Enhancement of COD removal in constructed wetlands treating saline wastewater: Intertidal wetland sediment as a novel inoculation

Journal of Environmental Management 249 (2019) 109398

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Research article

Enhancement of COD removal in constructed wetlands treating saline wastewater: Intertidal wetland sediment as a novel inoculation

T

Qian Wanga,1, Zhenfeng Caoa,1, Qian Liua, Jinyong Zhangb, Yanbiao Hua, Ji Zhanga, Wei Xua, ⁎ ⁎⁎ Qiang Konga,c, , Xunchao Yuana, QingFeng Chena, a College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250358, PR China b Enviromental Engineering Co., Ltd of Shandong Academy of Environmental Sciences, 50 Lishan Road, Jinan, 250014, Shandong, PR China c Department of Civil and Environmental Engineering, National University of Singapore, Singapore, 117576, Singapore

A R T I C LE I N FO

A B S T R A C T

Keywords: Saline wastewater Intertidal wetland sediment Constructed wetlands COD removal Salt-tolerant microorganisms

This study investigated intertidal wetland sediment (IWS) as a novel inoculation source for saline wastewater treatment in constructed wetlands (CWs). Samples of IWS (5–20 cm subsurface sediment), which are highly productive and rich in halophilic and anaerobic bacteria, were collected from a high-salinity natural wetland and added to CW matrix. IWS-supplemented CW microcosms that are planted and unplanted Phragmites australis were investigated under salty (150 mM NaCl: PA+(S) and CT+(S)) and non-salty (0 mM NaCl: PA+ and CT+) conditions. The chemical oxygen demand (COD) removal potential of IWS-supplemented CWs was compared with that of conventional CWs without IWS (PA(S) and CT(S), PA, and CT). Results showed that the COD removal rate was higher in PA+(S) (51.80% ± 3.03%) and CT+(S) (29.20% ± 1.26%) than in PA(S) (27.40% ± 3.09%) and CT(S) (27.20% ± 3.06%) at 150 mM NaCl. The plants' chlorophyll content and antioxidant enzyme activity indicated that the addition of IWS enhanced the resistance of plants to salt. Microbial community analysis showed that the dominant microorganisms in PA+(S) and CT+(S), namely, Anaerolineae, Desulfobacterales, and Desulfuromonadales, enhanced the organic removal rates via anaerobic degradation. IWS-induced Dehalococcoides, which is a key participant in ethylene formation, improved the plants’ stress tolerance. Several halophilic/tolerant microorganisms were also detected in the CW system with IWS. Thus, IWS is a promising inoculation source for CWs that treat saline wastewater.

1. Introduction Saline wastewater is produced in various ways. A large amount of NaCl is used in chemical synthesis, food processing, and leather refining (Lefebvre and Moletta, 2006; Shi et al., 2015). The tailwater produced by seawater utilization industries, such as marine aquaculture and seafood processing, is also characterized by high salinity and organic matter content (Fu et al., 2019; Liang et al., 2018a; Vymazal, 2014). Biological treatment techniques, which are more environment-friendly than traditional physical and chemical treatment methods, focus on the selection of halophytes and adaptation of conventional microorganisms to high-salinity environments (Liang et al., 2017; Xu et al., 2018). Constructed wetlands (CWs) are economical, highly efficient, and

environment-friendly infrastructures for treating contaminants via the synergistic effects of soil, macrophytes, and microorganisms (Wu et al., 2018; Zhang et al., 2018). CWs can be employed to treat saline wastewater (Wu et al., 2014; Liang et al., 2018b). Calheiros et al. (2012) studied the removal rates of high-salinity wastewater (2.2–6.6 g Cl−/L) in horizontal subsurface CWs with different salt-tolerant plants, such as Arundo and Sarcocornia. The chemical oxygen demand (COD), total phosphorus, and ammonia nitrogen (NH4+-N) removal rates reached 51%–80%, 40%–93%, and 31%–89%, respectively. In addition to salt-tolerant plants, microorganisms play an important role in pollutant degradation in CWs (Wang et al., 2016; Wu et al., 2015). Salt-tolerant microorganisms can adapt to high-salinity environments by releasing Na+ through the Na+/H+ reverse

⁎ Corresponding author. College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250358, PR China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (Q. Kong), [email protected] (Q. Chen). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.jenvman.2019.109398 Received 9 March 2019; Received in revised form 6 August 2019; Accepted 12 August 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.

