Publications by authors named "Anne Winding"

24 Publications

  • Page 1 of 1

Insights into the earthworm gut multi-kingdom microbial communities.

Sci Total Environ 2020 Jul 8;727:138301. Epub 2020 Apr 8.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark. Electronic address:

Earthworms are widely known to impact soil health, having a key role in nutrient cycling and are often referred to as soil engineers. They are vital for soil microbial assemblages particularly through their feeding and burrowing activity in soil. Earthworms feed on soil organic matter and litter, and the resulting casts alter the soil microbial community. However, the gut microbiome of earthworms remains less known. In this study, we used amplicon sequencing of the 16S rRNA gene for bacteria and 18S rRNA gene for eukaryotes to assess the gut community assemblages of earthworm species within three genera Aporrectodea, Allolobophora and Lumbricus that represent different life forms sharing the same habitat. The objective was to compare the gut microbiome profiles of eukaryotic and prokaryotic organisms to assess significance of earthworm life forms, and to explore the cross kingdom networks in an attempt to identify keystone species. We found a high eukaryotic diversity with a dominance of the SAR supergroup along with fungi and metazoan in the earthworm gut. The bacterial community were dominated by members of Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, and Verrucomicrobia. The eukaryotic and prokaryotic communities showed similar differences in alpha diversity, being lowest in Lumbricus herculeus. The beta diversity showed earthworm species as a key factor in shaping gut microbiomes with L. herculeus harboring distinct microbiomes compared to species of Aporrectodea caliginosa, A. longa, A. tuberculata and Allolobophora chlorotica. Cross kingdom networks showed high interactions between several protist and bacterial OTUs. In conclusion, this study suggested that the community assemblages of gut microbiomes were shaped by earthworm species and life form, and such assemblage consists of cross kingdom interactions among eukaryotes and prokaryotes.
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http://dx.doi.org/10.1016/j.scitotenv.2020.138301DOI Listing
July 2020

Land use as a driver for protist community structure in soils under agricultural use across Europe.

Sci Total Environ 2020 May 8;717:137228. Epub 2020 Feb 8.

Department of Environmental Science, Aarhus University, Roskilde, Denmark. Electronic address:

Soil biodiversity is threatened by intensification of land use. The consequences of different land use on belowground biodiversity remain insufficiently explored for soil protists. Alongside being abundant and extremely diverse in soil, protists provide many ecosystem services: key players in the microbial loop, turnover of organic matter and stimulation of plant growth-promoting rhizobacteria. However, we lack knowledge of effects of site, land use intensity and management on diversity of soil protists. Here we assessed protist communities in four European arable sites with contrasting land use intensities at each site: Lusignan, France; Moskanjci, Slovenia; Castro Verde, Portugal and Scheyern, Germany as well as two grassland sites: Hainich, Germany and Lancaster, UK. Each site has consistent agricultural management history of low and high land use intensities quantified in terms of land use index (LUI). We employed high-throughput sequencing of environmental DNA, targeting the V4 region of the 18S rRNA gene. By assigning the protist composition to trophic groups, we inspected for effects of management, and other biotic and abiotic variables. While overall protist richness was unaffected by LUI within sites, specific trophic groups such as plant pathogens and saprotrophs were affected. Effects on protist biome across land uses and sites were also observed. LUI sensitive taxa were taxonomically diverse in each plot, and their trophic groups responded in specific patterns to specific practices. The most abundant trophic group was phagotrophs (73%), followed by photoautotrophs (16%), plant pathogens (4%), animal parasites (2%) and saprotrophs (1%). Community compositions and factors affecting the structure of individual trophic groups differed between land uses and management systems. The agricultural management selected for distinct protist populations as well as specific functional traits, and the protist community and diversity were indeed affected by site, LUI and management, which indicates the ecological significance of protists in the soil food web.
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http://dx.doi.org/10.1016/j.scitotenv.2020.137228DOI Listing
May 2020

Soil bacteria and protists show different sensitivity to polycyclic aromatic hydrocarbons at controlled chemical activity.

FEMS Microbiol Lett 2019 09;366(17)

Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark.

