249 research outputs found
Modeling the fate of Escherichia coli O157:H7 and Salmonella enterica in the agricultural environment: current perspective
No full textThe significance of fresh vegetable consumption on human nutrition and health is well recognized. Human infections with Escherichia coli O157:H7 and Salmonella enterica linked to fresh vegetable consumption have become a serious public health problem inflicting a heavy economic burden. The use of contaminated livestock wastes such as manure and manure slurry in crop production is believed to be one of the principal routes of fresh vegetable contamination with E. coli O157:H7 and S. enterica at preharvest stage because both ruminant and nonruminant livestock are known carriers of E. coli O157:H7 and S. enterica in the environment. A number of challenge-testing studies have examined the fate of E. coli O157:H7 and S. enterica in the agricultural environment with the view of designing strategies for controlling vegetable contamination preharvest. In this review, we examined the mathematical modeling approaches that have been used to study the behavior of E. coli O157:H7 and S. enterica in the manure, manure-amended soil, and in manure-amended soil-plant ecosystem during cultivation of fresh vegetable crops. We focused on how the models have been applied to fit survivor curves, predict survival, and assess the risk of vegetable contamination preharvest. The inadequacies of the current modeling approaches are discussed and suggestions for improvements to enhance the applicability of the models as decision tools to control E. coli O157:H7 and S. enterica contamination of fresh vegetables during primary production are presented
Fate of Escherichia coli O157:H7 and Salmonella enterica in the manure-amended soil-plant ecosystem of fresh vegetable crops: a review
No full textEnterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica have been implicated in several disease outbreaks linked to consumption of fresh vegetables. Both ruminant and non-ruminant animals carry EHEC and S. enterica in their gastrointestinal tracts and can shed the pathogens in the faecal matter both in symptomatic and asymptomatic states. Application of animal waste in soil fertility management and irrigation of crops with contaminated waste water has been recognised as an important route through which EHEC and S. enterica can contaminate fresh vegetables during primary production. The behavior of E. coli O157:H7 and S. enterica in the agricultural environment has been extensively studied in the last decades. Several microbiological detection methods have been applied. This review therefore puts together current knowledge on the behavior of E. coli O157:H7 and S. enterica in the manure-amended soil-plant ecosystem of fresh vegetable crops during cultivation under various environmental conditions. The review focuses on methodological issues involved in undertaking survival studies and makes comparative analysis of experimental results obtained from studies conducted under controlled environmental conditions integrating results obtained from field experiments. Finally, a theoretical discussion on the potential likely impact of climate change on pre-harvest safety of field-cultivated vegetables is highlighted
Zinc toxicity to nitrification in soil and soilless culture can be predicted with the same biotic ligand model
No full textThe inhibitory effect of Zn on the nitrification process in ZnCl2 spiked soils (12 soils, pH range 4.8-7.5) was compared to toxic effects of Zn on the nitrification by Nitrosospira sp. in soilless solutions with varying pH (pH 6-8) and ionic composition. The nitrification was reduced by 20% at Zn solution concentrations (EC20) ranging between 7 and 1200 microM Zn in the soil pore water and between 5 and 150 microM Zn in the soilless solutions. Protective effects of H+, Ca2+, and Mg2+ against Zn2+ toxicity were observed in both systems. Zinc speciation was determined, and 60-90% of the Zn in the soils and 35-80% of the Zn in the soilless solutions was present as Zn2+. A biotic ligand model and a Freundlich-type model, incorporating the competition of Zn2+ ions with H+, Ca2+, and Mg2+ for binding on the biotic ligands, were used to model the results. The Zn2+ activities resulting in 20% reduction of the nitrification were well predicted using the same parameters for both (soil and soilless) systems, indicating that microorganisms in soil are exposed to zinc through the free zinc ion in soil pore water.Jelle Mertens, Fien Degryse, Dirk Springael, and Erik Smolder
Fysiologische en genetische responsen van de polycyclische aromatische koolwaterstof afbrekende Sphingomonas sp. LH128 naar abiotische stress
No full textPolycyclic aromatic hydrocarbons (PAHs) are widespread environmental organic contaminants and have toxic, carcinogenic and mutagenic properties. Microorganisms are used in bioremediation processes to remove or recycle organic pollutants such as PAHs. However, they often fail to efficiently degrade these pollutants when introduced into or present in a contaminated environment. This is mainly due to different abiotic orbiotic stress situations in the environment. This study aimed at exploring and understanding the abiotic stress response features and gene functions that are involved in stress mitigation in the phenanthrene-degrading Sphingomonas sp. LH128. The first part of the study focuses on identifying the physiological and genome-wide transcription response of LH128 biofilm in relation to its PAH-degrading activity when exposed to either an acute (4 h) or chronic (3 days) solute stress invoked by 450 mM NaCl. Biofilm morphology, oxygen consumption and phenanthrene mineralization activities were not significantly affected after