Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.     

Rock Biofertilizers with Acidithiobacillus on Sugarcane Yield and Nutrient Uptake in a Brazilian Soil

The experiment was conducted with the aim to evaluate the effects of biofertilizers with phosphate and potash rocks and soluble fertilizers (Triple super phosphate and potassium chloride) in chemical attributes of a Brazilian tableland soil grown with sugarcane. The experiment was arranged in a completely randomized factorial design 2 × 4 × 3 + 1, with four replicates. Two varieties of sugarcane, three sources of P and K mixture (natural apatite + natural biotite; P + K biofertilizers with Acidithiobacillus and P + K chemical fertilizers) were applied in four levels. A control treatment with no P and K fertilization (P₀K₀) was added for comparative purposes. Significant differences between varieties were observed in all analyzed parameters, with better results when applied the recommended levels of biofertilizers and chemical fertilizers. Stalk fresh matter increased with fertilizers and biofertilizers applications, especially when applied in levels near recommendation. Total N, total P and total K in stalk dry matter increased significantly when biofertilizers were applied. The results indicate potential use of biofertilizers that may be used as P source; however, long-term studies are necessary due to soil pH reductions and its possible adverse effects.     

Luminescence-Based Systems for Detection of Bacteria in the Environment

Abstract

The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase.

Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process.

In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.     

Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review

Evidence supporting a key role for anaerobic methane oxidation in the global methane cycle is reviewed. Emphasis is on recent microbiological advances. The driving force for research on this process continues to be the fact that microbial communities intercept and consume methane from anoxic environments, methane that would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments. Geochemical evidence for this process has been observed in numerous marine sediments along the continental margins, in methane seeps and vents, around methane hydrate deposits, and in anoxic waters. The anaerobic oxidation of methane is performed by at least two phylogenetically distinct groups of archaea, the ANME-1 and ANME-2. These archaea are frequently observed as consortia with sulfate-reducing bacteria, and the metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. The archaeal member of a consortium apparently oxidizes methane and shuttles reduced compounds to the sulfate-reducing bacteria. Despite recent advances in understanding anaerobic methane oxidation, uncertainties still remain regarding the nature and necessity of the syntrophic association, the biochemical pathway of methane oxidation, and the interaction of the process with the local chemical and physical environment. This review will consider the microbial ecology and biogeochemistry of anaerobic methane oxidation with a special emphasis on the interactions between the responsible organisms and their environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transient development of filamentous Thiothrix species in a marine sulfide oxidizing, denitrifying fluidized bed reactor

In this study, microscopic and molecular microbial analyses were integrated to characterize rapidly developing white filamentous tufts in a fluidized bed reactor used for nitrate removal from a marine recirculating fish culture system. Formation and rapid elongation of the tufts (often exceeding 50 mm day (-1)) was strongly correlated to transient elevated sulfide concentrations (>50 microM) in the reactor. The dominant bacterial constituents of these tufts were filamentous gram-negative bacteria with densely packed intracellular sulfur granules. Using 16S rRNA gene analysis and fluorescence in situ hybridization it was found that these filamentous bacteria represented a novel Thiothrix phylotype closely related (97% sequence identity) to a previously identified Thiothrix strain endogenous to the marine crustacean Urothoe poseidonis. In addition to filamentous morphotypes, rosette-shaped morphotypes of Thiothrix were also detectable within the tufts.     

Effects of Bioavailable Heavy Metal Species,Arsenic, and Acid Drainage from Mine Tailings on a Microbial Community Sampled Along a Pollution Gradient in a Freshwater Ecosystem

