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Article

The Effect and Mechanism of AQDS Promoting Anaerobic Cr(VI) Bio-Reduction Under a Sulfate-Rich Environment

by
Zhujun Wang
1,
Liuzhu Zhao
2,
Chunlin Huang
3,
Duyang Yao
1,
Yayi Wang
1 and
Min Wu
1,*
1
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Siping Road, Shanghai 200092, China
2
Shanghai Municipal Engineering Design Institute (Group) Co., Ltd., Zhongshan North Second Road, Shanghai 200092, China
3
Hefei Construction Quality and Safety Supervision Station, Huoqiu Road, Hefei 230061, China
*
Author to whom correspondence should be addressed.
Water 2025, 17(22), 3287; https://doi.org/10.3390/w17223287
Submission received: 22 October 2025 / Revised: 14 November 2025 / Accepted: 15 November 2025 / Published: 18 November 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

Hexavalent chromium (Cr(VI)) is a highly toxic and carcinogenic pollutant commonly found in industrial wastewater. To address the challenge of sulfate inhibition on biological methods for treating chromium-containing wastewater, this study investigated the enhancement effect and mechanism of anthraquinone-2,6-disulfonate (AQDS) on the anaerobic bio-reduction of Cr(VI). At an AQDS dosage of 30 mg/L, Cr(VI) reduction efficiency increased by 7.8-fold compared to the group with only sulfate. AQDS demonstrated remarkable performance of Cr(VI) bio-reduction by reducing intracellular Cr(VI) penetration, lowering reactive oxygen species (ROS) levels, and maintaining optimal NADH/NAD+ ratios. Importantly, AQDS restores Cr(VI) reduction efficiency by directing electron flow toward Cr(VI) reduction through enhanced extracellular electron transfer, thereby mitigating the competitive inhibitory effect of sulfate. It concluded that AQDS effectively enhances Cr(VI) bio-reduction, offering a promising strategy for the environmental remediation of Cr(VI)-contaminated wastewater under sulfate-rich conditions.

