Combined biochar and metal-immobilizing bacteria reduces edible tissue metal uptake in vegetables by increasing amorphous Fe oxides and abundance of Fe- and Mn-oxidising Leptothrix species.

In this study, a highly effective combined biochar and metal-immobilizing bacteria (Bacillus megaterium H3 and Serratia liquefaciens CL-1) (BHC) was characterized for its effects on solution Pb and Cd immobilization and edible tissue biomass and Pb and Cd accumulation in Chinese cabbages and radishes and the mechanisms involved in metal-polluted soils. In the metal-containing solution treated with BHC, the Pb and Cd concentrations decreased, while the pH and cell numbers of strains H3 and CL-1 increased over time. BHC significantly increased the edible tissue dry weight by 17-34% and reduced the edible tissue Pb (0.32-0.46 mg kg-1) and Cd (0.16 mg kg-1) contents of the vegetables by 24-45%. In the vegetable rhizosphere soils, BHC significantly decreased the acid-soluble Pb (1.81-2.21 mg kg-1) and Cd (0.40-0.48 mg kg-1) contents by 26-47% and increased the reducible Pb (18.2-18.8 mg kg-1) and Cd (0.38-0.39 mg kg-1) contents by 10-111%; while BHC also significantly increased the pH, urease activity by 115-169%, amorphous Fe oxides content by 12-19%, and relative abundance of gene copy numbers of Fe- and Mn-oxidising Leptothrix species by 28-73% compared with the controls. These results suggested that BHC decreased edible tissue metal uptake of the vegetables by increasing pH, urease activity, amorphous Fe oxides, and Leptothrix species abundance in polluted soil. These results may provide an effective and eco-friendly way for metal remediation and reducing metal uptake in vegetables by using combined biochar and metal-immobilizing bacteria in polluted soils.

Hunter-Gatherers Harvested and Heated Microbial Biogenic Iron Oxides to Produce Rock Art Pigment.

Red mineral pigment use is recognized as a fundamental component of a series of traits associated with human evolutionary development, social interaction, and behavioral complexity. Iron-enriched mineral deposits have been collected and prepared as pigment for use in rock art, personal adornment, and mortuary practices for millennia, yet little is known about early developments in mineral processing techniques in North America. Microanalysis of rock art pigments from the North American Pacific Northwest reveals a sophisticated use of iron oxide produced by the biomineralizing bacterium Leptothrix ochracea; a keystone species of chemolithotroph recognized in recent advances in the development of thermostable, colorfast biomaterial pigments. Here we show evidence for human engagement with this bacterium, including nanostructural and magnetic properties evident of thermal enhancement, indicating that controlled use of pyrotechnology was a key feature of how biogenic iron oxides were prepared into paint. Our results demonstrate that hunter-gatherers in this area of study prepared pigments by harvesting aquatic microbial iron mats dominated by iron-oxidizing bacteria, which were subsequently heated in large open hearths at a controlled range of 750 °C to 850 °C. This technical gesture was performed to enhance color properties, and increase colorfastness and resistance to degradation. This skilled production of highly thermostable and long-lasting rock art paint represents a specialized technological innovation. Our results contribute to a growing body of knowledge on historical-ecological resource use practices in the Pacific Northwest during the Late Holocene.Figshare link to figures: https://figshare.com/s/9392a0081632c20e9484.

Long Range Correlation in Redox Potential Fluctuations Signals Energetic Efficiency of Bacterial Fe(II) Oxidation.

Differentiating biotic and abiotic processes in nature remains a persistent challenge, specifically in evaluating microbial contributions to geochemical processes through time. Building on previous work reporting that biologically-influenced systems exhibit stronger long-range correlation than abiotic systems, this study evaluated the relationship between long-range correlation of redox potential and oxidation rates of circumneutral microaerophilic bacterial Fe(II) oxidation using a series of batch microcosms with bacteriogenic iron oxides (BIOS). Initial detrended fluctuation analysis (DFA) scaling exponents of the abiotic microcosms were lower (ca. 1.20) than those of the biotic microcosms (ca. 1.80). As Fe(II) oxidation proceeded, correlation strength decayed as a logistic function of elapsed reaction time, exhibiting direct dependence on the free energy of reaction. Correlation strength for all microcosms decayed sharply from strong correlation to uncorrelated fluctuations. The decay rates are greater for abiotic microcosms than biotic microcosms. The ΔGm relaxation edges for biotic microcosms were lower, indicating less remaining free energy for Fe(II) oxidation than abiotic systems, with the implication that biologically-catalyzed reactions are likely more energetically efficient than abiotic reactions. These results strengthen the case for employing novel DFA techniques to distinguish in situ microbial metabolic activity from abiotic processes, as well as to potentially differentiate metabolisms among different chemoautotrophs.

