Complete oxidative degradation of diclofenac via coupling free radicals and oxygenases of a micro/nanostructured biogenic Mn oxide composite from engineered Pseudomonas sp. MB04R-2.

Oxidative degradation can effectively degrade aromatic emerging contaminants (ECs). However, the degradability of lone inorganic/biogenic oxides or oxidases is typically limited when treating polycyclic ECs. Herein, we report a dual-dynamic oxidative system comprising engineered Pseudomonas and biogenic Mn oxides (BMO), which completely degrades diclofenac (DCF), a representative halogen-containing polycyclic EC. Correspondingly, recombinant Pseudomonas sp. MB04R-2 was constructed via gene deletion and chromosomal insertion of a heterologous multicopper oxidase cotA, allowing for enhanced Mn(II)-oxidizing activity and rapid formation of the BMO aggregate complex. Additionally, we characterized it as a micro/nanostructured ramsdellite (MnO2) composite using multiple-phase composition and fine structure analyses. Furthermore, using real-time quantitative polymerase chain reaction, gene knockout, and expression complementation of oxygenase genes, we demonstrated the central and associative roles of intracellular oxygenases and cytogenic/BMO-derived free radicals (FRs) in degrading DCF and determined the effects of FR excitation and quenching on the DCF degradation efficiency. Finally, after identifying the degraded intermediates of 2H-labeled DCF, we constructed the DCF metabolic pathway. In addition, we evaluated the degradation and detoxification effects of the BMO composite on DCF-containing urban lake water and on biotoxicity in zebrafish embryos. Based on our findings, we proposed a mechanism for oxidative degradation of DCF by associative oxygenases and FRs.

BPA degradation using biogenic manganese oxides produced by an engineered Escherichia coli with a non-blue laccase from Bacillus sp. GZB.

Bisphenol A (BPA), an endocrine disruptor that is often found in a variety of environmental matrixes, poses a serious health risk. One of the most effective methods for completely degrading BPA is biological oxidation. This study used a non-blue laccase to develop an engineer Escherichia coli strain for the synthesis of biogenic manganese oxides (BMO). The recombinant strain LACREC3 was utilized for the efficient production of BMO. The LACREC3 strain developed the erratic clumps of BMO after prolonged growth with Mn2+, as shown by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDS) tests. After 12 days of incubation under liquid culture conditions, a total of 51.97 ± 0.56% Mn-oxides were detected. The Brunauer-Emmett-Teller (BET) surface areas, X-ray diffraction (XRD), Fourier transform infrared (FT-IR), and X-ray photoelectron spectroscopy (XPS) experiments were further used to characterize the purified BMO. Data revealed that Mn(IV)-oxides predominated in the structure of BMO, which was amorphous and weakly crystalline. The BPA oxidation assay confirmed the high oxidation efficiency of BMO particle. BMO degraded 96.16 ± 0.31% of BPA in total over the course of 60 min. The gas chromatography and mass spectroscopy (GC-MS) identified BPA-intermediates showed that BPA might break down into less hazardous substances that were tested by Photobacterium Phosphoreum in an acute toxicity experiment. Thus, employing BMO generated by a non-blue laccase, this study introduces a new biological technique of metal-oxidation and organic-pollutant degradation.

Towards BioMnOx-mediated intra/extracellular electron shuttling for doxycycline hydrochloride metabolism in Bacillus thuringiensis.

Doxycycline hydrochloride (DCH) could be continuously removed by Bacillus thuringiensis S622 with the in-situ biogenic manganese oxide (BioMnOx) via oxidizing/regenerating. The DCH removal rate was significantly increased by 3.01-fold/1.47-fold at high/low Mn loaded via the integration of biological (intracellular/extracellular electron transfer (IET/EET)) and abiotic process (BioMnOx, Mn(III) and •OH). BioMnOx accelerated IET via activating coenzyme Q to enhance electrons transfer (ET) from complex I to complex III, and as an alternative electron acceptor for respiration and provide another electron transfer transmission channel. Additionally, EET was also accelerated by stimulating to secrete flavins, cytochrome c (c-Cyt) and flavin bounded with c-Cyt (Flavins & Cyts). To our best knowledge, this is the first report about the role of BioMnOx on IET/EET during antibiotic biodegradation. These results suggested that Bacillus thuringiensis S622 incorporated with BioMnOx could adopt an alternative strategy to enhance DCH degradation, which may be of biogeochemical and technological significance.