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PA+). The plants were collected from Nansi Lake at the beginning of March 2017. The roots and stems of the plants were carefully washed with tap water until the soil residue on the surface was removed to eliminate interference in the experiment. The plants were transplanted into the cells with normal and consistent growth. Prior to the experiments, the plants were cultivated in 10% Hoagland solution for two weeks (Hoagland and Arnon, 1950). The plants with normal and consistent growth were transplanted into the microcosms, with each group comprising three replicates. The salt concentration of the saline wastewater was adjusted to 150 mM NaCl, which is higher than the salt tolerance of indigenous microorganisms and is suitable for treating pretreated effluent from high-salinity wastewater (Liang et al., 2017).

transporter by changing the enzyme structure in cells and releasing sugar, amino acids, and metabolites to maintain osmotic balance. Therefore, these microorganisms show great potential in handling saline wastewater (Chen et al., 2019; Fu et al., 2018). Most studies on salt-tolerant microorganisms focused on the adaptability of microorganisms to high-salt stress and the screening of salt-tolerant microbial strains (Lefebvre et al., 2005). Only a few have applied salt-tolerant microorganisms to treat high-salinity wastewater, in which most of the microorganisms used are single strains that are screened or purified from saline environments. In contrast to many coculture treatments, single-strain treatments often cannot withstand environmental changes and extraneous species pollution, and thus cannot be widely used (Zhang et al., 2014). Intertidal wetland sediment (IWS) with considerable amounts of halophilic/tolerant microorganisms can be used as a potential inoculation source for biologically treated saline wastewater because of its high salinity. Shi et al. (2015) applied IWS (5–20 cm) as a substrate for the anaerobic biological treatment of pharmaceutical wastewater. This new approach achieved improved COD removal rate compared with the traditional anaerobic sludge. Thus, rich microbial diversity and salt-tolerant population ensure the optimal treatment of wastewater. CWs are plant–microorganism coupling treatment systems (Wang et al., 2018). When IWS is added into the CW systems, the combination of plants and halophilic/tolerant microorganisms will stabilize and optimize the treatment of saline wastewater (Li et al., 2019). In the current study, IWS (5–20 cm subsurface sediment) collected from a high-salinity natural wetland (Yellow River Delta in Dongying, Shandong Province, China) was analyzed and added into the CW matrix. IWS-supplemented CW microcosms that are planted and unplanted with Phragmites australis were investigated under salty (PA+(S) and CT+(S)) and non-salty (PA+ and CT+) conditions, and the outputs were compared with those of conventional CWs without IWS (PA(S) and CT(S), PA, and CT). Several treatment parameters, including COD removal rates, salt tolerance indexes of plants, and microbial community composition, were investigated and compared with the traditional CW system to provide a comprehensive understanding of the potential of IWS as an alternative for the CW treatment of saline wastewater.

2.2. Operation All CW microcosms were batch-operated for a hydraulic retention time of three days. The microcosms that were dried for 3 days to remove the bleach were then initially fed with tap water until substrate saturation. After a week, the CW microcosms were fed with 10% Hoagland nutrient solution for two weeks. The microcosms were then fed synthetic municipal sewage with a COD of 180 mg/L and an NH4+N concentration of 45 mg/L for several weeks until the system was established. The simulated wastewater contained saccharose, (NH4)2SO4, K2HPO4, MgSO4, FeSO4, CaCl2, and tap water. For the first 10 cycles, all cells were fed with non-saline wastewater to acclimatize the plants and microorganisms to the system. The steady state was reached when the plants grew new shoots and the microbial community divergence distinctly increased (Weber and Legge, 2011). The concentration of salt in the experimental cells (PA+(S), PA(S), CT+(S), and CT(S)) was then increased from a low salinity (0, 50, and 100 mM) to the appointed salinity (150 mM NaCl) at each cycle to buffer the severe effect of high salinity. The control cells (PA+, PA, CT+, and CT) were simultaneously fed with non-salty simulated wastewater. For each cycle, 4 L of synthetic saline wastewater was manually added into each cell to maintain the water level below the sand surface (Wang et al., 2015). Microbial communities stabilized after 75–100 days during system startup (Truu et al., 2009; Weber and Legge, 2011). Thus, the microcosms were precultured for 3 months before the experiment.

2. Materials and methods 2.3. Sampling 2.1. Site description and design On the third day of each cycle, microcosm effluents were collected and filtered. All samples were then stored in 120-mL sterile plastic bottles and immediately processed in the laboratory for chemical analysis. After the experiment, the plants were carefully removed from the microcosms and repeatedly rinsed with distilled water to separate the debris. The plant leaves were cut and chopped under sterile conditions before storing at −80 °C until further analysis. Soil samples were obtained from the upper layer (5–10 cm) of each microcosm in strict accordance with the five-spot sampling method using a soil sampler (Calheiros et al., 2010). The samples were collected in sterile 5-mL Eppendorf tubes and then stored at −80 °C until microbial analysis.

