Effects of sludge-amendment on mineralization of pyrene and microorganisms in sludge and soil.

Hydrophobic contaminants sorb to sludge in wastewater treatment plants and enter the soil environment when the sludge is applied to agricultural fields. The mineralization of pyrene was examined in soil, in sludge mixed homogeneously into soil, and in sludge-soil systems containing a lump of sludge. Sludge-amendment enhanced the mineralization of pyrene in the soil compared to soil without sludge, and the most extensive mineralization was observed when the sludge was kept in a lump. The number of protozoa, heterotrophic bacteria and pyrene-mineralizing bacteria was much higher in the sludge compared to the soil. The amendment of sludge did not affect the number of protozoa and bacteria in the surrounding soil, which indicated that organic contaminants in the sludge had a little effect on the number of protozoa and bacteria in the surrounding soil.

Development and application of a most-probable-number-pcr assay to quantify flagellate populations in soil samples.

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.

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis.

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

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.

An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.

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

> Abstract To 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 x 10(6) cells/g dry weight (dw) to a high of 5.3 x 10(7) 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 x 10(5) 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 x 10(6) 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 x 10(4) cells/g dw. Populations of sulfate reducers decreased with an increase in horizontal distance from the landfill ranging from a high of 9.0 x 10(3) 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 10(6) 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.

Notes on protozoa in agricultural soil with emphasis on heterotrophic flagellates and naked amoebae and their ecology.

Heterotrophic flagellates and naked amoebae are usually very numerous in agricultural soils; with numbers in the magnitude of 10,000 to 100,000 (active+encysted) cells per gram of soil. In 'hotspots' influenced by living roots or by dead organic material, the number may occasionally be as high as several millions per gram of soil. An exact enumeration of these organisms is virtually impossible. As they most often adhere closely to the soil particles, direct counting will underestimate numbers since the organisms will be masked. The method usually applied for enumeration of these organisms, the 'most probable number (MPN) method', is based on the ability of the organisms to grow on particular culture media. This method will in many cases underestimate the total protozoan number (active+encysted). It is uncertain how many of the heterotrophic flagellates and naked amoebae are actively moving and how many are encysted at a particular time; the 'HCl-method' which has usually been used to discriminate between active and encysted has proven to be highly unreliable. Despite the methodological difficulties many investigations of these organisms indicate that they play an important role in agricultural soils as bacterial consumers, and to a minor extent as consumers of fungi. Because of their small size and their flexible body they are able to graze bacteria in small pores in the soil in which larger organisms are precluded from coming. Key factors restricting the number and activity of heterotrophic flagellates and naked amoebae in soils seem to be water potential and soil structure and texture. In micro-cosm experiments, small heterotrophic flagellates and naked amoebae regulate the size and composition of the bacterial community. Bacterial activity seems to be stimulated by these organisms in most cases as well as the mineralization of carbon and nitrogen and possibly other mineral nutrients. In the rhizosphere of living plants the activity of protozoa has proven to stimulate uptake of nitrogen in pot experiments, and it has been hypothesized that organic matter liberated by plants in the root zone will stimulate bacterial and protozoan activity, leading to mineralization of organic soil nitrogen which is subsequently taken up by the plants.