Heavy metals transformation in arbuscular mycorrhizal assistant constructed wetlands

Shanshan Hu

Doctoral thesis

Abstract:

Bioremediation technology as an environment-friendly technology has focused more and more attention on heavy metal removal from wastewater (Srivastava and Majumder 2008; Mora-Ravelo et al. 2017). Among them, wetland plants play an important role in heavy metal removal from wastewater. Generally, wetland plants remove heavy metals from wastewater mainly through the accumulation, retention, and fixation process (Barya et al., 2020). However, the accumulation of heavy metals by wetland plants is limited, and excessive heavy metals in wastewater can easily result in stunted growth and sometimes death of the wetland plants (Deng et al., 2006; Yang and Ye, 2015). In order to enhance the wetland plants' resistance to heavy metals toxicity and improve heavy metal remediation, previous studies reported that AMF can establish symbiosis with the roots of some wetland plants in semi-aquatic and aquatic habitats in recent years (Calheiros et al., 2019; Dolinar and Gaberščik, 2010), which may provide an opportunity for AMF application in heavy metals remediation in CWs systems. Although AMF can be found in the roots of wetland plants, AMF colonization in CWs is affected by some factors such as phosphorus (Tang et al., 2001), wastewater quality (Wang et al., 2010), hydraulic condition (Vallino et al., 2014), CW types (Wu et al., 2001), and operation modes of CW (Li et al., 2011a). Phosphorus level in the rhizosphere is one of the main abiotic factors that affect AMF colonization in plants (Wang et al., 2010). The negative correlation between phosphorus availability and AMF colonization well-documented in terrestrial ecosystems, and may also prevail in aquatic habitats (Stevens et al., 2002). Tang et al. (2001) studied the effect of phosphorus availability on AMF colonization of T.angustifolia, indicating that the proportional colonization in the roots was significantly greater for plants grown at 1 and 10 uM phosphorus than for the 100 uM phosphorus treatment. Stevens et al. (2002) studied the phosphorus, AMF, and performance of the wetland plant (Lythrum salicaria) under inundated conditions, results showed that the levels of AMF colonization generally decreased with increasing phosphorus supply. Tang et al. (2001) also evaluated the effect of phosphorus availability on AMF colonization of T. angustifolia, indicating that AMF colonization was present in the three lowest phosphorus (1, 10, 100 uM ) treatments, but was absent from the 500 uM phosphorus control. The reduced colonization of AMF at high phosphorus concentrations may be due to the increased availability of phosphorus in the environment, which directly inhibited the growth and spread of external hyphae (Graham et al., 1982). Some researchers also found that the extent of AMF colonization of plants had no significant difference in wetlands under different soil phosphorus concentrations (Miller and Bever, 1999; Van Hoewyk et al., 2001). Cornwell et al. (2001) studied the occurrence of AMF in a phosphorus-poor wetland and mycorrhizal response to phosphorus fertilization, showing that T. latifolia and Carex lasiocarpa were nonmycorrhizal in phosphorus-poor condition. Hence it can be predicted that the AMF colonization in wetland ecosystems may be inhibited under high or low phosphorus concentrations. Overall, more attention should be paid to phosphorus concentration when AMF is used in wastewater treatment CWs. AMF colonization could be gradually decreased as the increase of pollutant (e.g. phosphorus, nitrogen, and heavy metals) concentrations in wastewater. Similar to the factor of phosphorus, nitrogen is also one of the main factors influencing AMF colonization in CWs. The nitrogen concentration can affect the allocation of carbon in plant roots to AMF. Generally, plants could reduce the carbon allocation toAMF under a high nitrogen concentration, thereby inhibiting the growth of AMF (Xu et al., 2016). Blanke et al. (2005) observed that plant roots can absorb nitrogen without the

