A Biotic Strategy for Enhanced Hexavalent Chromium Removal by Zero-Valent Iron under the Interference of Humic Acid

Abstract

Zero-valent iron (Fe⁰) has been extensively used in hexavalent chromium (Cr(VI)) removal from groundwater, but its treatment suffers from interference of humic acid (HA) and ferrochrome precipitate. In this study, a biotic Fe⁰ system was established to address these problems in Cr(VI) removal from HA-rich groundwater by introducing a combination of heterotrophic and hydrogen-autotrophic microorganisms. Due to the formation of HA-Fe complexes and ferrochrome precipitates on the Fe⁰ surface, the HA-abiotic Fe⁰ system obtained a slight Cr(VI) removal of 10.5%. While in the HA-biotic Fe⁰ system, heterotrophic microbes could effectively eliminate HA through biodegradation and decrease HA-Fe complex generation; autotrophic microbes used H₂ from iron corrosion as electron donors for their metabolism and promoted iron corrosion and active secondary mineral generation (e.g., magnetite and green rust) for Cr(VI) adsorption and reduction. Therefore, a much higher Cr(VI) removal of 84.9% was achieved. Additionally, increasing HA content and extra electron acceptors (e.g., sulfate and nitrate) both boosted Cr(VI) removal, further proving the role of heterotrophic microbes in biodegrading HA for enhanced Cr(VI) elimination. This work presented a feasible strategy to achieve efficient Cr(VI) removal with Fe⁰ by diminishing HA interference and ferrochrome precipitate passivation through the synergistic effect of heterotrophic and hydrogen-autotrophic microorganisms.

1. Introduction

Chromium is widely used in electroplating, leather tanning, and other industries [1]. Due to improper disposal and inadvertent leakage, a substantial amount of chromium has been released and entered groundwater systems [2]. In aquifers, chromium predominantly exists in two stable oxidation states: hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)) [3]. The Cr(VI) species, known for their carcinogenic, mutagenic, and teratogenic properties, manifest high solubility in aqueous solutions and thus are identified as paramount contaminants. In contrast, the Cr(III) species exhibit low solubility and mobility in aquifers and are essential elements for organisms [4]. Therefore, there is an urgent need to remove Cr(VI) by reducing it to Cr(III) in Cr(VI)-contaminated groundwaters.

The Cr(VI) removal methods in practice include chemical precipitation, photocatalysis, and electrochemical treatment, but they often involve secondary contamination, special reaction conditions, and are energy intensive [5,6]. Among various techniques, zero-valent iron (Fe⁰) has been widely used as an efficient material in permeable reactive barriers for Cr(VI) removal from groundwater [7]. In Fe⁰-H₂O systems, Cr(VI) is screened and reduced to Cr(III) with Fe⁰ being oxidized to Fe(II) and Fe(III). The resultant Fe(III) tends to react with Cr(III) to generate sparingly soluble Fe-Cr hydroxides. Passive Fe-Cr hydroxides formed on the Fe⁰ surface serve as a physical barrier limiting electron transfer from the iron core to the surface and ceasing further Fe⁰ corrosion (Equations (1)–(3)) [8].

HCrO₄⁻ + Fe⁰ + 3H₂O → Cr³⁺ + Fe³⁺ + 7OH⁻

(1)

(1-χ)Fe³⁺ + χCr³⁺ + 3H₂O → (Cr_χFe_1-χ)(OH⁻)₃(s) + 3H⁺

(2)

(1-χ)Fe³⁺ + χCr³⁺ + 2H₂O → Cr_χFe_1-χOOH(s) + 3H⁺

(3)

It is reported that incorporating hydrogen-autotrophic microbes into Fe⁰-H₂O systems is an effective strategy to promote Fe⁰ corrosion and biogenic iron mineral formation for enhanced Cr(VI) removal [9]. In biotic Fe⁰ systems, H₂ from Fe⁰ corrosion can serve as electron donors for autotrophic microbes (e.g., denitrifying, sulfate-reducing, and methanogenic bacteria) to reduce Cr(VI) (Equation (4)) [10,11].