Sediment and water samples representing a pollution gradient in a long, narrow lake polluted at one end by heavy metals, arsenic, and acid drainage from mine tailings, together with samples from an unpolluted reference lake, were analyzed to determine effects of pollutants on the microbial community of the polluted lake. Ribosomal ribonucleic acid, fatty acid, and phospholipid analyses, along with assays of CO₂ production, denitrification, and enzyme activities, were performed to characterize the microflora; and environmental conditions were defined by various physicochemical analyses, including determination of bioavailable metal species. Mine waste pollution fostered the growth of Holophagal Acidobacteria, green sulphur bacteria, and α-Proteobacteria but inhibited numerous other types of microorganisms, reducing the overall productivity, biomass, and biodiversity of the microflora. The beneficial effects imply toleration of pollutants, suppression of competing or antagonistic species, and utilization of biogenic sulphide; and the toxic effects are attributable to bioavailable metals, arsenic, and sulphuric acid produced by oxidation of sulphides. The bioavailability and toxicity of sediment-bound metals were evidently increased by acidification, elevation of sediment Eh, and inhibition of metal-immobilizing bacteria by pollutants but were decreased by metal-scavenging oxyhydroxides, sulphide, and organic matter. Metal toxicity also depended on specific metal properties (e.g., electronegativity), providing a basis for inferring mechanisms of toxicity and oxidation states of metals and explaining differences in relative toxicity. The pollutants harmed the ecosystem as a whole by inhibiting microorganisms that performed crucial ecological functions, notably oxygen-releasing photosynthesis, decomposition and humification of organic matter, nutrient recycling, and control of metal availability.     

Legionella pneumophila in the Environment: The Occurrence of a Fastidious Bacterium in Oligotrophic Conditions

Legionella pneumophila, a micro-organism encountered in aquatic environments, can cause serious intracellular infections among humans. Since the bacterium is ubiquitous in aquatic habitats, it appears to be impossible to prevent L. pneumophila from entering man-made water systems. However, many questions concerning the survival and/or growth in the environment, the partners and opponents of L. pneumophila remain unanswered. This review focuses on the factors governing the ecology of L. pneumophila, since there is considerable divergence and even contradiction in literature on its environmental requirements. A key question to be resolved is the discrepancy between the fastidious nature of L. pneumophila in axenic cultures (e.g. 400 mg l⁻¹ L-cysteine and 250 mg l^-1 ferric iron) and the nutritionally poor environments in which it is commonly detected. It is assumed that dense microbial communities, as occurring in sediments and biofilms – but not likely in surface and drinking water, – can provide the necessary growth requirements for L. pneumophila. However, most of the studies concerning L. pneumophila have led to the general opinion that the organism can only multiply in the aquatic environment as a parasite in certain protozoa. The discovery of the non-classical siderophore legiobactin also indicates that the iron requirement for survival and autonomous growth is not as high as has been assumed. It thus appears that in order to control Legionella in the environment, focus should be on the eradication of microbial hotspots in which L. pneumophila resides.     

Towards a Molecular Taxonomy for Protists: Benefits,Risks, and Applications in Plankton Ecology

The increasing use of genetic information for the development of methods to study the diversity, distributions, and activities of protists in nature has spawned a new generation of powerful tools. For ecologists, one lure of these approaches lies in the potential for DNA sequences to provide the only immediately obvious means of normalizing the diverse criteria that presently exist for identifying and counting protists. A single, molecular taxonomy would allow studies of diversity across a broad range of species, as well as the detection and quantification of particular species of interest within complex, natural assemblages; goals that are not feasible using traditional methods. However, these advantages are not without their potential pitfalls and problems. Conflicts involving the species concept, disagreements over the true (physiological/ecological) meaning of genetic diversity, and a perceived threat by some that sequence information will displace knowledge regarding the morphologies, functions and physiologies of protistan taxa, have created debate and doubt regarding the efficacy and appropriateness of some genetic approaches. These concerns need continued discussion and eventual resolution as we move toward the irresistible attraction, and potentially enormous benefits, of the application of genetic approaches to protistan ecology.     

Activity of a Methanotrophic Consortium Isolated from Landfill Cover Soil: Response to Temperature,pH, CO2, and Porous Adsorbent

A robust, naturally evolving methanotrophic community in landfill cover soil (LFCS) can be the simplest way to mitigate landfill methane emission. In this study, bacterial community composition in LFCS and methane oxidation potential of enriched methanotrophic consortium, in comparison to that of axenic Methylosinus sporium, was investigated. Growth and methane oxidation of the consortium was studied in liquid phase batch experiments under varying temperature (20–40°C), pH (5–10), headspace CO2, and in presence of porous adsorbent (1.3 cm³ sponge cubes). The 16S rRNA gene analysis revealed presence of both type-I and type-II methanotrophs along with few obligate methylotroph in LFCS. Though the optimal growth condition of the consortium was at 30°C and pH 7, it was more resilient in comparison to M. sporium. With increasing availability of porous adsorbent, methane consumption by the consortium was significantly improved (p < 0.001) reaching a maximum specific methane oxidation rate of 11.4 μmol mg^?1 biomass h^?1. Thus, inducing naturally thriving methanotrophs in LFCS is a better alternative to axenic methanotrophic culture in methane emission management.     

