Biosynthesis of silver nanoparticles using Burkholderia contaminans ZCC and mechanistic analysis at the proteome level.

The biogenic synthesis of silver nanoparticles (AgNPs) by microorganisms has been a subject of increasing attention. Despite extensive studies on this biosynthetic pathway, the mechanisms underlying the involvement of proteins and enzymes in AgNPs production have not been fully explored. Herein, we reported that Burkholderia contaminans ZCC was able to reduce Ag+ to AgNPs with a diameter of (10±5) nm inside the cell. Exposure of B. contaminans ZCC to Ag+ ions led to significant changes in the functional groups of cellular proteins, with approximately 5.72% of the (C-OH) bonds being converted to (C-C/C-H) (3.61%) and CO (2.11%) bonds, and 4.52% of the CO (carbonyl) bonds being converted to (C-OH) bonds. Furthermore, the presence of Ag+ and AgNPs induced the ability of extracellular electron transfer for ZCC cells via specific membrane proteins, but this did not occur in the absence of Ag+ ions. Proteomic analysis of the proteins and enzymes involved in heavy metal efflux systems, protein secretion system, oxidative phosphorylation, intracellular electron transfer chain, and glutathione metabolism suggests that glutathione S-transferase and ubiquinol-cytochrome c reductase iron-sulfur subunit play importance roles in the biosynthesis of AgNPs. These findings contribute to a deeper understanding of the functions exerted by glutathione S-transferase and ferredoxin-thioredoxin reductase iron-sulfur subunits in the biogenesis of AgNPs, thereby hold immense potential for optimizing biotechnological techniques aimed at enhancing the yield and purity of biosynthetic AgNPs.

Au(III)-induced extracellular electron transfer by Burkholderia contaminans ZCC for the bio-recovery of gold nanoparticles.

The biorecovery of gold (Au) by microbial reduction has received increasing attention, however, the biomolecules involved and the mechanisms by which they operate to produce Au nanoparticles have been not resolved. Here we report that Burkholderia contaminans ZCC is capable of reduction of Au(III) to Au nanoparticles on the cell surface. Exposure of B. contaminans ZCC to Au(III) led to significant changes in the functional group of cell proteins, with approximately 11.1% of the (C-C/C-H) bonds being converted to CO (8.1%) and C-OH (3.0%) bonds and 29.4% of the CO bonds being converted to (C-OH/C-O-C/P-O-C) bonds, respectively. In response to Au(III), B. contaminans ZCC also displayed the ability of extracellular electron transfer (EET) via membrane proteins and could produce reduced riboflavin as verified by electrochemical and liquid chromatography-mass spectrometric results, but did not do so without Au(III) being present. Addition of exogenous reduced riboflavin to the medium suggested that B. contaminans ZCC could utilize indirect EET via riboflavin to enhance the rate of reduction of Au(III). Transcriptional analysis of the riboflavin genes (ribBDEFH) supported the view of the importance of riboflavin in the reduction of Au(III) and its importance in the biorecovery of gold.

Potential of cadmium resistant Burkholderia contaminans strain ZCC in promoting growth of soy beans in the presence of cadmium.

Bioremediation of Cd contaminated environments can be assisted by plant-growth-promoting bacteria (PGPB) enabling plant growth in these sites. Here a gram-negative Burkholderia contaminans ZCC was isolated from mining soil at a copper-gold mine. When exposed to Cd(II), ZCC displayed high Cd resistance and the minimal inhibitory concentration was 7 mM in LB medium. Complete genome analysis uncovered B. contaminans ZCC contained 3 chromosomes and 2 plasmids. One of these plasmids was shown to contain a multitude of heavy metal resistance determinants including genes encoding a putative Cd-translocating PIB-type ATPase and an RND-type related to the Czc-system. These additional heavy metal resistance determinants are likely responsible for the increased resistance to Cd(II) and other heavy metals in comparison to other strains of B. contaminans. B. contaminans ZCC also displayed PGPB traits such as 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore production, organic and inorganic phosphate solubilization and indole acetic acid production. Moreover, the properties and Cd(II) binding characteristics of extracellular polymeric substances was investigated. ZCC was able to induce extracellular polymeric substances production in response to Cd and was shown to be chemically coordinated to Cd(II). It could promote the growth of soybean in the presence of elevated concentrations of Cd(II). This work will help to better understand processes important in bioremediation of Cd-contaminated environment.

