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Article

Enhanced Oilfield-Produced-Water Treatment Using Fe3+-Augmented Composite Bioreactor: Performance and Microbial Community Dynamics

by
Qiushi Zhao
1,2,
Chunmao Chen
1,*,
Zhongxi Chen
1,2,
Hongman Shan
2 and
Jiahao Liang
3,*
1
College of Chemical Engineering and Environment, China University of Petroleum-Beijing, Beijing 102249, China
2
Daqing Oilfield Design Institute Co., Ltd., Daqing 163712, China
3
Key Laboratory of Petrochemical Pollution Control of Guangdong Higher Education Institutes, School of Environmental Science and Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China
*
Authors to whom correspondence should be addressed.
Bioengineering 2025, 12(7), 784; https://doi.org/10.3390/bioengineering12070784
Submission received: 27 May 2025 / Revised: 11 July 2025 / Accepted: 14 July 2025 / Published: 19 July 2025
(This article belongs to the Topic Wastewater Treatment by Physical, Chemical, Photochemical, and Biological Processes, and Their Combinations)
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Abstract

The presence of recalcitrant organic compounds in oilfield-produced-water poses significant challenges for conventional biological treatment technologies. In this study, an Fe3+-augmented composite bioreactor was developed to enhance the multi-pollutant removal performance and to elucidate the associated microbial community dynamics. The Fe3+-augmented system achieved efficient removal of oil (99.18 ± 0.91%), suspended solids (65.81 ± 17.55%), chemical oxygen demand (48.63 ± 15.15%), and polymers (57.72 ± 14.87%). The anaerobic compartment served as the core biotreatment unit, playing a pivotal role in microbial pollutant degradation. High-throughput sequencing indicated that Fe3+ supplementation strengthened syntrophic interactions between iron-reducing bacteria (Trichococcus and Bacillus) and methanogenic archaea (Methanobacterium and Methanomethylovorans), thereby facilitating the biodegradation of long-chain hydrocarbons (e.g., eicosane and nonadecane). Further metabolic function analysis identified long-chain-fatty-acid CoA ligase (EC 6.2.1.3) as a key enzyme mediating the interplay between hydrocarbon degradation and nitrogen cycling. This study elucidated the ecological mechanisms governing Fe3+-mediated multi-pollutant removal in a composite bioreactor and highlighted the potential of this approach for efficient, sustainable, and adaptable management of produced water in the petroleum industry.

1. Introduction

As most global oilfields increasingly enter mid-to-late production stages, a significant portion of crude oil remains trapped within reservoir pores due to capillary, viscous, gravitational, and other hydrodynamic effects [1]. Chemical Enhanced Oil Recovery (CEOR) technologies—which inject chemical flooding agents to increase solution viscosity, expand sweep volume, and regulate fluid flow—have been established as the principal, economically viable strategies for recovering residual oil, with widespread application in countries such as China, the United States, and Canada [2,3]. However, the chemical flooding process generates chemical-flooding-produced water (CFPW) characterized by high concentrations of polymeric agents, emulsified oil droplets, and a diversity of complex organics [4]. These attributes result in markedly increased water viscosity and enhanced emulsion stability, rendering the composition of produced water increasingly complex and significantly challenging its effective treatment [5,6].
Traditional physicochemical processes, including coagulation–flocculation, air flotation, advanced oxidation, and membrane separation, have long been used for treating CFPW due to their rapid pollutant removal and reliability. However, these approaches often incur high operational costs, generate secondary pollutants, and show limited efficacy in degrading persistent organic compounds [7,8,9,10,11]. In contrast, biological treatment offers a promising green alternative for produced water management, known for its environmental compatibility, cost-effectiveness, and sustainability [12]. Biotechnologies such as fixed-bed hybrid bioreactors (FBHBRs) [13], biological-contact-oxidation reactors (BCORs) [14], membrane-aerated biofilm reactors (MABRs), and immobilized biological systems [15] have shown promising potential for oilfield-produced water treatment. By facilitating the formation of biofilms or immobilizing functional microorganisms, these technologies enhance the metabolic diversity and robustness of microbial communities, thereby enabling the effective degradation of complex organic pollutants present in the produced water. This capability is particularly crucial for maintaining high treatment efficiency under fluctuating operational conditions. Nevertheless, the inherently low biodegradability of CFPW remains a significant bottleneck that limits the overall efficiency of biological processes [16]. Thus, enhancing the biodegradation capacity is critical for advancing oilfield-produced-water treatment technologies.
Iron-based materials have recently attracted significant attention due to their multifunctional roles in environmental remediation [17]. Besides serving as an essential trace element for microbial growth, iron species facilitate pollutant degradation through redox pathways. For instance, zero-valent iron (Fe0) undergoes corrosion to continuously release H2 and Fe2+, supplying both electrons and hydrogen to methanogenic archaea, thereby promoting direct interspecies electron transfer (DIET) between syntrophic acetogens and methanogens, and methanogenesis [18]. Iron oxides, such as Fe3O4, can act as electron shuttles to further accelerate DIET and enhance the co-metabolic degradation of recalcitrant organics [19]. Furthermore, Fe2+/Fe3+ cycling activates persulfate and hydrogen peroxide to generate highly reactive SO4·− and ·OH radicals, sustaining the advanced oxidation of organics [20,21]. Numerous studies have confirmed that iron-based materials can substantially improve microbial degradation efficiency towards organic contaminants [22,23,24,25]. Notably, microbial processes enhanced by Fe3+ reduction have recently shown great promise, with Fe3+ addition substantially improving chemical oxygen demand (COD) and total nitrogen (TN) removal in conventional A2O systems [26,27,28,29]. These findings suggest that the integration of iron reduction in CFPW biotreatment processes may significantly augment pollutant-removal efficiencies.
This study aimed to (1) systematically evaluate the performance and application potential of an Fe3+-augmented composite bioreactor treating CFPW; (2) elucidate the interrelationship between pollutant degradation and iron ion transformation under Fe cycling; and (3) characterize the impacts of Fe cycling on microbial community structure and function. The results provided critical scientific support for the development and application of Fe-cycling-based biotreatment technologies for oilfield-produced water, while offering innovative insights for tackling the challenges associated with recalcitrant organic biodegradation in oilfield wastewater.

2. Materials and Methods

2.1. Oilfield-Produced Water

The actual oilfield-produced water used in this study was collected from the influent of a wastewater treatment facility at Daqing Oilfield. Detailed physicochemical properties are listed in Table 1.

2.2. Experimental Setup and Operating Procedure

The composite bioreactor used in this study consisted of two anaerobic zones, an anoxic zone, two aerobic zones, and a settling zone (Figure 1). Suspended biofilm carriers were installed in both the anaerobic and anoxic zones to promote microbial attachment and growth, with filling ratios of 30% and 35% for Anaerobic Zones 1 and 2, respectively, and 40% for the anoxic zone. In the aerobic zones, a combination of packing materials was positioned above the aeration system to enhance biofilm formation. The effective working volume of each reaction zone (Anaerobic Zone 1, Anaerobic Zone 2, Anoxic Zone, Aerobic Zone 1, and Aerobic Zone 2) was 48 L, resulting in a total effective reactor volume of 240 L (excluding the settling chamber).
During operation, the overall hydraulic retention time (HRT) of the composite bioreactor was maintained at 16 h. The oilfield-produced water flowed sequentially through the anaerobic, anoxic, and aerobic zones. Effluent from the aerobic zone was recycled to the anoxic zone at a ratio of 100%. Dissolved oxygen (DO) in the aerobic zone was maintained at 2–3 mg/L. To optimize nutrient balance and assess the effect of Fe3+ augmentation, the influent was supplemented with ferric chloride (Fe3+ concentration: 100 mg/L), urea (total nitrogen: 5 mg/L), and monopotassium phosphate (total phosphorus: 2 mg/L).