1. Introduction

With the development of industrialization, chromium (Cr) has been widely utilized for electroplating, tanning, dyes, and metallurgy, which would form plentiful wastewater containing Cr [1]. For example, the concentration of hexavalent chromium (Cr(VI)) in the electroplating industry typically ranges from 0.43 to 48.7 mg/L, and even reaches up to 200 mg/L in an extreme circumstance [2,3]. In addition, the Cr(VI) contained in waste residue can easily dissolve and enter natural waterbodies with overland runoff. Compared with trivalent chromium (Cr(III)), Cr(VI) has higher toxicity and stronger oxidizing capacity. Meanwhile, it can penetrate cell membranes to induce oxidative stress, gene mutations, apoptosis, and carcinogenesis, threatening ecological security and human health [4]. For this reason, Cr(VI) has been classified as a carcinogen by the World Health Organization, and its maximum level in drinking water is set at 100 μg/L by the United States Environmental Protection Agency [5].
Traditionally, Cr (VI) is removed by transforming a dissolved state into an insoluble state through chemical reduction, electrochemical reduction, or concentrating and then separating through electrodialysis, ion exchange, or activated carbon adsorption [6,7,8]. Despite their widespread adoption, these methods are often accompanied by significant drawbacks, including high operational costs, substantial energy consumption, and the generation of secondary toxic sludge requiring further treatment [9]. In comparison, bio-reduction has attracted intensive attention due to its environmental sustainability and cost-effectiveness in recent years [10,11]. This biological method demonstrates significant practical application potential in treating large volumes of industrial wastewater generated from processes such as electroplating and metal refining [12]. However, the anaerobic biological reduction of Cr(VI) is influenced by various co-existing anions including CO32−, HCO3−, SO42−, and NO3− [13]. Among them, sulfate has a similar structure to chromate, which causes that causes sulfate reduction to compete for electron donors with chromate reduction, resulting in relatively low efficiency and long reaction times for Cr(VI) reduction [14,15]. On the other hand, sulfuric acid is commonly used in processes such as polishing metal surfaces before electroplating, so that industrial wastewater is often heavily polluted with both hexavalent chromium (Cr(VI)) and sulfate [16]. Consequently, sulfate concentrations in electroplating effluents typically range from 200 to 300 mg/L [17]. Kim et al. reported that sulfates, as competing electron acceptors, reduce the number of electrons available for Cr(VI) reduction, thereby inhibiting its effective reduction [18]. Similarly, Jin et al. observed that Cr(VI) reduction was slightly inhibited when the sulfate concentration was 120 mg/L [19]. Therefore, high sulfate concentrations that coexist with Cr(VI) in wastewater have become an important challenge faced by promoting the biological reduction efficiency of Cr(VI).
Recent studies have confirmed that redox mediators have the ability to facilitate electron transfer from intracellular to extracellular environments through the reversible conversion of hydroquinone and quinone [20]. Among them, anthraquinone-2,6-disulfonate (AQDS), as a common humus analog, has been found to mediate effectively the bio-reduction of heavy metals, including Cr(VI) [21,22]. This phenomenon could be explained that an electron transport chain between microbial cells and extracellular metals was established by the reversible conversion between AQDS and AH2QDS (Figure 1). Firstly, electrons released from the oxidation of organic matters are transferred to the electron transport chain attached to the cell membrane. Then, AQDS receives the above electrons to be reduced to produce AH2QDS. Finally, AH2QDS will react with Cr(VI) to generate AQDS and Cr(III) via 10H+ + 3AH2QDS + 2CrO42−→3AQDS + 2Cr3+ + 8H2O [23]. Herein, the Cr(VI) reduction efficiency is significantly improved. In addition, AQDS can decrease the toxicity of metals to protect the microbial structure and function, and thereby maintain the growth and metabolism of microorganisms [24]. However, although the effect of AQDS on the Cr(VI) reduction has been reported, the role of AQDS in complex systems containing high-sulfate concentrations remains poorly understood.
In this study, thus, AQDS was added into the system with anaerobic Cr(VI) bio-reduction under sulfate-rich conditions, aiming to explore how AQDS mediates the competition for electrons between Cr(VI) reduction and sulfate reduction in depth. Firstly, the effect of AQDS concentration on Cr(VI) reduction was investigated. Subsequently, the effect of AQDS on Cr(VI) reduction in sulfur-rich environments was studied. Furthermore, the underlying mechanisms were elucidated by analyzing key intracellular indicators, including total intracellular chromium, reactive oxygen species (ROS) levels, and the NADH/NAD+ ratio. This study is expected to provide novel insights and methods for Cr(VI) removal under complex environmental conditions for future scientific research and engineering applications.

2. Materials and Methods

2.1. Sludge Acclimation

The utilized sludge was collected from the anaerobic digestion tank of a municipal wastewater treatment plant. To eliminate large particulate impurities, it was initially filtered by a sieve with a 1.5 mm aperture and washed three times with deionized water. Then, it was concentrated by discarding a portion of the supernatant. Finally, it was placed in a 4 °C refrigerator for subsequent use. The main properties of the anaerobic sludge are as follows: total COD (TCOD) 22950 ± 250 mg/L, soluble COD (SCOD) 646 ± 7 mg/L, total suspended solids (TSS) 4961 ± 122 mg/L, volatile suspended solids (VSS) 1760 ± 40 mg/L, and pH 8.5 ± 0.1.
The acclimation of functional bacteria responsible for Cr(VI) reduction was carried out in serum bottles. Adding 1 g/L of potassium acetate as a carbon source, 20 mg/L Cr(VI) solution, appropriate anaerobic sludge, and nutrient solution into each bottle. The components and corresponding concentrations of the nutrient solution are shown in Table 1. The pH was controlled at 8.5 with 1 M NaOH or HCl. All bottles were purged with nitrogen for 10 min, sealed with rubber stoppers, and placed in a thermostatic shaker at 35 ± 2 °C and 150 rpm for acclimation. Each acclimation period lasted two weeks. Between two adjacent periods, the supernatant was decanted after 2 h of settling, followed by the supplementation of potassium acetate, Cr(VI) solution, and nutrient solution. The chromium concentration was maintained at 20 mg/L at the beginning of each successive acclimation period. When the reduction rate of Cr(VI) attained 95%, the chromium-reducing bacteria were considered to be successfully acclimated.

