A combination of microbial electrolysis cells and bioaugmentation can effectively treat synthetic wastewater containing polycyclic aromatic hydrocarbon

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants composed of two or more benzene rings. They are well known for being recalcitrant and harmful in nature. PAHs are found in the environment from natural sources, such as volcanic activities, forestry, wildfires, and anthropogenic sources, including fossil fuel combustion, wood and waste burning, and coal mining (Bao et al. 2023). PAHs are among the most widely distributed persistent organic contaminants in environmental media, such as soil, water, and air (Ghosal et al. 2016). Therefore, it is urgent to develop efficient techniques for the remediation and amendment of sites contaminated with PAHs. Researchers have developed various technologies to eliminate PAHs in contaminated sites (Tomei & Daugulis 2013). Among those, physical and chemical methods (i.e., photocatalytic oxidation, chemical oxidation, thermal technologies, and soil washing) exhibit drawbacks such as high costs and severe secondary pollution (Kuppusamy et al. 2017). Compared to physical and chemical technologies, biodegradation techniques offer the advantages of cost-effectiveness and environmental friendliness (Ghosal et al. 2016).

The degradation of PAHs is a redox process, in which PAHs serve as an electron donor and undergo a series of oxidations. The generated electrons combine with the electron acceptor, leading to a reduction process. Common electron acceptors for PAH degradation include O₂, ⁠, ⁠, metal ions (i.e., Fe³⁺ and Mn⁴⁺), and CO₂. Therefore, the microbial degradation of PAHs can be categorized into aerobic degradation and anaerobic degradation. Anaerobic degradation can be further divided into degradation with electron acceptors and degradation without electron acceptors (under methanogenic conditions).

Although many PAHs can be biodegraded under aerobic conditions, most sediment environments contaminated with PAHs are anaerobic. Research has revealed that anaerobic microorganisms can completely degrade and mineralize PAHs, which significantly contributes to the alleviation of PAH inhibition in anaerobic environments (Coates et al. 1996). However, compared to aerobic degradation studies, studies on the anaerobic degradation of PAHs are relatively limited. Within this criteria, methanogenic conditions represent a significant pathway for the natural attenuation of PAHs as most natural environments lack electron acceptors (Chang et al. 2001; Zhang et al. 2012a; Roy et al. 2016; Gou et al. 2023).

The anaerobic biodegradation of PAHs relies on syntrophic collaboration between bacteria and methanogens. This process involves two steps: (1) bacteria hydrolyze PAHs to produce substances such as acetic acid and hydrogen and (2) archaea further degrade these intermediates to generate methane and carbon dioxide. Even with the acclimated microbial communities, the anaerobic degradation rate of PAHs remains slow. Under some circumstances, significant PAH removal in anaerobic microbial-catalyzed conditions occurred after 100 days (Chang et al. 2005; Zhang et al. 2012b). Hence, the sluggish growth of anaerobic microorganisms and the recalcitrance of PAHs limit the degradation efficiency. Researchers have attempted various endeavors to enhance the anaerobic degradation of PAHs, including biostimulation and bioaugmentation. Biostimulation involves the addition of external substances to enhance specific microbial metabolism and increase the degradation rate of PAHs. Examples include adding an electron acceptor, providing nutrients such as carbon source, nitrogen, and phosphorus, using surfactants to increase the solubility of hydrophobic pollutants, and introducing conductive materials (Bianco et al. 2020; Ferraro et al. 2021). Bioaugmentation relies on adding specific acclimated strains to assist the indigenous microbial community in degrading PAHs (Li et al. 2022). Due to the complexity of the anaerobic environment, integrated approaches were found to be more advantageous over singular methods regarding PAH removal in practical applications (Kuppusamy et al. 2017). Zhang et al. (2015) proposed a two-stage bioremediation method utilizing electron donors (acetate) and electron acceptors (⁠⁠) to remediate marine sediment contaminated with petroleum hydrocarbons. A higher petroleum hydrocarbon removal rate (72%) was acquired than the untreated control (20%).

Recently, bioelectrochemical systems have emerged as a prominent technology in pollution remediation (Ahmad et al. 2022; Lin et al. 2023). Installing a bioelectrochemical system to treat PAHs has been previously reported in microbial fuel cells, where PAHs oxidation couples with oxygen reduction to generate electricity (Kronenberg et al. 2017). However, in microbial electrolysis cells (MECs), the external voltage is supplied to empower electroactive microorganisms to carry out thermodynamically unfavorable PAH degradation reactions (Dolfing et al. 2009). Since PHA degradation is a redox process, a supply of external voltage could accelerate such a process, where the PHAs are degraded at the anode for electrons, CO₂, and H₂ generation, and methanogens receive electrons, CO₂, and H₂ for methane production. Hence, implementing MECs may represent an efficient strategy that could speed up the detoxification rate of PAH in conventional anaerobic digestion systems, but such a hypothesis requires thorough investigation.

