Application of humin-immobilized biocathode in a continuous-flow bioelectrochemical system for nitrate removal at low temperature
Introduction
The groundwater contamination with nitrate has emerged as a serious environmental problem, and has already been reported in many countries including China and the United States (Temkin et al., 2019; Zhang et al., 2021). Excess nitrate in groundwater can cause eutrophication of the aquatic systems; further, when used as a drinking water source, nitrate-contaminated groundwater poses threats to human health (causing methemoglobinemia, even cancers) due to the potential reduction of nitrate to nitrite (Li et al., 2018). Nitrate can persist in groundwater for decades, and in many cases, it is difficult to clean up (Li et al., 2018). Given these concerns, the World Health Organization (WHO) and United States Environmental Protection Agency (USEPA) have established the maximum contaminant levels of 11.3 mg NO3−-N/L and 10 mg NO3−-N/L for drinking water, respectively (Wang and Chu, 2016). In addition, considerable attention has been given to the development of viable technologies to remove nitrate from contaminated groundwater.
Biological denitrification removes nitrate by converting it to harmless nitrogen gas through the metabolism of denitrifying bacteria, and is considered to be more economical and feasible for the remediation of nitrate-contaminated groundwater compared to the physiochemical methods (e.g., ion exchange and reverse osmosis) (Rahimi et al., 2020). Heterotrophic biological denitrification using organic soluble electron donors (OSED, e.g., ethanol and acetate) is conventionally employed for nitrate-contaminated groundwater remediation (Rezvani et al., 2019; Zhang and Angelidaki, 2013). However, extensive dosages of the OSED may cause secondary pollution of groundwater by organic residues, limiting the lifespan of remediation (Sahinkaya et al., 2011). Thus, reducing the dose of OSED is one of the critical factors to increase the application feasibility of the heterotrophic denitrification process for nitrate removal from groundwater. Denitrifying bioelectrochemical system (BES), using cathodes as electron donors and denitrifying bacteria as biocatalysts (referred to as biocathodes) to achieve nitrate removal, may be an alternative way to overcome the limitations of conventional remediation technologies and reduce OSED usage (Liang et al., 2019; Wang et al., 2019). The presence of an appropriate electroactive biocatalysts responsible for electrode respiration is a prerequisite for employing denitrifying BES in groundwater bioremediation (Li and Yu, 2015). However, in some cases, there is a lack of suitable electroactive biocatalysts in groundwater (Wang et al., 2020). In addition, the biocathode-dependent denitrification process usually proceeds with a low nitrate reduction rate and incomplete overall nitrogen removal, probably due to the difficulty in forming a cathodic biofilm or poor electron delivery efficiency (Rosenbaum and Franks, 2014; Wang et al., 2019). Therefore, on-site applicable electroactive cathodic biofilms with high functional stability are being given high priority for scaling up the denitrifying BES for in-field applications.
Denitrifying bacteria are sensitive to low temperatures, and the optimal temperature range for their growth is 20–30 °C (Lu et al., 2014); a decrease in temperature may have negative effects on the microbial denitrification performance (Nordström and Herbert, 2017; Wang and Wang, 2012). For example, Wang and Wang (2012) found that the nitrate removal efficiency of a laboratory-scale denitrification reactor packed with biodegradable snack ware was decreased with an increased accumulation of nitrite when temperature decreased from 25 °C to 12 °C. However, in many areas (e.g., northern China, southeastern Canada), groundwater temperatures are normally below 10 °C (Biehler et al., 2020; Cheng et al., 2017). Therefore, various approaches have been used to improve low-temperature denitrification performance; these include: regulation of operational parameters (increasing hydraulic retention time (HRT); Zhou et al., 2018), and addition of dissolved redox mediators (e.g., anthraquinone-1,5-disulfonate (AQDS); Yuan et al., 2019). These methods could improve the denitrification performance to some extent at low temperatures. However, they also caused an increase in the remediation cost and secondary pollution.