This study linked growth inhibition of soil bacteria and protists to the chemical activity (a) of polycyclic aromatic hydrocarbons (PAHs) and compared the sensitivities of bacteria and protists. Passive dosing from pre-loaded silicone provided well-defined and constant a of PAHs in independent tests. Single-species growth inhibition with two bacterial (Pseuodomonas fluorescens DR54 and Sinorhizobium meliloti) and two protist (Cercomonas longicauda and Acanthamoeba castellanii) strains at maximum a (amax) of nine and four PAHs, respectively, showed no inhibition of PAHs with amax below 0.1 (pyrene and anthracene), while growth inhibition was observed for PAHs with amax above 0.1 (e.g. fluorene, fluoranthene and naphthalene). The bacteria were less sensitive than the protists. Soil bacterial community-level growth inhibition by naphthalene was in good agreement with single-species data, but also indicated the presence of sensitive bacteria that were inhibited by a below 0.05 and increasing pre-exposure time giving higher inhibition. The a of 50% inhibition (Ea50) was 0.434 and 0.329 for 0.5 and 4 h pre-exposure time, respectively. Invertebrates tended to be more sensitive than single-celled organisms tested here. This suggests that PAH exposure leads to differential toxicity in soil biota, which may affect soil food web structure and cycling of organic matter.
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http://dx.doi.org/10.1093/femsle/fnz214DOI Listing
September 2019

Inter-laboratory testing of the effect of DNA blocking reagent G2 on DNA extraction from low-biomass clay samples.

Sci Rep 2018 04 9;8(1):5711. Epub 2018 Apr 9.

Center for Permafrost (CENPERM) and Department of Geochemistry, Geological Survey of Denmark and Greenland, Copenhagen, Denmark.

Here we show that a commercial blocking reagent (G2) based on modified eukaryotic DNA significantly improved DNA extraction efficiency. We subjected G2 to an inter-laboratory testing, where DNA was extracted from the same clay subsoil using the same batch of kits. The inter-laboratory extraction campaign revealed large variation among the participating laboratories, but the reagent increased the number of PCR-amplified16S rRNA genes recovered from biomass naturally present in the soils by one log unit. An extensive sequencing approach demonstrated that the blocking reagent was free of contaminating DNA, and may therefore also be used in metagenomics studies that require direct sequencing.
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http://dx.doi.org/10.1038/s41598-018-24082-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890260PMC
April 2018

Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome.

Protist 2017 07 14;168(3):283-293. Epub 2017 Mar 14.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark. Electronic address:

Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays.
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http://dx.doi.org/10.1016/j.protis.2017.03.002DOI Listing
July 2017

Soil networks become more connected and take up more carbon as nature restoration progresses.

Nat Commun 2017 02 8;8:14349. Epub 2017 Feb 8.

NIOO-KNAW, Microbial Ecology, Droevendaalsesteeg 10, Wageningen 6708 PB, The Netherlands.

Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered.
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http://dx.doi.org/10.1038/ncomms14349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309817PMC
February 2017

Effects of Bacillus cereus Endospores on Free-Living Protist Growth.

Microb Ecol 2017 04 7;73(3):699-709. Epub 2016 Dec 7.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000, Roskilde, Denmark.

We studied the predator-prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.
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http://dx.doi.org/10.1007/s00248-016-0905-7DOI Listing
April 2017

Metagenomes provide valuable comparative information on soil microeukaryotes.

Res Microbiol 2016 Jun 26;167(5):436-50. Epub 2016 Mar 26.

Section of Microbiology, University of Copenhagen, 2100 Copenhagen, Denmark.

Despite the critical ecological roles of microeukaryotes in terrestrial ecosystems, most descriptive studies of soil microbes published so far focused only on specific groups. Meanwhile, the fast development of metagenome sequencing leads to considerable data accumulation in public repositories, providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure, including the DNA extraction method, the database choice and also the annotation procedure. While most studies on soil microeukaryotes are based on sequencing of PCR-amplified taxonomic markers (18S rRNA genes, ITS regions), this work represents, to our knowledge, the first report based solely on metagenomic microeukaryote DNA. Choosing the correct annotation procedure and reference database has proven to be crucial, as it considerably limits the risk of wrong assignments. In addition, a significant and pronounced effect of the DNA extraction method on the taxonomical structure of soil microeukaryotes has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods.
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http://dx.doi.org/10.1016/j.resmic.2016.03.003DOI Listing
June 2016

Comparison of three DNA extraction methods for recovery of soil protist DNA.