Effect of hydraulic retention time on electricity generation using a solid plain-graphite plate microbial fuel cell anoxic/oxic process for treating pharmaceutical sewage.

Treatment efficiency and electricity generation were evaluated using a solid plain-graphite plate microbial fuel cell (MFC) anoxic/oxic (A/O) process that treated pharmaceutical sewage using different hydraulic retention times (HRT). Short HRTs increased the volumetric organic loading rate, thereby reducing the MFC performance due to rapid depletion of the substrate (carbon/nitrogen source). The COD removal efficiency decreased from 96.28% at a HRT of 8 h to 90.67% at a HRT of 5 h. The removal efficiency of total nitrogen was reduced from 74.16% at a HRT of 8 h to 53.42% at a HRT of 5 h. A shorter HRT decreased the efficiency in treatment of the pharmaceutical products (PPs), which included acetaminophen, ibuprofen and sulfamethoxazole in an aerobic reactor because these antibiotic compounds inhibited the microbial activity of the aerobic activated sludge in the MFC A/O system. The average power density and coulombic efficiency values were 162.74 mW m-2 and 7.09% at a HRT of 8 h and 29.12 mW m-2 and 2.23% at a HRT of 5 h, respectively. The dominant bacterial species including Hydrogenophaga spp., Rubrivivax spp. and Leptothrix spp., which seem to be involved in PP biodegradation; these were identified in the MFC A/O system under all HRT conditions for the first time using next generation sequencing. Bacterial nanowires were involved in accelerating the transfer of electrons and served as mediators in the SPGRP biofilm. In conclusion, a SPGRP MFC A/O system at a HRT of 8 h gave better removal of COD, T-N and PPs, as well as generated more electricity.

Amino group in Leptothrix sheath skeleton is responsible for direct deposition of Fe(III) minerals onto the sheaths.

Leptothrix species produce microtubular organic-inorganic materials that encase the bacterial cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with inorganic material. Functional carboxyl groups of polysaccharides in these fibrils are considered to attract and bind metal cations, including Fe(III) and Fe(III)-mineral phases onto the fibrils, but the detailed mechanism remains elusive. Here we show that NH2 of the amino-sugar-enriched exopolymer fibrils is involved in interactions with abiotically generated Fe(III) minerals. NH2-specific staining of L. cholodnii OUMS1 detected a terminal NH2 on its sheath skeleton. Masking NH2 with specific reagents abrogated deposition of Fe(III) minerals onto fibrils. Fe(III) minerals were adsorbed on chitosan and NH2-coated polystyrene beads but not on cellulose and beads coated with an acetamide group. X-ray photoelectron spectroscopy at the N1s edge revealed that the terminal NH2 of OUMS1 sheaths, chitosan and NH2-coated beads binds to Fe(III)-mineral phases, indicating interaction between the Fe(III) minerals and terminal NH2. Thus, the terminal NH2 in the exopolymer fibrils seems critical for Fe encrustation of Leptothrix sheaths. These insights should inform artificial synthesis of highly reactive NH2-rich polymers for use as absorbents, catalysts and so on.

Biogenic nanosized iron oxides obtained from cultivation of iron bacteria from the genus Leptothrix.

A detailed investigation of nanostructured iron oxides/(oxy)hydroxides gathered after cultivation of bacteria from the genus Leptothrix as iron (II) oxidizers is presented. A specific type of medium is selected for the cultivation of the bacteria. Results for sediment powder and bio-film on glass substrate samples from the same media are discussed. XRD, Raman spectroscopy, SEM, and TEM images and PPMS measurements are used to prove the exact composition of the biogenic products and to interpret the oxidation process. Analysis of the data collected shows that around 80 % of the iron (II) from the growth medium has been transformed into iron (III) in the form of different (oxy)hydroxides, with the rest found to be in a mixed 2,5 valence in magnetite. Our investigation shows that the bio-film sample has a phase content different from that of the powdered biomass and that lepidocrocite (γ-FeOOH) is the predominant and the initial biogenic phase in both samples. Magnetite nanoparticles are a secondary product in the bio-film, part of which possesses a defective quasi-maghemite surface layer. In the powdered biomass, the oxidation steps are not fully completed. The initial products are non-stoichiometric and due to the mixed ferric and ferrous ions present, they develop into: (i) lepidocrocite (γ-FeOOH) as a basic sediment, (ii) magnetite (Fe3O4) and (iii) goethite (α-FeOOH) in small quantities. The average size of all iron-bearing particles is found to be below 30 nm. The magnetic measurements performed show a superparamagnetic behavior of the material at room temperature.