Abstract:

help of AMF under the high nitrogen concentration in the environment, which results in that the negative impact on plants caused by AMF absorption carbon from plant roots was higher than the contribution of AMF transporting nutrients to the plant roots. Some researchers also suggested that a higher incidence of AMF colonization in CWs was due to the less organic matter and lower nutrient availability as compared with those in the natural wetlands (Confer and Niering, 1992). Besides, some studies reported that AMF colonization (e.g. arbuscule numbers and spore densities) in roots of terrestrial plants were decreased significantly under high heavy metals contamination (Akhtar et al., 2019; Wu et al., 2019). However, Wu et al. (2020) evaluated the AMF effect on the growth and photosynthesis of P. australis under copper stress, indicating that AMF colonization levels (18-21%) had no significant difference between the Cr stress and non-heavy metal addition treatments. Ban et al. (2017) also found that AMF diversity in heavy metal polluted wetlands was higher than most of the records of upland habitats. In conclusion, wastewater quality has influences on the AMF colonization of wetland plant roots in CWs, but further study needs to be evaluated especially for the heavy metal removal in CWs. AMF can easily survive and colonize the roots of plants under aerobic conditions, this is why higher AMF colonization is detected in terrestrial plants (Ramírez-Viga et al., 2018; Xu et al., 2016). However, the soil moisture in CWs is usually under the saturated situation, which leads to the low oxygen content in the substrate, thus affecting AMF colonization in the roots of wetland plants (Brundrett, 2009; Stevens et al., 2011). Previous studies reported that AMF colonization in wetland plants gradually decreased with the increase of hydraulic loading rate (Miller, 2000; Wang et al., 2011). Despite that, increasing evidence demonstrates that AMF was observed in the roots of many wetland plants under different hydrologic sites around the world (Wang et al., 2018b; Wang et al., 2011; Xu et al., 2016). In addition, AMF can even be detected in the submerged wetland plants in some polluted (e.g. heavy metals) wetlands (Sudová et al., 2015; Wang et al., 2018b). Furthermore, some researchers revealed that hydraulic loading rate had no significant effect on AMF colonization, as well as the percentage of the root in AMF colonization was not related to the hydrologic category (Ipsilantis and Sylvia, 2007b; Turner et al., 2000). Moreover, Dolinar and Gaberščik (2010) found that once AMF symbiosis was established in the roots, the hydraulic loading rate did not affect their colonization. Therefore, it is necessary to evaluate whether AMF colonization can be affected by the hydraulic loading rate in CWs. AMF spore germination and hyphal growth can be influenced by oxygen content directly. Thus, the different CWs types (FWS CWs, HSSF CWs, and VSSF CWs) that affect AMF colonization in the roots of wetland plants are mainly related to the oxygen contents in each CWs system. The main pathways of oxygen transfer in FWS CWs are atmospheric diffusion and plant-mediated (well-developed aerenchyma in wetland plant roots) oxygen transfer (Xu et al., 2016). The limited oxygen-transfer capability in SSF CWs is determined. To solve this problem, some enhanced treatment CWs systems (e.g. aeration, tidal flow CWs, hybrid CWs) are constructed to provide sufficient oxygen for the respiration of microorganisms (e.g. bacteria and AMF), resulting in improving the pollutants removal (Austin et al., 2003; Li et al., 2019; Vymazal and Kröpfelová, 2015). Nevertheless, compared to the HSSF CWs, the VSSF CWs are also exposed to the atmosphere, easing to transfer of oxygen from the atmosphere, thereby leading to higher oxygen concentrations in the systems. Therefore, AMF should easy to colonize the roots of wetland plants in the VSSF CWs. Aeration, season, wastewater recirculation, alternation of wetting and drying, and intermittent operation can bring oxygen into CWs, which is beneficial to the growth, abundance, and diversity of AMF, thus enhancing pollutants purification in wastewater (Li et al., 2011b). Liang et al. (2018) studied the effects of AMF and soil nutrient addition on the growth of P. australis under different drying- rewetting cycles (1, 2, or 4 cycles), indicating that AMF increased leaf area and decreased belowground to aboveground biomass