J Microbiol Methods 2015 Aug 9;115:13-9. Epub 2015 May 9.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark. Electronic address:

The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism.
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http://dx.doi.org/10.1016/j.mimet.2015.05.011DOI Listing
August 2015

Co-transport of polycyclic aromatic hydrocarbons by motile microorganisms leads to enhanced mass transfer under diffusive conditions.

Environ Sci Technol 2014 Apr 25;48(8):4368-75. Epub 2014 Mar 25.

Department of Environmental Science, Aarhus University , P.O. Box 358, 4000 Roskilde, Denmark.

The environmental chemodynamics of hydrophobic organic chemicals (HOCs) are often rate-limited by diffusion in stagnant boundary layers. This study investigated whether motile microorganisms can act as microbial carriers that enhance mass transfer of HOCs through diffusive boundary layers. A new experimental system was developed that allows (1) generation of concentration gradients of HOCs under the microscope, (2) exposure and direct observation of microorganisms in such gradients, and (3) quantification of HOC mass transfer. Silicone O-rings were integrated into a Dunn chemotaxis chamber to serve as sink and source for polycyclic aromatic hydrocarbons (PAHs). This resulted in stable concentration gradients in water (>24 h). Adding the model organism Tetrahymena pyriformis to the experimental system enhanced PAH mass transfer up to hundred-fold (benzo[a]pyrene). Increasing mass transfer enhancement with hydrophobicity indicated PAH co-transport with the motile organisms. Fluorescence microscopy confirmed such transport. The effective diffusivity of T. pyriformis, determined by video imaging microscopy, was found to exceed molecular diffusivities of the PAHs up to four-fold. Cell-bound PAH fractions were determined to range from 28% (naphthalene) to 92% (pyrene). Motile microorganisms can therefore function as effective carriers for HOCs under diffusive conditions and might significantly enhance mobility and availability of HOCs.
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http://dx.doi.org/10.1021/es404793uDOI Listing
April 2014

Impact of activated carbon, biochar and compost on the desorption and mineralization of phenanthrene in soil.

Environ Pollut 2013 Oct 16;181:200-10. Epub 2013 Jul 16.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark.

Sorption of PAHs to carbonaceous soil amendments reduces their dissolved concentrations, limiting toxicity but also potentially biodegradation. Therefore, the maximum abiotic desorption of freshly sorbed phenanthrene (≤5 mg kg(-1)) was measured in three soils amended with activated carbon (AC), biochar or compost. Total amounts of phenanthrene desorbed were similar between the different soils, but the amendment type had a large influence. Complete desorption was observed in the unamended and compost amended soils, but this reduced for biochar (41% desorbed) and AC (8% desorbed). Cumulative amounts mineralized were 28% for the unamended control, 19% for compost, 13% for biochar and 4% for AC. Therefore, the effects of the amendments in soil in reducing desorption were also reflected in the extents of mineralization. Modeling was used to analyze key processes, indicating that for the AC and charcoal treatments bacterial activity did not limit mineralization, but rather desorption into the dissolved phase.
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http://dx.doi.org/10.1016/j.envpol.2013.06.026DOI Listing
October 2013

Comparing the desorption and biodegradation of low concentrations of phenanthrene sorbed to activated carbon, biochar and compost.

Chemosphere 2013 Feb 22;90(6):1767-78. Epub 2012 Aug 22.

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark.