2.3. Analytical Methods

2.3.1. Physicochemical Analysis

COD was determined using the potassium dichromate titration method [30]. Oil content and suspended solids (SS) were measured according to the China Petroleum Industry Standard SY/T 5329-2022. TN was quantified via alkaline potassium persulfate digestion followed by UV spectrophotometry (HJ 636-2012), and ammonia nitrogen (NH4+-N) was determined using Nessler’s reagent spectrophotometry (HJ 535-2009). UV absorbance at 254 nm (UV254) was measured as a supplementary indicator for assessing the presence of organic compounds with conjugated double bonds and aromatic structures, in accordance with the procedure specified in DB 37/T 4149-2020. Total iron and ferrous ion (Fe2+) concentrations were analyzed using phenanthroline spectrophotometry (HJ/T 345-2007).

2.3.2. Organic Composition Analysis

After 45 days of operation, water samples were collected to evaluate the removal of refractory organic compounds. Analytical measurements were conducted using a gas chromatograph–mass spectrometer (GC-MS, 7890A-5975C, Agilent Technologies Inc., Santa Clara, CA, USA), which was fitted with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies Inc., Santa Clara, CA, USA). Target analytes were isolated from the aqueous matrices via a liquid–liquid extraction protocol employing HPLC-grade dichloromethane as the solvent. The column oven was initially held at 80 °C for 1 min, subsequently ramped to 150 °C at a gradient of 7 °C min−1. The thermal program then increased the oven temperature to 300 °C at a rate of 10 °C min−1, with a final isothermal hold at 300 °C for 5 min. High-purity helium served as the carrier gas at a constant flow rate of 1.0 mL min−1, and 1.0 μL of each extract was injected for analysis. The mass spectrometer operated in the electron ionization (EI) mode, with a signal-to-noise ratio of 65:1 and a data acquisition rate of 2 Hz. Spectral resolution was maintained at 0.6 amu (FWHM) using a quadrupole mass analyzer (Agilent Technologies Inc., Santa Clara, CA, USA). Compound identification was achieved by comparing the acquired spectra to entries in the NIST08 mass spectral database.

2.3.3. Microbial Community Analysis

High-throughput sequencing was employed to characterize the bacterial and methanogenic archaeal communities in each reactor unit. At the end of the experimental period, biofilm samples were collected from Anaerobic Zone 1 (AN1), Anaerobic Zone 2 (AN2), Anoxic Zone (ANO), Aerobic Zone 1 (O1), and Aerobic Zone 2 (O2). Genomic DNA was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Inc., Norcross, GA, USA). The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Sequencing was performed on the Illumina MiSeq platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China), and bacterial community composition was analyzed via the Majorbio Cloud Platform (https://cloud.majorbio.com (accessed on 11 October 2023)).

2.3.4. Metagenomic Analysis

Metagenomic sequencing was performed using the Illumina HiSeq 4000 platform (Illumina Inc., San Diego, CA, USA), generating 223,675,084 raw reads from 5 samples. A non-redundant gene catalog comprising 1,055,539 genes with an average sequence length of 623.63 bp was constructed. Gene catalog sequences were aligned to the KEGG GENES database using BLASTP (version 2.2.28+, http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 17 October 2023)) with an e-value threshold of 1 × 10−5. Functional annotation was performed using KOBAS 2.0 (KEGG Orthology-Based Annotation System, Beijing, China). All bioinformatics analyses, including data processing and annotation, were conducted on the Majorbio Cloud Platform (https://cloud.majorbio.com (accessed on 17 October 2023)).

3. Results and Discussion

3.1. Pollutant Removal Performance of Fe3+-Augmented Bioreactor

Produced water from chemically enhanced oil recovery operations poses considerable treatment challenges due to its high content of emulsified hydrocarbons, SS, and refractory polymers. The treated oilfield-produced water is primarily intended for reinjection, with oil content and SS serving as critical quality parameters for reinjection suitability [31]. As shown in Figure 2A,B, the composite bioreactor demonstrated excellent removal of petroleum hydrocarbons, achieving an average oil-removal efficiency of 99.18 ± 0.91%, while effluent oil concentrations consistently remained below 1 mg/L, meeting reinjection standards. Notably, the anaerobic unit served as the primary functional zone for oil degradation, with an average removal efficiency of 96.22%. Regarding SS removal (Figure 2C,D), the overall removal efficiency reached 65.81 ± 17.55%.
In addition to conventional pollutants, residual polymers—particularly hydrolyzed polyacrylamide (HPAM), commonly applied in enhanced oil recovery—posed significant treatment challenges due to their high molecular weight, structural complexity, and emulsifying properties [32]. As shown in Figure 2E,F, the removal efficiency of polymers reached an average of 48.63 ± 15.15%, with the anaerobic unit identified as the major zone for polymer removal (mean removal: 48.49%). To assess the biodegradation of organic nitrogen in polymers, NH4+-N was selected as an indicator. As shown in Figure 2G,H, a total NH4+-N removal efficiency of 92.40% was achieved. The NH4+-N concentrations in the anaerobic effluent exceeded those in the influent, suggesting that microbial activity in the anaerobic unit successfully converted organically bound nitrogen from polymers into ammonia. This observation provided indirect evidence for the microbial-mediated depolymerization of nitrogenous macromolecules, potentially including HPAM, and highlights the functional role of Fe3+ in accelerating redox-driven bioconversions. The superior performance of the system was largely attributed to the bioaugmentation effect conferred by the Fe3+ addition, which served as an effective electron acceptor and stimulated the proliferation and activity of iron-reducing bacteria (FeRB) [33]. By enhancing anaerobic hydrolysis and acidogenesis, Fe3+ introduction facilitated the degradation of petroleum hydrocarbons and high-molecular-weight polymers. The composite bioreactor demonstrated superior oil removal efficiency (96.2%) compared to previously reported systems lacking Fe3+ augmentation, which exhibited removal efficiencies in the range of 73.97% to 86.3% [34,35]. This significant improvement confirms the synergistic benefits of Fe3+-mediated bioaugmentation in enhancing oil-degradation performance. These findings highlight the addition of Fe3+ as a viable strategy for enhancing bioreactor performance in treating refractory industrial wastewaters [28]. The Fe3+-augmented composite bioreactor offered a promising technological solution for the efficient treatment of oilfield-produced water.
As illustrated in Figure 3A, the composite bioreactor achieved an average COD removal efficiency of 57.72 ± 14.87%, with the anaerobic compartment accounting for 39.45% of the total removal, thereby functioning as the primary zone for organic mineralization. UV absorbance at 254 nm (UV254) is widely recognized as a sensitive indicator for humic-like substances and aromatic organic compounds in aquatic environments [36]. In the anaerobic zone, the UV absorbance decreased markedly from 1.94 ± 1.06 AU/cm in the influent to 0.95 ± 0.60 AU/cm, corresponding to a 51.09% reduction. The anoxic and aerobic units exhibited absorbance reductions of 29.51% and 37.30%, respectively. These findings demonstrate that the Fe3+-enhanced biological system, particularly the anaerobic unit, facilitated the efficient microbial degradation of humic-like substances and aromatic organic compounds.
GC-MS analysis (Figure 3C) revealed the presence of 75 organic contaminants in the influent, predominantly long-chain alkanes and alkenes (C7–C30), such as eicosane (32.62%), nonadecane (9.71%), phthalic acid, di(2-propylpentyl) ester (6.85%), 2,4-di-tert-butylphenol (4.39%), squalene (3.16%), heneicosane (2.69%), and heptadecane (2.32%). To systematically assess the degradation performance across the different reactor compartments, organic pollutants exhibiting peak intensities above 250,000 mV were selected as target analytes. Figure 3C shows a clear and progressive reduction in the number of analyte peaks above the 250,000 mV threshold along the treatment continuum from anaerobic to anoxic and, subsequently, to aerobic stages. This sequential decline underscored an effective stepwise attenuation of recalcitrant organic pollutants. Following the anaerobic phase, the total count of such peaks diminished to 19, signaling substantial contaminant removal at this stage of the treatment process. Notably, predominant compounds such as eicosane, phthalic acid di(2-propylpentyl) ester, nonadecane, squalene, and 2,4-di-tert-butylphenol showed substantial reductions in peak intensity, while easily biodegradable compounds, including 2-methyl-5-propyl-nonane and tetradecane, diminished below the selected threshold, indicating that the anaerobic process facilitated the breakdown of recalcitrant hydrocarbons into lower-molecular-weight intermediates [37]. Within the anoxic zone, structural destabilization of high-molecular-weight organics promoted their aggregation and recombination, resulting in the emergence or increased intensities of certain compounds. For example, phthalic acid di(2-propylpentyl) ester exhibited a higher peak intensity than its initial level, becoming a primary residual contaminant alongside eicosane and terephthalic acid 2-ethylhexyl octyl ester. Upon aerobic treatment, both the number and overall intensity of high-abundance organic peaks decreased considerably, with only four compounds—phthalic acid di(2-propylpentyl) ester, squalene, 1,3-benzenedicarboxylic acid bis(2-ethylhexyl) ester, and 2,4-di-tert-butylphenol—remaining above 250,000 mV. Squalene exhibited a higher peak intensity in the aerobic effluent than in the influent. The reaction of small-molecule degradation products with destabilized macromolecules may be responsible for the formation of recalcitrant compounds (>C20). Overall, the Fe3+-augmented composite bioreactor demonstrated pronounced efficiency in eliminating residual petroleum-derived organics from oilfield-produced water, with particularly high removal efficiencies observed for alkanes such as eicosane and nonadecane. Notably, the marked reduction in persistent compounds, exemplified by phthalic acid di(2-propylpentyl) ester, underscores the crucial role of Fe3+ in stimulating microbial bio-oxidation pathways targeting structurally complex organic pollutants. These findings not only validate the enhanced degradative capability of the Fe3+-mediated biological system for conventional hydrocarbon contaminants but also highlight their potential for the effective removal of recalcitrant organic species in oilfield-produced water.