Interaction between in vivo bioluminescence and extracellular electron transfer in Shewanella woodyi via charge and discharge.

Extracellular electron transfer (EET) and bioluminescence are both important for microbial growth and metabolism, but the mechanism of interaction between EET and bioluminescence is poorly understood. Herein, we demonstrate an exclusively respiratory luminous bacterium, Shewanella woodyi, which possesses EET ability and electron communication at the interface of S. woodyi and solid substrates via charge and discharge methods. Using an electro-chemiluminescence apparatus, our results confirmed that the FMN/FMNH2 content and the redox status of cytochrome c conjointly regulated the bioluminescence intensity when the potential of an indium-tin oxide electrode was changed. More importantly, this work revealed that there is an interaction between the redox reaction of single cells and bioluminescence of group communication via the EET pathway.

Effective methods for extracting extracellular polymeric substances from Shewanella oneidensis MR-1.

Extracellular polymeric substances (EPS) play crucial roles in bio-aggregate formation and survival of bacterial cells. To develop an effective but harmless method for EPS extraction from Shewanella oneidensis MR-1, five extraction methods, i.e. centrifugation (control), heating (40, 45, 50, and 60 °C), and treatments with H2SO4, ethylenediaminetetraacetic acid (EDTA) and NaOH, were examined, respectively. Results from scanning electron microscope and flow cytometric analyses indicate that MR-1 cells were severely broken by H2SO4, NaOH and heating temperature ≥45 °C. Proteins and polysaccharides in EPS extracted by heating at 40 °C were 7.12 and 1.60 mg g-1 dry cell, respectively. Although EDTA treatment had a relatively lower yield of EPS (proteins and polysaccharides yields of 5.15 and 1.30 mg g-1 dry cell, respectively), cell lysis was barely found after EPS extraction. Three peaks were identified from the three-dimensional excitation-emission matrix spectrum of each EPS sample, suggesting the presence of protein-like substances. Furthermore, the peak intensity was in good accordance with protein concentration measured by the chemical analysis. In short, heating (40 °C) and EDTA treatments were found the most suitable methods for EPS extraction considering the cell lysis and EPS content, composition and functional groups together.

Soil microbial community composition and nitrogen enrichment responses to the operation of electric power substation.

The surge in global energy demand mandates a significant expansion of electric power substations. Nevertheless, the ecological consequences of electric power substation operation, particularly concerning the electromagnetic field, on soil microbial communities and nitrogen enrichment remain unexplored. In this study, we collected soil samples from six distinct sites at varying distances from an electric power substation in Xintang village, southeastern China, and investigated the impacts of electromagnetic field on the microbial diversity and community structures employing metagenomic sequencing technique. Our results showed discernible dissimilarities in the fungal community across the six distinct sites, each characterized by unique magnetic and electric intensities, whereas comparable variations were not evident within bacterial communities. Correlation analysis revealed a diminished nitrogen fixation capacity at the site nearest to the substation, characterized by low moisture content, elevated pH, and robust magnetic induction intensity and electric field intensity. Conversely, heightened nitrification processes were observed at this location compared to others. These findings were substantiated by the relative abundance of key genes associated with ammonium nitrogen and nitrate nitrogen production. This study provides insights into the relationships between soil microbial communities and the enduring operation of electric power substations, thereby contributing fundamental information essential for the rigorous environmental impact assessments of these facilities.

Impact of soil surface properties on soil swelling of different soil layers in collapsing wall of Benggang.

