Elsevier

Water Research

Volume 241, 1 August 2023, 120115
Water Research

Long-term transformation of nanoscale zero-valent iron explains its biological effects in anaerobic digestion: From ferroptosis-like death to magnetite-enhanced direct electron transfer networks

https://doi.org/10.1016/j.watres.2023.120115Get rights and content

Highlights

  • Relation between transformation and biological effects of nZVI in AD was established.

  • The chronic effects of nZVI on methanogenesis were time-dependent.

  • Short-term exposure to nZVI induced bacterial death with ferroptosis-like hallmarks.

  • Formation of vivianite covering the surface of cells fortified membrane rigidity.

  • Magnetite-enhanced DIET facilitated cooperative behaviors for methanogenesis.

Abstract

Nanoscale zero-valent iron (nZVI) has been extensively used for environmental remediation and wastewater treatment. However, the biological effects of nZVI remain unclear, which is no doubt a result of the complexity of iron species and the dynamic succession of microbial community during nZVI aging. Here, the aging effects of nZVI on methanogenesis in anaerobic digestion (AD) were consecutively investigated, with an emphasis on deciphering the causal relationships between nZVI aging process and its biological effects. The addition of nZVI in AD led to ferroptosis-like death with hallmarks of iron-dependent lipid peroxidation and glutathione (GSH) depletion, which inhibited CH4 production during the first 12 days of exposure. With prolonged exposure time, a gradual recovery (12–21 days) and even better performance (21–27 days) in AD were observed. The recovery performance of AD was mainly attributed to nZVI-enhanced membrane rigidity via forming siderite and vivianite on the outer surface of cells, protecting anaerobes against nZVI-induced toxicity. At the end of 27-days exposure, the significantly increased amount of conductive magnetite simulated direct interspecies electron transfer among syntrophic partners, improving CH4 production. Metagenomic analysis further revealed that microbial cells gradually adapted to the aging of nZVI by upregulating functional genes related to chemotaxis, flagella, conductive pili and riboflavin biosynthesis, in which electron transfer networks likely thrived and the cooperative behaviors between consortium members were promoted. These results unveiled the significance of nZVI aging on its biological effects toward multiple microbial communities and provided fundamental insights into the long-term fates and risks of nZVI for in situ applications.

Introduction

Due to its intrinsic high reactivity, large surface areas and environmental friendliness, nanoscale zero-valent iron (nZVI) has been intensively used for in situ environmental remediation over the past decades (Xu et al., 2020a, 2020b; Phenrat et al., 2009). On the other hand, as a kind of nanoparticle, nZVI has attracted much attention in terms of nanotoxicology, and indeed, the adverse effects of nZVI on aquatic organisms, microorganisms and mammalian cells have been widely documented (Chen et al., 2012; Kotchaplai et al., 2017). Disruption of cell membrane structures, DNA damage and apoptosis related to the production of reactive oxygen species (ROS) are potential toxicity mechanisms of nZVI in a broad range of microbes (Lee et al., 2008; Yang et al., 2013). However, the majority of current studies have focused on the short-term effects of nZVI on pure cultures. In long-term application, nZVI certainly oxidizes over time (i.e., “ages”), which would likely change its bioavailability and toxicity (Phenrat et al., 2009; Chen et al., 2012). Several in vitro studies have shown that the bactericidal effect of pristine nZVI is greater than that of aged nZVI or nanoscale iron oxides (Lee et al., 2008; Auffan et al., 2008). Moreover, some bacteria preferentially thrive by utilizing (semi)conductive iron minerals for respiration in natural environments (Kato et al., 2010, 2012). Thus, the biological effects of nZVI, either toxic or beneficial to microbial communities, are supposedly associated with its aging process. Unfortunately, to date, the causal mechanisms linking the long-term transformation of nZVI with its biological effects are not yet defined.

Anaerobic digestion (AD) is considered to be the most widely used and cost-effective technology for high-strength wastewater treatment and renewable energy recovery (Li et al., 2019). Recently, nZVI was reported to be a promising candidate for elevating methane production in AD, improving the hydrolysis-acidification steps and/or promoting the hydrogenotrophic methanogenesis (HM) process (Feng et al., 2014; He et al., 2017). Nevertheless, the inhibition of methanogenesis was also observed due to the destroyed cell integrity and the rapid H2 accumulation caused by nZVI dissolution (Yang et al., 2013). Such contradictory effects of nZVI in AD may derive from the complex interactions between nZVI and microbial aggregates, which would not only alter nZVI reactivity but also affect the physiological status of cells (Auffan et al., 2008; Jin et al., 2015). For example, the interactions of nZVI with extracellular polymeric substances (EPSs) could reduce its inhibition of the AD process by avoiding the fast accumulation of H2 and restricting damage to cell integrity (He et al., 2020). Moreover, a rebound in cell number was observed after prolonged exposure to nZVI, which is attributed to the decreased membrane-fluidizing effect of fatty acids that hindering the interactions between cells and nZVI (Kotchaplai et al., 2017). However, previous studies mainly focused on the responsive mechanism of microbial cells at the single or sequential nZVI exposure endpoints and neglected the dynamic transformation processes of nZVI during long-term incubation. Unlike most aging studies of nZVI undertaken in pure water or simplified simulation conditions (Pullin et al., 2017; Sarathy et al., 2008), limited research in terms of the transformation of nZVI has been conducted in AD where more complex microbial communities are involved, which are closely correlated with the performance of methanogenesis.