Fe⁰ + 2H₂O → H₂↑ + Fe²⁺ + 2OH⁻

(4)

However, Fe⁰ in groundwater remediation inevitably suffers from interferences by coexisting geochemical constituents such as humic acid (HA) [12]. Humic acid is known for its strong binding affinity with Fe³⁺ to form HA-Fe complexes. These complexes readily turn into macroaggregates by association of hardness ions such as calcium [13]. Compared with iron oxides, the macroaggregates with low conductivity and thicker dimensions result in greater blockage of the Fe⁰ surface and stronger inhibitory effect on Cr(VI) removal [14]. The depletion of H₂ kinetically promotes Fe⁰ corrosion and secondary mineral production for Cr(VI) adsorption, reduction, and sequestration. However, under more complex geochemical conditions, HA preferentially occupies reaction sites on Fe⁰ or chelates Fe(II)/Fe(III) with its structural functional groups, such as hydroxyl and carboxyl groups. Therefore, bio-promoted Fe⁰ corrosion first requires breaking through the physical barrier formed by HA-Fe complexes, a task that cannot be accomplished by autotrophic microbes alone. Fortunately, it is reported that heterotrophic microbes such as denitrifying and sulfate-reducing bacteria can utilize HA as a carbon and energy source for their metabolic processes [15,16]. In addition, those microbes can show a reducing ability from the degradation of HA for Cr(VI) reduction [17]. Hence, incorporating heterotrophic microbes into a conventional biotic Fe⁰ system is expected to resolve the HA inhibition problem with an increase in Cr(VI) removal.

Theoretically, the collaboration between Fe⁰ and autotrophic and heterotrophic microbes integrates the inherent unique physicochemical properties of Fe⁰ with the complex biochemical characteristics of both microbial types. This integration has a potential to concurrently address inhibition and passivation issues [18,19]. However, heterotrophic microbes differ in biological behavior from autotrophic microbes. Further research is needed to determine whether a competitive or synergistic relationship exists between hydrogen-autotrophic and heterotrophic microbes when coexisting.

Therefore, this study aims to establish a ternary system combining Fe⁰ with heterotrophic and hydrogen-autotrophic microbes to investigate the removal of Cr(VI) from groundwater rich in humic acid (HA), thereby deepening our understanding of these complex chemical and biological processes. The Cr(VI) removal efficiency in abiotic Fe⁰, HA-abiotic Fe⁰, biotic Fe⁰, and HA-biotic Fe⁰ systems was compared. Solid-phase analysis was used to determine changes on the Fe⁰ surface and secondary mineral formation. Chromium species on the Fe⁰ surface were analyzed to describe the mechanism of chromium removal and immobilization. The impact of HA concentration and additional electron acceptors such as nitrate and sulfate on Cr(VI) removal was also investigated to further explore the role of microbes.

2. Materials and Methods

2.1. Materials and Microbes

Iron powder of 0.15 mm was purchased from Guangzhou Reagent Factory (Guangzhou, China) and directly used without any treatment. Other conventional chemicals such as K₂Cr₂O₇, humic acid, and CaCl₂ were used as received. They were of analytical purity from Aladdin (Shanghai, China).

Anaerobic sludge was taken from a municipal wastewater treatment plant. To obtain Cr(VI)-resistant mixed heterotrophic and hydrogen-autotrophic microbes, the acclimation of a mixed culture was conducted according to a previous study [20]. An anaerobic bioreactor with a volume of 5 L was used for acclimation, containing 5 g L⁻¹ volatile suspended solids (VSS) and 10 g L⁻¹ of granular iron. Influent Cr(VI) concentration was 25 mg L⁻¹, along with a nutrient medium containing (mg L⁻¹) glucose 400, K₂HPO₄ 40, NaNO₃ 300, Na₂SO₄ 300, MgCl₂ 35, NaHCO₃ 500, and trace metals (CoCl₂·5H₂O, CuCl₂·2H₂O, MnCl₂·4H₂O, NiCl₂·6H₂O, NH₄MO₃, and ZnSO₄, each at 0.5 mg L⁻¹). Influent retention time was 3 d. After 6 months of acclimation, when the removal rates of Cr(VI), NO₃⁻-N, and SO₄²⁻ reached 90%, this culture could be harvested. The cultivated sludge was collected, rinsed, and mixed with distilled water to obtain a bacterial suspension for batch experiments.

