Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Patterns of local, intercontinental and interseasonal variation of soil bacterial and eukaryotic microbial communities
    Gruyter, Johan De; Weedon, James T. ; Bazot, Stéphane ; Dauwe, Steven ; Fernandez-Garberí, Pere Roc ; Geisen, Stefan ; La Motte, Louis Gourlez De; Heinesch, Bernard ; Janssens, Ivan A. ; Leblans, Niki ; Manise, Tanguy ; Ogaya, Romà ; Löfvenius, Mikaell Ottosson ; Peñuelas, Josep ; Sigurdsson, Bjarni D. ; Vincent, Gaëlle ; Verbruggen, Erik - \ 2019
    FEMS microbiology ecology 96 (2019)3. - ISSN 0168-6496
    microbial ecology - protists - soil biogeography - soil microbial communities - spatio-temporal variability

    Although ongoing research has revealed some of the main drivers behind global spatial patterns of microbial communities, spatio-temporal dynamics of these communities still remain largely unexplored. Here, we investigate spatio-temporal variability of both bacterial and eukaryotic soil microbial communities at local and intercontinental scales. We compare how temporal variation in community composition scales with spatial variation in community composition, and explore the extent to which bacteria, protists, fungi and metazoa have similar patterns of temporal community dynamics. All soil microbial groups displayed a strong correlation between spatial distance and community dissimilarity, which was related to the ratio of organism to sample size. Temporal changes were variable, ranging from equal to local between-sample variation, to as large as that between communities several thousand kilometers apart. Moreover, significant correlations were found between bacterial and protist communities, as well as between protist and fungal communities, indicating that these microbial groups change in tandem, potentially driven by interactions between them. We conclude that temporal variation can be considerable in soil microbial communities, and that future studies need to consider temporal variation in order to reliably capture all drivers of soil microbiome changes.

    Microbiome dynamics of disease suppresive soils
    Gómez Expósito, Ruth - \ 2017
    Wageningen University. Promotor(en): F.P.M. Govers; J.M. Raaijmakers, co-promotor(en): J. Postma; I. de Bruijn. - Wageningen : Wageningen University - ISBN 9789463431774 - 267
    suppressive soils - soil suppressiveness - plant diseases - thanatephorus cucumeris - microbial ecology - soil microbiology - rhizosphere bacteria - soil bacteria - community ecology - soil fungi - transcriptomics - taxonomy - ziektewerende gronden - bodemweerbaarheid - plantenziekten - thanatephorus cucumeris - microbiële ecologie - bodemmicrobiologie - rizosfeerbacteriën - bodembacteriën - gemeenschapsecologie - bodemschimmels - transcriptomica - taxonomie

    Disease suppressive soils are soils in which plants do not get diseased from plant pathogens due to the presence (and activities) of the microbes present in the soil. Understanding which microbes contribute to confer suppression and through which mechanisms they can protect plants is crucial for a sustainable control of plant diseases. In the research conducted in this thesis, I first examined the role of Lysobacter species, previously associated with disease suppressive soils, in suppressing damping-off disease caused by the soil-borne fungal pathogen Rhizoctonia solani on sugar beet. The majority of the Lysobacter strains tested revealed a broad metabolic potential in producing a variety of enzymes and secondary metabolites able to suppress R. solani in vitro. However, any of these strains could consistently suppress damping-off disease when applied in soil bioassays. Their ability to promote plant growth was also tested for sugar beet, cauliflower, onion or Arabidopsis thaliana. Results indicated that any of the Lysobacter strains could consistently promote plant growth, neither via direct contact nor via volatile production. Second, I investigated whether the antagonistic activity of Lysobacter species could be triggered when applied as bacterial consortia, together with Pseudomonas and Streptomyces species. Although several bacterial combinations showed an increased antagonistic effect towards R. solani in vitro, no consistent effects were observed when these bacterial consortia were applied in vivo. Third, I investigated the dynamical changes in the bacterial community composition and functions occurring during the process of disease suppressiveness induction by performing whole community analyses using next-generation sequencing techniques. Results indicated that suppressiveness induction was most associated with changes in certain bacterial traits rather than changes in the bacteria community composition itself. Among the functions found as more active in suppressive soils were several ‘classic’ mechanisms of disease suppression, including competition for nutrients, iron and space and production of extracellular enzymes, indol-acetic-acid and hydrogen cyanide. Among the enzymes found in higher abundance in suppressive soil were these ones involved in the degradation of oxalic acid, a pathogenicity factor produced by pathogenic fungi to help infecting the host plant. Hence, I finally studied the role of bacteria able to produce enzymes able to degrade oxalic acid in suppressing R. solani disease. Enrichment of native oxalotrophic bacteria existing in soil, their isolation and further application into soil revealed that they could effectively suppress Rhizoctonia disease. Characterization of these oxalotrophic bacteria revealed that members within the Caulobacter and Nocardioides species could suppress R. solani disease by their own. Furthermore, the research done in this thesis highlights the importance of combining different techniques to unravel the mechanisms underlying disease suppression and the importance of studying function-over-phylogeny. Additionally, it also highlights the importance of organic amendments (such as oxalic acid) directly into soils in order to “engineer” the bacterial functions towards the control of diseases caused by R. solani.

    Assessing the effects of chemicals on aquatic microbial ecosystems
    Rocha Dimitrov, M. - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt; Paul van den Brink. - Wageningen : Wageningen University - ISBN 9789462576667 - 264
    aquatic ecosystems - microorganisms - macroinvertebrates - microbial ecology - aquatic fungi - chemicals - tebuconazole - fungicide residues - pesticides - marine sediments - toxicity - enrofloxacin - fluoroquinolones - zooplankton - phytoplankton - antibiotic resistance - periphyton - bacteria - ecological risk assessment - aquatische ecosystemen - micro-organismen - macroinvertebraten - microbiële ecologie - waterschimmels - chemicaliën - tebuconazool - fungicidenresiduen - pesticiden - mariene sedimenten - toxiciteit - enrofloxacine - fluoroquinolonen - zoöplankton - fytoplankton - antibioticaresistentie - perifyton - bacteriën - ecologische risicoschatting
    Explorations of soil microbial processes driven by dissolved organic carbon
    Straathof, A.L. - \ 2015
    Wageningen University. Promotor(en): Rob Comans; Ellis Hoffland. - Wageningen : Wageningen University - ISBN 9789462573277 - 146
    organische koolstof - bodemmicrobiologie - bodem - microbiële ecologie - bodembiologie - bodemchemie - organic carbon - soil microbiology - soil - microbial ecology - soil biology - soil chemistry

    Explorations of soil microbial processes driven by dissolved organic carbon

    Angela L. Straathof

    June 17, 2015, Wageningen UR

    ISBN 978-94-6257-327-7


    Dissolved organic carbon (DOC) is a complex, heterogeneous mixture of C compounds which, as a substrate, may influence various processes of the soil microbial community. Microbial respiration and volatile production are two such processes. These have both been linked to general disease suppression (GDS), a phenomenon in agricultural soils which inhibits pathogenic infestation in crops. The underlying hypothesis of this thesis is that the quality of DOC, via regulation of microbial processes, may be an important indicator of soil functions, including GDS. Properties of DOC quality include proportions of hydrophobic and hydrophilic fractions, and aromaticity. This thesis describes a high range in DOC fractions from various types of compost, which is often added to soil as an amendment to promote GDS. Differences in soil microbial respiration rates were attributed to differences in the composition of compost DOC added to soil in a laboratory incubation experiment. Compost DOC high in proportion of the hydrophilic (Hi) fraction promoted respiration rates. Depletion of the hydrophobic humic acid (HA) fraction was also observed. The relationship between DOC and microbial respiration was further explored in a survey of 50 arable soils. Both HA and Hi fractions of DOC that were found to be statistically, significantly related to respiration rates in these soils. Furthermore, in an assay measuring in vitro pathogen suppression by microbial volatile production, DOC concentration and microbial respiration were linked to growth suppression of Rhizoctonia solani, Fusarium oxysporum, and Pythium intermedium via multivariate regression modelling. This thesis provides evidence for the importance of DOC and DOC quality’s influence on microbial respiration and volatile production, thus supporting the hypothesis that DOC is a microbially-relevant soil chemical parameter, and potential indicator of general disease suppression in agricultural soils.

