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Remediation of resins-contaminated soil by the combination of electrokinetic and bioremediation processes
Journal Pre-proof Remediation of resins-contaminated soil by the combination of electrokinetic and bioremediation processes Jing Ma, Qi Zhang, Fu Chen, Qianlin Zhu, Yifei Wang, Gangjun Liu PII:
S0269-7491(19)37160-X
DOI:
https://doi.org/10.1016/j.envpol.2020.114047
Reference:
ENPO 114047
To appear in:
Environmental Pollution
Received Date: 2 December 2019 Revised Date:
14 January 2020
Accepted Date: 22 January 2020
Please cite this article as: Ma, J., Zhang, Q., Chen, F., Zhu, Q., Wang, Y., Liu, G., Remediation of resins-contaminated soil by the combination of electrokinetic and bioremediation processes, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2020.114047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
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Remediation of resins-contaminated soil by the combination of electrokinetic and
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bioremediation processes
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Jing Maa,1, Qi Zhangb,1, Fu Chena,b, *, Qianlin Zhua, Yifei Wangb, Gangjun Liuc
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221008, China
a
Low Carbon Energy Institute, China University of Mining and Technology, Xuzhou
10 11
b
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and Technology, Xuzhou 221008, China
School of Environmental Science and Spatial Informatics, China University of Mining
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c
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Melbourne 3000, Australia
Geospatial Science, College of Science, Engineering and Health, RMIT University,
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*Corresponding author. E-mail address: [email protected]
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1
Qi Zhang and Jing Ma contributed equally to this work.
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Abstract
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In this work, soil contaminated by petroleum resins was remediated by
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electrokinetic-bioremediation (EK-BIO) technology for 60 days. A microbial
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consortium, comprising Rhizobium sp., Arthrobacter globiformis, Clavibacter xyli,
29
Curtobacterium flaccumfaciens, Bacillus subtilis, Pseudomonas aeruginosa and
30
Bacillus sp., was used to enhance the treatment performance. The results indicate that
31
resin removal and phytotoxicity reduction were highest in the inoculated EK process,
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wherein 23.6% resins was removed from the soil and wheat seed germination ratio was
33
increased from 47% to around 90% after treatment. The microbial counts, soil basal
34
respiration and dehydrogenase activity were positively related to resins degradation, and
35
they could be enhanced by direct current electric field. After remediation, the C/H ratio
36
of resins decreased from 8.03 to 6.47. Furthermore, the structure of resins was analyzed
37
by Fourier-transform infrared spectroscopy, elemental analysis, and 1H nuclear magnetic
38
resonance (1H NMR) before and after treatment. It was found that the changes of the
39
structure of resins took place during EK-BIO treatment and finally led to the reduction
40
of aromaticity, aromaticity condensation and phytotoxicity.
41
Key words: Petroleum resins; phytotoxicity; dehydrogenase activity; seed germination;
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aromaticity
43 44 45
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1. Introduction
47
Soil petroleum contamination is one of the most serious soil pollution problems
48
among the existing environmental problems (Khan et al., 2018). When petroleum enters
49
the soil through various ways such as spills, leaks and accidents, soil organic matter
50
content increases, carbon:nitrogen (C:N) and carbon:phosphorus (C:P) ratios are
51
unbalanced, which leads to the loss of soil nutrients and the decline of metabolic
52
capacity in the soil.
53
Crude oil is an extremely complex mixture of hydrocarbons containing thousands
54
of components. On the basis of the common separation procedure, crude oil can be
55
grouped into four major classes: saturate hydrocarbon (saturates), aromatic hydrocarbon
56
(aromatics), resins and asphaltenes (SARA) (Bissada et al., 2016). Heavy crude oil
57
reserves has a large proportion in the total crude oil resources in China. For example, in
58
Liaohe Oilfield, the content of resins and asphaltenes is up to 34% and 12%,
59
respectively (Li et al., 2017).
60
As the most abundant heavy component of heavy oil, resins has been a difficult
61
problem in the process of petroleum-contaminated soil remediation. Resins is comprised
62
of many condensed ring units and abundant in polar substituents containing nitrogen,
63
sulfur and/or oxygen (Li et al., 2017). Resins has a high molecular weight and strong
64
polarity, and its degradation is abiotically and biotically difficult in the environment,
65
leading to the accumulation and potential environmental hazards. Therefore, the
66
removal of resins and the reduction of its toxicity in petroleum-contaminated soil have
3
67
become an urgent issue to be solved during remediation practice.
68
As a green and efficient remediation technology, electrokinetic-bioremediation
69
(EK-BIO) has been tested for remediation of various organic-contaminated soils
70
including petroleum-contaminated soil (Dong et al., 2013; Hassan et al., 2016; Zhang et
71
al., 2017). On the one hand, electrodialysis, electromigration, electrophoresis and
72
electrochemical oxidation reactions are involved in EK process. On the other hand, EK
73
technology can provide good living conditions for microbial populations, promoting
74
microbial activity and enhancing biodegradation of pollutants. However, due to its
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macromolecular characteristics, resins is difficult to degrade, and the improvement of
76
resins degradation by EK-BIO is limited. Thus, the degradation rate of resins is
77
insufficient to accurately and comprehensively describe the remediation effect.
78
Existing studies demonstrate that both photooxidation and microbial degradation
79
can significantly alter the molecular structure of resins (Akhmedbekova et al., 2009;
80
Minai-Tehrani et al., 2015). Fourier-transform infrared spectroscopy (FT-IR) analysis
81
revealed that, during photooxidation of resins, quinone structure was produced, the
82
content of carboxyl groups increased, and sulfoxide groups were oxidized
83
(Akhmedbekova et al., 2009). Nevertheless, these studies only focused on the structural
84
changes of resins, and whether the ecotoxicity has changed or not was unknown.
85
In this study, EK-BIO technology was used to remediate soil contaminated with
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resins, and the changes of molecular structure, bioavailability, ecotoxicity and soil
87
microbial activity were explored before and after the remediation process. This study
4
88
aimed to improve the index system of petroleum-contaminated soil remediation, and to
89
provide a scientific basis for the degradation of resins during the remediation process.
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2. Materials and methods
91
2.1. Soil samples
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An uncontaminated soil was collected from a field in the campus of China
93
University of Mining and Technology, Xuzhou, China. The soil was air-dried and sieved
94
through a 20-mesh sieve. The characteristics of the soil are: pH (1:2.5 water), 6.9; sand,
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63.6 wt.%; silt, 21.5 wt.%; clay, 14.9 wt.%; cation exchange capacity (CEC), 9.6
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cmol/kg; total organic carbon (TOC), 5.3 g/kg; particle size distribution: < 2 µm 18.4%,
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2–50 µm 58.6%, 50–2000 µm 23.0%. No petroleum contamination was detected in the
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soil.
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Resins fraction was extracted from a heavy crude oil (resins content 24%) through
100
the SARA method (Bissada et al., 2016). The oil was obtained from the Liaohe Oilfield,
101
China. Resins was dissolved into dichloromethane and then spiked into the soil to reach
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an initial content of about 4.5 g/kg after solvent evaporation and one month of
103
equilibrium.
104
2.2. Microbial culture
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A microbial consortium capable of degrading crude oil was isolated by selective
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enrichment from petroleum-contaminated soil in Liaohe Oilfield, China. The strains of
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the consortium were identified as Rhizobium sp., Arthrobacter globiformis, Clavibacter
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xyli, Curtobacterium flaccumfaciens, Bacillus subtilis, Pseudomonas aeruginosa and
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Bacillus sp. respectively by means of genetic methods. The microbial culture was
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inoculated into the soil at the ratio of 2.0 ×107 colony forming units (CFU) per g soil.
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2.3. Experimental methods
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Figure 1 shows the schematic design of the experimental setup used in this study.