2.2. Solution Preparation

Chromium stock solution was prepared by dissolving chemicals (K₂Cr₂O₇ 1 g L⁻¹, K₂HPO₄ 5 g L⁻¹, NH₄Cl 5 g L⁻¹, and NaHCO₃ 1 g L⁻¹) into deionized water. Humic acid (HA) stock solution of 1 M was prepared by dissolving HA in deionized water at pH 9 and filtered through a 0.45 µm membrane [21]. The concentration of HA was expressed as dissolved organic carbon (mg L⁻¹ as DOC). Calcium ion stock solution was prepared by dissolving CaCl₂ (100 mM) into deionized water.

Synthetic Cr(VI)-contaminated groundwater was prepared by diluting mixed stock solutions of Cr(VI), HA, and Ca²⁺ to target concentrations with deionized water that was deoxygenated by purging with nitrogen gas for 1 h before usage. The background electrolyte was 5 mM NaCl unless otherwise specified and 0.8 mM Ca²⁺, equivalent to 80 mg L⁻¹ as CaCO₃ hardness, representing moderately hard water. Initial pH was adjusted to 7.0 by 0.1 M NaOH and HCl. No buffer was used.

2.3. Batch Experiment

Batch experiments were conducted in a 300 mL conical flask containing 250 mL of synthetic Cr(VI)-contaminated groundwater and 50 mL headspace, with 10 mg L⁻¹ of Cr(VI) and 20 mg L⁻¹ of HA. The reaction was divided into three systems: (1) abiotic Fe⁰ (Fe⁰ only, including abiotic Fe⁰ and HA-abiotic Fe⁰), (2) microbes (mixed microbes, including microbe and HA-microbe), and (3) biotic Fe⁰ (Fe⁰+ mixed microbes, including biotic Fe⁰ and HA-biotic Fe⁰). The reactors were dosed with 1 g L⁻¹ Fe⁰ and/or 200 mg VSS L⁻¹ mixed microbes under N₂ protection. After sealing with rubber plugs, the reactors were set to react at 50 rpm and 25 °C. At certain time intervals (1–5 d), 3 mL of solution was withdrawn via a long needle and filtered through a 0.45 μm membrane for immediate analysis of Cr(VI), HA, and total Fe; 3 mL of nitrogen was injected into the flask to maintain pressure. The values presented in this study were averaged over triplicate experiments for each.

The effects of HA, nitrate, and sulfate were investigated based on the above benchmark experiment, ranging from 0 to 40 mg L⁻¹ (HA), 56 mg L⁻¹ (NO₃⁻-N), and 384 mg L⁻¹ (SO₄²⁻), respectively.

2.4. Analytical Methods

The concentration of Cr(VI) in solution was measured by the 1,5-diphenylcarbazide colorimetric method at a wavelength of 540 nm (UV-5200, Metash, Shanghai, China). The concentrations of HA, total Fe, and total Cr in solution were determined by a TOC analyzer (TOC-5000A, Shimadzu, Kyoto, Japan) and an atomic absorbance spectrometer (AAS, GGX-600, Beijing, China), respectively. The pH value was measured with a pH meter (PHS-3C, Leici, Shanghai, China).

After remediation, reacted Fe⁰ and unreacted Fe⁰ were freeze-dried for 1 d and collected through magnetic dressing. The above process was completed in an anaerobic chamber. All the collected samples were analyzed by scanning electron microscope/energy-dispersive X-ray (SEM/EDX, ZEISS Merlin, Oberkochen, Germany), X-ray diffraction (XRD, Shimadzu XRD-7000, Tokyo, Japan), and X-ray photoelectron spectroscopy (XPS, Shimadzu Axis Supra, Tokyo, Japan). The valence distribution of chromium was analyzed according to the method in the literature [22]. Detailed methods are provided in Text S1.