imposing acute or chronic solute stress. This is in agreement with the transcriptomic data since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to non-stressed condition. The strain maintained its catabolic activity and adapted to the solute stress byincreasing the degree of saturation of membrane fatty acids and increasing the expression of genes important for coping with solute stress, such as osmoregulatory genes. In the second part of the study, genes involved in survival and activity of the strain LH128 under solute and matric stress were identified by plasposon mutagenesis. About 5,000 mini-Tn5 plasposon mutants of strain LH128 were generated from which 11 and 14 mutants showed growth impairment when exposed to solute stress (450 mM NaCl) that is believed to simulate the osmotic stress in saline environments and matric stress (10% PEG6000, w/v) that is believedto simulate the dehydrating properties of low water-content habitats respectively. None of the disrupted genes in these mutants was shared between solute and matric stress affected mutants. One NaCl affected mutant (NA7E1) with a disruption in a gene encoding the outer membrane protein of Sphingomonas (OpsA) was affected at lower NaCl concentrations compared to other mutants. NA7E1 was also the only mutant that showed reduced growth and phenanthrene degradation in soil. It was suggested that opsA is a crucial gene for survival of strain LH128 in soil especially in case of reduced moisture content probably by mitigating effects of solute stress. The physiological response and phenanthrene catabolic activity of strain LH128 when inoculated in PAH-contaminated soil was then investigated in detail. Four hours after inoculation, the strain showsa rapid and large reduction (approximately 100-fold) in Colony Forming Units (CFU) after which the CFU number increases till a number similar to the inoculated cell density. However, microscopic counts of Gfp-containing and moving LH128 cells in soil at 4 h incubation corresponded with the initial culture cell density suggesting that the largest fraction of LH128 cells are alive but entered into a Viable But None-Culturable (VBNC)-like state. The VBNC-like cells were apparently not dormant butactive as suggested from the increase in the degree of saturation of membrane fatty acids and transcriptome analysis. Moreover, modeling and transcriptomics suggested that the VBNC-like cells were particularly active regarding PAH biodegradation. The latter showed highly increased expression of genes (up to 133-fold) involved in PAH degradation at 4 h incubation. In addition, genes involved in stress control showed increased expression suggesting that the largest fraction of cells experienced stress in soil. A similar approach was followed to identify the physiological and whole-genome transcription response of LH128 to long-term nutrient starvation. The results from microscopic counting of Gfp-containing cells and CFU suggested that the strain enters into VBNC-like state and survived starvation stress without drastic loss of viability. The strain adapted to starvation by reducing cell size and changingcell shape from rod to round morphology, and increasing the degree of saturation of membrane fatty acids. The strain also survived starvation stress by increasing the expression of gene functions involved in efflux systems, acquisition of stored foods such as by degradation of rRNA by ribonucleases and β-oxidation of fatty acids by acyl-CoA dehydrogenases and by decreasing the expression of many gene functions such asthose involved in ribosomal proteins biosynthesis, chromosomal replication, and cell division.In the last part of the thesis, a whole-cell bioreporter was successfully constructed in strain LH128 by means of a transcriptional fusion of the promoter region of phnA1f with gfp to detect PAH biodegradation activity and the bioavailable fraction of PAHs in soil. The newly developed reporter strain LH128PGFP detected the presence of PAHs in liquid culture and in PAH-contaminated soilboth on solid agar surface and in microcosms. Such a rapid and simple bioreporter-based diagnostic approach of detecting bioavailable PAHs can be helpful for policy makers in monitoring the effectiveness of bioremediation activities.In conclusion, Sphingomonas sp. LH128 shows several features of abiotic stress adaptation without compromising PAH degradation activity. Due to these attributes, LH128 and possibly other Sphingomonas strains can contribute to biodegradation of pollutantsin environments experiencing regular abiotic stresses and can be used for biosensor applications.status: Publishe
Microbiële ecologie van polycyclische aromatische koolwaterstof (PAK)-afbrekende Mycobacterium en Sphingomonas in PAK-vervuilde bodem
No full textPolycyclische aromatische koolwaterstoffen (PAKs) vormen een belangrijk e groep van hydrofobe vervuilende organische stoffen in de bodem. Door d e lage PAK-biobeschikbaarheid kan zelfs een bodem met een hoge PAK-vervu iling beschouwd worden als een oligotrofe omgeving voor micro-organismen . PAK-afbrekende bacteriën geïsoleerd uit PAK-vervuilde bodems behoren t ot een beperkt aantal genera, maar vooral Mycobacterium en&nb sp;Sphingomonas worden beschouwd als sleutelorganismen in PAK-afbr ekende gemeenschappen in de bodem. Recente studies tonen aan dat PAK-afb rekende mycobacteriën misschien beter aangepast zijn dan Sphingo monas aan de lage PAK-biobeschikbaarheid en oligotrofe condities in P AK-vervuilde bodems. De studie van de microbiële ecologie van PAK-afbrek ende Mycobacterium en Sphingomonas in PAK-vervuild e bodem is van belang in de context van natuurlijke bioattenuatie en bio stimulatie als bioremediatiestrategieën voor PAK-vervuilde bodem. Versch illende experimenten met