2.2. Experimental Procedure

2.2.1. Effect of AQDS on Cr(VI) Bio-Reduction

The experiments were carried out in 6 serum bottles with a volume of 250 mL. They were all filled with 200 mL mixed solution, which included the acclimated sludge, 1 g/L potassium acetate, and 20 mg/L K2Cr2O7. The AQDS concentration was set to 0, 10, 20, 30, 40, and 50 mg/L, respectively. The solution pH value in each bottle was controlled at 8.5 with 1 mol/L of NaOH and 1 mol/L of HCl. The mixture was purged with nitrogen for 10 min and then sealed with rubber stoppers. All bottles were put into a thermostatic shaker at 35 °C and 150 rpm in the dark. The samples were collected at 0, 12, 24, 36, 48, 60, and 72 h to determine the Cr(VI) concentration. All experiments were performed in triplicate. The reduction efficiency and reduction rate of Cr(VI) are calculated as shown in Equations (1) and (2).
C r ( V I )   r e d u c t i o n   e f f i c i e n c y = C 0 − C e C 0
C r ( V I )   r e d u c t i o n   r a t e = C t 1 − C t 2 t 2 − t 1
where C 0 is initial Cr(VI) concentration; C e is final Cr(VI) concentration; and C t i is Cr(VI) concentration at time t i .

2.2.2. Effect of AQDS on the SO42− Interference During the Cr(VI) Bio-Reduction

The experiments were carried out in 4 serum bottles with a volume of 250 mL, and they were named A, B, C, and D. Each bottle was filled with 200 mL mixed solution, which included the acclimated sludge, and 20 mg/L of K2Cr2O7. Bottle A was used as a control without SO42− and AQDS. Bottle B was added with 200 mg/L of SO42−. Bottle C was added with 30 mg/L of AQDS. Bottle D was added with 30 mg/L of AQDS and 200 mg/L of SO42−. To measure the Cr(VI) and SO42− concentration, the samples were collected at 0, 12, 24, 36, 48, 60, and 72 h. All experiments were performed in triplicate.

2.3. Chemical and Statistical Analysis

In the bio-reduction systems, the supernatant was sampled by using a 10 mL syringe periodically, and then instantly filtered through single-use Millipore filters with 0.45 μm pore size. According to the standard methods, pH, TCOD, SCOD, VSS, and TSS were analyzed [25]. The Cr(VI) concentration was determined by the diphenyl carbazide method using a UV/Vis spectrophotometer (UV-2550, SHIMADZU, Kyoto, Japan) at 540 nm [26]. After the samples were digested in an autoclave (DSX-302, SHENAN, Shenyang, China) for 30 min, the total Cr concentration was determined using ICP-MS (ICPMS7700, Agilent, Santa Clara, CA, USA). The determination of intracellular total Cr requires a pretreatment step before the digestion described above: A 30 mL sample of the sludge suspension was taken from the reactor and placed in a centrifuge tube, which was then centrifuged at 1600 rpm for 5 min. The supernatant was decanted, and the remaining sludge was resuspended in 30 mL of washing solution (1 mM EDTA, 0.1 M NaCl, pH 7.0). The suspension was then agitated at 150 rpm for 30 min to remove Cr adsorbed onto the cell surface. This was followed by centrifugation at 1600 rpm for 5 min, and the supernatant was discarded. This washing procedure was repeated once. Finally, the resulting sludge pellet was quantitatively transferred to an autoclave using ultrapure water for digestion [27,28]. The NADH and NAD+ levels were determined following the method described by Su et al. [29]. Intracellular ROS assay was performed according to Limbath et al. [30]. Data significance was evaluated using analysis of variance (ANOVA), with p < 0.05 considered statistically significant.

