Preparation for Denitrification and Phenotypic Diversification at the Cusp of Anoxia: a Purpose for N2O Reductase Vis-à-Vis Multiple Roles of O2.

Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O2 repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed "bet-hedging," where all cells synthesize the N2O reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O2 or N2O. To test this, cells were exposed to gradual O2 depletion or sudden anoxia and subsequent spikes of O2 and N2O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS, and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e--acceptor deprivation and show that O2 spiking leads to bet-hedging, while N2O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O spike had much lower respiration rates than those rescued by the O2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy-limited systems. IMPORTANCE Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2- to N2O. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O2 or N2O. We confirm such entrapment and the rescue of all cells by an N2O spike but only a fraction by an O2 spike. The results shed light on the role of O2 repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic nirS expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of N2O, which contribute to anthropogenic climate forcing.

Carbon-driven enrichment of the crucial nitrate-reducing bacteria in limed peat soil microcosms.

Bacteria of Dechloromonas were recognized as potential functional important denitrifiers in a long-term shell sand-amended peat soil. Different microcosms in a solid matrix and slurry systems with the addition of carbon and nitrogen sources, for example, clover leaves, glutamate and nitrate, were established. The bacterial community structures were analysed by pyrosequencing of the 16S rRNA gene to select the conditions for enriching bacteria of Dechloromonas. The results showed that a relatively even bacterial community in the initial soil shifted to communities dominated by a few types of nitrate-reducing bacteria after the incubation, which strongly responded to the carbon substrates addition and consumption. The bacteria of several genera including Dechloromonas, Pseudomonas, Clostridium, Aeromonas and Ferribacterium were significantly enriched after a certain period of time. The bacteria of Dechloromonas became one of the most predominant bacteria in the incubated community. Especially when added the mixed carbon substrates into the solid soil matrix, as high as 34% of abundance was detected. This study proved that the functional important bacteria from the genus of Dechloromonas could be enriched to an extremely high abundance by using proper culture condition which will benefit to the isolation or direct metagenomics study for Dechloromonas. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of key players in a microbial community is always of important. In this study, the functional important denitrifiers in a shell sand-amended peat soil were investigated. Using different carbon sources in the incubation, we found the bacteria from the genus of Dechloromonas were enriched to an abundance of higher than 34% with several other denitrifiers together. This work provides us helpful insights not only for knowing the diversity of denitrifiers in the studied peat soil, but also for understanding their response to the carbon sources and the culture conditions.

Microbial biomass, community structure and metal tolerance of a naturally Pb-enriched forest soil.

The effect of long-term elevated soil Pb levels on soil microbiota was studied at a forest site in Norway, where the soil has been severely contaminated with Pb since the last period of glaciation (several thousand years). Up to 10% Pb (total amount, w/w) has been found in the top layer. The microbial community was drastically affected, as judged from changes in the phospholipid fatty acid (PLFA) pattern. Specific PLFAs that were high in Pb-enriched soil were branched (especially br17:0 and br18:0), whereas PLFAs common in eukaryotic organisms such as fungi (18:2omega6,9 and 20:4) were low compared with levels at adjacent, uncontaminated sites. Congruent changes in the PLFA pattern were found upon analyzing the culturable part of the bacterial community. The high Pb concentrations in the soil resulted in increased tolerance to Pb of the bacterial community, measured using both thymidine incorporation and plate counts. Furthermore, changes in tolerance were correlated to changes in the community structure. The bacterial community of the most contaminated soils showed higher specific activity (thymidine and leucine incorporation rates) and higher culturability than that of control soils. Fungal colony forming units (CFUs) were 10 times lower in the most Pb-enriched soils, the species composition was widely different from that in control soils, and the isolated fungi had high Pb tolerance. The most commonly isolated fungus in Pb-enriched soils was Tolypocladium inflatum. Comparison of isolates from Pb-enriched soil and isolates from unpolluted soils showed that T. inflatum was intrinsically Pb-tolerant, and that the prolonged conditions with high Pb had not selected for any increased tolerance.

Removal of nitrogen in different wetland filter materials: use of stable nitrogen isotopes to determine factors controlling denitrification and DNRA.

