Analysis of in situ manganese(II) oxidation in the Columbia River and offshore plume: linking Aurantimonas and the associated microbial community to an active biogeochemical cycle

The Columbia River is a major source of dissolved nutrients and trace metals for the west coast of North America. A large proportion of these nutrients are sourced from the Columbia River Estuary, where coastal and terrestrial waters mix and resuspend particulate matter within the water column. As estuarine water is discharged off the coast, it transports the particulate matter, dissolved nutrients and microorganisms forming nutrient‐rich and metabolically dynamic plumes. In this study, bacterial manganese oxidation within the plume and estuary was investigated during spring and neap tides. The microbial community proteome was fractionated and assayed for Mn oxidation activity. Proteins from the outer membrane and the loosely bound outer membrane fractions were separated using size exclusion chromatography and Mn(II)‐oxidizing eluates were analysed with tandem mass spectrometry to identify potential Mn oxidase protein targets. Multi‐copper oxidase (MCO) and haem‐peroxidase enzymes were identified in active fractions. T‐RFLP profiles and cluster analysis indicates that organisms and bacterial communities capable of oxidizing Mn(II) can be sourced from the Columbia River estuary and nearshore coastal ocean. These organisms are producing up to 10 fM MnO₂ cell^?1 day^?1. Evidence for the presence of Mn(II)‐oxidizing bacterial isolates from the genera Aurantimonas, Rhodobacter, Bacillus and Shewanella was found in T‐RFLP profiles. Specific Q‐PCR probes were designed to target potential homologues of the Aurantimonas manganese oxidizing peroxidase (Mop). By comparing total Mop homologues, Aurantimonas SSU rRNA and total bacterial SSU rRNA gene copies, it appears that Aurantimonas can only account for ～1.7% of the peroxidase genes quantified. Under the broad assumption that at least some of the peroxidase homologues quantified are involved in manganese oxidation, it is possible that other organisms oxidize manganese via peroxidases.     

Sulfate Reduction Potential in Sediments in the Norilsk Mining Area,Northern Siberia

Abstract

The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a ³⁵SO₄ ^2? tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm^?3 day^?1. Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 10⁷ cells ml^?1. The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28°C vs. 4°C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4°C, but they were also found in mesophilic enrichments.     

Effects of Agronomic Treatments on Structure and Function of Ammonia-Oxidizing Communities

The aim of this study was to determine the effects of different agricultural treatments and plant communities on the diversity of ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing, was used to analyze 16S rRNA genes of ammonia oxidizers belonging to the β subgroup of the division Proteobacteria by use of DNA extracted from cultivated, successional, and native deciduous forest soils. Community profiles of the different soil types were compared with nitrification rates and most-probable-number (MPN) counts. Despite significant variation in measured nitrification rates among communities, there were no differences in the DGGE banding profiles of DNAs extracted from these soils. DGGE profiles of DNA extracted from samples of MPN incubations, cultivated at a range of ammonia concentrations, showed the presence of bands not amplified from directly extracted DNA. Nitrosomonas-like bands were seen in the MPN DNA but were not detected in the DNA extracted directly from soils. These bands were detected in some samples taken from MPN incubations carried out with medium containing 1,000 μg of NH₄⁺-N ml⁻¹, to the exclusion of bands detected in the native DNA. Cell concentrations of ammonia oxidizers determined by MPN counts were between 10- and 100-fold lower than those determined by competitive PCR (cPCR). Although no differences were seen in ammonia oxidizer MPN counts from the different soil treatments, cPCR revealed higher numbers in fertilized soils. The use of a combination of traditional and molecular methods to investigate the activities and compositions of ammonia oxidizers in soil demonstrates differences in fine-scale compositions among treatments that may be associated with changes in population size and function.     