Manganese oxidation and prokaryotic community analysis in a polycaprolactone-packed aerated biofilm reactor operated under seawater conditions.

Biogenic manganese oxides (BioMnOx) have been receiving increasing attention for the removal of environmental contaminants and recovery of minor metals from water environments. However, the enrichment of heterotrophic Mn(II)-oxidizing microorganisms for BioMnOx production in the presence of fast-growing coexisting heterotrophs is challenging. In our previous work, we revealed that polycaprolactone (PCL), a biodegradable aliphatic polyester, can serve as an effective solid organic substrate to enrich Mn-oxidizing microbial communities under seawater conditions. However, marine BioMnOx-producing bioreactor systems utilizing PCL have not yet been established. Therefore, a laboratory-scale continuous-flow PCL-packed aerated biofilm (PAB) reactor was operated for 238 days to evaluate its feasibility for BioMnOx production under seawater conditions. After the start-up of the reactor, the average dissolved Mn removal rates of 0.4-2.3 mg/L/day, likely caused by Mn(II) oxidation, were confirmed under different influent dissolved Mn concentrations (2.5-14.0 mg/L on average) and theoretical hydraulic retention time (0.19-0.77 day) conditions. The 16S rRNA gene amplicon sequencing analysis suggested the presence of putative Mn(II)-oxidizing and PCL-degrading bacterial lineages in the reactor. Two highly dominant operational units (OTUs) in the packed PCL-associated biofilm were assigned to the genera Marinobacter and Pseudohoeflea, whereas the genus Lewinella and unclassified Alphaproteobacteria OTUs were highly dominant in the MnOx-containing black/dark brown precipitate-associated biofilm formed in the reactor. Excitation-emission matrix fluorescence spectroscopy analysis revealed the production of tyrosine- and tryptophane-like components, which may serve as soluble heterotrophic organic substrates in the reactor. Our findings indicate that PAB reactors are potentially applicable to BioMnOx production under seawater conditions.

Insights into the synergistic removal mechanisms of thallium(I) by biogenic manganese oxides in a wide pH range.

The behavior and mechanism of thallium (Tl) adsorption by biogenic manganese oxides (BMnOx) are poorly understood. In this study, BMnOx was applied for Tl(I) removal from aqueous solution, and the adsorption interactions were systematically revealed for the first time. BMnOx was successfully prepared with high productivity by effectively oxidizing Mn(II) with a manganese oxide bacterium in an optimal Mn(II) concentration range of 4.0-28 mg/L. Compared with other adsorbents, the prepared BMnOx achieved high Tl(I) adsorption capacity over a wide pH range from 3.0 to 9.0 and high humic acid (HA) concentration (40 mg/L) interference. The experimental results were well depicted by pseudo-second-order kinetics and the Langmuir isotherm model, indicating that chemisorption played the dominant role during the adsorption process. The adsorption mechanisms were verified as synergetic interactions of oxidation-precipitation, electrostatic attraction, ion exchange and surface complexation. X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) results suggested that 19.46% of the highly toxic Tl(I) was transformed into the much less toxic product Tl2O3 after adsorption onto BMnOx. This study provides theoretical guidance for high-concentration Tl(I) decontamination from groundwater by biogenic manganese oxides.

Complete Degradation and Detoxification of Ciprofloxacin by a Micro-/Nanostructured Biogenic Mn Oxide Composite from a Highly Active Mn2+-Oxidizing Pseudomonas Strain.