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The setup mainly consists of a direct-current power supply, two electrode chambers;
114
two graphite electrodes, and a soil chamber. The assembly of EK test compartments was
115
made of Pyrex glass. The dimension of soil chamber was 25 cm × 12 cm × 12 cm, and 2
116
kg of dry soil was loaded. The dimension for electrode chambers is 10 cm × 10 cm × 10
117
cm. The electrode compartments contain a filter paper, a porous plate and a perforated
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graphite electrode. To prevent soil particles from penetrating into the electrolyte
119
solution reservoirs, the filter paper was placed between the soil and the porous stone and
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both were placed in front of the electrode. A direct-current power supply (RXN-303A,
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Zaoxin Instrument Co., Ltd., China) was used to maintain a constant voltage of 25 V,
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resulting in a voltage gradient of 1.0 V/cm. For all treatments, 0.01 M NaNO3 was used
123
as the supporting electrolyte. The soil was saturated with 0.01 M NaNO3 solution for 24
124
h before application of electrical field. Once the soil was loaded into the cell, the cell
125
assembly was completed. The experiments were conducted at a room temperature, and
126
all aqueous solutions were prepared using deionized water. To prevent anode
127
acidification and cathode alkalization, the electrolyte pH was controlled at around 6.9
128
(namely the background pH of soil) by an automatic pH controller.
129
Soil samples were moistened with an inorganic medium to provide 20% (w/w)
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130
moisture content for nonflooded conditions. The composition of the medium is as
131
follows: NaNO3 1.5 g/L, (NH4)2SO4 1.5 g/L, K2HPO4 1.0 g/L, KCl 0.5 g/L,
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MgSO4•7H2O 0.5 g/L, FeSO4•7H2O 0.01 g/L, CaCl2 0.002 g/L, pH 7.0. The medium
133
was autoclaved at 121 °C for 20 min at 0.11 MPa before use.
134
Table 1 summarizes the experimental setup involved in the experimental program.
135
CK was a baseline test conducted to evaluate the bioremediation potential of soil
136
microbial indigenous population. EK-BS was performed to evaluate the joint
137
performance of EK and indigenous microbial populations. BG was aimed to reveal the
138
combined influence of exogenous and indigenous microbes on pollutant removal with
139
no electricity. EK-BG was carried out to investigate the effect of exogenous microbes
140
on the EK system behavior.
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The experiments lasted for 60 days, carried out at room temperature (25±2 °C).
142
Soil moisture content was kept at around 20% by regular addition of sterile inorganic
143
medium (for EK-BS and EK-BG) or distilled water (for CK and BG). After each
144
experiment of 10 days, the soil was removed from the apparatus, cut into five equal
145
slices (S1–S5, from anode to cathode) and characterized for microbial parameters and
146
concentration of residual contaminants.
147
2.4. Analytical methods
148
The soil pH was determined by using a pH meter (model pHS-3B, Shanghai
149
Precision Scientific Instrument Co., Ltd., China) with a solid:water ratio of 1:2.5. TOC
150
was measured by catalytic oxidation on a TOC analyzer (Shimadzu TOC-VCPH, Kyoto,
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151
Japan). CEC was determined after leaching of <2 mm air-dried soil with 1 M
152
CH3COONH4 at pH 7.0. Soil particle size was analyzed using the pipette method
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(Miller and Miller, 1987).
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2.5. Extraction, determination and characterization of resins
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Soil samples were freeze-dried, ground and passed through 20-mesh sieve, and
156
then the soil samples were extracted on an accelerated solvent extractor system (ASE
157
300, Dionex, Sunnyvale, CA, USA) with dichloromethane. The extract was dried under
158
N2 flow to constant final weight, and then resins content was determined
159
gravimetrically.
160
Resins samples were subjected to a series of instrumental analyses including
161
Fourier-transform infrared spectroscopy (FT-IR), elemental analysis, and 1H nuclear
162
magnetic resonance (NMR). FT-IR spectra were collected on a Nicolet 6700 FT-IR
163
(Thermo Scientific Corp., Madison, WI, USA). C, H and N contents were determined
164
on a Carlo-Erba model 1106 elemental analyzer (Carlo Erba, Milano, Italy). 1H NMR
165
spectroscopic measurements were carried out with a Bruker 400 MHz Spectrometer
166
using tetramethylsilane (TMS) as an internal standard in chloroform-d (CDCl3).
167
2.6. Microbial activity analysis
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Soil bacteria were enumerated on nutrient agar plates through plate spread method
169
(five replicates per sample) (Lu and Zhang, 2014). The total number of colonies was
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counted after incubation at 28 °C in the dark for 2 days, and the results were expressed
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as CFU per gram of dry soil.
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Soil basal respiration was analyzed using the MicroResp™ method and 15 carbon
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sources in addition to water (basal respiration rate control) on a Spectra Max 190
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microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm (Drage et al.,
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2012).
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Microbial dehydrogenase activity (DHA) in soil samples was determined following
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the method of Lu et al. (2009), and results were expressed as µg TPF/(g soil•6 h).
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Controls were prepared with autoclaved samples (121 °C, 60 min) and treated like
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biotic samples.
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2.7. Seed germination experiments
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Wheat seed germination experiments were performed to analyze soil toxicity
182
changes before and after remediation. Plumped and uniform-sized seeds were selected,
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soaked in 3% H2O2 solution for 20 min, rinsed with sterile water, and then immersed in
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sterile water for 24 h under darkness before sowing. Fifty seeds were uniformly placed
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in petri dishes, covered with the prepared soils (about 1 cm thickness), and kept in a
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growth chamber for 10 days. The details have been described previously (Chen et al.,
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2019a).
188
2.8. Data analysis
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In this study, the results are reported as means ± standard deviations on the basis of
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dry weight. Statistical analysis of the results was performed with SPSS 11.0 for
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Windows (SPSS Inc.).
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3. Results and discussion
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3.1. Changes of soil physicochemical properties
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Figure 2 shows the variation of soil pH, moisture content and electroconductibility
195
in different soil sections after 60 days of treatments. In CK and BG, various indexes
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changed little. In EK-BS and EK-BG, the pH profile followed the trend with pH
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increasing from 6.7 near the anode (S1) to 7.1 near the cathode (S5) [Figure 2(A)].
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Moreover, no apparent difference was observed in soil pH between EK-BS and EK-BG.
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This stable pH can be ascribed to the pH control of electrolyte. Generally, electrolysis
200
reactions may cause a low pH at anode and a high pH at cathode due to the generation
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of H+ and OH– ions at anode and cathode, respectively (Chen et al., 2019b).
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The soil moisture content kept relatively stable across various sections among
203
different tests [Figure 2(B)]. Basically, the moisture content was in the range of 16–22%.
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Figure 2(C) shows the distribution of soil electroconductibility after 60 days. The initial
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soil electroconductibility was 1.7 mS/cm. Soil electroconductibility decreased from S1
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to S5 in EK-BS and EK-BG whereas kept constant in CK and BG, which followed the
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same pattern as the pH of soil sections [Figure 2(C)]. Nevertheless, the difference in the
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electroconductibility of soil sections was insignificant in EK-BS and EK-BG. The
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mobility of ions under EK conditions can result in the variation of soil
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electroconductibility. Soil electroconductibility depends on pH, and high pH may reduce
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electroconductibility because of metal precipitation, whereas a lower pH can lead to a
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higher electroconductibility value due to enhanced mobility of metal ions (Chen et al.,
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2019b). In this study, the supplement of inorganic medium to the soil could alleviate
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electroconductibility alteration caused by ion migration.
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3.2. Resins removal
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Figure 3 demonstrates resins removal in the soil sections after the 60-day treatment.
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Resins was not detected in electrolyte, indicating no migration of resins under electric
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field because of its high hydrophobicity and extremely low solubility. In CK and BG,
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the concentration of resins was found to be evenly distributed throughout the soil
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column. The application of EK enhanced resins removal in the soil as compared to CK
221
and BG (Figure 3). After 60 days, the average removal ratio of resins followed the order
222
of EK-BG (23.6%) > EK-BS (9.3%) > BG (6.5%) > CK (1.2%). It can be seen that the
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combined application of EK and microbial inoculant has an enhanced effect on resins
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degradation, and the degradation ratio was higher than that of EK or bioremediation
225
alone. The soil was artificially contaminated with resins, and effective resins-degrading
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microbial community could not evolve in a short time, thus resins removal was
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extremely low in CK (Figure 3). It is also found that the maximum removal in EK-BG
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and EK-BS was obtained adjacent to the electrodes, and the removal extents of resins
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would decrease with the distance to the electrodes. For example, the removal extent of
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resins in S3 was 15.2% and 6.4% in EK-BG and EK-BS, respectively, as compared to
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28.3% and 12.2% in S5 of EK-BG and EK-BS, respectively. These results demonstrate
232
that resins could be more favourably degraded towards electrodes, and the degradation
233
ability of microbes was evidently enhanced by the application of electric field.