2.5. Data Analysis

The removal rate of Cr(VI) was calculated according to Equation (5). The removal data were fitted with zero-, first-, and second-order kinetic models (Equations (6)–(8)).

R (%) = (C₀ − C_t)/C₀ × 100

(5)

C₀ − C_t = k₀t

(6)

−ln(C_t/C₀) = k₁t

(7)

(1/C_t) − (1/C₀) = k₂t

(8)

where C₀ and C_t are concentrations of Cr(VI) before and after reaction (mg L⁻¹); t is reaction time (d); k₀ − k₂ are zero-, first-, and second-order reaction rate constants (mg (L d)⁻¹, d⁻¹, and L (mg d)⁻¹).

3. Results and Discussion

3.1. Characterization

The surface morphology of reacted Fe⁰ is shown in Figure 1. Compared with the smooth surface of unreacted Fe⁰ (Figure 1a), the surface of abiotic Fe⁰ became rough with some attached granular precipitates, which could be iron oxides and Fe-Cr precipitates (Figure 1b). In the presence of HA, a large number of noncrystal aggregates with an apparently greater thickness were observed on the Fe⁰ surface in the HA-abiotic Fe⁰ system. Based on previous studies, these floccules were assigned to HA-Fe complexes [12,23]. They were further confirmed by the observation of higher Fe (86.4%) and Ca (1.6%) than the 82.6% and 1.2% on the Fe⁰ surface without HA in EDX analysis (

Table 1. EDX analysis of unreacted and reacted Fe⁰ samples.

Sample	Element (wt%)
	Fe	O	Cr	C	Si	Ca	Others
Unreacted Fe0	99.4	0.5	ND	0.1	ND	ND	ND
Abiotic Fe0	82.6	7.9	0.8	6.5	0.4	1.2	0.6
HA-abiotic Fe0	86.4	1.5	0.3	9.6	0.3	1.6	0.3
Biotic Fe0	72.8	10.6	3.8	11.0	0.1	0.2	1.5
HA-biotic Fe0	45.6	28.3	6.9	17.1	0.2	0.1	1.8

Note: ND: not detected.

). The formation of HA-Fe complexes blocked the Fe⁰ surface from further corrosion.

When microbes were introduced, flocculent precipitates disappeared with a sharp decrease in the proportion of Ca from 1.6% to 0.1% on the HA-biotic Fe⁰ surface (Figure 1e). Instead, various secondary minerals began to appear. Additionally, it is evident that the HA-biotic Fe⁰ also contained more secondary minerals than the biotic Fe⁰ without HA as shown in Figure 1d. This was attributed to the fact that heterotrophic microbes could effectively degrade HA and clean up HA-Fe complexes. More reaction sites were liberated and available on Fe⁰ to interact with autotrophic microbes promote iron corrosion and biogenic mineral production. Petal-like hexagonal plate minerals and honeycomb minerals were observed (Figure 1e). Their shapes were similar to carbonate green rust, an essential iron mineral with strong adsorption, reduction, and sequestration properties as reported by Legrand et al. [24]. As shown in Table 1, due to the formation of these minerals, the content of Fe showed a significant decrease in the HA-biotic Fe⁰ system from 72.8% to 45.6% with a sharp increase in O and Cr content from 10.6% and 3.8% to 28.3% and 6.9% as compared to the biotic Fe⁰ system. In addition, the Cr on the Fe⁰ surface in the HA-biotic Fe⁰ system was mainly Cr(III) according to the results presented in Figure 2b. This finding indicated severe iron corrosion and secondary mineral formation for Cr(VI) removal under the collaboration of heterotrophic and autotrophic microbes.