3.2. Microbial Community Structure

3.2.1. Bacterial Community Analysis

The bacterial diversity indices of all samples are summarized in Table 2. Sequencing coverage for each of the six samples exceeded 99.9%, affirming the comprehensiveness and representative quality of the community profiling. As shown in Figure 4A, a total of 310 operational taxonomic units (OTUs) were commonly observed across all bioreactor zones, with Anaerobic Zone 1 (AN1) exhibiting the highest number of unique OTUs. The elevated bacterial diversity not only enhanced the system’s capacity for the degradation of a broad range of pollutants but also improved its resilience and adaptability to toxic and high-strength contaminant shocks. A progressive decline in the relative abundance of Firmicutes and Chloroflexi was observed from anaerobic through anoxic to aerobic stages, accompanied by a marked enrichment of Proteobacteria (Figure 4B). The relative abundances of Proteobacteria across bioreactor compartments were 3.13% (AN1), 22.25% (AN2), 20.76% (ANO), 24.23% (O1), and 45.62% (O2), respectively. This distribution pattern suggested that Proteobacteria not only persisted through all stages but that their abundance was positively correlated with increased dissolved oxygen concentrations, culminating in their dominance in Aerobic Zone 2. The eventual predominance of Proteobacteria could be ascribed to their exceptional metabolic flexibility and ecological adaptability, which enable them to exploit a broad spectrum of organic substrates and efficiently perform aerobic metabolic processes. Additionally, many members of the Proteobacteria phylum are known to play key roles in nutrient cycling, including ammonia oxidation and denitrification, further supporting their competitive advantage under aerobic conditions [38].
As shown in Figure 4C, Trichococcus was the dominant genus in both the anaerobic and anoxic zones. This genus is well-adapted to anaerobic and anoxic environments and is recognized as a key fermentative bacterium with the capability to metabolize complex organic substrates into acetate and formate [39,40]. Moreover, Trichococcus has been identified as a pivotal functional genus involved in the biodegradation of high-molecular-weight polymers [41]. In the aerobic compartments, facultative anaerobes such as Clostridium sensu stricto 1 and Exiguobacterium became prominent, mediating the hydrolytic acidification of organic matter and petroleum hydrocarbons [41,42,43]. The presence of these bacteria under aerobic conditions was attributable to the stratified oxygen gradients within the biofilm, which created hypoxic microenvironments conducive to anaerobic metabolism. Pseudomonas was also abundant in the aerobic zone. The genus Pseudomonas demonstrated multifunctionality during wastewater treatment. Firstly, it was capable of synthesizing biosurfactants and accumulating intracellular poly-β-hydroxybutyrate (PHB) granules, thereby promoting the degradation of petroleum hydrocarbons, denitrification, and heterotrophic nitrification [44,45]. Secondly, it actively participated in Fe-mediated nitrogen cycling, particularly through the oxidation of NH4+-N [46,47].
Additionally, Fe3+ introduction enriched FeRB, such as Trichococcus [48], Bacillus [49], Clostridium sensu stricto 1 [50], and Desulfobulbus [51]. Trichococcus emerged as the dominant genus in the AN1, AN2, and ANO compartments (Figure 4C). This genus facilitated the reduction of Fe3+ to Fe2+ while simultaneously enhancing the degradation of organic pollutants through microbial electron transfer processes. Clostridium sensu stricto 1 gradually increased from AN2 to ANO and remained prevalent in O1, likely due to localized anaerobic/oxic microniches within the aerobic biofilm carriers, consistent with the detection of Fe2+ in aerobic effluent (Section 3.4). Bacillus and Desulfobulbus, though present at lower abundances, may contribute to ancillary processes such as sulfate reduction.
In addition, several iron-oxidizing bacteria (FeOB) were detected, including unclassified f__Rhodobacteraceae [52], Azospira [53], and Aliihoeflea [54]. Among these, unclassified __Rhodobacteraceae dominated the aerobic zones, playing a central role in mediating Fe2+ oxidation to Fe3+. Notably, Pseudomonas and Paracoccus, which possessed dual capabilities for iron reduction [55,56] and iron oxidation [57], were detected at low abundance in the anaerobic zones, where they likely played auxiliary roles in Fe3+ reduction. In contrast, their abundance increased markedly in the aerobic zones, where they were the predominant taxa responsible for Fe2+ oxidation. These findings reveal a distinct ecological succession and division of labor among key iron-cycling microorganisms in Fe3+-augmented systems. Overall, the Fe3+-augmented bioreactor fostered a targeted and dynamic microbial community structure that strongly supported the efficient removal of recalcitrant contaminants and the synergistic coupling of multiple biogeochemical processes.

3.2.2. Archaeal Community Analysis

In anaerobic environments, archaea and bacteria synergistically interact to facilitate petroleum hydrocarbon biodegradation [58,59]. As shown in Figure 5A,B, archaeal communities were predominantly distributed in the anaerobic and anoxic units. Euryarchaeota were the principal functional phylum throughout the bioreactor zones. Methanogens, including Methanothrix, Methanobacterium, Methanomethylovorans, and Methanospirillum, were ubiquitously distributed across all treatment units, demonstrating their metabolic versatility and syntrophic potential. Integrated analysis of pollutant removal efficiency (Figure 2) and the spatial distribution of microbial communities across bioreactor compartments revealed significant co-occurrence between methanogenic archaea and bacteria, particularly in the anaerobic and anoxic zones. This spatial association suggests functional complementarities that are critical for pollutant removal processes [60]. Specifically, Methanothrix and Methanomethylovorans in the anaerobic/anoxic units were known to facilitate DIET, therefore improving the efficiency of anaerobic digestion [61,62]. Methanobacterium, widely reported in petroleum-contaminated environments, demonstrated hydrocarbon-degrading potential [63,64]. Integrating the results of the oil removal (Figure 2A) and organic degradation characteristics (Figure 3C), it was inferred that Methanobacterium and Methanomethylovorans likely established syntrophic partnerships with dominant bacterial genera such as Trichococcus in the anaerobic zones, thereby enhancing the removal of petroleum hydrocarbons. This cooperative interaction among archaeal and bacterial communities supported the enhanced treatment performance observed in the Fe3+-augmented bioreactor.

