Mechanism of extracellular electron transport and reactive oxygen mediated Sb(III) oxidation by Klebsiella aerogenes HC10.

Microbial oxidation and the mechanism of Sb(III) are key governing elements in biogeochemical cycling. A novel Sb oxidizing bacterium, Klebsiella aerogenes HC10, was attracted early and revealed that extracellular metabolites were the main fractions driving Sb oxidation. However, linkages between the extracellular metabolite driven Sb oxidation process and mechanism remain elusive. Here, model phenolic and quinone compounds, i.e., anthraquinone-2,6-disulfonate (AQDS) and hydroquinone (HYD), representing extracellular oxidants secreted by K. aerogenes HC10, were chosen to further study the Sb(III) oxidation mechanism. N2 purging and free radical quenching showed that oxygen-induced oxidation accounted for 36.78% of Sb(III) in the metabolite reaction system, while hydroxyl free radicals (·OH) accounted for 15.52%. ·OH and H2O2 are the main driving factors for Sb oxidation. Radical quenching, methanol purification and electron paramagnetic resonance (EPR) analysis revealed that ·OH, superoxide radical (O2•-) and semiquinone (SQ-•) were reactive intermediates of the phenolic induced oxidation process. Phenolic-induced ROS are one of the main oxidants in metabolites. Cyclic voltammetry (CV) showed that electron transfer of quinone also mediated Sb(III) oxidation. Part of Sb(V) was scavenged by the formation of the secondary Sb(V)-bearing mineral mopungite [NaSb(OH)6] in the incubation system. Our study demonstrates the microbial role of oxidation detoxification and mineralization of Sb and provides scientific references for the biochemical remediation of Sb-contaminated soil.

Enhancing the nitrogen removal of anammox sludge by setting up novel redox mediators-mediated anammox process.

Anaerobic ammonium oxidizing (anammox) bacteria have been proven weak-electroactive. However, the impact of exogenous anthraquinone-2,6-disulfonate (AQDS) on the anammox activity, although it usually plays essential roles in the life activities of many other electroactive microorganisms, is still unknown. Therefore, this study further explored the influences of AQDS on the anammox activity and the interaction mechanism with anammox bacteria, as well as the behaviors of NH4+, NO2-, and NO3-. The results showed that exogenous AQDS increased the ammonium and total nitrogen removal rates by 12.8% and 10.7%, respectively. Interestingly, the conversion from NO2- to NO3- was significantly reduced after adding AQDS, resulting in a 40.1% reduction in NO3- production of anammox process. In this study, we found for the first time that anammox bacteria could not only carry out the conventional anammox process but also perform a weak redox mediator-mediated anammox process, which could achieve the 1:1 consumption of NH4+ and NO2-. The redox mediator-mediated anammox process was related to an endogenous redox mediator (ERM) synthesized and secreted by anammox bacteria, whose redox midpoint potential was around -0.26 V (vs. Ag/AgCl). After adding AQDS, not only the ERM-mediated anammox process was enhanced, but also two novel redox mediator-mediated anammox processes were introduced, including the AQDS-mediated anammox process and ERM-AQDS-mediated anammox process. These three redox mediator-mediated anammox processes significantly improved the nitrogen removal performance of anammox bacteria and reduced energy consumption. These findings will help reduce the dependence of anammox technology on NO2-, reduce the cost of subsequent treatment of NO3-, and provide new visions for optimizing and applying anammox technology.

The dual effect of disodium anthraquinone-2,6-disulfonate (AQDS) on the Cr(VI) removal by biochar: The enhanced electron transfer and the inhibited adsorption.