This study aimed to understand the feasibility of simultaneously reducing PAH and producing methane by MECs equipped with a carbon brush or nickel foam cathode. Naphthalene is one of the most abundant PAHs in nature, exhibiting high levels of contamination in aquatic environments. Naphthalene and methylated naphthalenes constitute approximately 95% of the total PAHs and have the highest solubility. Therefore, this study chose naphthalene as a representative PAH to investigate its degradation pathways and associated mechanisms. Furthermore, the introduction of bioaugmentation on PAH removal and methane production recovery was assessed. The biochemical mechanism of PAH reduction was elucidated by the methane yield, the composition of reducing products, microbial surface morphology, and community structure.

Each reactor received 100 mL of digested sludge as inoculum, followed by 50 mL of phosphate buffer supply to maintain pH stability within the reactor. The joints between the reactor components were sealed with glass glue to avoid leakage. Reactors were then purged with nitrogen gas for 5 min to remove dissolved oxygen in the liquid. Finally, the gas bag was emptied, and the reactors were placed in a temperature-controlled water bath set at 37 °C.

The naphthalene used in this study was purchased from Chengdu Stende Biotechnology Co. The naphthalene standard was pre-diluted to 1 mg/L with dichloromethane and n-hexane (chromatographically pure grades). The standard was stored in a sealed glass vial and placed in a cold room at 4 °C before use.

A multi-channel potentiostat (CHI 1040, China) was used for electrochemical experiments. The instrument comprises a digital signal generator, a multi-channel data acquisition system, and multiple channels. Before starting the electrochemical experiment, the output leads of the potentiostat were connected to the corresponding anode, cathode, and reference electrodes of MEC reactors, respectively. The potential of MEC-1, MEC-2, and MEC-3 was set to −0.5V (vs. SHE), −1V (vs. SHE), and −1V (vs. SHE), respectively.

The experiment was divided into four stages: Stage 1 (day 1–day 8): preliminary stage, where the anode and cathode were connected by wire to form an MFC. Acetate was supplied daily to enrich electroactive microorganisms. Stage 2 (day 9–day 32): power-on stage, where the anode and cathode were connected to the multi-channel potentiostat (Table 1). Stage 3 (day 33–day 50): naphthalene introduction stage, where, in total, 3 μg of naphthalene was introduced into the MECs and C2, respectively. Stage 4 (day 51–day 77): bioaugmentation stage, where bioaugmentation dosage from a methane-producing reactor was supplied to MECs and C2 to evaluate the removal efficiency of naphthalene and recovery efficiency of methane production (the bioaugmentation was conducted by adding in total 180 mL of digestate in the methane-producing reactor in 4 consecutive days, days 51–54, 45 mL/d). A detailed description of the bioaugmentation dosage can be seen in the Supplementary material.

Gas volume was measured daily with a 100 mL air-tight glass syringe. The compositions of biogas (H₂, N₂, CH₄, and CO₂) were analyzed using a gas chromatograph (GC8A, Shimadzu, Japan).

Volatile fatty acids (VFAs) were determined by a gas chromatograph (GC-2010 Plus, Shimadzu, Japan) equipped with an FID detector. Detection conditions were set: sample inlet temperature 230 °C, column temperature 60 °C, and detector temperature 250 °C.

Naphthalene and its decomposed intermediates were detected by gas chromatography–mass spectrometry (7890A-5975C, Agilent, USA). Qualitative analysis of naphthalene was based on retention time, characteristic ions, and the ratio of different ion abundances, while quantitative analysis was performed using an internal standard method.

The current during MEC operation was monitored by the multi-channel potentiostat (CHI 1040, China) and recorded as an I–T curve.

Total DNA from the samples was extracted using an E.Z.N.A.^® soil DNA kit (Omega Bio-tek, USA). The V3–V4 variable region of the bacterial 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) that had attached Barcode sequences. The archaeal 16S rRNA gene V3–V4 variable region was amplified using primers 524F (5′-TGYCAGCCGCCGCGGTAA-3′) and 958R (5′-YCCGGCGTTGAVTCCAATT-3′). The recovered products were quantified using the Quantus™ Fluorometer (Promega, USA).