234
Due to its high hydrophobicity, resins cannot be removed by means of
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235
electro-osmosis, -migration or -phoresis, such that the removal of resins in EK-BG and
236
EK-BS can be attributed to electrochemical oxidation. During the EK-BIO process,
237
resins can be not only directly degraded by electrochemical oxidation and microbes, but
238
also indirectly oxidatively broken down by chlorine, hypochlorite and hydrogen
239
peroxide, which are generated by anodic reaction and water electrolysis reaction (Zhang
240
et al., 2017). However, the chemical stability and microbial degradation resistance of
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resins resulted in a smaller removal extent in both EK-BG and EK-BS experiments. In
242
this study, the higher removal occurring adjacent to the electrodes indicates that the
243
strong electrochemical reactions appeared near the electrodes. An electric field can
244
improve the mass transfer between pollutants and microbes by dispersing them, leading
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to enhanced bioavailability of soil pollutants (Wick et al., 2007). The density of electric
246
field is a driving force for mass transfer in the electric field; thus, the closer to the
247
electrodes, the greater the mass transfer formed (Fan et al., 2015). A better opportunity
248
for interaction between pollutants and microbes in the vicinity of electrodes resulted in a
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higher resins removal extent. Resins constitutes a large and diverse class of organic
250
components, and the effectiveness of biodegradation or EK alone may be limited by
251
various factors. In this work, EK-BG showed a significant improvement in resins
252
removal relative to other treatments, signifying a synergistic effect between
253
electrokinetic and bioremediation. In addition, resins removal in EK-BG (23.6%) was
254
higher than the sum value of EK-BS (9.3%) and BG (6.5%), suggesting that
255
bioremediation was greatly stimulated by the electric field under the EK-BG treatment.
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256
This may be due to the higher microbial activity, or the enhanced interaction between
257
pollutants and microbes in EK-BG.
258
3.3. Phytotoxicity evaluation
259
Seed germination tests have been widely used for soil phytotoxicity evaluation
260
(Jiang et al., 2016). Table 2 reveals the inhibitory influences of resins on the
261
germination of wheat. The control soil without resins addition and the contaminated soil
262
before treatment had a germination of 98% and 47%, respectively.
263
It can be found from Table 2 that the germination ratio followed the order of
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EK-BG > EK-BS > BG > CK. Moreover, the germination ratio would increase with
265
approaching the electrodes. These situations were in agreement with that of resins
266
removal, namely higher removal corresponded to higher seed germination ratio. It is
267
also observed that the germination ratio of EK-BG was close to that of uncontaminated
268
soil, though EK-BG exhibited a low resins removal (averagely 23.6%). This indicates
269
that soil phytotoxicity can be efficiently reduced by resins degradation in EK-BG.
270
3.4. Microorganism and its activity in soil
271
Figure 4(A) shows the change of bacterial population in different regions of soil
272
specimen during remediation. The initial bacterial number (namely after one month of
273
equilibrium) was about 3.5×106 and 2.3×107 CFU/g soil in uninoculated (CK and
274
EK-BS) and inoculated (EK-BS and EK-BG) soils, respectively. As shown in Figure
275
4(A), the difference of bacterial density in different sampling points after remediation
276
was basically consistent with the trend of resins removal ratio. EK-BG showed the
13
277
highest bacterial density among the five cases. A general increase in soil bacterial
278
density was also observed relative to the initial situations [Figure 4(A)], which was due
279
to nutrient stimulation in this work. These findings suggest that the increase of
280
microbial populations was related to the decrease of soil resins content, and the growth
281
and reproduction of microbes could be promoted by direct current electric field. The
282
transport and distribution of microbes in soil column under electric field depend on
283
many factors such as electrophoresis, electroosmosis, and nutrient flow (Dong et al.,
284
2013). Because of their negatively charged surfaces, bacteria have a negative
285
electrokinetic potential and bacterial movement towards anode would occur because of
286
electrical attraction between anode and bacteria (electrophoresis) (Dong et al., 2013).
287
Moreover, bacteria may migrate toward cathode along with electroosmotic flow (EOF)
288
through soil (electroosmosis). Under electric field, ions and water in soil were migrated
289
toward the cathode by EOF, leading to favorable conditions for bacterial growth in the
290
region adjacent to the cathode. Thus, the final bacterial distribution in soil column came
291
from the competition between electrophoresis, electroosmosis, and nutrient flow.
292
Figure 4(B) exhibits the change of soil basal respiration in different regions of soil
293
specimen during remediation. Generally, the basal respiration values were in the order:
294
EK-BG > EK-BS > BG > CK >initial. The basal respiration was highest in S5 of
295
EK-BG [38.2 µg CO2-C/(g soil•h)], which was 2.3 and 1.8 times that of initial value
296
[16.6 µg CO2-C/(g soil•h)] and BG [21.2 µg CO2-C/(g soil•h)]. Soil basal respiration is
297
the sum of all metabolic processes that produce CO2, and it is a good indicator of
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298
organics decomposition and changes in soil environment under pollution stress.
299
DHA is recognized as an important index of soil microbial activity, being capable
300
of catalyzing the oxidation of organic compounds by transferring protons and electrons
301
(Garcia et al., 1997). The variations of DHA in the experiments before and after 60 days
302
of treatment are presented in Figure 4(C). The average DHA in EK-BG was higher than
303
that in other experiments after 60 days of treatment. At the end of the experiments, the
304
average DHA was 77.3, 52.1, 37.8 and 22.5 µg TPF/(g soil•6h) in EK-BG, EK-BS, BG
305
and CK, respectively. The soil DHA increased significantly after 60 days of treatment (P
306
< 0.01). Sampling site S3 was the least influenced by the electrochemical reaction and
307
was located in the weakest electric field intensity area; therefore, the soil DHA was
308
slightly lower than those at other sites with no significant differences. Sampling sites S1
309
and S5 were closer to the electrodes, and the generation of oxidizing and reducing zones
310
near the electrodes could provide beneficial conditions for the degraders (Lohner et al.,
311
2011).
312
It can be seen from the changes of bacterial density, basal respiration and DHA
313
[Figure 4(A), (B) and (C)] that the activity of microbes in the soil after remediation was
314
significantly increased, and the trend was basically consistent with the removal ratio of
315
resins, indicating that the ecotoxicity of the soil after remediation was effectively
316
reduced. Bacterial cell respiration can be enhanced by anodic oxygen generated by
317
electrolysis (Fan et al., 2015). The generation of anodic oxygen might elevate dissolved
318
oxygen level in the soil, and enhance the respiration of microbes and indirectly increase
15
319
the redox level of soil, thus stimulating DHA and improving the biodegradation of
320
resins.
321
3.5. FT-IR analysis of resins
322
FT-IR spectral analyses of resins are shown in Figure 5. Frequencies and probable
323
assignments of each peak of the spectra are shown in Table 3. It can be seen from Figure
324
5 that the peak intensities for the characteristic region of CH2 and CH3 groups (2920,
325
2853, 1461, 1376 and 756 cm–1 wavenumbers) declined, indicating the shortening of
326
resins carbon chains. The peak intensities at 1710 and 1215 cm–1 decreased obviously
327
after treatment, demonstrating the reduction of carboxyl and phenolic groups. The peak
328
intensities at 1605 and 868 cm–1 (the characteristic regions of aromatic and heterocyclic
329
rings) also declined, suggesting that the aromatic and heterocyclic rings of resins were
330
opened or degraded into saturated rings during remediation. A new peak occurred at
331
1030 cm–1 after remediation, indicating the formation of new functional groups such as
332
R–OH and aromatic–O–R.