ratio in the medium frequency of drying-rewetting cycles. Dolinar and Gaberščik (2010) showed that the highest level of AMF colonization in the roots of P. australis was obtained at the end of the growing season under an intermittent wetland. Xu et al. (2020) also evaluated the effects of aeration on the formation of AMF under a flooded state and copper oxide nanoparticle removal in VFSS CWs, founding that the highest AMF colonization with values of 67% and 21% for frequency and intensity in P. australis roots was observed in the treatment with intermittent aeration for 4 h day−1. Therefore, a suitable operation mode of CWs would improve the AMF colonization and enhance wastewater purification. Many studies have shown that AMF can significantly enhance the tolerance of terrestrial plants to heavy metals such as Zn, Cd, Cu, As, U, and radioactive elements, as well as can change the distribution of heavy metals in terrestrial plants (Bai et al., 2008; Wu et al., 2019; Wu et al., 2014b). In most cases, AMF can enhance the retention of heavy metals in terrestrial plant roots and reduce the transport of heavy metals to aboveground parts (Janeeshma and Puthur, 2020). Compared to the AMF application in the heavy metal contaminated soil system, few studies focus on the role of AMF in alleviating heavy metal toxicity in CWs. Recent studies have found that AMF can colonize the roots of wetland plants in heavy metals-contaminated CWs, this provides a chance that they may play an important role in wastewater purification (He et al., 2020; Weishampel and Bedford, 2006; Xu et al., 2016). Xu et al. (2020) studied the effects of aeration and AMF on copper oxide nanoparticle removal in VSSF CWs, the results observed that the impact of AMF inoculated treatment on Cu purification was better than that of non-inoculated controls. He et al. (2020) evaluated the Cd leaching from polluted soils under simulated heavy rainfall, showing that Cd concentrations of the water in the mycorrhizal and hyphal compartments were 38.6%-93.8% lower than those in the soil compartment. Meanwhile, Huang et al. (2018) studied the effects of AMF on the uptake, translocation, and distribution of cadmium in P. australis, revealing that Cd concentrations and contents in the roots of AMF inoculated P. australis at the Cd concentration of 20 mg/L were higher than that in the non-inoculated controls, while a contrasting tendency was observed in the stems of P. australis. Therefore, AMF may alleviate heavy metal toxicity in CWs by decreasing heavy metal concentration in wastewater and improve heavy metal accumulation in wetland plants. Although previous studies have proven that AMF could enhance heavy metal polluted wastewater purification in CWs, the mechanisms of AMF alleviating heavy metal toxicity in CWs are still unclear. Based on the AMF application in terrestrial ecosystems, we suggest that the AMF improve the tolerance of wetland plants to heavy metals in CWs mainly divided into direct and indirect influences. Direct influence mainly refers to the direct adsorption, absorption, and transformation of heavy metals by AMF. AMF mycelial cell wall components such as chitin, cellulose, and its derivatives can be combined with metal ions through carboxyl group, amino group, hydroxyl group, thiol group, and other groups, thus have a strong adsorption capacity for metals (Dhalaria et al., 2020; Wang et al., 2020). Moreover, AMF also have effects on the morphology and bioavailability of heavy metals in the rhizosphere, thereby affecting the uptake and transport of heavy metals by host plants (Wu et al., 2019). Xu et al. (2020) indicated that AMF in the CWs may release some active substances to change the rhizosphere microbial community, thereby promoting the absorption of heavy metals. Besides, Xu et al. (2016) also reported that some microorganisms (e.g. AMF) can accumulate heavy metals in cells to form amorphous mineral inclusions. On the other hand, indirect influence means that AMF enhance plant tolerance to heavy metals by promoting plant absorption of mineral nutrients (such as nitrogen, phosphorus) and enhancing plant production reactive oxygen species scavenging (e.g. decrease malondialdehyde and superoxide anion contents, increase antioxidant activities) (Ferrol et al., 2016). Chen et al. (2007) showed that AMF increased the uptake of heavy metal contents in plants, probably due to improving plant nutrition and increased biomass. Meanwhile, heavy metals could