    Community and genomic analysis of the human small intestine microbiota
    Bogert, B. van den - \ 2013
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Erwin Zoetendal. - S.l. : s.n. - ISBN 9789461736628 - 214
    darmmicro-organismen - dunne darm - dna microarrays - dna-sequencing - microbiële ecologie - immunomodulerende eigenschappen - intestinal microorganisms - small intestine - dna microarrays - dna sequencing - microbial ecology - immunomodulatory properties

    Our intestinal tract is densely populated by different microbes, collectively called microbiota, of which the majority are bacteria. Research focusing on the intestinal microbiota often use fecal samples as a representative of the bacteria that inhabit the end of the large intestine. These studies revealed that the intestinal bacteria contribute to our health, which has stimulated the interest in understanding their dynamics and activities. However, bacterial communities in fecal samples are different compared to microbial communities at other locations in the intestinal tract, such as the small intestine. Despite that the small intestine is the first region where our food and intestinal microbiota meet, we know little about the bacteria in the small intestine and how they influence our overall well-being. This is mainly attributable to difficulties in obtaining samples with the small intestine being located between the stomach and the large intestine. Therefore, the work in this thesis aimed at providing a better understanding of the composition and dynamics of the human small intestinal microbiota and to provide insight in the metabolic potential as well as immunomodulatory properties of some of its typical commensal inhabitants. Small intestinal samples used in the work described in this thesis were collected from ileostomy subjects, individuals that had their large intestine surgically removed and the end of the small intestine connected to an abdominal stoma, providing access to luminal content of the small intestine.

    Considering the importance of molecular techniques in contemporary ecological surveys of microbial communities, first of all, two technologies, barcoded pyrosequencing and phylogenetic microarray analysis were compared in terms of their capacity to determine the bacterial composition in fecal and small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, the use of different primer pairs in the amplification step was assessed in terms of its impact on the outcome of microbial profiling. The analyses revealed that the different primer pairs and the two profiling technologies provide overall similar results for samples of fecal and terminal ileum origin. In contrast, the microbial profiles obtained for small intestinal samples by barcoded pyrosequencing and phylogenetic microarray analysesdiffered considerably. This is most likely attributable to the constraints that are intrinsic to the use of the microarray to enable the detection of predefined microbiota members only, which is due to the probe design that is largely based on large intestinal microbiota communities. However, the pyrosequencing technology also allows for identification of bacteria that were not in advance known to inhabit our intestinal tract.

    The pyrosequencing technology was used as the method of choice to study the total and active small intestinal communities in ileostoma effluent samples from four different subjects through sequencing the 16S ribosomal RNA gene (rDNA) and ribosomal RNA (rRNA) contentcombined with metatranscriptome analysis by Illumina sequencing of cDNA derived from enriched mRNAof the same sample set to investigate the activities of the small intestinal bacteria. The composition of the small intestinal bacterial communities as assessed from rDNA, rRNA, and mRNA patterns appeared to be similar, indicating that the dominant bacteria in the small intestine are also highly active in this ecosystem. Streptococcusspp. were among the bacterial species that were detected in each ileostoma effluent sample, albeit that their abundance varied greatly between samples from the same subject as well as samples from different subjects. Veillonellaspp. frequently co-occurred with Streptococcus spp., indicating that the Streptococcusand Veillonellapopulations play a prominent role in the human small intestine ecosystem and their co-occurrence suggests a metabolic relation between these genera.

    Therefore, cultivation and molecular typing methodologies were employed to zoom-in on these groups, which revealed that the richness of the small intestinal streptococci strongly exceeded the diversity that could be estimated on basis of 16S rRNA analyses, and could be extended to the genomic lineage level (anticipated to resemble strain-level). From ileostoma samples 3 different Streptococcusspecies were recovered belonging to the S. mitisgroup, S. bovisgroup, and S. salivariusgroup, which could be further divided in 7 genomic lineages. Notably, the Streptococcuslineages that were isolated displayed distinct carbohydrate utilization capacities, which may imply that their growth and relative community composition may respond quite strongly to differences in the dietary intake of simple carbohydrates over time. This notion is in good agreement with the observation that the Streptococcuslineage populations fluctuated in time with only one Streptococcuslineage being cultivated from both ileostoma samples collected in a one-year time frame. Conversely, the cultivated Veillonellaisolates from samples during that same time-interval consistently encompassed a single lineage. Furthermore, this Veillonellalineage could be isolated from both the oral cavity as well as the ileostoma effluent. Analogously, three Streptococcuslineages that belong to a single phylotype also appeared to be present in bacterial communities from the oral cavity as well as the small intestine. These observations suggest the representatives of the Veillonellaand Streptococcusgenera that are encountered in the oral and small intestinal microbial ecosystems are closely related and indicate that the oral microbiota may serve as an inoculum for the upper GI tract.

    The metabolic capacity of 6 small intestinal Streptococcus lineages, that were obtained from a single ileostoma effluent sample, was further investigated through the determination of genomic sequences of these lineages. The small-intestinal Streptococcusgenomes were found to encode different carbohydrate transporters and the necessary enzymes to metabolize different sugars, which was in excellent agreement with what carbohydrates could be used by representative strains of the Streptococcuslineages.

    To further our understanding how the different streptococci as representatives of the dominant small intestinal bacterial populations may influence our immune system, human dendritic cells were stimulated with strains of the different Streptococcuslineages to study their immunomodulatory properties. The Streptococcuslineages differed significantly in their capacity to modulate cytokine responses of blood-monocyte derived immature dendritic cells. As Streptococcusand Veillonellafrequently co-occur in the small intestinal ecosystem, pair-wise combinations of strains of these two species were also tested for their combined immunomodulatory properties. This resulted in considerably different cytokine responses as those that could be predicted from the stimulations with either Streptococcusor Veillonella, indicating that it is not trivial to predict gut mucosal associated immune responses and thatthe composition of the intestinal microbiota as a whole may have a distinct influence on an individual’s immune status.

    In conclusion, the work described this thesis provides an expansion to the accumulating knowledge on the human intestine microbiota. Whereas most studies focus on the microbiota present in the distal regions of the intestinal tract, this study targeted the microbiota of the poorly proximal regions of the intestine and also addressed its capacity to interact with the local mucosal tissue. The data presented here can be exploited to guide the design of future studies that aim to elucidate the interplay between diet, microbiota and the mucosal tissues in the human small intestinal tract.