333
3.6. Elemental analysis of resins
334
Elemental analysis was conducted on the resins in S5 of EK-BG before and after
335
60 days of treatment. Results show that the mass fractions of C, H, and N were 74.86%,
336
9.315% and 0.848% respectively for the untreated sample, and 68.02%, 10.52% and
337
0.783% respectively for the treated sample. After remediation, the mass fraction of C
338
declined whilst that of H increased, and the C/H ratio decreased from 8.03 to 6.47. This
339
indicates that some unsaturated rings of resins were degraded into saturated ones,
16
340
leading to the decrease in unsaturation of resins. On the basis of the mass fractions of C,
341
H, and N, it can be calculated that the average chemical formula of resins was
342
C103H153N and C101H187N for the untreated and treated samples, respectively.
343
3.7. 1H NMR analysis
344
To study the structure changes of resins, 1H NMR analysis was performed on the
345
resins in S5 of EK-BG before and after 60 days of treatment. On the basis of the
346
assignment of protons in 1H NMR spectrum (Chen et al., 2009), the percentages of
347
integrated areas were obtained, as listed in Table 4. Aromaticity fA and aromaticity
348
condensation HAU/CA were obtained using Eqs. (1) and (2), as demonstrated (Chen et al.,
349
2010):
CT / H T − ( H α + H β + H γ / 2 H T ) CT / H T
350
fA =
351
H AU H A / H T + Hα / 2 H T = CA CT / H T − ( H α + H β + H γ ) / 2 H T
(1)
(2)
352
where CT and HT represent the total C and H, respectively, HT = HA+ Hα+ Hβ+ Hγ;
353
CT/HT is the C/H ratio.
354
It is easy to see from Table 4 that the percentage of HA and Hα decreased whilst
355
that of Hβ and Hγ increased after treatment, namely the percentage of H atoms in
356
aromatic rings declined. Moreover, the aromaticity and aromaticity condensation of
357
resins decreased after treatment. The decrease of the former could be caused by the
358
hydrogenation of unsaturated groups while that of the latter could be attributed to the
359
ring-opening of the aromatic system.
17
360
4. Conclusions
361
The present study has demonstrated that the efficiency of resins degradation was
362
stimulated by the electric field and bioaugmentation in the coupled technology of
363
EK-BIO, signifying a synergistic effect between electrokinetic and bioremediation. The
364
contribution of inoculated microbes was higher than that of indigenous counterparts in
365
the degradation of resins, and the contribution of electrokinetic was higher than that of
366
microbes in the degradation of resins. Microbial enumeration, dehydrogenase activity
367
and soil basal respiration were related to resins removal in the soil. The results of
368
instrumental analysis indicate that there were significant structural changes in the resins
369
molecules after undergoing EK-BG treatment. According to the components analysis, a
370
considerable proportion of resins still remained in the soil, though the soil phytotoxicity
371
significantly declined. Thus, a better understanding of the interactions among
372
bioremediation, electrokinetic and the variation in resins is necessary, as this may
373
provide a theoretical basis for the regulation of the EK-BIO process, helping to improve
374
the degradation level of resins.
375
Acknowledgement
376
This work was supported by the National Natural Science Foundation of China
377
(no.51974313, 41907405) and the Natural Science Foundation of Jiangsu Province
378
(no.BK20180641).
379
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380
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Bissada, K.A., Tan, J., Szymczyk, E., Darnell, M., Mei, M., 2016. Group-type
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Chen, Y., Yang, C., Wang, Y., 2010. Gemini catalyst for catalytic aquathermolysis of
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Chen, F., Zeng, S., Ma, J., Li, X., Zhang, S., Zhu, Q., 2019a. Interactions between
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Chen, F., Li, X., Ma, J., Qu, J., Yang, Y., Zhang, S., 2019b. Remediation of soil
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co-contaminated with decabromodiphenyl ether (BDE-209) and copper by enhanced
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Dong, Z.Y., Huang, W.H., Xing, D.F., Zhang, H.F., 2013. Remediation of soil
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co-contaminated with petroleum and heavy metals by the integration of electrokinetics
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Drage, S., Engelmeier, D., Bachmann, G., Sessitsch, A., Mitter, B., Hadacek, F., 2012.
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Fan, R., Guo, S., Li, T., Li, F., Yang, X., Wu, B., 2015. Contributions of electrokinetics
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Garcia, C., Hernandez, T., Costa, F., 1997. Potential use of dehydrogenase activity as an
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Hassan, I., Mohamedelhassan, E., Yanful, E.K., Yuan, Z.C., 2016. A review article:
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Zhang, M., Guo, S., Li, F., Wu, B., 2017. Distribution of ion contents and
459
microorganisms during the electro-bioremediation of petroleum-contaminated saline
460
soil. J. Environ. Sci. Health A 52, 1141–1149.
22
Table 1
The experimental setup used in this study.
Group
Abbreviation
Voltage (V)
Inoculation
Control blank Electrokinetic biostimulation Bioaugmentation Electrokinetic-bioaugmentation
CK EK-BS BG EK-BG
– 25 – 25
No No Yes Yes
1
Table 2 Wheat seed germination ratios in resins-spiked soils across various sections after 60 days of treatments. Treatment
S1
S2
S3
S5
S5
CK EK-BS BG EK-BG
48% 75% 62% 90%
48% 71% 62% 84%
48% 66% 62% 77%
48% 72% 62% 85%
48% 77% 62% 92%
2
Table 3
FT-IR frequencies for the investigated samples and assignment Wavenumber (cm–1)
Assignment
Probable group
3000–2850 1710 1605 1461 1376 1260, 1215 1190 1030 868, 812 756
νCH νC=O νC=C, βNH δ(σ)CH2 δCH3 νC–O, νC–N γC–O νC–O, νC–N γCH ρCH2
CH2, CH3 COOH, COOR Aromatic rings, heterocyclic rings, NHR –CH2– –CH3– COOH, aromatic–OH, aromatic–NH R1–O–R2 R–OH, aromatic–O–R, RCH2NH2 Aromatic rings, heterocyclic rings –CH2–
3
Table 4 Results of 1H NMR of resins in S5 of EK-BG before and after 60 days of treatment. Untreated Treated
HA(%)
Hα(%)
Hβ(%)
Hγ(%)
fA
HAU/CA
5.18 2.06
15.73 8.75
57.02 60.25
22.07 28.94
0.292 0.103
0.338 0.826
4
Figure 1. Schematic diagram of the experimental set-up for the remediation system.
1
CK
8
EK-BS
BG
EK-BG
(A)
Soil pH
6
4
2
0 S1
S2
S3
S4
S5
Soil section (from anode)
CK
25
EK-BS
BG
EK-BG
(B) Soil moisture content (%)
20
15
10
5
0 S1
S2
S3
S4
S5
Soil section (from anode)
CK
Soil electroconductibility (mS/cm)
8
EK-BS
BG
EK-BG
(C)
6
4
2
0 S1
S2
S3
S4
S5
Soil section (from anode)
Figure 2. Variation of (A) pH, (B) moisture content and (C) electroconductibility in soil sections across various tests after 60 days of treatments.
2
CK
30
EK-BS
BG
EK-BG
Resins removal (%)
25
20
15
10
5
0 S1
S2
S3
S4
S5
Soil section (from anode)
Figure 3. Resins removal in soil sections across various tests after 60 days of treatments.
3
(A)
Bacterial density (log CFU/g soil)
10
S1
S2
S3
S4
S5
8
6
4
2
0
Soil basal respiration [µg CO2-C/(g soil.h)
Initial uninoculated
40
(B)
Initial inoculated
S1
CK
EK-BS
BG
EK-BG
Tests
S2
S3
S4
S5
30
20
10
0 Initial uninoculated
Initial inoculated
CK
EK-BS
BG
EK-BG
Tests
100
(C)
S1
S2
S3
S4
S5
Soil DHA [µg TPF/(g soil.6h)
80
60
40
20
0 Initial uninoculated
Initial inoculated
CK
EK-BS
BG
EK-BG
Tests
Figure 4. Soil bacterial density (A), basal respiration (B), and DHA (C) before and after 60 days of remediation across various soil sections (S1-S5). For simplicity, only one value was demonstrated for CK and BG since the difference among various soil sections was negligible. 4
Figure 5. FT-IR spectral analysis of resins in S5 of EK-BG before and after 60 days of treatment.