Due to large specific surface area, abundant surface functional groups, and stable chemical structure, biochar has been widely used in many environmental fields, including the remediation of Cr pollution. Alternatively, electrochemically active organic matter (e-OM), which is prevalent in both natural environments and industrial wastewater, exerts an inevitable influence on the mechanisms underlying Cr(VI) removal by biochar. The synergistic interplay between biochar and e-OM in the context of Cr(VI) remediation remains to be fully elucidated. In this study, disodium anthraquinone-2,6-disulfonate (AQDS) was used as a model for e-OM, characterized by its quinone group's ability to either donate or accept electrons. We found that AQDS sped up the Cr(VI) removal process, but the enhancement effect decreased with the increase in pyrolysis temperature. With the addition of AQDS, the removal amount of Cr(VI) by BC300 and BC600 increased by 160.0% and 49.5%, respectively. AQDS could release more electrons trapped in the lower temperature biochar samples (BC300 and BC600) for Cr(VI) reduction. However, AQDS inhibited the Cr(VI) removal by BC900 due to the adsorption of AQDS on biochar surface. In the presence of the small molecule carbon source lactate, more AQDS was adsorbed onto the biochar surface. This led to an inhibition of the electron transfer between biochar and Cr(VI), resulting in an inhibitory effect. This study has elucidated the electron transfer mechanism involved in the removal of Cr(VI) by biochar, particularly in conjunction with e-OM. Furthermore, it would augment the efficacy of biochar in applications targeting the removal of heavy metals.

Arsenic release from microbial reduction of scorodite in the presence of electron shuttle in flooded soil.

Scorodite (FeAsO4·H2O) is a common arsenic-bearing (As-bearing) iron mineral in near-surface environments that could immobilize or store As in a bound state. In flooded soils, microbe induced Fe(III) or As(V) reduction can increase the mobility and bioavailability of As. Additionally, humic substances can act as electron shuttles to promote this process. The dynamics of As release and diversity of putative As(V)-reducing bacteria during scorodite reduction have yet to be investigated in detail in flooded soils. Here, the microbial reductive dissolution of scorodite was conducted in an flooded soil in the presence of anthraquinone-2,6-disulfonate (AQDS). Anaeromyxobacter, Dechloromonas, Geothrix, Geobacter, Ideonella, and Zoogloea were found to be the dominant indigenous bacteria during Fe(III) and As(V) reduction. AQDS increased the relative abundance of dominant species, but did not change the diversity and microbial community of the systems with scorodite. Among these bacteria, Geobacter exhibited the greatest increase and was the dominant Fe(III)- and As(V)-reducing bacteria during the incubation with AQDS and scorodite. AQDS promoted both Fe(III) and As(V) reduction, and over 80% of released As(V) was microbially transformed to As(III). The increases in the abundance of arrA gene and putative arrA sequences of Geobacter were higher with AQDS than without AQDS. As a result, the addition of AQDS promoted microbial Fe(III) and As(V) release and reduction from As-bearing iron minerals into the environment. These results contribute to exploration of the transformation of As from As-bearing iron minerals under anaerobic conditions, thus providing insights into the bioremediation of As-contaminated soil.

Mixture of Anthraquinone Sulfo-Derivatives as an Inexpensive Organic Flow Battery Negolyte: Optimization of Battery Cell.

Anthraquinone-2,7-disulfonic acid (2,7-AQDS) is a promising organic compound, which is considered as a negolyte for redox flow batteries as well as for other applications. In this work we carried out a well-known reaction of anthraquinone sulfonation to synthesize 2,7-AQDS in mixture with other sulfo-derivatives, namely 2,6-AQDS and 2-AQS. Redox behavior of this mixture was evaluated with cyclic voltammetry and was almost identical to 2,7-AQDS. Mixture was then assessed as a potential negolyte of anthraquinone-bromine redox flow battery. After adjusting membrane-electrode assembly composition (membrane material and flow field)), the cell demonstrated peak power density of 335 mW cm-2 (at SOC 90%) and capacity utilization, capacity retention and energy efficiency of 87.9, 99.6 and 64.2%, respectively. These values are almost identical or even higher than similar values for flow battery with 2,7-AQDS as a negolyte, while the price of mixture is significantly lower. Therefore, this work unveils the promising possibility of using a mixture of crude sulfonated anthraquinone derivatives mixture as an inexpensive negolyte of RFB.

AQDS Activates Extracellular Synergistic Biodetoxification of Copper and Selenite via Altering the Coordination Environment of Outer-Membrane Proteins.

