Subscriber access provided by NEW YORK UNIV
Article
High resolution dynamics of microbial communities during dissimilatory selenate reduction in an anoxic soil Ronald Ragudo Navarro, Tomo Aoyagi, Makoto Kimura, Hideomi Itoh, Yuya Sato, Yoshitomo Kikuchi, Atsushi Ogata, and Tomoyuki Hori Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505210p • Publication Date (Web): 28 May 2015 Downloaded from http://pubs.acs.org on June 7, 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 32
Environmental Science & Technology
1
High resolution dynamics of microbial communities during dissimilatory selenate
2
reduction in an anoxic soil
3 4
Ronald R. Navarro1, Tomo Aoyagi1, Makoto Kimura1, Hideomi Itoh2, Yuya Sato1,
5
Yoshitomo Kikuchi2, Atsushi Ogata1 and Tomoyuki Hori1*
6 7
1
8
Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Ibaraki 305-8569,
9
Japan
Environmental Management Research Institute, National Institute of Advanced
10
2
11
and Technology (AIST), Tsukisamu-higashi 2-17-2-1 Toyohira-ku, Sapporo, Hokkaido,
12
062-8517, Japan
Bioproduction Research Institute, National Institute of Advanced Industrial Science
13 14
R. Navarro, T. Aoyagi, and M. Kimura contributed equally to this work
15 16
*
17
Address: Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan
18
Phone: +81 29 849 1107; Fax: +81 29 861 8326; e-mail:
[email protected] Corresponding author: Tomoyuki Hori
1
ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 32
19
Abstract:
20
Selenate is one of the most common toxic metal compounds in contaminated soils. Its
21
redox status can be changed by microbial activity, thus affecting its water solubility and
22
soil mobility. However, current knowledge on microbial dynamics has been limited by
23
the low sensitivity of past isolation and identification protocols. Here, high-throughput
24
Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the
25
microorganisms in an anoxic contaminated soil after Se(VI) and acetate amendment. An
26
autoclaved soil with both chemicals and a live soil with acetate alone were used as
27
controls. Preliminary chemical analysis clearly showed the occurrence of biological
28
selenate reduction coupled with acetate oxidation. Principal coordinate analysis and
29
diversity indices of Illumina-derived sequence data showed dynamic succession and
30
diversification of the microbial community in response to selenate reduction. The
31
high-resolution phylogenetic analysis revealed that the relative frequency of an
32
operational taxonomic unit (OTU) from the genus Dechloromonas increased remarkably
33
from 0.2% to 36% as a result of Se(VI) addition. Multiple OTUs representing less
34
abundant microorganisms from the Rhodocyclaceae and Comamonadaceae families had
35
significant increases as well. This study demonstrated that these microorganisms are
36
concertedly involved in selenate reduction of the employed contaminated soil under
37
anoxic condition.
2
ACS Paragon Plus Environment
Page 3 of 32
Environmental Science & Technology
38
Introduction:
39
Selenium (Se) is a naturally-occurring element found in the earth’s crust. It is
40
essential to humans, with a daily requirement of 0.8 µg kg-1, but is at the same time
41
toxic when ingested beyond 5 µg kg-1.1 The ability of Se to substitute for sulfur atoms in
42
biological systems accounts for its toxicity.2 Excessive intake is commonly a result of
43
food and water being contaminated by various anthropogenic sources. Selenium may be
44
released by human activities such as mining when metal sulfide and phosphate ores
45
containing selenides are exposed to air and water. In such episodes, they are converted
46
to soluble selenite, Se(IV), and selenate, Se(VI), by natural oxidative weathering.