Isolation of Sphaerotilus-Leptothrix strains from iron bacteria communities in Tierra del Fuego wetlands.

Sheath-forming iron- and manganese-depositing bacteria belonging to the Sphaerotilus-Leptothrix group (SLG) are widespread in natural and artificial water systems. Known requirements for their growth include the presence of organic substrates and molecular oxygen. High concentrations of reduced iron or manganese, although not necessary for most species, make their growth a noticeable phenomenon. Such microbial communities have been studied mostly in the Northern Hemisphere. Here, we present descriptions of diverse ochre-depositing microbial communities in Tierra del Fuego, Argentina, using a combined approach of microscopical examination, clone library construction and cultivation focused on SLG bacteria. To date, only few SLG type strains are available. The present work increases the number and diversity of cultivated SLG bacteria by obtaining isolates from biofilms and sediment samples of wetlands in Tierra del Fuego. Thirty isolates were selected based on morphological features such as sheath formation and iron/manganese deposition. Five operational taxonomic units (OTUs) were deduced. Sequencing of 16S rRNA genes showed that one OTU is identical to the Leptothrix mobilis Feox-1(T) -sequence while the four remaining OTUs show similarity values related to previously described type strains. Similarity values ranged from 96.5% to 98.8%, indicating possible new species and subspecies.

Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

Bacterial nanometric amorphous Fe-based oxide: a potential lithium-ion battery anode material.

Amorphous Fe(3+)-based oxide nanoparticles produced by Leptothrix ochracea, aquatic bacteria living worldwide, show a potential as an Fe(3+)/Fe(0) conversion anode material for lithium-ion batteries. The presence of minor components, Si and P, in the original nanoparticles leads to a specific electrode architecture with Fe-based electrochemical centers embedded in a Si, P-based amorphous matrix.

Nano-micrometer-architectural acidic silica prepared from iron oxide of Leptothrix ochracea origin.

We prepared nano-micrometer-architectural acidic silica from a natural amorphous iron oxide with structural silicon which is a product of the iron-oxidizing bacterium Leptothrix ochracea. The starting material was heat-treated at 500 °C in a H2 gas flow leading to segregation of α-Fe crystalline particles and then dissolved in 1 M hydrochloric acid to remove the α-Fe particles, giving a gray-colored precipitate. It was determined to be amorphous silica containing some amount of iron (Si/Fe = ~60). The amorphous silica maintains the nano-microstructure of the starting material-~1-µm-diameter micrometer-tubules consisting of inner globular and outer fibrillar structures several tens of nanometer in size-and has many large pores which are most probably formed as a result of segregation of the α-Fe particles on the micrometer-tubule wall. The smallest particle size of the amorphous silica is ~10 nm, and it has a large surface area of 550 m(2)/g with micropores (0.7 nm). By using pyridine vapor as a probe molecule to evaluate the active sites in the amorphous silica, we found that it has relatively strong Brønsted and Lewis acidic centers that do not desorb pyridine, even upon evacuation at 400 °C. The acidity of this new silica material was confirmed through representative two catalytic reactions: ring-opening reaction and Friedel-Crafts-type reaction, both of which are known to require acid catalysts.

Acidic amorphous silica prepared from iron oxide of bacterial origin.

Microporous and mesoporous silica derived from biogenous iron oxide is an attractive catalyst for various organic reactions. Biogenous iron oxide contains structural silicon, and amorphous silica remains after iron oxide is dissolved in concentrated hydrochloric acid. The amorphous silica containing slight amounts of iron (Si/Fe = ∼150) is composed of ∼6-nm-diameter granular particles. The amorphous silica has a large surface area of 540 m(2)/g with micropores (1.4 nm) and mesopores (<3 nm). By using pyridine vapor as a probe molecule to evaluate the active sites in the amorphous silica, it was found that this material has strong Brønsted and Lewis acid sites. When the catalytic performance of this material was evaluated for reactions including the ring opening of epoxides and Friedel-Crafts-type alkylations, which are known to be catalyzed by acid catalysts, this material showed yields higher than those obtained with common silica materials.

