Phototrophic Fe(II)-oxidation in the chemocline of a ferruginous meromictic lake.

Precambrian Banded Iron Formation (BIF) deposition was conventionally attributed to the precipitation of iron-oxides resulting from the abiotic reaction of ferrous iron (Fe(II)) with photosynthetically produced oxygen. Earliest traces of oxygen date from 2.7 Ga, thus raising questions as to what may have caused BIF precipitation before oxygenic photosynthesis evolved. The discovery of anoxygenic phototrophic bacteria thriving through the oxidation of Fe(II) has provided support for a biological origin for some BIFs, but despite reports suggesting that anoxygenic phototrophs may oxidize Fe(II) in the environment, a model ecosystem of an ancient ocean where they are demonstrably active was lacking. Here we show that anoxygenic phototrophic bacteria contribute to Fe(II) oxidation in the water column of the ferruginous sulfate-poor, meromictic lake La Cruz (Spain). We observed in-situ photoferrotrophic activity through stimulation of phototrophic carbon uptake in the presence of Fe(II), and determined light-dependent Fe(II)-oxidation by the natural chemocline microbiota. Moreover, a photoferrotrophic bacterium most closely related to Chlorobium ferrooxidans was enriched from the ferruginous water column. Our study for the first time demonstrates a direct link between anoxygenic photoferrotrophy and the anoxic precipitation of Fe(III)-oxides in a ferruginous water column, providing a plausible mechanism for the bacterial origin of BIFs before the advent of free oxygen. However, photoferrotrophs represent only a minor fraction of the anoxygenic phototrophic community with the majority apparently thriving by sulfur cycling, despite the very low sulfur content in the ferruginous chemocline of Lake La Cruz.

Nutrient removal and microbial communities' development in a young unplanted constructed wetland using Bauxsol™ pellets to treat wastewater.

Municipal wastewater was treated over a six month period in an unplanted constructed wetland with a lower soil layer and an upper Bauxsol™ pellet layer. The interactions between Bauxsol™ pellets, soil, effluent and microbial communities demonstrated a positive influence on contaminant removal. Bauxsol™ treated effluent showed >95% phosphate removal and ~26% nitrogen removal during the trial. Substantial quantities of nitrate, trace-metals and Colwell P were bound to the pellets, whereas only ammonium was bound to the soil. The structure of microbial communities analysed by denaturing gradient gel electrophoresis (DGGE) showed distinct bacterial communities attached to Bauxsol™ pellets and soil owing to differences in geochemistry and micro-environmental conditions. Polymerase chain reaction (PCR) amplification of specific marker genes (i.e. bacterial and archaeal amoA genes, nosZ gene, and hzo gene) was used to evaluate the presence of microbial communities associated with nitrogen transformation. Data revealed the co-existence of aerobic ammonia-oxidising bacteria, anaerobic ammonia-oxidising bacteria (anammox) and denitrifiers attached to Bauxsol™ pellets and ammonia-oxidising bacteria and archaea attached to soil. This study successfully demonstrates that Bauxsol™ pellets are a suited alternative media for constructed wetland to treat wastewater effectively removing phosphate and serving as biomass support particles for bacterial communities associated with nitrogen-cycling.

Fungi, bacteria and soil pH: the oxalate-carbonate pathway as a model for metabolic interaction.

The oxalate-carbonate pathway involves the oxidation of calcium oxalate to low-magnesium calcite and represents a potential long-term terrestrial sink for atmospheric CO(2). In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non-sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.

Diversity of microbial communities in an attached-growth system using Bauxsol™ pellets for wastewater treatment.

Columns of Bauxsol™ pellets were used in a field experiment as biomass support particle for wastewater microbial communities. The attached microbial community structure was analysed using denaturing gradient gel electrophoresis (DGGE), targeting the 16S rDNA gene's V3 region. DGGE profiles showed that the type and composition of support particles used (i.e. Bauxsol™ pellets or gravel) had a significant impact on the attached bacterial communities (64% dissimilarity). In addition, ecological indices revealed a more heterogeneous bacterial community structure on the Bauxsol™ pellets. TOC/TN ratios post-experiment (6.5-9.3) suggested a good level of biological activity (i.e. active biofilm) in the Bauxsol™ columns. Moreover, Bauxsol™ pellets were mostly made of inorganic carbon, suggesting insoluble carbonate biomineralisation. Polymerase chain reaction (PCR) amplification of specific marker genes (i.e. bacterial and archaeal amoA genes, nosZ gene, and hzo gene) were used to identify the presence of attached bacterial communities associated with nitrogen transformation. The results along with geochemical data (i.e. up to 50% nitrogen removal) revealed co-existence of ammonia-oxidising bacteria, denitrifiers, and anammox organisms. This study conclusively demonstrates that microbial communities are well-adapted to Bauxsol™ pellets and bacterial communities involved in the nitrogen cycle are present.