modelsystemen en historisch PAK-vervuilde bodem s werden ontworpen om de omgevingsfactoren, die het gedrag en de gemeens chapsdynamiek van PAK-afbrekende Mycobacterium en Sph ingomonas in de bodem bepalen, beter te begrijpen met nadruk op het e ffect van concentratie, structuur en biobeschikbaarheid van de PAKs. Om de hypothese te bestuderen dat mycobacteriën beter aangepast zijn dan sphingomonads aan het gebruik van PAKs met lage biobeschikbaarheid, we rden mineralisatie-experimenten uitgevoerd met poreuze, synthetische mod elsystemen met gesorbeerd fenantreen. PAK-afbrekende Mycobacteri um stammen werden minder beïnvloed door de dalende PAK-biobeschikbaar heid vergeleken met Sphingomonas stammen. Toevoeging van mini male nutriënten had echter een voordeliger effect op de mineralisatie va n gesorbeerd fenantreen door Sphingomonas dan door My cobacterium. De resultaten tonen aan dat Mycobacterium bet er aangepast is aan het gebruik van gesorbeerd fenantreen, vergeleken me t Sphingomonas, vooral in het geval van arme nutriëntconditie s. Om de omgevingsfactoren te bestuderen die de Mycobacterium ge meenschap in PAK-vervuilde bodems bepalen, werd een relatie gelegd tusse n de aanwezigheid van Mycobacterium, de fysico-chemische bode meigenschappen en de PAK-afbraakcapaciteit in acht historisch PAK-vervui lde bodems met een verschillende graad van veroudering van de PAK-vervui ling. Er werd geen relatie gevonden tussen de aanwezigheid van M ycobacterium en bodemeigenschappen zoals kleigehalte en gehaltes aan totale organische koolstof, totale stikstof en minerale olie, maar er wa s wel een relatie tussen de aanwezigheid van Mycobacterium en de PAK-concentratie. Mycobacterium werd niet gedetecteerd in de twee bodems met de hoogste PAK-concentraties, maar was aanwezig in a lle andere bodems met blijkbaar de meest verouderde vervuilingen. Versch illen in relatieve mineralisatiekinetiek van vers toegevoegde fenantreen en pyreen konden opgemerkt worden tussen bodems met een verouderde PAK- vervuiling en bodems met een minder verouderde vervuiling. Deze verschil len konden verklaard worden door een verschillende PAK-afbrekende gemeen schap in beide bodemsoorten, mogelijk de Mycobacterium gemeen schap, maar ook door competitie voor mineralisatie tussen toegevoegde 14 C-gemerkte PAKs en natuurlijke PAKs aanwezig in de bodems met hoge PAK -concentraties. Tot besluit kan men stellen dat mycobacteriën misschien een rol spelen in de waargenomen mineralisatiecapaciteit voor pyreen en dat een hoge fenantreenconcentratie in PAK-vervuilde bodem kan leiden to t verlies van competitiviteit van PAK-afbrekende Mycobacterium</ > ten opzichte van andere PAK-afbrekers in de bodem. De hypothese werd bestudeerd dat, in analogie met vasthechting aan de su bstraatbron in modelsystemen, Mycobacterium vooral geassociee rd is met bodempartikels aangerijkt met PAKs. De kleifractie van bodem K3663 met een verouderde PAK-vervuiling bevatte de grootste hoeveelheid PAKs en vertoonde, in tegenstelling met de zand- en leemfracties, direc te mineralisatie-activiteit voor pyreen en fenantreen na toevoeging van 14C-gemerkte pyreen of fenantreen. Mycobacterium vertegenwoor digde ongeveer 10% van de bacteriën in de kleifractie, terwijl dit slech ts ongeveer 0.1% was in de zand- en leemfracties. Mycobacteriën konden b ovendien geïdentificeerd worden tussen de belangrijkste cultiveerbare PA K-afbrekende populaties in zowel de totale bodem als de kleifractie. De resultaten tonen aan dat PAK-afbrekende mycobacteriën vooral geassocieer d zijn met PAK-aangerijkte bodempartikels en vandaar ondervindt Mycobacterium ook in PAK-vervuilde bodem misschien voordelen verbonde n met vasthechting aan het substraat. Om te bestuderen of de gemeenschapssamenstelling van Mycobacteri um en Sphingomonas in de bodem beïnvloed wordt door PAK-bi obeschikbaarheid en veroudering van de PAK-vervuiling, werd hun gemeensc hapsdynamiek onderzocht in twee PAK-vervuilde bodems met een verschillen d verouderde PAK-vervuiling nadat de concentratie, structuur en biobesch ikbaarheid van de PAKs kunstmatig veranderd werden. In bodem K3663, aan gerijkt met fenantreen of pyreen om de PAK-biobeschikbaarheid te verhoge n, werd snelle biodegradatie van de toegevoegde PAKs waargenomen. Dit g ing samen met een significante stijging van het aantal fenantreen- en py reenafbrekers, met een kleine, transiënte daling in diversiteit van de&n bsp;Mycobacterium gemeenschap, en geen verandering in de S phingomonas gemeenschap. Een grote, transiënte verschuiving in de bac teriële gemeenschap, verbonden met de aangroei van γ-proteobac teriën zoals Pseudomonas, werd echter vastgesteld in de fenan treen-aangerijkte bodem, maar niet in de pyreen-aangerijkte bodem. In bo dem AndE met een minder verouderde PAK-vervuiling werd de biobeschikbare PAK-fractie geëxtraheerd door Tenax korrels om de PAK-biobeschikbaarhei d te doen dalen. De Sphingomonas gemeenschap in de geëxtrahee rde bodem ontwikkelde zich initieel tot een meer diverse gemeenschap, ma ar uiteindelijk daalde deze gemeenschap in omvang tot onder de detectiel imiet, terwijl mycobacteriën nooit werden gedetecteerd. Op lange termijn veranderde de bacteriële gemeenschap in een gemeenschap die gedomineerd werd door een γ-proteobacterie. Onze resultaten toonden aan d at veroudering van PAK-vervuiling in de bodem inderdaad de dynamiek van de algemene gemeenschap in de bodem beïnvloedt en mogelijk selecteert vo or verschillende PAK-afbrekende bacteriële populaties. De rol van < I>Mycobacterium en Sphingomonas in deze populaties was ech ter niet zo duidelijk. Om te bestuderen of Mycobacterium ook aanwezig is als een afb reker van hoog- moleculair-gewicht (HMW)-PAKs in een extreem zure PAK-vervuilde bodem, werden aanrijkingsculturen met pyreen