The results of XRD analysis are displayed in Figure 3. Only one iron diffraction peak was visible on unreacted Fe⁰. In addition to the strong iron peak, several weak peaks of lepidocrocite and magnetite were observed on abiotic Fe⁰, suggesting slow iron corrosion and mineral generation. Notably, diffraction peaks of iron minerals were hardly seen under the interference of HA due to the generation of HA-Fe complexes. However, the peak intensity of secondary minerals (e.g., lepidolite, magnetite, and green rust) increased on biotic Fe⁰ because of microbial activities. With HA, the intensities of those minerals further increased in the HA-biotic Fe⁰ system due to the clearance of HA-Fe complexes by heterotrophic microbes. Therefore, the collaboration of autotrophic and heterotrophic microbes alleviated HA inhibition and achieved promoted Fe⁰ corrosion for Cr(VI) removal.

3.2. Batch Experiment

Parallel experiments were carried out to investigate the performance of HA-abiotic Fe⁰ and HA-biotic Fe⁰ in Cr(VI) removal (Figure 4). A control system of HA was used to evaluate abiotic Cr(VI) loss with reaction time. The Cr(VI) concentration remained stable and slightly decreased by 1.3% within 5 d, primarily due to the chelation and reduction of Cr(VI) by HA [25]. For abiotic Fe⁰, a low Cr(VI) removal of 20.9% in 5 d was observed, mainly due to passive Fe-Cr hydroxide generation on the Fe⁰ surface (Figure 4a). In the HA-abiotic Fe⁰ system, it exhibited an even lower Cr(VI) removal of only 10.5%, which was inhibited by 50% as compared to the absence of HA. It was attributed to the deposition of HA-Fe complexes on the Fe⁰ surface. This result was further confirmed by a slight increase in total Fe concentration and HA utilization rate from 0.06 and 0.1 mg L⁻¹ in 1 d to 0.1 and 0.3 mg L⁻¹ in 5 d (Figure 1b,c, Figure 4b and Figure S1).

Unlike the abiotic Fe⁰ system, the microbe system exhibited enhanced behaviors in the presence of HA. Its Cr(VI) removal rate increased from 22.8% to 30.5% within 5 d at 20 mg L⁻¹ HA. The corresponding HA concentration decreased by 8.7 mg L⁻¹ since heterotrophic microbes were capable of degrading HA and obtained reductive ability from this process to reduce Cr(VI). Consequently, the introduction of mixed heterotrophic and autotrophic microbes caused a substantial improvement in Cr(VI) removal of 84.9% in the HA-biotic Fe⁰ system, 0.4 and 7.1 times higher than those of the biotic Fe⁰ (59.0%) and HA-abiotic Fe⁰ (10.5%) systems. Heterotrophic microbes transformed HA from an inhibitor into an ideal carbon source and electron donor. The degradation of HA released reaction sites on the Fe⁰ surface for autotrophic microbes. By consumption of H₂ from iron corrosion and CO₂ from HA biodegradation, autotrophic microbes promoted Fe⁰ corrosion and biogenic mineral production for enhanced Cr(VI) adsorption, reduction, and sequestration. Therefore, a significantly enhanced Cr(VI) removal was achieved in the HA-biotic Fe⁰ system. It indicated a synergistic interaction between heterotrophic and autotrophic microbes with Fe⁰ for enhanced Cr(VI) elimination.

As shown in Figure S1 and Figure 4b, the HA-biotic Fe⁰ system exhibited a significantly higher total Fe of 1.0 mg L⁻¹ after 2 d than those of the biotic Fe⁰ (0.2 mg L⁻¹) and HA-abiotic Fe⁰ (0.1 mg L⁻¹) systems with the highest HA utilization of 15.2 mg L⁻¹. The results also suggested a synergistic interaction between two types of microbes for promoted iron corrosion and Cr(VI) removal. Since iron ion was a dominant nutrient to satisfy the growth of heterotrophic microbes [26] and CO₂ produced from HA degradation was promptly consumed by autotrophic microbes, the HA-biotic Fe⁰ system exhibited a higher HA utilization as compared to the pure HA-microbe system. As the reaction progressed, the iron ion concentration dropped to 0.12 mg L⁻¹ due to the accumulation of secondary minerals on the Fe⁰ surface [27]. Additionally, promoted iron corrosion also led to an increase in pH value from 7.0 to 7.4 in the HA-biotic Fe⁰ system (Table S1), which was conducive to iron hydroxide generation and subsequent Cr(VI) sequestration.