Microbial Investigations in Opalinus Clay,an Argillaceous Formation under Evaluation as a Potential Host Rock for a Radioactive Waste Repository

Various deep, compact, sedimentary formations have been studied in recent years as potential host rock for a repository for high-level, long-lived radioactive waste. Considering that microbial activities may influence radionuclide chemistry and migration in such environments, we investigated the potential presence of microorganisms in the Opalinus Clay formation, from unperturbed sediment samples (i.e., not affected by gallery excavation and borehole drilling) recovered under aseptic conditions in the Mont Terri Underground Rock Laboratory (Switzerland). A combination of molecular biology techniques and a cultivation-based approach suggested the presence of a few sparse autochthonous microbial cells in the Opalinus Clay. For the first time, ribosomal RNA (rRNA) genes were sequenced from enrichment cultures from such samples. The results suggested that at least two of the bacterial strains isolated were likely unknown species of the Sphingomonas and Alicyclobacillus genera, as their fully-sequenced 16S-rRNA genes shared less than 97% similarity with validly published sequences. Early genetic divergence occurring after physical isolation of bacterial ancestors in the geosphere by the sedimentation process or following later geological events may have resulted in the generation of particular taxa in the subsurface.     

铁矿物强化厌氧氨氧化效能及其微生物机制研究进展

自然界中的氮循环与铁循环相互交联,参与氮循环的厌氧氨氧化（anaerobic ammonium oxidation,anammox）菌的生长代谢及活性发挥也与铁元素紧密关联。自然界广泛存在的铁矿物因具有运行成本低廉、稳定性好、二次污染小等优势,在污水处理领域得到广泛应用。在厌氧氨氧化脱氮系统中引入适量铁矿物,不仅有助于促进anammox菌和铁还原菌的富集,提高功能基因丰度和相关酶活性,还可能通过影响污泥浓度、血红素c含量、胞外聚合物含量和颗粒化程度,改善污泥性能和提高厌氧氨氧化系统的稳定性。同时,铁矿物具有促进体系多种氮素转化途径（如anammox、铁自养反硝化、铁氨氧化、异化硝酸盐还原成铵和反硝化）相耦合的潜能,可以提高anammox污水处理系统的总氮去除率。本文基于铁矿物在促进污水生物脱氮方面的良好性能及其在anammox系统中的变化,从脱氮效能、污泥特性、微生物特征及酶活性等方面,系统综述了铁矿物对厌氧氨氧化系统的强化作用机制,并从anammox菌对铁矿物的利用及铁元素的摄取角度展望了后续的研究方向,以期为铁矿物强化厌氧氨氧化系统的实际应用提供理论和技术指导。     

Effect of different media composition on the micropropagation of Erica andevalensis, a metal accumulator species growing in mining areas (SW Spain)

We describe a micropropagation and acclimatization protocol of Erica andevalensis Cabezudo & Rivera, using stem pieces collected from wild plants. This species always grows in soils enriched with metals such as Fe, Pb, Zn and in acid pH (2.5–4). Among the different media and hormone concentration tested, MS medium with ammonium nitrate concentration reduced to a quarter, 2 mg l⁻¹ indole-acetic acid (IAA) and 0.5 mg l⁻¹ kinetin was found to yield the best growth response in the plants. This medium produced an average of 3.6 ± 1.2 shoots and 16.9 ± 8.3 verticils per explant after 30 days of the onset of the culture. MS media achieved rooting without phytohormones. Rooted plantlets were successfully transferred to plastic containers with autoclaved perlite, watered with MS half-strength, and then to pots with commercial substrate after 2 weeks. New plants were produced in about 100 days. As far as we know, this is the first time that a replicable and complete micropropagation protocol for E. andevalensis has been developed. The plants looked healthy with no visual detectable phenotypic variations. The protocol provides a successful technique that could be used for the conservation of the species, as well as new researches on phenolics and triterpenoids compounds found in plant extracts.     