3.3. Metabolic Function Analysis

The primary pollutants in the produced water included residual petroleum hydrocarbons (PHs) and nitrogen-containing polymers. Key metabolic pathways involved in the degradation of PHs and nitrogenous pollutants (ko00071, ko00361, ko00362, ko00622, ko00623, ko00624, ko00625, ko00626, ko01220, and ko00910) were identified based on a review of the literature [65,66]. These pathways were subsequently used to elucidate the functional succession of microbial communities across various compartments of the bioreactor. As illustrated in Figure 6A,B, the predominant Level 3 KEGG pathways associated with PH degradation included “Metabolic pathways,” “Microbial metabolism in diverse environments,” “Benzoate degradation,” “Fatty acid degradation,” and “Biosynthesis of secondary metabolites.” The relative abundance of these functional pathways increased progressively along the treatment gradient (anaerobic → anoxic → aerobic). Analysis of key functional enzymes revealed that long-chain-fatty-acid CoA ligase (EC 6.2.1.3) and acetyl-CoA C-acetyltransferase (EC 2.3.1.9) were ubiquitously distributed, mediating the degradation of long-chain fatty acids and benzoate derivatives across all compartments. Carboxybiotin decarboxylase (EC 7.2.4.1) and carboxymuconolactone decarboxylase (EC 4.1.1.44) were predominantly enriched in the anaerobic unit. The carboxymuconolactone decarboxylase (EC 4.1.1.44) facilitated the cleavage of C–C, C–O, and C–N bonds via lyase activity to support the anaerobic degradation of aromatic and benzoate compounds. Furthermore, medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7) and enoyl-CoA hydratase (EC 4.2.1.17) were primarily present in the aerobic compartments, catalyzing fatty acid oxidation reactions. Recent studies have highlighted that petroleum hydrocarbons can significantly impact microbial nitrogen cycling, while core nitrogen-cycling microorganisms, in turn, contribute reciprocally to hydrocarbon degradation through bidirectional metabolic interactions [66,67,68].
As shown in Figure 6C,D, nitrogen-cycling-related metabolic pathways in the composite bioreactor were dominated by “Nitrogen metabolism,” “Metabolic pathways,” and “Microbial metabolism in diverse environments.” Long-chain-fatty-acid CoA ligase (EC 6.2.1.3) remained highly abundant within the ko00910 nitrogen cycle pathway, indicating a strong metabolic linkage between PH degradation and nitrogen transformation processes. Carbonic anhydrase (EC 4.2.1.1), functioning as a lyase, contributed to biological nitrogen metabolism via carbon–oxygen bond cleavage and hydrolytic activity. Nitrogenase (EC 1.18.6.1), primarily detected in the anaerobic compartments, played a dual role in facilitating both nitrogen metabolism and the degradation of chlorinated alkanes/alkenes.
A co-occurrence network analysis of predicted enzyme-coding genes was conducted to further elucidate the potential roles of specific enzymes in PH biodegradation and nitrogen cycling (Figure 6E). The network comprised 28 nodes, with clustering coefficient analysis identifying key enzymes, including long-chain-fatty-acid CoA ligase (EC 6.2.1.3), enoyl-CoA hydratase (EC 4.2.1.17), carboxymethylenebutenolidase (EC 3.1.1.45), and hydroxylamine reductase (EC 1.7.99.1). These enzymes primarily mediated the hydrolysis and cleavage of molecular bonds in organic pollutants and served as the primary functional enzymes in organic degradation.
To further elucidate the keystone enzymes mediating microbial coordination in organic pollutant degradation, the centrality metrics (degree centrality, closeness centrality, and betweenness centrality) were evaluated. The result revealed that aldehyde dehydrogenase (EC 1.2.1.3), carboxybiotin decarboxylase (EC 7.2.4.1), (S)-2-haloacid dehalogenase (EC 3.8.1.2), 3-oxoadipate enol-lactonase (EC 3.1.1.24), and glutamate dehydrogenase (EC 1.4.1.2) acted as pivotal hubs that bridge multiple nodes. These keystone enzymes functioned as critical connectors in the interactive networks, thereby playing critical roles in the synergistic degradation of petroleum hydrocarbons and nitrogenous contaminants.

3.4. Redox Transformation Analysis of Fe3+/Fe2+

To elucidate the redox transformation dynamics of Fe3+/Fe2+ within anaerobic compartments, Fe2+ production and Fe3+ consumption were quantified as key performance indicators (Figure 7B). As shown in Figure 7B, Fe2+ generation and Fe3+ consumption predominantly occurred in the anaerobic zones, with average values of 2.55 ± 2.30 mg/L and 5.17 ± 6.43 mg/L, respectively. This finding demonstrates that FeRB utilized electrons derived from organic matter oxidation to reduce Fe3+ to Fe2+ under anaerobic conditions, leading to progressive Fe2+ accumulation. Notably, the production rate of Fe2+ and the consumption of Fe3+ revealed a stoichiometric discrepancy over the operational period. Specifically, Fe2+ accumulation in the anaerobic compartment (AN) reached 2.55 mg/L, while net Fe2+ consumption occurred in the anoxic (−5.46 mg/L) and aerobic (−1.05 mg/L) units. Correspondingly, Fe3+ depletion was most evident in the anaerobic phase (5.17 mg/L), with substantially lower values measured in the anoxic (−2.80 mg/L) and aerobic (−0.63 mg/L) zones. These results clearly indicate that Fe2+ generation and Fe3+ reduction were predominantly confined to the anaerobic reactors. The observed stoichiometric discrepancy can be primarily attributed to the biofilm-mediated retention of Fe2+, as a significant fraction of the produced Fe2+ was adsorbed onto biofilm surfaces, thereby reducing its detectable concentration in the effluent. Additionally, microenvironmental heterogeneity within the biofilm facilitated the formation of local anaerobic microniches, enabling partial Fe3+ reduction by FeRB and leading to trace levels of Fe2+ in the aerobic effluent. Importantly, FeRB continuously oxidized organic substrates to capture electrons, thereby sustaining multi-stage organic removal through sequential iron redox cycling and microbial metabolic cascades.

3.5. Mechanistic Insights into Fe3+-Augmented Synergistic Degradation of Organic Pollutants

Based on the integrated analysis of Fe3+/Fe2+ redox transformation and contaminant degradation (Figure 2), a comprehensive model of Fe3+-mediated synergistic degradation pathways was proposed (Figure 8). Under Fe3+ supplementation, synergistic interactions between FeRB (including Trichococcus, Bacillus, and Clostridium sensu stricto 1) and methanogenic archaea (including Methanothrix, Methanobacterium, and Methanomethylovorans) markedly facilitated the oxidation of organic contaminants. The underlying mechanisms involved dissimilatory iron reduction, maleate addition, anaerobic hydroxylation, and carboxylation processes [69,70,71]. Within the anaerobic compartments, the breakdown of long-chain alkanes, aromatic hydrocarbons, and polyacrylamides was facilitated by the cleavage of C–C, C–O, and C–N bonds through metabolic pathways such as long-chain fatty acid degradation, benzoate catabolism, and ammonification. Fe3+ reduction (Fe3+ + e− → Fe2+) and amine dealkylation (R–NH2 → NH4+-N) were identified as the predominant reactions, jointly mediating the degradation of recalcitrant organic compounds. Microbial oxidation of organic substrates provided a continuous electron flow that enabled the simultaneous degradation of contaminants and biological nitrogen removal. In chemical-flooding-produced water, organic pollutants such as residual petroleum hydrocarbons and polymers act as primary electron donors. These substrates underwent progressive oxidation by hydrolytic-acidifying microorganisms, iron-reducing bacteria (FeRB), and denitrifying bacteria, during which electrons were released. These electrons served as reducing equivalents that enabled the parallel reduction of Fe3+ to Fe2+ and the stepwise reduction of nitrate and nitrite during denitrification. In the aerobic compartments, residual organic compounds underwent further transformation through mono-terminal oxidation, di-terminal oxidation, sub-terminal oxidation, ω-oxidation, and β-oxidation, while concurrent nitrification ensured a sustained supply of NO3−, reinforcing denitrification and contaminant removal in the anoxic environment. Under Fe3+-enriched conditions, the collective action of multiple bioactive enzymes played a decisive role in the bioconversion of organic pollutants in produced water. Notably, auxiliary enzymes (e.g., EC 1.2.1.3, EC 7.2.4.1, EC 3.8.1.2, EC 3.1.1.2, and EC 1.4.1.2) enhanced the catalytic activity of primary degradative enzymes (EC 6.2.1.3, EC 4.2.1.17, EC 3.1.1.45, and EC 1.7.99.1), collectively driving the bio-oxidation and mineralization of complex organics.