The occurrence of chemolitho-autotrophic nitrifiers in water-saturated grassland soils

Relatively high most probable number (MPN) counts of chemolithotrophic nitrite oxidizers were present in water-saturated soils compared with MPNs and activity of ammonia oxidizers. These high numbers of nitrite oxidizers were confirmed by fluorescent antibody counts and potential activity measurements. Application of different nitrite concentrations in the MPN procedure discriminated within the community of nitrite oxidizers and revealed a large number of nitrite-sensitive nitrite oxidizers and a subcommunity of nitrite-insensitive nitrite oxidizers. The size of this subcommunity was small but corresponded with the low numbers of ammonium oxidizers. Numbers of nitrite-sensitive nitrite oxidizers outnumbered the ammonia oxidizing bacteria by 2–4 orders of magnitude in these soils. The possibility is discussed that the fraction of the nitrite-insensitive cells was active as aerobic nitrite oxidizers, whereas the nitrite-sensitive cells represented an inactive group of nitrite oxidizers growing as heterotrophs or as anaerobes reducing nitrite. In this situation, both MPN enumerations at a low nitrite concentration and activity measurements could give false information about the size of the in situ nitrite-oxidizing community.     

Microbiological quality of raw milk produced in Estonia

Aims: The microbial quality of farm bulk‐tank raw milk produced in Estonia during years 2004–2007 was investigated. Methods and Results: Bulk‐tank milk samples were analysed for lactic acid bacteria count (LABC), psychrotrophic bacteria count (PBC), aerobic spore‐forming bacteria count (ASFBC), total bacterial counts using BactoScan and somatic cell count (SCC) using Fossomatic. Randomly selected psychrotrophic isolates were subjected to 16S–23S PCR‐ribotyping. LABC remained below 10⁴ CFU ml^?1 in most samples, while psychrotrophic micro‐organisms dominated in 60% of farms. PBC ranged from 4·2 × 10² to 6·4 × 10⁴ CFU ml^?1, and ASFBC varied from 5 to 836 CFU ml^?1. Conclusions: In general, the microbiological quality of the farm bulk‐tank milk was good – more than 91% of samples contained <50 000 CFU ml^?1, and SCC in the majority of samples did not exceed the internationally recommended limits. Genus Pseudomonas spp. was the dominating spoilage flora with Pseudomonas fluorescens as the prevailing species. Significance and Impact of the Study: Specific bacterial groups (LABC, PBC and ASFBC), not analysed routinely by dairies, were determined in bulk‐tank raw milk of numerous dairy farms during 4‐year period. Based on the survey, dairy plants can better control their supply chains and select farms (milk) for the production of specific products, i.e. milk with low PBC and high LABC for cheesemaking.     

Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism

Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and freshwater environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal medium with [3‐¹³C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23–0.29 h^?1) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo‐steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636–640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate. Biotechnol. Bioeng. 2009;102: 1161–1169. © 2008 Wiley Periodicals, Inc.     

Microbial diversity of Minnesota peatlands

Microbial diversity, numbers, and metabolic activities in Minnesota peatlands were investigated using a variety of microbial enrichment and enumeration procedures together with radioisotopic measurements of microbial degradative processes. Minnesota peatlands were shown to contain large microbial populations of wide metabolic diversity. Direct counts of bacteria using epifluorescence microscopy indicated bacterial populations of about 10⁸ ml^–1 of peatland water, irrespective of depth. Radioisotopic most-probable-number (MPN) counts of heterotrophs able to mineralize¹⁴C-labeled substrates to¹⁴CO₂ showed significant populations of glucose degraders (10⁴–10⁶ ml^–1) as well as degraders of benzoate (10²–10³ ml^–1), 2,4-dichlorophenoxyacetate (10²–10⁵ ml^–1), and sphagnum (10³–10⁷ ml^–1) in the various peatlands examined. The MPNs of NO₃ ^– reducers varied from 10³–10⁶ ml^–1, SO₄ ^– reducers from 10²–10³ ml^–1, methanogenic bacteria from 10³–10⁶ ml^–1, and methane oxidizers from 10³–10⁴ ml^–1, depending on sampling site and depth. Eighty pure cultures of aerobic bacteria and fungi were isolated from Minnesota peats. Most of those cultures tested were able to grow on at least 20 organic compounds (carbohydrates, aromatic molecules, hydrocarbons, etc.) as sole sources of carbon and energy. One isolate, aBacillus, was able to fix atmospheric N₂. Several of the isolates were able to mineralize¹⁴C-labeled lignin.     

