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Interconnection of key microbial functional genes for enhanced benzo[a]pyrene biodegradation in sediments by microbial electrochemistry Zaisheng Yan, Yuhong He, Haiyuan Cai, Joy D Van Nostrand, Zhili He, Jizhong Zhou, Lee R Krumholz, and He-Long Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00209 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017
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Interconnection of key microbial functional genes for
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enhanced benzo[a]pyrene biodegradation in sediments by
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microbial electrochemistry
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Zaisheng Yan a, Yuhong He a, Haiyuan Cai a, Joy D. Van Nostrand b, Zhili He b,
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Jizhong Zhou b,c,d , Lee R. Krumholz b and He-Long Jiang a,*
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a
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Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008,
State Key Laboratory of Lake Science and Environment, Nanjing Institute of
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China.
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b
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Norman, OK, USA.
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c
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94720, USA.
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d
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100084, China.
Department of Microbiology and Plant Biology, University of Oklahoma,
Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
Department of Environmental Science and Engineering, Tsinghua University, Beijing
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*
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Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing
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210008, China. Tel./fax:
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(H.L. Jiang).
Corresponding author.
Mail address: Nanjing Institute of Geography and
0086-25-8688 2208. E-mail:
[email protected] 23 24
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ABSTRACT:
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degradation of polycyclic aromatic hydrocarbons in sediments, but the mechanism of
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this process is poorly understood at the microbial functional gene level. Here, the use
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of SMFC resulted in 92% benzo[a]pyrene (BaP) removal over 970 days relative to
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54% in the controls. Sediment functions, microbial community structure and network
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interactions were dramatically altered by the SMFC employment. Functional gene
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analysis showed that c-type cytochromes genes for electron transfer, aromatic
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degradation genes and extracellular ligninolytic enzymes involved in lignin
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degradation were significantly enriched in bulk sediments during SMFC operation.
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Correspondingly, chemical analysis of the system showed that these genetic changes
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resulted in increases in the levels of easily oxidizable organic carbon and humic acids
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which may have resulted in increased BaP bioavailability and increased degradation
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rates. Tracking microbial functional genes and corresponding organic matter
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responses should aid mechanistic understanding of BaP enhanced biodegradation by
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microbial electrochemistry and development of sustainable bioremediation strategies.
Sediment microbial fuel cells (SMFCs) can stimulate the
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INTRODUCTION
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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants
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and pose a serious risk for both humans and ecosystems in which they are present 1-2.
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In aquatic environments, PAHs generally accumulate in sediments due to their strong
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hydrophobicity 3. High molecular weight (HMW) PAHs, such as benzo[a]pyrene
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(BaP), a five-ring compound, shows carcinogenicity, teratogenicity and acute toxicity
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4-5
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increasingly important to mitigate the associated risks.
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. Thus, removal of HMW-PAHs from contaminated sediments is becoming
PAHs, once entering into sediments, are subject to natural attenuation which 4
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involved biotic/abiotic degradation
. Microbial degradation under aerobic or
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anaerobic condition by microbes represents the major mechanism for the ecological
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recovery of PAHs-contaminated sites
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contaminants in sedimentary anoxic environments because of a lack of suitable
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electron acceptors 9-10.
2, 6-8
. Even so, PAHs often persist as
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To facilitate PAHs removal from sediments, biostimulation techniques involving
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addition of electron acceptors such as oxygen, nitrate, sulfate, ferric iron, or an
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electrode serving as the electron acceptor have been proposed 9, 11-12. The anode of the
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sediment microbial fuel cell (SMFC) as an electron acceptor for stimulating the
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degradation of aromatic hydrocarbon contaminants could be useful as it provides a
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low-cost, low-maintenance, continuous sink for electrons
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been successful using an electrode approach to improve the removal of hydrocarbons
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including diesel 13-14, petroleum 15, petroleum sludge 16 or PAHs 2, 10.
9-10
. Previous studies have
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The SMFC has been shown to alter the microbial community on the anode
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surface, and those distinctive microbial communities enriched on the anode biofilm
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played a key role in SMFC enhancement of PAH bioremediation 2, 14. However, it was 3
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still unknown how SMFC operation affected microbial functional structures and its
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possible relationship with the biogeochemical cycle in the bulk sediments during
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enhanced degradation of HMW-PAHs. The genetic determinants of all these processes
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and the mechanisms involved in their regulation are much less studied.