Laboratory incubations with varying O2 and NO3 concentrations were performed with a range of filter materials used in constructed wetlands (CWs). The study included material sampled from functioning CWs as well as raw materials subjected to laboratory pre-incubation. 15N-tracer techniques were used to assess the rates of denitrification versus dissimilatory nitrate reduction to ammonium (DNRA), and the relative role of nitrification versus denitrification in producing N2O. The N2O/(N2 + N2O) product ratio was assessed for the different materials. Sand, shell sand, and peat sustained high rates of denitrification. Raw light-weight aggregates (LWA) had a very low rate, while in LWA sampled from a functioning CW, the rate was similar to the one found in the other materials. The N2O/(N2 + N2O) ratio was very low for sand, shell sand and LWA from functioning CWs, but very high for raw LWA. The ratio was intermediate but variable for peat. The N2O produced by nitrification accounted for a significant percentage of the N2O accumulated during the incubation, but was dependent on the initial oxygen concentration. DNRA was significant only for shell sand taken from a functioning CW, suggesting that the establishment of active DNRA is a slower process than the establishment of a denitrifying flora.

Surface Attachment of Ammonia-Oxidizing Bacteria in Soil.

A BSTRACTIndigenous ammonia-oxidizing bacteria (AOB) in a clay loam soil were extremely difficult to release from soil particles compared to most heterotrophic bacteria; less than 1% of indigenous AOB (estimated as potential ammonia oxidation rate) were extractable by the dispersion-density-gradient centrifugation technique. This is at least 10-fold less than the extractability of heterotrophic bacteria. Urea applications to the same soil induced a 5-fold increase in the potential ammonia oxidation rate, and this resulted in a much higher percentage (8%) extractability of AOB. Thus, the newly grown AOB in the urea-treated soil were less strongly attached to the soil particles. The contrast suggests that the strong attachment of indigenous AOB is a gradual process taking place due to a long residence time (infrequent/slow cell division) compared to heterotrophic organisms. However, the contrast could also reflect differences in species composition of the original AOB community and those growing in response to urea inputs. Specific detection of AOB in extinction dilution cultures was done by PCR and sequencing of the products. Considerable diversity was found within the genus Nitrosospira, but severe problems with the specificity of the primers were observed. Two allegedly AOB specific PCR primers pairs were used: one specific for Nitrosospira (SPIRA) and one which should encompass all AOB within the beta- Proteobacteria (GAOB). Only 33% of the cultures that gave PCR products with GAOB also gave products with the SPIRA primer pair, suggesting the presence of AOB other than Nitrosospira. However, the phylogeny based on the sequencing placed all the cultures in various clusters of the Nitrosospira clade, suggesting that the SPIRA primers do not match all members of the Nitrosospira genus. The cultures obtained from the urea-treated soil were different from the others in giving PCR products only with the SPIRA primers and not with the GAOB. Since sequencing also here confirmed the presence of Nitrosospira, these observations suggest that the GAOB primers do not match all AOB species.

An evaluated improvement of the extinction dilution method for isolation of ammonia-oxidizing bacteria.

An improved method for isolation of ammonia-oxidizing bacteria (AOB) by the extinction dilution technique is described. It is important to prevent the growth of heterotrophic organisms, which may easily outnumber the AOB in mixed cultures. This was achieved by careful elimination of C sources in the medium and by sealing the cultures from contact with the atmosphere, thus excluding air-borne, volatile compounds which support growth of heterotrophs. The sealing of the cultures reduced the number of heterotrophs by a factor of 10, thus grossly increasing the chances of obtaining pure AOB cultures. Another important factor is to use actively growing 'late log' cultures during the final isolation step. This was achieved by adjusting the buffer capacity to ensure a clearly visible pH indicator shift at a stage when one-third to one-half of the ammonia had been oxidized. By this improved isolation procedure, AOB were isolated from three different locations: an arable soil, a lead-contaminated soil and an animal house. For an unknown reason, several attempts to isolate pure cultures from a forest soil were unsuccessful, despite the presence of AOB in the primary extinction dilution cultures. The isolates from soils were all Nitrosospira spp. For isolation of AOB from the animal house, two growth media were used, one containing ammonium sulfate, and one containing urea. From the cultures with ammonium sulfate, Nitrosomonas spp. were isolated, whereas Nitrosospira spp. were isolated from the cultures with urea as the main ammonia source. The identifications of all isolates are based on morphology and 16S rDNA sequences.