Carbonaceous soil amendments are applied to contaminated soils and sediments to strongly sorb hydrophobic organic contaminants (HOCs) and reduce their freely dissolved concentrations. This limits biouptake and toxicity, but also biodegradation. To investigate whether HOCs sorbed to such amendments can be degraded at all, the desorption and biodegradation of low concentrations of (14)C-labelled phenanthrene (≤5 μg L(-1)) freshly sorbed to suspensions of the pure soil amendments activated carbon (AC), biochar (charcoal) and compost were compared. Firstly, the maximum abiotic desorption of phenanthrene from soil amendment suspensions in water, minimal salts medium (MSM) or tryptic soy broth (TSB) into a dominating silicone sink were measured. Highest fractions remained sorbed to AC (84±2.3%, 87±4.1%, and 53±1.2% for water, MSM and TSB, respectively), followed by charcoal (35±2.2%, 32±1.7%, and 12±0.3%, respectively) and compost (1.3±0.21%, similar for all media). Secondly, the mineralization of phenanthrene sorbed to AC, charcoal and compost by Sphingomonas sp. 10-1 (DSM 12247) was determined. In contrast to the amounts desorbed, phenanthrene mineralization was similar for all the soil amendments at about 56±11% of the initially applied radioactivity. Furthermore, HPLC analyses showed only minor amounts (<5%) of residual phenanthrene remaining in the suspensions, indicating almost complete biodegradation. Fitting the data to a coupled desorption and biodegradation model revealed that desorption did not limit biodegradation for any of the amendments, and that degradation could proceed due to the high numbers of bacteria and/or the production of biosurfactants or biofilms. Therefore, reduced desorption of phenanthrene from AC or charcoal did not inhibit its biodegradation, which implies that under the experimental conditions these amendments can reduce freely dissolved concentration without hindering biodegradation. In contrast, phenanthrene sorbed to compost was fully desorbed and biodegraded.
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http://dx.doi.org/10.1016/j.chemosphere.2012.07.048DOI Listing
February 2013

Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen.

Environ Microbiol 2012 Aug 19;14(8):2034-47. Epub 2011 Dec 19.

Laboratory of Applied Micro and Nanotechnology (LAMINATE), National Veterinary Institute (VET), Technical University of Denmark (DTU), Hangøvej 2, DK-8200 Aarhus N, Denmark.

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.
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http://dx.doi.org/10.1111/j.1462-2920.2011.02655.xDOI Listing
August 2012

Factors affecting vegetable growers' exposure to fungal bioaerosols and airborne dust.

Ann Occup Hyg 2012 Mar 14;56(2):170-81. Epub 2011 Oct 14.

The National Research Centre for the Working Environment, Lersø Parkallé 105, DK-2100 Copenhagen Ø, Denmark.

We have quantified vegetable growers' exposure to fungal bioaerosol components including (1→3)-β-d-glucan (β-glucan), total fungal spores, and culturable fungal units. Furthermore, we have evaluated factors that might affect vegetable growers' exposure to fungal bioaerosols and airborne dust. Investigated environments included greenhouses producing cucumbers and tomatoes, open fields producing cabbage, broccoli, and celery, and packing facilities. Measurements were performed at different times during the growth season and during execution of different work tasks. Bioaerosols were collected with personal and stationary filter samplers. Selected fungal species (Beauveria spp., Trichoderma spp., Penicillium olsonii, and Penicillium brevicompactum) were identified using different polymerase chain reaction-based methods and sequencing. We found that the factors (i) work task, (ii) crop, including growth stage of handled plant material, and (iii) open field versus greenhouse significantly affected the workers' exposure to bioaerosols. Packing of vegetables and working in open fields caused significantly lower exposure to bioaerosols, e.g. mesophilic fungi and dust, than harvesting in greenhouses and clearing of senescent greenhouse plants. Also removing strings in cucumber greenhouses caused a lower exposure to bioaerosols than harvest of cucumbers while removal of old plants caused the highest exposure. In general, the exposure was higher in greenhouses than in open fields. The exposures to β-glucan during harvest and clearing of senescent greenhouse plants were very high (median values ranging between 50 and 1500 ng m(-3)) compared to exposures reported from other occupational environments. In conclusion, vegetable growers' exposure to bioaerosols was related to the environment, in which they worked, the investigated work tasks, and the vegetable crop.
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http://dx.doi.org/10.1093/annhyg/mer090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277710PMC
March 2012

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy.

FEMS Microbiol Lett 2011 Mar 12;316(1):16-22. Epub 2011 Jan 12.

