., 2010; Goslan et al., 2017; Hua et al., 2018a; Huang et
32
al., 2009; Plummer and Edzwald, 2001; Zhu et al., 2015). AOM, as a precursor to DBPs, can be
33
generally categorized into extra- (EOM) and intra-cellular organic matter (IOM). While EOM is
34
rapidly released via algal exudation in soluble organic form (Fogg et al., 1965), IOM is the
35
internal organic substances released by the rupture of the cell membrane (Tomlinson et al., 2016).
36
Nonetheless,
37
coagulation/sedimentation/filtration processes (Cheng and Chi, 2003; Henderson et al., 2010;
38
Takaara et al., 2007), and controlling AOM-derived DBPs during the algal bloom has thus become
AC C
EP
TE D
M AN U
SC
RI PT
20
they
both
are
difficult
to
effectively
3
be
removed
by
conventional
ACCEPTED MANUSCRIPT the greatest challenge for sustainable production of safe drinking water.
40
A better understanding of AOM physicochemical properties significantly benefits an effective
41
control of its derived DBPs. In general, probing fluorophores provide valuable information about
42
the possible composition and relatively quantitative content of AOM precursor (Chen et al., 2003;
43
Coble, 2007). The use of fluorescent excitation-emission matrices (EEM) as a characterization
44
tool further allows us to acquire more insights into AOM-derived DBPs (Fang et al., 2010; Hua et
45
al., 2018b; Li et al., 2012). On the other hand, molecular weight (MW) is another important
46
characteristic that also shows significant connection to the effectiveness of AOM treatment (Guo
47
et al., 2017; Liu et al., 2018) and its derived DBPs in particular (Hua et al., 2018a; Lui et al., 2012;
48
Pivokonsky et al., 2014; Zhou et al., 2015). As the common and effective approaches to trace
49
DBP formation, the characterization of AOM-DBP precursors based on fluorescent and MW
50
properties has attracted more and more attention. However, current research into AOM-DBP has
51
as yet not fully resolved such relationships, because AOM is a complex and heterogeneous
52
mixture varying in size, structure, and functionality (Fang et al., 2010; Hong et al., 2008; Hua et
53
al., 2017; Li et al., 2012; Lui et al., 2012). There is a critical need to elucidate the principal role of
54
fluorescent and MW properties and their relationship with the formation of AOM-derived DBPs,
55
where the existent knowledge has remained unclear.
56
To address this issue, heterogeneous AOM can be isolated into more homogeneous groups based
57
on molecular size. Fractionation by a serial ultrafiltration technique is commonly used to isolate
AC C
EP
TE D
M AN U
SC
RI PT
39
4
ACCEPTED MANUSCRIPT OM sample into different MW fractions (Henderson et al., 2008b; Hua and Reckhow, 2007;
59
Pivokonsky et al., 2014; Zhu et al., 2015). This study was aimed at characterizing the fractionated
60
AOM precursors based on MW, as a function of fluorescence, and to examine the relationship
61
between AOM fluorescent and MW properties with its derived DBP formation upon chlorination.
62
IOM-derived DBP was the central focus of this research because, in practice, IOM (i) contributes
63
a significant fraction to total AOM (>80%) (Tomlinson et al., 2016), (ii) is a more difficult
64
matter to be removed by conventional treatments (Pivokonsky et al., 2014; Takaara et al., 2007),
65
and (iii) importantly is a higher-yielding DBP precursor compared to EOM (Hua et al., 2018b;
66
Huang et al., 2009; Li et al., 2012; Plummer and Edzwald, 2001). The IOM was extracted from a
67
cyanobacterium, M. aeruginosa (MA), and a green alga, Chlorella sp. (CH). MA and CH were
68
selected because they are two commonly blooming algae that occur worldwide (Ndlela et al.,
69
2016; Tas and Gonulol, 2007). Furthermore, two major classes of carbonaceous DBPs, including
70
trihalomethanes (THMs) and haloacetic acids (HAAs), were investigated, as they are dominantly
71
found upon chlorination of IOM, compared to other DBPs, such as chloroacetones,
72
chloroacetonitriles, nitrosodimethylamine and chloronitromethanes (Li et al., 2012; Lui et al.,
73
2011; Wert and Rosario-Ortiz, 2013), and are regulated worldwide (Tomlinson et al., 2016).
74
2. Materials and methods
75
2.1 Algal cultivation and IOM extraction
76
M. aeruginosa and Chlorella sp. were isolated from a local reservoir in Matsu, Taiwan. They were
AC C
EP
TE D
M AN U
SC
RI PT
58
5
ACCEPTED MANUSCRIPT isolated into the unialgal culture by a commonly used protocol with agar plate streaking technique
78
and successive dilution method. Their axenic cultures were conducted in a thermostat
79
photobioreactor containing 6 L of modified BG11 and Chlorella medium. The conditions of algal
80
culture were followed our previous work (Hua et al., 2018b). Algal growth phases were
81
determined by monitoring the cell population every 2 days.
82
The algal suspensions with the volume of 500 mL from the late exponential phase of the two
83
species were selected for IOM extraction. Algal cells were separated from EOM by centrifugation
84
for 15 min, at 2700 G, at 4oC (Boeco Centrifuges U-320R, Germany) and then washed twice with
85
deionized (DI) water to remove the remaining EOM. The collected cells were lyophilized in a
86
freeze-drier (Pin-Chen Co., Taiwan) at –50°C and 2 × 10–4 Torr for 6 h. IOM was extracted
87
physically by gently grinding the lyophilized cells for 2 min with a mortar and pestle. After
88
grinding, IOM was extracted by re-suspending the ruptured cells in 100 mL of DI water and
89
shaking vigorously for 30 s using a vortex mixer. This suspension was then spun in a centrifuge
90
again, for 15 min, at 2700 G, at 4oC, prior to filtration through a 0.45 µm filter (Advantec, Japan)
91
to archive the IOM.
92
2.2 Molecular weight fractionation
93
The IOM water samples of the two species were separated into different MW fractions using a
94
series of centrifugal devices with membrane support (Macrosep® Advance Centrifugal Device,
95
Pall, USA), with MW cut-offs (MWCO) of 100, 30, 10, 3, and 1 kDa. Before using for IOM
AC C
EP
TE D
M AN U
SC
RI PT
77
6
ACCEPTED MANUSCRIPT isolation, each centrifugal device was washed twice by DI water at the identical centrifugation
97
conditions with samples. It is noticed that such centrifugation conditions could recover 95% of the
98
blank (DI water) for all MWCO. 20 mL of concentrated IOM samples (DOC ≈ 100 mg/L) was
99
loaded into the top of the centrifugal devices and spun at 2700 G for 30 min at 4oC. After the
100
serial fractionation, IOM samples were separated into six fractions with the MW ranges of F1
101
(>100 kDa), F2 (100–30 kDa), F3 (30–10 kDa), F4 (10–3 kDa), F5 (3–1 kDa), F6 (<1 kDa). This
102
process was duplicated, and the duplicates were used for other analyses and DBP formation tests.
103
All water samples were adjusted to pH 7 and stored at –20oC before use.
104
2.3 DOC and DON measurements
105
Dissolved organic carbon (DOC) concentration was determined using a TOC analyzer (TOC-L,
106
Shimadzu). Dissolved organic nitrogen (DON) content was measured with a DR/6000U
107
spectrophotometer (HACH Company, US). The DON content is the difference between the total
108
nitrogenous content (TNT Persulfate Digestion 10071 Method) and the total inorganic
109
nitrogenous content (Titanium Trichloride Reduction 10021 Method). All DOC and DON
110
measurements were conducted in duplicate for each MW fraction.
111
2.4 EEM spectra
112
Fluorescent spectra (or EEM spectra) of the original and fractionated IOM samples were analyzed
113
with a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA). Before
114
EEM analysis, water samples were adjusted to a DOC of 10 mg/L for comparison. EEM scanning
AC C
EP
TE D
M AN U
SC
RI PT
96
7
ACCEPTED MANUSCRIPT was carried out with excitation wavelengths ranged from 200 to 400 nm with 10 nm increments
116
and emission wavelengths ranging from 250 to 550 nm with 2 nm increments. The voltage of the
117
photomultiplier tube was set to 800 V for low light detection. DI water with DOC <0.2 mg/L was
118
used as a blank. The raw EEM intensities of each sample were subtracted for the blank (DI water)
119
and Rayleigh and Raman scattering to remove the background and interference. Four regional
120
fluorescent spectra were determined based on their excitation/emission wavelengths following
121
Chen et al., (2003). These represent the aromatic protein- (AP), soluble microbial product- (SMP),
122
humic- (HA), and fulvic-like (FA) substances. The average fluorescent intensity (AFI) of each
123
component was calculated based on the average intensity value of each selected region
124
respectively.
125
2.5 Characterization of chemical functional structure
126
The chemical functional groups of the fractionated IOM were analyzed with a High-Resolution
127
X-ray Photoelectron Spectrometer (XPS) (Physical Electronics, PHI 1600 spectrometer). Prior to
128
chemical analysis, samples were prepared by freeze-drying pretreatment for IOM solutions. XPS
129
survey was conducted by coupling with an Mg Kα X-ray source (1253.6 eV). Low-resolution
130
scanning was carried out from 0 to 1400 eV with a pass energy of 117.4 eV for elemental
131
determination. High-resolution surveys with pass energy set at 23.5 eV were also conducted for
132
the C, N, and O elements over the 278–298 eV, 391–411 eV, and 523–543 eV ranges, respectively.
133
The spectral deconvolution was performed by XPS Peak Fit program with the subtraction of
AC C
EP
TE D
M AN U
SC
RI PT
115
8
ACCEPTED MANUSCRIPT Shirley background and was optimized to achieve the R2 > 0.99 for C and O, and R2 > 0.90 for N.
135
2.6 DBP formation potential tests
136
For DBP formation potential (DBPFP) tests, water samples of different fractions (F1 to F6) and
137
original IOM were adjusted to DOC ≈ 5 mg/L and pH 7 ± 0.1. The tests were carried out by
138
spiking each sample with sodium hypochlorite (Cl2:DOC = 5:1) in headspace-free amber glass
139
bottles at room temperature (25 ± 1oC). After 7 days of chlorination, water samples were acidified
140
and quenched with ammonium chloride to obtain the THMFP and HAAFP. The THMFP were
141
measured by a coupling a purge and trap system (Model 4660, OI Analytical, Texas, USA) and a
142
gas chromatograph-mass spectrometer (GC-MS) (Agilent 6890 GC/5973 MSD) with a RTX-VOC
143
capillary column (60 m × 0.32 mm ID, 1.5 µm film thickness), following the United States
144
Environmental Protection Agency (US EPA) method 551.1. The HAAFP were measured by a gas
145
chromatography-electron capture detector (GC/ECD) (Agilent 6890 GC/micro ECD) with a
146
DB-1701 column (30 m × 0.25 mm ID, 0.25 µm film thickness), following US EPA method 552.3.