3. Results and Discussion

3.1. The Effect of Strengthening Cr(VI) Reduction by AQDS

AQDS plays a crucial role during the bio-reduction of Cr(VI) because it can accelerate the electron transfer between microorganisms and Cr(VI). The effect of AQDS concentration on bio-reduction of Cr(VI) is shown in Figure 2a. It was found that the final Cr(VI) concentration decreased significantly with the presence of AQDS. When AQDS concentrations were 0, 10, 20, 30, 40, and 50 mg/L, the Cr(VI) concentrations were 9.68, 5.96, 1.92, 0, 0.76, and 7.52 mg/L, respectively, after 72 h. The corresponding Cr(VI) reduction efficiencies were 51.6%, 70.2%, 90.4%, 100%, 96.2%, and 62.4%, respectively. Clearly, the Cr(VI) reduction efficiency was the highest when the AQDS concentration was 30 mg/L. At this moment, Cr(VI) was reduced completely, which increased by 93.80% compared with the control group. However, there was a decline in the efficiency of Cr(VI) reduction when AQDS concentration exceeded 30 mg/L. This might be because high concentrations of AQDS exhibited toxic effects on microorganisms, inhibiting cellular metabolism and growth [31]. For instance, Nevin et al. demonstrated that the excessive AQDS significantly suppressed Fe(III) reduction [32]. Herein, excess AQDS fails to fulfill its electron transport function [33,34]. In addition, AQDS itself had a negative impact on soil ecology and aquatic organisms, so that achieving pollutant removal at a relatively low AQDS concentration was a sustainable strategy. Therefore, the optimum AQDS concentration was chosen to be 30 mg/L.
The effect of AQDS on the average reduction rates of Cr(VI) at the time intervals of 0~12 h, 12~24 h, 24~36 h, 36~48 h,48~60 h, and 60~72 h is depicted in Figure 2b. It was found that the average reduction rate of Cr(VI) decreased with increasing AQDS concentration at 0~12 h, suggesting a delay during the anaerobic Cr(VI) bio-reduction with the presence of AQDS. This might be explained that the microorganisms in sludge required an adaptive process after adding AQDS, because exogenous quinones might have cytotoxicity to influence cell viability [35]. After 12 h, the average reduction rate of Cr(VI) in the group with AQDS was generally higher than that in the group without AQDS, which was related to the fact that AQDS can act as a redox mediator to accelerate electron transfer between microorganisms and Cr(VI) [36]. Similarly to the variation of Cr(VI) reduction efficiency, the average reduction rate of Cr(VI) increased with the AQDS concentration increasing from 0 to 30 mg/L, and decreased with the AQDS concentration increasing from 30 to 50 mg/L. When the AQDS concentration was 30 mg/L, the average reduction rates of Cr(VI) were 2.74, 6.12, and 3.20 times that of the control group without AQDS at 24~36 h, 36~48 h, and 48~60 h, respectively. Subsequently, Cr(VI) was reduced completely at 60~72 h, causing the average reduction rate to drop sharply.