als enige bron van koolstof en ene rgie opgezet met zure PAK-vervuilde bodem als inoculum. Aanrijkingscultu ren die groeiden op pyreen bij pH 2 werden verkregen uit twee zure PAK-v ervuilde bodems en werden inderdaad gedomineerd door een Mycobac terium stam. Echter, in tegenstelling tot snel-groeiende PAK-afbreken de mycobacteriën geïsoleerd bij neutrale pH, was de stam geïsoleerd in d eze studie een traag-groeiend Mycobacterium species. Dit spec ies was ook de dominante Mycobacterium in de oorspronkelijke zure bodems en dit toont aan dat de geïsoleerde Mycobacterium stam ook een rol speelt in biodegradatie van HMW-PAKs in de oorspronke lijke bodems. Tot besluit kan men stellen dat dit onderzoek bijgedragen heeft tot een beter begrip van de omgevingsfactoren die het gedrag en de gemeenschapsd ynamiek van PAK-afbrekende Mycobacterium en Sphingomo nas in de bodem bepalen. Mycobacteriën lijken beter aangepast te zijn aan lage PAK-biobeschikbaarheid, vergeleken met sphingomonads, en verta len deze aanpassing in een kolonisatie van vooral bodems met een veroude rde PAK-vervuiling. Een mogelijke strategie die een rol speelt in de ove rleving van Mycobacterium in de omgeving van een PAK-vervuild e bodem, is de vasthechting van Mycobacterium aan de substraa tbron, hetgeen ook het geval was in een lange termijn vervuilde bodem on derzocht in deze studie. Kunstmatige veranderingen in PAK-biobeschikbaar heid in bodems vertoonden echter niet altijd de verwachte gemeenschapsdy namiek van Mycobacterium en Sphingomonas, hetgeen erop wijst dat de omgevingsfactoren die hun dynamiek in de bodem bepalen , meer complex zijn. Uiteindelijk werd aangetoond dat zelfs in zure bode m, Mycobacterium een belangrijke afbreker van HMW-PAKs blijf t. De resultaten geven bijkomende ondersteuning van het belang van bacte riële genera zoals Mycobacterium als PAK-afbrekende specialis ten in PAK-vervuilde bodems en van hun uitzonderlijke eigenschappen om z ich aan te passen aan zon omgeving. Meer onderzoek om hun niche in PAK- vervuilde bodem te beschrijven en de rol van PAK-biobeschikbaarheid daar in, blijft belangrijk om het gedrag van PAKs in vervuilde bodems op lan ge termijn te begrijpen.status: Publishe
Multi-species bacteriële consortia die in staat zijn om het fenylureumherbicide linuron af te breken en hun ruimtelijke organizatie op vaste oppervlakken
No full textHet intensief gebruik van fenylureumherbiciden zoals linuron in de landbouw heeft geleid tot vervuiling van grond- en oppervlaktewater. Fenylureumherbiciden en hun afbraakproducten kunnen toxisch zijn voor waterorganismen en hormoonverstorende eigenschappen bezitten, en vormen zo een risico voor de werking van ecosystemen en de menselijke gezondheid. Bodembacteriën blootgesteld aan fenylureumherbiciden blijken echter de capaciteit te hebben ontwikkeld om deze componenten af te breken. Alzo kunnen fenylureumafbrekende bacteriële populaties aanwezig in landbouwbodems de migratie van herbicideresidus naar onderliggende waterlagen beperken. In deze thesis werd getracht om meer informatie te bekomen omtrent de samenstelling van linuronafbrekende bacteriële gemeenschappen in landbouwbodems, over hoe leden van linuronafbrekende multi-species consortia interageren wanneer ze opgegroeid worden op een vast oppervlak, en over hoe hun activiteit, structuur en samenstelling beïnvloed wordt door omgevingscondities. Bovendien werd getracht om enzymen te identificeren die een rol spelen in bacteriële linuronafbraak.In een eerste deel van de studie werden linuronmineraliserende culturen aangerijkt vanuit lange-termijn linuron-behandelde landbouwbodems van verschillende geografische oorsprong. De bekomen culturen bevatten steeds één of meerdere linuronafbrekende bacteriën samen met bacteriën die in staat waren om ofwel 3,4-dichlooraniline (3,4-DCA) ofwel N,O-dimethylhydroxylamine (N,O-DMHA), twee eerder beschreven metabolieten in de bacteriële linuronafbraakweg, af te breken. Op basis van 16S rRNA gensequenering werden de meeste linuronafbrekende stammen geïdentificeerd als Variovorax. Bovendien werd een linuronafbrekende Hydrogenophaga stam en Achromobacter stam geïsoleerd wat aantoont dat deze eigenschap niet enkel gelimiteerd is tot Variovorax. Isolaten die in staat waren om 3,4-DCA maar niet linuron af te breken behoorden tot de genera Variovorax, Cupriavidus en Afipia. Hyphomicrobia waren betrokken in de afbraak van N,O-DMHA. Hoewel verschillende isolaten in staat waren om individueel linuron volledig af te breken, resulteerde toevoeging van een mengsel van aminozuren en specifieke combinaties van linuron- en 3,4-DCA-afbrekende stammen in verhoogde linuronafbraaksnelheid. Deze resultaten suggereren dat in de bodem linuron doorgaans afgebroken wordt door multi-species synergetische consortia met karakteristieke samenstelling.In een tweede deel werd nagegaan of de verschillende leden van een linuronafbrekend multi-species consortium fysieke associaties aangingen wanneer ze werden opgegroeid als biofilm en gevoed werden met een medium dat metabole interacties stimuleert, en of de ruimtelijke en structurele organizatie van deze biofilms een invloed had op de linuronafbraakefficiëntie. Hiertoe werden de drie leden van een eerder beschreven linuronafbrekend consortium bestaande uit de linuron- en 3,4-DCA-afbreker Variovorax sp. WDL1, de 3,4-DCA-afbreker Comamonas testosteroni WDL7 en de N,O-DMHA-afbreker Hyphomicrobium sulfonivorans WDL6, opgegroeid als mono- of multi-species biofilms in flow cells die continu gevoed werden met selectieve of niet-selective media, en de resulterende biofilms werden geanalyseerd met confocale laser scanning microscopie (CLSM). In tegenstelling tot mono-species biofilms van WDL1 was de drieledige consortium biofilm in staat om linuron volledig