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Article Cite This: ACS Sens. XXXX, XXX, XXX−XXX
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Swift Acid Rain Sensing by Synergistic Rhizospheric Bioelectrochemical Responses Tian Li,† Xin Wang,*,† Qixing Zhou,† Chengmei Liao,† Lean Zhou,† Lili Wan,† Jingkun An,‡ Qing Du,† Nan Li,‡ and Zhiyong Jason Ren*,§
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MOE Key Laboratory of Pollution Processes and Environmental Criteria/Tianjin Key Laboratory of Environmental Remediation and Pollution Control/College of Environmental Science and Engineering, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China ‡ School of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China § Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States S Supporting Information *
ABSTRACT: Acid rain poses significant threats to crops and causes a decline in food production, but current monitoring and response to acid rain damage is either slow or expensive. The direct damage observation on plants can take several hours to days when the damage is irreversible. This study presents a real time bioelectrochemical monitoring approach that can detect acid rain damage within minutes. The rhizospheric bioelectrochemical sensor (RBS) takes advantage of the fast chain responses from leaves to roots, and then to the microbial electrochemical reactions in the rhizosphere. Immediate and repeatable current fluctuations were observed within 2 min after acid rain, and such changes were found to correspond well to the changes in rhizospheric organic concentration and electrochemical responses. Such correlation not only can be observed during acid rain events that can be remedied via rinsing, but it was also validated when such damage is irreversible, resulted in zero current, photosynthetic efficiency, and electrochemical signals. The alanine, aspartate, and glutamate metabolism and galactose metabolism in leaves and roots were inhibited by the acid rain, which resulted in the decrease of rhizodeposits such as fumaric acid, D-galactose, and D-glucose. These changes resulted in reduced electroactivity of anodic microorganisms, which was confirmed by a reduced redox current, a narrower spectrum in differential pulse voltammetry, and the loss of peak in the Bode plot. These findings indicate that the RBS process can be a simple, swift, and lowcost monitoring tool for acid rain that allows swift remediation measures, and its potential may be broadened to other environmental monitoring applications. KEYWORDS: acid rain, rhizospheric bioelectrochemical sensor, rhizodeposits, metabolites, rice plant
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calories consumed worldwide by humans, but unfortunately rice plants are widely grown in acid rain impacted regions such as Europe and Southeast Asia.7,8 Given the enormous risks posed by acid rain, real time monitoring and fast responses are critical to food production, especially in developing countries. Plants have been used as indicators for toxic compounds9,10 and direct observation of damage can be confirmed by yellowing leaves or slow growth, but such responses take several hours to days before such effects can be observed, while by then the plants have already suffered significant damage and are difficult to rescue. Fluorescence imaging techniques enable
cid rain has become a worldwide environmental problem as it causes widespread damages to plants, infrastructure, and the larger ecosystem.1 Acid rain is formed from SOx and NOx emitted to the atmosphere primarily from fossil fuel combustion, and it precipitates in the form of rain, snow, hail, dew, or fog that transports sulfur and nitrogen compounds from the high atmosphere to the ground. Studies have shown that acid rain directly inhibits plant growth, reduces photosynthetic rate, and destroys the ultrastructure of chloroplast, so it is a significant threat to crops and causes a decline in food production.2−4 Previous studies have shown that rice production was reduced by ∼20% when impacted by simulated acid rain, and the plant can die when hit on multiple occasions.5,6 Rice is among the most important grain for human nutrition and provides more than one-fifth of the © XXXX American Chemical Society
Received: May 13, 2018 Accepted: June 6, 2018