5
Highlights
Soil toxicity was significantly reduced after treatment
The structure of resins was altered during treatment
Soil microbial activity was positively related to resins degradation
I have made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work. I have drafted the work or revised it critically for important intellectual content; AND I have approved the final version to be published. I agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons who have made substantial contributions to the work reported in the manuscript, including those who provided editing and writing assistance but who are not authors, are named in the Acknowledgments section of the manuscript and have given their written permission to be named. If the manuscript does not include Acknowledgments, it is because the authors have not received substantial contributions from nonauthors.
Declaration of interests ☑ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0269-7491(19)37160-X
DOI:
https://doi.org/10.1016/j.envpol.2020.114047
Reference:
ENPO 114047
To appear in:
Environmental Pollution
Received Date: 2 December 2019 Revised Date:
14 January 2020
Accepted Date: 22 January 2020
Please cite this article as: Ma, J., Zhang, Q., Chen, F., Zhu, Q., Wang, Y., Liu, G., Remediation of resins-contaminated soil by the combination of electrokinetic and bioremediation processes, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2020.114047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
1
1
Remediation of resins-contaminated soil by the combination of electrokinetic and
2
bioremediation processes
3 4 5 6 7 8
Jing Maa,1, Qi Zhangb,1, Fu Chena,b, *, Qianlin Zhua, Yifei Wangb, Gangjun Liuc
9
221008, China
a
Low Carbon Energy Institute, China University of Mining and Technology, Xuzhou
10 11
b
12
and Technology, Xuzhou 221008, China
School of Environmental Science and Spatial Informatics, China University of Mining
13 14
c
15
Melbourne 3000, Australia
Geospatial Science, College of Science, Engineering and Health, RMIT University,
16 17
*Corresponding author. E-mail address: [email protected]
18
1
Qi Zhang and Jing Ma contributed equally to this work.
19 20 21 22 23 24
1
25
Abstract
26
In this work, soil contaminated by petroleum resins was remediated by
27
electrokinetic-bioremediation (EK-BIO) technology for 60 days. A microbial
28
consortium, comprising Rhizobium sp., Arthrobacter globiformis, Clavibacter xyli,
29
Curtobacterium flaccumfaciens, Bacillus subtilis, Pseudomonas aeruginosa and
30
Bacillus sp., was used to enhance the treatment performance. The results indicate that
31
resin removal and phytotoxicity reduction were highest in the inoculated EK process,
32
wherein 23.6% resins was removed from the soil and wheat seed germination ratio was
33
increased from 47% to around 90% after treatment. The microbial counts, soil basal
34
respiration and dehydrogenase activity were positively related to resins degradation, and
35
they could be enhanced by direct current electric field. After remediation, the C/H ratio
36
of resins decreased from 8.03 to 6.47. Furthermore, the structure of resins was analyzed
37
by Fourier-transform infrared spectroscopy, elemental analysis, and 1H nuclear magnetic
38
resonance (1H NMR) before and after treatment. It was found that the changes of the
39
structure of resins took place during EK-BIO treatment and finally led to the reduction
40
of aromaticity, aromaticity condensation and phytotoxicity.
41
Key words: Petroleum resins; phytotoxicity; dehydrogenase activity; seed germination;
42
aromaticity
43 44 45
2
46
1. Introduction
47
Soil petroleum contamination is one of the most serious soil pollution problems
48
among the existing environmental problems (Khan et al., 2018). When petroleum enters
49
the soil through various ways such as spills, leaks and accidents, soil organic matter
50
content increases, carbon:nitrogen (C:N) and carbon:phosphorus (C:P) ratios are
51
unbalanced, which leads to the loss of soil nutrients and the decline of metabolic
52
capacity in the soil.
53
Crude oil is an extremely complex mixture of hydrocarbons containing thousands
54
of components. On the basis of the common separation procedure, crude oil can be
55
grouped into four major classes: saturate hydrocarbon (saturates), aromatic hydrocarbon
56
(aromatics), resins and asphaltenes (SARA) (Bissada et al., 2016). Heavy crude oil
57
reserves has a large proportion in the total crude oil resources in China. For example, in
58
Liaohe Oilfield, the content of resins and asphaltenes is up to 34% and 12%,
59
respectively (Li et al., 2017).
60
As the most abundant heavy component of heavy oil, resins has been a difficult
61
problem in the process of petroleum-contaminated soil remediation. Resins is comprised
62
of many condensed ring units and abundant in polar substituents containing nitrogen,
63
sulfur and/or oxygen (Li et al., 2017). Resins has a high molecular weight and strong
64
polarity, and its degradation is abiotically and biotically difficult in the environment,
65
leading to the accumulation and potential environmental hazards. Therefore, the
66
removal of resins and the reduction of its toxicity in petroleum-contaminated soil have
3
67
become an urgent issue to be solved during remediation practice.
68
As a green and efficient remediation technology, electrokinetic-bioremediation
69
(EK-BIO) has been tested for remediation of various organic-contaminated soils
70
including petroleum-contaminated soil (Dong et al., 2013; Hassan et al., 2016; Zhang et
71
al., 2017). On the one hand, electrodialysis, electromigration, electrophoresis and
72
electrochemical oxidation reactions are involved in EK process. On the other hand, EK
73
technology can provide good living conditions for microbial populations, promoting
74
microbial activity and enhancing biodegradation of pollutants. However, due to its
75
macromolecular characteristics, resins is difficult to degrade, and the improvement of
76
resins degradation by EK-BIO is limited. Thus, the degradation rate of resins is
77
insufficient to accurately and comprehensively describe the remediation effect.
78
Existing studies demonstrate that both photooxidation and microbial degradation
79
can significantly alter the molecular structure of resins (Akhmedbekova et al., 2009;
80
Minai-Tehrani et al., 2015). Fourier-transform infrared spectroscopy (FT-IR) analysis
81
revealed that, during photooxidation of resins, quinone structure was produced, the
82
content of carboxyl groups increased, and sulfoxide groups were oxidized
83
(Akhmedbekova et al., 2009). Nevertheless, these studies only focused on the structural
84
changes of resins, and whether the ecotoxicity has changed or not was unknown.
85
In this study, EK-BIO technology was used to remediate soil contaminated with
86
resins, and the changes of molecular structure, bioavailability, ecotoxicity and soil
87
microbial activity were explored before and after the remediation process. This study
4
88
aimed to improve the index system of petroleum-contaminated soil remediation, and to
89
provide a scientific basis for the degradation of resins during the remediation process.
90
2. Materials and methods
91
2.1. Soil samples
92
An uncontaminated soil was collected from a field in the campus of China
93
University of Mining and Technology, Xuzhou, China. The soil was air-dried and sieved
94
through a 20-mesh sieve. The characteristics of the soil are: pH (1:2.5 water), 6.9; sand,
95
63.6 wt.%; silt, 21.5 wt.%; clay, 14.9 wt.%; cation exchange capacity (CEC), 9.6
96
cmol/kg; total organic carbon (TOC), 5.3 g/kg; particle size distribution: < 2 µm 18.4%,
97
2–50 µm 58.6%, 50–2000 µm 23.0%. No petroleum contamination was detected in the
98
soil.
99
Resins fraction was extracted from a heavy crude oil (resins content 24%) through
100
the SARA method (Bissada et al., 2016). The oil was obtained from the Liaohe Oilfield,
101
China. Resins was dissolved into dichloromethane and then spiked into the soil to reach
102
an initial content of about 4.5 g/kg after solvent evaporation and one month of
103
equilibrium.