af breken blijkbaar dankzij synergetische interacties. Deze biofilm vertoonde een heterogene structuur met onregelmatig oppervlak, en was opgebouwd uit geassocieerde WDL1, WDL7 en WDL6 cellen. Deze associatie werd niet waargenomen wanneer het consortium gevoed werd met een niet-selectieve koolstofbron zoals citraat. Dit wijst erop dat de ruimtelijke organisatie in de linurongevoede consortium biofilms de metabole interacties tussen de verschillende consortiumleden weerspiegelt. Bovendien was de structuur van zowel mono- als multi-species biofilms afhankelijk van de voorziene koolstofbron. Mogelijk draagt de specifieke structuur bij tot de efficiënte afbraak van linuron.In een derde deel werd onderzocht of de activiteit en stabiliteit van de drieledige linuronafbrekende consortium biofilms werd beïnvloed wanneer blootgesteld aan (i) alternatieve gemakkelijk afbreekbare koolstof- en/of stikstofbronnen naast linuron, en (ii) tijdelijke veranderingen in nutriëntenstatus (namelijk niet-selectief, en koolstof- en/of stikstofgebrek). De aanwezigheid van citraat of TSB (trypticase soy broth) in het medium naast linuron had een duidelijk negatief effect op de linuronafbraak door de consortium biofilms. Ook wanneer linuronafbrekende consortium biofilms tijdelijk geïrrigeerd werden met niet-selectieve media in afwezigheid van een C en/of N bron of met niet-selectieve C/N-rijke media, werd een negatief effect vastgesteld op de linuronafbraakactiviteit van de biofilms wanneer de voeding met het selectieve linuronbevattende medium hersteld werd. In geen enkel geval kon volledige linuronafbraak, zoals vastgesteld vóór blootstelling aan de alternatieve media, bereikt worden. De verminderde prestaties van de consortium biofilms kon gerelateerd worden aan veranderingen in biofilmstructuur en samenstelling van de gemeenschap. Elke conditie leidde tot een specifieke biofilmstructuur en samenstelling. Deze resultaten geven aan dat pesticide-afbrekende consortia georganizeerd in biofilms en hun afbraakactiviteit sterk afhankelijk zijn van de heersende nutriëntenstatus, en dat de ideale biofilmconfiguratie zoals geobserveerd onder selectieve condities waarschijnlijk niet voorkomt in natuurlijke situaties waar verschillende C en N bronnen simultaan voorhanden zijn en perioden van overvloed en schaarste zich afwisselen.In een laatste deel werd aan de hand van een differentiële proteïnenexpressie analyse getracht om enzymen te identificeren die een cruciale rol spelen in het metabolisme van linuron in Variovorax sp. WDL1. WDL1 werd opgegroeid in een heterotroof medium in aan- en afwezigheid van linuron en/of 3,4-DCA en geëxtraheerde proteïnen werden gescheiden met behulp van 2-D PAGE. Geselecteerde differentieel tot expressie gebrachte proteïnen werden vervolgens geïdentificeerd met nanoLC-ESI-MS/MS. In de cultuur waar 3,4-DCA aan werd toegevoegd, werd de opregulatie van verschillende proteïnen met aminozuursequenties gelijkend aan deze van bepaalde componenten (zoals TdnQ, TdnT, TdnA1) van het multicomponent aniline dioxygenase (AD) geïdentificeerd in aniline-afbrekende Proteobacteria, waargenomen. Bovendien werden meerdere TdnQ-gerelateerde proteïnen gedetecteerd, wat aangeeft dat WDL1 verschillende tdnQ genkopijen bezit en tot expressie brengt in aanwezigheid van 3,4-DCA. Meerdere opgereguleerde proteïnen in de linuron- en/of 3,4-DCA-gesupplementeerde culturen vertoonden geen significante of betrouwbare overeenkomst met gekende proteïnenfuncties. Deze proteïnen spelen mogelijk een rol in linuron- en/of 3,4-DCA-afbraak in WDL1. Tot op heden zijn de enzymen die de initiële transformatie van linuron tot 3,4-DCA katalyseren niet gekend en meer onderzoek is nodig om de exacte linuron- en 3,4-DCA-afbraakweg in Variovorax sp. WDL1 te ontrafelen.Ter conclusie draagt dit onderzoek bij tot de kennis van de samenstelling en werking van linuronafbrekende bacteriële bodemgemeenschappen. Bovendien werd de fysieke basis van species interacties en multi-species biofilmvorming en -dynamiek van een linuronafbrekend consortium toegelicht. Daarbij werd het belang van de samenstelling van de gemeenschap en biofilmopbouw in relatie tot de afbraak van een gehalogeneerde organische component benadrukt. Hoewel deze bevindingen gebaseerd zijn op observaties in artificiële systemen op laboratoriumschaal, kunnen ze desalnietemin bijdragen tot het begrip van species interacties en hun dynamiek onder natuurlijke omstandigheden. Bovendien werd voor de eerste maal de opregulatie van een mogelijk dichlooraniline dioxygenase aangetoond en op basis van de bekomen aminozuursequenties kunnen dichlooraniline afbraakgenen van WDL1 gekarakteriseerd worden.status: Publishe
Genetische karakterisering van 2,6-dichlorobenzamide (BAM) degradatie in Aminobacter sp. MSH1
No full text2,6-dichlorobenzamide (BAM) is a pesticide transformation product and a frequently detected groundwater micropollutant. Concentrations often exceed the EU drinking water threshold limit of 0.1 μg/L causing the costly closure of groundwater abstraction wells and the installation of expensive activated carbon filters in drinking water production plants. A promising alternative to treat BAM contaminated groundwater is the bioaugmentation of water treatment units (such as sand filters) with Aminobacter sp. MSH1. This strain has the rare ability to use BAM as sole source of carbon, nitrogen and energy resulting in its mineralization. The BAM metabolic pathway used by strain MSH1 and the genetics involved are however largely unknown and need to be characterized to evaluate the production of potentially toxic intermediates, define potential pathway bottlenecks, and develop monitoring tools. This work aims at the genetic characterization of BAM metabolism in Aminobacter sp. MSH1.