4. Conclusions

This study comprehensively investigated the performance and microbial community dynamics of an Fe3+-augmented composite bioreactor for the treatment of oilfield-produced water. Fe3+ supplementation, as a bioenhancement strategy, not only significantly improved the metabolic activity and syntrophic interactions of key functional microorganisms (e.g., FeRB and methanogenic archaea) in the anaerobic zones but also facilitated the efficient biodegradation of recalcitrant organic pollutants such as petroleum hydrocarbons and polymers. The anaerobic compartments, serving as the core biotreatment module in the composite bioreactor, achieved high removal efficiencies for oil (96.22%), polymers (48.49%), and COD (39.45%). Microbial community analysis revealed a pronounced enrichment of FeRB (e.g., Trichococcus and Bacillus) and their synergistic interactions with methanogenic archaea (e.g., Methanobacterium and Methanomethylovorans), underscoring the importance of microbial syntrophy in multi-pollutant removal under Fe3+ enhancement. Furthermore, functional gene prediction identified key enzymes—including long-chain-fatty-acid CoA ligase (EC 6.2.1.3), enoyl-CoA hydratase (EC 4.2.1.17), carboxymethylenebutenolidase (EC 3.1.1.45), and hydroxylamine reductase (EC 1.7.99.1)—that played central roles in the biodegradation of petroleum hydrocarbons and nitrogenous polymers. This study provides novel insights into the dynamic regulation of the microbial community structure and function under Fe3+ bioaugmentation in a composite bioreactor and elucidates a multi-mechanistic pathway for the synergistic degradation of refractory organic pollutants. The findings provide a technical basis for the development of green, efficient, and sustainable produced-water treatment solutions in the oil industry.