The kinetic analysis of Cr(VI) removal was conducted and the results are presented in Table S2. Based on these fitting outcomes, the pseudo second-order kinetic model outperformed other models. It indicated that Cr(VI) removal on the HA-biotic Fe⁰ surface primarily occurred through more efficient and plausible chemisorption. The corresponding reaction rate constant for the HA-biotic Fe⁰ system reached 0.110 L (mg d)⁻¹, 54 times higher than that of the HA-abiotic Fe⁰ system (0.002 L (mg d)⁻¹). This result demonstrated that Cr(VI) removal could be significantly enhanced by introducing mixed heterotrophic and autotrophic microbes into Fe⁰-H₂O systems in the presence of HA.

3.3. Valence Distribution of Chromium

The valence distribution of Cr after reaction was determined and the results are presented in Figure 2. Only 5.6% of Cr(VI) was reduced to Cr(III) on the HA-abiotic Fe⁰ surface, half of that in the abiotic Fe⁰ system without HA. It was attributed to the inhibitory of HA-Fe complexes that impeded electron transfer from Fe⁰ to surface-bonded Cr(VI). In contrast, the HA-microbe system showed an increase in Cr(VI) removal of 29.1% as compared to the microbe system without HA (21.4%). Owing to biological HA degradation by heterotrophic microbes, the reduction of Cr(VI) was improved, resulting in the predominance of Cr(III) in the adsorbed state (16.5% in solid phase and 12.9% in liquid phase) [28].

Obviously, the removal of Cr(VI) by Fe⁰ was substantially enhanced by the introduction of microbes in the synergistic system. Both biotic Fe⁰ and HA-biotic Fe⁰ systems showed higher Cr(VI) removal as compared to the other systems, which had an overall Cr(VI) reduction rate of 36.1% and 84.0%, respectively. The synergistic system exhibited a high degree of immobilized Cr(III) and low degree of soluble Cr(III), accounting for 34.4% and 1.7% in the biotic Fe⁰ system and 79.2% and 4.8% in the HA-biotic Fe⁰ system of the total chromium content, respectively. These results indicated that most of the Cr existed on Fe⁰ in the form of Cr(III), rather than in solution of the synergistic systems. Interestingly, the Cr(III) on HA-biotic Fe⁰ showed a much higher portion of 98.8% as compared to that of biotic Fe⁰ of 60%. It indicated that the HA-biotic Fe⁰ system not only had an increased ability to reduce Cr(VI) by biodegrading HA but also allowed more iron corrosion for Cr(VI) adsorption and reduction.

In conclusion, the HA-biotic Fe⁰ system could significantly enhance the immobilization of Cr(VI) by transforming Cr(VI) in solution to Cr(III) on the Fe⁰ surface and thus decreased the mobility, bioavailability, and toxicity of Cr(VI).

3.4. Effects of Humic Acid Concentration and Common Electron Acceptors

In order to further clarify the contribution of heterotrophic microbes in the HA-biotic Fe⁰ system, the effect of HA concentration was investigated (Figure 5a). As expected, the Cr(VI) removal increased with HA concentration. When the HA concentrations was elevated from 0 to 40 mg L⁻¹, the Cr(VI) removal rate increased from 59.0% to 94.6%. In contrast, a negative effect of HA was observed without microbes in the HA-abiotic Fe⁰ system. It showed a decrease in Cr(VI) removal from 20.9% to 6.9% as the initial HA concentration was increased from 0 to 40 mg L⁻¹ (Figure S3). The opposite results emphasized the crucial role of heterotrophic microbes in degrading HA to enhance Cr(VI) removal. In addition, the utilization rate of HA also increased from 1.5 to 28 mg L⁻¹ with HA concentration from 5 to 40 mg L⁻¹ (Figure 5d), probably due to the enhancement of biological activities of heterotrophic microbes by utilizing HA.