The experiments were performed in designed microcosm subsurface (SSF) CWs located in Shandong Normal University in Jinan, China (36 °N, 117 °E), which has a warm temperature monsoonal climate. The area is characterized by a mean temperature of 14.7 °C, an annual precipitation of 671 mm, and natural illumination. Four types of microcosm wetland systems were established using a subsurface flow design for saline wastewater treatment on March 4, 2017 (Fig. 1). The microcosm wetland systems were composed of polyvinylchloride (PVC) columns (diameter: 30 cm; height: 48 cm; volume: 33.9 L) with an effluent outlet at the bottom. A white PVC pipe with holes (diameter: 4 cm) was inserted into the center of the microcosm to measure the indexes, such as dissolved oxygen, in situ. The substrates were mainly composed of gravel (15 mm), river sand (particle size < 2 mm), and IWS. The experimental columns (PA+ and CT+) contained four layers from bottom to top, namely, 5 cm-deep gravel, 25 cm-deep river sand, 5 cm-deep IWS as the inoculation matrix, and 5 cm-deep river sand on top. The control columns (PA and CT) comprised two layers. The bottom 5 cm was filled with gravel to achieve a stable flow and then overlaid with 35 cm-deep river sand. The purchased gravel and river sand were continuously washed with running water to eliminate impurities absorbed on the surface. IWS with a 5–20 cm subsurface sediment depth was collected from the Yellow River Delta in Dongying, Shandong Province, China. P. australis, which is a typical wetland plant in North China, was planted at a density of 15 rhizomes per cell (PA and

2.4. Analysis 2.4.1. Plant growth and physiological index monitoring A) Chlorophyll content Plant samples were obtained from 10 leaf positions for accurate results. All samples were carefully washed, cut, and stored in 10-mL plastic centrifuge tubes. After grounding the samples with liquid nitrogen, a small amount of 80% acetone was added to the tubes and incubated in the dark for > 75 h. The supernatant was obtained and 2

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Fig. 1. Construction of four types of CW microcosm.

measured using an HACH DR 2800TM spectrophotometer (USA) at wavelengths of 663 nm and 645 nm. The total chlorophyll content was calculated using the following modified formula according to Cave et al. (1981):

MDA concentration = (ODmeasured − ODcontrol )/(ODs tan dard − ODblank ) × SC / P ,

(2)

where ODmeasured, ODcontrol, ODstandard, and ODblank are the absorbance values of the measured, control, standard, and blank samples, respectively; SC is the standard concentration (10 nmol/mL); P is the protein concentration (mg protein/mL).

Chlorophyll content = (8.05 × A663mm + 20.2 × A645mm ) × V × N /FW , (1) where A663mm and A645mm are the absorbance values at 663 mm and 645 mm, respectively; V is the total volume of the extraction liquid (μL); N is the dilution rate; and FW is the fresh weight of plant samples (g).

C) Catalase (CAT) and superoxide dismutase (SOD) activities The SOD and CAT activities in the plant samples were determined using the SOD and CAT Assay Kits provided by Jiancheng Biotechnology Co., Ltd. (Nanjing, China) and a visible light spectrophotometer at 405 and 550 nm wavelengths, respectively. Both activities (U/mg protein) were calculated using the following formulas (3) and (4) from detailed instructions of the SOD Assay Kit and CAT Assay Kit provided by Jiancheng Biotechnology Co., Ltd. (Nanjing, China), as described by Li et al. (2013):

B) Protein and malondialdehyde (MDA) content The plant samples were centrifuged and prepared as 10% tissue homogenates, and the protein concentration was determined using the Coomassie Brilliant Blue method (Bradford, 1976). The standard curve was constructed using 1 mg/mL protein solution with 0.15 mol/L NaCl. In accordance with the standard method, the propanediol in the product of lipid peroxidation was condensed with thiobarbituric acid (TBA), thereby forming a red substrate with a maximum absorption peak at 532 mm. The absorbance of red TBA-MDA complex was measured using an HACH DR 2800TM spectrophotometer at 532 nm and the MDA content (nmol/mg protein) was calculated according to the following equation (2) from detailed instructions of the MDA Assay Kit provided by Jiancheng Biotechnology Co., Ltd. (Nanjing, China), as described by Li et al. (2013):

SOD activity = (ODcontrast − ODmeasured )/ ODcontrast × 2V /(R × P ),

(3)

where ODcontrast and ODmeasured are the absorbance values of the contrast and measured samples, respectively; V is the total volume of the reaction liquid (mL); R is the volume of the sample (mL); and P is protein concentration (mg protein/mL).

CAT activity = (ODcontrast − ODmeasured ) × 271 × 1/(60 × R × P ),

(4)

where ODcontrast and ODmeasured are the absorbance values of the contrast 3

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et al., 2013). The results indicated that IWS demonstrates an anaerobic characteristic. The phylum Proteobacteria, which is the dominant bacterial group in conventional biotreatments, is rich in IWS, particularly in the classes Deltaproteobacteria (12.8%), Gammaproteobacteria (8.5%), and Alphaproteobacteria (8.2%). The anaerobic characteristic of IWS was confirmed by the large number of Deltaproteobacteria, which are commonly observed in anaerobic digestors (Wang et al., 2012). The abundance of Gammaproteobacteria and Bacteroidetes demonstrated the halophilic characteristics of IWS; both of which are generally discovered in saline environments (Sivasankar et al., 2019). These two groups of bacteria were highly productive, as indicated by the abundance of Cyanobacteria at the surface layer (35.5% at 0–5 cm and 0%–0.1% at 5–20 cm) and Rhodobacterales (class Alphaproteobacteria and phylum Proteobacteria); the latter is conducive to the rich nutrients in the intertidal environment. Gemmatimonadetes, which is found in activated sludge sewage treatment systems and can survive in aerobic and anaerobic environments, was also abundant (Wong et al., 2019). Other ubiquitous and abundant bacterial communities of soil, (e.g., Acidobacteria) were also abundant in IWS (Eichorst et al., 2018). In conclusion, IWS contained abundant halophilic and anaerobic bacteria and was highly productive, especially at 0–5 cm surface layer. This unique community structure suggested the biodegradation potential of IWS as an inoculation source for saline wastewater treatment.

