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Here, high-throughput Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the microorganisms in an anoxic contaminated soil afte...
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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

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Environmental Science & Technology

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High resolution dynamics of microbial communities during dissimilatory selenate

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reduction in an anoxic soil

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Ronald R. Navarro1, Tomo Aoyagi1, Makoto Kimura1, Hideomi Itoh2, Yuya Sato1,

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Yoshitomo Kikuchi2, Atsushi Ogata1 and Tomoyuki Hori1*

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1

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Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Ibaraki 305-8569,

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Japan

Environmental Management Research Institute, National Institute of Advanced

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2

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and Technology (AIST), Tsukisamu-higashi 2-17-2-1 Toyohira-ku, Sapporo, Hokkaido,

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062-8517, Japan

Bioproduction Research Institute, National Institute of Advanced Industrial Science

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R. Navarro, T. Aoyagi, and M. Kimura contributed equally to this work

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*

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Address: Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan

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Phone: +81 29 849 1107; Fax: +81 29 861 8326; e-mail: [email protected]

Corresponding author: Tomoyuki Hori

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Abstract:

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Selenate is one of the most common toxic metal compounds in contaminated soils. Its

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redox status can be changed by microbial activity, thus affecting its water solubility and

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soil mobility. However, current knowledge on microbial dynamics has been limited by

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the low sensitivity of past isolation and identification protocols. Here, high-throughput

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Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the

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microorganisms in an anoxic contaminated soil after Se(VI) and acetate amendment. An

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autoclaved soil with both chemicals and a live soil with acetate alone were used as

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controls. Preliminary chemical analysis clearly showed the occurrence of biological

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selenate reduction coupled with acetate oxidation. Principal coordinate analysis and

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diversity indices of Illumina-derived sequence data showed dynamic succession and

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diversification of the microbial community in response to selenate reduction. The

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high-resolution phylogenetic analysis revealed that the relative frequency of an

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operational taxonomic unit (OTU) from the genus Dechloromonas increased remarkably

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from 0.2% to 36% as a result of Se(VI) addition. Multiple OTUs representing less

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abundant microorganisms from the Rhodocyclaceae and Comamonadaceae families had

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significant increases as well. This study demonstrated that these microorganisms are

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concertedly involved in selenate reduction of the employed contaminated soil under

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anoxic condition.

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Introduction:

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Selenium (Se) is a naturally-occurring element found in the earth’s crust. It is

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essential to humans, with a daily requirement of 0.8 µg kg-1, but is at the same time

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toxic when ingested beyond 5 µg kg-1.1 The ability of Se to substitute for sulfur atoms in

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biological systems accounts for its toxicity.2 Excessive intake is commonly a result of

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food and water being contaminated by various anthropogenic sources. Selenium may be

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released by human activities such as mining when metal sulfide and phosphate ores

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containing selenides are exposed to air and water. In such episodes, they are converted

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to soluble selenite, Se(IV), and selenate, Se(VI), by natural oxidative weathering.

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Major efforts have been made during the past decades to mitigate soluble Se

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contamination in soils by evaluating the mechanism of their reduction back to the

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insoluble state for subsequent immobilization.3-6

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The reduction of soluble Se oxyanions is known to be mediated by both chemical

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and biological processes. In the former, metal compounds such as green rusts may play

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a vital role. 3-5 However, at the natural soil conditions, abiotic Se reduction is considered

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less likely to occur.6

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reduction. In fact, even under conditions that inhibit chemical reduction, biological

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transformations have been found to proceed rapidly over a wide range of salinities and

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pH.6 In the point of view of on-site soil remediation, biological anaerobic Se reduction

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through dissimilatory redox process is a promising approach1,7 because soluble and

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toxic Se(VI) migrate and ultimately partition into subsurface layers where oxygen

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supply is limited. Their further migration to underground aquifers may therefore be

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prevented. Dissimilatory reduction involves the oxidation of electron donors to

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transform soluble Se(VI) into less soluble zerovalent Se.6 The amendment of