Ciprofloxacin (CIP), as a representative broad-spectrum antibiotic, poses a major threat to human health and the ecological environment as a result of its abuse and emissions. In this study, a highly active Mn2+-oxidizing bacterium, Pseudomonas sp. CCTCC M2014168, was induced to form micro-/nanostructured biogenic Mn oxide (BMO) aggregates through continuous culturing with 1 mmoL-1 Mn2+. Following the characterization of Mn4+ oxides and the micro-/nanostructures by scanning electron microscopy, high-resolution transmission electron microscopy and X-ray diffraction assays, the BMO composites were subjected to CIP degradation and detoxification in laboratory trials. High-performance liquid chromatograph (HPLC) analysis identified that the BMO composites were capable of completely degrading CIP, and HPLC with a mass spectrometer (LC/MS) assays identified three intermediates in the degradation pathway. The reaction temperature, pH and initial ciprofloxacin concentration substantially affected the degradation efficiency of CIP to a certain extent, and the metal ions Mg2+, Cu2+, Ni2+ and Co2+ exerted significant inhibitory effects on CIP degradation. A toxicity test of the degradation products showed that CIP was completely detoxified by degradation. Moreover, the prepared BMO composite exhibited a high capacity for repeated degradation and good performance in continuous degradation cycles, as well as a high capacity to degrade CIP in real natural water.

Enrichment of marine manganese-oxidizing microorganisms using polycaprolactone as a solid organic substrate.

OBJECTIVE: Heterotrophic manganese (Mn)-oxidizing microorganisms responsible for biogenic manganese oxide (Bio-MnOx) production are fastidious. Their enrichment is not easily accomplished by merely adding a soluble organic substrate to non-sterile mixed cultures. The objective of this study was to evaluate polycaprolactone (PCL), an aliphatic polyester, as an effective solid organic substrate for the enrichment of marine Mn-oxidizing microorganisms. RESULTS: We successfully obtained marine microbial enrichment with the capacity for dissolved Mn removal and MnOx production using PCL as a solid organic substrate. The removal of dissolved Mn by the Mn-oxidizing enrichment culture followed first-order kinetics with a rate constant of 0.014 h-1. 16S rRNA gene amplicon sequencing analysis revealed that the Mn-oxidizing enrichment culture was highly dominated by operational taxonomic units related to the bacterial phyla Cyanobacteria, Planctomycetes, and Proteobacteria. CONCLUSIONS: Our data demonstrate that PCL can serve as a potential substrate to enrich Mn-oxidizing microorganisms with the ability to produce MnOx under marine conditions.

Efficient removal of 17α-ethinylestradiol from secondary wastewater treatment effluent by a biofilm process incorporating biogenic manganese oxide and Pseudomonas putida strain MnB1.

This study proposes a biofilm process to immobilize biogenic manganese oxide (BMO) and Pseudomonas putida MnB1 (BMO-MnB1), which shows excellent synergistic effects for 17α-ethinylestradiol (EE2) from secondary wastewater treatment effluent (WWTE). Modified granular activated carbon (M-GAC) was used as the packing carrier, inoculated with Pseudomonas putida MnB1 and Mn(II) to form the BMO-MnB1 biofilm. Feasibility tests were performed to compare the EE2 removal efficiency with that of the conventional biofilm process (BAC) for heterogeneous microbial communities. Results show that in the BAC, EE2 was removed mainly by adsorption, with biodegradation contributing only slightly to the overall performance. In contrast, the BMO-MnB1 biofilter outperformed the BAC. Furthermore, less than 4% of the total EE2 removed was extracted from the biofilter medium over 150 days of operation, confirming that EE2 was biodegraded by P. putida MnB1 or chemically oxidized by BMO. Our results suggest that BMO-MnB1 biofilm processes have high potential for practical applications in removal of endocrine disrupting compounds from wastewater effluent.

Effective stabilization of arsenic in contaminated soils with biogenic manganese oxide (BMO) materials.

