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Article
Ecological effects of combined pollution associated with e-waste recycling on the composition and diversity of soil microbial communities Jun Liu, Xiao-Xin He, Xue-Rui Lin, Wen-Ce Chen, Qi-Xing Zhou, Wensheng Shu, and Li-Nan Huang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5049804 • Publication Date (Web): 28 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
Environmental Science & Technology
1 2
Ecological effects of combined pollution associated with e-waste recycling on the
3
composition and diversity of soil microbial communities
4 5
Jun Liu,†,# Xiao-xin He,†,# Xue-rui Lin,‡ Wen-ce Chen,† Qi-xing Zhou,§ Wen-sheng Shu,†
6
Li-nan Huang*,†
7 8
†
9
Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution,
State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and
10
Sun Yat-sen University, Guangzhou 510275, PR China
11
‡
12
Guangzhou 510655, PR China
13
§
14
Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of
15
Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China
South China Institute of Environmental Sciences, Ministry of Environmental Protection,
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education),
16 17
#
18
*Corresponding author
19
College of Ecology and Evolution, Sun Yat-sen University, Guangzhou 510275, PR China
20
Tel.: +86 20 39332933
21
Fax: +86 20 39332944
22
E-mail: [email protected]
These authors contributed equally to this work
1
ACS Paragon Plus Environment
Environmental Science & Technology
23
ABSTRACT: The crude processing of electronic waste (e-waste) has led to serious
24
contamination in soils. While microorganisms may play a key role in remediation of the
25
contaminated soils, the ecological effects of combined pollution (heavy metals,
26
polychlorinated biphenyls and polybrominated diphenyl ethers) on the composition and
27
diversity of microbial communities remain unknown. In this study, a suite of e-waste
28
contaminated soils were collected from Guiyu, China, and the indigenous microbial
29
assemblages were profiled by 16S rRNA high-throughput sequencing and clone library
30
analysis. Our data revealed significant differences in microbial taxonomic composition
31
between the contaminated and the reference soils, with Proteobacteria, Acidobacteria,
32
Bacteroidetes and Firmicutes dominating the e-waste-affected communities. Genera
33
previously identified as organic pollutants-degrading bacteria, such as Acinetobacter,
34
Pseudomonas and Alcanivorax, were frequently detected. Canonical correspondence analysis
35
revealed that approximately 70% of the observed variation in microbial assemblages in the
36
contaminated soils was explained by eight environmental variables (including soil
37
physiochemical parameters and organic pollutants) together, among which moisture content,
38
decabromodiphenyl ether (BDE-209) and copper were the major factors. These results
39
provide the first detailed phylogenetic look at the microbial communities in e-waste
40
contaminated soils, demonstrating that the complex combined pollution resulting from
41
improper e-waste recycling may significantly alter soil microbiota.
42 43 44
2
ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
45
Environmental Science & Technology
INTRODUCTION
46
Discarded or unused electrical and electronic devices, generally referred to as electronic
47
waste (e-waste), are an increasingly important environmental problem worldwide. It has been
48
estimated that globally about 40 million tons of e-waste are generated each year,(1) with an
49
increasing rate of 4% per year.(2) The United States Environmental Protection Agency
50
estimates that only 15-20% of e-waste can be recycled while the remaining parts are often
51
disposed of in landfills.(3) However, e-waste typically contains high levels of toxic substances
52
such as organic pollutants (e.g. polychlorinated biphenyls (PCBs) and polybrominated
53
diphenyl ethers (PBDEs)) and heavy metals (e.g. lead, zinc, copper and cadmium);(2) if
54
disposed of improperly, these hazardous substances pose a serious threat to the environment
55
and human health.(2) Notably, most e-waste has been transported to developing countries, in
56
particular China, where these waste materials are processed extensively (e.g. burning and
57
scouring) for recovery of precious metals and the residues are improperly discarded.(4) These
58
practices have caused serious pollution problems for air, terrestrial and aquatic
59
environments.(4)
60
Microbes drive the bulk of Earth’s biogeochemical cycles and are crucial to the functioning
61
of virtually all ecosystems. However, microbes and especially their metabolic activities are
62
sensitive to environmental change. Numerous studies have demonstrated that environmental
63
pollution may cause drastic changes in microbial community composition and activity,(5, 6)
64
or have found significant correlations between microbial diversity and contamination
65
gradients.(7, 8) Typically, pollution could result in a decrease of microbial diversity and
66
enrichment of tolerant species via the process of environmental filtering; this in turn may
3
ACS Paragon Plus Environment
Environmental Science & Technology
67
affect their overall ecosystem function.(5) On the other hand, microorganisms may be actively
68
involved in the degradation and transformation of various pollutants and play a crucial role in
69
the remediation of polluted environments.(9)
70
In the past two decades, the influence of heavy metal contamination on soil microbial
71
communities has attracted much attention,(5, 7, 10, 11) and many species (e.g. Pseudomonas
72
spp., Acinetobacter spp. and Sphingomonas spp.) resistant and tolerant to heavy metals (e.g.