The purified products were then used for library construction with the NEXTFLEX Rapid DNA-Seq Kit, followed by sequencing on the MiSeq PE300 platform. Finally, based on the compelling data, operational taxonomic unit clustering and species classification analyses were performed on the Majorbio Cloud Platform.

Phylogenetic Investigation of Communities by Reconstruction (PICRUSt2) was used to predict the gene expression of the microbial community. Particularly, whole sequencing reads were annotated against the Kyoto Encyclopedia of Genes and Genomes databases using PICRUSt2 to predict gene functions. The potential function of microbial communities was expressed based on their taxonomic composition.

Scanning electron microscopy (SEM) was employed to observe the surface morphology of the biofilm. The carbon brush and nickel foam electrodes were washed three times with deionized water to remove surface impurities. Then, glutaraldehyde solution (2.5%) was added, and the electrodes were stored overnight at 4 °C. Subsequently, they were washed three times with deionized water and subjected to gradient dehydration in a series of ethanol solutions. The dehydrated samples were placed at −20 and −80 °C for 12 h each and then freeze-dried in a vacuum freeze dryer for another 12 h. Dried samples were cut using sterilized scissors, affixed to a specialized sample holder using nanometer adhesive, coated with gold using an ion sputter coater, and tested with SEM.

On day 33, naphthalene was introduced into the reactors, imposing pronounced inhibition in methane production, decreasing sharply from 169 ± 18–252 ± 20 mL/gCOD to approximately 25 mL/gCOD in 10 days (Figure 2(b)). Results obtained from this study differed from previous studies where the addition of phenanthrene (2–5 mg/L) stimulated methane production in anaerobic digestion (Chen et al. 2022; Yao et al. 2022). One possible reason could be the substrate (acetate) used in our study, where only syntrophic acetate oxidation bacteria and methanogens could be enriched. In comparison, more complex microbial guilds that possess potential PAH-degrading bacteria may use complex substrates such as sludge and food waste, providing high versatility in metabolic patterns when faced with PAH inhibition (Chen et al. 2022; Yao et al. 2022).

Besides methane production reduction, methane content decreased significantly from 60 ± 1–70 ± 2% to 40 ± 1% when naphthalene was introduced (Figure 2(a)), implying a substantial impact of naphthalene on the activity of methanogens. From day 35 to day 45, C2, MEC-1, and MEC-2 did not recover, while MEC-3, equipped with the nickel foam cathode, gradually showed improvement in terms of methane content and methane yield (Figure 2(b)). Methane content in MEC-3 was close to C1 (57 ± 1%) on day 45, implying a fast acclimation of methanogens on naphthalene inhibition in MEC-3. From day 45 onwards, gas production in the naphthalene-added reactors gradually retrieved, with MEC-3 obtaining the quickest recovery, followed by MEC-1 and MEC-2. While C2 presented the slowest recovery, it reached an average of 12.5% of methane production in Stage 2. Despite methane production in experimental groups showing certain recovery, the overall methane yield remained significantly lower than that of C1 with no naphthalene.

On day 51, bioaugmentation dosage was introduced in naphthalene-added groups to verify its effect on the alleviation of naphthalene inhibition. Results showed that after bioaugmentation, MEC-3 rapidly restored, with the highest daily methane production of 227 ± 2 mL/gCOD on day 75 (Figure 2(b)). Moreover, MEC-1 and MEC-2 gradually recovered, reaching methane production in C2, but lower than their corresponding methane production in Stage 2. In contrast, C2 showed the most sluggish methane recovery performance, reflected by a 50% recovery of methane production of C1 at the end of the experiment (Figure 2(b)). Nickel foam is an excellent cathode material due to its high conductivity, large surface area, low cost, and good stability. Li et al. (2019) reported a simultaneous heavy metal removal rate (Cr(VI): 1.72 g/(m^3. h) and maximum output power (702.86 mW/m²)) using nickel foam as the cathode. Moreover, Jeremiasse et al. (2010) claimed that the MEC equipped with the nickel foam cathode generated high current densities and H₂ production rates. More current output enables microorganisms to gain more reducing power, which is critical for recalcitrant naphthalene removal (Hao et al. 2020). Moreover, the H₂ production capacity associated with nickel foam may explain the highest methane production in MEC-3 in this study, as more CO₂ was converted into methane through hydrogenotrophic methanogenesis.

