Leptothrix Species Research Articles

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.

Metal(loid)-resistant bacteria reduce wheat Cd and As uptake in metal(loid)-contaminated soil

This study characterized the effect of the metal(loid)-resistant bacteria Ralstonia eutropha Q2-8 and Exiguobacterium aurantiacum Q3-11 on Cd and As accumulation in wheat grown in Cd- and As-polluted soils (1 mg kg−1 of Cd + 40 mg kg−1 of As and 2 mg kg−1 of Cd + 60 mg kg−1 of As). The influence of strains Q2-8 and Q3-11 on water-soluble Cd and As and NH4+concentration and pH in the soil filtrate were also analyzed. Inoculation with these strains significantly reduced wheat plant Cd (12–32%) and As (9–29%) uptake and available Cd (15–28%) and As (22–38%) contents in rhizosphere soils compared to the controls. Furthermore, these strains significantly increased the relative abundances of the arsM bacterial As metabolism gene and of Fe- and Mn-oxidizing Leptothrix species in rhizosphere soils. Notably, these strains significantly reduced water-soluble Cd and As concentrations and increased pH and NH4+ concentration in the soil filtrate. These results suggest that these strains increased soil pH and the abundance of genes possibly involved in metal(loid) unavailability, resulting in reduced wheat Cd and As accumulation and highlight the possibility of using bacteria for in situ remediation and safe production of wheat or other food crops in metal(loid)-polluted soils.

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.

Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cells

Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aqueous-phase inorganics, especially Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid organic–inorganic nature. To verify our assumption that the organic skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examined the sorption potential of chemically and enzymatically prepared protein-free organic sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% atomic percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform infrared spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5–10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, especially via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.

Direct Adherence of Fe(III) Particles onto Sheaths of Leptothrix sp. Strain OUMS1 in Culture

Leptothrix species, one of the Fe/Mn-oxidizing bacteria, oxidize Fe(II) and produce extracellular, microtubuar, Fe-encrusted sheaths. Since protein(s) involved in Fe(II) oxidation is excreted from Leptothrix cells, the oxidation from Fe(II) to Fe(III) and subsequent Fe(III) deposition to sheaths have been thought to occur in the vicinity or within the sheaths. Previously, Fe(III) particles generated in MSVP medium amended with Fe(II) salts by abiotic oxidation were directly recruited onto cell-encasing and/or -free sheaths of L. cholodnii SP-6. In this study, whether this direct Fe(III) adherence to sheaths also occurs in silicon-glucose-peptone (SGP) medium amended with Fe(0) (SGP + Fe) was investigated using another strain of Leptothrix sp., OUMS1. Preparation of SGP + Fe with Fe powder caused turbidity within a few hours due to abiotic generation of Fe(III) particles via Fe(II), and the medium remained turbid until day 8. When OUMS1 was added to SGP + Fe, the turbidity of the medium cleared within 35 h as Fe(III) particles adhered to sheaths. When primitive sheaths, cell-killed, cell-free, or lysozyme/EDTA/SDS- and proteinase K-treated sheath remnants were mixed with Fe(III) particles, the particles immediately adhered to each. Thus, vital activity of cells was not required for the direct Fe(III) particle deposition onto sheaths regardless of Leptothrix strains.

Use of Iron Powder to Obtain High Yields of Leptothrix Sheaths in Culture

The Leptothrix species, Fe-oxidizing bacteria, produce an extracellular, microtubular sheath with a complicated organic–inorganic hybrid nature. We have discovered diverse industrial functions for this material, e.g., electrode material for Li-ion batteries, catalyst enhancers, pigments, plant growth promoters, and plant protectants. To consistently obtain material with the qualitative and quantitative stability needed for industrial applications, we focused on developing an optimum culture system for sheath synthesis by the Leptothrix sp. strain OUMS1. Although we have used Fe plates as an Fe source in the liquid silicon-glucose-peptone medium (SGP), the plates do not yield a consistent quality or precise mass, and formation of Fe-encrusted sheath is restricted to a surface of the plates, which limits harvest yield. In this study, to obtain a high yield of sheaths, we cultured OUMS1 in SGP supplemented with Fe powders. The addition of Fe powders to the medium (up to 14.0 g/L) did not adversely influence growth of OUMS1. The final yield of sheaths was about 10-fold greater than in the Fe plate culture. The sheaths also maintained a microtubular form and crystalline texture similar to those produced on Fe plates in SGP. The results proved the usefulness of Fe powder for consistently high yields of Fe-encrusted sheaths of stable quality.