    Beheer van microbiele gemeenschappen en visgezondheid in recirculatiesystemen
    Rurangwa, E. ; Schram, E. ; Smidt, H. ; Verdegem, M.C.J. - \ 2011
    Aquacultuur 26 (2011)1. - ISSN 1382-2764 - p. 6 - 11.
    visteelt - hergebruik van water - diergezondheid - darmmicro-organismen - projecten - microbiële ecologie - aquacultuur - waterkwaliteit - fish culture - water reuse - animal health - intestinal microorganisms - projects - microbial ecology - aquaculture - water quality
    In dit artikel wordt de rol van de microbiële gemeenschap in de darm van vissen beschreven en wordt een overzicht gegeven van de belangrijkste huidige kennis en inzichten. Ook wordt een korte beschrijving gegeven van het ministerie van EL&I (voormalig LNV) gefinancierde project "Visgezondheid in RAS" en wordt aangestipt wat we verwachten te leren van dit project.
    Bodemweerbaarheid: hoe krijgen we er grip op?
    Os, G.J. van; Postma, J. - \ 2011
    Gewasbescherming 42 (2011)1. - ISSN 0166-6495 - p. 11 - 12.
    bodemkwaliteit - gezondheid - bodemfauna - fysische gewasbeschermingsmethoden - microbiële ecologie - gewasbescherming - soil quality - health - soil fauna - physical control - microbial ecology - plant protection
    Met de afnemende beschikbaarheid van chemische gewasbeschermingsmiddelen, is de land- en tuinbouw steeds meer aangewezen op de natuurlijke, ziekteonderdrukkende eigenschappen van de bodem. In een ziektewerende grond zal, ondanks de aanwezigheid van een ziekteverwekker, geen of weinig schade optreden in een vatbaar gewas. Het microbiële bodemleven is hierbij een belangerijke factor. Een rijk en divers bodemleven kan goede concurrenten of antagonisten tegen ziekteverwekkers bevatten. De samenstelling van het bodemleven is afhankelijk van de fysische en chemische eigenschappen van de bodem. Toevoeging van organische stof kan de fysische- en chemische variatie in de grond verhogen en daarmee ook de bodembiodiversiteit. Maar, zorgt meer organische stof ook altijd voor een hogere bodemweerbaarheid? Dit is onderzocht in het project TopSoil + (2005-2009)
    Shiga toxin-producing Escherichia coli in humans and the food chain in Bangladesh
    Islam, M.A. - \ 2009
    Wageningen University. Promotor(en): Marcel Zwietering, co-promotor(en): A.E. Heuvelink. - [S.l.] : S.n. - ISBN 9789085852919 - 183
    escherichia coli - bacteriële toxinen - microbiële ecologie - diarree - ziekteprevalentie - voedselketens - vee - bangladesh - escherichia coli - bacterial toxins - microbial ecology - diarrhoea - disease prevalence - food chains - livestock - bangladesh
    Shiga toxin-producing Escherichia coli (STEC) are significant pathogenic bacteria that can cause severe gastrointestinal diseases and also the hemolytic-uremic syndrome. Domestic ruminants appear to be the main reservoirs of these organisms. Although Bangladesh is an endemic zone for diarrhea caused by different enteric pathogens, no systematic study on STEC has yet been done there. We estimated the prevalence of STEC infections among diarrheal patients and the occurrences of STEC in the human food chain in Bangladesh. In addition, we evaluated methods for the isolation of STEC O157 from animal feces and foods. We found that the prevalence of STEC was low among diarrhoeal patients compared with other diarrheagenic pathogens. In contrast, there is a high prevalence of STEC including serogroup O157 in animal reservoirs and in the food chain. We concluded that the lack of STEC O157 infection among Bangladeshi population might be due to the protective immunity against these pathogens acquired by the frequent exposure to the antigens.
    Ecology and modelling of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cattle manure and soil
    Semenov, A.V. - \ 2008
    Wageningen University. Promotor(en): Ariena van Bruggen, co-promotor(en): Leo van Overbeek. - S.l. : S.n. - ISBN 9789085852452 - 212
    mest - bodem - escherichia coli - salmonella enteritidis - microbiële ecologie - voedselbesmetting - simulatiemodellen - bodembeheer - modelleren - manures - soil - escherichia coli - salmonella enteritidis - microbial ecology - food contamination - simulation models - soil management - modeling
    The number of food poisoning cases caused by enteropathogens has increased in recent years. A significant part of the outbreaks associated with the consumption of raw vegetables has been attributed to Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium. Bovine manure and slurry are the main environmental sources of these pathogens. Thus, reduction of the multiplication of E. coli O157:H7 and Salmonella serovar Typhimurium in cattle and their survival in manure and slurry are important tasks to minimize the risks of contamination of plant products and outbreaks of food-borne diseases. This thesis describes the influence of various environmental factors on survival of E. coli O157:H7 and Salmonella serovar Typhimurium in manure, slurry and soil amended with manure or slurry. The results presented in this thesis can be used for risk assessment of E. coli O157:H7 and Salmonella serovar Typhimurium in dairy farming systems and will help to identify and evaluate potential control strategies to minimize the chance of pathogen spread in the vegetable production chain.
    Onion thrips, Thrips tabaci, have gut bacteria that are closely related to the symbionts of the western flower thrips, Frankliniella occidentalis
    Vries, E.J. de; Wurff, A.W.G. van der; Jacobs, G. ; Breeuwer, J.A.J. - \ 2008
    Journal of Insect Science 8 (2008). - ISSN 1536-2442 - p. 1 - 11.
    microbial ecology - thysanoptera - transmission - insects - growth - evolutionary - association - resistance - wolbachia - termites
    It has been shown that many insects have Enterobacteriaceae bacteria in their gut system. The western flower thrips, Frankliniella occidentalis Pergande [Thysanoptera: Thripidae], has a symbiotic relation with Erwinia species gut bacteria. To determine if other Thripidae species have similar bacterial symbionts, the onion thrips, Thrips tabaci, was studied because, like F. occidentalis, it is phytophagous. Contrary to F. occidentalis, T. tabaci is endemic in Europe and biotypes have been described. Bacteria were isolated from the majority of populations and biotypes of T. tabaci examined. Bacteria were present in high numbers in most individuals of the populations studied. Like F. occidentalis, T. tabaci contained one type of bacterium that clearly outnumbered all other types present in the gut. This bacterium was identified as an Erwinia species, as was also the case for F. occidentalis. However, its biochemical characteristics and 16S rDNA sequence differed from the bacteria present in F. occidentalis.
    Community structure of actively growing bacterial populations in plant pathogen suppressive soil
    Hjort, K. ; Lembke, A. ; Speksnijder, A.G.C.L. ; Smalla, K. ; Jansson, J.K. - \ 2007
    Microbial Ecology 53 (2007)3. - ISSN 0095-3628 - p. 399 - 413.
    gradient gel-electrophoresis - 16s ribosomal-rna - bromodeoxyuridine immunocapture - plasmodiophora-brassicae - molecular-cloning - microbial ecology - chitinase gene - fungi - identification - amplification
    The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil
    Ecology and risk assessment of E. coli O157:H7 and Salmonella typhimurium in the primary production chain of lettuce
    Franz, E. - \ 2007
    Wageningen University. Promotor(en): Ariena van Bruggen, co-promotor(en): Aad Termorshuizen. - [S.l.] : S.n. - ISBN 9789085047285 - 216
    lactuca sativa - slasoorten - voedselbesmetting - escherichia coli - salmonella typhimurium - stalmest - risicofactoren - microbiële ecologie - bodem - biologische landbouw - biologische voedingsmiddelen - primaire productie - lactuca sativa - lettuces - food contamination - escherichia coli - salmonella typhimurium - farmyard manure - risk factors - microbial ecology - soil - organic farming - organic foods - primary production
    Survival of the green fluorescent protein-transformed human pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was studied in a laboratorysimulated lettuce production chain. Dairy cows were fed 3 different roughage types: high digestible grass silage + maize silage (6:4), low digestible grass silage and straw. Each was adjusted with supplemental concentrates to a high and low crude protein level. The pathogens were added to manure which was subsequently mixed (after 56 and 28 d for resp. E. coli O157:H7 and Salmonella serovar Typhimurium) with 2 pairs of organically and conventionally managed loamy and sandy soil. After another 14 d, iceberg-lettuce seedlings were planted and checked for pathogens after 21 d of growth. Survival data were fitted to a logistic decline function (exponential for E. coli O157: H7 in soil). Roughage type significantly influenced the decline rate of E. coli O157: H7 in manure with the fastest decline in manure from the pure straw diet and the slowest in manure from the grass-silage + maize-silage diet. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although decline was significantly faster in the manure derived from straw compared to the manure from the grass-silage + maize-silage diet. The pH and fiber content of the manure were significant explanatory factors and were positively correlated with the rate of decline. With E. coli O157:H7 there was a trend of faster decline in organic compared to conventional soils. No pathogens were detected in the edible lettuce parts. The results indicate that cattle diet and soil management are important factors with respect to the survival of human pathogens in the environment.
    Gastrointestinal microbiology
    Ouwehand, Arthur C. ; Vaughan, Elaine E. - \ 2006
    CRC Press - ISBN 9780824726416 - 410 p.
    intestinal microorganisms - digestive tract - microbial ecology

    This reference supplies a comprehensive and current overview of every aspect of gastrointestinal microbiota. Expertly written chapters cover conventional and molecular techniques for the study of differing microbial populations, as well as the analysis of microbial activity and interaction with host bodies. Illustrative and up-to-date, this source showcases how fluctuations in our diet, environment, and pharmaceutical intake affect microbial composition and behavior.