Additionally, during the operation of MEC, a workstation (CHI1040) was deployed to continuously monitor the current over time (referred to as the I–T curve, which can be seen in the Supplementary material, Figure S1; day 25, before the addition of naphthalene; day 65, after the addition of naphthalene). Accordingly, a minimal current of around 0.001–0.006 mA was recorded during MEC operation. Based on Faraday's laws of electrolysis, the amount of substances produced at the electrodes is proportional to the amount of electricity passing through the electrodes. Assume that 1 mol of H₂ was generated and 2 mol of electrons were required. Thus, 192970 C (Coulomb) was needed (1 mol electron carries 96485 C). According to the definition of Coulomb, Coulomb is equal to the electric charge delivered by a 1-ampere current in 1 s, defined as 1C = 1A × 1s. So, the MEC produces around 0.0864 C (minimum) to 0.5184 C (maximum) daily. It equals 4.5 × 10⁻⁷mol (minimum) and 2.7 × 10⁻⁶mol (maximum) of H₂. Based on the ideal gas law, it equals 0.01 mL (minimum) and 0.06 mL (maximum) of H₂ (at standard temperature and pressure, STP). Therefore, the cathodic hydrogen production is negligible in this study. The prevail of hydrogenotrophic methanogenesis in this study may come from the reinforcement of current as reducing power to stimulate the hydrolytic capacity of PAH-degrading bacteria and acetate-degrading bacteria through a syntrophic oxidation pathway to generate H₂. The H₂ was then utilized by hydrogenotrophic methanogens.

To sum up, adopting the MEC alone for naphthalene-containing wastewater disposal showed recovery in methane production to a certain extent. However, the total methane production was still lower than that of the naphthalene-free treatment (C1). After receiving a bioaugmentation dosage, methane production rapidly increased in all tested treatments. More specifically, methane content in MEC-1 and MEC-2 recovered quickly only after the bioaugmentation. Thus, it can be concluded that combined MEC and bioaugmentation could be viable strategies to alleviate naphthalene inhibition.

Studies using MECs to deal with PAHs are limited. Within such criteria, Ding et al. (2021) announced an improved naphthalene removal rate in MEC compared with open-circuit control (95.17% in MEC vs. 85.88% in open-circuit). Additionally, they affirmed a significantly higher live/dead bacteria ratio and biofilm thickness, suggesting that the growth of biofilms and microbial activities were simultaneously enhanced by electrical stimulation. Similarly, Luo et al. (2019) underlined the external voltage on PAH (nitrobenzene) removal. They claimed that a near-complete removal (99.6%) of nitrobenzene was realized under 0.8 V compared with a negligible removal rate (18.2%) at open-circuit control. Furthermore, Saidi et al. (2020) highlighted the superior performance of the nickel foam cathode on chlortetracycline removal, where a degradation yield of 99% was obtained.

Therefore, the MEC-AD can significantly promote the efficiency of naphthalene degradation compared with anaerobic digestion alone. In this study, however, the significance of bioaugmentation cannot be neglected, which can substantially accelerate the PAH removal process (Larsen et al. 2009; Ferraro et al. 2021). Therefore, a combination of MEC and bioaugmentation was proved the most prominent strategy for realizing the fast inhibition recovery brought by naphthalene. Otherwise, only MEC-3 with nickel foam showed the potential to recover within the short term (Figure 4).

During the degradation process of naphthalene in MEC-AD, it initially forms monoaromatic compounds, which then further mineralize into long-chain aliphatic compounds or aliphatic compounds with branched chains. Eventually, under the influence of anaerobic bacteria, they are transformed into CO₂ and methane. During the anaerobic degradation of PAHs, anaerobic microorganisms convert PAHs to benzoates first with the help of electron acceptors and then decompose the benzoates afterward. The ring-breaking of PAHs is a gradual process, which leads to the generation of intermediates containing monobenzene rings after the incomplete degradation of PAHs, such as dibutyl phthalate, 2,4-di-tert-butylphenol, 2,5-di-tert-butylphenol, 2-benzylthiophene, and 2-nitro-benzaldehyde. These compounds all contain a monobenzene ring structure, the leading cause of PAH degradation in anaerobic degradation. These compounds contain a monobenzene ring structure and a long carbon chain substituent structure. The presence of these intermediates indicates that naphthalene has been progressively degraded.

Microorganisms aggregate as biofilms, which serve as a self-protective mechanism for microorganisms against harsh environmental conditions, i.e., physical and chemical pressures, shear forces, and limited nutrient supply. Observing the electrodes of the MEC-AD reactor through SEM can further reveal the surface morphology of the electrodes. Sufficient electroactive biofilm on the electrodes is the prerequisite for the excellent performance of the MEC.

