Flemming Ekelund

Method for Spiking Soil Samples with Organic Compounds

ABSTRACT We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.

Distribution and Composition of Microbial Populations in a Landfill Leachate Contaminated Aquifer (Grindsted, Denmark).

A bstractTo investigate whether landfill leachates affected the microbial biomass and/or community composition of the extant microbiota, 37 samples were collected along a 305-m transect of a shallow landfill-leachate polluted aquifer. The samples were analyzed for total numbers of bacteria by use of the acridine orange direct count method (AODC). Numbers of dominant, specific groups of bacteria and total numbers of protozoa were measured by use of the most probable number method (MPN). Viable biomass estimates were obtained from measures of ATP and ester-linked phospholipid fatty acid (PLFA) concentrations. The estimated numbers of total bacteria by direct counts were relatively constant throughout the aquifer, ranging from a low of 4.8 × 106 cells/g dry weight (dw) to a high of 5.3 × 107 cells/g dw. Viable biomass estimates based on PLFA concentrations were one to three orders of magnitude lower with the greatest concentrations (up to 4 × 105 cells/g dw) occurring at the border of the landfill and in samples collected from thin lenses of clay and silt with sand streaks. Cell number estimates based on ATP concentrations were also found to be lower than the direct count measurements (<2.2 × 106 cells/g dw), and with the greatest concentrations close to the landfill. Methanogens (Archaea) and reducers of sulfate, iron, manganese, and nitrate were all observed in the aquifer. Methanogens were found to be restricted to the most polluted and reduced part of the aquifer at a maximum cell number of 5.4 × 104 cells/g dw. Populations of sulfate reducers decreased with an increase in horizontal distance from the landfill ranging from a high of 9.0 × 103 cells/g dw to a low of 6 cells/g dw. Iron, manganese, and nitrate reducers were detected throughout the leachate plume all at maximum cell numbers of 106 cells/g dw. Changes in PLFA profiles indicated that a shift in microbial community composition occurred with increasing horizontal distance from the landfill. The types and patterns of lipid biomarkers suggested that increased proportions of sulfate- and iron-reducing bacteria as well as certain microeukaryotes existed at the border of the landfill. The presence of these lipid biomarkers correlated with the MPN results. There was, however, no significant correlation between the abundances of the specific PLFA biomarkers and quantitative measurements of redox processes. The application of AODC, MPN, PLFA, and ATP analyses in the characterization of the extant microbiota within the Grindsted aquifer revealed that as distance increased from the leachate source, viable biomass decreased and community composition shifted. These results led to the conclusion that the landfill leachate induced an increase in microbial cell numbers by altering the subsurface aquifer so that it was conducive to the growth of methanogens and of iron-and sulfate-reducing bacteria and fungi.

Long-term manipulation of the microbes and microfauna of two subarctic heaths by addition of fungicide, bactericide, carbon and fertilizer

Nutrient availability is a major constraint to plant production and carbon storage in arctic ecosystems, but there are few studies coupling processes in the decomposer and microbial food web and the implications these processes have on the control of nutrient mineralization. We studied the relationship between microbial biomass and the abundance of microbivore and the role of grazing on nutrient turnover after annual addition of carbon (sucrose), fertilizer (NPK), fungicide (benomyl) and bactericides (streptomycin and penicillin) to two dwarf shrub communities, a low and a high altitude heath. After four years of repeated additions, we measured microbial biomass by fumigation-extraction and phospholipid fatty acid (PLFA) analysis, the fungal to bacterial biomass ratio by PLFA analysis and estimated the numbers of protozoa and nematodes, assigned into feeding groups. The fungal to bacterial ratio of nematode feeding groups was around 0.2, indicating a bacterial-based food web in both communities. The size of the microbial biomass did not change after the additions, except when the amount of available carbon was increased (low altitude heath) or when addition of carbon was combined with fertilizer (high altitude heath). In contrast, fertilizer but not carbon increased the number of microbivores. This suggests that the amount of available carbon and not grazing pressure controls the size of the microbial biomass. Furthermore, it suggests that the food quality, e.g. nutrient content of the micro-organisms, had a larger effect on the microbivore than the size of the microbial biomass. The addition of bactericides and fungicide did not significantly change the fungal to bacterial biomass ratio of the micro-organisms. We could not detect any effects of the bactericides. In contrast, the fungicide strongly decreased nematode density, least in the fungal feeders, probably due to increased abundance of the insensitive Aphelenchoides ssp.

Some Heterotrophic Flagellates from a Cultivated Garden Soil in Australia

Summary The flagellates of an Australian garden soil were studied by placing coverslips on wet soil and subsequently examining the coverslips by light microscopy. A number of genera and species were found which have not previously been reported from soil samples. Besides the three new species, Apusomonas australiensis sp. nov., Peltomonas hanelisp. nov., and Sciviamonas terricola gen. nov. sp. nov. they include species from the genera Amastigomonas, Cryptaulax, Paraphysomonas, and Protaspis. Among genera which have been reported from soils, we recorded a number of species previously unreported from soils: Petalomonas pusilla, Bicosoeca epiphytica, Bicosoeca mignotii, and Ancyromonas sigmoides. In addition, we extracted a number of forms which have been found in soil but which are usually not considered as members of the soil flagellate community. They are: Codosiga botrytis, Salpingoeca amphoridium, and Goniomonas truncata. Only a minority of the taxa recorded are thought of as common and widespread in soils, they include: Apusomonas proboscidea, and species of Cercomonas and Spumella. At least part of the difference between communities of flagellates from freshwaters and those of soils are due to different sampling methods and not only to the existence of different communities.