Perspectives on the Biogenesis of Iron Oxide Complexes Produced by Leptothrix, an Iron-oxidizing Bacterium and Promising Industrial Applications for their Functions

Leptothrix species, one of the Fe-/Mn-oxidizing bacteria, are ubiquitous in aqueous environments, especially at sites characterized by a circumneutral pH, an oxygen gradient and a source of reduced Fe and Mn minerals. Characteristic traits that distinguish the genus Leptothrix from other phylogenetically related species are its filamentous growth and ability to form uniquely shaped microtubular sheaths through the precipitation of copious amounts of oxidized Fe or Mn. The sheath is an ingenious hybrid of organic and inorganic materials produced through the interaction of bacterial exopolymers with aqueous-phase inorganics. Intriguingly, we discovered that Leptothrix sheaths have a variety of unexpected functions that are suitable for industrial applications such as material for lithium battery electrode, a catalyst enhancer, pottery pigment among others. This review focuses on the structural and chemical properties of the Leptothrix sheaths and their noteworthy functions that show promise for development of cost-effective, eco-friendly industrial applications.

Increased Abundance of Gallionella spp., Leptothrix spp. and Total Bacteria in Response to Enhanced Mn and Fe Concentrations in a Disturbed Southern Appalachian High Elevation Wetland

The Sorrento wetland hosts several Fe- and Mn-rich seeps that are reported to have appeared after the area was disturbed by recent attempts at development. Culture-independent and culture-based analyses were utilized to characterize the microbial community at the main site of the Fe and Mn seep. Several bacteria capable of oxidizing Mn(II) were isolated, including members related to the genera Bacillus, Lysinibacillus, Pseudomonas, and Leptothrix, but none of these were detected in clone libraries. Most probable number assays demonstrated that seep and wetland sites contained higher numbers of culturable Mn-oxidizing microorganisms than an upstream reference site. When compared with quantitative real time PCR (qPCR) assays of total bacteria, MPN analyses indicated that less than 0.01% of the total population (estimated around 109 cells/g) was culturable. Light microscopy and fluorescence in situ hybridization (FISH) images revealed an abundance of morphotypes similar to Fe- and Mn-oxidizing Leptothrix spp. and Gallionella spp. in seep and wetland sites. FISH allowed identification of Leptothrix-type sheath-forming organisms in seep samples but not in reference samples. Gallionella spp. and Leptothrix spp. cells numbers were estimated using qPCR with a novel primer set that we designed. Results indicated that numbers of Gallionella, Leptothrix or total bacteria were all significantly higher at the seep site relative to the reference site (where Gallionella was below detection). Interestingly, numbers of Leptothrix in the seep site were estimated at only 107 cells/g and were not statistically different in the late summer versus the late winter, despite dramatic changes in sheath abundance (as indicated by microscopy). qPCR also indicated that Gallionella spp. may represent up to 10% (3 × 108 cells/g) of the total bacteria in seep samples. These data corroborate clone library data from samples taken in October 2008, where 11 SSU rRNA sequences related to Gallionella spp. were detected out of 77 total sequences (roughly 10–15%), and where Leptothrix sequences were not detected. Analysis of this SSU rRNA clonal library revealed that a diverse microbial community was present at seep sites. At a 3% difference cutoff, 30 different operational taxonomic units were detected out of 77 sequences analyzed. Dominant sequence types clustered among the beta- and gamma- Proteobacteria near sequences related to the genera Ideonella, Rhodoferax, Methylotenera, Methylobacter, and Gallionella. Overall, results suggest that high metal concentrations at the seep sites have enriched for Fe- and Mn-oxidizing bacteria including organisms related to Gallionella and Leptothrix species, and that members of these genera coexist within a diverse microbial community.