Benggang is one of the most serious soil erosion problems in tropical and subtropical areas in southern China. Little work has been reported on the surface properties of soil colloidal particle and its influence on soil swelling of different soil layers in collapsing wall of Benggang. In this present work, the effects of sodium concentration on soil swelling, and the correlations between soil swelling rates and soil colloidal surface properties were comprehensively evaluated by carefully examining soil physicochemical properties and soil colloidal surface properties of red, sandy and detritus soil layers from a collapsing wall. Our results showed that the soil swelling rates of red, sandy and detritus soil layers all exponentially decreased with increasing initial water contents. The relationship between soil swelling rate and the thickness of shear plane showed an extremely significant negative correlation for red soil layer and no correlation for sandy and detritus soil layers. Moreover, the elevating sodium concentrations reduced the thickness of shear plane from 39.69 to 0.76 nm for red soil layer, followed from 22.56 to 0.79 nm for sandy soil layer and from 18.61 to 0.64 nm for detritus soil layer. These findings indicated that the soil particle interactions played a crucial role in the development and occurrence of Benggang. This work will be helpful in understanding the mechanisms of soil mass loss on the gully head and collapsing wall of Benggang.

Selenite reduced uptake/translocation of cadmium via regulation of assembles and interactions of pectins, hemicelluloses, lignins, callose and Casparian strips in rice roots.

Selenium (Se) can reduce cadmium (Cd) uptake/translocation via regulating pectins, hemicelluloses and lignins of plant root cell walls, but the detailed molecular mechanisms are not clear. In this study, six hydroponic experiments were set up to explore the relationships of uptake/translocation inhibition of Cd by selenite (Se(IV)) with cell wall component (CWC) synthesis and/or interactions. Cd and Se was supplied (alone or combinedly) at 1.0 mg L-1 and 0.5 mg L-1, respectively, with the treatment without Cd and Se as the control. When compared to the Cd1 treatment, the Se0.5Cd1 treatment 1) significantly increased total sugar concentrations in pectins, hemicelluloses and callose, suggesting an enhanced capacity of binding Cd or blocking Cd translocation; 2) stimulated the deposition of Casparian strips (CS) in root endodermis and exodermis to block Cd translocation; 3) stimulated the release of C-O-C (-OH- or -O-) and CO (carboxyl, carbonyl, or amide) to combine Cd; 4) regulated differential expression genes (DEGs) and metabolites (DMs) correlated with synthesis and/or interactions of CWSs to affect cell wall net structure to affect root cell division, subsequent root morphology and finally elemental uptake; and 5) stimulated de-methylesterification of pectins via reducing expression abundances of many DMs and DEGs in the Yang Cycle to reduce supply of methyls to homogalacturonan, and regulated gene expressions of pectin methylesterase to release carboxyls to combine Cd; and 6) down-regulated gene expressions associated with Cd uptake/translocation.

Antioxidant CeO2 doped with carbon dots enhance ammonia production by an electroactive Azospirillum humicireducens SgZ-5T.

Microbial nitrogen fixation is a fundamental process in the nitrogen cycle, providing a continuous supply of biologically available nitrogen essential for life. In this study, we combined cerium oxide-doped carbon dots (CeO2/CDs) with electroactive nitrogen-fixing bacterium Azospirillum humicireducens SgZ-5T to enhance nitrogen fixation through ammonium production. Our research demonstrates that treatment of SgZ-5T cells with CeO2/CDs (0.2 mg mL-1) resulted in a 265.70% increase in ammonium production compared to SgZ-5T cells alone. CeO2/CDs facilitate electron transfer in the biocatalytic process, thereby enhancing nitrogenase activity. Additionally, CeO2/CDs reduce the concentration of reactive oxygen species in SgZ-5T cells, leading to increased ammonium production. The upregulation of nifD, nifH and nifK gene expression upon incorporation of CeO2/CDs (0.2 mg mL-1) into SgZ-5T cells supports this observation. Our findings not only provide an economical and environmentally friendly approach to enhance biological nitrogen fixation but also hold potential for alleviating nitrogen fertilizer scarcity.