Author Contributions

Conceptualization, Q.Z. and C.C.; methodology, Q.Z. and Z.C.; formal analysis, Q.Z., C.C., H.S. and J.L.; investigation, Q.Z., Z.C. and H.S.; data curation, Q.Z. and C.C.; writing—original draft preparation, Q.Z. and J.L.; writing—review and editing, C.C.; visualization, Q.Z.; project administration, C.C. and H.S.; funding acquisition, C.C. and J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Scientific Research and Technology Development Project of China Petroleum and Natural Gas Co., Ltd.—Research on Green and Efficient Treatment Technology of Oilfield-Produced Water (grant number 2021DJ2602); the Science and Technology Program of Maoming, China (grant number 2022S041); and the Projects of Talents Recruitment of GDUPT, China (grant number RC-XJ2022000702).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Author Qiushi Zhao, Zhongxi Chen, Hongman Shan were employed by the company Daqing Oilfield Design Institute Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Wang, Y.; Han, X.; Li, J.; Liu, R.; Wang, Q.; Huang, C.; Wang, X.; Zhang, L.; Lin, R. Review on Oil Displacement Technologies of Enhanced Oil Recovery: State-of-the-Art and Outlook. Energy Fuels 2023, 37, 2539–2568. [Google Scholar] [CrossRef]
  2. Cao, H.; Li, Y.; Gao, W.; Cao, J.; Sun, B.; Zhang, J. Experimental investigation on the effect of interfacial properties of chemical flooding for enhanced heavy oil recovery. Colloids Surf. A Physicochem. Eng. Asp. 2023, 677, 132335. [Google Scholar] [CrossRef]
  3. Xue, L.; Liu, P.; Zhang, Y. Status and Prospect of Improved Oil Recovery Technology of High Water Cut Reservoirs. Water 2023, 15, 1342. [Google Scholar] [CrossRef]
  4. Wu, M.; Zhai, M.; Li, X. Adsorptive removal of oil drops from ASP flooding-produced water by polyether polysiloxane-grafted ZIF-8. Powder Technol. 2021, 378, 76–84. [Google Scholar] [CrossRef]
  5. Deng, J.; Zhang, X.; Gu, Z.; Tong, Y.; Meng, F.; Sun, L.; Liu, H.; Wang, Q. High-efficiency purification of alkali-surfactant-polymer flooding produced water by ultrasonication-ionic liquids combination: Performance and separation mechanism. Sep. Purif. Technol. 2025, 363, 132255. [Google Scholar] [CrossRef]
  6. Liu, J.; Liu, S.; Zhong, L.; Yuan, S.; Wang, Q.; Wei, C. Study on the emulsification characteristics of heavy oil during chemical flooding. Phys. Fluids 2023, 35, 53330. [Google Scholar] [CrossRef]
  7. Sun, X.; Wang, Q.; Yang, X.; Zhang, X.; Chen, X.; Zhao, D. Effects of polymer, surfactant and solid particle on the stability of wastewater produced from surfactant/polymer flooding. Colloids Surf. A Physicochem. Eng. Asp. 2024, 698, 134419. [Google Scholar] [CrossRef]
  8. Saththasivam, J.; Ogunbiyi, O.; Lawler, J.; Al-Rewaily, R.; Liu, Z. Evaluating dissolved air flotation for oil/water separation using a hybridized coagulant of ferric chloride and chitosan. J. Water Process Eng. 2022, 47, 102836. [Google Scholar] [CrossRef]
  9. Weschenfelder, S.E.; Fonseca, M.J.C.; Borges, C.P. Treatment of produced water from polymer flooding in oil production by ceramic membranes. J. Pet. Sci. Eng. 2021, 196, 108021. [Google Scholar] [CrossRef]
  10. Liu, Y.; Bu, F.; Chen, S.; Jiang, M. Investigating effect of polymer concentrations on separation performance of hydrocyclone by sensitivity analysis. Energy Sci. Eng. 2021, 9, 1202–1215. [Google Scholar] [CrossRef]
  11. Zhao, Y.; Chang, C.; Ji, H.; Li, Z. Challenges of petroleum wastewater treatment and development trends of advanced treatment technologies: A review. J. Environ. Chem. Eng. 2024, 12, 113767. [Google Scholar] [CrossRef]
  12. Mansour, M.S.M.; Abdel-shafy, H.I.; Ibrahim, A.M. Petroleum wastewater: Environmental protection, treatment, and safe reuse: An overview. J. Environ. Manag. 2024, 351, 119827. [Google Scholar] [CrossRef] [PubMed]
  13. Lusinier, N.; Seyssiecq, I.; Sambusiti, C.; Jacob, M.; Lesage, N.; Roche, N. A comparative study of conventional activated sludge and fixed bed hybrid biological reactor for oilfield produced water treatment: Influence of hydraulic retention time. Chem. Eng. J. 2021, 420, 127611. [Google Scholar] [CrossRef]
  14. Zhou, H.; Chen, C.; Zhou, S.; Bu, K.; Li, P.; Lin, X.; Jiang, L.; Zhang, C. Performance and microbial community analysis of a bio-contact oxidation reactor during the treatment of low-COD and high-salinity oilfield produced water. Bioresour. Technol. 2021, 335, 125267. [Google Scholar] [CrossRef] [PubMed]
  15. Nie, H.; Nie, M.; Diwu, Z.; Wang, L.; Yan, H.; Lin, Y.; Zhang, B.; Wang, Y. Biological treatment of high salinity and low pH produced water in oilfield with immobilized cells of P. aeruginosa NY3 in a pilot-scale. J. Hazard. Mater. 2020, 381, 121232. [Google Scholar] [CrossRef] [PubMed]
  16. Ghafoori, S.; Omar, M.; Koutahzadeh, N.; Zendehboudi, S.; Malhas, R.N.; Mohamed, M.; Al-Zubaidi, S.; Redha, K.; Baraki, F.; Mehrvar, M. New advancements, challenges, and future needs on treatment of oilfield produced water: A state-of-the-art review. Sep. Purif. Technol. 2022, 289, 120652. [Google Scholar] [CrossRef]
  17. Peng, Y.; He, S.; Wu, F. Biochemical processes mediated by iron-based materials in water treatement: Enhancing nitrogen and phosphorus removal in low C/N ratio wastewater. Sci. Total Environ. 2021, 775, 145137. [Google Scholar] [CrossRef]
  18. Li, Y.; Zhang, Z.; Tang, J.; Zhao, M.; Huang, Z.; Shi, W.; Ruan, W. The enhanced biological methanation performance in trickle-bed reactor by different continuous hydrogen supply from nano-zero valent iron corrosion. J. Water Process Eng. 2022, 50, 103292. [Google Scholar] [CrossRef]
  19. Li, Y.; Kong, W.; Liu, H.; Hong, Y.; Huang, T. Enhanced degradation of phenolic compounds in coal gasification wastewater by activated carbon-Fe3O4 nanoparticles coupled with anaerobic co-metabolism. Biochem. Eng. J. 2022, 189, 108717. [Google Scholar] [CrossRef]
  20. Tang, Y.; Dou, J.; Lu, Z.; Xu, J.; He, Y. Accelerating Fe2+/Fe3+ cycle via biochar to improve catalytic degradation efficiency of the Fe3+/persulfate oxidation. Environ. Pollut. 2023, 316, 120669. [Google Scholar] [CrossRef] [PubMed]
  21. Zhao, C.; Ran, Y.; Gong, Y.; Hong, C.; Xing, Y.; Sun, Y.; Wang, H.; Ling, W.; Wang, Y.; Feng, W.; et al. Study on the degradation performance of coking wastewater using an in-situ enhanced Fe2+/Fe3+ cycle dual-cathode Electro-Fenton system. J. Environ. Chem. Eng. 2024, 12, 114591. [Google Scholar] [CrossRef]
  22. Liang, D.; Yu, Z.; Wang, Y.; Zhang, Y.; Wang, R.; Hao, J.; Feng, Y. Differences in the efficiency and mechanisms of different iron-based materials driving synchronous nitrogen and phosphorus removal. Environ. Res. 2025, 268, 120706. [Google Scholar] [CrossRef] [PubMed]
  23. Zhu, B.; Yuan, R.; Wang, S.; Chen, H.; Zhou, B.; Cui, Z.; Zhang, C. Iron-based materials for nitrogen and phosphorus removal from wastewater: A review. J. Water Process Eng. 2024, 59, 104952. [Google Scholar] [CrossRef]
  24. Li, X.; Yuan, Y.; Huang, Y. Enhancing the nitrogen removal efficiency of a new autotrophic biological nitrogen-removal process based on the iron cycle: Feasibility, progress, and existing problems. J. Clean. Prod. 2021, 317, 128499. [Google Scholar] [CrossRef]
  25. Lu, X.; Zhong, Z.; Yan, R.; Zan, F.; Lou, W.; Liu, J.; Wu, X.; Zhang, B. La–Fe magnetic bentonite stimulated denitrifying phosphorus removal from low C/N wastewater in the A2/O process: Performance, microbial community, and potential mechanism. J. Clean. Prod. 2022, 373, 133746. [Google Scholar] [CrossRef]
  26. Wan, L.; Liu, H.; Wang, X. Anaerobic ammonium oxidation coupled to Fe(III) reduction: Discovery, mechanism and application prospects in wastewater treatment. Sci. Total Environ. 2022, 818, 151687. [Google Scholar] [CrossRef] [PubMed]
  27. Zhu, T.-T.; Lai, W.-X.; Zhang, Y.-B.; Liu, Y.-W. Feammox process driven anaerobic ammonium removal of wastewater treatment under supplementing Fe(III) compounds. Sci. Total Environ. 2022, 804, 149965. [Google Scholar] [CrossRef] [PubMed]
  28. Zhang, L.; Zhang, M.; You, S.; Ma, D.; Zhao, J.; Chen, Z. Effect of Fe3+ on the sludge properties and microbial community structure in a lab-scale A2O process. Sci. Total Environ. 2021, 780, 146505. [Google Scholar] [CrossRef] [PubMed]
  29. Sun, Y.; Sun, B.; Li, J.; Zhang, X. Fe3+ addition for enhancing the formation and stability of aerobic granular sludge to treat low-strength wastewater. J. Water Process Eng. 2025, 71, 107144. [Google Scholar] [CrossRef]
  30. Standard Methods for the Examination of Water and Wastewater; APHA: Washington, DC, USA, 2018.