73
copper, zinc and cadmium) have been isolated.(5, 12) Meanwhile, recent research has focused
74
on investigating bacterial community composition in soils polluted by toxic organic
75
pollutants(13-15) and obtaining in pure culture functional bacteria (e.g. Acinetobacter spp.,
76
Pseudomonas spp. and Alcanivorax spp.) capable of degrading PBDEs, PCBs and polycyclic
77
aromatic hydrocarbons.(16-18) Subsequently, the mechanisms of microbial degradation in
78
these bacteria have been studied, which will help improve degradative capabilities for the
79
bioremediation of polluted environments.(19-21) Furthermore, some laboratory studies have
80
examined both single and combined influences of individual metals or individual organic
81
compounds on soil microbial assemblages.(22-24) However, field studies with a specific
82
focus on the effects of combined pollution (e.g. PBDEs and heavy metals, and PCBs and
83
heavy metals) on the indigenous microorganisms have been very limited.(13, 15, 25)
84
Improper e-waste processing could result in heavy metal and toxic organic compound
85
contamination in soil.(2) However, the responses of soil microbiota as a whole community to
86
the combined pollution (including PCBs, PBDEs and heavy metals) and its ecological
87
consequences have not been explored. Here, we report an in-depth and comprehensive survey
88
of the microbial assemblages from a suite of soils contaminated by uncontrolled e-waste
4
ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
Environmental Science & Technology
89
recycling activities. Microbial populations were profiled by both Illumina high-throughput
90
sequencing and clone library analysis targeting the 16S rRNA genes that were then
91
taxonomically analyzed. Specifically, we aimed to elucidate the composition and diversity of
92
microbes in the e-waste affected soils and examine how microbial assemblages are shaped by
93
the environmental conditions.
94 95
MATERIALS AND METHODS
96
Site Description and Sample Collection. Guiyu, a town with a total area of 52 km2 and a
97
population of 400,000 in Guangdong Province, China, has been a booming e-waste
98
processing center since 1995.(26) Due to crude e-waste recycling activities in a large number
99
of simple household e-waste recycling workshops, the air, water and soil have been heavily
100
contaminated by toxic organic compounds and heavy metals.(2, 26) In November 2011,
101
representative contaminated soil samples were collected in triplicate at a depth of 0-10 cm
102
from two e-waste dumping sites (Dump-1 and Dump-2), two e-waste burning sites (Burn-1
103
and Burn-2), one acid stripping site (Acid-strip), one contaminated farmland site (Farmland)
104
and one contaminated mudflat site near the Lianjiang river (Mudflat) (Figure 1). For
105
comparison, triplicate samples were also collected from each of the five corresponding
106
reference soil types (Dump_ck, Burn_ck, Acid-strip_ck, Farmland_ck and Mudflat_ck) that
107
were well apart in the upstream direction from the contaminated sites and thus unlikely
108
affected by e-waste. All samples were kept in cooler boxes during sampling and then
109
transported to the laboratory where they were stored at 4 °C prior to processing (within 48 hrs)
110
for subsequent analyses (see below).