Identification of a Novel Chromate and Selenite Reductase FesR in Alishewanella sp. WH16-1.

A ferredoxin protein (AAY72_06850, named FesR) was identified to associate with chromate [Cr(VI)] resistance in Alishewanella sp. WH16-1. FesR and its similar proteins were phylogenetically separated from other reductase families. Unlike the reported Cr(VI) and selenite [Se(IV)] reductases, two 4Fe-4S clusters and one flavin adenine dinucleotide (FAD) -binding domain were found in the FesR sequence. The experiment in vivo showed that the mutant strain ΔfesR had lost partial Cr(VI) and Se(IV) reduction capacities compared to the wild-type and complemented strains. Furthermore, overexpression in Escherichia coli and enzymatic tests in vitro showed FesR were involved in Cr(VI) and Se(IV) reduction. 4Fe-4S cluster in purified FesR was detected by ultraviolet-visible spectrum (UV-VIS) and Electron Paramagnetic Resonance (EPR). The Km values of FesR for Cr(VI) and Se(IV) reduction were 1682.0 ± 126.2 and 1164.0 ± 89.4 µmol/L, and the Vmax values for Cr(VI) and Se(IV) reduction were 4.1 ± 0.1 and 9.4 ± 0.3 µmol min-1 mg-1, respectively. Additionally, site-directed mutagenesis and redox potential analyses showed that 4Fe-4S clusters were essential to FesR, and FAD could enhance the enzyme efficiencies of FesR as intracellular electron transporters. To the best of our knowledge, FesR is a novel Cr(VI) and Se(IV) reductase.

Three-dimensional self-supporting superstructured double-sided nanoneedles arrays of iron carbide nanoclusters embedded in manganese, nitrogen co-doped carbon for highly efficient oxygen reduction reaction.

Nitrogen- and transition metal-dual doped carbon materials with low cost and high catalytic performances are considered as one of promising alternatives for noble metal catalysts in acceleration of oxygen reduction reaction (ORR). In this work, three-dimensional (3D) self-supporting superstructures of iron carbide (Fe3C) nanoclusters entrapped in manganese (Mn)- and nitrogen (N)-dual doped carbon nanosheets covered with double-sided nanoneedles carbon arrays (Fe3C/Mn,N-NCAs) are simply synthesized by a coordination pyrolysis method, in which dicyandiamide mainly behaves as nitrogen source and 1-(2-pyridylazo)-2-naphthol (PAN) as carbon source. Integration of the unique 3D self-supporting superstructures and synergistic effects of the multi-compositions, the as-obtained catalyst displays appealing ORR performance such as the much positive onset potential (Eonset = 0.98 V vs. RHE) and half-wave potential (E1/2 = 0.88 V vs. RHE), as well as a just 10 mV negative shift in E1/2 after 2000 cycles, surpassing commercial Pt/C. This work provides some valuable perspectives for preparation of high-efficiency and low-cost non-noble metal ORR electrocatalysts in energy transformation and storage correlated systems.

Liposoluble quinone promotes the reduction of hydrophobic mineral and extracellular electron transfer of Shewanella oneidensis MR-1.

A large number of reaction systems are composed of hydrophobic interfaces and microorganisms in natural environment. However, it is not clear how microorganisms adjust their breathing patterns and respond to hydrophobic interfaces. Here, Shewanella oneidensis MR-1 was used to reduce ferrihydrite of a hydrophobic surface. Through Fe(II) kinetic analysis, it was found that the reduction rate of hydrophobic ferrihydrite was 1.8 times that of hydrophilic one. The hydrophobic surface of the mineral hinders the way the electroactive microorganism uses the water-soluble electron mediator riboflavin for indirect electron transfer and promotes MR-1 to produce more liposoluble quinones. Ubiquinone can mediate electron transfer at the hydrophobic interface. Ubiquinone-30 (UQ-6) increases the reduction rate of hydrophobic ferrihydrite from 38.5 ± 4.4 to 52.2 ± 0.8 µM·h-1. Based on the above experimental results, we propose that liposoluble electron mediator ubiquinone can act on the extracellular hydrophobic surface, proving that the metabolism of hydrophobic minerals is related to endogenous liposoluble quinones. Hydrophobic modification of minerals encourages electroactive microorganisms to adopt differentiated respiratory pathways. This finding helps in understanding the electron transfer behavior of the microbes at the hydrophobic interface and provides new ideas for the study of hydrophobic reactions that may occur in systems, such as soil and sediment.