Four stages, namely, hydrolysis, acidogenesis, acetogenesis and methanogenesis, are predominantly involved in canonical AD, in which syntrophic acetogenesis often governs the overall rate of anaerobic digestion process due to the slow and indirect way of interspecies electron transfer (IET) via H2/acetate (Huang et al., 2016; Rotaru et al., 2014). In contrast, as a faster and more specific electron transfer pathway, direct IET (DIET), in which species exchange electrons through electrical connections, including conductive pili, cytochromes (Cyt) and iron minerals, was demonstrated to be an alternative to IET in anaerobic methane-producing communities (Lovley, 2017; Shi et al., 2016). In comparison to IET, DIET can provide more energy benefits for syntrophic partners and accelerate reaction rate, finally enhancing methane production (Lovley, 2017). The transformation of nZVI might boost methane production, since some aging products, especially the magnetite, were conductive and probably participated in DIET (Kato et al., 2010; Suanon et al., 2016; Li et al., 2015). In addition, supplementation with nZVI or nanoscale iron oxides could biochemically improve the electron transfer by mediating electron transfer chain activity and the biosynthesis of fluvic acid in EPSs (He et al., 2020; You et al., 2021). Consequently, the transformation of nZVI may thrive in electron transfer networks, which affected the cooperative behaviors between consortium members and further facilitated methanogenesis (Kato et al., 2012; You et al., 2021). However, there is still a lack of detailed studies demonstrating DIET in syntrophic communities during the aging of nZVI in AD.

In this study, we aimed to investigate the dynamic relationships between transformation processes and biological effects of nZVI in AD, with an emphasize on the potential DIET-stimulation mechanism for methanogenesis during nZVI aging. To achieve this goal, the performance of ADs was consecutively estimated during long-term exposure to nZVI. Simultaneously, the aging products of nZVI as well as their interactions with anaerobes at different operation stages were characterized through multiple spectrum analysis and electron microscopy images. The dynamics of functional gene regulation, microbial metabolism and electron transfer potential were further quantified to explore the adaptative strategy of anaerobes in response to nZVI aging.

Section snippets

Anaerobic reactor operation and nZVI exposure experiments

Anaerobic sequencing batch reactors (AnSBRs) with a working volume of 4 L (Fig. S1A) were constructed to culture anaerobic sludge taken from the AD of Jiangxinzhou Municipal Wastewater Treatment Plant (Nanjing, China). The accumulation and cultivation processes were performed with the injection of synthetic wastewater (SW) and 17.19 g volatile solids (VS)/L into AnSBRs. A SW with glucose, NH4Cl and KH2PO4 as carbon, nitrogen and phosphorus sources was employed, and the specific components of SW

Chronic effects of nZVI on the performance of AD

COD removal and CH4 production are two decisive indicators that reflect the biological effects of nZVI on AD. Fig. 1A showed that the COD removal efficiency in R1 fluctuated between 65.61% and 80.56%, with an average value of 74.63% during the whole exposure period. After the addition of nZVI and 12 days of operation, the average COD removal efficiency decreased to 50.98%, indicating overall deterioration in the effluent performance in R2. After that, a gradual recovery to 80.63% at day 27 was

Conclusions

This study demonstrated that the effects of nZVI on methanogenesis were time dependent, which was attributed to the transformation of nZVI as well as the specific regulation of microbes in response to nZVI aging. When operating AD with nZVI, the nZVI-induced ferroptosis-like death to anaerobes could inhibit the methane production at initial exposure period, whereas a recovery and promotion of methane production were achieved with the aging of nZVI, as a result of the reinforced membrane

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgment

We are grateful for the grants for the project from National Natural Science Funds for Youth of China (Grant Nos. 42107386, 52009031), Key Program of National Natural Science Foundation of China (Grant No. 92047201), National Postdoctoral Program for Innovative Talents (Grant No. BX20190106) and PAPD.

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