The role of biogenic manganese oxide (BMO) materials on the stabilization of arsenic (As) in contaminated soil was investigated. Experimental results indicated that the addition of BMO was proved to be highly effective to stabilize As in soils. Bioavailable As content was decreased from 4.56 mg kg-1 in the control samples to 1.72-1.86 mg kg-1 in BMO-treated soils. X-ray absorption near edge structure (XANES) results confirmed that BMO was mainly responsible for oxidizing As(III) to As(V). Sequential extraction results indicated that the transformation of As fractions was from non-specifically adsorbed fraction to poorly-crystalline hydrous oxides fraction and residual fraction, which can decrease the risk of As in contaminated soils. Moreover, BMO had a higher efficiency in stabilizing As than two types of abiotic Mn oxides. High throughput sequencing analysis indicated that the bacterial community and diversity were significantly changed after BMO treatment. The abundance of Proteobacteria phylum, including Massilia, Phenylobacterium and Sphingomonas genera significantly increased with the increasing amount of BMO. These findings suggested that BMO can be considered as a low cost, high effectiveness and environmental friendliness material for the remediation of As contaminated soils.

Metal oxyanion removal from wastewater using manganese-oxidizing aerobic granular sludge.

As, Sb, and Cr are redox-sensitive and toxic heavy metal(loid)s, and redox reactions are usually involved in the treatment of substrates containing these elements. In this study, manganese-oxidizing aerobic granular sludge (Mn-AGS) was obtained by continuously adding Mn(II) to the sludge in a sequencing batch reactor (SBR). Morphological observations, and analyses of extracellular polymeric substances (EPS), Mn valence-states, and microbial communities were performed on the resulting sludge. After 50 days of cultivation, biogenic Mn(III,IV) oxides (bio-MnOx) accumulated up to approximately 25 mg Mn/g suspended solids (SS). X-ray photoelectron spectroscopy (XPS) revealed that the percentage of Mn(III,IV) was 87.6%. The protein (PN) component in EPS increased from 80.3 to 87.8 mg/g volatile suspended solids (VSS) during cultivation, which might be favorable for sludge granulation and heavy metal(loid) removal. Batch experiments showed that Mn-AGS was better at oxidizing As(III)/Sb(III) into less toxic As(V)/Sb(V) than traditional AGS. Remarkably, the results indicated that Mn-AGS did not oxidize Cr(III) but was able to reduce Cr(VI) into relatively harmless Cr(III). This work provided a new promising method with which to treat As(III), Sb(III), and Cr(VI) in wastewaters.

Manganese-mediated immobilization of arsenic by calcifying macro-algae, Chara braunii.

The restoration capability of charophyte Chara braunii was studied in arsenic-polluted water in the context of biogenic calcium and manganese depositions on the plant. In addition to calcite encrustation, formation of craterlike shape deposits of manganese oxides (MnOx) with diameters of 5-10 µm was detected on the cell walls of the plants grown in Mn-rich media. Relative proportions of arsenic taken up by the plant biomass to those incorporated into the calcium and manganese biominerals were determined using a modified sequential chemical extraction method. The mean total arsenic recovery from water reached its highest value at 375 mg kg-1 in treatment with HCO3- and high concentrations of Ca and Mn (40 and 2 mg L-1, respectively). The percentage of arsenic associated with the manganese deposits in the plants exposed to 0.5 mg L-1 As(III) increased from 16.3% to 51.7% of the total arsenic accumulation at low and high Mn levels (<0.05 and 2 mg L-1, respectively), that accounted for the highest Mn-bound arsenic contribution. Surface oxidation of As(III) by MnOx and subsequent precipitation-adsorption of the formed As(V) onto the evolving structure of MnOx could be a plausible mechanism for arsenic removal. The presence, and in some cases dominance of arsenic bound to the biogenic Ca and Mn deposits on the studied aquatic plant may contribute to preservation of arsenic in sediments in a less bioavailable form upon its senescence and decomposition.

Synergistic effects of biogenic manganese oxide and Mn(II)-oxidizing bacterium Pseudomonas putida strain MnB1 on the degradation of 17 α-ethinylestradiol.