A first part of this work focused on the identification and characterization of the initial catabolic step in BAM degradation, i.e., the conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA). Protein fractionation followed by mass spectrometry identified a protein, BbdA, that showed amidase activity converting BAM to 2,6-DCBA in crude cell extracts of Aminobacter sp. MSH1. The corresponding bbdA gene was identified in the draft genome sequence of strain MSH1 and the heterologous expression of BbdA in E. coli confirmed its activity of converting BAM to 2,6-DCBA. The bbdA gene was absent in spontaneous mutants of MSH1 that were defective in BAM degradation suggesting that there is no functional redundancy in strain MSH1 to degrade BAM. BbdA shows low amino acid sequence identity to other amidases and is predicted to contain a N-terminal domain involved in dimerization and forming a narrow substrate binding tunnel. bbdA is carried by an unusual IncP-1β plasmid, pBAM1 (40.6 kb), that lacks several genes for conjugation (trbE to trbP) conserved in other IncP-1 plasmids. Homologs of bbdA and plasmid pBAM1 were also present in other BAM mineralizing Aminobacter strains. BbdA displayed a broad substrate range (including benzamide and ortho-chlorobenzamide (OBAM)), a high optimal temperature (Topt = 62.5 °C) and a high affinity for BAM (KM = 0.71 μM). Moreover, differential proteomics and transcriptional reporter analysis suggested the constitutive expression of BbdA. Both the constitutive expression of BbdA and the high affinity of BbdA for BAM are of interest for strain MSH1 to degrade BAM at micropollutant concentrations.
The second part of this work covers the identification of gene functions involved in the downstream pathway of BAM metabolism, i.e., the further degradation of 2,6-DCBA. 2,6-DCBA degradation was found to be encoded by a second plasmid of 57.8 kb, pBAM2, that was identified as a repABC type plasmid. Due to the presence of several perfect repeats, the complete sequence of pBAM2 could not be determined but several genes with putative catabolic gene functions for chloro-aromatic catabolism (such as a dioxygenase, mono-oxygenase and dehalogenase) were identified. These catabolic genes are encoded by two gene clusters, i.e., bbdB1B2B3CDE and bbdFGHIJK. Expression of bbdD (encoding a putative mono-oxygenase) in a MSH1 mutant that lacked the ability to metabolize 2,6-DCBA resulted in the conversion of 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoic acid (3-OH-2,6-DCBA) indicating that further degradation of 2,6-DCBA starts with conversion to 3-OH-2,6-DCBA. Based on this reaction and the annotated gene functions of the catabolic genes found on pBAM2, a putative metabolic pathway of BAM degradation beyond 2,6-DCBA was proposed.
The third part aimed at acquiring insight into the regulation of 2,6-DCBA metabolism in Aminobacter sp. MSH1. The transcriptional activity of several possible promoters combined with adjacent regulatory genes on pBAM2 was evaluated by transcriptional fusions with a promoterless gfpmut3.1 gene in wild type MSH1 and spontaneous mutants lacking pBAM1 and/or pBAM2. The results revealed that P1, located in front of gene cluster bbdB1B2B3CDE, is constitutively transcribed. In contrast, P4, located in front of bbdFGHIJK, shows background transcriptional activity and is upregulated by the upstream and minus strand encoded repressor BbdR2 in the presence of an unknown 2,6-DCBA degradation intermediate. Increased transcription from the P4 region was only observed at concentrations of 1 mg/L BAM/2,6-DCBA or higher. However, the leaky transcription from the P4 region at lower concentrations ensures that no induction is needed for low level expression and thus that no threshold concentration for BAM degradation at micropollutant concentrations is created by lack of induction. Differential proteomics showed that up to 6.5 % of the proteome of MSH1 is significantly altered upon growth on BAM compared to growth on glucose. Differences in protein expression mainly related to environmental information processing (transporters) and metabolic processes, including carbohydrate and xenobiotic metabolic proteins. Furthermore, several stress associated proteins showed an increased abundance upon growth on BAM compared to growth on glucose indicating that MSH1 experiences a stress situation when grown on BAM.
We conclude that important steps were taken to unravel the genetics of BAM degradation in Aminobacter sp. MSH1. Key catabolic steps were identified and first knowledge was acquired on the regulation of the bbd genes. Together they provided insight into the BAM metabolic pathway, the possible formation of BAM degradation bottlenecks in MSH1, the evolutionary path of BAM degradation in Aminobacter spp. and defined targets for genetic improvement and molecular tools for monitoring purposes.status: Publishe
Thermocaproicibacter melissae gen. nov., sp. nov., a thermophilic chain-elongating bacterium, producing n-caproate from polymeric carbohydrates
No full textStrain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37–60 °C (optimum, 50–55 °C) and at pH 5.0–7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3–0.5×1.0–3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae , being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae . Pairwise average nucleotide identity and DNA–DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LMG 32615T=NCCB 100883T).sponsorship: MELiSSA Foundation|4000114262/15/NL/JCstatus: Publishe
De relatie tussen samenstelling en functionaliteit van de microbiële gemeenschap in laboschaal anaerobe vergistingsreactoren bestudeerd aan de hand van biologische replica’s
No full textAnaerobic digestion (AD) is the microbiological conversion of organic material under anaerobic conditions, leading to the formation of biomass, biogas and a nutrient rich digestate. The process is gaining an increasing interest because of its potential for simultaneous waste stabilisation and valorisation. Using AD, methane-rich biogas as well as valuable chemicals and a nutrient-rich residue can be produced from organic material in solid wastes and wastewater. Last decades, the demand for more sustainable waste management techniques, together with the urgent need for renewable energy sources, has led to an increasing interest and application of AD technologies, both in solid waste and wastewater treatments. Aside from the potential bioenergy and nutrient recovery, AD for wastewater treatment comprises several other advantages over conventional aerobic technology, including lower energy demand, small footprint and fewer excess sludge production. However, disadvantages include wash-out of the anaerobic biomass, long start-up periods and higher sensitivity to fluctuating process conditions. The first problem is largely countered by the development of methods that allow to concentrate the anaerobic biomass in the reactor. The second issue is mostly covered as well, since a more widespread implementation of AD has resulted in an increasing availability of active anaerobic sludge for inoculation of new AD systems, which allows to start-up within several weeks. The last, however, is a major reason why in many cases aerobic treatment is still chosen over anaerobic technology. An increasing knowledge on AD process operation has helped to increase reactor stability, but still, industries fear the complexity of the anaerobic process and its sensitivity to changing conditions, which limits the application of AD technology.