Development of a small-scale bioreactor method to monitor the molecular diversity and environmental impacts of bacterial biofilm communities from an acid mine drainage impacted creek

Shamokin Creek is a tributary of the Susquehanna River in central Pennsylvania that is heavily impacted by the acid mine drainage (AMD) caused by the oxidation of pyrite from the region's extensive anthracite coal mining industry. Recent studies have begun to characterize the microbial communities present in this and other AMD-impacted waters, but varying environmental conditions have complicated attempts to determine the ecological impacts of individual bacterial species within these communities. This study developed a small-scale biofilm reactor protocol that allowed us to simultaneously monitor the development of bacterial biofilm communities in AMD-impacted creek collected water using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes, while assessing the impacts that the developing biofilms were having on water quality. Our analysis confirmed that the diversity and composition of these small in situ biofilm communities could be monitored using molecular methods, and indicated the possible presence of many taxa frequently found in AMD environments, including Sulfobacillus, Nitrospira, Desulfovibrio, Geobacter, and Leptothrix species. A significant increase in the total sulfate was observed in the bioreactor, and as most likely due to the accumulation of sulfur-oxidizing bacteria such as Sulfobacillus in the biofilms. This system will allow us to study the microbial ecology of Shamokin Creek through controlled experiments that will ultimately integrate microscopic, molecular, physiological and chemical analyses, and that can be utilized to develop more effective and cost-efficient environmental remediation techniques for AMD-impacted areas.

Isolation of a Leptothrix Strain, OUMS1, from Ocherous Deposits in Groundwater

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as "biogenous iron oxide (BIOX)". The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

Occurrence of manganese-oxidizing microorganisms and manganese deposition during biofilm formation on stainless steel in a brackish surface water

Abstract Biofilm formation on 316L stainless steel was investigated in a pilotscale flow-through system fed with brackish surface water using an alternating flow/stagnation/flow regime. Microbial community analysis by denaturing gradient gel electrophoresis and sequencing revealed the presence of complex microbial ecosystems consisting of, amongst others, Leptothrix-related manganese-oxidizing bacteria in the adjacent water, and sulfur-oxidizing, sulfate-reducing and slime-producing bacteria in the biofilm. Selective plating of the biofilm indicated the presence of high levels of manganese-oxidizing microorganisms, while microscopic and chemical analyses of the biofilm confirmed the presence of filamentous manganese-precipitating microorganisms, most probably Leptothrix species. Strong accumulation of iron and manganese occurred in the biofilm relative to the adjacent water. No evidence of selective colonization of the steel surface or biocorrosion was found over the experimental period. The overall results of this study highlight the potential formation of complex microbial biofilm communities in flow-through systems thriving on minor concentrations of manganese.

Occurrence of manganese-oxidizing microorganisms and manganese deposition during biofilm formation on stainless steel in a brackish surface water

Biofilm formation on 316L stainless steel was investigated in a pilotscale flow-through system fed with brackish surface water using an alternating flow/stagnation/flow regime. Microbial community analysis by denaturing gradient gel electrophoresis and sequencing revealed the presence of complex microbial ecosystems consisting of, amongst others, Leptothrix-related manganese-oxidizing bacteria in the adjacent water, and sulfur-oxidizing, sulfate-reducing and slime-producing bacteria in the biofilm. Selective plating of the biofilm indicated the presence of high levels of manganese-oxidizing microorganisms, while microscopic and chemical analyses of the biofilm confirmed the presence of filamentous manganese-precipitating microorganisms, most probably Leptothrix species. Strong accumulation of iron and manganese occurred in the biofilm relative to the adjacent water. No evidence of selective colonization of the steel surface or biocorrosion was found over the experimental period. The overall results of this study highlight the potential formation of complex microbial biofilm communities in flow-through systems thriving on minor concentrations of manganese.