104
2.2. Microbial culture
105
A microbial consortium capable of degrading crude oil was isolated by selective
106
enrichment from petroleum-contaminated soil in Liaohe Oilfield, China. The strains of
107
the consortium were identified as Rhizobium sp., Arthrobacter globiformis, Clavibacter
108
xyli, Curtobacterium flaccumfaciens, Bacillus subtilis, Pseudomonas aeruginosa and
5
109
Bacillus sp. respectively by means of genetic methods. The microbial culture was
110
inoculated into the soil at the ratio of 2.0 ×107 colony forming units (CFU) per g soil.
111
2.3. Experimental methods
112
Figure 1 shows the schematic design of the experimental setup used in this study.
113
The setup mainly consists of a direct-current power supply, two electrode chambers;
114
two graphite electrodes, and a soil chamber. The assembly of EK test compartments was
115
made of Pyrex glass. The dimension of soil chamber was 25 cm × 12 cm × 12 cm, and 2
116
kg of dry soil was loaded. The dimension for electrode chambers is 10 cm × 10 cm × 10
117
cm. The electrode compartments contain a filter paper, a porous plate and a perforated
118
graphite electrode. To prevent soil particles from penetrating into the electrolyte
119
solution reservoirs, the filter paper was placed between the soil and the porous stone and
120
both were placed in front of the electrode. A direct-current power supply (RXN-303A,
121
Zaoxin Instrument Co., Ltd., China) was used to maintain a constant voltage of 25 V,
122
resulting in a voltage gradient of 1.0 V/cm. For all treatments, 0.01 M NaNO3 was used
123
as the supporting electrolyte. The soil was saturated with 0.01 M NaNO3 solution for 24
124
h before application of electrical field. Once the soil was loaded into the cell, the cell
125
assembly was completed. The experiments were conducted at a room temperature, and
126
all aqueous solutions were prepared using deionized water. To prevent anode
127
acidification and cathode alkalization, the electrolyte pH was controlled at around 6.9
128
(namely the background pH of soil) by an automatic pH controller.
129
Soil samples were moistened with an inorganic medium to provide 20% (w/w)
6
130
moisture content for nonflooded conditions. The composition of the medium is as
131
follows: NaNO3 1.5 g/L, (NH4)2SO4 1.5 g/L, K2HPO4 1.0 g/L, KCl 0.5 g/L,
132
MgSO4•7H2O 0.5 g/L, FeSO4•7H2O 0.01 g/L, CaCl2 0.002 g/L, pH 7.0. The medium
133
was autoclaved at 121 °C for 20 min at 0.11 MPa before use.
134
Table 1 summarizes the experimental setup involved in the experimental program.
135
CK was a baseline test conducted to evaluate the bioremediation potential of soil
136
microbial indigenous population. EK-BS was performed to evaluate the joint
137
performance of EK and indigenous microbial populations. BG was aimed to reveal the
138
combined influence of exogenous and indigenous microbes on pollutant removal with
139
no electricity. EK-BG was carried out to investigate the effect of exogenous microbes
140
on the EK system behavior.
141
The experiments lasted for 60 days, carried out at room temperature (25±2 °C).
142
Soil moisture content was kept at around 20% by regular addition of sterile inorganic
143
medium (for EK-BS and EK-BG) or distilled water (for CK and BG). After each
144
experiment of 10 days, the soil was removed from the apparatus, cut into five equal
145
slices (S1–S5, from anode to cathode) and characterized for microbial parameters and
146
concentration of residual contaminants.
147
2.4. Analytical methods
148
The soil pH was determined by using a pH meter (model pHS-3B, Shanghai
149
Precision Scientific Instrument Co., Ltd., China) with a solid:water ratio of 1:2.5. TOC
150
was measured by catalytic oxidation on a TOC analyzer (Shimadzu TOC-VCPH, Kyoto,
7
151
Japan). CEC was determined after leaching of <2 mm air-dried soil with 1 M
152
CH3COONH4 at pH 7.0. Soil particle size was analyzed using the pipette method
153
(Miller and Miller, 1987).
154
2.5. Extraction, determination and characterization of resins
155
Soil samples were freeze-dried, ground and passed through 20-mesh sieve, and
156
then the soil samples were extracted on an accelerated solvent extractor system (ASE
157
300, Dionex, Sunnyvale, CA, USA) with dichloromethane. The extract was dried under
158
N2 flow to constant final weight, and then resins content was determined
159
gravimetrically.
160
Resins samples were subjected to a series of instrumental analyses including
161
Fourier-transform infrared spectroscopy (FT-IR), elemental analysis, and 1H nuclear
162
magnetic resonance (NMR). FT-IR spectra were collected on a Nicolet 6700 FT-IR
163
(Thermo Scientific Corp., Madison, WI, USA). C, H and N contents were determined
164
on a Carlo-Erba model 1106 elemental analyzer (Carlo Erba, Milano, Italy). 1H NMR
165
spectroscopic measurements were carried out with a Bruker 400 MHz Spectrometer
166
using tetramethylsilane (TMS) as an internal standard in chloroform-d (CDCl3).
167
2.6. Microbial activity analysis
168
Soil bacteria were enumerated on nutrient agar plates through plate spread method
169
(five replicates per sample) (Lu and Zhang, 2014). The total number of colonies was
170
counted after incubation at 28 °C in the dark for 2 days, and the results were expressed
171
as CFU per gram of dry soil.
8
172
Soil basal respiration was analyzed using the MicroResp™ method and 15 carbon
173
sources in addition to water (basal respiration rate control) on a Spectra Max 190
174
microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm (Drage et al.,
175
2012).
176
Microbial dehydrogenase activity (DHA) in soil samples was determined following
177
the method of Lu et al. (2009), and results were expressed as µg TPF/(g soil•6 h).
178
Controls were prepared with autoclaved samples (121 °C, 60 min) and treated like
179
biotic samples.
180
2.7. Seed germination experiments
181
Wheat seed germination experiments were performed to analyze soil toxicity
182
changes before and after remediation. Plumped and uniform-sized seeds were selected,
183
soaked in 3% H2O2 solution for 20 min, rinsed with sterile water, and then immersed in
184
sterile water for 24 h under darkness before sowing. Fifty seeds were uniformly placed
185
in petri dishes, covered with the prepared soils (about 1 cm thickness), and kept in a
186
growth chamber for 10 days. The details have been described previously (Chen et al.,
187
2019a).
188
2.8. Data analysis
189
In this study, the results are reported as means ± standard deviations on the basis of
190
dry weight. Statistical analysis of the results was performed with SPSS 11.0 for
191
Windows (SPSS Inc.).
192
3. Results and discussion
9
193
3.1. Changes of soil physicochemical properties
194
Figure 2 shows the variation of soil pH, moisture content and electroconductibility
195
in different soil sections after 60 days of treatments. In CK and BG, various indexes
196
changed little. In EK-BS and EK-BG, the pH profile followed the trend with pH
197
increasing from 6.7 near the anode (S1) to 7.1 near the cathode (S5) [Figure 2(A)].
198
Moreover, no apparent difference was observed in soil pH between EK-BS and EK-BG.
199
This stable pH can be ascribed to the pH control of electrolyte. Generally, electrolysis
200
reactions may cause a low pH at anode and a high pH at cathode due to the generation
201
of H+ and OH– ions at anode and cathode, respectively (Chen et al., 2019b).
202
The soil moisture content kept relatively stable across various sections among
203
different tests [Figure 2(B)]. Basically, the moisture content was in the range of 16–22%.
204
Figure 2(C) shows the distribution of soil electroconductibility after 60 days. The initial
205
soil electroconductibility was 1.7 mS/cm. Soil electroconductibility decreased from S1
206
to S5 in EK-BS and EK-BG whereas kept constant in CK and BG, which followed the
207
same pattern as the pH of soil sections [Figure 2(C)]. Nevertheless, the difference in the
208
electroconductibility of soil sections was insignificant in EK-BS and EK-BG. The
209
mobility of ions under EK conditions can result in the variation of soil
210
electroconductibility. Soil electroconductibility depends on pH, and high pH may reduce
211
electroconductibility because of metal precipitation, whereas a lower pH can lead to a
212
higher electroconductibility value due to enhanced mobility of metal ions (Chen et al.,
213
2019b). In this study, the supplement of inorganic medium to the soil could alleviate
10
214
electroconductibility alteration caused by ion migration.