  31. Li, C.; Li, J.; Wang, N.; Zhao, Q.; Wang, P. Status of the treatment of produced water containing polymer in oilfields: A review. J. Environ. Chem. Eng. 2021, 9, 105303. [Google Scholar] [CrossRef]
  32. Wang, N.; Sun, X.; Zhao, Q.; Wang, P. Treatment of polymer-flooding wastewater by a modified coal fly ash-catalysed Fenton-like process with microwave pre-enhancement: System parameters, kinetics, and proposed mechanism. Chem. Eng. J. 2021, 406, 126734. [Google Scholar] [CrossRef]
  33. Pan, K.; Guo, T.; Liao, H.; Huang, Z.; Qian, Z.; Li, F.; Li, J. Adding iron shavings in activated sludge system to enhance removal of refractory organics and nitrogen for textile-dyeing wastewater. J. Environ. Chem. Eng. 2023, 11, 110999. [Google Scholar] [CrossRef]
  34. Zhang, X.; Wei, D.; Lu, Q.; Zhao, Q.; Ma, J.; Liu, G.; Ouyang, J.; Luo, E.; Li, C.; Wei, L. A study on the efficiency and microbial community of anaerobic/anaerobic/anoxic/oxic membrane bioreactor for treating saline alkali-surfactant-polymer flooding wastewater. J. Water Process Eng. 2025, 71, 107305. [Google Scholar] [CrossRef]
  35. Qian, G.; Liu, P.; Wei, L.; Mackey, H.; Hao, T. Can a compact biological system be used for real hydraulic fracturing wastewater treatment? Sci. Total Environ. 2022, 816, 151524. [Google Scholar] [CrossRef] [PubMed]
  36. Sato, Y.; Xiang, Y.; Cooper, P.; Cassol, G.S.; Luo, Y.; Zeng, Q.; Shang, C.; Ren, Z.J.; Chen, G. Evaluating UV254 absorbance reductions in landfill leachate for municipal sewage co-treatment through timed UV/electrooxidation. J. Hazard. Mater. 2023, 445, 130624. [Google Scholar] [CrossRef] [PubMed]
  37. Wang, Q.; Liang, J.; Zhang, S.; Yoza, B.A.; Li, Q.X.; Zhan, Y.; Ye, H.; Zhao, P.; Chen, C. Characteristics of bacterial populations in an industrial scale petrochemical wastewater treatment plant: Composition, function and their association with environmental factors. Environ. Res. 2020, 189, 109939. [Google Scholar] [CrossRef] [PubMed]
  38. Parab, A.S.; Ghose, M.; Manohar, C.S.; Gauns, M.U.; Paul, S. Metagenomic insights into bacterial dynamics and niche partitioning in response to varying oxygen gradients in the Arabian Sea oxygen minimum zone (OMZ). Reg. Stud. Mar. Sci. 2024, 78, 103768. [Google Scholar] [CrossRef]
  39. Li, Z.; Feng, Q.; Zhang, F.; Zhao, F.; Lu, M.; Qin, F.; Guo, R. Simultaneous denitrification enhancement and sludge reduction based on novel suspended carrier modified using activated carbon and magnetite at low carbon/nitrogen ratio. Bioresour. Technol. 2024, 395, 130360. [Google Scholar] [CrossRef] [PubMed]
  40. Wang, Y.; Wu, G. Leveraging anaerobic biodegradation of tetracycline in anaerobic digestion systems with different operational modes. Environ. Technol. Innov. 2023, 32, 103373. [Google Scholar] [CrossRef]
  41. Song, T.; Li, S.; Ding, W.; Li, H.; Bao, M.; Li, Y. Biodegradation of hydrolyzed polyacrylamide by the combined expanded granular sludge bed reactor-aerobic biofilm reactor biosystem and key microorganisms involved in this bioprocess. Bioresour. Technol. 2018, 263, 153–162. [Google Scholar] [CrossRef] [PubMed]
  42. Thontowi, A.; Yetti, E.; Yopi, Y. Medium Chain and Long Chain Alkanes Hydroxylase Producing Whole Cell Biocatalyst From Marine Bacteria. Ann. Bogor. 2018, 22, 12–19. [Google Scholar] [CrossRef]
  43. Chen, Q.; Li, J.; Liu, M.; Sun, H.; Bao, M. Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment. PLoS ONE 2017, 12, e0174445. [Google Scholar] [CrossRef] [PubMed]
  44. Liu, Y.; Wan, Y.Y.; Wang, C.; Ma, Z.; Liu, X.; Li, S. Biodegradation of n-alkanes in crude oil by three identified bacterial strains. Fuel 2020, 275, 117897. [Google Scholar] [CrossRef]
  45. Dai, H.; Sun, Y.; Wan, D.; Abbasi, H.N.; Guo, Z.; Geng, H.; Wang, X.; Chen, Y. Simultaneous denitrification and phosphorus removal: A review on the functional strains and activated sludge processes. Sci. Total Environ. 2022, 835, 155409. [Google Scholar] [CrossRef] [PubMed]
  46. Guo, Q.; Hu, H.-Y.; Shi, Z.-J.; Yang, C.-C.; Li, P.; Huang, M.; Ni, W.-M.; Shi, M.-L.; Jin, R.-C. Towards simultaneously removing nitrogen and sulfur by a novel process: Anammox and autotrophic desulfurization–denitrification (AADD). Chem. Eng. J. 2016, 297, 207–216. [Google Scholar] [CrossRef]
  47. Ding, B.; Li, Z.; Qin, Y. Nitrogen loss from anaerobic ammonium oxidation coupled to Iron(III) reduction in a riparian zone. Environ. Pollut. 2017, 231, 379–386. [Google Scholar] [CrossRef] [PubMed]
  48. Tang, Y.; Li, Y.; Zhang, M.; Xiong, P.; Liu, L.; Bao, Y.; Zhao, Z. Link between characteristics of Fe(III) oxides and critical role in enhancing anaerobic methanogenic degradation of complex organic compounds. Environ. Res. 2021, 194, 110498. [Google Scholar] [CrossRef] [PubMed]
  49. Zhang, S.; Song, C.; Wang, L.; Wang, M.; Zhang, D.; Tang, G. Exploring the promoting effect of nitrilotriacetic acid on hydroxyl radical and humification during magnetite-amended composting of sewage sludge. Bioresour. Technol. 2024, 403, 130863. [Google Scholar] [CrossRef] [PubMed]
  50. Wang, Z.; Liu, R.; Cui, Y.; Zhang, Z.; Jing, H.; Chen, Z.; Tian, S.; Yang, Z.; Liu, Y. Effects of Fe3O4 on production of even-carbon volatile fatty acids by anaerobic fermentation of kitchen waste under ultrasonic-alkali pretreatment: Performance, metabolic functions, and metabolic pathways. J. Environ. Chem. Eng. 2025, 13, 116615. [Google Scholar] [CrossRef]
  51. Hartman Wyatt, H.; Bueno de Mesquita Clifton, P.; Theroux Susanna, M.; Morgan-Lang, C.; Baldocchi Dennis, D.; Tringe Susannah, G. Multiple microbial guilds mediate soil methane cycling along a wetland salinity gradient. mSystems 2024, 9, e00936-23. [Google Scholar] [CrossRef] [PubMed]
  52. Huang, X.; Yang, Q.; Feng, J.; Yang, Z.; Yu, C.; Zhang, J.; Ling, J.; Dong, J. Introduction of exotic species Sonneratia apetala alters diazotrophic community and stimulates nitrogen fixation in mangrove sediments. Ecol. Indic. 2022, 142, 109179. [Google Scholar] [CrossRef]
  53. Wang, Y.; Wang, H.; Chen, H. Evaluating optimized volatile fatty acids production from carbon-rich wastewater during hydrolysis acidification process by Fe(Ⅱ) and Fe(Ⅲ) addition. J. Environ. Chem. Eng. 2023, 11, 110724. [Google Scholar] [CrossRef]
  54. Zhang, E.; Wu, S.; Liu, J.; Li, H.; Liu, X.; Lu, Y.; Ge, C.; Zhou, D. Activated carbon as a strong DOM adsorbent mitigates antimony and arsenic release in flooded mining-impacted soils. J. Hazard. Mater. 2024, 473, 134663. [Google Scholar] [CrossRef] [PubMed]
  55. Kang, Y.; Sun, H.; Gao, B.; Dang, J.; Zhang, M.; Li, M.; Dong, J.; Wu, H.; Zhang, J.; Guo, Z. Enhanced reduction of Cr(VI) in iron-carbon micro-electrolysis constructed wetlands: Mechanisms of iron cycle and microbial interactions. Chem. Eng. J. 2022, 439, 135742. [Google Scholar] [CrossRef]
  56. Luan, Y.-N.; Yin, Y.; Guo, Z.; Wang, Q.; Xu, Y.; Zhang, F.; Xiao, Y.; Liu, C. Partial nitrification-denitrification and enrichment of paracoccus induced by iron-chitosan beads addition in an intermittently-aerated activated sludge system. J. Environ. Manag. 2024, 353, 120189. [Google Scholar] [CrossRef] [PubMed]
  57. Bi, Y.; Gao, X.; Su, L.; Lei, Y.; Li, T.; Dong, X.; Li, X.; Yan, Z. Unveiling the impact of flooding and salinity on iron oxides-mediated binding of organic carbon in the rhizosphere of Scirpus mariqueter. Sci. Total Environ. 2024, 908, 168447. [Google Scholar] [CrossRef] [PubMed]
  58. Sierra-Garcia, I.N.; Belgini, D.R.B.; Torres-Ballesteros, A.; Paez-Espino, D.; Capilla, R.; Santos Neto, E.V.; Gray, N.; de Oliveira, V.M. In depth metagenomic analysis in contrasting oil wells reveals syntrophic bacterial and archaeal associations for oil biodegradation in petroleum reservoirs. Sci. Total Environ. 2020, 715, 136646. [Google Scholar] [CrossRef] [PubMed]
  59. Zhou, J.; Lin, W.-H.; Yu, Y.-L.; Dong, C.-D.; Zhang, H.; Hu, Z.; Kao, C.-M. Transitioning weathered oil fields towards new energy: A review on utilizing hydrogenotrophic methanogens for petroleum hydrocarbons remediation. J. Hazard. Mater. 2024, 477, 135279. [Google Scholar] [CrossRef] [PubMed]
  60. Gallardo-Altamirano, M.J.; Maza-Márquez, P.; Montemurro, N.; Rodelas, B.; Osorio, F.; Pozo, C. Linking microbial diversity and population dynamics to the removal efficiency of pharmaceutically active compounds (PhACs) in an anaerobic/anoxic/aerobic (A2O) system. Chemosphere 2019, 233, 828–842. [Google Scholar] [CrossRef] [PubMed]