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N2O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N2O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N2O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N2O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N2O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH4 oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH4 in soils.

Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria.

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20-30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 µm(3)) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 µm(3)) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.

Accumulation of radiocaesium in fungi.

The accumulation of radioactive Cs by fungi was studied by analysis of fruit bodies (n (total) = 205, n greater than or equal to 5 for 22 species) collected in 1988 in a Norwegian mountain area with high deposition of radiocaesium from the Chernobyl accident. To account for site variation, the radiocaesium content of soil and plants was determined for each sampling spot. The soil contained 5-600 kBq/m2 (median = 50 kbq/m2, 134Cs + 137Cs). The plant content ranged from 0.25 to 23 Bq/g dry weight (median = 3.1 Bq/g) and was positively correlated with radiocaesium concentration in the soil (r = 0.56) and negatively correlated with soil pH (r = -0.28). The ratio between radiocaesium content in fungi and that in plants at the same spot (F/P) differed among species: 25 species had F/P values between 30 and 270, 12 species had F/P values between 10 and 30, and the rest (16 species) had F/P values below 10 (only four samples had values below 1). The concentration of nonradioactive Cs in fruit bodies was positively correlated with their radiocaesium content. Certain species selectively accumulated one or several trace elements (V, Cd, Hg, Pb, Th).

Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups.

Viable counts of heterotropic soil bacteria were 3-5 times higher on low-nutrient agar media compared with a series of conventional agar media. Substantial amounts of monosaccharides and amino acids were present in solid media made from distilled water and agar powder, and a salt-solution agar medium (without organic substrates added) gave practically the same colony counts as the low nutrient soil extract agar medium. MPN values were comparable to or lower than plate counts. A search for slow-growing cells in the negative MPN tubes by fluorescence microscopical examination after 3 months incubation was negative.The viable counts were 2-4% of the total microscopical counts in different soils. Assuming that the colony-forming cells did not derive from the numerous "dwarf" cells present in soil, a calculated percent viability of the larger cells was about 10%. The ecological significance of the plate-counting technique is discussed.

The relationship between cell size and viability of soil bacteria.

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small ("dwarf") cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4µm, about 10% for cells with diameters between 0.4 and 0.6µm, and 30-40% for cells with diameters above 0.6µm. Only 10-20% of the viable cells with diameters <0.4µm increased their diameter to >0.4µm prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.

Separation and purification of bacteria from soil.

Bacteria were released and separated from soil by a simple blending-centrifugation procedure. The percent yield of bacterial cells (microscopic counts) in the supernatants varied over a wide range depending on the soil type. The superantants contained large amounts of noncellular organic material and clay particles. Further purification of the bacterial cells was obtained by centrifugation in density gradients, whereby the clay particles and part of the organic materials sedimented. A large proportion of the bacteria also sedimented through the density gradient, showing that they had a buoyant density above 1.2 g/ml. Attachment to clay minerals and humic material may account for this apparently high buoyant density. The percent yield of cells was negatively correlated with the clay content of the soils, whereas the purity was positively correlated with it. The cell size distribution and the relative frequency of colony-forming cells were similar in the soil homogenate, the supernatants after blending-centrifugation, and the purified bacterial fraction. In purified bacterial fraction from a clay loam, the microscopically measured biomass could account for 20 to 25% of the total C and 30 to 40% of the total N as cellular C and N. The amount of cellular C and N may be higher, however, owing to an underestimation of the cell diameter during fluorescence. A part of the contamination could be ascribed to extracellular structures as well as partly decayed cells, which were not revealed by fluorescence microscopy.

Buoyant densities and dry-matter contents of microorganisms: conversion of a measured biovolume into biomass.

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm and 7% dry weight to 1.084 g/cm and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.