5
ACS Paragon Plus Environment
Environmental Science & Technology
111
Physicochemical Analyses. Moisture content was determined by weighing subsamples
112
before and after oven-drying at 105 °C for 24 h. Samples were air-dried and sieved to
Article
Ecological effects of combined pollution associated with e-waste recycling on the composition and diversity of soil microbial communities Jun Liu, Xiao-Xin He, Xue-Rui Lin, Wen-Ce Chen, Qi-Xing Zhou, Wensheng Shu, and Li-Nan Huang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5049804 • Publication Date (Web): 28 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
Environmental Science & Technology
1 2
Ecological effects of combined pollution associated with e-waste recycling on the
3
composition and diversity of soil microbial communities
4 5
Jun Liu,†,# Xiao-xin He,†,# Xue-rui Lin,‡ Wen-ce Chen,† Qi-xing Zhou,§ Wen-sheng Shu,†
6
Li-nan Huang*,†
7 8
†
9
Conservation of Guangdong Higher Education Institutes, College of Ecology and Evolution,
State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and
10
Sun Yat-sen University, Guangzhou 510275, PR China
11
‡
12
Guangzhou 510655, PR China
13
§
14
Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of
15
Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China
South China Institute of Environmental Sciences, Ministry of Environmental Protection,
Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education),
16 17
#
18
*Corresponding author
19
College of Ecology and Evolution, Sun Yat-sen University, Guangzhou 510275, PR China
20
Tel.: +86 20 39332933
21
Fax: +86 20 39332944
22
E-mail: [email protected]
These authors contributed equally to this work
1
ACS Paragon Plus Environment
Environmental Science & Technology
23
ABSTRACT: The crude processing of electronic waste (e-waste) has led to serious
24
contamination in soils. While microorganisms may play a key role in remediation of the
25
contaminated soils, the ecological effects of combined pollution (heavy metals,
26
polychlorinated biphenyls and polybrominated diphenyl ethers) on the composition and
27
diversity of microbial communities remain unknown. In this study, a suite of e-waste
28
contaminated soils were collected from Guiyu, China, and the indigenous microbial
29
assemblages were profiled by 16S rRNA high-throughput sequencing and clone library
30
analysis. Our data revealed significant differences in microbial taxonomic composition
31
between the contaminated and the reference soils, with Proteobacteria, Acidobacteria,
32
Bacteroidetes and Firmicutes dominating the e-waste-affected communities. Genera
33
previously identified as organic pollutants-degrading bacteria, such as Acinetobacter,
34
Pseudomonas and Alcanivorax, were frequently detected. Canonical correspondence analysis
35
revealed that approximately 70% of the observed variation in microbial assemblages in the
36
contaminated soils was explained by eight environmental variables (including soil
37
physiochemical parameters and organic pollutants) together, among which moisture content,
38
decabromodiphenyl ether (BDE-209) and copper were the major factors. These results
39
provide the first detailed phylogenetic look at the microbial communities in e-waste
40
contaminated soils, demonstrating that the complex combined pollution resulting from
41
improper e-waste recycling may significantly alter soil microbiota.
42 43 44
2
ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
45
Environmental Science & Technology
INTRODUCTION
46
Discarded or unused electrical and electronic devices, generally referred to as electronic
47
waste (e-waste), are an increasingly important environmental problem worldwide. It has been
48
estimated that globally about 40 million tons of e-waste are generated each year,(1) with an
49
increasing rate of 4% per year.(2) The United States Environmental Protection Agency
50
estimates that only 15-20% of e-waste can be recycled while the remaining parts are often
51
disposed of in landfills.(3) However, e-waste typically contains high levels of toxic substances
52
such as organic pollutants (e.g. polychlorinated biphenyls (PCBs) and polybrominated
53
diphenyl ethers (PBDEs)) and heavy metals (e.g. lead, zinc, copper and cadmium);(2) if
54
disposed of improperly, these hazardous substances pose a serious threat to the environment
55
and human health.(2) Notably, most e-waste has been transported to developing countries, in
56
particular China, where these waste materials are processed extensively (e.g. burning and
57
scouring) for recovery of precious metals and the residues are improperly discarded.(4) These
58
practices have caused serious pollution problems for air, terrestrial and aquatic
59
environments.(4)
60
Microbes drive the bulk of Earth’s biogeochemical cycles and are crucial to the functioning
61
of virtually all ecosystems. However, microbes and especially their metabolic activities are
62
sensitive to environmental change. Numerous studies have demonstrated that environmental
63
pollution may cause drastic changes in microbial community composition and activity,(5, 6)
64
or have found significant correlations between microbial diversity and contamination
65
gradients.(7, 8) Typically, pollution could result in a decrease of microbial diversity and
66
enrichment of tolerant species via the process of environmental filtering; this in turn may
3
ACS Paragon Plus Environment
Environmental Science & Technology
67
affect their overall ecosystem function.(5) On the other hand, microorganisms may be actively
68
involved in the degradation and transformation of various pollutants and play a crucial role in
69
the remediation of polluted environments.(9)
70
In the past two decades, the influence of heavy metal contamination on soil microbial
71
communities has attracted much attention,(5, 7, 10, 11) and many species (e.g. Pseudomonas
72
spp., Acinetobacter spp. and Sphingomonas spp.) resistant and tolerant to heavy metals (e.g.