The effect of nitrate and sulfate on Cr(VI) removal in the HA-biotic Fe⁰ system is predicted in Figure 5b,c. The Cr(VI) removal rate was 84.9%, 89.9%, 95.3%, 73.8%, and 59.0% within 5 d when initial NO₃⁻-N concentrations were 0, 7, 14, 28, and 56 mg L⁻¹, respectively. The NO₃⁻-N concentration decreased to 0, 1.1, 3.4, 15.6, and 26.1 mg L⁻¹ after the reaction. Obviously, moderate quantities of NO₃⁻-N (7–14 mg L⁻¹) could serve as an efficient electron acceptor for heterotrophic microbes to metabolize HA and reduce Cr(VI). Meanwhile, the HA utilization rate increased steadily from 15 mg L⁻¹ to 19.5 mg L⁻¹ with increasing NO₃⁻-N concentration as shown in Figure 5b. However, when initial NO₃⁻-N concentrations were further elevated to 28 and 56 mg L⁻¹, the Cr(VI) removal decreased from 84.9% to 73.8% and 59.0% in 5 d. The decrease in Cr(VI) removal at this moment was because excessive nitrate competed with Cr(VI) for electron flow from biological HA degradation and Fe⁰ corrosion. A similar negative effect was observed in a nitrobenzene reduction by biotic Fe⁰. The nitrobenzene reduction rate decreased from 83.6 to 78.1 and 75.2% after a 12 d reaction time with an increase in initial nitrate concentration from 0 to 1 and 5 mmol L⁻¹.

Different from nitrate, sulfate exhibited a constantly positive effect on Cr(VI) removal in the HA-biotic Fe⁰ system. The Cr(VI) removal rate increased from 73.2% to 97% in 3 d as initial SO₄²⁻ concentration was increased from 0 to 384 mg L⁻¹ (Figure 5c). Meanwhile, the SO₄²⁻ concentration decreased from 48, 96, 192, and 384 to 1.5, 3.2, 98.2, and 141.3 mg L⁻¹, respectively. These results also indicated that heterotrophic microbes could utilize sulfate as electron donors to degrade HA and reduce Cr(VI). Theoretically, sulfate would be similar to nitrate as a common electron acceptor, competing with Cr(VI) for electrons [29]. However, the presence of sulfate constantly exhibited a significantly enhanced effect on Cr(VI) removal in the HA-biotic Fe⁰ system. The Cr(VI) removal rate increased from 84.9% to 99.8% with an increase in sulfate concentration from 0 to 384 mg L⁻¹. This result was consistent with the finding obtained by Yan et al. that the Cd(II) removal rate increased from 58.6% to 86.1% as initial sulfate concentration was elevated from 0 to 100 mg L⁻¹ in a biotic Fe⁰ system [30]. It appeared that sulfate promoted the performance of biotic Fe⁰ systems due to the formation of iron sulfide minerals, which not only counteracted the inhibitory effect of SO₄²⁻ but also substantially improved Cr(VI) removal by generating Cr sulfide precipitation.

In summary, the above results further demonstrated the important role of heterotrophic microbes to eliminate HA inhibition in the HA-biotic Fe⁰ system, and the presence of sulfate and an appropriate amount of nitrate accelerated the utilization of HA by heterotrophic microbes and enhanced the effectiveness in Cr(VI) removal.

