Enhanced Extracellular Electron Transfer in Magnetite-Mediated Anaerobic Oxidation of Methane Coupled to Humic Substances Reduction: The Pivotal Role of Membrane-Bound Electron Transfer Proteins.

Humic substances are organic substances prevalent in various natural environments, such as wetlands, which are globally important sources of methane (CH4) emissions. Extracellular electron transfer (EET)-mediated anaerobic oxidation of methane (AOM)-coupled with humic substances reduction plays an important role in the reduction of methane emissions from wetlands, where magnetite is prevalent. However, little is known about the magnetite-mediated EET mechanisms in AOM-coupled humic substances reduction. This study shows that magnetite promotes the reduction of the AOM-coupled humic substances model compound, anthraquinone-2,6-disulfonate (AQDS). 13CH4 labeling experiments further indicated that AOM-coupled AQDS reduction occurred, and acetate was an intermediate product of AOM. Moreover, 13CH313COONa labeling experiments showed that AOM-generated acetate can be continuously reduced to methane in a state of dynamic equilibrium. In the presence of magnetite, the EET capacity of the microbial community increased, and Methanosarcina played a key role in the AOM-coupled AQDS reduction. Pure culture experiments showed that Methanosarcina barkeri can independently perform AOM-coupled AQDS reduction and that magnetite increased its surface protein redox activity. The metatranscriptomic results indicated that magnetite increased the expression of membrane-bound proteins involved in energy metabolism and electron transfer in M. barkeri, thereby increasing the EET capacity. This phenomenon potentially elucidates the rationale as to why magnetite promoted AOM-coupled AQDS reduction.

Enhancing assimilatory sulfate reduction with ferrihydrite-humic acid coprecipitate in anaerobic sulfate-containing wastewater treatment.

Sulfide produced from dissimilatory sulfate reduction can combine with hydrogen to form hydrogen sulfide, causing odor issues and environmental pollution. To address this problem, ferrihydrite-humic acid coprecipitate was added to improve assimilatory sulfate reduction (ASR), resulting in a decrease in sulfide production (190.2 ± 14.6 mg/L in the Fh-HA group vs. 246.3 ± 8.1 mg/L in the Fh group) with high sulfate removal. Humic acid, adsorbed on the surface of ferrihydrite, delayed secondary mineralization of ferrihydrite under sulfate reduction condition. Therefore, more iron-reducing species (e.g. Trichococcus, Geobacter) were enriched with ferrihydrite-humic acid coprecipitate to transfer more electrons to other species, which led to more COD reduction, an increase in electron transfer capacity, and a decrease in the NADH/NAD+ ratio. Metagenomic analysis also indicated that functional genes related to ASR was enhanced with ferrihydrite-humic acid coprecipitate. Thus, the addition of ferrihydrite-humic acid coprecipitate can be considered as a promising candidate for anaerobic sulfate wastewater treatment.

Insights into feasibility and microbial characterizations on simultaneous elimination of dissolved methane from anaerobic effluents and nitrate/nitrite reduction in a conventional anoxic reactor with magnetite.

Discovery of nitrate/nitrite-dependent anaerobic methane oxidation (DAMO) challenges the conventional biological treatment processes, since it provides a possibility of simultaneously mitigating dissolved methane emissions from anaerobic effluents and reducing additional carbon sources for denitrification. Due to the slow growth of specialized DAMO microbes, this possibility has been just practiced with biofilms in membrane biofilm reactors or granular sludge in membrane bioreactors. In this study, simultaneous elimination of dissolved methane from anaerobic effluents and nitrate/nitrite reduction was achieved in a conventional anoxic reactor with magnetite. Calculations of electron flow balance showed that, with magnetite the eliminated dissolved methane was almost entirely used for nitrate/nitrite reduction, while without magnetite approximately 52 % of eliminated dissolved methane was converted to unknown organics. Metagenomic sequencing showed that, when dissolved methane served as an electron donor, the abundance of genes for reverse methanogenesis and denitrification dramatically increased, indicating that anaerobic oxidation of methane (AOM) coupled to nitrate/nitrite reduction occurred. Magnetite increased the abundance of genes encoding the key enzymes involved in whole reverse methanogenesis and Nir and Nor involved in denitrification, compared to that without magnetite. Analysis of microbial communities showed that, AOM coupled to nitrate/nitrite reduction was proceeded by syntrophic consortia comprised of methane oxidizers, Methanolinea and Methanobacterium, and nitrate/nitrite reducers, Armatimonadetes_gp5 and Thauera. With magnetite syntrophic consortia exchanged electrons more effectively than that without magnetite, further supporting the microbial growth.

Effects of ferroferric oxide on azo dye degradation in a sulfate-containing anaerobic reactor: From electron transfer capacity and microbial community.

