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Per vierkante meter bodem leven honderden wormen en insecten samen met kilometers aan schimmeldraden, vele miljoenen aaltjes en miljarden bacteriën. Onderzoek maakt steeds meer duidelijk van het precaire evenwicht ondergronds, en de grote invloed daarvan op het leven bovengronds. Het levert nieuwe strategieën op voor gewasbescherming.

Micro-organismen spelen een sleutelrol in bodemfuncties zoals de kringlopen van koolstof en stikstof. Voor een duurzame landbouw is het van belang dat deze kringlopen optimaal functioneren om verliezen van nutriënten zoveel mogelijk te voorkomen. Dit onderzoek geeft inzicht in de rol van bodemmicroben bij optimalisatie van de stikstofkringloop door toevoeging van zowel minerale kunstmest als verschillende kwaliteiten organisch materiaal.

De voorlopige resultaten zijn beschreven van de ontwikkeling van een indicator voor de functionele diversiteit van microbiele populaties met behulp van microtiterplaten van Biolog. Voor dit doel werden zowel aquatische als terrestrische milieu monsters getoetst. Het rapport beschrijft de experimentele procedure en de uitwerking van de meetgegevens. In het rapport worden twee biodiversiteitsindicatoren onderscheiden, berekend uit de log-logistische relatie tussen de gemeten activiteit en het aantal kolonie vormende eenheden (CFU). Een indicator is de helling in het buigpunt, de andere indicator is de logaritme van de CFU die nodig is om 50% van de maximale activiteit te bereiken. De functionaliteit van de indicatoren wordt bediscussieerd.

Thirty-nine Dutch laboratories and 33 laboratories of the EC participated in a fifth trial with reference materials for water microbiology. Reference materials with the test strains WR63 Enterococcus faecium and WR52 Staphylococcus warneri were used. The materials were analysed for total aerobic viable bacteria by a reference method and the national standard method, both with incubation at 22 and 37 degrees C. After statistically analysing the results, the repeatability (r) and reproducibility (R) were calculated. Very good results were found for r and R. Especially the values of r found with the material with strain WR63 were very close to the theoretical optimum.

Door zes vleeskeuringsdiensten en het RIVM werd gezamenlijk onderzoek verricht naar het in de Vleeskeuringswet omschreven "Bacteriologisch Onderzoek" (BO) bij slachtdieren. Doel was na te gaan of er verschillen zouden zijn in de aantallen positieve resultaten van het BO tussen de diensten bij toepassing op dieren met vergelijkbare afwijkingen. Gebleken is dat er soms aanzienlijke verschillen (33%) en soms zeer geringe verschillen (< 3%) voorkwamen. Aangezien de kiemen die bij een positief onderzoek geisoleerd worden meestal behoren tot micro- organismen met een zeer geringe betekenis vanuit volksgezondheidsoogpunt (Corynebacterium pyogenes en haemolytische streptococcen, niet zijnde groep A of B) en kiemen die in dat kader wel betekenis hebben (Samonella, Streptococcus suis) gemist worden door de voorgeschreven wijze van onderzoek, dient overwogen te worden of de keuring aanpassing behoeft.

Omwonenden van composteerbedrijven van GFT- en groenafval kunnen stankoverlast ervaren. Hierdoor kunnen omwonenden ongerust raken over de gezondheid. Gemeten concentraties chemische stoffen in de omgeving van deze bedrijven, zoals verschillende vluchtige organische stoffen, ammoniak en zwavelhoudende verbindingen, zijn zodanig laag dat het niet aannemelijk is dat gezondheidskundige grenswaarden worden overschreden. Over de gezondheidseffecten van bio-aerosolen (micro-organismen die worden verspreid door de lucht, zoals bacteriën en schimmels) die tijdens het composteerproces in de omgeving vrij kunnen komen, is weinig bekend. Vanwege de beperkt beschikbare studies is het vooralsnog niet mogelijk hierover conclusies te trekken. Dit blijkt uit een literatuuronderzoek van het RIVM naar de gezondheidsaspecten van wonen in de buurt van composteerbedrijven van GFT- en groenafval. Hierbij is het composteerproces beschouwd, de mogelijk schadelijke effecten van stoffen en bio-aerosolen, en welke gezondheidseffecten in de buurt van deze bedrijven voorkomen. Om meer duidelijkheid te krijgen over mogelijke risico's voor de gezondheid van bewoners in de buurt van composteerbedrijven zijn methoden nodig om de blootstelling aan bioaerosolen te meten. Daarnaast is het nuttig om een beoordelingskader vast te stellen op basis waarvan de mogelijke risico's van bioaerosolen kunnen worden getoetst. Hiervoor kan worden aangesloten bij een nog op te stellen beoordelingskader voor gezondheidsaspecten bij de intensieve veehouderij.

Micro-organismen zijn belangrijk voor de bodemvruchtbaarheid en voor de afbraak van organisch materiaal en vervuilingen in bodems en sedimenten. Door hun functie en alomtegenwoordige aanwezigheid kan de microflora dienen als een zeer milieurelevante indicator voor milieuverontreiniging. Microbiele testen moeten selectief gebruikt worden voor het vaststellen van kwaliteitsrichtlijnen voor bodem en sediment. Dit literatuuroverzicht geeft een evaluatie van microbiele toxiciteitstesten en een nieuwe methode om kwaliteitsrichtlijnen af te leiden. Het vervangen van gevoelige micro-organismen door andere resistente soorten kan ernstige ecologische gevolgen hebben. Dit gaat gepaard met het uitsterven van gewenste soorten en het woekeren van ongewenste soorten. De aanpassing van een levensgemeenschap aan een vervuiling moet dan ook gezien worden als het proces dat een vervuild ecosysteem verstoort. De resistente micro-organismen kunnen vaak specifieke ecologische functies niet vervullen. Het voorkomen van resistente soorten kan gebruikt worden als een gevoelige en ecologisch relevante indicator voor de achteruitgang door milieuvervuiling. Persistente toxische effecten op de microflora kunnen door zink, cadmium en koper worden veroorzaakt bij gehalten die lager liggen dan de huidige normwaarden van de Europese Gemeenschap. Na de evaluatie van de testen, wordt een nieuwe methode beschreven om bodem en sediment kwaliteitsrichtlijnen af te leiden uit microbiele testen. Microbiele toxiteitstesten kunnen gebruikt worden voor risico-evaluatie omdat micro-organismen tot de gevoeligste organismen voor effecten van verontreingingen behoren.

In zwemwater wat voldeed aan de normen voor microbiologische kwaliteit uit de huidige Zwemwaterrichtlijn 76/160/EEG werden micro-organismen aangetroffen die infectieziekten zoals gastro-enteritis of oorontsteking kunnen veroorzaken. Op basis van de strengere normen uit de herziene Zwemwaterrichtlijn 2006/7/EC werd de kwaliteit van dit zwemwater minder gunstig beoordeeld. Op twee officiele Nederlandse zwemlocaties, Katwijk-Noord en Vinkeveenseplassen (bij eiland 1), is de aanwezigheid van (potentieel) pathogene micro-organismen onderzocht en gerelateerd aan de huidige en de herziene Europese zwemwaternormen; voor de parameters uit de herziene Zwemwaterrichtlijn werden twee verschillende (toegestane) detectiemethoden toegepast. De ziekteverwekkende micro-organismen Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio, Cryptosporidium en Giardia werden op beide locaties aangetroffen. Campylobacter werd niet gevonden bij Katwijk-Noord, maar wel in de Vinkeveenseplassen. Salmonella, norovirus en enterovirus werden op geen van beide locaties aangetroffen, terwijl rotavirus eenmaal bij Katwijk-Noord en adenovirus eenmaal in de Vinkeveenseplassen werd aangetoond. Volgens de huidige Zwemwaterrichtlijn was de waterkwaliteit bij Katwijk-Noord 'uitstekend' en in de Vinkeveenseplassen 'goed'. Voor de parameters uit de herziene Zwemwaterrichtlijn bestond er verschil in beoordeling bij gebruik van de verschillende methoden: op basis van membraanfiltratie was de waterkwaliteit bij Katwijk-Noord 'goed', terwijl deze op basis van MPN resultaten 'aanvaardbaar' was. De zwemwaterkwaliteit in de Vinkeveenseplassen werd op basis van beide methoden als 'slecht' beoordeeld. Er zijn aanwijzingen verkregen dat de beoordeling van de waterkwaliteit op Nederlandse zwemlocaties volgens de herziene Zwemwaterrichtlijn afhankelijk is van de toegepaste detectiemethode (membraanfiltratie of MPN). Het voldoen aan de Europese Zwemwaterrichtlijn garandeert niet de afwezigheid van voor de mens ziekteverwekkende micro-organismen in zwemwater.

