Potential Contribution of Anammox to Nitrogen Loss from Paddy Soils in Southern China

The anaerobic oxidation of ammonium (anammox) process has been observed in diverse terrestrial ecosystems, while the contribution of anammox to N₂ production in paddy soils is not well documented. In this study, the anammox activity and the abundance and diversity of anammox bacteria were investigated to assess the anammox potential of 12 typical paddy soils collected in southern China. Anammox bacteria related to “Candidatus Brocadia” and “Candidatus Kuenenia” and two novel unidentified clusters were detected, with “Candidatus Brocadia” comprising 50% of the anammox population. The prevalence of the anammox was confirmed by the quantitative PCR results based on hydrazine synthase (hzsB) genes, which showed that the abundance ranged from 1.16 × 10⁴ to 9.65 × 10⁴ copies per gram of dry weight. The anammox rates measured by the isotope-pairing technique ranged from 0.27 to 5.25 nmol N per gram of soil per hour in these paddy soils, which contributed 0.6 to 15% to soil N₂ production. It is estimated that a total loss of 2.50 × 10⁶ Mg N per year is linked to anammox in the paddy fields in southern China, which implied that ca. 10% of the applied ammonia fertilizers is lost via the anammox process. Anammox activity was significantly correlated with the abundance of hzsB genes, soil nitrate concentration, and C/N ratio. Additionally, ammonia concentration and pH were found to be significantly correlated with the anammox bacterial structure.     

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis

We investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.     

Construction of an Efficient Biologically Contained Pseudomonas putida Strain and Its Survival in Outdoor Assays

Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn5 transposon bearing a fusion of the P_lac promoter to the gef killing gene and a fusion of the Pm promoter to the lacI gene plus the positive regulator of the Pm promoter, the xylS gene. This mini-Tn5 transposon was transferred to the chromosome of Pseudomonas putida CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide in the absence of this aromatic compound. The rate of killing escape was on the order of 10⁻⁸ per cell and per generation. This contained strain and an uncontained control strain were used in outdoor tests performed in the spring-summer and autumn-winter periods to determine their survival in planted and unplanted soils with and without 3-methylbenzoate. In unplanted soils the numbers of both the contained strain and the uncontained strain per gram of soil tended to decrease, but the numbers of the contained strain decreased faster in soils without 3-methylbenzoate. The decrease in the number of CFU per gram of soil was faster in the spring-summer period than in the autumn-winter period. In planted soils survival in the rhizosphere and survival in bulk soil were studied. In the rhizosphere the uncontained control strain tended to become established at levels on the order of 10⁵ to 10⁶ CFU/g of soil regardless of the presence of 3-methylbenzoate. In the bulk soil the numbers of bacterial cells were 2 to 3 orders of magnitude lower. In planted soils the contained strain tended to disappear, but this tendency was more pronounced in the absence of 3-methylbenzoate and occurred faster in the summer assay than in the winter assay. We found no evidence of dispersal of the test strains outside the experimental plots.     

Population Densities of Rhizobium japonicum Strain 123 Estimated Directly in Soil and Rhizospheres

Rhizobium japonicum serotype 123 was enumerated in soil and rhizospheres by fluorescent antibody techniques. Counting efficiency was estimated to be about 30%. Indigenous populations of strain 123 ranged from a few hundred to a few thousand per gram of field soil before planting. Rhizosphere effects from field-grown soybean plants were modest, reaching a maximum of about 2 × 10⁴ cells of strain 123 per g of inner rhizosphere soil in young (16-day-old) plants. Comparably slight rhizosphere stimulation was observed with field corn. High populations of strain 123 (2 × 10⁶ to 3 × 10⁶ cells per g) were found only in the disintegrating taproot rhizospheres of mature soybeans at harvest, and these populations declined rapidly after harvest. Pot experiments with the same soil provided data similar to those derived from the field experiments. Populations of strain 123 reached a maximum of about 10⁵ cells per g of soybean rhizosphere soil, but most values were lower and were only slightly higher than values in wheat rhizosphere soil. Nitrogen treatments had little effect on strain 123 densities in legume and nonlegume rhizospheres or on the nodulation success of strain 123. No evidence was obtained for the widely accepted theory of specific stimulation, which has been proposed to account for the initiation of the Rhizobium-legume symbiosis.     

Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism

Damping-off disease is caused by Rhizoctonia solani and leads to serious loss in many crops. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Optical micrographs, scanning electron micrographs, and the determination of hydrolytic enzymes were used to investigate the antagonism of Trichoderma harzianum SQR-T37 (SQR-T37) against R. solani. Experiments were performed in pots to assess the in vivo disease-control efficiency of SQR-T37 and bio-organic fertilizer. The results indicate that the mycoparasitism was the main mechanism accounting for the antagonistic activity of SQR-T37. In one experiment, the population of R. solani was decreased from 10⁶ internal transcribed spacer (ITS) copies per gram soil to 10⁴ ITS copies per gram soil by the presence of the antagonist. In this experiment, 45% of the control efficiency was obtained when 8 g of SQR-T37 hyphae per gram soil was applied. In a second experiment, as much as 81.82% of the control efficiency was obtained when bio-organic fertilizer (SQR-T37 fermented organic fertilizer, BIO) was applied compared to only 27.27% of the control efficiency when only 4 g of SQR-T37 hyphae per gram soil was applied. Twenty days after incubation, the population of T. harzianum was 4.12 × 10⁷ ITS copies per gram soil in the BIO treatment, which was much higher than that in the previous treatment (8.77 × 10⁵ ITS copies per gram soil), where only SQR-T37 was applied. The results indicated that SQR-T37 was a potent antagonist against R. solani in a mycoparasitic way that decreased the population of the pathogen. Applying BIO was more efficient than SQR-T37 application alone because it stabilized the population of the antagonist.     

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.     

Bacterial Community Structure in Two Permafrost Wetlands on the Tibetan Plateau and Sanjiang Plain,China

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10¹⁰ 16S rRNA gene copies per gram of wet soil in both wetlands, with 10⁸ pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.     

Specific Detection and Real-Time PCR Quantification of Potentially Mycophagous Bacteria Belonging to the Genus Collimonas in Different Soil Ecosystems

The bacterial genus Collimonas has the remarkable characteristic that it grows at the expense of living fungal hyphae under laboratory conditions. Here, we report the first field inventory of the occurrence and abundance of Collimonas in soils (n = 45) with naturally different fungal densities, which was performed in order to test the null hypothesis that there is a relationship between the presence of Collimonas and fungal biomass. Estimates of fungal densities were based on ergosterol measurements. Each soil was also characterized in terms of its physical and chemical properties and vegetation and management types. Culturable Collimonas was identified in plate-spread soil samples by its ability to clear colloidal chitin, in combination with a Collimonas-specific restriction fragment length polymorphism analysis of 16S rRNA PCR amplified from individual colonies. Using this approach, we found culturable collimonads only in (semi)natural grasslands. A real-time PCR assay for the specific quantification of Collimonas 16S rRNA in total soil DNA was developed. Collimonas was detectable in 80% of the soil samples, with densities up to 10⁵ cells g⁻¹ (dry weight) soil. The numbers of Collimonas cells per gram of soil were consistently lowest in fungus-poor arable soils and, surprisingly, also in fungus-rich organic layers of forest soils. When all soils were included, no significant correlation was observed between the number of Collimonas cells and ergosterol-based soil fungal biomass. Based on this result, we rejected our null hypothesis, and possible explanations for this were addressed.     