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces

Aims: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. Methods and Results: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤1 μg ml^?1, while 16 μg ml^?1 of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre‐enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre‐enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 μg ml^?1), S (400 μg ml^?1), R (30 μg ml^?1) and colistin (C, 100 U ml^?1) (assigning as BAM‐CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. Conclusion: BAM‐CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. Significance and Impact of the Study: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.     

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10⁸ pfu ml^?1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10⁶ and 10² cfu ml^?1). In contrast, broth inoculated with 10⁴ phage and 10² bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10⁵ cfu ml^?1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml^?1. Phages could be produced with titres of 10¹⁰ pfu ml^?1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.     

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml^?1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml^?1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml^?1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml^?1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml^?1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.     

Prevention of the accumulation of Alicyclobacillus in apple concentrate by restricting the continuous process running time

Aims: To study the accumulation of vegetative cells and endospores of Alicyclobacillus, as well as viable aerobic counts during the continuous production of apple juice concentrate. Methods and Results: Apples were processed for a continuous process running time of 108 h (processing rate 1·8–2·0 t h^?1) without clean‐in‐place (CIP) procedures in‐between different batches. Samples from single‐strength apple juice, concentrate after evaporation (±30°Brix), the final product (concentrate pasteurized at 102–104°C for 90 s) and condensate water (by‐product of the juice concentration process) were collected every 12 h. From 12 to 84 h of processing, vegetative Alicyclobacillus counts in single‐strength apple juice increased significantly (P < 0·05) from 1 to 3·15 log₁₀ CFU ml^?1. Accumulation patterns of vegetative cells in apple concentrate and the final product were similar from 24 to 84 h of processing, with the respective counts increasing from 0·13 to 1·63 and 0·01 to 1·69 log₁₀ CFU ml^?1. The highest Alicyclobacillus endospore counts in single‐strength juice, concentrate and the final product was at 84 h of processing with 1·32, 1·59 and 1·64 log₁₀ CFU ml^?1, respectively. Conclusions: Alicyclobacillus vegetative cells and endospores accumulate in fruit concentrates during a continuous process running time of 108 h. Significance and Impact of the Study: In conjunction with good manufacturing practices, fruit concentrate manufactures can minimize Alicyclobacillus accumulation in fruit concentrates by limiting the continuous process running time between clean‐ups to under 84 h.     

Flow‐injection determination of manganese (II) using surfactant enhanced diperiodatonickelate (IV)‐rhodamine 6G chemiluminescence

Chemiluminescence (CL) of the rhodamine 6‐G‐diperiodatonickelate (IV) (Rh6‐G‐Ni(IV) complex) in the presence of Brij‐35 was examined in an alkaline medium and implemented using flow‐injection analysis to analyze Mn(II) in natural waters. Brij‐35 was identified as the surfactant of choice that enhanced CL intensity by about 62% of the reaction. The calibration curves were linear in the range 1.7 × 10^?3 – 0.2 (0.9990, n = 7) and 8.0 × 10^?4 – 0.1 μg ml^?1 (0.9990, n = 7) with limits of detection (LODs) (S:N = 3) of 5.0 × 10^?4 and 2.4 × 10^?4 μg ml^?1 without and with using an in‐line 8‐hydroxyquinoline (8‐HQ) resin mini‐column, respectively. The sample throughput and relative standard deviation were 200 h^?1 and 1.7–2.2% in the range studied respectively. Mn(II) concentrations in certified reference materials and natural water samples was successfully determined. A brief discussion about the possible CL reaction mechanism is also given. In addition, analysis of V(III), Cr(III) and Fe(II) was also performed without and with using an in‐line 8–HQ column and selective elution of each metal ion was achieved by adjusting the pH of the sample carrier stream with aqueous HCl solution.     