Terrestrial Ecology, Department of Biology, University of Copenhagen, Copenhagen K, Denmark.

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.
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http://dx.doi.org/10.1111/j.1574-6968.2010.02182.xDOI Listing
March 2011

UVC fluencies for preventative treatment of Pseudomonas aeruginosa contaminated polymer tubes.

Biofouling 2010 Oct;26(7):821-8

DTU Fotonik, Roskilde, Denmark.

Exposing Pseudomonas aeruginosa biofilm grown on the inner surface of Teflon and silicone tubes to UVC light (265 nm) from light emitting diodes (LED) has previously been shown to substantially reduce biofilm growth. Smaller UVC fluencies were required to disinfect Teflon tubes compared to silicone tubes. Light propagation enhancement in tubes can be obtained if the refractive index of the intra-luminal saline solution is higher than that of the polymer. This condition is achieved by using Teflon tubes with a low refractive index (1.34) instead of the polymers with a high refractive index (1.40-1.50) normally used for tubing in catheter production. Determining whether or not UVC light exposure can disinfect and maintain the intra-luminal number of colony forming units (CFUs) at an exceedingly low level and thus avoid the growth and establishment of biofilm is of interest. The use of UVC diodes is demonstrated to be a preventative disinfection treatment on tubes made of Teflon, which enhances the UVC light propagation, and on tubes made of a softer material, ethylene vinyl acetate (EVA), which is suitable for catheters but much less suitable for UVC light propagation. Simulating an aseptic breach (∼10(3)-10(4) CFU ml(-1)), the UVC disinfection set-up was demonstrated using tubes contaminated with planktonic P. aeruginosa. After the tubes (10-20 cm) were inoculated with the bacterial solution for 3 h, they were emptied and filled with saline solutions (0.9-20%). Next UVC fluencies (0-21 mJ cm(-2)) were applied to the tubes 3 h after inoculation. Colony counts were carried out on liquid samples drawn from the tubes the first day after UVC treatment and liquid and surface samples were collected and analyzed 3-4 days later. A fluence of approximately 1.0 mJ cm(-2) was noted as being sufficient for no growth for a period of 3-4 days for the Teflon tubes. Determining the fluence threshold for the EVA tubes was not possible. Almost all of the UVC-treated EVA tubes were disinfected simply by filling the tubes with a saline solution. Direct UVC treatment of the contaminated EVA tubes revealed, however, that a fluence of 21 mJ cm(-2) killed the bacteria present in the tubes and kept them disinfected for a period of 3-4 days.
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http://dx.doi.org/10.1080/08927014.2010.520314DOI Listing
October 2010

Exposure to bioaerosols during the growth season of tomatoes in an organic greenhouse using Supresivit (Trichoderma harzianum) and Mycostop (Streptomyces griseoviridis).

Appl Environ Microbiol 2010 Sep 9;76(17):5874-81. Epub 2010 Jul 9.

The National Research Centre for the Working Environment, Lersø Parkalle 105, DK-2100 Copenhagen Ø, Denmark.

In working environments, especially in confined spaces like greenhouses, elevated concentrations of airborne microorganisms may become a problem for workers' health. Additionally, the use of microbial pest control agents (MPCAs) may increase exposure to microorganisms. The aim of this study was to investigate tomato growers' exposure to naturally occurring bioaerosol components [dust, bacteria, fungi, actinomycetes, (1-->3)-beta-D-glucans, and endotoxin] and MPCAs applied by drip irrigation. Airborne dust was collected with filter samplers and analyzed for microorganisms by plate counts and total counts using a microscope. Analysis of (1-->3)-beta-D-glucan and endotoxin content was performed by kinetic, chromatic Limulus amoebocyte lysate tests. The fungal strain (Trichoderma harzianum) from the biocontrol product Supresivit was identified by PCR analysis. Measurements were performed on the day of drip irrigation and 1 week, 1 month, and 3 months after the irrigation. T. harzianum from Supresivit could be detected only on the day of treatment. Streptomyces griseoviridis, an applied MPCA, was not detected in the air during this investigation. We found that bioaerosol exposure increases during the growth season and that exposure to fungi, bacteria, and endotoxin can reach levels during the harvest period that may cause respiratory symptoms in growers. The collected data indicate that MPCAs applied by drip irrigation do not become airborne later in the season.
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http://dx.doi.org/10.1128/AEM.00446-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935076PMC
September 2010

Effects of C60 fullerene nanoparticles on soil bacteria and protozoans.