Table 2 presents the alpha diversity of different samples through the Shannon and Simpson indexes. Among naphthalene-added samples, MEC-3 samples exhibited the highest alpha diversity indexes (Table 2). Previous studies have shown that there is a positive correlation between anaerobic digestion performance and alpha diversity. This correlation is reflected in higher Shannon or Simpson indexes (Ferguson et al. 2018). This also explains the superior methane production performance observed in MEC-3 (Mao et al. 2019).

In this study, the predominant archaeal genera are Methanobacterium and Bathyarchaeia, with varying shares in different groups (Figure 7). Remarkably, the presence of naphthalene significantly suppressed the relative abundance of Methanosaeta, decreasing from 30.4% in C2 to 9.5–12.9% in naphthalene-fed reactors. Previous studies have highlighted the vulnerability of Methanosaeta to disturbances, such as toxic compounds (Zhu et al. 2021).

In contrast, hydrogenotrophic methanogens, especially Bathyarchaeia, were found to be predominant in inhibitory conditions, particularly in MECs (23.4–24.5%). Hydrogenotrophic methanogens could work syntrophically with bacteria to survive harsh conditions (Capson-Tojo et al. 2020; Zhang et al. 2023). Song et al. (2019) affirmed that Bathyarchaeia played a critical role in the biodegradation of hydrolyzed polyacrylamide, a recalcitrant macromolecular pollutant. Additionally, the enrichment of Bathyarchaeia was owing to the bioaugmentation dosage used in this study, which was rich in Bathyarchaeia (Wang et al. 2023).

Based on 16s rRNA sequences, enzymes engaged in naphthalene and methane production were predicted to verify further the effect of combined MEC and bioaugmentation on naphthalene detoxification and methane recovery. PAHs such as naphthalene, phenanthrene, and pyrene undergo ring-opening and stepwise oxidation with the catalyzing enzymes encoded by PAH degradation genes (such as the nah gene). Then, intermediates enter the tricarboxylic acid cycle through the salicylic acid or phthalic acid pathways for complete degradation. Enzymes containing nah and phn genes were higher in MEC-3 over other treatments, which was in line with its highest naphthalene removal rate (Figure 4).

This study indicated faster PAH removal via MECs than conventional anaerobic digestion. Despite its potential implementation, challenges to successfully applying MECs in PAH removal still need to be clarified.

• (1) Cultivation of specific microbes on PAH metabolism. The bioaugmentation dosage used in this study was rich in hydrogenotrophic methanogens. Moreover, future studies should focus on screening and investigating the optimal cultivation condition of typical electroactive PAH-degrading bacteria (i.e., Geobacter sulfurreducens, Pseudomonas aeruginosa, Enterobacter cloacae, Shewanella sp., Rhodopseudomonas sp., Bacteriodetes sp., and Clostridium sp.) in MECs. Additionally, their associated metabolic pathway in MECs can be further revealed by, i.e., Metatranscriptomics and Metaproteomics.

• (2) Elucidate the degradation pathway of PAH in MECs. Unraveling the degradation pathway of PAH by the isotope method (i.e., ¹⁴C labeled PAH) could help understand the degradation dynamic, which could assist in observing the degradation bottleneck step during the MEC treatment of PAH.

• (3) Improve the electrode configuration. Electrode materials should feature high electrical conductivity, low internal resistance, good biocompatibility, large surface area, and reasonable mechanical strength and toughness. With the development of nanotechnology, more endeavors should be conducted to incorporate functional nanomaterials on carbon or metallic material for improved PAH removal.

This study evaluated the effect of MEC combined with bioaugmentation on the anaerobic biodegradation of PAHs. The naphthalene degradation efficiency in the nickel foam equipped MEC, supplied with bioaugmentation, achieved the highest naphthalene removal rate of 94.5%. This removal rate was significantly higher than that observed in an open-circuit (49.6%) with the same reactor setup. As a consequence, the highest recovered methane yield was obtained in the nickel foam cathode. The microbial community analysis suggested that norank_f__Prolixibacteraceae, Corynebacterium, norank_f__norank_o__SBR1031, and Bathyarchaeia were selectively enriched in MECs, especially in nickel foam MEC-3. The prosperity of these microbial guilds was responsible for improved PAH degradation and methane production. Our findings indicated a novel insight into the PAH degradation by MEC combined with bioaugmentation.

The authors would like to thank the National Natural Science Foundation of China for funding support (52300185).

Z.M., T.R., and L.Y. contributed to the study conception and design.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.