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DOI: 10.1021/acssensors.8b00401 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
the same wet weight, similar size, and healthy appearance. The RBSs were inoculated with mixed culture of microorganisms obtained from microbial fuel cells fed with acetate (operated over 1 year) in our laboratory. The reactors were first fed by the 1:1 (v/v) mixture of 50 mM phosphate buffer solution (PBS, Na2HPO4, 4.576 g/L; NaH2PO4, 2.132 g/L; NH4Cl, 0.31 g/L; and KCl, 0.13 g/L) and Hoagland’s solution with 1 g/L acetate amended as the carbon source. When the current density reached 0.50 A/m2, acetate was removed from the medium. All RBSs were then operated for over 1 week to stabilize the plant and microbial community. All experiments were carried out at 25 ± 1 °C with 24 h of illumination. Acid Rain Spray Experiment. Acid rain was simulated by adjusting the pH using concentrated H2SO4 and HNO3 at a ratio of 3:1 (v/v, by chemical equivalents).9 The solution was adjusted to pH 3.5 with deionized water. Simulated acid rain was sprayed at 1 h intervals on plant leaves, waited for 5 h, and then the leaves were washed using deionized water. This treatment was repeated for 3 times. Duplicate experiments are shown in Supporting Information. Plant Analysis. A portable Photosynthesis System (LI 6800, Lincoling, Nebraska, USA) was used to characterize the photosynthetic activity of rice leaves before and after acid rain, including photosynthetic efficiency, transpiration rate, intercellular CO 2 concentration, and stomatal conductance. The parameters were all measured with a light intensity of 800 μmol/m2 s. Rhizospheric total organic carbon (TOC) was measured using a Liquid TOC Analyzer (nulti N/C 3100, Jena, Germany) to show the changes of total rhizodeposits. Roots were carefully rinsed with pure water before moving to deionized water for rhizodeposit release. The rhizodeposit release was performed at the following conditions: temperature, 25 °C; humidity, 50%; light intensity, 100 μmol/m2 s; and time, 4 h. The water samples were filtered through a 0.22 μm membrane before TOC tests. Electrochemical Characterization. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted using a potentiostat (PGSTAT 302N, Metrohm, Switzerland) to provide the redox information before and after acid rain in RBS. The parameters for CV were as follows: equilibrium time, 200 s; scan rate, 1 mV/s; Einitial (Ei) = −0.6 V; and Efinal (Ef) = 0.2 V. The parameters for DPV were as follows: Ei = −0.6 V; Ef = 0.2 V; pulse height, 50 mV; pulse width, 300 ms; step height, 2 mV; step time, 500 ms; scan rate, 4 mV/ s.27 Electrochemical impedance spectroscopy (EIS) was measured at open circuit potential (disconnected for 1 h before each test) over a frequency range from 100 kHz to 0.005 Hz with a sinusoidal excitation of 10 mV.28 Nyquist plots were fitted according to the equivalent circuit reported previously.29 Measurements of Metabolic Activity. A gas chromatograph with quadruple mass spectroscopy (GC-MS; 6890N/5973, Agilent, USA) was employed to measure the metabolites at the rhizosphere of the rice plant before and after acid rain. The samples were first frozen in liquid nitrogen. A 4.5 mL mixed solution of methanol/chloroform/ water (volumetric ratio = 2.5:1:1) was used to extract rhizodeposits under the condition of an ice-bath. The homogenate was centrifuged for 10 min at 9000 g to collect the first supernatant. Then a 4 mL mixed solution was added into the lower sediment followed by a centrifugation to collect the second supernatant. 500 μL of ultrapure water was added into the mixed supernatant before centrifuging at 9000 g, 5 min. The supernatant was then filtered through a 0.22 μm membrane, while the lower liquid was filtered through a 5 cm silica gel column and dried via nitrogen blow-off. Methoxamine hydrochloride (20 mg/mL, 50 μL) and N-methyl-N-(trimethylsilyl) trifluoroacetamide (80 μL) were added as derivatives.30 The GC parameters were as follows: capillary-column chromatograph, HP-5MS; temperature, 6 min at 325 °C with speed of 15 °C/min after 80 °C for 2 min. Mass spectroscopy parameters were as follows: ion source temperature, 250 °C; full scan range, from 70 to 600 m/z. Finally, MetaboAnalyst was used to analyze the Kyto Encyclopedia of Genes and Genomes (KEGG).