The biotransformation of heavy metals in the environment is usually affected by co-existing pollutants like selenium (Se), which may lower the ecotoxicity of heavy metals, but the underlying mechanisms remain unclear. Here, we shed light on the pathways of copper (Cu2+) and selenite (SeO32-) synergistic biodetoxification by Shewanella oneidensis MR-1 and illustrate how such processes are affected by anthraquinone-2,6-disulfonate (AQDS), an analogue of humic substances. We observed the formation of copper selenide nanoparticles (Cu2-xSe) from synergistic detoxification of Cu2+ and SeO32- in the periplasm. Interestingly, adding AQDS triggered a fundamental transition from periplasmic to extracellular reaction, enabling 14.7-fold faster Cu2+ biodetoxification (via mediated electron transfer) and 11.4-fold faster SeO32- detoxification (via direct electron transfer). This is mainly attributed to the slightly raised redox potential of the heme center of AQDS-coordinated outer-membrane proteins that accelerates electron efflux from the cells. Our work offers a fundamental understanding of the synergistic detoxification of heavy metals and Se in a complicated environmental matrix and unveils an unexpected role of AQDS beyond electron mediation, which may guide the development of more efficient environmental remediation and resource recovery biotechnologies.

The combined enhancement of RL, nZVI and AQDS on the microbial anaerobic-aerobic degradation of PAHs in soil.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous persistent organic pollutants in soil, which have carcinogenic, teratogenic and mutagenic hazards. The effects of rhamnolipid (RL), nano zero-valent iron (nZVI), and anthraquinone-2,6-disulfonic acid (AQDS) on the degradation of PAHs in soil were studied. It was found that the treatment of 5 mg·kg-1RL + 1% nZVI +0.2 mmol·kg-1AQDS had the highest degradation rate. The degradation rate of total PAHs and HMW-PAHs was 72.81% and 79.47% respectively after 90 days. High-throughput sequencing showed that in RL + nZVI + AQDS enhanced soil, Clostridium, Geobacter, Anaeromyxobacter and Sphingomonas were the dominant species for anaerobic degradation of PAHs. Rhodococcus, Nocardioides, and Microvirga are the dominant species for aerobic degradation of PAHs. The activities of methyltransferase, dehydrogenase and catechol 1,2-dioxygenase in the anaerobic-aerobic degradation process of PAHs were consistent with the degradation process of PAHs, indicating the role of these enzymes in the degradation of PAHs. RL, nZVI, and AQDS combined enhanced microbial anaerobic-aerobic degradation has great application potential in remediation of PAHs-contaminated soil.

Metagenomic study of humic acid promoting the dechlorination of polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that degrade slowly in the environment. Humic acid (HA), the main component of soil organic matter, or more specifically, the quinone moieties in HA, is generally regarded as an "electron shuttle" between pollutants and microorganisms, which could promote microbial remediation of contamination. In this study, we examined the dechlorination of PCB153 by adding HA and anthraquinone-2,6-disulfonate (AQDS, a model compound of quinones) to systems containing PCB dechlorinators, analyzed the composition and functional gene network of the microbial community by metagenomics, and explored the role of HA by modifying or substituting carbon sources or electron donors. However, this study found that HA accelerated microbial dechlorination of PCBS, while AQDS did not. Moreover, HA without quinone activity still promoted dechlorination, but not without carbon source or electron donor. Metagenomic analysis showed that HA did not promote the growth of PCB dechlorinator (Dehalococcoides), but the transmembrane electron carriers in the HA group were higher than those in the AQDS group and the control group, so HA may have promoted the electron transport process. This study is helpful for microbial remediation of PCB contamination, and provides new insights into the role that HA plays in the biogeochemical cycle.

Reductive Transformation of 3-Nitro-1,2,4-triazol-5-one (NTO) by Leonardite Humic Acid and Anthraquinone-2,6-disulfonate (AQDS).

3-Nitro-1,2,4-triazol-5-one (NTO) is a major and the most water-soluble constituent in the insensitive munition formulations IMX-101 and IMX-104. While NTO is known to undergo redox reactions in soils, its reaction with soil humic acid has not been evaluated. We studied NTO reduction by anthraquinone-2,6-disulfonate (AQDS) and Leonardite humic acid (LHA) reduced with dithionite. Both LHA and AQDS reduced NTO to 3-amino-1,2,4-triazol-5-one (ATO), stoichiometrically at alkaline pH and partially (50-60%) at pH ≤ 6.5. Due to NTO and hydroquinone speciation, the pseudo-first-order rate constants (kObs) varied by 3 orders of magnitude from pH 1.5 to 12.5 but remained constant from pH 4 to 10. This distinct pH dependency of kObs suggests that NTO reactivity decreases upon deprotonation and offsets the increasing AQDS reactivity with pH. The reduction of NTO by LHA deviated continuously from first-order behavior for >600 h. The extent of reduction increased with pH and LHA electron content, likely due to greater reactivity of and/or accessibility to hydroquinone groups. Only a fraction of the electrons stored in LHA was utilized for NTO reduction. Electron balance analysis and LHA redox potential profile suggest that the physical conformation of LHA kinetically limited NTO access to hydroquinone groups. This study demonstrates the importance of carbonaceous materials in controlling the environmental fate of NTO.