be immobilized in the substrates through the extra radical mycelium, thus reducing the heavy metal concentrations in the water or soil. Kullu et al. (2020) showed that the extra radical mycelium of AMF acted as a plant root extension, which can reach beyond the root depletion zone and may absorb heavy metal from the soil. The extra radical mycelium of AMF can also secrete organic acids to activate mineral nutrients in the soil, which enhance plant absorption of mineral nutrients, thereby improving plant tolerance to heavy metals (Subramanian et al., 2009). Besides, Wu et al. (2019) showed that heavy metal preferential accumulation in intraradical fungal structures rather than in root cells, which decreases the heavy metals toxicity to host plants. In all, numerous studies focused on exploring the effects of AMF on detoxification of terrestrial plants to heavy metal stress, but the mechanisms of AMF alleviating the toxicity of wetland plants to heavy metals still need further study in wetland systems. To determine the optimum candidates for AMF colonization in roots of wetland plants under the wetland systems, we evaluated the effects of water depths (four water levels) on AMF colonization in different wetland plants (two wetland plant species). Although some studies indicated that AMF can promote wetland plant growth, increase chlorophyll contents in leaves, and facilitate nutrient uptake under abiotic stresses (e.g. heavy metal) (Fusconi and Mucciarelli, 2018; Wu et al., 2020), few studies focus on the AMF effect on the antioxidant response in wetland plants under heavy metal stress. Besides, heavy metals distribution and transformation in AMF assistant wetland systems are also unclear. To solve these problems, we investigated the influences of AMF on wetland plant growth and antioxidant response under heavy metal stress (three Cr concentrations) regarding two water levels to explore the relationships between AMF and physiological functions toward wetland plants. Furthermore, the impacts of AMF on heavy metal distribution and bioavailability in semi-aquatic habitats were determined to evaluate the capacity of AMF for promoting wastewater purification. The results are as follows: AMF colonization and physiological functions toward two wetland plants (P. arundinacea and S. sylvaticus) under different water depths (below the surface of sands: water levels of 5 cm, 9 cm, 11 cm, and fluctuating water depth (9-11 cm)) were studied. In this context, the best condition for AMF colonization and wetland plant growth were determined under the fluctuating water level. As expected, wetland plant species had no significant difference in AMF colonization under semi-aquatic systems. AMF formation in the roots of wetland plants was inhibited under high water depths, which indicated that AMF application in semi-aquatic or aquatic habitats (such as CWs systems) might be limited under high hydraulic loading rates. Meanwhile, the production of lipid peroxidation in wetland plants was determined under the low water depth (11 cm), resulting in the growth of wetland plants was stressed by water shortage. Moreover, physiological functions toward two wetland plants were improved by AMF inoculation under the fluctuating water level. Therefore, AMF application can be recommended for wetland systems under an optimum water level such as the fluctuating water depth. While high AMF colonization in the roots of wetland plants can be found in the semi-aquatic systems under the fluctuating water depth, the effect of AMF on wetland plants' growth under heavy metal stress needed to be investigated as well. For this reason, the influences of AMF on wetland plant (I. pseudacorus) growth and antioxidant response under various Cr stresses (Cr concentration of 0, 5, 25 mg/kg) at different water depths (low water depth and fluctuating water depth) were evaluated. Generally, Cr had a negative influence on wetland plant growth by increasing ROS and lipid peroxidation accumulation, but AMF inoculation can improve wetland plant growth and enhance ROS scavenging under Cr stress (especially under the low Cr stress). Meanwhile, AMF promoted the antioxidant response in wetland plants, with higher biomass, height, the contents of chlorophyll, SOD, POD, K, and P in AMF inoculated wetland plants were determined compared to the non-inoculated controls. Besides, the wetland plants' tolerance to Cr stress was also enhanced under the fluctuating water depth. In