147
THMFP and HAAFP were measured in duplicate with a standard deviation of less than 10%. In
148
this study, the total of THMFP and HAAFP was considered as carbonaceous DBPFP (C-DBPFP),
149
and the ratio of HAAFP/THMFP was calculated by dividing the amount of HAAFP to THMFP.
150
All samples were prepared with bromide-free water.
AC C
EP
TE D
M AN U
SC
RI PT
134
9
ACCEPTED MANUSCRIPT 2.7 Statistical analysis
152
SPSS Statistics 22.0 software (IBM, Armok, NY, USA) was used to compare the differences of
153
THMFP, HAAFP, and C-DBPFP. Canonical correspondent analysis (CCA) was carried out by
154
XLSTAT software (Anddinsoft, Paris, French) to examine the relationship among IOM-derived
155
DBP, MW, and fluorescent properties. Data from six MW fractions of two IOM (12 sample sites)
156
were applied for this test.
157
3. Results and Discussion
158
3.1 MW distributions by DOC and DON
159
Distribution of MW in IOM was characterized by the contribution of each fraction to the total
160
DOC and DON of two species, as shown in Fig. 1. Both MA- and CH-IOM had bimodal
161
distributions in which large MW (>100 kDa; F1) and small MW fractions (<10 kDa; F4, F5, and
162
F6) mostly contributed up to >90% of the total DOC, together with insignificant contributions of
163
fractions F2 and F3 (Fig. 2a). While the total DON of MA showed a similar distribution with the
164
DOC, only a large MW fraction F1 was found abundantly in CH-DON (Fig. 2b). Briefly, the
165
distributions of six fractions in IOM followed the order as F1 >F4≈F5≈F6 >F2≈F3, and fraction
166
F1 evidently made the greatest contributions to the total DOC and DON profiles of all IOM,
167
accounting for 33% and 26% in MA-IOM, and 42% and 64% in CH-IOM, respectively. This
168
likely results from the dominance of internal phycobiliproteins and chloroplast in MA and CH
169
cells, respectively (Glazer and Fang, 1973; Purton, 2001). Furthermore, this results further
AC C
EP
TE D
M AN U
SC
RI PT
151
10
ACCEPTED MANUSCRIPT supports previous work where the fractions with MW >100 kDa also contributed >50% of the
171
total DOC and DON from the IOM of Microcytic aeruginosa (Guo et al., 2017; Zhou et al., 2015),
172
suggesting the significance of large molecules (MW >100 kDa) in the MW distribution of IOM.
173
3.2 Fluorescent properties
174
Probing fluorescent properties provides valuable insights into the chemical compositions of OM
175
precursors and their DBP formation potential (Chen et al., 2003; Hua et al., 2018b). Fluorescent
176
properties of the two original IOMs were firstly characterized by EEM spectroscopy to provide
177
the general fluorescent spectra of the IOM before fractionation. Distinct differences in two were
178
found, as shown in Fig. S1, Supporting information. In particular, the original MA-IOM
179
consisted of a more complex matrix of all four fluorescent components: AP, SMP, HA, and FA,
180
and accounted for 20%, 16%, 33%, and 31% of the EEM intensity, respectively. Conversely, the
181
original CH-IOM was mainly constituted of AP and SMP, which contributed up to 66% and 22%
182
of the original intensity, with negligible contributions by HA and FA. These results concurred with
183
the findings of most current reports (Henderson et al., 2008b; Hua et al., 2017; Li et al., 2012),
184
indicating that the fluorescent characteristics of IOM are species-dependent.
185
Furthermore, EEM scanning was carried out on the six fractionated IOMs of the two species, and
186
their AFI values for four regional components were calculated for quantitative comparison. As
187
showed in Fig. 2, the AFI percentages of the four regional components, AP-, SMP-, HA-, and
188
FA-like, vary significantly within the reduction of MW. Reducing MW particularly caused a shift
AC C
EP
TE D
M AN U
SC
RI PT
170
11
ACCEPTED MANUSCRIPT from the dominance of AP and SMP to the dominance of HA and FA components. Comparing the
190
six fractions, AP and SMP were most abundant in large fraction F1, accounting for >75% of the
191
total AFI of the two species. The dominance of AP and SMP decreased significantly when the
192
MW decreased to <10 kDa (in F4, F5, and F6). In contrast, HA and FA became the dominant
193
components under this MW range, accounting for around 85% and 60% of the total AFI in cases
194
of F5 and F6 of both algal species (Fig. 2a). However, these results differ from those in Liu et al.,
195
(2018) who reported that AP (tryptophan and tyrosine-like) and SMP were the dominant
196
components in the small MW fraction of <5 kDa of the IOM derived from Microcytic aeruginosa.
197
This can possibly be attributed to the differences in the IOM extraction protocol between the two
198
studies. Nevertheless, there requires practical evidence to clarify the effects of extraction methods
199
on IOM properties. In brief, our EEM AFI results show a strong relationship between MW
200
variations and fluorescent properties of IOM in which large MW (>100 kDa) of IOM mainly
201
exhibits AP- and SMP-like fluorescence, whereas small MW (>10 kDa) of IOM is abundant in
202
HA- and FA-fluorescence.
203
3.3 THM and HAA formation potential
204
DBP formation tests were carried out for six MW fractions and the original IOM of MA (Fig. 3a)
205
and CH (Fig. 3b). As no bromide was included in the DBP tests, brominated-DBPs were
206
negligibly detected in this study. Trichloromethane (TCM) was the major THM observed (>98%
207
of total THM), while monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), and
AC C
EP
TE D
M AN U
SC
RI PT
189
12
ACCEPTED MANUSCRIPT trichloroacetic acid (TCAA) were the three dominantly obtained compounds in HAAs (>99% of
209
total HAAs). The results of formation tests show that the C-DBPs of the two species rapidly
210
reduced with the decrease of IOM MW from F1 to F6. The impact of MW reduction on
211
IOM-DBP yields was statistically more pronounced in HAAFP and C-DBPFP than in THMFP.
212
The largest amounts of C-DBPs of the six IOM fractions was found in the large MW fractions (F1
213
>100 kDa) of both species, which significantly were three-fold larger than those derived from the
214
smallest MW (F6). Furthermore, the total C-DBP produced by F1 was even larger than that of the
215
corresponding original IOM. These results indicate that the large molecules of IOM with MW
216
>100 kDa are the most important precursors of IOM-derived DBPs, and that IOM-derived DBPs
217
are strongly MW dependent.
218
Fig. 3 also shows a shift from a dominance of HAAs to a dominance of THMs with the decrease
219
of IOM MW. In particular, when the MW was <10 kDa (F4, F5, and F6), the dominance of THM
220
formation became more significant. The ratios of HAAFP/THMFP decreased from 2.5 to >1 with
221
the decrease of IOM-MW. This suggests that small molecules of IOM with MW <10 kDa are
222
likely favorable precursors to the formation of THMs, rather than HAAs.
223
By normalizing the DOC contributions in Fig. 1a, the distribution of each fractionated MW of
224
IOM to THMFP, HAAFP, and C-DBPFP was calculated, as shown in Fig. S2. As the F1 fractions
225
of the two species comprised the largest contribution to the total DOC associated with their
AC C
EP
TE D
M AN U
SC
RI PT
208
13
ACCEPTED MANUSCRIPT greatest DBP formation potential, F1 thus contributed the largest proportion to the formation of
227
THMs, HAAs, and C-DBPs among six MW fractions, accounting for 34–53%, 70–75%, and 54–
228
65%, respectively. Although small fractions with MW <10 kDa (F4, F5, and F6) also had high
229
contribution of the total DOC, of about 50–60% (Fig. 1a), their final contribution to DBP
230
formation was relatively insignificant (<20%) (Fig. S2) because they possessed low specific
231
DBPFPs. The explanation for the IOM-derived DBPs varying within MW will be discussed
232
below.
233
3.4 Functionality
234
XPS analysis was carried out for IOM fractions F1 (>100 kDa) and F6 (<1 kDa), as they were
235
extremely different in terms of MW, fluorescence and DBP formation potential. Their chemical
236
compositions and functional structures were used to further interpret the formation of
237
IOM-derived C-DBPs. Three essential elements of OM, C, O, and N, were investigated. Table 1
238
lists the contributions of each element and their relative ratios in the chemical structures of the F1
239
and F6 fractions. The percentages and ratios of the three elements were comparable for F1
240
fractions of the two species, while they were very different for F6 of both IOM. In particular, two
241
F1 were similarly constituted with high carbon content, which was relatively twice and
242
twelve-fold higher than that of the oxygen and nitrogen content, respectively (as shown in Table
243
1). Although the total amount of C and O content in fraction F6 of MA was similar to F1, F6
244
comprised only 2% of the N content. In contrast, fraction F6 of CH had a greater O content
AC C
EP
TE D
M AN U
SC
RI PT
226
14
ACCEPTED MANUSCRIPT (>50%) with a comparable amount of N, but significantly less C compared to its corresponding
246
F1.
247
Detail scanning for core-level spectra of each C1s, O1s, and N1s was further carried out for F1
248
and F6 fractions to further understand their functionality, as shown in Fig. S3 and Table 2. After
249
resolving the overlapped chemical bonding peaks in C1s, three corresponding groups were
250
generally detected in the large fractions F1 of the two species of algae. These included
251
aliphatic/aromatic C–C/C–H (283.92 ± 0.04 eV), alcohol C–O (285.24 ± 0.10 eV), and keto C =
252
O (286.57 ± 0.05 eV) (Ting et al., 1995). These C functional groups were also found in the small
253
MW fractions of F6 associated with the additions of carboxyl O–C = O groups (291.89 ± 0.01 eV)
254
and unknown groups located at 294.62 ± 0.01 eV. As shown in Table 2, fractions F1 of the two
255
species comprised of similar and large amounts of aliphatic/aromatic groups (55–57%) with the
256
absence of carboxylic and unknown groups. Conversely, fractions F6 constituted much less
257
aliphatic/aromatic contents with a greater amount carboxylic and unknown carbon, particularly in
258
the case of CH IOM, where only 25% of carbon bonding belonged to aliphatic/aromatic
259
compounds, but >50% whereas carboxylic and unknown carbon bonds accounted for >50% of the
260
carbon bonding. The O1s results mostly correspond with the C1s findings in which carboxylic
261
groups were mainly found in the fractions F6 of both species (>40%). For N1s, both F1 fractions
262
clearly showed higher contents of N–C bond compared to F6 fractions.
AC C
EP
TE D
M AN U
SC
RI PT
245
15
ACCEPTED MANUSCRIPT The determination of the composition and function of the two distinct fractions (F1 and F6)
264
further support the results given in Fig. 2, implying that (i) the dominance of AP- and SMP-like
265
fluorescent components in large MW fractions (>100 kDa; F1) of IOM is constituted by carbon-
266
and nitrogen-rich substances with considerable sites of C–C/C–H and N–C, and (ii) the abundance
267
of HA- and FA-like fluorescence in the small MW fractions (<1 kDa; F6) can be attributed to
268
carboxylic-based molecules that associates with either less N (for MA) or more O (for CH)
269
content in their chemical structures.