Patterns of sheath elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodnii.

Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class ß-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chemical perspective, the sheath can be described as a supermolecule composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, we attempted to detect sheath elongation through microscopic examination by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, we assumed that the newly secreted thiopeptidoglycan molecules are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidation in microcultures was also examined with respect to cultivation time. Results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.

An uncertain role for Cu(II) in stimulating Mn(II) oxidation by Leptothrix discophora SS-1.

In an effort to improve understanding of the role of Cu(II) in bacterial Mn(II) oxidation, a model Mn(II)-oxidizing bacterium, Leptothrix discophora SS-1, was grown in presence of toxic and non-toxic concentrations of Cu(II), Cd(II) and Mn(II). Mn(II)-oxidizing activity increased by 40% when cells were grown in the presence of 0.05 microM of Cu(II) and increased twofold at 0.18 microM Cu(II). Toxic levels of Cd(II) did not stimulate Mn(II) oxidizing activity, indicating that Mn(II) oxidation is not a response to metal toxicity. Stimulation by Cu(II) confirms the specific role of Cu(II) in Mn(II) oxidation. Comparison of transcript levels of the multicopper oxidase mofA gene in the presence and absence of added Cu(II) do not indicate a statistically significant change in mofA transcript levels in cultures supplemented with Cu(II). Thus, the exact role of Cu(II) in Mn(II) oxidation and its affect on mofA gene expression remain uncertain.

Virus removal by biogenic cerium.

The rare earth element cerium has been known to exert antifungal and antibacterial properties in the oxidation states +III and +IV. This study reports on an innovative strategy for virus removal in drinking water by the combination of Ce(III) on a bacterial carrier matrix. The biogenic cerium (bio-Ce) was produced by addition of aqueous Ce(III) to actively growing cultures of either freshwater manganese-oxidizing bacteria (MOB) Leptothrix discophora or Pseudomonas putida MnB29. X-ray absorption spectroscopy results indicated that Ce remained in its trivalent state on the bacterial surface. The spectra were consistent with Ce(III) ions associated with the phosphoryl groups of the bacterial cell wall. In disinfection assays using a bacteriophage as model, it was demonstrated that bio-Ce exhibited antiviral properties. A 4.4 log decrease of the phage was observed after 2 h of contact with 50 mg L(-1) bio-Ce. Given the fact that virus removal with 50 mg L(-1) Ce(III) as CeNO(3) was lower, the presence of the bacterial carrier matrix in bio-Ce significantly enhanced virus removal.

Microbiologically-influenced corrosion of orthodontic alloys: a review of proposed mechanisms and effects.

AIMS: To summarise the currently available evidence on the effects of microorganisms on the corrosion processes of biomaterials, with specific relevance to orthodontic alloys. METHODS: Factors related to the micro-environmental milieu, conditions, site characteristics, microbial species, enzymes and compounds released in response to their attachment to surfaces and formation of biofilms are analysed with respect to their contributory roles in corrosion. RESULTS: Application to orthodontics is projected from relevant evidence, albeit hypothetical due to the lack of extensive investigations of the subject. The micro-organisms involved and effects observed in orthodontic alloys are summarised. Finally, protective measures are proposed to minimise the potential for corrosion processes in orthodontic patients.

Atomic-scale structure of biogenic materials by total X-ray diffraction: a study of bacterial and fungal MnOx.

Biogenic materials are produced by microorganisms and are typically found in a nanophase state. As such, they are difficult to characterize structurally. In this report, we demonstrate how high-energy X-ray diffraction and atomic pair distribution function analysis can be used to determine the atomic-scale structures of MnO(x) produced by bacteria and fungi. These structures are well-defined, periodic, and species-specific, built of Mn-O(6) octahedra forming birnessite-type layers and todorokite-type tunnels, respectively. The inherent structural diversity of biogenic material may offer opportunities for practical applications.

Iron requirement for Mn(II) oxidation by Leptothrix discophora SS-1.