    The significance of microbial diversity in agricultural soil for disease suppressiveness
    Garbeva, P. - \ 2005
    Wageningen University; Leiden University. Promotor(en): J.A. van Veen; J.D. van Elsas, co-promotor(en): E.J.J. Lugtenberg; Lijbert Brussaard. - NWO - 169
    micro-organismen - bodemflora - microbiële ecologie - diversiteit - plantenziekten - bedrijfssystemen - bodembeheer - plantenziektebestrijding - microbiële diversiteit - microorganisms - soil flora - microbial ecology - diversity - plant diseases - farming systems - soil management - plant disease control - microbial diversity
    Prebiotics in piglet nutrition? Fermentation kinetics along the GI tract
    Awati, A.A. - \ 2005
    Wageningen University. Promotor(en): Martin Verstegen, co-promotor(en): B.A. Williams; M.W. Bosch. - Wageningen : S.n. - ISBN 9789085041641 - 143
    biggen - anti-infectieuze middelen - koolhydraten - fermentatie - kinetica - voedertoevoegingen - microbiële ecologie - varkensvoeding - voedingsfysiologie - piglets - antiinfective agents - carbohydrates - fermentation - kinetics - feed additives - microbial ecology - pig feeding - nutrition physiology

    Keywords: fermentation, gas production, piglets

    The generalized theory behind the carbohydrate to protein fermentation in the GIT is that in presence of fermentable carbohydrate substrate, microbes prefer to ferment carbohydrate source to derive energy and use the nitrogen available for their own growth. With this background information, it was hypothesized that inclusion of fermentable carbohydrates in the piglet diet will reduce the protein fermentation, which will be confirmed by reduced levels of ammonia and branched chain fatty acids in end product profile of the fermentation. The aim of this thesis was to study the effects of inclusion of fermentable carbohydrates in weaning piglets' diet, on GIT fermentation and any changes in microbial community composition and activity. Weaning process in an intensified pig production system brings many sudden changes in the environmental and physical factors in piglets' life. These sudden changes, especially in diet cause serious imbalance in the microbial community. Quicker stabilization and diversification of microbial community post weaning, is crucial in attending the gut health and reducing the risk of pathogenic infections by 'Colonization resistance: As part of this overall aim, the in vitro cumulative gas production technique was used to study the fermentation of selected fermentable substrates. While these substrates namely lactulose, inulin, wheat starch and sugar beet pulp (SBP) were included in test diet and their effect on GIT fermentation was studied in vivo. The combination of microbial community analysis based on fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) with nutritional analysis of fermentation end product profiles, was used in vivo and in vitro studies. In in vivo trials, emphasis was given on using combination of slow fermenting carbohydrate sources such as, SBP and wheat starch with fast fermenting lactulose and inulin. The hypothesis behind this approach was to induce carbohydrate fermentation along the GIT, by providing carbohydrate substrate for the microbiota in different parts of GIT. Especially by taking in to account the difference in the transit time of feed in the different parts of GIT, it was expected that fast fermenting lactulose and inulin would be fermented in small intestine while wheat starch somewhere in the beginning of the large intestine while, SBP will reach the distal part of colon. It was found that fermentation along the GIT was improved or in other words skewed more towards the carbohydrate fermentation in vivo. It was observed in vivo that inclusion of fermentable carbohydrates in the diet reduces the protein fermentation in the GIT and ammonia concentration in end product profile. This decrease was observed along the GIT and in time in faecal fermentation end product profiles post weaning. Microbial community analysis using fingerprinting techniques revealed that inclusion of fermentable carbohydrates stabilized and diversified microbial community in the ileum as well as in the colon by day 10 post weaning. This way, the prebiotic effects of fermentable carbohydrates was evidenced. -
    Effects of prebiotics, probiotics and synbiotics in the diet of young pigs
    Shim, S.B. - \ 2005
    Wageningen University. Promotor(en): Martin Verstegen, co-promotor(en): J.M.A.J. Verdonk. - s.l. : S.n. - ISBN 9789085041931 - 178
    biggen - probiotica - koolhydraten - anti-infectieuze middelen - voedertoevoegingen - microbiële ecologie - fermentatie - spijsverteringsstelsel - varkensvoeding - voer - diergezondheid - groei - voedingsfysiologie - piglets - probiotics - carbohydrates - antiinfective agents - feed additives - microbial ecology - fermentation - digestive system - pig feeding - feeds - animal health - growth - nutrition physiology

    Keywords: prebiotics, piglets, gut health
    Prebiotics are non-digestible carbohydrates that are not metabolized in the small intestine and fermented in the large intestine. Oligofructose are non-digestible oligosaccharides which may stimulate beneficial bacteria in the gut and may affect the gut ecosystem. Prebiotic effects will depend largely on their chemical structure (degree of polymerization). Dietary inclusion of probiotics in young pig diets may beneficially affect gut microbiota. Synbiotics, a combination of prebiotics and probiotics may also stimulate the gut ecosystem. The objective of this thesis was to evaluate the effects of pre-, pro- and synbiotics on the gut ecosystem, and some performance parameters. A series of in vivo and in vitro experiments were carried out using suckling and weaned piglets. The experimental results are discussed in this thesis. Overall, it was concluded that synbiotics, a combination of multi-strain probiotics and oligofructose, can positively affect performance especially feed intake, and can improve the gut health. However, we did not observe a clear synergistic effect compared to supplementing oligofructose or probiotics alone. A combination of high and low polymer inulin will probably be more beneficial for the intestinal ecosystem and health than using either high- or low polymer inulin alone. The present studies show that the pre-, pro- and synbiotic treatments affect gut microbiota and performance of young pigs.
    Food Fermentation
    Nout, M.J.R. ; Vos, W.M. de; Zwietering, M.H. - \ 2005
    Wageningen : Wageningen Academic Publishers - ISBN 9789076998831 - 217
    fermentatie - gefermenteerde voedingsmiddelen - zuursels - microbiële ecologie - voedselbiotechnologie - fermentation - fermented foods - starters - microbial ecology - food biotechnology
    The focus of this book is on state of the art technologies and scientific developments in academia and industry that contribute to the characterization and specification of fermentation starter microorganisms, to the present-day experimental approaches in product and process development and control, and to high throughput analytical techniques that facilitate the precise design of tailor-made fermented food products. Aspects covered include: microbial biodiversity of starter lactic acid bacteria, yeasts and moulds; product technology and functionality relating to flavour formation and control; health promoting aspects of foods and of probiotic and nutraceutical microbes; European legislation of fermented foods and ingredients; modelling and control of bacterial and fungal fermentation processes; and the relevance of ~omics (genomics, transcriptomics, proteomics, metabolomics) in starter design, metabolic control and safety assurance. This volume surely is an essential up-date for R&D professionals and advanced students of food science and technology.
    Organic matter decomposition in simulated aquaculture ponds
    Torres Beristain, B. - \ 2005
    Wageningen University. Promotor(en): Johan Verreth, co-promotor(en): Marc Verdegem. - [S.I.] : S.n. - ISBN 9789085041702 - 138
    visvijvers - visteelt - aquacultuur - organische verbindingen - decompositie - microbiële afbraak - microbiële ecologie - biomassa productie - recycling - fish ponds - fish culture - aquaculture - organic compounds - decomposition - microbial degradation - microbial ecology - biomass production - recycling

    Different kinds of organic and inorganic compounds (e.g. formulated food, manures, fertilizers) are added to aquaculture ponds to increase fish production. However, a large part of these inputs are not utilized by the fish and are decomposed inside the pond. The microbiological decomposition of the organic matter is a critical factor for water quality control and nutrient recycle. Usually, management practices are developed to control the survival and health of the cultured animals and to maintain good water quality. The microbiological processes are affected by these practices but usually unintentionally. A better control of culture conditions and sustainability of aquaculture ponds is possible with an improvement of the microbiological processes. The present thesis is divided in two parts, the first is a literature review of the microbial ecology of aquaculture ponds and the second is the description of a series of experiments in lab-sc ale aquaculture ponds.