A variety of bacterial species have been identified to facilitate Se

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contaminated soil by organic compounds as electron donors is thus a powerful approach

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for on-site remediation.8 Previous works have for instance employed acetate for this

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purpose7 since it is not fermented in anoxic environments9. Its use as carbon and energy

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source could positively confirm that Se(VI) disappearance is directly related to the

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microbially-mediated redox reactions described below: 10

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CH3COO-

+

H+

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CH3COO-

+

5H+

+ +

4 SeO422 SeO42-

 

2CO2 +

4SeO32-

2CO2

2Se0

+

+ +

2H2O 4H2O

(1) 11 (2)

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For a comprehensive understanding of biological remediation performance,

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detailed evaluation of key microorganisms as well as the changes in microbial

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community structure during the entire process is essential. This is important for the

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creation of optimal ecophysiological conditions for the microorganisms performing the

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decontamination.

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in-depth ecological characterization has been limited by the early culture-dependent

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methods. This is further complicated by the fact that anaerobic bacteria capable of

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dissimilatory Se reduction are difficult to culture.7 This explains why through close to

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three decades of related research, each work has reported novel Se-reducing isolates.

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Such lack of consistency in the identity of isolated microorganisms could indicate the

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richness of Se-reducers but on a negative note, this could also be a reflection of the

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limited capability of previous isolation and identification protocols.10 This has been

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improved by culture-independent methods relying on nucleic acids from soil

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microorganisms, such as PCR/DGGE of 16S rRNA genes, which were able to monitor

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the community-level changes, albeit at lower resolution.7,8 With the advent of

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high-throughput DNA sequencing technologies, 16S rRNA gene amplicon sequencing

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has provided much higher sequence diversity resolution than the conventional

Unfortunately, through many years of investigation in this field,

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molecular methods.12-16 For example, pyrosequencing has been used for monitoring

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shifts in microbial community in wastewaters during Se reduction under changing

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conditions.17 In this work, we have employed Illumina-based sequencing to characterize

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the transition of the soil microbiota during dissimilatory selenate reduction coupled with

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acetate oxidation. It is our hope that, with this technique, the higher resolution would

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reveal the involvement of hitherto unknown microorganisms during the biological

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remediation of Se-contaminated soil.

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Materials and Methods:

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Anaerobic incubation of sediment slurry with Se(VI) and acetate

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A soil sample with a known history of selenium contamination was used for the

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study. The analysis confirmed residual Se(VI) of 0.007 ppm (mg kg-1). A set of 36

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20-mL soil slurry samples were prepared by mixing a 5-g soil with distilled water, and

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placed in 50-mL serum vials and sealed with butyl rubber septa. The soil slurries were

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pre-incubated anaerobically at 25oC in the dark for more than one month without

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substrate addition to deplete most of the carbon sources and allow the stabilization of

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soil microbial communities. After pre-incubation, the headspace was flushed with N2

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gas (99.99% purity). Three runs were carried out involving (i) non-autoclaved or live

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soil amended with Se(VI) and acetate, (ii) live soil with acetate only, and (iii)

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autoclaved soil with Se(VI) and acetate. Soil sterilization was performed four times at

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121°C for 1 h. All samples were subjected to static incubation for 8 days at 25°C.

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Selenate (as Na2SeO4) was supplemented repeatedly at day 0, 2, 4, and 6 by the addition

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of 0.5 mL of 6 mM stock solution. This level of Se(VI) contamination, which is around

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0.15 mM per addition, was established based on a previous research of similar nature7 5

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as well as on our preliminary incubations runs. Acetate was initially added at a

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concentration of 3 mM, which is in excess of that theoretically required for Se(VI)

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reduction up to its zerovalent form. Three vials from each set of the live incubations

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were sacrificed after 0, 1, 2, 4, and 8 days for analysis. Sterilized slurries were taken at

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day 0 and day 8. It is important to emphasize that each sample was sacrificed prior to

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the next Se(VI) amendment so that the concentrations at the time just after amendment

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was not determined, except for that at day 0. These samples were centrifuged to separate

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the supernatant from the soil matter. The supernatant was further filtered through a

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cellulose acetate membrane (0.2 µm; Advantec, Tokyo, Japan). The collected

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supernatants and soils were stored at -80°C prior to chemical and microbiological

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analyses, respectively.