Solid-phase humin is a fraction of humic substances, which is redox active and not dissolved in any pH conditions (Lipczynska-Kochany, 2018). In a previous study, suspended humin particles were found to function as redox mediators and promote electron transfer from the electrode to the denitrifying bacterium Pseudomonas stutzeri (JCM20778), although it is not electroactive (Xiao et al., 2016). This study implies that humin may be an ideal redox mediator to be applied in the denitrifying BES to promote electron transfer, even if the denitrifying bacteria present in groundwater are not electroactive or are poorly electroactive. Thereby, humin may increase the application feasibility of BES for bioremediation as humic substances-utilizing denitrifying bacteria are abundant in diverse environments (Pham et al., 2020). However, whether humin could mediate the electron transfer from the electrode to the denitrifying biofilm has yet to be demonstrated. More recently, in a microcosm study, we found that humin, as an additional electron donor, could also enhance the low-temperature denitrification performance of a microbial consortium, but its intrinsic reducing capacity was limited and could not support the low-temperature denitrification for a long time (Xiao et al., 2021). Nonetheless, humin still has a significant potential as an economically feasible material for nitrate-contaminated groundwater remediation because of its non-toxic nature and wide distribution. However, these studies were all conducted in short-term (≤7 days) batch experiments and humin particles were present as suspensions. To enhance the applicability of humin-assisted electrode-driven denitrification, it is important to immobilize humin on the cathode surface (to prevent it from being flushed away by water flow) and to test its relative long-term denitrification performance under continuous nitrate feeding. To the best of our knowledge, the biocathode based long-term low-temperature denitrification is rarely reported.
In this study, a continuous-flow denitrifying BES with a solid-phase humin-immobilized biocathode (H-BioC) was developed. Humin was expected to function as a redox mediator and be persistently reduced on the cathode to continuously provide reducing power to a denitrifying biofilm. The denitrification performance in the BES at 5 °C (because the bioremediation rate is often assumed to be low or insignificant at groundwater temperatures of 5 °C or less (Bradley and Landmeyer, 2006; Peng et al., 2015)) was assessed by measuring the influent and effluent nitrate concentrations. Biofilm of H-BioC was characterized by electrochemical and molecular biological analyses. The mechanism of enhanced low-temperature denitrification compared to the control BES with unaltered cathode was also explored. This study provides an alternative strategy of applying the denitrifying BES for the remediation of groundwaters wherein the biocatalysts have poor electroactivity.
Section snippets
BES configuration
The BES used in this study was a single-chambered polypropylene equipment (cylinder-shaped, effective volume of 170 mL) with a working electrode (cathode: carbon fiber felt, 130 mm × 130 mm), a counter electrode (anode: graphite rod, diameter 5 mm), and a reference electrode (Ag/AgCl reference electrode, +200 mV vs. standard hydrogen electrode, SHE) (Supplementary information). Humin (same as that used in our recent study; Xiao et al., 2021) amounting to 2 g was immobilized on the cathode using
NO3−-N removal
The H-BioC and the unaltered cathode in the control BES were both evaluated for microbial denitrification. As shown in Fig. 1A and B, in phase I (days 1–80), the provided influent NO3−-N concentrations in both systems were approximately 28 mg/L, and the effluent NO3−-N and NO2−-N concentrations of the H-BioC (14.42 ± 0.84 mg/L and 1.46 ± 0.50 mg/L) were lower than that of the unaltered cathode (21.14 ± 0.98 mg/L and 6.44 ± 0.10 mg/L). No NH4+-N was detected and N2O–N production accounted for
Conclusions
The BES equipped with a humin-immobilized biocathode (H-BioC) enhanced the low-temperature denitrification performance because humin promoted biofilm formation and accelerated electron transfer on the biocathode. The BES with H-BioC maintained a stable and reproducible denitrification performance with low nitrite accumulation at 5 °C. The H-BioC attained a higher specific microbial denitrification rate than the unaltered cathode (1.55 g N/(mg protein·m3·day) vs. 0.39 g N/(mg protein·m3·day)),
Credit author statement
Dan Chen: Investigation, Writing - Original Draft, Funding acquisition; Lizhuang Yang: Investigation, Writing - Original Draft; Zhiling Li: Writing - Review & Editing, Supervision; Zhixing Xiao: Conceptualization, Resources, Writing - Review & Editing, Funding, acquisition, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (grant numbers 51908281 and 41807122); the Natural Science Foundation of Jiangsu Province, China (grant numbers BK20180719 and BK20180716); and the Open Research Fund of State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences (grant number KF 2017-20). The authors thank Xiayuan Wu and Lijun Fu for their assistance with electrochemical impedance
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