3.2. The Effect of AQDS on Chromium Reduction in Sulfate-Rich Environments

It was seen from Figure 3a that adding 200 mg/L sulfate impeded the bio-reduction of Cr(VI), leading to a notable decrease in the reduction efficiency of Cr(VI) from 64.73% to 12.65% after 72 h compared to the control group. The inhibition could be attributed to the structural similarity between CrO42− and SO42− so that sulfate acted as a competitive electron acceptor during the bio-reduction of Cr(VI) [14,37]. Previous research has established that approximately 63% of the electrons generated from organic oxidation were used to reduce sulfate in the presence of sulfate and chromate [18]. In addition, a continuous decline in sulfate content over time was observed in the control group (Figure 3c), reaching approximately half of the initial level after 72 h. It suggested that sulfate reduction gradually became the dominant reaction, and Cr (VI) reduction was hindered.
Considering that the total amount of Cr(VI) reduction in the experimental group with SO42− added was relatively low, the changes in the content of total intracellular chromium during the chromium reduction process in the other three groups were only investigated, and the results are depicted in Figure 3b. For the control group without AQDS, Cr(VI) was the first to enter cells by active transport and then biologically reduced in the cells. At 0~24 h, a large amount of Cr(VI) entered cells, aiming at preparing for its biological reduction, leading to an increase in total chromium content within cells [38]. After 24 h, the Cr(VI) that had not been reduced was excreted outside the cells, leading to a gradual decrease in total chromium content within cells. For the two experimental groups containing AQDS, the extracellular Cr(VI) reduction was enhanced, so the intracellular Cr(VI) reduction was relatively decreased, causing the Cr(VI) content entering the cells to decrease [39]. Therefore, compared with the control group, the total chromium content within cells clearly decreased. On the other hand, the total chromium content within cells increased from 0 to 24 h and decreased from 24 to 72 h in the presence of AQDS. This can be explained by the following reasons. At the initial reaction stage from 0 to 24 h, the microorganisms in sludge required an adaptive process after adding AQDS, and most of Cr(VI) entered cells by active transport and was biologically reduced, leading to an increase in total chromium content within cells. After 24 h, AQDS exhibited the role of the redox mediator and enhanced the extracellular Cr(VI) reduction, causing the Cr(VI) content entering the cells to decrease. Moreover, the extracellular Cr(III) generated by Cr(VI) reduction cannot be taken up by cells, and the unreduced Cr(VI) in the cells would be excreted, leading to a decrease in total chromium content within cells [40]. For the group with AQDS and sulfate, the total chromium content within cells was slightly higher compared with the group with AQDS. This was because sulfate reduction might receive the electrons released from the oxidation of AH2QDS, interfering with the extracellular Cr(VI) reduction. So, some Cr(VI) entered the cells and was then biologically reduced. The Cr(VI) entered the cell can be reduced in the cytoplasm, mitochondria, and nucleus. The formed Cr(III) will bind to proteins to form stable complexes, which remain intracellular in the cells [41]. So, there is still total chromium within cells at 72 h.
AQDS can act as an electron mediator to enhance electron transfer for the extracellular reduction of Cr(VI), resulting in a higher Cr(VI) removal rate [42]. Due to its electron transfer ability, AQDS can alleviate the inhibitory effect of sulfate. In bottle B, to which only sulfate was added, the Cr(VI) concentration at 72 h was 17.47 mg/L, and the Cr(VI) reduction efficiency was only 12.65% due to the inhibitory effect of sulfate. However, in bottle D, which contained both AQDS and sulfate, the Cr(VI) concentration at 72 h was 0.37 mg/L, and the Cr(VI) reduction efficiency increased to 98.15% (Figure 3a). It can likely be attributed to the fact that the addition of AQDS enabled more intracellular electrons to be transferred extracellularly for the reduction of hexavalent chromium instead of sulfate. As indicated by some studies, when sulfate-reducing bacteria reduce sulfate, the relevant enzyme systems are located within the cells [14,17]. As depicted in Figure 3c, the sulfate concentration exhibited a notable decline without AQDS, but only a marginal decrease of 19.73% was observed at 72 h in the presence of AQDS. The previous studies indicated that the intensely humified dissolved organic matter (DOM) and AQDS had an inhibitory effect on sulfate-reducing bacteria (SRB), which potentially contributed to the observed decrease in the sulfate reduction rate [36,43,44]. Recently, Xu et al. discovered that AQDS effectively inhibited the sulfate reduction process in simulated anaerobic bioreactors. This phenomenon was attributed to its ability to reduce the activity of key sulfate-reducing enzymes (Sat, Apr, and Dsr), decrease the relative abundance of SRB such as Desulfovirga and Desulfosporosinus, and downregulate the expression of genes associated with sulfate reduction [45]. Therefore, AQDS may mitigate the inhibitory effect of SO42− on anaerobic Cr(VI) bio-reduction by suppressing SRB activity and modifying electron transport pathways. Although the observed decrease in sulfate consumption (Figure 3c) and supporting literature provide strong circumstantial evidence for this mechanism, direct molecular verification of SRB abundance or specific enzyme activity remains a promising direction for future research to build upon the current scope.