Onderzocht is welke gezondheidsrisico's verbonden zijn aan het gebruik van oppervlaktewater als bron voor drinkwater. Hiervoor is gebruik gemaakt van een 'ketenbenadering', enerzijds omdat daarmee (zowel chemische als microbiologische) analytische beperkingen kunnen worden omzeild, anderzijds omdat hiermee beter inzicht te verkrijgen is in het effect van mogelijke maatregelen op een bepaald punt in de keten, nu of in de toekomst (prognose). De benadering is toegepast op drie anorganische microverontreinigingen, zes bestrijdingsmiddelen, drie desinfectiebijproducten en drie pathogene micro-organismen. Van deze stoffen en micro-organismen zijn eerst de emissies berekend. Daarna is op een aantal bestaande innamepunten voor de drinkwatervoorziening uit oppervlaktewater berekend wat het effect van de drinkwaterbereiding is op de concentraties. Ook de desinfectiebijproducten zijn in beschouwing genomen. Hierbij is gekeken naar zowel een viertal bestaande zuiveringssystemen als een hypothetische 'eenvoudige' zuivering. De resulterende concentraties in drinkwater zijn vergeleken met de, op gezondheidskundige overwegingen gebaseerde, maximaal gewenste concentraties. Waar mogelijk werden risico's berekend. Bij bepaalde typen zuiveringen leken de risico's van micro-organismen en desinfectiebijproducten hoger te zijn dan gewenst, zowel bij de bestaande als bij de 'eenvoudige' zuivering. Anorganische microverontreinigingen en bestrijdingsmiddelen komen volgens de berekeningen nergens in ongewenst hoge concentraties voor, zelfs niet bij de 'eenvoudige' zuivering. Geconcludeerd is dat de ketenbenadering goed werkt, zij het dat er (niet altijd kwantificeerbare) onzekerheden verbonden zijn aan de uitkomsten en de koppelingen tussen modellen en databases steeds handmatig gelegd moesten worden.

A novel method is described to study the effect of toxicants on the mineralization of natural and xenobiotic substrates at low concentrations. The method is ecologically relevant because it uses the in situ microflora mineralizing substrates at environmental concentrations and it does not allow unnatural rapid growth. It is shown that this method can be more sensitive than traditional methods which monitor the mineralization of substrates at high concentrations. Compared to the established earthworm toxicity test the method is more sensitive for 2,3,4,5-tetrachlorophenol and pentachlorophenol and less sensitive for 3-chlorophenol. Moreover it is much faster and it can be used for soils, subsoils and aquatic sediments. The sensitivity for toxicants varies widely between different soils and different substrates, which means that a toxicant should have a relatively low concentration to prevent effects on any of the soils and mineralization processes which occur in the environment.

The mineralization of 14-C Acetate was studied in bottles with fresh soil and groundwater. Addition of toxicants inhibited the formation of 14-CO2 and dose effect curves were obtained. The acetate mineralization was not inhibited by zinc, cadmium, K2CR2O7, chloropyrifos, and paraquat in an acid sandy soil at 1000 mg/kg dry soil. The IC10 is the toxicant concentration which inhibits 10% of the initial mineralization rate. The IC10 concentrations for 3,4-dichloroaniline, triphenyltin, and orthoxylene were 48, 96 and 730 mg/kg, respectively in the acid sandy soil. The IC10 of pentachlorophenol was measured in samples from the acid sandy soil and in several other soil and subsoil samples. The geometrical mean of the thirteen IC10 values was 16 mg pentachlorophenol/kg. A statistical method was used to calculate the PCP concentration above which 5% of the most sensitive acetate mineralizing communities in all soils are influenced. The best estimate of this concentration is 0.3 mg PCP/kg but to be on the safe side the 95% confidence level of this concentration is 25 mug/kg.

Een rekenmodel werd ontwikkeld om de verwijdering van micro-organismen door langzame zandfiltratie, een belangrijke zuiveringstap in de drinkwaterbereiding, te voorspellen onder verschillende bedrijfscondities. Deze bedrijfscondities zijn filtratiesnelheid, temperatuur, korrelgrootte en de Schmutzdecke, een slijmlaag op de zandfilters. De condities kunnen varieren afhankelijk van benodigde productiecapaciteit, weersomstandigheden en het drinkwaterbedrijf. Inzicht in de effecten van deze bedrijfscondities is van belang bij de vertaling tussen bedrijven of van literatuurgegevens naar bedrijf en daardoor ook van belang voor de wettelijk verplichte kwantitatieve microbiologische risicoschattingen. Het model werd ontwikkeld op grond van meetgegevens van voorgaand onderzoek op proefinstallatieschaal. De processen die de verwijdering van micro-organismen door langzame zandfiltratie bepalen, zijn hechting en zeefwerking. Ook het effect van de Schmutzdecke werd geevalueerd. De mate van hechting van een micro-organisme aan het zand is locatiespecifiek. Het model werd vervolgens gebruikt om de verwijdering van bacteriofaag MS2, E. coli en Cryptosporidium bij de vier drinkwaterbedrijven voor de voor deze bedrijven relevante bedrijfscondities te voorspellen en op basis daarvan onderzoeksvoorstellen op proefinstallatieschaal te formuleren om het model te valideren.

Regenwater opgevangen in reservoirs en toegepast voor onder andere toiletspoeling is vaak fecaal verontreinigd en bevat soms ziekteverwekkende bacterien. Om het infectierisico bij toepassing van dit water te kunnen schatten is aanvullend onderzoek nodig waarbij ziekteverwekkers worden gekwantificeerd en getypeerd en waarbij onderzocht wordt in welke mate gebruikers worden blootgesteld aan het besmette water.Regenwater is aanvankelijk onbesmet, maar bij afstromen langs oppervlakken en tijdens opslag in reservoirs kan besmetting optreden met micro-organismen die ziekte bij de mens kunnen veroorzaken. Dit kan gebeuren wanneer bijvoorbeeld vogelfeces van het dak wordt gespoeld of ratten of andere dieren toegang hebben tot het reservoir of open leidingen. Onderzoek van opgevangen hemelwater op vier verschillende locaties in Nederland toonde de aanwezigheid van de indicatoren voor fecale verontreiniging, bacterien van de coligroep, E. coli en enterococcen, in respectievelijk 28, 27 en 27 van de 28 onderzochte monsters aan. De potentieel ziekteverwekkende bacterien Campylobacter en Legionella pneumophila werden elk een maal op een locatie aangetroffen. Aeromonas en Clostridium perfringens, die ook ziekte bij de mens kunnen veroorzaken, werden in respectievelijk 20 en 23 van de 28 monsters gevonden. Salmonella en Vibrio werden op geen van de locaties aangetroffen. De aanwezigheid van ziekteverwekkende micro-organismen in regenwater toegepast voor toiletspoeling kan negatieve gevolgen voor de volksgezondheid hebben. Op basis van de verkregen resultaten is het nog niet mogelijk om het risico op het oplopen van een infectie bij blootstelling aan dit water te schatten omdat daarvoor nog aanvullende typerings- en blootstellingsgegevens nodig zijn.