and measured samples, respectively; 271 is the reciprocal of the standard curve's slope; R is the volume of the sample (mL); and P is the protein concentration (mg protein/mL). 2.4.2. Chemical analysis The COD content was detected using an HACH DR 2800TM spectrophotometer (USA) through the potassium dichromate method. The COD removal rates (%) were calculated using the difference in the concentration between the influent and effluent samples, as shown in the following equation according to Xu et al. (2018):

Removal rate = (1 − C1/C0) × 100%,

(5)

where C0 and C1 (mg/L) are the concentrations of the influent and effluent, respectively. 2.4.3. Microbial analyses The soil samples used for DNA extraction were obtained from each microcosm at equal heights (approximately 10–15 cm) and mixed as one composite sample for high-throughput sequencing to investigate the effect of salt stress on microbial community and diversity. The V3–V4 regions of the 16S rRNA gene were amplified on the basis of the Illumina MiSeq sequencing platform provided by Personal Biotechnology Co., Ltd. (Shanghai, China) using the primers 338f ( 5′-ACTCCTACGGGAGGCAGCA-3′) and 806r (5′-GGACTACHVGGGTWTCTAAT-3′). A total of 272,615 trimmed sequences with an average length of > 150 bp remained after raw sequence processing. The FLASH software (version 1.2.7) (http://ccb.jhu.edu/software/FLASH/) was used to pair double-ended sequences on the basis of overlapping bases. The trimmed sequences were obtained using QIIME (version 1.8.0) (http://qiime.org/) and grouped into operational taxonomic units (OTUs) at a 97% identity threshold. Thus, the sequencing results can represent bacterial community composition. The data were analyzed using the method described by Wang et al. (2015), and the output was presented using Krona software (https://github.com/marbl/Krona/ wiki) for to provide the interactive presentation of the community's taxonomic composition. Moreover, the taxonomy was assigned to OTUs using the SILVA database (Release 115) (http://www.arb-silva.de).

3.2. Analysis of the performance of CWs The effluent water parameters of the different CWs are described in Fig. 2. The COD removal rates of the planted microcosms (PA+ and PA) without additional NaCl were similar and notably higher than those of the unplanted microcosms (CT+ and CT). Thus, plants play an essential role in removing pollutants, as validated in previous studies (Al-Saedi et al., 2018; Türker, 2018). However, evident differences were observed between CT+ (80.40% ± 2.43%) and CT (70.30% ± 1.05%), which suggest that the addition of IWS positively affected the COD removal in CT + even without additional NaCl. Similar observations were reported by Hussain et al. (2018), who discovered that microbial inoculation enhanced the COD removal of CWs. In CWs with saline wastewater, the COD removal rates of the four groups showed the following trend: PA+(S) (51.80% ± 3.03%) > PA(S) (27.40% ± 3.09%), CT+(S) (29.70% ± 1.26%) > CT(S) (27.20% ± 3.06%). This condition suggested that regardless of the presence of plants, the addition of IWS can improve the COD removal for saline wastewater treatment in CWs. Calheiros et al. (2012) tested horizontal SSF CWs to polish high-salinity (2.2–6.6 g Cl/L) wastewaters and obtained a COD removal rate of 36% in the first bed. In contrast to previous results, the current findings indicate that IWS significantly enhanced the COD removal of saline wastewater in CWs (Ali et al., 2018; Saggaï et al., 2017). In particular, the removal of CT+ was higher than that of CT, indicating that IWS is essential for COD removal to enhance the community and diversity of microorganisms (Ahmadi et al., 2017; Shi et al., 2015). At salinity (150 mM), the maximum removal rate in PA+(S) was 51.8% ± 3.03%, which was approximately twice of that in PA(S), CT+(S), and CT(S). However, the salt stress harmed the purification capacity of CWs. Furthermore, the potential of IWS was more significant between PA+(S) and PA(S) than between CT+(S) and CT(S).