conclusion, AMF provided an opportunity to improve the wetland plants' tolerance to heavy metal stress in wetland systems. AMF had good potential in reducing oxidative damage of wetland plants under heavy metal stress. However, it is unclear whether AMF can modulate the heavy metal transformation in wetland systems. For this purpose, the impacts of AMF on heavy metal (Cr) distribution and bioavailability in semi-aquatic habitats were performed. It showed that AMF can regulate the Cr distribution in wetland plants by increasing the Cr accumulation in the roots of wetland plants and decreasing the transfer of Cr from roots to shoots. In addition, Cr concentrations and mass contents in water can also be decreased by AMF inoculation. Meanwhile, AMF can reduce the Cr bioavailability in substrates under the fluctuating water depth. Overall, the results indicated a chance for heavy metal removal in AMF assistant wetland systems. The application of microorganisms generally represents a viable solution for improving heavy metal remediation in semi-aquatic or aquatic habitats such as CWs systems. Although it is a challenge for AMF to colonize the roots of wetland plants due to the high water content in wetland habitats, the obtained results in this Ph.D. thesis have proven that high AMF conization (the intensity of mycorrhizal colonization of 15.6% - 49.7%) in the roots of wetland plants can be observed under the fluctuating water level, which may provide the basis for the application of AMF in tidal flow CWs. Moreover, AMF promoted the antioxidant response in wetland plants and enhanced the heavy metal remediation in semi-aquatic systems, which may establish a good starting point for the application of AMF in the removal of heavy metals from wastewater under semi-aquatic and aquatic habitats (e.g. CWs). The knowledge gained here thus provides an efficient and environmentally friendly new strategy for the treatment of wastewater in CWs technology. Although this thesis concluded that AMF can be applied in the CWs systems to purify heavy metal contaminated wastewater, this method is still in the preliminary research stage. Wu et al. (2016b) evaluated the cellular distribution and speciation of Cr in both extraradical mycelium and mycorrhizal roots exposed to Cr(VI) in terrestrial ecosystems by using field emission scanning electron microscopy equipped with energy dispersive X-ray spectrometer, scanning transmission soft X-ray microscopy, and X-ray absorption fine structure spectroscopy techniques, which revealed the mechanisms underlying Cr immobilization in cell walls and intraradical fungal structures in mycorrhizal roots of terrestrial plants. Compared to the effect of AMF on heavy metal remediation in terrestrial ecosystems, the mechanism of AMF promote the heavy metal distribution and transformation in wetland systems remains unclear. Therefore, it is necessary to reveal the microscopic migration and transformation processes of heavy metals in the AMF assistant wetland systems by using synchrotron radiation X-ray spectroscopy analysis technology. In addition, pot experiments were carried out in this thesis to evaluate the effect of AMF on wetland plant growth and heavy metal distribution. Thus, long-term pilot-scale and full-scale experiments could be required to directly investigate the influence of AMF on wastewater removal in wetland systems. Moreover, pollutants removal efficiencies in AMF assistant wetland systems are easily affected by some factors such as climate, nutrient concentrations (especially phosphorus contents), pH, hydraulic loading rates, and the size of the wetland system, which indicated that the application of AMF in wetland systems should consider these factors. In all, AMF inoculation is beneficial for heavy metal remediation in wetland habitats, but further studies are still needed to explore the effect of AMF on wastewater purification (e.g. nitrogen, phosphorus, organic carbon, and emerging pollutants removal) in CWs systems

Keywords

Arbuscular mycorrhizal fungi, Constructed wetlands, Heavy metals, Antioxidant response, Wetland plants

Language used: English

Date on which the thesis was submitted / produced: 8. 4. 2021

Thesis defence

• Supervisor: doc. Zhongbing Chen
• Reader: Martina Janoušková, Ing., externi, Rui S. Oliveira, externi

Czech University of Life Sciences Prague

Faculty of Environmental Sciences

Doctoral programme / field:
Environmental Sciences / Applied and Landscape Ecology