47
Major efforts have been made during the past decades to mitigate soluble Se
48
contamination in soils by evaluating the mechanism of their reduction back to the
49
insoluble state for subsequent immobilization.3-6
50
The reduction of soluble Se oxyanions is known to be mediated by both chemical
51
and biological processes. In the former, metal compounds such as green rusts may play
52
a vital role. 3-5 However, at the natural soil conditions, abiotic Se reduction is considered
53
less likely to occur.6
54
reduction. In fact, even under conditions that inhibit chemical reduction, biological
55
transformations have been found to proceed rapidly over a wide range of salinities and
56
pH.6 In the point of view of on-site soil remediation, biological anaerobic Se reduction
57
through dissimilatory redox process is a promising approach1,7 because soluble and
58
toxic Se(VI) migrate and ultimately partition into subsurface layers where oxygen
59
supply is limited. Their further migration to underground aquifers may therefore be
60
prevented. Dissimilatory reduction involves the oxidation of electron donors to
61
transform soluble Se(VI) into less soluble zerovalent Se.6 The amendment of
A variety of bacterial species have been identified to facilitate Se
3
ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 32
62
contaminated soil by organic compounds as electron donors is thus a powerful approach
63
for on-site remediation.8 Previous works have for instance employed acetate for this
64
purpose7 since it is not fermented in anoxic environments9. Its use as carbon and energy
65
source could positively confirm that Se(VI) disappearance is directly related to the
66
microbially-mediated redox reactions described below: 10
67
CH3COO-
+
H+
68
CH3COO-
+
5H+
+ +
4 SeO422 SeO42-
2CO2 +
4SeO32-
2CO2
2Se0
+
+ +
2H2O 4H2O
(1) 11 (2)
69
For a comprehensive understanding of biological remediation performance,
70
detailed evaluation of key microorganisms as well as the changes in microbial
71
community structure during the entire process is essential. This is important for the
72
creation of optimal ecophysiological conditions for the microorganisms performing the
73
decontamination.
74
in-depth ecological characterization has been limited by the early culture-dependent
75
methods. This is further complicated by the fact that anaerobic bacteria capable of
76
dissimilatory Se reduction are difficult to culture.7 This explains why through close to
77
three decades of related research, each work has reported novel Se-reducing isolates.
78
Such lack of consistency in the identity of isolated microorganisms could indicate the
79
richness of Se-reducers but on a negative note, this could also be a reflection of the
80
limited capability of previous isolation and identification protocols.10 This has been
81
improved by culture-independent methods relying on nucleic acids from soil
82
microorganisms, such as PCR/DGGE of 16S rRNA genes, which were able to monitor
83
the community-level changes, albeit at lower resolution.7,8 With the advent of
84
high-throughput DNA sequencing technologies, 16S rRNA gene amplicon sequencing
85
has provided much higher sequence diversity resolution than the conventional
Unfortunately, through many years of investigation in this field,
4
ACS Paragon Plus Environment
Page 5 of 32
Environmental Science & Technology
86
molecular methods.12-16 For example, pyrosequencing has been used for monitoring
87
shifts in microbial community in wastewaters during Se reduction under changing
88
conditions.17 In this work, we have employed Illumina-based sequencing to characterize
89
the transition of the soil microbiota during dissimilatory selenate reduction coupled with
90
acetate oxidation. It is our hope that, with this technique, the higher resolution would
91
reveal the involvement of hitherto unknown microorganisms during the biological
92
remediation of Se-contaminated soil.
93 94
Materials and Methods:
95
Anaerobic incubation of sediment slurry with Se(VI) and acetate
96
A soil sample with a known history of selenium contamination was used for the
97
study. The analysis confirmed residual Se(VI) of 0.007 ppm (mg kg-1). A set of 36
98
20-mL soil slurry samples were prepared by mixing a 5-g soil with distilled water, and
99
placed in 50-mL serum vials and sealed with butyl rubber septa. The soil slurries were
100
pre-incubated anaerobically at 25oC in the dark for more than one month without
101
substrate addition to deplete most of the carbon sources and allow the stabilization of
102
soil microbial communities. After pre-incubation, the headspace was flushed with N2
103
gas (99.99% purity). Three runs were carried out involving (i) non-autoclaved or live
104
soil amended with Se(VI) and acetate, (ii) live soil with acetate only, and (iii)
105
autoclaved soil with Se(VI) and acetate. Soil sterilization was performed four times at
106
121°C for 1 h. All samples were subjected to static incubation for 8 days at 25°C.
107
Selenate (as Na2SeO4) was supplemented repeatedly at day 0, 2, 4, and 6 by the addition
108
of 0.5 mL of 6 mM stock solution. This level of Se(VI) contamination, which is around
109
0.15 mM per addition, was established based on a previous research of similar nature7 5
ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 32
110
as well as on our preliminary incubations runs. Acetate was initially added at a
111
concentration of 3 mM, which is in excess of that theoretically required for Se(VI)
112
reduction up to its zerovalent form. Three vials from each set of the live incubations
113
were sacrificed after 0, 1, 2, 4, and 8 days for analysis. Sterilized slurries were taken at
114
day 0 and day 8. It is important to emphasize that each sample was sacrificed prior to
115
the next Se(VI) amendment so that the concentrations at the time just after amendment
116
was not determined, except for that at day 0. These samples were centrifuged to separate
117
the supernatant from the soil matter. The supernatant was further filtered through a
118
cellulose acetate membrane (0.2 µm; Advantec, Tokyo, Japan). The collected
119
supernatants and soils were stored at -80°C prior to chemical and microbiological
120
analyses, respectively.