Nutrient and trace-metal removal by Bauxsol pellets in wastewater treatment.

In this study, Bauxsol pellets packed in PVC columns were used to remove nutrients and trace-metals from municipal wastewater during a 6 months field trial. Bauxsol pellet columns showed a high phosphate removal rate via precipitation of PO(4)(3-) with Ca(2+) and Mg(2+) ions: at 90% in the 1st month; at 80% from the second to fifth months; and at 60% in the sixth month. Pellet bound total phosphorus and Colwell phosphorus were 7.3 g/kg and 2 g/kg and are about 20 times the concentrations found in most fertile soils. Trace-metals in effluents were bound, probably irreversibly under the columns' environmental conditions, to the Bauxsol minerals that have high surface area to volume ratios and high charge to mass ratios. Experimental results showed a complex nitrogen cycle operating within the Bauxsol pellet columns including anoxic nitrification, denitrification, and anammox processes. Although a transient pH spike, associated with the release of unreacted CaO from the cement binder used in the pellets, was observed, this may be readily corrected through post-treatment pH adjustment. Hence, the geochemistry of Bauxsol pellets can effectively remove and bind nutrients and trace-metals during wastewater treatment, and further research may show that saturated spent pellets can be used as fertilizer.

Minimising alkalinity and pH spikes from Portland cement-bound Bauxsol (seawater-neutralized red mud) pellets for pH circum-neutral waters.

Bauxsol reagents (powder, slurry, or pellet forms) are powerful tools in environmental remediation and water and sewage treatment However, when used in circum-neutral water treatments, cement-bound Bauxsol pellets produce a sustained pH and alkalinity spike due to the presence of unreacted CaO in the cement binder. This study developed a pellet treatment system to minimize the alkalinity/pH spike. The recipe for pelletization consisted of Bauxsol powder, ordinary Portland cement (OPC), hydrophilic fumed silica, aluminum powder, a viscosity modifier, and water. Several batches (including different ratios and sizes) were run using modified makeup waters (H(2)0 + CO(2) or NaHCO(3)) or curing brines (CO(2), NaHCO(3), or Mg/CaCl(2)). Alkalinity, pH stability, and slake durability tests were performed on pellets before and/or after curing. The best result for reducing the alkalinity/pH spike was obtained from a MgCl(2), CaCl(2) bath treatment using a Bauxsol:cement ratio of 2.8:1 (pH 8.28; alkalinity 75.1 mg/L) for a 100 g batch or 245:1 (pH 8.05; alkalinity 35.4 mg/L) for a 1 kg batch. Although brine curing does provide a control on pH/alkalinity release, the pellets may still contain unreacted CaO. Therefore, a freshwater rinse of pellets before treating circum-neutral waters is recommended as is the continued investigation of alternative pellet binders.

Cow dung extract: a medium for the growth of pseudomonads enhancing their efficiency as biofertilizer and biocontrol agent in rice.

Some pseudomands are being utilized as biofertilizers and biopesticides because of their role in plant growth promotion and plant protection against root parasites, respectively. Two strains of Pseudomonas, P. jessenii LHRE62 and P. synxantha HHRE81, recovered from wheat rhizosphere, have shown their potential in field bioinoculation tests under rice-wheat and pulse-wheat rotation systems. Normally, pseudomonads are cultivated on synthetic media-like King's B and used for inoculation on seeds/soil drench with talcum or charcoal as carrier material. Cow dung is being used for different purposes from the ancient time and has a significant role in crop growth because of the content in humic compounds and fertilizing bioelements available in it. Here, cow dung extract was tested as a growth medium for strains LHRE62 and HHRE81, in comparison with growth in King's B medium. The log phase was delayed by 2 h as compared to growth in King's B medium. The bacterial growth yield, lower in plain cow dung extract as compared to King's B medium, was improved upon addition of different carbon substrates. Growth of rice var. Pant Dhan 4 in pot cultures was increased using liquid formulation of cow dung extract and bacteria as foliar spray, compared to their respective controls. Biocontrol efficacy of the bioagents was assessed by challenging rice crop with Rhizoctonia solani, a sheath blight pathogen. The growth promotion and biocontrol efficiencies were more pronounced in the case of mixed inocula of strains LHRE62 and HHRE81.