215
3.2. Resins removal
216
Figure 3 demonstrates resins removal in the soil sections after the 60-day treatment.
217
Resins was not detected in electrolyte, indicating no migration of resins under electric
218
field because of its high hydrophobicity and extremely low solubility. In CK and BG,
219
the concentration of resins was found to be evenly distributed throughout the soil
220
column. The application of EK enhanced resins removal in the soil as compared to CK
221
and BG (Figure 3). After 60 days, the average removal ratio of resins followed the order
222
of EK-BG (23.6%) > EK-BS (9.3%) > BG (6.5%) > CK (1.2%). It can be seen that the
223
combined application of EK and microbial inoculant has an enhanced effect on resins
224
degradation, and the degradation ratio was higher than that of EK or bioremediation
225
alone. The soil was artificially contaminated with resins, and effective resins-degrading
226
microbial community could not evolve in a short time, thus resins removal was
227
extremely low in CK (Figure 3). It is also found that the maximum removal in EK-BG
228
and EK-BS was obtained adjacent to the electrodes, and the removal extents of resins
229
would decrease with the distance to the electrodes. For example, the removal extent of
230
resins in S3 was 15.2% and 6.4% in EK-BG and EK-BS, respectively, as compared to
231
28.3% and 12.2% in S5 of EK-BG and EK-BS, respectively. These results demonstrate
232
that resins could be more favourably degraded towards electrodes, and the degradation
233
ability of microbes was evidently enhanced by the application of electric field.
234
Due to its high hydrophobicity, resins cannot be removed by means of
11
235
electro-osmosis, -migration or -phoresis, such that the removal of resins in EK-BG and
236
EK-BS can be attributed to electrochemical oxidation. During the EK-BIO process,
237
resins can be not only directly degraded by electrochemical oxidation and microbes, but
238
also indirectly oxidatively broken down by chlorine, hypochlorite and hydrogen
239
peroxide, which are generated by anodic reaction and water electrolysis reaction (Zhang
240
et al., 2017). However, the chemical stability and microbial degradation resistance of
241
resins resulted in a smaller removal extent in both EK-BG and EK-BS experiments. In
242
this study, the higher removal occurring adjacent to the electrodes indicates that the
243
strong electrochemical reactions appeared near the electrodes. An electric field can
244
improve the mass transfer between pollutants and microbes by dispersing them, leading
245
to enhanced bioavailability of soil pollutants (Wick et al., 2007). The density of electric
246
field is a driving force for mass transfer in the electric field; thus, the closer to the
247
electrodes, the greater the mass transfer formed (Fan et al., 2015). A better opportunity
248
for interaction between pollutants and microbes in the vicinity of electrodes resulted in a
249
higher resins removal extent. Resins constitutes a large and diverse class of organic
250
components, and the effectiveness of biodegradation or EK alone may be limited by
251
various factors. In this work, EK-BG showed a significant improvement in resins
252
removal relative to other treatments, signifying a synergistic effect between
253
electrokinetic and bioremediation. In addition, resins removal in EK-BG (23.6%) was
254
higher than the sum value of EK-BS (9.3%) and BG (6.5%), suggesting that
255
bioremediation was greatly stimulated by the electric field under the EK-BG treatment.
12
256
This may be due to the higher microbial activity, or the enhanced interaction between
257
pollutants and microbes in EK-BG.
258
3.3. Phytotoxicity evaluation
259
Seed germination tests have been widely used for soil phytotoxicity evaluation
260
(Jiang et al., 2016). Table 2 reveals the inhibitory influences of resins on the
261
germination of wheat. The control soil without resins addition and the contaminated soil
262
before treatment had a germination of 98% and 47%, respectively.
263
It can be found from Table 2 that the germination ratio followed the order of
264
EK-BG > EK-BS > BG > CK. Moreover, the germination ratio would increase with
265
approaching the electrodes. These situations were in agreement with that of resins
266
removal, namely higher removal corresponded to higher seed germination ratio. It is
267
also observed that the germination ratio of EK-BG was close to that of uncontaminated
268
soil, though EK-BG exhibited a low resins removal (averagely 23.6%). This indicates
269
that soil phytotoxicity can be efficiently reduced by resins degradation in EK-BG.
270
3.4. Microorganism and its activity in soil
271
Figure 4(A) shows the change of bacterial population in different regions of soil
272
specimen during remediation. The initial bacterial number (namely after one month of
273
equilibrium) was about 3.5×106 and 2.3×107 CFU/g soil in uninoculated (CK and
274
EK-BS) and inoculated (EK-BS and EK-BG) soils, respectively. As shown in Figure
275
4(A), the difference of bacterial density in different sampling points after remediation
276
was basically consistent with the trend of resins removal ratio. EK-BG showed the
13
277
highest bacterial density among the five cases. A general increase in soil bacterial
278
density was also observed relative to the initial situations [Figure 4(A)], which was due
279
to nutrient stimulation in this work. These findings suggest that the increase of
280
microbial populations was related to the decrease of soil resins content, and the growth
281
and reproduction of microbes could be promoted by direct current electric field. The
282
transport and distribution of microbes in soil column under electric field depend on
283
many factors such as electrophoresis, electroosmosis, and nutrient flow (Dong et al.,
284
2013). Because of their negatively charged surfaces, bacteria have a negative
285
electrokinetic potential and bacterial movement towards anode would occur because of
286
electrical attraction between anode and bacteria (electrophoresis) (Dong et al., 2013).
287
Moreover, bacteria may migrate toward cathode along with electroosmotic flow (EOF)
288
through soil (electroosmosis). Under electric field, ions and water in soil were migrated
289
toward the cathode by EOF, leading to favorable conditions for bacterial growth in the
290
region adjacent to the cathode. Thus, the final bacterial distribution in soil column came
291
from the competition between electrophoresis, electroosmosis, and nutrient flow.
292
Figure 4(B) exhibits the change of soil basal respiration in different regions of soil
293
specimen during remediation. Generally, the basal respiration values were in the order:
294
EK-BG > EK-BS > BG > CK >initial. The basal respiration was highest in S5 of
295
EK-BG [38.2 µg CO2-C/(g soil•h)], which was 2.3 and 1.8 times that of initial value
296
[16.6 µg CO2-C/(g soil•h)] and BG [21.2 µg CO2-C/(g soil•h)]. Soil basal respiration is
297
the sum of all metabolic processes that produce CO2, and it is a good indicator of
14
298
organics decomposition and changes in soil environment under pollution stress.
299
DHA is recognized as an important index of soil microbial activity, being capable
300
of catalyzing the oxidation of organic compounds by transferring protons and electrons
301
(Garcia et al., 1997). The variations of DHA in the experiments before and after 60 days
302
of treatment are presented in Figure 4(C). The average DHA in EK-BG was higher than
303
that in other experiments after 60 days of treatment. At the end of the experiments, the
304
average DHA was 77.3, 52.1, 37.8 and 22.5 µg TPF/(g soil•6h) in EK-BG, EK-BS, BG
305
and CK, respectively. The soil DHA increased significantly after 60 days of treatment (P
306
< 0.01). Sampling site S3 was the least influenced by the electrochemical reaction and
307
was located in the weakest electric field intensity area; therefore, the soil DHA was
308
slightly lower than those at other sites with no significant differences. Sampling sites S1
309
and S5 were closer to the electrodes, and the generation of oxidizing and reducing zones
310
near the electrodes could provide beneficial conditions for the degraders (Lohner et al.,
311
2011).
312
It can be seen from the changes of bacterial density, basal respiration and DHA
313
[Figure 4(A), (B) and (C)] that the activity of microbes in the soil after remediation was
314
significantly increased, and the trend was basically consistent with the removal ratio of
315
resins, indicating that the ecotoxicity of the soil after remediation was effectively
316
reduced. Bacterial cell respiration can be enhanced by anodic oxygen generated by
317
electrolysis (Fan et al., 2015). The generation of anodic oxygen might elevate dissolved
318
oxygen level in the soil, and enhance the respiration of microbes and indirectly increase
15
319
the redox level of soil, thus stimulating DHA and improving the biodegradation of
320
resins.