Ter uitvoering van actiepunt NMW1a uit het Strategisch Plan van Aanpak biodiversiteit (SPA; LNV, VROM, V&W, OCW, EZ, BZ, 1995) en als onderdeel van actie N59 uit het NMP-2, is eerder een studie uitgevoerd naar de mogelijkheden om een indicatorsysteem op te stellen voor biodiversiteit van de bodem in relatie tot Life support functies (LSF). In een pilotproject is het indicatorsysteem vervolgens getest binnen de infrastructuur van het Landelijk Meetnet Bodemkwaliteit (LMB). Het indicatorsysteem, en de daaruit af te leiden bodembiologische indicator, bestaat uit een aantal deelindicatoren. De vragen van het onderzoek waren.- kan de gekozen indicatorset worden toegepast in een meetnet; hebben de deelindicatoren van de set voldoende onderscheidend vermogen; kunnen de resultaten worden ingepast in de huidige diagnostische en prognostische gereedschappen van het RIVM (Natuurplanner en EKI).De resultaten en conclusies uit de pilot zijn in dit beleidsgerichte rapport kort beschreven. Een meer gedetailleerde uitwerking van de onderzoeksresultaten verschijnt in het achtergrond-rapport. De pilot heeft aangetoond dat de geselecteerde deelindicatoren gevoelig en onderscheidend zijn voor verschillende combinaties van bodemtype en landgebruik. Als een mogelijke vorm voor het weergeven van de resultaten behaald met de bodembiologische indicator, is een AMOEBE gemaakt voor graslanden op zeeklei. Hierbij is gebruik gemaakt van een gekozen (voorlopige) referentie. De gegevens zijn verder geaggregeerd tot een Bodemkwaliteitsindex (BKX). Op deze wijze zou aan een ecologische bodembeoordeling vorm gegeven kunnen worden. Door systematisch onderzoek aan LSF-deelindicatoren in het Landelijk Meetnet Bodemkwaliteit (40 locaties per jaar) kan in 5 jaar een database opgebouwd worden waarmee responsrelaties voor bodemeigenschappen, systeemeigen- en systeemvreemde stoffen zijn af te leiden. Hiermee kan een LSF-module (prognostisch instrument) worden opgenomen in een decision support systeem als de Natuurplanner

The toxic effects of 5 toxicants on 5 anaerobic microbial processes in fresh water sediments were measured. Four 14-C labelled substrates were mineralized to 14-CO2 at concentrations from 1-4 mug/l in fresh sediment microcosms. The half-lives of the substrates acetate, benzoate and 4-monochlorophenol were 12-30, 24, and 84 min. respectively. The chloroform mineralization was much slower with a half-life of 4.5 - 12 days. The natural methane production in the sediment was also used to measure the toxic effects. A dangerous Concentration 5% (DC5) can be calculated from the toxicity data using methods previously adapted for single animal species toxicity tests. Above this toxicant concentration more than 5% of the possible mineralization reactions can be partly inhibited. The HC5 concentrations for benzene, pentachlorophenol, 1,2-dichloroethane, chloroform and zinc are 1, 0.39, 0.0004, 0.0001 and 270 mg/kg sediment dry weight respectively. The chlorinated alkanes are much more toxic for anaerobic micro-organisms than for aerobic fish.

Propionate, butyrate, acetate, hydrogen and formate are the major intermediates of organic matter degradation. Sulfate-reducing bacteria (SRB) contribute significantly to the consumption of these substrates in sulfate-rich marine sediments. In sulfate-depleted sediments, however, complete degradation of propionate or butyrate is only possible via syntrophic cooperation of acetogenic bacteria and methanogenic archaea. Despite that the predominance of SRB in sulfate-rich and methanogens in sulfate-depleted sediments was reported, recent studies showed that both types of microorganism could be present in upper and lower parts of marine sediments. In this thesis, propionate and butyrate conversions and the involved microbial community in sulfate, sulfate-methane transition and methane zone sediment of Aarhus Bay, Denmark were studied using sediment slurry incubations. Interspecies hydrogen transfer and coexistence during acetate degradation were investigated in mixed pure cultures. In Chapter 2, interspecies hydrogen transfer between aceticlastic Methanosaeta concilii and hydrogenotrophic microorganisms, Desulfovibrio vulgaris or Methanococcus maripaludis, was investigated. Additionally, coexistence of M. concilii and Desulfobacter latus growing on acetate under sulfidogenic conditions was studied. The results of Chapter 2 showed that D. vulgaris could reduce sulfate and grow on leaked hydrogen from M. concilii. Hydrogen leakage from M. concilii provides an explanation for biogeochemical zonation both for competitive (e.g. acetate) and non-competitive substrates (methyl compounds), and this indicates the possible coexistence of SRB and methanogens in sulfate-rich environments. In chapter 3 and 4, long term incubations were examined focusing on butyrate and propionate conversion and the microbial community dynamics in sediment slurry enrichments at different sulfate (o, 3 and 20 mM) concentrations and incubation temperatures (10°C and 25°C). Sulfate reduction is the dominant process for butyrate and propionate conversion in Aarhus Bay sediments. In the absence of sulfate, both substrates can be converted efficiently, indicating the presence of syntrophic communities throughout the sediment. The fluctuating methane concentrations and the enrichment of anaerobic methanotrophic archaea (ANME) during butyrate and propionate conversion at 10°C suggest the occurrence of anaerobic oxidation of methane (AOM) in sulfate-methane transition zone (SMTZ) of Aarhus Bay. The microbial community involved in butyrate and propionate conversions were investigated using next generation sequencing (NGS) of the 16S rRNA amplicon sequencing. The enriched sulfate-reducing bacteria at high sulfate concentration (20 mM) were different when butyrate and propionate were used as substrate. Desulfosarcina and Desulfobacterium dominate the butyrate-converting slurries (Chapter 3), whereas Desulfosarcina, Desulfobulbus and Desulforhopalus are the main SRB in propionate-converting slurries (Chapter 4). The increase in the relative abundance of Desulfobacteraceae and Desulfobulbaceae in SZ, SMTZ and MZ sediment slurries suggests the presence of sulfate reducers throughout the anoxic sediment column. In the absence of sulfate, Syntrophomonas and Cyrptanaerobacter become dominant which suggests their role in syntrophic butyrate and propionate conversion, respectively. These results were further supported in Chapter 6. The increase in the relative abundance of Syntrophomonas in the presence of sulfate (Chapter 3) and some members of Desulfobacteraceae (Chapter 4) in the absence of sulfate shows the metabolic flexibility of the microorganisms at different sulfate concentrations. Temperature has an impact on the microbial community (Chapter 4) and IPL composition (Chapter 5) in enrichment slurries. Cryptanaerobacter is dominant at 25°C, and, Desulfobacteraceae (Desulfofaba), especially Desulfobulbaceae members (Desulfobulbus, Desulforhopalus) become dominant at 10°C at 0 and 3 mM sulfate concentrations in propionate-amended enrichment slurries. In butyrate-amended slurries, Clostridiales have higher relative abundance at 10°C regardless of the sulfate concentration and the sediment depth which supports important role of Clostridiales in butyrate conversion in marine sediments. Archaeal community analyses revealed the dominance of hydrogenotrophic methanogens belonging to Methanomicrobiales in both butyrate- and propionate-converting slurries (Chapter 3 and 4) and enrichment cultures (Chapter 6) regardless of the sediment depth, the incubation temperature and the presence of sulfate, which indicate that they are the main syntrophic partners of butyrate and propionate degraders. The other syntrophic partner organisms are the aceticlastic methanogenic families: Methanosarcinaceae and Methanosaetaeceae. The presence of methane-oxidizing archaea (ANME-1b) in low temperature SMTZ slurries together with Desulfobacteraceae (Chapter 3 and 4) suggests the occurrence of anaerobic oxidation of methane (AOM) in SMTZ of Aarhus Bay. In conclusion, this thesis confirms the presence and activity of methanogens in sulfate-rich, and SRB in sulfate-depleted marine sediments; and their involvement in butyrate, propionate and acetate conversion. Novel bacterial and archaeal members enriched in the sediment slurries are likely involved in propionate, butyrate and acetate conversions at different depths of marine sediments in addition to known the cultured species.