Molecular detection of anammox bacteria in terrestrial ecosystems: distribution and diversity.

Anaerobic oxidation of ammonium (anammox) is recognized as an important process in the marine nitrogen cycle yet nothing is known about the distribution, diversity and activity of anammox bacteria in the terrestrial realm. In this study, we report on the detection of anammox sequences of Candidatus 'Brocadia', 'Kuenenia', 'Scalindua' and 'Jettenia' in marshes, lakeshores, a contaminated porous aquifer, permafrost soil, agricultural soil and in samples associated with nitrophilic or nitrogen-fixing plants. This suggests a higher diversity of anammox bacteria in terrestrial than in marine ecosystems and could be a consequence of the larger variety of suitable niches in soils. Anammox bacteria were not ubiquitously present but were only detected in certain soil types and at particular depths, thus reflecting specific ecological requirements. As opposed to marine water column habitats where Candidatus 'Scalindua' dominates anammox guilds, 'Kuenenia' and 'Brocadia' appear to be the most common representatives in terrestrial environments.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

Differences between bacterial communities in the gut of a soil-feeding termite (Cubitermes niokoloensis) and its mounds.

In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics.

White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition.

White lupins (Lupinus albus L.) respond to phosphate deficiency by producing special root structures called cluster roots. These cluster roots secrete large amounts of carboxylates into the rhizosphere, mostly citrate and malate, which act as phosphate solubilizers and enable the plant to grow in soils with sparingly available phosphate. The success and efficiency of such a P-acquisition strategy strongly depends on the persistence and stability of the carboxylates in the soil, a parameter that is influenced to a large extent by biodegradation through rhizosphere bacteria and fungi. In this study, we show that white lupin roots use several mechanisms to reduce microbial growth. The abundance of bacteria associated with cluster roots was decreased at the mature state of the cluster roots, where a burst of organic acid excretion and a drastic pH decrease is observed. Excretion of phenolic compounds, mainly isoflavonoids, induced fungal sporulation, indicating that vegetative growth, and thus potential citrate consumption, is reduced. In addition, the activity of two antifungal cell wall-degrading enzymes, chitinase and glucanase, were highest at the stage preceding the citrate excretion. Therefore, our results suggest that white lupin has developed a complex strategy to reduce microbial degradation of the phosphate-solubilizing agents.

How elevated pCO2 modifies total and metabolically active bacterial communities in the rhizosphere of two perennial grasses grown under field conditions.

The response of total (DNA-based analysis) and active (RNA-based analysis) bacterial communities to a pCO2 increase under field conditions was assessed using two perennial grasses: the nitrophilic Lolium perenne and the oligonitrophilic Molinia coerulea. PCR- and reverse transcriptase-PCR denaturing gradient gel electrophoresis analysis of 16S rRNA genes generated contrasting profiles. The pCO2 increase influenced mainly the active and root-associated component of the bacterial community. Bacterial groups responsive to the pCO2 increase were identified by sequencing of corresponding denaturing gradient gel electrophoresis bands. About 50% of retrieved sequences were affiliated to Proteobacteria. Our data suggest that Actinobacteria in soil and Myxococcales (Deltaproteobacteria) in root are stimulated under elevated pCO2.

Bacteria associated with spores of the arbuscular mycorrhizal fungi Glomus geosporum and Glomus constrictum.

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.

Examination of Gould's modified S1 (mS1) selective medium and Angle's non-selective medium for describing the diversity of Pseudomonas spp. in soil and root environments.