    In the first part, the role of the microorganisms in aquaculture ponds is reviewed, focusing on the decomposition of organic matter and its influence on pond dynamics. It was theoretically estimated that the addition of I kg of formulated feed would yield approximately 125 g bacterial biomass. This bacterial biomass is potentially a valuable nutrients source for higher trophic levels. Sedimentation and resuspension processes are important factors affecting the decomposition pathways. Both processes are directly related with the feeding rate and the stocking density applied. The rate of organic matter loading, environmental factors and pond management practices influence the functioning of algae-bacteria interactions, which are extremely important in pond processes. Included is a literature that describes commercial probiotic products that claim to solve: nutritious, water quality and pathogens problems in pond aquaculture, were analyzed. Alternative management practices to steer the decomposition process were also presented (Chapter 2).

    The second part describes all the experiments that were conducted in lab scale microcosm systems, simulating the conditions of an intensive fish-less aquaculture pond with daily feeding rates. In Chapter 3 the influence of aerobic and anaerobic conditions and the organic C/N ratios on the decomposition process is described. Under aerobic decomposition less organic carbon remained in the systems. The results from this experiment suggest that a C/N ratio ranging between the tested values (6.3 and 12.8) does not have a significant influence on the carbon mineralization in the short term " 50 days). However, a C/N ratio decrease was observed in all the treatments during the experimental period; this reduction was especially fast and steep under aerobic conditions. This decrease in C/N ratio of the organic matter might explain why in all treatments the rate of decomposition slowed down at the end of the experiment. The C/N ratio also determined the concentration of inorganic nitrogen compounds in the water. Higher concentrations were found for the richest protein diet treatments. No nitrification was measured even though oxygen and ammonia were present.

    Bacterial biomass production was quantified testing two formulated fish feed with different protein content under aerobic and anaerobic conditions (Chapter 4). The oxic status significantly influences the bacterial abundance, bacterial biomass, bacterial respiration and bacterial efficiency. More bacterial biomass was produced under aerobic conditions. The two diets did not influence significantly the bacterial growth. The bacterial abundance at the end of the experiment was 3.4 x 109 cells ml-1 in aerobic treatments and 1.9 x 109 cells m1-1 in anaerobic treatments. The remaining amount of carbon, fixed in bacterial biomass and expressed on a per area basis, was 19 g m-2 day-t for aerobic system and 8 g m-2 day-1 for anaerobic systems.

    In Chapter 5 the effect of the oxic-anoxic range on fish feed decomposition was investigated. Different ranges, from completely aerobic to completely anaerobic, were tested. To establish intermediate oxic levels the following treatments were used: 1) alternated flows of 02 or N2 at different periods and 2) maintaining the coexistence of aerobic and anaerobic layers while applying short resuspension events. Similar amounts of carbon were converted to CO2 under completely aerobic conditions and under the different ranges of aerobic-anaerobic conditions. Under anaerobic conditions much less carbon was converted into CO2. This means that actually only limited periods of oxic conditions (or resuspension) are needed to stimulate complete organic matter decomposition. From our results it appears that only 6h per day of aerobic conditions or only once mixing of aerobic and anaerobic layers (i.e. resuspension) per four days are needed to reach the same carbon mineralization as in continuous aerobic conditions. Very limited nitrification was observed in the completely aerobic treatment. Nitrification and denitrification were registered for all the systems when aerobic and anaerobic conditions coexisted in time or space. The highest nitrogen removal (around 70%) was found in the resuspension treatments (and 12 h O2 flow treatment).

    The use of controlled lab scale microcosm simulating intensive aquaculture ponds allowed us to follow the fate of carbon and nitrogen during particular decomposition processes. The results found in the different chapters are discussed in Chapter 6. Both the quality and the quantity of the organic matter influenced the decomposition process and its products. The use of high protein diets increased the concentration of nitrogen species affecting the water quality. The aerobic and anaerobic conditions determined the nutrients pathway (mineralized, assimilated or partially decomposed). More bacterial biomass was produced under aerobic conditions than under anaerobic. The coexistence of aerobic and anaerobic conditions stimulated organic matter decomposition; it avoided the accumulation of ammonia while maintaining good water quality conditions.

    A better understanding and control of the organic matter decomposition in aquaculture ponds is crucial. The anaerobic decomposition only becomes a problem when it predominates in the sediment, causing the aerobic-anaerobic interface to move up into the water column, and thus remains disconnected from the aerobic decomposition. Management practices that link aerobic and anaerobic processes can stimulate fish production by recycling carbon and nitrogen compounds. The recycling of surplus organic matter through bacterial processes, however, has a limit. Increasing fish pond productivity should come along with practices to stimulate the autotrophic and heterotrophic food webs, without exceeding the capacity of this aquatic system.

    Molecular microbial ecology manual
    Kowalchuk, G.A. ; Bruijn, F.J. de; Head, I.M. ; Akkermans, A.D.L. - \ 2004
    Dordrecht : Kluwer - ISBN 9781402021831
    microbiologie - microbiële ecologie - microbiële fysiologie - methodologie - micro-organismen - ecologie - biochemie - microbiology - microbial ecology - microbial physiology - methodology - microorganisms - ecology - biochemistry
    The field of microbial ecology has been revolutionized in the past two decades by the introduction of molecular methods into the toolbox of the microbial ecologist. This molecular arsenal has helped to unveil the enormity of microbial diversity across the breadth of the earth's ecosystems, and has revealed that we are only familiar with a very small minority of the organisms that carry out key microbial functions in diverse habitats. The Molecular Microbial Ecology Manual, Second Edition (MMEM-II) provides a detailed and user-friendly description of the methods that have made this revolution in microbial ecology possible. However, what is perhaps most exciting about MMEM-II is that it contains a large number of new chapters, highlighting the newest trends in microbial ecology research, which seek to provide more quantitative and statistically robust data, and means of coupling microbial identity and function. In addition, the majority of the proven methods described in MMEM's first version have undergone significant revisions to provide the most up-to-date applications available. The state-of-the-art methods described in MMEM-II have not only been provided by experts in the field, but in most cases by the laboratories that actually first developed and applied the methods, thus providing the MMEM-II user with unique first-hand tips and insight.
    Development of bacterial and bifidobacterial communities in feces of newborn babies
    Favier, C.F. ; Vos, W.M. de; Akkermans, A.D.L. - \ 2003
    Anaerobe 9 (2003). - ISSN 1075-9964 - p. 219 - 229.
    16s ribosomal-rna - gradient gel-electrophoresis - bottle-fed infants - human intestinal microflora - species-specific primers - formula-fed infants - fecal flora - microbial ecology - targeted probes - human-colon
    Microbial 16S rDNA from babies' fecal samples were amplified by PCR, and analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. PCR-DGGE profiles were used to follow in time the colonization of the intestine by bacteria. Four healthy babies, one baby who received antibiotics and their parents participated to the present study to determine the extent to which administration of antibiotics can modify the bacterial colonization of neonatal human gut and verify the influence of parental factors on the formation of the fecal bacterial community. In the healthy babies, Escherichia coli or bacteria belonging to Clostridium spp. were the initial colonizers rapidly followed by Bifidobacterium, Bacteroides, Clostridium, Streptococcus, Enterococcus and Actinomyces. Bifidobacterium species appeared already after five days in the breast-fed babies while there was a delay in the baby who received a formula based diet during only one day after birth. In each baby two or three bifidobacterial species including B. infantis were found. The observed variations in species were not associated with the feeding changes. The comparison of DGGE profiles of the babies and their parents patterns showed bands with equal migration suggesting a vertical transmission determined by genetic and environmental factors. The brief appearance of pioneer bacteria determined as being E coli and Enterococcus spp. in the profile from the. baby under antibiotic therapy, was succeeded by a small stable community consisting of Ruminococcus species. No Bifidobacterium sequences were detectable in this antibiotic-treated baby in spite of a partly breast-milk diet. (C) 2003 Elsevier Ltd. All rights reserved.
    Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by DGGE analysis of 16S rDNA
    Konstantinov, S.R. ; Zhu Wei-Yun, ; Williams, B.A. ; Tamminga, S. ; Vos, W.M. de; Akkermans, A.D.L. - \ 2003
    FEMS microbiology ecology 43 (2003)2. - ISSN 0168-6496 - p. 225 - 235.
    gastrointestinal-tract - microbial ecology - rna - populations - diversity - pcr - dietary - swine - genes - identification
    The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
    Molecular ecology of Frankia and other soil bacteria under natural and chlorobenzoate-stressed conditions
    Ramirez-Saad, H.C. - \ 1999
    Agricultural University. Promotor(en): W.M. de Vos; A.D.L. Akkermans. - S.l. : Ramirez Saad - ISBN 9789058080660 - 119
    bodembacteriën - frankia - microbiële ecologie - benzoaten - soil bacteria - frankia - microbial ecology - benzoates

    Microbial Ecology studies aim to describe and assess the behavior and activity of microorganisms in their natural environments (Brock 1966). Nowadays it is clear that the large number of existing microorganisms has surpassed our capabilities to rapidly characterise them by traditional culturing methods. This has resulted in a poor understanding of the structure and composition of microbial communities. As an alternative, microbial communities can be described on the basis of 16S rRNA sequence diversity, without the bias-introducing step of cultivation.