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Chemical analysis of supernatant

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The concentration of selenite ions was analyzed by an Inductively Coupled

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Plasma (ICP) spectrometer (SPS 3000S; Seiko Instruments, Tokyo, Japan) equipped

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with a hydride generator to reduce and transform selenite to H2Se before detection.18

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For the total Se analysis, pre-digestion of slurry supernatants in equal volume of 6M

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HCl solution under reflux for 15 min was employed in order to reduce all traces of

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selenate ions to selenite.19 The selenate ions were calculated from the difference

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between the total Se and selenite ion concentrations.

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The concentration of volatile fatty acids (VFAs), particularly acetate, in the

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slurry supernatant was measured by a high-pressure liquid chromatography (HPLC)

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(Alliance e26951, Waters) equipped with an RSpak KC-811 column (Shodex) and a

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photodiode array (2998, Waters). The carrier liquid was 10 mM H2SO4 and flow rate 6

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was 0.75 mL min-1. Standard acetate solutions of 0.5, 1 and 5 mM were prepared.

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Nitrate was analyzed by an Ion Chromatograph (Dionex DX-500) equipped

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with an IonPac AS-11 column, ED40 Electrochemical detector and Dionex ASRS 300

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suppressor. The flow rate of NaOH gradient (0 to 30 mM over 13 min) was 1.2 mL

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min-1. Standard nitrate solutions of 0.02, 0.05 and 0.1 mM were prepared. The limit of

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detection (LOD) was 0.02 mM.

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Total and ferrous irons were determined as described earlier.9 Briefly, 0.5 ml of

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the slurry samples were mixed with HCl at a final concentration of 0.5 M, and incubated

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for 16 hours. Fe(II) and hydroxylamine reducible Fe(III) were determined by the

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ferrozine method.

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Extraction of the genomic DNA from soil and subsequent PCR amplification

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Nucleic acids were extracted from the soil by a direct lysis protocol involving

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bead beating as described previously.20 Traces of RNA were digested with RNase (Type

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II-A; Sigma-Aldrich, Tokyo, Japan). The purified DNA was used as template for PCR

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amplification with a high-fidelity DNA polymerase (Q5; NEB, Tokyo, Japan). The V4

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region of 16S rRNA genes was amplified using the universal primers 515F and 806R.16

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Both these primers were modified to contain an Illumina adapter region, and the reverse

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primers were encoded with 12-bp barcode for the multiplex sequencing.15 The thermal

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conditions of PCR were as follows: initial denaturation at 98°C for 90 s, 35-40 cycles of

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denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 30

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s; and a final extension step at 72°C for 2 min. The non-amplification of DNAs obtained

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from the autoclaved incubations was confirmed by the absence of targeted bands in the

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agarose-gel during electrophoresis. 7

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High-throughput Illumina sequencing of 16S rRNA gene amplicons

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The PCR products were first purified using an AMPure XP kit (Beckman

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Coulter, Tokyo, Japan). The resulting DNA solution was subjected to agarose-gel

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electrophoresis and the target DNA band was excised. The recovery of DNA in the gel

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slice was performed with a QIAquick gel extraction kit (QIAGEN, Tokyo, Japan). The

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DNA concentration was determined spectrophotometrically with a Quant-iT PicoGreen

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dsDNA reagent and kit (Life Technologies, Tokyo, Japan). An appropriate amount of the

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16S rRNA gene segments and an internal control (PhiX Control V3; Illumina, Tokyo,

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Japan) was subjected to paired-end sequencing with a 300-cycle MiSeq reagent kit

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(Illumina) and a MiSeq sequencer (Illumina). Removal of PhiX, low-quality (Q