NanoFe3O4 as Solid Electron Shuttles to Accelerate Acetotrophic Methanogenesis by Methanosarcina barkeri.

Magnetite nanoparticles (nanoFe3O4) have been reported to facilitate direct interspecies electron transfer (DIET) between syntrophic bacteria and methanogens thereby improving syntrophic methanogenesis. However, whether or how nanoFe3O4 affects acetotrophic methanogenesis remain unknown. Herein, we demonstrate the unique role of nanoFe3O4 in accelerating methane production from direct acetotrophic methanogenesis in Methanosarcina-enriched cultures, which was further confirmed by pure cultures of Methanosarcina barkeri. Compared with other nanomaterials of higher electrical conductivity such as carbon nanotubes and graphite, nanoFe3O4 with mixed valence Fe(II) and Fe(III) had the most significant stimulatory effect on methane production, suggesting its redox activity rather than electrical conductivity led to enhanced methanogenesis by M. barkeri. Cell morphology and spectroscopy analysis revealed that nanoFe3O4 penetrated into the cell membrane and cytoplasm of M. barkeri. These results provide the unprecedented possibility that nanoFe3O4 in the cell membrane of methanogens serve as electron shuttles to facilitate intracellular electron transfer and thus enhance methane production. This work has important implications not only for understanding the mechanisms of mineral-methanogen interaction but also for optimizing engineered methanogenic processes.

Extracellular electron transfer of Enterobacter cloacae SgZ-5T via bi-mediators for the biorecovery of palladium as nanorods.

In nature, microbes use extracellular electron transfer (EET) to recover noble metals. Most attention has been paid to the biorecovery process occurring intracellularly and on the cell surface. In this work, we report that Pd nanorods could be biosynthesized by Enterobacter cloacae SgZ-5T in the extracellular space. This bacterium possesses both a direct EET pathway through membrane redox systems and an indirect EET pathway via the self-secreted electron carrier hydroquinone (HQ). When exposed to Pd(II), the bacteria adjusted their metabolic pathway and membrane-bound proteins to secrete riboflavin (RF). However, no HQ was detected in the supernatant in presence of Pd(II). No significant change was observed through metabolomic analysis regarding the abundance of HQ in presence of Pd(II) compared to Pd(II)-free supernatant. Similar results were also obtained through transcriptomic analysis of YqjG gene encoding glutathionyl-HQ reductase synthase. X-ray photoelectron spectroscopic evidence indicated that HQ may adsorb to the surface of Pd nanorods. Moreover, the gene encoding RF synthase (ribE) was up-regulated in the present of Pd(II), suggesting that this bioreduction process induced RF synthase, which had been shown in previous results. The UV-vis spectroscopy data demonstrated that the Pd(II) reduction rate was enhanced by 5%, 5.5% and 30% by the addition of 3.33 µM HQ, 3.33 µM RF and the both, respectively. All these results revealed that the bi-mediators secreted by bacteria were beneficial for biorecovery of Pd. This work is of significance for understanding metal biorecovery processes and natural biogeochemical processes.

Electrochemiluminescence for the identification of electrochemically active bacteria.