3.3. Effects of AQDS and SO42− on ROS

Under the stress of heavy metals, ROS generated from cellular activities can damage cell membranes, proteins, and DNA, which is a crucial factor for cellular damage [46]. As shown in Figure 4, the changes in intracellular ROS levels clearly indicated that the relative abundance of intracellular ROS in the presence of AQDS is consistently lower than that in the control group. Specifically, at 24 h, 48 h, and 72 h, the ROS levels in the control group were 1.16, 5.71, and 57.1 times higher than those in the bottle with AQDS, respectively. In addition, the ROS level in the bottle with AQDS was nearly undetectable at the end of the reaction. AQDS significantly reduced the Cr(VI)-induced ROS levels, thereby protecting cell activity. At this moment, the addition of AQDS led to a significantly higher rate of Cr(VI) reduction. This phenomenon might be explained that AQDS induced a fundamental transition from periplasmic Cr(VI) reduction to extracellular Cr(VI) reduction, greatly reducing the opportunity for Cr(VI) to enter the cell, and thus reducing the generation of ROS induced by Cr(VI) (Figure 3) [24,47]. In previous research, it has been discovered that AQDS has the ability to modify the chemical characteristics of the cell surface. Specifically, it can regulate the coordination environment of outer membrane proteins, thereby exerting an influence on the electron transfer process [24]. In the current study, such alterations in the cell surface chemistry might endow the cells with enhanced adaptability in the face of heavy metal stress. For instance, when Cr(VI) is present, AQDS may interact with specific proteins on the cell surface, which could modify the electron transfer route. As a result, electrons are more prone to participate in the reduction of Cr(VI) outside the cell.
Additionally, although the relative amount of ROS in the presence of both SO42− and AQDS was slightly higher than that when only AQDS was added within the initial 24 h, it remained lower compared to the blank control. This phenomenon can be attributed to the oxidative stress induced by AQDS on SRB, which triggers membrane depolarization and increases cell membrane permeability [43]. In the presence of sulfate, prior research indicates that the activity of SRB could be influenced [14]. The oxidative stress induced by AQDS on SRB might also disrupt the sulfate reduction process (Figure 3c). At the beginning of the reaction, the impact of AQDS on SRB may cause a temporary shift in the intracellular redox balance, leading to a slight increase in ROS generation, yet remaining lower than that of the control group. As the reaction progresses, the intracellular metabolic processes adapt to this alteration. Concurrently, the reduction of Cr(VI) and other potential detoxification mechanisms act in concert to gradually decrease the ROS level to a comparable level as when only AQDS is added. The significant decrease in intracellular ROS content after 24 h can be attributed to the conversion of intracellular Cr(VI) to Cr(III) or the efflux of Cr(VI) [24]. Therefore, AQDS can alleviate the oxidative stress induced by Cr(VI) and significantly restore the activity of bacterial cells, enabling them to perform Cr(VI) reduction more efficiently.
In summary, AQDS significantly reduces the level of ROS, allowing bacteria to maintain their activity and perform efficient reduction reactions under high-toxicity Cr(VI) exposure. The role of AQDS in the Cr(VI) reduction process is not limited to electron transfer but also has the unique function of reducing oxidative stress. It effectively reduces the level of ROS by transferring the Cr(VI) reduction reaction outside the cell, while restoring the activity of bacteria and enhancing the reduction effect of Cr(VI) [48]. The reduction of ROS is of great significance for the long-term survival of bacteria and the sustainable bioremediation of heavy metal pollution, further proving that AQDS can reduce intracellular oxidative stress and improve the detoxification efficiency of bacteria by changing the detoxification pathway.