The Microbial Ecology of a Hyper-Alkaline Spring,and Impacts of an Alkali-Tolerant Community During Sandstone Batch and Column Experiments Representative of a Geological Disposal Facility for Intermediate-Level Radioactive Waste

Naturally occurring hyper-alkaline springs and associated hyper-alkaline environments may have components that are analogous to a cement-based deep geological disposal facility (GDF) for intermediate level radioactive waste (ILW). Such high pH environments could give insights into the biogeochemical processes that could occur in the region of a GDF environment after the ingress of GDF-derived groundwater leads to the formation of a hyper-alkaline plume in the surrounding rock mass. This study focuses on the microbial community composition found at a highly alkaline spring near Buxton, Derbyshire, England, and the variation in community structure across spatially separated sample points of contrasting pH values (ranging from pH 7.5–13). Communities containing alkaliphilic and alkalitolerant bacteria were observed across the site by PCR amplification and 16S rRNA gene pyrosequencing and included members of the families Comamonadaceae and Xanthomonadaceae. At pH 13, the sequence library was dominated by Gammaproteobacteria of the families Pseudomonadaceae and Enterobacteriaceae. Bacterial communities from the site demonstrated the ability to reduce Fe(III) in microcosm experiments up to pH 11.5, suggesting the potential to reduce other metals and radionuclides of relevance to cement-encapsulated intermediate level radioactive waste (ILW) disposal. In laboratory column flow-through experiments, microbial communities present at the field site were also able to colonize crushed sandstone. Bacterial community composition varied between columns that had been supplied with alkali surface waters from the site amended with carbon (lactate and acetate, as proxies for products of cellulose degradation from ILW), and control columns that were not supplied with added carbon. Members of the family Clostridiaceae dominated the sequence library obtained from the carbon amended column inlet (45.8% of library), but became less dominant at the outlet (20.8%). Members of the family Sphingomonadaceae comprised 11.8% of the sequence library obtained from the control column inlet, but were not present in sediments collected from the column outlet, whereas the relative abundance of members of the family Comamonadaceae increased from the column inlet (35.2%) to the column outlet (57.2%). The spatial variation in community composition within the columns is indicative of discrete biogeochemical zonation in these flow-through systems.     

Electron transfer of dissolved organic matter and its potential significance for anaerobic respiration in a northern bog

We investigated electron transfer processes of dissolved organic matter (DOM) and their potential importance for anaerobic heterotrophic respiration in a northern peatland. Electron accepting and donating capacities (EAC, EDC) of DOM were quantified using dissolved H₂S and ferric iron as reactants. Carbon turnover rates were obtained from porewater profiles (CO₂, CH₄) and inverse modeling. Carbon dioxide was released at rates of 0.2–5.9 mmol m⁻² day⁻¹ below the water table. Methane (CH₄) formation contributed <10%, and oxygen consumption 2% to 40%, leaving a major fraction of CO₂ production unexplained. DOM oxidized H₂S to thiosulfate and was reduced by dissolved ferric iron. Reduction with H₂S increased the subsequently determined EDC compared to untreated controls, indicating a reversibility of the electron transfer. In situ redox capacities of DOM ranged from 0.2 to 6.1 mEq g⁻¹ C (EAC) and from 0.0 to 1.4 mEq g⁻¹ C (EDC), respectively. EAC generally decreased with depth and changed after a water table drawdown and rebound by 20 and −45 mEq m⁻², respectively. The change in EAC during the water table fluctuation was similar to CH₄ formation rates. In peatlands, electron transfer of DOM may thus significantly contribute to the oxidation of reduced organic substrates by anaerobic heterotrophic respiration, or by maintaining the respiratory activity of sulfate reducers via provision of thiosulfate. Part of the anaerobic electron flow in peat soils is thus potentially diverted from methanogenesis, decreasing its contribution to the total carbon emitted to the atmosphere.