Electrochemically active bacteria (EAB) use extracellular electron transfer (EET) to exchange electron with extracellular acceptors. Previous studies regarding the measurement of EAB were based on either extracellular reduction or oxidation. In this work, we developed a simple electrochemiluminescence (ECL) assay for the identification and detection of EAB. The results of this proposed method revealed that EET of EAB influenced the content of dissolved oxygen and the formation of Ru(bpy)32+• thus leading to qualitative changes of the ECL signal. EAB with the ability of extracellular reduction (such as Shewanella oneidensis MR-1) gave enhanced signal on ECL emission while those displaying the ability of extracellular oxidation (i.e., Sulfobacillus acidophilus) showed the opposite effect on ECL emission, but non-EAB (i.e., Escherichia coli) did not. These changes in ECL intensity were also proportional to the cell density that could be quantitatively detected in the concentration range of (1.1 ±â€¯1) × 105-212 ±â€¯2 CFU/mL (i.e. Shewanella oneidensis MR-1). Moreover, the measurement of the ability of EAB using this approach was in agreement with measurements using the dissimilatory Fe(III) reduction method. Compared to previous reports, this method displayed a continual and steady ECL signal that allowed accurate measurements of EAB. Most important, only a low cell density was needed in this Ru(bpy)32+ - based ECL method, which is beneficial for cell detection.

Flavins mediate extracellular electron transfer in Gram-positive Bacillus megaterium strain LLD-1.

The extracellular electron transfer (EET) mechanism of an isolated Gram-positive Bacillus megaterium strain (LLD-1), identified by 16S rRNA gene sequencing and physiological analysis, was investigated in the present study. The electrochemical activity of strain LLD-1 was confirmed by electrochemical E-t and amperometric I-t tests. Flavins in culture suspension from strain LLD-1 were further proved to be able to act as electron shuttles, strengthening the electron transfer from LLD-1 to the electrode. The output voltage and current output were increased 2.8 times and 3.7 times, respectively, by adding 100nM exogenetic flavins into microbial fuel cells inoculated with LLD-1. Electricity generation by LLD-1 from different carbon sources can be enhanced by adding 100nM exogenetic flavins. This study indicated that flavins were essential to the EET process of the Gram-positive strain LLD-1. Furthermore, a putative EET model for B. megaterium strain LLD-1 and even for Gram-positive bacteria was proposed.

Biodegradation of sulfadiazine in microbial fuel cells: Reaction mechanism, biotoxicity removal and the correlation with reactor microbes.

Sulfadiazine (SDZ) is a high priority sulfonamide antibiotic and was always detected in environmental samples. This study explored the removal of SDZ in microbial fuel cells (MFCs), in terms of MFC operation, degradation products, reaction mechanism, SDZ biotoxicity removal, and the correlation between microbial community and SDZ removal. SDZ would greatly impact the activity of reactor microbes, and longtime acclimation is required for the biodegradation of SDZ in MFCs. After acclimation, 10 mg/L of SDZ could be removed within 48 h. Liquid chromatographic-mass spectroscopic analysis showed that SDZ could be degraded into 2-aminopyrimidine, 2-amino-4-hydroxypyrimidine and benzenesulfinic acid. Compared with published SDZ biodegradation mechanism, we found that the sulfanilamide part (p-Anilinesulfonic acid) of SDZ would be degraded into benzenesulfinic acid in the system. The effects of background constituents on SDZ biodegradation were explored, and co-existed humic acid (HA) and fulvic acid (FA) could accelerate the removal of SDZ in MFCs. After analyzing the reactor microbial community and the removal of SDZ at different operation cycles, it was found that the relative abundance of Methanocorpusculum, Mycobacterium, Clostridium, Thiobacillus, Enterobacter, Pseudomonas, and Stenotrophomonas was highly correlated with the removal of SDZ throughout the experiment.

Electrogenerated chemiluminescence of bis[4-(dimethylamino)phenyl]squaraine.

For the first time, we reported the strong electrogenerated chemiluminescence of organic dye, bis[4-(dimethylamino)phenyl]squaraine (BDPSQ), a high quantum yield and light-stable species, which might find applications in ECL analysis and imaging.