  61. Zhang, M.; Han, Y.; Zeng, Y.; Wang, T.; Wang, Z.; Wu, Y.; Li, N.; Lobo, F.L.; Wang, X. Understanding the microbial processes on carbon brushes that accelerate methanogenesis of long-chain fatty acids in anaerobic digestion. Water Res. 2025, 273, 123084. [Google Scholar] [CrossRef] [PubMed]
  62. Yu, Y.; Lu, X. Start-up performance and granular sludge features of an improved external circulating anaerobic reactor for algae-laden water treatment. Saudi J. Biol. Sci. 2017, 24, 1222–1228. [Google Scholar] [CrossRef] [PubMed]
  63. Roy, A.; Sar, P.; Sarkar, J.; Dutta, A.; Sarkar, P.; Gupta, A.; Mohapatra, B.; Pal, S.; Kazy, S.K. Petroleum hydrocarbon rich oil refinery sludge of North-East India harbours anaerobic, fermentative, sulfate-reducing, syntrophic and methanogenic microbial populations. BMC Microbiol. 2018, 18, 151. [Google Scholar] [CrossRef] [PubMed]
  64. Cabral, L.; Giovanella, P.; Pellizzer, E.P.; Teramoto, E.H.; Kiang, C.H.; Sette, L.D. Microbial communities in petroleum-contaminated sites: Structure and metabolisms. Chemosphere 2022, 286, 131752. [Google Scholar] [CrossRef] [PubMed]
  65. Aubé, J.; Senin, P.; Bonin, P.; Pringault, O.; Jeziorski, C.; Bouchez, O.; Klopp, C.; Guyoneaud, R.; Goñi-Urriza, M. Meta-omics Provides Insights into the Impact of Hydrocarbon Contamination on Microbial Mat Functioning. Microb. Ecol. 2020, 80, 286–295. [Google Scholar] [CrossRef] [PubMed]
  66. Kong, L.; Xu, T.; Wang, Z.; Wen, X.; Jiao, Z.; Liu, J. Metagenomic analysis of petroleum biodegradation coupled to specific N-cycling process in oil-contaminated soil. Appl. Soil Ecol. 2024, 193, 105144. [Google Scholar] [CrossRef]
  67. Zhang, T.; Zhang, X.; Liu, Z.; Ou, Y.; Duan, X.; Wu, M. Sensitive indicator microorganisms and C,N-cycle processes in soil with different petroleum hydrocarbon pollution levels. Biochem. Eng. J. 2025, 220, 109752. [Google Scholar] [CrossRef]
  68. Ou, Y.; Wu, M.; Yu, Y.; Liu, Z.; Kang, H.; Hu, M.; Zhang, C.; Chen, X. Influence mechanisms underlying the degradation of petroleum hydrocarbons in response to various nitrogen dosages supplementation through metatranscriptomics analysis. J. Hazard. Mater. 2025, 487, 137074. [Google Scholar] [CrossRef] [PubMed]
  69. Zhao, N.; Hao, D.; Xuji, Z.; Tom, G.; Zuotao, Z.; Nan, Z.; Wang, H. Dissimilatory iron-reducing microorganisms: The phylogeny, physiology, applications and outlook. Crit. Rev. Environ. Sci. Technol. 2025, 55, 73–98. [Google Scholar] [CrossRef]
  70. Scherr, K.E.; Lundaa, T.; Klose, V.; Bochmann, G.; Loibner, A.P. Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation. J. Biotechnol. 2012, 157, 564–572. [Google Scholar] [CrossRef] [PubMed]
  71. Toth, C.R.A.; Gieg, L.M. Time Course-Dependent Methanogenic Crude Oil Biodegradation: Dynamics of Fumarate Addition Metabolites, Biodegradative Genes, and Microbial Community Composition. Front. Microbiol. 2018, 8, 2610. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic diagram of the experimental setup: (1) influent tank; (2) lift pump; (3) Anaerobic Zone 1; (4) Anaerobic Zone 2; (5) Anoxic Zone; (6) Aerobic Zone 1; (7) Aerobic Zone 2; (8) sedimentation zone; (9) recirculation pump; (10) aeration pump; (11) chemical dosing tank.
Figure 1. Schematic diagram of the experimental setup: (1) influent tank; (2) lift pump; (3) Anaerobic Zone 1; (4) Anaerobic Zone 2; (5) Anoxic Zone; (6) Aerobic Zone 1; (7) Aerobic Zone 2; (8) sedimentation zone; (9) recirculation pump; (10) aeration pump; (11) chemical dosing tank.
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Figure 2. Removal performance of oil content, SS, and polymers in the Fe3+-augmented composite bioreactor: (A) overall oil content removal; (B) oil content removal across treatment units; (C) overall SS removal; (D) SS removal across treatment units; (E) overall polymer removal; (F) polymer removal across treatment units; (G) overall NH4+-N removal; (H) NH4+-N removal across treatment units.
Figure 2. Removal performance of oil content, SS, and polymers in the Fe3+-augmented composite bioreactor: (A) overall oil content removal; (B) oil content removal across treatment units; (C) overall SS removal; (D) SS removal across treatment units; (E) overall polymer removal; (F) polymer removal across treatment units; (G) overall NH4+-N removal; (H) NH4+-N removal across treatment units.
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Figure 3. Organic matter removal performance: (A) COD removal; (B) UV254 reduction; (C) GC-MS spectrum of organic pollutants.
Figure 3. Organic matter removal performance: (A) COD removal; (B) UV254 reduction; (C) GC-MS spectrum of organic pollutants.
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Figure 4. Bacterial community composition: (A) Venn diagram; (B) phylum level; (C) genus level.
Figure 4. Bacterial community composition: (A) Venn diagram; (B) phylum level; (C) genus level.
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Figure 5. Archaeal community composition: (A) archaeal distribution; (B) phylum level; (C) genus level.
Figure 5. Archaeal community composition: (A) archaeal distribution; (B) phylum level; (C) genus level.
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Figure 6. Degradation pathways and key enzymes for PHs and nitrogenous pollutants: (A) top 15 KEGG pathways (Level 3) involved in PH degradation; (B) top 15 enzymes involved in PH degradation; (C) top 15 KEGG pathways (Level 3) involved in nitrogen cycling; (D) top 15 enzymes involved in nitrogen cycling; (E) co-occurrence network of the top 30 enzymes based on abundance.
Figure 6. Degradation pathways and key enzymes for PHs and nitrogenous pollutants: (A) top 15 KEGG pathways (Level 3) involved in PH degradation; (B) top 15 enzymes involved in PH degradation; (C) top 15 KEGG pathways (Level 3) involved in nitrogen cycling; (D) top 15 enzymes involved in nitrogen cycling; (E) co-occurrence network of the top 30 enzymes based on abundance.
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Figure 7. Variations in total iron, Fe3+, and Fe2+ concentrations: (A) concentration profiles of total iron, Fe3+, and Fe2+ across bioreactor compartments; (B) Fe3+ consumption and Fe2+ production.
Figure 7. Variations in total iron, Fe3+, and Fe2+ concentrations: (A) concentration profiles of total iron, Fe3+, and Fe2+ across bioreactor compartments; (B) Fe3+ consumption and Fe2+ production.
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Figure 8. Proposed mechanism underlying Fe3+-enhanced degradation of organic pollutants.
Figure 8. Proposed mechanism underlying Fe3+-enhanced degradation of organic pollutants.
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Table 1. Physicochemical properties of the oilfield-produced water.
Table 1. Physicochemical properties of the oilfield-produced water.
ParameterValue
COD (mg/L)492.69 ± 192.03
Oil content (mg/L)129.63 ± 92.57
SS content (mg/L)144.62 ± 44.29
Polymer concentration (mg/L)42.51 ± 13.24
NH4+-N (mg/L)15.17 ± 3.30
Viscosity (mPa∙s)0.70 ± 0.03
pH7.56 ± 0.05
Table 2. Bacterial diversity indices of sludge samples.
Table 2. Bacterial diversity indices of sludge samples.
SampleAceChaoCoverageShannonSimpsonSobs
AN1141614120.99393.510.171156
AN2151615060.99414.160.121259
ANO160716150.99303.590.211283
O1155515570.99334.380.051254
O2109110760.99584.240.04913
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Zhao, Q.; Chen, C.; Chen, Z.; Shan, H.; Liang, J. Enhanced Oilfield-Produced-Water Treatment Using Fe3+-Augmented Composite Bioreactor: Performance and Microbial Community Dynamics. Bioengineering 2025, 12, 784. https://doi.org/10.3390/bioengineering12070784

AMA Style

Zhao Q, Chen C, Chen Z, Shan H, Liang J. Enhanced Oilfield-Produced-Water Treatment Using Fe3+-Augmented Composite Bioreactor: Performance and Microbial Community Dynamics. Bioengineering. 2025; 12(7):784. https://doi.org/10.3390/bioengineering12070784

Chicago/Turabian Style

Zhao, Qiushi, Chunmao Chen, Zhongxi Chen, Hongman Shan, and Jiahao Liang. 2025. "Enhanced Oilfield-Produced-Water Treatment Using Fe3+-Augmented Composite Bioreactor: Performance and Microbial Community Dynamics" Bioengineering 12, no. 7: 784. https://doi.org/10.3390/bioengineering12070784

APA Style

Zhao, Q., Chen, C., Chen, Z., Shan, H., & Liang, J. (2025). Enhanced Oilfield-Produced-Water Treatment Using Fe3+-Augmented Composite Bioreactor: Performance and Microbial Community Dynamics. Bioengineering, 12(7), 784. https://doi.org/10.3390/bioengineering12070784

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