While biogenic manganese oxide (BMO) generated via the oxidation of Mn(II) by the Mn-oxidizing bacteria (MOB) have received attention, the relative roles of biological activity by MOB themselves were not clearly investigated. In this study, the synergistic effects of BMO and MOB Pseudomonas putida strain MnB1 on the degradation of 17α-ethinylestradiol (EE2) was investigated. Experiments with BMO in the presence of P. putida MnB1 showed 15-fold higher removal than that with BMO alone, suggesting that EE2 degradation was mediated by the biological activity of MOB as well as abiotic reaction by BMO. Trapping experiments with pyrophosphate (PP) proved that Mn(III) intermediate formed during the biological process from Mn (II) to Mn (IV) contribute much to the EE2 removal. Also, sharp decreases in EE2 removal were observed when microbial activity was inactivated by heat treatment or sodium azide. From this study, the EE2 removal mechanisms by BMO in the presence P. putida MnB1 are described as follows: (1) abiotic oxidation of EE2 by BMO occurs. (2) P. putida MnB1 indirectly oxidizes EE2 by transferring electrons from the Mn (III) intermediate. (3) P. putida MnB1 continuously re-oxidizes the Mn(II) released from the oxidative degradation of EE2 by BMO, generating new Mn(III)-intermediates or BMO.

Biogenic manganese oxides generated by green algae Desmodesmus sp. WR1 to improve bisphenol A removal.

Biogenic manganese oxides (BioMnOx) have attracted considerable attention as active oxidants, adsorbents, and catalysts. This study investigated the characteristics of algae-generated BioMnOx and determined its oxidative activity towards bisphenol A (BPA), an endocrine disrupter. Amorphous nanoparticles with a primary Mn valency of +3 were found in BioMnOx produced by Desmodesmus sp. WR1. The mechanism might be that algal growth created conditions favorable to Mn oxidation through increasing DO and pH. Initial Mn2+ concentrations of 6, 30, and 50mgL-1 produced a maximum of 5, 13, and 11mgL-1 of BioMnOx, respectively. Mn2+-enriched cultures exhibited the highest BPA removal efficiency (∼78%), while controls only reached about 27%. BioMnOx may significantly promote BPA oxidation in algae culture, enhancing the accumulation of substrates for glycosylation. Moreover, continuous BioMnOx increase and Mn2+ decrease during BPA oxidation confirmed Mn oxide regeneration. In conclusion, Mn oxide formation by microalgae has the potential to be used for environmental remediation.

The key role of biogenic manganese oxides in enhanced removal of highly recalcitrant 1,2,4-triazole from bio-treated chemical industrial wastewater.

The secondary effluent from biological treatment process in chemical industrial plant often contains refractory organic matter, which deserves to be further treated in order to meet the increasingly stringent environmental regulations. In this study, the key role of biogenic manganese oxides (BioMnOx) in enhanced removal of highly recalcitrant 1,2,4-triazole from bio-treated chemical industrial wastewater was investigated. BioMnOx production by acclimated manganese-oxidizing bacterium (MOB) consortium was confirmed through scanning electronic microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) analysis. Pseudomonas and Bacillus were found to be the most predominant species in acclimated MOB consortium. Mn2+ could be oxidized optimally at neutral pH and initial Mn2+ concentration below 33 mg L-1. However, 1,2,4-triazole removal by BioMnOx produced occurred optimally at slightly acidic pH. High dosage of both Mn2+ and 1,2,4-triazole resulted in decreased 1,2,4-triazole removal. In a biological aerated filter (BAF) coupled with manganese oxidation, 1,2,4-triazole and total organic carbon removal could be significantly enhanced compared to the control system without the participation of manganese oxidation, confirming the key role of BioMnOx in the removal of highly recalcitrant 1,2,4-triazole. This study demonstrated that the biosystem coupled with manganese oxidation had a potential for the removal of various recalcitrant contaminants from bio-treated chemical industrial wastewater.