AD process performance is directly linked with the structure and dynamics of the microbial reactor community. Therefore, a better understanding of the AD microbiome and the identification of microbial indicators of process performance and stability is considered a key research subject towards the optimisation, control and management of anaerobic reactors. Despite intensive efforts to identify microbial parameters that significantly correlate with process performance and stability, microbial-based control and management of AD reactors is still far from reality. The high community diversity in the AD microbiome, complex microbial interactions and a high degree of functional redundancy, which is the ability of different microbial communities to perform the same ecological functions, are considered major causes for the slow progression in this field. A thorough understanding of the relationship between community composition and functionality is currently hampered by the lack of biological replicates in well-controlled experimental conditions. Taking into account the high functional redundancy existing in AD communities, the variation in community assembly and its effect on reactor performance is difficult to grasp.
The overall objective of this study was (i) to get more insight into the factors that shape community assembly in the AD process, both at the taxonomic and functional level, (ii) to evaluate the impact on process performance and (iii) to indicate common determining species and functions. The focus was on the role of the inoculum community and the role of stress factors. In contrast to many other AD studies, a replicate approach was used in order to get insight into the variability of the community assembly process and the role of deterministic and stochastic factors in shaping AD communities. Community structures were studied using amplicon metagenomic sequencing of the 16S rRNA gene and transcript, to target the total and the active AD community, respectively. Functional profiles were retrieved by applying metaproteomics using high-resolution tandem mass spectrometry.
In a first chapter, taking into account the crucial role that the inoculum plays in industrial AD reactors, we examined the relationship between the inoculum community composition, process performance, and reactor community assembly. We questioned whether identical operational conditions would lead to similar assemblies at the taxonomic level despite using different starting communities, and examined the deterministic character of the assembly process. Moreover, we determined the contribution of the inoculum to reactor community assembly. To this end, we studied three sets of biologically replicated AD reactors inoculated with different communities, but operated identically, targeting both total and active communities. All reactors performed highly similar regarding volatile fatty acid and methane production. Community analyses showed reproducible total and active community compositions in replicate reactors indicating that particularly deterministic factors shape the AD community. However, between differently inoculated reactors we observed strong variation in community composition, indicating the role of inoculum composition in community shaping. In all three reactor sets, especially species that were low in abundance or even not detected in the inoculum contributed to the reactor communities, supporting the importance of functional redundancy and high diversity in inocula used for AD seeding. The careful start-up of the AD process using initially low organic loading rates likely contributed to the successful assembly of rare species into a novel cooperative AD community in the reactors.
In a second chapter, we used the same approach to explore how community assembly was affected by the presence of a stress factor under the form of phenol. Four inocula were used to start three replicate fed-batch reactors each, which received an increasing loading rate of phenol in addition to a dairy synthetic feed. Three of the inocula were sampled from AD systems that were previously exposed to phenolic compounds, to examine whether this pre-exposure affected success rate and community assembly. Phylotypes which explained reactor performance and community assembly were identified. The non-pre-exposed sludge developed successful and stable AD in all three replicates. In contrast, only one of the pre-exposed sludges developed stable AD, and this in only two of the three replicates. Well-performing reactors always showed phenol degradation, indicating that phenol removal was an essential asset for establishing phenol tolerance in the reactors. In well-performing stable reactors, replicate reactors inoculated with the same community, showed highly similar community compositions, indicating that phenol did not affect the deterministic character of the community assembly process. In contrast, in unstable reactors, community compositions showed more variation, even among reactors started from the same inoculum. The AD performance in stable reactors was explained by the proliferation of species belonging to Syntrophus, Cryptanaerobacter, Lactivibrio and Mesotoga, of which Syntrophus and Cryptanaerobacter are known for their phenol degrading capacity. these determining species were observed as rare species in the starting communities of all reactors, including those of unstable reactors. Our study showed that the use of pre-exposed sludge does not necessarily improve the tolerance of AD reactors to phenol, nor AD process performance on a phenol-rich influent. Moreover, it showed that start-up with phenol-impacted wastewaters is not always a reliable process. Comparison of total and active community compositions showed a stronger response to changing conditions and process disturbance in the active than in the total community, suggesting that RNA-based community analysis has the higher potential to provide an early indication of community structure responses.
Triggered by the large functional redundancy observed in the AD reactor communities, in a third chapter, we examined whether the functional profiles of the AD reactor communities showed less variability than the taxonomic profiles, and hence would be more suited to search for universal biomarkers determining the AD process. To this end, the metaproteomes from the dairy-fed replicate reactor communities were examined, and the observed similarities were compared to those at the taxonomic level. We found significantly higher similarity in the community's metaproteomic profiles compared to their taxonomic profiles, both at the DNA and the RNA level. This functional similarity supports the idea that robust microbial indicators would rather be function-related than taxonomic biomarkers. Highly abundant enzymes shared among all stable-performing dairy-fed reactors included several enzymes linked with the AD process.