270
3.5 Discussion
271
3.5.1 Variations of IOM-derived THMs and HAAs within MW
272
IOM-derived THMs and HAAs are strongly depended on MW properties. For IOM MW varying
273
from >100 kDa to <1 kDa, more significant levels of C-DBPs are formed for MW (>100 kDa),
274
but only low levels of C-DBPs are produced MW (<10 kDa). The differences in IOM-derived
275
DBPs with large and small MW are attributed to their different fluorescent compositions and
276
functional structures of the precursors. Large MW IOM is evidently constituted by AP- and
277
SMP-like components. These are carbon nitrogen-rich substances from the essential aromatic
278
proteins in algal cells, such as tryptophan, tyrosine, phenylalanine, and histidine (Fang et al.,
279
2010). Studies have reported that aromatic proteins and/or substances with AP- and SMP-like
280
components are high-yielding DBP precursors and produce substantial levels of both THMs and
281
HAAs because they have a large content of highly reactive carbons in their chemical structures for
AC C
EP
TE D
M AN U
SC
RI PT
263
16
ACCEPTED MANUSCRIPT chlorine incorporation (Hong et al., 2009; Hua et al., 2017; Hua et al., 2018b). Indeed, our results
283
show that the abundance of AP- and SMP-like components in the large MW fraction also
284
associates with the more available reactive carbon content (C–C/C–H) and electron-donating
285
functional groups (N–R2 and –OR) (Table 2). In contrast, the chemical structures of the small
286
MW fractions (<10 kDa), which are mainly presented in HA- and FA-like fluorescence, contain (1)
287
less amount of carbons, (2) a considerable number of carbonyl groups (COO–), which is
288
unfavorable for chlorine substitution, and (3) limited electron-donating functional groups. As the
289
OM has more available reactive carbon sites, the higher the degree of chlorine substitution
290
(Reckhow et al., 1990). Large MW IOM (>100 kDa) is thus a more important and higher-yielding
291
precursor for the formation of C-DBPs compared to small MW IOM (<10 kDa).
292
MW property of IOM also influences the ratio of THM and HAA formation, as shown in Fig. 3.
293
While MW fractions >10 kDa (F1, F2, and F3) preferentially follow the formation pathway of
294
HAAs, MW fractions at <10 kDa (F4, F5, and F6) tend to produce more THMs. It should be
295
noted that the THMs produced in this study were chiefly contributed by TCM, and HAAs were
296
accounted for DCAA and TCAA. During the chlorination of OM precursors, DCAA and TCAA
297
were predominantly formed when the chemical structure of intermediates was constituted by
298
readily oxidizable functional groups, i.e. carbon-carbon double bond, alcohol, and amine.
299
Otherwise, TCM is possibly formed as the final product (Hong et al., 2009; Hua et al., 2017;
300
Reckhow et al., 1990). As shown in Table 2, the functionality of small MW fractions is more
AC C
EP
TE D
M AN U
SC
RI PT
282
17
ACCEPTED MANUSCRIPT deficient in C = C, O–H, and N–C compared to large MW fractions. This results in a higher
302
tendency to undergo the formation of THMs rather than HAAs from small MW of IOM.
303
Combined with the fluorescent results, these results further support our previous findings (Hua et
304
al., 2017) that found that precursors containing AP- and SMP-like components are favorable
305
precursors of HAAs, when precursors with HA- and FA-like components tend to form THMs.
306
3.5.2 Fluorescent and MW dependence of IOM-derived THMs and HAAs
307
Our results convincingly demonstrate the dependence of IOM-derived THMs and HAAs on the
308
fluorescent and MW properties. CCA was used to explore these relationships, and Fig. 4 shows
309
the CCA ordination biplot for twelve MW fractions from two IOMs as sample sites, associated
310
with four EEM components as quantitative explanatory variables, and two DBPs as object points.
311
As can be seen in Fig.4, the CCA test separates the sample sites into two groups: group I of large
312
MW (F1; >100 kDa and F2; 100–30 kDa), and group II of small MW (F3, F4, F5, and F6; >30
313
kDa). Interestingly, it also divides THMs and HAAs closely associated with these two sample
314
groups, further confirming the dependence of THM and HAA formation on IOM MW properties.
315
Furthermore, the CCA biplot shows distinctly different directions of EEM components. While AP
316
and SMP components are closed and have a positive correlation with HAAs and two large MW
317
IOMs (group I), HA and FA components, by contrast, correlate mostly positively with THMs and
318
small MW IOMs (group II). These results are robust confirmation that IOM-derived THMs and
319
HAAs are strongly dependent on fluorescent and MW properties. Large MW IOM exhibiting AP-
AC C
EP
TE D
M AN U
SC
RI PT
301
18
ACCEPTED MANUSCRIPT and SMP-like fluorescence is favorable for the formation of HAAs, while small MW IOM with
321
HA- and FA-like fluorescence tends to form THMs. To the best of our knowledge, this study is the
322
first to elucidate the role of fluorescent and MW properties on the formation of IOM-derived
323
THMs and HAAs. Thus, the results from this study will provide useful information for the future
324
DBP research. Furthermore, since fluorescent and MW properties are common and important
325
characteristics of the water matrix, our findings imply that the control of DBPs derived from
326
algae-containing water can significantly be benefited by applying these adequate characterization
327
tools.
328
4. Conclusions
329
Our experimental and statistical results convincingly show the strong dependence of IOM-derived
330
THMs and HAAs on the fluorescent and MW properties of IOM precursors. Of the MW varying
331
from >100 kDa to <1 kDa, the large fraction of >100 kDa is the most important portion of IOM
332
precursors, which not only makes the greatest contribution to MW distribution, but also produces
333
and contributes the highest levels of THMs, HAAs, and C-DBPs. This evidently results from the
334
abundance of carbon-nitrogen rich substances in such large MW IOM, which contain a large
335
number of reactive carbon sites for chlorine substitution, and exhibits AP- and SMP-like
336
fluorescence. By contrast, although small MW fractions (>10 kDa) accounts for around half of the
337
total DOC of IOM, they are insignificant in contributing to the overall C-DBPs. Low THMs and
338
HAAs derived from small MW fractions are mainly attributed to the dominance of the carboxylic
AC C
EP
TE D
M AN U
SC
RI PT
320
19
ACCEPTED MANUSCRIPT group, and the deficiency of the conjugated system and amide content in their chemical structures.
340
The outcomes of this study provide more significant insights into the principle of IOM-derived
341
DBPs and potentially benefits the control of algae yielded DBPs in drinking water.
342
Acknowledgments
343
We greatly appreciate the assistance of Wouter Holleman for proofreading this paper.
344
References
345 346 347
Chen, W., Westerhoff, P., Leenheer, J.A. and Booksh, K., 2003. Fluorescence Excitation−Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci. Technol. 37 (24), 5701–5710.
348 349
Cheng W.P., Chi F.H. 2003. Influence of eutrophication on the coagulation efficiency in reservoir water. Chemosphere 53, 773–778.
350 351
Coble, P.G., 2007. Marine Optical Biogeochemistry: The Chemistry of Ocean Color. Chem. Rev. 107 (2), 402–418.
352
Fang, J., Yang, X., Ma, J., Shang, C. and Zhao, Q., 2010. Characterization of algal organic matter
353
and formation of DBPs from chlor (am) ination. Water Res. 44(20), 5897–5906.
354 355
Fogg, G., Nalewajko, C. and Watt, W., 1965. Extracellular products of phytoplankton photosynthesis. Proc. R. Soc. Lond. B. Biol. Sci. 162 (989), 517–534.
356 357
Glazer A.N., Fang S. 1973. Chromophore content of blue-green algal phycobiliproteins. J. Biol. Chem. 248, 659–662.
358 359 360
Goslan E.H., Seigle C., Purcell D., Henderson R., Parsons S.A., Jefferson B., 2017. Carbonaceous and nitrogenous disinfection by-product formation from algal organic matter. Chemosphere, 170, 1– 9.
361 362
Guo T., Yang Y., Liu R., Li X. 2017. Enhanced removal of intracellular organic matters (IOM) from Microcystic aeruginosa by aluminum coagulation. Sep.Purif. Technol. 189, 279–287.
363 364
Henderson, R., Parsons, S.A. and Jefferson, B., 2008a. The impact of algal properties and pre-oxidation on solid–liquid separation of algae. Water Res. 42(8–9), 1827–1845.
365 366
Henderson, R.K., Baker, A., Parsons, S.A. and Jefferson, B., 2008b. Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res. 42(13), 3435–
AC C
EP
TE D
M AN U
SC
RI PT
339
20
ACCEPTED MANUSCRIPT 367
3445.
368 369
Henderson, R.K., Parsons, S.A. and Jefferson, B., 2010. The impact of differing cell and algogenic organic
370 371 372
Hong H.C., Mazumder A., Wong M.H., Liang Y. 2008. Yield of trihalomethanes and haloacetic acids upon chlorinating algal cells, and its prediction via algal cellular biochemical composition. Water Res. 42, 4941–4948.
373 374
Hong H.C., Wong M.H., Liang Y. 2009. Amino Acids as Precursors of Trihalomethane and Haloacetic Acid Formation During Chlorination. Arch. Environ. Contam. Toxicol. 56, 638–645.
375
Hua, G. and Reckhow, D.A., 2007. Characterization of Disinfection Byproduct Precursors Based on
376
Hydrophobicity and Molecular Size. Environ. Sci. Technol. 41 (9), 3309–3315.
377 378
Hua, L.C., Lin, J.L., Chen, P.C. and Huang, C.P., 2017. Chemical structures of extra- and intra-cellular
379
Eng. J. 328, 1022–1030.
380 381
Hua, L.C., Lin, J.L., Zhao, S.J., and Huang, C.P., 2018a. Probing algogenic organic matter (AOM) by
382
Environ. 645: 71–78
383 384 385
Hua, L.C., Lin, J.L., Syue, M.Y., Huang, C.P. and Chen, P.C., 2018b. Optical properties of algogenic organic
386 387 388
Huang, J., Graham, N., Templeton, M.R., Zhang, Y., Collins, C. and Nieuwenhuijsen, M., 2009. A comparison of the role of two blue–green algae in THM and HAA formation. Water Res. 43(12), 3009–3018.
389 390
Li, L., Gao, N., Deng, Y., Yao, J. and Zhang, K., 2012. Characterization of intracellular & extracellular
391
byproducts and odor & taste compounds. Water Res. 46(4), 1233–1240.
392 393 394
Liu R., Guo T., Ma M., Yan M., Qi J., Hu C., 2018. Preferential binding between intracellular organic matters and Al13 polymer to enhance coagulation performance. J. Environ. Sci. (In press). https://doi.org/10.1016/j.jes.2018.05.011.