Simulating the synergy of electron donors and different redox mediators on the anaerobic decolorization of azo dyes: Can AQDS-chitosan globules replace the traditional redox mediators?

During anaerobic treatment of azo dye wastewater, the decolorization efficiency is low and dissolved redox mediators (RMs) added to the system are easy lost. In order to solve these issues, immobilized RMs have been a hot area of research. In this study a novel immobilized RM material, disodium anthraquinone-2,6-disulfonate (AQDS)-chitosan globules, which is natural, highly efficient and environmentally friendly, was prepared. Compared with natural immobilized RMs (activated carbon) and dissolved RMs (AQDS), it can be considered that it has a significant strengthening effect on the anaerobic biological degradation and decolorization of azo dye wastewater. An electron donor (ED, glucose) or RM (AQDS solution) was dosed into an anaerobic reactor to determine the enhancing effect and appropriate concentration for the decolorization treatment. The results indicate that a certain concentration of ED or RM [300 mg/L (1.667 mmol/L) glucose or 200 µmol/L AQDS solution] can improve effectively the anaerobic biological degradation and decolorization effect of azo dye wastewater. While by adding both 300 mg/L (1.667 mmol/L) glucose and 300 µmol/L AQDS (the concentrations were the initial reactive concentrations) together the decolorization efficiency was improved further. At the same time, the synergy of ED (glucose) and RM (AQDS solution) on the anaerobic decolorization of azo dye was simulated by the central combination design. A mathematical model for the decolorization efficiency has been established. According to this model, the hydraulic retention time of the best decolorization speed and efficiency has been obtained.

Effects of the antibiotics trimethoprim (TMP) and sulfamethoxazole (SMX) on granulation, microbiology, and performance of aerobic granular sludge systems.

This work assessed the effect of the antibiotics trimethoprim (TMP) and sulfamethoxazole (SMX) on the granulation process, microbiology, and organic matter and nutrient removal of an aerobic granular sludge (AGS) system. In addition, after the maturation stage, the impact of the redox mediator anthraquinone-2,6-disulfonate (AQDS) (25 µM) on the biotransformation of the antibiotics was evaluated. The reactor R1 was maintained as a control, and the reactor R2 was supplemented with TMP and SMX (200 µg L-1). The ability to remove C, N, and P was similar between the reactors. However, the structural integrity of the AGS was impaired by the antibiotics. Low TMP (∼30%) and SMX (∼60%) removals were achieved when compared to anaerobic or floccular biomass aerobic systems. However, when the system was supplemented with AQDS, an increase in the removal of TMP (∼75%) and SMX (∼95%) was observed, possibly due to the catalytic action of the redox mediator on cometabolic processes. Regarding the microbial groups, whereas Proteobacteria and Bacterioidetes increased, Planctomycetes decreased in both reactors. However, TMP and SMX presence seemed to inhibit or favor some genera during the formation of the granules, possibly due to their bactericidal action.

Enhancement of nitrite reduction and enrichment of Methylomonas via conductive materials in a nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane-oxidizing (n-damo) process has a promising prospect in anaerobic wastewater treatment, utilizing methane as the sole electron source to remove nitrite. However, the metabolic activity of n-damo bacteria is too low for practical application. This study aimed to stimulate n-damo process by introducing conductive nano-magnetite and/or electron shuttle anthraquinone-2,6-disulfonate (AQDS), and also set a comparative treatment of adding insulated ferrihydrite. The results showed that the nitrite reduction rate was enhanced the most significantly in treatment with nano-magnetite, approximately 1.6 times higher than that of the control without any supplement. While ferrihydrite application showed an adverse effect on n-damo process. The well-known aerobic methane oxidizer Methylomonas spp. was found to be enriched under n-damo condition with the supplementation of nano-magnetite and/or AQDS, but abundance of n-damo bacteria did not exhibit significant increase. It was hypothesized that Methylomonas spp. could be survived under anaerobic n-damo condition using oxygen produced by n-damo bacteria for the self-growth, and the nitrite reduction could be promoted through the enhancement of microbial interspecies electron transfer triggered by the introduction of conductive materials. It opens a new direction for the stimulation of n-damo activity, which needs more evidences to verify the hypothetic mechanism.