Occurrence of thermotolerant Campylobacter species in surface waters of a Mediterranean area and in its prevailing pollution sources

Aims: To determine the prevalence of Campylobacter in surface waters of a highly populated Mediterranean area. Methods and Results: Surface water and wastewater samples were collected from an area in the north‐east of Spain during a 2‐year study. All the samples were analysed using the MPN method and Multiplex PCR to quantify and identify Campylobacter. It was detected in 82% of the samples from the Llobregat River with a mean of 1·3 MPN 100 ml^?1. The lowest counts were obtained in summer. Campylobacter coli was the predominant species in this river. The bacteria were isolated from marsh water but not from seawater samples. The highest counts of campylobacters were found in poultry wastewater where Camp. jejuni was the predominant species, as in urban sewage. In pig slurry, Camp. coli was the only species detected. Conclusions: Campylobacter jejuni and Camp. coli are present and widely distributed in the surface water of the studied area. The two species co‐exist, with Camp. coli being predominant. In river water, campylobacter counts presented a seasonal distribution. No relationship with faecal indicators was found. Significance and Impact of the Study: This study provides the first data on the occurrence and concentrations of thermotolerant campylobacter species in surface water in a Mediterranean area.     

New approach to use ethidium bromide monoazide as an analytical tool

Aims: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real‐time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. Methods and Results: We performed a low‐dose double EMA treatment. Live or heat‐dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 μg ml^?1 of EMA followed by a treatment with 10 μg ml^?1 of EMA without washing, and direct real‐time PCR. We observed that DNA amplification from 10⁷ cells ml^?1 of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 10²–10³ cells ml^?1 of viable cells could be detected. When a 3‐h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 10⁰–10² cells ml^?1. We compared the low‐dose double‐treated EMA‐PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. Conclusions: We concluded that the newly developed low‐dose double‐treated EMA‐PCR is a very effective tool for live Ent. sakazakii detection in PIF. Significance and Impact of the Study: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.     

Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf,Antarctica

Marine sediments of the Ross Sea, Antarctica, harbor microbial communities that play a significant role in the decomposition, mineralization, and recycling of organic carbon (OC). In this study, the cell densities within a 153‐cm sediment core from the Ross Sea were estimated based on microbial phospholipid fatty acid (PLFA) concentrations and acridine orange direct cell counts. The resulting densities were as high as 1.7 × 10⁷ cells mL^?1 in the top ten centimeters of sediments. These densities are lower than those calculated for most near‐shore sites but consistent with deep‐sea locations with comparable sedimentation rates. The δ¹³C measurements of PLFAs and sedimentary and dissolved carbon sources, in combination with ribosomal RNA (SSU rRNA) gene pyrosequencing, were used to infer microbial metabolic pathways. The δ¹³C values of dissolved inorganic carbon (DIC) in porewaters ranged downcore from ?2.5‰ to ?3.7‰, while δ¹³C values for the corresponding sedimentary particulate OC (POC) varied from ?26.2‰ to ?23.1‰. The δ¹³C values of PLFAs ranged between ?29‰ and ?35‰ throughout the sediment core, consistent with a microbial community dominated by heterotrophs. The SSU rRNA gene pyrosequencing revealed that members of this microbial community were dominated by β‐, δ‐, and γ‐Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Among the sequenced organisms, many appear to be related to known heterotrophs that utilize OC sources such as amino acids, oligosaccharides, and lactose, consistent with our interpretation from δ¹³C_PLFA analysis. Integrating phospholipids analyses with porewater chemistry, δ¹³C_DIC and δ¹³C_POC values and SSU rRNA gene sequences provides a more comprehensive understanding of microbial communities and carbon cycling in marine sediments, including those of this unique ice shelf environment.     