    In the present thesis a molecular analysis is given of two ecosystems that harbour several uncultured bacteria. The first part of the thesis is focused on the detection and characterisation of Frankia in actinorhizal nodules and soil. Elucidation of the actual diversity within the family Frankiaceae was hampered by the inability to obtain isolates from all known actinorhizal plants. So far, the Nod +/Fix +Frankia symbionts in root nodules of plants from the families Coriariaceae, Datiscaceae, Rosaceae and Rhamnaceae (with exceptions reported by Carú 1993, Carú et al . 1990, and Carrasco et al . 1995) have resisted isolation. Best opportunities to characterise those uncultured endophytes require molecular methods that relay heavily on an easy and efficient technique to extract DNA from the respective actinorhizal nodules. Chapter 2 describes the techniques to isolate DNA from root nodules of different actinorhizal plants such as Casuarina sp, Alnus sp and Ceanothus sp. The procedure has also been successfully applied by Wolters et al . (1997b) in the minuscule ineffective nodules on Alnus glutinosa .

    Several attempts to characterise the uncultured endophytes from Coriaria sp. and Datisca sp. plants pointed on the one hand, to the presence in those actinorhizae of Frankia -related actinomycetes. This assumption was based mainly on the repeated isolation from those nodules of Nod -/Fix -Frankia -like strains (Hafeez 1983, Mirza et. al . 1994b, c). On the other hand, the effective (Fix +), non-isolated symbionts showed to be phylogenetically closely related (Mirza et al. 1994a), forming a separate lineage within the genus, in spite of the distant geographical distribution of the plants (Nick et al. 1992).

    The work described in Chapter 3 is focussed on the localisation and phylogenetic position of the nitrogen-fixing Frankia and Nod -/Fix -actinomycetes, both present in root nodules of the Mexican actinorhizal plant Ceanothus caeruleus . Application of the TGGE technique allowed localising the Nod -/Fix -actinomycete in the outer layers of the C. caeruleus nodules. Similar bacteria were also detected in Hippophaë rhamnoides nodules induced with soil inoculum that was collected in the vicinity of the former plant. The fact that a second nodule inhabitant was commonly present in these nodules containing recalcitrant endophytes may allow some speculations about their possible role in the symbiosis. However, it seems worthwhile to apply the same TGGE methodology to other actinorhizal nodules, even to those containing Frankia strains that are easy-to-isolate (i.e. Elaeagnus,Casuarina, Alnus spp.), since the detection of Frankia -related actinomycetes, in addition to the Fix +endophyte, would provide further evidence about the need for their presence. Coincidentally, the Nod -/Fix -isolates from Coriaria, Datisca and Ceanothus are phylogenetically related, pointing again to a certain specificity for their presence in the nodules. This relatedness has also been supported by analysis of low molecular weight RNA (i.e. 5S rRNA and tRNA's) using staircase electrophoresis (Velázquez et al. 1998).

    The 16S rDNA sequence from the non-isolated Fix +endophyte in C. caeruleus root nodules (Chapter 3), was the first full sequence obtained from a field-collected Ceanothus symbiont. Parsimony and phylogenetic distance analyses grouped it within the Dryas cluster that originally contained only the uncultured endophytes from Dryas , Coriaria and Datisca as proposed by Normand et al. (1996). Benson et al . (1996) redefined this cluster by adding other uncultured endophytes present in Ceanothus griseus (Rhamnaceae), Purshia tridentata and Dryas drummondii (Rosaceae) root nodules. Since the determined partial 16S rDNA sequences were almost identical, they suggested that the Frankia diversity from these actinorhizal plant families might be low. However, Clawson et al. (1998) demonstrated that Frankia isolates obtained from several genera within the Rhamnaceae (i.e. Talguenea , Colletia , Discaria, Retanilla and Trevoa ) were phylogenetically different than those in Ceanothus, grouping in the Elaeagnus cluster. These findings were consistent with morphological differences of the endophytes in planta , since the vesicles found in the Ceanothus symbionts resemble more to those in the Rosaceae, while all the latter host plants in the Rhamnaceae family have endophytes like those in Elaeagnus .

    The results reported in the first part of the thesis have demonstrated that TGGE and sequence analysis of 16S rDNA provide an accurate picture for the identification of recalcitrant endophytes in root nodules of actinorhizal plants. It has also been demonstrated that besides the N 2 -fixing endophyte, root nodules of C. caeruleus also harbour Frankia -related actinomycetes. Since these have also been observed in other actinorhizae, a further study is needed to understand the possible function of these co-symbionts.

    The work described in the second part of the thesis was addressing the changes occurring under chlorobenzoate stress in the soil bacterial community and other selected groups of bacteria present in peat soil collected from a natural Alnus glutinosa stand. A combination of culturing and non-culture based approaches was used for the assessment. Among the latter approaches, the possibilities offered by TGGE were exploited in several ways. Profiling of complex communities and subsequent analysis of specific bacterial groups has been one of the major applications of TGGE (Felske et al . 1996). With this approach, major population shifts induced by either 3CBA or 2,5DCB were detected in the uncultured bacterial community (Chapter 4). Although only the former compound was readily metabolised in soil, both xenobiotics promoted similar changes. Several bacterial populations were reduced or suppressed, while few others were enriched in time, as assessed by shifts in the TGGE banding patterns of the total bacterial community.

    To characterise the soil-enriched bacterial populations, 3CBA-degrading enrichment cultures were obtained and their composition was addressed by TGGE. Further isolation attempts were directed by this means to prove that the isolated strains were indeed the same enriched organisms as detected in soil. One of the enrichment cultures contained two of the soil-enriched bacteria as predominant components. Although isolation was not achieved, both bacteria were identified as belonging to the genus Burkholderia . The bacterial group detected as predominantly enriched in both spiked soils was not present in any of the enrichment cultures, suggesting that the microorganisms belonging to this groups are either unable to degrade 3CBA or not growing under the used culturing conditions. In any case their fitness to the soil conditions imposed by the addition of chlorobenzoates was high, but the mechanisms involved were not elucidated. These bacteria were also identified as Burkholderia by partial 16S rDNA sequence analysis (Chapter 4).

    The diversity (H) and the equitability (J) indices are important parameters used by ecologists to assess the species richness and the species evenness, respectively, within a community. As the estimation of such indices relies heavily on species definition and individuals enumeration, their application in microbial ecology studies is seldomly possible. Furthermore, assessment of H and J in uncultured bacterial communities must rely on the interpretation of community fingerprints, which should provide means to distinguish between species or operational taxonomic units (OTU), and to estimate their abundance. TGGE community profiling offers both possibilities, and the community changes occurring in the model soil system were evaluated with this original approach (Chapter 5).

    In addition, H and J indices were also estimated for the fluorescent pseudomonads group, a selected culturable fraction of the bacterial community. OTU recognition was addressed by using TGGE as a ribotype-fingerprinting technique for the isolated fluorescent pseudomonads. Estimation of H and J at the community level without culturing by TGGE profiling, and at the group level by a combination of culturing and TGGE ribotyping should allow to address and compare the population changes occurring, since the target molecule used in both TGGE was the same. Such comparison was only partially possible since most of the bands corresponding to the fluorescent pseudomonads could not be assessed in the community profiles. However, estimation of H and J indices indicated a clear reduction of species richness and individuals abundance in the uncultured community, which was related to the presence of chlorobenzoates in soil. Evaluation of population shifts by indexed values as H and J proved to be a useful means for analysing the community structure in time, and may be used to assess short and long-term responses of a bacterial community to environmental perturbations.