3.4. Effects of AQDS and SO42− on NADH

The NADH can serve as an electron carrier to facilitate the transfer of reducing equivalents between redox reactions that take place inside the cell and play a crucial role in maintaining the cellular antioxidant defenses [49]. It was displayed in Figure 5a that the NADH concentration in the bottle with AQDS exhibited a lower level than that without AQDS, irrespective of the presence of SO42−. Specifically, both the bottle with AQDS and the bottle with AQDS and SO42− exhibited approximately a 50% decrease in NADH concentration within 72 h. It is essential to maintain the equilibrium between oxidation and reduction rates of NADH/NAD+ to preserve numerous fundamental cellular processes [50]. As depicted in Figure 5b, AQDS caused a decrease in the NADH/NAD+ ratio. This phenomenon can be attributed to the quinone moiety of AQDS, which acts as an electron acceptor from NADH, leading to its conversion into NAD+ and subsequently reducing the concentration of NADH. Meanwhile, the NADH consumption levels in the group with AQDS and the group with AQDS and sulfate were basically the same. This may be attributed to the coupled effects of SO42− inhibitory action on Cr(VI) bioreduction and the competitive inhibition of SO42− reduction by AQDS, directing electrons primarily toward AQDS reduction rather than SO42− and Cr(VI) reduction. Consequently, Cr(VI) removal was predominantly achieved through AQDS-mediated chemical reduction pathways. It is concluded from these results that AQDS can serve as a redox mediator to accept electrons from NADH and transfer them to extracellular Cr(VI), thereby facilitating the reduction of extracellular Cr(VI) and reducing intracellular chromium accumulation. Furthermore, this process enables NADH regeneration for an appropriate NADH/NAD+ ratio in chromium-reducing bacteria, thereby enhancing the anaerobic bio-reduction of Cr(VI).

3.5. Possible Mechanism and Limitations

In this study, AQDS significantly enhanced the biological reduction efficiency of Cr(VI), maintaining good performance even under sulfate-rich conditions. Its enhancement mechanism primarily involves two aspects. Firstly, AQDS acts as an efficient redox mediator, promoting the extracellular electron transfer process. It captures electrons generated from microbial metabolism and directly reduces Cr(VI) to Cr(III) extracellularly, thereby reducing the transport of Cr(VI) into the cell. This decreases intracellular chromium accumulation and ROS levels, effectively alleviating toxic stress. Secondly, the addition of AQDS inhibited the sulfate reduction reaction, which may be attributed to its role as an electron acceptor in the electron transport chain. Herein, AQDS can compete with organic matters with SRB, and has an inhibitory effect on the activities of some SRB, thereby diverting more electron flow towards the Cr(VI) reduction pathway. Even so, the promoting effect of AQDS on Cr(VI) reduction was dose-dependent, and excessive concentrations (>30 mg/L) exhibited an inhibitory effect, presumably due to its own biotoxicity. On the other hand, the Cr(VI) concentration was set at 20 mg/L in this study, so the applicability of AQDS under higher Cr(VI) concentrations requires further verification. Therefore, in the future, the issues worth paying attention to include the optimization of AQDS dosing strategy, the exploration of the mechanisms of the interaction between chromium-reducing bacteria and SRB, and the evaluation of the long-term operational stability and economic feasibility in high-load treatment systems.

4. Conclusions

The anaerobic reduction efficiency of Cr(VI) was enhanced significantly by AQDS, which not only served as a redox mediator to enhance electron transfer but also mitigated the inhibitory effects of sulfate on Cr(VI) reduction. Under a sulfate-rich environment (200 mg/L SO42−), the Cr(VI) reduction efficiency was improved from 12.65% to 98.15% (7.8 times) when the initial AQDS concentration was 30 mg/L. In addition, the addition of AQDS significantly decreased intracellular ROS levels and maintained the NADH/NAD+ ratio, thereby enhancing Cr(VI) reduction efficiency. Furthermore, to develop its practical application, the long-term stability of Cr(VI) reduction in the presence of AQDS in continuous-flow bioreactors such as UASB should be studied, and the interaction between AQDS conversion, sulfate reduction, and Cr(VI) reduction needs to be elucidated from the perspective of molecular biology in future work.

Author Contributions

Writing—original draft preparation, Z.W.; supervision, L.Z.; resources, C.H.; methodology, D.Y.; Conceptualization, Y.W.; writing—review and editing, M.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Key Research and Development Program of China, grant number 2021YFC3200604.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

Author Liuzhu Zhao was employed by Shanghai Municipal Engineering Design Institute. Author Chunlin Huang was employed by Hefei Construction Quality and Safety Supervision Station. 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.