2.4.4. Data analysis The data were analyzed using Origin 8.5, whereas the statistical analyses of the correlation among different variables were performed using IBM SPSS Statistics 21. The significant correlations of COD removal rates, plant growth, and physiological indexes and microbe values among different groups were determined via ANOVA. In all tests, the differences and correlations were statistically significant when P < 0.05. 3. Results 3.1. Microbial analysis of IWS Fig. S1 and Table 1 demonstrate the distributions and proportions of the dominant bacteria (> 2% sequence percentage) in the IWS at the 0–20 cm layer. Seven groups of bacteria rich in the IWS were determined, namely, Chloroflexi (33.3%), Proteobacteria (32.5%), Cyanobacteria (8.9%), Actinobacteria (5.4%), Gemmatimonadetes (4%), Acidobacteria (3.7%), and Bacteroidetes (3.1%). Chloroflexi is generally discovered in sediment and is involved in carbon cycling, organohalide respiration, and acetogenesis with adenosine triphosphate (ATP) formation by phosphorylation at the substrate level (Yao et al., 2017). In this study, the bacterial groups rich in IWS were mainly of the family Anaerolineaceae (28.6%), which plays an important role in the anaerobic degradation of hydrocarbons, such as n-alkanes (Sutton et al., 2013; Yan et al., 2018). In addition to Chloroflexi, Actinobacteria and Bacteroidetes, which are phyla that are commonly discovered in typical anaerobic processes, have abundance of IWS (Zhang et al., 2018; Zheng

3.3. Analysis of the salt resistance of plants Plants demonstrate a sensitive physiological response to salt concentration. In this study, the chlorophyll and MDA contents, as well as the SOD and CAT activities, were measured to characterize the resistance of plants to salt stress (Table 2). Chlorophyll content is elemental for normal plant growth and development because of its role in photosynthesis and signaling (Muñoz and Munné-Bosch, 2017). The overall chlorophyll content significantly decreased with the addition of 150 mM NaCl, demonstrating that the 4

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Table 1 Proportion (%) of main communities in different soil layers of the IWS at phylum and class level. Taxonomy

Bacteria Names

Average

0–5 cm

5–10 cm

10–15 cm

15–20 cm

phylum

Chloroflexi Proteobacteria Cyanobacteria Actinobacteria Gemmatimonadetes Acidobacteria Bacteroidetes Anaerolineae Gemmaproteobacteria Deltaproteobacteria Alphaproteobacteria Chloroplast Cyanobacteria Acidimicrobiia BD2-11 terrestrial group

33.3 32.5 8.9 5.4 4.0 3.7 3.1 28.6 12.8 8.5 8.2 5.9 3.0 2.6 2.5

16.0 26.3 35.5 6.5 3.5 0.4 5.5 15.3 12.6 2.1 11.1 23.4 12.1 1.6 3.2

30.2 43.6 0.1 6.0 2.4 3.6 4.1 26.4 14.1 13.5 10.1 0.1 – 3.1 1.4

41.8 32.8 0.1 3.8 4.3 4.6 1.9 37.5 17.4 9.3 4.2 – – 1.6 2.5

45.3 27.3 – 5.4 5.9 6.4 0.9 35.3 7.2 9.3 7.3 – – 4.1 3.1

class

that of PA(S), suggesting that the cell membrane damage caused by salinity was minimal in the former. The results confirmed that IWS addition plays a considerable role in the resistance of plants to salinity. The SOD and CAT in plants, which are located in the microbodies (e.g., peroxisomes), play a major role in the detoxification of H2O2 generated by environmental stresses (e.g., temperature, salt, and metals) and O− produced by physiological conditions (e.g., respiration) (Kao et al., 2018). In the current study, SOD and CAT activities increased with the addition of salt. These activities were higher in PA + than in PA, especially at high salinity. Thus, IWS can enhance the plants’ salt tolerance by inducing antioxidant enzyme activity to protect plastids and modulate H2O2 generation (Martí et al., 2009). 3.4. Comparative analysis of microbial communities 3.4.1. Microbial community diversity As demonstrated in Table 3, the Chao 1 and ACE indexes were calculated to reveal microbial diversity and richness (Fu et al., 2019). In addition, the Shannon and Simpson indexes, which respectively indicate microbial diversity and bacterial group distribution, were obtained. Based on the indexes, the microbial diversity of PA + remained high even under high-salt stress, whereas that of PA decreased with the addition of salt. Therefore, IWS introduced a suitable environment for microorganisms. In CT, the microbial diversity dramatically decreased with salinity; the values were significantly lower than those of CT+, which demonstrates the advantage of IWS. This result corroborates the pivotal role of IWS in promoting microbial community richness and diversity (Shi et al., 2015).

Fig. 2. The COD removal rates in different CWs (%). Note: Means ± SE, N = 12. Means within each CW indicate significant difference of the results at p = 0.05 level.

salinity disrupted the physiological functions (e.g., photosynthesis) by destroying the chlorophyll. This observation is consistent with the findings of previous studies (Agastian et al., 2000). The chlorophyll content of PA+ was significantly higher than that of PA, regardless of the addition of NaCl. However, a substantial enhancement in chlorophyll content between PA+ and PA was detected in salty CWs. In conclusion, IWS inoculation improved the stress tolerance of plants by improving photosynthesis. MDA, as a credible indicator of oxidative stress, can aggravate cell membrane damage through liposome peroxidation (Abdel and Tran, 2016). Table 2 illustrates that the membrane damage caused by salt stress was enhanced and the MDA accumulated due to salinity's detrimental effect. The MDA content of PA+(S) was evidently lower than

3.4.2. Microbial community structures Fig. 3 demonstrates the relative abundance of the dominant bacterial phyla in the four types of CWs. For the planted groups, Chloroflexi (35%) exhibited the highest relative abundance in PA+, which was 1.5 times more than that of PA under non-salty conditions. With the addition of salt, the relative abundance of Chloroflexi in PA+(S) increased to 46% at 150 mM NaCl, which was 6.6 times more than that of PA(S).