In the last chapter, we performed a quantitative metaproteomic analysis of the phenol-amended reactors in order to determine specific functional markers related with stable anaerobic phenol degradation. In addition, we compared the metaproteomes of the phenol-fed communities with those obtained from communities in dairy-fed reactors without phenol in order to determine effects of phenol at the metaproteome level. From the set of shared abundant enzymes in dairy-fed reactors, only lactate dehydrogenase was not detected in any of the phenol-fed reactors. Furthermore, in several reactors which had experienced high phenol concentrations acetyl-CoA synthetase, a key enzyme related to acetoclastic methanogenesis, was not detected, suggesting a shift from acetoclastic to hydrogenotrophic methanogenesis after phenol toxicity. The metaproteomic analysis of phenol-fed reactors provided evidence for the anaerobic degradation of phenol via the benzoyl-CoA pathway and further degradation to acetyl-CoA.
In conclusion, this study showed variability in AD community's functional, but mostly taxonomic profiles, using lab-scale replicate reactors under well- controlled conditions and applying a multi-omics approach including amplicon metagenomics and metaproteomics. These results will further elucidate the microbial 'black box' of anaerobic reactors and evaluate the possibilities for microbial-based control and management in the future.status: Publishe
Microbiële ecologie van PAK-afbrekende micro-organismen bij variabele PAK-biobeschikbaarheid en nutriëntcondities
No full textPolycyclic aromatic hydrocarbons (PAHs) are widespread in the environment due to anthropogenic and natural processes. Removal of PAHs in soils is primarly mediated through biodegradation by soil microbial communities. PAH‑degrading bacterial strains belonging to genera such as Mycobacterium, Sphingomonas and Pseudomonas are often isolated from the same soil sample. However, bacteria belonging to these genera have distinct (PAH) carbon source preferences and physiological characteristics, of which some have been related to improvement of PAH-bioavailability and survival in oligotrophic conditions. The apparent co-existence of different PAH-degrading bacteria in the same soil raises questions about the microscale comportment of distinct bacteria with similar ecological functions. It can be questioned whether different PAH‑degrading bacteria which apparently inhabit the same macro‑environment, (i) occupy the same or different microniches within that macro‑environment, (ii) whether those organisms socialize or compete for niches in a micro‑environment, (iii) which environmental and bacterial characteristics define these niches and interactions, and (iv) how bacterial microscale comportment and interactions affect PAH-biodegradation in the system. To respond to those questions, the (growth) behavior of PAH-degrading bacterial strains belonging to three different genera, i.e., Mycobacterium sp. VM552, Sphingomonas sp. LH128 and Pseudomonas putida OUS82 was monitored by means of confocal laser scanning microscopy (CLSM) during colonization of a continuously irrigated flow chamber containing solid PAH as the sole carbon source in single species and multispecies systems. All three strains can use phenanthrene as the sole source of carbon and energy. In addition, strain VM552 can use pyrene for growth. A non‑invasive method to fluorescently mark Mycobacterium cells with the nucleic acid stain Syto62®, was developed and validated, to allow CLSM-visualization of Mycobacterium sp. VM552. The three PAH‑degrading strains showed a distinct growth behavior at the microscale when colonizing flow chambers containing phenanthrene as single PAH source in single species systems. It was suggested that the strains only kept dissolution maximal by effective removal of aqueous Phen, without extra bioavailability promoting properties. Only minor differences in PAH-removal efficiency existed between the three strains. In systems containing phenanthrene and inoculated with two or all three strains, the microscale comportment of all strains was clearly affected by the presence of one or both of the opponent PAH‑degrading strains. Bilateral inhibition of strains VM552 and OUS82 in VM552/OUS82 dual species systems was clearly observed, resulting into a decreased phenanthrene removal rate. In contrast, in VM552/LH128 dual species systems, strains VM552 and LH128 rather seemed to socialize in a highly controlledmanner, resulting in stratified biofilms, however without affecting phenanthrene removal rates. Similarly, in LH128/OUS82 dual species systems, LH128 and OUS82 were organized into a stratified structure pointing towards a commensal relationship, and in this case, enhanced phenanthrene removal was observed. VM552/LH128/OUS82 triple species systems also showed a stratified organization similar to that observed in VM552/LH128 dual species systems. The results show that the microscale behavior of a PAH‑degrader and its degradation efficiency does not only depend on its own physiological characteristics and the applied environmental conditions but also on interactions with other PAH-degrading strains and the identity of the opponent strain(s). Moreover, the observed interactions seem to affect Phen-removal from the system.In systems containing strains LH128 and/or OUS82, the presence of a readily degradable carbon source (citrate) in addition to solid phenanthrene, clearly affected their colonization behavior in a negative way. As a result, decreased rates of phenanthrene removal were observed. The results suggest that the presence of a more readily available carbon source in addition to PAHs, as is often the case in PAH-polluted soils, can affect (commensal) interactions between PAH‑degraders and PAH‑degradation negatively.In a last experiment set-up, the growth behavior of the strains was examined in single, dual and triple species containing either (i) pyrene or (ii) phenanthrene and pyrene in order to find out how the provision of a second PAH source, i.e., pyrene, which can only be utilized by strain VM552, affected system functionality. In pyrene containing systems, VM552 was clearly found to increase the diffusive flux of pyrene above its dissolution rate, which might have been mediated by attachment of VM552 cells to the pyrene surface. In dual and triple species systems containing only pyrene, VM552 was clearly the dominant strain, showing a similar growth behavior and pyrene degradation efficiency. In systems containing both phenanthrene and pyrene, VM552 also became the dominant strain, and the interactive behavior between both strains as observed in the presence of phenanthrene, had dramatically changed. Also, both phenanthrene or pyrene degradation was at certain point inhibited. The results suggest that even in a slightly complex system, specialized niches are available, thereby enabling a level of socialization between phylogenetically diverse bacterial strains and a maintenance of system functionality, and allowing strains to thrive in conditions in which they would theoretically be outcompeted. Overall, the results suggest that in an environmental setting as in soil, bacteria with a similar ecosystem service, i.e. PAH‑biodegradation, will interact at the microscale and that such microscale interactions will contribute to and affect community shaping and functioning.status: Publishe
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