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting

Aims:  Zero‐valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. Methods:  A field‐scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c. 8·5 log CFU 100 ml^?1 of Escherichia coli O157:H12 was introduced to all three column treatments in 20‐l doses. Filtered waters were subsequently overhead irrigated to ‘Tyee’ spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. Results:  ZVI filters inactivated c. 6 log CFU 100 ml^?1E. coli O157:H12 during filtration on day 0, significantly (P < 0·05) more than S filter (0·49 CFU 100 ml^?1) when compared to control on day 0 (8·3 log CFU 100 ml^?1). On day 0, spinach plants irrigated with ZVI‐filtered water had significantly lower E. coli O157 counts (0·13 log CFU g^?1) than spinach irrigated with either S‐filtered (4·37 log CFU g^?1) or control (5·23 log CFU g^?1) water. Soils irrigated with ZVI‐filtered water contained E. coli O157:H12 populations below the detection limit (2 log CFU g^?1), while those irrigated with S‐filtered water (3·56 log CFU g^?1) were significantly lower than those irrigated with control (4·64 log CFU g^?1). Conclusions:  ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. Significance and Impact of the Study:  Zero‐valent ion treatment may be a cost‐effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.     

Methane Oxidation and the Competition for Oxygen in the Rice Rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O₂ with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently <5 μM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters

Aims: To better understand and manage the fate and transport of Salmonella in agricultural watersheds, we developed a culture‐based, five tube–four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters. Methods and Results: The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media for Salmonella and a TaqMan confirmation step. This method has determined the density of Salmonella in 20‐l samples of pond inflow and outflow streams as low as 0·1 MPN l^?1 and a low 95% confidence level 0·015 MPN l^?1. Salmonella densities ranged from not detectable to 0·55 MPN l^?1 for pond inflow samples and from not detectable to 3·4 MPN l^?1 for pond outflow samples. Salmonella densities of pond inflow samples were associated with densities of Escherichia coli and faecal enterococci that indicated stream contamination with faeces and with nondetectable pond outflow densities of the faecal indicator bacteria. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of pond inflow (mean flux of 2·5 l s^?1) and outflow (mean flux of 5·6 l s^?1). Fluxes of Salmonella ranged from 100 to greater than 10⁴ MPN h^?1. Conclusions: This is a culture‐based method that can detect small numbers of Salmonella in environmental waters of watersheds containing animal husbandry and wildlife. Significance and Impact of the Study: Applying this method to environmental waters will improve our understanding of the transport and fate of Salmonella in agricultural watersheds, and can be the basis of valuable collections of environmental Salmonella.     

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.     

Molecular characterization of activated sludge from a seawater‐processing wastewater treatment plant

The prokaryotic community composition of activated sludge from a seawater‐processing wastewater treatment plant (Almeria, Spain) was investigated by using the rRNA approach, combining different molecular techniques such as denaturing gradient gel electrophoresis (DGGE), clone libraries and in situ hybridization (FISH and CARD‐FISH). Most of the sequences retrieved in the DGGE and the clone libraries were similar to uncultured members of different phyla. The most abundant sequence recovered from Bacteria in the clone library corresponded to a bacterium from the Deinococcus–Thermus cluster (almost 77% of the clones), and the library included members from other groups such as the Alpha, Gamma and Delta subclasses of Proteobacteria, the Bacteroidetes and Firmicutes. Concerning the archaeal clone library, we basically found sequences related to different orders of methanogenic Archaea, in correspondence with the recovered DGGE bands. Enumeration of DAPI (4′,6‐diamidino‐2‐phenylindole) stained cells from two different activated sludge samples after a mechanical flocculation disruption revealed a mean cell count of 1.6 × 10⁹ ml^?1. Around 94% of DAPI counts (mean value from both samples) hybridized with a Bacteria specific probe. Alphaproteobacteria were the dominant bacterial group (36% of DAPI counts), while Beta‐, Delta‐ and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes contributed to lower proportions (between 0.5–5.7% of DAPI counts). Archaea accounted only for 6% of DAPI counts. In addition, specific primers for amplification of the amoA (ammonia monooxygenase) gene were used to detect the presence of Beta, Gamma and archaeal nitrifiers, yielding positive amplifications only for Betaproteobacteria. This, together with negative in situ hybridizations with probes for well‐known nitrifiying bacteria, suggests that nitrification is performed by still undetected microorganisms. In summary, the combination of the three approaches provided different and complementary pictures of the real assemblage composition and allowed to get closer to the main microorganisms involved in key processes of seawater‐processing activated sludge.