Redox mediator, microaeration, and nitrate addition as engineering approaches to enhance the biotransformation of antibiotics in anaerobic reactors.

The present work assessed some engineering approaches, such as the addition of the redox mediator anthraquinone-2,6-disulfonate (AQDS) (50 and 100 µM), microaeration (1 mL air min-1), and nitrate (100-400 mg L-1), for enhancing the biotransformation of the antibiotics sulfamethoxazole (SMX) and trimethoprim (TMP) (200 µg L-1 each) in anaerobic reactors operated at a short hydraulic retention time (7.4 h). Initially, very low removal efficiencies (REs) of SMX and TMP were obtained under anaerobic conditions (∼6%). After adding AQDS, the anaerobic biotransformation of these antibiotics significantly improved, with an increase of approximately 70% in the REs with 100 µM of AQDS. Microaeration also enhanced the biotransformation of SMX and TMP, especially when associated with AQDS, which provided REs above 70%, particularly for TMP (∼91% with 1 mL air min-1 and 50 µM of AQDS). Concerning nitrate, the higher the added concentration, the higher the REs of the antibiotics (∼86% with 400 mg L-1). Therefore, all the assessed approaches were demonstrated to be very effective in improving the limited biotransformation of SMX and TMP in anaerobic reactors, ensuring REs comparable to those found in higher-cost wastewater treatment technologies, such as conventional activated sludge, membrane bioreactors, and hybrid processes.

Assessing the Effect of Humic Substances and Fe(III) as Potential Electron Acceptors for Anaerobic Methane Oxidation in a Marine Anoxic System.

Marine anaerobic methane oxidation (AOM) is generally assumed to be coupled to sulfate reduction, via a consortium of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). ANME-1 are, however, often found as single cells, or only loosely aggregated with SRB, suggesting they perform a form of AOM independent of sulfate reduction. Oxidized metals and humic substances have been suggested as potential electron acceptors for ANME, but up to now, AOM linked to reduction of these compounds has only been shown for the ANME-2 and ANME-3 clades. Here, the effect of the electron acceptors anthraquinone-disulfonate (AQDS), a humic acids analog, and Fe3+ on anaerobic methane oxidation were assessed by incubation experiments with anoxic Black Sea water containing ANME-1b. Incubation experiments with 13C-methane and AQDS showed a stimulating effect of AQDS on methane oxidation. Fe3+ enhanced the ANME-1b abundance but did not substantially increase methane oxidation. Sodium molybdate, which was added as an inhibitor of sulfate reduction, surprisingly enhanced methane oxidation, possibly related to the dominant abundance of Sulfurospirillum in those incubations. The presented data suggest the potential involvement of ANME-1b in AQDS-enhanced anaerobic methane oxidation, possibly via electron shuttling to AQDS or via interaction with other members of the microbial community.

Enhancement of anaerobic degradation of petroleum hydrocarbons by electron intermediate: Performance and mechanism.

A quinone-respiring strain capable of degrading multitudinous petroleum hydrocarbons was isolated by selective medium and identified as Bacillus sp. (named as C8). Maximum 76.7% of total petroleum hydrocarbons (TPH) were degraded by the biosurfactant-mediated C8 with the aid of nitrate and electron intermediate (anthraquinone-2,6-disulphonate, AQDS). The quantitative real-time PCR results of several intracellular key functional genes suggested that AQDS could participate in the transformation of intermediates and accelerate the electron transfer in the degradation of TPH and nitrate, thereby eliminating the accumulation of nitrite and increasing the degradation efficiency of TPH. A strengthening mechanism, which promoted electron transport in the anaerobic denitrification degradation of petroleum hydrocarbons by quinone-respiring strain with the aid of electron intermediate, was proposed. The influencing factors were evaluated by using response surface methodology, and the TPH removal was positively related to temperature but negatively to pH.

A Membrane-Bound Cytochrome Enables Methanosarcina acetivorans To Conserve Energy from Extracellular Electron Transfer.

Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in ArchaeaIMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.

Evaluation and correction on quinones' quantification errors: Derived from the coexistence of different quinone species and pH-sensitive feature.

Quinones are becoming an essential tool for refractory organics treatment, while their quantification may be not well-considered. In this paper, two kinds of potential errors in quantification were evaluated in multiple pH conditions. They were derived from the coexistence of oxidized/reduced quinone species (Type I) and pH-sensitive feature (Type II), respectively. These errors would remarkably influence the accuracy of quantification while they haven't been emphasized. Thus, to elaborate the relationship between the two types of errors and the absorbance or pH conditions, three typical quinones [Anthraquinone-1-sulfonate (α-AQS), anthraquinone-2,6-disulfonate (AQDS) and lawsone] were selected and their acid dissociation coefficients (pKa) as well as UV-Vis spectra were determined. Results revealed that, for Type I, the relative error (RE) of α-AQS concentration would exceed the limit (5%) when reduced α-AQS was below 48% of total α-AQS. Similar results were found for lawsone. However, the RE can be eliminated by the equation established in this paper. For Type II, the pH-sensitive feature was related to the pKa values of quinones. Absorbances of α-AQS and lawsone would change remarkably with pH variation. Therefore, a model for correction was established. Analog data showed high consistency with experimental data [r = 0.995 (n = 25, p < 0.01) and r = 0.997 (n = 36, p < 0.01), for lawsone and α-AQS respectively]. Especially, the determination of AQDS concentrations was noticed to be pH-independent at 437 nm under pH 4.00 to 9.18 conditions. Based on these features, a comprehensive data solution was proposed for handling these errors.

Promoting nitrogen removal during Fe(III) reduction coupled to anaerobic ammonium oxidation (Feammox) by adding anthraquinone-2,6-disulfonate (AQDS).

Feammox, i.e., Fe(III) reduction coupled to anaerobic ammonium oxidation, is a potential alternative to ammonium removal in natural and artificial ecosystems. However, the efficiency of Feammox is quite low to restrain its practical application in wastewater/solid disposal. In this study, three batch experiments, including control (Fe2O3/AQDS-free), Fe2O3 group (25 mM Fe2O3 only) and AQDS-Fe2O3 group (25 mM Fe2O3 and 0.6 mM AQDS), were conducted in 200 mL serum vials to explore whether AQDS can promote Feammox. Results showed that the nitrogen removal efficiency of the AQDS-Fe2O3 group was 82.6%, compared with 64.3% of the Fe2O3 group and 46.0% in the control. AH2QDS, the reduced state of AQDS, was detected in the AQDS-Fe2O3 group. Another experiment indicated that AH2QDS was oxidized back to AQDS by Fe2O3. These results suggested that AQDS/AH2QDS had been serving as electron shuttles between ammonium and Fe2O3 to successively forward the oxidation of NH4+. X-ray diffraction analysis showed that new Fe(III) species were found in the systems, implying that a Fe(II)/Fe(III) cycle also occurred. In agreement, both iron-reducing and oxidizing bacteria were detected in the systems.

Inhibitory effects of quinoid redox mediators on a denitrifying culture.

Denitrification with p-cresol as the electron source was studied in the presence of three quinones at different molar concentrations (0-2 mM): menadione (MEN), alizarine (ALZ) and anthraquinone-2,6-disulfonate (AQDS). Results showed that denitrifying yields were not altered (0.9), but the substrates' consumption efficiency was mainly affected when adding MEN. In the presence of ALZ and MEN, specific consumption rates decreased 40% for p-cresol and 90% for nitrate. The sludge previously exposed to quinones was submitted to recovering denitrifying studies using acetate and p-cresol. After exposing to AQDS and ALZ, the metabolic activity of denitrifying sludge was completely recovered. The previous exposition to any MEN concentration resulted in a very low metabolic recuperation. The results show that MEN and ALZ have a marked inhibitory effect on substrates' consumption and the AQDS does not affect at all. The evidence suggests that MEN modifies the transport system of substrates and ALZ forms a complex with molybdenum. A model based on oxido-reduction potentials of the enzymes involved points out that the influence of quinones tested appears to be more associated with quinone moiety structural properties and hydrophobicity.

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances.

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH2QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH2QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13C,15N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.