395
Lui Y.S., Hong H.C., Zheng G.J.S., Liang Y. 2012. Fractionated algal organic materials as
396 397
precursors of disinfection by-products and mutagens upon chlorination. J. Hazard. Mater. 209–210, 278–284. 21
M AN U
SC
RI PT
matter (AOM) characteristics on the coagulation and flotation of algae. Water Res. 44 (12), 3617–3624.
algogenic organic matters as precursors to the formation of carbonaceous disinfection byproducts. Chem.
TE D
size-exclusion chromatography to predict AOM-derived disinfection by-product formation. Sci. Total.
matter within the growth period of Chlorella sp. and predicting their disinfection by-product formation. Sci.
AC C
EP
Total. Environ. 621, 1467–1474.
algae organic matters (AOM) of Microcystic aeruginosa and formation of AOM-associated disinfection
ACCEPTED MANUSCRIPT Lui Y.S., Qiu J.W., Zhang Y.L., Wong M.H., Liang Y. 2011. Algal-derived organic matter as precursors of disinfection by-products and mutagens upon chlorination. Water Res. 45, 1454–1462.
400 401
Ndlela L.L., Oberholster P.J., Van W.J.H., Cheng P.H. 2016. An overview of cyanobacterial bloom occurrences and research in Africa over the last decade. Harmful Algae 60, 11–26.
402
Pivokonsky, M., Safarikova, J., Baresova, M., Pivokonska, L. and Kopecka, I., 2014. A comparison of the
403 404
character of algal extracellular versus cellular organic matter produced by cyanobacterium, diatom and green
405 406
Plummer, J.D. and Edzwald, J.K., 2001. Effect of Ozone on Algae as Precursors for Trihalomethane and
407
Purton S. 2001. Algal Chloroplasts. e LS. https://doi.org/10.1038/npg.els.0000316.
408 409
Reckhow, D.A., Singer, P.C. and Malcolm, R.L., 1990. Chlorination of humic materials: byproduct formation
410 411
Takaara T., Sano D., Konno H., Omura T. 20017. Cellular proteins of Microcystis aeruginosa inhibiting coagulation with polyaluminum chloride. Water Res. 41, 1653–1658.
412 413
Tas B., Gonulol A., 2007. An ecologic and taxonomic study on phytoplankton of a shallow lake, Turkey. J.Environ. Biol. 28, 439–445.
414
Ting Y.P., Teo W.K., Soh C.Y., 1995. Gold uptake by Chlorella vulgaris. J. Appl. Phycol. 7, 97–100.
415 416
Tomlinson, A., Drikas, M. and Brookes, J.D., 2016. The role of phytoplankton as pre-cursors for disinfection by-product formation upon chlorination. Water Res. 102, 229–240.
417 418
Wert, E.C. and Rosario-Ortiz, F.L., 2013. Intracellular Organic Matter from Cyanobacteria as a Precursor for
419 420
Zhou, S., Zhu, S., Shao, Y. and Gao, N., 2015. Characteristics of C-, N-DBPs formation from algal organic
421 422
Zhu, M., Gao, N., Chu, W., Zhou, S., Zhang, Z., Xu, Y. and Dai, Q., 2015. Impact of pre-ozonation on
423
Microcystis aeruginosa. Ecotoxicol. Environ. Saf. 120, 256–262.
alga. Water Res. 51 (0), 37–46.
M AN U
SC
Haloacetic Acid Production. Environ. Sci. Technol. 35(18), 3661–3668.
RI PT
398 399
EP
TE D
and chemical interpretations. Environ. Sci. Technol. 24 (11), 1655–1664.
AC C
Carbonaceous and Nitrogenous Disinfection Byproducts. Environ. Sci. Technol. 47 (12), 6332–6340.
matter: Role of molecular weight fractions and impacts of pre-ozonation. Water Res. 72, 381–390.
disinfection by-product formation and speciation from chlor(am)ination of algal organic matter of
22
ACCEPTED MANUSCRIPT
Tables
N% 5.5 2.0 5.5 4.1
O% 25.5 29.3 27.7 50.2
12.6 34.0 12.3 11.3
SC
C% MA-IOM F1 >100 kDa 69.0 F6 <1 kDa 68.7 CH-IOM F1 >100 kDa 66.8 F6 <1 kDa 45.7 Note: Sum of C%, N%, and O% was 100%.
RI PT
Table 1 Contribution of C, O, and N elements in the large MW fraction F1 and small MW F6 from the IOM of M. aeruginosa (MA) and Chlorella sp. (CH), and their relative ratios. C/N C/O Origin MW Fraction Element percentage (%) 2.7 2.3 2.4 0.9
AC C
EP
TE D
M AN U
Table 2 Chemical functional groups of C, O, and N for the fractionated F1 and F6 of the IOMs from M. aeruginosa (MA) and Chlorella sp. (CH). Functional Binding Contribution of each functional group (%) group energy (eV) MA CH F1 (>100 kDa) F6 (<1 kDa) F1 (>100 kDa) F6 (<1 kDa) C1s functional groups C-C/C-H 283.92 ± 0.04 57.3 57.5 54.9 24.9 C-O 285.24 ± 0.10 29.1 14.1 27.4 13.0 C=O 286.57 ± 0.05 13.6 8.2 17.7 6.7 O-C=O 291.89 ± 0.01 14.0 36.4 Unknown 294.62 ± 0.01 6.2 19.0 O1s functional groups C=O 530.10 ± 0.51 38.5 26.8 27.0 23.4 O-C/O-H 530.97 ± 0.37 53.2 32.8 42.0 35.0 O-C=O 532.05 ± 0.52 8.4 27.9 31.1 34.6 O-C=O* 534.42 ± 0.23 12.6 7.0 N1s functional groups N-C 398.86 ± 0.26 97.0 87.1 93.1 87.1 N-H 401.48 ± 0.97 3.0 12.9 6.9 12.9 Note: O-C=O* chemical shift of carboxylic group.
ACCEPTED MANUSCRIPT
(a)
(b)
<1 3-1 10-3 30-10 100-30 >100
60
20
0
MA
CH
MA
80
60
40
20
0
CH
EP
TE D
Fig. 1 Distributions of each MW fraction in (a) DOC and (b) DON of IOM derived from M. aeruginosa (MA) and Chlorella sp. (CH). MW unit is in kDa
AC C
2 3 4
M AN U
40
RI PT
80
100
SC
100
Distribution in DON (%)
Figures
Distribution in DOC (%)
1
ACCEPTED MANUSCRIPT 100
(a)
RI PT
80
60
40
SC
AFI percentage (%)
(b)
AP SMP HA FA
0 Original >100 100-30 30-10
<1
Original >100 100-30 30-10
10-3
1-3
<1
Apparent MW fraction (kDa)
TE D
Fig. 2 Variations of calculated regional AFI percentages within the MW of (a) MA-IOM and (b) CH-IOM (DOC = 10 mg/L; pH 7).
EP
6 7
1-3
Apparent MW fraction (kDa)
AC C
5
10-3
M AN U
20
ACCEPTED MANUSCRIPT 160
THM HAA C-DBP
120 100 80 60 40 20
> 100
30-10
10-3
3-1
<1
Original
THM HAA C-DBP
120 100 80 60 40 20
> 100
100-30
30-10
10-3
3-1
<1
Original
Apparent MW faction (kDa)
Apparent MW faction (kDa)
SC
Fig. 3 Variations of THMFP, HAAFP, and C-DBPFP derived from each MW fraction and the
TE D
M AN U
original (a) MA-IOM and (b) CH-IOM. Chlorination conditions: DOC ≈ 5 mg/L, Cl2:DOC = 5:1, pH 7, 7 days, 25oC.
EP
10 11
100-30
AC C
9
(b) CH IOM
0
0
8
140
RI PT
140
(a) MA IOM
Specific C-DBPFP (µ µ g/ mg-C)
Specific C-DBPFP (µ µ g/ mg-C)
160
ACCEPTED MANUSCRIPT
C-DBPs
MW fractions
EEM components MA-IOM_F3
Group I
0.1
RI PT
0.2
CH-IOM_F3 CH-IOM_F2
SC
MA-IOM_F1
0
MA-IOM_F2 CH-IOM_F1
-0.1
SMP AP
MA-IOM_F4 MA-IOM_F5
THMs MA-IOM_F6
FA HA
TE D
-0.2
CH-IOM_F5 CH-IOM_F6
CH-IOM_F4
M AN U
CCA2
HAAs
Group II
-0.4 -1
-0.8
-0.6
AC C
-1.2
EP
-0.3
-0.4
-0.2
0
0.2
0.4
0.6
0.8
CCA1
Fig. 4 CCA ordination biplot showing the relationships among fluorescent EEM components (AP, SMP, HA, FA) as explanatory variables, MW fractions of two IOM as sample sites (12 samples), and THMs as well as HAAs as response variables
1
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
IOM-derived THMs and HAAs from M. aeruginosa and Chlorella sp. were examined. Roles of fluorescent and MW properties of IOM on its THMs and HAAs formation were examined. IOM-derived THMs and HAAs strongly depend on fluorescent and MW properties.
AC C
• • •
S0043-1354(18)30848-0
DOI:
https://doi.org/10.1016/j.watres.2018.10.051
Reference:
WR 14162
To appear in:
Water Research
Received Date: 25 August 2018 Revised Date:
17 October 2018
Accepted Date: 20 October 2018
Please cite this article as: Hua, L.-C., Chao, S.-J., Huang, C., Fluorescent and molecular weight dependence of THM and HAA formation from intracellular algogenic organic matter (IOM), Water Research (2018), doi: https://doi.org/10.1016/j.watres.2018.10.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Fluorescent and molecular weight dependence of THM and HAA
SC
RI PT
formation from intracellular algogenic organic matter (IOM)
M AN U
Lap-Cuong Hua, Shu-Ju Chao, Chihpin Huang*
EP
TE D
Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwan
AC C
Submitted to Water Research
*Corresponding author: Prof. Chihpin Huang TEL: +886-3-5712121-55507, FAX: +886-3-5725958 Email: [email protected] 1
ACCEPTED MANUSCRIPT Abstract
2
This study (i) examined the formation of two major carbonaceous disinfection by-products
3
(C-DBPs), trihalomethanes (THMs) and haloacetic acids (HAAs), during the chlorination of
4
intracellular algogenic organic matter (IOM) extracted from two commonly blooming algae M.
5
aeruginosa (MA) and Chlorella sp. (CH), and (ii) investigated the roles and relationships of
6
fluorescent and molecular weight (MW) properties on/with IOM-derived THMs and HAAs. The
7
extracted IOM samples were separated into different MW fractions by centrifugal devices with
8
membrane support with MW cut-offs of 100, 30, 10, 3, and 1 kDa. We observed an overall
9
reduction of C-DBPs with a decrease of IOM-MW from >100 kDa to <1 kDa. Of six fractionated
10
IOM, a large fraction (>100 kDa) contributed the largest amount to the MW distribution of IOM,
11
accounting for 33 and 42% of the total dissolved organic carbon (DOC) of MA and CH, respectively.
