To investigate how cell elongation impacts extracellular electron transfer (EET) of electroactive microorganisms (EAMs), the division of model EAM Shewanella oneidensis (S. oneidensis) MR-1 is engineered by reducing the formation of cell divisome. Specially, by blocking the translation of division proteins via anti-sense RNAs or expressing division inhibitors, the cellular length and output power density are all increased. Electrophysiological and transcriptomic results synergistically reveal that the programmed cell elongation reinforces EET by enhancing NADH oxidation, inner-membrane quinone pool, and abundance of c-type cytochromes. Moreover, cell elongation enhances hydrophobicity due to decreased cell-surface polysaccharide, thus facilitates the initial surface adhesion stage during biofilm formation. The output current and power density all increase in positive correction with cellular length. However, inhibition of cell division reduces cell growth, which is then restored by quorum sensing-based dynamic regulation of cell growth and elongation phases. The QS-regulated elongated strain thus enables a cell length of 143.6 ± 40.3 µm (72.6-fold of that of S. oneidensis MR-1), which results in an output power density of 248.0 ± 10.6 mW m-2 (3.41-fold of that of S. oneidensis MR-1) and exhibits superior potential for pollutant treatment. Engineering cellular length paves an innovate avenue for enhancing the EET of EAMs.

Anaerobic ammonium oxidation (anammox) is an energy-efficient method for nitrogen removal that opens the possibility for energy-neutral wastewater treatment. Research on anammox over the past decade has primarily focused on its implementation in domestic wastewater treatment. However, emerging studies are now expanding its use to novel biotechnological applications and wastewater treatment processes. This review highlights recent advances in the anammox field that aim to overcome conventional bottlenecks, and explores novel and niche-specific applications of the anammox process. Despite the promising results and potential of these advances, challenges persist for their real-world implementation. This underscores the need for a transition from laboratory achievements to practical, scalable solutions for wastewater treatment which mark the next crucial phase in the evolution of anammox research.

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. \'Methanoperedens manganicus\', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. \'M. manganicus\' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. \'M. manganicus\', and the total archaea population in the microbial community suggested that Ca. \'M. manganicus\' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. \'M. manganicus\'. We tested this hypothesis by adding humic acids to a Ca. \'M. manganicus\' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

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The slow rate of electron transfer and the large consumption of carbon sources are technical bottlenecks in the biological treatment of wastewater. Here, we first proposed to domesticate aerobic denitrifying bacteria (ADB) from heterotrophic to autotrophic by electricity (0.6 V) under zero organic carbon source conditions, to accelerate electron transfer and shorten hydraulic retention time (HRT) while increasing the biodegradation rate. Then we investigated the extracellular electron transfer (EET) mechanism mediated by this process, and additionally examined the integrated nitrogen removal efficiency of this system with composite pollution. It was demonstrated that compared with the traditional membrane bioreactor (MBR), the BEC displayed higher nitrogen removal efficiency. Especially at C/N = 0, the BEC exhibited a NO3--N removal rate of 95.42 ± 2.71 % for 4 h, which was about 6.5 times higher than that of the MBR. Under the compound pollution condition, the BEC still maintained high NO3--N and tetracycline removal (94.52 ± 2.01 % and 91.50 ± 0.001 %), greatly superior to the MBR (10.64 ± 2.01 % and 12.00 ± 0.019 %). In addition, in-situ electrochemical tests showed that the nitrate in the BEC could be directly converted to N2 by reduction using electrons from the cathode, which was successfully demonstrated as a terminal electron acceptor.