Abbreviations

The following abbreviations are used in this manuscript:
AQDSanthraquinone-2,6-disulfonate
ROSreactive oxygen species
SRBsulfate-reducing bacteria
TCODTotal Chemical Oxygen Demand
SCODSoluble Chemical Oxygen Demand
TSSTotal Suspended Solids
VSSVolatile Suspended Solids
NADHNicotinamide Adenine Dinucleotide (reduced form)
NAD+Nicotinamide Adenine Dinucleotide (oxidized form)

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Water 17 03287 g001
Figure 1. The mechanism by which AQDS stimulates Cr(VI) bio-reduction.
Figure 1. The mechanism by which AQDS stimulates Cr(VI) bio-reduction.
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Figure 2. (a) Effect of AQDS concentration on Cr(VI) concentration and (b) the Cr(VI) reduction rate in different time intervals in the supernatant of anaerobic sludge.
Figure 2. (a) Effect of AQDS concentration on Cr(VI) concentration and (b) the Cr(VI) reduction rate in different time intervals in the supernatant of anaerobic sludge.
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Figure 3. (a) Effect of sulfate on reduction of Cr(VI) promoted by AQDS, (b) AQDS and sulfate on total chromium in cells and (c) AQDS on sulfate content in reduction of Cr(VI) by anaerobic sludge (Bottle A: control group without SO42− and AQDS; Bottle B: 200 mg/L SO42−; Bottle C: 30 mg/L AQDS; Bottle D: 30 mg/L AQDS and 200 mg/L SO42−).
Figure 3. (a) Effect of sulfate on reduction of Cr(VI) promoted by AQDS, (b) AQDS and sulfate on total chromium in cells and (c) AQDS on sulfate content in reduction of Cr(VI) by anaerobic sludge (Bottle A: control group without SO42− and AQDS; Bottle B: 200 mg/L SO42−; Bottle C: 30 mg/L AQDS; Bottle D: 30 mg/L AQDS and 200 mg/L SO42−).
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Figure 4. Effects of AQDS and sulfate on intracellular ROS during anaerobic reduction of Cr(VI).
Figure 4. Effects of AQDS and sulfate on intracellular ROS during anaerobic reduction of Cr(VI).
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Figure 5. Effects of AQDS and sulfate on NADH (a) and NADH/NAD+ (b).
Figure 5. Effects of AQDS and sulfate on NADH (a) and NADH/NAD+ (b).
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Table 1. The components of the nutrient solution.
Table 1. The components of the nutrient solution.
ComponentConcentration (mg/L)
Basic MaterialsK2HPO4·3H2O750
NaH2PO4·H2O400
NH4H2PO4250
MgCl2·6H2O83
CaCl2·2H2O10
Trace MaterialsFeCl2·4H2O2
MnCl2·4H2O0.5
ZnCl2·7H2O0.05
AlCl3·6H2O0.09
CuCl2·2H2O0.03
CoCl2·6H2O2
H3BO30.05
NiCl2·6H2O0.05
Na2MoO4·2H2O0.03
NaSeO3·5H2O0.05
Na2WO4·2H2O0.05
Na2EDTA0.3
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Wang, Z.; Zhao, L.; Huang, C.; Yao, D.; Wang, Y.; Wu, M. The Effect and Mechanism of AQDS Promoting Anaerobic Cr(VI) Bio-Reduction Under a Sulfate-Rich Environment. Water 2025, 17, 3287. https://doi.org/10.3390/w17223287

AMA Style

Wang Z, Zhao L, Huang C, Yao D, Wang Y, Wu M. The Effect and Mechanism of AQDS Promoting Anaerobic Cr(VI) Bio-Reduction Under a Sulfate-Rich Environment. Water. 2025; 17(22):3287. https://doi.org/10.3390/w17223287

Chicago/Turabian Style

Wang, Zhujun, Liuzhu Zhao, Chunlin Huang, Duyang Yao, Yayi Wang, and Min Wu. 2025. "The Effect and Mechanism of AQDS Promoting Anaerobic Cr(VI) Bio-Reduction Under a Sulfate-Rich Environment" Water 17, no. 22: 3287. https://doi.org/10.3390/w17223287

APA Style

Wang, Z., Zhao, L., Huang, C., Yao, D., Wang, Y., & Wu, M. (2025). The Effect and Mechanism of AQDS Promoting Anaerobic Cr(VI) Bio-Reduction Under a Sulfate-Rich Environment. Water, 17(22), 3287. https://doi.org/10.3390/w17223287

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