3.5. Immobilization Mechanism

The results of XPS spectra are illustrated in Figure 6. In the HA-abiotic Fe⁰ system, the iron surface exhibited a high proportion of 41.7% C-O and 6.9% C=O in C 1s and O 1s due to the deposition of HA-Fe complexes on the Fe⁰ surface via hydroxyl and carboxyl groups (Figure 1 and Figure 6a,b) [31]. After the introduction of microbes, there was a significant decrease in the proportions of C-O (19.5%) and C=O (5.7%) on the HA-biotic Fe⁰ surface due to the biodegradation of HA by heterotrophic microbes. Meanwhile, a portion of C-O transformed to 44.6% Fe-O-H, suggesting that ferrous minerals predominantly replaced HA-Fe complexes after their degradation (Figure 4b). In the Fe 2p spectra, the presence of 89% Fe(III) on HA-abiotic Fe⁰ confirmed the formation of HA-Fe(III) species as an insulated barrier hindering electron transfer [32,33]. By utilizing HA and accelerating iron corrosion, both heterotrophic and autotrophic microbes significantly stimulated reactive mineral generation and the Fe(II) proportion increased to 60.4% on the HA-biotic Fe⁰ surface. This evidence corroborated the generation of Fe(II)-bearing minerals, such as magnetite and green rust, as shown in Figure 3. These minerals favored the adsorption and reduction of Cr(VI). In the Cr 2p spectra, similar to the results in Figure 2b, the deconvoluted peaks of HA-abiotic Fe⁰ allocated 43.8% to Cr(VI) and 56.2% to Cr(III), whereas only the characteristic peaks of Cr(III) were observed on the HA-biotic Fe⁰ surface. The synergistic interaction between Fe⁰ and microbes promoted Cr(VI) reduction and Cr(III) distribution into more stable forms on the iron surface, such as Cr_xFe_1-x(OH)₃ and ionic-bonded Cr₂O₃/FeCr₂O₄.

Based on the above discussion, potential pathways to enhance Cr(VI) removal by the combined action of heterotrophic microbes to relieve HA inhibition and hydrogen-autotrophic microbes to promote iron corrosion were hypothesized (Figure 6e): (1) biodegradation of HA by heterotrophic microbes: as a carbon source and an electron acceptor for heterotrophic microbes, HA biodegradation not only renewed reaction sites by eliminating HA-Fe complexes but also stimulated biological activities to reduce Cr(VI); (2) bio-amended iron corrosion by hydrogenotrophic microbes: after the first step of exposing the iron surface, hydrogenotrophic microbes obtained energy by consuming H₂ from Fe⁰ corrosion to reduce Cr(VI) and promoted iron corrosion; (3) biomineralization by microbes: the activities of mixed microbes induced the formation of biominerals, such as green rust. These Fe(II)-containing minerals caused a substantial reduction of adsorbed Cr(VI). Surface-bonded Cr(III) entered the lattice of iron oxides to form Cr-Fe precipitates with low bioavailability and mobility.

4. Conclusions

In the present study, a synergistic Fe⁰ system combined with heterotrophic and hydrogen-autotrophic microbes was established for enhanced Cr(VI) removal under interference of HA. Heterotrophic microbes were employed to degrade humic acid (HA) accumulated on the Fe⁰ surface for hydrogen-autotrophic microbes to promote iron corrosion. This innovative strategy effectively alleviated the inhibitory effects of HA and ferrochrome precipitate. Heterotrophic microbes utilized HA as additional energy to reduce Cr(VI), while autotrophic microbes promoted iron corrosion by consuming H₂ from iron corrosion and generated highly reactive minerals on Fe⁰, such as magnetite and green rust, resulting in a remarkably enhanced Cr(VI) removal in the HA-biotic Fe⁰ system. The valence distribution of Cr in both solid and liquid phases demonstrated that Cr(VI) was transported to the Fe⁰ surface and reduced to Cr(III). The Cr(III) was subsequently immobilized by sequestering into iron oxides to form Fe-Cr hydroxides with low solubility and mobility. Additional HA and common electron acceptors, such as nitrate and sulfate, improved the performance of HA-biotic Fe⁰ in Cr(VI) removal, further emphasizing the crucial role of heterotrophic microbes in alleviating HA inhibition. The results expanded our understanding of the environmental behavior of microbes in the coexistence of Cr(VI), Fe⁰, and HA. This will contribute to comprehending the chemical and biochemical mechanisms involved in actual treatment processes for high-potential heavy-metal-contaminated environments.