The goals of this thesis are to increase the understanding of microbial metabolism and to functionally (re-)design microbial systems using Genome- Scale Metabolic models (GSMs). GSMs are species-specific knowledge repositories that can be used to predict metabolic activities for wildtype and genetically modified organisms. Chapter 1 describes the assumptions associated with GSMs, the GSM generation process, common GSM analysis methods, and GSM-driven strain design methods. Thereby, chapter 1 provides a background for all other chapters. In this work, there is a focus on the metabolically versatile bacterium Pseudomonas putida (chapters 2,3,4,5,6), but also other model microbes and biotechnologically or societally relevant microbes are considered (chapters 3,4,6,7,8).GSMs are reflections of the genome annotation of the corresponding organism. For P. putida, the genome annotation that GSMs have been built on is more than ten years old. In chapter 2, this genome annotation was updated both on a structural and functional level using state-of-the-art annotation tools. A crucial part of the functional annotation relied on the most comprehensive P. putida GSM to date. This GSM was used to identify knowledge gaps in P. putida metabolism by determining the inconsistencies between its growth predictions and experimental measurements. Inconsistencies were found for 120 compounds that could be degraded by P. putida in vitro but not in silico. These compounds formed the basis for a targeted manual annotation process. Ultimately, suitable degradation pathways were identified for 86/120 as part of the functional reannotation of the P. putida genome.For P. putida there are 3 independently generated GSMs, which is not uncommon for model organisms. These GSMs differ in generation procedure and represent different and complementary subsets of the knowledge on the metabolism of the organism. However, the differing generation procedures also makes it extremely cumbersome to compare their contents, let alone to combine them into a single consensus GSM. Chapter 3 addresses this issue through the introduction of a computational tool for COnsensus Metabolic Model GENeration (COMMGEN). COMMGEN automatically identifies inconsistencies between independently generated GSMs and semi-automatically resolves them. Thereby, it greatly facilitates a detailed comparison of independently generated GSMs as well as the construction of consensus GSMs that more comprehensively describe the knowledge on the modeled organism.GSMs can predict whether or not the corresponding organism and derived mutants can grow in a large variety of different growth conditions. In comparison, experimental data is extremely limited. For example, BIOLOG data describes growth phenotypes for one strain in a few hundred different media, and genome-wide gene essentially data is typically limited to a single growth medium. In chapter 4 GSMs of multiple Pseudomonas species were used to predict growth phenotypes for all possible single-gene-deletion mutants in all possible minimal growth media to determine conditionally and unconditionally essential genes. This simulated data was integrated with genomic data on 432 sequenced Pseudomonas species, which revealed a clear link between the essentiality of a gene function and the persistence of the gene within the Pseudomonas genus.Chapters 5 and 6 describe the use of GSMs to (re-)design microbial systems. P. putida is, despite its acknowledged versatile metabolism, an obligate aerobe. As the oxygen-requirement limits the potential applications of P. putida, there have been several experimental attempts to enable it to grow anaerobically, which have so far not succeeded. Chapter 5 describes an in silico effort to determine why P. putida cannot grow anaerobically using a combination of GSM analyses and comparative genomics. These analyses resulted in a shortlist of several essential and oxygen-dependent processes in P. putida. The identification of these processes has enabled the design of P. putida strains that can grow anaerobically based on the current understanding of P. putida metabolism as represented in GSMs.Efficient microbial CO2 fixation is a requirement for the biobased community, but the natural CO2 fixation pathways are rather inefficient, while the synthetic CO2 fixation pathways have been designed without considering the metabolic context of a target organism. Chapter 6 introduces a computational tool, CO2FIX, that designs species-specific CO2 fixation pathways based on GSMs and biochemical reaction databases. The designed pathways are evaluated for their ATP efficiency, thermodynamic feasibility, and kinetic rates. CO2FIX is applied to eight different organisms, which has led to the identification of both species-specific and general CO2 fixation pathways that have promising features while requiring surprisingly few non-native reactions. Three of these pathways are described in detail.In all previous chapters GSMs of relatively well-understood microbes have been used to gain further insight into their metabolism and to functionally (re-)design them. For complex microbial systems, such as algae (chapter 7) and gut microbial communities (chapter 8), GSMs are similarly useful, but substantially more difficult to create and analyze. Algae are widely considered as potential centerpieces of a biobased economy. Chapter 7 reviews the current challenges in algal genome annotation, modeling and synthetic biology. The gut microbiota is an incredibly complex microbial system that is crucial to our well-being. Chapter 8 reviews the ongoing developments in the modeling of both single gut microbes and gut microbial communities, and discusses how these developments will enable the move from studying correlation to causation, and ultimately the rational steering of gut microbial activity.Chapter 9 discusses how the previous chapters contribute to the research goals of this thesis. In addition, it provides an extensive discussion on current GSM practices, the issues associated therewith, and how these issues can be tackled. In particular, the discussion focuses on issues related to: (i) The inability to distinguish between biological difference and GSM generation artifacts when using multiple GSMs, (ii) The lack of continuous GSM updates, (iii) The mismatch between what GSM predictions and experimental data represent, (iv) The need for standardization in GSM evaluation, and (v) The lack of experimental validation of GSM-driven strain design for metabolic engineering.

Within this project, two new methods of the control of pathogens were investigated. New methods are: a. use of local bacteria that are isolated from soils or growing substrates; and b. bacteria that are present within the plant. By using local antagonists, already present in growing substrates or within plants in the greenhouse, the chance is higher that antagonist can be successfully used against local pathogens. Bacteria that were isolated from soil of growers were assessed on their antagonistic potential in lab trials against Pythium ultimum, Meloidogyne spp. and Rhizobium rhizogenes and Fusarium solani and F. oxysporum. Finally, the effect of antagonists against Pythium and Meloidogyne was evaluated in pot trials in the greenhouse. All antagonists diminished brown colourization symptoms in stems caused by Pythium. Alcaligenues sp., Bacillus sp. en two unidentified species diminished root damage and Alcaligenues sp. as well as Bacillus also reduced also the number of offspring of Meloidogyne spp. within the roots. The use of local microorganisms offers a sustainable-, new solution to control pathogens. In this study, it was shown that Proteinase inhibitor 2 (PINII), Glucanase (LeGluB) and Chitinase (LeChi3) can be used in tomato to investigate the influence of antagonists or endophytes on the plant defence. Binnen dit project worden twee nieuwe methoden van plantversterking tegen ziekten en plagen onderzocht: a. het gebruik van bacteriën die al op het bedrijf aanwezig zijn; en b. bacteriën die in de plant aanwezig zijn. Door het gebruik van bacteriën uit de plant of teelt substraat is de kans groter dat antagonisten succesvol ingezet kunnen worden. Bacteriën werden in vitro getoetst tegen Pythium ultimum uit chrysant, tegen het wortelknobbelaaltje Meloidogyne incognita en tegen de schadelijke bacterie Rhizobium rhizogenes uit tomaat en tegen Fusarium solani en F. oxysporum uit Amaryllis. Vervolgens werden antagonisten van Pythium en Meloidogyne getoetst in potproeven met grond afkomstig van de glastuinbouw bedrijven. Alle isolaten verminderde de bruin-verkleuring symptomen van Pythium in de stengel van chrysant. Alcaligenues sp., Bacillus sp. en twee niet-geïdentificeerde soorten gaven een onderdrukking van de wortelschade van Meloidogyne spp. terwijl Alcaligenues sp. en Bacillus sp. ook een vermindering gaf van het aantal nakomelingen. De inzet van lokaal micro-leven tegen ziekten en plagen biedt een duurzame-, en nieuwe oplossing voor de verkleining van het beschikbare middelenpakket. Uit de studie blijkt dat Proteïnase remmer 2 (PINII), Glucanase (LeGluB) en Chitinase (LeChi3) gebruikt kunnen worden als merkers in tomaat voor onderzoek naar het effect van antagonisten en endofieten op de plantafweer.

