Article pubs.acs.org/est
Electron Acceptor-Dependent Respiratory and Physiological Stratifications in Biofilms Yonggang Yang,†,‡ Yinbo Xiang,‡ Guoping Sun,†,‡ Wei-Min Wu,§ and Meiying Xu*,†,‡ †
Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, China 510070 ‡ State Key Laboratory of Applied Microbiology Southern China, Guangzhou, China 510070 § Department of Civil & Environmental Engineering, Center for Sustainable Development & Global Competitiveness, William and Cloy Codiga Resource Recovery Research Center, Stanford University, Stanford, California 94305-4020, United States S Supporting Information *
ABSTRACT: Bacterial respiration is an essential driving force in biogeochemical cycling and bioremediation processes. Electron acceptors respired by bacteria often have solid and soluble forms that typically coexist in the environment. It is important to understand how sessile bacteria attached to solid electron acceptors respond to ambient soluble alternative electron acceptors. Microbial fuel cells (MFCs) provide a useful tool to investigate this interaction. In MFCs with Shewanella decolorationis, azo dye was used as an alternative electron acceptor in the anode chamber. Different respiration patterns were observed for biofilm and planktonic cells, with planktonic cells preferred to respire with azo dye while biofilm cells respired with both the anode and azo dye. The additional azo respiration dissipated the proton accumulation within the anode biofilm. There was a large redox potential gap between the biofilms and anode surface. Changing cathodic conditions caused immediate effects on the anode potential but not on the biofilm potential. Biofilm viability showed an inverse and respiration-dependent profile when respiring with only the anode or azo dye and was enhanced when respiring with both simultaneously. These results provide new insights into the bacterial respiration strategies in environments containing multiple electron acceptors and support an electron-hopping mechanism within Shewanella electrode-respiring biofilms.
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INTRODUCTION Anaerobic microbial respiration is a central driving force in Earth’s subsurface biogeochemical cycles and various bioremediation processes.1,2 The electron acceptors respired by bacteria can be generally divided into two forms, that is, soluble electron acceptors such as nitrate, sulfate, and dissolved oxygen and solid electron acceptors such as Fe(III) and Mn(IV), which primarily exist as minerals. Both forms of electron acceptors are ubiquitous in most natural environments. Therefore, understanding how bacteria cells attached to the surface of solid electron acceptors interact with other ambient soluble electron acceptors is fundamental to understanding the overall biogeochemical processes. Addition of favorable electron acceptors such as Fe(III) or nitrate has been considered to be a promising strategy to stimulate the bioremediation of contaminated sites.3−5 Bioelectrochemical systems (BES), representatively microbial fuel cells (MFCs), provide an anode as a solid electron acceptor for bacteria (exoelectrogens) to generate electricity and perform bioremediation in contaminated environments simultaneously.6,7 Moreover, BES provide a novel and quantifiable tool to understand electron transfer mechanisms between bacteria and solid electron acceptors such as electrodes, © 2014 American Chemical Society
minerals, or their bacteria partners. An important concern with BES application is the ubiquity of alternative electron acceptors (e.g., O2, nitrate, sulfate, or CO2) in complicated environmental systems.8−10 Theoretically, the presence of alternative electron acceptors may dissipate the electron flow toward anode and affect the performance of BES. However, counterintuitively, several studies have reported that different bacterial respirations could occur simultaneously or even be reciprocally enhanced in BES,9,11,12 suggesting that microbial electron transfer networks within field-applied BES could be more complicated than anticipated. Anode-respiring biofilms play a key role in BES. The electron transfer mechanism in anode-respiring biofilm has been intensively investigated yet remains under debate.13−15 Two bacteria−electrode electron transfer models have been proposed recently, electron-hopping and metal-like conductivity.14,15 The physicochemical microenvironments (e.g., pH, redox potential, and diffusion efficiency) and physiological Received: Revised: Accepted: Published: 196
September 17, 2014 December 11, 2014 December 11, 2014 December 11, 2014 DOI: 10.1021/es504546g Environ. Sci. Technol. 2015, 49, 196−202
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Environmental Science & Technology
recorder (Keithley 2700, module 7702). The reactors were operated in triplicate for each experimental condition. Chemical and Physiological Analysis. Absorption at 520 nm was used to monitor the changes in amaranth concentration as previously reported.20,21 Protein content was quantified to evaluate the cell growth of planktonic cells and biofilm as described before because azo dye can interfere with optical assay and colony count due to its red color and its inhibition on cell division.9 Confocal laser scanning microscopy (CLSM) was used to analyze the biofilm structure and cellular activity.22−24 Before CLSM analysis, biofilm on the anode was sampled and dipped in sterilized PBS to remove loosely attached planktonic cells or debris on biofilms. The sample was then stained with LIVE/DEAD BacLight staining kit (Molecular Probes, Invitrogen) and observed under CLSM (LSM 700, Zeiss). The staining kit was developed based on bacteria membrane permeability and is commonly used to distinguish bacteria with high (green) or low (red) growth activity rather than determine if the cells are living or dead.25 Randomly sampled view fields were observed and analyzed for each anode biofilm. To obtain three-dimensional (3D) structure information, the biofilm sample was observed under the “Stack” model of the Zen software (Zeiss). Specific viability of each biofilm layer was analyzed and presented as the ratio of viable to total biofilm cells based on pixel counting.9,22 Total viability of a biofilm sample was the averaged viability of each biofilm layer. Microelectrode Analysis. The dissolved oxygen, pH, and redox potential profiles within bulk liquid and anode biofilm were determined using a microelectrode system (Unisense, Denmark) (Figure S2, Supporting Information) before amaranth was exhausted.26,27 Prior to analysis, the rubber stoppers of the anode chambers of the MFCs were replaced with parafilm through which the microelectrodes (tip diameter, 25 μm) were inserted and stepped down toward the anode surface using SensorTrace Pro (v.3.2.7) software. A microscope (300× , Supereyes) was used to monitor the positions of the microelectrode tips.
properties (e.g., transcription, viability, and metabolites) within anode-respiring biofilms are spatially stratified due to electron transfer toward the electrode.16−19 These findings provide critical information for understanding the electron transfer mechanism within anode-respiring biofilm as well as mineralrespiring biofilm in natural environments. To date, however, most studies on BES have been performed using anode as the sole electron acceptor, so little information is available on how the presence of multiple electron acceptors will affect the anode biofilms. In this study, we tested the azo dye amaranth, a chemical widely found in textile wastewater, as a representative alternative electron acceptor to examine its impact on anode biofilm in MFCs. The MFCs were inoculated with Shewanella decolorationis, a biofilm-forming exoelectrogen. In the MFCs, the alternative electron acceptor was reduced simultaneously with current generation. A spatially divided respiration of the planktonic and biofilm cells occurred within the anode chamber. Electron acceptor-dependent chemical and physiological stratification were observed within anode biofilm, suggesting a stratified and flexible electron transfer pattern within Shewanella biofilm in the presence of multiple electron acceptors.
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MATERIALS AND METHODS Bacterial Strain. The S. decolorationis strain S12T (CCTCC M203093T = IAM 15094T) was isolated from activated sludge from a textile-printing wastewater treatment plant and preserved in our laboratory.20,21 This strain can use amaranth (Figure S1, Supporting Information) or Fe(III) as electron acceptor and form biofilms on the anode of MFCs.9,20,21 A single colony of strain S12 from a Luria−Bertani (LB) plate was picked and inoculated into sterilized LB broth, and the culture was aerobically incubated overnight at 30 °C. The cells were separated from the culture by centrifugation (6000g) for 2 min and then washed twice using sterilized phosphate buffer solution (PBS) to remove residual nutrients. The washed cells were used as inoculum of MFCs. MFCs and Operations. Dual-chamber MFCs were assembled as previously described.9 Plain graphite plates (2 cm × 3 cm × 0.2 cm) were used as anodes and cathodes. An Ag/AgCl electrode (+0.197 V vs standard hydrogen electrode) was used as a reference electrode to the anode. The anode and cathode chambers were separated with a piece of Nafion 115 membrane (7.1 cm2). After being assembled and sterilized (115 °C for 20 min), the anode chamber (120 mL) was filled with 100 mL of LM medium (12.8 g/L of Na2HPO4, 3 g/L of KH2PO4, 0.5 g/L of NaCl, 1.0 g/L of NH4Cl, and lactate 10 mM, pH 6.8). To stimulate biofilm growth, 0.05% (w/v) yeast extract was added to the medium. Different concentrations of azo dye amaranth (0, 2, 5, 7, and 10 mM) were added into the anode chambers as the alternative electron acceptor, unless otherwise stated. The catholyte contained 100 mL of PBS and 50 mM of potassium ferricyanide. After inoculation (2%, v/v), the anode chamber was bubbled with N2 to remove headspace air and dissolved oxygen and then was sealed with rubber stoppers. MFCs were operated at 30 °C with closed external circuits including 1000 Ω external resistors. A set of an equal number of MFCs with open circuits were operated in parallel as normal anaerobic (NA) control reactors. Abiotic reactors containing different amaranth concentration were also operated to evaluate the effects and possible electrochemical reduction of amaranth. Current generation was monitored with a data
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RESULTS AND DISCUSSION Simultaneous Azo Reduction and Current Generation. In either MFCs or NA reactors, amaranth was completely reduced within 28 h (Figure 1A), except for the MFCs containing 10 mM of amaranth where 0.7 mM of amaranth remained due to exhaustion of the electron donor. No amaranth reduction was detected in the abiotic reactors. Amaranth reduction rates in the MFCs were generally lower than those in the NA reactors containing the same amaranth concentration, indicating that current generation redirected some of the electron flow away from azo reduction. Moreover, the amaranth reduction rate in all reactors increased with the amaranth concentration even though the azo dye concentration used in our study (up to 10 mM) was higher than those used in other reports,31 indicating a relatively high azo reducing capacity of this strain. Figure 1B shows the current generation of MFCs with or without amaranth (complete current generation period is shown in Figure S3, Supporting Information). The amaranthfree MFCs generated a current of 0.01 mA upon inoculation, while the MFCs with amaranth had a lag phase of about 8 h before current generation (Figure 1B), indicating that the planktonic cells preferred amaranth as electron acceptor rather than the anode. Accompanied with azo reduction, the current generation in amaranth-containing MFCs increased rapidly 197
DOI: 10.1021/es504546g Environ. Sci. Technol. 2015, 49, 196−202
Article
Environmental Science & Technology
Figure 2. Growth of S. decolorationis with different electron acceptors. (A) Change in protein concentrations as indicator of planktonic cells in MFCs with or without amaranth (5 mM) or NA reactors. (B) Protein contents on anodes from MFCs with or without amaranth or NA reactors after 32 h.
Figure 1. Simultaneous amaranth reduction and current generation. (A) Reduction of different concentrations of amaranth in MFCs (solid lines) and NA reactors (dashed lines). Inset shows amaranth reducing rates in MFCs and NA reactors. (B) Current generations in MFCs containing different concentrations of amaranth.
reduction and current generation contributed to the biofilm growth. Differences in the biofilm biomass suggests that azo reduction accounted for a major portion of the biofilm biomass in amaranth-containing MFCs while current generation accounted for a smaller portion. These biomass differences were consistent with the expected differences based on the Gibbs free energy calculations, which show that the free energy generated from amaranth reduction may be over 3-fold higher (−61.8 J, calculated with eq S1, Supporting Information) than that generated from anode respiration (−17.7 J, calculated with eq S2, Supporting Information) in the MFCs containing 5 mM of amaranth. pH and Redox Potential Profiles in Biofilms. Measurement with an O2 microelectrode confirmed that the bulk liquid was anoxic during experiments (Figure S5, Supporting Information). A pH microelectrode measurement (Figure 3A, B) showed that the bulk liquid pH decreased with cell growth, that is, amaranth-containing MFC (6.43) < NA reactor (6.55) < amaranth-free MFCs (6.65), indicating acidic metabolites were secreted during Shewanella extracellular respiration. The greater increase in current generation in amaranth-containing MFCs would also decrease pH as bacteria electrode respiration is a proton-releasing reaction. A decreasing pH gradient (about 0.02) was observed in the biofilm in amaranth-free MFCs, indicating protons accumulation within the anode-respiring biofilm as predicted previously.35 The pH gradient observed in this study was smaller than those (0.08 to 1 unit) observed in other anode-respiring biofilms,17,36 which could be due to a thinner biofilm or the relatively high diffusion efficiency of Shewanella biofilms.37 In contrast with the anode-respiring biofilm, the pH gradient decreased or disappeared in the
after the lag phase and was nearly 2-fold higher than in amaranth-free MFCs (i.e., 0.3, 0.34, 0.29, and 0.28 mA for MFCs containing 2, 5, 7, and 10 mM amaranth, respectively, versus 0.13 mA for amaranth-free MFCs). The enhanced current generation in the amaranth-containing MFCs could be attributed to the electron mediator role of the naphthols generated from amaranth reduction.9 MFCs containing 5 mM amaranth generated the highest current, while a decreased metabolic activity of S. decolorationis cells was observed at higher amaranth concentrations due to toxicity (Figure S4, Supporting Information).32 Therefore, 5 mM of amaranth was used as the alternative electron acceptor in subsequent experiments. Planktonic and Biofilm Cell Growth. The protein-based planktonic biomass in amaranth-containing MFCs was approximately twice that of amaranth-free MFCs but showed no significant difference to that in NA reactors with the same concentration of amaranth (Figure 2A). The results indicated that anode respiration did not show significant influence on planktonic cell growth, that is, that the planktonic cell growth was primarily driven by amaranth reduction rather than electrode respiration. This is consistent with previous reports that S. decolorationis current generation was biofilm-dominated, and the anode-respiring S. decolorationis cells preferred to attach on the anode surface rather than be suspended in the culture liquid.33,34 Unlike the planktonic cells, the protein content of the anode biofilm in the amaranth-containing MFCs was significantly higher than that of amaranth-free MFCs (P < 0.01) and NA reactors (P < 0.05) (Figure 2B), indicating that both azo 198
DOI: 10.1021/es504546g Environ. Sci. Technol. 2015, 49, 196−202
Article
Environmental Science & Technology
Figure 3. pH and redox potential profiles above anodes in different reactors. (A) pH profiles from the liquid surface to the anode surface. (B) Magnification of pH profiles within 95 μm above anode surface. (C) Redox potential profiles from the liquid surface to anode surface. (D) Magnification of the potential profiles within 95 μm above anode surface. The 0 μm depth represents the biofilm−anode interface, and the dashed lines indicate the approximate biofilm−liquid interface (red for amaranth-respiring biofilm; green for anode-respiring biofilm; blue for the biofilm respiring both). The interfaces were determined by the real time microscope monitoring (300×) and CLSM biofilm structure observation.
predicted that the redox potential within anode-respiring biofilm containing electron mediators would first decrease and then increase as a function of the redox state of electron mediators.35 The prediction is suitable for Shewanella biofilm as Shewanella anode respiration is dominated by electron mediators, that is, flavins.38 The potential within anoderespiring biofilm continuously decreased to −340 mV then dramatically increased to 102 mV at the anode surface within several micrometers (