12
It also had the highest-yielding potential to produce significant levels of THMs and HAAs, and total
13
C-DBPs over other small MW fractions. Although small MW fractions (>10 kDa) contributed
14
around 50% of the total DOC, they made an insignificant contribution (>20%) to the THMs, HAAs,
15
and overall C-DBPs. Furthermore, the decrease of IOM MW caused a shift from the domination of
16
HAA formation to THM formation, especially when MW was <10 kDa. By canonical correspondent
17
analysis, the relationship of IOM-derived THMs and HAAs with IOM properties was examined. In
18
particular, large fractions of IOM, exhibiting aromatic protein- (AP) and soluble microbial product-
19
(SMP) like fluorescence, are favorable for the formation of HAAs, whereas small MW fractions of
AC C
EP
TE D
M AN U
SC
RI PT
1
2
ACCEPTED MANUSCRIPT IOM with HA- and FA-like fluorescence preferentially tends to form THMs. Our findings evidently
21
show the strong dependence of IOM-derived THMs and HAAs on the fluorescent and MW
22
properties. Therefore, the characterization of MW and fluorescent properties can provide the
23
advantages in the control of algae-derived DBPs upon the chlorination of eutrophic water.
24
Keywords: algogenic organic matter; DBPs; chlorination; fluorescence; molecular weight
25
1. Introduction
26
Bloom of algae in an aquatic environment is a critical problem in water treatment utilities. It
27
frequently causes a dramatic deterioration of water quality by increasing turbidity, taste and odor,
28
and algal toxins in the water column (Henderson et al., 2008a; Tomlinson et al., 2016).
29
Importantly, algal bloom releases a significant content of algogenic organic matter (AOM) that
30
has been proved to be a major precursor of disinfection by-products (DBPs) upon
31
chlor(am)ination or ozonation (Fang et al., 2010; Goslan et al., 2017; Hua et al., 2018a; Huang et
32
al., 2009; Plummer and Edzwald, 2001; Zhu et al., 2015). AOM, as a precursor to DBPs, can be
33
generally categorized into extra- (EOM) and intra-cellular organic matter (IOM). While EOM is
34
rapidly released via algal exudation in soluble organic form (Fogg et al., 1965), IOM is the
35
internal organic substances released by the rupture of the cell membrane (Tomlinson et al., 2016).
36
Nonetheless,
37
coagulation/sedimentation/filtration processes (Cheng and Chi, 2003; Henderson et al., 2010;
38
Takaara et al., 2007), and controlling AOM-derived DBPs during the algal bloom has thus become
AC C
EP
TE D
M AN U
SC
RI PT
20
they
both
are
difficult
to
effectively
3
be
removed
by
conventional
ACCEPTED MANUSCRIPT the greatest challenge for sustainable production of safe drinking water.
40
A better understanding of AOM physicochemical properties significantly benefits an effective
41
control of its derived DBPs. In general, probing fluorophores provide valuable information about
42
the possible composition and relatively quantitative content of AOM precursor (Chen et al., 2003;
43
Coble, 2007). The use of fluorescent excitation-emission matrices (EEM) as a characterization
44
tool further allows us to acquire more insights into AOM-derived DBPs (Fang et al., 2010; Hua et
45
al., 2018b; Li et al., 2012). On the other hand, molecular weight (MW) is another important
46
characteristic that also shows significant connection to the effectiveness of AOM treatment (Guo
47
et al., 2017; Liu et al., 2018) and its derived DBPs in particular (Hua et al., 2018a; Lui et al., 2012;
48
Pivokonsky et al., 2014; Zhou et al., 2015). As the common and effective approaches to trace
49
DBP formation, the characterization of AOM-DBP precursors based on fluorescent and MW
50
properties has attracted more and more attention. However, current research into AOM-DBP has
51
as yet not fully resolved such relationships, because AOM is a complex and heterogeneous
52
mixture varying in size, structure, and functionality (Fang et al., 2010; Hong et al., 2008; Hua et
53
al., 2017; Li et al., 2012; Lui et al., 2012). There is a critical need to elucidate the principal role of
54
fluorescent and MW properties and their relationship with the formation of AOM-derived DBPs,
55
where the existent knowledge has remained unclear.
56
To address this issue, heterogeneous AOM can be isolated into more homogeneous groups based
57
on molecular size. Fractionation by a serial ultrafiltration technique is commonly used to isolate
AC C
EP
TE D
M AN U
SC
RI PT
39
4
ACCEPTED MANUSCRIPT OM sample into different MW fractions (Henderson et al., 2008b; Hua and Reckhow, 2007;
59
Pivokonsky et al., 2014; Zhu et al., 2015). This study was aimed at characterizing the fractionated
60
AOM precursors based on MW, as a function of fluorescence, and to examine the relationship
61
between AOM fluorescent and MW properties with its derived DBP formation upon chlorination.
62
IOM-derived DBP was the central focus of this research because, in practice, IOM (i) contributes
63
a significant fraction to total AOM (>80%) (Tomlinson et al., 2016), (ii) is a more difficult
64
matter to be removed by conventional treatments (Pivokonsky et al., 2014; Takaara et al., 2007),
65
and (iii) importantly is a higher-yielding DBP precursor compared to EOM (Hua et al., 2018b;
66
Huang et al., 2009; Li et al., 2012; Plummer and Edzwald, 2001). The IOM was extracted from a
67
cyanobacterium, M. aeruginosa (MA), and a green alga, Chlorella sp. (CH). MA and CH were
68
selected because they are two commonly blooming algae that occur worldwide (Ndlela et al.,
69
2016; Tas and Gonulol, 2007). Furthermore, two major classes of carbonaceous DBPs, including
70
trihalomethanes (THMs) and haloacetic acids (HAAs), were investigated, as they are dominantly
71
found upon chlorination of IOM, compared to other DBPs, such as chloroacetones,
72
chloroacetonitriles, nitrosodimethylamine and chloronitromethanes (Li et al., 2012; Lui et al.,
73
2011; Wert and Rosario-Ortiz, 2013), and are regulated worldwide (Tomlinson et al., 2016).
74
2. Materials and methods
75
2.1 Algal cultivation and IOM extraction
76
M. aeruginosa and Chlorella sp. were isolated from a local reservoir in Matsu, Taiwan. They were
AC C
EP
TE D
M AN U
SC
RI PT
58
5
ACCEPTED MANUSCRIPT isolated into the unialgal culture by a commonly used protocol with agar plate streaking technique
78
and successive dilution method. Their axenic cultures were conducted in a thermostat
79
photobioreactor containing 6 L of modified BG11 and Chlorella medium. The conditions of algal
80
culture were followed our previous work (Hua et al., 2018b). Algal growth phases were
81
determined by monitoring the cell population every 2 days.
82
The algal suspensions with the volume of 500 mL from the late exponential phase of the two
83
species were selected for IOM extraction. Algal cells were separated from EOM by centrifugation
84
for 15 min, at 2700 G, at 4oC (Boeco Centrifuges U-320R, Germany) and then washed twice with
85
deionized (DI) water to remove the remaining EOM. The collected cells were lyophilized in a
86
freeze-drier (Pin-Chen Co., Taiwan) at –50°C and 2 × 10–4 Torr for 6 h. IOM was extracted
87
physically by gently grinding the lyophilized cells for 2 min with a mortar and pestle. After
88
grinding, IOM was extracted by re-suspending the ruptured cells in 100 mL of DI water and
89
shaking vigorously for 30 s using a vortex mixer. This suspension was then spun in a centrifuge
90
again, for 15 min, at 2700 G, at 4oC, prior to filtration through a 0.45 µm filter (Advantec, Japan)
91
to archive the IOM.
92
2.2 Molecular weight fractionation
93
The IOM water samples of the two species were separated into different MW fractions using a
94
series of centrifugal devices with membrane support (Macrosep® Advance Centrifugal Device,
95
Pall, USA), with MW cut-offs (MWCO) of 100, 30, 10, 3, and 1 kDa. Before using for IOM
AC C
EP
TE D
M AN U
SC
RI PT
77
6
ACCEPTED MANUSCRIPT isolation, each centrifugal device was washed twice by DI water at the identical centrifugation
97
conditions with samples. It is noticed that such centrifugation conditions could recover 95% of the
98
blank (DI water) for all MWCO. 20 mL of concentrated IOM samples (DOC ≈ 100 mg/L) was
99
loaded into the top of the centrifugal devices and spun at 2700 G for 30 min at 4oC. After the
100
serial fractionation, IOM samples were separated into six fractions with the MW ranges of F1
101
(>100 kDa), F2 (100–30 kDa), F3 (30–10 kDa), F4 (10–3 kDa), F5 (3–1 kDa), F6 (<1 kDa). This
102
process was duplicated, and the duplicates were used for other analyses and DBP formation tests.
103
All water samples were adjusted to pH 7 and stored at –20oC before use.
104
2.3 DOC and DON measurements
105
Dissolved organic carbon (DOC) concentration was determined using a TOC analyzer (TOC-L,
106
Shimadzu). Dissolved organic nitrogen (DON) content was measured with a DR/6000U
107
spectrophotometer (HACH Company, US). The DON content is the difference between the total
108
nitrogenous content (TNT Persulfate Digestion 10071 Method) and the total inorganic
109
nitrogenous content (Titanium Trichloride Reduction 10021 Method). All DOC and DON
110
measurements were conducted in duplicate for each MW fraction.
111
2.4 EEM spectra
112
Fluorescent spectra (or EEM spectra) of the original and fractionated IOM samples were analyzed
113
with a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA, USA). Before
114
EEM analysis, water samples were adjusted to a DOC of 10 mg/L for comparison. EEM scanning
AC C
EP
TE D
M AN U
SC
RI PT
96
7
ACCEPTED MANUSCRIPT was carried out with excitation wavelengths ranged from 200 to 400 nm with 10 nm increments
116
and emission wavelengths ranging from 250 to 550 nm with 2 nm increments. The voltage of the
117
photomultiplier tube was set to 800 V for low light detection. DI water with DOC <0.2 mg/L was
118
used as a blank. The raw EEM intensities of each sample were subtracted for the blank (DI water)
119
and Rayleigh and Raman scattering to remove the background and interference. Four regional
120
fluorescent spectra were determined based on their excitation/emission wavelengths following
121
Chen et al., (2003). These represent the aromatic protein- (AP), soluble microbial product- (SMP),
122
humic- (HA), and fulvic-like (FA) substances. The average fluorescent intensity (AFI) of each
123
component was calculated based on the average intensity value of each selected region
124
respectively.