Environ Toxicol Chem 2008 Sep;27(9):1895-903

Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, University of Aarhus, Frederiksborgvej 399, DK-4000 Roskilde, Denmark.

Nanotechnology should produce numerous new materials in the coming years. Because of the novel design of nanomaterials with new physicochemical characteristics, their potential adverse impact on the environment and human health must be addressed. In the present study, agglomerates of pristine C60 fullerenes (50 nm to microm-size) were applied to soil at 0, 5, 25, and 50 mg/kg dry soil to assess their effect on the soil microbiota by measuring total respiration; biomass, number, and diversity of bacteria; and total number and diversity of protozoans during 14 d. Respiration and microbial biomass were unaffected by the fullerenes at any time, whereas the number of fast-growing bacteria was decreased by three- to fourfold just after incorporation of the nanomaterial. Protozoans seemed not to be very sensitive to C60, because their number decreased only slightly in the beginning of the experiment. With polymerase chain reaction and denaturing gradient gel electrophoresis analysis of eubacteria and kinetoplastids from the soil, however, a difference between the fullerene treatments and nonamended controls was demonstrated. The fullerenes did not induce more than 20 to 30% of relative dissimilarity (with both bacteria and protozoans) between treatments, but this effect was persistent throughout the experiment. It therefore is recommended that fullerene nanomaterial not be spread deliberately in the environment and that their ecotoxicology be further clarified.
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http://dx.doi.org/10.1897/07-375.1DOI Listing
September 2008

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73.

Microb Ecol 2009 Apr 31;57(3):501-9. Epub 2008 Oct 31.

Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, University of Aarhus, Roskilde, Denmark.

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.
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http://dx.doi.org/10.1007/s00248-008-9455-yDOI Listing
April 2009

The use of microorganisms in ecological soil classification and assessment concepts.

Ecotoxicol Environ Saf 2005 Oct;62(2):230-48

Department of Environmental Chemistry and Microbiology, National Environmental Research Institute (NERI), Frederiksborgvej 399, P.O. Box 358, 4000 Roskilde, Denmark.

Microbial communities are integral parts of soil and their activity is very important to the functioning of soil. Therefore, microorganisms should be included in soil quality classification and assessment concepts. The challenges of using microbial indicators are to identify the best choice among the many techniques available to assess soil quality and to convert the information obtained from the microbial indicator into a form relevant for policy makers. In this article, we present a wide range of possible microbial indicators, some of them standardized; each provides slightly different information on soil quality. Experience with the use of indicators for assessment of microbial communities and soil quality is discussed. At present, as many microbial indicators as possible should be included to gain experience. At a minimum, measures of microbial biomass, respiration, and N mineralization and a community profiling method (e.g., DGGE, PLFA, or CLPP) should be included.
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http://dx.doi.org/10.1016/j.ecoenv.2005.03.026DOI Listing
October 2005

Microbial community-level toxicity testing of linear alkylbenzene sulfonates in aquatic microcosms.