Further studies on the distribution of a nuclear-polyhedrosis virus of the fall webworm,Hyphantria cunea,in soil

The median infectious concentration (IC₅₀) of a nuclear-polyhedrosis virus of the fall webworm, Hyphantria cunea, in soil was 7.14×10⁷ polyhedra per gram of soil. The number of virus-killed larvae equivalent to IC₅₀ decreased with the advance of the larval instar from 22 first-instar to 0.04 seventh-instar larvae per gram of soil. The degree of virus contamination of the surface soil in an area where the virus disease had occurred naturally in host populations was estimated to be as high as IC₅₀ on the basis of soil bioassays. Nearly 1000 virus-killed seventh-instar larvae/m² were necessary to obtain such extensive soil contamination. The possibility of the occurrence of so many virus-killed cadavers in the study area was discussed in relation with the past records of epizootics and host population trends. The distribution of the fallen leaves of large trees favorable to the fall webworm, such as plane and poplar trees, correlated with that of the nuclear-polyhedrosis virus in the soil.     

Abundance and Diversity of Viruses in Six Delaware Soils

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 10⁹ to 4.17 × 10⁹ g⁻¹ dry weight) than in agricultural soils (8.7 × 10⁸ to 1.1 × 10⁹ g⁻¹ dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.     

Enhancement of Population Densities of Pseudomonas putida PpG7 in Agricultural Ecosystems by Selective Feeding with the Carbon Source Salicylate

Sodium salicylate (1,000 μg/ml) was delivered through a drip irrigation system to agricultural field soils planted to tomato and infested with Pseudomonas putida PpG7, the host of the salicylate catabolic plasmid NAH7. In nonfumigated soils infested with approximately 10³ CFU of PpG7 per g in the top 30 cm, population densities were increased up to 112-fold within 14 days of the initial application of salicylate compared with the densities in the respective nonamended soils. Mean season-long population densities of PpG7 in the top 30 cm of soil were significantly increased (P < 0.01) from 216 CFU/g in nonamended soils to 1,370 CFU/g in salicylate-amended soils. In the respective rhizosphere soils, mean population densities of PpG7 were significantly increased (P < 0.01) from 92 to 2,066 CFU/cm of root. Soil fumigation interacted (P < 0.01) with salicylate amendment and further increased the mean population densities of PpG7 in nonrhizosphere soil by an additional 5,689 CFU/g of soil. This fumigation effect was not detected in rhizosphere soils. The effect of salicylate in increasing population densities of PpG7 in soil also was affected by inoculum level, field site, and soil depth. Proportionate differences were greater in soils infested with approximately 10³ CFU of PpG7 per g than in comparable soils infested with 10⁵ CFU/g. In low-inoculum soils, increases from salicylate amendments were 26- and 29-fold in rhizosphere and nonrhizosphere soils, respectively, and in high-inoculum soils, the respective increases were 5.6- and 5-fold. No increases of fungi able to utilize salicylate were detected in soils amended with salicylate. However, soil fumigation with metham-sodium significantly reduced (P < 0.01) population densities of fungal salicylate utilizers in rhizosphere and nonrhizosphere soils.     

DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement     

Cultural and Environmental Factors Affecting the Longevity of Escherichia coli in Histosols

The survival of Escherichia coli in organic soils (Histosols) was examined. The death rate of this organism in Pahokee muck was less than that observed in Pompano fine sand. The number of viable E. coli cells found in the muck was approximately threefold greater than that found in the sand following 8 days of incubation. The initial population of the coliform affected the death rate. The rate of loss of viability varied 100-fold when the population size decreased from 2.5 × 10⁷ to 3.4 × 10⁴. Other factors affecting the viability of E. coli in muck were aerobic versus anaerobic growth of the organism and moist versus flooded conditions in the soil. The greatest survival of the coliform was noted with anaerobically grown cells amended to flooded soil. That the observed decrease in E. coli viability in soil was the result of biotic factors was demonstrated with amendment of sterile soil with E. coli. When 1.1 × 10⁵ bacteria per g of soil were added to sterile muck, a population of 3.0 × 10⁷ organisms per g of soil developed over a 10-day period. The role of the protozoa in eradication of the coliform from the muck was indicated by a sixfold increase in the protozoan population in natural soil amended with E. coli. Higher organic matter content in a Histosol compared with a mineral soil resulted in an increased survival of the fecal coliforms. Biotic factors are instrumental in the decline in coliform populations, but the potential for growth of the coliform in the organic soil could extend the survival of the organism.     

Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils

Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5×10³ copy numbers per gram of soil, but increased by over a thousand fold to 10⁷ copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.     

Distribution of rare actinomycetes in Japanese soils

The distribution of rare actinomycetes in 237 soil samples from various locations throughout Japan was investigated using a special isolation medium, HV agar.The populations (colony forming units) of these actinomycetes per gram of dried soil were Microtetraspora 6 × 10³, Saccharomonospora 1.7 × 10⁴, Dactylosporangium 5.4 × 10⁴, Streptosporangium 1.2 × 10⁵, Microbispora 1.4 × 10⁵, Nocardioforms 1.9 × 10⁵, and Micromonospora 6.8 × 10⁵. Streptomycetes 2.2 × 10⁶, and Unidentified actinomycetes 0.9 × 10⁶ were also observed.Their distributions seemed to be associated with environmental factors such as soil type (Land Use Classification), soil pH, humus content, and the characteristics of the humic acid. In general, the largest populations were found in soils of cultivated fields, which were rich in humus and had pH values between 6.5–7.0.However, the distribution of some genera in cultivated field soils (154 samples) was remarkable. The numbers of Microbispora and Streptosporangium were the largest in humus-rich acidic (pH 5.0–6.05) soils with low humic acid Δ log K values (black colored humic acid). Saccharomonospora was found most frequently in relatively humus-poor alkaline (pH 7.0–7.5) soils having higher Δ log K values (brown humic acid).Dactylosporangium and Microtetraspora, Saccharomonospora, and Micromonospora were most frequently isolated from mountainous forest soils, level-land forest or cultivated field soils, and pasture soils, respectively.     

Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 10⁷ ± 1.9 × 10⁷ cells ml⁻¹. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.     

Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R ²?=?1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0?~?5?×?10⁸ and 0?~?5?×?10⁷ cells per gram of soil, respectively (n?=?5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract^?1 as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA^?1) at ≥5?×?10⁵ MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y?=?a?·?x) revealed excellent quantitative agreement between the two technologies (a?=?0.98, R ²?=?0.97 in the CupMBT set and a?=?0.90, R ²?=?0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.     

Enumeration of Thiobacilli within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil

The Lemoine tailings of Chibougamau, Quebec, Canada, were deposited as a pH-neutral mineral conglomerate consisting of aluminum-silicates, iron-aluminum-silicates, pyrite, chalcopyrite, and sphalerite. These tailings are colonized by an active population of Thiobacillus ferrooxidans which is localized to an acid zone occupying 40% of the tailings' surface. This population peaked at 7 × 10⁸ most probable number per gram of tailings during July and August 1990 and extended to a depth of 40 cm from the surface. Examination of samples over this depth profile by transmission electron microscopy and electron dispersive spectroscopy revealed a microbially mediated mineral transition from sulfides (below 40 cm) to chlorides and phosphates (at the surface). Silicate minerals were unaltered by microbial action. Transmission electron microscopy showed a tight association between Thiobacillus species and the sulfide minerals, which helps account for their prominence in tailings environments. Accurate enumeration of T. ferrooxidans from tailings required the disruption of their bonding to the mineral interface. Vortexing of a 10% aqueous suspension of the tailings material prior to most-probable-number analysis best facilitated this release. Even though heavy metals were highly mobile under acidic conditions at the Lemoine tailings, it was evident by transmission electron microscopy and electron dispersive spectroscopy that they were being immobilized as bona fide fine-grain minerals containing iron, copper, chlorine, phosphorus, and oxygen on bacterial surfaces and exopolymers. This biomineralization increased with increasing bacterial numbers and was most evident in the upper 3 cm of the acidic zone.     

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus)

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10³ and 0.5 × 10² cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10⁴, 3.1 × 10⁶, and 2.3 × 10⁹ viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10³ viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10³ viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts. Correspondence to: Thomas Andtid