In this study, a 13-mer antimicrobial peptide (RRWRIVVIRVRRC) named by E6 was used as an enhancer of a green biocide to mitigate the biocorrosion of EH36 ship steel. Results show that a low concentration of E6 (100 nM) alone was no-biocidal and could not resist the Desulfovibrio vulgaris adhesion on the EH36 steel surface. However, E6 enhanced the bactericidal effect of tetrakis hydroxymethyl phosphonium sulfate (THPS). When E6 and THPS were both added to the bacteria and steel system, both the sessile D. vulgaris cells and biocorrosion rate of EH36 steel decreased significantly. Compared with the 80 ppm THPS alone treatment, the combination of 100 nM E6 + 80 ppm THPS led to an extra 1.6-log reduction in the sessile cell count. Fewer sessile D. vulgaris cells led to a lower extracellular electron transfer (EET) rate, directly resulting in 78% and 83% decreases in weight loss and pit depth of EH36 steel, respectively. E6 saved more than 50% of THPS dosage in this work to achieve a similar biocorrosion mitigation effect on EH36 steel.

Manganese-redox-mediated nitrogen transformation is promising for ammonium wastewater treatment. However, due to the limited contact between insoluble Mn and the microbe, extracellular electron transfer (EET) inefficiencies become a technical bottleneck in the technical practical application. To overcome this obstacle, humic acid (HA) was introduced to synthesize manganese-humic acid complex (Mn-HA) to increase Mn solubility. The TIN (Total Inorganic Nitrogen) removal rate constant k was 3.18, 1.08, 3.56, 1.13 and 1.05 times higher than CK (Control group) at 10, 15, 20, 40 and 60 mg/L influent nitrate in the MH group, respectively. Mn-HA was inferred to stimulated the nitrogen removal by providing more reaction active sites, bridging Mn-O bonds to transfer electrons and playing a redox role in the respiratory chain. A Mnammox-NDMO (manganese oxide reduction-coupled ammonium oxidation - nitrate/nitrite- dependent manganese oxidation) bacteria consortium was enriched in MH group, containing Mnammox bacteria Geothrix, Geobacter and NDMO bacteria Pseudomonas and Bacillus.

A comparison was conducted between pre-culture bacteria (PCB) and heat treatment anaerobic granular sludge (HTAGS) for hydrogen production, and it was found that hydrogen molar yield (HMY) of PCB was 21-35% higher than that of HTAGS. The addition of biochar increased hydrogen production in both cultivation methods by acting as an electron shuttle to enhance extracellular electron transfers of Clostridium and Enterobacter. On the other hand, Fe3O4 did not promote hydrogen production in PCB experiments but had a positive effect on HTAGS experiments. This was due to the fact that PCB was mainly composed of Clostridium butyricum, which could not reduce extracellular iron oxide, resulting in a lack of respiratory driving force. In contrast, HTAGS retained a significant amount of Enterobacter, which possess the ability of extracellular anaerobic respiration. Different pretreatment methods of inoculum resulted in significant changes in the sludge community, thus exerting a noticeable impact on biohydrogen production.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

UNASSIGNED: Electron shuttles (ESs) play a key role in extracellular electron transfer (EET) in Shewanella oneidensis MR-1. However, the quantification relationship between ES concentration, biofilm formation, and biocurrent generation has not been clarified.
UNASSIGNED: In this study, 9,10-anthraquinone-2-sulfonic acid (AQS)-mediated EET and biofilm formation were evaluated at different AQS concentrations in bioelectrochemical systems (BESs) with S. oneidensis MR-1.
UNASSIGNED: Both the biofilm biomass (9- to 17-fold) and biocurrent (21- to 80-fold) were substantially enhanced by exogenous AQS, suggesting the dual ability of AQS to promote both biofilm formation and electron shuttling. Nevertheless, biofilms barely grew without the addition of exogenous AQS, revealing that biofilm formation by S. oneidensis MR-1 is highly dependent on electron shuttling. The biofilm growth was delayed in a BES of 2,000 μM AQS, which is probably because the redundant AQS in the bulk solution acted as a soluble electron acceptor and delayed biofilm formation. In addition, the maximum biocurrent density in BESs with different concentrations of AQS was fitted to the Michaelis-Menten equation (R 2 = 0.97), demonstrating that microbial-catalyzed ES bio-reduction is the key limiting factor of the maximum biocurrent density in BESs. This study provided a fundamental understanding of ES-mediated EET, which could be beneficial for the enrichment of electroactive biofilms, the rapid start-up of microbial fuel cells (MFCs), and the design of BESs for wastewater treatment.