Table 2 Plant physiological indexes during the dosing period (N = 3). Salt

Type

Plant physiological indexes Chlorophyll content (μg/g)

None Salty

PA+ PA PA+(S) PA (S)

a

1541.72 ± 289.79 1498.67 ± 230.17a 965.87 ± 89.21a 506.78 ± 102.43b

MDA concentration (nmol/mg protein) 0.54 0.50 1.62 2.99

± ± ± ±

c

0.01 0.05c 0.35b 0.56a

SOD activity (U/mg protein) 457.45 458.06 604.53 653.84

a, b, c, d: different letters at the same rows means significant difference at p = 0.05 level. 5

± ± ± ±

c

9.51 8.46c 9.25b 10.02a

CAT activity (U/mg protein) 1.11 1.32 2.26 1.56

± ± ± ±

0.04d 0.05c 0.03a 0.02b

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Desulfuromonadales in planted CWs with IWS. In CT+, the relative abundance of Anaerolineaceae was still high but slightly decreased, which indicated that the lack of plants negatively affected the microbial abundance and activity under salt stress. For CWs without IWS, the phyla Proteobacteria, Actinobacteria, and Saccharibacteria were dominant. In PA, the relative abundance of Pseudomonas, which is a dominant genus of the phylum Proteobacteria, was 38%. In CT, the uncultured_bacterium of Saccharibacteria was dominant, accounting for 19% of the composition. The classes Pseudomonadaceae and Micrococcaceae, which respectively accounts for 14% and 5% in CT, also played an important role in the microbial structure. Fig. S2 shows the analysis of the dominant bacteria at the genus level. Numerous Anaerolinea species, which can retain moisture and degrade organic carbon matter (Ahmadi et al., 2017), were detected in PA+(S) and CT+(S), whereas numerous Dehalococcoides species were found in PA+(S) and PA+. In addition, the detected Dehalococcoides strains are all hydrogenotrophs that require specific organic chloride as electron acceptors for energy metabolism (Mao et al., 2019). The substrate of the Dehalococcoides strains consists of numerous priority pollutants (e.g., chloroethenes) (He et al., 2007). These strains are the key participants in ethylene formation, which might be the hormonal factor in the interaction between plants and rhizosphere microorganisms. Abundant halophiles, which can exhibit extreme halotolerance, such as Marinobacter, Microbacteriaceae, and Gracilimonas, were found in CWs with IWS (Han et al., 2003). In summary, for CWs with IWS, anaerobic and halophilic microorganisms were dominant, whereas for CWs without IWS, aerobic and facultative anaerobic microorganisms were abundant.

Table 3 Richness and diversity estimation of microbial communities in the microcosms from the pyrosequencing analysis. Salt

Sample

Simpson

Chao1

ACE

Shannon

None

PA+ PA CT+ CT PA+ (S) PA (S) CT+ (S) CT (S)

0.997,271 0.988,555 0.996,189 0.992,052 0.997,877 0.988,876 0.995,593 0.961,642

2938.57 2092.63 1919.00 2239.57 2476.25 1989.62 2436.70 2422.10

3132.88 2167.94 1919.30 2319.69 2595.90 2083.75 2582.34 2556.33

10.14 9.22 9.74 9.46 10.03 8.57 9.76 8.40

Salty

Fig. 3. The dominant bacteria analysis at phyla level.

4. Discussion COD degradation in CWs as well as the interactions between plants and rhizosphere bacteria (Fig. 5) was discussed in this study.

Hence, Chloroflexi introduced by IWS acclimated to the high-salt stress conditions in PA+, which is consistent with the accumulation of Chloroflexi in mudflat sediment reported by Yan et al. (2018). The relative abundance of Chloroflexi in PA+(S) was twice of that in CT+(S), indicating that the plants positively affected the acclimatization of Chloroflexi. However, several differences, such as relative abundance of Saccharibacteria in PA(S) and CT(S), were observed in the groups without IWS. This strain affects the degradation of organic compounds like saccharose under aerobic, nitrate-reducing, and anaerobic conditions (An et al., 2018). At salinity, Proteobacteria showed a significant increase in relative abundance at 59% and became the most dominant bacteria. In the unplanted CWs, a considerable amount of Chloroflexi bacteria were observed, especially in CT+(S), which exhibited 2.9 times more than that in CT(S). Saccharibacteria was the dominant phyla in CT(S). Acidobacteria accounted for 12% in CT+(S), and these strains participate in the anaerobic degradation of cellulose under anoxic conditions (Jing et al., 2019). Although the COD removal rate was evidently lower in unplanted CWs than in planted CWs, the differences remained between the unplanted CWs with and without IWS. The investigations of specific taxa within the top three dominant phyla (Proteobacteria, Actinobacteria, and Chloroflexi) are shown in Fig. 4. The relative abundance of the family Anaerolineaceae (under phylum Chloroflexi) in PA+(S) was distinctly enhanced and became dominant under salt stress, reaching up to 42% at 150 mM salt concentration. Sherry et al. (2013) discovered that the family Anaerolineaceae plays a role in the hydrocarbon degradation under anaerobic conditions. PA+(S) is abundant in Deltaproteobacteria (under phylum Proteobacteria), accounting for 15%. These bacteria are rich in marine sediments and are important for pollutant removal. The relative abundances of the orders Desulfobacterales and Desulfuromonadales (class Deltaproteobacteria) in PA+(S) were increased to 15% and 9% at 150 mM NaCl, respectively. The CWs without IWS contained trace amounts of those groups of bacteria, indicating that salt stress can induce the growth of Anaerolineaceae, Desulfobacterales, and