125
2.5 Characterization of chemical functional structure
126
The chemical functional groups of the fractionated IOM were analyzed with a High-Resolution
127
X-ray Photoelectron Spectrometer (XPS) (Physical Electronics, PHI 1600 spectrometer). Prior to
128
chemical analysis, samples were prepared by freeze-drying pretreatment for IOM solutions. XPS
129
survey was conducted by coupling with an Mg Kα X-ray source (1253.6 eV). Low-resolution
130
scanning was carried out from 0 to 1400 eV with a pass energy of 117.4 eV for elemental
131
determination. High-resolution surveys with pass energy set at 23.5 eV were also conducted for
132
the C, N, and O elements over the 278–298 eV, 391–411 eV, and 523–543 eV ranges, respectively.
133
The spectral deconvolution was performed by XPS Peak Fit program with the subtraction of
AC C
EP
TE D
M AN U
SC
RI PT
115
8
ACCEPTED MANUSCRIPT Shirley background and was optimized to achieve the R2 > 0.99 for C and O, and R2 > 0.90 for N.
135
2.6 DBP formation potential tests
136
For DBP formation potential (DBPFP) tests, water samples of different fractions (F1 to F6) and
137
original IOM were adjusted to DOC ≈ 5 mg/L and pH 7 ± 0.1. The tests were carried out by
138
spiking each sample with sodium hypochlorite (Cl2:DOC = 5:1) in headspace-free amber glass
139
bottles at room temperature (25 ± 1oC). After 7 days of chlorination, water samples were acidified
140
and quenched with ammonium chloride to obtain the THMFP and HAAFP. The THMFP were
141
measured by a coupling a purge and trap system (Model 4660, OI Analytical, Texas, USA) and a
142
gas chromatograph-mass spectrometer (GC-MS) (Agilent 6890 GC/5973 MSD) with a RTX-VOC
143
capillary column (60 m × 0.32 mm ID, 1.5 µm film thickness), following the United States
144
Environmental Protection Agency (US EPA) method 551.1. The HAAFP were measured by a gas
145
chromatography-electron capture detector (GC/ECD) (Agilent 6890 GC/micro ECD) with a
146
DB-1701 column (30 m × 0.25 mm ID, 0.25 µm film thickness), following US EPA method 552.3.
147
THMFP and HAAFP were measured in duplicate with a standard deviation of less than 10%. In
148
this study, the total of THMFP and HAAFP was considered as carbonaceous DBPFP (C-DBPFP),
149
and the ratio of HAAFP/THMFP was calculated by dividing the amount of HAAFP to THMFP.
150
All samples were prepared with bromide-free water.
AC C
EP
TE D
M AN U
SC
RI PT
134
9
ACCEPTED MANUSCRIPT 2.7 Statistical analysis
152
SPSS Statistics 22.0 software (IBM, Armok, NY, USA) was used to compare the differences of
153
THMFP, HAAFP, and C-DBPFP. Canonical correspondent analysis (CCA) was carried out by
154
XLSTAT software (Anddinsoft, Paris, French) to examine the relationship among IOM-derived
155
DBP, MW, and fluorescent properties. Data from six MW fractions of two IOM (12 sample sites)
156
were applied for this test.
157
3. Results and Discussion
158
3.1 MW distributions by DOC and DON
159
Distribution of MW in IOM was characterized by the contribution of each fraction to the total
160
DOC and DON of two species, as shown in Fig. 1. Both MA- and CH-IOM had bimodal
161
distributions in which large MW (>100 kDa; F1) and small MW fractions (<10 kDa; F4, F5, and
162
F6) mostly contributed up to >90% of the total DOC, together with insignificant contributions of
163
fractions F2 and F3 (Fig. 2a). While the total DON of MA showed a similar distribution with the
164
DOC, only a large MW fraction F1 was found abundantly in CH-DON (Fig. 2b). Briefly, the
165
distributions of six fractions in IOM followed the order as F1 >F4≈F5≈F6 >F2≈F3, and fraction
166
F1 evidently made the greatest contributions to the total DOC and DON profiles of all IOM,
167
accounting for 33% and 26% in MA-IOM, and 42% and 64% in CH-IOM, respectively. This
168
likely results from the dominance of internal phycobiliproteins and chloroplast in MA and CH
169
cells, respectively (Glazer and Fang, 1973; Purton, 2001). Furthermore, this results further
AC C
EP
TE D
M AN U
SC
RI PT
151
10
ACCEPTED MANUSCRIPT supports previous work where the fractions with MW >100 kDa also contributed >50% of the
171
total DOC and DON from the IOM of Microcytic aeruginosa (Guo et al., 2017; Zhou et al., 2015),
172
suggesting the significance of large molecules (MW >100 kDa) in the MW distribution of IOM.
173
3.2 Fluorescent properties
174
Probing fluorescent properties provides valuable insights into the chemical compositions of OM
175
precursors and their DBP formation potential (Chen et al., 2003; Hua et al., 2018b). Fluorescent
176
properties of the two original IOMs were firstly characterized by EEM spectroscopy to provide
177
the general fluorescent spectra of the IOM before fractionation. Distinct differences in two were
178
found, as shown in Fig. S1, Supporting information. In particular, the original MA-IOM
179
consisted of a more complex matrix of all four fluorescent components: AP, SMP, HA, and FA,
180
and accounted for 20%, 16%, 33%, and 31% of the EEM intensity, respectively. Conversely, the
181
original CH-IOM was mainly constituted of AP and SMP, which contributed up to 66% and 22%
182
of the original intensity, with negligible contributions by HA and FA. These results concurred with
183
the findings of most current reports (Henderson et al., 2008b; Hua et al., 2017; Li et al., 2012),
184
indicating that the fluorescent characteristics of IOM are species-dependent.
185
Furthermore, EEM scanning was carried out on the six fractionated IOMs of the two species, and
186
their AFI values for four regional components were calculated for quantitative comparison. As
187
showed in Fig. 2, the AFI percentages of the four regional components, AP-, SMP-, HA-, and
188
FA-like, vary significantly within the reduction of MW. Reducing MW particularly caused a shift
AC C
EP
TE D
M AN U
SC
RI PT
170
11
ACCEPTED MANUSCRIPT from the dominance of AP and SMP to the dominance of HA and FA components. Comparing the
190
six fractions, AP and SMP were most abundant in large fraction F1, accounting for >75% of the
191
total AFI of the two species. The dominance of AP and SMP decreased significantly when the
192
MW decreased to <10 kDa (in F4, F5, and F6). In contrast, HA and FA became the dominant
193
components under this MW range, accounting for around 85% and 60% of the total AFI in cases
194
of F5 and F6 of both algal species (Fig. 2a). However, these results differ from those in Liu et al.,
195
(2018) who reported that AP (tryptophan and tyrosine-like) and SMP were the dominant
196
components in the small MW fraction of <5 kDa of the IOM derived from Microcytic aeruginosa.
197
This can possibly be attributed to the differences in the IOM extraction protocol between the two
198
studies. Nevertheless, there requires practical evidence to clarify the effects of extraction methods
199
on IOM properties. In brief, our EEM AFI results show a strong relationship between MW
200
variations and fluorescent properties of IOM in which large MW (>100 kDa) of IOM mainly
201
exhibits AP- and SMP-like fluorescence, whereas small MW (>10 kDa) of IOM is abundant in
202
HA- and FA-fluorescence.
203
3.3 THM and HAA formation potential
204
DBP formation tests were carried out for six MW fractions and the original IOM of MA (Fig. 3a)
205
and CH (Fig. 3b). As no bromide was included in the DBP tests, brominated-DBPs were
206
negligibly detected in this study. Trichloromethane (TCM) was the major THM observed (>98%
207
of total THM), while monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), and
AC C
EP
TE D
M AN U
SC
RI PT
189
12
ACCEPTED MANUSCRIPT trichloroacetic acid (TCAA) were the three dominantly obtained compounds in HAAs (>99% of
209
total HAAs). The results of formation tests show that the C-DBPs of the two species rapidly
210
reduced with the decrease of IOM MW from F1 to F6. The impact of MW reduction on
211
IOM-DBP yields was statistically more pronounced in HAAFP and C-DBPFP than in THMFP.
212
The largest amounts of C-DBPs of the six IOM fractions was found in the large MW fractions (F1
213
>100 kDa) of both species, which significantly were three-fold larger than those derived from the
214
smallest MW (F6). Furthermore, the total C-DBP produced by F1 was even larger than that of the
215
corresponding original IOM. These results indicate that the large molecules of IOM with MW
216
>100 kDa are the most important precursors of IOM-derived DBPs, and that IOM-derived DBPs
217
are strongly MW dependent.
218
Fig. 3 also shows a shift from a dominance of HAAs to a dominance of THMs with the decrease
219
of IOM MW. In particular, when the MW was <10 kDa (F4, F5, and F6), the dominance of THM
220
formation became more significant. The ratios of HAAFP/THMFP decreased from 2.5 to >1 with
221
the decrease of IOM-MW. This suggests that small molecules of IOM with MW <10 kDa are
222
likely favorable precursors to the formation of THMs, rather than HAAs.
223
By normalizing the DOC contributions in Fig. 1a, the distribution of each fractionated MW of
224
IOM to THMFP, HAAFP, and C-DBPFP was calculated, as shown in Fig. S2. As the F1 fractions
225
of the two species comprised the largest contribution to the total DOC associated with their
AC C
EP
TE D
M AN U
SC
RI PT
208
13
ACCEPTED MANUSCRIPT greatest DBP formation potential, F1 thus contributed the largest proportion to the formation of
227
THMs, HAAs, and C-DBPs among six MW fractions, accounting for 34–53%, 70–75%, and 54–
228
65%, respectively. Although small fractions with MW <10 kDa (F4, F5, and F6) also had high
229
contribution of the total DOC, of about 50–60% (Fig. 1a), their final contribution to DBP
230
formation was relatively insignificant (<20%) (Fig. S2) because they possessed low specific
231
DBPFPs. The explanation for the IOM-derived DBPs varying within MW will be discussed
232
below.
233
3.4 Functionality
234
XPS analysis was carried out for IOM fractions F1 (>100 kDa) and F6 (<1 kDa), as they were
235
extremely different in terms of MW, fluorescence and DBP formation potential. Their chemical
236
compositions and functional structures were used to further interpret the formation of
237
IOM-derived C-DBPs. Three essential elements of OM, C, O, and N, were investigated. Table 1
238
lists the contributions of each element and their relative ratios in the chemical structures of the F1
239
and F6 fractions. The percentages and ratios of the three elements were comparable for F1
240
fractions of the two species, while they were very different for F6 of both IOM. In particular, two
241
F1 were similarly constituted with high carbon content, which was relatively twice and
242
twelve-fold higher than that of the oxygen and nitrogen content, respectively (as shown in Table
243
1). Although the total amount of C and O content in fraction F6 of MA was similar to F1, F6
244
comprised only 2% of the N content. In contrast, fraction F6 of CH had a greater O content
AC C
EP
TE D
M AN U
SC
RI PT
226
14
ACCEPTED MANUSCRIPT (>50%) with a comparable amount of N, but significantly less C compared to its corresponding
246
F1.
