Peter Roslev

Stimulation by ammonium-based fertilizers of methane oxidation in soil around rice roots.

Methane is involved in a number of chemical and physical processes in the Earths atmosphere, including global warming. Atmospheric methane originates mainly from biogenic sources, such as rice paddies and natural wetlands; the former account for at least 30% of the global annual emission of methane to the atmosphere. As an increase of rice production by 60% is the most appropriate way to sustain the estimated increase of the human population during the next three decades, intensified global fertilizer application will be necessary: but it is known that an increase of the commonly used ammonium-based fertilizers can enhance methane emission from rice agriculture. Approximately 10–30% of the methane produced by methanogens in rice paddies is consumed by methane-oxidizing bacteria associated with the roots of rice; these bacteria are generally thought to be inhibited by ammonium-based fertilizers, as was demonstrated for soils and sediments. In contrast, we show here that the activity and growth of such bacteria in the root zone of rice plants are stimulated after fertilization. Using a combination of radioactive fingerprinting and molecular biology techniques, we identify the bacteria responsible for this effect. We expect that our results will make necessary a re-evaluation of the link between fertilizer use and methane emissions, with effects on global warming studies.

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of 13C-Labeled Cell Lipids

ABSTRACT Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [14C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [13C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The 13C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The 13C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in 13C, suggesting that actinomycetes metabolized phenanthrene in this soil. The 13C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess 13C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [13C]phenanthrene.

State of the art molecular markers for fecal pollution source tracking in water

Most environmental waters are susceptible to fecal contamination from animal and/or human pollution sources. To attenuate or eliminate such contamination, it is often critical that the pollution sources are rapidly and correctly identified. Fecal pollution source tracking (FST) is a promising research area that aims to identify the origin(s) of fecal pollution in water. This mini-review focuses on the potentials and limitations of library independent molecular markers that are exclusively or strongly associated with fecal pollution from humans and different animals. Fecal-source-associated molecular markers include nucleic acid sequences from prokaryotes and viruses associated with specific biological hosts, but also sequences such as mitochondrial DNA retrieved directly from humans and animals. However, some fecal-source-associated markers may not be absolutely specific for a given source type, and apparent specificity and frequency established in early studies are sometimes compromised by new studies suggesting variation in specificity and abundance on a regional, global and/or temporal scale. It is therefore recommended that FST studies are based on carefully selected arrays of markers, and that identification of human and animal contributions are based on a multi-marker toolkit with several markers for each source category. Furthermore, future FST studies should benefit from increased knowledge regarding sampling strategies and temporal and spatial variability of marker ratios. It will also be important to obtain a better understanding of marker persistence and the quantitative relationship between marker abundance and the relative contribution from individual fecal pollution source types. A combination of enhanced pathogen screening methods, and validated quantitative source tracking techniques could then contribute significantly to future management of environmental water quality including improved microbial risk assessment.

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

ABSTRACT Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. TwoNitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC50], 6 to 38 mg liter−1) was affected much less by LAS than the growth rate and viability (EC50, 3 to 14 mg liter−1) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter−1); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH4+ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge

Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

ABSTRACT Most heterotrophic bacteria assimilate CO2 in various carboxylation reactions during biosynthesis. In this study, assimilation of 14CO2 by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient 14CO2 during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of 14C-labeled organic substrates. Experiments with E. coli showed that 14CO2 was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO2-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [14C]oleic acid. However, the new HetCO2-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO2−, whereas the addition of O2 or NO3− initiated growth, as indicated by detectable 14CO2 assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO2-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO2-MAR results were supported by stable isotope analysis of 13C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of 13CO2. In conclusion, the novel HetCO2-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark

OBJECTIVES The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). METHODS The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. RESULTS Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. CONCLUSIONS Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Formation of nonculturable Escherichia coli in drinking water

Aims:  To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division.

Dynamics of a pasture soil microbial community after deposition of cattle urine amended with [13C]urea.

ABSTRACT Within grazed pastures, urine patches are hot spots of nitrogen turnover, since dietary N surpluses are excreted mainly as urea in the urine. This short-term experiment investigated 13C uptake in microbial lipids after simulated deposition of cattle urine at 10.0 and 17.1 g of urea C m−2. Confined field plots without or with cattle urine amendment were sampled after 4 and 14 days, and soil from 0- to 5-cm and 10- to 20-cm depths was analyzed for content and composition of phospholipid fatty acids (PLFAs) and for the distribution of urea-derived 13C among individual PLFAs. Carbon dioxide emissions were quantified, and the contributions derived from urea were assessed. Initial changes in PLFA composition were greater at the lower level of urea, as revealed by a principal-component analysis. At the higher urea level, osmotic stress was indicated by the dynamics of cyclopropane fatty acids and branched-chain fatty acids. Incorporation of 13C from [13C]urea was low but significant, and the largest amounts of urea-derived C were found in common fatty acids (i.e., 16:0, 16:1ω7c, and 18:1ω7) that would be consistent with growth of typical NH4+-oxidizing (Nitrosomonas) and NO2−-oxidizing (Nitrobacter) bacteria. Surprisingly, a 20‰ depletion of 13C in the cyclopropane fatty acid cy17:0 was observed after 4 days, which was replaced by a 10 to 20‰ depletion of that in cy19:0 after 14 days. Possible reasons for this pattern are discussed. Autotrophic nitrifiers could not be implicated in urea hydrolysis to any large extent, but PLFA dynamics and the incorporation of urea-derived 13C in PLFAs indicated a response of nitrifiers which differed between the two urea concentrations.