4.1. COD enhancement via microbial anaerobic degradation Given the abundant anaerobic and halophilic bacteria in CWs with IWS, the acclimated soil bacteria were beneficial in resisting adverse environmental stress, and the COD removal was evidently enhanced. In planted CWs with IWS, Chloroflexi, especially the family Anaerolineaceae, accounted for a large proportion and played an important role in the hydrocarbon degradation under anaerobic conditions (Liang et al., 2016). The anaerobic degradation of organic matter includes four sequential microbe-mediated stages, namely, hydrolysis, fermentation (acidogenesis), acetogenesis (dehydrogenation), and methanogenesis (acetoclastic or hydrogenotrophic) (Vanwonterghem et al., 2014). The results showed that Anaerolineaceae predominated, indicating that the syntrophic cooperation with the archaea Methanosaeta was likely involved in the methanogenic degradation of organic matter (Chen et al., 2019; Liang et al., 2016). The COD removal rate of PA + at 150 mM remained at approximately 51.8%. Hence, with increased salt concentration, the IWS induced the accumulation of abundant Anaerolineaceae, which is crucial for the increased COD removal rate of PA + under high-salt stress. The relative abundances of the orders Desulfobacterales and Desulfuromonadales (class Deltaproteobacteria) were increased under high-salt conditions. Desulfobacterales species, which are enriched in marine sediments, served as efficient H2 scavengers in anoxic salty sediments and promoted anaerobic degradation by oxidizing H2 (Dyksma et al., 2018). The improvement of acetogenesis (dehydrogenation) is essential to maintain the energetically favorable anaerobic degradation of organic matter, as well as the production of CH4 (Zhuang et al., 2018). The order Desulfuromonadales assumes a rod-shaped morphology and utilizes the acetate involved in the reduction of Fe(III), thereby facilitating fermentation (Greene et al., 2009). Hence, the accumulation of Desulfobacterales and Desulfuromonadales in PA+(S) at high salt 6

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Fig. 4. The dominant bacteria (account for more than 5% of all) analysis of the phyla Proteobacteria, Actinobacteria and Chloroflexi.

Saccharibacteria, was lower than that of anaerobic degrading bacteria (more than 60% in total) in PA(S) and CT(S) in IWS-supplemented CWs. Moreover, both bacteria can degrade organics through aerobic pathways (Devatha and Pavithra, 2019). The potential of microbial degradation activities is influenced by microbial abundance, diversity, and activity (Raiesi and Sadeghi, 2019). The activity of aerobic microbes can be inhibited by the changes in enzyme types and activity due to the addition of salt (Meena et al., 2019). Thus, the microbial aerobic degradation of COD in PA(S) and CT(S) can be limited by the inhibited microbial activity of aerobic microbes.

concentrations promotes a considerable COD degradation rate through anaerobic degradation (Zhuang et al., 2018). The accumulation of Anaerolineaceae, Desulfobacterales, and Desulfuromonadales suggests that the addition of IWS can enhance the COD removal in CWs through anaerobic degradation under high-salt conditions. In CT+(S), Anaerolineaceae remained detectable but was not dominant, indicating that the lack of plants negatively affected the microbial abundance and activity under salt stress. In PA(S) and CT(S), the COD removal rates were 27.4% ± 3.09% and 27.2% ± 3.06%, respectively, which were almost half of that of PA+(S). Moreover, anaerobic and salt-tolerant microorganisms were also present in minimal count. The relative abundance of aerobic or facultative anaerobic degrading bacteria, such as Pseudomonas and

Fig. 5. Role of plant microorganisms in a constructed wetland treating saline wastewater. 7

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4.2. Interaction between plant and rhizosphere bacteria