247
Detail scanning for core-level spectra of each C1s, O1s, and N1s was further carried out for F1
248
and F6 fractions to further understand their functionality, as shown in Fig. S3 and Table 2. After
249
resolving the overlapped chemical bonding peaks in C1s, three corresponding groups were
250
generally detected in the large fractions F1 of the two species of algae. These included
251
aliphatic/aromatic C–C/C–H (283.92 ± 0.04 eV), alcohol C–O (285.24 ± 0.10 eV), and keto C =
252
O (286.57 ± 0.05 eV) (Ting et al., 1995). These C functional groups were also found in the small
253
MW fractions of F6 associated with the additions of carboxyl O–C = O groups (291.89 ± 0.01 eV)
254
and unknown groups located at 294.62 ± 0.01 eV. As shown in Table 2, fractions F1 of the two
255
species comprised of similar and large amounts of aliphatic/aromatic groups (55–57%) with the
256
absence of carboxylic and unknown groups. Conversely, fractions F6 constituted much less
257
aliphatic/aromatic contents with a greater amount carboxylic and unknown carbon, particularly in
258
the case of CH IOM, where only 25% of carbon bonding belonged to aliphatic/aromatic
259
compounds, but >50% whereas carboxylic and unknown carbon bonds accounted for >50% of the
260
carbon bonding. The O1s results mostly correspond with the C1s findings in which carboxylic
261
groups were mainly found in the fractions F6 of both species (>40%). For N1s, both F1 fractions
262
clearly showed higher contents of N–C bond compared to F6 fractions.
AC C
EP
TE D
M AN U
SC
RI PT
245
15
ACCEPTED MANUSCRIPT The determination of the composition and function of the two distinct fractions (F1 and F6)
264
further support the results given in Fig. 2, implying that (i) the dominance of AP- and SMP-like
265
fluorescent components in large MW fractions (>100 kDa; F1) of IOM is constituted by carbon-
266
and nitrogen-rich substances with considerable sites of C–C/C–H and N–C, and (ii) the abundance
267
of HA- and FA-like fluorescence in the small MW fractions (<1 kDa; F6) can be attributed to
268
carboxylic-based molecules that associates with either less N (for MA) or more O (for CH)
269
content in their chemical structures.
270
3.5 Discussion
271
3.5.1 Variations of IOM-derived THMs and HAAs within MW
272
IOM-derived THMs and HAAs are strongly depended on MW properties. For IOM MW varying
273
from >100 kDa to <1 kDa, more significant levels of C-DBPs are formed for MW (>100 kDa),
274
but only low levels of C-DBPs are produced MW (<10 kDa). The differences in IOM-derived
275
DBPs with large and small MW are attributed to their different fluorescent compositions and
276
functional structures of the precursors. Large MW IOM is evidently constituted by AP- and
277
SMP-like components. These are carbon nitrogen-rich substances from the essential aromatic
278
proteins in algal cells, such as tryptophan, tyrosine, phenylalanine, and histidine (Fang et al.,
279
2010). Studies have reported that aromatic proteins and/or substances with AP- and SMP-like
280
components are high-yielding DBP precursors and produce substantial levels of both THMs and
281
HAAs because they have a large content of highly reactive carbons in their chemical structures for
AC C
EP
TE D
M AN U
SC
RI PT
263
16
ACCEPTED MANUSCRIPT chlorine incorporation (Hong et al., 2009; Hua et al., 2017; Hua et al., 2018b). Indeed, our results
283
show that the abundance of AP- and SMP-like components in the large MW fraction also
284
associates with the more available reactive carbon content (C–C/C–H) and electron-donating
285
functional groups (N–R2 and –OR) (Table 2). In contrast, the chemical structures of the small
286
MW fractions (<10 kDa), which are mainly presented in HA- and FA-like fluorescence, contain (1)
287
less amount of carbons, (2) a considerable number of carbonyl groups (COO–), which is
288
unfavorable for chlorine substitution, and (3) limited electron-donating functional groups. As the
289
OM has more available reactive carbon sites, the higher the degree of chlorine substitution
290
(Reckhow et al., 1990). Large MW IOM (>100 kDa) is thus a more important and higher-yielding
291
precursor for the formation of C-DBPs compared to small MW IOM (<10 kDa).
292
MW property of IOM also influences the ratio of THM and HAA formation, as shown in Fig. 3.
293
While MW fractions >10 kDa (F1, F2, and F3) preferentially follow the formation pathway of
294
HAAs, MW fractions at <10 kDa (F4, F5, and F6) tend to produce more THMs. It should be
295
noted that the THMs produced in this study were chiefly contributed by TCM, and HAAs were
296
accounted for DCAA and TCAA. During the chlorination of OM precursors, DCAA and TCAA
297
were predominantly formed when the chemical structure of intermediates was constituted by
298
readily oxidizable functional groups, i.e. carbon-carbon double bond, alcohol, and amine.
299
Otherwise, TCM is possibly formed as the final product (Hong et al., 2009; Hua et al., 2017;
300
Reckhow et al., 1990). As shown in Table 2, the functionality of small MW fractions is more
AC C
EP
TE D
M AN U
SC
RI PT
282
17
ACCEPTED MANUSCRIPT deficient in C = C, O–H, and N–C compared to large MW fractions. This results in a higher
302
tendency to undergo the formation of THMs rather than HAAs from small MW of IOM.
303
Combined with the fluorescent results, these results further support our previous findings (Hua et
304
al., 2017) that found that precursors containing AP- and SMP-like components are favorable
305
precursors of HAAs, when precursors with HA- and FA-like components tend to form THMs.
306
3.5.2 Fluorescent and MW dependence of IOM-derived THMs and HAAs
307
Our results convincingly demonstrate the dependence of IOM-derived THMs and HAAs on the
308
fluorescent and MW properties. CCA was used to explore these relationships, and Fig. 4 shows
309
the CCA ordination biplot for twelve MW fractions from two IOMs as sample sites, associated
310
with four EEM components as quantitative explanatory variables, and two DBPs as object points.
311
As can be seen in Fig.4, the CCA test separates the sample sites into two groups: group I of large
312
MW (F1; >100 kDa and F2; 100–30 kDa), and group II of small MW (F3, F4, F5, and F6; >30
313
kDa). Interestingly, it also divides THMs and HAAs closely associated with these two sample
314
groups, further confirming the dependence of THM and HAA formation on IOM MW properties.
315
Furthermore, the CCA biplot shows distinctly different directions of EEM components. While AP
316
and SMP components are closed and have a positive correlation with HAAs and two large MW
317
IOMs (group I), HA and FA components, by contrast, correlate mostly positively with THMs and
318
small MW IOMs (group II). These results are robust confirmation that IOM-derived THMs and
319
HAAs are strongly dependent on fluorescent and MW properties. Large MW IOM exhibiting AP-
AC C
EP
TE D
M AN U
SC
RI PT
301
18
ACCEPTED MANUSCRIPT and SMP-like fluorescence is favorable for the formation of HAAs, while small MW IOM with
321
HA- and FA-like fluorescence tends to form THMs. To the best of our knowledge, this study is the
322
first to elucidate the role of fluorescent and MW properties on the formation of IOM-derived
323
THMs and HAAs. Thus, the results from this study will provide useful information for the future
324
DBP research. Furthermore, since fluorescent and MW properties are common and important
325
characteristics of the water matrix, our findings imply that the control of DBPs derived from
326
algae-containing water can significantly be benefited by applying these adequate characterization
327
tools.
328
4. Conclusions
329
Our experimental and statistical results convincingly show the strong dependence of IOM-derived
330
THMs and HAAs on the fluorescent and MW properties of IOM precursors. Of the MW varying
331
from >100 kDa to <1 kDa, the large fraction of >100 kDa is the most important portion of IOM
332
precursors, which not only makes the greatest contribution to MW distribution, but also produces
333
and contributes the highest levels of THMs, HAAs, and C-DBPs. This evidently results from the
334
abundance of carbon-nitrogen rich substances in such large MW IOM, which contain a large
335
number of reactive carbon sites for chlorine substitution, and exhibits AP- and SMP-like
336
fluorescence. By contrast, although small MW fractions (>10 kDa) accounts for around half of the
337
total DOC of IOM, they are insignificant in contributing to the overall C-DBPs. Low THMs and
338
HAAs derived from small MW fractions are mainly attributed to the dominance of the carboxylic
AC C
EP
TE D
M AN U
SC
RI PT
320
19
ACCEPTED MANUSCRIPT group, and the deficiency of the conjugated system and amide content in their chemical structures.
340
The outcomes of this study provide more significant insights into the principle of IOM-derived
341
DBPs and potentially benefits the control of algae yielded DBPs in drinking water.
342
Acknowledgments
343
We greatly appreciate the assistance of Wouter Holleman for proofreading this paper.
344
References
345 346 347
Chen, W., Westerhoff, P., Leenheer, J.A. and Booksh, K., 2003. Fluorescence Excitation−Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci. Technol. 37 (24), 5701–5710.
348 349
Cheng W.P., Chi F.H. 2003. Influence of eutrophication on the coagulation efficiency in reservoir water. Chemosphere 53, 773–778.
350 351
Coble, P.G., 2007. Marine Optical Biogeochemistry: The Chemistry of Ocean Color. Chem. Rev. 107 (2), 402–418.
352
Fang, J., Yang, X., Ma, J., Shang, C. and Zhao, Q., 2010. Characterization of algal organic matter
353
and formation of DBPs from chlor (am) ination. Water Res. 44(20), 5897–5906.
354 355
Fogg, G., Nalewajko, C. and Watt, W., 1965. Extracellular products of phytoplankton photosynthesis. Proc. R. Soc. Lond. B. Biol. Sci. 162 (989), 517–534.
356 357
Glazer A.N., Fang S. 1973. Chromophore content of blue-green algal phycobiliproteins. J. Biol. Chem. 248, 659–662.
358 359 360
Goslan E.H., Seigle C., Purcell D., Henderson R., Parsons S.A., Jefferson B., 2017. Carbonaceous and nitrogenous disinfection by-product formation from algal organic matter. Chemosphere, 170, 1– 9.
361 362
Guo T., Yang Y., Liu R., Li X. 2017. Enhanced removal of intracellular organic matters (IOM) from Microcystic aeruginosa by aluminum coagulation. Sep.Purif. Technol. 189, 279–287.
363 364
Henderson, R., Parsons, S.A. and Jefferson, B., 2008a. The impact of algal properties and pre-oxidation on solid–liquid separation of algae. Water Res. 42(8–9), 1827–1845.
365 366
Henderson, R.K., Baker, A., Parsons, S.A. and Jefferson, B., 2008b. Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res. 42(13), 3435–
AC C
EP
TE D
M AN U
SC
RI PT
339
20
ACCEPTED MANUSCRIPT 367
3445.
368 369
Henderson, R.K., Parsons, S.A. and Jefferson, B., 2010. The impact of differing cell and algogenic organic
370 371 372
Hong H.C., Mazumder A., Wong M.H., Liang Y. 2008. Yield of trihalomethanes and haloacetic acids upon chlorinating algal cells, and its prediction via algal cellular biochemical composition. Water Res. 42, 4941–4948.