Anaerobic decolorization of azo dye in sulfate-containing wastewater has been regarded as an economical and effective method, but it is generally limited by the high concentration of azo dye and accumulation of toxic intermediates. To address this problem, Fe3O4 was added to one of the anaerobic reactors to investigate the effects on system performances. Results showed that AO7 removal rate, COD removal rate, and sulfate reduction were enhanced with the addition of Fe3O4 under various influent AO7 concentrations (153 mgCOD/L - 1787 mgCOD/L). According to the proposed pathway for the degradation of AO7, more intermediates (2-hydroxy-1,4-naphthoquinone, phthalide, 4-methylphenol) were produced in the presence of Fe3O4. The electron transfer capacity of sludge was also increased since Fe3O4 could stimulate to secrete humic acid-like organics in EPS. Microbial analysis showed that iron-reducing bacteria like Clostridium and Geobacter were also enriched, which were capable of azo dye and aromatic compounds degradation.

Continuously feeding fenton sludge into anaerobic digesters: Iron species change and operating stability.

Fenton sludge generated from the Fenton process contains a large number of ferric species and organic pollutants, which need to be properly treated before discharge. In this study, Fenton sludge as an Fe(III) source for dissimilatory iron reduction (DIR) was continuously added with increasing dosage into an anaerobic digester to enhance the treatment. Results showed continuously feeding Fenton sludge to the anaerobic digester did not deteriorate the performance and increased methane production and COD removal rate by 2.2 folds and 14.0%, respectively. The Fe content of sludge in the digester increased from 40.25 mg/g (dry weight) to 131.53 mg/g after continuously feeding for 77days, and then declined to 109.17 mg/g when the feeding was stopped. Mass balance analysis showed that 20.5 to 48.4% of Fe in the Fenton sludge was released to the effluent. After experiment, the ratio of reducible Fe species to the total Fe was 75.1%, which maintained the high activity in DIR. Microbial community analysis showed that iron-reducing bacteria were enriched with the addition of Fenton sludge and the sludge in the digester had a higher conductivity and capacitance to strengthen the electron transfer of DIR. All results suggested that feeding Fenton sludge into anaerobic digesters was a feasible method to dispose of Fenton sludge as well as to enhance the performance of anaerobic digestion.

Glycol/glycerol-fed electrically conductive aggregates suggest a mechanism of stimulating direct interspecies electron transfer in methanogenic digesters.

The possibility of stimulating direct interspecies electron transfer (DIET) within aggregates of methanogenic digesters respectively with ethanol, glycol, and glycerol as a primary substrate was investigated to better understand the mechanisms of alcohol compounds stimulating DIET. Aggregates fed with ethanol, glycol, and glycerol were electrically conductive (10.4-19.4 uS/cm), with a temperature dependence of metallic-like conductivity. Close examination of transmission electron microscope images observed the potential interspecies connected networks assembled by filaments within these aggregates. Further investigations via metatranscriptomics found that, genes for electrically conductive pili (e-pili) (Log2FPKM, 9.39-10.96) and c-type cytochromes (8.90-9.64) were highly expressed within aggregates. Glycerol-fed aggregates exhibited the highest gene expression for e-pili, while glycol-fed aggregates exhibited the highest gene expression for c-type cytochromes. Methanothrix species were dominant and metabolically active within aggregates. Genes encoding the enzymes involved in carbon dioxide reduction were highly expressed in Methanothrix species, suggesting that they participated in DIET. In addition, transcript abundance of genes encoding alcohol dehydrogenase and NADH-quinone oxidoreductase in alcohol dehydrogenation closely associated with NADH/NAD+ transformation within glycol- and glycerol-fed aggregates was generally higher than that within ethanol-fed aggregates. These results, and the fact that NADH/NAD+ transformation was very linked to the ATP synthesis complex that further supported the formation of extracellular electrical connection components, e-pili and membrane-bound multi-heme c-type cytochromes (MHCs), provided a possibility that alcohol compounds comprised of hydroxy groups could stimulate DIET and more hydroxy groups comprised were better for this stimulation.

Independent of direct interspecies electron transfer: Magnetite-mediated sulphur cycle for anaerobic degradation of benzoate under low-concentration sulphate conditions.

The aim of this study was to investigate the primary mechanism of magnetite promoting anaerobic degradation of aromatic compounds under the low-concentration sulphate conditions. Under influent conditions of benzoate at 50 mM-chemical oxygen demand (COD) and sulphate at 15 mM, magnetite promoted benzoate degradation (77.1% vs 56.3%), while the effluent sulphate concentration was slightly higher than that without magnetite (1.6 mM vs 0.7 mM), inconsistent with functional gene prediction that both sulphate respiration and sulphur compound respiration were relatively more active in the presence of magnetite. Remarkably, X-ray diffraction showed that, signal related to Fe3O4 faded away and finally was replaced by FeSO4 and FeS, indicating that magnetite participated in benzoate degradation coupled to sulphate reduction via dissimilatory Fe(III) reduction. Further X-ray photoelectron spectroscopy showed that, signal related to S0 was only detected with magnetite, suggesting the possibility of re-oxidation of sulphide to elemental sulphur coupled to Fe(III) reduction. This was further supported by the increase in abundance of Desulfuromonas acetexigens capable of growing on Fe(III). In addition, magnetite specially enriched the chemolithotrophic sulphur-disproportionating microbes, Desulfovibrio aminophilus, which might proceed the disproportionation of elemental sulphur to sulphate and sulphide to achieve a sulphur cycle for benzoate degradation.