    Chapter 6 describes the changes in the total frankiae in soil and in the fraction of the population that is able to produce root nodules in Alnus glutinosa seedlings . Culture-independent approaches based on the most probable number concept were used, one in combination with a Frankia -specific PCR detection and another in combination with a plant-nodulation bioassay. After 15 days of incubation in the presence of chlorobenzoates both fractions of the soil Frankia populations were reduced in more than one order of magnitude, while the populations in the unspiked control soil were not affected. The results indicated that 3CBA and 2,5DCB both had a negative effect on the size of the native Frankia population from the used peat forest soil. This negative effect was also evident during in vitro experiments using Frankia strains isolated from Alnus sp. The presence of 1 mM 3CBA in the culture medium, in addition to the normal carbon source, resulted in reduction or suppression of biomass yield.

    The influence of Alnus glutinosa on the dechlorination of 3CBA by Pseudomonas sp. strain B13 was assessed in hydroponic cultures. It was expected that root exudates could enhance the dechlorination activity of Pseudomonas B13. When the bacteria were incubated in the presence of an alder plant, only a slight increase in the dechlorination rate of 3CBA was registered in comparison to the control without plant. The main observed effect in the alder plants appears to be a protection against 3CBA toxicity, as the alders inoculated with Pseudomonas B13 showed a better survival rate and grew more vigorously than the non-inoculated plants.

    The toolbox for microbial ecology studies is increasing constantly by means of developing new techniques or by adapting foreign tools into the field, such as indices to evaluate species diversity and eveness. Although the information provided by these community parameters facilitates comparisons and assessment of changes, their suitability to evaluate bacterial communities is still uncomplete. Estimation of diversity indices requires the recognition of bacterial species as discrete units, and this condition is far from real in natural environments. Species-independent approaches to evaluate diversity must be developed, that consider the bacterial diversity as a continuous range of phylogenetically related taxonomic units.

    In conclusion, the work described in the first part of the thesis strengthen the current phylogenetic division of the Frankiaceae, by adding new evidence supporting two of the already described clusters ( Dryas cluster, Nod -/Fix -cluster). Although the current taxonomic status of the latter cluster must be better evaluated in order to assess its pertinence to the genus Frankia . In addition, the common occurrence of Nod -/Fix -Frankia -like actinomycetes in nodules containing recalcitrant endophytes, mainly from the Dryas cluster, was also demonstrated for Ceanothus actinorhiza. In the second part, the suitability and versatility of molecular tools such as TGGE were demonstrated by their application in community profiling and estimation of bacterial diversity. Community changes occurring in stressed and unstressed soil systems were easily detected and assessed by this means.

    In addition, specific populations such as the Burkholderia -like bacteria that strongly reacted to the addition of chlorobenzoates to soil were further characterised. Moreover, TGGE was shown to be a fast ribotyping technique that may enable its use in combination with the community profiles to address shifts of specific groups within bacterial communities. It is tempting to suggest that this general approach will be of importance to direct the isolation of hitherto uncultured bacteria from soil.

    Detectie en ecologie van de bruinrot-bacterie: Recente resultaten van het onderzoek naar de detectie en ecologie van Ralstonia (Pseudomonas) solanacearum (ras 3), de veroorzaker van bruinrot in aardappel
    Kastelein, P. ; Wolf, J.M. van der; Vuurde, J.W.L. van; Griep, R.A. ; Schots, A. ; Elsas, J.D. van - \ 1998
    Gewasbescherming 29 (1998)2. - ISSN 0166-6495 - p. 39 - 41.
    plantenziekten - plantenziekteverwekkende bacteriën - solanum tuberosum - aardappelen - microbiële ecologie - oogstschade - diagnostische technieken - pseudomonas - nederland - plant diseases - plant pathogenic bacteria - solanum tuberosum - potatoes - microbial ecology - crop damage - diagnostic techniques - pseudomonas - netherlands
    Ecologie en beheersing van bitterzoet in relatie tot bruinrot
    Kempenaar, C. ; Groeneveld, R.M.W. ; Lotz, L.A.P. ; Wenneker, M. ; Janse, J.D. - \ 1998
    Gewasbescherming 29 (1998)4. - ISSN 0166-6495 - p. 119 - 123.
    plantenziekten - plantenziekteverwekkende bacteriën - solanum tuberosum - aardappelen - microbiële ecologie - oogstschade - diagnostische technieken - pseudomonas - nederland - plant diseases - plant pathogenic bacteria - potatoes - microbial ecology - crop damage - diagnostic techniques - netherlands
    Bitterzoet wordt beschouwd als een belangrijke factor in de overleving van de bruinrotbacterie, veroorzaker van bruinrot in aardappel, in oppervlaktewater in Nederland. Onderzoek heeft geleid tot verbeterde inzichten in de ecologie en beheersmogelijkheden van bitterzoet langs watergangen
    Microorganisms in Foods 6: Microbial Ecology of Food Commodities.
    Schothorst, M. van - \ 1997
    London : Blackie Academic & Professional - ISBN 9780751404302 - 615
    voedselindustrie - voedseltechnologie - micro-organismen - bacteriën - classificatie - taxonomie - dierlijke producten - bacteriologie - kiemgetal - voedselinspectie - microbiologie - voedingsmiddelen - voedselbewaring - groenteproducten - opslag - plantaardige producten - microbiële ecologie - methodologie - biologische technieken - experimenten - uitrusting - voedselmicrobiologie - voedselproducten - landbouwproducten - voedselbesmetting - toxische stoffen - xenobiotica - haccp - food industry - food technology - microorganisms - bacteria - classification - taxonomy - animal products - bacteriology - bacterial count - food inspection - microbiology - foods - food preservation - vegetable products - storage - plant products - microbial ecology - methodology - biological techniques - experiments - equipment - food microbiology - food products - agricultural products - food contamination - toxic substances - xenobiotics - haccp
    HACCP in de luchtvaartcatering.
    Beumer, R.R. ; Vrouwenvelder, Th. ; Brinkman, E. - \ 1994
    Voedingsmiddelentechnologie 27 (1994)10. - ISSN 0042-7934 - p. 13 - 16.
    luchttransport - luchtvaartuig - dierlijke producten - kiemgetal - bacteriologie - controle - gemaksvoedsel - engineering - voedselinspectie - voedselmicrobiologie - voedselbewaring - voedingsmiddelen - bedrijfsvoering - microbiële ecologie - monitoring - voorgekookt voedsel - voorbereide voedingsmiddelen - ruimtevlucht - statistiek - kwaliteitscontroles - queuing theory - haccp - air transport - aircraft - animal products - bacterial count - bacteriology - control - convenience foods - engineering - food inspection - food microbiology - food preservation - foods - management - microbial ecology - monitoring - precooked foods - prepared foods - space flight - statistics - quality controls - queuing theory - haccp
    16S rRNA as molecular marker in ecology of Frankia
    Hahn, D. - \ 1990
    Agricultural University. Promotor(en): A.J.B. Zehnder; A.D.L. Akkermans. - S.l. : Hahn - 126
    frankia - stikstofbindende bacteriën - symbiose - rhizobium - betulaceae - microbiële ecologie - frankia - nitrogen fixing bacteria - symbiosis - rhizobium - betulaceae - microbial ecology

    The research described in this thesis focusses on the role of biotic factors encountered with the establishment of the symbiosis between black alder plants ( Alnus glutinosa ) and introduced Frankia strains. A selection of plant clones and Frankia strains that gave optimal nodulation and nitrogen fixation in forestry was made. For this reason nodulation tests with increasing complexity were set up. An attempt was made to investigate whether introduced strains behaved differently on plants grown under axenic and non-axenic conditions. Since Frankia strains were difficult to identify by conventional techniques, special attention was given to the development of new molecular techniques for identification of the strains at the nucleic acid level.