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Within sediments, organic matter (OM) was an important factor influencing the 3, 17
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degradation of PAHs
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was controlled by the interaction between PAHs and sediment OM
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components of sediment OM have different effects on PAHs transformation in
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sediments. For example, labile OM such as cyanobacteria-derived OM could
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dramatically enhance pyrene and BaP degradation, which was related to higher
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bioavailability and PAH-degrading bacteria number with a result of biostimulation
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and priming effect by labile carbon 3, 20.
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. Many studies have shown that the bioavailability of PAHs 3, 17-19
. Complex
The electrode-driven microbial process not only altered sediment OM 21-22
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characteristics, but also enhanced the decomposition of OM
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sediment OM around the electrodes had an increased level of aromatics with a higher
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humic acid (HA) fraction, a larger molecular size, and index of humification 10, 21. The
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presence of dissolved OM such as HA is currently considered a valid biostimulation
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strategy to speed up PAHs bioremediation
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degradation of PAHs in sediments depended on the exposure regime
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lower biodegradation via binding between PAHs and HA, but also might act as
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carriers of PAH compounds to induce PAHs flux to the degrading bacteria
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Additionally, HA can serve as an electron acceptor and redox mediator for microbial
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respiration in sediments
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complex.
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. It was found that
23-24
. However, the effect of HA on 25
. HA could
25-26
.
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. So, the role of HA in HMW-PAHs biodegradation is
In this study, we hypothesized that: (1) SMFC employment will influence the 4
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interactions of microbial functional groups in bulk sediments; (2) carbon cycling and
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organic remediation functional genes in sediments will be enriched during SMFC use
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and closely related to BaP biodegradation. To test these hypotheses, both GeoChip
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and community sequencing were used to analyze functional genes and microbial
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community structures involved in organic carbon transformation and bioremediation.
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GeoChip 5.0 contains about 167,000 50-mer oligonucleotide probes covering
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∼395,000 coding sequences from >1,590 functional genes involved in carbon cycling,
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organic remediation, electron transfer and other environmental processes
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allows us to analyze the functional composition, structure, and dynamics of microbial
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communities.
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, and
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MATERIALS AND METHODS
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Sediment and Water Samples. Surface sediments (at 0-10 cm depth) and
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overlying water were collected from East Taihu Lake (30°58′ N, 120°22′ E) in China,
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and transported to the laboratory immediately. After sieving at 2 mm, bulk samples of
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surface sediment were mixed and homogenized. BaP was added to the sediments, by
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mixing a stock solution (1000 mg L-1 of BaP in acetonitrile; 98% purity, Alfa Acsar
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Co., UK), with wet sediments followed by mechanical mixing
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concentration of BaP of 1000 µg kg−1 dry sediments, much higher than the probable
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effect level (PEL) according to sediment quality guidelines
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acetonitrile was allowed to maximize removal by volatilization in the fume hood.
2-3
to a final
29
. The cosolvent
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Experimental Design and Sampling Procedures. Plexiglass columns with
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approximately 4-L volume (12 cm × 35 cm, diameter × height) were used as sediment
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microcosms to perform the biodegradation experiments in the dark at 25 °C. Each 5
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microcosm contained 1600 g wet sediment and 1.2 L surface water. SMFCs were
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installed in the sediment microcosms as described in previous studies
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anode composed of a graphite felt cylinder (6.4 cm × 8 cm × 0.5 cm, diameter ×
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height × thickness) was buried 5 cm below the sediment surface. The cathode,
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composed of round graphite felt (9.5 cm × 0.5 cm, diameter × thickness), was placed
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6 cm above the sediment in the water column, and the overlying water was
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continuously bubbled with air to maintain dissolved oxygen concentration above 5 mg
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L-1. All connections were made with water-tight connectors using marine-grade wire
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and sealed with silver conductive epoxy and marine epoxy. The voltage signal
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between the anode and cathode across an external load of 100 Ω was measured using
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a multimeter (model 2700, Keithley Instruments, Cleveland, OH, USA). Details of
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electrochemical
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Supplementary Information (SI). In addition, the control treatment was applied to
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determine natural attenuation of BaP in sediment without SMFC employment. Each
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experimental condition was performed in triplicate.
measurement
and
SMFC
performance
were
2, 10, 30
. The
described
in
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During experiments, sediment samples (10 g) were taken from each microcosm
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at days 60, 90, 180, 450 and 970. The following physical and chemical parameters
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were measured: BaP, easily oxidizable organic carbon (EOC), total carbon (TC),
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humic acid (HA), total nitrogen (TN), and total phosphate (TP). More detailed
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information of geochemical analysis and initial properties of the sediment sample are
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provided in the SI and Table S1. Aliquots (5 g) of sediment were stored at -80 °C for
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molecular analysis. At the end of experiments, anode biofilm samples were collected
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from sediment microcosms. The anode electrodes were rinsed with sterile water to
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remove visible sediments on the surface, and then a sterile razor blade was used to
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scrape the electrodes vigorously to acquire a complex consisting of carbon felt and 6
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electrode-associated microbes for subsequent DNA extraction 2.