Micro-organismen spelen een sleutelrol in bodemfuncties zoals de kringlopen van koolstof en stikstof. Voor een duurzame landbouw is het van belang dat deze kringlopen optimaal functioneren om verliezen van nutriënten zoveel mogelijk te voorkomen. Dit onderzoek geeft inzicht in de rol van bodemmicroben bij optimalisatie van de stikstofkringloop door toevoeging van zowel minerale kunstmest als verschillende kwaliteiten organisch materiaal.

Undefined mixed complex starter cultures are broadly used in Gouda-type cheese production due to their robustness to phage predation, resilience for changes in environmental conditions and aroma compounds production ability during ripening. These microbial communities of lactic acid bacteria prior their isolation and deposition in starter culture collections were continuously used at the farm-level production facilities. Thus, one can consider undefined mixed complex starters as domesticated microbial communities. The process of domestication was facilitated by humans who have been continuously repeating successful fermentations using part of previous batch as inoculum (i.e. back-slopping). Therefore, a term ‘community breeding’ can describe this human-driven domestication of microbial communities. Community breeding of a model complex starter Ur led to establishment of a simple two-species composition of Lactococcus lactis and Leuconostoc mesenteroides represented by, in total, 8 genetic lineages. At the same time, this simple microbial community displays a high degree of intraspecies diversity, presumably caused by evolutionary processes of horizontal gene transfer, genome decay and mutations. Such diversity at strain level is particularly interesting in the context of continuous bacteriophage predation pressure present in this microbial community. It is thought that constant-diversity (CD) dynamics, based on the ‘kill-the-winner’ principles, is operational in Ur starter at the strain level. According to CD model, the fittest strain(s), which feed on the most abundant substrate, will be selected against due to density-dependent phage predation. The control of the fittest strain abundance by bacteriophages opens space for differentiation of strains via eco-evolutionary feedbacks. In particular, strains of complex starter culture not only adapted to quickly acidify milk (via efficient consumption of lactose and protein to peptides degradation), but concurrently, to consume other substrates present in milk. In addition, throughout the process of community breeding microbe-microbe interactions between community members have evolved. These interactions have led to division of metabolic labor among strains present in the culture, and eventually to better starter microbial community functioning. The aim of this thesis was to investigate the factors impacting the formation of compositionally and functionally stable undefined mixed complex starter cultures to further use this knowledge in steering its functionality, and potentially in developing new strategies for robust starter culture design. To facilitate this study, well-characterized Ur culture strain isolates were used to systematically reconstitute the starter culture into multi-strain blends with increasing level of strain and genetic lineage diversity. The investigation of factors such as phage predation, level of strain and genetic lineage diversity as well as environmental conditions, was performed during experimental evolution studies in milk. The functionality of the (evolved) starter cultures was tested in an adapted lab-scale MicroCheese model system. The specific approach used in each of the research chapters is described below in more detail. Strains isolated from Ur starter culture were characterized in terms of their resistance against bacteriophages isolated from the same starter (Chapter 2). This test confirmed high diversity in phage resistance among strains belonging to different genetic lineages as well as among strains of the same lineage. Next, selected strains, which represented different levels of bacteriophage predation: resistant, moderately resistant, sensitive and no detectable sensitivity, were mixed in simple blends containing 4 strains representing 3 genetic lineages of Ur starter (3 such blends were designed). These blends were exposed to phage predation (one phage per blend) at the onset of prolonged sequential propagation experiment or propagated without phage addition (control). Throughout the serial propagation the genetic lineage composition was monitored. During the propagation of control blends we detected quick domination of a single lineage. This dominating lineage contained strains sensitive to phages. Genetic lineage level composition of the phage-challenged blends was much more dynamic suggesting the impact of phage predation. The relatively low strain diversity introduced in these blends was not high enough to sustain maximal diversity at the level of lineages. Chapter 3 describes a study using defined blends with higher complexity by extending the number of strains used. In total, 24 strains representing all 8 Ur starter lineages were exposed in sequential propagation experiment to a cocktail of 3 phages isolated from Ur starter. The propagation in milk of this multi-strain blend was executed for more than 500 generations and the abundance of genetic lineages was monitored throughout. Similarly as in the simple blends experiment, control blends were not exposed to bacteriophages. In control blends we observed a domination of one genetic lineage upon serial propagation, which resembles a periodic-selection-like (PS) behavior, where the fittest strains are dominating the microbial community and in result genetic-lineage diversity is being substantially reduced. In contrast, the composition of phage-challenged blends was again more dynamic than in control blends. In one of the phage-challenged blends behavior characteristic for a constant-diversity dynamics model was observed; throughout the serial transfer experiment, genetic lineage diversity was maintained by the presence of phage predation at relatively high level. In case of the second phage-challenged blend, due to a stochastic event, which likely caused a reduction in phage pressure, we observed a gradual recovery of the fittest strains, which again resembled a periodic-selection behavior. Therefore, phage predation, among other factors, can lead to shifts in microbial community population dynamics resulting in alternative stable states. The experimental evolution approach, resembling traditional process of back-slopping, was used in a Long-term experimental evolution of Undefined Mixed Starter Culture (LUMSC) study described in Chapter 4. The aim of this study was to investigate the compositional and functional stability ascribed to the undefined mixed Ur starter during enclosed prolonged propagation without any possible external influx of bacterial or phage material. Surprisingly, during this 1000-generation long experiment the enforced conditions of specific incubation temperature and propagation regime resulted in enrichment of previously not detected strain of Lactococcus laudensis. This strain was found to consume a by-product of metabolism of another strain present in the community, in particular, D-mannitol produced by Le. mesenteroides. Thus, a new putative interaction in the microbial community of the complex starter culture was found. This new interaction and the possible ability of L. laudensis to efficiently use peptides released by caseinolytic L. lactis ssp. cremoris resulted in a relatively high abundance of L. laudensis in all evolved LUMSC cultures. The high abundance of L. laudensis had a certain effect on the functionality of the cultures. The aroma profiles of model lab-scale milli-cheeses manufactured with LUMSC cultures, showed significant differences in formation of esters and alcohols when compared to cheeses produced with the original Ur starter. Moreover, L. laudensis strain was not only under the radar of previously used culture-dependent and culture-independent methods, but as well, under the radar of phage predation continuously present throughout the LUMSC experiment. This observation sheds new light on the possibility of how a strain can emerge to relatively high abundance in an enclosed serially propagated microbial community operating in accordance with CD dynamics model. Finally, the aspect of adaptation to environmental conditions was addressed by the study of an adjunct strain of Lactobacillus helveticus DSM 20075 described in Chapter 5. The aim was to develop a strain with increased autolytic capacity in conditions resembling the cheese matrix to possibly improve cheese ripening. The approach used here was based on a previously reported study, where the incubation of Lactococcus lactis MG1363 at high temperature resulted in spontaneous mutations causing stable heat-resistant and, in some cases, salt-hypersensitive phenotypes. In present study, after incubation of the Lb. helveticus DSM 20075 adjunct at different high temperatures (45-50 °C), heat-sensitive variants were recovered from plates. These variants were further characterized in terms of their growth rates at elevated temperatures (42-45 °C) and their autolytic capacity in low pH buffer with addition of NaCl. One of the variants (V50) showed substantially increased intracellular lactate dehydrogenase enzyme activity in the buffer suggesting its increased autolytic capacity. Next, both wild type and variant V50 were tested as adjuncts in lab-scale model milli-cheeses to determine their possible impact on the cheese aroma profiles. Indeed, adjunct strains, both WT and the variant, impacted the aroma profiles by producing benzaldehyde. In case of the variant strain the relative abundance of this compound was 3-fold higher. The applied strategy of incubating Lb. helveticus DSM20075 at high temperature resulted in specific, but different than in case of L. lactis MG1363, mutations suggesting another, yet to be elucidated, mechanisms for increasing the autolytic capacity of industrially-relevant strains. The approach of high-temperature incubation can be applied in dairy industry for the selection of (adjunct) cultures targeted at accelerated cheese ripening and aroma formation. In conclusion, the work presented in this thesis highlights the importance of co-evolution of strains in compositional and functional stability of the complex undefined mixed starter culture. In particular, the factors such as heterogeneity of bacteriophage resistance among highly related strains, microbe-microbe interactions and division of metabolic labor are crucial for optimal functioning of a complex starter microbial community. Further investigation of the factors impacting the composition of starter cultures is crucial to steer the functionality in a desired direction. With straightforward methods, such as changing the incubation temperature or the propagation regime it is possible to induce shifts in strain composition and thereby obtain cultures with new characteristics. Moreover, experimental evolution studies with microbial communities used in food fermentation can lead to the discovery of new strains with potentially new characteristics. Additionally, the study of microbial communities of starter cultures not only delivers industrially applicable knowledge but also reveals the action of basic principles in microbial ecology and evolution.