73
copper, zinc and cadmium) have been isolated.(5, 12) Meanwhile, recent research has focused
74
on investigating bacterial community composition in soils polluted by toxic organic
75
pollutants(13-15) and obtaining in pure culture functional bacteria (e.g. Acinetobacter spp.,
76
Pseudomonas spp. and Alcanivorax spp.) capable of degrading PBDEs, PCBs and polycyclic
77
aromatic hydrocarbons.(16-18) Subsequently, the mechanisms of microbial degradation in
78
these bacteria have been studied, which will help improve degradative capabilities for the
79
bioremediation of polluted environments.(19-21) Furthermore, some laboratory studies have
80
examined both single and combined influences of individual metals or individual organic
81
compounds on soil microbial assemblages.(22-24) However, field studies with a specific
82
focus on the effects of combined pollution (e.g. PBDEs and heavy metals, and PCBs and
83
heavy metals) on the indigenous microorganisms have been very limited.(13, 15, 25)
84
Improper e-waste processing could result in heavy metal and toxic organic compound
85
contamination in soil.(2) However, the responses of soil microbiota as a whole community to
86
the combined pollution (including PCBs, PBDEs and heavy metals) and its ecological
87
consequences have not been explored. Here, we report an in-depth and comprehensive survey
88
of the microbial assemblages from a suite of soils contaminated by uncontrolled e-waste
4
ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
Environmental Science & Technology
89
recycling activities. Microbial populations were profiled by both Illumina high-throughput
90
sequencing and clone library analysis targeting the 16S rRNA genes that were then
91
taxonomically analyzed. Specifically, we aimed to elucidate the composition and diversity of
92
microbes in the e-waste affected soils and examine how microbial assemblages are shaped by
93
the environmental conditions.
94 95
MATERIALS AND METHODS
96
Site Description and Sample Collection. Guiyu, a town with a total area of 52 km2 and a
97
population of 400,000 in Guangdong Province, China, has been a booming e-waste
98
processing center since 1995.(26) Due to crude e-waste recycling activities in a large number
99
of simple household e-waste recycling workshops, the air, water and soil have been heavily
100
contaminated by toxic organic compounds and heavy metals.(2, 26) In November 2011,
101
representative contaminated soil samples were collected in triplicate at a depth of 0-10 cm
102
from two e-waste dumping sites (Dump-1 and Dump-2), two e-waste burning sites (Burn-1
103
and Burn-2), one acid stripping site (Acid-strip), one contaminated farmland site (Farmland)
104
and one contaminated mudflat site near the Lianjiang river (Mudflat) (Figure 1). For
105
comparison, triplicate samples were also collected from each of the five corresponding
106
reference soil types (Dump_ck, Burn_ck, Acid-strip_ck, Farmland_ck and Mudflat_ck) that
107
were well apart in the upstream direction from the contaminated sites and thus unlikely
108
affected by e-waste. All samples were kept in cooler boxes during sampling and then
109
transported to the laboratory where they were stored at 4 °C prior to processing (within 48 hrs)
110
for subsequent analyses (see below).
5
ACS Paragon Plus Environment
Environmental Science & Technology
111
Physicochemical Analyses. Moisture content was determined by weighing subsamples
112
before and after oven-drying at 105 °C for 24 h. Samples were air-dried and sieved to