FEMS Microbiol Ecol 2004 Aug;49(2):229-41

Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

Complex microbial communities may serve as ideal and ecologically relevant toxicity indicators. We here report an assessment of frequently used methods in microbial ecology for their feasibility to detect toxic effects of the environmentally important surfactant linear alkylbenzene sulfonate (LAS) on microbial communities in lake water and treated waste water. The two microbial communities were evaluated for changes in community structure and function over a period of 7 weeks in replicated aquatic microcosms amended with various levels of LAS (0, 0.1, 1, 10 or 100 mg l(-1)) and inorganic nutrients. In general, the two communities behaved similarly when challenged with LAS. Following lag periods of 1-3 weeks, LAS was degraded to non-toxic substances. Denaturing gradient gel electrophoresis of 16S rRNA gene fragments and [3H]leucine incorporation were the most sensitive assays with effect levels of 0-1 and 1-10 mg LAS l(-1), respectively. Community-level physiological profiles and pollution-induced community tolerance determinations using Biolog microplates demonstrated less sensitivity with effect levels of 10-100 mg LAS l(-1). Total cell counts and net uptake of inorganic N and P were unaffected even at 100 mg LAS l(-1). Interestingly, different microbial communities developed in some replicate microcosms, indicating the importance of stochastic events for community succession. We conclude that microbial community-level toxicity testing holds great promise and suggest a polyphasic approach involving a range of independent methods targeting both the structure and function of the tested microbial communities.
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http://dx.doi.org/10.1016/j.femsec.2004.03.006DOI Listing
August 2004

Non-target effects of bacterial biological control agents suppressing root pathogenic fungi.

FEMS Microbiol Ecol 2004 Feb;47(2):129-41

Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Roskilde, Denmark.

Microorganisms have knowingly been used during the last century to control plant diseases. During the last decades, research and application of biological control agents (BCAs) as a pest control strategy have gained increasing attention. This review focuses specifically on non-target effects of bacterial BCAs that are used to suppress root pathogenic fungi. It attempts to critically evaluate the strengths and weaknesses of non-target effect studies published to date and relate them to the success of the BCA in fungal pathogen control. Significant non-target effects of BCAs have indeed been observed, but these are generally small in scale and limited to a growth season, and have not been proven to affect soil health. We discuss these studies and point out what we believe are notable deficiencies. Among the modes of disease suppression by BCAs, antibiotic production is believed to be of major importance. But assurances that in situ antibiotic production actually occurs in environmental samples are lacking in the non-target effect studies. Also the effectiveness of the BCA on the target pathogen, the absence of appropriate controls for inoculation effects, and the presence of pathogenic fungi are missing in most studies. In future non-target effect studies we recommend focusing on proven effective BCAs and clearly distinguishing effects of antimicrobial compounds from effects of general microbial activity.
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http://dx.doi.org/10.1016/S0168-6496(03)00261-7DOI Listing
February 2004

Microbial succession in the rhizosphere of live and decomposing barley roots as affected by the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil.

FEMS Microbiol Ecol 2003 Apr;43(3):383-92

Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Frederiksborgvej 399, 4000 Roskilde, Denmark.

Abstract The protocol used in the present study was a long-term mesocosm experiment where the microbial succession around live barley roots and subsequent decomposing roots was assessed after seed coating with either the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil. Four diversity measures were used: community level physiological profiles (CLPP), Bacteria-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), actinomycete-specific PCR-DGGE and phospholipid fatty acid (PLFA), as well as total cell counts, colony-forming units (CFU) and culturable spore formers, and spore counts of the Bacillus cereus group. Analysis of non-treated plants provided a baseline description of the natural microbial succession from which effects of the treatments could be evaluated. A microbial succession occurred both in the rhizosphere and around decomposing roots, shown with all three diversity measures. A clear response to root death was found, and a clear distinction between root tip and root base samples. Using the recommended concentration of imazalil and a realistic number of DR54-BN14 for seed coating, transient, initial effects of both treatments on the microbial communities were observed at the root base with the PLFA analysis only. The lack of lasting significant side effects of DR54-BN14 is in agreement with an initial fast reduction in culturable DR54-BN14.
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http://dx.doi.org/10.1111/j.1574-6941.2003.tb01079.xDOI Listing
April 2003

Linking of microorganisms to phenanthrene metabolism in soil by analysis of (13)C-labeled cell lipids.

Appl Environ Microbiol 2002 Dec;68(12):6106-13

National Environmental Research Institute, Department of Environmental Chemistry and Microbiology, DK-4000 Roskilde, Denmark.

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [(14)C]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 [(13)C]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 (13)C-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 (13)C-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 (13)C, suggesting that actinomycetes metabolized phenanthrene in this soil. The (13)C-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 (13)C was recovered in the 18:2omega6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [(13)C]phenanthrene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC134424PMC
http://dx.doi.org/10.1128/AEM.68.12.6106-6113.2002DOI Listing
December 2002