The increasing prevalence of antibiotic resistance in pathogenic bacteria and the potential future implications for human and animal morbidity and mortality, health-care costs and economic losses pose an urgent worldwide problem. As a result, exploration of alternative strategies to combat antibiotic resistant bacteria have intensified over the last decades. The work described in this thesis focused on the study of naturally occurring antimicrobial peptides (AMPs) and other bioactive molecules produced by bacteria as potential alternatives to prevent or treat infections with pathogenic bacteria. A large part of the thesis aimed to increase knowledge about the role of the microbiota (the collection of microbes present at a certain location of the body) of the oral cavity or small intestine in the abundance of Streptococcus suis, a pathogenic bacteria that mostly causes disease in young pigs. We identified commensal bacteria that displayed strong and selective antagonism against this S. suis. Several bacteria that showed strong growth inhibition of S. suis in the lab through the production of AMPs were isolated and characterised. This thesis increased the understanding of the role of host- and microbiota-derived biologically active small molecules in microbe-microbe and microbe-host interplay. Such knowledge may contribute to the development of novel therapeutic solutions to treat antibiotic resistant bacteria, such as beneficial microbial communities (i.e. next-generation probiotics) or biotechnological applications of natural or modified AMPs.

Per vierkante meter bodem leven honderden wormen en insecten samen met kilometers aan schimmeldraden, vele miljoenen aaltjes en miljarden bacteriën. Onderzoek maakt steeds meer duidelijk van het precaire evenwicht ondergronds, en de grote invloed daarvan op het leven bovengronds. Het levert nieuwe strategieën op voor gewasbescherming.

Fermented products have high nutritional value and constitute an important part of the Chinese dietary profile; they are also gaining popularity throughout the world. Daqu is a traditional natural fermentation starter culture that has a significant impact on the quality and flavour of Chinese liquor and vinegar. A review of the literature was conducted focusing on the classification, composition, and manufacture of Daqu. The review provided a preliminary understanding of the link between the fermentation process and the characteristics of the final Daqu product. Then the occurrence, levels, and diversity of microorganisms were studied in different types of Daqu produced by various fermentation processes. The results showed that Bacillus licheniformis and Saccharomycopsis fibuligera were present in all the tested samples of Daqu. Regional comparisons showed Staphylococcus gallinarum and Staphylococcus saprophyticus in southern Daqu. The fungi Sm. fibuligera and Lichtheimia ramosa were found in low/medium-temperature Daqu and Thermomyces lanuginosus occurred in high-temperature Daqu. In order to study the functionality of Daqu and the contribution of the predominant microorganisms to alcoholic fermentation, the mesophilic and thermophilic bacteria and spores, Enterobacteriaceae, lactic acid bacteria, yeasts, and moulds present in the core and outer portions of Fen-Daqu were isolated. The isolates were identified by culture-dependent sequencing of rRNA genes (16S rRNA for bacteria; 18S rRNA, 26S rRNA, and ITS rRNA for fungi). A succession of fungi, lactic acid, and Bacillus spp. was associated with prevailing acidity, moisture content, and temperature during Daqu fermentation. The predominant species in fermentation were B. licheniformis, Pediococcus pentosaceus, Lactobacillus plantarum, Pichia kudriavzevii, Wickerhamomyces anomalus, Sacchromyces cerevisiae, and Sm. fibuligera. One strain of the each of the above-mentioned predominant species, with the highest starch degrading ability and alcohol tolerance, was selected and used in different combinations to perform alcoholic fermentation. Metabolite composition differed significantly between various fermentation trials. S. cerevisiae provided superior ethanol production. Sm. fibuligera and B. licheniformis provided the amylolytic activity that converted starch and polysaccharides into fermentable sugars. Finally, W. anomalus was found to be an important contributor to formation of the liquor aroma. Understanding the microbial diversity and functional activity, as well as the production dynamics and safety of Daqu will enable commercial producers to improve and/or scale-up traditional processes and enhance product quality and safety, thus facilitating entry into international markets.