A common form of biocatalysis of Mn(II) oxidation results in the formation of biogenic Mn(III, IV) oxides and is a key reaction in the geochemical cycling of Mn. In this study, we grew the model Mn(II)-oxidizing bacterium Leptothrix discophora SS-1 in media with limited iron (0.1 microM iron/5.8 mM pyruvate) and sufficient iron (0.2 microM iron/5.8 mM pyruvate). The influence of iron on the rate of extracellular Mn(II) oxidation was evaluated. Cultures in which cell growth was limited by iron exhibited reduced abilities to oxidize Mn(II) compared to cultures in medium with sufficient iron. While the extracellular Mn(II)-oxidizing factor (MOF) is thought to be a putative multicopper oxidase, Mn(II) oxidation in the presence of zero added Cu(II) was detected and the decrease in the observed Mn(II) oxidation rate in iron-limited cultures was not relieved when the medium was supplemented with Cu(II). The decline of Mn(II) oxidation under iron-limited conditions was not accompanied by siderophore production and is unlikely to be an artifact of siderophore complex formation with Mn(III). The temporal variations in mofA gene transcript levels under conditions of limited and abundant iron were similar, indicating that iron limitation did not interfere with the transcription of the mofA gene. Our quantitative PCR results provide a step forward in understanding the regulation of Mn(II) oxidation. The mechanistic role of iron in Mn(II) oxidation is uncertain; the data are consistent with a direct requirement for iron as a component of the MOF or an indirect effect of iron resulting from the limitation of one of many cellular functions requiring iron.

Manganese removal during bench-scale biofiltration.

As biological manganese (Mn) removal becomes a more popular water treatment technology, there is still a large gap in understanding the key mechanisms and range of operational characteristics. This research aimed to expand on previous bench-scale experiments by directly comparing small filtration columns inoculated with indigenous biofilms from a Mn filtration plant and filtration columns inoculated with a liquid suspension of Leptothrix discophora SP-6. Batch tests found that in the absence of manganese oxidizing bacteria Mn was not removed by air alone, whereas a mixed population and Leptothrix strain achieved greater than 90% removal of Mn. The bench-scale biofiltration experiments found that biological filters can be inoculated with a pure culture of L. discophora SP-6 and achieve a similar removal of indigenous biofilm. While Mn oxidizing bacteria (MOB) seem to be necessary for the auto-catalytic nature of these biological filters, Mn removal is achieved with a combination of adsorption to Mn oxides and biological oxidation. Additionally, it was demonstrated that biological Mn removal is possible over a broader "field of activity" (e.g., Mn removal occurred at a pH level as low as 6.5) than has previously been reported. The ability of this treatment technology to work over a broader range of influent conditions allows for more communities to consider biological treatment as an option to remove Mn from their drinking water.

Manganese-oxidizing bacteria mediate the degradation of 17α-ethinylestradiol.

Manganese (II) and manganese-oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17α-ethinylestradiol (EE2) at trace concentrations. Mn(2+)-derived higher oxidation states of Mn (Mn(3+), Mn(4+)) by Mn(2+)-oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn(2+)-dependent degradation of EE2 was found to be a slow process, which requires multi-fold excess of Mn(2+) and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2-derived degradation products were shown to no longer exhibit undesirable estrogenic activity.

Control of ferrous iron oxidation within circumneutral microbial iron mats by cellular activity and autocatalysis.

Ferrous iron (Fe2+) oxidation by microbial iron mat samples, dominated by helical stalks of Gallionella ferruginea or sheaths of Leptothrix ochracea, was examined. Pseudo-first-order rate constants for the microbial mat samples ranged from 0.029 +/- 0.004 to 0.249 +/- 0.042 min(-1) and correlated well with iron content (R2 = 0.929). Rate constants for Na azide-treated (1 mM) samples estimated autocatalytic oxidation by iron oxide stalks or sheaths, with values ranging from 0.016 +/- 0.008 to 0.062 +/- 0.006 min(-1). Fe2+ oxidation attributable to cellular activities was variable with respect to sampling location and sampling time, with rate constants from 0.013 +/- 0.005 to 0.187 +/- 0.037 min(-1). Rates of oxidation of the same order of magnitude for cellular processes and autocatalysis suggested that bacteria harnessing Fe2+ as an energy source compete with their own byproducts for growth, not chemical oxidation (under conditions where aqueous oxygen concentrations are less than saturating). The use of cyclic voltammetry within this study for the simultaneous measurement of Fe2+ and oxygen allowed the collection of statistically meaningful and reproducible data, two factors that have limited aerobic, circumneutral, Fe2+ -oxidation rate studies.