373 374
Hong H.C., Wong M.H., Liang Y. 2009. Amino Acids as Precursors of Trihalomethane and Haloacetic Acid Formation During Chlorination. Arch. Environ. Contam. Toxicol. 56, 638–645.
375
Hua, G. and Reckhow, D.A., 2007. Characterization of Disinfection Byproduct Precursors Based on
376
Hydrophobicity and Molecular Size. Environ. Sci. Technol. 41 (9), 3309–3315.
377 378
Hua, L.C., Lin, J.L., Chen, P.C. and Huang, C.P., 2017. Chemical structures of extra- and intra-cellular
379
Eng. J. 328, 1022–1030.
380 381
Hua, L.C., Lin, J.L., Zhao, S.J., and Huang, C.P., 2018a. Probing algogenic organic matter (AOM) by
382
Environ. 645: 71–78
383 384 385
Hua, L.C., Lin, J.L., Syue, M.Y., Huang, C.P. and Chen, P.C., 2018b. Optical properties of algogenic organic
386 387 388
Huang, J., Graham, N., Templeton, M.R., Zhang, Y., Collins, C. and Nieuwenhuijsen, M., 2009. A comparison of the role of two blue–green algae in THM and HAA formation. Water Res. 43(12), 3009–3018.
389 390
Li, L., Gao, N., Deng, Y., Yao, J. and Zhang, K., 2012. Characterization of intracellular & extracellular
391
byproducts and odor & taste compounds. Water Res. 46(4), 1233–1240.
392 393 394
Liu R., Guo T., Ma M., Yan M., Qi J., Hu C., 2018. Preferential binding between intracellular organic matters and Al13 polymer to enhance coagulation performance. J. Environ. Sci. (In press). https://doi.org/10.1016/j.jes.2018.05.011.
395
Lui Y.S., Hong H.C., Zheng G.J.S., Liang Y. 2012. Fractionated algal organic materials as
396 397
precursors of disinfection by-products and mutagens upon chlorination. J. Hazard. Mater. 209–210, 278–284. 21
M AN U
SC
RI PT
matter (AOM) characteristics on the coagulation and flotation of algae. Water Res. 44 (12), 3617–3624.
algogenic organic matters as precursors to the formation of carbonaceous disinfection byproducts. Chem.
TE D
size-exclusion chromatography to predict AOM-derived disinfection by-product formation. Sci. Total.
matter within the growth period of Chlorella sp. and predicting their disinfection by-product formation. Sci.
AC C
EP
Total. Environ. 621, 1467–1474.
algae organic matters (AOM) of Microcystic aeruginosa and formation of AOM-associated disinfection
ACCEPTED MANUSCRIPT Lui Y.S., Qiu J.W., Zhang Y.L., Wong M.H., Liang Y. 2011. Algal-derived organic matter as precursors of disinfection by-products and mutagens upon chlorination. Water Res. 45, 1454–1462.
400 401
Ndlela L.L., Oberholster P.J., Van W.J.H., Cheng P.H. 2016. An overview of cyanobacterial bloom occurrences and research in Africa over the last decade. Harmful Algae 60, 11–26.
402
Pivokonsky, M., Safarikova, J., Baresova, M., Pivokonska, L. and Kopecka, I., 2014. A comparison of the
403 404
character of algal extracellular versus cellular organic matter produced by cyanobacterium, diatom and green
405 406
Plummer, J.D. and Edzwald, J.K., 2001. Effect of Ozone on Algae as Precursors for Trihalomethane and
407
Purton S. 2001. Algal Chloroplasts. e LS. https://doi.org/10.1038/npg.els.0000316.
408 409
Reckhow, D.A., Singer, P.C. and Malcolm, R.L., 1990. Chlorination of humic materials: byproduct formation
410 411
Takaara T., Sano D., Konno H., Omura T. 20017. Cellular proteins of Microcystis aeruginosa inhibiting coagulation with polyaluminum chloride. Water Res. 41, 1653–1658.
412 413
Tas B., Gonulol A., 2007. An ecologic and taxonomic study on phytoplankton of a shallow lake, Turkey. J.Environ. Biol. 28, 439–445.
414
Ting Y.P., Teo W.K., Soh C.Y., 1995. Gold uptake by Chlorella vulgaris. J. Appl. Phycol. 7, 97–100.
415 416
Tomlinson, A., Drikas, M. and Brookes, J.D., 2016. The role of phytoplankton as pre-cursors for disinfection by-product formation upon chlorination. Water Res. 102, 229–240.
417 418
Wert, E.C. and Rosario-Ortiz, F.L., 2013. Intracellular Organic Matter from Cyanobacteria as a Precursor for
419 420
Zhou, S., Zhu, S., Shao, Y. and Gao, N., 2015. Characteristics of C-, N-DBPs formation from algal organic
421 422
Zhu, M., Gao, N., Chu, W., Zhou, S., Zhang, Z., Xu, Y. and Dai, Q., 2015. Impact of pre-ozonation on
423
Microcystis aeruginosa. Ecotoxicol. Environ. Saf. 120, 256–262.
alga. Water Res. 51 (0), 37–46.
M AN U
SC
Haloacetic Acid Production. Environ. Sci. Technol. 35(18), 3661–3668.
RI PT
398 399
EP
TE D
and chemical interpretations. Environ. Sci. Technol. 24 (11), 1655–1664.
AC C
Carbonaceous and Nitrogenous Disinfection Byproducts. Environ. Sci. Technol. 47 (12), 6332–6340.
matter: Role of molecular weight fractions and impacts of pre-ozonation. Water Res. 72, 381–390.
disinfection by-product formation and speciation from chlor(am)ination of algal organic matter of
22
ACCEPTED MANUSCRIPT
Tables
N% 5.5 2.0 5.5 4.1
O% 25.5 29.3 27.7 50.2
12.6 34.0 12.3 11.3
SC
C% MA-IOM F1 >100 kDa 69.0 F6 <1 kDa 68.7 CH-IOM F1 >100 kDa 66.8 F6 <1 kDa 45.7 Note: Sum of C%, N%, and O% was 100%.
RI PT
Table 1 Contribution of C, O, and N elements in the large MW fraction F1 and small MW F6 from the IOM of M. aeruginosa (MA) and Chlorella sp. (CH), and their relative ratios. C/N C/O Origin MW Fraction Element percentage (%) 2.7 2.3 2.4 0.9
AC C
EP
TE D
M AN U
Table 2 Chemical functional groups of C, O, and N for the fractionated F1 and F6 of the IOMs from M. aeruginosa (MA) and Chlorella sp. (CH). Functional Binding Contribution of each functional group (%) group energy (eV) MA CH F1 (>100 kDa) F6 (<1 kDa) F1 (>100 kDa) F6 (<1 kDa) C1s functional groups C-C/C-H 283.92 ± 0.04 57.3 57.5 54.9 24.9 C-O 285.24 ± 0.10 29.1 14.1 27.4 13.0 C=O 286.57 ± 0.05 13.6 8.2 17.7 6.7 O-C=O 291.89 ± 0.01 14.0 36.4 Unknown 294.62 ± 0.01 6.2 19.0 O1s functional groups C=O 530.10 ± 0.51 38.5 26.8 27.0 23.4 O-C/O-H 530.97 ± 0.37 53.2 32.8 42.0 35.0 O-C=O 532.05 ± 0.52 8.4 27.9 31.1 34.6 O-C=O* 534.42 ± 0.23 12.6 7.0 N1s functional groups N-C 398.86 ± 0.26 97.0 87.1 93.1 87.1 N-H 401.48 ± 0.97 3.0 12.9 6.9 12.9 Note: O-C=O* chemical shift of carboxylic group.
ACCEPTED MANUSCRIPT
(a)
(b)
<1 3-1 10-3 30-10 100-30 >100
60
20
0
MA
CH
MA
80
60
40
20
0
CH
EP
TE D
Fig. 1 Distributions of each MW fraction in (a) DOC and (b) DON of IOM derived from M. aeruginosa (MA) and Chlorella sp. (CH). MW unit is in kDa
AC C
2 3 4
M AN U
40
RI PT
80
100
SC
100
Distribution in DON (%)
Figures
Distribution in DOC (%)
1
ACCEPTED MANUSCRIPT 100
(a)
RI PT
80
60
40
SC
AFI percentage (%)
(b)
AP SMP HA FA
0 Original >100 100-30 30-10
<1
Original >100 100-30 30-10
10-3
1-3
<1
Apparent MW fraction (kDa)
TE D
Fig. 2 Variations of calculated regional AFI percentages within the MW of (a) MA-IOM and (b) CH-IOM (DOC = 10 mg/L; pH 7).
EP
6 7
1-3
Apparent MW fraction (kDa)
AC C
5
10-3
M AN U
20
ACCEPTED MANUSCRIPT 160
THM HAA C-DBP
120 100 80 60 40 20
> 100
30-10
10-3
3-1
<1
Original
THM HAA C-DBP
120 100 80 60 40 20
> 100
100-30
30-10
10-3
3-1
<1
Original
Apparent MW faction (kDa)
Apparent MW faction (kDa)
SC
Fig. 3 Variations of THMFP, HAAFP, and C-DBPFP derived from each MW fraction and the
TE D
M AN U
original (a) MA-IOM and (b) CH-IOM. Chlorination conditions: DOC ≈ 5 mg/L, Cl2:DOC = 5:1, pH 7, 7 days, 25oC.
EP
10 11
100-30
AC C
9
(b) CH IOM
0
0
8
140
RI PT
140
(a) MA IOM
Specific C-DBPFP (µ µ g/ mg-C)
Specific C-DBPFP (µ µ g/ mg-C)
160
ACCEPTED MANUSCRIPT
C-DBPs
MW fractions
EEM components MA-IOM_F3
Group I
0.1
RI PT
0.2
CH-IOM_F3 CH-IOM_F2
SC
MA-IOM_F1
0
MA-IOM_F2 CH-IOM_F1
-0.1
SMP AP
MA-IOM_F4 MA-IOM_F5
THMs MA-IOM_F6
FA HA
TE D
-0.2
CH-IOM_F5 CH-IOM_F6
CH-IOM_F4
M AN U
CCA2
HAAs
Group II
-0.4 -1
-0.8
-0.6
AC C
-1.2
EP
-0.3
-0.4
-0.2
0
0.2
0.4
0.6
0.8
CCA1
Fig. 4 CCA ordination biplot showing the relationships among fluorescent EEM components (AP, SMP, HA, FA) as explanatory variables, MW fractions of two IOM as sample sites (12 samples), and THMs as well as HAAs as response variables
1
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
IOM-derived THMs and HAAs from M. aeruginosa and Chlorella sp. were examined. Roles of fluorescent and MW properties of IOM on its THMs and HAAs formation were examined. IOM-derived THMs and HAAs strongly depend on fluorescent and MW properties.
AC C
• • •