Summary Climate change is generally recognized as a major threat to humans and the environment. With respect to food production, climate change does not only affect crop production or food security, but possibly also effects on food safety by affecting the prevalence and levels of bacteria, fungi or other pests and pesticides. Fresh-cut or ready-to-eat leafy vegetables (e.g. lettuce and spinach) are increasingly consumed because they are promoted as part of a healthy diet. Such leafy green vegetables (LGVs) are identified as the fresh produce commodity group of highest concern from a microbiological safety perspective, because they are often grown in the open field and therefore vulnerable to contamination and contact with (faeces of) wildlife. Moreover, they are grown and consumed in large volumes and often consumed raw. Bacteria, such as Salmonella spp. and pathogenic Escherichia coli strains are the main pathogens causing foodborne disease through LGVs. A major knowledge gap is understanding how climate change may directly or indirectly affect the contamination of LGVs. This primarily relates to the current lack of methods and tools to link climate data and climate change scenarios to food safety. My thesis aims to quantify the impacts of climate change on microbial safety of pre-harvested LGVs. To achieve this, I reviewed the literature and synthesised major impacts of climate change on contamination sources and pathways of foodborne pathogens (focussing on Escherichia coli O157 and Salmonella spp.) on pre-harvested LGVs (Chapter 2). Subsequently, I developed a statistical model that identifies the weather and management variables that are associated with the LGVs contamination with generic E. coli using regression analysis (Chapter 3). To apply suitable climate data to this statistical model to assess future impacts, I have prepared a tool to downscale coarse climate and climate change data for local food safety scenario analysis (Chapter 4). Finally, I applied the downscaled data to the statistical model and used multi-criteria scenario analysis to explore future food safety (Chapter 5). E.coli is used as a hygienic indicator in this thesis to study microbial safety of LGVs. Its presence is indicative for an increased pathogen presence probability. E. coli and many foodborne bacteria share the same contamination pathways and climate change is expected to similarly impact on both bacteria. Hygienic status is therefore used in my thesis as a proxy for the microbial safety of LGVs. The major result of the literature review in Chapter 2 is that the impact of climate change on LGV contamination depends on the resulting local balance of the positive and negative impacts. The review shows that the interactions between climate change and contamination are real but poorly understood. Therefore, integrative quantitative modelling approaches with scenario analyses and additional laboratory experiments are needed. With this knowledge background, mixed effect logistic regression and linear regression models were developed to identify the climate and management variables that are associated with the presence and concentration of E. coli on LGVs (Chapter 3). These models used E. coli data of 562 lettuce and spinach samples taken between 2011 and 2013 from 23 open-field farms from Belgium, Brazil, Egypt, Norway and Spain. Weather and agriculture management practices together had a systematic influence on E.coli presence and concentration. Temperature explained most of the observed variation on E. coli prevalence and concentration on LGVs. Minimum temperature of the sampling day (odds ratio [OR] 1.47), region and application of inorganic fertilizer explained a significant amount of variation in E. coli prevalence. Maximum temperature on three days before sampling and region best explained the variation in E. coli concentration (R2= 0.75). Region is a variable masking many management variables including use of rain water, surface water, manure, inorganic fertilizer and spray irrigation. Climate variables and E. coli presence and concentration are positively related. The results indicate that climate change will have an impact on microbiological safety of LGVs. These impacts can be directly through an increasing temperature, but also indirectly through changes in irrigation water type, fertilizer type and irrigation method. Therefore, climate change and farm management should be considered more systematically in an integrated way in future studies on fresh produce safety. To prepare climate data for local food safety scenario analysis, a climate data downscaling tool was presented and demonstrated (Chapter 4). Coarse gridded data from two general circulation models, HadGEM2-ES and CCSM4, were selected and downscaled using the ‘Delta method’ with quantile-quantile correction for the Belgium meteorological station in Ukkel. Observational daily temperature and precipitation data from 1981 to 2000 were used as a reference period for this downscaling. Data were provided for four future representative concentration pathways (RCPs) for the periods 2031–2050 and 2081–2100. These RCPs are radiative forcing scenarios for which future climate conditions are projected. The climate projections for these RCPs show that both temperature and precipitation will increase towards the end of the century in Ukkel. The climate change data were subsequently used with Ratkowsky's bacterial growth model to illustrate how projected climate data can be used for projecting bacterial growth in the future. In this example, the future growth rate of Lactobacillus plantarum and the number of days that the bacteria are able to grow are both projected to increase in Ukkel. This example illustrates that this downscaling method can be applied to assess future food safety. This downscaling tool is relatively straightforward compared to other more complex downscaling tools, so the food safety researchers can easily understand and apply it to their impact studies. With the statistical model (Chapter 3) and downscaled climate data (Chapter 4), a multi-criteria scenario analysis tool was developed to explore future food safety using pre-harvest spinach in Spain as an example (Chapter 5). The future E. coli concentrations on spinach were projected to change in RCP 8.5 and RCP 2.6 by the end of the century in Spain. The E. coli concentration was projected to increase between 0.2 log10 CFU/g and 0.3 log10 CFU/g (depending on the climate scenarios and management options applied) due to higher temperature by the end of the century compared to the concentrations by the end of the last century. This comparison assumed no changes in agricultural management practices. This tool can be used to help selecting the best management practices considering climate change and other indicators. The pioneering research presented in my thesis brought new methods and tools, and another mind set to food safety research. The climate-change data downscaling tool provides detailed temporal and spatial climate data for climate scenario analysis in food safety assessment studies. The multi-criteria scenario analysis tool provides a platform to study changes in weather or climate, and management impacts on future food safety. This tool also allows for inclusion of different stakeholders’ perspectives or interests and supports their decision making processes. Moreover, the thesis presents a statistical model that can be used to study the relationship between climate and E. coli contamination. My thesis quantified the impacts of climate change on microbial safety of pre-harvested LGVs contaminated with generic E. coli for the first time. With one degree increase in minimum temperature of the sampling day, the odds of having E. coli presence on LGVs increase by a factor of 1.5. The mean E. coli concentrations are also expected to increase. Climate change should not be ignored in food safety management and research.

Microorganisms capable of performing anaerobic oxidation of methane (AOM) coupled to sulphate reduction have high doubling times which make their enrichment difficult. However, due to higher energy gain, they might be rapidly enriched using alternative electron acceptors. In chapter 2, it was shown that up to 50 times higher conversion rates were obtained with thiosulphate when compared to sulphate. However, it was also presented that thiosulphate was not be exclusively used by microorganisms that reduce it, but that it was also disproportionated into sulphate and sulphide (Chapter 2). In Chapter 3, a 5 litre membrane bioreactor was fed not only with methane and sulphate but also with acetate and thiosulphate. As previous experiments using these additional substrates had allowed to obtain either faster conversion rates or enrichment of methanotrophic microorganisms; it was expected that AOM rates in the reactor would increase relatively fast. However, the microorganisms that were enriched were not AOM related. They microbial community that showed the highest activity rates in the reactor was comprised by thiosulphate disproportionated bacteria and green sulphur bacteria. The former disproportionated thiosulphate into sulphate and sulphide while the latter converted the sulphide into elemental sulphur. Chapter 4, unlike the previous chapters, focused on studying the occurrence of AOM in a fresh water ecosystem. Such system was located next to a natural gas source which captured methane for domestic purposes. It was presented how, with the different electron acceptors added, AOM and trace methane oxidation occurred. However, net AOM was only measured in the presence of sulphate as electron acceptor. Furthermore, the microorganisms that were enriched in the presence of methane and sulphate were also detected. There are several hypotheses which attempted to explain the AOM coupled to sulphate reduction. One of them indicates that it is a process that involves two microorganisms working in a syntrophic relationship. The first microorganism would convert the methane into carbon dioxide and pass the electrons to the second one. Consequently, the second microorganism would convert the sulphate into hydrogen sulphide. In such a structure, the way that electrons are released by the conversion performed by the first microorganism is unknown. It is possible, that electrons are not transfer via electron shuttles or chemical compounds, but that they are transferred directly from one microorganism to the other. A methodology which could be used to determine if the AOM consortia uses direct electron transfer mechanisms was evaluated in Chapter 5. Different kinds of granular biomass were used for this evaluation and, the granule types that would potentially be capable of using direct electron transfer mechanisms were detected.