Abstract Studies on the diversity of environmental culturable Pseudomonas populations are dependent on the isolation procedure. This procedure includes the use of selective media which may influence the recovery of strains and thus the diversity described. In this study, we assessed the use of two agar isolation media for describing the diversity of soil- and root-inhabiting Pseudomonas associated with the perennial grass Molinia coerulea. A total of 382 Pseudomonas strains were recovered on either non-selective Angle's medium, or on Gould's modified S1 (mS1) Pseudomonas-selective medium. Their diversity was assessed by restriction analysis of PCR (polymerase chain reaction)-amplified 16S-23S rDNA internal transcript spacer sequences. The comparison of mS1- and Angle-recovered populations showed that the use of mS1 selective medium led to an underestimation of both Pseudomonas counts and diversity, especially in the soil environment.

Is the contribution of bacteria to terrestrial carbon budget greatly underestimated?

Some commonly found species of soil bacteria use low molecular weight organic acids as their sole source of carbon and energy. This study shows that acids such as citrate and oxalate (produced in large amounts by fungi and plants) can rapidly be consumed by these bacteria. Two strains, Ralstonia eutropha and Xanthobacter autotrophicus, were cultured on acetate- and citrate-rich media. The resulting CO2 and/or HCO3- reacted with calcium ions to precipitate two polymorphs of calcium carbonate (CaCO3), calcite and vaterite, depending on the quantity of slime produced by the strains. This production of primary calcium carbonate crystals by oxalate- and citrate-degrading bacteria from soil organic carbon sources highlights the existence of an important and underestimated potential carbon sink.

Specific PCR amplification for the genus Pseudomonas targeting the 3' half of 16S rDNA and the whole 16S-23S rDNA spacer.

A PCR protocol was developed for the selective amplification of a segment of the ribosomal RNA operon in Pseudomonas strains. Two specific conserved sequences suitable for PCR priming were identified in the middle of the 16S rDNA and at the very beginning of the 23S rDNA respectively. As a result, amplified region includes the 3' half of the 16S rDNA with the whole 16S-23S rRNA Internal Transcripted Spacer (ITS1) sequence. The specificity of the primer set was checked on sequence databases and validated on collection strains and on one hundred soil bacterial isolates. Our results showed that both collection, soil-inhabiting Pseudomonas and some Pseudomonas-related Azotobacter DNAs could be amplified. This specific PCR for the detection of Pseudomonas strains was in good agreement with colony hybridisation using a Pseudomonas-specific probe. The targeted segment is relevant for a characterisation at the species (16S rDNA) as well as at the infraspecific (ITS1) levels. This PCR-based approach offers promising potential for the characterisation of environmental Pseudomonas populations.

nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass.

Rhizosphere associative dinitrogen fixation could be a valuable source of nitrogen in many nitrogen limited natural ecosystems, such as the rhizosphere of Molinia coerulea, a hemicryptophytic perennial grass naturally occurring in contrasted oligonitrophilic soils. The diversity of the dinitrogen-fixing bacteria associated with this environment was assessed by a cloning-sequencing approach on the nifH gene directly amplified from environmental DNA extracts. Seventy-seven randomly picked clones were analysed. One type of NifH sequence was dominant in both roots and surrounding soil, and represented 56% of all retrieved sequences. This cluster included previously described environmental clones and did not contain any NifH sequences similar to cultivated diazotrophs. The predominance of few NifH sequence types in the roots and the rhizosphere of Molinia coerulea indicate that the plant environment mediates a favourable niche for such dinitrogen-fixing bacteria.

Isolation and characterization of a new type of aerobic, oxalic acid utilizing bacteria, and proposal of Oxalicibacterium flavum gen. nov., sp. nov.

A mesophilic, aerobic oxalic acid utilizing yellow-pigmented bacterium has been isolated from litter of oxalate producing plants in the region of Izmir (Turkey). It is motile by means of 1-3 polar flagella. Optimal growth occurred between 25-30 degrees C at pH 6.9. The G+C content of DNA is 62-64 mol % (Tm). Based on its morphological and biochemical features the organism belongs to the genus Pseudomonas, but differs from all the previously described species. The taxonomic relationships among strains described as or previously tentatively assigned to the genus Pseudomonas were investigated using numerical classification, DNA base composition and DNA-DNA hybridization. 16S rDNA sequences were determined for the strain TA17. On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed to classify TA17T in a new genus and species for which the name Oxalicibacterium flavum gen. nov., sp. nov. is proposed. The type strain is TA17T (= NEU98T, = LMG 21571T).