Quantitative estimation of flagellate community structure and diversity in soil samples.

Heterotrophic flagellates occur in nearly all soils and, in most cases, many different species are present. Nevertheless, quantitative data on their community structure and diversity are sparse, possibly due to a lack of suitable techniques. Previous studies have tended to focus on either total flagellate numbers and biomass, or the identification and description of flagellate species present. With the increased awareness of the role of biodiversity and of food web interactions, the quantification of species within the community and their response to environmental change is likely to become more important. The present paper describes a modification of the most probable number method that allows such a quantification of individual flagellate morphotypes in soil samples. Observations were also made on the biomass of flagellate morphotypes in soil. 20 to 25 morphotypes of heterotrophic flagellates were detectable per gram of two different arable soils, which were treated experimentally to test the technique. One of the soils was fumigated with chloroform vapour for different lengths of time (0, 0.5, 2 or 24 hours); this led to a reduction in the number of morphotypes, in the Shannon diversity index and in the evenness. The other soil was planted with wheat, and while rhizosphere soils contained the same morphotypes as bulk soil, the abundance of individual morphotypes was significantly different and the Shannon diversity index in rhizosphere soils was significantly higher. Soil influenced by an elevated CO2 level likewise differed significantly in morphotype abundance when compared to soil exposed to ambient levels of CO2. The technique recovered more than 80% of the discernible morphotypes and could also be used to quantify amoebal and ciliate communities in a similar way.

Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

Spatial Distribution and Successional Pattern of Microbial Activity and Micro-Faunal Populations on Decomposing Barley Roots

1. With the current trend in agricultural practice to increase the importance of indigenous and added organic matter in the build-up of soil fertility, a better understanding of the dynamics of decomposition activity around organic resources is required. In this study the changes in microbial activity and populations of protozoa and nematodes were followed in decomposing barley root material buried in soil cores, and in three soil fractions with increasing distance from the root material. 2. Respiratory activity was maximal during the first week and decreased throughout the experiment, and at the last sampling after 392 days no significant effect of the roots on respiration was observed. 3. Following root addition microbial activity (dehydrogenase activity and potential denitrification rate) increased rapidly in the root material and in soil up to 1.8 mm from the roots. Microbial activity peaked after 4 days followed by a peak in protozoan numbers after 2 weeks and a peak in the number of nematodes after 6 weeks. 4. Growth potential of bacteria, as indicated by enzyme synthesis during a potential denitrification assay, and average bacterial cell size was larger on the roots than in the soil suggesting a more active bacterial biomass in this fraction. 5. The root effect was very local, and was limited mainly to the soil fraction adjacent to the roots; soil 1.8-5.4 mm from the roots and soil more than 5.4 mm from the roots was hardly affected by the resource. 6. These results demonstrate distinct successional patterns in the microbial food web with a sequence of population development from micro-organisms to protozoa and nematodes.

Population Dynamics of Active and Total Ciliate Populations in Arable Soil Amended with Wheat

ABSTRACT Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.

Buried alive – germination of up to a century-old marine protist resting stages

Lundholm N., Ribeiro S., Andersen T.J., Koch T., Godhe A., Ekelund F. and Ellegaard M. 2011. Buried alive – germination of up to a century-old marine protist resting stages. Phycologia 50: 629–640. DOI: 10.2216/11-16.1 We report on the survival and germination of up to a century-old marine protist resting stages naturally preserved in sediments from Koljö Fjord on the west coast of Sweden. This work has focused on germination of dinoflagellate cysts, but diatom resting stages were also observed. We record the longest known survival of dormant dinoflagellate cells. We individually isolated more than 1200 cysts of the three most abundant dinoflagellate taxa: Pentapharsodinium dalei, Lingulodinium polyedrum and Scrippsiella spp. Germination success decreased with core depth, and all successful germinations took place within the first 2 wk of incubation. Pentapharsodinium dalei had the highest germination success rate, with a maximum of up to 80% in 28-yr-old sediment, and could successfully germinate from core sediments dated to 1920 ± 12. Scrippsiella spp. cysts with cell contents occurred down to c. 90-yr-old sediment and could germinate from down to ca. 40-yr-old sediments, with a maximum germination rate of 50–60% in recent sediments. Cysts of L. polyedrum germinated frequently down to 20 yr and rarely to c. 80 yr, with a maximum of 20–50% germination success in recent sediments. Cyst isolation under cooled conditions rather than at room temperature resulted in a significantly higher germination success in P. dalei, while no effect was observed for L. polyedrum. The time elapsed since slicing of the core affected survival of L. polyedrum cysts negatively, most likely due to the effect of oxygen. The long-term survival potential of benthic resting stages that we report here has important implications, as viable resting stages accumulated in bottom sediments can be transported back to the water column by, for example, bioturbation and human-mediated sediment dredging. Hence, the sediment may to a higher degree than previously considered play a role as seed bank. This is important in a changing climate and might have particularly severe impacts in the case of harmful species.

Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.