immediate detection of a plant stress situation, but the need for expensive equipment greatly limits its applications.11,12 Electrical resistivity (ER) has been used for the quantification and visualization of the plant and soil water relationship, but the data are hard to interpret and difficult to report using online real time platforms.13 Other instruments such as remote sensing tools that were used to monitor plant status showed low sensitivity.14,15 In this context, bioelectrochemical signals may provide a new approach for acid rain monitoring, because it provides fast electrochemical signals that reflect the biochemical reactions between leave, root, and rhizo-microcosm. Plant roots secrete organic matter including carbohydrates, amino acids, amides, aliphatic acids, aromatic acids, and fatty acids throughout their whole life,16,17 and the behavior of such activities can be coupled with organic consumption by rhizosphere microbes and converted to electrical signals reflecting the microbial extracellular electron transfer (EET) process.18,19 Previous studies demonstrated that rhizosphere microbes could produce electrical current in devices called plant microbial fuel cells (PMFCs) by degrading the organic excretes of the rhizodeposits, and theoretically any concentration changes of the bioavailable substrates can be reflected in current fluctuation.20−22 In addition, the milliwatt level of power that is produced by the PMFC can power a wireless sensor for the monitoring of the nutrient level of the plant in smart agriculture. This study presents a new approach to use the electrochemical signals produced by the rhizosphere microbes. Such signals can be used as an early warning sensing system for acid rain and other environmental hazards. We hypothesize that such a device can provide electrochemical responses minutes after the event, which allows fast response to minimize the damage. Three-electrode rhizospheric bioelectrochemical sensors (RBS) were designed to investigate the response mechanism of the leaf-root-microcosm under the stress of acid rain. The rice plant was used as the model grain crop due to its popularity and vulnerability. This process was investigated using combined electrochemical methods, photosynthesis, and leaf-root metabolic analyses. Moreover, the mechanisms of how the plant biochemical stress level can be translated through leaf-root-microorganism to electrical signals were characterized.
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EXPERIMENTAL SECTION
RBS Construction and Operation. Three-electrode reactors were designed to provide a stable redox environment at the anodes (working electrodes). Reactors were constructed by a cylindrical chamber (19.2 cm in length and 8 cm in diameter) with an effective volume of 500 mL and 44.2 cm2 of cross section area.23 In order to maintain an anaerobic environment and avoid the flowback of acid rain to rhizosphere, reactors were sealed by a plastic cover. The working electrode (WE) was a carbon mesh (42 cm2) connected to a titanium wire.24 The counter electrode (CE) was a 1 cm2 Pt sheet with a reference electrode (RE) of Ag/AgCl (3.5 M KCl, 0.197 V versus standard hydrogen electrode). WE, RE, and CE were fixed to the reactor as previously described.25 The rice plants were fixed on the top of the reactors with roots close to WEs. Three electrodes were connected to a multichannel potentiostat (CHI 1000C, Chenhua Instruments, Shanghai, China). The potential of WEs was poised at 0 V (versus Ag/AgCl) except as noted. The rice seeds (14ZJ02, Oryza sativa japonica) were bought from Tianjin Academy of Agricultural Sciences. The rice plants were precultured in a hydroponics equipment using Hoagland’s solution.26 After 7 days, rice plants were transferred into the parallel reactors with B
DOI: 10.1021/acssensors.8b00401 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
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RESULTS AND DISCUSSION Current Decline and Recovery after Acid Rain Events in RBS. After 7 d of growth solely on Hoagland’s solution, RBS reached a stable current plateau of 2.1 ± 0.3 A/m2 (Figures 1
increased after the second acid rain (Figure S2), probably due to the stress responses in the rhizosphere. This is reflected by the metabolic shifts observed in metabolite analysis (Figure 5). The current density dropped below 0.5 A/m2 after the third acid rain, accompanied by withering yellow leaves, indicating permanent plant damage (Figure 1). When the rice plants were removed from the RBS, no current was observed, confirming that the rhizodeposits were the only source of current production. The fast response (