121 122
Chemical analysis of supernatant
123
The concentration of selenite ions was analyzed by an Inductively Coupled
124
Plasma (ICP) spectrometer (SPS 3000S; Seiko Instruments, Tokyo, Japan) equipped
125
with a hydride generator to reduce and transform selenite to H2Se before detection.18
126
For the total Se analysis, pre-digestion of slurry supernatants in equal volume of 6M
127
HCl solution under reflux for 15 min was employed in order to reduce all traces of
128
selenate ions to selenite.19 The selenate ions were calculated from the difference
129
between the total Se and selenite ion concentrations.
130
The concentration of volatile fatty acids (VFAs), particularly acetate, in the
131
slurry supernatant was measured by a high-pressure liquid chromatography (HPLC)
132
(Alliance e26951, Waters) equipped with an RSpak KC-811 column (Shodex) and a
133
photodiode array (2998, Waters). The carrier liquid was 10 mM H2SO4 and flow rate 6
ACS Paragon Plus Environment
Page 7 of 32
Environmental Science & Technology
134
was 0.75 mL min-1. Standard acetate solutions of 0.5, 1 and 5 mM were prepared.
135
Nitrate was analyzed by an Ion Chromatograph (Dionex DX-500) equipped
136
with an IonPac AS-11 column, ED40 Electrochemical detector and Dionex ASRS 300
137
suppressor. The flow rate of NaOH gradient (0 to 30 mM over 13 min) was 1.2 mL
138
min-1. Standard nitrate solutions of 0.02, 0.05 and 0.1 mM were prepared. The limit of
139
detection (LOD) was 0.02 mM.
140
Total and ferrous irons were determined as described earlier.9 Briefly, 0.5 ml of
141
the slurry samples were mixed with HCl at a final concentration of 0.5 M, and incubated
142
for 16 hours. Fe(II) and hydroxylamine reducible Fe(III) were determined by the
143
ferrozine method.
144 145
Extraction of the genomic DNA from soil and subsequent PCR amplification
146
Nucleic acids were extracted from the soil by a direct lysis protocol involving
147
bead beating as described previously.20 Traces of RNA were digested with RNase (Type
148
II-A; Sigma-Aldrich, Tokyo, Japan). The purified DNA was used as template for PCR
149
amplification with a high-fidelity DNA polymerase (Q5; NEB, Tokyo, Japan). The V4
150
region of 16S rRNA genes was amplified using the universal primers 515F and 806R.16
151
Both these primers were modified to contain an Illumina adapter region, and the reverse
152
primers were encoded with 12-bp barcode for the multiplex sequencing.15 The thermal
153
conditions of PCR were as follows: initial denaturation at 98°C for 90 s, 35-40 cycles of
154
denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 30
155
s; and a final extension step at 72°C for 2 min. The non-amplification of DNAs obtained
156
from the autoclaved incubations was confirmed by the absence of targeted bands in the
157
agarose-gel during electrophoresis. 7
ACS Paragon Plus Environment
Environmental Science & Technology
158
Page 8 of 32
High-throughput Illumina sequencing of 16S rRNA gene amplicons
159
The PCR products were first purified using an AMPure XP kit (Beckman
160
Coulter, Tokyo, Japan). The resulting DNA solution was subjected to agarose-gel
161
electrophoresis and the target DNA band was excised. The recovery of DNA in the gel
162
slice was performed with a QIAquick gel extraction kit (QIAGEN, Tokyo, Japan). The
163
DNA concentration was determined spectrophotometrically with a Quant-iT PicoGreen
164
dsDNA reagent and kit (Life Technologies, Tokyo, Japan). An appropriate amount of the
165
16S rRNA gene segments and an internal control (PhiX Control V3; Illumina, Tokyo,
166
Japan) was subjected to paired-end sequencing with a 300-cycle MiSeq reagent kit
167
(Illumina) and a MiSeq sequencer (Illumina). Removal of PhiX, low-quality (Q