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DNA Extraction, 454 Pyrosequencing and GeoChip Hybridization. Sediment
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DNA for 454 pyrosequencing was extracted with a PowerSoil®DNA Isolation Kit
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(MO BIO) according to the manufacturer’s instructions. DNA quality was assessed by
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260 nm/280 nm and 260/230 nm ratios, and DNA concentration was estimated with a
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Nanodrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, USA).
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Roche GS-FLX 454 pyrosequencing was conducted by Meiji Biotechnology
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Company (Shanghai, China). Nucleotide sequences were all deposited in the
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GenBank database (accession number: SRP109064). Details of amplicon preparation,
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sequencing and data analysis are described in SI.
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Sediment DNA for GeoChip analysis was extracted using a freeze-grinding
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mechanical lysis approach in order to get a large fragment of DNA for direct
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hybridization as described previously
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(Santa Clara, CA, USA) and used to analyze the functional structure of the microbial
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communities and functional gene abundances. Signal intensities were measured on the
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basis of scanned images. A floating signal to noise ratio was used so that thermophile
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probes accounted for 5% of the positive probes. Details for template amplification,
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labelling and hybridization, image processing and GeoChip data pre-processing are
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described in SI.
31
. GeoChip 5.0 was synthesized by Agilent
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BaP Bioavailability and Enumeration of PAH-Degrading Microorganisms. An
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aqueous-based mild extraction technique utilizing hydroxypropyl-β-cyclodextrin
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(HPCD) was adapted to evaluate the change of PAHs bioavailability in sediments 3,
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and HA-binding experiments (binding isotherms) were performed with a fixed HA 7
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concentration of 25 mg L-1 and increasing PAH concentrations from 0 to 75% of their
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water solubility, as described in a previous report with a slight modification
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additional details provided in the SI.
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and
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Bacterial counts from triplicate bulk sediment samples were performed using a
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most probable number (MPN) method 2, with details for aerobic and anaerobic
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cultivation provided in the SI.
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Network Construction and Network Characterization Analysis. To understand
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the interactions among different microbial communities and their responses to SMFC
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addition, a random matrix theory (RMT)-based approach was used to discern
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functional and phylogenetic molecular ecological networks (f/pMENs) in sediment
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microbial communities based on GeoChip data and community sequence data 32. The
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data sets were uploaded to the open-accessible network analysis pipeline (Molecular
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Ecological Network Analysis Pipeline, MENAP, http://ieg2.ou.edu/ MENA), and the
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networks were constructed using random matrix theory (RMT)-based methods 33.
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Cytoscape 3.2.1 software was used to visualize the network graphs
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. Other
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information about genes, e.g., taxonomy, relative abundance, and edge information,
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e.g., weights and positive and negative correlations, was also imported into the
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software and visualized in the network figures. Since we are interested in the impact
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of SMFC employment on network interactions, the f/pMENs were constructed
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separately for the SMFC treatment and control treatments. Various indexes, including
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average degree (connectivity), nodes, edges, average clustering coefficient, average
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geodesic distance, and modularity were used to describe the properties of individual
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nodes in the network and the overall topologies or structures of the different networks.
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Statistical Analysis. Pre-processed data (e.g. GeoChip, 454 pyrosequencing) were
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analyzed as follows: (1) microbial diversity index using the two-tailed t-test; (2)
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hierarchical clustering for microbial community structure and composition; (3)
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analysis of similarity, permutational multivariate analysis of variance using distance
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matrices and multiresponse permutation procedure analysis of difference of microbial
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communities; (4) percentage change of functional genes within SMFCs were
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calculated using the following formula: (TS-TC)×100/TC, where TC and TS were the
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average signal intensities of genes detected by GeoChip 5.0 in the control treatment
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samples and the SMFC treatment samples, respectively; and (5) Mantel and
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redundancy analysis and the corresponding partial analyses, were used to link the
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functional structure of microbial communities with environmental variables.
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RESULTS
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Effects of SMFC Employment on Sediment Chemical Properties. During 970
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days of biostimulation, the sediment BaP content was significantly decreased (P