321
3.5. FT-IR analysis of resins
322
FT-IR spectral analyses of resins are shown in Figure 5. Frequencies and probable
323
assignments of each peak of the spectra are shown in Table 3. It can be seen from Figure
324
5 that the peak intensities for the characteristic region of CH2 and CH3 groups (2920,
325
2853, 1461, 1376 and 756 cm–1 wavenumbers) declined, indicating the shortening of
326
resins carbon chains. The peak intensities at 1710 and 1215 cm–1 decreased obviously
327
after treatment, demonstrating the reduction of carboxyl and phenolic groups. The peak
328
intensities at 1605 and 868 cm–1 (the characteristic regions of aromatic and heterocyclic
329
rings) also declined, suggesting that the aromatic and heterocyclic rings of resins were
330
opened or degraded into saturated rings during remediation. A new peak occurred at
331
1030 cm–1 after remediation, indicating the formation of new functional groups such as
332
R–OH and aromatic–O–R.
333
3.6. Elemental analysis of resins
334
Elemental analysis was conducted on the resins in S5 of EK-BG before and after
335
60 days of treatment. Results show that the mass fractions of C, H, and N were 74.86%,
336
9.315% and 0.848% respectively for the untreated sample, and 68.02%, 10.52% and
337
0.783% respectively for the treated sample. After remediation, the mass fraction of C
338
declined whilst that of H increased, and the C/H ratio decreased from 8.03 to 6.47. This
339
indicates that some unsaturated rings of resins were degraded into saturated ones,
16
340
leading to the decrease in unsaturation of resins. On the basis of the mass fractions of C,
341
H, and N, it can be calculated that the average chemical formula of resins was
342
C103H153N and C101H187N for the untreated and treated samples, respectively.
343
3.7. 1H NMR analysis
344
To study the structure changes of resins, 1H NMR analysis was performed on the
345
resins in S5 of EK-BG before and after 60 days of treatment. On the basis of the
346
assignment of protons in 1H NMR spectrum (Chen et al., 2009), the percentages of
347
integrated areas were obtained, as listed in Table 4. Aromaticity fA and aromaticity
348
condensation HAU/CA were obtained using Eqs. (1) and (2), as demonstrated (Chen et al.,
349
2010):
CT / H T − ( H α + H β + H γ / 2 H T ) CT / H T
350
fA =
351
H AU H A / H T + Hα / 2 H T = CA CT / H T − ( H α + H β + H γ ) / 2 H T
(1)
(2)
352
where CT and HT represent the total C and H, respectively, HT = HA+ Hα+ Hβ+ Hγ;
353
CT/HT is the C/H ratio.
354
It is easy to see from Table 4 that the percentage of HA and Hα decreased whilst
355
that of Hβ and Hγ increased after treatment, namely the percentage of H atoms in
356
aromatic rings declined. Moreover, the aromaticity and aromaticity condensation of
357
resins decreased after treatment. The decrease of the former could be caused by the
358
hydrogenation of unsaturated groups while that of the latter could be attributed to the
359
ring-opening of the aromatic system.
17
360
4. Conclusions
361
The present study has demonstrated that the efficiency of resins degradation was
362
stimulated by the electric field and bioaugmentation in the coupled technology of
363
EK-BIO, signifying a synergistic effect between electrokinetic and bioremediation. The
364
contribution of inoculated microbes was higher than that of indigenous counterparts in
365
the degradation of resins, and the contribution of electrokinetic was higher than that of
366
microbes in the degradation of resins. Microbial enumeration, dehydrogenase activity
367
and soil basal respiration were related to resins removal in the soil. The results of
368
instrumental analysis indicate that there were significant structural changes in the resins
369
molecules after undergoing EK-BG treatment. According to the components analysis, a
370
considerable proportion of resins still remained in the soil, though the soil phytotoxicity
371
significantly declined. Thus, a better understanding of the interactions among
372
bioremediation, electrokinetic and the variation in resins is necessary, as this may
373
provide a theoretical basis for the regulation of the EK-BIO process, helping to improve
374
the degradation level of resins.
375
Acknowledgement
376
This work was supported by the National Natural Science Foundation of China
377
(no.51974313, 41907405) and the Natural Science Foundation of Jiangsu Province
378
(no.BK20180641).
379
References
380
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22
Table 1
The experimental setup used in this study.
Group
Abbreviation
Voltage (V)
Inoculation
Control blank Electrokinetic biostimulation Bioaugmentation Electrokinetic-bioaugmentation
CK EK-BS BG EK-BG
– 25 – 25
No No Yes Yes
1
Table 2 Wheat seed germination ratios in resins-spiked soils across various sections after 60 days of treatments. Treatment
S1
S2
S3
S5
S5
CK EK-BS BG EK-BG
48% 75% 62% 90%
48% 71% 62% 84%
48% 66% 62% 77%
48% 72% 62% 85%
48% 77% 62% 92%
2
Table 3
FT-IR frequencies for the investigated samples and assignment Wavenumber (cm–1)
Assignment
Probable group
3000–2850 1710 1605 1461 1376 1260, 1215 1190 1030 868, 812 756
νCH νC=O νC=C, βNH δ(σ)CH2 δCH3 νC–O, νC–N γC–O νC–O, νC–N γCH ρCH2
CH2, CH3 COOH, COOR Aromatic rings, heterocyclic rings, NHR –CH2– –CH3– COOH, aromatic–OH, aromatic–NH R1–O–R2 R–OH, aromatic–O–R, RCH2NH2 Aromatic rings, heterocyclic rings –CH2–
3
Table 4 Results of 1H NMR of resins in S5 of EK-BG before and after 60 days of treatment. Untreated Treated
HA(%)
Hα(%)
Hβ(%)
Hγ(%)
fA
HAU/CA
5.18 2.06
15.73 8.75
57.02 60.25
22.07 28.94
0.292 0.103
0.338 0.826
4
Figure 1. Schematic diagram of the experimental set-up for the remediation system.
1
CK
8
EK-BS
BG
EK-BG
(A)
Soil pH
6
4
2
0 S1
S2
S3
S4
S5
Soil section (from anode)
CK
25
EK-BS
BG
EK-BG
(B) Soil moisture content (%)
20
15
10
5
0 S1
S2
S3
S4
S5
Soil section (from anode)
CK
Soil electroconductibility (mS/cm)
8
EK-BS
BG
EK-BG
(C)
6
4
2
0 S1
S2
S3
S4
S5
Soil section (from anode)
Figure 2. Variation of (A) pH, (B) moisture content and (C) electroconductibility in soil sections across various tests after 60 days of treatments.
2
CK
30
EK-BS
BG
EK-BG
Resins removal (%)
25
20
15
10
5
0 S1
S2
S3
S4
S5
Soil section (from anode)
Figure 3. Resins removal in soil sections across various tests after 60 days of treatments.
3
(A)
Bacterial density (log CFU/g soil)
10
S1
S2
S3
S4
S5
8
6
4
2
0
Soil basal respiration [µg CO2-C/(g soil.h)
Initial uninoculated
40
(B)
Initial inoculated
S1
CK
EK-BS
BG
EK-BG
Tests
S2
S3
S4
S5
30
20
10
0 Initial uninoculated
Initial inoculated
CK
EK-BS
BG
EK-BG
Tests
100
(C)
S1
S2
S3
S4
S5
Soil DHA [µg TPF/(g soil.6h)
80
60
40
20
0 Initial uninoculated
Initial inoculated
CK
EK-BS
BG
EK-BG
Tests
Figure 4. Soil bacterial density (A), basal respiration (B), and DHA (C) before and after 60 days of remediation across various soil sections (S1-S5). For simplicity, only one value was demonstrated for CK and BG since the difference among various soil sections was negligible. 4
Figure 5. FT-IR spectral analysis of resins in S5 of EK-BG before and after 60 days of treatment.
5
Highlights
Soil toxicity was significantly reduced after treatment
The structure of resins was altered during treatment
Soil microbial activity was positively related to resins degradation
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Declaration of interests ☑ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: