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Environmental Technology
ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: https://www.tandfonline.com/loi/tent20
Enhanced phosphorus reduction in simulated
eutrophic water: a comparative study of
submerged macrophytes, sediment microbial fuel
cells, and their combination
Peng Xu, Enrong Xiao, Dan Xu, Juan Li, Yi Zhang, Zhigang Dai, Qiaohong Zhou
& Zhenbin Wu
To cite this article: Peng Xu, Enrong Xiao, Dan Xu, Juan Li, Yi Zhang, Zhigang Dai, Qiaohong Zhou & Zhenbin Wu (2018) Enhanced phosphorus reduction in simulated eutrophic water: a
comparative study of submerged macrophytes, sediment microbial fuel cells, and their combination, Environmental Technology, 39:9, 1144-1157, DOI: 10.1080/09593330.2017.1323955
To link to this article: https://doi.org/10.1080/09593330.2017.1323955
Accepted author version posted online: 26 Apr 2017.
Published online: 13 May 2017. Submit your article to this journal
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Enhanced phosphorus reduction in simulated eutrophic water: a comparative
study of submerged macrophytes, sediment microbial fuel cells, and their
combination
Peng Xua,b, Enrong Xiao a, Dan Xua,c, Juan Lia,c, Yi Zhanga, Zhigang Daia, Qiaohong Zhouaand Zhenbin Wua
a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People’s Republic of China;bUniversity of Chinese Academy of Sciences, Beijing, People’ Republic of China;cCollege of Resources and Environmental Engineering, Wuhan University of Technology, Wuhan, People’s Republic of China
ABSTRACT
The phosphorus reduction in water column was attempted by integrating sediment microbial fuel cells (SMFCs) with the submerged macrophyte Vallisneria spiralis. A comparative study was conducted to treat simulated water rich in phosphate with a control and three treatments: SMFC alone (SMFC), submerged macrophytes alone (macophyte), and combined macrophytes and fuel cells (M-SMFC). All treatments promoted phosphorus flux from the water column to sediments. Maximum phosphorus reduction was obtained in proportion to the highest stable phosphorus level in sediments in M-SMFC. For the initial phosphate concentrations of 0.2, 1, 2, and 4 mg/L, average phosphate values in the overlying water during four phases decreased by 33.3% (25.0%, 8.3%), 30.8% (5.1%, 17.9%), 36.5% (27.8%, 15.7%), and 36.2% (0.7%, 22.1%) for M-SMFC (macrophyte, SMFC), compared with the control. With macrophyte treatment, the obvious phosphorus release from sediments was observed during the declining period. However, such phenomenon was significantly inhibited with M-SMFC. The electrogenesis bacteria achieved stronger phosphorus adsorption and assimilation was significantly enriched on the closed-circuit anodes. The higher abundance of Geobacter and Pseudomonas in M-SMFC might in part explain the highest phosphorus reduction in the water column. M-SMFC treatment could be promising to control the phosphorus in eutrophic water bodies.
ARTICLE HISTORY
Received 17 November 2016 Accepted 23 April 2017
KEYWORDS
Sediment microbial fuel cell; Vallisneria spiralis; coupled system; phosphorus reduction; anode bacterial community
1. Introduction
Phosphorus (P) is considered a major contributing factor to eutrophication in aquatic ecosystems [1]. The release of P from sediments to the overlying water is the essen-tial cause of the failure to eliminate eutrophication once the input of external P to a water body is controlled [2]. Iron hydroxides serve as potent repositories of P in sedi-ments. Their dissolution by iron-reducing bacteria (IRB) often results in release of P into the water column [3], which is likely to occur under anaerobic conditions. Therefore, improving the low sediment potential to inhibit phosphate (PO34−) regeneration and release is always a key issue in control of eutrophication [4,5].
The sediment adsorption is often defined as the strong interaction between P dissolved in the water column and that on fine sediment particles containing Fe/Al oxides (hydroxides) and calcium minerals [6]. Ligand exchange with OH groups, electrostatic attrac-tion, and ion exchange are generally accepted as the main three types of adsorptive modes [7]. The Fe(III)
hydroxides have large surface area and positive charge, and are often the key agents of sediment adsorption at the sediment–water interface. In current management practice, they are often used to reduce the P concen-tration in the water body [8]. Therefore, enhancing the oxidation of Fe2+ to Fe3+ to increase the Fe3+ ratio in sediments is a significant approach for improving the adsorption of P.
Sediment microbial fuel cells (SMFCs) include an anode embedded in anaerobic sediments and a cathode suspended in the aerobic water column. They have attracted considerable attention for controlling eutrophication of water bodies. In SMFCs, bacteria mediate the transfer of electrons from carbon sources in the sediments to the anode, thereby generating elec-tric current and facilitating degradation of organic matter (OM) in sediments [9]. The electrode inserted into the sediments diverts the flux of electrons from Fe(III) oxides to the surface of the anode, preventing P from dis-solving and being released [10]. The competition
© 2017 Informa UK Limited, trading as Taylor & Francis Group
CONTACT Enrong Xiao [email protected] State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of
Sciences, Wuhan 430072, People’s Republic of China
VOL. 39, NO. 9, 1144–1157
between anode and Fe3+ as the electron acceptors for the electrons produced by extracellular bacteria, such as the IRB, could lead to an increase of Fe3+content in sediments. In addition, the produced electron flow from anode to cathode across the external circuit, causing an increase of sediment Eh [11], may also have effects on the increase of ratio of Fe2+over Fe3+. There-fore, an increase of Fe3+ content in sediments due to SMFC presence could enhance the sediment adsorption with increased P flux from the overlying water to sedi-ments to form more stable P in sedisedi-ments [12]. However, the temporarily fixed P on sediments remains prone to release if the anaerobic condition returns due to interruption in the generated power. Therefore, how to further improve and maintain aerobic conditions in the sediments is essential when using SMFC to control P in shallow lake ecosystems.
The submerged macrophytes are a major functional group in aquatic ecology, which bridge the water –sedi-ment interface and could influence the dynamic equili-brium of P by changing dissolved oxygen (DO), pH, and Eh of the sediments and the overlying water. The submerged macrophytes often have ability to remove P from water and sediments through tissue assimilation and enhancement of P adsorption on sediments [13]. However, the P removal appears to be dependent on morphology, a conclusion has been supported by pre-vious studies. For instance, Ceratophyllum demersum, with characters of high surface area:volume ratio and biomass in overlying water, has been proved to remove P more effectively in overlying water compared with other submerged macrophytes [14,15]. However, the C. demersum had no significant effect on P removal in the pore water and no influence on the inorganic phosphorus (IP) in sediments due to the lack of true roots [15]. Potamogeton crispus with weak roots could not alter the sediment Eh and P adsorption [16]. Vallis-neria spiralis as a widespread and dominant species has large biomass, root/shoot ratio, and strong radial oxygen loss (ROL) [17], would function well for P control both in water bodies and sediments. Plentiful IP could be assimilated by V. spiralis from water and sedi-ments [14]. Mobilization of PO34− from water bodies on sediments may be promoted when Eh is increased by release of oxygen from roots, allowing formation of more Fe(III) hydroxides in the sediment rhizosphere (root zone) [18,19]. Consequently, it is natural to think of combining V. spiralis with SMFC, based on their common potential for enhancing the sediment Eh and sediment adsorption of P from water bodies. Actually, the root exudates of hygrophytes have been reported to improve the power production of SMFCs [20,21]. In other work, coupled M-SMFC was also used to treat
wastewater and harvest power simultaneously [22,23]. However, little information is available on changes in the P flux in sediment–water systems when submerged macrophytes and SMFC are combined.
Hence, the aim of this study was to determine whether the coupled M-SMFC technology could control sediment P release sustainably, and promote P flux from water bodies to sediments. This was determined by comparing the performance of P migration and trans-formation in a water–sediment system using three treat-ments: submerged macrophyte V. spiralis alone (macrophyte), SMFC alone (SMFC), and combined macro-phytes with SMFC (M-SMFC), respectively.
2. Materials and methods 2.1. Materials preparation
Fresh sediments collected from West Lake (Hangzhou, China, 30°23′N 120°14′E) were homogenized, after which macrofauna and macrophyte debris was removed. The submerged macrophyte V. spiralis, heavily used in phytoremediation, was employed in this study. Well-grown V. spiralis individuals with similar morphology were chosen to transplant into the tanks.
2.2. Experimental set-up
Twelve tanks of the same size (35 × 35 × 65 cm) and volume (79.6 L) were designed and allocated to a control set and three treatment sets in triplicates. The control simulated a eutrophic lake and contained only sediments and water. The three treatments were SMFC only (SMFC), only submerged macrophytes, V. spiralis (macrophyte), and the combination of SMFC and macro-phytes (M-SMFC).
In each tank, three Rhizon SMS samplers (Shanghai Safe Biotech Co., Ltd., China) were installed at heights of 2, 7, and 12 cm from the bottom. Graphite felt 0.5 cm thick was used as electrodes. Three pieces of graphite felt of identical dimensions (35 × 15 cm) were connected by titanium wire and fixed vertically with equal spacing for use as the anode. For the cathode, graphite felt with the same surface area (35 × 45 cm) as the anode was placed vertically in the overlying water and the top 5 cm was exposed to the air. The dis-tance between the centers of the anode and cathode electrodes was 32.5 cm. The identical electrodes were set in the control and macrophyte tanks, but with an open circuit to determine the sediment Eh and to collect sediment microbial samples conveniently. Then, each tank was filled with 10 kg of sediments, which accounted for 23% of the total volume. The submerged
macrophytes used in this experiment were collected in May from a shallow water area belonging to West Lake with abundant fast-growing V. spiralis. The living con-ditions of V. spiralis were as follows: total nitrogen (TN) and total phosphorus (TP) levels of water bodies were 1.2 and 0.07 mg/L, respectively, OM content of sedi-ments was 14.1% (dry weight). Well-grown seedlings of V. spiralis with similar root lengths (8–10 cm), number of green leaves (6–8), and 15-cm leaf lengths were selected [24]. Twelve sets of prepared V. spiralis individ-uals were transplanted into the sediments in four equidi-stant lines in the macrophyte and M-SMFC treatments. Afterwards, 55 L of tap water was added to every tank. Then, copper wires were connected with the titanium wire to form circuits through an external resistance of 100Ω for the SMFC and M-SMFC treatments. In addition, four lamps were positioned above the 12 tanks to artifi-cially illuminate the macrophytes using a 12:12-h light/ dark cycle. Water loss due to evaporation and sampling was mainly replenished with distilled water. Figure 1
shows the experimental arrangement.
Experiments were performed in static mode in the temperature range of 20−35°C. In order to clarify the migration pathways of PO34− in overlying water and to investigate the influence of initial concentration on the migration quantity of PO34− under electrogenesis in SMFC and M-SMFC, the whole experiment was divided into four phases, according to the use of different initial PO3−4 concentrations (0.2, 1, 2, and 4) mg/L in the overlying water. These concentrations were achieved by adding KH2PO4 respectively at the start of phase I
(days 1−55), phase II (days 68−104), phase III (days 117 −153), and phase IV (days 161−197).
2.3. Sampling and analysis
The cathode potential and voltage were recorded every five minutes by paperless recorders (R6000, Shanghai Jisame Electric Co., Ltd). The Ag/AgCl(S) reference elec-trode (200 mV against a standard hydrogen elecelec-trode,
218, Shanghai Leici Co., Ltd) was installed near the cathode to measure the cathode potential; the anode potential was calculated by subtracting the voltage from the cathode potential. The current (I ) was calcu-lated according to Ohm’s law: U = I R, where U is voltage and R is external resistance. The current density was calculated with the function of anodic surface area. A Hach portable water quality analyser (HQ30d, Hach Company, USA) was used to monitor online, the par-ameters of DO, pH, and temperature (T ) in the tanks before sampling. The overlying water samples collected by siphon every nine days were used to determine the TP and phosphate (PO3−4 ) concentrations. Three layers of pore water were sampled using Rhizon SMS samplers to measure PO3−4 and dissolved total phosphorus (DTP) on days 1, 28, 55, 77, 104, 135, 161, and 197. Dissolved organic phosphorus (DOP) concentration was calculated as PO34−subtracted from DTP (DOP= DTP − PO34−). The indexes detailed above were tested using standard lab-oratory procedure methods [25]. As for the pretreated sediments, air-drying was used to determine the water content (wt%) of pretreated sediments; the OM content was determined by weighing the sample before and after combustion at 550°C for 4 h; and the pH was measured using a pH meter (PHS-3C, China). Quantitative analysis of elements in the pretreated sedi-ments was determined using X-ray fluorescence spec-trometer (Axios advanced, PANalytical B.V., The Netherlands). During the operation, sediment samples were collected from each tank using a miniature cylind-rical sediment sampler at the end of each phase. Three separate sediment columns were mixed homogeneously to serve as one sample. TP was determined according to the standard measurements and testing harmonized protocol [26]. P forms including P loosely adsorbed (NH4Cl-P), P bound Fe(III)-hydroxides and Mn
com-pounds (BD-P), P absorbed to Al oxides (NaOH-P), and acid soluble P (HCl-P) according to the methods of Pet-tersson et al. [27] and Hupfer et al. [28] Organic P (OP) was obtained by subtracting the sum of the four
fractions from TP. All analytical determinations were per-formed in triplicate and averaged.
Sediments on the anode surface collected at the end of experiment were firstly removed by rinsing with sterile water, followed by scraping the carbon felts using a sterile razor blade to get the anode-associated biofilms. Four biofilm samples from the anodes of the control, SMFC, macrophyte, and M-SMFC tanks were used to extract Genomic DNA with an E.Z.N.A.® DNA Isolation Kit (Omega Biotek, USA). Illumina high-throughput sequencing, using extremely pure genomic DNA (A260/ A280≈ 1.8) was conducted by Hengchuang Biotechnol-ogy Company (Shenzhen, China). Similar sequences were clustered into operational taxonomic units (OTUs) based on 3% dissimilarity. Based on these clusters, lists of OTUs were generated in QIIME using a cut-off of 0.03 through random selections of the minimum sequences of all samples. The phylum, class, order, family, and genus levels of taxonomic classification were performed based on sequences from Ribosomal Database Project, with a bootstrap cut-off of 80%. Cluster and Treeview programs were used to construct a taxon heatmap.
2.4. Statistical analysis
The results of this study were given in mean ± standard deviation. The significance of differences among differ-ent treatmdiffer-ents was evaluated using two-way analysis of variance (time and treatment as two factors), using SPSS software, where P < .05 was defined as significant difference.
3. Results and discussion
3.1. Power generation performances of SMFCs and M-SMFCs
Figure 2(a) shows the electrical current densities in SMFC and M-SMFC; a similar trend during 197 days of oper-ation was observed. Current density of SMFC gradually increased during phases I and II, followed by significant decreases that started from phase III. M-SMFC performed significant increases of current density during the first 86 days, after which constant decreases were observed. The maximum current densities for SMFC and M-SMFC were 18.11 and 12.12 mA/m2, respectively, and occurred on days 117 and 86, respectively. Han et al. [17] studied quantitative imaging of ROL from V. spiralis roots with a fluorescent planar optode. The results showed that oxygen release estimated from the rhizosphere of V. spiralis is much higher than that estimated for many other macrophyte species. Therefore, at the start of
phase II, the significantly lower generation of current density in M-SMFC (compared with SMFC) might be related to biological and chemical oxygen reduction due to the ROL from V. spiralis, when the electrons gen-erated by electrogenesis bacteria were transferred to oxygen instead of the anodes [29].
The sediment Eh in the control was relatively stable from −453 to −375 mV during the whole operation (Figure 2(b)). In SMFC, the sediment Eh gradually decreased from an initial value of 248 mV, and a minimum Eh of −43 mV was achieved at the start of phase III. This was followed by a significant increase after-wards. These results suggested that the sediment Eh was significantly enhanced by electrogenesis, and that the anaerobic condition was converted to anoxic condition [11,30]. The ROL from V. spiralis also resulted in elevating the sediment Eh when the average Eh of 74 and 152 mV were achieved by SMFC and M-SMFC, respectively, during the whole operation. The highest sediment Eh
Figure 2.Changes in current (a), sediment Eh (b), and the par-ameters of DO (c), pH (d), and T (e) in overlying water during four phases.
was achieved with M-SMFC, implying a synergistic inter-action between the anodes and ROL.
3.2. P mobility and influencing factors in sediment–water system
The mass balance of P in the system was determined to assess the mobility between water and sediments. During the four phases of operation, the corresponding increments of TP in the sediments were basically equal to the TP content reduction in the overlying water (Figures 3(b) and4(a)). TP decreases irrespective of the initial concentration in the overlying water were mostly attributed to the migration of PO3−4 from water to sedi-ments. This might have been driven by chemical
adsorption to form insoluble sediments with iron, alu-minium, and calcium compounds (Table 1) [6,7], by microbial assimilation [31], and by the concentration gra-dient. With increase of the initial PO34− concentration, despite the decrease in the removal rate, the quantity migrating was increased (Table 2). The decrease of P in the overlying water did not significantly influence the P concentration in the pore water (Figure 5). The concen-trations of DTP and PO3−4 in the pore water decreased as the depth increased, whereas the DOP concentration (in vertical profile) lacked a specific trend. The PO3−4 was the main form of DTP, especially in the upper and middle layers. A sustained drop of DTP (PO3−4 ) in the pore water during the first period might also have resulted from adsorption and assimilation of P by sediments. The
Figure 3.Changes in PO3−4 (a) and TP (b) concentrations in overlying water over time at different initial concentrations.
constant increase of DTP (PO3−4 ) that followed might be associated with decreased P adsorption by sediments with increased temperature (Figure 2(e)) [32]. Another possible explanation is that the release of PO34−resulted from the constant OP mineralization in sediments (Figure 4(b)). The NaOH-P and NH4Cl-P did not change signifi-cantly in the control sediments during the experiment (Figure 6(b,d)), implying that the PO34− from artificial addition in the overlying water and mineralization of OP were mainly adsorbed onto calcium minerals and Fe(III) hydroxides to form HCl-P and BD-P with increasing trends (Figure 6(a,c)).
3.3. Effects of SMFC on P mobility between water and sediments
Electrogenesis had significant effects on P removal from the overlying water during phases II to IV (Figure 3and
Table 2) (P < .05). The increase in the amount of flux with SMFC, compared with the control, was also significantly improved with increase of the initial PO3−4 concen-tration. The PO34− in the middle and bottom layers of pore water was also lowered under electrogenesis during the whole operation (Table 3) (P < .05). These results suggested that a higher migration rate (flux) from water column to sediments was present in SMFC, and that the process was more significant in water bodies with higher PO34− content. The installation of SMFC could enhance the microbial oxidation of Fe2+ to Fe3+ in sediments if the sediment Eh were signifi-cantly increased [12]; furthermore, the introduction of an anode as an electron acceptor in sediments could also lead to an increase of the availability of Fe(III) hydroxides when the IRB transfer electrons to the anode instead of to Fe(III) hydroxides [10]. This would result in increased PO34− bound with [Fe(OH)3]x, a
colloid with high-specific surface area and stability. Another possible explanation is increased microbial activity, especially of the polyphosphate-accumulating organisms (PAOs) in sediments. During phase I, SMFC had no effect on P removal from the overlying water, whereas significantly lower PO34− was observed during electrogenesis in the bottom layer of the pore water (Figure 5(b)). This implied that the influence of electro-genesis on the PO3−4 in the pore water was greater than that in the overlying water when the oxidation
layer was not thoroughly formed. The PO3−4 level in the upper layer of pore water was not influenced signifi-cantly by electrogenesis. This might have been because the higher oxygen level is not conducive to the growth of IRB, so that iron oxidation/reduction reaction does not occur readily at the sediment–water interface under aerobic conditions [33].
The lower DOP concentration in the pore water (middle and bottom layers), and the OP in the sediments in SMFC suggested facilitated decomposition of OP with concomitant generation of electricity, which was consist-ent with previous observations [9,10,12]. The stronger mineralization of OP in sediments and the flux of PO3−4 from the overlying water, followed by adsorption, led directly to the higher BD-P and HCl-P under electrogen-esis. More stable HCl-P in SMFC might also be related to the alkalization of the overlying water (Figure 2(d)), when co-precipitation of P and calcite was promoted in the water column. Compared with the control, BD-P in SMFC increased by 8.6%, 12.1%, 21.6%, and 26.4% on days 55, 104, 153, and 197, respectively. Slight increases of HCl-P in SMFC were 3.7% and 4.6% observed on days 153 and 197. During the whole oper-ation, the influence of SMFC on NaOH-P and NH4Cl-P
was not significant.
3.4. Effects of V. spiralis on P mobility between water and sediments
In this study, the V. spiralis in macrophyte treatment played a significant role in PO3−4 removal in the upper and middle layers of pore water during phases I–III. It is often assumed that the submerged macrophytes obtain P for vegetative growth primarily from the sedi-ment pore water [13]. Besides the root uptake, the pro-moted P adsorption to oxidized sediments might also result in significantly enhanced removal of PO3−4 by V. spiralis from the pore water if the sediment Eh were significantly elevated. Racchetti et al. [19] found that higher oxygen level and Eh within the rhizosphere of V. spiralis were able to maintain the oxic conditions in the sediments, allowing the formation of iron plaques on the roots, followed by precipitation of PO3−4 with Fe (III) hydroxides from the pore water. ROL from V. spiralis could also be validated by the significant decreases of OP in sediments and of DOP from pore water (upper
Table 1.Properties of pretreated sediments used for experiments.
pH OM (dt%) Water content (wt%)
Compound concentrations (dt%)
SiO2 Al2O3 Fe2O3 CaO K2O MgO
7.30 ± 0.13 13.33 ± 0.58 40.0 ± 4.0 56.95 15.16 3.97 1.90 1.67 1.10
Notes: The data of pH, OM, and water content was given in mean ± standard deviation. dt% and wt% represent the percentage of dry-weight and wet-weight, respectively.
and middle layers) when a suitable niche for the miner-alization of OP by aerobic bacteria was formed. The insig-nificant effect on PO34− removal in the bottom layer of pore water by the macrophyte might be attributed to the majority of root lengths being mainly 1–10 cm. Han et al. [17] suggested that the measured oxygen pen-etration depths by V. spiralis from root tips were just several millimetres with extension of the incubation period. In the overlying water, assimilation by leaves also significantly facilitated the removal of TP/PO3−4 during phase I [14]. The insignificant effect on PO34− removal during phase II suggested that uptake by the leaves of V. spiralis was finite and limited. Therefore, the following significantly stronger P removal during phase III might also be due to increased sediment Eh and the affinity of P in sediments under V. spiralis, when 26.3% (27.8%) of TP (PO34−) was reduced by macro-phyte compared with the control.
The insignificant PO34−removals in the water column by macrophyte during phase IV (Figures 3(a) and 5(b)) were mainly attributed to the macrophyte decay. The life cycle for most submerged macrophytes are usually completed in several months, the debris of V. spiralis in macrophyte might have resulted in the accumulation of OP in sediments (from 495.3 to 521.7 mg/kg) during phase IV and of DOP in the bottom layer of pore water due to precipitation [34]. Wang et al. [35] found that the addition of submerged macrophyte Myriophyllum spicatum in sediments could increase the alkaline phos-phatase activity of sediments, the OP mineralization was then facilitated, followed by significant PO34− release from sediments to water. In this study, the same mech-anism might also have driven the PO34−release. Besides, the alkalization of the overlying water in macrophyte might have increased the HCl-P in sediments, especially during phase III (Figure 6(c)). The co-precipitation of PO34− with Ca2+ is easy to occur under alkaline conditions, followed by the formation of deposit of Ca5(PO4)3OH as the main component [36]. The elevated
pH (Figure 2(d)), owning to intense photosynthesis by periphyton and the plant, could result in CaCO3
supersaturation. This would promote P removal by co-precipitation [37,38]. However, in the last phase of operation (phase IV), a decrease of HCl-P was observed (from 535.9 to 506.3 mg/kg). The Ca-P may have been re-dissolved with autogenous release of Ca-P to the water column, when the sediments were acidified due to acid degradation of debris [39]. In addition, the increased internal OP was bound to result in stronger mineralization, which might result in saturated plant uptake capacity. Furthermore, increased microbial and chemical consumption of oxygen in organically richer sediments could minimize the thickness of oxic layers
Table 2. The initial, final, and mean values of TP and PO 3− 4 in overlying water during four phases of operation. Para meters Phase I (1 –55 day s) Phase II (68 –1 04 d ays) Phase III (117 –153 days) Phase IV( 161 –1 97 d ays) C ontrol SMFC Ma croph yte M-SMFC Cont rol SMF C Macrop hyte M-SMFC Contro l SMFC Macroph yte M-SMFC Co ntrol SMFC Ma crophyt e M-SMFC Initia l TP (mg /L) 0.22 0.23 0.23 0.22 1.38 1.38 1.38 1.38 2. 62 2. 62 2.62 2.62 4.78 4.78 4.78 4.78 Fin al TP (mg /L) 0.06 0.06 0.03 0.03 0.17 0.06 0.14 0.06 0. 75 0. 42 0.14 0.06 2.94 1.73 2.30 0.90 Re (%) 72.73 73.91 86.96 86.36 87.68 95.65 8 9.86 9 5.65 71. 37 83. 97 94.66 97.71 38.50 63.81 51.88 81.17 Rq (mg) 8.80 9.35 11.00 10.45 66.55 72.60 6 8.20 7 2.60 102. 85 121. 00 136.40 140.80 101.20 167.75 136.40 213.40 Me an TP (mg /L) 0.13 ± 0.08a 0.14 ± 0.07a 0.10 ± 0.08b 0. 09 ± 0.08b 0.6 2 ± 0. 48a 0.4 9 ± 0. 53b 0.58 ± 0.5 0a 0.44 ± 0.5 5b 1.52 ± 0.74a 1.30 ± 0.87b 1.12 ± 1.00c 0.96 ± 1.04d 3 .58 ± 0.74a 2 .67 ± 1.26b 3. 57 ± 0 .90a 2. 21 ± 1 .57c RR e (%) 23.1 30.8 21.0 6.5 2 9.0 14. 5 26.3 36.8 25.4 0.3 38.3 Initia l P O 3− 4 0.20 0.20 0.20 0.20 1.00 1.00 1.00 1.00 2. 00 2. 00 2.00 2.00 4.00 4.00 4.00 4.00 Fin al PO 3− 4 0.03 0.04 0.03 0.02 0.06 0.02 0.05 0.01 0. 53 0. 30 0.06 0.02 2.07 1.32 1.70 0.63 Re (%) 85.00 80.00 85.00 90.00 94.00 98.00 9 5.00 9 9.00 73. 50 85. 00 97.00 99.00 48.25 67.00 57.50 84.25 Rq (mg) 9.35 8.80 9.35 9.90 51.70 53.90 5 2.25 5 4.45 80. 85 93. 50 106.70 108.90 106.15 147.40 126.50 185.35 Me an PO 3− 4 (mg /L) 0.12 ± 0.07a 0.11 ± 0.07a 0.09 ± 0.07b 0. 08 ± 0.08b 0.3 9 ± 0. 39a 0.3 2 ± 0. 41b 0.37 ± 0.4 0a 0.27 ± 0.4 3b 1.15 ± 0.57a 0.97 ± 0.67b 0.83 ± 0.77c 0.73 ± 0.80d 2 .71 ± 0.83a 2 .11 ± 1.12b 2. 69 ± 0 .89a 1. 73 ± 1 .37c RR e (%) 8.3 25.0 33.3 17.9 5.1 3 0.8 15. 7 27.8 36.5 22.1 0.7 36.2 Note s: Re, Rq, and RRe stand for th e remov al efficiency, remov al quantity , and remo val efficiency com pared with control d uring each phase, respect iv ely. As for th e mean values, n = 5 for each phas e, the data were given in mean ± standar d deviation, different lower case letters den ote a significant difference.
around roots, thus attenuating the effects of plants on sediment Eh and precipitation of PO34− with Fe(III) hydroxides [40].
The facilitated adsorption and mineralization in macrophyte enhanced the BD-P in sediments, which is
consistent with the results of Di Luca et al. [41] when Typha domingensis with developed roots was used to remove high concentrations of P from water. The reduction of TP content in macrophyte was mainly from decreased NH4Cl-P and NaOH-P, which implied
Figure 5.DTP (a), PO34−(b), and DOP (c) in vertical three layers of pore water changed over time, u, m, and b represent the upper (0– 5 cm), middle(5–10 cm), and bottom layers (10–15 cm), respectively.
Figure 6.BD-P (a), NaOH-P (b), HCl-P (c), and NH4Cl-P (d) in sediments changed over time. BD-P, NaOH-P, HCl-P, and NH4Cl-P represent
that these two species could be easily available to V. spiralis as necessary nutrients.
3.5. Effects of coupled M-SMFC on P mobility between water and sediments
In the overlying water, the V. spiralis played a significant part in PO3−4 removal during phases I and III, whereas sig-nificant removal by SMFC was observed in phases II–IV. Therefore, SMFC and V. spiralis played a complementary role and the coupled M-SMFC performed significant PO3−4 removal in all phases (Figure 3 andTable 2). The defects of V. spiralis and SMFC constrained by plant growth and electrogenesis on PO3−4 removal were over-come by the coupled system. The maximum PO34− removal was achieved by M-SMFC when both V. spiralis and SMFC played significant roles during the high-power production phase (III). In the pore water, V. spiralis played significant roles in PO34− removal in the upper and middle layers of pore water, whereas sig-nificant effects were achieved by SMFC in the middle and bottom layers. Most of the PO3−4 removal in the pore water achieved by M-SMFC was in the middle layer (Table 3). These results suggest that it is feasible to enhance PO34− removal from the water column by using the combined system. Besides the plant uptake, the maximum generation of stable BD-P and HCl-P in sediments (Figure 6(a,c)) suggested the enhanced PO3−4 flux from water to sediments compared with the macro-phyte and SMFC treatments. Compared with the control, 13.6%, 14.2%, 25.6% (6.2%), and 25.0% (6.9%) of BD-P (HCl-P) were increased in M-SMFC on days 55, 104, 153, and 197, respectively. The simultaneous introduction of electron acceptors with the anode and macrophyte ROL generated the highest sediment Eh, implying the most Fe(III) hydroxides accumulation, and thereby pro-moting P adsorption. The similarity of SMFC and V. spiralis in improving the alkalinity of the overlying water might result in the maximum HCl-P in sediments despite that pH differences between macrophyte and M-SMFC were not significant in phases I–III (Figure 2
(d)). Furthermore, the presence of macrophytes might promote the growth of rhizosphere microorganisms and influence the microbial community [42], especially the microorganisms involved in the iron redox cycle and polyphosphate accumulation, thus indirectly affect-ing P adsorption. The lowest internal OP was achieved in M-SMFC. Besides the effects of electrogenesis and oxygen on mineralization, the increased OH−in the over-lying water could also compete with PO3−4 for metal-organic bond sites, and promote the mineralization of OP through increased bacterial activity in the sediments [43]. Table 3. Characteristics of TP (a), PO 3− 4 (b), and DOP (c) in three layers of pore water. Para meters Uppe r layer (5 –10 cm) Middle layer (5 –10 cm) Bottom layer (10 –15 cm) Co ntrol SMF C Macroph yte M-SMFC Contro l SMFC Macroph yte M-SMFC Contro l SMFC Ma croph yte M-SMFC DTP 0. 284 ± 0.083a 0.273 ± 0.08 8a 0.232 ± 0.102 b 0.236 ± 0.108b 0.247 ± 0.05 5a 0.211 ± 0.084b 0.201 ± 0. 083b 0.156 ± 0.095c 0.177 ± 0.066 a 0.147 ± 0.063b 0.1 69 ± 0 .065a 0.145 ± 0. 070b RRe (%) 3.7 18.2 16.7 14.6 18.6 3 6.8 1 6.9 4.5 18.1 PO 3− 4 0. 189 ± 0.076a 0.181 ± 0.07 7ab 0.155 ± 0.089 c 0.160 ± 0.092c 0.143 ± 0.05 4a 0.118 ± 0.070b 0.122 ± 0. 054b 0.098 ± 0.066c 0.097 ± 0.049 a 0.079 ± 0.047bc 0.0 93 ± 0 .044ab 0.078 ± 0. 046c RRe (%) 4.4 17.8 15.6 17.5 14.7 3 1.5 1 8.6 4.1 19.6 DOP 0. 095 ± 0.017a 0.093 ± 0.02 0ab 0.077 ± 0.017 bc 0.077 ± 0.018c 0.104 ± 0.04 2a 0.094 ± 0.035b 0.079 ± 0. 050c 0.059 ± 0.036d 0.081 ± 0.040 a 0.068 ± 0.041b 0.0 75 ± 0 .045ab 0.067 ± 0. 052b RRe (%) 2.3 19.1 19.1 9.6 24.0 4 3.3 1 6.0 7.4 17.3 Note s: n = 8 for parame ters, the data were given in mean ± standar d deviation, RRe sta nds for th e remova l efficiency com pared wit h cont rol, dif ferent lowe r case letter s denotes a signi ficant d ifference .
How to extend the life cycle of submerged macro-phytes to effectively avoid the OM accumulation in sedi-ments to control P in aquatic ecosystems is an important issue in the restoration of aquatic plants. Generally speaking, the growth of submerged macrophytes planted in fertile sediments with a high level of OM can be inhibited by lower redox potential, which is fol-lowed by decay [44]. Zhou et al. [45] once found that the closed-circuit SMFC could facilitate the growth of the submerged macrophyte Potamogeton malaianus. In our study, despite that the biomass of V. spiralis was not significantly increased with closed circuit, it seemed to be that the biological activity of V. spiralis in M-SMFC was maintained during the last phase of oper-ation when the Eh was elevated under elecctrogenesis. Furthermore, Song et al. [46] showed that employment of the bioanode could effectively enhance decompo-sition of macrophyte litter in sediments. Therefore, com-pared with SMFC, the V. spiralis in M-SMFC still had significant effect on the PO3−4 removal from the overlying water and from the middle layer of the pore water, in the last phase of operation (P < .05). This was due to inhibited OP accumulation in sediments under electrogenesis, along with accumulations of HCl-P and BD-P in sedi-ments. Although SMFC showed potential for eutrophica-tion control of water bodies when the flux of PO3−4 to sediments was facilitated, the temporarily mobilized
BD-P in sediments was still prone to be desorbed under low Eh, if induced by interrupted generation of power, which would facilitate eutrophication [3]. At this time, the introduction of submerged macrophytes V. spiralis could maintain the oxic condition in the sedi-ments, thus reducing the potential risk of endogenous P release from SMFC.
3.6. Influence of microorganisms on P mobility Compared with control, the electrogenesis influenced the bacterial community on the anode surfaces (Figure 7). Bacteria with power generation abilities were abun-dant on the surface of anodes in SMFC and M-SMFC. Bacillus [47], of phylum Firmicutes, has high electrochemi-cal activity and was abundant on the anodes of SMFC, but rarely on those of M-SMFC. Further examples of selective enrichment on the M-SMFC anodes include Geobacter [48,49] and Pseudomonas [50] affiliated to Pro-teobacter, Tolumonas [51] and Bacteroides [52] affiliated to Firmicutes and Bacteroidetes, respectively, are also well-known exoelectrogens. The differences in the elec-trogenic bacterial community might be related to effects by V. spiralis on the physicochemical properties of sediments: more short-chain fatty acids generated from roots and cellulose hydrolysis from litter decompo-sition. Geobacter directly utilizes acetate and propionate
to generate electricity [47,53]. Bacteroidetes, which degrades cellulose, was enriched on the surface of anodes in M-SMFC, indirectly suggesting higher content of the short-chain acids generated [54]. Clostri-dium, which includes a variety of carbohydrate-ferment-ing organisms, has been reported to have syntrophic interactions with Geobacter for electricity generation from cellulose [55]. The higher enrichments of Geobacter, Bacteroidetes, and Clostridium on the anodes of M-SMFC also implied an inhibited accumulation of OP in sedi-ments subjected to electrogenesis.
Geobacter, which also has the ability of indirect elec-tron transfer, can utilize the small molecular organic acid such as lactate, acetate, and propionate as electron donors to reduce Fe3+to Fe2+, resulting in power gener-ation by electron transfer to anodes [48,56,57]. Enhanced P adsorption from the water column in M-SMFC might be related to the higher abundance of Geobacter. Bond et al. [49] found that the stronger P cycles were related to iron redox cycles. In fact, microbial ferric ion reduction and ferrous ion oxidation in the rhizosphere might be coupled. According to Zhao et al. [58] denitrifiers could oxidize Fe2+ to Fe3+ and release electrons to reduce nitrate to nitrite [24]. The enrichment of denitrification bacteria such as Bacillus and Pseudomonas on the anode surface implied a strong microbial oxidation of Fe2+ to Fe3+ with the closed circuit, generating more Fe3+bound with P. The Pseudomonads-dominated iron-oxidizing bacteria were also enriched at the anode in a lithotrophic microbial fuel cell studied by Nguyen et al. [59] Meanwhile, the higher abundance of Pesudomonas implied greater Fe3+ in M-SMFC than in SMFC, which might also lead to a decrease in power generation due to the transfer of electrons generated from carbon sources to Fe3+, instead of to the anode.
Fluctuating Eh levels in the sediments, especially at the sediment–water interface, benefited the growth of PAOs with characteristic anaerobic release and aerobic uptake of P [31,60]. Bacillus and Pseudomonas genera (as PAOs) were significantly enriched on anodes of SMFC and M-SMFC, respectively [61,62], and absent when the circuit was open. Previous studies showed that P release was trig-gered by adding simple organic compounds, such as glucose, acetic acid, and propionic acid, in anaerobic phase [63]. More release of P in anaerobic phase resulted in increased assimilation in aerobic phase and enhanced P removal [64]. The fermentation end products can be used by exoelectrogenic bacteria on the anodes to produce current [65], which might stimulate the higher abundance and diversity of PAOs in sediments with intensified P uptake. More short-chain fatty acids in the rhizosphere in M-SMFC might facilitate the assimilation of P from the water column, especially in the declining period of
V. spiralis. The microbiological mechanisms for this remain unclear because the bacteria responsible for P removal have not been isolated or identified so far.
4. Conclusions
1. SMFC enhanced the formation of BD-P and HCl-P in sediments by elevating the sediment Eh and pH in the overlying water, facilitating the flux of PO34− from the water column to sediments in all the oper-ation phases. Such a process was especially significant when the sediments were thoroughly oxidized. The quantity of facilitated PO34−migration from overlying water to sediments was significantly enhanced with increase of the initial PO34− concentration. The PO34− concentration in pore water was significantly reduced in the middle (0–5 cm) and bottom (5– 10 cm) layers.
2. V. spiralis achieved significant reduction of PO34− in the overlying water mainly through direct uptake during early colonization. During its period of robust growth, a steady decrease in PO3−4 resulted from increases of the sediment Eh and the pH in the over-lying water. There was greater generation of BD-P and HCl-P during the period of robust growth, and OP accumulation in sediments and HCl-P release from sediments during the period of decline. Compared with SMFC, V. spiralis performed significantly more PO34− removal from the upper and middle layers of pore water.
3. In the M-SMFC, formation of HCl-P and BD-P was most promoted at the highest sediment Eh and maintained relatively high pH in the overlying water, which might account for the lowest PO3−4 in the water column. Fur-thermore, the OP accumulation in sediments and HCl-P release observed in the macrophyte treatment were inhibited during electrogenesis.
4. Higher abundance of exoelectrogens such as Geobac-ter (involved in the iron redox cycle), Bacillus, and Pseudomonas known as PAOs and iron-oxidizing bac-teria might be related to stronger P fluxes in closed circuits. V. spiralis enhanced the abundance of Geo-bacter and Pseudomonas, which might explain the greatest reduction of P content in the water column. Combined treatment by submerged macro-phytes and SMFC appears to offer a promising way to control eutrophication in water bodies.
Acknowledgements
The authors would like to thank Drs Feng He, Dong Xu, Biyun Liu and Ms Liping Zhang, Lei Zeng, Qingwei Lin, Fenli Min for
the experimental help (Institute of Hydrobiology, Chinese Academy of Sciences).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the National Natural Science Foundation of China [grant number 51308530]; Major Science and Technology Program for Water Pollution Control and Treatment [grant number 2012ZX07101007-005]; National Key Research and Development Plan of China [grant number 2016YFC0500403-03]; and Key Research Program of the Chinese Academy of Sciences [grant number KFZD-SW-302-02].
ORCID
Enrong Xiao http://orcid.org/0000-0002-3375-8358
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