    Inoculation tests

    Initially, plant material of two physiologically different ecotypes of Alnus glutinosa, the forest ecotype "Bentheim" and the pioneer ecotype "Weerribben", respectively, was selected. Using tissue culture techniques plant material of both ecotypes was cloned in order to obtain genetically identical plants (Chapter 2). These micropropagated plants were used to set up a standardized inoculation system under axenic conditions in order to study the genetically determined nodulation ability and nitrogen-fixing capacity of Frankia strains and to select superior Frankia strains as source of inoculum (Chapter 3). The usefulness of selected Frankia strains as inoculum was further tested under more practical conditions, in perlite as model environment for nitrogen-limited conditions and in two soils, representing natural environments with different nutritional factors and different microbial populations. The results of the inoculation tests under axenic conditions were confirmed by studies under greenhouse conditions.

    The performance of the symbiosis was effected by many variables, e.g. the plant genotype, the Frankia strain and environmental conditions. The influence of the environmental conditions became more pronounced when plants were grown on either a sandy loam ("Bentheim") or a peat ("Weerribben") soil and inoculated with Frankia strains. Plant growth was positively influenced, e.g. by mycorrhizal fungi in "Bentheim" soil, or negatively influenced, e.g. by oomycetes in "Weerribben" soil. The effects of inoculation with Frankia on plant growth remained minimal. The establishment of the introduced Frankia strain was also dependent on the soil conditions. The introduced spore(-) Frankia strain was only able to compete with the natural spore(+) population of the "Weerribben" soil. Introduction of this strain to "Bentheim" soil did not show any establishment of the introduced strain. In contrast to the sandy loam of "Bentheim" which was rich in nutrients, the peat of "Weerribben" was a representative of poor soils. It could therefore be used for feasability studies in inoculation programmes. The use of pure cultures of Frankia as inoculum instead of soil or crushed nodules, has the advantage to prevent the contamination of the plant with root pathogens. Pure cultures did not result in a better symbiosis.

    Atypical Frankia strains

    Screening of several isolates obtained from nodules of both alder ecotypes indicated the existence of atypical, ineffective Frankia strains. The alder clones used showed variable resistance against infection of the ineffective strains (Chapter 4). When compared with growth after the addition of a single strain dual inoculation of typical, effective Frankia strains and an ineffective Frankia strain to both alder clones showed growth increment of the plants (Chapter 5). The growth enhancing effect of the ineffective Frankia strain was not paralleled by increased number of nodules. Nothing is known yet about the growth stimulation by atypical Frankia strains. The results indicate that simultaneous inoculation of different Frankia strains to Alnus plants can be profitable for the host plant.

    Ribosomal RNA

    Because the ineffective Frankia strains lacked morphological and physiological characteristics of typical Frankia strains and because nodule formation on actinorhizal plants might be reduced or even absent, detection of the ineffective strains and studies on their competitive abilities were quite difficult. Reliable markers which could be used to detect both types of Frankia in nodules and in soil without reisolation had not been available at that moment. An attempt was made to find specific markers in a molecule which was commonly used to unravel evolutionary relationships: the 16S ribosomal RNA. New sequencing techniques allowed the rapid determination of total or almost total 16S rRNA sequences. Total 16S rRNA sequences indicated the presence of conserved and variable regions. Conserved regions had been used to investigate quantitative evolutionary relationships amoung bacteria. The conserved regions of the total 16S rRNA sequence of the effective Frankia strain Ag45/Mut15 were compared with aligned sequences of other actinomycetes and used to determine the position of the family Frankiaceae in the phylogenetic tree of the actinomycetes (Chapter 6).

    Analyses of variable regions of 16S rRNA of closely related organisms indicated sufficient variation, despite the fact that DNA/DNA homology studies suggested these two species might actually be one and the same. Large differences in DNA/DNA homology studies of Frankia which were also obtained between strains of one compatibility group suggested chances on large variation within the variable regions of different strains. Sequence analyses of variable regions of 16S rRNA of two ineffective Frankia strains (i.e. AgB1.9 and AgW1.1) and the effective strain Ag45/Mut1 5, all belonging to the Alnus -compatibility group, showed large differences in base composition. These sequences were used to design complementary synthetic oligonucleotides that could act as specific probes in hybridization experiments. The specificity of these probes was shown in hybridization experiments against immobilized rRNA from 23 Frankia strains belonging to different compatibiliy groups and of several related soil actinomycetes. The probes were able to distinguish between Nif +and Nif -strains, between several Nif -strains and between several Alnus compatible Nif +strains and strain AgKG'84/4 also belonging to the Alnus -compatibility group (Chapter 7). Strong strain specific sequences, however, were not obtained. The design of oligonucleotide probes opens up the possibility to investigate competitive abilities of selected strains under defined conditions, e.g. in model systems with perlite and defined Frankia strains. The question whether competition studies under these controlled conditions are ecologically relevant needs further investigations because little basic knowledge on Frankia population dynamics is yet available. The application of probes to identify introduced strains in soil remains restricted, due to the low specificity for strains. Up to now we are not able to design reliable strain specific probes that can be used to follow the establishment of introduced Frankia strains in natural environments. A much more . promising application of probes towards rRNA is concerned with the development of a genus-specific oligonucleoticle probe against Frankia (Chapter 9) that theoretically enables quantitative detection of total Frankia populations.

    RNA extraction

    The application of oligonucleoticle probes in the detection of specific Frankia strains does not only depend on specificity of the probes but also on the development of a reliable isolation method for target sequences. Ribosomal RNA is preferable to DNA as target because of its relative abundance in large amounts in metabolically active cells. Actinorhizal nodules represent enrichments of Frankia, which are metabolically highly active and consequently contain large amounts of Frankia RNA. Our investigations resulted in the development of a rapid RNA extraction method that was sensitive enough to investigate strain composition also from very small nodules or lobes (Chapter 8). The detection of target sequences, however, remained limited by the design of specific probes and the ratios of different target sequences in one sample. For reliable signal expression in hybridization experiments quite similar amounts of target sequences per sample were needed.

    So far, the usefulness of rRNA sequences as targets for oligonucleoticle probes was only shown in combination with pure cultures of Frankia (Chapter 7) or in metabolically highly active enrichments, e.g. nodules (Chapter 8). Terrestrial environments like soil contain populations of many different microorganisms. These populations normally grow under suboptimal nutrition conditions. Bacteria adapt to these conditions by forming special starvation cells, which are metabolically inactive and contain only low amounts of rRNA. The starvation respons often results in viable, but non-culturable populations. The recalcitrant character of Frankia, which are difficult to isolate, makes it a useful model microorganism of soil bacteria. The application of oligonucleoticle probes for detection of Frankia in soil depends on the development of an extraction method for RNA. RNA directly isolated from soil as target for Frankia specific oligonucleoticle probes was useful in detection of Frankia (Chapter 9). Quantification of the obtained signals, however, is still unreliable because Frankia is a hyphae forming organism. It is also quite difficult to correlate cell numbers (theoretical estimation) to the amount of RNA. The concentration of these molecules in an organism is a function of the activity of the individual cell. Quantification of hybridization signals therefore depends on the availability of basic information of the metabolic activity of Frankia cells in soil. This information, however, is very difficult to obtain for recalcitrant microorganisms like Frankia. It is much easier for other microorganisms, e.g. for Streptomyces . Streptomyces spores are quite easy to isolate from soil and the establishment of Streptomyces cells, i.e. as spores or as mycelium in soil, is well studied. Quantification based on hybridization signals must be possible when this basic knowledge is available. In case of Frankia methods that enable quantification must still be developed. Similar to Streptomyces these quantification methods for Frankia can be of direct character, e.g. quantitative extraction of spores, or of indirect character, e.g. determination of mycelium by phage counts.

    The development of rapid and sensitive methods to detect Frankia on the basis of rRNA sequences opens up new ways to study other recalcitrant microorganisms in the environment. This molecular approach in microbial ecology can definitely further be explored when the advantages of rRNA as stable target and the rapid extraction of RNA from soil can be combined with in vitro amplification methods commonly used with DNA or mRNA. Promising approaches can also be expected in in situ studies using hybridization signal intensity of fluorescent dye labelled oligonucleotides and the amount of rRNA as criterium for bacterial activity.

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