This PhD study has evaluated hydrochars derived from biowastes as adsorbents for pathogen removal in water treatment. Pathogen removal experiments were conducted by carrying out breakthrough analysis using a simple sand filtration set-up. Glass columns packed by 10 cm sand bed supplemented with minor amount of hydrochar (1.5 %, w/w) were flushed with artificial ground water seeded with test microorganisms at an upward flow rate of 1 mL / min. Either back flushing or deionized water flushing was performed at pathogen retaining columns in order to investigate the pathogen removal mechanism of hydrochar-amended sand columns.Two home-made two-step reverse transcription-quantitative polymerase chain reaction assays were developed in order to quantify rotavirus in the samples from virus removal experiments. Since the total cost of the assays was mainly determined by the cost of reverse transcriptase, two reverse transcriptases with the lowest consumer price were employed. The efficiencies of home-made assays were comparable to a selected reference commercial kit in analyzing both environmental and laboratory samples, while the total cost of home-made assays was 11 times less. Though home-made assays necessitate more manual operations and time, the low-cost aspect might be appealing in those settings where the expenditure for consumables inhibits laboratories in their functioning.Hydrochars produced via hydrothermal carbonization of agricultural residue from maize or stabilized sewage sludge were evaluated for adsorptive removal of Escherichia coli. In order to improve the removal capacity, the hydrochars were activated by being suspended in 1M KOH solution (5 g hydrochar / L) for 1 h at room temperature. The activation improved the Escherichia coli removal efficiency from ~70 to ~90 %. In addition, successive detachment experiments carried out by performing back flushing or deionized water flushing into sand columns supplemented with maize-derived hydrochar indicated that the strength of Escherichia coli attachment increased by KOH activation. Also, Escherichia coli removal of sewage sludge-derived hydrochar was evaluated in larger column with 50 cm filter bed for 30 days of intermittent operation. 3 pairs of columns packed with either sand, raw hydrochar or activated hydrochar showed average removal efficiency of 36.5% ± 10.1 (n=60), 24.4% ± 10.5 (n=56) and 91.2% ± 7.5 (n=60), respectively. Idle time of filtration unit did not affect the Escherichia coli removal efficiency of hydrochar-amended columns. The results from material characterization attributed the enhancement in Escherichia coli removal induced from the KOH activation to development of macroporous surface with increased hydrophobicity and surface charge. It was apparent that the activation with the KOH solution removed tar-like substances from hydrochar surface, resulting in exposure of hydrophobic core and development of rough surface structure. Also, deposition of K+ ion in hydrochar was observed, which might have increased the surface charge.The removal of human pathogenic rotavirus and adenovirus was investigated using hydrochar derived from stabilized sewage sludge or swine faecal waste. Throughout virus removal experiments, rotavirus and adenovirus showed comparable removal. At 1 mL / min flow rate, raw hydrochar (without KOH activation) derived from either feedstock showed mean virus removal efficiency from 2 to 3 > log removal (99 - 100 %). Also flow rates of 2.5 and 5 mL / min were tested using faecal waste-derived hydrochar. The virus removal efficiency remained still high (2.1 log - 3 log) at the elevated flow rates. We speculated that the improvement in virus removal derived from hydrochar supplement is induced by provision of extra hydrophobic surfaces in sand column media. Regardless the type of packing material, successive deionized water flushing into virus-retaining columns released more rotavirus than adenovirus, indicating larger role of the secondary energy minimum in rotavirus retention. It was remarkable, because both types of viruses are similar in their shape and size. This observation provides evidence that virus transport-retention behaviour could be mainly determined by surface characteristics of virus rather than its size and shape. In this sense, the use of model virus needs to be carefully considered when performing water treatment or pathogen transport experiments.Successful removal of pathogenic virus using faecal waste-derived hydrochar highlights the potential of hydrothermal carbonization technology in less developed regions where modern water-sanitation systems are not affordable. Faecal waste, one of the most important pathogen sources, can be completely sanitized in elevated temperature and pressure during hydrothermal carbonization process. Moreover, the resulting hydrochar can be utilized at water or wastewater treatment. Despite general low-cost aspect of hydrothermal carbonization such as less energy dependency than dry pyrolysis and utilization of waste as a feedstock, the need for high-pressure reactor might hamper the implementation of the technology. Development of localized low-cost reactor, evaluation of hydrochar in its use at agriculture and/or energy production and overall economic analysis are recommended.&nbsp

Microorganisms capable of performing anaerobic oxidation of methane (AOM) coupled to sulphate reduction have high doubling times which make their enrichment difficult. However, due to higher energy gain, they might be rapidly enriched using alternative electron acceptors. In chapter 2, it was shown that up to 50 times higher conversion rates were obtained with thiosulphate when compared to sulphate. However, it was also presented that thiosulphate was not be exclusively used by microorganisms that reduce it, but that it was also disproportionated into sulphate and sulphide (Chapter 2). In Chapter 3, a 5 litre membrane bioreactor was fed not only with methane and sulphate but also with acetate and thiosulphate. As previous experiments using these additional substrates had allowed to obtain either faster conversion rates or enrichment of methanotrophic microorganisms; it was expected that AOM rates in the reactor would increase relatively fast. However, the microorganisms that were enriched were not AOM related. They microbial community that showed the highest activity rates in the reactor was comprised by thiosulphate disproportionated bacteria and green sulphur bacteria. The former disproportionated thiosulphate into sulphate and sulphide while the latter converted the sulphide into elemental sulphur. Chapter 4, unlike the previous chapters, focused on studying the occurrence of AOM in a fresh water ecosystem. Such system was located next to a natural gas source which captured methane for domestic purposes. It was presented how, with the different electron acceptors added, AOM and trace methane oxidation occurred. However, net AOM was only measured in the presence of sulphate as electron acceptor. Furthermore, the microorganisms that were enriched in the presence of methane and sulphate were also detected. There are several hypotheses which attempted to explain the AOM coupled to sulphate reduction. One of them indicates that it is a process that involves two microorganisms working in a syntrophic relationship. The first microorganism would convert the methane into carbon dioxide and pass the electrons to the second one. Consequently, the second microorganism would convert the sulphate into hydrogen sulphide. In such a structure, the way that electrons are released by the conversion performed by the first microorganism is unknown. It is possible, that electrons are not transfer via electron shuttles or chemical compounds, but that they are transferred directly from one microorganism to the other. A methodology which could be used to determine if the AOM consortia uses direct electron transfer mechanisms was evaluated in Chapter 5. Different kinds of granular biomass were used for this evaluation and, the granule types that would potentially be capable of using direct electron transfer mechanisms were detected.

Als gevolg van verplichte verwerking van een deel van de mestoverschotten zal de komende jaren het transport en het gebruik van producten uit mestverwerking toenemen. Dit brengt mogelijk hygiënische risico’s voor mensen en dieren met zich mee door verspreiding van ziektekiemen, o.a. via lozing op het oppervlaktewater. In opdracht van de Provincie Noord-Brabant en waterschap Aa en Maas is een onderzoek uitgevoerd met het doel inzicht te krijgen in het effect van de verschillende processtappen van mestverwerking op het overleven van ziekteverwekkende bacteriën en virussen en in de mogelijke hygiënische risico’s gerelateerd aan distributie en het gebruik van de eindproducten van mestverwerking. Hiervoor werden in de processtromen van praktijkinstallaties de concentraties gemeten van een aantal ziekteverwekkende micro-organismen en mogelijke indicatoren daarvoor. Het betrof de volgende bacteriën en virussen: Escherichia coli, enterococcen, Salmonella, Clostridium difficile, MRSA, ESBLE.coli, somatische colifagen en Hepatitis E virus. Uit de resultaten komt naar voren dat bij mechanische scheiding de micro-organismen zich sterk in de vaste fractie concentreren en dat mineralenconcentraat uit omgekeerde osmose iets lagere concentraties aan micro-organismen bevat dan de drijfmest waaruit het geproduceerd wordt. Door vergisting lijkt de concentratie aan micro-organismen af te nemen. Hygiënisering door middel van compostering en door verhitting resulteert in vrijwel steriele producten. Hierbij moet worden opgemerkt dat beide technieken slechts in twee monsters, afkomstig van één installatie, zijn onderzocht. Het permeaat (effluent) na omgekeerde osmose is microbiologisch nagenoeg schoon; lozing van dit product op het oppervlaktewater vormt daarom naar verwachting geen gezondheidsrisico. Het onderzoek heeft inzicht verschaft in het effect van processtappen op de concentratie van microorganismen in de producten uit mestverwerking. De risico’s van de doses microbiële verontreinigingen die via bemesting met deze producten, m.n. vaste fractie en mineralenconcentraat, worden toegediend moet verder worden onderzocht.