5. Applications and prospects

In salt-supplemented CWs, the plants were subjected to severe salt stress. The activities of antioxidant enzymes in plants increased, indicating that the plants undergo a series of physiological changes in response to salt stress (Abdel and Tran, 2016). The microbial community structure also showed that the rhizosphere microbial communities were significantly different. A significant accumulation of halophilic and hormone-related bacteria (e.g., Dehalococcoides) was detected in PA+(S). Plants respond to environmental stresses by changing their root exudates, which can affect the root microbiome (Qin et al., 2016). Profitable microbiome members (Fig. 5) can aid plants in absorbing the available nutrients and signal when the plants are under adverse conditions (Sasse et al., 2018). The rhizosphere is the surrounding soil zone that contains roots and a mixture of stimulants and selective metabolites secreted by the roots. This mixture can regulate rhizosphere microflora and thus coordinate different biotic and abiotic responses in the root environment (Bakker et al., 2018). Consequently, plants can send signals and selectively enrich the microbiota that can protect them and change the characteristics of microbial activity in soil (Koziol et al., 2018). This mechanism of soil inheritance will aid the future generations of plants to survive against the stress that triggered such a response. Certain plant-protected microorganisms, such as Dehalococcoides provided by IWS, are significantly assembled in the rhizosphere. Dehalococcoides can completely dechlorinate tetrachloroethylene and trichloroethylene as nontoxic ethylene (Tabernacka et al., 2017). The hormone-dependent metabolites produced by plant roots play an important role in the interaction between the plant and the soil microbiome (Bakker et al., 2018). Plant growth and development generally require specific signal integration, and the ethylene receptors and transcription factors participate in the salt tolerance of plants at varying degrees. Achard et al. (2006) stated that independent salt-activated ethylene signaling pathways regulate the plant's resistance to adverse environments (e.g., high salinity). As a result, the ethylene induced by salt stress regulates the salt tolerance of plants. IWS introduced abundant halophiles, which assembled around the rhizosphere and were responsible for pollutant degradation. Marinobacter can exhibit extreme halotolerance, and Microbacteriaceae can survive in up to 10% NaCl (Rizzo et al., 2016). The salt-tolerant genus Gracilimonas is a facultative anaerobic bacterium that assists in the anaerobic degradation under high salinity (Lu et al., 2017). In general, an enzyme can be destroyed if it exceeds its appropriate salt concentration limit. High salinity can improve osmotic pressure, result in plasmolysis, and restrict the normal growth of microorganisms (He et al., 2017). However, in this study, the IWS modified by seawater, which includes a halophile-formed salt tolerance mechanism, can accommodate the level of osmotic pressure. IWS can accumulate high K+ and Cl− concentrations, which can adjust the osmotic pressure to resist salt stress (Beales, 2010). Compatible solutes are accumulated as protectants to resist high-salinity environments, which is the main salt tolerance mechanism of halophilic bacteria. Certain common compatible solutes, such as betaine, glutamic acid, and proline, can improve intracellular water activity, compensate for the imbalance of internal and external osmotic pressures caused by high NaCl concentration, and considerably alleviate high-salt stress on cells (Sudmalis et al., 2018). Halophiles demonstrate the transport system of catalytically compatible solute absorption, which is either a primary or secondary property that is coupled with ATP hydrolysis. This system can absorb compatible solutes, maintain protein stability, and implement osmotic pressure regulation, thereby enabling the microorganisms to survive (Numan et al., 2018). The naturalized halophiles introduced by the IWS survived in the CWs to treat saline wastewater.

In this study, IWS was introduced into CWs as a novel inoculation source. This new approach achieved better COD removal than the traditional CWs. In addition, this approach is more economical and environmentally friendly than traditional physical and chemical treatment methods because IWS is inexpensive and readily available. This method is suitable for treating high-salinity and high-organic-matter tailwater produced by seawater utilization industries (e.g., marine aquaculture and seafood processing) in developing countries, such as China, Thailand, Laos, Cambodia, and Vietnam. Large-scale CWs with IWS can be built near sizable seawater farms for deep wastewater treatment or near the coastline for ecological restoration. Small distributed CW systems are suitable for seafood processing factories or markets to handle tailwater and landscape effects. Biological control must be conducted to create favorable environmental conditions for plants and microbes and achieve stable control of CW removal rates. Future studies should focus on the mechanism of the interactions between plants and microbes and the environmental factors that regulate coupling effects. 6. Conclusion This work proposes an effective method to enhance the performance of CWs in the treatment of saline wastewater by introducing IWS as an inoculation source. IWS, which is highly productive and contains rich halophilic and anaerobic bacteria, enhanced COD removal in CWs for saline wastewater treatment. In addition, plant stress tolerance was also improved through the accumulation of halophilic and hormone-related bacteria. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 51608315 and 51708340), Key Research and Development Program of Shandong Province, PR China (No. 2019GSF109103), International Postdoctoral Exchange Fellowship Program (No. 20180063), Special Financial Grant from the China Postdoctoral Science Foundation (No. 2015T80738) and National Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07101001). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2019.109398. References Abdel, L.A.A., Tran, L.P., 2016. Impacts of priming with silicon on the growth and tolerance of maize plants to alkaline stress. Front. Plant Sci. 7, 243. https://doi.org/10. 3389/fpls.2016.00243. Achard, P., Cheng, H., De Grauwe, L., Decat, J., Schoutteten, H., Moritz, T., Van Der Straeten, D., Peng, J., Harberd, N.P., 2006. Integration of plant responses to environmentally activated phytohormonal signals. Science 311, 91–94. https://doi.org/ 10.1126/science.1118642.

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